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EP1581478A1 - Inhibiteurs de l'enzyme dhodh et leur procede d'identification - Google Patents

Inhibiteurs de l'enzyme dhodh et leur procede d'identification

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Publication number
EP1581478A1
EP1581478A1 EP03813575A EP03813575A EP1581478A1 EP 1581478 A1 EP1581478 A1 EP 1581478A1 EP 03813575 A EP03813575 A EP 03813575A EP 03813575 A EP03813575 A EP 03813575A EP 1581478 A1 EP1581478 A1 EP 1581478A1
Authority
EP
European Patent Office
Prior art keywords
atom
compound
alkyl
group
dhodh
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03813575A
Other languages
German (de)
English (en)
Inventor
Johann Leban
Bernd Kramer
Roland Baumgartner
Katharina Aulinger-Fuchs
Stefan Tasler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
4SC AG
Original Assignee
4SC AG
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Filing date
Publication date
Priority claimed from EP03028137A external-priority patent/EP1541198A1/fr
Application filed by 4SC AG filed Critical 4SC AG
Priority to EP03813575A priority Critical patent/EP1581478A1/fr
Publication of EP1581478A1 publication Critical patent/EP1581478A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/40—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/57—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C233/60—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/04—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
    • C07C2601/08—Systems containing only non-condensed rings with a five-membered ring the ring being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
    • C07C2601/10—Systems containing only non-condensed rings with a five-membered ring the ring being unsaturated

Definitions

  • the present invention relates to a polypeptide which comprises the ligand binding domain of human dihydroorotate dehydrogenase (DHODH), the crystalline forms of this polypeptide complexed with new antiproliferative agents and the use of these crystalline forms to determine the three dimensional structure of the ubiquinone binding site of DHODH complexed with the ligands.
  • DHODH human dihydroorotate dehydrogenase
  • the invention also refers to the use of the three dimensional structure of the ubiquinone binding site of DHODH in methods of designing and/or identifying potential inhibitors of dihydroorotate dehydrogenase (DHODH), for example, compounds which are inhibitors of the ubiquinone binding site, for example, compounds which inhibit the binding of a native substrate to the ubiquinone binding site of DHODH.
  • DHODH dihydroorotate dehydrogenase
  • Inhibitors of DHODH, an enzyme of the pyrimidine biosynthesis, and pharmaceutical compositions containing them are useful, for example, for the treatment of rheumatoid arthritis (RA). Its treatment with usual medications as for example non-steroid anti- inflammatory agents is not satisfactory.
  • RA rheumatoid arthritis
  • the DHODH inhibiting leflunomide [EP 780128, WO 97/34600] is the first medicament of this class of compounds (leflunomides) for the treatment of RA.
  • Leflunomide has immunomodulatorial as well as anti-inflammatorial properties [EP 217206, DE 2524929].
  • DHODH catalyzes the synthesis of pyrimidines, which are necessary for cell growth.
  • An inhibition of DHODH inhibits the growth of (pathologically) fast proliferating cells, whereas cells which grow at normal speed may obtain their required pyrimidine bases from the normal metabolic cycle.
  • the most important types of cells for the immune response, the lymphocytes use exclusively the synthesis of pyrimidines for their growth and react particularly sensitively to DHODH inhibition.
  • Substances that inhibit the growth of lymphocytes are important medicaments for the treatment of auto-immune diseases.
  • WO 99/45926 is a further reference that discloses compounds which act as inhibitors of DHODH.
  • a further object of the present invention is to provide alternative effective agents which can be used for the treatment of diseases which require the inhibition of DHODH.
  • Structure, 2000, Vol. 8, No. 1, pages 25-33 the structure of human DHODH in complex with the antiproliferative agents brequinar and leflunomide are described.
  • Structure, 2000, Vol. 8, No. 1, pages 1227-1238 crystal structures of DHODH B and its product complex are determined.
  • Pharmaceutical Reasearch, 1998, Vol. 15, No. 2, pages 286-295, and in Biochemical Pharmacology, 1990, Vol. 40, No. 4, pages 709-714 the structure-activity relationship of leflunomide and quinoline carboxylic acid analogues is analyzed.
  • the present invention relates to a polypeptide comprising the ligand binding domain of human dihydroorotate dehydrogenase (DHODH), crystalline forms of this polypeptide complexed with a ligand, and the three dimensional structure of the polypeptide, including the three dimensional structure of the ubiquinone binding site of DHODH
  • DHODH human dihydroorotate dehydrogenase
  • the present invention provides a method of determining the three dimensional structure of a crystalline polypeptide comprising the ubiquinone binding site of DHODH complexed with the ligands.
  • the method comprises the steps of (1) obtaining a crystal of the polypeptide comprising the ubiquinone binding site of DHODH complexed with a ligand; (2) obtaining x-ray diffraction data for said crystal; and (3) solving the crystal structure of said crystal by using said x-ray diffraction data and the atomic coordinates for the DHODH complex with the ligand.
  • the invention further relates to a method of identifying a compound which is a potential inhibitor of DHODH.
  • the method comprises the steps of (1) obtaining a crystal of the polypeptide comprising the ubiquinone binding site of DHODH complexed with a ligand; (2) obtaining the atomic coordinates of the polypeptide in said crystal; (3) using said atomic coordinates to define the ubiquinone binding site of DHODH complexed with a ligand; and (4) identifying a compound which fits the ubiquinone binding site.
  • the method can further include the steps of obtaining or synthesizing the compound to inhibit at least one biological activity of DHODH, such as enzymatic activity.
  • the method of identifying a potential inhibitor of DHODH comprises the step of determining the ability of one or more functional groups and/or moieties of the compound, when present in, or bound to, the ubiquinone binding site of DHODH; to interact with one or more subsites of the ubiquinone binding site of DHODH.
  • the ubiquinone binding site of DHODH is defined by the atomic coordinates of a polypeptide comprising the ubiquinone binding site of DHODH. If the compound is able to interact with a preselected number or set of subsites, or has a calculated interaction energy with a desired or preselected range, the compound is identified as a potential inhibitor of DHODH.
  • the human DHODH enzyme is composed of two domains, namely a large C-terminal domain (Met78 to C-terminus) and a small N-terminal domain (Met30 to Leu68), connected by an extended loop.
  • the large C-terminal domain can be described best as an / ⁇ -barrel fold with a central barrel of eight parallel ⁇ strands surrounded by eight a helices.
  • the redox site formed by the substrate binding site and the site of the cofactor flavine mononucleotide (FMN), is located on this large C-terminal domain.
  • the small N- terminal domain on the other hand, consists of two a helices, al and al, connected by a short loop.
  • This small N-terminal domain contains the binding site for the cofactor ubiquinone.
  • the helices al and al span a slot of about 10 x 20 A in the so-called hydrophobic patch, with the short ⁇ l- 2 loop at the narrow end of that slot.
  • the slot forms the entrance to a tunnel that ends at the FMN cavity nearby the l- ⁇ 2 loop.
  • This tunnel is narrowing towards the proximal redox site and ends with several charged or polar sidechains (Gln47, His56, Tyr356, Thr360 and Argl36). It is evident that ubiquinone which can easily diffuse into the mitochondrial inner membrane uses this tunnel to approach the FMN cofactor for a redox reaction.
  • the structural knowledge mentioned above can be used to design potential inhibitors of the human DHODH activity targeting the tunnel mentioned above and competing with ubiquinone for the ubiquinone binding site.
  • Potential inhibitors were co- crystallized with human DHODH (Met30 to Arg396) and the three dimensional structures were solved by protein X-ray crystallography techniques, ten of the solved structures being three dimensional structures of human DHODH (Met30 to Arg396) in complex with compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. These crystal structures were solved at atomic resolution and the binding modes of the ten compounds were analyzed.
  • the structural formulars of the co-crystallized compounds are given below.
  • Each subsite includes molecular functional groups or moieties capable of forming stabilizing interactions with complementary functional groups or moieties of an inhibitor.
  • a hydrogen bond is formed between a hydrogen atom covalently bond to an electronegative element (proton donor or hydrogen bond donor) and a lonely electron pair of a second electronegative atom (proton acceptor or hydrogen bond acceptor). Hydrogen bonds typically occur when the hydrogen bond donor and the hydrogen bond acceptor are separated by about 2.5 A and 3.5 A.
  • Stabilizing hydrophobic or lipophilic interactions occur if two groups or moieties with hydrophobic/lipophilic character, for example, aliphatic chains or aromatic systems, are separated by distances close to their van der Waals radii.
  • the method of identifying a potential inhibitor of DHODH comprises the step of determining the ability of one or more functional groups and/or moieties of the compound, when present in the ubiquinone binding site, to interact with one or more subsites of the ubiquinone binding site.
  • the ubiquinone binding site is defined by the atomic coordinates of a polypeptide comprising the ubiquinone binding site of DHODH. If the compound is able to interact with a preselected number or set of subsites, the compound is identified as a potential inhibitor of DHODH.
  • a functional group or moiety of the compound is said to "interact" with a subsite of the ubiquinone binding site if it participates in an energetically favourable, or stabilizing, interaction with one or more complementary moieties within the subsite.
  • Two chemical moieties are "complementary" if they are capable, when suitably positioned, of participating in an attractive, or stabilizing, interaction, such as an electrostatic or an van der Waals interaction.
  • the attractive interaction is an ion- ion, a salt bridge, ion-dipole, dipole-dipole, hydrogen bond, pi-pi or hydrophobic interaction.
  • An extreme case of attractive interaction is the formation of a covalent bond by a chemical reaction between the test compound and the enzyme.
  • a negatively charged moiety and a positively charged moiety are complementary because, if suitably positioned, they can form a salt bridge.
  • a hydrogen bond donor and a hydrogen bond acceptor are complementary if suitably positioned.
  • the groups capable of hydrogen bond formation are selected from halogen, such as fluorine, chlorine, bromine and iodine, NO 2 , haloalkyl, haloalkyloxy, CN, hydroxyl, amino, hydroxylamine, hydroxamic acid, carbonyl, carbonic acid, sulfonamide, amide, sulfone, sulfonic acid, alkylthio, alkoxy, ester, hydroxyalkylamino group, and other groups including a heteroatom having at least one lone pair of electrons, such as groups containing trivalent phosphorous, di- and tetravalent sulfur, oxygen and nitrogen atoms;
  • halogen such as fluorine, chlorine, bromine and iodine
  • NO 2 haloalkyl, haloalkyloxy
  • CN hydroxyl, amino, hydroxylamine, hydroxamic acid, carbonyl, carbonic acid, sulfonamide, amide, sulfone
  • hydrophobic groups are selected from groups, such as linear, branched or cyclic alkyl groups; linear, branched or cyclic alkenyl groups; linear, branched or cyclic alkynyl groups; aryl groups, such as mono- and polycyclic aromatic hydrocarbyl groups and mono- and polycyclic heteroaryl groups;
  • negatively charged groups are selected from groups, such as carboxylate, sulfonamide, sulfamate, boronate, vanadate, sulfonate, sulfinate and phosphonate groups.
  • groups such as carboxylate, sulfonamide, sulfamate, boronate, vanadate, sulfonate, sulfinate and phosphonate groups.
  • a given chemical moiety can contain one or more of these groups.
  • Subsite 1 Hydrophobic pocket; interacting chemical moieties: H; Residues involved: Leu 42; Met 43; Leu 46; Ala 55; Ala 59; Phe 98; Met 111; Leu 359; Pro 364;
  • Non-hydrogen atoms which interact with H Leu 42 CB, CG, GDI, CD2; Met 43 SD, CE; Leu 46 CB, CG, CD1, CD2; Ala 55 CB; Ala 59 CA, CB; Phe 98 CG, GDI, CD2, CE1, CE2; Met 111 SD, CE; Leu 359 CA, CB, CG, CD1, CD2; Pro 364 CB, CD, CG;
  • R ⁇ is independently H, -CO 2 R -CONHR ⁇ -CR ⁇ O, -SO 2 NR", -NR ⁇ -CO-haloalkyl, -N0 2 , -NR"-SO 2 -haloalkyl, -NR"-S0 2 -alkyl, -SO 2 -alkyl, -NR ⁇ -CO-alkyl, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, -OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl; R ⁇ is independently hydrogen, haloalkyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl; R ⁇ is preferably F, CI, Br
  • Subsite 2 First anion binding site; interacting with HB, N, HB and N, HB and HB, or N and N;
  • Residues involved Gin 47; Arg 136; one conserved water molecule
  • Non-hydrogen atoms which interact with HB and N Glu 47 OE1, NE2; Arg 136 NE, NH1,
  • the group is selected from halogen, such as fluorine, chlorine, bromine and iodine, NO 2 , haloalkyl, haloalkyloxy, CN, hydroxyl, amino, hydroxylamine, hydroxamic acids, carbonyl, carbonic acid, sulfonamide, amide, sulfone, sulfonic acid, alkylthio, alkoxy, such as methoxy, ester, hydroxyalkylamino, carboxylate, tetrazole, sulfonamide, sulfamate, boronate, vanadate, sulfonate, sulfinate and phosphonate group, more preferably from a carboxylate, sulfonamide, sulfamate, sulfonate, carbonyl or carbonic acid group.
  • halogen such as fluorine, chlorine, bromine and iodine, NO 2 , haloalkyl, haloalky
  • Subsite 3 Second anion binding site; interacting with HB, N, HB and N, HB and HB, or N and N;
  • Residues involved His 56; Tyr 356; Tyr 147 (interacting via a conserved water molecule); Non-hydrogen atoms which interact with HB and N: His 56 N, ND1; Tyr 356 OH; Tyr 147 OH (interacting via a conserved water molecule); preferably for one or two hydrogen bond formations with subsite 2 the group is selected from halogen, such as fluorine, chlorine, bromine and iodine, NO 2 , haloalkyl, haloalkyloxy, CN, hydroxyl, amino, hydroxylamine, hydroxamic acids, carbonyl, carbonic acid, sulfonamide, amide, sulfone, sulfonic acid, alkylthio, alkoxy, such as methoxy, ester, hydroxyalkylamino, carboxylate, tetrazole, sulfonamide, sulfamate, boronate, vanadate, sulfonate, sulf
  • Subsite 4 Remote hydrophobic pocket; interacting chemical moieties: H;
  • Non-hydrogen atoms which interact with H Pro 52 CB, CG, CD; Val 134 CB, CGI, CG2; Val 143 CB, CGI, CG2; Thr 360 CG2; FMN C7M, C8M;
  • the group is selected from such as linear, branched or cyclic C ⁇ -C 6 -alkyl groups; such as methyl, ethyl, propyl, butyl, tert.
  • the core is selected from cyclic alkyl groups; cyclic alkenyl groups; cyclic alkynyl groups; aryl groups, such as mono- and polycyclic aromatic hydrocarbyl groups and mono- and polycyclic heteroaryl groups; more preferably it is selected from mono-, or bicyclic aromatic or non-aromatic ring systems, most preferably from 5-membered mono-, or bicyclic aromatic or non-aromatic ring systems, such as trans-cyclopentan-l,2-diyl, trans-cyclohexan-l,2-diyl, cis-cyclopentan-l,2-diyl, cis-cyclohexan-l,2-diyl, 1- cyclopenten-l,2-diyl, 2-cyclopenten-l,2-diyl, 3-cyclopenten-l,2-diyl, 4-
  • the bridge is selected from -NH; -O; -CO-NH; -NH-CO; -NH-CO-NH; alkyl; -O-CH 2 ; -CH 2 -O; -O-CH 2 -CH 2 ; -CH 2 -CH 2 -O; -NH-CH 2 ; -CH 2 -NH; -NH-CH 2 -CH 2 ; -CH 2 - CH 2 -NH; -CH 2 -CO-NH; -CH 2 -NH-CO;
  • Subsite 5 Solvent anchor; interacting chemical moieties: HB
  • Non-hydrogen atoms which interact with HB Met 30 O, SD, CE; Tyr 38 OH, CE2, CD2;
  • Leu 67 O preferably for the hydrogen bond formation with subsite 5, the group is selected from F, CI, Br, I, CF 3 , OCF 3 , or OCH 3
  • Subsite 6 Solvent anchor; interacting chemical moieties: H;
  • Non-hydrogen atoms which interact with H Leu 68 CB, CG, GDI, CD2;
  • the group is selected from such as linear, branched or cyclic C ⁇ -C 6 -alkyl groups; such as methyl, ethyl, propyl, butyl, tert. butyl, linear, branched or cyclic Ci-C ⁇ -alkenyl groups; linear, branched or cyclic C1- 5 - alkynyl groups; aryl groups, such as mono- and bi aromatic hydrocarbyl groups, such as -
  • an alkyl group denotes a linear or branched Ci- -alkyl, preferably a linear or branched chain of one to five carbon atoms, a linear or branched Ci— C 6 -alkenyl or a linear or branched -C ⁇ -alkinyl group, which can optionally be substituted by one or more substituents R ⁇ preferably by halogen;
  • R is independently H, -CO 2 R", -CONHR", -CR ⁇ O, -SO 2 NR -NR ⁇ -CO-haloalkyl, -NO 2 , -NR"-SO 2 -haloalkyl, -NR -SO 2 -alkyl, -SO 2 -alkyl, -NR ⁇ -CO-alkyl, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, -OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl;
  • R" is independently hydrogen, haloalkyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl;
  • a cycloalkyl group denotes a non-aromatic ring system containing four to eight carbon atoms, preferably four to eight carbon atoms, wherein one or more of the carbon atoms in the ring can be substituted by a group X, X being as defined above;
  • the C 4 -C 8 -cycloalkyl residue may be selected from the group comprising -cyclo-C H 7 , -cyclo-CsH 9 , -cyclo-CgH ⁇ , -cyclo-C 7 H 13 , -cyclo-CgHis;
  • an alkoxy group denotes an O-alkyl group, the alkyl group being as defined above; the alkoxy group is preferably a methoxy, ethoxy, isopropoxy, t-butoxy or pentoxy group;
  • an alkylthio group denotes an S-alkyl group, the alkyl group being as defined above.
  • an haloalkyl group denotes an alkyl group which is substituted by one to five halogen atoms, the alkyl group being as defined above; the haloalkyl group is preferably a -C(R 10 ) 3 , -CR 10 (R 10' ) 2 , -CR 10 (R 10' )R 10" , -C 2 (R 10 ) 5 , -CH 2 -C(R 10 ) 3 , -CH 2 -CR 10 (R 10' ) 2 , -CH 2 - CR 10 (R 10' )R 10'" , -C 3 (R 10 ) 7 or -C 2 l -C(R 10 h, wherein R 10 , R 10' , R 10" represent F, CI, Br or I, preferably F;
  • a hydroxyalkyl group denotes an HO-alkyl group, the alkyl group being as defined above;
  • an haloalkyloxy group denotes an alkoxy group which is substituted by one to five halogen atoms, the alkyl group being as defined above; the haloalkyloxy group is preferably a -OC(R 10 ) 3) -OCR 10 (R 10' ) 2 , -OCR 10 (R 10' )R 10" , -OC 2 (R 10 ) 5 , -OCH 2 -C(R 10 ) 3 , -OCH 2 - CR 10 (R 10' ) 2 , -OCH 2 -CR 10 (R 10' )R 10" , -OC 3 (R 10 ) 7 or -OC 2 H 4 -C(R 10 ) 3 , wherein R 10 , R 10' ,
  • R 10' represent F, CI, Br or I, preferably F;
  • a hydroxyalkylamino group denotes an (HO-alkyl) 2 -N- group or HO-alkyl-NH- group, the alkyl group being as defined above;
  • an alkylamino group denotes an HN-alkyl or N-dialkyl group, the alkyl group being as defined above;
  • a heteroaryl group denotes a 5- or 6-membered heterocyclic group which contains at least one heteroatom like O, N, S.
  • This heterocyclic group can be fused to another ring.
  • this group can be selected from a thiazol-2-yl, thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, l,2,4-oxadiazol-3-yl, l,2,4-oxadiazol-5-yl, l,2,4-thiadiazol-3-yl, l,2,4-thiadiazol-5-yl, l,2,5-oxadiazol-3-yl, l,2,5-oxadiazol-4-yl, l,2,5-thiadiazol-3-yl, 1-imidazolyl, 2-imidazolyl, l,2,5-thiadiazol-4-yl, 4-imidazolyl
  • the present invention provides DHODH inhibitors, and methods of use thereof, which are capable of binding to the ubiquinone binding site of DHODH, for example, compounds wich are identified as inhibitors of DHODH or which are designed by the methods described above to inhibit DHODH.
  • the invention includes compounds which interact with one or more, preferably two or more, and more preferably, three or more of DHODH subsites 1 to 6.
  • an inhibitor of DHODH should have a core-unit and interact with subsite 1, 2, 3 and 5 or an inhibitor of DHODH should have a core-unit and interact with subsite 1, 2 and 5, or an inhibitor of DHODH should have a core-unit and interact with subsite 1, 3 and 5.
  • an inhibitor of DHODH should have a core-unit and interact with subsite 1, 2 and 3, or an inhibitor of DHODH should have a core-unit and interact with subsite 1 and 3.
  • Fig. 1 the spatial arrangement of the subsites is depicted schematically. subsite 2
  • the three dimensional structure published by Shenpig et al. shows human DHODH(Met30-Arg396) in complex with brequinar and the leflunomide metabolite A771726, respectively.
  • the main interaction in the binding of brequinar to DHODH is the formation of a salt bridge between the carboxy group of brequinar and the sidechain of Argl36.
  • the salt bridge is formed between the carboxylic group and the atoms NE, NH1 or NH2. More precisely, the above mentioned subsite 2, the first anion binding site, is addressed in this kind of interaction. In the following, this type of interactioned will be termed "brequinar-like binding mode".
  • non-brequinar-like binding mode is characterized by a number of hydrogen bonds formed between the ligand and protein residues belonging to subsite 3. In particular this residues are His 56, Tyr 356 and Tyr 147. Non-hydrogen atoms involved in the formation of hydrogen bonds are N and NDl of His 56, the oxygen of the hydroxyl group of Tyr 356 and the oxygen of the hydroxyl group of Tyr 147. The latter interaction involves a conserved water molecule bridging the space between the carboxyl function of the ligand molecule and the hydroxyl group of the tyrosine residue 147.
  • both a brequinar-like and a non-brequinar-like binding mode can be utilized.
  • the carboxy group of compounds 2, 6 and 10 forms hydrogen bonds to the sidechains of residues Gin 47 and Arg 136.
  • the non-brequinar-like binding mode the five membered ring of compounds 2, 6 and 10 containing the carboxy group is rotated by almost 180 degrees and forms hydrogen bonds to residues His 56 and Tyr 356.
  • Non-hydrogen atoms involved in the formation of hydrogen bonds are N and NDl of His 56 and the oxygen of the hydroxyl group of Tyr 356.
  • the compounds 2, 3 and 4 are particularly interesting for a structure-activity- relationship (SAR) analysis. These molecules differ only in the degree of ring substitution (see structures above). Clearly, one can observe a correlation between the number of fluorinated positions at the aromatic ring in the middle of the molecules and the corresponding IC50 values. The higher the number of ring substituents the lower the IC 50 . Interestingly compound 2 and compound 3 display both the brequinar-like and non- brequinar-like binding mode in the crystal structure (see table 27). It is quite reasonable to speculate whether the ring substituents exhibit a steering effect on the five membered ring and by such facilitate the formation of the more favourable brequinar-like binding mode. Therefore, the presence of both binding modes might explain the increased affinity of this compounds.
  • SAR structure-activity- relationship
  • Table 27 Relation of inhibitor binding mode and degree of ring substitutions. Structures of the compounds are shown above.
  • a similar structure-activity-relationship can be deduced from the crystal structures of humann DHODH in complex with compounds 9 and 10. These compounds carry a sulfur atom at an ortho position with respect to the carboxylic group in the five membered ring.
  • Compound 10 is single substituted with fluorine at the biaryl ring system, whereas compound 9 bears two substituents.
  • compound 9 exhibits a pure brequinar- like binding mode whereas compound 10 shows both alternatives.
  • the sulfur atom in the ortho position on the five membered ring can favourably interact with the protein's subsite 4 (remote hydrophobic pocket).
  • the activity data correlate to a very high degree with the presence of a particular binding mode (Table 28). Obviously, not only the degree of ring subsitution but also ring planarity might contribute to the formation of a particular binding mode.
  • Table 28 Relation of inhibitor binding mode and degree of ring substitutions. Structures of the compounds are shown above.
  • the invention further provides a method of designing a compound which is a potential inhibitor of DHODH.
  • the method includes the steps of (1) identifying one or more functional groups capable of interacting with one or more subsites of the ubiquinone binding site of DHODH; and (2) identifying a scaffold which presents the functional group or functional groups identified in step 1 in a suitable orientation for interacting with one or more subsites of the ubiquinone binding site of DHODH.
  • the compound which results from attachment of the identified functional groups or moieties to the identified scaffold is a potential inhibitor of DHODH.
  • the DHODH ubiquinone binding site is, generally, defined by the atomic coordinates of a polypeptide comprising the DHODH ubiquinone binding site.
  • the invention provides a new three dimensional structure of a crystalline polypeptide comprising the ubiquinone binding site of DHODH complexed with the ligands.
  • This structure enables the rational development of inhibitors of DHODH by permitting the design and/or identification of molecular structures having features which facilitate binding to the ubiquinone binding site of DHODH.
  • the methods of use of this structure disclosed herein thus, permit more rapid discovery of compounds which are potentially useful for the treatment of conditions which are mediated, at least in part, by DHODH activity.
  • the polypeptide preferably comprises the ubiquinone binding site of a mammalian DHODH. More preferably the polypeptide comprises the ubiquinone binding site of human DHODH.
  • the polypeptide is a polypeptide of the present invention, as described above.
  • the polypeptide can be crystallized using methods known in the art, such as the methods described in Structure, 2000, Vol. 8, No. 1, pages 25-33, to afford polypeptide crystals which are suitable for x-ray diffraction studies.
  • a crystalline polypeptide/ligand complex can be produced by co-crystallizing the polypeptide with a solution including the ligand.
  • the atomic coordinates of the polypeptide and the ligand can be determined, for example, by x-ray crystallography using methods known in the art.
  • the data obtained from the crystallography can be used to generate atomic coordinates, for example, of the polypeptide and ligand, if present.
  • solution and refinement of the x- ray crystal structure can result in the determination of coordinates for some or all of the non-hydrogen atoms.
  • the atomic coordinates of the polypeptide can be used, as is known in the art, to generate a three-dimensional structure of the ubiquinone binding site of DHODH. This structure can then be used to assess the ability of any given compound, preferably using computer-based methods, to fit into the ubiquinone binding site.
  • the atomic coordinates of the polypeptide/ligand complex can be used, as is known in the art, to generate a three-dimensional structure of the ligand in its binding conformation.
  • This structure can then be used to assess the ability of any given compound, preferably using computer-based methods, to exhibit a similar spatial orientation and electrostatic and/or van der Waals interactions as the ligand and therefore, to fit into the addressed binding site.
  • a compound fits into the ubiquinone binding site if it is of suitable size and shape to physically reside in the ubiquinone binding site, that is if it has a shape which is complementary to the ubiquinone binding site and can reside in the ubiquinone binding site without significant unfavorable sterical or van der Waals interactions.
  • the compound includes one or more functional groups and/or moieties which interact with one or more subsites within the ubiquinone binding site.
  • Computational methods for evaluating the ability of a compound to fit into the ubiquinone binding site, as defined by the atomic coordinates of the polypeptide are known in the art, and representative examples are provided below.
  • the method of identifying a potential inhibitor of DHODH comprises the step of determining the ability of one or more functional groups and/or moieties of the compound, when present in the DHODH ubiquinone binding site, to interact with one or more subsites of the DHODH ubiquinone binding site.
  • the DHODH ubiquinone binding site is defined by the atomic coordinates of a polypeptide comprising the DHODH ubiquinone binding site. If the compound is able to interact with a preselected number of subsites, the compound is identified as a potential inhibitor of DHODH.
  • the method of identifying a potential inhibitor of DHODH comprises the steps of (1) identifying the size and shape of the ligand co-crystallized in the polypeptide/ligand complex and/or identifying functional groups or moieties of the ligand which are capable to form stabilizing interactions with the polypeptide, and (2) by comparison with these, identifying one or more functional groups and/or moieties of any given compound which have similar size and shape as the cocrystallized ligand and/or are capable to form one or more interactions to the polypeptide in a similar manner as the co- crystallized ligand. If a compound exhibits one or more of these features, the compound is identified as a potential inhibitor of DHODH.
  • a functional group or moiety of the compound is said to "interact" with a subsite of the DHODH ubiquinone binding site if it participates in an energetically favourable, or stabilizing, interaction with one or more complementary moieties within the subsite, as defined above.
  • a functional group or moiety of the compound is said to interact in a "similar” manner as the co-crystallized ligand if one or more, preferably two or more of its functional groups or moieties capable of forming the attractive interactions mentioned above can be superimposed on those functional groups or moieties of the co-crystallized ligand capable of forming the attractive interactions.
  • the superposition can be performed based on the identity of atoms, and/or the identity or similarity of functional groups, and/or the similarity of molecular shape and/or the identity or similarity of interaction possibilities.
  • an -OH group of a compound and an -NH group of the cocrystallized ligand may interact in the same way, namely as hydrogen bond donors, with a hydrogen bond acceptor atom suitably positioned in the enzyme. Therefore, the -OH group and the -NH group are said to have similar interaction properties, and a molecule containing an -OH group may be superimposed onto a molecule carrying an -NH group at the corresponding position.
  • the assessment of interactions between (1) the test compound and the DHODH ubiquinone binding site and (2) the superposition of a test compound and the cocrystallized ligand employ computer-based computational methods, such as those known in the art, in which, for the first case, possible interactions of a compound with the protein, as defined by atomic coordinates, are evaluated with respect to interaction strength by calculating the interaction energy upon binding the compound to the protein.
  • the superposition of a test compound and the cocrystallized ligand is performed according to the identity of atoms, and/or the identity or similarity of functional groups, and/or the similarity of molecular shape and/or the identity or similarity of interaction possibilities in a process termed alignment. Matching atoms / functional groups / shape / interaction possibilities are evaluated and summarized to an alignment score enabling the ranking of the tested molecules.
  • Compounds which have calculated interaction energies within a preselected range or which otherwise, in the opinion of the computational chemist employing the method, have the greatest potential as DHODH inhibitors can then be provided, for example, from a compound library or via synthesis, and assayed for the ability to inhibit DHODH.
  • the interaction energy for a given compound generally depends upon the ability of the compound to interact with one or more subsites within the protein catalytic domain.
  • the atomic coordinates used in the method are the atomic coordinates set forth in Figs. 2, 3 and 4. It is to be understood that the coordinates set forth in Figs. 2, 3 and 4 can be transformed, for example, into a different coordinate system, in ways known to those of skill in the art without substantially changing the three dimensional structure represented thereby.
  • a moiety of the compound can interact with a subsite via two or more individual interactions.
  • a moiety of the compound and a subsite can interact if they have complementary properties and are positioned in sufficient proximity and in a suitable orientation for a stabilizing interaction to occur.
  • the possible range of distances for the moiety of the compound and the subsite depends upon the distance dependence of the interaction, as known in the art.
  • a hydrogen bond typically occurs when a hydrogen bond donor atom, which bears a hydrogen atom, and a hydrogen bond acceptor atom are separated by about 2.5 A and about 3.5 A. Hydrogen bonds are well known in the art. Generally, the overall interaction, or binding, between the compound and the ubiquinone binding site will depend upon the number and strength of these individual interactions.
  • the ability of a test compound to interact with one or more subsites of the ubiquinone binding site can be determined by computationally evaluating interactions between functional groups, or moieties, of the test compound and one or more amino acid side chains and/or backbone atoms in the ubiquinone binding site.
  • a compound which is capable of participating in stabilizing interactions with a preselected number of subsites, preferably without simultaneously participating in significant destabilizing interactions is identified as a potential inhibitor of DHODH.
  • Such a compound will interact with one or more subsites, preferably with two or more subsites and, more preferably, with three or more subsites.
  • the invention further provides methods of designing a compound which is a potential inhibitor of DHODH.
  • the first method includes the steps of (1) identifying one or more functional groups capable of interacting with one or more subsites of the DHODH ubiquinone binding site; and (2) identifying a scaffold which presents the functional group or functional groups identified in step 1 in a suitable orientation for interacting with one or more subsites of the DHODH ubiquinone binding site.
  • the compound which results from attachment of the identified functional groups or moieties to the identified scaffold is a potential inhibitor of DHODH.
  • the DHODH ubiquinone binding site is, generally, defined by the atomic coordinates of a polypeptide comprising the DHODH ubiquinone binding site, for example, the atomic coordinates set forth in Figs. 2, 3 and 4.
  • the second method comprises the steps of (1) identifying one or more functional groups or moieties capable of interacting in a similar way as one or more functional groups or moieties of the co-crystallized ligand, and (2) identifying a scaffold which presents the functional group or functional groups identified in step 1 in a suitable orientation for interacting in a similar way as one or more functional groups or moieties of the cocrystallized ligand.
  • the compound which results from attachment of the identified functional groups or moieties to the identified scaffold is a potential inhibitor of DHODH.
  • the co-crystallized ligand is, generally, defined by the atomic coordinates of a ligand complexed in the polypeptide comprising the DHODH ubiquinone binding site, for example, the atomic coordinates set forth in Figs. 2, 3 and 4.
  • Suitable methods can be used to identify chemical moieties, fragments or functional groups which are capable of interacting favorably with a particular subsite or sets of subsites. These methods include, but are not limited to: interactive molecular graphics; molecular mechanics; conformational analysis; energy evaluation; docking; database searching; virtual high-throughput screening (US 422303, DE 10009479, EP 1094415, US 693731, US 885893, US 885517); structural alignment; functional group alignment; interaction-point alignment; pharmacophore modeling; de novo design; property estimation and descriptor-based database searching. These methods can also be employed to assemble chemical moieties, fragments or functional groups into a single inhibitor molecule. These same methods can also be used to determine whether a given chemical moiety, fragment or functional group is able to interact favorably with a particular subsite or sets of subsites.
  • the design of potential DHODH inhibitors begins from the general perspective of three-dimensional shape and electrostatic complementarity for the ubiquinone binding site, and subsequently, interactive molecular modeling techniques can be applied by one skilled in the art to visually inspect the quality of the fit of a candidate molecule into the binding site.
  • Suitable visualization programs include SYBYL (Tripos Inc., St. Louis, MO), MOLOC (Gerber Molecular Design, Basel), RASMOL (Sayle et al. Trends Biochem. Sci. 20:374-376 (1995)) and MOE (Chemical Computing Group Inc., Montreal).
  • a further embodiment of the present invention utilizes a database searching program which is capable of scanning a database of small molecules of known three-dimensional structure for candidates which fit into the target protein site.
  • Suitable software programs include 4SCan ® (US 422303, DE 10009479, EP 1094415, US 693731, US 885893, US 885517), FLEXX (Rarey et al., J. Mol. Biol. 261:470-489 (1996)), and UNITY (Tripos Inc., St. Louis, MO).
  • 4SCan ® was developed to scan/screen large virtual databases up to several millions of small molecules in a reasonable time-frame.
  • a further embodiment of the present invention utilizes a database searching program which is capable of scanning a database of small molecules of known three-dimensional structure for candidates which align properly with the co-crystallized ligand, both in shape and interaction properties.
  • Suitable software programs include 4SCan ® (US 422303, DE 10009479, EP 1094415, US 693731, US 885893, US 885517) and FLEXS (Lemmen et al., J. Med. Chem 41:4502-4520 (1998)).
  • 4SCan ® is capable of aligning large virtual databases up to several millions of small molecules in a reasonable time-frame. It is not expected that the molecules found in the search will necessarily be leads themselves, since a complete evaluation of all interactions will necessarily be made during the initial search.
  • GRID Garford J. Med. Chem. 28:849-857 (1985) has produced a computer program, GRID, which seeks to determine regions of high affinity for different chemical groups (termed probes) on the molecular surface of the binding site. GRID hence provides a tool for suggesting modifications to known ligands that might enhance binding.
  • a range of factors including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, conformational strain, ligand flexibility and cooperative motions of ligand and enzyme, all influence the binding effect and should be taken into account in attempts to design bioactive inhibitors.
  • Yet another embodiment of a computer-assisted molecular design method for identifying inhibitors of DHODH comprises searching for fragments which fit into a binding region subsite and link to a pre-defined scaffold.
  • the scaffold itself may be identified in such a manner.
  • a representative program suitable for the searching of such functional groups and monomers include LUDI (Boehm, J. Comp. Aid. Mol. Des. 6:61-78 (1992)) and MCSS (Miranker et al., Proteins 11: 314-328 (1991)).
  • Yet another embodiment of a computer-assisted molecular design method for identifying inhibitors of DHODH comprises the de novo synthesis of potential inhibitors by algorithmic connection of small molecular fragments that will exhibit the desired structural and electrostatic complementarity with the active site of the enzyme.
  • the methodology employs a large template set of small molecules which are iteratively pierced together in a model of the DHODH ubiquinone binding site. Programs suitable for this task include GROW (Moon et al. Proteins 11:314-328 (1991)) and SPROUT (Gillet et al. J. Comp. Aid. Mol. Des. 7:127 (1993)).
  • the suitability of inhibitor candidates can be determined using an empirical scoring function, which can rank the binding affinities for a set of inhibitors.
  • an empirical scoring function can rank the binding affinities for a set of inhibitors.
  • a compound which is identified by one of the foregoing methods as a potential inhibitor of DHODH can then be obtained, for example, by synthesis or from a compound library, and assessed for the ability to inhibit DHODH in vitro.
  • Such an in vitro assay can be performed as is known in the art, for example, by contacting DHODH in solution with the test compound in the presence of the substrate and cofactor of DHODH and ubiquinone. The rate of substrate transformation can be determined in the presence of the test compound and compared with the rate in the absence of the test compound. Suitable assays for DHODH biological activity are described below, the teachings of each of which are hereby incorporated by reference herein in their entity.
  • An inhibitor identified or designed by a method of the present invention can be a competitive inhibitor, an uncompetitive inhibitor or a noncompetitive inhibitor with respect to ubiquinone.
  • table 25 the structures of the highest ranking compounds of the combinatorial library are shown.
  • the consensus score of each molecule is calculated by the summation of the two predicted 4SCan ® activity scores for the two different structures of the ubiquinone binding site.
  • the compounds of the present invention can be used for a variety of human and animal diseases, preferably human diseases, where inhibition of the pyrimidine metabolism is beneficial.
  • diseases are: - fibrosis, uveitis, rhinitis, asthma or arthropathy, in particular, arthrosis
  • These immunological events also include a desired modulation and suppression of the immune system; - all types of autoimmune diseases, in particular rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, multiple sclerosis, insulin dependent diabetes mellitus and non-insulin dependent diabetes mellitus, and lupus erythematoidis, ulcerative colitis, Morbus Crohn, inflammatory bowel disease, as well as other chronic inflammations, chronic diarrhea;
  • autoimmune diseases in particular rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, multiple sclerosis, insulin dependent diabetes mellitus and non-insulin dependent diabetes mellitus, and lupus erythematoidis, ulcerative colitis, Morbus Crohn, inflammatory bowel disease, as well as other chronic inflammations, chronic diarrhea;
  • the compounds according to the invention and medicaments prepared therewith are generally useful for the treatment of cell proliferation disorders, for the treatment or prophylaxis, immunological diseases and conditions (as for instance inflammatory diseases, neuroimmunological diseases, autoimmune diseases or other).
  • the compounds of the present invention are also useful for the development of immunomodulatory and anti-inflammatory medicaments or, more generally, for the treatment of diseases where the inhibition of the pyrimidine biosynthesis is beneficial.
  • the compounds of the present invention are also useful for the treatment of diseases which are caused by malignant cell proliferation, such as all forms of hematological and solid cancer. Therefore the compounds according to the invention and medicaments prepared therewith are generally useful for regulating cell activation, cell proliferation, cell survival, cell differentiation, cell cycle, cell maturation and cell death or to induce systemic changes in metabolism such as changes in sugar, lipid or protein metabolism. They can also be used to support cell generation poiesis, including blood cell growth and generation
  • prohematopoietic effect after depletion or destruction of cells, as caused by, for example, toxic agents, radiation, immunotherapy, growth defects, malnutrition, malabsorption, immune dysregulation, anemia and the like or to provide a therapeutic control of tissue generation and degradation, and therapeutic modification of cell and tissue maintenance and blood cell homeostasis.
  • diseases and conditions include but are not limited to cancer as hematological (e.g. leukemia, lymphoma, myeloma) or solid tumors (for example breast, prostate, liver, bladder, lung, esophageal, stomach, colorectal, genitourinary, gastrointestinal, skin, pancreatic, brain, uterine, colon, head and neck, ovarian, melanoma, astrocytoma, small cell lung cancer, glioma, basal and squameous cell carcinoma, sarcomas as Kaposi's sarcoma and osteosarcoma), treatment of disorders involving T-cells such as aplastic anemia and DiGeorge syndrome, Graves' disease.
  • hematological e.g. leukemia, lymphoma, myeloma
  • solid tumors for example breast, prostate, liver, bladder, lung, esophageal, stomach, colorectal, genitourinary, gastrointestinal, skin, pan
  • Leflunomide was previously found to inhibit HCMN replication in cell culture.
  • Ocular herpes is the most common cause of infectious blindness in the developed world. There are about 50.000 cases per year in the US alone, of which 90% are recurrences of initial infections. Recurrences are treated with antivirals and corticosteroids. Cytomegalovirus, another herpes virus, is a common cause of retinal damage and blindness in patients with aids.
  • the compounds of the present invention can be used alone or in combination with other antiviral compounds such as ganciclovir and foscarnet to treat such diseases.
  • the compounds of the present invention can further be used for diseases that are caused by protozoal infestations in humans and animals.
  • Such veterinary and human pathogenic protozoas are preferably intracellular active parasites of the phylum Apicomplexa or Sarcomastigophora, especially Trypanosoma, Plasmodia, Leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida, Amoebia, Coccidia and Trichomonadia.
  • These active substances or corresponding drugs are especially suitable for the treatment of Malaria tropica, caused by Plasmodium falciparum, Malaria tertiana, caused by Plasmodium vivax or Plasmodium ovale and for the treatment of Malaria quartana, caused by Plasmodium m ⁇ lariae.
  • Toxoplasmosis caused by Toxoplasma gondii
  • Coccidiosis caused for instance by Isospora belli
  • intestinal Sarcosporidiosis caused by Sarcocystis suihominis
  • dysentery caused by Entamoeba histolytica
  • Cryptosporidiosis caused by Cryptosporidium parvum
  • Chargas disease, caused by Trypanosoma cruzi, sleeping sickness, caused by Trypanosoma brucei rhodesiense or gambiense, the cutaneous and visceral as well as other forms of Leishmaniosis.
  • veterinary pathogenic protozoa like Theileria parva, the pathogen causing bovine East coast fever, Trypanosoma congolense congolense or Trypanosoma vivax vivax, Trypanosoma brucei brucei, pathogens causing ⁇ agana cattle disease in Africa, Trypanosoma brucei evansi causing Surra , Babesia bigemina, the pathogen causing Texas fever in cattle and buff alos, Babesia bovis, the pathogen causing European bovine Babesiosis as well as Babesiosis in dogs, cats and sheep, Sarcocystis ovicanis and ovifelis pathogens causing Sarcocystiosis in sheep, cattle and pigs, Cryptosporidia, pathogens causing Cryptosporidioses in cattle and birds, Eimeria and Isospora species, pathogens causing Coccidio
  • the use of the compounds of the present invention is preferred in particular for the treatment of Coccidiosis or Malaria infections, or for the preparation of a drug or feed stuff for the treatment of these diseases.
  • This treatment can be prophylactic or curative.
  • the compounds of the present invention may be combined with other anti-malaria agents.
  • the compounds of the present invention can further be used for viral infections or other infections caused for instance by Pneumocystis carinii.
  • the cDNA encoding for an N-terminally truncated human DHODH(Met30- Arg396) was amplified by the polymerase chain reaction (PCR) from a human liver cDNA bank (Invitrogen, Groningen).
  • PCR polymerase chain reaction
  • the following primers were used to amplify the DHODH gene form the cDNA bank: DHODH-V: 5'-GGA ATT CCA TAT GGC CAC GGG AGA TGA GCG-3'
  • DHODH-R 5 '-GCG CGG ATC CTC ACC TCC GAT GAT CTG C-3 '
  • the underlined sequence regions encode for the cutting sites of the restriction enzymes Ndel (DHODH-V) and BamHI (DHODH-R), respectively.
  • the primers are designed such that subcloning using the Ndel and BamHI restriction sites into a ⁇ ET-19b vector is possible.
  • the amplified DNA bands were purified and isolated from an agarose gel (QIAquick PCR purification kit). The band showed the expected length of 1.2 kb.
  • the isolated PCR fragment was subcloned into a TOPO vector (Invitrogen, Groningen) according to the protocol outlined in the TOPT TA Cloning Kit.
  • the TOPO vector including the ligated PCR fragment was digested with the restriction enzymes Ndel and BamHI (New England Biolabs Inc.) to produce sticky ends. Finally, the fragment was cloned into the Ndel/BamHI sites of a pET-19b vector (Novagen, Madison, WI). This vector produced the human DHODH(Met30-Arg396) as an N-terminal ten histidine fusion protein (hislO-hDHODH(Met30-Arg396)). The vector was transformed into chemical competent E.coli BL21(DE3)Gold cells (Stratagene, LaJolla, CA). Cells were stored as glycerol stocks at -80°C until further use.
  • the cells were harvested by centrifugation for 15 min in a JA-10 Beckmann rotor at 5000 rpm at 4°C. The cell pellet was stored until further use at -20°C.
  • the pellets of 4 x 800 mL expression were thawed on ice and resuspended in 100 mL lysisbuffer containing 50 mM HEPES at pH 7.7, 300 mM NaCl, 10% glycerol, 10% bugbuster (Novagen, lOx), two tablets of protease-inhibitor mix (Complete Tabletes EDTA-free, Roche) and 1% triton X-100.
  • the cell suspension was incubated under gentle rocking for 20 min at room temperature.
  • the resulting suspension was centrifuged in a JA-25.50 rotor (Beckmann) at 25.000 rpm for 1 hour at 4°C.
  • the supernatant was loaded onto a Ni-NTA-column (resin was from Quiagen, column adapter from Pharmacia).
  • the column had a bed volume of 3 mL and was equilibrated with 5 column volumes (CV) of starting buffer (50 mM HEPES pH 7.7; 300 mM NaCl; 10% glycerol and 10 mM imidazole).
  • the sample was loaded with a flow rate of 1 mL/min at 4°C using a BioRad Econopump.
  • the column was mounted on a BioRad BioLogic-LP chromatography system and washed with 5 - 10 CVs of 50 mM HEPES pH 7.7, 300 mM NaCl, 10% glycerol, 10 mM imidazole and 10 mM N,N- dimethylundecylamin-N-oxide (C11DAO) at a rate of 1 mL/min.
  • Another more stringent washing step was performed by applying step gradients consisting of the above washing buffer containing 20 mM and 50 mM imidazole, respectively.
  • the hanging drops were incubated against 0.5 mL reservoir of 0.1 M acetate pH 4.8, 2.4 - 2.6 M ammonium sulfate and 30% glycerol.
  • the crystallization conditions were screened by variation of pH versus ammonium sulfate concentration using a small grid screen (see figure 5):
  • Figure 5 Minimal grid screen used for crystallization trails.
  • Crystals usually appeared as small cubes within three days. They usually reached a full size of 0.2 x 0.2 x 0.2 mm within three to four weeks. The protein crystallized in the space group P3 2 21. Crystals were harvested with pre-mounted loops of size 0.5 mm
  • a total of 55 frames, 65 frames, 96 frames, 62 frames, 120 frames, 60 frames, 100 frames and 100 frames (1° each) were collected from human DHODH(Met30-Arg396) crystals co-crystallized with compound 3, 4, 5, 6, 7, 8, 9 and 10 respectively.
  • the crystals were maintained at a temperature of 100 K during data collection.
  • the indexing and integration of the reflection intensities were performed with the program MOSFLM (Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763.).
  • Data were scaled and merged with SCALA and reduced to structure factor amplitudes with TRUNCATE, both from the CCP4 program suite (Collaborative Computational Project, Number 4 (1994). Acta Cryst.
  • the structure for the human DHODH (Met30-Arg396) in complex with compound 1 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FMN) and one acetate molecule which was present under the crystallization conditions.
  • FMN cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX protocols. Finally, SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.). The resulting experimental electron density was so excellent that the conformation of compound 1 could be interpreted unambiguously.
  • a pdb file for compound 1 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 1 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), two sulfate ions (SO4), one molecule of compound 1 (INH) and 153 water molecules (TIP) (see figure 2).
  • the model is well refined and has very good geometry.
  • the refinement process which included data from 12.0 - 2.35 A resulted in an R-factor of 18.5 % and a free R-factor of 21.7%. With the exception of glycine residues, 92.4 % (278) of the residues are located in the most favoured region of the ramachandran plot and 7.6 % (22) cluster in the additional allowed regions.
  • Table 13 summarizes the refinement statistics for the inhibitor compound 1 in complex with human DHODH. Nalues in parentheses give the R-factor and R free -factors, respectively, for the last resolution bin ranging from 2.50 to 2.35. The ⁇ -terminal His tag could not be detected in the electron density map.
  • the structure for the human DHODH (Met30-Arg396) in complex with compound 2 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FM ⁇ ) and one acetate molecule which was present under the crystallization conditions.
  • FM ⁇ cofactor flavinmononucleotide
  • MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally a SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • Pdb files for the compound 2 in conformation A and B were created using the program MOE (Chemical Computing Group Inc., MOE 2002.02) . Both compounds were energy minimized and built into the electron density manually. Topology and parameter files for compound 2 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol., 273, 371-376). After an additional round of model building and water picking using CNX, another complete round of refinement was performed.
  • the final model included the human DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), four sulfate ions (SO4), one molecule of compound 2 (INH) either in conformation A or conformation B and 250 water molecules (TIP) (see figures 3 and 4).
  • the models are well refined and show very good geometry.
  • the refinement process which included data from 12.0 - 2.4 A resulted in an R-factor of 17.5 % and a free R-factor of 21.1% for conformation A complex and an R-factor of 17.6 % and a free R-factor of 21.6% for conformation B complex, respectively.
  • the structure for the human DHODH(Met30-Arg396) in complex with compound 3 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FMN) and one acetate molecule which was present under the crystallization conditions.
  • FMN cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-f actor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • Topology and parameter files for compound 3 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol., 273, 371-376). After an additional round of model building and water picking using CNX, another complete round of refinement was performed.
  • the final model included the human DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), two acetate molecules (ACT), two sulfate ions (SO4), one molecule of compound 3 (INH) either in conformation A or conformation B and 263 water molecules (WAT). Residues which are missing the coordinate file due to very poor electron density are listed in the header of the pdb files.
  • the structure for the human DHODH(Met30-Arg396) in complex with compound 4 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FMN) and one acetate molecule which was present under the crystallization conditions.
  • FMN cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the resulting experimental electron density was so excellent that the conformation of the inhibitor compound 4 could be interpreted unambiguously.
  • the electron density around the five- membered ring carrying the carboxy group clearly showed the carboxy group in contact with residues His 56 and Tyr 356 in non-brequinar-like conformation.
  • a pdb file for compound 4 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 4 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH (Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), one sulfate ion (SO4), one molecule of compound 4 (L H) and 192 water molecules (TIP).
  • the model is well refined and shows very good stereochemical geometry.
  • the refinement process which included data from 19.9 - 2.15 A resulted in an R-factor of 20.1 % and a free R-factor of 22.1%. Except for non-glycine and non-proline residues 91.6% of the residues are located in the most favoured region of the ramachandran plot and 8 % and 0.3 % cluster in the additional allowed or generously allowed regions, respectively. There are no residues in the disallowed region.
  • Table 16 summarizes the refinement statistics for compound 4 in complex with human DHODH. Nalues in parentheses give the R-factor and R f r ee -factors, respectively, for the last resolution bin ranging from 2.28 to 2.15.
  • DHODH/compound 5 complex The structure for the human DHODH(Met30-Arg396) in complex with compound 5 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FMN) and one acetate molecule which was present under the crystallization conditions.
  • FMN cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing, protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the resulting experimental electron density was so excellent that the conformation of the inhibitor compound 5 could be interpreted unambiguously.
  • the electron density around the five- membered ring carrying the carboxy group clearly showed the carboxy group in contact with residues His 56 and Tyr 356 in non-brequinar-like conformation.
  • the protein's active site discriminates between the S- and R-enantiomere. Inspection of the corresponding electron density unequivocally shows the presences of the R-enantiomere only.
  • a pdb file for compound 5 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 5 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), two sulfate ions (SO4), one molecule of compound 5 (INH) and 287 water molecules (TLP).
  • the model is well refined and shows very good stereochemical geometry.
  • the refinement process which included data from 25.5 - 2.2 A resulted in an R-factor of 18.3 % and a free R-factor of 20.9 %.
  • DHODH/compound 6 complex The structure for the human DHODH(Met30-Arg396) in complex with compound 6 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G. ⁇ db was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FM ⁇ ) and one acetate molecule which was present under the crystallization conditions.
  • FM ⁇ cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the resulting experimental electron density was so excellent that the conformation of the inhibitor compound 6 could be interpreted unambiguously.
  • the electron density around the five- membered ring carrying the carboxy group clearly showed that the inhibitor molecule adopts both the brequinar and non-brequinar binding mode.
  • the carboxy group is in contact with both anion binding sites.
  • the protein's active site discriminates between the S- and R-enantiomere. Inspection of the corresponding electron density unequivocally shows the presences of the R-enantiomere only.
  • a pdb file for compound 6 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 6 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH (Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), one sulfate ion (SO4), one molecule of compound 6 (INH) and 312 water molecules (TIP).
  • DHODH/compound 7 complex The structure for the human DHODH(Met30-Arg396) in complex with compound 7 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FM ⁇ ) and one acetate molecule which was present under the crystallization conditions.
  • FM ⁇ cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the resulting experimental electron density was so excellent that the conformation of the inhibitor compound 7 could be interpreted unambiguously.
  • the electron density around the five- membered ring carrying the carboxy group clearly showed the carboxy group in contact with residues His 56 and Tyr 356 in non-brequinar-like conformation addressing subsite 3.
  • a hydroxy group at 3-position at the five membered ring was introduced creating a stereo center at this position.
  • the racemic mixture was used for crystallization experiments.
  • Analysis of the electron density reveals the presence of both enantiomeres. interestingly only the R-enantiomere is able to form additional contacts to the side chains of residues Gln47 and Argl36 and to a conserved water molecule.
  • a pdb file for compound 7 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 7 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), two sulfate ions (SO4), one molecule of compound 7 (INH) and 229 water molecules (TIP).
  • the model is well refined and shows very good stereochemical geometry.
  • the refinement process which included data from 17.0 - 2.0 A resulted in an R-factor of 17.5 % and a free R-factor of 20.4 % for the R-form and S-form. Except for non-glycine and non-proline residues 92.3 % of the residues are located in the most favoured region of the ramachandran plot and 7.7 % cluster in the additional allowed regions. There are no residues in the disallowed region.
  • Table 19 summarizes the refinement statistics for compound 7 in complex with human DHODH. Nalues in parentheses give the R-factor and R free -factors, respectively, for the last resolution bin ranging from 2.13 to 2.0.
  • the structure for the human DHODH(Met30-Arg396) in complex with compound 8 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FM ⁇ ) and one acetate molecule which was present under the crystallization conditions.
  • FM ⁇ cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the resulting experimental electron density was so excellent that the conformation of the inhibitor compound 8 could be interpreted unambiguously.
  • the electron density around the five- membered ring carrying the carboxy group clearly showed the carboxy group in contact with residues His 56 and Tyr 356 in non-brequinar-like conformation addressing subsite 3.
  • a hydroxy group at 5-position at the five membered ring was introduced creating a stereo center at this position.
  • the racemic mixture was used for crystallization experiments. Analysis of the electron density reveals that both enantiomeres fit into the electron density.
  • the R-enantiomere appears to be positioned in a more favourable position to form interactions with subsite 3 whereas in the S-enantiomere the hydroxy group protrudes into the direction of subsite 4 (remote hydrophobic pocket) in a less favourable manner.
  • a pdb file for compound 8 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 8 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), five sulfate ions (SO4), one molecule of compound 8 (INH) and 218 water molecules (TIP).
  • the model is well refined and shows very good stereochemical geometry.
  • the refinement process which included data from 19.0 - 1.8 A resulted in an R-factor of 18.2 % and a free R-factor of 19.6 % for the R-form and S-form (statistics are given only for R-form) .
  • the structure for the human DHODH(Met30-Arg396) in complex with compound 9 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FMN) and one acetate molecule which was present under the crystallization conditions.
  • FMN cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the resulting experimental electron density was so excellent that the conformation of the inhibitor compound 9 could be interpreted unambiguously.
  • the electron density around the five- membered ring carrying the carboxy group clearly showed the carboxy group in contact with residues Gin 47 and Arg 136 and a conserved water molecule in a unique brequinar- like conformation addressing subsite 2 only.
  • the sulfur atom of the five membered ring comes into close contact to Val 134 and Val 143 which form in part subsite 4 (remote hydrophobic pocket).
  • a pdb file for compound 9 was created using the program MOE (Chemical Computing Group Inc., MOE 2002.02). After energy minimization the compound was built into the electron density manually. Topology and parameter files for compound 9 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol. 273, 371-376). After an additional round of model building and water picking using CNX another complete round of refinement was performed.
  • the final model included the DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate molecule (ACT), five sulfate ions (SO4), one molecule of compound 9 (INH) and 291 water molecules (TIP).
  • the model is well refined and shows very good stereochemical geometry.
  • the refinement process which included data from 17.2 - 2.0 A resulted in an R-factor of 18.1 % and a free R-factor of 20.0 %. Except for non-glycine and non-proline residues 92.1 % of the residues are located in the most favoured region of the ramachandran plot and 7.9 % cluster in the additional allowed regions.
  • Table 21 summarizes the refinement statistics for compound 9 in complex with human DHODH. Values in parentheses give the R-factor and R f r ee -factors, respectively, for the last resolution bin ranging from 2.13 to 2.0.
  • the structure for the human DHODH(Met30-Arg396) in complex with compound 10 was solved using the method of molecular replacement (MR).
  • MR molecular replacement
  • the free accessible pdb entry lD3G.pdb was used as a search model.
  • the ligands brequinar and DDQ as well as all of the water molecules were removed prior to the MR search.
  • the search model included the polypeptide chain of hDHODH(Met30-Arg396), one molecule of orotate, one molecule of the cofactor flavinmononucleotide (FMN) and one acetate molecule which was present under the crystallization conditions.
  • FMN cofactor flavinmononucleotide
  • the MR model was subjected to rigid body refinement and a slow cooling simulated annealing protocol using a maximum likelihood target to remove model bias (Accelrys Inc. CNX program suite, CNX2002). Additionally, an individual b-factor refinement was carried out using standard CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron density maps were calculated and displayed together with the protein model in the program O (DatOno AB; Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47, 110-119.).
  • the pdb files for the compound 10 in conformation A and B were created using the program MOE (Chemical Computing Group Inc., MOE 2002.02) . Both compounds were energy minimized and built into the electron density manually. Topology and parameter files for compound 10 were created using the program Xplo2d (Uppsala Software Factory; Kleywegt, G.M.(1997) J. Mol. Biol., 273, 371-376). After an additional round of model building and water picking using CNX, another complete round of refinement was performed.
  • MOE Computer Computing Group Inc., MOE 2002.02
  • the final model included the human DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN), one orotate molecule (ORO), two acetate molecules (ACT), four sulfate ions (SO4), one molecule of compound 10 (LNH) either in conformation A or conformation B and 226 water molecules (TIP). Residues which are missing the coordinate file due to very poor electron density are listed in the header of the pdb files. The models are well refined and show very good geometry. The refinement process which included data from 19.5 - 1.8 A resulted in an R-factor of 19.5% and a free R-factor of 20.5% for the complex in conformation A and for the complex in conformation B, respectively.
  • R-factors indicate that non of the conformers A and B represent a preferred conformation. Except for non-glycine and non-proline residues 91.6% are located in the most favoured region of the ramachandran plot and 8.4 % cluster in the additional allowed regions. There are no residues in the disallowed region.
  • Table 22 summarizes the refinement statistics for compound 10 in complex with human DHODH. Values in parentheses give the R-factor and R f r ee -factors, respectively, for the last resolution bin ranging from 1.91 to 1.8.
  • ORIGX2 0.000000 1.000000 0.000000 0.00000
  • ORIGX3 0.000000 0.000000 1.000000 0.00000
  • ATOM 2872 OH2 TIP 33 48. 477 40. 642 9.325 1.00 18.06 ATOM 2873 OH2 TIP 34 48. 151 60. 664 -5.676 1.00 38.87
  • ATOM 3080 OH2 TIP 292 20, .724 36, .629 8. .924 1. .00 43, .74
  • ATOM 3118 024 INH 1 49. .215 40. .225 -0. ,791 0. .00 20, .42
  • ATOM 109 CA MET A 43 50. ,781 47. ,701 -8. ,243 1. ,00 28. ,07
  • ATOM 110 CB MET A 43 49. ,559 48. ,057 -7. ,389 1. 00 24. ,63
  • ATOM 139 CA GLN A 47 50, .669 41 .990 -10 .329 1, .00 40 .49 ATOM 140 CB GLN A 47 49, .254 42 .446 -9, .957 1, .00 40, .67
  • ATOM 148 CA GLY A 48 51. ,260 41. ,634 -14. ,061 1. 00 39. ,68
  • ATOM 180 C PRO A 52 50. ,054 35. .993 -4.996 1.00 25.59

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Abstract

L'invention concerne des composés de formule générale (I) ou des sels ou isomères de ces composés. Dans ladite formule (I), A représente un système cyclique non aromatique comportant 4 à 8 chaînons. Cette invention concerne également des composés de formule (II) ou des sels ou isomères de ces composés. Dans ladite formule (II), A représente un système cyclique non aromatique comportant 3 à 8 chaînons, et lorsque r = 0, il n'y a pas de liaison double entre les atomes de carbone portant les substituants -CZ1- et -CZ2-. La présente invention concerne en outre des composés de formule (III) dans laquelle A représente un système cyclique hétéroaromatique à 5 chaînons. Selon l'invention, lesdits composés peuvent se fixer sur le site de fixation ubiquinone de l'enzyme DHODH.
EP03813575A 2002-12-23 2003-12-17 Inhibiteurs de l'enzyme dhodh et leur procede d'identification Withdrawn EP1581478A1 (fr)

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EP03028137A EP1541198A1 (fr) 2003-12-05 2003-12-05 Composés cycloalkyles utilisés comme agents anti-inflammatoires, immunomodulateurs et anti-proliferatifs
EP03813575A EP1581478A1 (fr) 2002-12-23 2003-12-17 Inhibiteurs de l'enzyme dhodh et leur procede d'identification
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