AP353A - New peptides derivatives. - Google Patents
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- AP353A AP353A APAP/P/1992/000457A AP9200457A AP353A AP 353 A AP353 A AP 353A AP 9200457 A AP9200457 A AP 9200457A AP 353 A AP353 A AP 353A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
- C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
- C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
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- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/022—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2
- C07K5/0222—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2 with the first amino acid being heterocyclic, e.g. Pro, Trp
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention relates to new competitive inhibitors of
Description
Title: NEW PEPTIDES DERIVATIVES
Abstract The invention relates to new competitive inhibitors of thrombin, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic diseases, according to the formula,*
A61K 3//02 ¢0 tA
CO
Formula I
-(CH2)n-B wherein A represents a methylene group, an ethylene group or a propylene group, which may be substituted or Continued Overleaf
Documents cited: EP-4346078 EP-542525 A2 US-4713369 EP-530167 Al
EP-50J203 Al EP-235692 A2 US-49066’59 US-4395401 (56)
Inventors continued
RUTH ELVY BYLUND
Forellgatan 60
S-421 58 Vastra Frolunda
SWEDEN
Abstract Continued
A represents -CH2-O-CH2—# —CH2-S—CH2-» -CH2~SO-CH2~, or
A represents -CH2-O-, -CH2-S-, -CH2-SO-, with the heteroatom functionality in position 4, or n is an integer 2 to 6; and
B represents -N(R^)-C(NH)-NH21 wherein R^ is H or a methyl group, or
B represents -S-C(NH)-NH2, or -C(NH)-NH2·
Further described is new use in synthesis of pharmaceutical compounds of a compound of the formula;
NH
DESCRIPTION
This invention relates to new competitive inhibitors of thrombin, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, e.g. following angioplasty and coronary bypass operations.
The invention also relates to novel use of a compound as a starting material in synthesis of a serine protease inhibitor. Furthermore the invention relates to a novel structural fragment in a serine protease inhibitor.
BACKGROUND
Blood coagulation is the key process involved in both haemostasis (i.e. prevention of blood loss from a damaged vessel) and thrombosis (i.e. the pathological occlusion of a blood vessel by a blood clot) . Coagulation is the result of a complex series of enzymatic reactions, where one of the final steps is conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin plays a central role in coagulation. It activates platelets, it converts fibrinogen into fibrin monomers, which polymerise spontaneously into filaments, and it activates factor XIII, which in turn crosslinks the polymer to insoluble fibrin. Thrombin further activates factor V and factor VIII in a positive feedback reaction. Inhibitors of thrombin are therefore expected to be effective
BAD
ORIGINAL ft anticoagulants by inhibition of platelets, fibrin formation and fibrin stabilization. By inhibiting the positive feedback mechanism they are expected to excert inhibition early in the chain of events leading to coagulation and thrombosis.
PRIOR ART
Inhibitors of thrombin based on the amino acid sequence around the cleavage site for the fibrinogen Aa chain were first reported by Blomback et al in J. Clin. Lab. Invest. 24, suppl 107, 59, (1969), who suggested the sequence Phe-Val-Arg (P9-P2-P1, herein referred to as the P3-P2-P1 sequence) to be the best inhibitor.
In US 4,346,078 (Richter Gedeon Vegyeszeti Gyar R T, priority date 7.10.1980) and in Peptides 1983 by Walter de Gruyter ά Co, Berlin, pp 643-647, S. Bajusz et al described the thrombin inhibitor H-DPhe-Pro-Agm, a dipeptidyl derivative with an aminoalkyl guanidine in the Pl-position.
S. Bajusz et. al. also reported in J. Med. Chem. 1990, 33, 1729-1735 and in EP-A2-0,185,390 (Richter Gedeon Vegyeszeti Gyar R T) (priority date 21.12.84) that replacing the agmatine with an arginine aldehyde gave a thrombin inhibitor which had much higher potency.
The reason for the increased activity of this thrombin inhibitor is thought possibly to be due to interaction of the aldehyde function with the Ser-OH in the active site of the enzyme forming a hemiacetal. It is not concievable to have the same type of interaction in the dipetide derivative H-DPhe-Pro-Agm since it does not have an amino acid derivative with a carbonyl group in the Pl-position.
In other work in the thrombin inhibitor field, inhibitors of serine proteases that are based on electrophilic ketones instead of aldehydes in the Pl-position include the
Λ
BAD ORIGINAL f ollowing :
E. N. Shaw et al·. (Research Corporation) US-4,318,904 (priority date 25.04.80) describing peptide chloro-methyl ketones e.g. H-DPhe-Pro-Arg-Cr^Cl.
M. Szelke and D.M. Jones in EP-A1-0,118,280, (priority date 4.3.83) describing compounds derived from the P^ - ?2 ' pentapeptide sequence of the fibrinogen Aa chain in which the scissile P^ - Pj_' peptide bond was replaced with the -CO-Ch^-moiety, forming a keto isostere to the corresponding peptides .
M. Kolb et. al. (Merrell-Dow) EP-A2-0,195,212 (Priority date 4.2.85) describing peptidyl α-keto esters and amides.
B. Imperial! and R.H. Abeles, Biochemistry 1986. 25. 3760 describing peptidyl fluoroalkyl ketones.
D. Schirlin et al. (Merrell-Dow) EP-A1-0,362,002 (priority date 1.9.88) describing fluoroalkylamide ketones.
P. Bey et al., (Merrell-Dow) EP-A2-0,364,344 (priority date 1.9.88) describing α,β,δ- triketo compounds.
Ueda et al., Biochem. J. 1990, 265, 539 also describing peptidyl fluoroalkyl ketones.
Inhibitors of thrombin based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof have been reported by A.D Kettner et al. (Du Pont)
EP-A2-0,293,881 (priority dates 5.6.87 and 6.4.88).
An object of the present invention is to provide novel and potent thrombin inhibitors with competitive inhibitory activity towards their enzyme i.e. causing reversible inhibition. A further object is to obtain inhibitors which
BAD ORIGINAL are orally bioavailable and selective in inhibiting thrombin over other serine proteases. Stability, duration of action, and low toxicity at therapeutic dosages are still further objects of the invention.
DISCLOSURE OF THE INVENTION
Compounds
Compounds of the invention relate to the peptide sequence of human fibrinogen Aa chain representing modified sub-sites Pg, and P-j_:
P9
H-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-
-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-ValAccording to the invention it has been found that compounds of the general Formula I, either as such or in the form of physiologically acceptable salts, and including stereoisomers, are potent inhibitors of thrombin:
R3x
R2
Formula I
BAD ORIGINAL Q wherein :
A represents a methylene group, or 5
A represents an ethylene group and the resulting 5-membered ring may or may not carry one or two fluorine atoms, a hydroxy group or an oxo group in position 4, or may or may not be unsaturated, or 10
A represents -CH2-O-, -CH2-S-, -CH2-SO-, with the heteroatom functionality in position 4, or
A represents a n-propylene group and the resulting 6-membered ring may or may not carry in position 5 one fluorine atom, a hydroxy group or an oxo group, carry two fluorine atoms in one of positions 4 or 5 or be unsaturated in position 4 and 5, or carry in position 4 an alkyl group with 1 to 4 carbon atoms, or
A represents -CH2-O-CH2-, -CH2-S-CH2-, -CH2-SO-CH2-;
represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or R^^OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is H or an alkyl group having 1 to 4 carbon atoms or an alkylene group having 2-3 carbon atoms intramolecularly bound alpha to the carbonyl group in R1, or
R·*· represents R-^ooC-l, 4-phenyl-CH2-, where R1^ is h or an alkyl group having 1 to 4 carbon atoms, or
R1 represents R-^-NH-CO-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and where R13 is H or an alkyl group having 1 to 4 carbon atoms or -CH2COORI2 where R1^ lg as defined above, or
BAD ORIGINAL &
R1 represents R12COC-CH2-OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and where R is as defined above, or
Rx represents CH3SO2-/ or
R1 represents R^OCOCO- where R1^ £s as defined above, or
R1 represents -CH2PO(OR14)2, --CH2SO3H or
-CH2-(5-(IH)-tetrazolyl) where R14 is, individually at each occurrence, H, methyl or ethyl;
R3 represents H or an alkyl group having 1 to 4 carbon atoms or R21OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is possibly substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R22-(CH2)p-, wherein p = 0-2 and R22 is methyl, phenyl,
OH, COOR21, and R21 is H or an alkyl group having 1 to 4 carbon atoms.
/ m is 0, 1 or 2, R3 represents a cyclohexyl group and R4 represents H, or m is 1 and R3 represents a cyclohexyl or phenyl group and R4 forms an ethylene bridge together with R1, or m is 1 and R3 and R4 each represents a cyclohexyl or phenyl group;
R^ represents H or an alkyl group having 1 to 4 carbon atoms;
n is an integer 2 to 6; and
B represents -N(R6)-C(NH)-NH2, wherein R6 is H or a methyl group, or bad original $
B represents -S-C(NH)-ΝΗ2, or -C(NH)-NH2.
An alkyl group may be straight or branched unless specified otherwise. Alkyl groups having 1 to 4 carbon atoms are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl. When, unsaturation is referred to, a carboncarbon double bond is intended. Abbreviations are listed at the end of this specification.
According to a preferred embodiment the invention relates to compounds of Formula I, wherein represents R^^OOC-alky1-, where the alkyl group has 1 to 4 carbon atoms and R11 is H.
Of those compounds, the compounds where A is ethylene and R5 is H or an alkyl group having 1 to 4 carbon atoms, particularly those where R5 is H are preferred.
Of the compound of Formula I, those compounds where is cyclohexyl, m is 1 or 2, particularly m is 1 and R^ is H constitute another preferred subclass.
Another preferred group of compounds are the compounds where A is n-propylene and the resulting 6-membered ring may or may not carry in position 4 an alkyl group with 1 to 4 carbon atoms, and is H or an alkyl group having 1 to 4 carbon atoms, particularly those where R^ is H.
According to another preferred embodiment n is 3.
Compounds of Formula I having S-configuration on the a-amino acid in the P2-position are preferred ones, of those compounds also having R-configuration on the α-amino acid in the P3-position are particularly preferred ones.
Preferred compounds of the invention are:
BAD ORIGINAL $
Cexpound
7
8 9
13
17
18
21 22
23
27
0 28
33
H - i h) .ΓΟ*Agm .Ve - ί R' Cha - Pro - Agm
HO- (CH2 ) 3 ” '-R) Cha-Pro-Agm
HOOC-CH2-(R)Cha-Pro-Agm 1PrOOC-CH2- (R)Cha-Pro-Agm
HOOC-CH2-(Me)(R)Cha-Pro-Agm
HOOC-(R,S)CH(Me)-(R)Cha-Pro-Agm
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/a HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/b HOOC-(RorS)CH(nPr) - (R)Cha-Pro-Agm/a HOOC-(RorS)CH(nPr) -(R)Cha-Pro-Agm/b HOOC-(RorS)CH( Ph)-(R)Cha-Pro-Agm/b HOOC-(R,S)CH(CH2CH2Ph)-(R)Cha-Pro-Agm HOOC-(RorS)CH(CH2CH2Ph)-(R)Cha-Pro-Agm/a HOOC-CH2-CH2-(R)Cha-Pro-Agm EtOOC-CO-(R)Cha-Pro-Agm (R,S)Bla-(R)Cha-Pro-Agm
HOOC-(RorS)CH(CH2CH2Ph)-(R)Cha-Pro-Agm/b
H-(R)Cha-Pro-Nag nBu-(R)Cha-Pro-Nag
HO-(CH2)3-(R)Cha-Pro-Nag
HOOC-CH2-(R)Cha-Pro-Nag
EtOOC-CH2-(R)Cha-Pro-Nag 1PrOOC-CH2-(R)Cha-Pro-Nag tBuOOC-CH2-(R)Cha-Pro-Nag
HOCC-CH2-OOC-CH2-(R)Cha-Pro-Nag
H2N-CO-CH2-(R)Cha-Pro-Nag
HCOC-CH2-NH-CO-CH2-(R)Cha-Pro-Nag (HOOC-CH2)2 * (R) Cha-Pro-Nag
HOOC-CH2-(Me)(R)Cha-Pro-Nag
HOOC-CH2-(nBu)(R)Cha-Pro-Nag
HOOC-(R,S)CH(Me)- (R)Cha-Pro-Nag
HOOC-(RorS)CH(Me)- (R)Cha-Pro-Nag/a
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/b
EtOOC-(R,S)CH(Me)-(R)Cha-Pro-Nag
BAD ORIGINAL £
HOOC-(RorS)CH{nPr) -{R) Cha - Pro-Nag/a HOOC-(R)CH(CH2-CH)-(R)Cha-Pro-Nag HOOC-(R, S)CHI Ph)-(R)Cha-Pro-Nag HOOC-(S)CH(CH2CH9Ph)-R) Cha - Pro-Nag HOOC-(R)CH(CH2CH2Ph)- (R) Cha - Pro-Nag HOOC-CH2-CHo- (R)Cha - Pro-Nag RcOOC-CH2-CH2-(R)Cha-Pro-Nag HOOC-(CH2)3-(R)Cha-Pro-Nag EtOOC-(CH2)3-(R)Cha-Pro-Nag HOOC-CO-(R)Cha-Pro-Nag
MeOOC-CO-(R)Cha-Pro-Nag (R,S)Bla-(R)Cha-Pro-Nag
HOOC-(R,S)CH(CH2COOH)-'R) Cha-Pro-Nag MeOOC-(R,S)CH(CH2COOMe)- (R) Cha - Pro-Nag HOOC-Ph-4-CH2-(R)Cha-Pro-Nag (HO)2P(O)-CH2-(R)Cha-Pro-Nag
EtO(HO)P(0)-CH2-(R)Cha-Pro-Nag (EtO)2 P{O)-CH2-(R)Cha-Pro-Nag
HOOC-CH2-(R)Cha-Pro-Mag
H-(R, S)Pro(3-Ph)-Pro-Agm
H-(R,S)Pro(3-(trans)Ch)-Pro-Agm HOOC-CH2-(R,S) Pro (3 - (trans ) Ph)-Pro-Agm HOOC-CH2- (R, S) Pro ( 3 - (trans) Ph) -Pro-Nag HOOC-CH2-(R)Cha-Pic-Agm
HOOC-CH2-(Me)(R)Cha-(R,S)Pic-Agm HOOC-(R,S)CH(Me)-(R)Cha-Pic-Agm HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/a HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/b HOOC-CH2-CH2-(R)Cha-Pic-Agm H-(R)Cha-Pic-Nag
Me-(R)Cha-(R,S)Pic-Nag
HOOC-CH2-(R)Cha-Pic-Nag
MeOOC-CH2-(R)Cha-Pic-Nag 1PrOOC-CH2-(R)Cha-Pic-Nag
HOOC-CH2-(Me)(R)Cha-(RorS)Pic-Nag/b HOOC-(R, S)CH(Me)-(R)Cha-(R,S)Pic-Nag HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/c
BAD ORIGINAL fl
81 82
HOOC-(RorS)CH(Me)- (R)Cha-(RorS)Pic-Nag/d
HOOC-CH2-CH2-(R)Cha-Pic-Nag
HOOC-CH2-(R)Cha-(R,S)Mor-Agm
HOOC-CH2-(R)Cha-(RorS)Mor-Nag
H-(R)Cha-Aze-Nag
HOOC-CH2-(R)Cha-Aze-Nag
H-(R)Cha-Pro(5-(S)Me)-Nag
HOOC-CH2-(R)Cha-Pro(5-(S)Me)-Nag
HOOC-CH2-(R)Cha-(RorS)Pic(4,5-dehydro)-Nag/b HOOC-CH2-(R)Cha-Pic(4-(S)Me)-Nag HOOC-CH2-(R)Cha-(R)Pic(4-(R)Me)-Nag HOOC-CH2-(R)Cgl-Pic-Nag H-(R)Hoc-Pro-Nag
HOOC-CH2-(R)Hoc-Pro-Nag
HOOC-CH2-(R)Hoc-Pic-Nag
HOOC-CH2-(R)Dph-Pic-Nag
HOOC-CH2-(R)Dch-Pic-Nag
HOOC-CH2-(R)Cha-Pro(5-(R,S)Me)-Nag
H-(R)Cha-Pic(4-(R)Me)-Nag
HOOC-CH2-(R)Cha-Pic(4-(R)Me)-Nag
HOOC-CH2-(R)Cha-Pic(6-(S)Me)-Nag
Of those compounds, the compounds having Example Nos. 4, 6, 9, 22, 30, 34, 59, 63, 67, 73, 80 and 82 are particularly preferred, and of those the following compounds are most preferred:
0 HOOC-CH2- (Me) (R) Cha-Pro-Nag
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/b
HOOC-CH2-(R)Cha-Pic-Nag
In the above tables of compounds, the letters /a, /b, /c and /d refer to a substantially pure stereoisomer at the carbon atom denoted RorS. The stereoisomer canbe identified for
BAD ORIGINAL &
each compound with reference to the expenme R,S refers to a mixture of stereoisomers.
mbodiment the invention relates to a compound of the formula:
NH
Hoi·* n I’ll I - NH2 2 β · as a starting material in synthesis of a serine protease inhibitor, and in particular in synthesis of a thrombin inhibitor. It can be used as such or having the guanidino group either mono protected at the δ-nitrogen or diprctected at the δ-nitrogens or the γ, δ-nitrogens, preferably with a protective group such as benzyloxy carbonyl. Protection of the noragmatine derivatives is carried out by methods known in the art for guanidino compounds. This compound is named noragmatine or “Nag herein. The compound has been previously disclosed inter alia as a hair bleaching accelerator in GB 1,599,324 (Henkel, priority date 5.2.1977). The structural fragment of the formula
II
C—HN
NH
the noragmatine fragment renders a serine protease inhibitor, and in particular a thrombin inhibitor valuable.
BAD ORIGINAL ft
Medical and cr.ariraceutical use
In a further embodiment the invention relates to treatment, in a human or animal organism, of conditions where inhibition of thrombin is required. The compounds of the invention are expected to be useful in particular in animals including man in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. It is furthermore expected to be useful in situations where there is an undesirable excess of the thrombin without signes of hypercoagulability. Disease states in which the compounds have a potential utility, in treatment and/or prophylaxis, include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis. Further, the compounds have expected utility in prophylaxis of atherosclerotic diseases such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds are expected to be useful together with thrombolytics in thrombotic diseases, in particular myocardial infarction. Further, the compounds have expected utility in prophylaxis for re-occlusion after thrombolysis, percutaneous trans-luminal angioplasty (PTCA) and coronary bypass operations. Further, the compounds have expected utility in prevention of re-thrombosis after microsurgery. Further, the compounds * are expected to be useful in anticoagulant treatment in connection with artificial organs and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in haemodialysis and disseminated intravascular cpagulation.
A further expected utility is in rinsing of catheters and mechanical devises used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro.
BAD ORIGINAL ft
Pharmaceutical preparations
The compounds of- che Formula I will normally be administered by che oral, rectal, dermal, nasal or parenteral route in the form of pharmaceutical preparations comprising the active ingredient either as a free base or a pharmaceutical acceptable non-toxic acid addition salt, e.g. the hydrochloride, hydrobromide, lactate, acetate, citrate, ptoluenesulfonate, trifluoroacetate and the like in a pharmaceutically acceptable dosage form.
The dosage form may be a solid, semisolid or liquid preparation prepared by per se known techniques. Usually the active substance will constitute between 0.1 and 99 % by weight of the preparation, more specifically between 0.1 and 50 % by weight for preparations intended for parenteral administration and between 0.2 and 75 % by weight for preparations suitable for oral administration.
Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
Preparation
A further objective of the invention is the mode of preparation of the compounds. The compounds of Formula I may be prepared by coupling of an N-terminally protected amino acid or dipeptide or a preformed, N-terminally alkylated protected dipeptide to a compound
H2N-(CH2)n-X wherein n is as defined with Formula I and X is an unprotected or protected guanidino group or a protected amino
BAD ORIGINAL group, or a group transferable into an amino group, where the amino group is subsequently transferred into a guanidino group .
The coupling is accordingly done by one cf the following methods :
Method I
Coupling of an N-terminally protected dipeptide, prepared by standard peptide coupling, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula
BAD ORIGINAL wherein r\ R4 , R-*, n, m and A are as defined in Formula I,
Rb is H or alkyl, Wj is an amino protecting group such as tertiarybutoxy carbonyl and benzyloxy carbonyl and X is -MH-C(MH)NH?, -NH-C(NH)NH-W2, -N(W2)-C(NH)NH-W2,
-NH-C(NW?)MH-W2 or -NH-W2, where W2 is an amine protecting group such as tertiarybutoxy carbonyl or benzyloxy carbonyl, or X is a masked amino group such as azide, giving the protected peptide. The final compounds can be made in any of the following ways, depending on the nature of the X- group used: Removal of the protecting group(s) (when X=
-NH-C(MH)NH2, -N(W2)-C(NH)NH-W2, -NH-C(NW2)NH-W2 or NH-C(NH)NH-W2), or a selective deprotection of the Wj- group (e.g when X= -NH-C(NH)NH-W2,-N(W2)-C(NH)NH-W2, -NH-C(NW2)NHW2, W2 in this case must be orthogonal to wp followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/unmasking of the terminal alkylamino function (X= NH-W2 , W2 in this case must be orthogonal to or X= a masked aminogroup, such as azide) followed by a guanidation reaction, using standard methods, of the free amine and deprotection of the Wj_-group.
Method II
Coupling of an N-terminally protected amino acid, prepared by standard methods, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula
BAD ORIGINAL &
H2N-(CH2)n-X
R5
wherein W^, A , R^ and X are as defined above followed by deprotection of the W-j.-group and coupling with the N-terminal amino acid, in a protected form, leading to the protected peptide described in Method I or III, depending on the choice of the substitution pattern on the nitrogen of the N-terminal amino acid used in the coupling. The synthesis is then continued according to Method I or Method III to give the final peptides.
Method III
Coupling of a preformed N-terminally alkylated and protected dipeptide, prepared by standard peptide coupling, with either a protected or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked aminogroup at
BAD ORIGINAL ft the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula
wherein R^ , R3 , R^, R^, n, m, A and X are defined as above provided that R is other than H and W3 is an acyl protecting group such as trifluoroacyl.
The final compounds can be made in any of the following ways depending on the nature of the X-group used: Removal of protecting groups (when X = NH-C(NH)NH2, NH-C(NH)NH-W2,
N(w2)-C(NH)NH-W2, NH-C(NW2)NH-W2 or NH-W^ or a selective deprotection/unmasking of the terminal alkylamino function (X = NH-W2, W2 in this case must be orthogonal to W3 or X = a masked amino group such as azide) followed by a guanidation
BAD ORIGINAL ft deprotection of che W3 group.
DETAILED DESCRIPTION OF THE INVENTION
The following description is illustrative of aspects of the invention.
EXPERIMENTAL PART
Synthesis of the compounds of the invention is illustrated m Schemes I to VI appended hereto.
General Experimental Procedures.
The ^H NMR and 13C nmr measurements were performed on BRUKER AC-P 300 and BRUKER AM 500 spectrometers, the former operating at a frequency of 500.14 MHz and a 13C freguency of 125.76 MHz and the latter at ^H and -^C freguency of 300.13 MHz and 75.46 MHz respectively.
The samples were 10-50 mg dissolved in 0.6 ml of either of the following solvents; CDC13 (isotopic purity > 99.8%, Dr. Glaser AG Basel), CD^OD (isotopic purity > 99.95%, Dr. Glaser AG Basel) or D2O (isotopic purity > 99.98%, Dr. Glaser AG Basel) .
The 1H and ^3C chemical shift values in CDCI3 and CD3OD are relative to tetramethylsilane as an external standard. The ^H chemical shifts in D2O are relative to the sodium salt of 3 - (trimethylsilyl)-d4-propanoic acid and the 13C chemical shifts in D2O are referenced relative to 1,4-dioxane (67.3 ppm), both as external standard. Calibrating with an external standard may in some cases cause minor shift differences compared to an internal standard, however, the difference in 1H chemical shift is less than 0.02 ppm and in 13C less than 0.1 ppm.
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The “H NMR spectrum of peptide sequences containing a proline residue frequently exhibits two sets of resonances. This corresponds to the existence of to contributing conformers with respect to the rotation around the amide bond, where proline is the N-part of the amide bond. The conformers are named cis and trans. In our compounds the sequences (R)Cha-Pro- and -(R)Cha-Pic- often give rise to a cis-trans equilibrium with one conformer as the preponderant conformer (>90%). In those cases only the chemical shifts of the major rotamer is reported.
Thin-Layer Chromatography was carried out on commercial Merck Silicagel 60^254 coated glass or aluminium plates. Visualization was by a combination of UV-light, followed by spraying with a solution prepared by mixing 372 ml of EtOH(95%), 13.8 ml of concentrated H2SO4, 4.2 ml of concentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde or phosphomolybdic acid reagent (5-10 w.t % in EtOH(95%)) and heating.
Flash chromatography was carried out on Merck Silicagel 60 (40-63 mm, 230-400 mesh) under pressure of N2 ·
Reversed phase high-performance liquid chromatography (in the Examples referred to as RPLC) was performed on a Waters M-590 instrument equipped with three reverse phase Kromasil 100,C8 columns (Eka-Nobel) having different dimensions for analytical (4.6 mm x 250 mm), semipreparative (l x 250 mm) and preparative ( 2“ x 500 mm) chromatography detecting at 226 nm.
Freeze-drying was done on a Leybold-Heraeus, model Lyovac GT 2, apparatus.
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Protection Procedures
Boc-(R)Cha-OH
To a solution of H-(R)Cha-OH, 21.55 g (125.8 mmol;, in 130 mi 1 M NaOH and 65 ml THF was added 30 g (137.5 mmol) of (3oc)?0 and the mixture was stirred for 4.5 h at room temperature.
The THF was evaporated and an additional 150 ml of water was added. The alkaline aqueous phase was washed twice with EtOAc, then acidified with 2 M KHSO4 and extracted with 3 x 150 ml of EtOAc. The combined organic phase was washed with water, brine and dried (Na2SO4). Evaporation of the solvent afforded 30.9 g (90.5 %) of the title compound as a white solid.
Z-(R)Cha-OH
The same procedure as described in Bodanszky M. and Bodanszky
A. “ The Practice of Peptide Synthesis, Springer-Verlag,
1984, p. 12, was used starting from H-(R)Cha-OH.
BOC-(Me)Phe-OH
Prepared in the same way as Boc-(R)Cha-OH from Me-(R)Phe-OH.
Boc-(R,S)Pro (3-(trans) Ph)-OH
To a well stirred solution of 2.0 g (8.8 mmol, 1 eq.)
H-(R,S)Pro(3-(trans)Ph)-OH x HCl (Prepared as described in
J. Org. Chem., 55, p. 270-75 , 1990 and J. Org. Chem., 39, 1710-1716, 1974), in 17.6 ml of 1 N NaOH, 12 ml of H2O and 12 ml of THF at +5 °C was added 2.33 g (Boc)20 (10.7 mmol, 1.2 eq.). The reaction was allowed to reach room temperature and the stirring was continued for an additional 18 h. The organic solvent was evaporated and 50 ml of H2O was added to the residue. The basic water phase was washed with 2x50 ml of EtOAc and acidified with 2 M KHSO^ (pH about 1). The acidic
water phase was extracted with 4x75 ml of EtOAc and the combined organic phase was washed with 1x40 ml of H2O, 1x40 ml of brine and dried (MgSO4). Evaporation of the solvent gave 2.0 g (78 %) of pure product as a white solid.
1H-NMR (CDCI3, 500 MHz, mixture of two rotamers): δ 1.4 and
1.5 (2s, 9H) , 2.0-2.1 (m, IH) , 2.3-2.4 (m, IH), 3.45-3.88 (m, 3H), 4.3 and 4.45 (2d, IH), 7.2-7.4 (m, 5H).
Boc-(R,S)Pro(3-Ph)-OH
Prepared as above starting from a cis/trans mixture of H-(R,S)Pro(3-Ph)-OH.
BOC-(R)Dph-OH
Prepared according to the method described by K. Hsieh et.al. in J. Med. Chem., 32, p. 898 (1989) from H-(R)Dph-OH.
BOC-(R)Hop-OH
Prepared by the same procedure as described for Boc-(R)Cha-OH starting from H-(R)Hop-OH.
1H-NMR (300 MHz, CDC13): δ 1.45 (s, 9H), 2.00 (m, IH), 2.22 (m, IH), 2.75 (bt, 2H), 4.36 (bs, IH), 5.05 (bs, IH), 7.157.33 (m, 5H).
Deprotection Procedures.
(a) The protected peptide was dissolved in EtOH (95%) and hydrogenated over 5 % Pd/C at atmospheric pressure in the presence of an excess of TFA or HOAc (> 2 eq.) for about
1-4 h. The catalyst was filtered off, the solvent evaporated and the final peptide (TFA or HOAc salt) was isolated as a
BAD ORIGINAL ft white powder atter freeze drying (H2O) (b) The same as in (a) except that EtOH/H2O lca:5/l) was used as solvent.
(c) The same procedure as in (a) but MeOH was used as solvent.
(d) The same procedure as in (a) but 2 M HCl was used as acid to give the HCl-salt.
(e) Hydrolysis of esters, an illustrative example:
EtOOC-CH2-(R)Cha-Pro-Nag x 2 HOAc (0.4 mmol) was dissolved in
1.5 ml of MeOH and 1.2 ml (1.2 mmol) of 1M NaOH was added at room temperature. After 3 h the methanol was evaporated and an excess HOAc was added to the residue and the mixture was freeze dried and purified by RPLC (CH^CN/0.1 M NH^OAc (70/30)). The pure product was obtained as a powder in 73 % yield after freeze drying from water.
(f) Cleavage of t-butyl esters, an illustrative example:
The t-butyl ester was dissolved in an excess of TFA. After stirring for 2 h at room temperature the TFA was evaporated. Purification by treatment with activated charcoal in water-ethanol was followed by freeze drying from water giving the desired compounds.
Preparation of Starting Materials.
H-Pic-OEt x HCl
L-Pipecolinic acid, 4.0 g (0.031 mol), was slurried in 100 ml of abs. ethanol and HCl (g) was briefly bubbled through until a clear solution was obtained. It was cooled in an ice bath and 17 ml of thionyl chloride was added dropwise over 15 min.
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The ice bath was removed and the mixture was refluxed for 2.5 h. The solvent was evaporated and the product was obtained as its hydrochloride salt in a quantitative yield.
1H-NMR v 3 0 0 MHz, D2O) : 2.3-2.5 (m, IH), 3.1-3 δ 1.33 (t, 3H), 1.3-2.1 (m, 5H), (m, IH), 3.5-3.7 (m, IH), 4.14 (dd,
IH), 4.44 (q, 2H).
H-Pic-OMe x HC1
Prepared in the same way as described for H-Pic-OEt x HC1 by replacing EtOH with MeOH.
H-Aze-OEt X HCl
Prepared in the same way as described for H-Pic-OEt x HCl from H-Aze-OH.
Η-Pic (4- (S)Me)-OEt X HCl
Prepared in the same way as described for H-Pic-OEt x HCL from Η-Pic (4 - (S) Me)-OH (purchased from Synthelec, Lund, Sweden).
H-(R)Pic(4-(R)Me)-OEt x HCl
Prepared in the same way as described for H-Pic-OEt x HCl from H-(R)Pic(4-(R)Me)OH (purchased from Synthelec, Lund, Sweden).
H-(R)Dph-OH
Prepared by the general method given by A. Evans et. al. in JACS, 112, 4011 (1990) .
H-(R,S) Pic (4,5-dehydro)-OEt
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Η-(R,S) Pic (4,5-dehydro)-OH, 3.05 g (13.1 mmol) (Prepared according to the procedure by Burgstahler et. al. J.
Org. Chem, 25, 4, p. 489-92 (1960), was dissolved in 75 ml EtOH/HCl (saturated) and the mixture was refluxed for 5 hours. The solvent was evaporated and the remming residue was dissolved in water, made alkaline with sodium hydroxide (aq) and extracted three times with ethylacetate. Drying (Na2SO4) and carefull evaporation gave 2,05g (71%) of the title compound.
^H-NMR (CDC13): δ 1.28 (t, 3H), 1.88 (bs, NH) 2.2-2.4 (m, 2H), 3.45 (bs, 2H), 3.57 (dd, IH), 4.21 (q, 2H), 5.68-5.82 (m, 2H).
Boc-(R)Cgl-OH
Boc-(R) Pgl-OH was hydrogenated over 5% Rh/A^O-^ in MeOH at 5 Mpa. Filtration and evaporation of the solvent gave the title compound which was used without further purification.
1H-NMR (300 MHz,CDCl3): δ 0.9-1.7 (m, 20H), 4.0-4.2 (m, IH),
5.2 (d, IH).
BOC-(R)Dch-OH
Boc-(R)Dph-OH, 0.75 g (2.2 mmol), was dissolved in 25 ml of MeOH and a catalytic amount of 5% Rh/Al2O3 was added. The mixture was hydrogenated at 5 Mpa, 50°C for 40 h, filtered and evaporated to give 0.72 g (93%) of the thitle compound.
1H-NMR (CDC13) : δ 0.9-2.0 (m, 32H) , thereof 1.45 (bs, 9H) ,
4.55 (bd) and 4.9 (bd); two rotamers integrating for a total of IH, 5.7-6.1 (broad, NH).
H-{R)Pro(5-(S)Me)-OMe
Prepared according to the procedure given by B. Gopalan et.al. in J. Org. Chem., 51, 2405, (1986).
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H-Mor-OH
Prepared according to the method of K. Nakajima. et al. Bull. Chem. Soc. Jpn., 51 (5), 1577-78, 1978 and ibid 60,
2963-2965, 1937.
H-Mor-OEt X HCl
Prepared in the same way as H-Pic-OEt x HCl from H-Mor-OH.
Boc-(R)Cha-OSu
Boc-(R)Cha-OH (1 eq.), HOSu (1.1 eq) and DCC or CME-CDI (1.1 eq) were dissolved in aceconitrile (about 2.5 ml/mmol acid) and stirred at room temperature over night. The precipitate formed during the reaction was filtered off, the solvent evaporated and the product dried in vacuo. (When CME-CDI was used in the reaction the residue, after evaporation of the CH-jCN, was dissolved in EtO^c and the organic phase washed with water and dried. Evaporation of the solvent gave the title compound).
-’H-NMR (500 MHz, CDCl-j, 2 rotamers ca: 1:1 ratio) δ 0.85-1.1 (m, 2H), 1.1-1.48 (m, 4H), 1.5-1.98 (m, 16H; thereof 1.55 (bs, 9H)), 2.82 (bs, 4H), 4.72 (bs, IH, major rotamer), 4.85 (bs, IH, minor).
Boc-(Me)(R)Cha-OSu (i) Boc-(Me)(R)Cha-OH
A solution of 11,9 g (42.6 mmol) Boc-(Me)(R)Phe-OH in 150 ml MeOH was hydrogenated over 5% Rh/A^O^ at 0,28 Mpa for 24 h. Filtration of the catalyst and evaporation of the solvent gave the product as a white solid (95 % yield) wich was used in the next step without further purification.
BAD ORIGINAL ^H-NMR (500 MHz, CDCl-j, mixture of two rotamers ca: 1/' 1) . δ 0.3-1.1 (m, 2H) , 1.1-1.9 (m, 20H, thereof 1.47 and 1.45 (s, 3H)), 2.82 and 2.79 (s, total 3H) , 4.88 and 4.67 (m, total 1H! .
(ii) 3oc-(Me)(R)Cha-OSu
Prepared in the same way as described for Boc-(R)Cha-OSufrom Boc-(Me)(R)Cha-OH.
Boc-(R)Cha-Pro-OSu (i) Boc-(R)Cha-Pro-OH
H-(S)Pro-OH (680 mmol) was dissolved in 0.87M sodium hydroxide (750 ml). Boc-(R)Cha-OSu (170 mmol) dissolved in DMF (375 ml) was added dropwise during 20 min. The reaction mixture was stirred at room temperature for 20 h. The mixture was acidified (2M KHSO^) and extracted three times with ethyl acetate. The organic layers were combined and washed three times with water and once with brine. After drying over sodium sulphate and evaporation of the solvent, the syrupy oil was dissolved in diethyl ether, the solvent evaporated and finally the product dried in vacuo to yield
Boc-(R)Cha-Pro-OH as a white powder in almost quantitative yield.
1H-NMR (500 MHz, CDCl^, minor rotamer 10%) δ 0.8-1.05 (m,
2H), 1.05-1-55 (m, 15H; thereof 1.5 (bs, 9H)), 1.55-1.8 (m,
| 5H), 1.8-2.15 | (m, | 3H), 2.47 | (m, | IH), 3.48 (m, IH), 3.89 (m, | ||
| IH), 4. | 55 | (m, | 2H) | , 5.06 (m, | IH) | ; minor rotamer signals 2.27 |
| (m, IH) | , 3 | .58 | (m, | IH), 4.33 | (m, | IH), 5.0 (m, IH) |
(ii) Boc-(R)Cha-Pro-OSu
Prepared in the same way as described for Boc-(R)Cha-OSufrom Boc-(R)Cha-Pro-OH.
BAD ORIGINAL ^H-NMR (500 MHz, CDC13, 2 rotamers, 5:1 ratio) δ 0.78-1.05 (m, 2H), 1.05-1.83 (m, 20H; thereof 1.43 (bs, 9H)), 1.83-2.26 (m, 3H) , 2.32 (m, 1H) , 2.72-2.9 (m, 4H), 3.2 (m, IH, minor rotamer), 3.52 (m, IH, major), 3.68 (m, IH, minor rotamer),
3.89 (m, 1H, major), 4.31 (bq, IH, minor rotamer), 4.56 (bq,
IH, major), 4.71 (bt, IH, major rotamer), 4.93 (bt, IH, minor), 5.22 (bd, IH, major rotamer), 5.44 (bd, IH, minor).
Z-(R)Cha-Pro-OSu
Prepared in the same way as Boc-(R)Cha-Pro-OSu from Z-(R)Cha-OH.
Boc-(R)Cha-Pic-OSu (i) Boc-(R)Cha-Pic-OEt
Boc-(R)Cha-OH, 6.3 g (0.023 mol), was dissolved in 150 ml of 20 CH2CI2· The solution was cooled in an ice bath and 6.3 g (0.047 mol) of N-hydroxybenzotriazole and 11.2 g (0.0265 mol) of CME-CDI were added. The ice bath was removed after 15 min and the reaction mixture was stirred for 4 h at room temperature. The solvent was evaporated and the residue dissolved in
150 ml of DMF and cooled in an ice bath. H-Pic-OEtxHCl, 4.1 g (0.021 mol) was added and the pH adjusted to approximately 9 by addition of N-methylmorpholine. The ice bath was removed after 15 min and the reaction mixture was stirred for 3 days. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with dilute KHSO4 (aq), NaHCO3 (aq) and water. The organic layer was dried (Na2SO4) and evaporated to give 7.7 g (89 %) of Boc-(R)Cha-Pic-OEt which was used without further purification.
^H-NMR (500 MHz, CDC13, 2 rotamers, 3:1 ratio ) δ 0.7-1.0 (m, 2H), 1.1-1.9 (m, 29H; thereof 1.28 (t, 3H)), 1.45 (bs, 9H), 2.01 (bd, IH, major rotamer), 2.31 (bd, IH), 2.88 (bt, IH, ··»*» , ; BAD ORIGINAL minor ), 3.3 0 (bt, 1H, ma]or) , 3.80 (bd, 1H, ma jor), 4.15-4.3 (m, 2H) , 4.5-4.7 (m, 2H, minor), 4.77 (bq, IH, major), 4.90 (bd, IH, minor),· 5.28 (bd, 1H, major), 5.33 (bd,lH, major).
(ii) Boo-(R)Cha-Pic-OH
Boc-(R)Cha-Pic-OEt, 5.6 g (0.014 mol), was mixed with 100 ml of THE, 100 ml of water and 7 g of LiOH. The mixture was stirred at room temperature overnight. The THF was evaporated and the aqueous solution was acidified with KHSO4 (aq) and extracted three times with ethyl acetate. The combined organic phase was washed with water, dried (Na2SO4) and evaporated to give 4.9 g (94 %) of Boc-(R)Cha-Pic-OH which was used without further purification. The compound can be crystallized from diisopropyl ether/hexane.
1H-NMR (500 MHz, CDC13, 2 rotamers, 3.5:1 ratio) δ 0.8-1.1 (m, 2H), 1.1-2.1 (m, 27H; thereof 1.43 (s, 9H, major rotamer) , 1.46 (s, 9H, minor')), 2.33 (bd, IH) , 2.80 (bt, IH, minor), 3.33 (bt, IH, major), 3.85 (bd, IH, major), 4.57 (bd, IH, minor), 4.68 (m, IH, minor), 4.77 (bq, IH, major), 5.03 (bs, IH, minor), 5.33 (bd, IH, major), 5.56 (m, IH, major).
(iii) Boc-(R)Cha-Pic-OSu
Boc-(R)Cha-Pic-OH (1 g, 2.6 mmol) was dissolved in DMF (15 ml) at room temperature and then cooled to - 18°C, a temperature which was maintained during the additions of the reactants. Hydroxy succinimid (0.60 g, 5.2 mmol) was added and the reaction mixture was stirred for a few minutes until the crystals were dissolved. Dicyclohexyl carbodiimid (0.56 g, 2.7 mmol) dissolved in DMF (10 ml) and precooled was added dropwise to the rection mixture. After a few minutes at -18°C the reaction mixture was put into a water bath at 20°C for 2 h under stirring. The solvent was evaporated, ethyl acetate (40 ml) was added and the precipitated urea was filtered off.
BAD ORIGINAL A
The organic phase was washed once with water, twice with 0.3 M KHSO4, twice with diluted NaHCO-j, once with water, once with brine and dried (Na2SO4). The solvent was evaporated and the product dried in vacuo to yield 1.16 g (93%) of the product. According to ^H-NMR the product contained two diastereoisomers (epimers in Pic, S/R) in a ratio of 95/5.
^H-NMR (300 MHz, CDCl^, major diastereomer) δ 0.7-2.0 (m, 27H; thereof 1.46 (bs, 9H)) , 2.29 (bd, IH), 2.85 (bs, 4H),
3.40 (m, IH), 4.5-4.8 (m, IH) , 5.1-5.4 (m, IH), 5.70 (bd, LH, major).
Boc-(R)Cha-Mor-OSu
Prepared in the same way as Boc-(R)Cha-Pic-OSu from H-Mor-OEt x HCl except that CH^CN was used as solvent insted of DMF in the formation of the OSu-ester.
Boc-(Me)(R)Cha-Pro-OSu
Prepared in the same way as Boc-(R)Cha-Pro-OSu from Boc-(Me)(R)Cha-OH.
Boc-(Me)(R)Cha-Pic-OSu
Prepared in the same way as Boc-(R)Cha-Pic-OSu from
Boc-(Me)(R)Cha-OH.
Boc-(R,S)Pro(3-Ph)-Pro-OSu
Prepared in the same way as Boc-(R)Cha-Pro-OSu from
Boc-(R, S) Pro (3-Ph)-OH.
Boc-(R,S) Pro (3-(trans) Ph)-Pro-OSu (i) Boc-(R,S)Pro(3-(trans)Ph)-Pro-OBn
BAD ORIGINAL
To a slurry of 1.0 g of Boc-(R,S) Pro (3 - (trans) Ph)-OH (3.43 mmol, 1 eq.), 1.04 g of H-Pro-OBn x HCl (4.29 mmol, 1.25 eq.), 0.04 g of HOBt (0.24 mmol, 0.07 eq.) in 15 ml DMF was added 1.83 g of CME-CDI (4.29 nmol, 1.25 eq.) and 0.525 ml of MMM ( 4.73 mmol, 1.38 eq.) at room temperature. After stirring an additional 4 days the solvent was evaporated and the residue taken up in 200 ml EtOAc. The organic phase was washed with 2x40 ml of H2O, 2x25 ml of IM KHSO4, 2x25 ml of IM NaOH, 2x25 ml of H2O and dried (MgSO4). Evaporation of the solvent and flash chromathography (CH2Cl2/MeOH, 97/3) gave the pure product (44% yield) as a ca: 1:1 mixture of diastereomers.
(ii) Boc-(R,S)Pro (3 - (trans) Ph)-Pro-OH
The benzyl ester from the previous step was removed by hydrogenation over 5 % Pd/C in EtOH at atmospheric pressure for 4 h. Filtration and evaporation gave the pure product as a ca: 1:1 mixture of diastereomers in quantitative yield.
^H-NMR (CDCl3, 500 MHz, two diastereomers each consisting of two rotamers): δ 1.3-2.4 ( m + 4s from the Boc groups, total 14H), 2.5-2.9 (m, total IH), 3.2-3.9 <m, total 5H), 4.3-4.65 (m, total 2H), 7.2-7.5 (m, 5H).
(iii) Boc-(R,S)Pro (3- (trans) Ph)-Pro-OSu
Prepared according to the procedure described for Boc-(R)Cha-OSu from Boc-(R,S)Pro(3-(trans)Ph)-Pro-OH.
Boc-IR,S)Pro(3-(trans)Ch)-Pro-OSu (i) Boc-(R,S) Pro(3- (trans)Ch)-Pro-OH
Boc-(R,S)Pro(3-(trans)Ph)-Pro-OH was hydrogenated over 5 %
Rh/Al2O3 in methanol together with a small amount of HOAc for 7 days at 0,34 Mpa. Filtration of the catalyst, evaporation
of Che solvent and flash chromatograpy (CH2CI2/MeOH, 94/6) gave Che pure product as a white solid (mixture of two diastereomers).
iii) Boo - (R,S) Pro (3 - (trans )Ch)-Pro-OSu
Prepared according to the procedure described for
Boc-(R)Cha-OSu from Boc-(R,S)Pro(3-(trans)Ch)-Pro-OH.
Boc-(R)Hoc-Pro-OH (i) Boc-(R) Hoc-OH
Boc-(R)Hop-OH, 3.2 g (11.46 mmol) was dissolved in methanol (75 ml) . Rhodium on activated aluminium oxide (Rh/A^O-j), 0,5 g was added and the mixture stirred in hydrogen atmosphere at 0.41 MPa for 18 h. The catalyst was filtered off through celite and the solvent evaporated giving the product in almost quantitative yield.
1H-NMR (500 MHz, CDCI3): δ 0.90 (m, 2H), 1.08-1.33 (m, 6H), 1.43 (s, 9H) , 1.60-1.74 (m, 6H) , 1.88 (bs, IH) , 4.27 (bs,
IH) .
(ii) Boc-(R)Hoc-OSu
Prepared in the same way as described for Boc-(R)Cha-OSu from Boc-(R)Hoc-OH.
(iii) Boc-(RjHoc-Pro-OH
Prepared in the same way as described for Boc-(R)Cha-Pro-OH from Boc-(R)Hoc-OSu.
^•H-NMR (500 MHz, CDCI3): δ 0.80-0.94 (m, 2H) , 1.05-1.36 (m, 7H), 1.36-1.48 (bs, 9H), 1.48-1.78 (m, 7H), 1.98-2.14 (m,
2H) , 2.34 (m, IH) , 3.48 (m, IH), 3.85 (m, IH), 4.43 (m, IH) ,
BAD ORIGINAL ft
4.52 ( bd, IH), 5.26 (bd, IH), signals of a minor rotamer appears at: δ 1.92, 2.25, 3.58, 4.20 and 4.93.
Boc-(R)Hoc-Pic-OH (i) Boc-(R)Hoc-Pre-OMe
Prepared the same way as described for Boc-(R)Cha-Pic-OEt from Boc-(R)Hoc-OH and H-Pic-OMe x HCl.
(ii) Boc-(R)Hoc-Pic-OH
Prepared in the same way as described for Boc-(R)Cha-Pic-OH from Boc-(R)Hoc-Pic-GMe.
1H-NMR (500 MHz, CDC13): 5 0.82-0.97 (m, 2H) , 1.10-1.36 (m,
| 7H) , | 1.36-1.50 | (bs, 9H) | 1, 1.50-1.82 | (m, 11H) , 2.35 (bd, | IH) |
| 3.28 | (bt. IH), | 3.85 (bd, IH) 4,63 | (m, IH), 5.33 (bs, IH | ) , | |
| 5.44 | (bd, IH), | signals | of a minor | rotameter appears at: | δ |
| 1.88, | 2.80, 4. | 25, 4.55 | and 4.97. |
Boc-(R)Cha-Aze-OH
Prepared in the same way as described for Boc-(R)Cha-Pic-OH from H-Aze-OEt X HCL.
Boc-(R)Cha-Pic(4-(S)Me)-OH
Prepared in the same way as described for Boc-(R)Cha-Pic-OH from H-Pic(4-(S)Me)-OEt x HCl except that CH2CI2 was used as solvent.
BOC-(R)Cha-(R)Pic(4-(R)Me)-OSu (i) Boc-(R)Cha-(R)Pic(4-(R)Me)-OEt
Prepared in the same way as described for Boc-(R)Cha-Pic-OEt from H-(R)Pic(4-(R)Me)-OEt x HCl.
BAD ORIGINAL (ii) Boc-(R)Cha-(R)Pic(4-(R)Me)-OH
Prepared by using the deprotection (e) on the prduct (i) above .
(in) Boc - (R)Cha-(R) Pic (4 - (R)Me)OSu
Prepared in the same way as described for Boc-(R)Cha-Pic-OSu from Boc-(R)Cha-(R)Pic(4-(R)Me)-OH .
Boc-(R)Cha-(R,S)Pic(4,5-dehydro)-OH
Prepared according to the procedure described for Boc-(R)ChaPic-OH from H-(R,S)Pic(4,5-dehydro)-OEt.
Boc-(R)Cgl-Pic-OH (i) Boc-(R)Cgl-Pic-OMe
Pivaloyl chloride (1.000 mL, 8.1 mmol) was added to a solution of Boc-(R)Cgl-OH (2.086 g, 8.1 mmol) and triethyl amine (1.13 mL, 8.1 mmol) in toluene (25 mL) and DMF (5 mL) . A mixture of H-Pic-OMe x HCl (1.46 g, 8.1 mmol) and triethyl amine (1.13 mL, 8.1 mmol) in DMF (20 mL) was subsequently added at ice bath temperature. The reaction mixture was slowly allowed to warm up to room temperature and after 24 h it was diluted with water and extracted with toulene. After washing with 0.3 M KHSO4, 10% Na2CO3 and brine the solvent was removed in vacuo to give 2.52 g (81%) of colorless oil which was used without further purification.
'H-NMR (500 MHz, CDCI3, 2 rotamers, 5:1 ratio) δ 0.8-1.8 (m, 25H) , 2.25 (d, IH) , 2.75 (t, IH, minor rotamer) , 3.3 (t, IH) , 3.7 (s, 3H), 3.85 (d, IH), 4.3 (t, IH, minor rotamer), 4.54.6 (m, IH), 5.25 (d, IH), 5.30 (d, IH).
bad ORIGINAL (ii) Boc-ίR)Cgl-Pic-OH
Prepared according to the procedure for hydrolysis of Boc(R)Cha-Pic-OEt using the product from (i) above. The product was crystallized from di-isopropyl ether and hexane.
-'-H-NMR (500 MHz, CDCl^, 2 rotamers, 5:1 ratio) δ 0.8-1.8 (m, 25H) , 2.3 (d, IH) , 2.8 (t, IH, minor rotamer), 3.3 (t, IH) ,
3.9 (d, IH) , 4.4 (t, IH, minor), 4.5-4.6 (m, IH), 5.1 (s, IH, minor rotamer), 5.3 (d, IH), 5.40 (d, IH).
Boc-(R)Dph-Pic-OH
Prepared in the same way as described for Boc-(R)Cha-Pic-OH from Boc-(R)Dph-OH.
Boc-(R)Dch-Pic-OH
Prepared in the same way as described for Boc-(R)Cha-Pic-OH from Boc-(R)Dch-OH.
Boc-(R)Cha-Pro(5-(S)Me)-OH
Prepared in the same way as described for Boc-(R)Cha-Pic-OH from Η-Pro (5-(S) Me)-OMe.
Boc-Nag(Z) (i) N-Bensyloxycarbonyl-O-methyl isourea
To a stirred solution of concentrated aqueous NaOH (2.8 L,
50% w/w, 19.1 M, 53 mol) and water (32 L) at 18° C was added in two portions O-methylisourea hemisulphate (1.7 kg, 94%, 13.0 mol) and O-methylisourea hydrogensulphate (1.57 kg, 99%, 9.0 mol). The reaction mixture was cooled to 3-5° C. Benzyl chloroformiate (3.88 kg, 92%, 20.9 mol) was added over a 20 minutes period under cooling and vigorous stirring. The
BAD ORIGINAL ft reaction temperature went from 3 to 8° C during the addition of Z-Cl. The addition funnel was rinsed with 5 litres of water which was added to the reactor. The reaction mixture was stirred at 0-3° C for 18 h, filtered and 5 the crystals was washed with cooled (3° C) water (10 L). Vacuum drying 25° C, 10-20 mbar) for 48 h gave 3.87 kg (89%) of the title compound as a white crystalline powder.
(ii) Boc-Nag(Z)
To a stirred solution Boc-NH-(CH2)3-NH2 x HCl (prepared according to Mattingly P.G., Synthesis, 367 (1990)) (3.9 kg,
18.5 mol) in iso-propanol (24 kg) at 60-70° C was added in portions over a 30 minutes period KHCO3 (4.2 kg, 42 mol). A slow evolution of CO2 (g) occurs. The mixture was stirred for another 30 minutes followed by addition in portions over a 30 minutes period N-bensyloxycarbony1-O-methyl isourea (3.74 kg, 18.0 mol). The reaction mixture was stirred at 65-70° C for
16 h, cooled to 20° C and filtered. The precipitate was washed with iso-propanol (10 + 5 L). The combined filtrates was concentrated at reduced pressure keeping the heating mantle not warmer than 65-70° C. When approximately 45 litres was distilled off EtOAc (90 L) was added. The reaction mixture was cooled to 20-25° C, washed with water (10 and 5 L) and brine (5 L), and dried with
Na2SO4 (2 kg). After stirring the rection mixture was filtered and the filter cake was washed with EtOAc (11 and 7 L). The combined filtates were concentrated at reduced pressure keeping the heating mantle not wanner than
40-50° C. When approximately 90 litres of EtOAc was distilled off, toluene (25 L) was added and the evaporation continued. After collection of approximately another 18 litres of destiliate, toulene (20 L) was added under vigorous stirring and the resulting mixture was cooled to -1 to 0° C and gently stirred over night (17 h). The crystal slurry was filtered and the product was washed with cooled toluene (10 and 5 L).
BAD ORIG»nal
Vacuum drying (10-20 mbar,
40° C) for 24 h gave 4.83 kg (13.8 mol, 76%) of Boc-Nag(Z).
1H-NMR (300 MHz, CDC13>: δ 1.41 (s, 9H), 1.6-1.7 (m, 2H), 3.0-3.3 (m, 4H), 4.8-5.0 (bs, IH), 5.10 (s, 2H), 7.2-7.4 (m, 5H) .
Boc-Agm(Z) (i) Boc-Agm
To a slurry of 14.95 g (65.5 mmol, 1 eq.) of agmatine sulphate (Aldrich), 13.7 ml of Et3N (98.25 mmol, 1.5 eq.),
165 ml of H2O and 165 ml of THF was added 21.5 g (98.25 mmol, 1.5 eq.) of (Boc)20 during 5 minutes ac room temperature. The mixture was stirred vigorously over night, evaporated to dryness and the residue was washed wich 2x100 ml of Et2O to give Boc-Agm as a white powder which was used without further purification in the next step.
(ii) Boc-Agm(Z)
To a cold (+5°C) slurry of the crude Boc-Agm from the previous step (ca: 65.5 mmol) in 180 ml of 4N NaOH and 165 ml of THF was added 24 ml (169 mmol, 2.5 eq) of benzyl chloroformate during 10 minutes. After stirring at room temperature for 4 h methanol (150 ml) was added and the stirring was continued for an additional 20 h at room temperature. The organic solvent was evaporated and 200 ml of H2O was added to the residue. The basic water phase was extracted with 1x300 ml and 2x200 ml of EtOAc. The combined organic phases was washed with H2O (2x100ml), brine (1x100 ml) and dried (MgSO4). Evaporation of the solvent and flash chromathography (CH2Cl2/MeOH, a stepwise gradient of 97/3, 95/5 and 9/1 was used) gave 14.63 g (58%) of pure Boc-Agm(Z) as a white powder.
A
BAD ORIGINAL Cj} 1H-MMR (CDC13/ 500 MHz): δ 1.35-1.40 (m, 2H), 1.45 (s, 9H) ,
1.5-1.6 (m, 2H) , 3.0-3.2 (m, 4H) , 4.65 (bs, IH) , 5.1 (s, 2H) ,
7.25-7.40 (m, 5H).
i3C-?MR (CDC13, 75.5 MHz): δ 25.44, 27.36, 23.21, 65.33, 79.15, 127.47, 12/.66, 128.14, 137.29, 156.47, 161.48,
163.30.
Boc-NH-(CH2)3-N3
Prepared according co the method described by Mattingly P.
G., in Synthesis 1990, 367.
Z-NH-(CH2)2-NH2
To a cold solution of 6 g ethylene diamine (0.1 mol) and 22 ml triethyl amine in 20 ml of chloroform was added 2.5 g of Z-OSu dissolved in 5 ml of chloroform. The mixture was allowed to reach room temperature and left over night under stirring. Filtration, evaporation of the solvent and flash chromatography (C^C^/MeOH(NH3-saturated) , 95/5) gave 0.9 g (46 %) of the title compound.
1H-NMR (300 MHz, CDC13): δ 1.27 (s, 2H), 2.85 (t, 2H), 3.24 (q, 2H) , 5.14 (s, 2H), 7.22-7.40 (m, 5H) .
Agro x HCl
Prepared from Agm x H2SO4 (Aldrich) by exchanging the hydrogen sulphate ion for chloride on an ion exchange column.
H-Nag(Z) x 2 HCl
Prepared by bubbling HCl(e) into a solution of Boc-Nag(Z) in EtOAc followed by evaporation of the Solvent.
bad ORIGINAL
BnOOC-CH2-NH-CO-CH2-Br
To a solution of· p-TsOH x H-Gly-OBn (5 mmol) and triethyl amine (5 mmol) in 10 ml of CH2C12 was added 2-bromoacetic acid (5 mmol) dissolved in 10 ml of CH2C12 and dicyclohexyl carbodiimide (5 mmol). The mixture was stirred at room temperature over night and filtered. The organic phase was washed twice with 0.2 M KHSO4, 0.2 M NaOH, brine and dried. Evaporation and flash chromatography (CH2Cl2/MeOH, 95/5) gave a quantitative yield of the desired compound.
1H-NMR (300 MHz, CDC13) : 5 = 3.89 (s, 2H), 4.05-4.11 (d,
2H), 5.19 (s, 2H), 7.06 (bs, IH), 7.3-7.4 (m, 5H).
BnOOC-CH2-OCO-CH2-Br
A mixture of 2.8 g (0.020 mmol) bromoacetic acid, 4.2 g (0.020 mmol) of benzyl bromoacetate and 2.0 g (0.020 mmol) of trietylamine in 25 ml of EtOAc was refluxed for 3 h. It was diluted with more EtOAc and cooled. The solution was washed with dilute HCl and thereafter with NaHCO3(aq) and finally with water. Drying (Na2SO4) and evaporation followed by flash chromatography (heptane/etylacetate, 75/25) gave the title compound in 26 % yield.
1H-NMR (500 MHz, CDCl3): 6 3.95 (s, 2H) , 4.75 (S, 2H) , 5.23 (s, 2H), 7.35-7.45 (m, 5H).
BnO-(CH2)3-OTf
Propanediol monobenzyl ether (0.83 g, 5 mmol) was dissolved in dry pyridine (0.6 g, 7 mmol) and dichloromethane (20 ml) and cooled to -15°C. Triflic anhydride, precooled to -15°C, was added and the reaction mixture stirred for 45 min under which the temperature was allowed to rize to 15°C. The solvent was evaporated and the product dissolved in hexane/ethyl acetate 4:1 (10 ml) and filtered through silica.
BAD ORIGINAL
Finally the solvent was evaporated and the product dried in vacuo to yield 0.95 g (64%) of 1-benzyloxy
3-trifluoromethanesulfonylpropane which was used directly (see Example 21) .
1H-NMR (500 MHz, CDCl3): δ 2.12 (m, 2H), 3.6 (t, 2H), 4.51 (s, 2H), 4.72 (t,2H), 7.22-7.42 (m, 5H) .
BnO-(CH2)2-CHO
Prepared by Swern oxidation (described by D. Swern et al., J. Org. Chem., 1978, 2480-82) of BnO-(CH2)3-OH .
1H-NMR (300 MHz, CDCI3): δ 2.63 (dt, 2H), 3.80 (t, 2H), 4.51 (s, 2H), 7.30 (m, 5H), 9.76 (bt, IH).
Br-(S)CH(CH2OBn)-COOBa (i) Br-(S)CH(CH2OBn)-COOH
O-Benzylserine (3.9 g, 19 mmol) in water (10 ml) was added to a solution of sodium bromide (11 g, 107 mmol) in water (20 ml) and sulphuric acid (2 g, 20 mmol). The reaction mixture was cooled to -10°C and NaNO2 (1.73 g, 25 mmol) was added under vigorous stirring. Another portion of water was added to the thick mixture followed, after a few minutes, by H2SO4 (1 g, 10 mmol). The mixture was stirred at ambient temperature over night after which it was extracted twice with EtOAc (100ml) . The combined organic phase was washed twice with water and once with brine and dried (Na2SO4). Evaporation of the solvent gave 3.7 g (75%) of the title compound as a yellow oil which was pure enough to use directly in the next step.
(i i)Br-{S)CH(CH2OBn)-COOBn
To a solution of the crude product from (i) above (2.6 g, 10
BAD ORIGINAL ft mmol) in dry benzene (25 ml) was added oxalyl chloride (2.6 g, 20.5 mmoi) and molecular sieves (4 A, 1 g). The mixture was stirred at ambient temperature under an atmosphere of Argon for 18 h. The molecular sieves was removed by filtration and the solvent evaporated. The slightly yellow residue was dissolved in CH3CN (10 ml) and benzyl alcohol (1 g, 9.2 mmol) was added. The mixture was stirred at ambient temperature for 5 h. The solvent was evaporated and the residue dissolved in Et2<0 and washed once with 1 M NaOH, water, brine and dried (Na2SO4) Evaporation of the solvent followed by flash chromatography (CH2CI2/MeOH, 95/5) gave 1.3 g (67 %) of the desired compound.
1H-NMR (500 MHz, CDC13): δ 3.82 (dd, IH), 3.99 (dd, 1H) , 4.38 (dd, IH) , 4.56 (s, 2H), 5.23 (s, 2H) , 7.23-7.46 (m, 5H) .
Working Examples
Example 1
H-(R)Cha-Pro-Agm x 2 HOAc (i) Boc-(R)Cha-Pro-Agm x HOAc
Boc-(R)Cha-Pro-OSu (1.7 mmol) and agmatine dihydrochloride (2.0 mmol, 1.18 eq) was dissolved in DMF/^O 95:5 (35 ml). Triethyiamine was added to adjust the pH to about 10 and the solution was stirred at room temperature for 2 days. The solution was evaporated (5 mm Hg/ 60 °C) until dryness and the crude product was purified by RPLC (CH3CN/NH4OAc (0.1 M), 38:62). The desired compound was obtained as a white powder after freeze-dryiftfs 1H NMR (500 MHz, g©Gl3/DMSO-dg 5:2, Two rotamers, 9:1 δ (major rotafft^f) : 0.75-0.90 (m, 2H), 1.1-2.05 (m, 19H),
1.35 (s, 9H) 2s|S=1.14 (m, 4H), 3.37 (q,IH), 3.76 (m, IH),
4.20 (m,lH), 4,33 (dd, IH), 6.30 (d, IH), 7.05-7.80 (broad m,
BAD ORIGINAL Λ
5H) , 8.67 (broad d, IH) .
Exchange broadened signals of the minor rotamer are unambiguously observed at δ 3.44 (m, IH), 3.62 (m, IH), 4.10 (m,
IH) , 4.64 (m, IH) , 5.56 (d, IH), 9.08 (m, IH) .
(ii) H-(R)Cha-Pro-Agm x 2 HOAc
A solution of Boc-(R)Cha-Pro-Agm (0.2 mmol) in TFA (2ml) was stirred at room temperature for 4.5 h. The solvent was evaporated and the remaining oil was subjected to RPLC (CH^CN/NH^OAc (0.1 M) , 25:75). The diacetate salt was obtained as a white powder after repeated freeze-drying.
NMR (500.13 MHz, D2O): δ 0.80-0.95 (m, 2H), 1.00-1.21 (m, 3H) , 1.32 (m, IH), 1.40-1.78 (m,12H), 1.83-2.00 (m, 2H) , 1.90 (s, acetate), 2.20(m, IH) , 3.06-3.14(m. 4H), 3.50(m, IH), 3.67(m, IH), 4.20-4.30(m, 2H).
l^c nmr (75.6 MHz, D2O) : 9uanidine: δ 157.4; carbonyl carbons: δ 169.9, 174.5.
Example 2
Me-(R)Cha-Pro-Agm x 2 HOAc (i) Boc-(Me)(R)Cha-Pro-Agm
To a solution of 479.6 mg (1 mmol, 1 eq.) of
Boc-(Me) (R) Cha-Pro-OSu and 500 ml of NMM in 16 ml DMF/^O (15/1) was added 166.5 mg (1.2 mmol, 1.2 eq.) of Agm x HCl at room temperature. The reaction was stirred an additional 70 h and the solvent was evaporated to give a crude product as an oil. This was used without purification in the next step.
(ii) Me-(R)Cha-Pro-Agm x 2 HOAc
BAD ORIGINAL Q
The crude oil from the previous step was dissolved in 10 ml TFA/CH2C12 (1:4) at room temperature. After stirring for 2 h 25 min the solvent was evaporated and the crude product was purified with RPLC (CH3CN/NH4OAc(0.1M) , 35/65) to give the desired product as a white powder after freeze-drying.
1H-NMR (500 MHz, D2O): δ 0.93-1.05 (m, 2H), 1.10-1.29 (m,
3H), 1.33-1.43 (m, IH) , 1.50-1.80 (m, 12H), 1.88-2.10 (m, 2H,
1.92 (s, acetate), 2.27-2.36 (m, IH), 2.68 (s, 3H), 3.15-3.23 (m, 3H), 3.24-3.31 (m, IH) , 3.57-3.66 (m, IH) , 3.76-3.83 (m, IH), 4.28 (t, IH), 4.39 (dd, IH).
l^c-NMR (125.76 MHz, D2O): guanidine: δ 157.24; carbonyl carbons: δ 174.03, 168.24.
Example 3
HO-(CH2)3-(R)Cha-Pro-Agm x 2 HCl (i) Boc-(R)-Cha-Pro-Agm(Z)
Boc-Agm(Z) (1 eq) was dissolved in TFA/CH2C12 (1:4, ca: 6 ml/mmol) and stirred at room temperature for ca: 2 h. The solvent was evaporated and the product dissolved together with Boc-(R)Cha-Pro-OSu (1 eq) in DMF (ca: 1 ml/mmol), the pH was adjusted with NMM to ca: 9 and the mixture was stirred at room temperature for 20 h. The solvent was evaporated in vacuo, the crude product dissolved in CH2C12 and washed three times with water and once with brine. After drying (sodium sulphate) the solvent was evaporated and the product flash chromatographed (CH2Cl2/MeOH) affording Boc-(R)Cha-Pro-Agm(Z) as a white powder.
(ii) H-(R)Cha-Pro-Agm(Z)
Boc-(R)Cha-Pro-Agm(Z) was dissolved in TFA/CH2C12 (1:4, ca: 6 ml/mmol) and stirred at room temperature for 2 h. The solvent
BAD ORIGINAL was evaporated, the product dissolved in 0.2M NaOH (20 ml/mmol) and extracted twice with dichloromethane. The organic layers were combined and washed with brine, dried (sodium sulphate) and the solvent evaporated to yield H-(R)Cha-Pro-Agm(Z) as a white powder.
.( iii) BnO- (CH2 ) 3 - (R)Cha-Pro-Agm(Z)
H-(R)Cha-Pro-Agm(Z) (1 mmol) was dissolved in methanol (10 ml). Triethylammonium hydrochloride (lmmol), sodium cyanoborohydride (0.7 mmol) and thereafter BnO-(CH2)2-CHO (1.05 mmol) were added and the reaction mixture stirred at room temperature over night. The solvent was evaporated and the crude product was dissolved in ethyl acetate, washed twice with water, once with brine and dried over sodium sulphate. The solvent was evaporated and the crude product was purified by flash chromatography (EtOAc/MeOH).
(iv) HO-(CH2)3-(R)Cha-Pro-Agm x 2 HCI
Prepared by using deprotection procedure (d) on the product (iii) above.
^H-NMR (500 MHz, D2O) : δ 0.72 (m, minor rotamer), 0.84 (m, minor rotamer), 0.87-1.03 (m, 2H), 1.03-1-26 (m, 3H) ,
| 1.28-1.40 | (bs, | IH) , | 1.44 | -1.80 | (m, | UH) , | 1.80-1. | 95 (bs, | 3H) , |
| 1.95-2.10 | (bs, | 2H) , | 2.28 | (m, | IH) , | 3.04 | (m, IH), | 3.08-3 | .27 (m, |
| 5H), 3.58 | (bs, | IH) , | 3.67 | (bs, | 2H) | , 3.78 | (m, IH) | , 4.12 | (bd, |
minor rotamer), 4.30 (m,lH), 4.37 (m, IH).
13C-NMR (125 MHz, D2O): guanidine: δ 157.26; carbonyl carbons: δ 174.06, 168.36.
Example 4
HOOC-CH2-(R)Cha-Pro-Agm x HOAc
BAD ORIGINAL A
General Procedure for the alkylation of the N-terminal.
This procedure is described in more general terms and will be referred to in the Examples below together with the alkylating agent used in each specific Example.
The peptide to be alkylated (1 eq) and the alkylating agent (1.1-1.2 eq) were dissolved in acetonitrile (ca 10 ml/mmol). Potassium carbonate (2.0-2.2 eq) was added and the reaction mixture stirred at 50-60°C until the starting material was consumed (TLC, usually 1-5 h). Filtration, evaporation of the solvent and flash chromatography (CH2Cl2/MeOH,
CH2Cl2/MeOH(NH3-saturated) or EtOAc/MeOH, ca 9/1) gave the alkylated product after evaporation of the solvent.
(i) BnOOC-CH2-(R)Cha-Pro-Agm(Z)
Prepared from H-(R)Cha-Pro-Agm(Z) (See Example 3) and Br-CH2COOBn according to the procedure described above.
(ii) HOOC-CH2-(R)Cha-Pro-Agm x HOAc
Prepared by using the deprotection procedure (b) on the product (i) above.
1H-NMR (300 MHz, MeOD): δ 0.9-1.1 (m, 2H), 1.1-2.3 (m, 19H) 1.95 (s, acetate), 3.1-3.2 (m, 4H) , 3.2-3.65 (m, 3H), 3.85 (m, IH), 4.0 (bt, IH), 4.35 (dd, IH).
13C-NMR (75 MHz, D2O) : guanidine: δ 157.55; carbonyl carbons: δ 168.71, 171.37 and 174.3.
Example 5
Ipr-OOC-CHj-(R)Cha-Pro-Agm x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See
BADORIGINAL ft
Example 3) and Br-CH2COO1Pr followed by deprotection procedure (b) gave the title compound.
ΧΗ-ΝΜΚ (500 MHz, MeOD): δ 0.85-1.05 (m, 2H), 1.1-1.35 (m, 9H; thereof 1.23 (d, 3H), 1.25 (d, 3H) ) , 1.35-2.02 (m, 14H) 1.92 (s, acetate), 2.08 (m, IH), 2.2 (m, IH), 3.07-3.45 (m, cH) ,
3.55 (m, IH), 3.7-3.8 (m, 2H), 4.3 (dd, IH) , 5.05 (m, IH) .
^c-NMR (125 MHz, D2O) : guanidine: δ 157.39; carbonyl carbons: δ 171.10, 172.76 and 174.44.
Example 6
HOOC-CH2-(Me)(R)Cha-Pro-Agm x 2 TFA (i) Me-(R)Cha-Pro-Agm(Z)
Prepared from Boc-(Me)(R)Cha-Pro-OSu in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(Me)(R)Cha-Pro-Agm x 2 TFA
Alkylation as in Example 4 using Me-(R)Cha-Pro-Agm(Z) and Br-CH2COOBn followed by deprotection procedure (b) gave the title compound.
1H-NMR (300 MHz, D2O) : δ 0.9-1.35 (m, 6H) , 1.5-2.2 (m, 14H) ,
2.25-2.45 (m, IH), 3.12 (s, 3H), 3.15-3.35 (m, 4H), 3.6-3.75 (m, IH), 3.8-3.95 (m, IH), 4.22 (apparent bs, 2H), 4.45 (m, IH), 4.6 (bt, IH).
13C-NMR (75.47 MHz, D2O) : guanidine: δ 157.52; carbonyl carbons: δ 173.86, 168.79, 167.38.
BAD ORIGINAL &
Example 7
HOOC-(R,S)CH(Me)-(R)Cha-Pro-Agm x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) and Br-CH(Me)COOBn followed by deprotection procedure (a) gave the title compound as a mixture of two diastereomers.
Example 8
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/a x HOAc
Obtained by separating the diastereomers formed in Example 7 using RPLC (CH^CN/NH^OAc (0.1M), 1/4). This diastereomer came out first of the two from the column.
^H-NMR (500 MHz, D2O; 2 rotamers ca: 5:1 ratio): δ 0.74 (m,
| minor rotamer), | 1.01 (m, | 2H) , | 1.10-1.33 (m, 3H) | , 1.48- 1.88 |
| (m, 15H; thereof | 1.51 (d, | 3H) | ), 1.92-2.12 (m, | 3H) 1.96 (s, |
| acetate), 2.30 ( | m, IH), 3 | .20 | (m, 3H), 3.38 (m, | IH), 3.47 (q, |
| minor rotamer), | 3.53-3.68 | (m, | 2H), 3.73 (m, IH) | , 4.20 (d, |
| minor rotamer), | 4.33 (m, | IH) , | 4.38 (m, IH), 4.51 (d, minor |
rotamer).
13C-NMR (125 MHz, D2O): guanidine: δ 157.38; carbonyl carbons: δ 174.11, 173.45, 168.64.
Example 9
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/b x HOAc
The diastereomer that came out after the first one from the column in the separation in Example 8 is the title compound above.
^H-NMR (500 MHz, D2O, 2 rotamers ca 9:1 ratio): δ 0.88 (m,
BAD ORIGINAL <£ minor rotamer), 1.05 (m, 2H), 1.12-1.33 ;m, 3H), 1.42 (bs,
IH) , 1.50-1.88 (m, 15H; thereof 1.55 (d, 3H)), 1.93- 2.13 (m, 3H) 1.95 (s, acetate), 2.30 (m, IH), 2.40 (m, minor rotamer),
3.22 tt, 2H), 3.28 (t, 2H), 3.64 (m, IH), 3.70 (q, IH), 3.93 (t, minor rotamer), 4.35 (t, IH), 4.41 (id, IH).
Example 10
HOOC-(RorS)CH(nPr)-(R)Cha-Pro-Agm/a x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) and Br-CH(nPr)COOEt and deprocection procedure (e) followed by deprotection procedure (b) gave
HOOC-(R,S)CH(nPr)- (R)Cha-Pro-Agm. The title compound was obtained by separating the diastereomers by RPLC (CH-jCN/NH^OAc (0.1 Μ) , 1/4) and freeze drying (H2O) after evaportion of the solvent. This diastereomer came out first of the two from the column.
| 1H-NMR (300 MHz, MeOD): | δ 0 | .8-1.1 (m, 5H; | thereof | 0.92 | (t, |
| 3H) ) , 1.1-2.1 (m, 22H) 1 | .95 | (s, acetate), | 2.2 (m, | IH) , | |
| 3.1-3.35 (m, 5H), 3.48 | (m, | IH), 3.88 (m, | IH), 4. | 0 (m, | IH) |
4.4 (dd, IH).
13C-NMR (75 MHz, D2O): guanidine: δ 157.50; carbonyl carbons: δ 168.55 and 174.16.
Example 11
HOOC-(RorS)CH(nPr)-(R)Cha-Pro-Agm/b x HOAc
The other diastereomer from the separation in Example 10 which came out after the first one from the column is the title compound above.
•H-NMR (500 MHz, MeOD) : δ 0.85-1.05 (m, 5H; thereof 0.95 (t, 3H) ) 1.1-2.08 (m, 22H) 1.9 (s, acetate), 2.14 (m, IH),
BAD ORIGINAL &
3.1-3.4 (m, 5H), 3.45 (m, IH), 3.62 (m, IH), 3.80 (m, 1H),
4.34 (dd, IH).
♦13C-NMR (75 MHz, D2O): guanidine: δ 157.53; carbonyl carbons: 5 δ 169.01 and 174.27.
Example 12
HOOC-(RorS)CH(Ph)-(R)Cha-Pro-Agm/b x HOAc (i) t'BuOOC- (RorS)CH(Ph) - (R)Cha-Pro-Agm(Z)
A mixture of H-(R)Cha-Pro-Agm(Z) (See Example 3) (0.55 mmol), tert. butyl-(R,S) phenyl bromoacetate (0.66 mmol), K2CO3 (1.4 mmol) in CH-jCN (10 ml) was stirred at room temperature for 28 h and an additional 5 h at 60° C . The diastereomeric mixture (ca: 3:1, according to NMR) was filtered and evaporated. The remaining oil was twice subjected to flash chromatography (CH2Cl2/MeOH, 92/8), which resulted in a complete separation of the two diastereomers (R^=0.36 (minor isomer) and 0.27 (major isomer), respectively).
•'H NMR of major isomer (500.13 MHz, CDCl^): δ 0.79 (quart,IH), 0.90 (quart,IH), 1.06-1.70 (m, Η), 1.37 (s,9H),
1.85-2.03 (m,3H), 2.20 (m,IH), 3.10-3.24 (m,3H), 3.25-3.38 (m,2H), 3.42 (m,lH), 3.53 (m,lH), 4.30 (s,lH), 4.49 (dd,lH), 1 5.08 (s,2H), 7.19-7.40 (m,10H); broad NH signals are observed • in the region 6.7-8.6.
I {
(ii) HOOC-(RorS)CH(Ph)-(R)Cha-Pro-Agm/b x HOAc
The major isomer (50 mmol) and thioanisole (0.5 mmol) dissolved in TFA was kept at room temperature for 8 h. After evaporation (0.1 mm Hg) for 5 h, the remaining oil was . 35 purified on RPLC (CH3CN/NH4OAc (0.1 Μ), 2:3) to give the ΐ title compound after evaporation of the solvent and ♦
freeze-drying.
BAD ORIGINAL &
rH NMR (500.13 MHz, MeOD) : δ 0.85-1.01 (m, 2H) , 1.13-1.38
| (m, 4H), 1.53-2.05 (m, 14H), 1.92 (s, | acetate) | 2.18 | (m, | 1H) , |
| 3.08-3.26 (m, 3H), 3.32-3.45 (m, 2H), | 3.64 (m, | 1H) , | 3 . 93 | (t, |
| 1H), 4.37 (dd, 1H), 4.43 (5,1H), 7.28- | 7.50 (m, | 5H) . | ||
| 1-3C NMR (125.6 MHz, MeOD) : guanidine: | δ 158.7; | carbonyl | ||
| carbons: δ 173.8, 174.7, 177.0. | ||||
| Example 13 | ||||
| HOOC-(R,S)CH(CH2CH2Ph)-(R)Cha-Pro-Agm | X HOAC |
Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) and Br-CH(CH2-CH2-Ph)COOEt and deprotection procedure (a) followed by deprotection procedure (e) gave HOOC-(R,S)CH(CH2-CH2-Ph)-(R)Cha-Pro-Agm.
Example 14
HOOC- (RorS)CH(CH2CH2Ph) - (R)Cha-Pro-Agm/a x 2 TFA
The title compound was obtained by separating the diastereomers obtained in Example 13 by RPLC (CH3CN/NH4OAc (0.1 Μ), 2/3) and freeze drying (H2O/TFA) after evaportion of the solvent. This diastereomer came out first of the two from
| the column is | the | title | compound | above. | |
| 1H-NMR (500 MHz, MeOD): | δ 0.93-1 | .11 (m, 2H), 1.24 (m | , 1H) , | ||
| 1.29-1.40 (m, | 2H) , | 1.52 | -1.85 (m, | 11H), 1.89-2.11 (m, | 4H) , |
| 2.14-2.32 (m, | 3H.) , | 2.83 | (t, 2H), | 3.14 (t, 2H), 3.24 | (t, 2H), |
| 3.50 (q, 1H), | 3 .70 | (m, | 1H), 4.00 | (t, 1H), 4.36-4.42 | (m, 2H), |
| 7.17-7.31 (m, | 5H) . |
. 13C-NMR (125 MHz, MeOD): guanidine: δ 158.66; carbonyl ί 35 carbons: δ 168.08, 171.53, 174.16.
i t
Example 15 ί
< X *
BAD ORIGINAL fi
HOOC-CH2-CH2-(R)Cha-Pro-Agm x HOAc (ι) 3nOOC-CH2-CH2-(R)Cha-Pro-Agm(Z)
Benzyl acrylate (1.1 eq) and H-(R)Cha-Pro-Agm(Z) tSee Example
3) (1 eq) were dissolved in ethanol (20 ml/mmol) and stirred at room temperature for 20 h. The solvent was evaporated and
| the crude product purified by flash chromatography | |||||
| (CH2Cl2/MeOH(NH3-saturated) evaporated and the product | , 95/5) . dried in | Finally vacuo . | the | solvent was | |
| 1H-NMR (500 MHz, CDCl3): δ | 0.7-0.95 | (m, 2H), | . 1. | 0-1.5 (m, | |
| 10H) , 1.5-1.75 (m, 5H) , 1. | 75-1.92 ( | m, 2H), | 2 . 0 | (m, IH) , | 2 .17 |
| (bs, IH), 2.45 (m, 2H), 2.63 (m, IH) | , 2.79 | (m, | IH), 2.97 | -3.25 | |
| (m, 4H), 3.33 (m, 2H) , 3.52 | (bt, IH) | , 4.45 | (bd, | IH) , | |
| 4.95-5.12 (m, 4H), 7.13-7.4 | (m, 10H) | . |
(ii) HOOC-CH2-CH2-(R)Cha-Pro-Agm x HOAc
Prepared by using the deprotection procedure (a) on the product (i) above.
1H-NMR (500 MHz, D2O) : δ 0.88 (m, 2H), 1.00-1.23 (m, 3H) ,
1.33 (bs, IH), 1.42- 1.72 (m, 11H), 1.78- 2.00 (m, 3H) 1.94 (s, acetate), 2.18 (m, IH), 2.52 (m, 2H), 3.03-3.20 (m, 6H) ,
3.50 (m, IH), 3.72 (m, IH), 4.23 (m, IH), 4.30 (m, IH).
13C-NMR (125 MHz, D2O): guanidine: δ 157.25; carbonyl carbons: δ 178.07, 173.96, 168.24.
Example 16
EtOOC-CO-(R)Cha-Pro-Agm x HOAc (i) EtOOC-CO-(R)Cha-Pro-Agm(Z)
To a cold (-10° C) solution of H-(R)Cha-Pro-Agm(Z) (See
BAD ORIGINAL ft
Example 3) (0.46 g, 0.89 mmol) and MMM (199 mg, 1.97 mmol) m ml of THF was added Cl-COCOOEt (134 mg, 0.98 mmol) dissolved in 3 ml of THF. The mixture was keept at -10° C for one hour after which it was stirred at room temperature for another hour. The solvent was evaporated and the residue was dissolved in ethyl acetate. The organic phase was washed twice with water and dried (Na2SO4). Evaporation of the solvent and crystallization from EtOAc gave 0.275 g (50%) of the title compound as white crystals.
(ii) EtOOC-CO-(R)Cha-Pro-Agm x HOAc
Prepared by using the deprotection procedure (b) on the product (i) above.
1H-NMR (300 MHz, MeOD): δ 0.9-2.25 (m, 24H; thereof 1.17 (t, 3H)) 1.90 (s, acetate), 3.1-3.25 (m, 4H), 3.5-3.65 (m, 3H; thereof 3.59 (q,2H)), 3.88 (m, IH) , 4.35 (m, IH), 4.69 (dd,
IH) .
l^c-NMR (75.5 MHz, MeOD): guanidine: δ 157.56 and carbonyl carbons: δ 159.21, 160.74, 172.81, 174.56.
Examp1e 17 25 (R,S)Bla-(R)Cha-Pro-Agm x 2 TFA
Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) and α-bromo butyrolacton followed by deprotection procedure (a) gave the title compound as a mixture of two diastereomers.
1H-NMR (500 MHz, D2O, mixture of diastereomers ca: 1/1): δ
| 0.93- 1.06 | (m, | 2H), 1.09-1.30 | (m, 3H), 1.37-1.49 | (m, IH), | |
| 35 1.50-1.87 | (m, | 11H), 1.89-2.10 | (m, 3H), 2.24-2 | .36 | (m, IH), |
| 2.44-2.56 | (m, | IH), 2.72-2.85 | (m, IH), 3.10-3. | 30 | (m, 4H), |
| 3.56-3.65 | (m, | IH), 3.75-3.84 | (m, IH), 4.2-5.0 | (m | , 5H, |
bad original $ partially hidden by the H-O-D signal).
*^C-NMR ¢125.76.MHz, /0) guanidine: δ 157.34 (peaks overlapping); carbonyl carbons: δ 174.34, 173.90, 173.62,
167.88, 167.58 (two peaks are overlapping).
Example 18
HOOC-(RorS)CH(CH2CH2Ph)-(R)Cha-Pro-Agm/b x 2 TFA
The title compound was obtained by treating the diastereomer in Example 13 by the same way as described in Example 14. This diastereomer came out after the first one from the
| column . | ||||
| LH-NMR | (500 MHz, MeOD): | δ 0.95-1.06 | (m, 2H), 1.14-1.40 (m, | |
| 4H), 1. | 48-1.84 (m, | 11H) | , 1.87-2.30 | (m, 6H), 2.72-2.90 (m, |
| 2H), 3. | 12-3.32 (m, | 4H) , | 3.52 (m, IH) | 1, 3.72 (m, IH) , 4.04 (dd, |
| IH), 4. | 27 (t, IH), | 4.37 | (dd, IH), 7, | .17-7.32 (m, 5H). |
| 13c-nmr | (125 MHz, | MeOD) | : guanidine: | δ 158.68; carbonyl |
| carbons | : δ 168.14, | 171. | 46, 174.03. |
Example 19 25
H-(R)Cha-Pro-Nag x 2 HOAc (i) Z-(R)Cha-Pro-NH-(CH2)3-NH(Boc)
To a solution of Z-(R)Cha-Pro-OSu (1 mmol) in 1 ml of DMF at 0 °C was added H2N-(CH2)3-NH(Boc) (See Preparation of starting material) dissolved in 1 ml of DMF and the pH was adjusted to ca: 9 with NMM. The reaction was stirred at room temperature for 3 days after wich it was poured out on water.
The aqueous phase was extracted four times with EtOAc. The combined organic phase was washed twice with 0.3 M KHSO4, 0.2 M NaOH, brine and dried. Evaporation and flash
BAD ORIGINAL chromachography (EtOAc/ petroleum ether, 4/1) gave the title compond in 59 % yield.
(ιι) Z - ( P.) Cha-Pro-NH-(CH2 ) 3-NH2
Z-(R)Cha-Pro-NH-(GH2)3-NH(Boc) (0.6 mmol) was dissolved in CH2C12 (8 ml). TFA (2 ml) was added and the reaction mixture was stirred for 1 h. The solvent was evaporated and the residue was dissolved in CH2C12, washed twice with 0.2 M NaOH and dried (Na2SO4). Evaporation of the solvent gave che amine in 93 % yield.
'•H-NMR (500 MHz,CDC13): δ 0.79-1.03 (m, 2H) , 1.05-1.75 (m, 15H), 1.84-2.08 (m, 4H), 2.36 (m, IH), 2.66 (m, 2H), 3.25 (m, 2H) , 3.43 (q, IH), 3.85 (m, IH), 4.45 (m, IH), 4.56 (d, IH) 5.09 (m, 2H), 5.35 (d, IH), 7.30-7.45 (m, 5H).
(iii) Z-(R)Cha-Pro-Nag x HOAc
Z-(R)Cha-Pro-NH-(CH2)3-NH2 (0.55 mmol, 1 eq) was dissolved in DMF (2 ml) and the pH adjusted with triethylamine to 8-9.
3,5-Dimethyl-l-pyrazolylformamidinium nitrate (0.55 mmol, 1 eq) dissolved in DMF (1 ml) was added and the reaction mixture stirred at room temperature for three days. The solvent was evaporated, the crude product freeze-dried (H2O) and purified with RPLC (CH-jCN/NH^OAc (0.1M), 4/6) to give the title compound in 93 % yield after evaporation of the solvent and freeze-drying (H2O).
(iv) H-(R)Cha-Pro-Nag x 2 HOAc
Prepared by using the deprqfeection procedure (a) on the product (iii) above.
^H-NMR (500 MHz, D2O) : δ 0.82-1.03 (m, 2H) , 1.03-1.28 (m, 3H)
1.35 (m, IH), 1.53-1.82 (m, 9H), 1.82-2.05 (m, 3H) 1.89 (s, acetate), 2.24 (m, IH), 3.15 (t, 2H), 3.23 (q, 2H), 3.55 (m,
BAD ORIGINAL ft
IH), 3.72 (m, IH), 4.27-4.34 <m, 2H).
i3C-NMR (125 MHz, D2O): guanidine: δ 157.37; carbonyl carbons: δ 169.31, 174.52.
Example 20 nBu-(R)Cha-Pro-Nag x 2 HOAc (i) H-(R)Cha-Pro-Nag(Z)
Prepared from Boc-(R)Cha-Pro-OSu and Boc-Nag(Z) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
1H-NMR (500 MHz, CDC13): δ 0.8-1.03 (m, 2H), 1.10-1.50 (m,
6H) , 1.60-1.83 (m, 8H) , 1.87-2.20 (m, 3H) , 3.15 (m, IH) , 3.25 (m, 2H) , 3.42 (m, 2H) , 3.63 (dd, IH) , 3.70 (m, IH), 4.36 (bs, IH), 5.07 (s, 2H), 7.22-7.43 (m, 5H).
(ii) nBu-(R)Cha-Pro-Nag(Z)
H-(R)Cha-Pro-Nag(Z) (0.5 g, 1 mmol) was dissolved in methanol (10 ml). Triethylammonium hydrochloride (0.1 g, lmmol), sodium cyanoborohydride (44 mg, 0.7 mmol) and thereafter butyric aldehyde (76 mg, 1.05 mmol) were added and the reaction mixture stirred at room temperature for 20 h. The solvent was evaporated and the crude product was dissolved in ethyl acetate, washed twice with water, once with brine and dried over sodium sulphate. The solvent was evaporated and the crude product was purified by flash chromatography (EtOAc/EtOH/Et^N, 88/10/2) . Finally the solvent was evaporated and the product dried in vacuo to yield 0.22 g (40 %) of nBu-(R)Cha-Pro-Nag(Z).
1H-NMR (500 MHz, CDCI3): δ 0.82-1.0 (m, 5H; thereof 0.88 (t, 3H)), 1.08-1.49(m, 10H), 1.58-1.8 (m, 7H), 1.88-2.22 (m,
3H), 2.4 (m, IH), 2.5 (m, IH), 3.05 (bs, IH), 3.3 (m, IH) ,
BAD ORIGINAL
3.4-3.23 (m, 3H), 3.73 (m, IH), 4.42 (bs, IH), 5.1 (s, 2H) ,
7.25-7.43 (m, 5H).
(iii) --3u-(R)Cha-Pro-Nag x 2 HOAc 5
Prepared by using the deprotection procedure (a) on the product (ii) above.
1H-NMR (300 MHz, D2O): δ 0.94 (t, 2H), 1.10-1.31 (m, 3H),
1.38 (m, 3H), 1.55-1.88 (m, L1H), 1.88-2.15 (m, 3H) 1.95 (s, acetate), 2.34 (m, IH), 2.95 (m, IH), 3.08 (m, IH), 3.24 (t, 2H), 3.30 (m, 2H) , 3.66 (m, IH), 3.82 (m, IH), 4.32 (t, IH),
4.41 (dd, IH).
13C-NMR (125 MHz, D2O): guanidine: δ 157.40; carbonyl carbons: δ 180.39, 174.28, 168.55.
Example 21
HO-(CH2)3-(R)Cha-Pro-Nag x 2 TFA (i) BnO-(CH2)3 - (R)Cha-Pro-Nag(Z)
1-Benzyloxy 3-trifluoromethanesulfonylpropane (See Prep, of
Starting Materials) (0.5 g, 1 mmol) and H-(R)Cha-Pro-Nag(Z) (See Example 20) were dissolved in tetrahydrofurane (10 ml). Potassium carbonate (0.28 g, 2 mmol) was added and the reaction mixture was stirred at room temperature for two hours. The solvent was evaporated and the crude product extracted with ethyl acetate/water. The organic phase was washed once with aqueous sodium hydrogen carbonate, once with water and once with brine. After drying over sodium sulphate the solvent was evaporated and the crude product flash chromatographed (CH2CH2/MeOH(NH3-saturated), 95:5). Finally the solvent was evaporated and the product dried in vacuo to yield 0.29 g (45%) of the title compound.
bad ORIGINAL £
| iH-NMR | (500 | MHz, CDC13): | δ 0 . | .77-1.03 (m, | 2H), 1.03-2.18 | (m, |
| 19H), 2 | . 52 | (m, IH), 2.64 | (m, | IH), 3.03 I | lbs, IH), 3.1-3.6 | (m |
| 7H), 3. | 6 6 ( | m, IH), 4.41 ( | bs , | IH), 4.46 l | (3, 2H), 5.08 (s, | 2H |
| 7.2-7.4 | (m, | 5H), 7.55 (m, | IH) |
(ii) HO-(CH2)3-(R)Cha-Pro-Nag x 2 TFA
Prepared by using the deprotection procedure (a) on the product (i) above.
1H-NMR (500 MHz, D2O): δ 1.00 (bs, 2H) , 1.10-1.32 (m, 3H) ,
1.40 (bs, IH), 1.55-2.15 (m, 14H), 2.30 (m, IH), 3.05-3.35 (m, 6H), 3.57-3.75 (m, 3H), 3.81 (bs, IH), 4.35 (bs, IH),
4.42 (bs, IH).
Examp1e 22
HOOC-CH2-(R)Cha-Pro-Nag x HOAc (i) H-(R)Cha-Pro-NH-(CH2)3-N3
Prepared in the same way as H-(R)Cha-Pro-Agm(Z) (See Example
3) starting from Boc-(R,Cha-Pro-OSu and Boc-NH-(CH2)3-N3 (replacing Boc-Agm(Z)).
(ii) EtOOC-CH2-(R)Cha-Pro-NH-(CH2)3-NH2 x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-NH-(CH2)3-N3 and EtOOC-CH2-Br followed by deprotection procedure (a) to reduce the azide gave the title compound.
(iii) EtOOC-CH2-(R)Cha-Pro-Nag x HOAc
The same procedure as described in Example 19 (iii) for Z-(R)Cha-Pro-Nag was used to accomplish the guanidation of the amine from (ii) above. The title compound was obtained in a pure form after RPLC (CH3CN/NH4OAc (0.1M), 3/7) evaporation
BAD ORIGINAL £ of the solvent and freeze drying (H20).
(iv) HOOC-CH9 - (R·) Cha-Pro-Nag x HOAc
Prepared by using the deprotection procedure (e) on the product (iii) above.
1H-NMR (500 MHz, D2O): δ 0.99 (m, 2H), 1.09-1.30 (m, 3H), 1.44 (m, IH), 1.59-2.09 (m, 12H) 1.92 (s, acetate), 2.29 (m,
IH), 3.20 (t, 2H), 3.28 (m, 2H) , 3.52-3.63 (m, 3H) , 3.76 (tn, IH), 4.38 (dd, IH), 4.42 (t, IH).
13C-NMR (125 MHz, D2O): guanidine: δ 157.43; carbonyl carbons: δ 168.72, 171.36, 174.35.
Example 23
EtOOC-CH2-(R)Cha-Pro-Nag x HOAc
Prepared according to example 22 (iii).
| 1H-NMR (300 thereof 1.38 2.39 (m, IH) | MHz, D2O,) (t, 3H)), , 3.31 (t, | : δ 1.07 (m, 2H), 1.17-1.59 (m, 7H; | |||
| 1.60-2.24 2H), 3.39 | (m, (t, | 12H) 2.04 (s, acetate) | |||
| 2H), 3. | 63-3.90 (m, 4H) | ||||
| 25 4.12 (t, IH) | , 4.36 (q, | 2H), 4.46 | (dd, | IH) . | |
| 13C-NMR (75 | MHz, D2O,) | : guanidine | : δ | 157.37; | carbonyl |
carbons: δ 173.73, 175.09, 175.70.
Example 24 iPrOOC-CH2-(R)Cha-Pro-Nag x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See 35 Example 20) and Br-CH2COO1Pr followed by deprotection procedure (b) gave the title compound.
bad original
| iH-NMR ( | 500 MHz, MeOD) | : δ 0.85-1.05 (m, 2H), | 1.1-2 | . 15 ι m, |
| 2 2H; thereof 1.23 (d, | 3H), 1.25 (d, 3H)), 1. | 92 (s, | acecate) | |
| 2.2 (m, | IH), 3.10-3.35 | (m, 5H) , 3.4 (m, IH), | 3 . 55 | (m, IH), |
| 2.65-3.3 | (m, 2H), 4.28 | (dd, IH), 5.03 (m, IH | ) . | |
| i3C-NMR | (125 MHz, D2O) | : guanidine: δ 157.39; | carbonyl | |
| carbons : | δ 170.40, 172 | .00 and 174.50. | ||
| Example | 25 |
^uOOC-CHj-(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag (Z) (See Example 20) and 3r-CHoCOOtBu followed by deprotect ion procedure (b) gave the title compound.
l-H-NMR (300 MHz, MeOD): δ 0.9-1.15 (m, 2H) , 1.15-2.15 km,
25H; thereof 1.55 (bs, 9H)), 2.3 (m, IH), 3.15-3.45 (m, 4H),
3.55 (m, IH), 3.7-3.95 (m, 3H), 4.3- 4.4 (m, 2H).
•^c-NMR (75 MHz, D2O): guanidine: δ 157.55; carbonyl carbons: δ 166.55, 168.13 and 174.33.
Example 26
HOOC-CH2-OOC-CH2-(R)Cha-Pro-Nag x HOAc (i) BnOOC-CH2-OOC-CH2-(R)Cha-Pro-Nag(Z)
H-(R) Cha-Pro-Nagr (Z) (See Example 20), 0.20 g (0.40 mmol), was mixed with 0.115 g (0.40 mmol) of benzyloxycarbonylmethyl bromoacetate, 55 mg of K-^CO^ (0.40 mmol) and 5 ml of CH^CN. The mixture was stirred »t room temperature for 6 h. The solvent was evaporated and the crude product chromatographed (CH2Cl2/MeOH, 9/1) to give 0.20 g (71%) of the desired compound after evaporation of the solvent.
BAD ORIGINAL d (ii) HOOC-CH2-OOC-CH2- (R)Cha-Pro-Mag x HOAc
Prepared by using the deprotection procedure (a) on the product (i) above.
r□ 1H-NMR (500 MHz, MeOD): δ 0.85-1.1 (m, 2H) , 1.1-1.6 (m, 3H) ,
1.6-2.15 (m, 10H) 1.99 (3, acetate), 2.23 (m, IH), 3.1-3.4 (m, 4H), 3.45-3.65 (m, 4H), 3.7-3.9 (m, 3H) , 4.34 (m,IH),
4.48 (dd, 2H) .
^c-NMR (125 MHz, MeOD), guanidine: δ 158.8; carbonyl carbons: δ 176.1, 175.2,174.9, 173.1.
Examp1e 27
H2N-CO-CH2-(R)Cha-Pro-Nag x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20) and CI-CH2CONH2, in the presence of a catalytic (10 mol%) amount of KI in the reaction, followed by deprotection procedure (a) gave the title compound.
1H-NMR (500 MHz, D2O): δ 1.02 (m, 2H), 1.12-1.34 (m, 3H),
1.46 (m, IH), 1.61-2.13 (m, 9H) 1.99 (s, acetate), 2.34 (m,
IH), 3.25 (t, 2H), 3.33 (t, 2H), 3.60-3.82 (m, 4H), 4.22 (t, IH), 4.41 (dd, IH).
13C-NMR (75 MHz, D2O,): guanidine: δ 157.5; carbonyl carbons: δ 168.94, 169.40, 174.43.
Example 28
HOOC-CH2-NH-CO-CH2-(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20) and Br-CH2CONHCH2COOBn (See Prep, of starting materials) followed by deprotection procedure (a) gave the
BAD ORIGINAL &
ό ύ t-cle compound.
| iH-;CIR (50 | 0 MH | z, MeOD): | δ 1. | 01 (m, 2H), 1.15-1.38 im, | 3H) , | ||
| 1 .47 | (m, 1 | H) , | 1.64-2.13 | (m, | 12H), 2.27 (m, IH), 3.17- | 3.25 , m | |
| 3H) , | 3 . 37 | 1. m, | IH) , | 3 . 51 | tm, | 1H) , 3.83 (m, 1H) , 3.83 ( | 3 , 2 H 1 , |
| 3.93-4.06 | (m, | 2H; , | 4.35 | -4.45 | (m, 2H). |
^c-NMR (75 MHz, MeOD): guanidine: δ 158.71; carbonyl carbons: δ 166.94, 168.35, 172.44, 174.17.
Example 29 (HOOC-CH2)2-(R)Cha-Pro-Nag x HOAc (i) (EtOOC-CH2)2-(R) Cha-Pro-NH-(CH2)3-NH2 x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-NH-(CH2)3-N3 (See Example 22) and Br-CH2COOEt (10 eq. was used to accomplish the dialkylation) followed by deprotection procedure (a) gave the title compond.
(ii) (EtOOC-CH2)2-(R)Cha-Pro-Nag x HOAc
The same procedure as described in Example 19 (iii) for Z-(R)Cha-Pro-Nag was used to accomplish the guanidation of the amine above. Purification of the compound was made with RPLC (CH3CN/NH4OAc (0.1M), 4:6) (iii) (HOOC-CH2)2-(R)Cha-Pro-Nag x HOAc
The hydrolysis of the < -ter groups was made according to deprotection procedure (e) using a double amount of NaOH. The final compond was obtained pure after RPLC (CH3CN/NH4OAc (0.1M), 2:8), evaporation of the solvent and freeze drying bad original 4 ^H-NMR (300 MHz,'D2O): δ 0.92-1.49 (m, 6H) , 1.60-2.54 (m,
10Η) 2.05 (5, acetate), 3.25-3.50 (m, 4H), 3.65-4.03 (m, 6H; thereof 3.95 (s, 4H)), 4.49 (m, IH), 4.71 (m, IH; partly hidden by the H-O-D peak).
l-'C-NMR (75 MHz, D2O) : guanidine: δ 157.64; carbonyl carbons: δ 168.62, 171.39, 174.30.
Example 30
HOOC-CH2-(Me)(R)Cha-Pro-Nag x 2 TFA (i) Me-(R)Cha-Pro-Nag(Z)
Prepared from Boc-(Me)(R)Cha-Pro-OSu and Boc-Nag(Z) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(Me)(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using Me-(R)Cha-Pro-Nag(Z) and Br-CH2COOBn followed by deprotection procedure (b) gave the title compound.
1H-NMR (500 MHz, D2O) : δ 0.8-1.06 (m, 2H), 1.08-1.27 (m, 4H) ,
1.55-2.10 (m, 12H), 2.30 (m, IH), 3.04 (s, 3H), 3.14-3.33 (m, 4H), 3.63 (m, IH), 3.81 (m, IH), 4.13 (apparent bs, 2H), 4.38 (br.dd, IH), 4.56 (bt, IH).
13C-NMR (125.76 MHz, D2O> : guanidine: δ 157.40; carbonyl carbons: δ 174.05, 168.83, 167.44.
Example 31
BAD original
HOOC-CH2-(nBu)(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using Bu-(R)Cha-Pro-Nag(Z) ( See Example 20) and 3r-CH2COOBn followed by deprotection procedure (a) gave the title compound.
| 1H-NMR (500 MHz, | D2O) : δ 0.78-0.38 | (m | , 3H), 0 | .88-1.02 | (m, |
| 2H) , 1.02-1.23 (m, 4H) , 1.23-1.38 | 2H), 1. | 45-1.84 | (m, | ||
| 11H), 1.84-2.10 ( | m, 3H), 2.24 (m, | IH) | , 3.05-3 | . 18 (m, | 3H) , |
| 3.18-3.38 (m, 3H) | , 3.57 (m, IH), 3 | . > ! | (m, IH) | , 4.05-4 | . 2 5 |
| 2H), 4.32 (m, IH) | , 4.50 (m, IH). | ||||
| 13C-NMR (125 MHz, | D2O) : guanidine : | δ | 159.17 ; | carbonyl | |
| carbons: δ 175.66 | , 171.13, 169.31. | ||||
| Example 32 | |||||
| HOOC-(R,S)CH(Me)- | (R)Cha-Pro-Nag x | HOAc | |||
| Alkylation as in | Example 4 using H | - (R | )Cha-Pro | -Nag(Z) | ( See |
Example 20) and Br-CH(Me)COOBn followed by deprotection procedure (a) gave the title compound as a mixture of two diastereomers.
Example 33
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/a x HOAc
Obtained by separating the diastereomers formed in Example 32 using RPLC (CH3CN/NH4OAc (0.1M), 1/4) followed by evaporation of the solvent. This diastereomer came out first of the two from the column.
^H-NMR (300 MHz, D2O, 2 rotamers ca: 9:1 ratio): δ 0.78 (m, minor rotamer), 1.07 (m, 2H) , 1.17-1.42 (m, 3H), 1.48-1.64 (m, 4H; thereof 1.56 (d, 3H)), 1.64-1.95 (m, 9H) , 1.95- 2.20 (m, 3H) 2.00 (s, acetate), 2.37 (m, IH), 3.28 (t, 2H), 3.38
BAD ORIGINAL (t, 2H), 3.53 (m, minor rotamer), 3.63 (m, 2H), 3.77 (m, IH)
4.24 (d, minor rotamer), 4.35-4.50 (m, 2H), 4.60 (d, minor rotamer).
Example 34
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/b x HOAc
The title compound was obtained by using the same procedure as described in Example 33 on the compound formed in Example 32. This diastereomer came out after the first one from the column .
1H-NMR (300 MHz, D2O, 2 rotamers ca: 9:1 ratio): δ 0.95 (m, minor rotamer), 1.12 (m, 2H), 1.22-1.40 (m, 3H), 1.40-1.67 (m, 4H; thereof 1.60 (d, 3H)), 1.67-2.00 (m, 9H), 2.00-2.25 (m, 3H) 2.03 (s, acetate), 2.40 (m, IH), 3.25-3.48 (m, 4H), 3.66-3.84 (m, 2H), 3.93 (m, IH), 4.38 (m, IH), 4.50 (m, IH) ,
4.93 (m, minor rotamer).
l^c-NMR (75.5 MHz, D2O): δ 157.42; carbonyl carbons: δ 168.05, 171.99, 174.04.
Example 35
EtOOC-(R,S)CH(Me)-(R)Cha-Pro-Nag x 2 TFA
Prepared in the same way as described for Example 22 using EtOOC-CH(Me)-Br instead of Br-CH2-COOEt in the alkylation.
•^H-NMR (500 MHz, MeOD, 2 diastereomers ca: 2.5:1 ratio and 4 rotamers): δ 0.88-2.43 (m, 25H), 3.1-4.55 (m, 11H) .
l^c-NMR (75 MHz, MeOD): guanidine: δ 158.65; carbonyl carbons: δ 174.33, 170.66, 168.20.
BM>OWe|NAL
Example 3 6
HOOC-(RorS)CH(nPr)-(R)Cha-Pro-Nag/a x HOAc
Alkylation as in Example 4 using H-iR)Cha-Pro-Nag(Z) (See Example 20) and Br-CH(nPr)COOEt and deprotection procedure (e) followed by deprotection procedure (b) gave
HOOC-(R,S)CH(nPr)-(R)Cha-Pro-Agm. The title compound was obtained by separating the diastereomers (this diastereomer came out first of the two from the column) by RPLC (CH3CM/NH4OAc (0.1 M) , 1/4) and freeze drying (H2O) after evaportion of the solvent.
3H-NMR (500 MHz, MeOD): 5 0.85 -1.05 (m, 5H; thereof 0.95 (t, 3H)), 1.1-2.05 (m, 20H) 1.95 (s, acetate), 2.18 (m, IH), 3.15-3.3 (m, 4H), 3.35 (m, IH), 3.46 (m, IH), 3.85 (m, IH), 4.04 (m, IH), 4.33 (dd, IH).
13C-NMR (125 MHz, MeOD): guanidine: δ 158.73; carbonyl carbons: δ 171.63, 174.43 and 176.73.
Example 37
HOOC-(R)CH(CH2-OH)-(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20) and Br-(S)CH(CH2-OBn)-COOBn followed by deprotection procedure (a) gave the title compound.
1H-NMR (300 MHz, D2O): δ 0.75-1.56 (m, 7H), 1.56-2.30 (m, 11H), 2.40 (m, IH), 3.15-3.55 (m, 4H) , 3.55-4.60 (m, 7H) .
Example 38
HOOC-(R,S)CH(Ph)-(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See
BAD ORIGINAL A
Example 20) and Br-CH(Ph)COO^Bu and deprotection procedure (a) followed by (f) gave the title compound as a mixture of two diastereomers.
1H-NMR (300 MHz, MeOD): δ 0.8-1.1 (m, 2H), 1.1-2.13 (m, 16H),
2.26 (m, IH), 3.04-3.35 (m, 5H), 3.45 (m, IH), 3.~ (m, 1H) ,
4.35 (m, IH), 4.85 (s, IH, one isomer), 5.05 (s, In, the other isomer), 7.4-7.6 (m, 5H), 7.75 (bt, IH).
^c-NMR (75 MHz, D2O): guanidine: δ 158.68; carbonyl carbons: δ 174.39, 174.15 and 170.5, 170.06 and 168.32, 167.78.
Example 39
HOOC-(S)CH(CH2CH2Ph)-(R)Cha-Pro-Nag x HOAc
Alkylation as in Example 21«using H-(R)Cha-Pro-Nag(Z)(See Example 20) and TfO-(R)CH(CH2CH2Ph)-COOEt and deprotection procedure (e) followed by (a) gave the title compound.
1H-NMR (300 MHz, MeOD): δ 0.77-1.05 (m, 2H), 1.05-1.35 (m,
5H) , 1.35-2.16 (m, 14H) 1.88 (s, acetate), 2.71 (t, 2H) , 3.07-3.53 (m, 7H), 3.73 (m, IH), 4.32 (m, IH), 7.03-7.25 (m, 5H) .
13C-NMR (75 MHz, MeOD): guanidine: δ 158.71; carbonyl carbons: δ 174.15, 177.31, 182.61.
Example 40
HOOC-(R)CH(CH2CH2Ph)-(R) Cha-Pro-Nag x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20) and Br-CH(CH2CH2Ph)COOEt followed by deprotection procedure (a) and (e) gave HOOC-(R,S)CH(CH2~CH2“Ph)(R)Cha-Pro-Nag. The title compound was obtained by separating
BAD ORiG^blAL s the two diastereomers with RPLC (CH3CN/NH4OAc (0.1 M), 2/3) and freeze drying (H2O) after evaportion of the solvent.
1H-NMR (300 MHz, MeOD): δ 0 1.43-2.30 (m, 16H) 1.96 (s, <m, 3H) , 3.28-3.66 (m, 3H) , (dd, 1H), 7.11-7.28 (m, 5H) (m, 2H), 1.10-1 acetate), 2.70 (m, 3.84 (m, 1H), 4.14 (m, 3H), 2H), 3.06-3 (bt, 1 ri), 4 12C-NMR (75 MHz, MeOD): guanidine: δ 158.66
Example 41
HOOC-CH2-CH2-(R)Cha-Pro-Nag x HOAc (i) EtOOC· CH2CH2' (R)Cha-Pro-NH-(CH2)3-NH2
Alkylation as described in Example 15 using
H-(R) Cha-Pro-NH-(CH2 ) 3-N-j instead of H-(R) Cha-Pro-Agm ( Z ) followed by deprotection procedure (a) gave the title compound .
(ii) Et-OOC-CH2-CH2-(R)Cha-Pro-Nag x HOAc
Guanidation of the amine above in the same way as described in Example 19 for Z-(R)Cha-Pro-Nag gave the title compound (ii) .
(iii) HOOC-CH2-CH2-(R)Cha-Pro-Nag x HOAc
Prepared by using the deprotection procedure (e) on the product (ii) above.
^H-NMR (500 MHz, D2O) : δ 1.12 (m, 2H) , 1.22-1.48 (m, 3H) , 1.54 (bs, 1H), 1.70- 2.37 (m, 12H) 2.14 (s, acetate), 2.53 (m,
1H), 2.70 (bs, 2H), 3.15 (t,lH), 3.25-3.55 (m, 5H) , 3.75 (m, 1H), 3.93 (m, 1H), 4.43 (t, 1H), 4.52 (m, 1H).
BAD ORIGINAL fi
Example 42
EtOOC-CH2-CH2-(R)Cha-Pro-Nag x HOAc
Prepared according to Example 41 (ii).
1H-NMR (500 MHz, D2O): δ 0.97 (m, 2H), 1.11-1.39 (m,7H; thereof 1.30 (t,3H)), 1.50 (t, 2H), 1.62-1.76 (m,5H), 1.76-2.14 (m, 5H) 1.93 (s, acetate), 2.29 (m, IH) , 2.62 (c,
2H), 2.77-2.94 (m, 2H), 3.23 (t, 2H), 3.32 (t, 2H) , 3.60-3.37 (m, 3H), 4.20 (q, 2H) , 4.36 (dd, IH).
13C-NMR (125 MHz, D2O): guanidine: δ 157.39; carbonyl carbons: δ 182.05, 175.13, 175.02.
Example 43
HOOC-(CH2)3-(R)Cha-Pro-Nag x 2 HOAc (i) Et-OOC-CH=CH-CH2-(R)Cha-Pro-Nag(Z)
H-(R)Cha-Pro-Nag(Z) (See Example 20) (1 eq) and ethyl
3-bromocrotonate (1.1 eq) were dissolved in acetonitrile (15 ml/mmol). Potassium carbonate was added and the reaction mixture stirred at room temperature for 2 h. After filtration and evaporation of the solvent, the crude product was purified by flash chromatography (C^C^/MeOH) . Finally the solvent was evaporated and product dried in vacuo.
1H-NMR (500 MHz, CDCI3): δ 0.73-1.0 (m, 2H), 1.0-1.4 (m, 8H; thereof 1.33 (t, 3H)), 1.43-2.15 (m, 12H), 2.96 (bs, IH),
3.12 (dd, IH), 3.16-3.48 (m, 6H), 3.56 (m, IH) , 4.15 (q, 2H),
4.35 (bs, IH), 5.03 (s, IH), 6.0 (d, IH), 6.85 (dt, IH), 7.05 (bs, IH), 7.17-7.2 .m, 5H), 7.5 (bs, IH).
(ii) EtOOC-(CH (R)Cha-Pro-Nag x 2 TFA badowg‘nal
Prepared by using the deprotection procedure (a) on the product ii) above.
(iii) HOCC - (CH2 ) 3 - ( R) Cha - Pro -Mag ;< 2 HOAc q
-/
Prepared by using the deprotection procedure (e) on the product ίii) above.
| 1H-NMR (5( | DO MHz, D2O): δ | 1.02 | (bs, | 2H) , | 1.08-1.32 | (m, | 3H) | |
| 10 1.42 | (bs , | IH), 1.55-2.15 | (m, | 14H) | 1 . 92 | (s, acetate), 2 | .33 | |
| (bs , | 3H) , | 3.00 (bs, IH), | 3 . 07 | (bs , | IH) , | 3 .18-3.40 | (m, | 4H) |
| 3 . 62 | (bs, | IH), 3.82 (bs, | IH) , | 4.33 | ( bs , | IH), 4.40 | (bs , | IH |
13C-NMR (125 MHz, D20): guanidine: δ 157.42; carbonyl 15 carbons: δ 181.87, 174.34, 163.64.
Example 44
EtOOC-(CH2)3-(R)Cha-Pro-Nag x 2 TFA
Prepared according to Example 43 (ii).
1H-NMR (300 MHz, MeOD/D2O): δ 0.63-1.30 (m, 9H; thereof 1.02 (t, 3H)), 1.30-1.97 (m, 14H), 2.06 (bs, IH), 2.28 (m, 2H),
2.72-3.20 (m, 6H) , 3.36 (m, IH), 3.60 (m, IH), 3.94 (m, 2H) ,
4.06 (m, IH), 4.17 (m, IH).
13C-NMR (75 MHz, MeOD/D2O): guanidine: δ 158.10; carbonyl carbons: δ 175.40, 174.23, 168.54.
Example 45
HOOC-CO-(R)Cha-Pro-Nag x HOAc (i) EtOOC-CO-(R)Cha-Pro-Nag(Z)
H-(R)Cha-Pro-Nag(Ζ) , 0.50 g (0.97 mmol) was dissolved in 0.54
BAD ORIGINAL A
9 ml triethyl amine and 8 ml of CH2C12. Ethyl oxalylchloride, 0.146 g (1.07 mmol) dissolved in 2 ml of CH->C12 was added while the temperature rose from 22-28°C and the reaction was stirred at room temperature for 2 h. The organic phase was washed twice with water, dried (Na2SO4) and flash chromathographed (EtOAc/EtOH(99%), 9/1) to give 92 mg (15 %) of the title compund.
(ii) HOOC-CO-(R)Cha-Pro-Nag x HOAc
Using the deprotection procedure (b) followed by (e) gave the title compound.
1H-NMR (300 MHz, MeOD) : δ 0.88-1.14 (m, 2H) , 1.15-1.5 (m,
4H), 1.5-2.3 (m, 13H) 1.9 (s, acetate), 3.1-3.43 (m, 4H), 3.6 (m IH), 4.05 (m, IH), 4.43 (dd, IH), 4.5 (m, IH).
X3C-NMR (75 MHz, D2O) : guanidine: δ 157.57; carbonyl carbons: δ 165.94, 173.95, 174,85 and 181.22.
Example 46
MeOOC-CO-(R)Cha-Pro-Nag x HOAc (i) MeOOC-CO-(R)Cha-Pro-Nag(Z)
The methyl ester was obtained by transesterification of EtOOC-CO-(R)Cha-Pro-Nag(Z) (See Example 45) on the column during flash chromatography when EtOAc/MeOH(9:1) was used as eluent. Yield 55%.
(ii) MeOOC-CO-(R)Cha-Pro-Nag x HOAc
Prepared by using the deprotection procedure (b) on the 35 product (i) above.
XH-NMR (300 MHz, MeOD): δ 0.9-1.1 (m, 2H), 1.1-2.3 (m, 17H) badowg‘nal
1.9 ,3, acetate), 3.12-3.4 (m, 4H) , 3.52 -3.67 (m, 2H),3.y (3,
3H) , 4.35 Im, LH) , 4.65 (m, IH) .
O-NMR (7tMHc, D20) : guanidine: δ 157.52; carbonyl carbons :
δ 159.11, 161.20 173.17 and 174.90.
Example 47 (R,S)Bla-(R)Cha-Pro-Nag x 2 TFA
Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20) and α-bromo butyrolacton followed by deprotection procedure (a) gave the title compound as a mixture of two diastereomers.
'-H-NMR (300 MHz, D2O, mixture of diastreomers): δ 1.0-1.43 (m, 5H), 1.45-1.60 (br.s, IH), 1.64-2.28 (m, 12H), 2.31-2.50 (m, IH), 2.30-2.98 (m, IH) , 3.23-3.46 (m, 4H), 3.66-3.79 (m, IH), 3.82-3.96 (m, IH), 4.33-5.08 (m, 5H, partially hidden by the H-O-D signal).
Examp1e 48
HOOC-(R,S)CH(CH2COOH)-(R)Cha-Pro-Nag x HOAo (i) BnOOC-(R,S)CH(CH2COOBn)- (R)Cha-Pro-Nag (Z)
H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.21 g (0.42 mmol), and 0.12 g (0.42 mmol) of dibenzyl maleate were dissolved in 10 ml of CH3CN. The mixture was refluxed over night, evaporated and flash chromatographed (CH2Cl2/MeOH, 94/6). Evaporation of the solvent gave the desired compound in 22 % yield.
(ii) HOOC-(R,S)CH(CH2COOH)-(R)Cha-Pro-Nag x HOAc
Prepared by using the deprotection procedure (a) on the product (i) above.
feAD ORIGINAL ^H-NMR (500 MHz, MeOD): δ 0.9-2.4 (m, 19H), 2.00 (s, acetate) 2.7-3.0 (m, 2H) , 3.1-3.6 (m, 5H) , 3.75-3.9 (m, 2H),
4.2-4.5 (m, 2H) .·
Example 49
MeOOC-(R,S)CH(CH2COOMa)-(R)Cha-Pro-Nag x HOAc (i) MeOOC-(R,S)CH(CH2COOMe)- (R)Cha-Pro-Nag(Z)
H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.21 g (0.42 mmol), and 0.24 g (1.7 mmol) of dimethyl maleate were dissolved in 15 ml of MeOH. The mixture was refluxed over night, evaporated and flash chromatographed (CH2Cl2/MeOH, 9/1). Evaporation of the solvent gave the desired compound in 45% yield.
(ii) MeOOC-(R,S)CH(CH2COOMe)-(R)Cha-Pro-Nag x HOAc
Prepared by using the deprotection procedure (c) on the product (i) above.
1H-NMR (500 MHz, MeOD): 5 0.85-1.1 (m, 2H), 1.15-2.3 (m,
17H), 1.91 (s, acetate) , 2.6-2.8 (m, 2H) , 3.1-3.5 (m, 5H) ,
3.5-3.8 (m, 10H; thereof 4 singlets 3.66, 3.68, 3.71, 3.73),
4.29 (m, IH).
Example 50
HOOC-Ph-4-CH2-(R)Cha-Pro-Nag x 2 TFA (i) tBuOOC-Ph-4^CH2-(R)Cha-Pro-NH-(CH2)3-N3
H-(R)Cha-Pro-NH-(CH2) 3-N3 (See Example 22)-, 0.39 g (1.1 mmol) and 0.33 g (1.2 mmol) of tertiarybutyl p-bromomethylbenzoate were dissolved in 10 ml of CH3CN and 0.19 g (2.4 mmol) of K2CO3 was added. The mixture was refluxed over night and evaporated. The crude product was flash chromatographed
BAD ORIGINAL &
(CH-iCl-ι/MeOH, 92:8) to give 0.50 g (34%) of the title comper.d.
( 11) - 3uOOC - Ph-4 -CH2 - (R) Cha-Pro-MH- (CH->) 3 -NH7
To a solution of 0.60 g .1.8 mmol) of bis-phenylthio stanna.ne, 0.20 g (1.8 mmol) of thiophenol and 0.18 g (1.8 mmol) of triethyl amine in 50 ml of CH2CI2 at 0°C was added 0.50 g (0.92 mmo1) of tBuOOC-?h-4-CH') - (R) Cha-Pro-NH-(CH? ) 3-N3 . The mixture was stirred at 0°C for 30 min. and at room temperature for 4 h.
It was then diluted with CH2CI2 and washed with aqueous sodium bicarbonate and subsequently 3 times with 2% H2O2· The organic layer was extracted with dilute HCl. The combined acidic water phase was washed with EtOAc and subsequently made alkaline with NaOH(aq). The aqueous layer was extracted twice with ethyl acetate. The combined organic layer was dried (Na2SO4) and evaporated. Plash chromatography (C^C^/MeOH(NH3-saturated) , 8:2) gave 0.12g (26%) of the title compound.
(iii) HOOC-Ph-4-CH2-(R)Cha-Pro-Nag x 2 TFA
Guanidation of the amine above in the same way as described in Example 19 for Z-(R)Cha-Pro-Nag followed by deprotection procedure (f) gave the title compound.
•’H-NMR (500 MHz, MeOD) : δ 0.9-1.5 (m, 7H) , 1.4-1.9 (m, 9H) , 1.95-2.1 (m, 2H), 2.16 (m, IH), 2.32 (m, IH), 3.2-3.3 (m,
3H), 3.41 (pentet, IH), 3.53 (m, IH), 3.77 (m, IH), 4.2-4.3 (m, 3H), 4.42 (dd, IH), 7,15 (d, 2H), 8.10 (d, 2H).
13C-NMR (125 MHz, MeOD), guanidine: δ 160.8; carbonyl carbons: δ 174.3, 168.9, 168.2.
bad original
Example 51 (HO)2P(0)-CH2-(R)Cha-Pro-Nag x HOAc (EtO)2?O-CH2-(R)Cha-Pro-Nag(Z) (See Example 53), 60 mg (92 mmol), was dissolved in 3 ml of CH^CN. Trimethylsilyl bromide, 0.15 ml, was added and the mixture was left at room temperature for 21 h. After evaporation and NMR analysis it was found that some ester remained. The crude material was again dissolved in 3 ml of CH3CN and 0.15 ml of trimethylsilyl bromide was added. After 5 h the mixture was evaporated and purified with RPLC (CH3CN/NH4OAc (0.1M),
30:70) to give the final compound after filtration, evaporation and freeze drying in 8 % yield.
1H-NMR (500 MHz, MeOD): δ 0.8-1.1 (m, 2H), 1.15-1.4 (m, 4H),
1.5-1.9 (m, 10H), 1.9-2.1 (m, 4H) 1.96 (s, acetate), 2.20 (m, IH), 2.95 (m, IH), 3.0-3.2 (m, 3H), 3.4-3.5 (m, 2H), 4.09 (m, IH), 4.39 (bd, IH), 4.59 (m) IH).
l^C-NMR (125 MHz, MeOD): guanidine: δ 158.6; carbonyl carbons: δ 174.2, 170.6
Example 52
EtO(HO)P(O)-CH2-(R)Cha-Pro-Nag x 2 HOAc (i) (EtO) (HO)PO-CH2-(R)Cha-Pro-Nag(Z) .
(EtO)2PO-CH2-(R)Cha-Pro-Nag(Z) (See Example 53), 50 mg (77 mmol) was dissolved in 2 ml of EtOH and 2 ml 2 M NaOH. The mixture was stirred over night and evaporated. The crude material was purified with RPLC (CH3CN/NH4OAc (0.1M), 30:70) to give the title compound after filtration and evaporation of the solvent.
(ii) (EtO)(HO)PO-CH2-(R)Cha-Pro-Nag x 2 HOAc
BAD ORIGINAL
Prepared by using deprotection procedure (c) on the product (11 above .
'-H-NMR (500 MHz, MeOD): δ 0.9-1.1 (m, 2H), 1.15-1.35 (m, 6H; thereof 1.23 (t, 3H)), 1.35-1.5 (m, 2H), 1.5-1.6 (m, IH),
1.65-1.8 (m, 6H) , 1.9-2.1 (m, 3H) 1.95 (s, acetate), 2.19 (m, IH) , 2.8-3.0 (m, 2H), 3.1-3.25 (m, 2H), 3.27 (m, IH) , 3.36 (m, IH), 3.48 (m, IH), 3.9-4.05 (m, 4H) , 4.36 (bd, IH) .
l^c-NMR (125 MHz, MeOD): guanidine: δ 158.6; carbonyl carbons: δ 175.0, 174.7
Example 53 (EtO)2P(Ο)-CH2-(R)Cha-Pro-Nag x HOAc (i) (EtO)2PO-CH2-(R)Cha-Pro-Nag(Z).
H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.2 g (0.40 mmol), was dissolved in 5 ml of THF and 0.11 g (0.80 mmol) of potassium carbonate and 0.12 g (0.40 mmol) diethyl triflylmethylphosphonate were added. The mixture was stirred at room temperature for 2 h. The reaction was worked up with water and extraction of the aqueous layer three times with EtOAc. The combined organic layer was dried (Na2SO4) and evaporated to yield 0.14 g (53%) of the title compound.
(ii) (EtO)2PO-CH2-(R)Cha-Pro-Nag x HOAc
Prepared by using the deprotection procedure (c)on the product (i) above.
'•H-NMR (500 MHz, MeOD): δ 0.85-1.05 (m, 2H) , 1.15-1.3 (m,
5H) , 1.34 (t, 6H) , 1.5-1.85 (m, 8H) , 1.9-2.05 (m, 311) 1.91 (s, acetate), 2.10 (m, IH), 2.22 (m, IH), 2.90 (dd, IH) , 3.05 (dd, IH), 3.1-3.3 (m, 3H), 3.42 (m, IH), 3.53 (m, IH), 3.71 (dd, IH), 3.82 (m, IH), 4.1-4.2 (m, 4H), 4.28 (dd, IH).
BAD ORIGINAL ft 13C-NMR (125 MHz, MeOD), guanidine: δ 158.7; carbonyl carbons: δ 176.1, 175.1.
Example 54 5
HOOC-CH2-(R)Cha-Pro-Mag x HOAc (i) H-(R) Cha-Pro-NH-(CH2)2-NH(Z>
Prepared from Boc-(R)Cha-Pro-OSu and H2N-(CH2)2-NH(Z) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) EtOOC-CH2-(R)Cha-Pro-NH-(CH2)2-NH2 x H0Ac
Alkylation as in Example 4 followed by deprotection procedure (a) gave the title compound.
(iii) HOOC-CH2-(R)Cha-Pro-Mag x HOAc
Guanidation of the amine above in the same way as described in Example 19 for Z-(R)Cha-Pro-Nag followed by deprotection procedure (e) gave the title compound after purification by RPLC (CH^CN/NH^OAc (0.1M), 1/4) and freeze drying(H2O).
1H-NMR (300 MHz, D2O) 3H), 1.52 (m, IH), 1. 2.31-2.47 (m, IH) , 3. 3H), 3.85 (m, IH) , 4.
: δ 0.90-1.18 (m, 63-2.20 (m, 10H) 44 (m, 2H) , 3.50 46-4.54 (m, 2H).
2H), 1.19-1.43 (m, 2.06 (s, acetate), (m, 2H), 3.60-3.75 (m, 13C-NMR (75 MHz, δ 168.80, 171.41,
D2O): guanidine 174.81.
δ 157.82; carbonyl carbons:
Example 55
H-(R,S)Pro (3-Ph)-Pro-Agm x 2 TFA
Prepared from Boc-(R,S)Pro(3-Ph)-Pro-OSu (See Prep.
of
BAD ORIGINAL ft starting materials) rn the same way as described for
H-(R)Cha-Pro-Agm(Z) in Example 3 followed by deprotection procedure (b).
'•H-NMR (500 MHz, D2Q, mixture of two diastereomers with unknown relative stereochemistry): 5 1.0-1.8 (m, 7H), 2.0-2.5 (m, 3H), 2.8-4.3 (m, 10H), 4.56 (d, IH, major), 4.90 (d, IH, major), 7.2-7.5 (m, 5H).
13C-NMR (125.76 MHz, D2O) : guanidine: δ 157.36 (minor and major); carbonyl carbons: δ 174.1 (major), 174.0 (minor),
167.8 (major), 167.0 (minor).
Example 56
H-(R, S)Pro(3-(trans)Ch)-Pro-Agm x 2 TFA
Prepared from Boc-(R,S)Pro(3-(trans)Ch)-Pro-OSu (See Prep, of starting materials) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3 followed by deprotection procedure (b).
'H-NMR (500 MHz, D2O, mixture of two diastereomers, ratio . 1.8/1): δ 0.95-1.32 (m 5H) , 1.35-1.46 (m, IH), 1.50-1.92 (m, 10H) , 1.93-2.15 (m, 4H), 2.23-2.43 (m, 2H), 3.15-3.30 (m,
4H) , 3.35-3.50 (m, 2H) , 3.57-3.68 (m, IH) , 3.74-3.82 (m, IH) ,
4.34-4.41 (m, IH), 4.51 (d, IH, minor), 4.48 (d, IH, major).
13C-NMR (125.76 MHz, D2O): guanidine: δ 157.36 (minor and major), carbony1 carbons : δ 17 4.34 (major), 174.07 (minor), 168.94 (minor and major).
Example 57
HOOC-CH2-(R,S)Pro(3-(trans)Ph)-Pro-Agm x 2 TFA (i) H-(R,S)Pro(3-(trans)Ph)-Pro-Agm(Z)
BAD ORIGINAL
Prepared from Boc-(R,S) Pro (3 - (trans) Ph)-Pro-OSu (See Prep, of starting materials) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(R,S) Pro (3 - (trans) Ph)-Pro-Agm x 2 TEA
Alkylation as in Example 4 using Br-CH2COOBn followed by deprotection procedure (b) gave the title compound as a mixture of two diastereomers.
3H-NMR (500 MHz, MeOD, mixture of two diastereomers, ratio ca: 1.1/1): δ 1.40-1.80 (m, 6H) , 1.85-2.05 (m, IH), 2.10-2.30 (m, IH), 2.50-2.65 (m, 2H), 3.10-3.40 (m, 6H), 3.50-3.70 (m, 2H), 3.9-4.40 (m, 4H), 4.63 (d, IH, major), 4.67 (d, IH, minor), 7.30-7.60 (m, 5H).
13C-NMR (125.76 MHz, D2O): guanidine: δ 157.52 (both isomers); carbonyl carbons: δ 173.87, 173.73, 169.12, 168.94, 167.21, 167.00.
Example 58
HOOC-CH2-(R,S)Pro(3-(trans)Ph)-Pro-Nag x 2 TFA (i) H-(R, S) Pro(3-(trans)Ph)-Pro-Nag(Z)
Prepared from Boc-(R,S)Pro(3-(trans)Ph)-Pro-OSu (See Prep, of starting materials) and Boc-Nag(Z) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(R,S)Pro(3-(trans)Ph)-Pro-Nag x 2 TFA
Alkylation as in Example 4 using Br-CH2COOBn followed by deprotection procedure (b) gave the title compound as a mixture of two diastereomers.
^H-NMR (500 MHz, MeOD, mixture of two diastereomers, ratio
BAD ca: 1.5/1): δ 1.40-1.85 (m, 4H) , 1.90-2.00 (m, IH) , 2.10-2.30 (m, IH) , 2.45-2.70 (m, 2H) , 3.08-3.46 (m, 6H) , 3.57-3.70 2H) , 3.90-4.0 (m, IH) , 4.32-4.40 (m, IH), 4.04 and 4.29 (A3-quartet, 2H, major), 4.16 and 4.37 (A3-quartet, 2H, minor), 4.60 (d, IH, major), 4.64 (d, IH, minor), 7.3-~.6 5H) .
13C-NMR (125.76 MHz, D2O): guanidine: δ 157.48 (both isomers); carbonyl carbons: δ 173.90, 173.71, 169.01, 168.94, 167.07 (both isomers).
Example 59
HOOC-CH2-(R)Cha-Pic-Agm x 2 TFA (i) H-(R)Cha-Pic-Agm(Z)
Prepared from Boc-(R)Cha-Pic-OSu (See Prep, of starting materials) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(R)Cha-Pic-Agm x 2 TFA
Alkylation as in Example 4 using Br-CH2COOBn followed by deprotection procedure (a) gave the title compound.
1H-NMR (300 MHz, MeOD): δ 1.02 (m, 2H), 1.13-2.00 (m, 20H),
2.24 (bd, IH), 3.12-3.45 (m, 5H), 3.71 (bd, IH), 3.87 (s,
2H), 4.65 (bt, IH), 5.06 (m, IH).
13C-NMR (75 MHz, D2O): guanidine: δ 157.47; carbonyl carbons: δ 169.42, 170.03, 172.71.
Example 60
HOOC-CH2-(Me)(R)Cha-(R,S)Pic-Agm x HOAc
J3AD ORIGINAL J (i) Me-(R)Cha-(R,S)Pic-Agm(Z)
Prepared from Boc-(Me)(R)Cha-Pic-OSu in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(Me)(R)Cha-(R,S)Pic-Agm x HOAc
Alkylation as in Example 4 using Br-CH2COOBn followed by deprotection procedure (b) gave the title compound.
Comment: An epimerization of Pic occured somewhere during the synthesis .
The 1h-NMR spectrum is complex consisting of two 15 diastereomers ca: 1:1 ratio and rotamers thereof.
1H-NMR (500 MHz, MeOD): 5 0.75-2.15 (several m, 20Η) 1.95 (bs, acetate), 2.2-2.7 (6H, two distinct sets of signals are observed in the ratio of ca: 1:1; thereof 2.35 and 2.55 (s,
3H)), 3.0-3.5 (m, 6H) , 3.9-4.17 (m, 2H; thereof 4.14 (dd)),
4.4-4.5 (m, IH) , 4.97-5.15 (twobdd, IH).
l^c-NMR (75MHz, D2O) : guanidine: δ 157.50; carbonyl carbons: δ 169.65, 170.01, 170.54, 172.67, 172.89.
Example 61
HOOC- (R, S) CH(Me) - (R) Cha-Pic-Agm x TFA
Alkylation as in Example 4 using H-(R)Cha-Pic-Agm(Z) (See Example 59) and Br-CH(Me)COOBn followed by deprotection procedure (a) gave the title compound as a mixture of two diastereomers.
Example 62
HOOC-(RorS)CH(Me) - (R)Cha-Pic-Agm/a x 2 TFA
bad original
Obtained by separating the diastereomers formed in Example 01 using RPLC (CH^CN/NH^OAc (0.1M), 1/3) followed by evaporation of the solvent and freeze-drying from H2O/TFA. This diastereomer came out first of the two from the column.
^H-NMR (300 MHz, D2O, 2 rotamers minor rotamer), 0.75-1.0 (m, 2H), (m, 20H; thereof 1.57 (d, 3H)), 2 rotamer), 3.03-3.32 (m, 5H), 3.59 rotamer), 3.98 (q, IH) , 4.30-4.50 (m, IH), 4.95 (s, IH).
l^c-NMR (75 MHz, D2O): guanidine: δ 172.26 (2 carbons), 169.92.
Example 63
| ca: 5:1 ratio): δ 0 | .70 (m, |
| 1.0-1.28 (m, 3H), | 1.28-1.83 |
| .14 (bd, IH), 2.92 | (t, minor- |
| (bd, IH), 3.85 (q, | minor |
| (m, minor rotamer) | , 4.54 |
| δ 157.39; carbonyl | carbons : |
HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/b x 2 TFA
The title compound was obtained by using the same procedure as described in Example 62 on the compound formed in Example 61. This diastereomer came out after the first one from the column .
-^H-NMR (500 MHz, D2O, 2 rotamers ca: 5:1 ratio): δ 0.72 (m, minor rotamer), 0.82 (m, minor rotamer), 0.97 (m, 2H), 1.0-1.23 (m, 3H), 1.23-1.40 (m, 2H), 1.40-1.83 (m, 18H; thereof 1.63 (d, 3H) ) , 2.11 (d, IH), 2.17 (d, minor rotamer), 2.92 (t, minor rotamer), 3.05-3.25 (m, 4H), 3.29 (t, IH) ,
3.74 (d, IH), 4.02 (q, IH), 4.34 (d, minor rotamer), 4.41 (dd, minor rotamer), 4.52 (t, IH), 4.95 (s, IH).
l^c-NMR (125 MHz, D2O): guanidine: δ 154.68; carbonyl carbons: δ 169.81, 169.60, 167.36.
Example 64 bad ORIGINAL
HOOC-CH2-CH2-(R)Cha-Pic-Agm x 2 TFA
Prepared from H-(R)Cha-Pic-Agm(Z) (See Example 59) in the same way as described for HOOC-CH2-CH2-(R)Cha-Pro-Agm in
Example 15 using 1.2 eq. of benzylacrylate instead of 1.1 eq.
1H-NMR (500 MHz, D20, 2 rotamers ca: 4:1 ratio): δ 0.70-0.90 (m, minor rotamer), 0.90-1.0 (m, 2H), 1.05-1.25 (m, 3H),
1.30-1.45 (m, 2H) , 1.45-1.85 (m, 15H), 2.1 (bd, IH), 2.2 (bd, minor rotamer), 2.75 (t, 2H), 2.95 (t, minor rotamer),3.1-3.4 (m, 7H), 3.75 (bd, IH), 4.55 (t, IH), 4.95 (m, IH).
13C-NMR (75 MHz, D2O): guanidine: δ 157.48; carbonyl carbons: δ 170.10, 172.58, 174.75.
Example 65
H-(R)Cha-Pic-Nag x 2 TFA (i) Boc-(R)Cha-Pic-Nag(Z) (ia) Prepared by starting from Boc-(R)Cha-Pic-OSu by using the same procedure as described for Boc-(R)Cha-Pro-Agm(Z) in Example 3.
(ib) Prepared by starting from Boc-(R)Cha-Pic-OH
Diphenylphosphoryl azide (0.432 ml, 2 mmol) was added to a stirred solution of Boc-(R)Cha-Pic-OH (765 mg, 2 mmol) in 5 ml DMF at -10 °C. After 10 minutes H-Nag(Z) x 2 HCl (600 mg,
2.1 mmol, see Preparation of Starting Materials) in 5 ml DMF and triethylamine (615 mg, 4.4 mmol) was added. The reaction mixture was kept in an ice bath for 3 h and then at room temperature for 12 h after which it was poured out in water.
Extraction of the water phase with EtOAc followed by drying (MgSO4) of the organic phase and evaporation of the solvent in vacuo gave 1.18 g (96 %) of the product· as a mixture of bad original &
diastereomers (Epimers in Pic) in a ratio of 97:3 (RS/RR) ίic) Starting from Boc-(R)Cha-Pic-OH
EDC hydrochloride (4.2 g, 21.9 mmol) was added at -15° C to a stirred solution of Boc-(R)Cha-Pic-OH (8 g, 20.9 mmol), DMAP (10.6 g, 88 mmol) and H-Nag-(Z) x 2 HCl (6.3 g, 19.5 mmol, see Preparation of Starting Materials) in acetonitrile. The reaction mixture was allowed to warm up to +15° C during 16 h. The solvent was removed in vacuo an the residue was dissolved in ethyl acetate. Washing with water, 0.3 M XHSO.,, 0,3 M NaHCO-j, water and brine followed by drying (Na-jSO^) and evaporation of the solvent gave 11.9 g (92.5%) of the product as a mixture of diastereomers (Epimers in Pic) in a ratio of 98/2 (RS/RR).
1H-NMR (500 MHz, CDC13): δ 0.85-2.0 (m,29H; thereof 1.40 (bs, 9H)) , 2.46 (bd, IH) , 3.1-3.4 (m, 5H), 3.92 (bd, IH) , 4.53 (bq, IH), 5.10 (s, 2H), 5.22 (bs, IH), 5.29 (bd, IH), 6.7-7.2 (b, 3H) , 7.25-7 .45 (m, 5H) .
13C-NMR (125 MHz, CDC13): guanidine δ 156.9; carbonyl carbons: δ 173.6, 170.3, 163.7, 161.7.
(ii) H-(R)Cha-Pic-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3, starting from Boc-(R)Cha-Pic-Nag(Z).
1H-NMR (500 MHz, CDCI3): 6 0.8-2.0 (m, 22H), 2.24 (bd, IH),
3.1-3.4 (m, 5H), 3.72 (bd, IH), 3.84 (bq, IH), 5.05 (bd, IH), 5.08 (s, 2H), 7.3-7.5 (m, 5H).
(iii) H-(R)Cha-Pic-Nag x 2 TFA
Prepared by using the deprotection procedure (a) on the product (ii) above.
BAD ORIGINAL 1H-NMR (500 MHz, MeOD): δ 0.9-1.1 <m, 2H) , 1.2-2.0 (m, 18H) , 2.32 (bd, IH), 3.20 (t, 2H), 3.30 (t, 2H), 3.36 (m, IH,, 3.69 (bd, IH), 4.49 (dd, IH), 5.05 (bd, IH).
l^C-NMR (125 MHz, MeOD): guanidine: 5 158.7; carbonyl carbons: δ 172.7, 171.4
Example 66
Me-(R)Cha-(R,S) Pic-Nag x 2 TFA (i) Me-(R)Cha-(R,S)Pic-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) 15 in Example 3 staring from Boc-(Me)(R)Cha-Pic-OSu and BocNag(Z). An epimerization of Pic occured during the synthesis and the product was obtained as mixture of two diastereomers.
(ii) Me-(R)Cha-(R,S)Pic-Nag x 2 TFA
Prepared by using deprotection procedure (b).
The ^H-NMR spectrum is complex consisting of two diastereomers ca: 4:1 ratio and rotamers thereof.
1H-NMR (500 MHz, MeOD): δ 0.8-1.08 (m, 2H), 1.15-2.4 (several m, 19H) , 2.6-2.75 and 2.9-2.95 (several s, 3H) 3.1-3.6 (several m, 5H), 3.75-4.1 (several m, IH) 4.4-4.7 (several m, IH), 5.05-5.15 (two dd, IH).
l^c-NMR (125 MHz, D2O) : guanidine: δ 154.84; carbonyl carbons: δ 167.60 and 169.99.
Example 67 35
HOOC-CH2-(R)Cha-Pic-Nag (1) BnOGC-CH2(R)Cha-Pic-Nag ( Z)
Alkylation as in Example 4 using H-(R)Cha-Pic-Nag(Z) ;See Example 65) and Br-CH-)COOSn gave the title compund.
XH-NMR (500 MHz, CDC13): δ 0.8-1.0 (m, 2H), 1.1-1.7 (m, 19H',
1.79 (bd, IH) , 2.3-2.5 (m, 2H; thereof 2.38 (bd, IH)), 3.00 (bt, IH), 3.1-3.4 (m, 5H; thereof 3.38 (d, IH)) 3.58 (d, IH? ,
3.6- 3.7 (m, 2H) , 5.06 (dd, 2H) , 5.07 (s, 2H) , 5.16 (bs, IH, ,
6.7- 7.1 (b, IH), 7.15 (bs, LH), 7.2-7.4 (m, 10H).
i3C-NMR (125 MHz, CDC13) guanidine and carbonyl carbons: δ 176.0, 173.6, 170.8, 163.3, 161.7.
(iia) HOOC-CH2-(R)Cha-Pic-Nag x 2 HCl
Deprotection procedure (a) followed by purification with RPLC using CH3CN/0.1 M NH^OAc , 1/3 as eluent, evaporation at 40-50θ C and freeze drying gave the title compund as the acetate. Treatment with a 20-fold excess of hydrochloric acid, evaporation and renewed freeze drying gave the bis-hydrochloride of the desired compound.
XH-NMR (500MHz, D2O, mixture of two rotamers) : δ 0.7-2.0 (m, 20H), 2.17 (bd, IH), 2.95 (t, minor rotamer), 3.17 (t, 2H) ,
3.25-3.35 (m, 3H), 3.72 (bd, IH), 3.86 (dd, minor rotamer),
3.90 (s, 2H), 4.72 (t, IH), 4.99 (bs, IH).
X3C-NMR (75 MHz, D2O); guanidine δ 157.4; carbonyl carbons δ
169.9, 170.2, 173.0.
(iib) HOOC-CH2-(R)Cha-Pic-Nag x 2 HBr
BnOOC-CH2-(R)Cha-Pic-Nag(Z) was dissolved in 1Pr-OH/H2O (95/5) and hydrogenated over 5% Pd/C at atmospheric pressure in the presence of HBr (2.2 eq.). The catalyst was filtered off and the solvent evaporated to give a yellow oil
BAD ORIGINAL (Alternatively, the acid can be added after hydrogenation and filtration). Crystallisation from ^-Pr-OH (or EtOH)/EtOAc (1/1) gave the title compound as a white crystalline powder.
^H-NMR (500 MHz, D2O, mixture of two rotamers): δ 1.15-2.0 (m, 20H), 2.30 (bd, IH) , 3.30 (m, 2H), 3.40-3.50 (m, 3H) , 3.85-3.90 (m, IH), 3,95 (apparent s, 2H), 4.75-4.85 (m, IH, partially hidden by the H-O-D line), 5.10 (bs, IH).
l^C-NMR (125 MHz, D2O): guanidine: δ 157.6; carbonyl carbons: δ 169.7, 170.2, 173.0.
Example 68
MeOOC-CH2-(R)Cha-Pic-Nag x 2 TFA
The methyl ester MeOOC-CH2-(R)Cha-Pic-Nag(Z) was obtained by trans esterification of 1PrOOC-CH2-(R)Cha-Pic-Nag(Z) (See Example 69) on the column ddring flash chromatography when
CH2Cl2/MeOH was used as eluent. The title compound was obtained by the deprotection procedure (a).
1H-NMR (500 MHz, MeOD); 6 0.95-1.15 (m, 2H), 1.2-1.6 (m, 6H),
1.65-2.0 (m, 13H), 2.25 (bd, IH), 3.21 (t, 2H), 3.30 (t, 2H),
3.37 (m, IH), 3.71 (m, IH), 3.83 (s, 3H), 3.97 (dd, 2H), 4.67 (bt, IH), 5.05 (bs, IH).
^3C-NMR (125 MHz, MeOD), guanidine: δ 158.0; carbonyl carbons: δ 173.0, 171.1, 168.3.
Example 69 iPrOOC-CH2-(R)Cha-Pic-Nag X 2 TFA
Alkylation as described in Example 4 using
H-(R)Cha-Pic-NagiZ) (See Example 65) and Br-CH2-COOiPr followed by deprotection procedure (a) gave the title bad original compound .
^H-NMR (500 MHz, MeOD) : δ 0.95-1.1 (m, 2H), 1.15-1.6 (m,
12H; thereof 1.25 (d, 3H), 1.28 (d, 3H)), 1.65-1.95 (m, 12H), 2.28 (bd, IH), 3.21 (t, 2H), 3.30 (t,2H), 3.36 (m, IH) , 3.93 (dd, 2H), 4.67 (t, IH), 5.04 (bs, IH) , 5.11 (pentet, IH) .
13C-NMR (125 MHz, MeOD), guanidine: δ 157.9; carbonyl carbons: δ 173.1, 171.0, 168.3.
Example 70
HOOC-CH2-(Me)(R)Cha-(RorS)Pic-Nag/b x 2 TFA
Alkylation as described in Example 4 using
Me-(R)Cha-(R,S)Pic-Nag(Z) (See Example 66) and Br-CH?-COOBn followed by deprotection procedure (b) gave
HOOC-CH2-(Me)(R)Cha-(R,S)Pic-Nag. The two diastereomers where separated by RPLC (CH3CN/NH4OAc, 1:3) followed by freeze-drying from H2O/TFA. This diastereomer came out last of the two from the column.
2H-NMR (500 MHz, MeOD): δ 0.9-1.1 (m, 2H), 1.15-1.35 (m, 4H),
1.4-1.55 (m, 2H), 1.6-1.85 (m, 12H), 2.3 (m, IH), 2.85 (s,
3H), 3.15-3.45 (m, 5H), 3.65 (bs, 2H), 4.0 (m, IH), 4.65 (m, IH), 5.08 (dd, IH).
13C-NMR (75 MHz, D2O): guanidine: δ 157.65; carbonyl carbons: δ 169.86 and 172.48.
Example 71
HOOC-(R,S)CH(Me)-(R)Cha-(R,S)Pic-Nag x 2 TFA
Alkylation as described in Example 4 using H-(R)ChaPic-Nag(Z) (See Example 65) and Br-CH(Me)-COOBn followed by deprotection procedure (a) gave the title compound as a
BAD ORIGINAL mixture of four diastereomers.
Example 72
HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/c x 2 TFA
Obtained by separating the diastereomers formed in Example 71 using RPLC (CH3CN/NH4OAc (0.1M), 1/4) followed by evaporation and freeze-drying from H2O/TFA. This diastereomer came out as the third one of the four from the column.
'•H-NMR (300 MHz, D2O, 2 rotamers ca: 5:1 ratio): δ 0.88 (m, minor rotamer), 0.98-1.63 (m, 7H) , 1.63-2.02 (m, 16H; thereof 1.68 (d,3H), 2.28 (m, IH), 3.10 (t, minor rotamer), 3.25-3.50 (m, 5H; thereof 3.33 (t,2H) and 3.43 (t, 2H)), 3.82 (bd, IH) , 4.02 (q, IH), 4.55 (d, minor rotamer), 4.65 (d, minor rotamer), 4.72 (m, IH), 5.10 (m, IH).
Example 73
HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/d x 2 TFA
Obtained by separating the diastereomers formed in Example 71 using RPLC (CH3CN/NH4OAc (0.1 Μ), 1:4) followed by evaporation and freeze-drying from H2O/TFA. This diastereomer came out last of the four diastereomers from the column.
'H-NMR (500 MHz, D2O, 2 rotamers ca: 5:1 ratio): δ 0.80 (m, minor rotamer), 0.90 (m, minor rotamer), 1.03 (m, 2H), 1.10-1.33 (m, 3H), 1.42 (m, 2H), 1.51-1.92 (m, 16H; thereof 1.57 (d, 3H)), 2.18 (d, IH), 2.24 (d, minor rotamer) , 2.98 (t, minor rotamer), 3.21 (t, 2H), 3.28-3.40 (m, 3H; thereof 3.44 (t, 2H)), 3.82 (d, IH), 4.02 (q, IH), 4.42 (d, minor rotamer), 4.50 (t, minor rotamer), 4.62 (t, IH), 4.67 (s, minor rotamer), 5.03 (s, IH).
Example 74 r
BAD .ORlGlNA*33
HOOC-CH2-CH2-(R)Cha-Pic-Nag x 2 TFA
Prepared from H-(R)Cha-Pic-Nag(Z) (See Example 65) in the same wa/ as described for HOOC-CH2-CH-> - ( R j Cha-Pro-Agm in Example 15 using 1.2 eq. of benzylacrylate insted of 1.1 eq.
1H-NMR (500 MHz, D2O, rotamers ca: 4:1 ratio): δ 0.7-0,9 (m, minor rotamer) , 0.9-1.0 (m, 2H) , 1.05-1.3 (m, 3H) ,
1.3-1.45 (m, 2H) , 1.5-1.8 (m, 13H) , 2.10 (d, IH) , 2.20 (d, minor rotamer), 2.75 (t, 2H), 2.95 (t, minor rotamer), 3.15 (t, 2H), 3.2-3.35 (m, 5K), 3.75 (d, IH) , 4.55 (t, IH) , 4.95 (m, IH).
'3C-NMR (75 MHz, D2O) : guanidine: δ 157.57 ; carbonyl carbons: 5 170.16, 172.32, 174.75.
Example 75
HOOC-CH2-(R)Cha-(R,S)Mor-Agm x 2 TFA (i) H-(R)Cha-Mor-Agm(Z)
Prepared from Boc-(R)Cha-Mor-OSu (See Prep, of starting materials) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(R)Cha-(R,S)Mor-Agm x 2 TFA
Alkylation as in Example 4 using Br-CH2COOBn followed by deprotection procedure (b) gave the title compound. An epimerization of Mot had occured somewhere during the synthesis and a mixture of about 9:1 of two diastereomers was observed in the final product.
^H-NMR (300 MHz, MeOo;: δ 0.92-1.95 (m, 17 Η), 3.12-3.39 (m, 4H) , 3.44-4.05 (m, 7H) , 4.37 (d, IH), 4.63 (m, IH), 4.79 (bd, IH) .
BAD ORIGINAL i3C-NMR (75.47 MHz, MeOD): guanidine: δ 158.63; carbonyl carbons: δ 170.87, 170.82, 169.08 others: δ 69.06, 67.01 (C-O-C).
Example 76
HOOC-CH2-(R)Cha-(RorS)Mor-Nag x 2 TFA (i) H-(R)Cha-Mor-Nag(Z)
Prepared from Boc-(R)Cha-Mor-OSu (See Prep, of starting materials) and Boc-Nag(Z) in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.
(ii) HOOC-CH2-(R)Cha-(RorS)Mor-Nag x 2 TFA
Alkylation as described in Example 4 using Br-C^COOBn followed by deprotection procedure (b) gave the title compound.
1H-NMR (300 MHz, MeOD): δ 0.92-1.13 (m, 2H) , 1.15-1.42 (m,
3H) , 1.50 (br.s, IH), 1.62-1.95 (m, 9H) , 3.14-3.40 (m, 4H),
3.46-4.13 (m, 7H), 4.41 (d, IH), 4.63 (m, IH), 4.80 (br.d,
IH) .
’3C-NMR (75.47 MHz, MeOD): guanidine: δ 158.68; carbonyl carbons: δ 171.19, 170.90, 169.46. others: δ 68.81, 67.00 (C-O-C).
Example 77
H-(R)Cha-Aze-Nag x 2 HOAc (i) Boc-(R)Cha-Aze-Nag(Zi
Prepared from Boc-(R)Cha-Aze-OH in the same way as described for Boc-(R)Cha-Pic-Nag(Z) according to Example 65 (ic).
BAD ORIGINAL &
(ιι: Η-(R) Cha-Aze-Nag(Ζ)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z iSee Example 3).
(in) H-(R)Cha-Aze-Nag x 2 HOAc
Prepared by using the deprotection procedure (a) on the product (ii) above.
| 'H-NMR (300 MHz, D2O) : δ 0.85-1.10 im, | 2H), 1.10-2.04 (m, |
| 13H) 1.95 (s, acetate), 2.20-2.37 (m, | IH), 2.60-2.82 (m, IH |
| 3.15-3.40 (m, 4H), 3.96-4.15 (m, 2H) , | 4.18-4.30 (m, IH), |
| 4.30-4.42 (m, IH) , signals of a minor | rotamer appears at: δ |
| 0.70, 3.90 and 5.10. |
'•3C-NMR (75 MHz, D2O) : guanidine: δ 167.39 and carbonyl carbons: δ 170.22 and 172.38.
Example 78
HOOC-CH2-(R)Cha-Aze-Nag x HOAc (i) BnOOC-CH2-(R)Cha-Aze-Nag(Z)
Prepared from H-(R)Cha-Aze-Nag(Z) (See Example 77) according to the procedure described in Example 4.
(ii) HOOC-CH2-(R)Cha-Aze-Nag x HOAc
Prepared by using the the deprotection (a) on the product (i) above.
'H-NMR (500 MHz, MeOD): δ 0.90-1.10 (m, 2H) , 1.15-2.00 (m,
13H) 1.95 (s, acetate), 2.20-2.30 (m, IH), 2.58-2.70 (m, IH),
3.17-3.30 (m, 4H), 3.35-3.50 (m, 2H), 3.55-3.68 (m, IH),
BAD ORIGINAL
4.13-4.20 (m, IH), 4.30-4.38 (m, IH), 4.05-4.77 (m, IH), signals of minor rotamer appears at: δ 3.75, 3.98, 4.03 and 5 . 23 .
ljC-MMR (75 MHz, D2O): guanidine: δ 157.40 and carbonyl carbons: δ 169.16·, 171.92 and 172.13.
Example 79
H-(R)Cha-Pro(5-(S)Me)-Nag x 2 HCl (i: 3oc-(R)Cha-Pro(5-(S)Me)-Nag(Z)
The same procedure as described for the coupling between Boc(R)Cha-OH and H-Pic-OEt x HCl (See Preparation of Starting Macerials) was used to accomplish the coupling between Boc(?.) Cha-Pro ( 5-(S)Me) -OH and H-Nag(Z) x 2 HCl.
(ii) H-(R)Cha-Pro(5-(S)Me)-Nag(Z)
The same procedure as described for the synthesis of H-(R)Cgl-Pic-Nag (Z) (See Example 84 (ii) was used.
(iii) H-(R)Cha-Pro(5-(S) Me)-Nag x 2 HCl
Prepared by using the deprotection procedure (d) on the product (ii) above.
1H-NMR (300 MHz, D2O): δ 1.0-2.3 (m, 21H); thereof 1.47 (d, 3H), 2.4-2.55 im, IH) , 3.3-3.6 (m, 4H) , 4.30 (bt, IH) , 4.38 (dd, IH), 4.47 (bt, IH) .
l^c-NMR (75 MHz, D2O): guanidine: δ 157.6 carbonyl carbons: δ 174.6, 169.6.
bad ORIGINAL
Hxample 3ι
HOOC-CH2-(R)Cha-Pro(5-(S)Me)-Nag x HOAc
Alkylation as in Example 4 using H-(R)Cha-Pro(5-'3) Me)-Nag (z)
| (See Example 79) .and | Br-CH2 _COOBn followed by | deprotect ion | |
| procedure (a) gave the tide compound. | |||
| 1H-NMR | (300 MHz, D2O) | : δ 0.9-1.9 (m, 19H); thereof 1.34 ίbd | |
| 3H), 1. | 93 is, acetate | ), 2.0-2.2 (m, 3H) , 2.34 | (m, 1H), 3.1- |
| 3.5 (m, | 7H) , 3.97 (m, | IK) , 4.20 (m, IHi , 4.31 | ( be , 1H) . |
| 13c-nmr | (75 MHz, D->0) | : guanidine: δ 157.4. | |
| Examp1e | 91 |
HOOC-CH2-(R)Cha-(RorS)Pic(4,5-dehydro)-Nag/b x HOAc (i) Boc-(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z)
Prepared from Boc-(R)Cha-(R,S) Pic (4,5-dehydro)-OH in the same way as described for Boc-(R)Cha-Pic-Nag(Z) (See Example 65 (ic)) .
(ii) H-(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) ( See Example 3) .
(iii) BnOOC-CH2-· (R) Cha - (R, S)Pic(4,5-dehydro) -Nag(Z)
Prepared from H-(R)Cha-(R,S) Pic (4,5-dehydro)-Nag (Z) according to the procedure described in Example 4.
(iv) HOOC-CH2-(R)Cha-(RorS)Pic(4,5-dehydro)-Nag/b x HOAc
A mixture of 356 mg (0.539 mmol) of BnOOC-CH2-(R)Cha-(R,S)
BAD ORIGINAL π in
Pic(4,5-dehydro)-Nag(Ζ), 10.3 mL trifluoroaceticacid and 3.4 ml tioanisole was stirred at room temperature for 3.5 h.
Water was added and the mixture was washed twice with CH^Ci evaporation of the solvent gave HOOC-CH2-(R)Cha-(R,S)Picn, dehydro)-Nag. The title compound was obtained by separating the diastereomers by RPLC (CH2CN/NH4OAc (0.1 M), 3/7) and freeze drying (H2O) after evaporation of the solvent. The diastereomer came out last of the two from the column.
1H-NMR (300 MHz, D2O) δ 0.85-1.95 (m, 15H), 2.50-2.80 (m,
2H), 3.25 (t, 2H), 3.35 (t, 2H), 3.55 (bs, 2H), 3.85-4.6 im, 3H), 4.92 (minor rotamer), 5.30 (d, IH), 5.85-6.1 (m, 2H), 33C-NMR (75 MHz, D2O): guanidine: δ 157.59; carbonyl carbons:
δ 171.46, 172.53, 173.03.
Example 82
HOOC-CH2-(R)Cha-Pic(4-(S)Me)-Nag x 2 HCl (i) Boc-(R)Cha-Pic (4-(S)Me)-Nag(Z)
Prepared from Boc-(R)Cha-Pic(4-(S)Me)-OH in the same way as described for Boc-(R)Cha-Pic-Nag(Z) according to method (ic) in Example 65.
(ii) H-(R)Cha-Pic(4-(S)Me)-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (See Example 3) .
(iii) BnOOC-CH2-(R)Cha-Pic(4-(S)Me)-Nag(Z)
Prepared from H-(R)Cha-Pic(4-(S)Me)-Nag(Z) according to the procedure described in Example 4.
baDORIG‘nAL &
(iv! HOOC-CH2-(R)Cha-Pic(4- (S) Me)-Nag x 2 HCl
Precared by using the deprotection procedure (d) on the pre duct (111) above.
LH-NMR (500 MHz, D2O): δ 0.95-2.05 (m, 22H; thereof 1.05 (d, 3H) ), 2.30-2.38 (bd, IH) , 3.28-3.36 im, 2H) 3.36-3.50 (m,
3H) , 3.85-3.95 (m, IH) , 3.98 (s, 2H), 4.70-4.90 (m, IH; partly hidden behind the HOD signal), 5.22-5.27 (d, IH), signal of a minor roatmer appears at 5 0.93, 3.13 and 4.57.
-C-NMR (125 MHz, D2O): guanidine: δ 157.58; carbonyl carbons: δ 170.12, 170.32 and 172.82.
Example 83
HOOC-CH2-(R)Cha-(R)Pic(4-(R)Me)-Nag x 2 HCl ( i) Boc-(R)Cha-(R)Pic(4-(R)Me)-Nag(Z)
Prepared from Boc-(R)Cha-(R)Pic (4-(R)Me)-OSu and Boc-Nag(Z) in the same way as described for Boc-(R)Cha-Pro-Agm(Z) (See Examp1e 3) .
( ii) H-(R)Cha-(R)Pic(4-(R)Me)-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (See Example 3) .
(iii) BnOOC-CH2-(R)Cha-iR)Pic(4-(R)Me)-Nag(Z)
Prepared from H-(R)Cha-(R)Pic(4-(R)Me)-Nag(Z) according to the procedure described in Example 4.
(iv) HOOC-CH2-(R)Cha-(R)Pic(4-(R)Me)-Nag x 2 HCl
Prepared by using the deprotection procedure (d) on the
BAD ORIGINAL product (111) above.
“H-NMR (500 MHz, D2O): δ 1.00-2.05 (m, 22H), 2.18-2.26 (bd, IH) , 3.28 -3.3 6 (m, 2H) , 3.36-3.55 (m, 3H), 3.85-4.05 (m, 3H) ,
4.70-4.90 im, IH; partly hidden behind the HOD signal), 5.255.30 ;d, IH), signals of minor rotamer apppears at: δ 2.40, 2.90, 4.10, 4.42, 4.55 and 5.23.
^3C-NMR (125 MHz, D2O) : guanidine: δ 157.56: carbonyl 10 carbons: δ 169.69, 169.84 and 173.20.
Example 84
HOOC-CH2-(R)Cgl-Pic-Nag x 2 HCI (i) See-(R)Cgl-Pic-Mag(Z)
Prepared from Boc-(R)Cgl-Pic-OH in the same way as described for Boc-(R)Cha-Pic-Nag(Z) according to method (ic) in Example
0 6 5 .
1H-NMR (300 MHz, CDC13): δ 0.9-1.8 (m, 27H), 2.4 (d, IH),
3.1- 3.3 (m, 5H), 3.9 (d, IH), 4.2 (t, IH), 5.1 (s, 2H) , 5.2 (bd, 2H) , 6.7-7.4 (m, 9H) .
(ii) H-(R)Cgl-Pic-Nag(Z)
Gaseous hydrogen chloride was bubbled through a solution of Boc-(R)Cgl-Pic-Nag(Z) (1.38 g, 2.22 mmol) in ethyl acetate (25 ml). After 10 minutes the solvent was evaporated and the residue was dissolved in ethyl acetate and 10% Na2CO3.
The organic phase was separated, washed with brine and dried (MgSO4). Evaporation of the solvent gave 1.02 g (92%) of the title compound.
1H-NMR (300 MHz, MeOD): 6 1.0-1.9 (m, 18H), 2.2-2.3 (m, IH),
3.2- 3.3 (m, 5H), 3.6 (d, IH), 3.8-3.9 (bd, IH), 4.2 (t, IH), bad original
4.7-4.3 (bs, 5H), 5.1 (s, 2H), 5.2 (s, IH), 7.2-7.3 (tn, 5 H) .
(nr) 3nOOC-CH2-(R) Cgl - Pic -Nag (Z)
A solution of the triflate ester of benzyl glycolate (291 mg, 0.98 mmol) in CH2C12 (2 ml) was added at -25° C to a stirred mixture of H-(R)Cgl-Pic-Nag (Z) (0.52 g, 1.04 mmol) and K2COq (494 mg, 3.58 mmol) in acetonitrile (5 ml) and CH2C17 ι1 ml'. The temperature was allowed ro reach room temperature during a couple of hours and after 5 days the reaction mixture was diluted with water and extracted with EtOAc and toluene. Drying of the organic phase (MgSO^) and concentration of she solution gave 319 mg (47%) of colorless crystals .
1H-NMR (500 MHz, CDCI3): δ 1.0-1.1 (m, IH) , 1.1-1.3 (m, 4H) ,
1.35-1.6 (m, 5H), 1.6-1.85 (m, 8H), 1.8-2.2 (bs, IH), 2.222.5 (m, 2H), 2.9 (t, IH), 3.1-3.5 (m, 6H) , 3.6-3.7 (m, 2H) , 5.0-5.1 (m, 4H), 5.2 (s, IH), 6.5-7.4 (m, 13H) .
(iv) HOOC-CH2-(R)Cgl-Pic-Nag x 2 HCI
BnOOC-CH2-(R)Cgl-Pic-Nag (Z) (319 mg, 0.49 mmol) was dissolved by heating in isopropanol (50 ml) and water (5 ml) and hydrogenated for 24 h over 10% Pd/C (228 mg). After filtration and evaporation of the solvent and susequent dissolution in dilute hydrochloric acid followed by freeze drying, the peptide (223 mg, 91%) was isolated as a white powder .
1H-NMR (500 MHz, D2O): δ 1.1-2.1 (m, 18H) 2.3 (d, IH), 3.3 (t, 2H), 3.4 (t, 3H), 3.85-4.05 (m, 3H), 4.6 (d, IH), 5.15 (S, IH).
-'-^C-NMR (75 MHz, D2O) : guanidine: δ 157.43 carbonyl carbons: δ 169.2, 172.94.
Examp1e 85
BAD ORIGINAL
H-(R)Hoc-Pro-Nag χ 2 TFA (i) Boc-(R)Hoc-Pro-Nag(Z)
Prepared from Boc-(R)Hoc-Pro-OH in the same way as described for Boc-(R)Cha-Pic-Nag(Z) according to Example 65 (ic).
(ii) H-(R)Hoc-Pro-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Pro-AgrmZ) ( See Example 3) .
(iii) H-(R)Hoc-Pro-Mag x TFA
Prepared by using the deprotection procedure (a) on the product (ii) above.
1H-NMR (300 MHz, D2O): δ 0.90-1.05 (m, 2H), 1.16-1.48 (m,
6H), 1.48-1.84 (m, 6H) , 1.84-2.24 (m, 6H), 2.40 (m, IH),
3.25-3.45 (m, 4H), 3,74 (m, IH) , 3.85 (m, IH), 4.42 (m, IH),
4.51 (m, IH) .
Examp1e 86
HOOC-CH2-(R)Hoc-Pro-Nag x HOAc (i) BnOOC-CH2-(R)Hoc-Pro-Nag(Z)
Prepared from H-(R)Hoc-Pro-Nag(Z) (See Example 85) according 30 to the procedure described in Example 4.
(ii) HOOC-CH2-(R)Hoc-Pro-Nag x HOAc
Prepared by using the deprotection procedure (a) on the 35 product (i) above.
1H-NMR (300 MHz, D2O): δ 0.76-0.97 (m, 2H), 1.00-1.37 (m,
BAD ORIGINAL &
| 6 H i , 1.50-2.12 (m, 12H) | 1.89 (s, acetate), 2.27 (m, | IH) , |
| 3.::-1.33 (m, 4H), 3.41 | (bs, 2H), 3.51 (m, IH), 3.77 | (m, IH) |
| 4.12 m, IH), 4.37 (m, | IH) . | |
| L3C-?2<R (75 MHz, D2O) : | guanidine: δ 157.4; carbonyl | carbons |
| δ 1?.3, 173 .9, 174.5. | ||
| Example 37 |
HOOC-CH2-(R)Hoc-Pic-Nag x HOAc (i) Boc-(R)Hoc - Pic-Mag (Z)
Prepared from Boc-(R)Hoc-Pic-OH in the same way as described for Boc-(R)Cha-Pic-Mag (Z) according co method (ic) in Example 65 .
(ii) H-(R)Hoc-Pic-Nag (Z)
Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (See Example 3) .
(iii) BnOOC-CH2-(R)Hoc-Pic-Nag(Z)
Prepared according to the procedure described in Example 4.
(iv) HOOC-CH2-(R)Hoc-Pic-Nag x HOAc
Prepared by using the deprotection procedure (a) on the product (iii) above.
| XH-NMR (300 MHz | , D2O): | δ 0.75-0.95 | (m, 2H), | 1.00-1.30 | (m, |
| 6H), 1.30-1.50 | (m, 2H), | 1.50-1.82 | (m, 12H) , | 1.82-1.95 | (bs, |
| acetate), 2.23 | (bd, IH) | , 3.08-3.32 | (m, 6H), | 3.52 (bs, | 2H) , |
3.77 (bd, IH), 4.50 (bs, IH), 5.00 (bs, IH).
Example 88
BAD ORIGINAL
HOOC-CH2-(R)Dph-Pic-Nag x 2 HCl l·!) Boc-(RiDph-Pic-Nag(Z)
Prepared from Boc-(R)Dph-Pic-OH in the same way as described for Boc-(R) Cha-Pic-Nag (Z) (See Example 65 (ic)).
(ii) H-(R)Dph-Pic-Nag(Z)
Prepared in the same way as described for H-(R)Cgl-Pic-Nag(Z) (See Example 34 (ii)) .
( iii) BnOOC-Ch’2 - (R) Dph-Pic-Nag (Z)
Prepared from H-(R)Dph-Pic-Nag(Z) according to the procedure described in Example 4.
(iv) HOOC-CH2-(R)Dph-Pic-Nag x 2 HCl
Prepared by using the deprotection procedure (d) on the product (iii) above.
1H-NMR (500 MHz, D2O) : δ 0.46 (m, IH) , 1.2-1.35 (m, 2H) , 1.45 (m, IH), 1.53 (m, IH), 1.89 (pentet, 2H), 2.03 (bd, IH) , 3.24 (bt, IH), 3.29 (t, 2H), 3.38 (t, 2H), 3.72 (d, IH), 3.78 (d, IH), 3.79 (m, IH), 4.68 (d, IH), 4.89 (m, IH), 5.73 (d, IH) ,
7.4-7.6 (m, 6H), 7.65 (t, 2H), 7.81 (d, 2H).
Example 89
HOOC-CH2-(R)Dch-Pic-Nag x HOAc (i) Boc-(R)Dch-Pic-Nag(Z)
Prepared from Boc-(R)Dch-Pic-OH in the same way as described for Boc-(R)Cha-Pic-Nag(Z) (in Example 65 (ic).
BAD ORIGINAL &
100 (ii) Η-(R)Dch-Pic-Nag (Z)
Prepared in the same way as described for H-(R)Cgl-Pic-Nag(Z) (in Example 84 (ii).
(in ) BnOOC-CH2- (R) Dch-Pic-Nag (Z)
Prepared from H-(R)Dch-Pic-Nag (Z) according to the procedure described in Example 4.
(iv) HOOC-CH2-(R)Dch-Pic-Nag x HOAc
Prepared by using the deprotection procedure (a) on the product (iii) above.
1H-NMR (500 MHz, D20): δ 1-2-2.0 (m, 30H), 2.09 (s, acetate), 2.30 (bd, IH), 3.32 (t, 2H), 3.4-3.5 (m, 3H), 3.65 (d, IH), 3.70 (d, IH), 3.86 (bd, IH), 4.86 (m, IH), 5.09 (m, IH).
l^c-NMR (125 MHz, D2O): guanidine: δ 159.4, carbonyl carbons: δ 172.5, 173.3, 174.9.
Example Pl
Solution for parenteral administration
A solution is prepared from the following ingredients:
HOOC-CH2-(R)Cha-Pic-Nag x 2HBr 5 g
Sodium chloride for injection 9 g
Acetic acid 3 g
Water for inj. up to 1000 ml
The active constituent, the sodium chloride and the acetic acid are dissolved in the water. The pH is adjusted with 2 M NaOH to pH 3-7. The solution is filtered through a sterile 0.2 gm filter and is aseptically filled into sterile
BAD ORIGINAL
101 ampoules .
Example P2 o Tablets for oral administration
1000 tablets are prepared from the following ingredients:
Thrombin inhibitor 100 g 10 Lactose 200 g
Polyvinyl pyrrolidone 30 g Microcrysta.line cellulose 30 g Magnesium stearate 6 g
The active constituent and lactose are mixed with an aqueous solution of polyvinyl pyrrolidone. The mixture is dried and milled to form granules. The microcrystalline cellulose and then the magnesium stearate are then admixed. The mixture is then compressed in a tablet machine giving 1000 tablets, each containing 100 mg of active constituent.
Biology
Determination of thrombin clotting time and ICgqTT:
Human thrombin (T 6769, Sigma Chem Co) in buffer solution, pH 7.4, 100 μΐ, and inhibitor solution, 100 μΐ, were incubated for one min. Pooled normal citrated human plasma, 100 μΐ, was then added and the clotting time measured in an automatic device (KC 10, Amelung).
The clotting time in seconds was plotted against the inhibitor concentration, and the ΙΟςθΤΤ was determined by interpolation.
IC^qTT is the concentration of inhibitor that doubles the , thrombin clotting time for human plasma. pICggTT is the
BAD ORIGINAL ii»
102
-leg 10 of IC5QTT in mol/1. The preferred compounds of the invention have an ρΙΟ^θΤΤ in the range 6.6 - 8.2.
Cezerminazion of Activated Partial Thromboplastin Time (APTT)
APTT was determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors were added to the plasma (10 μΐ inhibitor solution to 90 μΐ plasma) and APTT was determined in the mixture by use of the coagulation analyser KC10 (Amelung) according to the instructions of the reagent producer. The clotting time in seconds was plotted against the inhibitor concentration in pi asma and the IC^qAPTT was determined by interpolation.
IC^qAPTT is defined as the concentration of inhibitor in plasma that doubled the Activated Partial Thromboplastin Time. pIC50APTT is the -log 10 of IC5oAPTT in mol/1. Those of the preferred compounds of the invention that were tested showed a pIC^qAPTT of 5.1 - 6.4.
ABBREVIATIONS
| Agm = | Agmatine |
| Agm ( Z) = | ω-Ν-benzyloxycarbonyl agmatine |
| AAj_ = | Amino acid 1 |
| aa2 = | Amino acid 2 |
| Aze = | (S)-Azetidin-2-carboxylic acid |
| Bla = | α-substituted butyrolactone |
| Boc = | tertiary butoxy carbonyl |
| Brine = | saturated water/NaCl solution |
| Bu = | butyl |
| Bn = | benzyl |
| Cgl = | (S)-Cyclohexyl glycine |
| Ch = | cyclohexyl |
| Cha = | (S)-β-cyclohexyl alanine |
| CME-CDI = | 1-Cyclohexyl-3 - (2-morpholinoethyl) |
carbodiimide metho-p-toluenesulfonate
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103
| DCC = | dicyclohexyl carbodiimide |
| Dch = | (S)-Dicyclohexyl alanine |
| DMAP = | Ν,Ν-dimethyl amino pyridine |
| DMF = | dimethyl formamide |
| 5 DMSO = | dimethyl sulphoxide |
Dph = (S)-Diphenyl alanine
EDC = 1- (3-Dimetylaminopropyl)-3-ethylcarbodiimide hydrochloride
| Et = | ethyl | |
| 10 | EtOAc = | ethyl acetate |
| HOAC = | acetic acid | |
| HOBt = | N-hydroxy benzotriazole | |
| Hoc = | (S)-Homocyclohexyl alanine | |
| Hop = | (3)-Homophenyl alanine | |
| 15 | HOSu = | N-hydroxysuccinimide |
| Mag = | miniagmatine | |
| Me = | methyl | |
| Mor = | ( S)-morpholine-2-carboxylic acid | |
| Mpa = | mega pascal | |
| 20 | Nag = | noragmatine |
| Nag(Z) = | δ-Ν-benzyloxycarbonyl-noragmatine | |
| NMM = | N-methyl morpholine | |
| Pgl = | (S)-phenyl glycine | |
| Ph = | phenyl | |
| 25 | Phe = | (S)-phenyl alanine |
| Pic = | (S)-pipecolinic acid | |
| Pr = | propyl | |
| Pro = | (S)-proline | |
| RPLC = | reverse phase high- performance liquid | |
| 30 | chromatography | |
| Tf = | trifluoromethyl sulphonyl | |
| TFA = | trifluoracetic acid | |
| THF = | tetrahydrofuran | |
| p-TsOH = | para-toluenesulfonic acid | |
| 35 | Val = | (S)-valine |
| Z = | benzyloxy carbonyl |
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104
Prefixes η, 3, 1 and t have their usual meanings: normal, iso, sec and tertiary.
BAD original J}
105
Scheme I (Example 3-18,20-21,24-28,30-34,36-40,43-49,
51-53,57-64 and 67-93)
Boc-AApAAg-OH
Coupling with H-Nag(Z) or H-Agm(Z).
Boc-AA1-AA2-Nag(Z) (or Agm(Z))
1. Deprotection of the N-terminal.
2. Reaction of the N-terminal with an electrophile (See each specific example , for detailed information).
AA, - H-(R)Cha-OH, Me-(R)Cha-OH, H-(R,S)Pro(3-(trans)Ph)-OH, H-(R)Hoc-OH, H-(R)Cgl-OH, H-(R)Dph-OH, H-(R)Dch-OH
AA2- H-Pro-OH, H-Pic-OH, H-Mor-OH, H-Aze-OH, H-Pic(4-{S)Me)-OH H-Pic(4-(R)Me)-OH. H-(R,S)Pic(4,5-dehydro)-OH, H-(R)Pic(4-(R)Me)-OH, H-Pro(5-(R,S)Me)-OH, H-Pro(5-(S)Me)-OH, H-Pic(6-{S)Me)-OH
The N terminal group in the final compound «
H, HO-(CH2)3-, nBu-. HOOC-CHj, MeOOC-CHr, 'PiOOC-CHr, ’BuOOC-CHz-,
HOOC-CH(Me)-, HOOC-CHfPr)·, HOOC-CH(Ph)-, HOOC-CH(CH2CH2Ph)HOOC-CH2CH2-, HOOC-CHgCHgCHg-tEtOOC-CHgCHjCHj-, Bla,
HOOC-CHgOOOCHj-, EtOOC-CO. MeOOC-CO, HOOC-CO·. HaNOC-ChV
HOOC-Cl-KCHaCOOH)-, MeOOC-CHiCHaCOOMe), HOOC-CHyNH-CGCHr,
HOOC-CH(CH2OH)-, (HO)2P(O)-CH2-i EtO(HO)P(O)-CHjj-, (EtO)2P(O)-CH2-,
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106
Scheme II ( Example 55,56,65 and 66 )
Boc-(R,S)Pro(3-Ph)-OH
1. H-Pro-OBn, HOBt, NMM.DMF
2. H2, Pd/C
3. HOSu.CME-CDI.CH3CN
H-Agm(Z),NMM
Boc-(R,S)Pro(3-Ph)-Pro-OSu DMF.r.t
Boc-(R,S)Pro(3-Ph)-Pro-Agm(Z)
1. TFA,CH2CI2
2. H2,Pd/C
H-(R,S)Pro(3-Ph)-Pro-Agm Example 55
Boc-(R,S)Pro(3-(trans)Ph)-OH
Rh/AI2O3,H2i
HOAc, MeOH
Boc-(R)Cha-OH
- Boc-(R,S)Pro(3-(trans)Ch)-OH
See Example 55 (above) +
H-(R,S)Pro(3-(trans)Ch)>Pro-Agm
1. HOBt,CME-CDI,CH^ Example 56
2. HCIxH-Pic-OEt,NMM.DMF
3. LiOH,THF,HgO
4. HOSu.DCC.DMF
Boc-(R)Cha-Pic-OSu or
Boc-(Mej(R)Cha-(R,S)Pic-OSu
1. HOBt,CME-CDI,CH2Cl2
2. HCIxH-Pic-OEt,NMM.DMF
3. LiOH,THF,H2O
4. HOSu.DCC.DMF
Boc-(Me)(R)Cha-OH
H-Nag(Z),NMM,
DMF.r.t.
Boc-(R)Cha-Pic-Nag(Z) or
Boc-(Me)(R)Cha-(R,S)Pic-Nag(Z)
1. TFA
2. H2,Pd/C
H-(R)Cha-Pic-Nag (Example 65) or
Me-(R)Cha-(R,S)PiC’Nag Example 66
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107
Scheme III (Example 1 and 2)
H-AgmxHCI,Et3N, Boc-(R)Cha-Pro-OSu DMF.f.t.
Boc-(R)Cha-Pro-Agm
TFA
H-(R)Cha-Pro-Agm Example 1
H-AgmxHCI.NMM
Boc-(Me)(R)Cha-Pro-OSu -►- Boc-(Me)(R)Cha-Pro-Agm
DMF.r.l.
TFA
Me-(R)Cha-Pro-Agm Example 2
Scheme IV (Example 19)
H2N-(CH2)3-NH(Boc)
Z-(R)Cha-Pro-OSu -► Z^RJCha-Pro-NH-iCHJa-NHtBoc)
NMM.DMF.r.t.
TFA
Z-<R)Cha-Pro-NH-(CH2)3-NH2
1. 3,5-Dimethyl-l-pyrazolylformamidiniiim nitrate ,Et3N,DMF
2. H2,Pd/C
H-(R)Cha-Pro-Nag Example 19
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108
Scheme V (Example 54)
Boc-(R)Cha-Pro-OSu
H2N-(CH2)2-NH(Z),
NMM.DMF.r.t.
Boc-(R)Cha-Pro-NH-{CH2)2-NH(Z)
1. TFA
2. BrCH2COOEt,CH3CN,K2CO3
EtOOC-CH2-(R)Cha-Pro-NH-(CH2)2-NH(Z)
1. H2,Pd/C
2. 3,5-Dimethyl-1-pyrazolylformamidinium nitrate,Et3N,DMF
3. NaOH/EtOH
HOOC-CH2-(R)Cha-Pro-Mag Example 54
BAD ORIGINAL \ / Jo
109
Scheme VI (Example 22,23,29,35,41,42 and 50)
HOOC-CHj-(R)Cha-Pro-Nag Example 22
NaOH/EtOH
EtOOC-CH2-(R)Cha-Pro-Nag Example 23
1. BrCH2COOEt.K2CO3,CH3CN
2. H;.Pd/C
3. 3,5-Dimethyl-1-pyrazolylformamidinium nitrate,Et3N.DMF (HOOC-CH^^RjCha-Pro-Nag Example 29
NaOH/EtOH (EtOOC-CH2)2-(R)Cha-Pro-Nag
1. BrCH2COOEt(excess),K2CO3,CH3CN ‘
2. H2,Pd/C
3. 3.5-Dimethyl-1-pyrazolylformamidinium nitrate,Et3N,DMF
NMM.DMF
-H-(R)Cha-Pro-NH-(CH2)3-N3
Boc-(R)Cha-Pro-OSu
1. BrCH(Me)COOEt,K2CO3,CH3CN
2. H2,Pd/C
3. 3,5-DimethyM-pyrazolyl' tormamidinium nitrate,Et3N,DMF EtOOC-(R.S)CH(Me)-(R)Cha-Pro-Nag
Example 35
1. CHj-CHCOOELEtOH
2. H2,Pd/C
3. 3,5-Dimethyl-l-pyrazolyl(ormamidimum nitrate, Et3N,DMF
H-(R)Cha-Pro-NH-(CH2)3-N3
1. lBuOOC-Ph-4-CH2Br,K2CO3,CH3CN
2. bis-phenylthio stannane,PhSH,Et3N
3. 3,5-Dimethyl-1-pyrazolyl(crmamidinium nitrate,Et3N,DMF
EtOOCCHsjCHyCRJCha-Pro-Nag (Example 42 NaOH/EtOH
HOOCCH2CH2-(R)Cha-Pro-Nag
Example 41 'BuOOC-Ph-4-CH^fRjCha-Pro-Nag
TFA
HOOC-PM-CH^RlCha-Pro-Nag Example 50
Claims (33)
1. A compound of the general formula
Formula I wherein :
A represents a methylene group, or
5 A represents an ethylene group and the resulting 5-membered ring is unsubstituted, substituted by one or two fluorine atoms, a hydroxy group or an oxo group in position 4, or may or may not be saturated, or
A represents -CH2-O-, -CH2-S-, -CH2-SO~, with the heteroatom 10 functionality in position 4, or
A represents a n-propylene group wherein the resulting 6membered ring is unsubstituted, substituted in position 5 by one fluorine atom, a hydroxy group or an oxo group, in one of positions 4 or 5 by two fluorine atoms, unsaturated in position 4 and 5, or substituted in position 4 by an alkyl group with 1 to 4 carbon atoms, or
EeAD-ORlG^1
111
A represents -CH2-O-CH2~, -CH2-S-CH2-, -CH2-SO-CH2-;
Rl represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2 or 3 carbon atoms or RnOOCalkyl-, wherein the alkyl group has 1 to 4 carbon atoms and R“ is H or an alkyl group having 1 to 4 carbon atoms or an alkylene group having 2 or 3 carbon atoms intramolecularly bound alpha to the carbonyl group in R1, or
R1 represents R12OOC-l, 4-pheny 1-CH,-, wherein R12 is H or an alkyl group having 1 to 4 carbon atoms, or
R1 represents Rl3-NH-CO-alky 1-, wherein the alkyl group has 1 to 4 carbon atoms, which is unsubstituted or is substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms, and wherein R13 is H or an alkyl group having 1 to 4 carbon atoms or -CH2COOR12, wherein R12 is as defined above, or
R1 represents R12OOC-CH2-OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms, which is unsubstituted or substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms, and R12 is as defined above, or
R* represents CH3SO2-, or
R* represents RI2OCOCO- wherein R*2 is as defined above, or
R* represents -CH2PO (OR14) 2, -CH2SO3H or -CH2-(5-(IH)-tetrazolyl) wherein each R14, which may be the same or different, represents H, methyl or ethyl;
R2 represents H or an alkyl group having 1 to 4 carbon atoms or R21OOC-alkyl-, wherein the alkyl group has l to 4 carbon atoms, and is unsubstituted or substituted in the position
BAD ORIGINAL &
112 which is alpha to the carbonyl group, and the alpha substituent is a group R22-(CH2)p-, wherein p is 0 to 2 and R22 is methyl, phenyl, OH, COOR21, and R21 is H or an alkyl group having 1 to 4 carbon atoms;
m is 0, 1 or 2, R2 3 represents a cyclohexyl group and R4 represents H, or m is 1 and R3 represents a cyclohexyl or phenyl group and R4 forms an ethylene bridge together with R1, or m is 1 and R3 and R4 each represent a cyclohexyl or phenyl group;
R5 represents H or an alkyl group having 1 to 4 carbon atoms;
n is an integer 2 to 6; and
B represents -N (R6)-C (NH)-NH2, wherein R6 is H or a methyl group, or
B represents -S-C(NH)-NH2, or -C(NH)-NH3, either the compound as such or in the form of a physiologically acceptable salt and including stereoisomers.
2. A compound according to claim 1 wherein R* represents RuOOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and R“ is H.
3. A compound according to claim 2 wherein A is ethylene.
4. A compound according to claim 2 wherein A is npropylene and the resulting 6-membered ring is unsubstituted or substituted in position 4 by an alkyl group with 1 to 4 carbon atoms.
BAD ORIGINAL &
113
5. A compound according to any one of the preceding claims wherein m is 1 or 2, R3 is cyclohexyl, and R4 is H.
6. A compound according to any one of the preceding claims wherein n is 3.
7. A compound according to any one of the preceding claims having S-configuration on the α-amino acid in the P2 position .
8. A compound according to claim 7 having R-configuration on the α-amino acid in the P3 position.
9. A compound selected from
H-(R)Cha-Pro-Agm
Me-(R)Cha-Pro-Agm
HO-(CH2)3-(R) Cha-Pro-Agm ‘PrOOC-CH-,- (R) Cha-Pro-Agm
HOOC-(R,S)CH(Me)-(R)Cha-Pro-Agm
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/a HOOC- (RorS) CH (nPr) - (R) Cha-Pro-Agm/a HOOC-(RorS)CH (nPr)-(R)Cha-Pro-Agm/b HOOC-(RorS)CH(Ph)-(R)Cha-Pro-Agm/b HOOC- (R, S) CH (CH2CH2Ph) - (R) Cha-Pro-Agm HOOC- (RorS) CH (CH2CH2Ph) - (R) Cha-Pro-Agm/a HOOC-CH2-CH2- (R) Cha-Pro-Agm
EtOOC-CO-(R)Cha-Pro-Agm (R,S)Bla-(R)Cha-Pro-Agm
HOOC- (RorS) CH (CH2CH2Ph) — (R) Cha-Pro-Agm/b
H-(R)Cha-Pro-Nag
Bu- (R)Cha-Pro-Nag
HO-(CHJ3-(R)Cha-Pro-Nag
Et00C-CH2- (R) Cha-Pro-Nag 'PrOOC-CH2- (R) Cha-Pro-Nag *Buooc-ch2- (R) Cha-Pro-Nag
BAD ORIGINAL
114
HOOC-CH2-OOC-CH2- (R) Cha-Pro-Nag
H2N-CO-CH2- (R) Cha-Pro-Nag
HOOC-CH2-NH-CO-CH2- (R) Cha-Pro-Nag (HOOC-CH2) 2- (R) Cha-Pro-Nag
HOOC-CH2-(nBu) (R) Cha-Pro-Nag
HOOC-(R,S)CH(Me)-(R)Cha-Pro-Nag
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/a
EtOOC-(R,S)CH(Me)-(R)Cha-Pro-Nag
HOOC- (RorS) CH (nPr) - (R) Cha-Pro-Nag/a
HOOC- (R) CH (CH2-OH) - (R) Cha-Pro-Nag
HOOC-(R,S)CH(Ph)-(R)Cha-Pro-Nag
HOOC- (S) CH (CH2CH2Ph) - (R) Cha-Pro-Nag
HOOC- (R) CH (CH2CH2Ph) - (R) Cha-Pro-Nag
HOOC-CH2-CH2- (R) Cha-Pro-Nag
EtOOC-CH2-CH2- (R) Cha-Pro-Nag
HOOC- (CH3),- (R) Cha-Pro-Nag
EtOOC- (CH2),- (R) Cha-Pro-Nag
HOOC-CO-(R)Cha-Pro-Nag
MeOOC-CO-(R)Cha-Pro-Nag (R,S)Bla-(R)Cha-Pro-Nag
HOOC- (R, S) CH (CH2COOH) - (R) Cha-Pro-Nag
MeOOC- (R, S) CH (CH2COOMe) - (R) Cha-Pro-Nag
HOOC-Ph-4-CH2- (R) Cha-Pro-Nag (HO) 2P(O) -CH2- (R) Cha-Pro-Nag
EtO (HO) P (0) -CH2- (R) Cha-Pro-Nag (EtO) 2P (0) -CH2- (R) Cha-Pro-Nag
HOOC-CH2- (R) Cha-Pro-Nag
H-(R,S)Pro(3-Ph)-Pro-Agm
H-(R,S)Pro(3-(trans)Ch)-Pro-Agm
HOOC-CH2- (R, S) Pro (3- (trans) Ph) -Pro-Agm
HOOC-CH2- (R, S) Pro (3- (trans) Ph) -Pro-Nag
HOOC-CH2- (Me) (R) Cha- (R, S) Pic-Agm
HOOC-(R,S)CH(Me)-(R)Chu-Pic-Agm
HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/a
HOOC-CH2-CH2- (R) Cha-Pic-Agm
BAD ORIGINAL $
115
H-(R)Cha-Pic-Nag
Me-(R)Cha-(R,S)Pic-Nag
MeOOC-CH2- (R) Cha-Pic-Nag 'PrOOC-CH2- (R) Cha-Pic-Nag
HOOC-CH2- (Me) (R) Cha- (RorS) Pic-Nag/b
HOOC- (R, S) CH (Me) - (R) Cha- (R, S) Pic-Nag
HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/c
HOOC-CH2-Ch2- (R) Cha-Pic-Nag
HOOC-CH2- (R) Cha- (R, S) Mor-Agm
HOOC-CH2- (R) Cha- (RorS) Mor-Nag
H-(R)Cha-Aze-Nag
HOOC-CH2- (R) Cha-Aze-Nag
H-(R)CharPro(5-(S)Me)-Nag
HOOC-CH2- (R) Cha- (RorS) Pic (4,5-dehydro) -Nag/b
HOOC-CH,- (R) Cha- (R) Pic (4 - (R) Me) -Nag
HOOC-CH2- (R) Cgl-Pic-Nag
H-(R)Hoc-Pro-Nag
HOOC-CH2- (R) Hoc-Pro-Nag
HOOC-CH2- (R) Hoc-Pic-Nag
HOOC-CH2- (R) Dph-Pic-Nag
HOOC-CH2- (R) Dch-Pic-Nag
HOOC-CH2- (R) Cha-Pro (5- (R, S) Me) -Nag
HOOC-CH2- (R) Cha-Pic (4- (R) Me) -Nag
H-(R)Cha-Pic(4-(R)Me)-Nag and
HOOC-CH2- (R) Cha-Pic (6- (S) Me) -Nag either as such or in the form of a physiologically acceptable salt and including stereoisomers.
10. A compound selected from
HOOC-CH2-(R) Cha-Pro-Agm
HOOC-CH2-(Me) (R) Cha-Pro Agm
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/b
HOOC-CH2- (R) Cha-Pro-Nag
HOOC-CH2- (R) Cha-Pic-Agm
BAD ORIGINAL &
116
HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/b
HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/d
HOOC-CH2- (R) Cha-Pro(5-(S)Me) -Nag and
HOOC-CH,- (R) Cha-Pic(4- (S) Me) -Nag either as such or in the form of a physiologically acceptable salt and including stereoisomers.
11. The compound
HOOC-CH2-(Me) (R)Cha-Pro-Nag, either as such or in the form of a physiologically acceptable salt and including stereoisomers.
12. The compound
HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/b, either as such or in the form of a physiologically acceptable salt and including stereoisomers.
13. The compound
HOOC-CH, - (R) Cha-Pic-Nag either as such or in the form of a physiologically acceptable salt and including stereoisomers.
14. A compound according to claim 1 specifically identified herein, either as such or in the form of a physiologically acceptable salt or stereoisomer.
15. A process for preparing a compound according to any of claims 1 to 14, which process comprises coupling of an N-terminally protected amino acid or dipeptide or a preformed, N-terminally alkylated protected dipeptide to a compound
H2N-(CHJ „-X wherein n is an integer 2 to 6 and X is an unprotected or
BAD ORIGINAL ft
117 protected guanidi.no group or a protected amino group, or a group convertible into an amino group, where the amino group is subseguently converted into a guanidino group, and if desired forming a physiologically acceptable salt, and 5 in those cases where the reaction results in a mixture of stereoisomers, these are optionally separated by standard techniques, and if desired a single stereoisomer is isolated.
16. A process for preparing a compound according to any of claims 1 to 14, which process comprises:
10 a) (Method I) Coupling of an N-terminally protected dipeptide with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain as shown in the formula:
BAD ORIGINAL
118 wherein R3, R4, R5, n, m and A are as defined in Formula I, R4 is H or alkyl, W, is an amino protecting group such as tertiarybutoxy carbonyl and benzyloxy carbonyl, and X is -NH-C (NH) NH2, -NH-C(NH)NH-W2 -N(W2) -C(NH)NH-W2,
5 -NH-C(NW2)-NHW2 or -NH-W2, wherein W2 is an amine protecting group such as tertiarybutoxy carbonyl or benzyloxy carbonyl, or X is a masked amino group such as azide, giving the protected peptide, or
b) (Method II) Coupling of an N-terminally protected amino 10 acid, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain as shown in the formula
OH
H2N-(CH2)n-X
R5
N A \z 0 NH-(CH2)nbad original $
119 wherein W,, A, Rs and X are as defined above followed by deprotection of the W,-group and coupling with the N-terminal amino acid, in a protected form, or
c) (Method III) Coupling of a preformed N-terminally 5 alkylated and protected dipeptide, with either a protected or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked aminogroup at the terminal end of the alkyl chain as shown in the formula
W,
R2'
R' \ 4 (CHR )m
R5 ,N A \z :n‘
Λ„
H2N-(CH2)n-X , R5, n, m, A and X are defined as above is other than H and W3 is an acyl protecting wherein R2, R3, R4 provided that R2
BAD ORIGINAL ft
120 group such as trifluoroacyl, and converting the coupled product thereby obtained into a compound of general formula I as defined in claim 1 and if desired forming a physiologically acceptable salt, and, where the reaction results in the preparation of a mixture of stereoisomers, optionally separately the stereoisomer mixture and isolating a single stereoisomer.
17. A process according to claim 16 wherein the coupled product is converted into the desired final compounds in any of the following ways, depending on the nature of the Xgroup used:
Removal of the protecting group(s) (when X= -NH-C (NH) NH2, NH-C(NH) NH-W2, -N(W2)-C(NH)NH-W3, -NH-C (NW2) NH-W2) or a selective deprotection of the W, group (e.g. when X= -NH-C(NH)NH-W2, -N(W2)-C(NH)NH-W2, -NH-C(NW2)NH-W2, W2 in this case must be orthogonal to W,) followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/unmasking of the terminal alkylamino function (X= NH-W2, W2 in this case must be orthogonal to W, and W3, respectively, or X= a masked aminogroup, such as azide) followed by a guanidation reaction, in conventional manner, of the free amine and deprotection of the W,- or W3-group, respectively, and if desired forming a physiologically acceptable salt, and in those cases where the reaction results in a mixture of stereoisomers, these are optionally separated by standard chromatographic or re-crystallisation techniques, and if desired a single steregjsomer is isolated.
18. A process for preparing a compound as claimed in any one of claims 1 to 14 substantially as described in any one of the foregoing Examples.
bad original $
121
19. A compound according to any one of claims 1 to 14 prepared by the process claimed in any one of claims 15 to
18.
20. A compound of the formula:
5 either as such or in the form of a salt, or having the guanidino group either mono protected at the 6-nitrogen or diprotected at the <5-nitrogens or the y, 6-nitrogens, for use as a starting material in synthesis of a serine protease inhibitor, and in particular in synthesis of a thrombin
10 inhibitor.
21. A compound according to claim 20 which is 6-Nbenzyloxycarbonyl-noragmatine either as such or in the form of a salt, or having protection at the 6-nitrogen or ynitrogen.
15
22. A compound according to claim 20 which is a-Ntert.butyloxycarbony1-6-N-benzyloxycarbonyl-noragmatine either as such or in the form of a salt, or having protection at the 6-nitrogen or y-nitrogen.
23. A compound according to any one of claims 20 to 22,
20 wherein the serine protease inhibitor is a peptidic compound.
24. A compound 6-N-benzyloxycarbonyl-noragmatine either as such or in the form of a salt, or having further protection at the 6-nitrogen or y-nitrogen.
bad or«g|NAL
122
25. A compound according to claim 24 which is 5-Nbenzy1oxycarbony1-noragmatine.
26. A compound of α-Ν-tert.butyloxy carbony1-6-Nbenzyloxycarbonyl-noragmatine either as such or in the form of a salt, or having protection at the 6-nitrogen or ynitrogen.
27. A compound according to claim 26 which is a-Ntert. buty loxy car bony 1- <5-N-benzy loxycar bony l-noragmatine.
28. A compound according to any one of claims 1 to 14 for use in therapy.
29. A compound according to claim 28 for use as an anticoagulant or antithrombotic agent.
30. A pharmaceutical preparation comprising an effective amount of a compound as claimed in any one of claims 1 to 14 together with one or more pharmaceutically acceptable carriers.
31. A pharmaceutical preparation according to claim 30 for use as an anticoagulant or antithrombotic agent.
32. Use of a compound according to any one of claims 1 to 14 for the manufacture of a medicament for inhibition of thrombin in a human or animal organism.
33. Use of a compound according to any one of claims 1 to 14 for the manufacture of a medicament for treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues in a human or animal organism.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9103612A SE9103612D0 (en) | 1991-12-04 | 1991-12-04 | NEW PEPTIDE DERIVATIVES |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AP9200457A0 AP9200457A0 (en) | 1993-01-31 |
| AP353A true AP353A (en) | 1994-08-14 |
Family
ID=20384531
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| APAP/P/1992/000457A AP353A (en) | 1991-12-04 | 1992-12-04 | New peptides derivatives. |
Country Status (27)
| Country | Link |
|---|---|
| US (4) | US5614499A (en) |
| EP (1) | EP0618926B1 (en) |
| JP (1) | JP3306826B2 (en) |
| CN (1) | CN1076199A (en) |
| AP (1) | AP353A (en) |
| AT (1) | ATE190066T1 (en) |
| AU (2) | AU670052B2 (en) |
| CA (1) | CA2125175C (en) |
| CZ (2) | CZ333895A3 (en) |
| DE (1) | DE69230727T2 (en) |
| EE (1) | EE9400455A (en) |
| FI (1) | FI115770B (en) |
| HU (1) | HUT70431A (en) |
| IL (1) | IL103910A0 (en) |
| IS (1) | IS3954A (en) |
| MA (1) | MA22729A1 (en) |
| MX (1) | MX9206938A (en) |
| NO (1) | NO311361B1 (en) |
| NZ (2) | NZ246106A (en) |
| SE (1) | SE9103612D0 (en) |
| SI (1) | SI9200363A (en) |
| SK (1) | SK63194A3 (en) |
| TN (1) | TNSN92109A1 (en) |
| TW (1) | TW223078B (en) |
| WO (1) | WO1993011152A1 (en) |
| YU (1) | YU104592A (en) |
| ZA (1) | ZA929099B (en) |
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| EP0648780A1 (en) * | 1993-08-26 | 1995-04-19 | Bristol-Myers Squibb Company | Heterocyclic thrombin inhibitors |
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| ZA951618B (en) * | 1994-03-04 | 1996-08-27 | Lilly Co Eli | Antithrombotic agents |
| US5707966A (en) * | 1994-03-04 | 1998-01-13 | Eli Lilly And Company | Antithrombotic agents |
| US5726159A (en) * | 1994-03-04 | 1998-03-10 | Eli Lilly And Company | Antithrombotic agents |
| US5484772A (en) * | 1994-03-04 | 1996-01-16 | Eli Lilly And Company | Antithrombotic agents |
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- 1992-11-24 ZA ZA929099A patent/ZA929099B/en unknown
- 1992-11-27 IL IL103910A patent/IL103910A0/en unknown
- 1992-12-01 SK SK631-94A patent/SK63194A3/en unknown
- 1992-12-01 DE DE69230727T patent/DE69230727T2/en not_active Expired - Lifetime
- 1992-12-01 CZ CZ953338A patent/CZ333895A3/en unknown
- 1992-12-01 NZ NZ246106A patent/NZ246106A/en not_active IP Right Cessation
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- 1992-12-01 CA CA002125175A patent/CA2125175C/en not_active Expired - Fee Related
- 1992-12-01 EP EP92924993A patent/EP0618926B1/en not_active Expired - Lifetime
- 1992-12-01 HU HU9401474A patent/HUT70431A/en unknown
- 1992-12-02 MA MA23019A patent/MA22729A1/en unknown
- 1992-12-02 MX MX9206938A patent/MX9206938A/en unknown
- 1992-12-02 US US07/984,884 patent/US5614499A/en not_active Expired - Lifetime
- 1992-12-03 IS IS3954A patent/IS3954A/en unknown
- 1992-12-03 TN TNTNSN92109A patent/TNSN92109A1/en unknown
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1995
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