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EP1513522A2 - Methods of treating conditions associated with an edg receptor - Google Patents

Methods of treating conditions associated with an edg receptor

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Publication number
EP1513522A2
EP1513522A2 EP03710713A EP03710713A EP1513522A2 EP 1513522 A2 EP1513522 A2 EP 1513522A2 EP 03710713 A EP03710713 A EP 03710713A EP 03710713 A EP03710713 A EP 03710713A EP 1513522 A2 EP1513522 A2 EP 1513522A2
Authority
EP
European Patent Office
Prior art keywords
alkyl
substituted
heteroaryl
edg
cιo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03710713A
Other languages
German (de)
French (fr)
Inventor
Geetha Shankar
David Solow-Cordero
Juliet V. Spencer
Charles Gluchowski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SRI International Inc
Original Assignee
SRI International Inc
Stanford Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SRI International Inc, Stanford Research Institute filed Critical SRI International Inc
Publication of EP1513522A2 publication Critical patent/EP1513522A2/en
Withdrawn legal-status Critical Current

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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
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    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
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    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/4164—1,3-Diazoles
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    • A61K31/425—Thiazoles
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/428—Thiazoles condensed with carbocyclic rings
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
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Definitions

  • the present invention relates generally to methods of modulating biological activity mediated by an Edg-2, an Edg-3, an Edg-4 or an Edg-7 receptor. More specifically, the present invention provides compounds and compositions, which may be used to selectively modulate, e.g., antagonize an Edg-2, an Edg-3, an Edg-4 or an Edg-7. The present invention also provides methods for making these compounds.
  • Lysophospholipids include compounds such as lysophosphatidic acid ("LPA”), sphingosine- 1-phosphate (“SIP”), lysophosphatidylcholine and sphingosylphosphorylcholine and are important second messengers that can activate particular cell surface transmembrane G-protein coupled receptors known as endothelial gene differentiation (“Edg”) receptors.
  • LPA lysophosphatidic acid
  • SIP sphingosine- 1-phosphate
  • Edg endothelial gene differentiation
  • S1P1 (Edg 1), S1P3 (Edg 3), S1P2 (Edg 5), and S1P5 (Edg 8) belong to one structural cluster and LPA1 (Edg 2), LPA2 (Edg 4) and LPA3 (Edg 7) are members of a second structural cluster (Goetzl, B. J., and Lynch, K. R. 2000, Ann. N. Y. Acad. Sci. 905:1-357).
  • Edg-1 human Edg-1, GenBank Accession No. AF233365
  • Edg-3 human Edg-3, GenBank Accession No. X83864
  • Edg-5 human Edg-5, GenBank Accession No. AF034780
  • Edg-6 human Edg-6, GenBank Accession No. AJ000479
  • Edg- 8 human Edg-8, GenBank Accession No. AF3 17676 receptors are activated by SIP, while LPA activates Edg-2 (human Edg-2, GenBank Accession No., U78 192), Edg-4 (human Edg-4, GenBank Accession Nos. AF233092 or AFO1 1466) and Edg-7 (human Edg-7, GenBank Accession No.
  • the present invention addresses these and other needs by providing compounds that modulate the Edg-4 (LPA2) receptor (e.g, human Edg-4, GenBank Accession Nos. AF233092 or AFO1 1466). Such compounds preferably selectively bind or otherwise modulate the Edg-4 receptor.
  • LPA2 Edg-4
  • the present invention provides methods for modulating (antagonizing or agonizing) Edg-4 receptor mediated biological activity.
  • the present invention also provides methods for using Edg-4 modulators (antagonists or agonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("AERDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous bums) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention.
  • diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer
  • acute lung diseases such as asthma, surface epithelial cell
  • the present invention provides compounds and compositions that can, for example, be used in modulating Edg-4 receptor mediated biological activity or treating or preventing diseases such as those mentioned above.
  • the present invention still further provides methods for synthesizing the compounds.
  • the present invention provides a method of modulating an Edg-4 receptor mediated biological activity in a cell. A cell expressing the Edg-4 receptor is contacted with an amount of an Edg-4 receptor modulator sufficient to modulate the Edg-4 receptor mediated biological activity.
  • the present invention provides a method for modulating Edg-4 receptor mediated biological activity in a subject.
  • an amount of of the Edg-2 receptor effective to modulate the Edg-2 receptor mediated biological activity is administered to the subject.
  • the present invention also provides compounds (agonists or antagonists) that modulate Edg-4 receptor mediated biological activity.
  • the agonists or antagonists are compounds of structural formula (I) and can be utilized as part of the methods of the present invention:
  • R] is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted dialkylamino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino;
  • A is NR 2 , O or S
  • R is hydrogen, alkyl or substituted alkyl
  • B and C are independently alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl.
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (IV):
  • each of Ri, R 2 , R , R or R 5 is independently -H, -halo, -NO , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -(CH 2 ) m N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 ,
  • each R 5 and Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(Ci-C, 0 )alkyl(Ci-Cio)alkyl, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(C 1 -C 10 )alkyl,
  • C 10 )alkyl ), -CO 2 (C,-C 10 )alkyl, -(C,-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl,
  • X and Y are each independently C or N; and Z is O, S, C or N, wherein if Z is O or S, then R is an electron pair;
  • R t and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 2 and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R 3 and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • the modulator is a compound of structural formula
  • each of Ri, R 2 , R 3 , R 4 or R 5 is independently -H, -halo, - ⁇ O 2 , -CN, -OH,
  • each R( is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • the agonists or antagonists are compounds of structural formula (VI):
  • each of Ri, R 2 , R 3 , R4 or R 5 is independently -H, -halo, -NO 2 , -CN, -OH,
  • R 5 or R ⁇ is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C 1 -C 10 )alkyl(C,-C 10 )alkyl, -O(d-C 10 )alkyl, -C(O)(d-C 10 )alkyl,
  • X, Y and Z are independently O, S, C or N, wherein if X, Y or Z is O or S, R ⁇ is an electron pair; Ri and R 2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • Ri and R 5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (VII):
  • each of Ri, R 2 , R 3 , R 4 , R 5 , R 7 or R 8 is independently -H, -halo, -NO 2 , -CN,
  • each R 5 or Rs is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • C 10 )alkyl or -SO 2 NH 2 ;
  • m is independently an integer ranging from 0 to 8;
  • p is independently an integer ranging from 0 to 5;
  • X is O, S, C or N, wherein if X is O or S, Ri is an electron pair;
  • Y and Z are independently N or C, wherein if Y or Z is N, R ⁇ and R 2 are each an electron pair.
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (VIII):
  • each of Ri, R 2 , R 3 , R , R 5 , R 7 , R 8 , R 9 or R ⁇ 0 is independently -H, -halo, -NO 2 , -CN, -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C ⁇ 0 )alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(d-C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5
  • each Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • o cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -phenyl, naphthyl, -(C 3 -C 10 )heterocycle, -CO 2 (CH 2 ) m (C ⁇ -C ⁇ o)alkyl, -CO 2 (CH 2 ) m H, -NHC(O)NH(d-C ⁇ 0 )alkyl, -OC(O)O(C ⁇ - C 10 )alkyl, or -SO 2 NH 2 ; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and X and Y are independently O, S or N, wherein if X or Y is O or S, R and R JO are an electron pair.
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (IX):
  • each of Ri, R 2 , R 3 , Rj , R 5 , R 7 , R 8 , R or Rio is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C 1 -C, 0 )alkyl, -(C 2 - C ⁇ o)alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicy
  • each R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C 10 )alkyl(C ⁇ -C 10 )alkyl, -O(d-C 10 )alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl,
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (X):
  • each of R,, R 2 , R 3 , R4 , R 5 or R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 ,
  • each R 5 or R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • R] and R 2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 2 and R 3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 4 and R 7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • the agonists or antagonists that can be utilized as part of the methods of the present are compounds of structural formula (XI):
  • each of Ri, R 2 , R 3 , R , R 5 , R 7 or R 8 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -(CH 2 ) m N(R 5 )(R 5 ), -O CH,) ⁇ , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -C(OH)R 5 , -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C ⁇ o)alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ 0 )cycloalkyl, -(C 8 -C ⁇ 4 )
  • each R 6 is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C 1 -C 10 )alkyl(C,-C ⁇ o)alkyl, -O(C 1 -C ⁇ 0 )alkyl, -C(O)(C 1 -C 10 )alkyl, -C(O)NH(CH 2 ) m (d-C, 0 )alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((d-C, 0 )alkyl(d- C,o)alkyl), -CO 2 (C,-do)alkyl, -(C,-C 10 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ 0 )cycloalkyl,
  • Ri and R 2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 2 and R 3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 3 and R 4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R 7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 8 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and Ri and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (XII):
  • each of Ri, R 2 , R 3 , R 4 , R 5 or R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -(CH 2 ) m N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -C(OH)R 5 , -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C ⁇ o)alkynyl, -(C 3 -C 10 )cycloalkyl, -(C 8 -C M )bic
  • each R 5 or R is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C 1 -C 10 )alkyl(C ⁇ -C 1 o)alkyl, -O(C 1 -C 1 o)alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl, -C(O)NH(CH 2 ) m (d-C 10 )alkyl, -OCF 3 , -benzyl, -C ⁇ 2 (CH 2 ) m CH((C,-C ⁇ o)alkyl(C ⁇ - C 10 )alkyl), -CO 2 (C ⁇ -C,o)alkyl, -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl,
  • R 3 or R 4 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring; Ri or R 2 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring; and R 2 or R 4 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring.
  • the present invention provides compounds that modulate the Edg-7 (LPA3) receptor (e.g., human Edg-7, GenBank Accession No. AF127138). Such compounds selectively bind or otherwise modulate the Edg-7 receptor.
  • LPA3 Edg-7
  • the compounds that can, for example, be used to modulate Edg-7 receptor mediated biological activity or to treat or prevent diseases such as those discussed above.
  • the agonists or antagonists are compounds of structural formula (XIII) and can be utilized in the methods of the present invention:
  • X is NR 3 , S or O
  • Ri is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, amino, carbamoyl, substituted carbamoyl, oxo, thiono or -NR 4 ;
  • R 2 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkyloxy, substituted alkyloxy, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, aryloxycarbonyl, substituted aryloxycarbonyl, arylalkyl, substituted arylalkyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted
  • R 3 is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkylsulfonyl, substituted alkylsulfonyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl or substituted heteroalkyl;
  • R 4 is alkyl, substituted alkyl, acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, heteroaryl substituted heteroaryl, cycloheteroalkyl, substituted cycloheteroalkyl, and
  • R 5 and R are independently hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylthio, substituted alkylthio, alkoxycarbonyl, substituted alkoxycarbonyl, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or optionally along with the carbon to which they are attached form an aryl, substituted aryl, cycloalkyl, substituted
  • the present invention also provides compounds (agonists or antagonists) that can, for example, be used to modulate Edg-7 receptor mediated biological activity or to treat or prevent diseases such as those discussed above.
  • the agonists or antagonists can be utilized in the methods of the present invention and are compounds of structural formula (XIV):
  • each of Ri, R 2 , R 3 R 4 and R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C-C ⁇ o)alkyl, -(C 2 -C ⁇ o)alkenyl, -(C 2 -C l0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C] 4 )bicycloalkyl, -(C 5 -C ⁇ 0 )cyclo
  • each R 5 and R 6 is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, ⁇ (0,-0 10 )3 ⁇ 1(0,-0 10 )3 ⁇ 1, -0(0,-0 10 )3 !, -C(O)(C ⁇ -C 10 )alkyl, -C(O)NH(CH 2 ) m (C,-C, 0 )alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C,-C ⁇ 0 )alkyl(C,- C,o)alkyl), -CO 2 (C,-C, 0 )alkyl, -(C ⁇ -C, 0 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C -C ⁇ o)cycl
  • R 3 R 4 and R 7 can also be an electron such that when two groups are on adjacent carbon atoms they form a double bond; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • the agonists or antagonists can be utilized in the methods of the present invention and are also compounds of structural formula (XV):
  • each of R,, R 2 , R 3 R 4 and R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C ⁇ 0 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(d-C ⁇ o)cycloalkyl, -(C 8 -C ⁇
  • R 3 is -H, -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C ⁇ o)alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl, -(C ⁇ )heteroaryl, -(C 5 -C ⁇ o)heteroaryl, -naphthyl, -(C
  • each R 5 and R* is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C, 0 )alkyl(C,-C,o)alkyl, -O(C,-C ⁇ 0 )alkyl, -C(O)(C,-C ⁇ o)alkyl,
  • the present invention provides methods for modulating Edg-7 receptor mediated biological activity.
  • the present invention also provides methods for using Edg-7 modulators (i.e., agonists and antagonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous burns) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention.
  • ARDS adult respiratory distress syndrome
  • the present invention provides compounds and compositions for use in modulating Edg-7 receptor mediated biological activity or treating or preventing diseases such as those mentioned above as well as methods for synthesizing the compounds.
  • the present invention provides a method of modulating
  • the present invention provides a method for modulating
  • the present invention provides compounds that modulate the Edg-2 (LPAl) receptor (e.g., human Edg-2, GenBank Accession No., U78192). Such compounds preferably selectively bind or otherwise modulate the EDG-2 receptor.
  • the present invention provides methods for modulating (antagonizing or agonizing) Edg-2 receptor mediated biological activity.
  • the present invention also provides methods for using Edg-2 modulators (agonists and antagonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous burns) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention.
  • diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer
  • acute lung diseases such as ovarian cancer, peritoneal cancer, end
  • the present invention provides compounds and compositions that can, for example, be used in modulating Edg-2 receptor mediated biological activity or treating or preventing diseases such as those mentioned above.
  • the present invention still further provides methods for synthesizing the compounds.
  • the present invention provides a method of modulating an Edg-2 receptor mediated biological activity in a cell. A cell expressing the Edg-2 receptor is contacted with an amount of an Edg-2 receptor modulator sufficient to modulate the Edg-2 receptor mediated biological activity.
  • the present invention provides a method for modulating Edg-2 receptor mediated biological activity in a subject.
  • an amount of a modulator of the Edg-2 receptor effective to modulate the Edg-2 receptor mediated biological activity is administered to the subject.
  • the present invention also provides compounds (agonists or antagonists) that modulate Edg-2 receptor mediated biological activity.
  • the agonists or antagonists are compounds of structural formula (XX) and can be utilized as part of the methods of the present invention: (XX)
  • P, Q and R are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl.
  • the agonists or antagonists can be utilized as part of the methods of the present invention and are compounds of structural formula (XXIII):
  • each of R,, R 2 and R 3 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C, 0 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C,o)alkynyl, -(C 3 -C,o)cycloalkyl, -(C 8 -C, 4 )bicycloalkyl, -(C 5 -C ⁇ o)cyclo alkenyl
  • each of R 5 and R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C ⁇ o)alkyl(C,-C,o)alkyl, -O(C ⁇ -C,o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH 2 ) m (C,-C,o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C,-C, 0 )alkyl(C ⁇ -
  • Ri and X or R 2 and Y can together form a double bond.
  • the agonists or antagonists are compounds that can be utilized as part of the methods of the present invention and are of structural formula (XVII):
  • each of Ri and R 2 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C, 0 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C, 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C, 0 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C,o)cycloalkyl, -(C 8 -C, 4 )bicycloalkyl, -(C 5 -C,o)cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -(C 5 -C,o)heteroaryl, -naphthyl, -(C
  • each R 5 and R 6 is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • each of Ri and R 2 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -NH(aryl), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-do)alkyl, -(C 2 -C, 0 )alkenyl, -aryl, -(C 2 -C,o)alkynyl, -(C 3 -C, 0 )cycloalkyl, -(C 3 -C ⁇ o)cycloalkyl(aryl), -(C 8 -C, 4
  • each R 5 and R 6 is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • R, and R 2 can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two R 6 groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • the present invention provides compounds that modulate the S1P3 or Edg-3 receptor (e.g. human Edg-3, GenBank Accession No. X83864). Such compounds preferably selectively bind or otherwise modulate the Edg-3 receptor. In one embodiment, the present invention provides methods for modulating
  • Edg-3 receptor mediated biological activity The present invention also provides methods for using Edg-3 modulators (i.e., agonists or antagonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS”), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous burns) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention.
  • diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer
  • acute lung diseases
  • the present invention provides methods for using Edg-3 modulators (i.e., agonists or antagonists) in treating or preventing disorders such as, but not limited to, vasoconstriction in cerebral arteries, autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia.
  • Edg-3 modulators i.e., agonists or antagonists
  • disorders such as, but not limited to, vasoconstriction in cerebral arteries, autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular ne
  • Edg-3 agonists and antagonists can be used to treat vascular occlusive disorders.
  • activation of Edg-3 receptors by using an Edg-3 agonist will result in increased vasoconstriction which is beneficial in conditions such as migraine headaches.
  • Inhibition of Edg-3 by an Edg3 antagonist will be beneficial in conditions such as a stroke, a subarachnoid hemorrhage, or a vasospasm such as a cerebral vasospasm.
  • the present invention provides a method of modulating an Edg-3 receptor mediated biological activity in a cell.
  • a cell expressing the Edg-3 receptor is contacted with an amount of an Edg-3 receptor modulator sufficient to modulate the Edg-3 receptor mediated biological activity.
  • the present invention provides a method for modulating an Edg-3 receptor mediated biological activity in a subject. In such a method, an amount of a modulator of the Edg-3 receptor effective to modulate an Edg-3 receptor mediated biological activity is administered to the subject.
  • the present invention also provides compounds and compositions for use in modulating (i.e., agonizing or antagonizing) Edg-3 receptor mediated biological activity or treating or preventing diseases such as those mentioned above as well as methods for synthesizing the compounds.
  • the Edg-3 receptor modulators are compounds of structural formula (XXXI) :
  • n 0 or 1 ;
  • o 0, 1, 2, 3 or 4;
  • X is C, NR 7 O or S
  • Y is C, NR 8 O or S
  • R is either absent or hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamin
  • R 2 , R 3 and R 4 are independently hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalky
  • each R 5 is independently, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, azido, carboxy, carbamoyl, substituted carbamoyl, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substitute
  • R and R 8 are independently absent, hydrogen, alkyl, substituted alkyl, acyl or substituted acyl.
  • Edg-3 receptor modulators are compounds of structural formula (XXXII):
  • each of R,, R 2 and R 3 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C ⁇ o)alkyl, -(C 2 -C ⁇ o)alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C,o)cycloalkyl, -(C 8 -C, 4 )bicycloalkyl, -(C 5 -C,o)cycloalkenyl, -
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C, 0 )alkynyl,
  • each R 5 and R is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C,o)alkyl(C ⁇ -C ⁇ o)alkyl, -O(C,-C,o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH 2 ) m (C,-C 10 )alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C,-C,o)alkyl(C,- C, 0 )alkyl), -CO 2 (C,-C, 0 )alkyl, -(C,-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C,o)cyclo
  • X is O, S, C(R 5 )(R 5 ) or N(R 5 );
  • Ri, R 2 or R 3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • Edg-3 receptor modulators are compounds of structural formula (XXXIII):
  • each of R,, R 2 and R 3 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C 10 )alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C ]0 )alkynyl, -(C 3 -C,o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C, 0 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C, 4 )bicycloalkyl, -(C 5 -C,o)cycloalkenyl, -(C 5 )heteroaryl, -(C )heteroaryl, -(C 5 -C,o)heteroaryl, -naphthyl, -(C 3
  • each R 5 and Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(d-C ⁇ o)alkyl(C,-C,o)alkyl, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH 2 ) m (d-C, 0 )alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C ⁇ -C, 0 )alkyl(C ⁇ - C ⁇ o)alkyl), -CO 2 (C ⁇ -C ⁇ o)alkyl, -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C -C ⁇ o)cycloal
  • Ri, R 2 or R 3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two R 6 groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • Fig. 1 illustrates the selectivity of 101 for the Edg-4 receptor
  • Fig. 2 illustrates a dose response curve for Edg-4 antagonists 101, 103 and 105;
  • Fig. 3 illustrates a dose response curve for 101 and LPA in HTC rat hepatoma cells transfected with human Edg-4 receptors
  • Fig. 4 illustrates a dose response curve for 101 in OV202 human ovarian cancer cells
  • Fig. 5 illustrates a dose response curve for 101 in CaOV-3 human ovarian cancer cells
  • Fig. 6 illustrates the inhibition of VEGF production by 101 in CaOV-3 human ovarian cancer cells
  • Fig. 7 illustrates the inhibition of IL-8 production by 101 in CaOV-3 human ovarian cancer cells
  • Fig. 8 illustrates the inhibition of LPA-stimulated proliferation by 101 in CaOV-3 human ovarian cancer cells
  • Fig. 9 illustrates the inhibition of LPA-stimulated chemotaxis by 103 in
  • Fig. 10 illustrates the lack of inhibition of S IP-stimulated migration by 103 in human umbilical vein endothelial cells
  • Fig. 11 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonists 101, 103, 107 and 113 in HTC rat hepatoma cells transfected with human Edg-4;
  • Fig. 12 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonists 101, 103, 107 and 113 in HTC rat hepatoma cells transfected with pooled rat Edg-4 clones
  • Fig. 13 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonists 101, 103, 107 and 113 in HTC rat hepatoma cells transfected with pooled mouse Edg-4 clones;
  • Figure 14 illustrates the efficacy of 101 in suppressing the tumor growth as tested by in vivo Z-chamber study
  • Figure 15 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonist 125 in HTC cells;
  • Figure 16 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonist 125 in CaOV-3 cells
  • Fig. 17 illustrates the selectivity of 701 for the Edg-7 receptor
  • Fig. 18 illustrates the inhibition of LPA-stimulated calcium mobilization by 703 in HT-1080 human fibrosarcoma cells
  • Fig. 19 illustrates the selectivity of 201 for the Edg-2 receptor
  • Fig. 20 illustrates the selectivity of 203 for the Edg-2 receptor
  • Fig. 21 illustrates the inhibition of LPA-stimulated calcium mobilization in immortalized ovarian surface epithelial cells by Edg-2 antagonist 203;
  • Fig. 22 illustrates the effects of 203 on the inhibition of cAMP production by LPA
  • Fig. 23 illustrates the inhibition of LPA-stimulated calcium mobilization in A431 human epitheloid carcinoma cells by Edg-2 antagonist 201 ;
  • Fig. 24 illustrates the inhibition of LPA-stimulated calcium mobilization in A431 human epitheloid carcinoma cells by Edg-2 antagonists 201 and 203, but not Edg-4 antagonist, 101;
  • Figure 25 illustrates the selectivity of 301 for the Edg-3 receptor
  • Figure 26 illustrates the inhibition of S IP-stimulated calcium mobilization by
  • “Compounds of the invention” refers generally to any modulator of the LPA2 or Edg-4 receptor (e.g. human Edg-4, GenBank Accession Nos. AF23 3092 or AFOl 1466) and includes any Edg-4 receptor modulator encompassed by generic formulae disclosed herein and further includes any species within those formulae whose structure is disclosed herein.
  • “Compounds of the invention” also refers generally to any modulator of the Edg-7 receptor (e.g. human Edg-7, GenBank Accession No. AF 127138) encompassed by generic formulae disclosed herein and further includes any species within those formulae whose structure is disclosed herein.
  • Compounds of the invention also refers generally to any modulator of the LPAl or Edg-2 receptor (human Edg-2, GenBank Accession No., U78192) and includes any Edg-2 receptor modulator encompassed by generic formulae disclosed herein and further includes any species within those formulae whose structure is disclosed herein.
  • Compounds of the invention also refers generally to any modulator of the S1P3 or Edg-3 receptor (e.g., human Edg-3, GenBank Accession No. X83864) and includes any Edg-3 receptor modulator encompassed by generic formulae disclosed herein and further includes any specific Edg-3 receptor modulator within those formulae whose structure is disclosed herein.
  • the compounds of the invention may be identified either by their chemical structure and/or chemical name.
  • the chemical structure is determinative of the identity of the compound.
  • the compounds of the invention may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers.
  • stereoisomers such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers.
  • the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures.
  • Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan.
  • the compounds of the invention may also exist in several tautomeric forms including, but not limited to, the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
  • the compounds of the invention also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature.
  • isotopes examples include, but are not limited to, 2 H, 3 H, ,3 C, 14 C, 15 N, ,8 0, 17 0, 31 P, 32 P, 35 S, 18 F and 36 C1. Further, it should be understood that when partial structures of the compounds of the invention are illustrated, brackets indicate the point of attachment of the partial structure to the rest of the compound.
  • Composition of the invention refers to at least one compound of the invention and a pharmaceutically acceptable vehicle, with which the compound is administered to a patient.
  • the compounds of the invention are administered in isolated form, which means separated from a synthetic organic reaction mixture.
  • Alkyl refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne.
  • Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop- 1 -en- 1-yl, prop-l-en-2-yl, prop-2- en-l-yl (allyl), cycloprop-1-en-l-yl; cycloprop-2-en-l-yl, prop-1-yn-l-yl, prop-2-yn- 1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-l-yl, 2-methyl- propan-2-yl, cyclobutan-1-yl, but- 1 -en- 1-yl, but-l-en-2-yl, 2-methyl-prop-l -en- 1-yl, but-2-en-l-
  • alkyl is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used. Preferably, an alkyl group comprises from 1 to 20 carbon atoms.
  • Alkanyl refers to a saturated branched, straight-chain or cyclic alkyl group derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane.
  • Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl fiec-butyl), 2-methyl-propan-l-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yi, etc.; and the like.
  • Alkenyl refers to an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene.
  • the group may be in either the cis or trans conformation about the double bond(s).
  • Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop- 1 -en- 1-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl; butenyls such as but- 1 -en- 1-yl, but-l-en-2-yl, 2-methyl-prop-l - en-l-yl, but-2-en-l-yl , but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-1,3- dien-2-yl, cyclobut-1-en-l-yl, cyclobut-l-en-3-yl, cyclobuta-l,3-dien-l-yl, etc.; and the like.
  • Alkynyl refers to an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne.
  • Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop- 1-yn- 1-yl, prop-2-yn- 1-yl, etc.; butynyls such as but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc and the like.
  • Acyl refers to a radical -C(O)R, where R is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
  • Representative examples include, but are not limited to formyl, acetyl, cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.
  • Acylamino refers to a radical -NR'C(O)R, where R' and R are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein.
  • Representative examples include, but are not limited to, formylamino, acetylamino, cylcohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino and the like.
  • Alkylamino means a radical -NHR where R represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylamino, ethylamino, 1-methylethylamino, cydohexyl amino and the like.
  • Alkoxy refers to a radical -OR where R represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.
  • Alkoxycarbonyl refers to a radical -C(O)-alkoxy where alkoxy is as defined herein.
  • Alkylarylamino refers to a radical -NRR' where R represents an alkyl or cycloalkyl group and R' is an aryl as defined herein
  • Alkylsulfonyl refers to a radical -S(O) 2 R where R is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl and the like.
  • Alkylsulfinyl refers to a radical -S(O)R where R is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfmyl and the like.
  • Alkylthio refers to a radical -SR where R is an alkyl or cycloalkyl group as defined herein that may be optionally substituted as defined herein. Representative examples include, but are not limited to methylthio, ethylthio, propylthio, butylthio, and the like.
  • Amino refers to the radical -NH .
  • Aryl refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
  • Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as- indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene,
  • Arylalkyl refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with an aryl group.
  • Typical arylalkyl groups include, but are not limited to, benzyl, 2- phenylethan-1-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2- naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like.
  • arylalkanyl arylalkenyl and/or arylalkynyl
  • an arylalkyl group is (C 6 -C 0 ) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C ⁇ -C 10 ) and the aryl moiety is (C 6 -C 20 ).
  • Arylalkyloxy refers to an -O-arylalkyl radical where arylalkyl is as defined herein.
  • Arylamino means a radical -NHR where R represents an aryl group as defined herein.
  • Aryloxycarbonyl refers to a radical -C(O)-O-aryl where aryl is as defined herein.
  • Carbamoyl refers to the radical -C(O)N(R) 2 where each R group is independently hydrogen, alkyl, cycloalkyl or aryl as defined herein, which may be optionally substituted as defined herein.
  • Carboxy means the radical -C(O)OH.
  • Cyano means the radical -CN.
  • Cycloalkyl refers to a saturated or unsaturated cyclic alkyl group. Where a specific level of saturation is intended, the nomenclature “cycloalkanyl” or “cycloalkenyl” is used. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and the like. In a preferred embodiment, the cycloalkyl group is (C -C ⁇ 0 ) cycloalkyl, more preferably (C 3 -C 6 ) cycloalkyl.
  • Cycloheteroalkyl refers to a saturated or unsaturated cyclic alkyl group in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom.
  • Typical heteroatoms to replace the carbon atom(s) include, but are not limited to, N, P, O, S, Si, etc. Where a specific level of saturation is intended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used.
  • Typical cycloheteroalkyl groups include, but are not limited to, groups derived from dioxanes, dioxolanes, epoxides, imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine, tetrahydrofuran, tetrahydropyran and the like.
  • Cvcloheteroalkyloxycarbonyl refers to a radical -C(O)-OR where R is cycloheteroalkyl is as defined herein.
  • Dialkylamino means a radical -NRR' where R and R' independently represent an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to dimethylamino, methyl ethylamino, di-(l- methylethyl)amino, (cyclohexyl)(methyl)amino, (cyclohexyl)(ethyl)amino, (cyciohexyl)(propyl)amino, and the like.
  • Halo means fluoro, chloro, bromo, or iodo.
  • Haloalkyl means an alkyl radical substituted by one or more halo atoms wherein alkyl and halo is as defined herein.
  • Heteroalkyloxy means an -O-heteroalkyl group where heteroalkyl is as defined herein.
  • Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkynyl refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with the same or different heteroatomic groups.
  • Heteroaryl refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system.
  • Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyrid
  • the heteroaryl group is between 5-20 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred.
  • Preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.
  • Heteroaryloxy refers to an -O-heteroarylalkyl radical where heteroarylalkyl is as defined herein.
  • Heteroaryloxycarbonyl refers to a radical -C(O)-OR where R is heteroaryl as defined herein.
  • Heteroarylalkyl refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used. In preferred embodiments, the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20 membered heteroaryl.
  • Haldroxy refers to the radical -OH.
  • Leaving group has the meaning conventionally associated with it in synthetic organic chemistry, i.e., an atom or a group capable of being displaced by a nucleophile and includes halo (such as chloro, bromo, and iodo), alkoxycarbonyl (e.g., acetoxy), aryloxycarbonyl, mesyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g, 2,4-dinitrophenoxy), methoxy, N,O-dimethylhydroxylamino, and the like.
  • halo such as chloro, bromo, and iodo
  • alkoxycarbonyl e.g., acetoxy
  • aryloxycarbonyl mesyloxy, tosyloxy, trifluoromethanesulfonyloxy
  • aryloxy e.g, 2,4-dinitrophenoxy
  • methoxy N,O-d
  • “Pharmaceutically acceptable” means approved by a regulatory agency of the
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzen
  • “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
  • Patient includes humans.
  • human and “patient” are used interchangeably herein.
  • Preventing or “prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
  • Prodrug refers to a pharmacologically inactive derivative of a drug molecule that requires a transformation within the body to release the active drug.
  • prodrugs are designed to overcome pharmaceutical and/or pharmacokinetically based problems associated with the parent drug molecule that would otherwise limit the clinical usefulness of the drug.
  • Promoiety refers to a form of protecting group that when used to mask a functional group within a drug molecule converts the drug into a prodrug.
  • the promoiety will be attached to the drug via bond(s) that are cleaved by enzymatic or non-enzymatic means in vivo. Ideally, the promoiety is rapidly cleared from the body upon cleavage from the prodrug.
  • Protecting group refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in Green et al, "Protective Groups in Organic
  • amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ”), tert-butoxycarbonyl ("Boc”), trimethylsilyl ("TMS”), 2-trimethylsilyl-ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro- veratryloxycarbonyl (“NVOC”) and the like.
  • hydroxy protecting groups include, but are not limited to, those where the hydroxy group is either acylated or alkylated such as benzyl, and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
  • Substituted refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s).
  • Sulfonylamino refers to a radical -NR'S(O 2 )R, where R' and R are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein.
  • “Therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
  • Thiocyanato refers to the radical -SCN.
  • Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discemible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder.
  • the present invention provides a method of modulating an LPA2 or Edg-4 receptor (e.g., human Edg-4, GenBank Accession Nos. AF233092 or AFOl 1466) mediated biological activity.
  • a cell expressing the Edg-4 receptor is contacted with an amount of an Edg-4 receptor agonist or antagonist sufficient to modulate the Edg-4 receptor mediated biological activity.
  • Edg-4 is a G protein coupled receptor ("GPCR").
  • the Edg-4 (LPA2) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-2 (LPAl) and Edg-7 (LPA3), binds lysophosphatidic acid (“LPA").
  • LPA G protein coupled receptor
  • the Edg-4 receptor is a human receptor.
  • the Edg-4 receptor may be expressed by recombinant DNA methods well known to those of skill in the art.
  • Particularly useful cell types for expressing and assaying Edg-4 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells).
  • Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA).
  • DNA encoding Edg-4 is well known (e.g, human Edg-4, GenBank Accession Nos. AF233092 or AFOl 1466) and can be transfected into human or mammalian cells according to methods known to those of skill in the art.
  • DNA encoding human Edg-4 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
  • GP-293 Cell Line
  • DNA encoding Edg-4 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the ewv gene to produce an ecotropic envelope.
  • Both methods i.e., co-transfection with a packaging vector or use of EcoPack-293 enable the production of an ecotropic envelope for viral packaging, and can thus advantageously be used to transfect rat and mouse cells.
  • AmphoPack-293 cell line can be used (Clontech Labs, Palo Alto, CA).
  • Edg-4 receptors include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB- 453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells and OV202 human ovarian cancer cells (ATCC, Manassas, VA; Vec
  • cells which express the Edg-4 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulation of Edg-4 receptor activity, regardless of the local environment.
  • cells that express the Edg-4 receptor are grown in vitro (i.e., are cultured).
  • cells that express the Edg-4 receptor are in vivo (i.e., are part of a complex organism).
  • the cells, in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, endothelial cells, carcinoma cells, pheochromocytoma cells, myoblast cells, platelet cells and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, 0V202 human ovarian cells, HTC rat hepatoma cells, CAOV-3 human ovarian cancer cells, MDA-MB-453 breast cancer cells, MDA-MB-231 breast cancer cells, HUVEC, A431 human epitheloid carcinoma cells and HT-1080 human fibrosarcoma cells.
  • an Edg-4 receptor mediated biological activity is modulated in a subject or in an animal model.
  • a therapeutically effective amount of a modulator of the Edg-4 receptor is administered to the subject or an animal.
  • the subject or animal is in need of such treatment.
  • the biological activity mediated by the Edg-4 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation or actin polymerization.
  • the biological activity mediated by the Edg-4 receptor includes, but is not limited to, apoptosis, angiogenesis, inhibition of wound healing, inflammation, cancer invasiveness or atherogenesis.
  • the biological activity mediated by the Edg-4 receptor is cell proliferation, which may lead to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colon cancer or prostrate cancer. In one embodiment, cell proliferation is stimulated by LPA.
  • the biological activity mediated by the Edg-4 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS”) and acute inflammatory exacerbation of chronic lung diseases like asthma.
  • ARDS adult respiratory distress syndrome
  • chronic lung diseases like asthma.
  • compounds that block Edg-4 can be potentially effective immunosuppressive agents because activated T cells have Edg-4 receptors (Zheng et al, 2000, FASEB J 14:2387-2389).
  • Edg-4 antagonists may be useful in a variety of autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-4 antagonists can be used in organ transplantation.
  • the modulator exhibits selectivity for the Edg-4 receptor.
  • the modulator exhibits at least about 5 to about 200 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
  • Inhibitory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.25 (Example 25), 6.27 (Example 27) and 6.28 (Example 28) respectively.
  • inhibitory selectivity can be measured by a calcium mobilization assay.
  • Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art.
  • the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to other non-Edg receptors, including, but not limited to, other GPCRs, ion channels, growth factor receptors and the like.
  • the modulator exhibits at least about 63 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
  • the modulator exhibits at least about 30 fold inhibitory selectivity for Edg-4 relative to other Edg receptors. In still another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
  • the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits at least about 63 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits at least about 30 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors. In still another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits activating selectivity for the Edg-4 receptor.
  • the modulator exhibits at least about 5 to about 200 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • Activating selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.13 (Example 13), 6.15 (Example 15) and 6.16 (Example 16) respectively.
  • activating selectivity can be measured by a calcium mobilization assay.
  • Other assays suitable for determining activating selectivity would be known to one of skill in the art.
  • the modulator exhibits at least about 200 fold activating selectivity for Edg-4 relative to other non-Edg receptors, including, but not limited to, other GPCRs, ion channels, growth factor receptors and the like. In another embodiment, the modulator exhibits at least about 63 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • the modulator exhibits at least about 30 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • the modulator exhibits at least about 10 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • the agonist modulator exhibits at least about 5 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors. In yet another embodiment, the modulator exhibits at least about 63 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits at least about 30 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits at least about 10 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator exhibits at least about 5 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 10 fold activating selectivity for Edg-4 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 10 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the modulator of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
  • the Edg-4 modulator is not a lipid.
  • the modulator of Edg-4 receptor mediated biological activity does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester.
  • the modulator of the Edg-4 receptor is not a phospholipid.
  • phospholipid includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of greater 10 carbon atoms or greater, any N-acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al, 1996, J.
  • the modulator is also not a compound of structural formula:
  • X is O or S
  • R 20 is alkyl, substituted alkyl, aryl, substituted aryl or halo
  • R 2 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
  • R 2 is hydrogen, alkyl or substituted alkyl
  • R 24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; or alternatively R 23 and R 24 form a cycloalkyl ring (International Application No: WO 01/60819).
  • the modulator is not any compound of the formula below:
  • R 2 o, R 2 ⁇ and R 24 are as previously defined.
  • the modulator is not any compound disclosed in International Application No: WO
  • the Edg-4 modulator may be a biomolecule such as a nucleic acid, protein,
  • the Edg-4 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof.
  • the Edg-4 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
  • the Edg-4 modulator may also be an organic molecule of molecular weight less than 750 daltons. In one embodiment, the molecular weight is about 200 to about
  • the molecular weight is about 200 to about 750 daltons. In yet another embodiment, the molecular weight is about 200 to about 500 daltons. Preferably, the molecular weight is about 300 to about 500 daltons.
  • the modulator may, for example, facilitate inhibition of the Edg-4 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the Edg-4 receptor, interference with Edg-4 or LPA biosynthesis, covalent modification of either LPA or the Edg-4 receptor, or may otherwise interfere with Edg-4 mediated signal transduction.
  • the agonist or antagonist binds to the Edg-4 receptor with a binding constant between about 10 ⁇ M and about 1 fM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 10 ⁇ M and about 1 nM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 1 ⁇ M and about 1 nM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the modulator binds to the Edg-4 receptor with a binding constant better (i.e., less) than about 10 nM.
  • the modulator is a compound of structural formula (I):
  • Ri is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted dialkylamino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino;
  • A is NR 2 , O or 5;
  • R 2 is hydrogen, alkyl or substituted alkyl
  • B and C are independently alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl.
  • R is alkyl, substituted alkyl, substituted aryl, substituted aryl, arylalkyloxy or substituted sulfonylamino. More preferably, R ⁇ is substituted alkyl. Even more preferably, Ri is substituted haloalkyl. Most preferably, Ri is substituted trifluoroalkyl (preferably, trifluoroalkanyl).
  • Ri has the structural formula (II):
  • R is haloalkyl or substituted haloalkyl
  • R 4 is oxo or thiono; and R 5 and R ⁇ are independently hydrogen, halo, alkyl or substituted alkyl.
  • R 3 is fluoroalkyl
  • R 4 is oxo and R 5 and R are independently hydrogen, halo or alkyl. More preferably, R is trifluoromethyl, R 4 is oxo and R 5 and R ⁇ 5 are independently hydrogen, chloro or methyl.
  • R 5 and Re are hydrogen. In another preferred embodiment, R 5 is hydrogen and R is chloro or methyl.
  • X is O
  • A is NR 2 and R 2 is hydrogen.
  • B and C are alkyl, substituted alkyl, independently, aryl, substituted aryl, heteroaryl or substituted heteroaryl. More preferably, B and C are independently indolo, substituted indolo, imidazolo, substituted, imidazolo, pyrazolo, substituted pyrazolo, phenyl or substituted phenyl. Even more preferably, B is heteroaryl or substituted heteroaryl and C is aryl or substituted aryl. Most preferably, B is pyrazolo or substituted pyrazolo and C is phenyl or substituted phenyl.
  • the modulator is a compound of structural formula (III):
  • R 7 is hydrogen, alkyl, substituted alkyl or halo
  • R 8 is hydrogen, carbamoyl or substituted carbamoyl
  • R 9 , Rio and Rn are independently hydrogen, alkoxy, substituted alkoxy, halo or optionally, R 9 and Rio together with the carbons to which they are attached form a [1,3] dioxolane ring.
  • Preferred modulators include compounds of the structural formula shown below:
  • the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (IV):
  • each of R,, R 2 , R 3 , R 4 or R 5 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -(CH 2 ) m N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 ,
  • each R 5 and Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • C ⁇ o)alkyl -CO 2 (C ⁇ -C, 0 )alkyl, -(d-C, 0 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ o)cyclo alkyl, -(C 8 -C] )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl,
  • X and Y are each independently C or N;
  • Z is O, S, C or N, wherein if Z is O or S, then R 3 is an electron pair;
  • Ri and R 2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 2 and R 3 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 4 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • Another embodiment of the present invention is directed to compounds of structural formula (V), which can be utilized for the purpose of this invention:
  • each of Ri, R 2 , R , R-jor R 5 is independently -H, -halo, -NO 2 , -CN, -OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C ⁇ 0 )alkyl, -(C 2 -C ⁇ o)alkenyl, -(C 2 -C ⁇ o)alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl,
  • each Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C ⁇ o)alkyl(C ⁇ -C ⁇ o)alkyl, -O(C,-C ⁇ o)alkyl, -C(O)(d-C,o)alkyl, -C(O)NH(CH 2 ) m (C ⁇ -C ⁇ o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((d-C,o)alkyl(d- C,o)alkyl), -CO 2 (C ⁇ -C ⁇ o)alkyl, -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ 0 )cycloalkyl, -(C 8 -C
  • Ri and R or R 2 and R 3 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • R ⁇ and R 2 are independently aryl, substituted aryl, heteroaryl or substituted heteroaryl.
  • R 2 is indole, and R 3 and R 4 are hydrogen.
  • the modulator is a compound of structural formula (VI):
  • each of R], R 2 , R 3 , R or R 5 is independently -H, -halo, -NO 2 , -CN, -OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C 10 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C ⁇ o)alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl, -
  • Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C,o)alkyl(CrC,o)alkyl, -O(d-C 10 )alkyl, -C(O)(C,-C, 0 )alkyl,
  • X, Y and Z are independently O, S, C or N, wherein if X, Y or Z is O or S, Ri is an electron pair;
  • Ri and R 2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 3 and Rj can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; Ri and R can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
  • R and R 5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • Ri and R 2 together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring. In a more specific embodiment,
  • Ri and R 4 together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • Illustrative modulators of the invention include, but are not limited to, the following compound:
  • the modulator is a compound of structural formula (VII):
  • each of R ⁇ , R 2 , R 3 , Rt , R 5 , R 7 or R 8 is independently -H, -halo, - ⁇ O 2 , -CN, -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C 10 )alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C 14 )bic
  • each R 5 or Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C ⁇ o)alkyl(C ⁇ -C ⁇ o)alkyl, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl, -C(O)NH(CH 2 ) m (C,-Ci 0 )alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C 1 -C 10 )alkyl(C 1 - C ⁇ o)alkyl), -CO 2 (C 1 -C, 0 )alkyl, -(C,-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl
  • X is O, S, C or N, wherein if X is O or S, Ri is an electron pair;
  • Y and Z are independently N or C, wherein if Y or Z is N, Ri and R 2 are each an electron pair.
  • Illustrative modulators of the invention include:
  • the modulator is a compound of structural formula (VIII):
  • each of Ri, R 2 , R 3 , R 4 , R 5 , R , R 8 , R 9 or Rio is independently -H, -halo, -NO 2 , -CN, -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -
  • each R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • C,o)alkyl -CO 2 (C ⁇ -C 10 )alkyl, -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl,
  • X and Y are independently O, S or N, wherein if X or Y is O or S, R 9 and Rio are an electron pair.
  • R 7 is a substituted or unsubstituted aryl.
  • Egd-4 modulators includes:
  • the modulator is a compound of structural formula
  • each of Ri, R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 or Rio is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C, 0 )alkyl, -(C 2 - C 10 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicy
  • each Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C,o)alkyl(d-C ⁇ o)alkyl, -O(C,-C 10 )alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl,
  • C ⁇ o)alkyl or -SO 2 NH 2 ;
  • m is independently an integer ranging from 0 to 8; and
  • p is independently an integer ranging from 0 to 5.
  • R 2 is a substituted alkyl, and one or more of R 5 , R 7 , R 8 , R 9 and R]o are halos.
  • R 2 is a halo-substituted alkyl.
  • R 2 is CF .
  • Specific examples of the modulators include:
  • the modulator is a compound of structural formula (X):
  • each of Ri, R 2 , R 3 , R 4 , R 5 or R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C C ⁇ 0 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)
  • each R 5 or Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • Ci 0 alkyl, or -SO 2 NH 2 ;
  • m is independently an integer ranging from 0 to 8;
  • p is independently an integer ranging from 0 to 5;
  • Ri and R 2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 3 and R 4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • R 3 and R 7 are substituted or unsubstituted aryls.
  • An illustrative modulator of the invention includes:
  • the modulator is a compound of structural formula (XI):
  • each of Ri, R 2 , R 3 , * , R 5 , R 7 or R 8 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -(CH 2 ) m N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 ,
  • each R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C,o)a ⁇ kyl(C,-C,o)alkyl, -O(C,-C 10 )alkyl, -C(O)(C,-C ⁇ 0 )alkyl,
  • C 10 )alkyl ), -CO 2 (C ⁇ -C 10 )alkyl, -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl,
  • C 10 )alkyl or -SO 2 NH 2 ;
  • m is independently an integer ranging from 0 to 8;
  • p is independently an integer ranging from 0 to 5;
  • Ri and R 2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 2 and R 3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 3 and t can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R and R 7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R 7 and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
  • R ⁇ and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • R 2 and R 3 together form a 5-membered ring.
  • Illustrative examples of the modulators of the invention include:
  • Another illustrative compound of the invention has the following structure:
  • the modulator is a compound of structural formula (XII):
  • each of Ri, R 2 , R 3 , R 4 , R 5 or R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -(CH 2 ) m N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -C(OH)R 5 , -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C l0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C M
  • each R 5 or Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • m is independently an integer ranging from 0 to 8;
  • p is independently an integer ranging from 0 to 5;
  • R 3 or can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring.
  • R ⁇ or R 2 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring;
  • R 2 or R can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring.
  • modulators of the invention include:
  • the compounds depicted in Scheme 1 are compounds of structural formula (I).
  • compounds of structural formula (I) may be made by the route depicted in Scheme 1.
  • Condensation of commercially available thiosemicarbazide 1 with acetophenone 3 in the presence of acid, (e.g., acetic acid) provides thiosemicarbazone 5.
  • acid e.g., acetic acid
  • strong base e.g., lithium diisopropylamide
  • the compounds depicted in Scheme 2 are compounds of structural formula (IX).
  • compounds of structural formula (IX) may be made by the route depicted in Scheme 2.
  • Unsubstituted or substituted pyridyl hydrazine 2 is reacted with unsubstituted or substituted benzoic acid 1 in the presence of l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1), 4- methylmorpholine and 1-hydroxybenzotriazole hydrate (HOBt), in anhydrous 1 :1 dichloromethane/acetonitrile.
  • Phosphorous oxychloride is then added to the solution of resulting compound 3 in toluene, and the compound of formula (IX) 4 is obtained.
  • Illustrative compounds 145, 147, 149, 151, 153, 155, 157 and 159 are commercially available from Specs (http//:www.specsnet.com); compounds 163, 165 and 167 are available from Chemdiv (http//www.chemdiv.com); compound 161 is available from Tripos (http://www.tripos.com); and compound 169 is available for purchase from Comgenex (http://www.comgenex.com).
  • the present invention provides a method of modulating an LPA3 or Edg-7 receptor (e.g. human Edg-7, GenBank Accession No. AF127138) mediated biological activity.
  • a cell expressing the Edg-7 receptor is contacted with an amount of an Edg- 7 receptor agonist or antagonist sufficient to modulate the Edg-7 receptor mediated biological activity.
  • the agonists or antagonists can be utilized in the methods of the present invention and are compounds of structural formula (XIII):
  • X is NR 3 , S or O
  • R] is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, amino, carbamoyl, substituted carbamoyl, oxo, thiono or -NR 4 ;
  • R 2 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkyloxy, substituted alkyloxy, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, aryloxycarbonyl, substituted aryloxycarbonyl, arylalkyl, substituted arylalkyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted
  • heteroaryloxy substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or R is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkylsulfonyl, substituted alkylsulfonyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl or substituted heteroalkyl;
  • R 4 is alkyl, substituted alkyl, acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, heteroaryl substituted heteroaryl, cycloheteroalkyl, substituted cycloheteroalkyl, and
  • R 5 and R ⁇ s are independently hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylthio, substituted alkylthio, alkoxycarbonyl, substituted alkoxycarbonyl, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or optionally along with the carbon to which they are attached form an aryl, substituted aryl, cycloalky
  • the modulator is a compound of structural formula:
  • Ri is oxo, thiono or NJL t , more preferably, Rj is oxo or R t .
  • R 4 is acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl or substituted carbamoyl.
  • t is substituted carbamoyl..
  • R 2 is acyl, substituted acyl, acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, aryloxycarbonyl, substituted aryloxycarbonyl, carbamoyl, substituted carbamoyl, or
  • R 2 is substituted alkoxycarbonyl or
  • R 5 and R 6 along with the carbon to which they are attached form a cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl ring. More preferably, R 5 and Re along with the carbon to which they are attached form a substituted cycloheteroalkyl ring.
  • the agonists or antagonists are also compounds of structural formula (XIV):
  • each of Ri, R 2 , R 3 ⁇ and R is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C 10 )alkyl, -(C 2 -C l0 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -Cj 4 )bicycloalkyl, -(C 5 -C ⁇ o)cyclo
  • each R 5 and R is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C ⁇ o)alkyl(C,-C ⁇ 0 )alkyl, -O(C,-C ⁇ o)alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl, -C(O)NH(CH 2 ) m (d-C 10 )alkyl, -OCF 3 , -benzyl, -C0 2 (CH 2 ) m CH((Ci-Cio)alkyl(Ci- C ⁇ o)alkyl), -CO 2 (C ⁇ -C 10 )alkyl, -(C,-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl
  • Ri, R 2 , R 3 R and R taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring; Ri , R 2 , R 3 R and R 7 can also be an electron such that when two groups are on adjacent carbon atoms they form a double bond; two R ⁇ groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • the modulator is a compound of structural formula (XV):
  • each of Ri, R 2 , R 3 R 4 and R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C ⁇ o)alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkeny
  • each R 5 and R is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C ⁇ o)aIkyl(C ⁇ -C ⁇ o)alkyl, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl, -C(O)NH(CH 2 ) m (d-C 10 )alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C 1 -C 10 )alkyl(d- C ⁇ o)alkyl), -C0 2 (C ⁇ -C ⁇ o)alkyl, -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(
  • X is C, or N
  • Ri, R 2 , R R 4 and R 7 taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring;
  • Ri, R , R 3 R 4 and R 7 can also be an electron such that when two groups are on adjacent carbon atoms they form a double bond; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • the compound is of formula:
  • the modulator is of structural formula (XVI):
  • each of Ri, R 2 , R 3 R 4 and R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-Co)alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl
  • each R 5 and Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C ⁇ o)alkyl(C ⁇ -C,o)alkyl, -O(d-d 0 )alkyl, -C(O)(C,-C, 0 )alkyl, -C(O) H(CH 2 ) m (C,-C ⁇ o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((d-do)alkyl(d- C ⁇ o)alkyl), -CO 2 (C,-C ⁇ 0 )alkyl, -(C ⁇ -C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C, 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl,
  • the a modulator is of structural formula (XVII):
  • each of Ri, R 2> R 3 R 4 and R 7 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ 0 )cycloalkyl, -(C 8 -C] 4 )bicycloalkyl, -(C 5 -C !
  • each R 5 and Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C,o)alkyl(C 1 -C 1 o)alkyl, -O(C,-C,o)alkyl, -C(O)(d-C 10 )alkyl, -C(O)NH(CH 2 ) m (C,-C,o)alkyl, -OCF 3 , -benzyl, C 10 )alkyl), -CO 2 (d-C 10 )alkyl, -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C 1 o)cyclo
  • the modulator is of formula:
  • Edg-7 receptor is a G protein coupled receptor ("GPCR").
  • GPCR G protein coupled receptor
  • the Edg-7 (LPA3) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-2 (LPAl) and Edg-4 (LPA2), binds lysophosphatidic acid (“LPA”).
  • LPA lysophosphatidic acid
  • the Edg-7 receptor is a human receptor.
  • the Edg-7 receptor may be expressed by recombinant DNA methods well known to those of skill in the art.
  • Particularly useful cell types for expressing and assaying Edg-7 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells).
  • Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA).
  • DNA encoding Edg-7 (human Edg-7, GenBank accession AFOl 1466) can be transfected into human or mammalian cells according to methods known to those of skill in the art.
  • DNA encoding human Edg-7 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
  • DNA encoding Edg-7 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the env gene to produce an ecotropic envelope.
  • Both methods i.e., co-transfection with a packaging vector or use of EcoPack-293
  • AmphoPack-293 cell line can be used (Clontech Labs, Palo Alto, CA).
  • Edg-7 receptors include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB- 453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells and OV202 human ovarian cancer cells (ATCC, Manassas, VA; Vec Technologies Inc. (Rensselaer, NY); Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA).
  • cells which express the Edg-7 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulation of Edg-7 receptor activity, regardless of the local environment.
  • cells that express the Edg-7 receptor are grown in vitro (i.e., are cultured).
  • cells that express the Edg-7 receptor are in vivo (i.e., are part of a complex organism).
  • the cells in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, carcinoma cells, pheochromocytoma cells, myoblast cells, endothelial cells, platelet cells and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, OV202 human ovarian cells, hepatoma cells (e.g.
  • HTC, Rh7777, HepG2 SKOV3 human ovarian cancer cells, CAOV-3 human ovarian cancer cells, HEY human ovarian cancer cells, HTC rat hepatoma cells, CAOV-3 human ovarian cancer cells, MDA- MB-453 breast cancer cells, MDA-MB-231 breast cancer cells, A431 human epitheloid carcinoma cells and HT-1080 human fibrosarcoma cells.
  • an Edg-7 receptor mediated biological activity is modulated in a subject or in an animal model.
  • a therapeutically effective amount of an modulator of the Edg-7 receptor is administered to the subject or animal.
  • the subject or an animal is in need of such treatment.
  • the biological activity mediated by the Edg-7 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation or actin polymerization.
  • the biological activity mediated by the Edg-7 receptor also includes, but is not limited to, apoptosis, angiogenesis, inhibition of wound healing, inflammation, cancer invasiveness or atherogenesis.
  • the biological activity mediated by the Edg-7 receptor is cell proliferation, which may lead to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer or prostrate cancer.
  • cell proliferation is stimulated by LPA.
  • the biological activity mediated by the Edg-7 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS”) and acute inflammatory exacerbation of chronic lung diseases like asthma.
  • ARDS adult respiratory distress syndrome
  • chronic lung diseases like asthma.
  • compounds that block Edg-7 can be potentially effective immunosuppressive agents because activated T cells have Edg-7 receptors (Zheng et al, 2000, FASEB J 14:2387-2389).
  • Edg-7 antagonists may be useful in a variety of autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-7 antagonists can be used in organ transplantation.
  • the modulator exhibits inhibitory selectivity for the Edg-7 receptor.
  • the modulator can exhibit at least about 200 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
  • Inhibitory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.4 (Example 4), 6.6 (Example 6) and 6.7 (Example 7) respectively.
  • Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art.
  • Preferred assays include the calcium mobilization assay of Section 6.5.
  • the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-7 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors. In a preferred embodiment, an modulator of cell proliferation exhibits at least about 100 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 20 fold inhibitory selectivity for Edg-7 relative to other Edg receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 10 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the modulator exhibits stimulatory selectivity for the Edg-7 receptor.
  • the modulator can exhibit at least about 200 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
  • Stimulatory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.5 (Example 5), 6.7 (Example 7) and 6.8 (Example 8) respectively.
  • Other assays suitable for determining stimulatory selectivity would be known to one of skill in the art.
  • Preferred assays include the calcium mobilization assay of Section 6.5.
  • the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator exhibits at least about 10 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors. In still another embodiment, the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the modulator exhibits at least about 10 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • an modulator of cell proliferation exhibits at least about 100 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 20 fold stimulatory selectivity for Edg-7 relative to other Edg receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 10 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
  • the Edg-7 modulator is not a lipid. In another embodiment, the Edg-7 modulator does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester. In another embodiment, the Edg-7 modulator is not a phospholipid.
  • phospholipid includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of greater 10 carbon atoms or greater, any N- acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al., 1996, J.
  • the Edg-7 modulator is not a compound of structural formula (XVIII):
  • X is O or S
  • R 2 o is alkyl, substituted alkyl, aryl, substituted aryl or halo
  • R 21 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
  • R 23 is hydrogen, alkyl or substituted alkyl
  • R 24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl
  • R 2 and R 24 form a cycloalkyl ring (International Application No: WO 01/60819).
  • the modulator is not any compound of the formula below:
  • R 0 , R 2 ⁇ and R 24 are as previously defined.
  • the modulator is not any compound disclosed in International Application No: WO 01/60819.
  • the modulator is a agonist of the Edg-7 receptor.
  • the modulator can be a weaker agonist than the natural agonist and may compete with the natural agonist for the binding site.
  • the modulator is antagonist of the Edg-7 receptor.
  • the Edg-7 modulator may be a biomolecule such as a nucleic acid, protein, (i.e., an enzyme or an antibody) or oligosaccharide or any combination thereof.
  • the Edg-7 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof.
  • the Edg-7 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
  • the Edg-7 modulator may also be an organic molecule of molecular weight less than 750 daltons.
  • the molecular weight is about 200 to about 1000 daltons.
  • the molecular weight is about 200 to about 750 daltons.
  • the molecular weight is about 200 to about 500 daltons.
  • the molecular weight is about 300 to about 500 daltons.
  • the modulator may, for example, facilitate inhibition of the Edg-7 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the
  • the agonist or antagonist binds to the Edg-7 receptor with a binding constant between about 10 ⁇ M and about 1 fM.
  • the modulator binds to the Edg-7 receptor with a binding constant between about 10 ⁇ M and about 1 nM.
  • the modulator binds to the Edg-7 receptor with a binding constant between about 1 ⁇ M and about 1 nM.
  • the modulator binds to the Edg-7 receptor with a binding constant between about 100 nM and about 1 nM.
  • the modulator binds to the Edg-7 receptor with a binding constant between about 10 nM and about 1 nM.
  • the modulator binds to the Edg-7 receptor with a binding constant better (i.e., less) than about 10 nM.
  • the compounds of the invention may be obtained via the synthetic methods illustrated in Schemes 3 and 4.
  • Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods.
  • Other methods for synthesis of the compounds described herein are either described in the art or will be readily apparent to the skilled artisan in view of general references well-known in the art (See e.g., Green et al, "Protective Groups in Organic Chemistry", (Wiley, 2 nd ed. 1991); Harrison et al, "Compendium of Synthetic Organic Methods", Vols.
  • analogues of 5 may be prepared simply by using different amines 1 and or isocyanates 3.
  • compounds other than the isocyanate 3 depicted may be reacted with amine 1 to provide compounds of the invention.
  • a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (I) other than those depicted above.
  • indolone 15 which may be alkylated, arylated, acylated or sulfonated by treatment with appropriate compounds to provide indolone 17.
  • acid and salt e.g., acetic acid and sodium acetate
  • indolone 15 which may be alkylated, arylated, acylated or sulfonated by treatment with appropriate compounds to provide indolone 17.
  • the alkylation, arylation, acylation or sulfontion can take place at either or both of the location indicated with a dashed bond.
  • analogues of 17 may be prepared simply by using different alkylation, arylation, acylation or sulfontion agents.
  • a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (I) other than those depicted above.
  • Illustrative compounds 709, 713, 715 and 723 are also commercially available from Specs (http//www.specs.net); illustrative compound 719 is commercially available from Asinex; illustrative compound 725 is commercially available from Tripos. Illustrative compounds 701, 711, and 719 are commercially available from Asinex. Illustrative compounds 717 is available from Labotest. Illustrative compound 727 is commercially available from Chemdiv. Illustrative compounds 729, 733, 735, and 737 are commercially available from Specs.
  • the present invention provides a method of modulating an LPAl or Edg-2 receptor (e.g., human Edg-2, GenBank Accession No., U78192) mediated biological activity.
  • a cell expressing the Edg-2 receptor is contacted with an amount of an Edg-2 receptor agonist or antagonist sufficient to modulate the Edg-2 receptor mediated biological activity.
  • the Edg-2 receptor is a G protein coupled receptor.
  • the Edg-2 (LPAl) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-4 (LPA2) and Edg-7 (LPA3), binds lysophosphatidic acid ("LPA").
  • the Edg-2 receptor is a human receptor.
  • the Edg-2 receptor may be expressed by recombinant DNA methods well known to those of skill in the art.
  • Particularly useful cell types for expressing and assaying Edg-2 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells).
  • Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA).
  • DNA encoding Edg-2 is well known (e.g., human Edg-2, GenBank Accession No., U78192) and can be transfected into human or mammalian cells according to methods known to those of skill in the art.
  • DNA encoding human Edg- 2 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
  • DNA encoding Edg-2 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the env gene to produce an ecotropic envelope.
  • Both methods i.e. co-transfection with a packaging vector or use of EcoPack-293
  • AmphoPack-293 cell line can be used (Clontech Labs, Palo Alto, CA).
  • Edg-2 receptors include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB- 453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells and OV202 human ovarian cancer cells (ATCC, Manassas, VA; Vec Technologies Inc. (Rensselaer, NY); Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA).
  • cells which express the Edg-2 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulation of the Edg-2 receptor activity regardless of the local environment.
  • cells that express the Edg-2 receptor are grown in vitro (i.e., are cultured).
  • cells that express the Edg-2 receptor are in vivo (i.e., are part of a complex organism).
  • the cells, in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, carcinoma cells, pheochromocytoma cells, myoblast cells, endothelial cells, platelet cells and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, OV202 human ovarian cell, HTC rat hepatoma cells, CAOV-3 and SKOV-3 human ovarian cancer cells, MDA-MB-453 breast cancer cells, MDA-MB-231 breast cancer cells, A431 human epitheloid carcinoma cells and HT-1080 human fibrosarcoma cells.
  • an Edg-2 receptor mediated biological activity is modulated in a subject or in an animal model.
  • a therapeutically effective amount of an modulator of the Edg-2 receptor is administered to the subject or an animal.
  • the subject or animal is in need of such treatment.
  • the biological activity mediated by the Edg-2 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of c AMP formation or actin polymerization.
  • the biological activity mediated by the Edg-2 receptor includes, but is not limited to, apoptosis, angiogenesis, inhibition of wound healing, inflammation, cancer invasiveness or atherogenesis.
  • the biological activity mediated by the Edg-2 receptor is cell proliferation, which may lead to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colon cancer or prostrate cancer. In one embodiment, cell proliferation is stimulated by LPA.
  • the biological activity mediated by the Edg-2 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS”) and acute inflammatory exacerbation of chronic lung diseases like asthma.
  • ARDS adult respiratory distress syndrome
  • chronic lung diseases like asthma.
  • compounds that block Edg-2 can be potentially effective immunosuppressive agents because activated T cells have Edg-2 receptors (Zheng et al, 2000, FASEB J 14:2387-2389).
  • Edg-2 antagonists may be useful in a variety of autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-2 antagonists can be used in organ transplantation.
  • the modulator exhibits selectivity for the Edg-2 receptor.
  • the modulator exhibits at least about 5 to about 200 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
  • Inhibitory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.8 (Example 8), 6.10 (Example 10) and 6.11 (Example 11) respectively.
  • inhibitory selectivity can be measured by a calcium mobilization assay.
  • Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art.
  • the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to other non-Edg receptors, GPCRs, growth factor receptors, ion channels and the like.
  • the modulator exhibits at least about 40 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
  • the modulator exhibits at least about 12 fold inhibitory selectivity for Edg-2 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator exhibits at least about 40 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator exhibits at least about 12 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator exhibits at least about 5 inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • an modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors. In another embodiment, the modulator exhibits activating selectivity for the
  • the modulator exhibits at least about 5 to about 200 fold activating selectivity for Edg-2 relative to other Edg receptors.
  • Activating selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.8 (Example 8), 6.10 (Example 10) and 6.11 (Example 11) respectively.
  • activating selectivity can be measured by a calcium mobilization assay.
  • Other assays suitable for determimng activating selectivity would be known to one of skill in the art.
  • the modulator exhibits at least about 200 fold activating selectivity for Edg-2 relative to other non-Edg receptors, GPCRs, growth factor receptors, ion channels and the like.
  • the modulator exhibits at least about 40 fold activating selectivity for Edg-2 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 12 fold activating selectivity for Edg-2 relative to other Edg receptors.
  • the modulator exhibits at least about 5 fold activating selectivity for Edg-2 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator exhibits at least about 40 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator exhibits at least about 12 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors. In still another embodiment, the modulator exhibits at least about 5 activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • an modulator of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-2 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold activating selectivity for Edg-2 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
  • the Edg-2 modulator is not a lipid.
  • the modulator of Edg-2 modulator does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester.
  • the Edg-2 modulator is not a phospholipid.
  • phospholipid includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of 10 carbon atoms or greater, dioctyl glycerol, any N-acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al., 1996, J.
  • the Edg-2 modulator is not a compound of the formula:
  • X is O or S
  • R 2 o is alkyl, substituted alkyl, aryl, substituted aryl or halo
  • R 21 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
  • R 2 is hydrogen, alkyl or substituted alkyl
  • R 24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl
  • R 23 and R 24 form a cycloalkyl ring (International Application No: WO 01/60819).
  • the modulator is not any compound of the formula below:
  • R 2 o, R 2 ⁇ and R 24 are as previously defined.
  • the modulator is not any compound disclosed in International Application No: WO 01/60819.
  • the Edg-2 modulator is an agonist of the Edg-2 receptor.
  • such an embodiment provides an Edg-2 modulator that is an agonist, but is a weaker agonist than a natural Edg-2 agonist (e.g., LPA) and as such, may compete with the natural agonist for Edg-2 binding site, resulting in a net inhibition of Edg-2 receptor activity.
  • a natural Edg-2 agonist e.g., LPA
  • the modulator is antagonist of the Edg-2 receptor.
  • the Edg-2 modulator may be a biomolecule such as a nucleic acid, protein (e.g., an enzyme, an antibody or a soluble Edg-2 receptor polypeptide) or oligosaccharide or any combination thereof.
  • the Edg-2 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof.
  • the Edg-2 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
  • the Edg-2 modulator may also be a small organic molecule.
  • a small organic molecule exhibits a molecular weight about 200 to about 1000 daltons, about 200 to about 750 daltons, 200 to about 500 daltons, or about 300 to about 500 daltons.
  • the small organic molecule can be orally administered to a subject.
  • the small organic molecule is capable of crossing the blood-brain barrier.
  • the modulator may, for example, facilitate inhibition of the Edg-2 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the Edg-2 receptor, interference with Edg-2 or LPA biosynthesis, covalent modification of either LPA or the Edg-2 receptor, or may otherwise interfere with Edg-2 mediated signal transduction.
  • the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 10 ⁇ M and 1 fM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 10 ⁇ M and about 1 nM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 1 ⁇ M and about 1 nM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the agonist or antagonist binds to the Edg-2 receptor with a binding constant better (i.e., less) than about 10 nM.
  • the Edg-2 modulator has the structural formula (XX):
  • P, Q and R are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl.
  • Q is cycloheteroalkyl, substituted cycloheteroalkyl and P and R are independently aryl or substituted aryl.
  • Q is cycloheteroalkyl or substituted cycloheteroalkyl and P and R are independently phenyl or substituted phenyl.
  • Q is heteroaryl or substituted heteroaryl and P and R are independently aryl or substituted aryl.
  • Q is heteroaryl or substituted heteroaryl and P and R are independently phenyl or substituted phenyl.
  • the Edg-2 modulator has the structural formula (XXI):
  • n 1, 2 or 3;
  • R ⁇ and R 2 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cyano, cyanato, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, oxo or thiono;
  • R 3 and R are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halo or thio;
  • B is NR 5 , O or S
  • R 5 is hydrogen, alkyl, substituted alkyl, alkylamino, substituted alkylamino, alkoxy, substituted alkoxy, amino, cyano, dialkylamino, substituted dialkylamino or hydroxy;
  • a and C are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl.
  • Ri and R 2 are independently hydrogen, alkyl substituted alkyl, oxo or thiono. More preferably, Ri and R 2 are independently oxo or thiono.
  • R 3 and R are independently hydrogen or alkyl.
  • a and C are independently aryl, substituted aryl, heteroaryl or substituted heteroaryl. More preferably, A and C are aryl or substituted aryl. Most preferably, A and C are phenyl or substituted phenyl.
  • B is NR 5 and R 5 is hydrogen, alkyl or hydroxy.
  • n is 1
  • Ri and R 2 are oxo
  • R and R 4 are hydrogen
  • B is NR 5 and R 5 is hydroxy.
  • n is 1 , Ri and R 2 are oxo, R 3 and R t are hydrogen, B is NR 5 , R 5 is hydroxy, A and B are aryl or substituted aryl.
  • n is 1 , Ri and R 2 are oxo, R 3 and R 4 are hydrogen, B is NR 5 , R 5 is hydroxy, A and B are phenyl or substituted phenyl.
  • the Edg-2 modulators can also include the following compounds.
  • the modulator has the structural formula (XXII):
  • R 3 ⁇ is hydrogen, alkyl or substituted alkyl
  • R 32 is hydrogen, alkyl or substituted alkyl
  • R 3 is aryl, substituted aryl, heteroaryl or substituted heteroaryl
  • R 34 is aryl, substituted aryl, heteroaryl or substituted heteroaryl.
  • R 31 and R 2 are alkyl.
  • R 3 and R 3 are aryl or substituted aryl.
  • R 3 ⁇ and R 32 are alkyl and R 3 and R 34 are aryl or substituted aryl.
  • R 3 and R 34 are phenyl or substituted phenyl.
  • R 3 ⁇ and R 2 are methyl or ethyl and R 33 and R 4 are phenyl or substituted phenyl.
  • the Edg-2 modulator has the structural formula (XXIII):
  • each of R R 2 and R 3 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C 10 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -d 0 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C
  • each R 5 and R ⁇ is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C ⁇ o)alkyl(C 1 -C ⁇ o)alkyl, -O(C,-C ⁇ o)alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl, -C(O)NH(CH 2 ) m (C 1 -C,o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((d-C 10 )alkyl(d- C ⁇ o)alkyl), -CO 2 (d-C ⁇ 0 )alkyl, -(C,-C ⁇ 0 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C -C ⁇ o)cycloalkyl, -(C
  • Ri and R 2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
  • X and Y are both O.
  • Ri is -N(OH)aryl.
  • the Edg-2 modulator has the structural formula (XXIV):
  • each of Ri and R 2 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C ⁇ o)alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C )
  • each of R 5 and Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C ⁇ -C ⁇ o)alkyl(C ⁇ -C ⁇ o)alkyl, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH 2 ) m (d-do)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C 1 -C 10 )alkyl(d- C ⁇ o)alkyl), -CO 2 (d-C 10 )alkyl, -(d-C, 0 )alkyl, -(C 2 -C, 0 )alkenyl, -(C 2 -C 10 )alkynyl, -(C -C 10 )cycloalkyl, -(C 8 -C
  • Ri is -H
  • R 2 and R 3 are independently -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C ⁇ o)alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C ⁇ 0 )cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C !
  • each of R 5 and R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -O(C r C ⁇ o)alkyl, -C(O)(C,-do)alkyl, -C(O)NH(CH 2 ) m (C,-C ⁇ o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C ⁇ -C 10 )alkyl(C,-C,o)alkyl), -CO 2 (C,-C ⁇ 0 )alkyl, -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C ]0 )alkynyl, -(C 3 -C ⁇ 0 )cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C
  • each Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(Ci-C, 0 )alkyl, -C(O)NH(CH 2 ) m (C ⁇ -d 0 )alkyl, -OCF 3 , -benzyl,
  • -CO 2 (CH 2 ) m CH((d-C 1 o)alkyl(C,-C 1 o)alkyl), -CO 2 (d-C 1 o)alkyl, -(C 1 -C 10 )alkyl, -(C 2 -C ⁇ o)alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C 10 )cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -phenyl, naphthyl, -(C 3 -C, 0 )heterocycle, -CO 2 (CH 2 ) m (d-C 10 )alkyl, -CO 2 (CH 2 ) m H, -NHC(O)(d
  • the Edg-2 modulators can also mclude the following compounds:
  • a prefened Edg-2 modulator is a compound of the formula below:
  • the Edg-2 modulators have the structural formula (XXV):
  • each of R! and R 2 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -NH(aryl), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -aryl, -(C 2 -C 10 )alkynyl, -(C 3 -C 10 )cycloalkyl, -(C 3 -d 0 )cycloalkyl(aryl), -(C 8 -C 14
  • each R 5 and R ⁇ 5 is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • C ⁇ o)alkyl -CO 2 (d-C l0 )alkyl, -(C,-C l0 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C 1 o)cycloalkenyl, -(C 5 )heteroaryl,
  • -N C(aryl), -OC(O)O(d-C 10 )alkyl, or -SO 2 NH 2 ;
  • Ri and R 2 can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • the Edg-2 modulator can also preferably be a compound of the structural formula (XXVI):
  • R is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH,
  • each R 5 and Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(d-do)alkyl(C ⁇ -C 10 )alkyl, -O(C,-C, 0 )alkyl, -C(O)(C ⁇ -C ⁇ o)alkyl,
  • R 7 is -CO 2 H, -C(O)(d-C, 0 )alkyl, -C(O)NH(CH 2 ) m (d-C ⁇ 0 )alkyl, -benzyl, -CO 2 (CH2) m CH((C 1 -C 1 o)alkyl(C 1 -do)alkyl), -CO 2 (C 1 -C 1 o)alkyl, -(C 1 -C 1 o)alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C 3 -C 10 )cycloalkyl, -(C 8 -C M )bicycloalkyl, -(C 5 -C]o)cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -phenyl, naphth
  • the Edg-2 modulators can also include the following compounds:
  • the Edg-2 modulators of the invention have the structural formula (XXVII):
  • each of R, and R 2 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -do)alkyl, -(C 2 -C ⁇ 0 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C -C ⁇ o)cycloalkyl, -(C 8 -C] 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -(C 5 -do)heteroaryl, -naphthyl, -(C 3 -do)heter
  • each R and Re is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • Ri is -NH(aryl).
  • the Edg-2 modulator has the structural formula (XXVIII):
  • each of Ri, R 2 and R 4 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 ,
  • -benzyl -CO 2 CH(R 5 )(R 5 ), -(d-C 10 )alkyl, -(C 2 -C ⁇ o)alkenyl, -(d-C ⁇ alkynyl, -(C 3 -do)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl,
  • each R 5 and Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C ⁇ o)alkyl(C ⁇ -C ⁇ o)alkyl, -O(C ⁇ -C ⁇ o)alkyl, -C(O)(C ⁇ -C 10 )alkyl, -C(O)NH(CH 2 ) m (C ⁇ -C ⁇ o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C,-do)alkyl(d- C,o)alkyl), -CO 2 (C ⁇ -C ⁇ 0 )alkyl, -(C ⁇ -C 10 )alkyl, -(C 2 -C ⁇ o)alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C !
  • the Edg-2 modulators can also include the following compounds:
  • Michael addition of aromatic amine 3 to maleic anhydride 1 provides amide 5, which may be cyclized (e.g., sodium acetate and acetic anhydride) to yield imide 7.
  • Michael addition of phenylhydroxylamine which may be prepared from nitrobenzene by partial reduction (e.g., Zn, NH 4 C1) gave the desired disubstituted imide 11.
  • a large number of analogues of 11 may be prepared simply by using different amines 3 and or hydroxylamines other than phenyl hydroxylamine.
  • acylated phenol 21 e.g., NBS, AIBN
  • bromide 23 which may be converted to ketone 25 (CrCl 2 ) by internal acyl transfer.
  • Ketone 25 may be acylated to provide benzoate 27, (e.g., benzoyl chloride, dimethylaminopyridine) which can undergo cyclization to give pyrazole 29 (e.g., ethylhydrazine, acetic acid).
  • benzoate 27 e.g., benzoyl chloride, dimethylaminopyridine
  • pyrazole 29 e.g., ethylhydrazine, acetic acid
  • the present invention provides a method of modulating an SI P3 or Edg-3 receptor (e.g., human Edg-3, GenBank Accession No. X83864) mediated biological activity.
  • a cell expressing the Edg-3 receptor is contacted with an amount of an Edg- 3 receptor agonist or antagonist sufficient to modulate an Edg-3 receptor mediated biological activity.
  • Edg-3 is a G protein coupled receptor ("GPCR").
  • GPCR G protein coupled receptor
  • the Edg-3 (S1P3) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-1 (SlPl), Edg-5 (S1P2), Edg-6 (S1P4) and Edg-8 (S1P5), binds sphingosine- 1-phosphate (“SIP").
  • SIP sphingosine- 1-phosphate
  • the Edg-3 receptor is a human receptor.
  • the Edg-3 receptor may be expressed by recombinant DNA methods well known to those of skill in the art.
  • Particularly useful cell types for expressing and assaying Edg-3 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells).
  • Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA).
  • DNA encoding Edg-3 is well known (e.g., human Edg-3, GenBank Accession
  • DNA encoding human Edg- 3 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
  • DNA encoding Edg-3 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the env gene to produce an ecotropic envelope.
  • Both methods i.e., co-transfection with a packaging vector or use of EcoPack-293
  • AmphoPack-293 cell line can be used (Clontech, Palo Alto, CA).
  • a number of natural cell lines naturally express Edg-3 receptors. These include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB-453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells, OV202 human ovarian cancer cells, Hela human cervical adenocarcinoam cells, HEK293 human embryonic kidney cells, NIH 3T3 mouse fibroblast cells (ATCC, Manassas, VA; Vec Technologies Inc., Rensselaer, NY; Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA).
  • CaOV-3 human ovarian cancer cells include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB-453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells, OV202 human ovarian cancer cells, Hela human cervical adenocarcinoam cells, HEK293 human embryonic kidney cells,
  • cells which express the Edg-3 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulating, e.g., agonizing or antagonizing, Edg-3 receptor activity, regardless of the local environment.
  • cells that express the Edg-3 receptor are grown in vitro (i.e., are cultured).
  • cells that express the Edg-3 receptor are in vivo (i.e., are part of a complex organism).
  • the cells in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, carcinoma cells, pheochromocytoma cells, myoblast cells, platelet cells, endothelial cells, keratinocytes and fibrosarcoma cells.
  • the cells in which the invention may be practiced include, but are not limited to, OV202 human ovarian cells, HTC rat hepatoma cells, CAOV-3 and SKOV-3 human ovarian cancer cells, MDA-MB-453 breast cancer cells, MDA-MB- 231 breast cancer cells, HUVEC, Hela human cervical adenocarcinoam cells, HEK293 human embryonic kidney cells, NIH 3T3 mouse fibroblast cells, A431 human epitheloid carcinoma cells, and HT-1080 human fibrosarcoma cells.
  • an Edg-3 receptor mediated biological activity is modulated in a subject or in an animal model.
  • a therapeutically effective amount of an modulator of the Edg-3 receptor is administered to the subject or animal.
  • the subject or animal is in need of such treatment.
  • the biological activity mediated by the Edg-3 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation or actin polymerization.
  • the biological activity mediated by the Edg-3 receptor also includes, but is not limited to, apoptosis, angiogenesis, wound healing, inflammation, expression of endogenous protein growth factors, cancer invasiveness or atherogenesis.
  • the biological activity mediated by the Edg-3 receptor is cell proliferation, which may lead to enhancement of wound healing; alternatively, it may lead ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer or prostrate cancer.
  • cell proliferation is stimulated by SIP.
  • the biological activity mediated by the Edg-3 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS”) and acute inflammatory exacerbation of chronic lung diseases like asthma.
  • ARDS adult respiratory distress syndrome
  • the present invention provides methods for using Edg-3 antagonists in treating or preventing disorders such as, but not limited to, vasoconstriction in cerebral arteries, autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia.
  • Edg-3 antagonists can also be used in organ transplantation. Without intending to be bound by any particular mechanism or theory of action, Edg-3 antagonists are believed to be potentially effective immunosuppresive agents because activated T cells express the Edg-3 receptor.
  • Edg-3 agonists and antagonists can be used to treat vascular occlusive disorders.
  • activation of Edg-3 receptors by using an Edg-3 agonist can result in increased vasoconstriction, which is beneficial in conditions such as migraine headaches.
  • Inhibition of Edg-3 by an Edg3 antagonist can be beneficial in conditions such as a stroke, a subarachnoid hemorrhage, or a vasospasm such as a cerebral vasospasm.
  • the modulator exhibits inhibitory selectivity for the Edg-3 receptor. In one embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. Inhibitory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.4 (Example 4), 6.6 (Example 6) and 6.7 (Example 7) respectively. Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art. Preferred assays include the calcium mobilization assay of Section 6.4. In another embodiment, the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator exhibits about 5 fold to about 200 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. In still another embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits about 5 fold to about 200 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator of cell proliferation exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits stimulatory selectivity for the Edg-3 receptor. In one embodiment, the modulator exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to other Edg receptors. Stimulatory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.4 (Example 4), 6.6 (Example 6) and 6.7 (Example 7) respectively. Other assays suitable for determining stimulatory selectivity would be known to one of skill in the art. Preferred assays include the calcium mobilization assay of Section 6.4. In another embodiment, the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-3 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator exhibits about 5 fold to about 200 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors. In still another embodiment, the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator exhibits about 5 fold to about 200 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
  • the modulator of cell proliferation exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the modulator of cell proliferation exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
  • the Edg-3 modulator is not a lipid.
  • the modulator of Edg-3 receptor mediated biological activity does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester.
  • the modulator of the Edg-3 receptor is not a phospholipid.
  • phospholipid includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of greater 10 carbon atoms or greater, dioctyl glycerol, any N-acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al., 1996, J.
  • the modulator is also not a compound of structural formula (XXXIV):
  • X is O or S
  • R 2 o is alkyl, substituted alkyl, aryl, substituted aryl or halo
  • R 21 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
  • R 23 is hydrogen, alkyl or substituted alkyl
  • R 24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl
  • R 23 and R 24 form a cycloalkyl ring (International Application No: WO 01/60819).
  • the modulator is not any compound of the formula below:
  • R 2 o, R 2 ⁇ and R 24 are as previously defined.
  • the modulator is not any compound disclosed in International Application No: WO 01/60819.
  • the modulator can be an agonist of the Edg-3 receptor.
  • the agonist can be a weaker agonist than the natural agonist and may compete with the natural agonist for the binding site.
  • the modulator can be an antagonist of the Edg-3 receptor.
  • the Edg-3 modulator can be a biomolecule such as a nucleic acid, protein, (e.g., an enzyme, an antibody, or a soluble Edg-3 receptor polypeptide) or oligosaccharide or any combination thereof.
  • the Edg-3 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof.
  • the Edg-3 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
  • the Edg-3 modulator may also be a small organic molecule.
  • the Edg-3 modulator can be an organic molecule of molecular weight less than 750 daltons.
  • the molecular weight can be about 200 to about 1000 daltons.
  • the molecular weight can be about 200 to about 750 daltons.
  • the molecular weight can be about 200 to about 600 daltons.
  • the molecular weight is about 300 to about 500 daltons.
  • the small organic molecule can be orally administered to a subject. In other embodiments, the small organic molecule is capable of crossing the blood-brain barrier.
  • the modulator may, for example, facilitate inhibition of the Edg-3 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the Edg-3 receptor, interfering with Edg-3 or LPA biosynthesis, covalently modifying either the LPA or the Edg-3 receptor, or otherwise interfering with Edg-3 mediated signal transduction.
  • the modulator binds to the Edg-3 receptor with a binding constant between about 10 ⁇ M and about 1 fM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 10 ⁇ M and about 1 nM.
  • the modulator binds to the Edg-3 receptor with a binding constant between about 1 ⁇ M and about 1 nM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the modulator binds to the Edg-3 receptor with a binding constant better (i.e., less) than about 10 nM. In certain embodiments, the modulator is a compound of structural formula
  • n 0 or l
  • o is O, 1, 2, 3 or 4;
  • X is C, NR 7 O or S
  • Y is C, NR 8 O or S
  • Ri is either absent or hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamin
  • R 2 , R 3 and R are independently hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl
  • each R 5 is independently, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, azido, carboxy, carbamoyl, substituted carbamoyl, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substitute
  • R 7 and R 8 are independently absent, hydrogen, alkyl, substituted alkyl, acyl or substituted acyl.
  • the modulator is a compound of structural formula (XXXV) or structural formula (XXXVI):
  • R ⁇ is either absent or hydrogen, acyl, substituted acyl, acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylamino, substituted alkylamino, alkylarylamino, substituted alkylarylamino, arylamino, substituted arylamino, arylalkyloxy, substituted arylalkyloxy, carbamoyl, substituted carbamoyl, dialkylamino, substituted dialkylamino, heteroalkyl, or substituted heteroalkyl.
  • R t is either absent or acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, arylamino substituted arylamino, or carbamoyl, substituted carbamoyl.
  • Ri is either absent or acylamino, substituted acylamino, arylamino or substituted arylamino.
  • R 2 , R 3 and R 4 are independently alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, carbamoyl, substituted carbamoyl, dialkylamino, substituted dialkylamino, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl.
  • R 2 , R 3 and R 4 are independently alkyl, substituted alkyl, acylamino, substituted acylamino, aryl, substituted aryl, arylamino, substituted arylamino, carbamoyl or substituted carbamoyl. More preferably, R 2 , R 3 and R 4 are independently alkyl, substituted acylamino, aryl, substituted arylamino or substituted carbamoyl.
  • each R 5 is independently, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, amino, aryl, substituted aryl, azido, carboxy, carbamoyl, substituted carbamoyl, cyano, halo, hydroxyl, nitro or thio.
  • each R 5 is independently, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, azido, carboxy, carbamoyl, substituted carbamoyl, cyano, halo, hydroxyl, nitro or thio.
  • R 7 and R 8 are independently absent, hydrogen, alkyl.
  • the modulator is a compound of structural formula (XXXII):
  • each of Ri, R 2 and R 3 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-do)alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C ⁇ 0 )alkynyl, -(C -do)cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C )he
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C -C ⁇ o)cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -(C 5 -do)heteroaryl, -naphthyl, -(C 3 -C ⁇ o)hetero
  • each R 5 and R is independently -halo, -NO 2 , -CN, -OH, -CO 2 H,
  • -CO 2 (CH 2 ) m H, -NHC(O)(C ⁇ -C 10 )alkyl, -NHC(O)NH(C ⁇ -C ⁇ o)alkyl, -NH(aryl), -N C(aryl), -OC(O)O(d-C 10 )alkyl, or -SO 2 NH 2 ;
  • X is O, S, C(R 5 )(R 5 ) or N(R 5 );
  • R] R 2 or R 3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • the modulator is a compound of structural formula
  • the Edg-3 receptor modulator is a compound of structural formula (XXXVIII):
  • the Edg-3 receptor modulator has the structural formula (XXXIII):
  • each of R,, R 2 and R 3 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 , -(CH 2 ) m OH, -N(R 5 )(R 5 ), -O(CH 2 ) m R 5 , -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -OCF 3 , -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C ⁇ -C ⁇ o)alkyl, -(C 2 -C,o)alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C -C ⁇ o)cycloalkenyl, -(C
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(d-do)alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C 14 )bicycloalkyl, -(C 5 -do)cycloalkenyl, -(Cs)heteroaryl, -(C 6 )heteroaryl, -(C 5 -C ⁇ o)heteroaryl, -naphthyl, -(C 3 -C ⁇ 0 )he
  • each R 5 and Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C,-C,o)alkyl(C ⁇ -C ⁇ o)alkyl, -O(C ⁇ -C,o)alkyl, -C(O)(C,-C ⁇ o)alkyl, -C(O)NH(CH 2 ) m (C,-C,o)alkyl, -OCF 3 , -benzyl, -CO 2 (CH 2 ) m CH((C,-C 10 )alkyl(C 1 - C 10 )alkyl), -CO 2 (C,-C 10 )alkyl, -(d-C 10 )alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -C 10 )alkynyl, -(C 3 -C 1 o)cycloalkyl,
  • X is O, S, or N(R 5 ); Ri, R 2 or R 3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging
  • each of Ri , R 2 , R 3 R t , R 7 and R 8 is independently -H, -halo, -NO 2 , -CN, -C(R 5 ) 3 ,
  • R 3 is -H -C(R 5 ) 3 , -(CH 2 ) m OH, -C(O)R 5 , -C(O)NR 5 R 5 , -C(O)NH(CH 2 ) m (R 5 ), -benzyl, -CO 2 CH(R 5 )(R 5 ), -(C,-do)alkyl, -(C 2 -C 10 )alkenyl, -(C 2 -do)alkynyl, -(C 3 -C ⁇ o)cycloalkyl, -(C 8 -C ⁇ 4 )bicycloalkyl, -(C 5 -C ⁇ o)cycloalkenyl, -(C 5 )heteroaryl, -(C 6 )heteroaryl, -(C 5 -C ⁇ heteroaryl, -naphthyl, -(C 3 -C ⁇ o)he
  • each R 5 and Re is independently -H, -halo, -NO 2 , -CN, -OH, -CO 2 H, -N(C 1 -C 10 )alkyl(d-do)alkyl, -O(d-C, 0 )alkyl, -C(O)(C,-C 10 )alkyl,
  • Ri and R2, R2 and R 3 , R 3 and R4, R 4 and R 7 ⁇ or R and R taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two Re groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
  • Preferred Edg-3 modulators include the following compounds:
  • Preferred Edg-3 receptor modulators also include the following compounds:
  • Prefened Edg-3 receptor modulators also include the following compounds:
  • the compounds of the invention may be obtained via the synthetic methods illustrated in Schemes 7, 8, and 9.
  • Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods.
  • Other methods for synthesis of the compounds described herein are either described in the art or will be readily apparent to the skilled artisan in view of general references well-known in the art (See e.g., Green et al, "Protective Groups in Organic Chemistry", (Wiley, 2 nd ed. 1991); Harrison et al, “Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt, Germany; Feiser et al, "Reagents for
  • amide 301 Condensation of propiophenone 1 with isatin 3 (i.e., Pfitzinger reaction, KOH, ethanol, heat) provided quinoline 5, which was then acylated with fluoroamine 301 (i.e., carbodiimide, HOBT) to give amide 301.
  • fluoroamine 301 i.e., carbodiimide, HOBT
  • amide 301 analogues of amide 301 may be made by simply reacting substituted propiophenones with substituted isatins and/or acylating the resulting quinoline with different arylamines.
  • the compounds depicted in Schemes 8 and 9 are compounds of structural formula (XXXII).
  • compounds of structural formula (XXXII) may be made by the route depicted in Scheme 2. Reaction of amine 1 with isocyanate 3 in the presence of organic solvents, (e.g., benzene) provides substituted urea 5.
  • organic solvents e.g., benzene
  • a large number of analogues of 5 may be prepared simply by using different amines 1 and/or isocyanates 3.
  • a wide variety of compounds other than the isocyanate 3 depicted may be reacted with amine 1 to provide compounds of the invention.
  • a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (XXXII) other than those depicted above.
  • indolone 15 which may be alkylated, arylated, acylated or sulfonated by treatment with appropriate compounds to provide indolone 17.
  • acid and salt e.g., acetic acid and sodium acetate
  • indolone 15 which may be alkylated, arylated, acylated or sulfonated by treatment with appropriate compounds to provide indolone 17.
  • the alkylation, arylation, acylation or sulfontion can take place at either or both of the location indicated with a dashed bond.
  • analogues of 17 may be prepared simply by using different alkylation, arylation, acylation or sulfontion agents.
  • a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (II) other than those depicted above.
  • Illustrative compounds 305, 307, and 309 are commercially available from
  • Illustrative compounds 301, 311, 313, 315, 319, and 121 are commercially available from Asinex.
  • Compound 317 is commercially available from Chemdiv. 5.9.
  • Therapeutic Uses of the Compounds of the Invention may be used to treat diseases, including but not limited to, ovarian cancer (Xu et al, 1995, Biochem. J. 309 (Pt 3):933-940; Xu et al, 1998, JAMA 280 (8):719-723; Goetzl et al, 1999, Cancer Res.
  • peritoneal cancer endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma (Chilton et al, 1996, J Exp Med 183:2235-45; Arbibe et al, 1998, J Clin. Invest 102:1152-60) surface epithelial cell injury, (e.g., transcomeal freezing or cutaneous bums (Liliom et al, 1998, Am. J.
  • ARDS adult respiratory distress syndrome
  • a compound and/or composition of the invention is administered to a patient, preferably a human, in need of treatment for a disease which includes but is not limited to, the diseases listed above.
  • the compounds and/or compositions of the invention can be administered to a patient, preferably a human, as a preventative measure against diseases or disorders such as those described above.
  • the compounds and/or compositions of the invention can be administered as a preventative measure to a patient having a predisposition, which includes but is not limited to, the diseases listed above.
  • the compounds and/or compositions of the invention may be used for the prevention of one disease or disorder and concurrently treating another disease (e.g., preventing cancer and treating cardiovascular diseases). It is well within the capability of those of skill in the art to assay and use the compounds and/or compositions of the invention to treat diseases, such as the diseases listed above.
  • the compounds and/or compositions of the invention may be advantageously used in medicine, including human medicine.
  • compounds and compositions of the invention are useful for the treatment or prevention of diseases, which include but are not limited to, cancers, including, but not limited to, ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, acute lung diseases, including, but not limited to, adult respiratory distress syndrome (ARDS) and acute inflammatory exacerbation of chronic lung diseases such as asthma; surface epithelial cell injury, including, but not limited to, transcomeal freezing or cutaneous bums; cardiovascular diseases, including, but not limited to, ischemia and arthesclerosis.
  • diseases include but are not limited to, cancers, including, but not limited to, ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uter
  • compounds and/or compositions of the invention When used to treat or prevent disease or disorders, compounds and/or compositions of the invention may be administered or applied singly, in combination with other agents.
  • the compounds and/or compositions of the invention may also be administered or applied singly, in combination with other pharmaceutically active agents, including other compounds and/or compositions of the invention.
  • the current invention provides methods of treatment and prophylaxis by administration to a patient of a therapeutically effective amount of a composition or compound of the invention.
  • the patient may be an animal, is more preferably a mammal, and most preferably a human.
  • the present compounds and/or compositions of the invention are preferably administered orally.
  • the compounds and/or compositions of the invention may also be administered by any other convenient route, for example, by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local.
  • Various delivery systems are known, (e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc.) that can be used to administer a compound and/or composition of the invention.
  • Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the prefened mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition. In most instances, administration will result in the release of the compounds and/or compositions of the invention into the bloodstream.
  • This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of the diseases listed above.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • a compound and/or composition of the invention may also be administered directly to the lung by inhalation.
  • a compound and or composition of the invention may be conveniently delivered to the lung by a number of different devices.
  • a Metered Dose Inhaler which utilizes canisters that contain a suitable low boiling propellant, (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas) may be used to deliver compounds of the invention directly to the lung.
  • a suitable low boiling propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas
  • a Dry Powder Inhaler (“DPI”) device may be used to administer a compound and/or composition of the invention to the lung.
  • DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient.
  • DPI devices are also well known in the art.
  • a popular variation is the multiple dose DPI (“MDDPI”) system, which allows for the delivery of more than one therapeutic dose.
  • MDDPI multiple dose DPI
  • capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch for these systems.
  • Liquid spray devices Another type of device that may be used to deliver a compound and/or a composition of the invention to the lung is a liquid spray device.
  • Liquid spray systems use extremely small nozzle holes to aerosolize liquid drug formulations that may then be directly inhaled into the lung.
  • a nebulizer is used to deliver a compound and/or composition of the invention to the lung.
  • Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled (see e.g., Verschoyle et al, British J. Cancer 1999, 80, Suppl. 2, 96, which is herein incorporated by reference).
  • Examples of nebulizers include devices supplied by Sheffield/Systemic Pulmonary Delivery Ltd. (See, Armer et al, United States Patent No. 5,954,047; van der Linden et al, United States Patent No. 5,950,619; van der Linden et al, United States Patent No. 5,970,974), Aventis and Batelle Pulmonary Therapeutics.
  • an electrohydrodynamic (“EHD”) aerosol device is used to deliver a compound and/or composition of the invention to the lung.
  • EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions (see e.g., Noakes et al, United States Patent No. 4,765,539).
  • EHD aerosol devices may more efficiently deliver drugs to the lung than other pulmonary delivery technologies.
  • the compounds of the invention can be delivered in a vesicle, in particular a liposome (see Langer, Science 1990, 249:1527-1533; Treat et al, in "Liposomes in the Therapy of Infectious Disease and Cancer," Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); see generally “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).
  • the compounds of the invention can be delivered via sustained release systems, preferably oral sustained release systems.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit Ref Biomed. Eng. 14:201; Saudek et al, N. Engl. J Med. 1989, 321 :574).
  • polymeric materials can be used (see “Medical Applications of Controlled Release,” Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); “Controlled Drug Bioavailability,” Drug Product Design and
  • polymeric materials are used for oral sustained release delivery.
  • enteric-coated preparations can be used for oral sustained release administration.
  • osmotic delivery systems are used for oral sustained release administration (Verma et al, Drug Dev. Ind. Pharm.
  • a controlled-release system can be placed in proximity of the target of the compounds and/or composition of the invention, thus requiring only a fraction of the systemic dose (see, e.g. Goodson, in "Medical Applications of Controlled Release,” supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled-release systems discussed in Langer, 1990, Science 249:1527-1533 may also be used.
  • compositions contain a therapeutically effective amount of one or more compounds of the invention, preferably in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle, so as to provide the form for proper administration to a patient.
  • the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile.
  • Water is a prefened vehicle when the compound of the invention is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.
  • Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents or pH buffering agents.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • compositions comprising a compound of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of compounds of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the pharmaceutically acceptable vehicle is a capsule (see e.g., Grosswald et al, United States Patent No. 5,698,155).
  • suitable pharmaceutical vehicles have been described in the art (see Remington's Pharmaceutical Sciences, Philadelphia College of Pharmacy and Science, 17th Edition, 1985).

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Abstract

The present invention provides a method of modulating an Edg-2, Edg-3, Ed-4 or Edg7 receptor mediated biological activity in a cell. A cell expressing the Edg-2, Edg-3, Edg-4 or Edg 7 receptor is contacted with a modulator of the Edg-2, Edg-3, Ed-4 or Edg 7 receptor sufficient to modulate receptor mediated biological activity. In another aspect, the present invention provides a method for modulating an Edg-2, Edg-3, Ed-4 or Edg-7 receptor mediated biological in a subject. A therapeutically effective amount of a modulator of the Edg-2, Edg-3, Ed-4 or Edg7 receptor is administered to the subject.

Description

v
METHODS OF TREATING CONDITIONS ASSOCIATED WITH AN EDG RECEPTOR
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of priority of U.S. Provisional Application Nos. 60/350,445, 60/350,446, 60/350,447, 60/350,448, all filed January 18, 2002, the contents of which are hereby incorporated by reference in their entireties.
1. FIELD OF INVENTION The present invention relates generally to methods of modulating biological activity mediated by an Edg-2, an Edg-3, an Edg-4 or an Edg-7 receptor. More specifically, the present invention provides compounds and compositions, which may be used to selectively modulate, e.g., antagonize an Edg-2, an Edg-3, an Edg-4 or an Edg-7. The present invention also provides methods for making these compounds.
2. BACKGROUND OF THE INVENTION
Recent studies have revealed a complex biological role for cell membrane phospholipids, which were previously believed to have only a structural function. Following cell activation, membrane phospholipids may be metabolized to eicosanoids and lysophospholipids, which are important regulators of cellular function and behavior. Lysophospholipids include compounds such as lysophosphatidic acid ("LPA"), sphingosine- 1-phosphate ("SIP"), lysophosphatidylcholine and sphingosylphosphorylcholine and are important second messengers that can activate particular cell surface transmembrane G-protein coupled receptors known as endothelial gene differentiation ("Edg") receptors.
Two quite distinct subfamilies of GPCRs bind LPA and SIP specifically and transduce diverse cellular signals by associating with one or more G proteins. Based on amino acid sequence identities, S1P1 (Edg 1), S1P3 (Edg 3), S1P2 (Edg 5), and S1P5 (Edg 8) belong to one structural cluster and LPA1 (Edg 2), LPA2 (Edg 4) and LPA3 (Edg 7) are members of a second structural cluster (Goetzl, B. J., and Lynch, K. R. 2000, Ann. N. Y. Acad. Sci. 905:1-357). Members of both subfamilies range in size from 351 to 400 amino acids, and are encoded by chromosomes 1, 9 or 19. The amino acid sequence of S1P4 (Edg 6) lies between those of the two major clusters by amino acid sequence identity (Graler et al, 1998, Genomics 53, 164-169). Edg-6, a novel G-protein-coupled receptor related to receptors for bioactive lysophospholipids, is specifically expressed in lymphoid tissue. (Graler et al, 1998, Genomics 53, 164- 169). Currently, there are three known Edg receptors specifically activated by LPA (LPAl or Edg 2, LPA2 or Edg 4 and LPA3 or Bdg 7) and five known SIP receptors specifically activated by SIP (S1P1 or Edg 1, S1P2 or Edg 5, S1P3 or Edg 3, S1P4 or Edg 6, and SIPS or Edg 8).
Edg-1 (human Edg-1, GenBank Accession No. AF233365), Edg-3 (human Edg-3, GenBank Accession No. X83864), Edg-5 (human Edg-5, GenBank Accession No. AF034780), Edg-6 (human Edg-6, GenBank Accession No. AJ000479) and Edg- 8 (human Edg-8, GenBank Accession No. AF3 17676) receptors are activated by SIP, while LPA activates Edg-2 (human Edg-2, GenBank Accession No., U78 192), Edg-4 (human Edg-4, GenBank Accession Nos. AF233092 or AFO1 1466) and Edg-7 (human Edg-7, GenBank Accession No. AF127 138) receptors. Although, all three LPA receptors (i.e., Edg-2, Edg-4 and Edg-7) bind LPA, compounds, which discriminate between these receptors have been identified (Im et al, 2000, Mol. Pharmacol. 57 (4):753-759). Further, Edg 2, Edg-4 and Edg-7 appear to exhibit significant pharmacological differences (Bandoh et al, 2000, FEBS Lett. 478:159- 165). Importantly, Edg receptors are believed to mediate critical cellular events such as cell proliferation and cell migration, which makes these receptors attractive therapeutic targets. However, currently known compounds, which bind to LPA, are almost exclusively phospholipids (e.g, LPA and SIP, analogs of LPA and SIP, dioctyl glycerol, etc). Most of these phospholipids compounds fail to effectively discriminate between different Edg receptors and have poor physicochemical properties, which limits their potential use as pharmaceutical agents. Thus, there exists a need for compounds, which are not phospholipids that bind or otherwise regulate Edg receptors and can also selectively bind to a specific Edg receptor.
3. SUMMARY OF THE INVENTION
In a first aspect, the present invention addresses these and other needs by providing compounds that modulate the Edg-4 (LPA2) receptor (e.g, human Edg-4, GenBank Accession Nos. AF233092 or AFO1 1466). Such compounds preferably selectively bind or otherwise modulate the Edg-4 receptor. The present invention provides methods for modulating (antagonizing or agonizing) Edg-4 receptor mediated biological activity. The present invention also provides methods for using Edg-4 modulators (antagonists or agonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("AERDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous bums) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention. Further, the present invention provides compounds and compositions that can, for example, be used in modulating Edg-4 receptor mediated biological activity or treating or preventing diseases such as those mentioned above. The present invention still further provides methods for synthesizing the compounds. In one aspect, the present invention provides a method of modulating an Edg-4 receptor mediated biological activity in a cell. A cell expressing the Edg-4 receptor is contacted with an amount of an Edg-4 receptor modulator sufficient to modulate the Edg-4 receptor mediated biological activity.
In another aspect, the present invention provides a method for modulating Edg-4 receptor mediated biological activity in a subject. In such a method, an amount of of the Edg-2 receptor effective to modulate the Edg-2 receptor mediated biological activity is administered to the subject.
The present invention also provides compounds (agonists or antagonists) that modulate Edg-4 receptor mediated biological activity. The agonists or antagonists are compounds of structural formula (I) and can be utilized as part of the methods of the present invention:
X
RX A— B— C
(I)
or a pharmaceutically available solvate or hydrate thereof, wherein:
R] is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted dialkylamino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino;
X= O or S;
A is NR2, O or S;
R is hydrogen, alkyl or substituted alkyl; and
B and C are independently alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl.
In another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (IV):
(IV)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R , R or R5 is independently -H, -halo, -NO , -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5,
-C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5- C10)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -(C5-Cι0)cycloheteroaryl, -(C3-C6)cycloheteroalkyl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -NR5R5, =NR5, -(C1-C10)alkylNHC(O)(CH2)mR5, -(C3-C1o)cycloheteroalkyl(R5)m, -(CH2)mR5, -(C-CoJalkylNRsRs, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Ci-C,0)alkyl(Ci-Cio)alkyl, -O(Cι-Cιo)alkyl, -C(O)(C1-C10)alkyl,
-C(O)NH(CH2)m(C1-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C,o)alkyl(C,-
C10)alkyl), -CO2(C,-C10)alkyl, -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl,
-(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C10)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-C10)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(C1-C10)alkyl, -NHC(O)NH(Cι.-Cι0)alkyl, -OC(O)O(d-
Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X and Y are each independently C or N; and Z is O, S, C or N, wherein if Z is O or S, then R is an electron pair;
Rt and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R3 and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In another embodiment, the modulator is a compound of structural formula
(V):
(N) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 or R5 is independently -H, -halo, -ΝO2, -CN, -OH,
-N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl, -(C -C 10)cycloalkyl, -(C8-C 1 )bicycloalkyl, -(C5-C ] 0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C10)heteroaryl, -naphthyl, -(C -Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5,-OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R(, is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(C1-Cι0)alkyl(C1-C,0)alkyl, -O(C1-C10)alkyl, -C(O)(C,-C10)alkyl, -C(O)NH(CH2)m(C1-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cιo)alkyl(Cι- C10)alkyl), -CO2(C1-C,o)alkyl, -(Cι-C10)alkyl, -(C2-C10)alkenyl, -(C2-Cιo)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cto)cycloalkenyl, -(C )heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle,
-CO2(CH2)m(C,-C10)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C10)alkyl, -NHC(O)NH(C Cι0)alkyl, -OC(O)(C,-Cιo)alkyl, -OC(O)O(C1-C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and Ri and R2 or R2 and R3 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In yet another embodiment, the agonists or antagonists are compounds of structural formula (VI):
(VI) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 or R5 is independently -H, -halo, -NO2, -CN, -OH,
-N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-do)alkyl, -(C2-C10)alkenyl, -(C2-Cι0)alkynyl, -(C3-C10)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -indole, -naphthyl, -(C -Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5,-OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
R5 or Rό is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C1-C10)alkyl(C,-C10)alkyl, -O(d-C10)alkyl, -C(O)(d-C10)alkyl,
-C(O)NH(CH2)m(d-Cι0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C10)alkyl(C1- C10)alkyl), -CO2(Cι-C10)alkyl, -(C C10)alkyl, -(C2-C10)alkenyl, -(C2-Cιo)alkynyl, -(C -Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-C]0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C -Cι0)heterocycle, -CO2(CH2)m(d-C10)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C10)alkyl, -NHC(O)NH(d- C10)alkyl, -OC(O)(C,-C10)alkyl, -OC(O)O(d-C,0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X, Y and Z are independently O, S, C or N, wherein if X, Y or Z is O or S, R\ is an electron pair; Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
Ri and R5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R and R5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (VII):
(VII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5, R7 or R8 is independently -H, -halo, -NO2, -CN,
-(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-CI0)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C10)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C10)heteroaryl, -naphthyl, -(C -Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5,
-(C1-C10)alkylNHC(O)(CH2)mR5, -(C1-d0)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 or Rs is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(CrC1o)alkyl(C1-d0)alkyl, -O(C1-C10)alkyl, -C(O)(C Cιo)alkyl,
-C(O)NH(CH2)m(d-do)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(C1-
C,0)alkyl), -CO2(d-Cι0)alkyl, -(d-C10)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl,
-(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(d-C10)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-Cι0)alkyl, -NHC(O)NH(d-Cι0)alkyl, -OC(O)O(d-
C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X is O, S, C or N, wherein if X is O or S, Ri is an electron pair; and
Y and Z are independently N or C, wherein if Y or Z is N, R\ and R2 are each an electron pair.
In another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (VIII):
(VIII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R , R5, R7, R8, R9 or Rι0 is independently -H, -halo, -NO2, -CN, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-Cι0)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(d-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C1-C1o)alkylNHC(O)(CH2)mR5, -(C1-C,o)alkylNR5R5>-OC(O)(CH2)IT1CHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5) -S(O)2NHR5, or
wherein;
each Re is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(C1-Cι0)alkyl(d-C1o)alkyl, -O(d-C10)alkyl, -C(O)(C1-C10)alkyl, -C(O)NH(CH2)m(Cι-Cι0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(d- C10)alkyl), -CO2(d-Cι0)alkyl, -(d-do)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C!o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C10)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)NH(d-Cι0)alkyl, -OC(O)O(Cι- C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and X and Y are independently O, S or N, wherein if X or Y is O or S, R and RJO are an electron pair.
In yet another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (IX):
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, Rj , R5, R7, R8, R or Rio is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C1-C,0)alkyl, -(C2- Cιo)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-C,o)alkylNHC(O)(CH2)mR5, -(C,-C1o)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-C10)alkyl(Cι-C10)alkyl, -O(d-C10)alkyl, -C(O)(Cι-Cιo)alkyl,
-C(O)NH(CH2)m(C,-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-CI0)alkyl(Cι- C10)alkyl), -CO2(C,-Cιo)alkyl, -(d-Cιo)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C -do)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(d-C10)alkyl, -CO2(CH2)mH, -NHC(O)(d-Cιo)alkyl, -NHC(O)NH(Cι-C,0)alkyl, -OC(O)O(d- Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; and p is independently an integer ranging from 0 to 5.
In yet another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (X):
(X) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2, R3, R4 , R5 or R7 is independently -H, -halo, -NO2, -CN, -C(R5)3,
-(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C,0)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-C10)cycloalkyl, -(C8-Cι )bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(d-C10)alkylNHC(O)(CH2)mR5, -(C,-C10)alkylNR5R5, -CO2H, -(C,-C10)alkylC(O)NH(CH2)mR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or R is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(Cι-Cιo)alkyl(Cι-C,o)alkyl, -O(C1-Cι0)alkyl, -C(O)(C1-Cι0)alkyl,
-C(O)NH(CH2)m(d-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((d-Cio)alkyl(Ci- C,0)alkyl), -CO2(d-Cι0)alkyl, -(d-C10)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl,
-(C -C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(C1-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-C10)alkyl, -NHC(O)NH(C,-Cι0)alkyl, -OC(O)O(d-
Cι0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
R] and R2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R4 and R7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring. In another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present are compounds of structural formula (XI):
(XI) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R , R5, R7 or R8 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O CH,)^, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cι0)cycloalkenyl, -(C )heteroaryl, -(C6)heteroaryl, -(C5-C10)heteroaryl, -(C5-Cιo)cycloheteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C1-C1o)alkylNHC(O)(CH2)mR5, -(d-Cιo)alkylNR5R5; -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R6 is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C1-C10)alkyl(C,-Cιo)alkyl, -O(C1-Cι0)alkyl, -C(O)(C1-C10)alkyl, -C(O)NH(CH2)m(d-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((d-C,0)alkyl(d- C,o)alkyl), -CO2(C,-do)alkyl, -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(Cδ)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-C10)alkyl, -NHC(O)NH(d-Cι0)alkyl, -OC(O)O(C,-
Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; Ri and R2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R3 and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R8 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and Ri and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (XII):
(XII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5 or R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-Cιo)alkynyl, -(C3-C10)cycloalkyl, -(C8-CM)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -(C5-Cιo)cycloheteroaryl, -(C3-C6)cycloheteroalkyl, -naphthyl, -(C3-C10)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -NR5R5, =NR5, -(C1-C,0)alkylNHC(O)(CH2)mR5, -(C3-d0)cycloheteroalkyl(R5)m, -(CH2)mR5, -(C,-C10)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or R is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C1-C10)alkyl(Cι-C1o)alkyl, -O(C1-C1o)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(d-C10)alkyl, -OCF3, -benzyl, -Cθ2(CH2)mCH((C,-Cιo)alkyl(Cι- C10)alkyl), -CO2(Cι-C,o)alkyl, -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C]0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(d-C,o)alkyl, -NHC(O)NH(C,-C10)alkyl, -OC(O)O(Cι- Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
R3 or R4 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring; Ri or R2 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring; and R2 or R4 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring.
In a second aspect, the present invention provides compounds that modulate the Edg-7 (LPA3) receptor (e.g., human Edg-7, GenBank Accession No. AF127138). Such compounds selectively bind or otherwise modulate the Edg-7 receptor.
The compounds (agonists or antagonists) that can, for example, be used to modulate Edg-7 receptor mediated biological activity or to treat or prevent diseases such as those discussed above. The agonists or antagonists are compounds of structural formula (XIII) and can be utilized in the methods of the present invention:
(XIII) or a pharmaceutically available solvate or hydrate thereof, wherein:
X is NR3, S or O;
Ri is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, amino, carbamoyl, substituted carbamoyl, oxo, thiono or -NR4; and
R2 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkyloxy, substituted alkyloxy, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, aryloxycarbonyl, substituted aryloxycarbonyl, arylalkyl, substituted arylalkyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or
R5
R6
R3 is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkylsulfonyl, substituted alkylsulfonyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl or substituted heteroalkyl;
R4 is alkyl, substituted alkyl, acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, heteroaryl substituted heteroaryl, cycloheteroalkyl, substituted cycloheteroalkyl, and
R5 and R are independently hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylthio, substituted alkylthio, alkoxycarbonyl, substituted alkoxycarbonyl, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or optionally along with the carbon to which they are attached form an aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl ring.
The present invention also provides compounds (agonists or antagonists) that can, for example, be used to modulate Edg-7 receptor mediated biological activity or to treat or prevent diseases such as those discussed above. The agonists or antagonists can be utilized in the methods of the present invention and are compounds of structural formula (XIV):
(XIV) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3 R4 and R7is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C-Cιo)alkyl, -(C2-Cιo)alkenyl, -(C2-Cl0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C,-C,o)alkylNHC(O)(CH2)mR5, -(C,-Cιo)alkylNR5R5; -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein each R5 and R6 is independently -H, -halo, -NO2, -CN, -OH, -CO2H, ^(0,-010)3^1(0,-010)3^1, -0(0,-010)3 !, -C(O)(Cι-C10)alkyl, -C(O)NH(CH2)m(C,-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-Cι0)alkyl(C,- C,o)alkyl), -CO2(C,-C,0)alkyl, -(Cι-C,0)alkyl, -(C2-C10)alkenyl, -(C2-Cι0)alkynyl, -(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(Cι-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(C Cιo)alkyl, or -SO2NH2; X is CH2, C=O, O, S, SO2, C, or NR5; Ri , R2> R3 R4 and R7 taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring;
Ri, R2> R3 R4 and R7 can also be an electron such that when two groups are on adjacent carbon atoms they form a double bond; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5. The agonists or antagonists can be utilized in the methods of the present invention and are also compounds of structural formula (XV):
(XV) or a pharmaceutically available solvate or hydrate thereof, wherein; each of R,, R2, R3 R4 and R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-Cι0)alkyl, -(C2-C,0)alkenyl, -(C2-Cι0)alkynyl, -(d-Cιo)cycloalkyl, -(C8-Cι )bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(RS), -(C,-Cιo)alkylNHC(O)(CH2)mR5, -(C,-Cιo)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H, -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(d-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(Cδ)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(Cι-Cιo)alkylNHC(O)(CH2)mR5, -(Cι-Cι0)alkylNR5R5) -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R* is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-C,0)alkyl(C,-C,o)alkyl, -O(C,-Cι0)alkyl, -C(O)(C,-Cιo)alkyl,
-C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(C,- C,o)alkyl), -CO2(Cι-C,0)alkyl, -(Cι-C,0)alkyl, -(C2-C,o)alkenyl, -(C2-C,o)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C, )bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(Ci-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(Ci-C,0)alkyl, -NHC(O)NH(C,-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-Cιo)alkyl, or -SO2NH2;
X, Y, and Z are each independently C=O, O, S, C, or N; wherein if X, Y, or Z is O or S, Ri is an electron pair; Ri, R2> R3 ^ and R7 taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring; two Re groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5. The present invention provides methods for modulating Edg-7 receptor mediated biological activity. The present invention also provides methods for using Edg-7 modulators (i.e., agonists and antagonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous burns) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention. Further, the present invention provides compounds and compositions for use in modulating Edg-7 receptor mediated biological activity or treating or preventing diseases such as those mentioned above as well as methods for synthesizing the compounds. In one aspect, the present invention provides a method of modulating
(antagonizing or agonizing) an Edg-7 receptor mediated biological activity in a cell. A cell expressing the Edg-7 receptor is contacted with an amount of an Edg-7 receptor modulator of the invention sufficient to modulate the Edg-7 receptor mediated biological activity. In a second aspect, the present invention provides a method for modulating
Edg-7 receptor mediated biological activity in a subject. In such a method, an amount of a modulator of the Edg-7 receptor of the invention effective to modulate the Edg-7 receptor mediated biological activity is administered to the subject. In a further aspect, the present invention provides compounds that modulate the Edg-2 (LPAl) receptor (e.g., human Edg-2, GenBank Accession No., U78192). Such compounds preferably selectively bind or otherwise modulate the EDG-2 receptor. In certain embodiments, the present invention provides methods for modulating (antagonizing or agonizing) Edg-2 receptor mediated biological activity. The present invention also provides methods for using Edg-2 modulators (agonists and antagonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous burns) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention. Further, the present invention provides compounds and compositions that can, for example, be used in modulating Edg-2 receptor mediated biological activity or treating or preventing diseases such as those mentioned above. The present invention still further provides methods for synthesizing the compounds. In one aspect, the present invention provides a method of modulating an Edg-2 receptor mediated biological activity in a cell. A cell expressing the Edg-2 receptor is contacted with an amount of an Edg-2 receptor modulator sufficient to modulate the Edg-2 receptor mediated biological activity.
In another aspect, the present invention provides a method for modulating Edg-2 receptor mediated biological activity in a subject. In such a method, an amount of a modulator of the Edg-2 receptor effective to modulate the Edg-2 receptor mediated biological activity is administered to the subject.
The present invention also provides compounds (agonists or antagonists) that modulate Edg-2 receptor mediated biological activity. In certain aspects, the agonists or antagonists are compounds of structural formula (XX) and can be utilized as part of the methods of the present invention: (XX)
or a pharmaceutically available salt, hydrate or solvate thereof wherein:
P, Q and R are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl. In other aspects, the agonists or antagonists can be utilized as part of the methods of the present invention and are compounds of structural formula (XXIII):
(XXIII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,o)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-Cιo)cyclo alkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-C,o)alkylNHC(O)(CH2)mR5, -(C,-C,0)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each of R5 and R is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-Cιo)alkyl(C,-C,o)alkyl, -O(Cι-C,o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(Cι-
Cιo)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-Cι0)alkynyl,
-(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(d-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Ci-C,0)alkyl, -NHC(O)NH(C,-C,o)alkyl, -OC(O)O(C,-
Cιo)alkyl, or -SO2NH2;
X, Y, and Z are each independently C(R5)( R5), C(O), O, C(S), S, C=N(R5), or NR3; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5; Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
Ri and X or R2 and Y can together form a double bond.
In other aspects, the agonists or antagonists are compounds that can be utilized as part of the methods of the present invention and are of structural formula (XVII):
(XVII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(Cι-C1o)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(d-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl),
-heterocylcoalkyl, -(Cι-C,o)alkylNHC(O)(CH2)mR5, -(C,-Cιo)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R6 is independently -H, -halo, -NO2, -CN, -OH, -CO2H,
-N(C1-C1o)alkyl(d-Cιo)alkyl, -O(Cι-Cι0)alkyl, -C(O)(Cι-C,o)alkyl, -C(O)NH(CH2)m(Cι-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cιo)alkyl(Cι- C10)alkyl), -CO2(Cι-C,o)alkyl, -(d-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(d-Ci0)alkyl, -CO2(CH2)mH, -NHC(O)(d-C10)alkyl, -NHC(O)NH(Cι-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-C,0)alkyl, or -SO2NH2; two R groups on adjacent carbon atoms can also be joined to form a 5 or 6- membered acyclic or heterocyclic ring or a 6 membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5. In yet other aspects, the agonists or antagonists are compounds of structural formula (XXV) and can be utilized as part of the methods of the present invention: (XXV)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -NH(aryl), -benzyl, -CO2CH(R5)(R5), -(d-do)alkyl, -(C2-C,0)alkenyl, -aryl, -(C2-C,o)alkynyl, -(C3-C,0)cycloalkyl, -(C3-Cιo)cycloalkyl(aryl), -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5- C10)heteroaryl, -naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, - NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(C,-C,o)alkylNHC(O)(CH2)mR5, -(C,-do)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R6 is independently -H, -halo, -NO2, -CN, -OH, -CO2H,
-N(C,-Cιo)alkyl(C,-C10)alkyl, -O(C,-do)alkyl, -C(O)(C,-do)alkyl, -C(O)NH(CH2)m(C,-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-Cιo)alkyl(C,- C,0)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,0)alkyl, -(C2-C10)alkenyl, -(C2-Cιo)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C10)alkyl, -NHC(O)NH(C,-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-Cιo)alkyl, or -SO2NH2;
R, and R2 can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two R6 groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In another aspect, the present invention provides compounds that modulate the S1P3 or Edg-3 receptor (e.g. human Edg-3, GenBank Accession No. X83864). Such compounds preferably selectively bind or otherwise modulate the Edg-3 receptor. In one embodiment, the present invention provides methods for modulating
Edg-3 receptor mediated biological activity. The present invention also provides methods for using Edg-3 modulators (i.e., agonists or antagonists) in treating or preventing diseases such as ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma, surface epithelial cell injury, (e.g., transcorneal freezing or cutaneous burns) and cardiovascular diseases (e.g., ischemia) in a subject in need of such treatment or prevention.
In a another aspect, the present invention provides methods for using Edg-3 modulators (i.e., agonists or antagonists) in treating or preventing disorders such as, but not limited to, vasoconstriction in cerebral arteries, autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-3 antagonists can also be used in organ transplantation. In yet another embodiment, Edg-3 agonists and antagonists can be used to treat vascular occlusive disorders. For example, activation of Edg-3 receptors by using an Edg-3 agonist will result in increased vasoconstriction which is beneficial in conditions such as migraine headaches. Inhibition of Edg-3 by an Edg3 antagonist will be beneficial in conditions such as a stroke, a subarachnoid hemorrhage, or a vasospasm such as a cerebral vasospasm. (PCT WO 01/69252 Al). In still other aspects, the present invention provides a method of modulating an Edg-3 receptor mediated biological activity in a cell. A cell expressing the Edg-3 receptor is contacted with an amount of an Edg-3 receptor modulator sufficient to modulate the Edg-3 receptor mediated biological activity. In yet other aspects, the present invention provides a method for modulating an Edg-3 receptor mediated biological activity in a subject. In such a method, an amount of a modulator of the Edg-3 receptor effective to modulate an Edg-3 receptor mediated biological activity is administered to the subject. The present invention also provides compounds and compositions for use in modulating (i.e., agonizing or antagonizing) Edg-3 receptor mediated biological activity or treating or preventing diseases such as those mentioned above as well as methods for synthesizing the compounds.
In certain embodiments, The Edg-3 receptor modulators are compounds of structural formula (XXXI) :
or a pharmaceutically available solvate or hydrate thereof, wherein:
n = 0 or 1 ;
o is 0, 1, 2, 3 or 4;
X is C, NR7 O or S;
Y is C, NR8 O or S;
R, is either absent or hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl;
R2, R3 and R4 are independently hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl;
each R5 is independently, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, azido, carboxy, carbamoyl, substituted carbamoyl, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, halo, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl, hydroxyl, nitro or thio; and
R and R8 are independently absent, hydrogen, alkyl, substituted alkyl, acyl or substituted acyl.
In other embodiments, Edg-3 receptor modulators are compounds of structural formula (XXXII):
(XXXII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-Cιo)alkyl, -(C2-Cιo)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,0)heteroaryl, -naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(Cι-C,o)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-C,0)alkynyl,
-(C3-Cιo)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(Cό)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(Cι-C,o)alkylNHC(O)(CH2)mR5, -(Cι-C,o)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(Cι-Cιo)alkyl, -O(C,-C,o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH2)m(C,-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,o)alkyl(C,- C,0)alkyl), -CO2(C,-C,0)alkyl, -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-C,0)alkyl, or -SO2NH2;
X is O, S, C(R5)(R5) or N(R5);
Ri, R2 or R3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In yet other embodiments, Edg-3 receptor modulators are compounds of structural formula (XXXIII):
R3 R2 (XXXIII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C10)alkyl, -(C2-Cι0)alkenyl, -(C2-C]0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(Cι-Cιo)alkylNHC(O)(CH2)mR5, -(Cι-C,0)alkylNR5R5,
-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C )heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(Cι-Cιo)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(d-Cιo)alkyl(C,-C,o)alkyl, -O(Cι-Cιo)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH2)m(d-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-C,0)alkyl(Cι- Cιo)alkyl), -CO2(Cι-Cιo)alkyl, -(Cι-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-C,0)alkynyl, -(C -Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(Ci-Ci0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-C10)alkyl, or -SO2NH2; X is O, S, or N(R5);
Ri, R2 or R3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two R6 groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
4. BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 illustrates the selectivity of 101 for the Edg-4 receptor; Fig. 2 illustrates a dose response curve for Edg-4 antagonists 101, 103 and 105;
Fig. 3 illustrates a dose response curve for 101 and LPA in HTC rat hepatoma cells transfected with human Edg-4 receptors; Fig. 4 illustrates a dose response curve for 101 in OV202 human ovarian cancer cells;
Fig. 5 illustrates a dose response curve for 101 in CaOV-3 human ovarian cancer cells;
Fig. 6 illustrates the inhibition of VEGF production by 101 in CaOV-3 human ovarian cancer cells;
Fig. 7 illustrates the inhibition of IL-8 production by 101 in CaOV-3 human ovarian cancer cells;
Fig. 8 illustrates the inhibition of LPA-stimulated proliferation by 101 in CaOV-3 human ovarian cancer cells; Fig. 9 illustrates the inhibition of LPA-stimulated chemotaxis by 103 in
CaOV-3 human ovarian cancer cells;
Fig. 10 illustrates the lack of inhibition of S IP-stimulated migration by 103 in human umbilical vein endothelial cells;
Fig. 11 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonists 101, 103, 107 and 113 in HTC rat hepatoma cells transfected with human Edg-4;
Fig. 12 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonists 101, 103, 107 and 113 in HTC rat hepatoma cells transfected with pooled rat Edg-4 clones; Fig. 13 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonists 101, 103, 107 and 113 in HTC rat hepatoma cells transfected with pooled mouse Edg-4 clones;
Figure 14 illustrates the efficacy of 101 in suppressing the tumor growth as tested by in vivo Z-chamber study; Figure 15 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonist 125 in HTC cells;
Figure 16 illustrates a dose response inhibition curve of LPA induced calcium mobilization by the Edg-4 antagonist 125 in CaOV-3 cells;
Fig. 17 illustrates the selectivity of 701 for the Edg-7 receptor; Fig. 18 illustrates the inhibition of LPA-stimulated calcium mobilization by 703 in HT-1080 human fibrosarcoma cells;
Fig. 19 illustrates the selectivity of 201 for the Edg-2 receptor;
Fig. 20 illustrates the selectivity of 203 for the Edg-2 receptor; Fig. 21 illustrates the inhibition of LPA-stimulated calcium mobilization in immortalized ovarian surface epithelial cells by Edg-2 antagonist 203;
Fig. 22 illustrates the effects of 203 on the inhibition of cAMP production by LPA;
Fig. 23 illustrates the inhibition of LPA-stimulated calcium mobilization in A431 human epitheloid carcinoma cells by Edg-2 antagonist 201 ;
Fig. 24 illustrates the inhibition of LPA-stimulated calcium mobilization in A431 human epitheloid carcinoma cells by Edg-2 antagonists 201 and 203, but not Edg-4 antagonist, 101;
Figure 25 illustrates the selectivity of 301 for the Edg-3 receptor; and Figure 26 illustrates the inhibition of S IP-stimulated calcium mobilization by
303 in mouse NIH 3T3 fibroblast cells.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Definitions
"Compounds of the invention" refers generally to any modulator of the LPA2 or Edg-4 receptor (e.g. human Edg-4, GenBank Accession Nos. AF23 3092 or AFOl 1466) and includes any Edg-4 receptor modulator encompassed by generic formulae disclosed herein and further includes any species within those formulae whose structure is disclosed herein. "Compounds of the invention" also refers generally to any modulator of the Edg-7 receptor (e.g. human Edg-7, GenBank Accession No. AF 127138) encompassed by generic formulae disclosed herein and further includes any species within those formulae whose structure is disclosed herein. "Compounds of the invention" also refers generally to any modulator of the LPAl or Edg-2 receptor (human Edg-2, GenBank Accession No., U78192) and includes any Edg-2 receptor modulator encompassed by generic formulae disclosed herein and further includes any species within those formulae whose structure is disclosed herein. "Compounds of the invention" also refers generally to any modulator of the S1P3 or Edg-3 receptor (e.g., human Edg-3, GenBank Accession No. X83864) and includes any Edg-3 receptor modulator encompassed by generic formulae disclosed herein and further includes any specific Edg-3 receptor modulator within those formulae whose structure is disclosed herein. The compounds of the invention may be identified either by their chemical structure and/or chemical name. Ifthe chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound. The compounds of the invention may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The compounds of the invention may also exist in several tautomeric forms including, but not limited to, the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. The compounds of the invention also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incoφorated in the compounds of the invention include, but are not limited to, 2H, 3H, ,3C, 14C, 15N, ,80, 170, 31P, 32P, 35S, 18F and 36C1. Further, it should be understood that when partial structures of the compounds of the invention are illustrated, brackets indicate the point of attachment of the partial structure to the rest of the compound.
"Composition of the invention" refers to at least one compound of the invention and a pharmaceutically acceptable vehicle, with which the compound is administered to a patient. When administered to a patient, the compounds of the invention are administered in isolated form, which means separated from a synthetic organic reaction mixture.
"Alkyl" refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop- 1 -en- 1-yl, prop-l-en-2-yl, prop-2- en-l-yl (allyl), cycloprop-1-en-l-yl; cycloprop-2-en-l-yl, prop-1-yn-l-yl, prop-2-yn- 1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-l-yl, 2-methyl- propan-2-yl, cyclobutan-1-yl, but- 1 -en- 1-yl, but-l-en-2-yl, 2-methyl-prop-l -en- 1-yl, but-2-en-l-yl , but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-l-en-1- yl, cyclobut-l-en-3-yl, cyclobuta-l,3-dien-l-yl, but- 1-yn- 1-yl, but-l-yn-3-yl, but-3- yn-l-yl, etc.; and the like.
The term "alkyl" is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions "alkanyl," "alkenyl," and "alkynyl" are used. Preferably, an alkyl group comprises from 1 to 20 carbon atoms.
"Alkanyl" refers to a saturated branched, straight-chain or cyclic alkyl group derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl fiec-butyl), 2-methyl-propan-l-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yi, etc.; and the like.
"Alkenyl" refers to an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop- 1 -en- 1-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl; butenyls such as but- 1 -en- 1-yl, but-l-en-2-yl, 2-methyl-prop-l - en-l-yl, but-2-en-l-yl , but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-1,3- dien-2-yl, cyclobut-1-en-l-yl, cyclobut-l-en-3-yl, cyclobuta-l,3-dien-l-yl, etc.; and the like. "Alkynyl" refers to an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop- 1-yn- 1-yl, prop-2-yn- 1-yl, etc.; butynyls such as but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc and the like.
"Acyl" refers to a radical -C(O)R, where R is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein. Representative examples include, but are not limited to formyl, acetyl, cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.
"Acylamino" refers to a radical -NR'C(O)R, where R' and R are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein. Representative examples include, but are not limited to, formylamino, acetylamino, cylcohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino and the like.
"Alkylamino" means a radical -NHR where R represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylamino, ethylamino, 1-methylethylamino, cydohexyl amino and the like.
"Alkoxy" refers to a radical -OR where R represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.
"Alkoxycarbonyl" refers to a radical -C(O)-alkoxy where alkoxy is as defined herein.
"Alkylarylamino" refers to a radical -NRR' where R represents an alkyl or cycloalkyl group and R' is an aryl as defined herein
"Alkylsulfonyl" refers to a radical -S(O)2R where R is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl and the like.
"Alkylsulfinyl" refers to a radical -S(O)R where R is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfmyl and the like.
"Alkylthio" refers to a radical -SR where R is an alkyl or cycloalkyl group as defined herein that may be optionally substituted as defined herein. Representative examples include, but are not limited to methylthio, ethylthio, propylthio, butylthio, and the like.
"Amino" refers to the radical -NH .
"Aryl" refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as- indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. Preferably, an aryl group comprises from 6 to 20 carbon atoms.
"Arylalkyl" refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2- phenylethan-1-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2- naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used. Preferably, an arylalkyl group is (C6-C 0) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (Cι-C10) and the aryl moiety is (C6-C20). "Arylalkyloxy" refers to an -O-arylalkyl radical where arylalkyl is as defined herein.
"Arylamino" means a radical -NHR where R represents an aryl group as defined herein.
"Aryloxycarbonyl" refers to a radical -C(O)-O-aryl where aryl is as defined herein.
"Azido" refers to the radical -N .
"Carbamoyl" refers to the radical -C(O)N(R)2 where each R group is independently hydrogen, alkyl, cycloalkyl or aryl as defined herein, which may be optionally substituted as defined herein.
"Carboxy" means the radical -C(O)OH.
"Cyanato" means the radical -OCN.
"Cyano" means the radical -CN.
"Cycloalkyl" refers to a saturated or unsaturated cyclic alkyl group. Where a specific level of saturation is intended, the nomenclature "cycloalkanyl" or "cycloalkenyl" is used. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and the like. In a preferred embodiment, the cycloalkyl group is (C -Cι0) cycloalkyl, more preferably (C3-C6) cycloalkyl.
"Cycloheteroalkyl" refers to a saturated or unsaturated cyclic alkyl group in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atom(s) include, but are not limited to, N, P, O, S, Si, etc. Where a specific level of saturation is intended, the nomenclature "cycloheteroalkanyl" or "cycloheteroalkenyl" is used. Typical cycloheteroalkyl groups include, but are not limited to, groups derived from dioxanes, dioxolanes, epoxides, imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine, tetrahydrofuran, tetrahydropyran and the like.
"Cvcloheteroalkyloxycarbonyl" refers to a radical -C(O)-OR where R is cycloheteroalkyl is as defined herein.
"Dialkylamino" means a radical -NRR' where R and R' independently represent an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to dimethylamino, methyl ethylamino, di-(l- methylethyl)amino, (cyclohexyl)(methyl)amino, (cyclohexyl)(ethyl)amino, (cyciohexyl)(propyl)amino, and the like.
"Halo" means fluoro, chloro, bromo, or iodo.
"Haloalkyl" means an alkyl radical substituted by one or more halo atoms wherein alkyl and halo is as defined herein.
"Heteroalkyloxy" means an -O-heteroalkyl group where heteroalkyl is as defined herein.
"Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkynyl" refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with the same or different heteroatomic groups. Typical heteroatomic groups include, but are not limited to, -O-, -S-, -O-O-, -S-S-, -O-S-, -NR'-, =N-N=, -NN-, -N-N-NR-, -PH-,-P(O)2-, -O-P(O)2-, -S(O)-, -S(O)2-, -SnH2- and the like, wherein R' is hydrogen, alkyl, substituted alkyl, cycloallcyl, substituted cycloalkyl, aryl or substituted aryl.
"Heteroaryl" refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoiine, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. Preferably, the heteroaryl group is between 5-20 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred. Preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.
"Heteroaryloxy" refers to an -O-heteroarylalkyl radical where heteroarylalkyl is as defined herein.
"Heteroaryloxycarbonyl" refers to a radical -C(O)-OR where R is heteroaryl as defined herein.
"Heteroarylalkyl" refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used. In preferred embodiments, the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20 membered heteroaryl.
"Hydroxy" refers to the radical -OH.
"Leaving group" has the meaning conventionally associated with it in synthetic organic chemistry, i.e., an atom or a group capable of being displaced by a nucleophile and includes halo (such as chloro, bromo, and iodo), alkoxycarbonyl (e.g., acetoxy), aryloxycarbonyl, mesyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g, 2,4-dinitrophenoxy), methoxy, N,O-dimethylhydroxylamino, and the like. "Nitro" refers to the radical -NO2.
"Oxo" refers to the divalent radical =O.
"Pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
"Pharmaceutically acceptable salt" refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chiorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l -carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N- methylglucamine and the like.
"Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
"Patient" includes humans. The terms "human" and "patient" are used interchangeably herein. "Preventing" or "prevention" refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
"Prodrug" refers to a pharmacologically inactive derivative of a drug molecule that requires a transformation within the body to release the active drug. Typically, prodrugs are designed to overcome pharmaceutical and/or pharmacokinetically based problems associated with the parent drug molecule that would otherwise limit the clinical usefulness of the drug.
"Promoiety" refers to a form of protecting group that when used to mask a functional group within a drug molecule converts the drug into a prodrug. Typically, the promoiety will be attached to the drug via bond(s) that are cleaved by enzymatic or non-enzymatic means in vivo. Ideally, the promoiety is rapidly cleared from the body upon cleavage from the prodrug.
"Protecting group" refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in Green et al, "Protective Groups in Organic
Chemistry", (Wiley, 2nd ed. 1991) and Harrison et al, "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996). Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro- veratryloxycarbonyl ("NVOC") and the like. Representative hydroxy protecting groups include, but are not limited to, those where the hydroxy group is either acylated or alkylated such as benzyl, and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
"Substituted" refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, -X, -R,4, -OS, =O, -ORH, -SRj4, S", =S -NR,4, Rι5, =NR,4,-CX3, -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O\ -S(O)2OH, - S(O)2Rι4, -OS(O2)O", -OS(O)2R14, -P(O)(O)2, -P(O)(OR,4)(O), -OP(O)(0R]4)(ORι5), -C(O)RH, -C(S)Ri4, -C(O)0R14, -C(O)NR14R15, -C(O)O", -C(S)OR,4, - NRι6C(O)NR,4R,5, -NR16C(S)NRi4R,5, -NRl7C(NR16)NRι4R,5 and -C(NR,6)NR,4R15, where each X is independently a halogen; each Rι4, Rι5, Rι6 and Rj are independently hydrogen, alkyl, substituted alkyl, aryl, substituted alkyl, arylalkyl, substituted alkyl, cycloalkyl, substituted alkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, -NRι5Rι , -C(O)R15 or -S(O)2R]5 or optionally Rι5 and Rι9 together with the atom to which they are both attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and Rι5 and R] are independently hydrogen, alkyl, substituted alkyl, aryl, substituted alkyl, arylalkyl, substituted alkyl, cycloalkyl, substituted alkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroallcyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.
"Sulfonylamino" refers to a radical -NR'S(O2)R, where R' and R are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein.
"Therapeutically effective amount" means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
"Thio" refers to the radical -SH.
"Thiocyanato" refers to the radical -SCN.
"Thiono" refers to the divalent radical =S.
"Treating" or "treatment" of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discemible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treating" or "treatment" refers to delaying the onset of the disease or disorder.
Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with the preferred embodiments, it will be understood that it is not intended to limit the invention to those preferred embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
5.2. The Use of the Edg-4 Compounds of the Invention
The present invention provides a method of modulating an LPA2 or Edg-4 receptor (e.g., human Edg-4, GenBank Accession Nos. AF233092 or AFOl 1466) mediated biological activity. A cell expressing the Edg-4 receptor is contacted with an amount of an Edg-4 receptor agonist or antagonist sufficient to modulate the Edg-4 receptor mediated biological activity.
Those of skill in the art will appreciate that Edg-4 is a G protein coupled receptor ("GPCR"). The Edg-4 (LPA2) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-2 (LPAl) and Edg-7 (LPA3), binds lysophosphatidic acid ("LPA"). Preferably, the Edg-4 receptor is a human receptor.
The Edg-4 receptor may be expressed by recombinant DNA methods well known to those of skill in the art. Particularly useful cell types for expressing and assaying Edg-4 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells). Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA).
DNA encoding Edg-4 is well known (e.g, human Edg-4, GenBank Accession Nos. AF233092 or AFOl 1466) and can be transfected into human or mammalian cells according to methods known to those of skill in the art. For example, DNA encoding human Edg-4 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA). Alternatively, DNA encoding Edg-4 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the ewv gene to produce an ecotropic envelope. Both methods (i.e., co-transfection with a packaging vector or use of EcoPack-293) enable the production of an ecotropic envelope for viral packaging, and can thus advantageously be used to transfect rat and mouse cells. For use in human and other mammalian cells, AmphoPack-293 cell line can be used (Clontech Labs, Palo Alto, CA).
In addition, a number of natural cell lines naturally express Edg-4 receptors. These include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB- 453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells and OV202 human ovarian cancer cells (ATCC, Manassas, VA; Vec
Technologies Inc. (Rensselaer, NY); Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA).
Those of skill in the art will appreciate that cells which express the Edg-4 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulation of Edg-4 receptor activity, regardless of the local environment. In one preferred embodiment, cells that express the Edg-4 receptor are grown in vitro (i.e., are cultured). In another preferred embodiment, cells that express the Edg-4 receptor are in vivo (i.e., are part of a complex organism). The cells, in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, endothelial cells, carcinoma cells, pheochromocytoma cells, myoblast cells, platelet cells and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, 0V202 human ovarian cells, HTC rat hepatoma cells, CAOV-3 human ovarian cancer cells, MDA-MB-453 breast cancer cells, MDA-MB-231 breast cancer cells, HUVEC, A431 human epitheloid carcinoma cells and HT-1080 human fibrosarcoma cells.
In another aspect, an Edg-4 receptor mediated biological activity is modulated in a subject or in an animal model. A therapeutically effective amount of a modulator of the Edg-4 receptor is administered to the subject or an animal. Preferably, the subject or animal is in need of such treatment.
The biological activity mediated by the Edg-4 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation or actin polymerization. Preferably, the biological activity mediated by the Edg-4 receptor includes, but is not limited to, apoptosis, angiogenesis, inhibition of wound healing, inflammation, cancer invasiveness or atherogenesis. Most preferably, the biological activity mediated by the Edg-4 receptor is cell proliferation, which may lead to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colon cancer or prostrate cancer. In one embodiment, cell proliferation is stimulated by LPA.
In another embodiment, the biological activity mediated by the Edg-4 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS") and acute inflammatory exacerbation of chronic lung diseases like asthma.
In yet another embodiment, compounds that block Edg-4 can be potentially effective immunosuppressive agents because activated T cells have Edg-4 receptors (Zheng et al, 2000, FASEB J 14:2387-2389). Edg-4 antagonists may be useful in a variety of autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-4 antagonists can be used in organ transplantation.
In one embodiment, the modulator exhibits selectivity for the Edg-4 receptor. For example, the modulator exhibits at least about 5 to about 200 fold inhibitory selectivity for Edg-4 relative to other Edg receptors. Inhibitory selectivity, can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.25 (Example 25), 6.27 (Example 27) and 6.28 (Example 28) respectively. In a preferred embodiment, inhibitory selectivity can be measured by a calcium mobilization assay. Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art.
In some embodiments, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to other non-Edg receptors, including, but not limited to, other GPCRs, ion channels, growth factor receptors and the like.
In other embodiments, the modulator exhibits at least about 63 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 30 fold inhibitory selectivity for Edg-4 relative to other Edg receptors. In still another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
In one embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In yet another embodiment, the modulator exhibits at least about 63 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In another embodiment, the modulator exhibits at least about 30 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors. In still another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In a preferred embodiment, the modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In another embodiment, the modulator exhibits activating selectivity for the Edg-4 receptor. For example, the modulator exhibits at least about 5 to about 200 fold activating selectivity for Edg-4 relative to other Edg receptors. Activating selectivity, can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.13 (Example 13), 6.15 (Example 15) and 6.16 (Example 16) respectively. In a preferred embodiment, activating selectivity can be measured by a calcium mobilization assay. Other assays suitable for determining activating selectivity would be known to one of skill in the art.
In one embodiment, the modulator exhibits at least about 200 fold activating selectivity for Edg-4 relative to other non-Edg receptors, including, but not limited to, other GPCRs, ion channels, growth factor receptors and the like. In another embodiment, the modulator exhibits at least about 63 fold activating selectivity for Edg-4 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 30 fold activating selectivity for Edg-4 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 10 fold activating selectivity for Edg-4 relative to other Edg receptors.
In one embodiment, the agonist modulator exhibits at least about 5 fold activating selectivity for Edg-4 relative to other Edg receptors.
In still another embodiment, the modulator exhibits at least about 200 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors. In yet another embodiment, the modulator exhibits at least about 63 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In another embodiment, the modulator exhibits at least about 30 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 10 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 5 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In a preferred embodiment, of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-4 relative to other Edg receptors. In another embodiment, the modulator of cell proliferation exhibits at least about 10 fold activating selectivity for Edg-4 relative to other Edg receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 10 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
In one embodiment, the Edg-4 modulator is not a lipid. In another embodiment, the modulator of Edg-4 receptor mediated biological activity does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester. In another embodiment, the modulator of the Edg-4 receptor is not a phospholipid. The term "phospholipid" includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of greater 10 carbon atoms or greater, any N-acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al, 1996, J. Lipid Research 391; Lilliom et al, 1996, Molecular Pharmacology 616, Hooks et al, 1998, Molecular Pharmacology 188; Fischer et al, 1998, Molecular Pharmacology 979; Heise et al, 2001, Molecular Pharmacology 1173; Hopper et al, 1999, J. Med. Chem. 42 (6):963-970; Tigyi et al, 2001 , Molecular Pharmacology 1161).
In another embodiment, the modulator is also not a compound of structural formula:
or a pharmaceutically available salt thereof, wherein:
X is O or S;
R20 is alkyl, substituted alkyl, aryl, substituted aryl or halo;
R2] is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
R2 is hydrogen, alkyl or substituted alkyl;
R24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; or alternatively R23 and R24 form a cycloalkyl ring (International Application No: WO 01/60819).
In another embodiment, the modulator is not any compound of the formula below:
wherein R2o, R2ι and R24 are as previously defined. In yet another embodiment the modulator is not any compound disclosed in International Application No: WO
01/60819. The Edg-4 modulator may be a biomolecule such as a nucleic acid, protein,
(i.e., an enzyme or an antibody) or oligosaccharide or any combination thereof.
Alternatively, the Edg-4 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof. The Edg-4 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
The Edg-4 modulator may also be an organic molecule of molecular weight less than 750 daltons. In one embodiment, the molecular weight is about 200 to about
1000 daltons. In another embodiment, the molecular weight is about 200 to about 750 daltons. In yet another embodiment, the molecular weight is about 200 to about 500 daltons. Preferably, the molecular weight is about 300 to about 500 daltons.
Without wishing to be bound by any particular theory or understanding, the modulator may, for example, facilitate inhibition of the Edg-4 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the Edg-4 receptor, interference with Edg-4 or LPA biosynthesis, covalent modification of either LPA or the Edg-4 receptor, or may otherwise interfere with Edg-4 mediated signal transduction.
In one embodiment, the agonist or antagonist binds to the Edg-4 receptor with a binding constant between about 10 μM and about 1 fM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 10 μM and about 1 nM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 1 μM and about 1 nM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the modulator binds to the Edg-4 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the modulator binds to the Edg-4 receptor with a binding constant better (i.e., less) than about 10 nM.
In a specific embodiment, the modulator is a compound of structural formula (I):
(I)
or a pharmaceutically available solvate or hydrate thereof, wherein:
Ri is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted dialkylamino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino;
X=O or S;
A is NR2, O or 5;
R2 is hydrogen, alkyl or substituted alkyl; and
B and C are independently alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl.
Preferably, R, is alkyl, substituted alkyl, substituted aryl, substituted aryl, arylalkyloxy or substituted sulfonylamino. More preferably, R\ is substituted alkyl. Even more preferably, Ri is substituted haloalkyl. Most preferably, Ri is substituted trifluoroalkyl (preferably, trifluoroalkanyl).
In a preferred embodiment, Ri has the structural formula (II):
(II) wherein:
R is haloalkyl or substituted haloalkyl;
R4 is oxo or thiono; and R5 and R^ are independently hydrogen, halo, alkyl or substituted alkyl.
Preferably, R3 is fluoroalkyl, R4 is oxo and R5 and R are independently hydrogen, halo or alkyl. More preferably, R is trifluoromethyl, R4 is oxo and R5 and R<5 are independently hydrogen, chloro or methyl.
In one preferred embodiment, R5 and Re are hydrogen. In another preferred embodiment, R5 is hydrogen and R is chloro or methyl.
Preferably in any of the above embodiment, X is O, A is NR2 and R2 is hydrogen. In another preferable version of the above embodiments, B and C are alkyl, substituted alkyl, independently, aryl, substituted aryl, heteroaryl or substituted heteroaryl. More preferably, B and C are independently indolo, substituted indolo, imidazolo, substituted, imidazolo, pyrazolo, substituted pyrazolo, phenyl or substituted phenyl. Even more preferably, B is heteroaryl or substituted heteroaryl and C is aryl or substituted aryl. Most preferably, B is pyrazolo or substituted pyrazolo and C is phenyl or substituted phenyl.
In a more specific embodiment, the modulator is a compound of structural formula (III):
(III) wherein:
R7 is hydrogen, alkyl, substituted alkyl or halo; R8 is hydrogen, carbamoyl or substituted carbamoyl; and R9, Rio and Rn are independently hydrogen, alkoxy, substituted alkoxy, halo or optionally, R9and Rio together with the carbons to which they are attached form a [1,3] dioxolane ring.
Preferred modulators include compounds of the structural formula shown below:
139
137
In another embodiment, the agonists or antagonists that can be utilized as part of the methods of the present invention are compounds of structural formula (IV):
(IV) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2, R3, R4 or R5 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5,
-C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-Cιo)alkenyl, -(C2-Cιo)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -(C5-Cιo)cycloheteroaryl, -(C3-C6)cycloheteroalkyl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -NR5R5, =NR5, -(C, -Cιo)alkylNHC(O)(CH2)mR5, -(C3-C10)cycloheteroalkyl(R5)m, -(CH2)mR5, -(d-C,0)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(Cι-C10)alkyl(Cι-Cιo)alkyl, -O(Cι-C10)alkyl, -C(O)(d-Cι0)alkyl,
-C(O)NH(CH2)m(Cι-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cι0)alkyl(d-
Cιo)alkyl), -CO2(Cι-C,0)alkyl, -(d-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cyclo alkyl, -(C8-C] )bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(Cό)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(C,-Cιo)alkyl, -NHC(O)NH(Cι-Cιo)alkyl, -OC(O)O(d-
Cι0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X and Y are each independently C or N; and
Z is O, S, C or N, wherein if Z is O or S, then R3 is an electron pair;
Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; R2 and R3 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R and R4 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
Some illustrative examples of the modulators of this embodiment include:
Another embodiment of the present invention is directed to compounds of structural formula (V), which can be utilized for the purpose of this invention:
(V) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R , R-jor R5 is independently -H, -halo, -NO2, -CN, -OH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-Cι0)alkyl, -(C2-Cιo)alkenyl, -(C2-Cιo)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C1o)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5,-OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(Cι-Cιo)alkyl, -O(C,-Cιo)alkyl, -C(O)(d-C,o)alkyl, -C(O)NH(CH2)m(Cι-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((d-C,o)alkyl(d- C,o)alkyl), -CO2(Cι-Cιo)alkyl, -(Cι-Cιo)alkyl, -(C2-C,0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cι0)alkyl, -CO2(CH2)mH, -NHC(O)(d-Cι0)alkyl, -NHC(O)NH(Cι- C10)alkyl, -OC(O)(d-Cι0)alkyl, -OC(O)O(Cι-Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and
Ri and R or R2 and R3 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring. In a specific embodiment, R\ and R2 are independently aryl, substituted aryl, heteroaryl or substituted heteroaryl. In a more specific embodiment, R2 is indole, and R3 and R4 are hydrogen.
Illustrative examples of the modulators of this embodiment include:
121 and its (+) and (-) enantiomers.
In another embodiment, the modulator is a compound of structural formula (VI):
R2 Rs
(VI) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R], R2, R3, R or R5 is independently -H, -halo, -NO2, -CN, -OH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-C,0)alkenyl, -(C2-Cιo)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5,-OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(CrC,o)alkyl, -O(d-C10)alkyl, -C(O)(C,-C,0)alkyl,
-C(O)NH(CH2)m(d-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(C,-
Cιo)alkyl), -CO2(d-C,o)alkyl, -(d-C10)alkyl, -(C2-C]0)alkenyl,
-(C2-Cιo)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C!o)cycloalkenyl,
-(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(C1-C10)alkyl, -CO2(CH2)mH, -NHC(O)(d-C10)alkyl, -NHC(O)NH(d-
C10)alkyl, -OC(O)(C,-C10)alkyl, -OC(O)O(Cι-Cl0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X, Y and Z are independently O, S, C or N, wherein if X, Y or Z is O or S, Ri is an electron pair;
Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R3 and Rj can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; Ri and R can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R and R5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In a specific embodiment, Ri and R2 together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring. In a more specific embodiment,
R\ and R2 together, and R3 and R4 together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring. In an even more specific embodiment, Ri and
R2 together, and R3 and R together form a 6-membered substituted or unsubstituted cyclic or aromatic ring. Even more specifically, Ri and R2, and R3 and ^ form a 6- membered substituted aromatic or cyclic ring. Illustrative modulators of the invention include, but are not limited to, the following compound:
In a specific embodiment, the modulator is a compound of structural formula (VII):
(NH) or a pharmaceutically available solvate or hydrate thereof, wherein; each of R\, R2, R3, Rt , R5, R7 or R8 is independently -H, -halo, -ΝO2, -CN, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-C1o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-do)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(d-C10)alkylNHC(O)(CH2)mR5, -(d-C10)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(Cι-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(C,-Ci0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(C1- Cιo)alkyl), -CO2(C1-C,0)alkyl, -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-do)heterocycle, -CO2(CH2)m(C1-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(CrC,o)alkyl, -NHC(O)NH(C,-C10)alkyl, -OC(O)O(Cι- C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X is O, S, C or N, wherein if X is O or S, Ri is an electron pair; and
Y and Z are independently N or C, wherein if Y or Z is N, Ri and R2 are each an electron pair. Illustrative modulators of the invention include:
157
165 167
In another specific embodiment, the modulator is a compound of structural formula (VIII):
(VIII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5, R , R8, R9 or Rio is independently -H, -halo, -NO2, -CN, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-C,0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5,
-(C,-C1o)alkylNHC(O)(CH2)mR5, -(C1-C1o)alkylNR5R5) -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(Cι-Cιo)alkyl(Cι-Cιo)alkyl, -O(Cι-C,o)alkyl, -C(O)(C1-Cιo)alkyl,
-C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-Cιo)alkyl(d-
C,o)alkyl), -CO2(Cι-C10)alkyl, -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl,
-(C3-Cι0)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(C1-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(d-C10)alkyl, -OC(O)O(d-
C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and
X and Y are independently O, S or N, wherein if X or Y is O or S, R9 and Rio are an electron pair.
In another embodiment, R7 is a substituted or unsubstituted aryl. An illustrative example of these Egd-4 modulators includes:
125
In another embodiment, the modulator is a compound of structural formula
(IX)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5, R7, R8, R9 or Rio is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C,0)alkyl, -(C2- C10)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-C,o)alkylNHC(O)(CH2)mR5, -(Cι-C10)alkylNR5R5,-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(d-Cιo)alkyl, -O(C,-C10)alkyl, -C(O)(Cι-Cιo)alkyl,
-C(O)NH(CH2)m(Cι-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(Cι-
C10)alkyl), -CO2(d-C10)alkyl, -(d-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl,
-(C3-C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-C10)alkyl, -NHC(O)NH(C,-C10)alkyl, -OC(O)O(d-
Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; and p is independently an integer ranging from 0 to 5.
In a specific embodiment, R2 is a substituted alkyl, and one or more of R5, R7, R8, R9 and R]o are halos. In a more specific embodiment, R2 is a halo-substituted alkyl. In an even more specific embodiment, R2 is CF . Specific examples of the modulators include:
127 129
131
In another specific embodiment, the modulator is a compound of structural formula (X):
(X) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5 or R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C Cι0)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(Cι-do)alkylNHC(O)(CH2)mR5, -(C1-C,o)alkylNR5R5, -CO2H, -(Cι-C10)alkylC(O)NH(CH2)mR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H,
-N(C,-Cιo)alkyl(Cι-Cιo)alkyl, -O(C,-Cιo)alkyl, -C(O)(C1-Cιo)alkyl,
-C(O)NH(CH2)m(d-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(C1- C,o)alkyl), -CO2(d-C10)alkyl, -(d-C10)alkyl, -(C2-C,0)alkenyl, -(C2-Cιo)alkynyl,
-(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C -do)heterocycle, -CO2(CH2)m(Cι-Cι0)alkyl,
-CO2(CH2)mH, -NHC(O)(d-C10)alkyl, -NHC(O)NH(d-Cι0)alkyl, -OC(O)O(Cι-
Ci0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
Ri and R2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R3 and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R and R7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring. In a specific embodiment, R3 and R7 are substituted or unsubstituted aryls. An illustrative modulator of the invention includes:
151
In yet another specific embodiment, the modulator is a compound of structural formula (XI):
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, * , R5, R7 or R8 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5,
-C(O)NH(CH2)m(Rs), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -(C5-Cio)cycloheteroaryl, -naphthyl, -(C3-C!o)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(Cι-do)alkylNHC(O)(CH2)mR5, -(C,-C10)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-C,o)aιkyl(C,-C,o)alkyl, -O(C,-C10)alkyl, -C(O)(C,-Cι0)alkyl,
-C(O)NH(CH2)m(d-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cιo)alkyl(d-
C10)alkyl), -CO2(Cι-C10)alkyl, -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl,
-(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-C1o)alkyl, -CO2(CH2)mH, -NHC(O)(d-C10)alkyl, -NHC(O)NH(C1-Cιo)alkyl, -OC(O)O(d-
C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
Ri and R2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R3 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R3 and t can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; R and R7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R7 and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R\ and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In another embodiment, R2 and R3 together form a 5-membered ring. In a more specific embodiment, R2 and R3 together, and R7 and R8 together form a 5- membered ring. Illustrative examples of the modulators of the invention include:
153
Another illustrative compound of the invention has the following structure:
In another specific embodiment, the modulator is a compound of structural formula (XII):
(XII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5 or R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C10)alkyl, -(C2-C10)alkenyl, -(C2-Cl0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-CM)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -(C5-Cιo)cycloheteroaryl, -(C3-C6)cycloheteroalkyl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -NR5R5, =NR5, -(d-do)alkylNHC(O)(CH2)mR5, -(C3-C10)cycloheteroalkyl(R5)m, -(CH2)mR5, -(d-C10)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H,
-N(d-do)alkyl(Cι-C1o)alkyl, -O(C1-Cιo)alkyl, -C(O)(C1-C1o)alkyl, -C(O)NH(CH2)m(Cι-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C,0)alkyl(C,-
C10)alkyl), -CO2(d-C,0)alkyl, -(d-Cιo)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl,
-(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C -C!o)heterocycle, -CO2(CH2)m(d-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(d-Cι0)alkyl, -NHC(O)NH(C,-C10)alkyl, -OC(O)O(Cι- Cι0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
R3 or can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring. R\ or R2 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring;
R2 or R can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring. Illustrative examples of the modulators of the invention include:
5.3. Synthesis of the Edg-4 Compounds of the Invention
Certain compounds of the invention may be obtained via the synthetic methods illustrated in Schemes 1 and 2. Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods. Other methods for synthesis of the compounds described herein are either described in the art or will he readily apparent to the skilled artisan in view of general references well-known in the art (See e.g., Green et al, "Protective Groups in Organic Chemistry", (Wiley, 2nd ed. 1991); Harrison et al, "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt, Germany; Feiser et al, "Reagents for
Organic Synthesis," Volumes 1-17, Wiley Interscience; Trost et al, "Comprehensive Organic Synthesis," Pergamon Press, 1991; "Theilheimer's Synthetic Methods of Organic Chemistry," Volumes 1-45, Karger, 1991; March, "Advanced Organic Chemistry," Wiley Interscience, 1991; Larock "Comprehensive Organic Transformations," VCH Publishers, 1989; Paquette, "Encyclopedia of Reagents for Organic Synthesis," John Wiley & Sons, 1995) and may be used to synthesize the compounds of the invention. Accordingly, the methods presented in Schemes 1 and 2 herein are illustrative rather than comprehensive.
Scheme 1
The compounds depicted in Scheme 1 are compounds of structural formula (I). Generally, compounds of structural formula (I) may be made by the route depicted in Scheme 1. Condensation of commercially available thiosemicarbazide 1 with acetophenone 3 in the presence of acid, (e.g., acetic acid) provides thiosemicarbazone 5. In the presence of strong base, (e.g., lithium diisopropylamide) ring formation takes place to form amine 7. Condensation of amine 7 with acetoacetate 9 provides the butyramide 11, which may be alkylated or acylated with an activated urea derivative to provide butyramide 13 (R8= alkyl, or -C(0)NHR2o, where R2ois alkyl). Those of skill in the art will appreciate that a wide variety of esters other than the acetoacetate 9 depicted may be condensed with amine 7 to provide compounds of the invention. Further the skilled artisan will appreciate that a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (I) other than those depicted above. Scheme 2
The compounds depicted in Scheme 2 are compounds of structural formula (IX). Generally, compounds of structural formula (IX) may be made by the route depicted in Scheme 2. Unsubstituted or substituted pyridyl hydrazine 2 is reacted with unsubstituted or substituted benzoic acid 1 in the presence of l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1), 4- methylmorpholine and 1-hydroxybenzotriazole hydrate (HOBt), in anhydrous 1 :1 dichloromethane/acetonitrile. Phosphorous oxychloride is then added to the solution of resulting compound 3 in toluene, and the compound of formula (IX) 4 is obtained.
The skilled artisan will appreciate that a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (IX) other than those depicted above.
Illustrative compounds 145, 147, 149, 151, 153, 155, 157 and 159 are commercially available from Specs (http//:www.specsnet.com); compounds 163, 165 and 167 are available from Chemdiv (http//www.chemdiv.com); compound 161 is available from Tripos (http://www.tripos.com); and compound 169 is available for purchase from Comgenex (http://www.comgenex.com).
5.3. The Use of the Edg-7 Compounds of the Invention
The present invention provides a method of modulating an LPA3 or Edg-7 receptor (e.g. human Edg-7, GenBank Accession No. AF127138) mediated biological activity. A cell expressing the Edg-7 receptor is contacted with an amount of an Edg- 7 receptor agonist or antagonist sufficient to modulate the Edg-7 receptor mediated biological activity.
The agonists or antagonists can be utilized in the methods of the present invention and are compounds of structural formula (XIII):
or a pharmaceutically available solvate or hydrate thereof, wherein:
X is NR3, S or O;
R] is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, amino, carbamoyl, substituted carbamoyl, oxo, thiono or -NR4; and
R2 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkyloxy, substituted alkyloxy, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, aryloxycarbonyl, substituted aryloxycarbonyl, arylalkyl, substituted arylalkyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,
.Rs
R6
heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or R is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkylsulfonyl, substituted alkylsulfonyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl or substituted heteroalkyl;
R4 is alkyl, substituted alkyl, acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, heteroaryl substituted heteroaryl, cycloheteroalkyl, substituted cycloheteroalkyl, and
R5 and R<s are independently hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylthio, substituted alkylthio, alkoxycarbonyl, substituted alkoxycarbonyl, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or optionally along with the carbon to which they are attached form an aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl ring.
In a specific embodiment, the modulator is a compound of structural formula:
Preferably, Ri is oxo, thiono or NJLt, more preferably, Rj is oxo or Rt. In one embodiment, R4 is acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl or substituted carbamoyl. Preferably, t is substituted carbamoyl.. In another embodiment, R2 is acyl, substituted acyl, acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, aryloxycarbonyl, substituted aryloxycarbonyl, carbamoyl, substituted carbamoyl, or
Preferably, R2 is substituted alkoxycarbonyl or
Preferably, R5 and R6 along with the carbon to which they are attached form a cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl ring. More preferably, R5 and Re along with the carbon to which they are attached form a substituted cycloheteroalkyl ring. In another embodiment, the agonists or antagonists are also compounds of structural formula (XIV):
(XIV) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3 ^ and R is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-Cl0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cj4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C1-C1o)alkylNHC(O)(CH2)mR5, -(CI-C,o)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein each R5 and R is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(C,-Cι0)alkyl, -O(C,-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(d-C10)alkyl, -OCF3, -benzyl, -C02(CH2)mCH((Ci-Cio)alkyl(Ci- Cιo)alkyl), -CO2(Cι-C10)alkyl, -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(Cό)heteroaryl, -phenyl, naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)m(C1-Cio)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(d-C10)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-Cιo)alkyl, or -SO2NH2; X is CH2, CO, O, S, SO2, C, orNR5;
Ri, R2, R3 R and R taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring; Ri , R2, R3 R and R7 can also be an electron such that when two groups are on adjacent carbon atoms they form a double bond; two Rό groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In another embodiment, the modulator is a compound of structural formula (XV):
(XV) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3 R4 and R7is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-Cιo)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C1-C10)alkylNHC(O)(CH2)mR5, -(C,-C10)alkylNR5R5) , -S(O)2N(R5)C(O)NH(heteroaryl), -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, - OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein each R5 and R is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)aIkyl(Cι-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(d-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(d- Cιo)alkyl), -C02(Cι-Cιo)alkyl, -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(Cι-Cι0)alkyl, -CO2(CH2)mH, -NHC(O)(d-C10)alkyl, -NHC(O)NH(Cι-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-Cι0)alkyl, or -SO2NH2;
X is C, or N;
Ri, R2, R R4 and R7 taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring;
Ri, R , R3 R4 and R7 can also be an electron such that when two groups are on adjacent carbon atoms they form a double bond; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In a specific embodiment, the compound is of formula:
737
In another embodiment, the modulator is of structural formula (XVI):
(XVI) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3 R4 and R7is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-Co)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-do)heterocycle, -CO2(CH2)mRs, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(CrC10)alkylNHC(O)(CH2)mR5, -(Cι-do)alkylNR5R5,
-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
each R5 and Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C,-Cιo)alkyl(Cι-C,o)alkyl, -O(d-d0)alkyl, -C(O)(C,-C,0)alkyl, -C(O) H(CH2)m(C,-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((d-do)alkyl(d- Cιo)alkyl), -CO2(C,-Cι0)alkyl, -(Cι-C10)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(d-C,o)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-Cιo)alkyl, or -SO2NH2;
X, Y, and Z are each independently C=O, O, S, C, or N; wherein if X, Y, or Z is O or S, Ri is an electron pair; Ri , R2, R3 R and R7 taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring; two Re groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5. In another embodiment, the a modulator is of structural formula (XVII):
(XVII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2> R3 R4 and R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-C!o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C1-C10)alkylNHC(O)(CH2)πιR5, -(d-C1o)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
each R5 and Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-C,o)alkyl(C1-C1o)alkyl, -O(C,-C,o)alkyl, -C(O)(d-C10)alkyl, -C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, C10)alkyl), -CO2(d-C10)alkyl, -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C1o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-C,o)alkyl, -NHC(O)NH(d-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-Cιo)alkyl, or -SO2NH2;
X, Y, and Z are each independently C=O, O, S, C, or N; wherein if X, Y, or Z is O or S, Ri is an electron pair; Ri, R2, R3 R4 and R taken in any combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6- membered aromatic ring; two R6 groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In a specific embodiment, the modulator is of formula:
Those of skill in the art will appreciate that Edg-7 receptor is a G protein coupled receptor ("GPCR"). The Edg-7 (LPA3) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-2 (LPAl) and Edg-4 (LPA2), binds lysophosphatidic acid ("LPA"). Preferably, the Edg-7 receptor is a human receptor.
The Edg-7 receptor may be expressed by recombinant DNA methods well known to those of skill in the art. Particularly useful cell types for expressing and assaying Edg-7 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells). Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA). DNA encoding Edg-7 (human Edg-7, GenBank accession AFOl 1466) can be transfected into human or mammalian cells according to methods known to those of skill in the art. For example, DNA encoding human Edg-7 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
Alternatively, DNA encoding Edg-7 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the env gene to produce an ecotropic envelope. Both methods (i.e., co-transfection with a packaging vector or use of EcoPack-293) enable the production of an ecotropic envelope for viral packaging, and can thus advantageously be used to transfect rat and mouse cells. For use in human and other mammalian cells, AmphoPack-293 cell line can be used (Clontech Labs, Palo Alto, CA).
In addition, a number of natural cell lines naturally express Edg-7 receptors. These include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB- 453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells and OV202 human ovarian cancer cells (ATCC, Manassas, VA; Vec Technologies Inc. (Rensselaer, NY); Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA).
Those of skill in the art will appreciate that cells which express the Edg-7 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulation of Edg-7 receptor activity, regardless of the local environment. In one preferred embodiment, cells that express the Edg-7 receptor are grown in vitro (i.e., are cultured). In another preferred embodiment, cells that express the Edg-7 receptor are in vivo (i.e., are part of a complex organism).
The cells in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, carcinoma cells, pheochromocytoma cells, myoblast cells, endothelial cells, platelet cells and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, OV202 human ovarian cells, hepatoma cells (e.g. HTC, Rh7777, HepG2), SKOV3 human ovarian cancer cells, CAOV-3 human ovarian cancer cells, HEY human ovarian cancer cells, HTC rat hepatoma cells, CAOV-3 human ovarian cancer cells, MDA- MB-453 breast cancer cells, MDA-MB-231 breast cancer cells, A431 human epitheloid carcinoma cells and HT-1080 human fibrosarcoma cells.
In a second aspect, an Edg-7 receptor mediated biological activity is modulated in a subject or in an animal model. A therapeutically effective amount of an modulator of the Edg-7 receptor is administered to the subject or animal. Preferably, the subject or an animal is in need of such treatment.
The biological activity mediated by the Edg-7 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation or actin polymerization. Preferably, the biological activity mediated by the Edg-7 receptor also includes, but is not limited to, apoptosis, angiogenesis, inhibition of wound healing, inflammation, cancer invasiveness or atherogenesis. Most preferably, the biological activity mediated by the Edg-7 receptor is cell proliferation, which may lead to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer or prostrate cancer. In one embodiment, cell proliferation is stimulated by LPA.
In another embodiment, the biological activity mediated by the Edg-7 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS") and acute inflammatory exacerbation of chronic lung diseases like asthma.
In yet another embodiment, compounds that block Edg-7 can be potentially effective immunosuppressive agents because activated T cells have Edg-7 receptors (Zheng et al, 2000, FASEB J 14:2387-2389). Edg-7 antagonists may be useful in a variety of autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-7 antagonists can be used in organ transplantation.
In one embodiment, the modulator exhibits inhibitory selectivity for the Edg-7 receptor. For example, the modulator can exhibit at least about 200 fold inhibitory selectivity for Edg-7 relative to other Edg receptors. Inhibitory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.4 (Example 4), 6.6 (Example 6) and 6.7 (Example 7) respectively. Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art. Preferred assays include the calcium mobilization assay of Section 6.5.
In another embodiment, the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-7 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
In still another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In still another embodiment, the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In still another embodiment, the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In still another embodiment, the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors. In a preferred embodiment, an modulator of cell proliferation exhibits at least about 100 fold inhibitory selectivity for Edg-7 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 20 fold inhibitory selectivity for Edg-7 relative to other Edg receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 10 fold inhibitory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In another embodiment, the modulator exhibits stimulatory selectivity for the Edg-7 receptor. For example, the modulator can exhibit at least about 200 fold stimulatory selectivity for Edg-7 relative to other Edg receptors. Stimulatory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.5 (Example 5), 6.7 (Example 7) and 6.8 (Example 8) respectively. Other assays suitable for determining stimulatory selectivity would be known to one of skill in the art. Preferred assays include the calcium mobilization assay of Section 6.5.
In another embodiment, the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 10 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
In still another embodiment, the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors. In still another embodiment, the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In still another embodiment, the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In still another embodiment, the modulator exhibits at least about 10 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In a preferred embodiment, an modulator of cell proliferation exhibits at least about 100 fold stimulatory selectivity for Edg-7 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 20 fold stimulatory selectivity for Edg-7 relative to other Edg receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 10 fold stimulatory selectivity for Edg-7 relative to Edg-2 and Edg-4 receptors.
In one embodiment, the Edg-7 modulator is not a lipid. In another embodiment, the Edg-7 modulator does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester. In another embodiment, the Edg-7 modulator is not a phospholipid. The term "phospholipid" includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of greater 10 carbon atoms or greater, any N- acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al., 1996, J. Lipid Research 391; Lilliom et al, 1996, Molecular Pharmacology 616, Hooks et al, 1998, Molecular Pharmacology 188; Fischer et al, 1998, Molecular Pharmacology 979; Heise et al. , 2001 , Molecular Pharmacology 1173; Hopper et al. , 1999, J.Med. Chem . 42 (6):963-970; Tigyi et al, 2001, Molecular Pharmacology 1161).
In another embodiment, the Edg-7 modulator is not a compound of structural formula (XVIII):
(XVIII) or a pharmaceutically available salt thereof, wherein:
X is O or S;
R2o is alkyl, substituted alkyl, aryl, substituted aryl or halo;
R21 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
R23 is hydrogen, alkyl or substituted alkyl;
R24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl;
or alternatively R2 and R24 form a cycloalkyl ring (International Application No: WO 01/60819). In another embodiment, the modulator is not any compound of the formula below:
wherein R 0, R2ι and R24 are as previously defined. In yet another embodiment the modulator is not any compound disclosed in International Application No: WO 01/60819.
In one preferred embodiment, the modulator is a agonist of the Edg-7 receptor. The modulator can be a weaker agonist than the natural agonist and may compete with the natural agonist for the binding site. In another preferred embodiment, the modulator is antagonist of the Edg-7 receptor. The Edg-7 modulator may be a biomolecule such as a nucleic acid, protein, (i.e., an enzyme or an antibody) or oligosaccharide or any combination thereof. Alternatively, the Edg-7 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof. The Edg-7 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
The Edg-7 modulator may also be an organic molecule of molecular weight less than 750 daltons. In one embodiment, the molecular weight is about 200 to about 1000 daltons. In another embodiment, the molecular weight is about 200 to about 750 daltons. In yet another embodiment, the molecular weight is about 200 to about 500 daltons. Preferably, the molecular weight is about 300 to about 500 daltons.
Without wishing to be bound by any particular theory or understanding, the modulator may, for example, facilitate inhibition of the Edg-7 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the
Edg-7 receptor, interference with Edg-7 or LPA biosynthesis, covalent modification of either LPA or the Edg-7 receptor, or may otherwise interfere with Edg-7 mediated signal transduction. In one embodiment, the agonist or antagonist binds to the Edg-7 receptor with a binding constant between about 10 μM and about 1 fM. In another embodiment, the modulator binds to the Edg-7 receptor with a binding constant between about 10 μM and about 1 nM. In another embodiment, the modulator binds to the Edg-7 receptor with a binding constant between about 1 μM and about 1 nM. In another embodiment, the modulator binds to the Edg-7 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the modulator binds to the Edg-7 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the modulator binds to the Edg-7 receptor with a binding constant better (i.e., less) than about 10 nM.
5.4 Synthesis of the Edg-7 Compounds of the Invention
The compounds of the invention may be obtained via the synthetic methods illustrated in Schemes 3 and 4. Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods. Other methods for synthesis of the compounds described herein are either described in the art or will be readily apparent to the skilled artisan in view of general references well-known in the art (See e.g., Green et al, "Protective Groups in Organic Chemistry", (Wiley, 2nd ed. 1991); Harrison et al, "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt, Germany; Feiser et al, "Reagents for Organic Synthesis," Volumes 1-17, Wiley Interscience; Trost et al, "Comprehensive Organic Synthesis," Pergamon Press, 1991; "Theilheimer's Synthetic Methods of Organic Chemistry," Volumes 1-45, Karger, 1991; March, "Advanced Organic Chemistry," Wiley Interscience, 1991; Larock "Comprehensive Organic Transformations," VCH Publishers, 1989; Paquette, "Encyclopedia of Reagents for Organic Synthesis," John Wiley & Sons, 1995) and may be used to synthesize the compounds of the invention. Accordingly, the methods presented in Schemes 3 and 4 herein are illustrative rather than comprehensive. Scheme 3
The compounds depicted in Schemes 3 and 4 are compounds of structural formula (I). Generally, compounds of structural formula (I) may be made by the route depicted in Scheme 1. Reaction of amine 1 with isocyanate 3 in the presence of organic solvents, (e.g., benzene) provides substituted urea 5.
Those of skill in the art will appreciate that a large number of analogues of 5 may be prepared simply by using different amines 1 and or isocyanates 3. In addition, those of skill in the art will appreciate that a wide variety of compounds other than the isocyanate 3 depicted may be reacted with amine 1 to provide compounds of the invention. Further the skilled artisan will appreciate that a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (I) other than those depicted above.
Scheme 4
-R
As shown in Scheme 4 above, addition of pseudothiohydantoin 11 to isatin 13 in the presence of acid and salt (e.g., acetic acid and sodium acetate) provides indolone 15 which may be alkylated, arylated, acylated or sulfonated by treatment with appropriate compounds to provide indolone 17. The alkylation, arylation, acylation or sulfontion can take place at either or both of the location indicated with a dashed bond. Those of skill in the art will appreciate that a large number of analogues of 17 may be prepared simply by using different alkylation, arylation, acylation or sulfontion agents. Further the skilled artisan will appreciate that a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (I) other than those depicted above.
Illustrative compounds 709, 713, 715 and 723 are also commercially available from Specs (http//www.specs.net); illustrative compound 719 is commercially available from Asinex; illustrative compound 725 is commercially available from Tripos. Illustrative compounds 701, 711, and 719 are commercially available from Asinex. Illustrative compounds 717 is available from Labotest. Illustrative compound 727 is commercially available from Chemdiv. Illustrative compounds 729, 733, 735, and 737 are commercially available from Specs.
5.5 The Use of the Edg-2 Compounds of the Invention The present invention provides a method of modulating an LPAl or Edg-2 receptor (e.g., human Edg-2, GenBank Accession No., U78192) mediated biological activity. A cell expressing the Edg-2 receptor is contacted with an amount of an Edg-2 receptor agonist or antagonist sufficient to modulate the Edg-2 receptor mediated biological activity. Those of skill in the art will appreciate that the Edg-2 receptor is a G protein coupled receptor. The Edg-2 (LPAl) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-4 (LPA2) and Edg-7 (LPA3), binds lysophosphatidic acid ("LPA"). Preferably, the Edg-2 receptor is a human receptor. The Edg-2 receptor may be expressed by recombinant DNA methods well known to those of skill in the art. Particularly useful cell types for expressing and assaying Edg-2 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells). Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA).
DNA encoding Edg-2 is well known (e.g., human Edg-2, GenBank Accession No., U78192) and can be transfected into human or mammalian cells according to methods known to those of skill in the art. For example, DNA encoding human Edg- 2 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
Alternatively, DNA encoding Edg-2 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the env gene to produce an ecotropic envelope. Both methods (i.e. co-transfection with a packaging vector or use of EcoPack-293) enable the production of an ecotropic envelope for viral packaging, and can thus advantageously be used to transfect rat and mouse cells. For use in human and other mammalian cells, AmphoPack-293 cell line can be used (Clontech Labs, Palo Alto, CA).
In addition, a number of natural cell lines naturally express Edg-2 receptors. These include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB- 453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells and OV202 human ovarian cancer cells (ATCC, Manassas, VA; Vec Technologies Inc. (Rensselaer, NY); Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA).
Those of skill in the art will appreciate that cells which express the Edg-2 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulation of the Edg-2 receptor activity regardless of the local environment. In one preferred embodiment, cells that express the Edg-2 receptor are grown in vitro (i.e., are cultured). In another prefened embodiment, cells that express the Edg-2 receptor are in vivo (i.e., are part of a complex organism).
The cells, in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, carcinoma cells, pheochromocytoma cells, myoblast cells, endothelial cells, platelet cells and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, OV202 human ovarian cell, HTC rat hepatoma cells, CAOV-3 and SKOV-3 human ovarian cancer cells, MDA-MB-453 breast cancer cells, MDA-MB-231 breast cancer cells, A431 human epitheloid carcinoma cells and HT-1080 human fibrosarcoma cells.
In a second aspect, an Edg-2 receptor mediated biological activity is modulated in a subject or in an animal model. A therapeutically effective amount of an modulator of the Edg-2 receptor is administered to the subject or an animal. Preferably, the subject or animal is in need of such treatment.
The biological activity mediated by the Edg-2 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of c AMP formation or actin polymerization. Preferably, the biological activity mediated by the Edg-2 receptor includes, but is not limited to, apoptosis, angiogenesis, inhibition of wound healing, inflammation, cancer invasiveness or atherogenesis. Most preferably, the biological activity mediated by the Edg-2 receptor is cell proliferation, which may lead to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colon cancer or prostrate cancer. In one embodiment, cell proliferation is stimulated by LPA.
In another embodiment, the biological activity mediated by the Edg-2 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS") and acute inflammatory exacerbation of chronic lung diseases like asthma.
In yet another embodiment, compounds that block Edg-2 can be potentially effective immunosuppressive agents because activated T cells have Edg-2 receptors (Zheng et al, 2000, FASEB J 14:2387-2389). Edg-2 antagonists may be useful in a variety of autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-2 antagonists can be used in organ transplantation.
In one embodiment, the modulator exhibits selectivity for the Edg-2 receptor. For example, the modulator exhibits at least about 5 to about 200 fold inhibitory selectivity for Edg-2 relative to other Edg receptors. Inhibitory selectivity, can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.8 (Example 8), 6.10 (Example 10) and 6.11 (Example 11) respectively. In a preferred embodiment, inhibitory selectivity can be measured by a calcium mobilization assay. Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art.
In another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to other non-Edg receptors, GPCRs, growth factor receptors, ion channels and the like.
In another embodiment, the modulator exhibits at least about 40 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 12 fold inhibitory selectivity for Edg-2 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
In still another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 40 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 12 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 5 inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors. In a preferred embodiment, an modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-2 relative to other Edg receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 200 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors. In another embodiment, the modulator exhibits activating selectivity for the
Edg-2 receptor. For example, the modulator exhibits at least about 5 to about 200 fold activating selectivity for Edg-2 relative to other Edg receptors. Activating selectivity, can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.8 (Example 8), 6.10 (Example 10) and 6.11 (Example 11) respectively. In a prefened embodiment, activating selectivity can be measured by a calcium mobilization assay. Other assays suitable for determimng activating selectivity would be known to one of skill in the art. In another embodiment, the modulator exhibits at least about 200 fold activating selectivity for Edg-2 relative to other non-Edg receptors, GPCRs, growth factor receptors, ion channels and the like.
In another embodiment, the modulator exhibits at least about 40 fold activating selectivity for Edg-2 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 12 fold activating selectivity for Edg-2 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 5 fold activating selectivity for Edg-2 relative to other Edg receptors.
In still another embodiment, the modulator exhibits at least about 200 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 40 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator exhibits at least about 12 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors. In still another embodiment, the modulator exhibits at least about 5 activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In a prefened embodiment, an modulator of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-2 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 5 fold activating selectivity for Edg-2 relative to other Edg receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 200 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 5 fold activating selectivity for Edg-2 relative to Edg-4 and Edg-7 receptors.
In one embodiment, the Edg-2 modulator is not a lipid. In another embodiment, the modulator of Edg-2 modulator does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester. In another embodiment, the Edg-2 modulator is not a phospholipid. The term "phospholipid" includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of 10 carbon atoms or greater, dioctyl glycerol, any N-acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al., 1996, J. Lipid Research 391; Lilliom et al, 1996, Molecular Pharmacology 616, Hooks et al, 1998, Molecular Pharmacology 188; Fischer et al, 1998, Molecular Pharmacology 979; Heise et al, 2001, Molecular Pharmacology 1173; Hopper et al, 1999, J.Med. Chem. 42 (6):963-970; Tigyi et al. , 2001 , Molecular Pharmacology 1161).
In another embodiment, the Edg-2 modulator is not a compound of the formula:
or a pharmaceutically available salt thereof, wherein:
X is O or S;
R2o is alkyl, substituted alkyl, aryl, substituted aryl or halo;
R21 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
R2 is hydrogen, alkyl or substituted alkyl;
R24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl;
or alternatively R23 and R24 form a cycloalkyl ring (International Application No: WO 01/60819). In another embodiment, the modulator is not any compound of the formula below:
wherein R2o, R2ι and R24 are as previously defined. In yet another embodiment the modulator is not any compound disclosed in International Application No: WO 01/60819.
In one embodiment, the Edg-2 modulator is an agonist of the Edg-2 receptor. In one aspect, such an embodiment provides an Edg-2 modulator that is an agonist, but is a weaker agonist than a natural Edg-2 agonist (e.g., LPA) and as such, may compete with the natural agonist for Edg-2 binding site, resulting in a net inhibition of Edg-2 receptor activity.
In another prefened embodiment, the modulator is antagonist of the Edg-2 receptor. The Edg-2 modulator may be a biomolecule such as a nucleic acid, protein (e.g., an enzyme, an antibody or a soluble Edg-2 receptor polypeptide) or oligosaccharide or any combination thereof. Alternatively, the Edg-2 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof. The Edg-2 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules. The Edg-2 modulator may also be a small organic molecule. In particular embodiments, such a small organic molecule exhibits a molecular weight about 200 to about 1000 daltons, about 200 to about 750 daltons, 200 to about 500 daltons, or about 300 to about 500 daltons. In a particularly preferred embodiment, the small organic molecule can be orally administered to a subject. In another prefened embodiment, the small organic molecule is capable of crossing the blood-brain barrier.
Without wishing to be bound by any particular theory or understanding, the modulator may, for example, facilitate inhibition of the Edg-2 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the Edg-2 receptor, interference with Edg-2 or LPA biosynthesis, covalent modification of either LPA or the Edg-2 receptor, or may otherwise interfere with Edg-2 mediated signal transduction.
In one embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 10 μM and 1 fM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 10 μM and about 1 nM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 1 μM and about 1 nM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the agonist or antagonist binds to the Edg-2 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the agonist or antagonist binds to the Edg-2 receptor with a binding constant better (i.e., less) than about 10 nM.
Preferably, the Edg-2 modulator has the structural formula (XX):
(XX)
or a pharmaceutically available salt, hydrate or solvate thereof wherein:
P, Q and R are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl. In one prefened embodiment, Q is cycloheteroalkyl, substituted cycloheteroalkyl and P and R are independently aryl or substituted aryl. In another prefened embodiment, Q is cycloheteroalkyl or substituted cycloheteroalkyl and P and R are independently phenyl or substituted phenyl. In still another prefened embodiment, Q is heteroaryl or substituted heteroaryl and P and R are independently aryl or substituted aryl. In still another prefened embodiment, Q is heteroaryl or substituted heteroaryl and P and R are independently phenyl or substituted phenyl. In a prefened embodiment, the Edg-2 modulator has the structural formula (XXI):
(XXI)
wherein:
n is 1, 2 or 3;
X = N or CH;
R\ and R2 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cyano, cyanato, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, oxo or thiono;
R3 and R are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halo or thio;
B is NR5, O or S;
R5 is hydrogen, alkyl, substituted alkyl, alkylamino, substituted alkylamino, alkoxy, substituted alkoxy, amino, cyano, dialkylamino, substituted dialkylamino or hydroxy; and
A and C are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl. Preferably, Ri and R2 are independently hydrogen, alkyl substituted alkyl, oxo or thiono. More preferably, Ri and R2 are independently oxo or thiono. In one preferred embodiment, R3 and R are independently hydrogen or alkyl. Preferably, A and C are independently aryl, substituted aryl, heteroaryl or substituted heteroaryl. More preferably, A and C are aryl or substituted aryl. Most preferably, A and C are phenyl or substituted phenyl.
In one embodiment, B is NR5 and R5 is hydrogen, alkyl or hydroxy. In another embodiment, n is 1 , Ri and R2 are oxo, R and R4 are hydrogen, B is NR5 and R5 is hydroxy.
In another preferred embodiment, n is 1 , Ri and R2 are oxo, R3 and Rt are hydrogen, B is NR5, R5 is hydroxy, A and B are aryl or substituted aryl. In still another preferred embodiment, n is 1 , Ri and R2 are oxo, R3 and R4 are hydrogen, B is NR5, R5 is hydroxy, A and B are phenyl or substituted phenyl.
The Edg-2 modulators can also include the following compounds.
In another preferred embodiment, the modulator has the structural formula (XXII):
(XXII) wherein:
R3ι is hydrogen, alkyl or substituted alkyl;
R32 is hydrogen, alkyl or substituted alkyl;
R 3 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; and
R34 is aryl, substituted aryl, heteroaryl or substituted heteroaryl. In one embodiment, R31 and R 2 are alkyl. In another embodiment, R3 and R3 are aryl or substituted aryl. In still another embodiment, R3ι and R32 are alkyl and R 3 and R34 are aryl or substituted aryl. In still another embodiment, R 3 and R34 are phenyl or substituted phenyl. In still another embodiment, R3ι and R 2 are methyl or ethyl and R33 and R 4 are phenyl or substituted phenyl.
In another embodiment, the Edg-2 modulator has the structural formula (XXIII):
X'ZNY
R-I R2
(XXIII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-C,0)alkenyl, -(C2-d0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-C10)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5,
-(Cι-C10)alkylNHC(O)(CH2)mR5, -(d-C10)alkylNR5R5) -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Rό is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(C1-Cιo)alkyl, -O(C,-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(C1-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((d-C10)alkyl(d- Cιo)alkyl), -CO2(d-Cι0)alkyl, -(C,-Cι0)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-do)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C!o)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(d-Cιo)alkyl, -OC(O)O(C,- Cιo)alkyl, or -SO2NH2;
X, Y, and Z are each independently C(R5)( R5), C(O), O, C(S), S, C=N(R5), or NR3; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5; and
Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
In one prefened embodiment, X and Y are both O. In another prefened embodiment, Ri is -N(OH)aryl.
In a prefened embodiment, the Edg-2 modulator has the structural formula (XXIV):
(XXIV) wherein:
each of Ri and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-C,0)alkenyl, -(C2-Cιo)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C )heteroaryl, -(Cό)heteroaryl, -(C -Cιo)heteroaryl, -naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(d-C,o)alkylNHC(O)(CH2)mR5, -(d-C10)alkylNR5R5; -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each of R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(Cι-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH2)m(d-do)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(d- Cιo)alkyl), -CO2(d-C10)alkyl, -(d-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl, -(C -C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C10)heterocycle, -CO2(CH2)m(Cι-Cι0)alkyl, -CO2(CH2)mH, -NHC(O)(C,-Cιo)alkyl, -NHC(O)NH(d-C10)alkyl, -OC(O)O(d- Cιo)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5; Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and Ri and X or R2 and Y can together form a double bond.
Preferably, Ri is -H, and R2 and R3 are independently -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-Cιo)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C!o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -(C1-C1o)alkylNHC(O)(CH2)mR5, -(C1-C1o)alkylNR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, or
wherein;
each of R5 and R is independently -halo, -NO2, -CN, -OH, -CO2H, -O(CrCιo)alkyl, -C(O)(C,-do)alkyl, -C(O)NH(CH2)m(C,-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-C10)alkyl(C,-C,o)alkyl), -CO2(C,-Cι0)alkyl, -(Cι-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-C]0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C10)heterocycle, -CO2(CH2)m(d-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,- C10)alkyl, -NΗC(O)NH(Cι-Cιo)alkyl, -OC(O)O(d-C,0)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5. More preferably, R\ is -H and R2 and R3 are
wherein;
each Re is independently -halo, -NO2, -CN, -OH, -CO2H, -O(Cι-Cιo)alkyl, -C(O)(Ci-C,0)alkyl, -C(O)NH(CH2)m(Cι-d0)alkyl, -OCF3, -benzyl,
-CO2(CH2)mCH((d-C1o)alkyl(C,-C1o)alkyl), -CO2(d-C1o)alkyl, -(C1-C10)alkyl, -(C2-Cιo)alkenyl, -(C2-C10)alkynyl, -(C3-C10)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)m(d-C10)alkyl, -CO2(CH2)mH, -NHC(O)(d- Cιo)alkyl, -NHC(O)NH(d-C,o)alkyl, -OC(O)O(d-C10)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5.
The Edg-2 modulators can also mclude the following compounds:
201 205
A prefened Edg-2 modulator is a compound of the formula below:
In another embodiment, the Edg-2 modulators have the structural formula (XXV):
(XXV)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R! and R2is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -NH(aryl), -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C10)alkenyl, -aryl, -(C2-C10)alkynyl, -(C3-C10)cycloalkyl, -(C3-d0)cycloalkyl(aryl), -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C )heteroaryl, -(C6)heteroaryl, -(C5- Cιo)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, - NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl,
-(C1-C1o)alkylNHC(O)(CH2)mR5, -(Cι-C1o)alkylNR5R5ι -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R<5 is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(Cι-Cιo)alkyl(Cι-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl,
-C(O)NH(CH2)m(Ci-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,o)alkyl(Cι-
Cιo)alkyl), -CO2(d-Cl0)alkyl, -(C,-Cl0)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-C1o)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(d-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(C1-C10)alkyl, -NH(aryl),
-N=C(aryl), -OC(O)O(d-C10)alkyl, or -SO2NH2;
Ri and R2 can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5. The Edg-2 modulator can also preferably be a compound of the structural formula (XXVI):
O
R NHR7
(XXVI)
or a pharmaceutically available solvate or hydrate thereof, wherein;
R, is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH,
-N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3,
-benzyl, -CO2CH(R5)(R5), -(d-Cιo)alkyl, -(C2-C10)alkenyl, -(C2-CI0)alkynyl, -(C3-C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, - OC(O)aryl, -(C,-C10)alkylNHC(O)(CH2)mR5, -(C,-Cιo)alkylNR5R5)- OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(d-do)alkyl(Cι-C10)alkyl, -O(C,-C,0)alkyl, -C(O)(Cι-Cιo)alkyl,
-C(O)NH(CH2)m(Cι-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cι0)alkyl(C C10)alkyl), -CO2(C,-Cιo)alkyl, -(d-Cl0)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-do)heterocycle, -CO2(CH2)m(C]-do)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-C10)alkyl, -NHC(O)NH(d-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-C,0)alkyl, or -SO2NH2;
R7 is -CO2H, -C(O)(d-C,0)alkyl, -C(O)NH(CH2)m(d-Cι0)alkyl, -benzyl, -CO2(CH2)mCH((C1-C1o)alkyl(C1-do)alkyl), -CO2(C1-C1o)alkyl, -(C1-C1o)alkyl, -(C2-C10)alkenyl, -(C2-Cι0)alkynyl, -(C3-C10)cycloalkyl, -(C8-CM)bicycloalkyl, -(C5-C]o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(C1-C10)alkyl, -CO2(CH2)mH, Ri and R2 can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two R<5 groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
The Edg-2 modulators can also include the following compounds:
In one embodiment, the Edg-2 modulators of the invention have the structural formula (XXVII):
(XXVII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R, and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-do)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-do)heterocycle, -OC(O)aryl, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(Cι-C1o)alkylNHC(O)(CH2)mR5, -(Cι-do)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C -Cιo)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-do)heteroaryl, -naphthyl, -(C3-do)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl),
-heterocylcoalkyl, -(d-Cιo)alkylNHC(O)(CH2)mR5, -(Cι-Cιo)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R and Re is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(C1-C1o)alkyl(d-C1o)alkyl, -O(C,-C,o)alkyl, -C(O)(Cι-CIO)alkyl,
-C(O)NH(CH2)m(d-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,o)alkyl(C,- C10)alkyl), -CO2(C,-C10)alkyl, -(d-C,0)alkyl, -(C2-Cιo)alkenyl, -(C2-C10)alkynyl, -(C3-do)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(C1-CI0)alkyl, -NHC(O)NH(Ci-Cio)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-C10)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In one preferred embodiment, R3 is -N=C(aryl). In another prefened embodiment, Ri is -NH(aryl).
In a preferred embodiment, the Edg-2 modulator has the structural formula (XXVIII):
(XVIII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2 and R4is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3,
-benzyl, -CO2CH(R5)(R5), -(d-C10)alkyl, -(C2-Cιo)alkenyl, -(d-C^alkynyl, -(C3-do)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)mR5,
-N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, - OC(O)aryl, -(C1-Cι0)alkylNHC(O)(CH2)mR5, -(C,-do)alkylNR5R5, -
OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5,
-S(O)2NHR5, or
wherein;
each R5 and Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C,-Cιo)alkyl(Cι-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-C10)alkyl, -C(O)NH(CH2)m(Cι-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-do)alkyl(d- C,o)alkyl), -CO2(Cι-Cι0)alkyl, -(Cι-C10)alkyl, -(C2-Cιo)alkenyl, -(C2-C10)alkynyl, -(C3-C!o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-do)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Ci-Cio)alkyl, -CO2(CH2)mH, -NHC(O)(d-Cιo)alkyl, -NHC(O)NH(d-C10)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-C,0)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
The Edg-2 modulators can also include the following compounds:
227 229
5.6 Synthesis of the Edg-2 Compounds of the Invention
Compounds of the invention, and intermediates thereof, are commercially available or can be prepared by well-known synthetic methods. Schemes 5 and 6 exemplify synthetic methods for preparing compounds of the invention. Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods. Other methods for synthesis of the compounds described herein are either described in the art or will be readily apparent to the skilled artisan in view of general references well-known in the art (See e.g., Green et al, "Protective Groups in Organic Chemistry", (Wiley, 2nd ed. 1991); Harrison et al, "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt, Germany; Feiser et al, "Reagents for Organic Synthesis," Volumes 1-17, Wiley Interscience; Trost et al, "Comprehensive Organic Synthesis," Pergamon Press, 1991; "Theilheimer's Synthetic Methods of Organic Chemistry," Volumes 1- 45, Karger, 1991; March, "Advanced Organic Chemistry," Wiley Interscience, 1991; Larock "Comprehensive Organic Transformations," VCH Publishers, 1989; Paquette, "Encyclopedia of Reagents for Organic Synthesis," John Wiley & Sons, 1995) and may be used to synthesize the compounds of the invention. Accordingly, the methods presented in Schemes 5 and 6 herein are illustrative rather than comprehensive. In addition, compounds of formula 227, 229, and 231 are commercially available from Chemical Diversity Labs, Inc. (San Diego, CA).
The compounds depicted in Schemes 1 and 2 are compounds of structural formula (II). Compounds of structural formula (II) may be made by the route depicted in these Schemes.
Scheme 5
7
11
As shown above in Scheme 5, Michael addition of aromatic amine 3 to maleic anhydride 1 provides amide 5, which may be cyclized (e.g., sodium acetate and acetic anhydride) to yield imide 7. Michael addition of phenylhydroxylamine, which may be prepared from nitrobenzene by partial reduction (e.g., Zn, NH4C1) gave the desired disubstituted imide 11. Those of skill in the art will appreciate that a large number of analogues of 11 may be prepared simply by using different amines 3 and or hydroxylamines other than phenyl hydroxylamine.
Scheme 6
As shown in Scheme 6 above, free radical bromination of acylated phenol 21 (e.g., NBS, AIBN) gave bromide 23, which may be converted to ketone 25 (CrCl2) by internal acyl transfer. Ketone 25 may be acylated to provide benzoate 27, (e.g., benzoyl chloride, dimethylaminopyridine) which can undergo cyclization to give pyrazole 29 (e.g., ethylhydrazine, acetic acid). Those of skill in the art will appreciate that many analogues may be simply prepared by using a different acylating agent or by staring with a different acyl phenol.
5.7 The Use of the Edg-3 Compounds of the Invention
The present invention provides a method of modulating an SI P3 or Edg-3 receptor (e.g., human Edg-3, GenBank Accession No. X83864) mediated biological activity. A cell expressing the Edg-3 receptor is contacted with an amount of an Edg- 3 receptor agonist or antagonist sufficient to modulate an Edg-3 receptor mediated biological activity.
Those of skill in the art will appreciate that Edg-3 is a G protein coupled receptor ("GPCR"). The Edg-3 (S1P3) receptor is encoded by an endothelial differentiation gene and along with related receptors, Edg-1 (SlPl), Edg-5 (S1P2), Edg-6 (S1P4) and Edg-8 (S1P5), binds sphingosine- 1-phosphate ("SIP"). Preferably, the Edg-3 receptor is a human receptor.
The Edg-3 receptor may be expressed by recombinant DNA methods well known to those of skill in the art. Particularly useful cell types for expressing and assaying Edg-3 include, but are not limited to, HTC4 (rat hepatoma cells), RH7777 (rat hepatoma cells), HepG2 (human hepatoma cells), CHO (Chinese hamster ovary cells) and HEK-293 (human embryonic kidney cells). Particularly useful vectors for expressing G-protein receptors include, but are not limited to, pLXSN and pCMV (Clontech Labs, Palo Alto, CA; Invitrogen Corporation, Carlsbad, CA). DNA encoding Edg-3 is well known (e.g., human Edg-3, GenBank Accession
No. X83864) and can be transfected into human or mammalian cells according to methods known to those of skill in the art. For example, DNA encoding human Edg- 3 can be co-transfected with a standard packaging vector, such as those described above, which provides an ecotropic envelope for viral replication, into a packaging cell line such as GP-293 (Clontech Labs, Palo Alto, CA).
Alternatively, DNA encoding Edg-3 can be transfected into the EcoPack-293 cell line which has, in addition to gag and pol, the env gene to produce an ecotropic envelope. Both methods (i.e., co-transfection with a packaging vector or use of EcoPack-293) enable the production of an ecotropic envelope for viral packaging, and can thus advantageously be used to transfect rat and mouse cells. For use in human and other mammalian cells, AmphoPack-293 cell line can be used (Clontech, Palo Alto, CA).
A number of natural cell lines naturally express Edg-3 receptors. These include, but are not limited to, CaOV-3 human ovarian cancer cells, MDA-MB-453 and MDA-MB-231 breast cancer cells, HT-1080 human fibrosarcoma, HUVEC cells, OV202 human ovarian cancer cells, Hela human cervical adenocarcinoam cells, HEK293 human embryonic kidney cells, NIH 3T3 mouse fibroblast cells (ATCC, Manassas, VA; Vec Technologies Inc., Rensselaer, NY; Dr. Edward Goetzl, University of California, San Francisco, San Francisco, CA). Those of skill in the art will appreciate that cells which express the Edg-3 receptor may grown in vitro or may be part of a complex organism such as, for example, a mammal. It is contemplated that the methods of the current invention will be applicable to modulating, e.g., agonizing or antagonizing, Edg-3 receptor activity, regardless of the local environment. In one prefened embodiment, cells that express the Edg-3 receptor are grown in vitro (i.e., are cultured). In another prefened embodiment, cells that express the Edg-3 receptor are in vivo (i.e., are part of a complex organism).
The cells in which the method of the invention may be practiced include, but are not limited to, hepatoma cells, ovarian cells, epithelial cells, fibroblast cells, neuronal cells, cardiac myocytes, carcinoma cells, pheochromocytoma cells, myoblast cells, platelet cells, endothelial cells, keratinocytes and fibrosarcoma cells. More specifically, the cells in which the invention may be practiced include, but are not limited to, OV202 human ovarian cells, HTC rat hepatoma cells, CAOV-3 and SKOV-3 human ovarian cancer cells, MDA-MB-453 breast cancer cells, MDA-MB- 231 breast cancer cells, HUVEC, Hela human cervical adenocarcinoam cells, HEK293 human embryonic kidney cells, NIH 3T3 mouse fibroblast cells, A431 human epitheloid carcinoma cells, and HT-1080 human fibrosarcoma cells.
In a second aspect of the invention, an Edg-3 receptor mediated biological activity is modulated in a subject or in an animal model. A therapeutically effective amount of an modulator of the Edg-3 receptor is administered to the subject or animal. Preferably, the subject or animal is in need of such treatment.
The biological activity mediated by the Edg-3 receptor may include, for example, calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation or actin polymerization. Preferably, the biological activity mediated by the Edg-3 receptor also includes, but is not limited to, apoptosis, angiogenesis, wound healing, inflammation, expression of endogenous protein growth factors, cancer invasiveness or atherogenesis. Most preferably, the biological activity mediated by the Edg-3 receptor is cell proliferation, which may lead to enhancement of wound healing; alternatively, it may lead ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer or prostrate cancer. In one embodiment, cell proliferation is stimulated by SIP. In another embodiment, the biological activity mediated by the Edg-3 receptor may include increasing fatty acids levels (e.g., free fatty acids and lyso- phosphatidylcholine) which may lead to acute lung diseases, such as adult respiratory distress syndrome ("ARDS") and acute inflammatory exacerbation of chronic lung diseases like asthma.
In yet another embodiment, the present invention provides methods for using Edg-3 antagonists in treating or preventing disorders such as, but not limited to, vasoconstriction in cerebral arteries, autoimmune and related immune disorders, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomerulonephritis, chronic thrombocytopenic purpura, and autoimmune hemolytic anemia. Additionally, Edg-3 antagonists can also be used in organ transplantation. Without intending to be bound by any particular mechanism or theory of action, Edg-3 antagonists are believed to be potentially effective immunosuppresive agents because activated T cells express the Edg-3 receptor.
In yet another embodiment, Edg-3 agonists and antagonists can be used to treat vascular occlusive disorders. For example, activation of Edg-3 receptors by using an Edg-3 agonist can result in increased vasoconstriction, which is beneficial in conditions such as migraine headaches. Inhibition of Edg-3 by an Edg3 antagonist can be beneficial in conditions such as a stroke, a subarachnoid hemorrhage, or a vasospasm such as a cerebral vasospasm.
In certain aspects, the modulator exhibits inhibitory selectivity for the Edg-3 receptor. In one embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. Inhibitory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.4 (Example 4), 6.6 (Example 6) and 6.7 (Example 7) respectively. Other assays suitable for determining inhibitory selectivity would be known to one of skill in the art. Preferred assays include the calcium mobilization assay of Section 6.4. In another embodiment, the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-3 relative to other Edg receptors.
In another embodiment, the modulator exhibits about 5 fold to about 200 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. In still another embodiment, the modulator exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits at least about 100 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits about 5 fold to about 200 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In a preferred embodiment, the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to other Edg receptors. In still another embodiment, the modulator of cell proliferation exhibits at least about 5 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 20 fold inhibitory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In other aspects, the modulator exhibits stimulatory selectivity for the Edg-3 receptor. In one embodiment, the modulator exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to other Edg receptors. Stimulatory selectivity can be measured by assays such as a calcium mobilization assay or a migration and/or invasion assay or a proliferation assay, for example, as described in Section 6.4 (Example 4), 6.6 (Example 6) and 6.7 (Example 7) respectively. Other assays suitable for determining stimulatory selectivity would be known to one of skill in the art. Preferred assays include the calcium mobilization assay of Section 6.4. In another embodiment, the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-3 relative to other Edg receptors. In another embodiment, the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
In another embodiment, the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
In still another embodiment, the modulator exhibits about 5 fold to about 200 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
In still another embodiment, the modulator exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors. In still another embodiment, the modulator exhibits at least about 100 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits at least about 200 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator exhibits about 5 fold to about 200 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In a prefened embodiment, the modulator of cell proliferation exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
In another embodiment, the modulator of cell proliferation exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to other Edg receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 5 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In still another embodiment, the modulator of cell proliferation exhibits at least about 20 fold stimulatory selectivity for Edg-3 relative to Edg-1, Edg-5, Edg-6 and Edg-8 receptors.
In certain embodiments, the Edg-3 modulator is not a lipid. In certain embodiments, the modulator of Edg-3 receptor mediated biological activity does not contain a phosphate group such as a phosphoric acid, a cyclic phosphate ester or a linear phosphate ester. In certain embodiments, the modulator of the Edg-3 receptor is not a phospholipid. The term "phospholipid" includes all phosphate (both phosphate esters and phosphoric acids) containing glycerol derivatives with an alkyl chain of greater 10 carbon atoms or greater, dioctyl glycerol, any N-acyl ethanolamide phosphate derivative (both phosphate esters and phosphoric acids), LPA, SIP or any of their analogues (both phosphate esters and phosphoric acids) (see, e.g., Bandoh, et al, 2000, FEBS Lett. 428, 759; Bittman et al., 1996, J. Lipid Research 391; Lilliom et al, 1996, Molecular Pharmacology 616, Hooks et al, 1998, Molecular Pharmacology 188; Fischer et al, 1998, Molecular Pharmacology 979; Heise et al, 2001 , Molecular Pharmacology 1173; Hopper et al, 1999, J.Med. Chem. 42 (6):963- 970; Tigyi et al, 2001, Molecular Pharmacology 1161).
In certain embodiments, the modulator is also not a compound of structural formula (XXXIV):
(XXXIV)
or a pharmaceutically available salt thereof, wherein:
X is O or S;
R2o is alkyl, substituted alkyl, aryl, substituted aryl or halo;
R21 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
R23 is hydrogen, alkyl or substituted alkyl;
R24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl;
or alternatively R23 and R24 form a cycloalkyl ring (International Application No: WO 01/60819). In certain embodiments, the modulator is not any compound of the formula below:
wherein R2o, R2ι and R24 are as previously defined. In yet another embodiment the modulator is not any compound disclosed in International Application No: WO 01/60819.
In certain embodiments, the modulator can be an agonist of the Edg-3 receptor. The agonist can be a weaker agonist than the natural agonist and may compete with the natural agonist for the binding site. In other embodiments, the modulator can be an antagonist of the Edg-3 receptor.
In certain aspects, the Edg-3 modulator can be a biomolecule such as a nucleic acid, protein, (e.g., an enzyme, an antibody, or a soluble Edg-3 receptor polypeptide) or oligosaccharide or any combination thereof. Alternatively, the Edg-3 modulator may be oligomers or monomers of the above biomolecules such as amino acids, peptides, monosaccharides, disaccharides, nucleic acid monomers, dimers, etc., or any combination thereof. The Edg-3 modulator may also be a synthetic polymer or any combination of synthetic polymer with biomolecules including monomers or oligomers of biomolecules.
The Edg-3 modulator may also be a small organic molecule. In certain embodiments, the Edg-3 modulator can be an organic molecule of molecular weight less than 750 daltons. In other embodiments, the molecular weight can be about 200 to about 1000 daltons. In other embodiments, the molecular weight can be about 200 to about 750 daltons. In yet other embodiments, the molecular weight can be about 200 to about 600 daltons. In certain prefened embodiments, the molecular weight is about 300 to about 500 daltons. In certain embodiments, the small organic molecule can be orally administered to a subject. In other embodiments, the small organic molecule is capable of crossing the blood-brain barrier.
Without wishing to be bound by any particular theory or understanding, the modulator may, for example, facilitate inhibition of the Edg-3 receptor through direct binding to the LPA binding site of the receptor, binding at some other site of the Edg-3 receptor, interfering with Edg-3 or LPA biosynthesis, covalently modifying either the LPA or the Edg-3 receptor, or otherwise interfering with Edg-3 mediated signal transduction. In one embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 10 μM and about 1 fM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 10 μM and about 1 nM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 1 μM and about 1 nM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 100 nM and about 1 nM. In another embodiment, the modulator binds to the Edg-3 receptor with a binding constant between about 10 nM and about 1 nM. Preferably, the modulator binds to the Edg-3 receptor with a binding constant better (i.e., less) than about 10 nM. In certain embodiments, the modulator is a compound of structural formula
(XXXI):
or a pharmaceutically available solvate or hydrate thereof, wherein:
n = 0 or l;
o is O, 1, 2, 3 or 4;
X is C, NR7 O or S;
Y is C, NR8 O or S;
Ri is either absent or hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl;
R2, R3 and R are independently hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl;
each R5 is independently, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, azido, carboxy, carbamoyl, substituted carbamoyl, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, halo, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl, hydroxyl, nitro or thio; and
R7and R8 are independently absent, hydrogen, alkyl, substituted alkyl, acyl or substituted acyl. In other embodiments, the modulator is a compound of structural formula (XXXV) or structural formula (XXXVI):
In one embodiment, R\ is either absent or hydrogen, acyl, substituted acyl, acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylamino, substituted alkylamino, alkylarylamino, substituted alkylarylamino, arylamino, substituted arylamino, arylalkyloxy, substituted arylalkyloxy, carbamoyl, substituted carbamoyl, dialkylamino, substituted dialkylamino, heteroalkyl, or substituted heteroalkyl. Preferably, Rt is either absent or acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, arylamino substituted arylamino, or carbamoyl, substituted carbamoyl. More preferably, Ri is either absent or acylamino, substituted acylamino, arylamino or substituted arylamino.
In another embodiment, R2, R3 and R4 are independently alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, carbamoyl, substituted carbamoyl, dialkylamino, substituted dialkylamino, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl. Preferably, R2, R3 and R4 are independently alkyl, substituted alkyl, acylamino, substituted acylamino, aryl, substituted aryl, arylamino, substituted arylamino, carbamoyl or substituted carbamoyl. More preferably, R2, R3 and R4 are independently alkyl, substituted acylamino, aryl, substituted arylamino or substituted carbamoyl. In another embodiment, each R5 is independently, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, amino, aryl, substituted aryl, azido, carboxy, carbamoyl, substituted carbamoyl, cyano, halo, hydroxyl, nitro or thio. Preferably, each R5 is independently, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, azido, carboxy, carbamoyl, substituted carbamoyl, cyano, halo, hydroxyl, nitro or thio. Preferably, R7and R8 are independently absent, hydrogen, alkyl.
In still other embodiments, the modulator is a compound of structural formula (XXXII):
(XXXII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(d-do)alkyl, -(C2-C10)alkenyl, -(C2-Cι0)alkynyl, -(C -do)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C )heteroaryl, -(C6)heteroaryl, -(C -Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mRs, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(C,-C1o)alkylNHC(O)(CH2)mR5, -(Cι-C1o)alkylNR5R5l -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C -Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-do)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH )mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(Cι-Cιo)alkylNHC(O)(CH2)mR5, -(C1-Cιo)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(CrC,o)alkyl(C,-C,o)alkyl, -O(Cι-C,0)alkyl, -C(O)(C1-Cιo)alkyl, -C(O)NH(CH2)m(Cι-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-Cι0)alkyl(C1-
C10)alkyl), -CO2(d-Cιo)alkyl, -(d-C10)alkyl, -(C2-C,0)alkenyl, -(C2-d0)alkynyl,
-(C3-C1o)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-C]o)cycloalkenyl, -(C )heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-C10)alkyl, -NHC(O)NH(Cι-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-C10)alkyl, or -SO2NH2;
X is O, S, C(R5)(R5) or N(R5);
R], R2 or R3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
In another embodiment, the modulator is a compound of structural formula
(XXXVII):
(XXXVII) or a pharmaceutically available solvate or hydrate thereof, wherein:
Ri is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted alkyl amino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino; and X = O or S.
In yet other embodiments, the Edg-3 receptor modulator is a compound of structural formula (XXXVIII):
(XXXVIII) or a pharmaceutically available solvate or hydrate thereof, wherein:
Ri is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted alkyl amino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino; and X = O.
In yet other embodiments, the Edg-3 receptor modulator has the structural formula (XXXIII):
R3 R2
(XXXIII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-C,o)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C -Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-do)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C1-C1o)alkylNHC(O)(CH2)mR5, -(d-C10)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(d-do)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-do)cycloalkenyl, -(Cs)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(d-C10)alkylNHC(O)(CH2)mR5, -(Cι-C10)alkylNR5R5,
-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(Cι-Cιo)alkyl, -O(Cι-C,o)alkyl, -C(O)(C,-Cιo)alkyl, -C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C10)alkyl(C1- C10)alkyl), -CO2(C,-C10)alkyl, -(d-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-C1o)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-do)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C -C!o)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(d-Cιo)alkyl, -NHC(O)NH(Cι-C10)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-Cιo)alkyl, or -SO2NH2; X is O, S, or N(R5); Ri, R2 or R3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5. In still other embodiments, the Edg-3 receptor modulator is a compound of structural formula (XXXIX):
(XXXIX) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri , R2, R3 Rt, R7 and R8 is independently -H, -halo, -NO2, -CN, -C(R5)3,
-(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-C!o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C,-do)alkylNHC(O)(CH2)mR5, -(C1-C1o)alkylNR5R5) -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C,-do)alkyl, -(C2-C10)alkenyl, -(C2-do)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C ^heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl),
-heterocylcoalkyl, -(d-Cιo)alkylNHC(O)(CH2)mR5, -(C,-do)alkylNR5R5s -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5) -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Re is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C1-C10)alkyl(d-do)alkyl, -O(d-C,0)alkyl, -C(O)(C,-C10)alkyl,
-C(O)NH(CH2)m(C1-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C10)alkyl(C1- C,o)alkyl), -CO2(d-C10)alkyl, -(d-Cιo)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-C!o)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C10)alkyl, -NHC(O)NH(d-Cιo)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(d-Cι0)alkyl, or -SO2NH2; X is O, S, or N(R5);
Ri and R2, R2 and R3, R3 and R4, R4 and R7ι or R and R taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two Re groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
Preferred Edg-3 modulators include the following compounds:
Preferred Edg-3 receptor modulators also include the following compounds:
303 301 Prefened Edg-3 receptor modulators also include the following compounds:
319
5.8 Synthesis of the Edg-3 Compounds of the Invention
The compounds of the invention may be obtained via the synthetic methods illustrated in Schemes 7, 8, and 9. Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods. Other methods for synthesis of the compounds described herein are either described in the art or will be readily apparent to the skilled artisan in view of general references well-known in the art (See e.g., Green et al, "Protective Groups in Organic Chemistry", (Wiley, 2nd ed. 1991); Harrison et al, "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt, Germany; Feiser et al, "Reagents for
Organic Synthesis," Volumes 1-17, Wiley Interscience; Trost et al, "Comprehensive Organic Synthesis," Pergamon Press, 1991; "Theilheimer's Synthetic Methods of Organic Chemistry," Volumes 1-45, Karger, 1991; March, "Advanced Organic Chemistry," Wiley Interscience, 1991; Larock "Comprehensive Organic Transformations," VCH Publishers, 1989; Paquette, "Encyclopedia of Reagents for Organic Synthesis," John Wiley & Sons, 1995) and may be used to synthesize the compounds of the invention. Accordingly, the methods presented in 7, 8 and 9 herein are illustrative rather than comprehensive.
The route described in Scheme 7 may used to synthesize compounds of Formula (XXXI).
Scheme 7
301
Condensation of propiophenone 1 with isatin 3 (i.e., Pfitzinger reaction, KOH, ethanol, heat) provided quinoline 5, which was then acylated with fluoroamine 301 (i.e., carbodiimide, HOBT) to give amide 301. Those of skill in the art will appreciate that a wide variety of analogues of amide 301 may be made by simply reacting substituted propiophenones with substituted isatins and/or acylating the resulting quinoline with different arylamines.
Scheme 8
The compounds depicted in Schemes 8 and 9 are compounds of structural formula (XXXII). Generally, compounds of structural formula (XXXII) may be made by the route depicted in Scheme 2. Reaction of amine 1 with isocyanate 3 in the presence of organic solvents, (e.g., benzene) provides substituted urea 5. Those of skill in the art will appreciate that a large number of analogues of 5 may be prepared simply by using different amines 1 and/or isocyanates 3. In addition, those of skill in the art will appreciate that a wide variety of compounds other than the isocyanate 3 depicted may be reacted with amine 1 to provide compounds of the invention. Further the skilled artisan will appreciate that a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (XXXII) other than those depicted above.
Scheme 9
As shown in Scheme 3 above, addition of pseudothiohydantoin 11 to isatin 13 in the presence of acid and salt (e.g., acetic acid and sodium acetate) provides indolone 15 which may be alkylated, arylated, acylated or sulfonated by treatment with appropriate compounds to provide indolone 17. The alkylation, arylation, acylation or sulfontion can take place at either or both of the location indicated with a dashed bond. Those of skill in the art will appreciate that a large number of analogues of 17 may be prepared simply by using different alkylation, arylation, acylation or sulfontion agents. Further the skilled artisan will appreciate that a wide variety of conventional synthetic methods may be used to synthesize compounds of structural Formula (II) other than those depicted above. Illustrative compounds 305, 307, and 309 are commercially available from
Specs. Illustrative compounds 301, 311, 313, 315, 319, and 121 are commercially available from Asinex. Compound 317 is commercially available from Chemdiv. 5.9. Therapeutic Uses of the Compounds of the Invention The compounds and or compositions of the present invention may be used to treat diseases, including but not limited to, ovarian cancer (Xu et al, 1995, Biochem. J. 309 (Pt 3):933-940; Xu et al, 1998, JAMA 280 (8):719-723; Goetzl et al, 1999, Cancer Res. 59 (20):5370-5375), peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer and prostrate cancer; acute lung diseases, adult respiratory distress syndrome ("ARDS"), acute inflammatory exacerbation of chronic lung diseases such as asthma (Chilton et al, 1996, J Exp Med 183:2235-45; Arbibe et al, 1998, J Clin. Invest 102:1152-60) surface epithelial cell injury, (e.g., transcomeal freezing or cutaneous bums (Liliom et al, 1998, Am. J. Physiol 274 (4 Pt 1): C1065-C1074)), cardiovascular diseases, (e.g., ischemia (Karliner et al, 2001, J. Mol Cell Cardiol. 33 (9):1713-1717) and athescierosis (Siess et al, 1999, Proc. Natl. Acad. Sci. U.S.A 96 (12):6931-6936; Siess et al, 2000, IUBMB.B Life 49 (3):167-171)). In accordance with the invention, a compound and/or composition of the invention is administered to a patient, preferably a human, in need of treatment for a disease which includes but is not limited to, the diseases listed above. Further, in certain embodiments, the compounds and/or compositions of the invention can be administered to a patient, preferably a human, as a preventative measure against diseases or disorders such as those described above. Thus, the compounds and/or compositions of the invention can be administered as a preventative measure to a patient having a predisposition, which includes but is not limited to, the diseases listed above. Accordingly, the compounds and/or compositions of the invention may be used for the prevention of one disease or disorder and concurrently treating another disease (e.g., preventing cancer and treating cardiovascular diseases). It is well within the capability of those of skill in the art to assay and use the compounds and/or compositions of the invention to treat diseases, such as the diseases listed above.
5.10. Therapeutic/Prophylactic Administration
The compounds and/or compositions of the invention may be advantageously used in medicine, including human medicine. As previously described in Section 5.4 above, compounds and compositions of the invention are useful for the treatment or prevention of diseases, which include but are not limited to, cancers, including, but not limited to, ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, acute lung diseases, including, but not limited to, adult respiratory distress syndrome (ARDS) and acute inflammatory exacerbation of chronic lung diseases such as asthma; surface epithelial cell injury, including, but not limited to, transcomeal freezing or cutaneous bums; cardiovascular diseases, including, but not limited to, ischemia and arthesclerosis.
When used to treat or prevent disease or disorders, compounds and/or compositions of the invention may be administered or applied singly, in combination with other agents. The compounds and/or compositions of the invention may also be administered or applied singly, in combination with other pharmaceutically active agents, including other compounds and/or compositions of the invention.
The current invention provides methods of treatment and prophylaxis by administration to a patient of a therapeutically effective amount of a composition or compound of the invention. The patient may be an animal, is more preferably a mammal, and most preferably a human.
The present compounds and/or compositions of the invention, which comprise one or more compounds of the invention, are preferably administered orally. The compounds and/or compositions of the invention may also be administered by any other convenient route, for example, by infusion or bolus injection, by absoφtion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, (e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc.) that can be used to administer a compound and/or composition of the invention. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. The prefened mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition. In most instances, administration will result in the release of the compounds and/or compositions of the invention into the bloodstream.
In specific embodiments, it may be desirable to administer one or more compounds and/or composition of the invention locally to the area in need of treatment. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of the diseases listed above.
In certain embodiments, it may be desirable to introduce one or more compounds and/or compositions of the invention into the central nervous system by any suitable route, including intraventricular, intrathecal and epidural injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
A compound and/or composition of the invention may also be administered directly to the lung by inhalation. For administration by inhalation, a compound and or composition of the invention may be conveniently delivered to the lung by a number of different devices. For example, a Metered Dose Inhaler ("MDI"), which utilizes canisters that contain a suitable low boiling propellant, (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas) may be used to deliver compounds of the invention directly to the lung.
Alternatively, a Dry Powder Inhaler ("DPI") device may be used to administer a compound and/or composition of the invention to the lung. DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient. DPI devices are also well known in the art. A popular variation is the multiple dose DPI ("MDDPI") system, which allows for the delivery of more than one therapeutic dose. For example, capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch for these systems.
Another type of device that may be used to deliver a compound and/or a composition of the invention to the lung is a liquid spray device. Liquid spray systems use extremely small nozzle holes to aerosolize liquid drug formulations that may then be directly inhaled into the lung.
In one embodiment, a nebulizer is used to deliver a compound and/or composition of the invention to the lung. Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled (see e.g., Verschoyle et al, British J. Cancer 1999, 80, Suppl. 2, 96, which is herein incorporated by reference). Examples of nebulizers include devices supplied by Sheffield/Systemic Pulmonary Delivery Ltd. (See, Armer et al, United States Patent No. 5,954,047; van der Linden et al, United States Patent No. 5,950,619; van der Linden et al, United States Patent No. 5,970,974), Aventis and Batelle Pulmonary Therapeutics.
In another embodiment, an electrohydrodynamic ("EHD") aerosol device is used to deliver a compound and/or composition of the invention to the lung. EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions (see e.g., Noakes et al, United States Patent No. 4,765,539). EHD aerosol devices may more efficiently deliver drugs to the lung than other pulmonary delivery technologies.
In another embodiment, the compounds of the invention can be delivered in a vesicle, in particular a liposome (see Langer, Science 1990, 249:1527-1533; Treat et al, in "Liposomes in the Therapy of Infectious Disease and Cancer," Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); see generally "Liposomes in the Therapy of Infectious Disease and Cancer," Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).
In yet another embodiment, the compounds of the invention can be delivered via sustained release systems, preferably oral sustained release systems. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit Ref Biomed. Eng. 14:201; Saudek et al, N. Engl. J Med. 1989, 321 :574).
In another embodiment, polymeric materials can be used (see "Medical Applications of Controlled Release," Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); "Controlled Drug Bioavailability," Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol Sci. Rev. Macromol Chem. 1983, 23:61; see also Levy et al, Science 1985, 228: 190; Dunng et al, Ann. Neurol 1989, 25:351; Howard et al, JNeurosurg. 1989, 71 : 105). In a prefened embodiment, polymeric materials are used for oral sustained release delivery. In another embodiment, enteric-coated preparations can be used for oral sustained release administration. In still another embodiment, osmotic delivery systems are used for oral sustained release administration (Verma et al, Drug Dev. Ind. Pharm. 2000, 26:695-708). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the compounds and/or composition of the invention, thus requiring only a fraction of the systemic dose (see, e.g. Goodson, in "Medical Applications of Controlled Release," supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in Langer, 1990, Science 249:1527-1533 may also be used.
5.11. Compositions of the Invention
The present compositions contain a therapeutically effective amount of one or more compounds of the invention, preferably in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle, so as to provide the form for proper administration to a patient. When administered to a patient, the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile. Water is a prefened vehicle when the compound of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents or pH buffering agents. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
Pharmaceutical compositions comprising a compound of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of compounds of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable vehicle is a capsule (see e.g., Grosswald et al, United States Patent No. 5,698,155). Other examples of suitable pharmaceutical vehicles have been described in the art (see Remington's Pharmaceutical Sciences, Philadelphia College of Pharmacy and Science, 17th Edition, 1985).
For topical administration compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration. Systemic formulations may be made in combination with a further active agent that improves mucociliary clearance of airway mucus or reduces mucous viscosity. These active agents include, but are not limited to, sodium channel blockers, antibiotics, N-acetyl cysteine, homocysteine and phospholipids.
In a preferred embodiment, the compounds of the invention are formulated in accordance with routine procedures as a composition adapted for intravenous administration to human beings. Typically, compounds of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer. For injection, a compound of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. When necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. When the compound of the invention is administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the compound of the invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions may contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cheny coloring agents and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selectively permeable membranes sunounding an osmotically active driving compound are also suitable for orally administered compounds of the invention. In these later platforms, fluid from the environment sunounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade. For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol) oils, alcohols, slightly acidic buffers between pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 mM, etc). Additionally, flavoring agents, preservatives, coloring agents, bile salts, acylcamitines and the like may be added.
For buccal administration, the compositions may take the form of tablets, lozenges, etc. formulated in conventional manner.
Liquid drug formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically mclude a compound of the invention with a pharmaceutically acceptable vehicle. Preferably, the pharmaceutically acceptable vehicle is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon. Optionally, another material may be added to alter the aerosol properties of the solution or suspension of compounds of the invention. Preferably, this material is liquid such as an alcohol, glycol, polyglycol or a fatty acid. Other methods of formulating liquid drug solutions or suspension suitable for use in aerosol devices are known to those of skill in the art (see, e.g., Biesalski, United States Patent No. 5,112,598; Biesalski, United States Patent No. 5,556,611). A compound of the invention may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, a compound of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, a compound of the invention may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. When a compound of the invention is acidic, it may be included in any of the above-described formulations as the free acid, a pharmaceutically acceptable salt, a solvate or hydrate. Pharmaceutically acceptable salts substantially retain the activity of the free acid, may be prepared by reaction with bases and tend to be more soluble in aqueous and other protic solvents than the conesponding free acid form.
5.12. Methods of Use And Doses
A compound of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended purpose. The compounds of the invention or compositions thereof, are administered or applied in a therapeutically effective amount for use to treat or prevent diseases or disorders including but not limited to, ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, acute lung diseases, (e.g., adult respiratory distress syndrome (ARDS) and asthma) surface epithelial cell injury (e.g., transcomeal freezing and cutaneous bums) and cardiovascular diseases such as ischemia and arthesclerosis.
The amount of a compound of the invention that will be effective in the treatment of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known in the art as previously described. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The amount of a compound of the invention administered will, of course, be dependent on, among other factors, the subject being treated, the weight of the subject, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
For example, the dosage may be delivered in a pharmaceutical composition by a single administration, by multiple applications or controlled release. In a preferred embodiment, the compounds of the invention are delivered by oral sustained release administration. Preferably, in this embodiment, the compounds of the invention are administered twice per day (more preferably, once per day). Dosing may be repeated intermittently, may be provided alone or in combination with other drugs and may continue as long as required for effective treatment of the disease state or disorder.
Suitable dosage ranges for oral administration are dependent on the potency of the, but are generally about 0.001 mg to about 200 mg of a compound of the invention per kilogram body weight. Dosage ranges may be readily determined by methods known to the skilled artisan.
Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 mg to about 100 mg per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to about 1 mg/kg body weight. Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight and comprise active ingredient in the range of about 0.5% to about 10% by weight. Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual or intracerebral administration are in the range of about 0.00 1 mg to about 200 mg per kilogram of body weight. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. Animal model systems include, but are not limited to, human tumor xenografts in nude mice. Such animal models and systems are well known in the art (Andersson et al, 2000, Acta Oncl 39:741-745; Chatzistamou et al, 2001, J. Clin. Endocrinol Metab, 86:2 144- 2152).
The compounds of the invention are preferably assayed in vitro and in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of a specific compound of the invention or a combination of compounds of the invention is preferred for reducing convulsion. The compounds of the invention may also be demonstrated to be effective and safe using animal model systems.
Preferably, a therapeutically effective dose of a compound of the invention described herein will provide therapeutic benefit without causing substantial toxicity. Toxicity of compounds of the invention may be determined using standard pharmaceutical procedures and may be readily ascertained by the skilled artisan. The dose ratio between toxic and therapeutic effect is the therapeutic index. A compound of the invention will preferably exhibit particularly high therapeutic indices in treating disease and disorders. The dosage of a compound of the inventions described herein will preferably be within a range of circulating concentrations that include an effective dose with little or no toxicity.
5.13. Combination Therapy
In certain embodiments, the compounds of the invention can be used in combination therapy with at least one other therapeutic agent. The compound of the invention and the other therapeutic agent can act additively or, more preferably, synergistically. In a prefened embodiment, a compound of the invention is administered concunently with the administration of another therapeutic agent. In another prefened embodiment, a composition comprising a compound of the invention is administered concunently with the administration of another therapeutic agent, which can be part of the same composition as the compound of the invention or a different composition. In another embodiment, a composition comprising a compound of the invention is administered prior or subsequent to administration of another therapeutic agent. Other therapeutic agents, which may be used with the compounds and/or compositions of the invention, include but are not limited to, agonists and antagonists of Edg-2, Edg-3, Edg-4 or Edg-7, drugs used to treat cardiovascular diseases and/or cancer such as, alkylating agents (e.g., cyclophosphamide, melphalan, chlorambucil), platinum compounds (e.g., cisplatin, carboplatin), anthracyclines (e.g., doxorubicin, epirubicin), taxanes (e.g., paclitaxel, docetaxel), chronic oral etoposide, topotecan, gemcitabine, hexamethylamine, methotrexate, and 5-fluorouracil.
5.14. Assays One of skill in the art can use the following assays to identify Edg-2, Edg-3,
Edg-4 or Edg-7 agonists or antagonists.
5.14.1. Intracellular Calcium Measurement Assays Specific assays for Edg-2, Edg-3, Edg-4 or Edg-7 receptor activity are known to those of skill in the art. For example, cells expressing Edg-2, Edg-3, Edg-4 or Edg- 7 receptors can be contacted with a membrane-permeant calcium sensitive dye such as Fluo-4 AM or a proprietary calcium dye loading kit (e.g., FLIPR Calcium Assay kit, Molecular Devices, Sunnyvale, CA). Intracellular calcium is capable of binding to the dye and emitting fluorescent radiation when illuminated at the appropriate wavelength. The cells can thus be illuminated an appropriate wavelength for the dye and any emitting light can be captured by a cooled CCD camera. Changes in fluorescence indicate changes in intracellular calcium resulting from the activation of an Edg-2, Edg-3, Edg-4 or Edg-7 receptor. Such changes can be measured advantageously in whole cells in "real-time" (Berridge et al, Nature Reviews 2000, 1:11-21).
Other methods of measuring intracellular calcium are known to those of skill in the art. For instance, a commonly used technique is the expression of receptors of interest in Xenopus laevis oocytes followed by measurement of calcium activated chloride currents (see Weber, 1999, Biochim Biophys Acta 142 1:213-233). In addition, several calcium sensitive dyes are available for the measurement of intracellular calcium. Such dyes can be membrane permeant or not membrane permeant. Examples of useful membrane permeant dyes include acetoxymethyl ester forms of dyes that can be cleaved by intracellular esterases to form a free acid, which is no longer membrane permeant and remains trapped inside a cell. Dyes that are not membrane permeant can be introduced into the cell by microinjection, chemical permeabilization, scrape loading and similar techniques (Haughland, 1993, in "Fluorescent and Luminescent Probes for Biological Activity" ed. Mason, W.T. pp 34-43; Academic Press, London; Haughland, 1996, in "Handbook of Fluorescent Probes and Research Chemicals", sixth edition, Molecular Probes, Eugene, OR).
5.14.2. IL-8 and VEGF Assays
The levels of interleukin-8 ("IL-8") and vascular endothelial growth factor ("VEGF") are important markers for the proliferative potential, angiogenic capacity and metastatic potential of a tumor cell line. Specific assays for IL-8 and VEGF are known to those of skill in the art. For example, IL-8 and VEGF assays can be performed by techniques that include, but are not limited to, a standard enzyme-linked inimunosorbent assay ("ELISA"). In a standard ELISA, the cells can be cultured, for example, in a 96 well format, serum starved overnight, and treated with LPA or SIP. Dose ranges would be known to one of skill in the art. For example, the doses can range from 0.1-10 μM in serum free medium. Cell supernatants can then be collected to measure the amount of IL-8 or VEGF secreted.
Methods to measure the amount of IL-8 or VEGF secreted are known to one of skill in the art. In one method, an anti-IL-8 or anti -VEGF capture antibody can be adsorbed on to any surface, for example, a plastic dish. Cell supernatants containing IL-8or VEGF can then be added to the dish and any method known in the art for detecting antibodies can be used to detect the anti-IEIL-8 or anti-VEGF antibody. In one embodiment, an anti-IL-8 or anti-VEGF biotinylated detection antibody and streptavidin-HRP can be used for detection via the addition of a substrate solution and colorimetric reading using a microtiter plate reader. The level of IL-8 or VEGF can be interpolated by non-linear regression analysis from a standard curve.
5.14.3. Migration and Invasion Assays Migration and invasion assays are known to one of skill in the art. For example, migration assays can be designed to measure the chemotactic potential of the cell line, or its movement toward a concentration gradient of chemoattractants, such as, but not limited to, LPA or SIP. Invasion assays can be designed, for example, to evaluate the ability of the cell line to pass through a basement membrane, a key feature of metastasis formation.
Specific assays, known to one of skill in the art include a modified Boyden Chamber assay in which a cell suspension can be prepared in serum free medium and added to the top chamber. The concentration of cells to be added, for example, about 105 cells/ml is known to one of skill in the art. An appropriate dose of a chemoattractant can then be added to the bottom chamber. Following an incubation period, the number of cells invading the lower chamber can be quantified by methods known in the art. In one embodiment, Fluoroblok filter inserts can be used and the number of cells migrating to the lower chamber can be quantified by staining the filter inserts and detecting the fluorescence by any means known in the art. The level of fluorescence may be conelated with the number of migrating cells.
5.14.4. Proliferation Assay Proliferation assays quantitate the extent of cellular proliferation in response to a stimulant, which, in the case of Edg-2, Edg-3, Edg-4 or Edg-7 receptors, may be LPA or SIP. Cells can be plated and treated with the stimulant (e.g., LPA or SIP) with or without any serum starvation. Stimulant doses may range from 0.1 to 10 μM and in any event may be readily determined by those of skill in the art. Typically, the cells can be treated for a period of a few hours to a few days before cellular proliferation is measured.
Specific methods to determine the extent of cell proliferation are known to one of skill in the art. For example, one method is bioluminescent measurement of ATP, which is present in all metabolically active cells. ATP can be extracted by addition of Nucleotide Releasing Reagent and its release can be monitored by the addition of the ATP Monitoring Reagent. An enzyme, such as luciferase, which catalyzes the formation of light from ATP and luciferin, can be used to quantitate the amount of ATP present. 5.14.5. Cyclic AMP Assay
Because cAMP acts a second messenger in cell signaling, activating protein kinases that in rum phosphorylate enzymes and transcription factors, cAMP concentration is frequently indicative of the activation state of downstream signaling pathways. For GPCRs like the Edg receptors, coupling via a Gui pathway results in inhibition of adenylyl cyclase activity, the key enzyme involved in breakdown of ATP and formation of cAMP. Thus, assays can be designed to measure inhibition of adenylyl cyclase activity, by first stimulating cAMP formation. One example of a compound, which stimulates cAMP formation is forskolin. Forskolin bypasses the receptor and directly activates adenylyl cyclase. Under these conditions, activation of a Gαi coupled receptor will inhibit forskolin-stimulated cAMP, and an antagonist at such a receptor will reverse the inhibition.
This assay can be performed by any means known to one of skill in the art. For example, cells can be plated and treated with or without any serum starvation. The cells may be initially treated with a compound, such as forskolin, to induce cAMP production. This is followed by the addition of an Edg-2, Edg-3, Edg-4 or Edg- 7 stimulator, for example, LPA or SIP. The dose of stimulator required is well known in the art, and could be in the range from 0.1-10 αM in serum free medium. Following an incubation period, the cells are lysed and the level of cAMP is determined.
The cAMP assay can be performed by any means known to one of skill in the art, for example, by performing a competitive immunoassay. Cell lysates can be added to a plate precoated with anti-cAMP antibody, along with a cAMP-AP conjugate and a secondary anti-cAMP antibody. Detection can be performed by any appropriate means, including, but not limited to, using a substrate solution and chemiluminescent readout.
6. EXAMPLES
The invention is further defined by reference to the following examples, which describe in detail preparation of compounds and compositions of the invention and assays for using compounds and compositions of the invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention. 6.1. Example 1 : Synthesis of 4,4,4-trifluoro-3-oxo-N-(5-phenyl-2H-pyrazol-3- vD-butyramide (101)
Ethyl 4,4,4-trifluoroacetoacetate (3.45 mL, 23.6 mmol) and acetic acid (5.2 mL) were added to 5-phenyl-lH-pyrazol-3-ylamine (2.5 g, 15.7 mmol). The reaction mixture was heated for 2.5 hours at 120°C, cooled to room temperature, concentrated in vacuo and purified by flash chromatography on silica gel
(chloro form/methanol/concentrated aqueous ammonium hydroxide) to provide 3.35 g (72% yield) of 101 as a white solid. 1H NMR (300 MHz, OMSO-d6) δ: 12.8 (s, IH), 10.6 (s, IH), 7.85 (m, 2H), 7.30 (m, 3H), 6.92 (s, IH), 3.04 (m, IH), 2.72 (m, IH). APCI-MS: m z = 298 [d3H,10F3N3O2+ H]. Melting range: 318.6-321.1°C (decomposed).
6.2. Example 2: Synthesis of N- [5-(3,4-dichloro-phen yl)-2H-pyrazol-3-yll-4,4,4- trifluoro-3-oxo-butyramide (103)
Thiosemicarbazide (1.15 g, 12.6 mmol) was added to 3', 4'- dichloroacetophenone (2.0 g, 10.6 mmol) in acetic acid (.12 mL) and ethanol (21 mL) (Dimmock et al, 1991, Ewr. J. Med. Chem. 26:529). The reaction mixture was stined for 4 days at room temperature, concentrated in vacuo and the resultant oil was taken up in chloroform. The chloroform solution was washed successively with saturated aqueous sodium bicarbonate, water and brine, dried with sodium sulfate and concentrated in vacuo to give 2.47 g (89%) of the thiosemicarbazone as a white solid. 1H NΕVIIR (OMS0-d6) δ: 9.7 (br, IH), 8.37 (s, IH), 8.28 (m, IH), 8.22 (s, IH), 7.89 (m, IH), 7.13 (m, IH), 2.24 (s, 3H).
The thiosemicarbazone of 3',4'-dichloroacetophenone (2.5g, 9.43 mmol) was added to a solution of lithium diisopropylamide (39.6 nimol) in THF (20 mL) at 0 °C (Beam, et al, 1997, J. Heterocyclic Chem. 34:1549). After two hours at 0 °C, aqueous hydrochloric acid (63 mL, 3N) was added and the reaction mixture was heated for 1 hour at 100 °C, poured into ice water (200 mL) and neutralized with solid sodium bicarbonate. Extraction of the aqueous mixture with chloroform followed by flash column chromatography on silica gel (4-7% methanol in methylene chloride) provided 1.55 g (72%) of 5-(3,4-dichloro-phenyl)-lH-pyrazol-3-ylamine as a tan foam. Η NMR (300 MHz, OMS0-d6) δ: 11.8 (br, IH), 7.8 (s, IH), 7.6 (m, 2H), 5.8 (s, IH), 4.8 (br, 2H). CI-MS: m/z = 228 [C9H7C12N3 + H].
Finally, following the procedure of Example 1, 5-(3,4-dichloro-phenyl)-lH- pyrazol-3 -ylamine (0.25 g, 1.10 mmol) was reacted with ethyl 4,4,4- trifluoroacetoacetate (0.16 mL, 1.10 mmol) to provide 67 mg (17%) of 103 as a white solid. 1H NMR (300 MHz, OMS0-d6) δ: 13.07 (s, IH), 10.78 (s, IH), 8.23 (s, IH), 7.88 (m, IH), 7.76 (m, IH), 7.27 (s, IH), 3.06 (m, IH), 2.77 (m, IH). CI-MS: m/z = 366 [Cι3H8Cl2F3N302+ H].
6.3. Example 3: Synthesis of 4,4-4-trifluoro-N-[5-(4-methoxy-phenyl)-2- pyrazol-3-vH-3-oxo-butyramide (105) Following the procedure of Example 1 , 5-(4-methoxy-phenyl)-2H-pyrazol-3- ylamine (0.20 g, 1.05 mmol) (Beam, et al, 1991, J. Heterocyclic Chem. 34:1549; Grandin, 1971, Bull. Chim. Soc. Fr. 4002) was reacted with ethyl 4,4,4- trifluoroacetoacetate (0.23 mL, 1.60 mmol) to provide 177mg (51%) of 105 as a tan solid. 1H NMR (300 MHz, OMSO-d6) δ: 12.62 (s, IH), 10.57 (s, IH), 7.77 (m, 2H), 6.92 (m, 3H), 3.70 (s, 3H), 2.93 (m, IH), 2.68 (m, IH). APCI-MS: m/z = 328 [C14H12F3N303 + H]. Melting Range: 307-310 °C (decomposed).
6.4. Example 4: Synthesis of 4,4-4-trifIuoro-N-15-(4-fluoro-phenyl)2H-pyrazol
3-yll-3-oxo-butyramide (107)
Following the procedure of Example 1, 5-(4-fluoro-phenyl)-2H-pyrazol-3- ylamine (0.30 g, 1.69 mmol) (Beam, et al, 1997, J. Heterocyclic Chem. 34:1549; Joshi et al, 1979, J. Heterocyclic Chem. 16:1141) was reacted with ethyl 4,4,4- trifluoroacetoacetate (0.37 mL, 2.54 mmol) to provide 205 mg (39%) of 107 as a white solid. 1H NMR (300 MHz, OM50-d6) δ: 12.78 (s, IH), 10.62 (s, IH), 7.83 (m, 2H), 7.32 (m, 2H), 6.96 (s, IH), 2.93 (m, IH), 2.70 (m, IH). APCI-MS: m/z = 316 [Cι3H9F4N3O2 + H]. Melting Range: 308-310 °C (decomposed).
6.5. Example 5: synthesis of 2-chIoro-4,4,4-trifluoro-3-oxo-N-(5-phenyl-2H- pyrazol-3-yl)-butyramide (109)
Following the procedure of Example 1, 5-phenyl-lH-pyrazol-3-ylamine (0.25 g, 1.57 mmol) was reacted with ethyl 2-chloro-3-keto-4,4,4-trifluorobutyrate (515 mg, 2.36 mmol) to provide 219 mg (42%) of 109 as a white solid. 1H ΝMR (300 MHz, OMSO-d6) δ: 12.95 (s, IH), 11.03 (s, IH), 7.77 (m, 2H), 5.39 (m, 4H), 4.42 (s, IH). CI-MS: m/z = 332 [C13H9ClF3Ν3O2 + H]. Melting Range: 259-261 °C (decomposed). 6.6. Example 6: Synthesis of N-[5-(3,5-dimethoxy-phenyl)-2H-pyrazol-3-yll-
4,4,4-trifluoro-3-oxo-butyramide (111) Thiosemicarbazide (1.9 g, 20.8 mmol) was added to 3', 5'- dimethoxyacetophenone (2.5 g, 13.9 mmol) following the procedure of Example 2 to give 3.5 g (100%) of the thiosemicarbazone as a white solid. 1H NMR (300 MHz, DMSO- δ: 10.2 (s, IH), 8.3 (s, IH), 7.9 (s, IH), 7.0 (s, 2H), 6.5 (s, IH), 3.7 (s, 6H), 2.3 (s, 3H). The thiosemicarbazone of 3',5'-dimethoxyacetophenone (3.5 g, 13.9 mmol) was reacted with base following the procedure of Example 2, to give 2.3 g (75%) of 5-(3,5-dimethoxy-phenyl)-2H-pyrazol-3-ylamine (Beam, et al, 1997, J. Heterocyclic Chem. 34:1549). 1H NMR (300 MHz, DMSO-^) δ: 11.9 (br, IH), 6.8 (s, 2H), 6.4 (s, IH), 5.8 (m, IH), 4.7 (br, 2H), 3.8 (s, 6H). Finally, following the procedure of Example 1 , reaction of 5-(3,5-dimethoxy- phenyl)-2H-pyrazol-3-ylamine (0.30 g, 1.37 mmol) with ethyl 4,4,4- trifluoroacetoacetate (0.30 mL, 2.05 mmol) provided 261 mg (53%) of 111 as a white solid. 1H NMR (300 MHz, DMSO-^) 6: 12.83 (s, IH), 10.78 (s, IH), 7.14 (s, 2H), 7.08 (s, IH), 6.52 (s, IH), 3.77 (s, 6H), 2.99 (m, IH), 2.78 (m, IH). APCI-MS: m/z = 358 [C15H14F3N3O4 + H]. Melting Range: 119-121 °C.
6.7. Example 7: Synthesis of 4,4,4-trifluoro-N-[5-(3-methoxy-phenyl)-2H-
PVrazol-3-yIl-3-oxo-butyramide (113) Following the procedure of Example 1, 5-(3-methoxy-phenyl)-2H-pyrazol-3- ylamine (0.32 g, 1.70 mmol) (Beam et al, 1997, J. Heterocyclic Chem. 34:1549; Bruni et al, 1993, J. Pharm. Sci. 82:480) was reacted with ethyl 4,4,4-trifluoroacetoacetate (0.37 mL, 2.54 mmol) to provide 183 mg (33%) of 113 as a white solid. Η NMR (300 MHz, DMSO-Oό) δ: 12.81 (s, IH), 10.68 (s, IH), 7.54 (s, IH), 7.42(m, 2H), 7.06 (s, IH), 6.96 (m, IH), 3.78 (s, 3H), 2.98 (m, IH), 2.76 (m, IH). APCI-MS: m/z = 328 [Cι4H12F3N3O3 + H]. Melting Range: 107-110 °C.
6.8. Example 8: Synthesis ofN-(5-benzo[3]dioxol-5-vI-2H-pyrazol-3-yl)-4,4,4- trifluoro-3-oxo-butyramide (115)
The thiosemicarbazone of 3',4'-(methylenedioxy) acetophenone (2.6 g, 11.1 mmol) was prepared following the procedure of Example 2 (Dimmock et al, 1991, Ewr. J. Med. Chem. 26:529). Reaction of the thiosemicarbazone with base following the procedure of Example 2 provided 0.37 g (16%) of 5-benzo[l,3]dioxol-5-yl-2H- pyrazol-3 -ylamine as an orange foam (Beam, et al, 1997, J. Heterocyclic Chem. 34:1549). 1H NMR (300 MHz, DMSO-rf*) δ: 11.7 (br, IH), 7.2 (s, IH), 7.0 (m, 2H), 6.0 (s, 2H), 5.7 (s, IH), 4.7 (br, 2H). CI-MS m/z 204 [C,0H9N3O2 + H]. Then following the procedure of Example 1, 5-benzo[l,3]dioxol-5-yl-2H- pyrazol-3-ylamine (0.36 g, 1.77 mmol) was reacted with ethyl 4,4,4- trifluoroacetoacetate (0.39 mL, 2.66 mmol) to provide 164mg (27%) of 115 as a white solid. Η NMR (300 MHz, DMSO-α ) δ: 12.69 (s, IH), 10.67 (s, IH), 7.48 (s, IH), 7.36 (m, IH), 7.02 (m, 2H), 6.09 (s, 2H), 2.96 (m, IH), 2.77 (m, IH). APCI-MS: m/z = 342 [C14H10F3N3O4 + H]. Melting Range: 128-130 °C.
6.9. Example 9: Synthesis of 4,4,4-trifluoro-2-methyl-3-oxo-N-f5-phenyl-2H-
Pyrazol-3-yll-butyramide (117)
Following the procedure of Example 1, 5-phenyl-IH-pyrazol-3-ylamine (0.25 g, 1.57 mmol) was reacted with ethyl 2-methyl-4,4,4-trifluoroacetoacetate (0.47, 2.36 mmol) to provide 75 mg (15%) of 117 as a white solid. 1H NMR (300 MHz, DMSO- d6) 6: 12.67 (s, IH), 10.51 (s, IH), 7.78 (m, 2H), 7.41 (m, 3H), 6.58 (s, IH), 2.64 (m, IH), 1.15 (m, 3H). CI-MS: m/z = 312 [C14Hι2F3N3O2 + H]. Melting Range: 286-288 °C (decomposed).
6.10. Example 10: Synthesis of 3-phenyl-5-(4,4,4-trifIuoro-3-oxo-butyrylamino)- pyrazole-1 -carboxylic acid allylamide (119)
4,4,4-trifluoro-3-oxo-N-(5-phenyl-2H-pyrazol-3-yl)-butyramide (101) was reacted with allyl isocyanate (0.09 mL, 1.0 mmol) in DMF (1 mL) at room temperature for 3 hours. Concentration in vacuo and purification by flash column chromatography on silica (chloro form/methanol concentrated aqueous ammonium hydroxide) provided 190 mg (98%) of 119 as a white solid. Η NMR (390 MHz,
DMSO-d6) δ: 9.87 (s, IH), 8.89 (m, IH), 8.12 (m, 2H), 7.43 (m, 3H), 7.32 (s, IH), 5.88 (m, IH), 5.13 (m, 2H), 3.87 (m, 2H), 3.22 (m, IH), 2.91 (m, IH). CI-MS: m/z= 381 [C,7H,5F3N403 + H]. Melting Range: 114-116 °C.
6.11. Example 11: Synthesis of N-[5-(2-Bromophenyl)-2H-pyrazole-3-yll-4,4,4- trifluoro-3-oxo-butyramide (133) A mixture of 2-bromoacetophenone (2 ml, 14 mmol), thiosemicarbazide (2g, 22 mmol), acetic acid (0.17 ml) and methanol (29 ml) was stined for 16.5 hours at room temperature. The mixture was concentrated in vacuo to obtain 2- bromoacetophenone thiosemicarbazone (3.39 g, 85%). 2-bromoacetophenone thiosemicarbazone was identified by NMR and was used without further purification.
Next, a solution of 2-bromoacetophenone thiosemicarbazone (3.39 g, 12.5 mmol) in tetrahydrofuran (63 ml) was added drop-wise to lithium diisopropylamide (2M in tetrahydrofuran, 37.3 ml, 74.6 mmol) at 0°C under nitrogen atmosphere. After the reaction was completed (as analyzed by TLC), the mixture was quenched with hydrochloric acid (3N, 83 ml), and the organic layer was dried and concentrated in vacuo. Following a silica gel chromatography (5-7.5 % methanol/dichloromethane), 3-amino-5-(2'-bromophenyl)-2H-pyrazole (0.688 g, 23 %) was obtained as a brown oil and identified by NMR. Then a mixture of 3-amino-5-(2'-bromophenyl)-2H-pyrazole (0.68 g, 2.86 mmol), ethyl-4,4,4-trifluoroacetoacetate (0.63 ml, 4.29 mmol) and acetic acid (1 ml) was stirred at reflux for 1.5 hours, then cooled to room temperature and concentrated in vacuo. The residue was azeotroped with toluene (70 ml) and the crude product was chromatographed twice on silica gel (20-40 % CMA/dichloromethane; CMA = 80:18:2 chloroform:methanol:ammonium hydroxide). A tan solid 133 (0.264 g, 25 %) was obtained: mp 128-131°C; 1H NMR (300 MHz, DMSO- <j) δ. 12.68 (s, IH), 10.72 (s, IH), 7.73 (d, IH), 7.54 (d, IH), 7.41 (m, IH), 6.55 (s, 2H), 2.83 (q, 2H); APCI MS m/z 376 [C13H9BrF3N3O2 + H]+.
6.12. Example 12: Synthesis of N-f5-(2,,4,-dimethoxyphenyl)-2H-pyrazole-3-yll-
4,4,4-trifluoro-3-oxo-butyramide (135)
A mixture of 2,4-dimethoxyacetophenone (2.5 g, 13.9 mmol), thiosemicarbazide (1.9 g, 20.8 mmol), acetic acid (0.159 ml) and methanol (28 ml) was heated for five days with stirring. The mixture was cooled to room temperature and concentrated in vacuo to obtain 2,4-dimethoxyacetophenone thiosemicarbazone (4.29 g, >100%) as a tan solid. 2,4-dimethoxyacetophenone thiosemicarbazone was identified by NMR and was used without further purification.
Next, a solution of 2,4-dimethoxyacetophenone thiosemicarbazone (4.29 g, 16.9 mmol) in tetrahydrofuran (85 ml) was added drop-wise to lithium diisopropylamide (2M in tetrahydrofuran, 59 ml, 199 mmol) at ambient temperature under nitrogen atmosphere. After the reaction was completed (as analyzed by TLC), the mixture was quenched with hydrochloric acid (4N, 113 ml), and the organic layer was dried and concentrated in vacuo. Following a silica gel chromatography (5-7.5 % methanol/dichloromethane), 3-amino-5-(2',4'-dimethoxyphenyl)-2H-pyrazole (2.36 g, 64 %) was obtained as a yellow-brown solid and identified by NMR.
Then a mixture of 3-amino-5-(2',4'-dimethoxyphenyl)-2H-pyrazole (0.3 g, 1.37 mmol) , ethyl-4,4,4-trifluoroacetoacetate (0.3 ml, 2.05 mmol) and acetic acid (0.5 ml) was stined at reflux for 1.75 hours, then cooled to room temperature and concentrated in vacuo. The residue was azeotroped with toluene (70 ml) and the resulting residue was chromatographed on silica gel (20-40 % CMA/dichloromethane; CMA = 80:18:2 chloroform:methanol:ammonium hydroxide), followed by a second silica gel chromatography (30-45 % methanol/dichloromethane). A white solid 135 (0.245 g, 50 %) was obtained: mp 125-128°C; 1H NMR (300 MHz, OMSO-d6) δ 12.35 (s, IH), 10.61 (s, IH), 7.46 (d, IH), 6.65 (s, IH), 6.58 (d, IH), 6.45 (s, IH), 3.82 (s, 3H), 3.75 (s, 3H), 2.81 (q, 2H); CI MS m z 358 [C]5H14F3N3O4 + H]+.
6.13. Example 13: Synthesis of 4,4,4-trifluoro-3-oxo-N-(5-thiophen-2-yl-2H-
PVrazol-3-vDbutyramide (137)
A mixture of 2-amino-5-(2'-thienyl)-2H-pyrazole (0.25 g, 1.52 mmol), ethyl- 4,4,4-trifluoroacetoacetate (0.332 ml, 2.28 mmol) and acetic acid (0.5 ml) was stirred at reflux for 1.5 hours and cooled to room temperature. The mixture was concentrated in vacuo and the residue was chromatographed on silica gel (20-40 % CMA/dichloromethane; CMA = 80:18:2 chloroform:methanol:ammonium hydroxide). A white solid 137 (0.332 g, 72 %) was obtained: mp 259-261°C; 1H NMR (300 MHz, DMSO- δ 12.95 (bs, IH), 10.78 (bs, IH), 7.75 (d, 2H), 7.15 (s, IH), 7.08 (s, IH), 3.01 (d, IH), 2.78 (d, IH); CI MS m/z 304 [d !H8F3N3O2S + H]+.
6.14. Example 14: Synthesis of 4,4,4-trifluoro-3-oxo-N-(5-thiophen-3-yl-2H- pyrazol-3-yl)butyramide (139)
A mixture of 3-acetylthiophene (2.5 g, 19.8 mmol), thiosemicarbazide (2.7 g, 29.7 mmol), acetic acid (0.226 ml) and methanol (40 ml) was stirred at 70°C for 14 hours and cooled to room temperature. The mixture was concentrated in vacuo, and 3-acetylthiophene thiosemicarbazone (3.92 g, 100 %) was obtained as a pale yellow solid. The compound was identified by NMR.
Next, a solution of 3-acetylthiophene thiosemicarbazone (2 g, 10.1 mmol) in tetrahydrofuran (50 ml) was added drop-wise to lithium diisopropylamide (2M in tetrahydrofuran, 30 ml, 60 mmol) at 0°C under nitrogen atmosphere. After the reaction was completed (as analyzed by TLC), the mixture was qhenched with hydrochloric acid (3N, 67 ml), and the organic layer was dried using magnesium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel (4- 8 % methanol/dichloromethane), and 3-amino-5-(3-thienyl)-2H-pyrazole (1 g, 60%) was obtained as a brown oil. The compound was identified by NMR. Then a mixture of 3-amino-5-(3-thienyl)-2H-pyrazole (0.9 g, 5.45 mmol), ethyl-4,4,4-trifluoroacetoacetate (1.2 ml, 8.2 mmol) and acetic acid (2 ml) was stined for 1.5 hours at reflux, and then cooled to room temperature. The mixture was concentrated in vacuo and azeotroped with toluene. The residue was chromatographed on silica gel (20-60 % CMA/dichloromethane; CMA = 80:18:2 chloroform:methanol:ammonium hydroxide). A tan solid 139 (0.119 g, 7.2 %) was obtained: mp 251-253°C; 1H NMR (300 MHz, DMSO- 6) δ 12.84 (s, IH), 10.61 (s, IH), 8.19 (s, IH), 7.75 (s, IH), 7.70 (s, IH), 7.12 (s, IH), 3.01 (d, IH), 2.79 (d, IH); CI MS m/z 304 [CnH8F3N3O2S + H]+.
6.15. Example 15: Synthesis of 4,4,4-trifluoro-3-oxo-N-(5-pyridin-4-yl-2H- pyrazol-3-yl)butyramide (141) Hydrazine (0.6 ml, 19 mmol) was added to a mixture of cyanoacetyl-4- pyridine (1.39 g, 9.52 mmol) in acetic acid (6 ml). The addition resulted in an exotherm and the mixture was heated for 2.5 hours. The mixture was then cooled to room temperature and diluted with 37 ml of water. Concentrated hydrochloric acid (0.16 ml) was added, and the mixture was heated for 0.5 hour. The mixture was again cooled to room temperature and filtered. N-(5-pyridin-4-yl-2H-pyrazol-3- yl)acetamide (1 g, 50 %) was obtained as an orange solid. The compound was identified by NMR and mass spectral analyses.
Next, a mixture of N-(5-pyridin-4-yl-2H-pyrazol-3-yl)acetamide (1 g, 4.95 mmol) and hydrochloric acid (IN, 20 ml) was heated for 4 hours, and then cooled to room temperature and filtered with water wash. The filtrate was neutralized with saturated sodium bicarbonate and extracted three times with methylene chloride. The combined extracts were dired using sodium sulfate and concentrated in vacuo. 3-amino-5-(4-pyridyl)-2H-pyrazole (0.122 g, 15 %) was obtained as a yellow solid. The compound was identified by NMR. Then a mixture of 3-amino-5-(4-pyridyl)-2H-pyrazole (0.122 g, 0.76 mmol), ethyl-4,4,4-trifluoroacetoacetate (0.167 ml, 1.14 mmol) and acetic acid (0.5 ml) was stined at reflux for 1.5 hours and then cooled to room temperature. The mixture was diluted with toluene and concentrated in vacuo. The residue was chromatographed twice on silica gel (25-75 % CMA/dichloromethane then 40 % CMA/dichloromethane; CMA = 80:18:2 chloroform:methanol:ammonium hydroxide). A yellow solid 141 (48.8 mg, 21.6 %) was obtained: mp 364-366°C; Η NMR (300 MHz, OMSO-d6) δ 13.13 (s, IH), 10.76 (s, IH), 8.69 (s, 2H), 7.98 (s, 2H), 7.25 (s, IH), 3.17 (d, IH), 2.80 (d, IH); CI MS m/z 299 [C12H9F3N4O2 + H]+.
6.16. Example 16: Synthesis of 5-p-ToIyl-lH-imidazole-2-thiol (143)
2-oxo-2-p-tolyl-ethylammonium chloride was prepared according to the procedures described in Synthesis, pp. 615-618 (1990). Starting from 2-bromo-4'- methylacetophenone (Aldrich), a substitution reaction by sodium diformylamide (TCI-US), followed by an acidic hydrolysis, was performed to provide 95 % yield. 2-oxo-2-p-tolyl-ethylammonium chloride (37.74 g, 0.203 mol), KSCN (Acros,
21.84 g, 0.225 mol, 1.1 equivalent) in glacial acetic acid (500 ml) were stined at 120- 125°C (oil bath temperature) for 2 hours (J. Ind. Chem. Soc, 58:1117-1118 (1981)). The content was then cooled to room temperature, and water (500 ml) was added. The mixture was chilled with an ice bath for 1 hour. The solid product was collected by suction filtration, washed with water, and air-dried. 5-p-tolyl-lH-immidazole-2- thiol was obtained as a yellow solid (37.17 g, 96 %): 1H NMR (300 MHz, OMSO-d6) δ 12.47 (s, IH), 12.10 (s, IH), 7.56 (m, 2H), 7.32 (s, IH), 7.18 (m, 2H), 2.29 (s, 3H); APCI-MS m z 191 [C10H10N2S + H]+; m.p. 266-267°C.
6.17. Example 17: Synthesis of 5-(lH-Indol-3-vn-6-phenyl-4.5-dihydro-2H- fl,2,41triazin-3-one (121)
Compound 121 was synthesized using the procedures disclosed in Russ, J. Org. Chem. 36: 626-628 (200). A mixture of the 6-phenyl-l,2,4-triazin(2H)one (0.097 g, 0.56 mmol), indole (0.066 g, 0.56 mmol) and acetic acid (2 ml) was stined at reflux for 12 hours. The acetic acid was removed in vacuo. Water (10 ml) was added to form a white precipitate, and the precipitate was filtered and washed with water. Recrystallization from methanol provided 5-(lH-Indol-3-yl)-6-phenyl-4,5- dihydro-2H-[l,2,4]triazin-3-one (0.14 g, 86%) as a white solid: mp 281 °C; Η NMR (500 MHz, CD3OD) δ 7.70 (t, 3H), 7.32 (d, IH), 7.28 (d, 3H), 7.19 (s, IH), 7.10 (t, IH), 7.05 (t, IH), 6.02 (s, IH); ESI MS m/z 291 [C H14N4O+H]+.
6.18. Example 18: Resolution of (+) and (-) Isomers of 5-(lH-Indol-3-yl)-6- phenyl-4,5-dihvdro-2H-[l,2,41triazin-3-one (121)
The (+) and (-) isomers of compound 121 were resolved by a chromatographic method. The chromatography was done using Chiralpak AD 50x500 mm column and 60:40 2-propanol/hexane at a flow rate of 118 ml/min. (+)-5-(lH-Indol-3-yl)-6-phenyl-4,5-dihydro-2H-[l,2,4]triazin-3-one (0.043 g,
[α]25 D +78.5° (c 0.169, THF)) was recovered as a white solid: mp 281°C; !H NMR (500 MHz, CD3OD) δ 7.73 (t, 3H), 7.35 (d, IH), 7.29 (s, 3H), 7.20 (s, IH), 7.11 (t, IH), 7.05 (t, IH), 6.03 (s, IH); ESI MS m/z 291 [C17H14N4O+H]+.
(-)-5-(lH-Indol-3-yl)-6-phenyl-4,5-dihydro-2H-[l,2,4]triazin-3-one (0.038 g, [α]25o -71.5° (c 0.186, THF)) was also recovered from the same chromatography as a white solid: mp 281°C; 1H NMR (500 MHz, CD3OD) δ 7.76 (m, 3H), 7.36 (d, IH), 7.28 (s, 3H), 7.21 (s, IH), 7.16 (t, IH), 7.09 (t, IH), 6.04 (s, IH); ESI MS m/z 291 [C17Hι4N4O+H]+.
6.19 Example 19: Synthesis of l-(2,6-dichlorophenyl)-6,7-dimethoxy-l,4- dihydro-2H-isoquinolin-3-one
3,4-Dimethoxyphenyl acetonitrile (3.54 g, 20 mmol) was added to polyphosphoric acid (11.1 g) preheated to 130°C. After 1 hour, 2,6- dichlorobenzaldehyde (1.75 g, 20 mmol) was added. The resulting mixture was stined for 12 hours and cooled to ambient temperature. Following an addition of water (50 ml), concentrated ammonium hydroxide was added. The mixture was allowed to stand for 18 hours. The solids were filtered, then stirred at reflux in sodium hydroxide (1.35 M, 50 ml) for 2 hours. The mixture was filtered while hot, and the solid was washed with water and dried. The solid was chromatographed (silica gel, 5 to 50 % ethyl acetate/dichloromethane) to provide l-(2,6- dichlorophenyl)-6,7-dimethoxy-l,4-dihydro-2H-isoquinolin-3-one (0.305 g, 9 %) as a slightly yellow solid: m.p. 228-229°C; 1H NMR (500 MHz, CD3OD) δ 7.47 (bs, 2H),
7.36 (t, IH), 6.79 (s, IH), 6.70 (s, IH), 6.38 (s, IH), 3.87 (s, 3H), 3.69 (d, 2H), 3.62
(s, 3H); ESI-MS m/z 352 [Cι7H,5C,2NO3 + H]+.
6.20 Example 20: Synthesis of 3-(2-chloro-6-fluorophenyl)-6-trifluoromethyI- fl,2,41triazolo[4,3-alpyridine (127) l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1, 1.32 g, 6.91 mmol), 4-methylmorpholine (0.76 ml, 6.91 mmol), 1 -hydroxybenzotriazole hydrate (HOBt, 0.132 g, 0.98 mmol) and 5-trifluoromethylpyridyl hydrazine (68%, 1.5 g, 5.76 mmol) were added to 2-chloro- 6-fluorobenzoic acid (1.0 g, 5.76 mmol) in anhydrous 1:1 dichloromethane/acetonitrile (40 ml) at 0 °C. The ice bath was removed and the mixture was stined at ambient temperature for 60 hours. The reaction mixture was concentrated in vacuo, diluted with dichloromethane (120 ml), washed with water (three times, 30 ml each) and brine (40 ml), dried with magnesium sulfate, and concentrated in vacuo. Following a flash silica gel chromatography (5-33% ethyl acetate/dichloromethane), 2-chloro-6-fluorobenzoic acid-N-(5-trifluoromethylpyridin- 2-yl)hydrazide (0.542 g, 28%) was obtained as a green solid. The compound was identified by NMR spectral analysis.
Next, phosphorous oxychloride (3.0 ml, 32.4 mmol) was added to a solution of 2-chloro-6-fluorobenzoic acid-N-(5-trifluoromethyl-pyridin-2-yl)hydrazide (0.542 g, 1.62 mmol) in anhydrous toluene (40 ml). The mixture was stirred at reflux for 18 hours. The mixture was then poured into cold sodium hydroxide (2M, 100ml), extracted with ethyl acetate, washed with water (35 ml) and brine (35 ml), dried with magnesium sulfate. Extra solvent was removed in vacuo. Following a flash silica gel chromatography (5-50% ethyl acetate/dichloromethane), 3-(2-chloro-6-fluorophenyl)- 6-trifluoromethyl-[l,2,4]triazolo[4,3-a]pyridine 127 (0.117 g, 23%) was obtained as a light yellow solid: mp 170-171°C; Η NMR (500 MHz, CD3OD) δ 8.60 (s, IH), 8.10 (d, IH), 7.80 (m, 2H), 7.65 (d, IH), 7.45 (t, IH); ESI MS m/z 316 [Cι3H6ClF4N3 + H]+.
6.21 Example 21: Synthesis of 3-(2,3-dichlorophenyl)-6-trifluoromethyl H,2,41triazolor4,3-alpyridine (129) l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1, 1.325 g, 6.91 mmol), N-methylmorpholine (0.76 ml, 6.91 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 0.177 g, 1.31 mmol) and -trifluoromethylpyridyl hydrazine (68%, 1.0 g, 3.84 mmol) were added to 2,3- dichlorobenzoic acid (1.0 g, 5.76 mmol)in anhydrous 1:1 dichloromethane/acetonitrile (40 ml) at 0°C. The ice bath was removed and the mixture was stirred at ambient temperature for 60 hours. The reaction mizture was then concentrated in vacuo, diluted with dichloromethane (100 ml), washed with water (three times, 30 ml each) and brine (40 ml), dried over magnesium sulfate, and concentrated in vacuo.
Following a flash silica gel chromatography (5-20% ethyl acetate/dichloromethane), 2,3-dichlorobenzoic acid-N-(5-trifluoromethyl-pyridin-2-yl)hydrazide (0.6 g, 40%) was obtained as a solid. The compound was identified by NMR spectral analysis.
Next, phosphorous oxychloride (3.2 ml, 34.3 mmol) was added to a solution of 2,3-dichlorobenzoic acid-N-(5-trifluoromethylpyridin-2-yl)hydrazide (0.6 g, 1.71 mmol) in anhydrous toluene (40 ml). The resulting mixture was stirred at reflux for 21 hours. The mixture was then poured into cold aqueous sodium hydroxide (2M, 100 ml), extracted with ethyl acetate, washed with water (40 ml) and brine (40 ml), dried over magnesium sulfate, and concentrated in vacuo. Following a flash silica gel chromatography (5-50% ethyl acetate/dichloromethane), a yellow solid was obtained. The yellow solid was again chromatographed on silica gel (5-33% ethyl acetate/hexanes), and 3-(2,3-dichlorophenyl)-6-trifluoromethyl-[l,2,4]triazolo[4,3- ajpyridine 129 (245 mg, 45%) was obtained as an off-white solid: mp 100-101°C; Η NMR (500 MHz, CD3OD) δ 8.59 (s, IH), 8.08 (d, IH), 7.95 (d, IH), 7.79 (d, IH), 7.72 (d, IH), 7.67 (m, IH); ESI MS m/z 332 [C13H6C12F3N3 + H]+.
6.22 Example 22: Synthesis of 3-(2,6-dichlorophenyl)-6-trifluoromethyl- fl,2,41triazolo[4,3-alpyridine (131) l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1,
1.325 g, 6.91 mmol), 4-methylmorpholine (0.76 ml, 6.91 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 0.177 g, 1.31 mmol) and 5-trifluoromethylpyridyl hydrazine (68%, 1.0 g, 3.84 mmol) were added to 2,6- dichlorobenzoic acid (1.0 g, 5.76 mmol) in anhydrous 1 :1 dichloromethane/acetonitrile (40 ml) at 0°C. The ice bath was removed and the mixture was stined at ambient temperature for 60 hours. The reaction mixture was concentrated in vacuo, diluted with dichloromethane (100 ml), washed with water (three times, 30 ml each) and brine (40 ml), dried with magnesium sulfate, and concentrated in vacuo. Following a flash silica gel chromatography (5-50% ethyl acetate/dichloromethane), 2,6-dichlorobenzoic acid-N-(5-trifluoromethylpyridin-2- yl)hydrazide (0.732 g, 36%) was obtained as a solid. The compound was identified by NMR spectral analysis.
Next, phosphorous oxychloride (3.9 ml, 41.8 mmol) was added to a solution of 2,6-dichlorobenzoic acid-N-(5-trifluoromethyl-pyridin-2-yl)hydrazide (0.732 g, 2.09 mmol) in anhydrous toluene (40 ml). The mixture was stined at reflux for 18 hours. The mixture was then poured into cold aqueous sodium hydroxide (2M, 100ml), extracted with ethyl acetate, washed with water (40 ml) and brine (40 ml), dried over magnesium sulfate, and concentrated in vacuo. Following a flash silica gel chromatography (5-25% ethyl acetate/hexanes), 3-(2,6-dichlorophenyl)-6- trifluoromethyl-[l,2,4]triazolo[4,3-a]pyridine 131 (0.1 g, 14%) was obtained as an off-white solid: mp 104-105°C; Η NMR (500 MHz, CD3OD) δ 8.61 (s, IH), 8.10 (d, IH), 7.78 (d, IH), 7.72 (s, 3H); ESI MS m/z 332 [Cι3H6Cl2F3N3 + H]+.
6.23 Example 23: Inhibition of the Edg-4 Receptor by Compound 101 Figure 1 demonstrates that compound 101 specifically inhibited the Edg 4 receptor. Compound 101 did not inhibit LPA-stimulated calcium increases in HTC cells expressing Edg 2 or Edg 7 receptors and also did not inhibit S IP-stimulated calcium increases in HTC cells expressing Edg 1, Edg 3, Edg 5, or Edg 8 receptors. When tested with the Edg 4 receptor, compound 101 almost completely blocked the LPA response in concentrations between about 1 μM and about 10 μM. Figure 2 shows that compound 103 has a 2-3 fold greater potency than compound 101, while compound 105 is less potent than compound 101.
Figure 3 illustrates a dose response to LPA using varying concentrations of 101 (0-10 μM) in HTC cells expressing human Edg 4 receptors. The data suggests that inhibition by compound 101 may be ineversible, as demonstrated by the inability of LPA to overcome inhibition by compound 101 at concentrations as high as 10 μM. Figure 4 demonstrates that compound 101 retained its activity when tested on endogenous Edg 4 receptors from human ovarian cancer cells (OV202). LPA- stimulated calcium responses in these cells was almost completely inhibited by 10 μM of compound 101. The calcium mobilization assays were conducted as described in
Section 6.25 (Example 25).
Compound 101 also inhibited LPA-stimulated calcium response in another human ovarian cancer cell line, CaOV3, in a non-competitive mode (Figure 5). In this instance, the LPA response was not completely inhibited, because these cells express other LPA receptors (Edg 2 and Edg 7).
Vascular Endothelial Growth Factor, ("VEGF"), is a potent mitogenic and highly angiogenic factor that causes vascular permeability, which leads to ascites formation. Furthermore, VEGF is tumor-specific. Plasma VEGF levels are significantly elevated in patients with various tumors, including prostate and ovarian cancer (George et al, 2001, Clin Cancer Res 7:1932-1936; Hu et al, 2001, Natl.
Cancer Inst. 93 (10):762-767). Therefore, the ability of Edg-receptor antagonists to block VEGF secretion from tumor cells is a particularly relevant secondary assay for potential anti -tumor therapies. Figure 6 shows that compound 101 completely blocked LPA-stimulated VEGF production in CaOV3 human ovarian cancer cells. The VEGF assays were conducted as described in Section 6.26 (Example 26).
Ovarian cancer cells are known to increase IL-8 secretion (Schwartz et al,
2001, Gynecol Oncol. 81 (2):291-300). Further, expression of IL-8 has been conelated with cell metastatic potential (Singh et al, 1994, Cancer Res. 54(12):3242- 3247). In addition to blocking production of VEGF, compound 101 also completely blocked the production of IL-8 in CaOV3 human ovarian cancer cells (Figure 7). The
IL-8 assays were conducted as described in Section 6.26 (Example 26).
Since LPA is a potent mitogen, it was important to establish whether blocking
Edg 4 in human ovarian cancer cells would also block proliferation. Compound 101 (10 μM) effectively abolished LPA-stimulated proliferation of CaOV3, human ovarian cancer cells over a period of 24 hours (Figure 8). The proliferation assays were conducted as described in Section 6.28 (Example 28).
LPA-stimulated chemotaxis is another important marker for angiogenesis and metastasis. Figure 9 demonstrates that LPA stimulated chemotaxis in CaOV3 human ovarian cancer cells was effectively blocked by Edg 4 antagonist 103.
6.24 Example 24: Selective Inhibition of the Edg-4 Receptor by Compounds
101 and 103 Selectivity of illustrative compounds 101 and 103 for Edg 4 was demonstrated in several ways. Compound 101 did not demonstrate any inhibitory activity at any of the other Edg receptors tested (Figure 1). In addition, compound 101 did not demonstrate any significant activity at various targets tested, including other Edg receptors, GPCRs, ion channels, and enzymes (Table 1). Compound 103 did not inhibit SIP induced chemotaxis in HUVEC cells, although it did inhibit LPA- stimulated chemotaxis in CaOV3 cells (Figure 10), which is mediated by the Edg-4 receptor. Table 1 demonstrates the selectivity of compounds 101 and 103 for Edg-4 relative to other Edg receptors. In addition, radioligand binding assays were conducted as described in Section 6.30 (Example 30).
(Measurements of IC50, unit = μM)
Table 1 illustrates the selectivity of illustrative compounds of the invention for Edg-4 relative to other Edg receptors. Similarly, selectivity exhibited by other compounds of the invention for Edg-4 receptors is summarized in Table 2. As shown in Table 2, the compounds of this invention show much higher efficacy for Edg-4 receptors than other Edg receptors.
Table 2: Selectivity of Various Compounds for Edg-4 Receptors
6.25 Example 25: Synthesis of Compound 701
2-chlorobenzenesulfonyl isocyanate (0.13 mL, 0.89 mmol) was added to a solution of ethyl 2-amino-4, 5, 6, 7-tetrahydrobenzo[B]thiophene-3-carboxylate (0.20 g, 0.89 mmol) in benzene (2 mL) at room temperature. After 2.5 hours, the reaction mixture was filtered to provide 310 mg (79%) of 701 as a white solid. Η NMR (300 MHz, CDC13) δ: 11.68 (s, IH), 8.33 (m, IH), 7.92 (br s, IH), 7.57 (m, 2H), 7.43 (m, IH), 4.39 (m, 2H), 2.73 (m, 2H), 2.58 (m, 2H), 1.75 (m, 4H), 1.38 (m, 3H). CI-MS: m/z = 443 [Cι8H19ClN2O5S2 + H]. Melting Range: 222-225 °C.
6.26 Example 26: Synthesis of 3-(2-Imino-4-oxothiazolidin-5-ylidene)-l-(toluene- 4-solfonylVl ,3-dihydro-indol-2-one (7031
A mixture of pseudothiohydantoin (789 mg, 6.80 mmol), isatin (1.00 g, 1 equiv.) and sodium acetate (1.67 g, 3 equiv.) in glacial acetic acid (20 mL) was stined at reflux overnight. The mixture was then cooled to room temperature and the precipitates were collected by suction filtration and washed with water. The solids were further triturated with hot methanol. 3-(2-Imino-4-oxo-thiazolidin-5-ylidene)- 1 ,3-dihydroindol-2-one was obtained as a red solid (1.47 g, 88% yield). 1H NMR (300 MHz, OMSO-d6) δ 11.07 (s, IH), 9.56 (s, IH), 9.30 (s, IH), 8.97 (m, IH), 7.34 (m, IH), 7.05 (m, IH), 6.93 (m, IH). APCI-MS: m z 246 [CπH7N3O2S + H]+ M.P. 345 °C (decomposed).
3-(2-Imino-4-oxothiazolidin-5-ylidene)-l,3-dihydro-indol-2-one (113 mg, 0.461 mmol) in dry DMF (5 mL) was cooled in an ice bath. NaH (60% in mineral oil, 28 mg, 0.691 mmol, 1.5 equiv.) was added in one portion. The cold bath was removed and the stirring continued overnight. /?-toluenesulfonyl chloride (97 mg, 0.51 nmuol, 1.1 equiv.) was then added and the reaction mixture was stined for another 12 hours. DMF solvent was removed by co-evaporation with toluene. The residue was taken up with ethyl acetate (30 mL) and suction filtered. The filter cake was found to be mostly unreacted starting materials along with j9-toluenesulfonic acid. The liquid filtrate was concentrated and subject to flash column chromatography over silica gel using methanol/dichloromethane (1 :10). 703 was obtained as a red solid (15 mg, 8% yield). 1H NMR (300 MHz, acetone-do) δ 8.90 (m, IH), 7.86 (m, 2H), 7.42- 7.48 (m, 3H), 7.10-7.15 (m, IH), 7.057.08 (m, IH), 2.45 (s, 3H). APCI-MS: m/z 400 [C,8H,3N304S2 + H]+.
6.27. Example 27: Synthesis of Compound 729: 2,3-bis-(4-Methoxyphenyl) quinoxaline-6-carboxylic acid
A mixture of 3,4-diaminobenzoic acid (0.153 g, 1.00 mmol), 4,4'- dimethoxybenzil (0.271 g, 1.00 mmol) and acetic acid (6 mL) was stirred at reflux for 12 h, cooled to room temperature and poured into water (75 mL). The resultant solid was taken up in aqueous sodium hydroxide (2M) and washed with dichloromethane; the aqueous layer was acidified and the resultant solid was recrystallized from methanol to afford 2,3-bis-(4-methoxyphenyl)quinoxaline-6-carboxylic acid ( 0.171 g, 44% yield) as a yellow solid: mp 284-285 °C; 1H NMR (500 MHz, Acetone-J6) δ 8.79 (s, IH), 8.39 (d, IH), 8.21 (d, IH), 7.61 (d, 4H), 6.97 (d, 4H), 3.89 (s, 7H); ESI MS m z 387 [C23H18N2O4 + H]+.
6.28. Example 28: Synthesis of Compound 731
(a) 2,4-Diethyl-10,10-dioxo-10H-10λ6-thioxanthen-9-one
Hydrogen peroxide ( 3.0 mL, 29.0 mmol) was added 1 mL at a time to a refluxing solution of 2,4-diethyl-thioxanthen-9-one (0.504 g, 1.88 mmol) in acetic acid (-10 mL) and allowed to stir for 2 h. The reaction was cooled to room temperature and allowed to stand 18 h. The reaction was filtered and the resulting yellow, highly viscous liquid was washed with dichloromethane and methanol, then reduced in vacuo. 2,4-Diethyl-10,10-dioxo-10H-10λ6-thioxanthen-9-one (0.381 g, 67% yield) was obtained as a yellow solid after recrystallization from ethanol and was identified on the basis of NMR spectral analysis.
(b) 2,4-Diethyl-l 0,10-dioxo-l OH-10λ6-thioxanthen-9-one
Zinc amalgam was formed via the addition of zinc (0.956 g, 14.6 mmol) to mercury(II) chloride (0.125 g, 0.46 mmol). To this mixture water (10 mL) was added slowly. Hydrochloric acid (0.25-0.50 mL) was added and the mixture stined for five minutes. The mixture was decanted and the zinc amalgam was covered with acetic acid (10 mL) and hydrochloric acid ( 2 mL), resulting in an exotherm. 2,4-Diethyl- 10,0-dioxo-10H-10λ6-thioxanthen-9-one (0.350 g, 1.17 mmol) was added; the mixture was stined at reflux for 2 h and cooled to room temperature. The mixture was decanted into water (50 mL), which resulted in a pink viscous liquid and a white precipitate; the suspension was extracted with dichloromethane (3 x 30 mL). The combined organic layers were dried (magnesium sulfate), filtered and concentrated in vacuo. Flash column chromatography (silica gel, 9:1, hexanes/ethyl acetate) afforded 2,4-diethyl-9H-thioxanthene 10,10-dioxide (0.190 g, 57%) as a clear oil: 1H NMR (300 MHz, CDC13) δ 8.09 (d, IH), 7.45 (m, 3H), 7.08 (d, 2H), 4.23 (s, 2H), 3.25 (q, 2H), 2.65 (q, 2H), 1.37 (t, 3H), 1.24 (t, 3H); APCI MS m/z 287 [C17H,8O2S + H]+. 6.29 Example 29. Synthesis of Compound 733
(a) 2,4-Dimethylthioxanthen-9-one
1,3-Dimethylbenzene (2.0 mL, 16.3 mmol) was added to a suspension of 2- mercapto- benzoic acid disulfide (0.950 g, 3.10 mmol) in concentrated sulfuric acid (10 mL); the resultant mixture was stirred at 50-55 °C for 1 h. The mixture was cooled and water (150 mL) was added. The resultant solid was filtered and slurried in aqueous sodium hydroxide (0.5 M), filtered and washed with water to afford 2,4- dimethylthioxanthen-9-one (0.565 g, 76% yield), which was identified by NMR spectral analysis.
(b) 2,4-Dimethyl-10,10-dioxo-10H-10λ6-thioxanthen-9-one
Hydrogen peroxide (30% wt., 3.0 mL, 29.0 mmol) was added dropwise to a solution of 2,4-dimethylthioxanthen-9-one (0.535 g, 2.23 mmol) in acetic acid (-10 mL) at reflux; the resultant mixture was stined 2 h at reflux. The reaction was cooled to ambient temperature, added to water (200 mL) and allowed to stand 48 h. Filtration afforded 2,4-dimethyl-10,10-dioxo-10H-10λ6-thioxanthen-9-one (0.450 g, 74%) as a yellow solid, which was identified by NMR spectral analysis.
(c) 2,4-Dimethyl-9-?7-thioxanthene 10,10-dioxide
Water (10 mL) was added slowly to zinc amalgam, formed from the addition of zinc (1.53 g, 23.4 mmol) to mercury(II) chloride (0.167 g, 0.64 mmol). Hydrochloric acid (0.25-0.50 mL) was added and the mixture stined for five min. The mixture was decanted and the zinc amalgam covered with acetic acid (10 mL) and hydrochloric acid (2 mL), resulting in an exotherm. 2,4-Dimethyl-10,10-dioxo- 10H-10λ6-thioxanthen-9-one (0.440 g, 1.62 mmol) was added and the reaction stined at reflux for 2 h. After cooling to room temperature, the mixture was decanted into water (50 mL), which resulted in a pink viscous liquid and a white precipitate being formed; the suspension was extracted with dichloromethane (3 x 30 mL). The combined organic layers were washed with aqueous sodium hydroxide (IM), dried (magnesium sulfate), filtered and concentrated in vacuo. The resultant solid was recrystallized twice from ethanol to afford 2,4-dimethyl-9H-thioxanthene-10,10- dioxide (0.205 g, 49% yield) as a pink crystalline solid: mp 103-105 °C; 1H NMR (300 MHz, CDC13) δ 8.09 (d, IH), 7.46 (m, 3H), 7.04 (d, 2H), 4.20 (s, 2H), 2.78 (s, 3H), 2.35 (s, 3H); APCI MS m/z 259 [C]5H14O2S + H]+.
6.30. Example 30: Compound 705: 4-MethyKV-[4-oxo-5-(2-oxo-l,2- dihydroindol-3-ylidene)thiazolidin-2-ylidenel-benzenesulfonamide
NaOAc, HOAc reflux A mixture of 2H-indole-2,3-dione (97.0 mg, 0.655 mmol), 4-methyl-N-(4-oxo- thiazolidin-2-ylidene)benzenesulfonamide (177 mg, 0.665 mmol), sodium acetate (161 mg, 1.97 mmol) and acetic acid (5 mL) was heated to reflux, stined for 15 h and cooled to ambient temperature. The resultant suspension was filtered and washed with water; the filtrate was triturated with hot methanol to afford 4-methyl-N-[4-oxo-5-(2- oxo- 1 ,2-dihydroindol-3-ylidene)thiazolidin-2-ylidene]benzenesulfonamide (0.167 g, 64%) as a red solid: mp 320 °C dec; 1H NMR (500 MHz, Acetone- d6) δ 10.69 (bs, IH), 10.23 (bs, IH), 8.97 (d, IH), 7.91 (d, 2H), 7.50 (d, 3H), 7.19 (t, IH), 7.09 (d, IH), 2.49 (s, 3H); ESI MS m/z 400 [Cι8H13N3O4S2 + H]+.
6.31. Example 31: Compound 707: 4-Bromo-2-[2-(4-chlorophenylimino)-4- oxothiazolidin-5-ylidenemethyl]phenoxyacetic acid
Methyl 4-bromo-2-[2-(4-chlorophenylimino)-4-oxothiazolidin-5- ylidenemethyljphenoxyacetate (0.147 g, 0.305 mmol) was added to a solution of potassium hydroxide in methanol (3%, 20 mL) at ambient temperature; the resultant mixture was stined for 18 h. Water (20 mL) was added and the methanol was removed by reduced pressure. The residue was cooled to 0 °C and aqueous hydrochloric acid (2M) was added to pH ~3. The solid was filtered and recrystallized from 95%o ethanol to afford 4-bromo-2-[2-(4-chlorophenylimino)-4-oxothiazolidin-5- ylidenemethyl]phenoxyacetic acid (0.133 g, 93% yield) as a yellow solid: mp 272 °C dec; Η NMR, for a 1 :1 mixture of E- and Z-isomers, (500 MHz, DMSO-d*) δ 7.91 (s, IH), 7.83 (s, IH), 7.79 (d, 2H), 7.75 (s, IH), 7.54 (m, 2H), 7.48 (m, AH), 7.35 (s, IH), 7.16 (d, 2H), 6.98 (dd, 2H , 4.80 (s, 2H), 4.79 (s, 2H), 3.40 (bs, 4H); ESI MS m/z 467 [C18H12BrClN2O4S + H]+.
6.32. Example 32: Synthesis of Compound 727: 4-Oxo-2-[(l- pentylhexylidene)hvdrazono]thiazolidin-5-ylacetic acid
The thiosemicarbazone (1.229 g, 5.05 mmol, Coll. Czech. Chem. Comm. 1997, 62, 124) was added to a solution of maleic anhydride (0.492 g, 5.02 mmol) in chloroform (45 mL); the mixture was stined at reflux for 1.5 h. The mixture was cooled and concentrated in vacuo. The residue was added to saturated ammonium chloride and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were extracted with aqueous sodium hydroxide (0.5 M, 2 x 50 mL). The combined aqueous layers were treated with hydrochloric acid (IM, ~ 75 mL), to pH 5-6. The mixture was extracted with ethyl acetate (3 x 30 mL); the organic layers were combined and concentrated in vacuo. The resultant solid was recrystallized from acetonitrile to afford 4-oxo-2-[(l-pentylhexylidene)hydrazono]thiazolidin-5-ylacetic acid (0.477 g, 28%) as a white solid: mp 134-135 °C; 1H NMR (300 MHz, CDC13) δ 4.23 (dd, IH), 2.85 (dd, IH), 2.81 (dd, IH), 2.40 (t, 2H), 2.26 (t, 2H), 1.49 (m, 6H), 1.39 (m, 6H), 0.86 (m, 6H); ESI-MS m/z 342 [Ci6H27N3O3S + H]+.
6.33. Example 33: Selective Inhibition of the Edg-7 Receptor by Compound 701
701 and 703 are representatives of a series of compounds that demonstrate inhibition of Edg-7 stimulated LPA responses. The compounds were tested in HTC cells expressing human Edg 7 receptors, as well as in HT1080 human fibrosarcoma cell lines that naturally express Edg 7 receptors. The rat hepatoma cell line, HTC, does not express any detectable levels of any of the known Edg receptors. Therefore, HTC proved to be a useful system because Edg-7 could be tested in isolation when recombinantly introduced into these cells. The compounds are tested in this recombinant system first, and subsequently tested in cell lines expressing Edg-7 (in addition to other Edg receptors).
Figure 17 demonstrates that 701 specifically inhibit the Edg-7 receptor. 701 did not inhibit LPA-stimulated calcium increases in HTC cells expressing Edg-2 or Edg-4 receptors and also did not inhibit S IP-stimulated calcium increases in HTC cells expressing Edg-1, Edg-3, Edg-5, or Edg-8 in concentrations as high as 10 μM. Figure 18 demonstrates that another Edg-7 antagonist, 703, exhibited selectivity for Edg 7 when tested on HT1080 cells. The LPA-stimulated calcium mobilization was blocked by 703, but not by the Edg 4 antagonist, 4,4,4-trifluoro-3- oxo-N-(5-phenyl-2H-pyrazol-3-yl)-butyramide, suggesting that in these cells, the LPA response is predominantly Edg 7 driven. The calcium mobilization assays were conducted as described in the examples below.
Selectivity of 701 for Edg-7 is also demonstrated in Tables 1 and 2. 701 did not demonstrate any significant activity at various targets tested, including other Edg receptors, GPCRs, and ion channels. Table 3 demonstrates the selectivity of 701 for
Edg-7 relative to other Edg receptors and Table 4 is a list of targets, including GPCRs and ion channels, for which 701 showed no significant activity in radioligand binding assays. The radioligand binding assays were conducted as described the examples below.
6.34 Example 34: Synthesis of l-(2-Ethoxyphenyl)-3-(hvdroxyphenylamino)- pyrrolidine-2,5-dione (201)
N-(2-ethoxyphenyl)maleimide was first prepared in two steps from maleic anhydride and ø-phenetidine in 64% overall yield (Rangnekar et al, 1986, Ind. J.
Chem. 1986, 25B:342-344). N-Phenylhydroxylamine was prepared from reduction of nitrobenzene and used without purification (Bordwell et al, 1996, J. Am. Chem. Soc. 118:8777-8781).
N-Phenylhydroxylamine (1.90 g, ca. 70% purity, 12.2 mmol) was added to a solution of N-(2-ethoxyphenyl)maleimide (1.80 g, 8.27 mmol) in dry toluene (20 mL) and the mixture was stined at room temperature for 12 hours. Solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel (ethyl acetate/hexanes (1 :4, 1 :3, and 1:1)) to provide the desired compounds as an off- white solid (2.53 g, 93% yield). Η ΝMR (300 MHz, CDC13) δ 7.28 - 7.40 (m, 3H), 6.98 - 7.22 (m, 6H), 5.78 (s, 0.5H), 5.61 (s, 0.5H), 4.90 (m, IH), 4.08 (m, 2H), 3.12 (m, lH), 2.85 (m, IH), 1.35 (m, 3H). APCI-MS: m/z 309 [Cι8H18Ν2O4 + H - H2O]+. M.p. 174-175 °C.
6.35 Example 35: Synthesis of 2-(l-Ethyl-3-methvI-5-phenyl-lH-pyrazol-4-yl)- phenol (203) o-Hydroxyphenylacetone was prepared from o-tolyl acetate in a two-step process. The first step was the benzylic bromination (Loukiala et al, 1997, Acta Chem. Scand. 51 :1162-1166), followed by intramolecular acetyl migration (Ledoussal et al, 1987, Tetrahedron 43:5841-5852) in 57% yield. o-Hydroxyphenylacetone (5.89 g, 39.2 mmol), DMAP (4.79g, 39.2 mmol, 1 equivalent) and pyridine (3.2 mL, 1 equivalent) were dissolved in dry THF (200 mL). The solution was cooled with an ice bath and benzoyl chloride (5.5 mL, 1.2 equivalents) was then added slowly via syringe. The mixture was stined at room temperature overnight and then concentrated in vacuo. The residue was diluted with ethyl acetate (400 mL) and washed with saturated aqueous NH4C1 (100 mL x 3), dried with Na2SO4 and evaporated. Flash column chromatography over silica gel using ethyl acetate/hexanes (1 :10, 1 :8, 1 :6, 1:4, and 1:3) gave benzoic acid 2-(2- oxopropyl)phenyl ester as a white crystalline solid (7.12 g, 71% yield). Η NMR (300 MHz, CDC13) δ 8.16-8.20 (m, 2H), 7.63-7.68 (m, IH), 7.50-7.55 (m, 2H), 7.22-7.44 (m, 4H), 3.67 (s, 2H), 2.12 (s, 3H).
Finally, benzoic acid 2-(2-oxopropyl)phenyl ester (20.33 g, 79.95 mmol), ethylhydrazine oxalate (15.01 g, 99.94 mmol, 1.25 equivalents), and acetic acid (37 mL) in ethylene glycol (70 mL) were stirred at 200-205 °C (oil bath temperature) for 90 minutes and the mixture was cooled to room temperature. (Dzvinchuk et al, 1995, Russ. J. Org. Chem. 31:218-220). Water (500 mL) was added and the content was chilled with an ice bath. A white precipitate was collected by suction filtration and further subjected to flash chromatography on silica gel (acetone/CH2Cl2 (1:10, 1:8)) to provide the desired compound as a white solid (13.30 g, 60% yield). 1H NMR (300 MHz, CDC13) δ 7.33-7.35 (m, 3H), 7.18-7.21 (m, 3H), 6.98-7.01 (m, IH), 6.83-6.86 (m, 2H), 4.99 (s, IH), 4.12 (m, 2H), 2.21 (s, 3H), 1.44 (m, 3H). APCI-MS: m/z 279 [C18Hι8N2O + H]+. M.p. 164 - 165 °C. Calculated: C, 77.67; H, 6.52; N, 10.06. Found: C, 77.41; H, 6.49; N, 10.04.
6.36. Example 36: Synthesis of Compound 211: l-(2-Ethoxyphenyl)-3-[(4- fluorophenyl)hvdroxylaminolpyrrolidine-2,5-dione
A mixture of 2'-ethoxyphenylmaleimide (0.115 g, 0.529 mmol) and 4- fluorophenylhydroxylamine (70%, 0.192 g, 1.058 mmol) in toluene (6 mL) was stined at ambient temperature. The mixture was concenfrated in vacuo and the residue chromatographed (silica gel, 10 to 50% ethyl acetate/hexanes) to afford l-(2- ethoxyphenyl)-3-[(4-fluorophenyl)hydroxylamino]pyrrolidine-2,5-dione (0.153 g, 84%) as a light yellow solid: mp 123-124 C; 1H NMR (500 MHz, CDC13) d 7.41 (m, IH), 7.17 (m, 3H), 7.03 (m, 4H), 5.99 (s, 0.5H), 5.78 (s, 0.5H), 4.80 (m, IH), 4.05 (m, 2H), 3.17 (m, IH), 2.85 (m, IH), 1.36 (t, 3H); ESI MS m/z 327 [C] 8H17FN2O4 + H - H2O]+.
6.37. Example 37: Synthesis of Compound 215: 5-(4-Bromophenyl)-3-(4- methoxyphenyl)-2-phenyl-trfl ιy-3-c -3a-tetrahvdropyrrolof3,4-<flisoxazole-4,6- dione and Compound 117: 5-(4-Bromophenyl)-3-(4methoxyphenyl)-2-phenyl- c>jy-3-c/y-3a-tetrahvdropyrrolo[3,4-<flisoxazole-4,6-dione and Compound 113: 5; (4-Bromophenyl)-3-(4-methoxyphenyl)-2-phenyl-3-3a-tetrahydropyrrolo[3,4- d isoxazole-4,6-dione
(a) C-(4-Methoxybenzyl)-N-phenylnitrone
Reference: J. Org. Chem. 1989, 54(21), 5176-80. A mixture of nitrobenzene (1.03 mL, 10.0 mmol), /røra-anisaldehyde (1.22 mL, 10.0 mmol), triphenylphosphine (100 mg), methanesulfonic acid (0.10 mL) and ethanol (10 mL) was purged with nitrogen gas. Platinum on carbon (100 mg, 5%,
50%) water) was added and a balloon filled with hydrogen gas was connected to the reaction vessel. The resultant mixture was stined under hydrogen atmosphere at room temperature for 18 h. The mixture was purged with nitrogen gas and filtered through
Celite with cold ethanol (15 ml). Water (20 mL) was added to the filtrate at 0 °C and the resultant suspension was filtered to afford the title compound (1.08 g, 47.6% yield) as a yellow solid, which was identified on the basis of NMR and mass spectral analysis.
A mixture of C-(4-methoxybenzyl)-N-phenylnitrone (0.300 g, 1.32 mmol), N- (4-bromophenyl)maleimide (0.333 g, 1.32 mmol) and benzene (2.6 mL) was stined overnight at 40 °C. The resultant suspension was cooled to room temperature and diluted with methylene chloride until a homogeneous mixture was achieved. The mixture was chromatographed (silica gel, 20% ethyl acetate/hexanes). 5-(4-Bromophenyl)-3-(4-methoxyphenyl)-2-phenyl-tra«5'-3-ew-3a- tetrahydropyrrolo[3,4- ]isoxazole-4,6-dione eluted first and was isolated as a tan solid (0.254 g, 40.1%): mp 203-205 °C dec; 1H NMR (300 MHz, CDC13) δ 7.50 (d, 4H), 7.32 (d, 2H), 7.20 (d, 2H), 7.04 (m, 3H), 6.60 (d, 2H), 5.75 (s, IH), 5.15 (d, IH), 4.06 (d, IH), 3.86 (s, 3H); APCI MS m/z 481 [C24H19BrN2O4 + H]+.
5-(4-Bromophenyl)-3-(4methoxyphenyl)-2-phenyl-cts-3-ct.s-3a- tetrahydropynolo[3,4-<JJisoxazole-4,6-dione eluted second and was isolated as a tan solid (0.198 g, 31.3% yield): mp 206-208 °C dec;1H NMR (300 MHz, CDC13) δ 7.61 (d, 2H), 7.39 (d, 2H), 7.25 (d, 2H), 7.15 (d, 3H), 7.03 (d, 2H), 6.90 (d, 2H), 5.30 (d, IH), 4.90 (d, IH), 4.08 (t, IH), 3.78 (s, 3H); APCI MS m/z 481 [C24H19BrN2O4 + H]+.
Illustrative compound 213 is formed by mixing 215 and 217 to form a 1:1 mixture.
6.38. Example 38: Synthesis of Compounds 207 and 209: Separation of
Enantiomers of Compound 201
Separation of enantiomers of l-(2-Ethoxyphenyl)-3-(hydroxyphenylamino)- pynolidine-2,5-dione was achieved by a chiral separation using Chiracel OD (50 x 500 mm) column (Eluent: 100% ethanol; flow rate: 60.0 mL/min; monitoring wavelength: 260 nm). The enantiomer which eluted first under the listed conditions was defined as 'Enantiomer A', while the one which eluted later was defined as 'Enantiomer B'. Compound 107 (Enantiomer A) Alpha-D = -32deg (c=0.15, methanol). Compound 109 (Enantiomer B) Alpha-D = +5.1deg (c=0.45, methanol).
6.39 Example 39: Inhibition of the Edg-2 Receptor by Compound 201
Figure 19 demonstrates compound 201 specifically inhibit the Edg 2 receptor. Compound 201 did not inhibit LPA-stimulated calcium increases in HTC cells expressing Edg-4 or Edg-7 receptors and also did not inhibit SlP-stimulated calcium increases in HTC cells expressing Edg 1, Edg 3, Edg 5, or Edg 8 receptors.
Figure 20 demonstrates that compound 203 specifically inhibited the Edg 2 receptor. Compound 203 did not inhibit LPA-stimulated calcium increases in HTC cells expressing Edg 4 or Edg 7 receptor and also did not inhibit SlP-stimulated calcium increases in HTC cells expressing Edg 1, Edg 3, Edg 5, or Edg 8 receptors. Figure 21 demonstrates that Edg-2 inhibitor 203 inhibited LPA-stimulated calcium mobilization in immortalized ovarian surface epithelial cells. LPA- stimulated calcium mobilization in these cells was almost completely inhibited by 10 μM 203. However, the same concentration of Edg-4 antagonist, 4,4,4-trifluoro-3-oxo- N-(5-phenyl-2H-pyrazol-3-yl)-butyramide 101, had little to no effect on calcium mobilization. The calcium mobilization assays were conducted as described in Section 6.8 (Example 8).
Figure 22 demonstrates that Edg-2 antagonist 203 reverses the inhibitory effects of LPA on forskolin-stimulated cAMP production, whereas Edg-4 antagonist 101 had little to no effect on reversing LPA inhibition of cAMP production. The cAMP assays were conducted as described in Section 6.12 (Example 12).
Fig. 23 illustrates a dose response to LPA using varying concentrations of compound 201 (0-1 OμM) to demonstrate the inhibition of LPA-stimulated calcium mobilization in A431 human epitheloid carcinoma cells by Edg-2 antagonist 201. The calcium mobilization assay was conducted as described in Section 6.8 (Example 8).
Fig. 24 illustrates the inhibition of LPA-stimulated calcium mobilization in A431 human epitheloid carcinoma cells by Edg-2 antagonists 201 and 203, but not Edg-4 antagonist, 101. The calcium mobilization assays were conducted as described in Section 6.8 (Example 8).
6.40 Example 40: Selective Inhibition of the Edg-2 Receptor by Compounds 201 and 203
Selectivity of compounds 201 and 203 for Edg-2 was demonstrated in several ways. First, compounds 201 and 203 did not demonstrate any inhibitory activity at any of the other Edg receptors tested (Figures 19 and 20). Second, compounds 201 and 203 did not demonstrate any significant activity at various targets tested, including other GPCRs, ion channels, and enzymes (Tables 5 and 6). Table 5 demonstrates the selectivity of compounds 201 and 203 for Edg-2 relative to other Edg receptors and Table 6 is a list of targets, including GPCRs and ion channels, for which compound 201 showed no significant activity in radioligand binding assays. The radioligand binding assays were conducted as described in Section 6.13 (Example 13). 6.41 Example 41: Synthesis of 3-methyl-2-phenyl-quinoline-4carboxylic acid 4- fluoro-benzylamide (Compound 301)
3-Methyl-2-phenylquinoline-4-carboxylic acid was prepared by a Pfitzinger reaction (J. Org. Chem. 1950, 15:511-516). Propiophenone (3.3 mL, 1.2 equiv.) was added to a solution of isatin (3.00 g, 20.0 mmol) in ethanol (95%, 30 mL), followed by the addition of KOH pellets (86%, 3.91g, 3 equiv.). The light brown mixture was stined at 80 °C (oil bath temperature) for 40 hours. Solvent was removed in vacuo and the resulting solid residue was dissolved in water (50 mL), extracted with ether (30 mL X 2) to remove any un-reacted starting materials. The aqueous layer was then cooled with an ice bath and acidified with concentrated aqueous HCl until pH - 3. The white precipitate was collected by suction filtration, washed with water and dried in a vacuum oven (30 °C) ovemight. The white solid was essentially pure carboxylic acid (5.10g, 97% yield) and was used for the next step without further purification. Η NMR (300 MHz, DMSO-tfό) δ 7.50-8.08 (m, 9H), 2.46 (s, 3H). 3-Methyl-2-phenylquinoline-4-carboxylic acid (1.00 g, 3.80 mmol) was then dissolved in dry THF/CH3CN (70 mL/30 mL). 1 -Hydroxy-7-azabenzotriazole (1.31 g, 9.60 mmol, 2.5 equiv), 4-fluorobenzylamine (0.67 mL, 5.85 mmol, 1.50 equiv) were then added and the mixture was cooled in an ice bath. EDC HC1 (1.15 g, 5.99 mmol, 1.58 equiv.) was added in one portion. The ice bath was removed 15 minutes later and the reaction mixture was stined at room temperature overnight. Organic solvents were removed in vacuo, the residue was taken up in dichloromethane (240 mL), washed with water (60 mL X 2) and brine (150 mL), dried with Na2SO4 and evaporated. Flash column chromatography over silica gel (ethyl acetate/dichloromethane (1 :20, 1 :10)) gave 301 as a white crystalline solid (1.15 g, 82% yield). 1H NMR (300 MHz, CDC 13) δ 8.12 (m, IH), 7.77 (m, IH), 7.69 (m, IH), 7.39-7.58 (m, 8H), 7.08 (m, 2H), 6.20 (m, IH), 4.76 (m, 2H), 2.41 (s, 3H). APCI-MS: m/z 371 [C24H19FN20 + H]+. M.P. 190-191 °C.
6.42 Example 42: Alternate Synthesis of Compound 301 2-chlorobenzenesulfonyl isocyanate (0.13 mL, 0.89 mmol) was added to a solution of ethyl 2-amino-4, 5, 6, 7-tetrahydrobenzo[B]thiophene-3-carboxylate (0.20 g, 0.89 mmol) in benzene (2 mL) at room temperature. After 2.5 hours, the reaction mixture was filtered to provide 310 mg (79%) of 301 as a white solid. Η NMR (300 MHz, CDC13) δ: 11.68 (s, IH), 8.33 (m, IH), 7.92 (br s, IH), 7.57 (m, 2H), 7.43 (m, IH), 4.39 (m, 2H), 2.73 (m, 2H), 2.58 (m, 2H), 1.75 (m, 4H), 1.38 (m, 3H). CI-MS: m/z = 443 [Cι8H19ClN2O5S2 + H]. Melting Range: 222-225 °C.
6.43 Example 44: [2-(2,4,6-Trimethyl-phenylamino)-benzo[l,31-dioxol-2-yll- carbamic acid ethyl ester (303)
303 is commercially available and was obtained from Asinex, Moscow, Russia (Vendor number BAS 0246895
6.44 Example 43: Selective Inhibition of the Edg-3 Receptor by Compound 301
301 and 303 are representatives of a series of compounds that demonstrate inhibition of Edg-3 stimulated SIP responses. The compounds were tested in HTC cells expressing human Edg-3 receptors, as well as in NIH 3T3 cell lines that naturally express Edg-3 receptors. The rat hepatoma cell line, HTC, does not express any detectable levels of any of the known Edg receptors. Therefore, HTC proved to be a useful system because Edg 3 could be tested in isolation when recombinantly introduced into these cells. The compounds were tested in this recombinant system first, and subsequently tested in cell lines expressing Edg-3 (in addition to other Edg receptors). Figure 25 demonstrates that 301 specifically inhibited Edg-3 receptors. 301 did not inhibit LPA-stimulated calcium increases in HTC cells expressing Edg-2, Edg-4 or Edg-7 receptors and also did not inhibit SlP-stimulated calcium increases in HTC cells expressing Edg-1, Edg-5, or Edg-8 in concentrations as high as 20 μM. Figure 26 demonstrates that another Edg-3 antagonist, 303, blocked S1P- stimulated calcium responses, suggesting that in these cells, the SIP response is predominantly Edg 3 driven. The calcium mobilization assay was conducted as described in Section 6.4 (Example 4).
Selectivity of 301 for Edg-3 is also demonstrated in Tables 7 and 7. 301 did not demonstrate any significant activity at various targets tested, including other Edg receptors, GPCRs, and ion channels. Table 7 demonstrates the selectivity of 301 for Edg-3 relative to other Edg receptors. Table 8 is a list of targets, including GPCRs and ion channels, against which 101 (10 μM) showed no activity in standard binding assays. The radioligand binding assays were conducted as described in Section 6.9 (Example 9). 6.45 Example 45: Intracellular Calcium Measurement Assays
LPA receptors such as Edg-2, Edg-4 and Edg-7, couple to calcium effector pathways, and result in increases in intracellular calcium following receptor activation (An et al, Molecular Pharmacology, 54:881-888, 1998, incorporated herein by reference). This biological response lends itself to a very efficient, high-throughput screen using a Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices, Sunnyvale, CA). The FLIPR system is a real-time, cell-based assay system with continuous fluorescence detection using a cooled CCD camera. The FLIPR system was used to developing an Edg receptor screen.
Rat hepatoma cells stably expressing Edg-4 receptor were plated on 384-well plates and loaded with a calcium dye loading kit (Molecular Devices, Sunnyvale, CA) for 1 hour at room temperature. Cells were then placed on the FLIPR354 (Molecular Devices, Sunnyvale, CA) and excited by an argon laser at 488 nm. The data for the entire 384-well plate was updated every second. An integrated robotic pipettor allowed for simultaneous compound addition into each individual well in the plate.
Figures 11 through 13 demonstrate the LPA induced calcium mobilization by the compounds 101, 103, 107 and 113. When the rat hepatoma cells were transfected by human Edg-4, all of the compounds completely inhibited LPA-stimulated calcium responses at 5-10 μM (Figure 11). However, the inhibition varied when the rat hepatoma cells were transfected with pooled rat Edg-4 clones (Figure 12) or pooled mouse Edg-4 clones (Figure 13). Similar tests for compound 125 in HTC cells and CaOV-3 cells are shown in Figures 15 and 16, respectively.
The FLIPR system was also used to developing an Edg-7 receptor screen. Rat hepatoma cells stably expressing Edg-7 receptor were plated on 384-well plates and loaded with a calcium dye loading kit (Molecular Devices, Sunnyvale, CA) for 1 hour at room temperature. Cells were then placed on the FLIPR384 (Molecular Devices, Sunnyvale, CA) and excited by an argon laser at 488 nm. The data for the entire 384- well plate was updated every second. An integrated robotic pipettor allowed for simultaneous compound addition into each individual well in the plate.
The FLIPR system was again used to developing an Edg-2 receptor screen. Rat hepatoma cells stably expressing Edg-2 receptor were plated on 384-well plates and loaded with a calcium dye loading kit (Molecular Devices, Sunnyvale, CA) for 1 hour at room temperature. Cells were then placed on the FLIPR384 (Molecular Devices, Sunnyvale, CA) and excited by an argon laser at 488 nm. The data for the entire 384-well plate was updated every second. An integrated robotic pipettor allowed for simultaneous compound addition into each individual well in the plate.
6.46 Example 46: Intracellular Calcium Measurement Assays
SIP receptors such as Edg-3, also couple to calcium effector pathways, and result in increases in intracellular calcium following receptor activation (An, 1998, J. Cell Biochem. Supp 30-31 :147-157). This biological response lends itself to a very efficient, high-throughput screen using a Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices, Sunnyvale, CA). The FLIPR system is a real-time, cell-based assay system with continuous fluorescence detection using a cooled CCD camera. The FLIPR system was used to developing an Edg-3 receptor screen. Rat hepatoma cells stably expressing Edg-3 receptor were plated on 384-well plates and loaded with a calcium dye loading kit (Molecular Devices, Sunnyvale, CA) for 1 hour at room temperature. Cells were then placed on the FLIPR384 (Molecular Devices, Sunnyvale, CA) and excited by an argon laser at 488 nm. The data for the entire 384-well plate was updated every second. An integrated robotic pipettor allowed for simultaneous compound addition into each individual well in the plate.
6.47 Example 47: IL-8 and VEGF Assays
IL-8 and VEGF assays were performed by standard enzyme-linked immunosorbent assay ("ELISA") techniques. Cells were cultured in a 96 well format, serum starved overnight, and treated with LPA or SIP (doses range from 0.1-10 μM in serum free medium) for 24 hours. Cell supernatants were then collected to measure the amount of IL-8 secreted.
The assay was a standard sandwich ELISA in which an anti-IL-8 or VEGF capture antibody was adsorbed to a plastic dish. Cell supernatants containing IL-8 or VEGF were added to the dish, and then an anti-IL-8/VEGF biotinylated detection antibody and streptavidin-HRP were added. Detection was via the addition of a substrate solution and colorimetric reading using a microtiter plate reader. The level of IL-8 or VEGF was interpolated by nonlinear regression analysis from a standard curve.
All reagents were from R&D Systems, Minneapolis, MN: MAB208 and AF- 293-NA (capture antibody for IL-8 and VEGF respectively), BAF2O8 and BAF-293 (detection Ab for IL-8 and VEGF respectively), 208-UL-010 and 293-VE-010 (recombinant human IL-8 protein standard and recombinant human VEGF protein standard respectively), DY998 (streptavidin-HRP), DY999 (substrate solution).
6.48 Example 48: Migration and Invasion Assays
Cells were plated in a 24 well format using Fluoroblok filter insert plates (8 μM pore size) or Fluoroblok matrigel coated filter insert plates (Becton Dickinson, San Diego, CA). The assay was a modified Boyden Chamber assay in which a cell suspension (1 x 105 cells/ml) was prepared in serum free medium and added to the top chamber. LPA or SIP (doses ranged from 0.1-10 μM in serum free medium) was added to the bottom chamber. Following a 20-24 hour incubation period, the number of cells migrating or invading into the lower chamber was quantitated by transferring the filter insert into a fresh 24-well plate containing 4 μg/ml calcein AM (Molecular Probes, Sunnyvale, CA) in Hank's Balanced Salt Solution and staining for one hour. Detection was via fluorescent readout at 450 nm excitation/530 nm emission using a fluorimeter. The level of fluorescence correlated with cell number.
For most cells types, no further manipulation was required. For CaOV3 human ovarian cancer cells, however, it was necessary that the cells be serum starved ovemight prior to preparing the cell suspension. In addition, the filter inserts were coated with a solution of 1 mg/ml rat-tail Collagen I (BD, SanDiego, CA).
6.29 Example 49: Proliferation Assay
Cells were plated in a 96 well foimat. Treatments were performed directly without any serum starvation, and typically included LPA or SIP doses in a range from 0.1-10 μM in semm free medium. Cells were treated for 24-48 before the extent of cellular proliferation was measured.
The assay was performed using the ViaLight HS kit from BioWhittaker, Rockland, ME, which is based upon the bioluminescent measurement of ATP that is present in all metabolically active cells. The reaction utilized an enzyme, luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light intensity was linearly related to the ATP concentration, which correlated with cell number.
Measurement of cell proliferation required the extraction of ATP by the addition of Nucleotide Releasing Reagent, followed by the addition of the ATP Monitoring Reagent (both provided in kit). Detection was via chemiluminescence using the EG&G Berthold Luminometer, Gaithersburg, MD.
6.50 Example 50: cAMP Assay Cells were plated in a 96 well format. Treatments were performed directly without any serum starvation. The cells were treated with forskolin to induce cAMP production, followed by LPA or SIP doses in the range from 0.1-10 μM in semm free medium. Following a 30-minute incubation period, the cells were lysed and the level of cAMP was determined. The cAMP assay was performed using the Tropix cAMP-Screen (Applied
BioSystems, Foster City, CA). The screen is a competitive immunoassay that utilizes a 96 well assay plate precoated with an anti-cAMP antibody. Cell lysates were added to the precoated plate, along with a cAMP-AP conjugate and a secondary anti-cAMP antibody. Detection was performed using a substrate solution and chemiluminescent readout. The level of chemiluminescence was inversely proportional to the level of cAMP and was calculated from a standard curve.
6.51 Example 51: Pharmacology Profiling (Selectivity Assays) In order to test the selectivity of compounds, various enzyme assays as well as radioligand binding assays were performed using numerous non-Edg receptor targets as listed below.
Enzyme Assays: 1. Phosphodiesterase PDE1: (Nicholson et al, 1991, Trends Pharmacol. Sci.
12:19-27).
Source: Bovine heart
Substrate: 1.01 μM [3H]cAMP+cAMP
Vehicle: 1 % DMSO Pre-Incubation Time/Temp: None;
Incubation Time/Temp: 20 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 5 mM MgC12, 2 mM CaC12, 10 unit Calmodulin, pH 7.5
Quantitation Method: Quantitation of [3H] adenosine Significance Criteria: > 50% of max stimulation or inhibition
2. Phosphodiesterase PDE2: (Nicholson et al, 1991, Trends Pharmnacol. Sci. 12:19-27). Source: Human platelets
Substrate: 25.1 μM [3H]cAMP + cAMP Vehicle: 1 % DMSO
Pre-Incubation Time/Temp: None Incubation Time/Temp: 20 minutes at 25 °C Incubation Buffer: 50 mM Tris-HCl, 5 mM MgC12, pH 7.5
Quantitation Method: Quantitation of [3H] adenosine Significance Criteria: > 50% of max stimulation or inhibition
3. Phosphodiesterase PDE3: (Nicholson et al, 1991, Trends Pharmacol. Sci. 12:19-27).
Source: Human platelets Substrate: 1.01 μM [3H]cAMP + cAMP Vehicle: 1 % DMSO Pre-Incubation Time/Temp: None Incubation Time/Temp: 20 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 5 mM MgC12, pH 7.5 Quantitation Method: Quantitation of [ Hjadenosine Significance Criteria: > 50% of max stimulation or inhibition
4. Phosphodiesterase PDE4: (Cortijo et al, 1993)
Source: Human U937 cells
Substrate: 1.01 μM {3H]cAMP + cAMP
Vehicle: 1 % DMSO
Pre-Incubation Time/Temp: None Incubation Time/Temp: 20 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 5 mM MgC12, pH 7.5
Quantitation Method: Quantitation of [3H] adenosine
Significance Criteria: a 50% of max stimulation or inhibition 5. Phosphodiesterase PDE5: (Nicholson et al, 1991, Trends Pharmacol. Sci. 12:19-27).
Source: Human platelets Substrate: 100 μM [3H]cGMP + cGMP Vehicle: 1 % DMSO
Pre-Incubation Time/Temp: None Incubation Time/Temp: 20 minutes at 25 °C Incubation Buffer: 50 mM Tris-HCl, 5 mM MgC12, pH 7.5 Quantitation Method: Quantitation of [3H] guanosine Significance Criteria: a 50% of max stimulation or inhibition
6. Phosphodiesterase PDE6: (Gillespie and Beavo, 1989) Source: Bovine retinal rod outer segments
Substrate: 100 μM [3H]cGMP + cGMP Vehicle: 1 % DMSO
Pre-Incubation Time/Temp: None
Incubation Time/Temp: 20 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 5 mM MgC12, pH 7.5
Quantitation Method: Quantitation of [3H]gnanosine Significance Criteria: > 50% of max stimulation or inhibition
7. Phospholipase PLA2-I (Katsumata et α/.,1986, Anal. Biochem., 154:676- 681).
Source: Porcine pancreas Substrate: 0.03 μCi l-Palmitoyl-2-{l -14C]oleoyl-3- phosphatidylcholine
Vehicle: 1 % DMSO
Pre-Incubation Time/Temp: 5 minutes at 37 °C
Incubation Time/Temp: 5 minutes at 37 °C
Incubation Buffer: 0.1 M glycine-NaOH, 20 M EDTA, pH 9.0 Quantitation Method: Quantitation of [14C]oleate
Significance Criteria: > 50% of max stimulation or inhibition
8. Phospholipase PLA2-II (Katsumata et al, 1986, Anal. Biochem. 154:676-
681). Source: Crotalus atrox
Substrate: 0.03 μCi l-Palmitoyl-2-[l-14C]oleoyl-3-phosphatidylcholine Vehicle: 1 % DMSO
Pre-Incubation Time/Temp: 5 minutes at 37°C Incubation Time/Temp: 5 minutes at 37°C
Incubation Buffer: 0.1 M glycine-NaOH, 20 M EDTA, pH 9.0 Quantitation Method: Quantitation of [14C]oleate Significance Criteria: >50% of max stimulation or inhibition
9. Phospholipase PLC (Hergenrother et al, 1995, Anal. Biochem. 229:313-
316).
Source: Bacillus cereus
Substrate: 400 μM 1 ,2-Dihexanoyl sn-glycerol-3-phosphocholine
Vehicle: 1 % DMSO Pre-Incubation Time/Temp: 10 minutes at 37°C
Incubation Time/Temp: 5 minutes at 37 °C Incubation Buffer: 0.1 M 3,3-dimethylglutaric acid, pH 7.3 Quantitation Method: Spectrophotometric quantitation of phosphorylcholine
Radioligand Binding Assays:
1. Adenosine Ai (Liebert et al, 1992, Biochem. Biophys. Res. Commun. 187:919-926).
Source: Human recombinant CHO cells
Ligand: 1 nM 3H DPCPX Vehicle: 0.4 % DMSO
Incubation Time/Temp: 90 minutes at 25 °C
Incubation Buffer: 20 mM HEPES pH 7.4, 10 mM MgCl2, 100 mM NaCl
Nonspecific Ligand: 100 μM R(-)-PIA Bmax: 2.7 pmol/mg Protein*
Specific Binding: 85% *
Quantitation Method: Radioligand Binding
Significance Criteria: O 50%> of max stimulation or
Quantitation Method: Radioligand Binding inhibition Significance Criteria: > 50% of max stimulation or inhibition
2. Adenosine A2A (Varani et al, 1996, Br. J. Pharmacol. 117:1693-1701) Source: Human recombinant HEK-293 cells Ligand: 0.05 μM 3H CGS-21680
Vehicle: 0.4 % DM50
Incubation Time/Temp: 90 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, pH 7.4. 10 mM MgCl2, 1 mM EDTA, 2 U/mL adenosine deaminase Nonspecific Ligand: 50 μM NEC A
I : 0.064 μM *
Bmax: 7 pmol/mg Protein*
Specific Binding: 85% *
Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
3. Adrenergic a!A (Michel et al, 1989, Br. J. Pharmacol. 98:883-889). Source: Wistar Rat submaxillary gland
Ligand: 0.25 nM 3H Prazosin Vehicle: 0.4 % DMSO
Incubation Time/Temp: 60 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4
NonSpecific Ligand: 10 μM Phentolamine
K^ O.H nM* Bmax: 0.18 pmol/mg Protein*
Specific Binding: 90% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
4. Adrenergic ajA (Uhlcn et al, 1994, J. Pharmacol. Exp. Ther. 271 :1558)
Source: Human recombinant insect Sf9 cells Ligand: l nM 3H MK-912 Vehicle: 0.4 % DMSO Incubation Time/Temp: 60 minutes at 25 °C Incubation Buffer: 75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA Nonspecific Ligand: 10 μM WB-4 101 IQ: 0.06 nM*
Bmax: 4.6 pmollmg Protein* Specific Binding: 95% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
5. Adrenergic βi, (Feve et al, 1994, Proc. Natl. Acad. Sci. USA 91:5677- 5681)
Source: Human recombinant Rex 16 cells
Ligand: 0.3 nM 12 I Cyanopindolol
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 2 hours at 25 °C Incubation Buffer: 50mM Tris-HCl, 5 mM EDTA, 1.5 mM CaCl2, 120 mM
NaCl, 1.4 mM ascorbic acid, lOmg/L BSA, pH 7.4
NonSpecific Ligand: 100 μM S(-)-Propranolol
IQ: 0.041 nM *
Bmax: 0.072 pmol/mg Protein* Specific Binding: 95% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
6. Adrenergic β2 (McCrea and Hill, 1993, Brit. J Pharmacol. 110:619-626). Source: Human recombinant CHO-NBRl cells
Ligand: 0.2 nM 3H CCGP-12177 Vehicle: 0.4 % DMSO Incubation Time/Temp: 60 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 0.5 mM EDTA, 5.0 mM MgCl2, 120 mM NaCl, pH 7.4
Nonspecific Ligand: 10 μM ICI-118551 K,: 0.44nM*
Bmax: 0.437 pmol/mg Protein* Specific Binding: 95% * Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
7. Adrenergic, Norepinephrine Transporter (Galli et al, 1995, J. Exp. Biol. 198:2197-2212).
Source: Human recombinant MDCK cells
Ligand: 0.2 nM 125I RTI-55
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 3 hours at 4°C Incubation Buffer: 50 mM Tris-HCl, 100 mM NaCl, 1 μM leupeptin, 10 μM
PMSF, pH 7.4
NonSpecific Ligand: 10 μM Desipramine
Kd: 0.024 μM *
Bmax: 2.5 pmol/mg Protein* Specific Binding: 75% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
8. Calcium Channel Type L, Dihydropyridine (Ehlert et al, 1982, Life Sci. 30:2191-2202).
Source: Wistar Rat cerebral cortex
Ligand: 0.1 nM 3H Nitrendipine
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 90 minutes at 25 °C Incubation Buffer: 50 mM Tris-HCl, pH 7.7
NonSpecific Ligand: 1 μM Nitrendipine
Bmax 0.23 pmol/mg Protein*
Specific Binding: 91% * Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
9. Cannabinoid CBj (Felder et al, 1995, Mol. Pharmacol. 48:443-450). Source: Human recombinant HEK-293 cells Ligand: 8 nM 3H WlN-55,212-2 Vehicle: 0.4 % DMSO Incubation Time/Temp: 90 minutes at 37 °C Incubation Buffer: 50 mM Hepes, pH 7.0, 5mg/mL BSA NonSpecific Ligand: 10 μM WIN-55,212-2
IQ: 0.3 μM *
Bma : 2.4 pmol/mg Protein* Specific Binding: 70% * Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
10. Dopamine Di (Dearry et al, 1990, Nature 347:72-76). Source: Human recombinant CHO cells
Ligand: 1.4 nM 3H SCH-23390 Vehicle: 0.4 % DMSO
Incubation Time/Temp: 2 hours at 37 °C
Incubation Buffer: 50mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.4 mM ascorbic acid, 0.001% BSA
NonSpecific Ligand: 10 μM (+)-Butaclamol Kd: 1.4 nM*
Bmax: 0.63 pmol/mg Protein*
Specific Binding: 95% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
11. Dopamine D2L (Bunzo et al, 1988, Nature 336:783-787). Source: Human recombinant CHO cells
Ligand: 0.16 nM 3H Spiperone Vehicle: 0.4 % DMSO Incubation Time/Temp: 2 hours at 25 °C
Incubation Buffer: 50mM Tris-HCl, pH 7.4, 150mM NaCl, 1.4mM ascorbic acid, 0.00 1% BSA
NonSpecific Ligand: 10 μM Haloperidol IQ: 0.08 nM* Bmax: 0.48 pmol/mg Protein*
Specific Binding: 85% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
12. GABAA, Agonist Site (Enna and Snyder, 1976, Mol Pharmacol. 13:442- 453). Source: Wistar Rat brain (minus cerebellum)
Ligand: 1 nM 3H Muscimol
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 10 minutes at 4°C
Incubation Buffer: 50 mM Tris-HCl, pH 7.4 NonSpecific Ligand: 0.1 μM Muscimol
I : 3.8 nM*
Bmax: 1.8 pmol/mg Protein*
Specific Binding: 90% *
Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
13. Glutamate, NMDA, Phencyclidine (Goldman et al, 1985, FEBS Lett. 190:333-336).
Source: Wistar Rat cerebral cortex Ligand: 2 nM 3H Idazoxan
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 30 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 0.5mM EDTA, pH 7.4
NonSpecific Ligand: 0.1 μM MK-801 (Dizolcipine) Kd: 4 nM*
Bma : 0.78 pmol/mg Protein*
Specific Binding: 94% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition 14. Histamine H Central (Hill et al, 1978, J. Neurochem. 31:997-1004). Source: Guinea pig cerebellum Ligand: 1.75 nM 3H Pyrilamine Vehicle: 0.4 % DMSO
Incubation Time/Temp: 60 minutes at 25 °C Incubation Buffer: 50 mM K-Na phosphate buffer pH 7.4 at 25°C NonSpecific Ligand: 1 μM Pyrilamine BmaX: 0.198 pmol/mg Protein*
Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
15. Imidazoline I2, Central (Brown et al, 1990, Br. J. Pharmacol. 99:803-
809).
Source: Wistar Rat cerebral cortex
Ligand: 2 nM 3H Idazoxan
Vehicle: 0.4% DMSO Incubation Time/Temp: 30 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, 0.5mM EDTA, pH 7.4 at 25°C
NonSpecific Ligand: 1 μM Idazoxan
Kd: 4nM*
Bmax: 0.14 pmol/mg Protein* Specific Binding: 85% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
16. Muscarinic M2 (Delmendo et al, 1989, Br. J. Pharmacol. 96:457-464). Source: Human recombinant insect Sf9 cells
Ligand: 0.29 nM 3H N-Methylscopolamine (NMS)
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 60 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, pH 7.4 10 mM MgCl2, ImM EDTA NonSpecific Ligand: 1 μM Atropine Kd: 0.16 nM*
Bmax: 4.9 pmol/mg Protein* Specific Binding: 96% * Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
17. Nicotinic Acetylcholine, Central (Pabreza et al, 1991, Mol. Pharmacol. 39:9-12). Source: Wistar Rat brain
Ligand: 2 nM 3H Cytisine
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 75 minutes at 4 °C
Incubation Buffer: 50 mM Tris-HCl, 120 mM NaCl, 5mM KCl, 1 mMMgCl2, 2.5 mM CaCl2, pH 7.4
NonSpecific Ligand: 100 μM Nicotine
IQ: I nM *
Bmax: 0.026 pmol/mg Protein*
Specific Binding: 90% * Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
18. Opiate μ (Wang et al, 1994, FEBS Lett. 338:217-222). Source: Human recombinant CHO-K1 cells Ligand: 0.6 nM 3H Diprenorphine
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 60 minutes at 25 °C
Incubation Buffer: 50 mM Tris-HCl, pH 7.4
NonSpecific Ligand: 10 μM Naloxone Kd: 0.41 nM *
Bmax: 3.8 pmol mg Protein*
Specific Binding: 90% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50%> of max stimulation or inhibition 19. Phorbol Ester (Ashendel, 1985, Biochem. Biophys. Acta 822:219-242). Source: ICR Mouse brain Ligand: 3 nM 3H PDBu Vehicle: 0.4 % DMSO
Incubation Time/Temp: 60 minutes at 25 °C
Incubation Buffer: 20 mM Tris-HCl, containing 5 mM CaCl2, pH 7.5 at 25 °C NonSpecific Ligand: 1 M PDBu IQ: 8.7nM* Bmax: 26 pmol/mg Protein*
Specific Binding: 80% * Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
20. Platelet-Derived Growth Factor (PDGF) (Williams et al, 1984, J. Biol.
Chem. 259:5287-5294).
Source: Mouse 3T3 cells
Ligand: 0.02 nM 125I PDGF
Vehicle:0.4 % DMSO Incubation Time/Temp: 45 minutes at 25 °C
Incubation Buffer: HBSS, 2 mg/ml BSA, 1 mM MgCl2, 1 mM CaCl2
NonSpecific Ligand: 0.1 nM PDGF
IQ: 0.012 nM*
Bmax: 3100 R/cell Receptor/cell* Specific Binding: 88% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
21. Potassium Channel [KATP] (Gaines et al, 1988, J. Biol. Chem. 263:2589-2592).
Source: Syrian hamster pancreatic beta cells HIT-T15
Ligand: 5 nM 3H Glyburide
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 2 hours at 25 °C Incubation Buffer: 50 mM MOPS, 0.1 mM CaCl2, pH 7.4 NonSpecific Ligand: 10 μM Glyburide IQ: 0.64nM* Bmax: 1 pmol/mg Protein* Specific Binding: 90% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
22. Sigma ff! (Ganapathy et al, 1999, Pharmacol. Exp. Ther. 289:251-260). Source:Human Jurkat cells TIB-152
Ligand: 8 nM 3H Haloperidol
Vehicle: 0.4 % DMSO
Incubation Time/Temp: 4 hours at 25 °C
Incubation Buffer: 5 mM K2HPO4/KH2P04 buffer pH 7.5 NonSpecific Ligand: 10 μM Haloperidol
IQ: 5.8nM*
Bmax: 0.71 pmol/mg Protein*
Specific Binding: 80% *
Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
23. Sigma σ2 (Hashimoto and London, 1993, Eur. J. Pharmacol. 236:159-163 Source: Wistar Rat brain
Ligand: 3 nM 3H Ifenprodil Vehicle: 0.4 % DMSO
Incubation Time/Temp: 60 minutes at 37 °C
Incubation Buffer: 50 mM Tris-HCl, pH 7.4
NonSpecific Ligand: 10 μM Ifenprodil
IQ: 4.8 nM* BmaX: 1.3 pmol/mg Protein *
Specific Binding: 85% *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition 24. Sodium Channel, Site 2 (Catterall et al, 1981, J. Biol. Chem. 256:8922- 8927.
Source: Wistar Rat brain
Ligand: 1.5 nM 3H Batrachotoxinin A 20-μ-Benzoate Vehicle: 0.4 % DMSO
Incubation Time/Temp: 30 minutes at 37 °C
Incubation Buffer: 50 mM Tris-HCl, pH 7.4 at 25°C, 50 mM Hepes, 130 mM choline-Cl, 5.4 mM KCl, 0.8 mM MgSO4.7H20, 5.5 mM glucose, 40 μg/ml LqTx
NonSpecific Ligand: 100 μM Veratridine Kd: 0.013 μM *
Bmax: 0.88 pmol/mg Protein *
Specific Binding: 85%, *
Quantitation Method: Radioligand Binding
Significance Criteria: > 50% of max stimulation or inhibition
25. Vascular Endothelial Growth Factor (VEGF) (Gitay-Goren et al, 1996, J. Biol. Chem. 271:5519-5523).
Source: Human umbilical vein endothelial cells
Ligand: 0.1 μM 125I VEGF165 Vehicle: 0.4 % DMSO
Incubation Time/Temp: 3 hours at 25 °C
Incubation Buffer: Buffer 1: M199 medium, 20% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, 4 mM L-glutamate, 15 mM Hepes, pH 7.4.
Buffer 2: Buffer 1 containing 1 μg/ml Heparmn and 0.1% gelatin NonSpecific Ligand: 3 nM VEGFι65
Kd: 0.035 nM *
Bmax: 8900 R/cell Receoptors/cell*
Specific Binding: 85% *
Quantitation Method: Radioligand Binding Significance Criteria: > 50% of max stimulation or inhibition
* Historical Values
6.31 Example 31: In Vivo Z-Chamber Study The Z-chamber assay is a fibrin-based in vivo assay, wherein fibrin and thrombin are added through a port in a two-sided chamber sealed by a nylon mesh. The chamber is implanted in the subcutaneous space of an animal and harvested for evaluation. Fibrin matrices are formed in normal wound healing and are used by tumors to sustain growth, thus Z-chambers are designed to study, for example, angiogenesis, wound healing, and tumor growth. In addition, their design is useful, for example, in studies of localized gene expression, stem cell, adenoviral, and tissue generation.
The efficacy of 101 in an in vivo tumor model was examined by Z-chamber® (SRI, Menlo Park, CA) study. Each tumor chamber consisted of 150-160 μl cell suspension made by suspending 190 million cells in 20 ml fibrin (4 mg/ml). Following the introduction of cell suspension, 2 units of thrombin was added into each chamber and the mixture was allowed to gel for 5-7 minutes before implantation. Rats were anesthetized with Nembutal (35 mg/kg). The skin of rats was surgically prepared with 70 % alcohol. Two incisions (approximately 2 cm in length) were made on the back, one over the mid vertebral and the other over the lower vertebral region. Pockets were made in the subcutaneous fascia lateral to the incisions by blunt dissection with the help of scissors, and the chambers were placed deep into these pockets. The incision wounds were later closed with an autoclip stapling device.
101 was dissolved in a 1 to 1 cremophor: ethanol solution and diluted 4 times with 5 % dextrose on water. Animals with tumor chambers were injected daily with 50 mg/kg 101 or 5 mg/kg taxol every other day as a positive control. Tumor chambers were harvested on day 16 post implantation. Chambers were cleared of all fascia, and tissue in each chamber was fixed in 10 % formalin, paraffin embedded and stained with hematoxylin and eosin. The tumor thickness was measured and compared to a negative control, i.e., chambers with no treatment, and the positive treatment. Four rats were used per each group. The results are summarized in Figure 14. These studies demonstrate the efficacy of illustrative compounds of the invention in reducing tumor thickness in an in vivo model. Particularly, illustrative compounds of the invention are effective in modulating biological activities of Edg-4, for example, inhibiting cell proliferation in an in vivo model. Finally, it should be noted that there are alternative ways of implementing the present invention. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.
All publications and patents cited herein are incoφorated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:
1. A method of modulating an Edg-4 receptor mediated biological activity comprising contacting a cell expressing the Edg-4 receptor with an amount of a modulator of the Edg-4 receptor sufficient to modulate the Edg-4 receptor mediated biological activity wherein the modulator is not a phospholipid.
2. A method of modulating an Edg-4 receptor mediated biological activity in a subject comprising administering to the subject a therapeutically effective amount of a modulator of the Edg-4 receptor wherein the modulator is not a phospholipid.
3. The method of Claim 1 or 2, wherein the modulator is an agonist.
4. The method of Claim 1 or 2, wherein the modulator is an antagonist.
5. The method of Claim 1 or 2, wherein the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
6. The method of Claim 1 or 2, wherein the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
7. The method of Claim 1 or 2, wherein the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
8. The method of Claim 1 or 2, wherein the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
9. The method of Claim 1 or 2, wherein the biological activity is cell proliferation.
10. The method of Claim 9, wherein the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
11. The method of Claim 9, wherein the modulator exhibits at least about 10 fold inhibitory selectivity for Edg-4 relative to other Edg receptors.
12. The method of Claim 9, wherein the modulator exhibits at least about 200 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
13. The method of Claim 9, wherein the modulator exhibits at least about
10 fold inhibitory selectivity for Edg-4 relative to Edg-2 and Edg-7 receptors.
14. The method of Claim 9, wherein cell proliferation leads to ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colon cancer or prostrate cancer.
15. The method of Claim 9, wherein cell proliferation is stimulated by LPA.
16. The method of Claim 1 or 2, wherein the biological activity is calcium mobilization, VEGF synthesis, IL-8 synthesis, platelet activation, cell migration, phosphoinositide hydrolysis, inhibition of cAMP formation, increasing the level of fatty acids, actin polymerization, apoptosis, angiogenesis, inhibition of wound healing, inflammation, expression of endogenous protein growth factors, cancer invasiveness, regulation of autoimmunity or atherogenesis.
17. The method of Claim 1 or 2 wherein the modulator binds to the Edg-4 receptor with a binding constant of at least about 1 μM.
18. The method of Claim 1 or 2 wherein the modulator binds to the Edg-4 receptor with a binding constant between about 1 μM and 100 nM.
19. The method of Claim 1 or 2, wherein the modulator is a nucleic acid, peptide or carbohydrate.
20. The method of Claim 1 or 2, wherein the modulator is an organic molecule of molecular weight of less than 750 daltons.
21. The method of Claim 1 , wherein the cell is a HTC hepatoma cell, an ovarian cell, an epithelial cell, a fibroblast cell, a neuronal cell, a Xenopus laevis oocyte cell, a carcinoma cell, a pheochromocytoma cell, a myoblast cell, a platelet cell or a fibrosarcoma cell.
22. The method of Claim 21, wherein the cell is 0V202 human ovarian cell, a HTC rat hepatoma cell, SKOV3 and CAOV-3 human ovarian cancer cells,
MDA-MB-453 breast cancer cell, MDA-MB-23 1 breast cancer cell, HUVEC cells A43 1 human epitheloid carcinoma cell or a HT-1 080 human fibrosarcoma cell.
23. The method of Claim 1 or 2, wherein the modulator is a compound of stmctural formula (I):
(I)
or a pharmaceutically available solvate or hydrate thereof, wherein:
Ri is hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkylarylamino, substituted alkylarylamino, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylamino, substituted arylamino, arylalkyl, substituted arylalkyl, dialkylamino, substituted dialkylamino, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl sulfonylamino or substituted sulfonylamino;
X=0 or S; A is NR2, O or S;
R2 is hydrogen, alkyl or substituted alkyl; and
B and C are independently alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl.
24. The method of Claim 23, wherein R\ is alkyl, substituted alkyl, aryl, substituted aryl, arylalkyloxy or substituted sulfonylamino.
25. The method of Claim 23, wherein R\ is substituted alkyl.
26. The method of Claim 23, wherein Ri is substituted haloalkyl.
27. The method of Claim 23, wherein R\ is substituted trifluoroalkyl.
28. The method of Claim 23, wherein Ri has the stmctural formula (II):
(II)
wherein:
R is haloalkyl or substituted haloalkyl;
R is oxo or thiono; and
R5 and R are independently hydrogen, halo, alkyl or substituted alkyl.
29. The method of Claim 28, wherein R is fluoroalkyl, R4 is oxo and R and Re are independently hydrogen, halo or alkyl.
30. The method of Claim 28, wherein R3 is trifluoromethyl, R4 is oxo and R5 and R are independently hydrogen, chloro or methyl.
31. The method of Claim 28, wherein R5 and Re are hydrogen.
32. The method of Claim 28 wherein R5 is hydrogen and Re is chloro or methyl.
33. The method of Claim 23, 28 or 30 wherein X is 0, A is NR2 and R2 is hydrogen.
34. The method of Claim 23, 28 or 30 wherein B and C are independently, aryl, substituted aryl, heteroaryl or substituted heteroaryl.
35. The method of Claim 23, 28 or 30 wherein B and C are independently indolo, substituted indolo, imidazolo, substituted, imidazolo, pyrazolo, substituted pyrazolo, phenyl or substituted phenyl.
36. The method of Claim 23, 28 or 30 wherein B is heteroaryl or substituted heteroaryl and C is aryl or substituted aryl.
37. The method of Claim 23, 28 or 30 wherein B is pyrazolo or substituted pyrazolo and C is phenyl or substituted phenyl.
38. The method of Claim 23, wherein the modulator is a compound of structural formula (III):
(III)
wherein: R7 is hydrogen, alkyl, substituted alkyl or halo;
R8 is hydrogen, carbamoyl or substituted carbamoyl; and
R , Rio and R\ \ are independently hydrogen, alkoxy, substituted alkoxy, halo or P.9 and P.iotogether with the carbons to which they are attached form a [1,3] dioxolane ring.
39. The method of Claim 23, wherein the modulator is compound of the formula:
139
137
40. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (IV):
(IV) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, ^ or R5 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(C1-C,0)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -(C5-Cιo)cycloheteroaryl, -(C3-C6)cycloheteroalkyl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -NR5R5, =NR5, -(C, -C10)alkylNHC(O)(CH2)mR5, -(C3-C10)cycloheteroalkyl(R5)m, -(CH2)mR5, -(Cι-Cι0)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C10)alkyl(C,-C10)alkyl, -O(Cι-C,0)alkyl, -C(O)(C,-C,0)alkyl,
C10)alkyl), -CO^d-C^alkyl, -(Cι-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl,
-(C3-Cι0)cycloalkyl, -(C8-C14)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C -Cι0)heterocycle, -CO2(CH2)m(Cι-Cι0)alkyl, -CO2(CH2)mH, -NHC(O)(C1-C,0)alkyl, -NHC(O)NH(Cι-C,0)alkyl, -OC(O)O(Cι-
Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X and Y are each independently C or N; and Z is O, S, C or N, wherein if Z is O or S, then R3 is an electron pair;
Ri and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R3 and t can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
41. The method of Claim 40, wherein the modulator is a compound of the following formula:
42. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (V):
or a pharmaceutically available solvate or hydrate thereof, wherein:
each of R, , R2, R3, ^ or R5 is independently -H, -halo, -NO2, -CN, -OH,
-N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-C]0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5,-OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R^ is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(C1-C1o)alkyl(Cι-C10)alkyl, -O(C1-C,o)alkyl, -C(O)(C1-C,0)alkyl, -C(O)NH(CH2)m(C,-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(Cι-
Ci0)alkyl), -CO2(C,-Cιo)alkyl, -(C,-Cι0)alkyl, -(C2-C10)alkenyl,
-(C2-Ci0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C[4)bicycloalkyl, -(C5-Cι0)cycloalkenyl,
-(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C1o)heterocycle,
-CO2(CH2)m(C1-C,0)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C10)alkyl, -NHC(O)NH(C,-
C10)alkyl, -OC(O)(Cι-Cιo)alkyl, -OC(O)O(C,-C10)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and
Ri and R or R2 and R3 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
43. The method of Claim 42, wherein Ri and R2 are independently aryl, substituted aryl, heteroaryl or substituted heteroaryl.
44. The method of Claim 42, wherein R2 is indole and R and R4 are hydrogen.
45. The method of Claim 42, wherein the modulator is a compound of the following formula:
or its (+) and (-) enantiomers.
46. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (VI):
(VI) or a pharmaceutically available solvate or hydrate thereof, wherein:
each of Ri, R2, R3, R4or R5 is independently -H, -halo, -NO2, -CN, -OH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-Cι0)alkenyl, -(C2-C10)alkynyl, -(C3-C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C10)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5,-OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
R6 is independently -halo, -NO2, -CN, -OH, -CO2H, -O(C,-C10)alkyl, -C(O)(C1-C10)alkyl,
-C(O)NH(CH2)m(C,-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-C10)alkyl(C,- C10)alkyl), -CO2(C,-C10)alkyl, -(C,-Cι0)alkyl, -(C2-Cι0)alkenyl, -(C2-Ci0)alkynyl, -(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C )heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)m(C,-C10)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,0)alkyl, -NHC(O)NH(d- Ci0)alkyl, -OC(O)(CrCιo)alkyl, -OC(O)O(C,-Cι0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X, Y and Z are independently O, S, C or N, wherein if X, Y or Z is O or S, Ri is an electron pair; Ri and R2 or can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; Ri and R5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
R and R5 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
47. The method of Claim 46, wherein Ri and R2 together form a 5-, 6- or
7-membered substituted or unsubstituted cyclic or aromatic ring.
48. The method of Claim 46, wherein: R] and R2 together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R3 and t together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
49. The method of Claim 46, wherein: R\ and R2 together form a
6- membered substituted or unsubstituted cyclic or aromatic ring; and R and R4 together form a 6-membered substituted or unsubstituted cyclic or aromatic ring.
50. The method of Claim 46, wherein: Ri. and R2 form a 6-membered substituted cyclic or aromatic ring, and R and R form a 6-membered substituted cyclic or aromatic ring.
51. The method of Claim 46, wherein the modulator is a compound of the following formula:
52. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (VII):
(VII) or a pharmaceutically available solvate or hydrate thereof, wherein:
each of R], R2, R , R , R5, R7 or R8 is independently -H, -halo, -NO2, -CN, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C10)alkyl, -(C2-C10)alkenyl, -(C2-C10)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C -Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C1-C,o)alkylNHC(O)(CH2)mR5, -(C,-Cιo)alkylNR5R5; -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C1-C1o)alkyl(C1-C10)alkyl, -O(C1-C10)alkyl, -C(O)(C1-C10)alkyl, -C(O)NH(CH2)m(C,-C10)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(C,- C,o)alkyl), -CO2(C,-C,0)alkyl, -(Cι-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C10)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C -C,o)heterocycle, -CO2(CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-C,0)alkyl, -OC(O)O(C,- C,0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
X is O, S, C or N, wherein if X is O or S, R, is an electron pair; and
Y and Z are independently N or C, wherein if Y or Z is N, R, and R2 are each an electron pair.
53. The method of Claim 52, wherein the modulator is a compound of the following formula:
54. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (VIII):
(VIII) or a pharmaceutically available solvate or hydrate thereof, wherein:
each of R,, R2, R , R4 , R5, R7, R8, R9 or R,o is independently -H, -halo, -NO2, -CN, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C -C,o)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-C1o)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5j -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R6 is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(C,-C,o)alkyl, -O(C,-C,0)alkyl, -C(O)NH(CH2)m(C,-Cιo)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,o)alkyl(C,- C,o)alkyl), -CO2(C,-C,0)alkyl, -(C Cιo)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Ci0)heterocycle5 -CO2(CH2)m(Cι-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-Cι0)alkyl, -OC(O)O(C,- C,o)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; and X and Y are independently O, S or N, wherein if X or Y is O or S, R9 and R,o are an electron pair.
55. The method of Claim 54, wherein R7 is substituted or unsubstituted aryl.
56. The method of Claim 54, wherein the modulator is a compound of the following formula:
57. The method of Claim 1 or 2, wherein the modulator is a compound of stmctural formula (IX):
(IX) or a pharmaceutically available solvate or hydrate thereof, wherein:
each of Ri, R2, R3, * , R5, R7, Rs, 9 or R)0 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-C,0)alkyl, -(C2- Cιo)alkenyl, -(C2-C,0)alkynyl, -(C3-C10)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-Cιo)alkylNΗC(O)(CH2)mR5, -(Cι-Cιo)alkylNR5R5>-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(C,-Cιo)alkyl(C,-C,o)alkyl, -O(C,-Cιo)alkyl, -C(O)(C C,o)alkyl, -C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C10)alkyl(Cι- C,o)alkyl), -CO2(C,-C,0)alkyl, -(C,-Cι0)alkyl, -(C2-C,0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cι0)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Co)heterocycle, -CO2(CH2)m(C,-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(Ci-C10)alkyl, -NHC(O)NH(C,-C,0)alkyl, -OC(O)O(Cι- Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; and p is independently an integer ranging from 0 to 5.
58. The method of Claim 57, wherein R2 is a substituted alkyl, and one or more of R5, R7, R8, R and Rio are halos.
58. The method of Claim 57, wherein R2 is a halo-substituted alkyl.
59. The method of Claim 57, wherein R is -CF3.
60. The method of Claim 57, wherein the modulator is a compound of the following formula:
131
61. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (X):
(X) or a pharmaceutically available solvate or hydrate thereof, wherein:
each of R,, R2, R3, R4 , R5 or R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-Cι0)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-C,0)cycloalkyl, -(C8-Cι )bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-C,0)alkylNHC(O)(CH2)mR5, -(Cι-Cιo)alkylNR5R5, -CO2H, -(C,-C,o)alkylC(O)NH(CH2)mR5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or Re is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(C,-Cιo)alkyl(Cι-C,o)alkyl, -O(Cι-C,0)alkyl, -C(O)(C,-Cιo)alkyl,
-C(O)NH(CH2)m(C,-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(C,-
C,o)alkyl), -CO2(C,-C,0)alkyl, -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl,
-(C -Cιo)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)m(Cι-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-Cι0)alkyl, -NHC(O)NH(C,-C,0)alkyl, -OC(O)O(Cι-
Cιo)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5; Ri and R2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
RA and R7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
62. The method of Claim 61, wherein R3 and R7 are substituted or unsubstituted aryls.
63. The method of Claim 61, wherein the modulator is a compound of the following formula:
64. The method of Claim 1 or 2, wherein the modulator is a compound of stmctural formula (XI):
(XI) or a pharmaceutically available solvate or hydrate thereof, wherein:
each of R,, R2, R3, R4 , R5, R7 or R8 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(Cι-Cιo)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,0)heteroaryl, -(C5-C,0)cycloheteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(Cι-C,o)alkylNHC(O)(CH2)mR5, -(Cι-C,0)alkylNR5R5>-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(Cι-Cιo)alkyl(Cι-C,o)alkyl, -O(C,-C,o)alkyl, -C(O)(C,-Cιo)alkyl,
-C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-Cι0)alkyl(C1- C,0)alkyl), -CO2(Cι-Cι0)alkyl, -(Cι-C,0)alkyl, -(C2-Cι0)alkenyl, -(C2-Cι0)alkynyl,
-(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-Cι0)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)m(C,-Cιo)alkyl,
-CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-C,0)alkyl, -OC(O)O(C,-
C,0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
R, and R2 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R2 and R can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R3 and R4 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring;
R and R7 can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; R7 and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring; and
Ri and Rs can optionally together form a 5-, 6- or 7-membered substituted or unsubstituted cyclic or aromatic ring.
65. The method of Claim 64, wherein R2 and R3 together form a
5-membered ring.
66. The method of Claim 64, wherein R2 and R3 together form a 5- membered ring, and R and R8 together form a 5 -membered ring.
67. The method of Claim 64, wherein the modulator is a compound of the following formula:
68. The method of Claim 64, wherein R2 is a substituted or unsubstituted piperazine moiety.
69. The method of Claim 64, wherein the modulator is a compound of the following formula:
70. The method of Claim 1 or 2, wherein the modulator is a compound of structural formula (XII):
(XII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of Ri, R2, R3, R4 , R5 or R7 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -(CH2)mN(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -C(OH)R5, -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-Cι0)alkyl, -(C2-Cιo)alkenyl, -(C2-Cι0)alkynyl, -(C3-C,0)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5- Cιo)cycloalkenyl, -(C )heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -(C5-Cιo)cycloheteroaryl, -(C3-C6)cycloheteroalkyl, -naphthyl, -(C3-C10)heterocycle, -CO2(CH2)mR5, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -NR5R5, =NR5, -(Cι-C,o)alkylNHC(O)(CH2)mR5, -(C3-Cιo)cycloheteroalkyl(R5)m, -(CH2)mR5, -(Cι-Cι0)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 or R6 is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(C,-C,o)alkyl, -O(C,-C,o)alkyl, -C(O)(C,-Cιo)alkyl, -C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C,o)alkyl(C1- C,0)alkyl), -CO2(C,-C,o)alkyl, -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(Cs)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO (CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(Cι-Cιo)alkyl, -NHC(O)NH(C,-C,o)alkyl, -OC(O)O(C,- C,0)alkyl, or -SO2NH2; m is independently an integer ranging from 0 to 8; p is independently an integer ranging from 0 to 5;
R3 or R can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring;
R, or R2 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring; and R2 or R4 can optionally form a substituted or unsubstituted cyclic, aromatic, heterocyclic, heteroaryl or cycloheteroalkyl ring.
71. The method of Claim 70, wherein the modulator is a compound of the following formula:
or
72. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (I)-(XII).
73. A method for treating or preventing ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, adult respiratory distress syndrome (ARDS), asthma, transcomeal freezing, cutaneous bums, ischemia or arthesclerosis in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (I)-(XII).
74. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (I)-(XII) and one or more agonists or antagonists of an LPA receptor.
75. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (I)-(XII) and one or more dmgs useful in treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases.
76. A method of modulating an Edg-7 receptor mediated biological activity comprising contacting a cell expressing the Edg-7 receptor with an amount of a modulator of the Edg-7 receptor sufficient to modulate the Edg-7 receptor mediated biological activity wherein the modulator is not a phospholipid.
77. A method of modulating an Edg-7 receptor mediated biological activity in a subject comprising administering to the subject a therapeutically effective amount of an modulator of the Edg-7 receptor wherein the modulator is not a phospholipid.
78. The method of Claim 76 or 77, wherein the modulator is a compound of stmctural formula (XIII):
or a pharmaceutically available solvate or hydrate thereof, wherein:
X is NR3, S or O;
R, is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, amino, carbamoyl, substituted carbamoyl, oxo, thiono or -NR4; and
R2 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkyloxy, substituted alkyloxy, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, amino, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, aryloxycarbonyl, substituted aryloxycarbonyl, arylalkyl, substituted arylalkyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or
R3 is hydrogen, alkyl, substituted alkyl, alkylthio, substituted alkylthio, alkylsulfonyl, substituted alkylsulfonyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl or substituted heteroalkyl;
R is alkyl, substituted alkyl, acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, heteroaryl substituted heteroaryl, cycloheteroalkyl, substituted cycloheteroalkyl, and
R5 and R^ are independently hydrogen, alkyl, substituted alkyl, acylamino, substituted acylamino, alkylthio, substituted alkylthio, alkoxycarbonyl, substituted alkoxycarbonyl, alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl, substituted alkylsulfinyl, arylalkyloxy, substituted arylalkyloxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl or optionally along with the carbon to which they are attached form an aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl ring.
79. The method of Claim 78, wherein the modulator is a compound according to the stmctural formula:
80. The method of Claim 78, wherein X is S or NR3.
81. The method of Claim 78, wherein R, is oxo, thiono or NR .
82. The method of Claim 81, where R4 is acyl, substituted acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkoxycarbonyl, substituted alkoxycarbonyl, carbamoyl or substituted carbamoyl.
83. The method of Claim 81 , where R4 is substituted carbamoyl..
84. The method of Claim 78, wherein R2 is acyl, substituted acyl, acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, alkylsulfonyl, substituted alkylsulfonyl, aryloxycarbonyl, substituted aryloxycarbonyl, carbamoyl, substituted carbamoyl, or
85. The method of Claim 78, wherein R2 is substituted alkoxycarbonyl or
86. The method of Claim 85, where R5 and Re along with the carbon to which they are attached form a cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl ring.
87. The method of Claim 85, where R5 and Re along with the carbon to which they are attached form a substituted cycloheteroalkyl ring.
88. The method of Claim 78, wherein the modulator is compound of the formula:
89. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XIII).
90. A method for treating or preventing ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, adult respiratory distress syndrome (ARDS), asthma, transcomeal freezing, cutaneous bums, ischemia or arthesclerosis in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XIII).
91. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XIII).
92. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XIII), and one or more dmgs useful in treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases.
93. A method of modulating an Edg-2 receptor mediated biological activity comprising contacting a cell expressing the Edg-2 receptor with an amount of an modulator of the Edg-2 receptor sufficient to modulate the Edg-2 receptor mediated biological activity wherein the modulator is not a lipid, phospholipid or a compound of the stmctural formula:
or a pharmaceutically available salt thereof, wherein:
X is O or S;
R20 is alkyl, substituted alkyl, aryl, substituted aryl or halo;
R2, is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl; R23 is hydrogen, alkyl or substituted alkyl;
R2 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; and
or alternatively R23 and R24 from a cycloalkyl ring.
94. A method of modulating an Edg-2 receptor mediated biological activity in a subject comprising administering to the subject a therapeutically effective amount of an modulator of the Edg-2 receptor wherein the modulator is not a phospholipid wherein the modulator is not a lipid, a phospholipid or a compound of the stmctural formula (I):
or a pharmaceutically available salt thereof, wherein:
X is O or S;
R2o is alkyl, substituted alkyl, aryl, substituted aryl or halo;
R2, is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
R23 is hydrogen, alkyl or substituted alkyl;
R24 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; and
or alternatively R23 and R24 from a cycloalkyl ring.
95. The method of Claim 93 or 94 wherein the modulator is a compound of stmctural formula (XXII):
P-
(XXII) or a pharmaceutically available salt, hydrate or solvate thereof wherein:
P, Q and R are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl.
96. The method of Claim 95 wherein the modulator is a compound of stmctural formula (XXIII) :
(XXIII)
wherein:
n is 1, 2 or 3;
X = N or CH;
R, and R2 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cyano, cyanato, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, oxo or thiono;
R3 and R4 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halo or thio; B is NR5, O or S;
R is hydrogen, alkyl, substituted alkyl, alkylamino, substituted alkylamino, alkoxy, substituted alkoxy, amino, cyano, dialkylamino, substituted dialkylamino or hydroxy; and
A and C are independently aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl or substituted heteroaryl.
97. The method of Claim 96, wherein Ri and R2 are independently hydrogen, alkyl substituted alkyl, oxo or thiono.
98. The method of Claim 96, wherein R, and R2 are independently oxo or thiono.
99. The method of Claim 96, wherein R3 and R are independently hydrogen or alkyl.
100. The method of Claim 96, wherein A and C are independently aryl, substituted aryl, heteroaryl or substituted heteroaryl.
101. The method of Claim 96, wherein A and C are aryl or substituted aryl.
102. The method of Claim 96, wherein A and C are phenyl or substituted phenyl.
103. The method of Claim 96, wherein B is NR5 and R5 is hydrogen, alkyl or hydroxy.
104. The method of Claim 96, wherein n is 1, R, and R2 are oxo, R3 and ^ are hydrogen, B is NR5 and R5 is hydroxy.
105. The method of Claim 96, wherein n is 1, R, and R2 are oxo, R3 and R are hydrogen, B is NR5, R5 is hydroxy, A and B are aryl or substituted aryl.
106. The method of Claim 96, wherein n is 1 , R, and R2 are oxo, R3 and R4 are hydrogen, B is NR5, R5 is hydroxy, A and B are phenyl or substituted phenyl.
107. The method of Claim 96, wherein the modulator has the formula:
108. The method of Claim 95, wherein Q is cycloheteroalkyl, substituted cycloheteroalkyl and P and R are independently aryl or substituted aryl.
109. The method of Claim 95, wherein Q is cycloheteroalkyl or substituted cycloheteroalkyl and P and R are independently phenyl or substituted phenyl.
110. The method of Claim 95, wherein Q is heteroaryl or substituted heteroaryl and P and R are independently aryl or substituted aryl.
111. The method of Claim 95, wherein Q is heteroaryl or substituted heteroaryl and P and R are independently phenyl or substituted phenyl.
112. The method of Claim 95, wherein the modulator has the stmctural formula (XXIV):
(XXIV)
wherein:
R3, is hydrogen, alkyl or substituted alkyl;
R 2 is hydrogen, alkyl or substituted alkyl;
R33 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; and
R34 is aryl, substituted aryl, heteroaryl or substituted heteroaryl.
113. The method of Claim 112, wherein R3, and R 2 are alkyl.
114. The method of Claim 112, wherein R3 and R34 are aryl or substituted aryl.
115. The method of Claim 112, wherein R3, and R32 are alkyl and R33 and R 4 are aryl or substituted aryl.
116. The method of Claim 112,wherein R33 and R 4 are phenyl or substituted phenyl.
117. The method of Claim 112, wherein R , and R32 are methyl or ethyl and
R33 and R 4 are phenyl or substituted phenyl.
118. The method of Claim 95, wherein the modulator has the formula:
119. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXIII) or (XXIV).
120. A method for treating or preventing ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, adult respiratory distress syndrome (ARDS), asthma, transcomeal freezing, cutaneous bums, ischemia or arthesclerosis in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXIII) or (XXIV).
121. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXIII) or (XXIV) and one or more agonists or antagonists of an Edg-2 receptor.
122. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXIII) or (XXIV) and one or more dmgs useful in treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases.
123. A method of modulating an Edg-2 receptor mediated biological activity comprising contacting a cell expressing the Edg-2 receptor with an amount of an modulator of the Edg-2 receptor sufficient to modulate the Edg-2 receptor mediated biological activity wherein the modulator is of the stmctural formula (XXII):
(XXII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C -C,o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,0)heteroaryl, -naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-Cιo)alkylNHC(O)(CH2)mR5, -(C,-Cι0)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-Cι0)alkyl(C,- C,o)alkyl, -O(C,-C,0)alkyl, -C(O)(C,-C10)alkyl, -C(O)NH(CH2)m(C,-C,0)alkyl, - OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cι0)alkyl(Cι-Cιo)alkyl), -CO2(C,-C,0)alkyl, -(C,- C,o)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl, -(C3-C,0)cycloalkyl, -(C8-C]4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)m(C,-Cι0)alkyl, -CO2(CH2)mH, -NHC(O)(C,-Cιo)alkyl, -NHC(O)NH(C,-C,o)alkyl, -OC(O)O(C,- C,o)alkyl, or -SO2NH2;
X, Y, and Z are each independently C(R5)( R5), C(O), O, C(S), S, C=N(R5), or NR3; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5; R, and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring; and R, and X or R2 and Y can together form a double bond.
124. A method of modulating an Edg-2 receptor mediated biological activity comprising contacting a cell expressing the Edg-2 receptor with an amount of an modulator of the Edg-2 receptor sufficient to modulate the Edg-2 receptor mediated biological activity wherein the modulator has the stmctural formula (XXIII):
(XXIII) wherein:
each of R, and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl,
-(C -Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C -C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -(C,-C,0)alkylNHC(O)(CH2)mR5, -(C,-C,0)alkylNR5R5j -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Ci-C,0)alkyl(Ci-Cio)alkyl, -O(C,-Cιo)alkyl, -C(O)(C,-C,0)alkyl,
-C(O)NH(CH2)m(C,-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C1-C10)alkyl(C1- C,0)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,0)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C]0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C -Co)heterocycle, -CO2(CH2)m(C,-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(Cι-C,0)alkyl, -OC(O)O(C,- C,o)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; each p is independently an integer ranging from 0 to 5; and R, and R2 can optionally together form a 5-, 6-, or 7-membered substituted or unsubstituted cyclic or aromatic ring.
125. The method of Claim 123 or 124 wherein the modulator is a compound of stmctural formula:
201 205
126. A method of modulating an Edg-2 receptor mediated biological activity comprising contacting a cell expressing the Edg-2 receptor with an amount of an modulator of the Edg-2 receptor sufficient to modulate the Edg-2 receptor mediated biological activity wherein compound of the stmctural formula (XXI):
(XXI)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R, and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C3-C10)heterocycle, -OC(O)aryl, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(C,-Cιo)alkylNHC(O)(CH2)mR5, -(C1-Cι0)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C10)alkenyl, -(C2-C,0)alkynyl, -(C -C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C -C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(C,-Cιo)alkylNHC(O)(CH2)mR5, -(Cι-Cιo)alkylNR5R5j -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R is independently -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(C,-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(C1-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(C1- C,o)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(Cs)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C1-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(C,-C,0)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
127. A method of modulating an Edg-2 receptor mediated biological activity in a subject comprising administering to the subject a therapeutically effective amount of an modulator of the Edg-2 receptor wherein the modulator a compound of the stmctural formula (XXII):
(XXII) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R^s independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, - OC(O)aryl, -(C,-C10)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5> -
OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein;
each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C1o)alkyl(C1-C,0)alkyl, -O(C,-C,o)alkyl, -C(O)(C1-C,o)alkyl, -C(O)NH(CH2)m(Cι-C,0)alkyl, -OCF3, C,o)alkyl), -CO2(C,-Cι0)alkyl, -(Cι-Cι0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)m(C,-Cι0)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(C,-C,0)alkyl, or -SO2NH2; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
128. The method of Claim 95 wherein the modulator is a compound selected from the group consisting of:
227 229
231
129. A method of modulating an Edg-2 receptor mediated biological activity comprising contacting a cell expressing the Edg-2 receptor with an amount of an modulator of the Edg-2 receptor sufficient to modulate the Edg-2 receptor mediated biological activity wherein compound of the stmctural formula (XXI):
(XXI)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R, and R2 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -NH(aryl), -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -aryl, -(C2-Cιo)alkynyl, -(C3-C,0)cycloalkyl, -(C3-C,0)cycloalkyl(aryl), -(C8-Cι4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5- C10)heteroaryl, -naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, - NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(C,-Cι0)alkylNHC(O)(CH2)mR5, -(C,-C,0)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C1o)alkyl(C,-C,o)alkyl, -O(C,-C,o)alkyl, -C(O)(C,-Cιo)alkyl, -C(O)NH(CH2)m(C,-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(C1- C,0)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-Cl0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(Cs)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(C,-Cιo)alkyl, -CO2(CH2)mH, -NHC(O)(C,-Cιo)alkyl, -NHC(O)NH(C,-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(C,-C10)alkyl, or -SO2NH2; X is O, S, Or NRe; R, and R2 can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two e groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
130. A method of modulating an Edg-2 receptor mediated biological activity in a subject comprising administering to the subject a therapeutically effective amount of an modulator of the Edg-2 receptor wherein the modulator a compound of the stmctural formula (XXII):
O
R NHR7
(XXII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
R, is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH,
-N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3,
-benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C1o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C3-C,o)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, - OC(O)aryl, -(C,-C10)alkylNHC(O)(CH2)mR5, -(C,-Cιo)alkylNR5R5, - OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H,
-N(Cι-Cιo)alkyl(C,-C,o)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl,
-C(O)NH(CH2)m(C,-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-C,o)alkyl(C,-
C,0)alkyl), -CO2(Cι-C,0)alkyl, -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,o)alkynyl, -(C3-Cιo)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl,
-(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)m(C,-C,0)alkyl,
-CO2(CH2)mH, -NHC(O)(Cι-C,0)alkyl, -NHC(O)NH(C,-C10)alkyl, -NH(aryl),
-N-C(aryl), -OC(O)O(C,-C,0)alkyl, or -SO2NH2;
R7 is -CO2H, -C(O)(C,-C,o)alkyl, -C(O)NH(CH2)m(C,-C,0)alkyl, -benzyl, -CO2(CH2)mCH((C1-C,o)alkyl(C1-C,o)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,0)alkyl,
-(C2-Cιo)alkenyl, -(C2-C,o)alkynyl, -(C3-C,0)cycloalkyl, -(C8-C,4)bicycloalkyl,
-(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl,
-(C3-C,0)heterocycle, -CO2(CH2)m(C,-C10)alkyl, -CO2(CH2)mH;
R, and R can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6- membered aromatic ring; two R groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
131. The method of Claim 95 wherein the modulator is a selected from the group consisting of:
219 221
132. A method of modulating an Edg-3 receptor mediated biological activity comprising contacting a cell expressing the Edg-3 receptor with an amount of an modulator of the Edg-3 receptor sufficient to modulate the Edg-3 receptor mediated biological activity wherein the modulator is not a phospholipid.
133. A method of modulating an Edg-3 receptor mediated biological activity in a subject comprising administering to the subject a therapeutically effective amount of an modulator of the Edg-3 receptor wherein the modulator is not a phospholipid.
134. The method of Claim 132 or 133, wherein the modulator is a compound of stmctural formula (XXXI):
or a pharmaceutically available solvate or hydrate thereof, wherein:
n = 0 or 1 ; o is 0, 1, 2, 3 or 4;
X is C, NR7 O or S;
Y is C, NR8 O or S;
R, is either absent or hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl;
R2, R3 and R4 are independently hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, arylsulfonyl, substituted arylsulfonyl, carboxy, carbamoyl, substituted carbamoyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl;
each R5 is independently, alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkylthio, substituted alkylthio, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, arylalkyloxy, substituted arylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylsulfonyl, substituted arylsulfonyl, azido, carboxy, carbamoyl, substituted carbamoyl, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, dialkylamino, substituted dialkylamino, halo, heteroaryloxy, substituted heteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl, hydroxyl, nitro or thio; and
R7and R8 are independently absent, hydrogen, alkyl, substituted alkyl, acyl or substituted acyl.
135. The method of Claim 134, wherein the modulator is a compound of stmctural formula (XXXV) or (XXXVI):
136. The method of Claim 135, wherein R, is either absent or hydrogen, acyl, substituted acyl, acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylamino, substituted alkylamino, alkylarylamino, substituted alkylarylamino, arylamino, substituted arylamino, arylalkyloxy, substituted arylalkyloxy, carbamoyl, substituted carbamoyl, dialkylamino, substituted dialkylamino, heteroalkyl, or substituted heteroalkyl.
137. The method of Claim 134, wherein R, is either absent or acylamino, substituted acylamino, alkoxycarbonyl, substituted alkoxycarbonyl, arylamino substituted arylamino, or carbamoyl, substituted carbamoyl.
138. The method of Claim 134, wherein R, is either absent or acylamino, substituted acylamino, arylamino or substituted arylamino.
139. The method of Claim 134, wherein R2, R3 and R4 are independently alkyl, substituted alkyl, acyl, substituted acyl, acylamino, substituted acylamino, alkylamino, substituted alkylamino, alkoxycarbonyl, substituted alkoxycarbonyl, alkylarylamino, substituted alkylarylamino, aryl, substituted aryl, arylalkyl, substituted arylalkyl, arylamino, substituted arylamino, carbamoyl, substituted carbamoyl, dialkylamino, substituted dialkylamino, heteroaryl, substituted heteroaryl, heteroalkyl, or substituted heteroalkyl.
140. The method of Claim 134, wherein R , R3 and R are independently alkyl, substituted alkyl, acylamino, substituted acylamino, aryl, substituted aryl, arylamino, substituted arylamino, carbamoyl or substituted carbamoyl.
141. The method of Claim 134, wherein R2, R3 and R are independently alkyl, substituted acylamino, aryl, substituted arylamino or substituted carbamoyl.
142. The method of Claim 134, wherein each R5 is independently, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, amino, aryl, substituted aryl, azido, carboxy, carbamoyl, substituted carbamoyl, cyano, halo, hydroxyl, nitro or thio.
143. The method of Claim 134, wherein each R5 is independently, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, azido, carboxy, carbamoyl, substituted carbamoyl, cyano, halo, hydroxyl, nitro or thio.
144. The method of Claim 134, wherein R7and R8 are independently absent, hydrogen, alkyl.
145. The method of Claim 134, wherein the modulator is a compound of the formula:
146. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, or cardiovascular diseases in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXXI).
147. A method for treating or preventing ovarian cancer, peritoneal cancer, endometrial cancer, cervical cancer, breast cancer, colorectal cancer, uterine cancer, stomach cancer, small intestine cancer, thyroid cancer, lung cancer, kidney cancer, pancreas cancer, prostrate cancer, adult respiratory distress syndrome (ARDS), asthma, transcomeal freezing, cutaneous bums, ischemia or arthesclerosis in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXXI).
148. A method for treating or preventing vasoconstriction, autoimmune disorders or vascular occlusive disorders in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXXI).
149. A method for treating or preventing vasoconstriction in cerebral arteries, systemic lupus erythematosus (SLE), rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn's disease, Behcet's disease, chronic glomemlonephritis, chronic thrombocytopenic purpura, autoimmune hemolytic anemia, migraine headache, stroke, subarachnoid hemorrhage, or a vasospasm in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXXI).
150. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, cardiovascular diseases, vasoconstriction, autoimmune disorders or vascular occlusive disorders in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of stmctural formula (XXXI) and one or more agonists or antagonists of an Edg receptor.
151. A method for treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, cardiovascular diseases, vasoconstriction, autoimmune disorders or vascular occlusive disorders in a patient comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of structural formula (XXXI) and one or more drugs useful in treating or preventing cancers, acute lung diseases, acute inflammatory exacerbation of chronic lung diseases, surface epithelial cell injury, cardiovascular diseases, vasoconstriction, autoimmune disorders or vascular occlusive disorders.
152. The method of Claim 132 or 133, wherein the modulator is a compound of stmctural formula (XXXII):
(XXXII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,0)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-Cιo)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -(Cι-C,0)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5; -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C-C^alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl,
-(C3-C,o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,o)heteroaryl, -naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(Cι-Cιo)alkylNHC(O)(CH2)mR5, -(Cι-Cιo)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Re is independently -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(C1-C,0)alkyl, -O(C,-C10)alkyl, -C(O)(C,-Cιo)alkyl, -C(O)NH(CH2)m(C,-C,0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C,0)alkyl(C,- C,o)alkyl), -CO2(C,-C10)alkyl, -(C,-C,0)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C -C,0)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(Cs)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(C,-C,0)alkyl, or -SO2NH2; X is O, S, C(R5)(R5) or N(R5); Rj, R2 or R3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
153. The method of Claim 132 or 133 wherein the modulator is selected from the group consisting of:
154. The method of Claim 132 or 133, wherein the modulator is a compound of stmctural formula (XXXIII): (XXXIII)
or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2 and R3 is independently -H, -halo, -NO2, -CN, -C(R5)3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cι0)heteroaryl, -naphthyl, -(C -C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C,-C1o)alkylNHC(O)(CH2)mR5, -(Cι-C,0)alkylNR5R5> -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C10)alkynyl, -(C3-C,o)cycloalkyl, -(C8-Cι4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,0)heteroaryl, -naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(C,-Cιo)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5; -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and Rό is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(C,-C,o)alkyl(C1-Cιo)alkyl, -O(C,-C,o)alkyl, -C(O)(C,-C,o)alkyl, -C(O)NH(CH2)m(C,-C,o)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((C,-C10)alkyl(C,- C,o)alkyl), -CO2(C,-C,0)alkyl, -(C,-C,o)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,0)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)m(C,-C,0)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C,o)alkyl, -NHC(O)NH(C,-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(Cι-C,0)alkyl, or -SO2NH2;
X is O, S, or N(R5);
R,, R2 or R3 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two Rό groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
155. The method of Claim 132 or 133, wherein the modulator is a compound of stmctural formula (XXXIV):
(XXXIV) or a pharmaceutically available solvate or hydrate thereof, wherein;
each of R,, R2, R R4, R and R8 is independently -H, -halo, -NO2, -CN, -C(R )3, -(CH2)mOH, -N(R5)(R5), -O(CH2)mR5, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -OCF3, -benzyl, -CO2CH(R5)(R5), -(C,-C,0)alkyl, -(C2-Cι0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-Co)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-C,0)heteroaryl, -naphthyl, -(C3-Cι0)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -heterocylcoalkyl, -C(S)N(R5)(R5), -(C,-C1o)alkylNHC(O)(CH2)mR5, -(C,-C,o)alkylNR5R5, -OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
R3 is -H -C(R5)3, -(CH2)mOH, -C(O)R5, -C(O)NR5R5, -C(O)NH(CH2)m(R5), -benzyl, -CO2CH(R5)(R5), -(C-Co)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(C6)heteroaryl, -(C5-Cιo)heteroaryl, -naphthyl, -(C3-Cιo)heterocycle, -CO2(CH2)mR5, -N(OH)aryl, -NHC(O)R5, -NHC(O)OR5, -NHC(O)NHR5, -N=C(aryl), -heterocylcoalkyl, -(Cι-C,0)alkylNHC(O)(CH2)mR5, -(C1-C,o)alkylNR5R5,
-OC(O)(CH2)mCHR5R5, -CO2(CH2)mCHR5R5, -OC(O)OR5, -SR5, -S(O)R5, -S(O)2R5, -S(O)2NHR5, or
wherein; each R5 and R^ is independently -H, -halo, -NO2, -CN, -OH, -CO2H, -N(Cι-Cιo)alkyl(C,-Cιo)alkyl, -O(Cι-Cιo)alkyl, -C(O)(Cι-Cιo)alkyl, -C(O)NH(CH2)m(C,-Cι0)alkyl, -OCF3, -benzyl, -CO2(CH2)mCH((Cι-Cιo)alkyl(C,- C,0)alkyl), -CO2(C,-Cι0)alkyl, -(C,-C10)alkyl, -(C2-C,0)alkenyl, -(C2-C,0)alkynyl, -(C3-C,o)cycloalkyl, -(C8-C,4)bicycloalkyl, -(C5-C,o)cycloalkenyl, -(C5)heteroaryl, -(Cδ)heteroaryl, -phenyl, naphthyl, -(C3-C,0)heterocycle, -CO2(CH2)m(C,-C,o)alkyl, -CO2(CH2)mH, -NHC(O)(C,-C10)alkyl, -NHC(O)NH(C,-C,0)alkyl, -NH(aryl), -N=C(aryl), -OC(O)O(C,-C,o)alkyl, or -SO2NH2; X is O, S, or N(R5); R, and R2, R2 and R3, R3 and R4, R4 and R7> or R7 and R8 taken in combination can form one or more substituted or unsubstituted 5 or 6 membered cyclic or heterocyclic rings or a 6-membered aromatic ring; two Re groups on adjacent carbon atoms can together form a 5 or 6 membered cyclic or heterocyclic ring or a 6-membered aromatic ring; each m is independently an integer ranging from 0 to 8; and each p is independently an integer ranging from 0 to 5.
156. The method of Claim 132 or 133 wherein the modulator has is selected from the group consisting of:
319
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