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  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen 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
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  • C07C271/40—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings
  • C07C271/42—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/44—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
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  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
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  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
  • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
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  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
  • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
• • C—CHEMISTRY; METALLURGY
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
  • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
  • C07D295/182—Radicals derived from carboxylic acids
  • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
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  • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
  • C07D295/20—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carbonic acid, or sulfur or nitrogen analogues thereof
  • C07D295/205—Radicals derived from carbonic acid
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/04—Systems containing only non-condensed rings with a four-membered ring

Definitions

• This invention relates to novel N-substituted 3,4-diamino-3-cyclobutene-l,2- dione derivatives which inhibit leukocyte adhesion mediated by interaction of the ⁇ 4 ⁇ . integrin (VLA-4) with its counterreceptor VCAM-1, and their use for the treatment of inflammatory and autoimmune diseases.
• VLA-4 ⁇ 4 ⁇ . integrin
• VLA-4 (also referred to as ⁇ ⁇ , integrin and CD49d/CD29), first identified by Hemler and Takada (Hemler and Takada, European Patent Application, Publication No. 330, 506, published August 30, 1989) is a member of the ⁇ , integrin family of cell surface receptors, each of which comprises two subunits, an ⁇ chain and a ⁇ , chain. There are at least nine ⁇ , integrins, all sharing the same ⁇ , chain and each having a distinct ⁇ chain. These nine receptors all bind a different complement of the various cell matrix molecules such as fibronectin, laminin and collagen. VLA-4, for example, binds to fibronectin.
• VLA-4 is unique among ⁇ , integrins in that it also binds non-matrix molecules that are expressed by endothelial and other cells. These non-matrix molecules include VCAM-1, which is expressed on cytokine-activated human umbilical vein endothelial cells in culture. Distinct epitopes of VLA-4 are responsible for fibronectin and VCAM- 1 binding activities and each activity has been shown to be inhibited independently (Elices, et al, Cell, 60:577-584 (1990)).
• Intercellular adhesion mediated by VLA-4 and other cell surface receptors is associated with a number of inflammatory responses.
• activated vascular endothelial cells express molecules that are adhesive for leukocytes.
• the mechanics of leukocyte adhesion to endothelial cells involves, in part, the recognition and binding of cell surface receptors on leukocytes to the corresponding cell surface molecules on endothelial cells. Once bound, the leukocytes migrate across the blood vessel wall to enter the injured site and release chemical mediators to combat infection.
• adhesion receptors of the immune system see, for example. Springer (Springer, Nature, 346:425-434 (1990)) and Osborn (Osborn, Cell, 62:3-6 (1990)).
• Inflammatory brain disorders such as multiple sclerosis (MS) and meningitis
• MS multiple sclerosis
• meningitis are examples of central nervous system disorders in which the endothelium / leukocyte adhesion mechanism results in destruction to otherwise healthy brain tissue.
• BBB blood brain barrier
• the leukocytes release toxic mediators that cause extensive tissue damage resulting in impaired nerve conduction and paralysis.
• tissue damage also occurs via an adhesion mechanism resulting in migration or activation of leukocytes.
• tissue damage also occurs via an adhesion mechanism resulting in migration or activation of leukocytes.
• an adhesion mechanism mediated by an adhesion mechanism include asthma (Pretolani, et al., J. Exp. Med., 180:795 (1994); Abraham, et al., Clin.
• each receptor/ligand interaction is rapidly reversible; however, during the process of cell adhesion, multiple ⁇ 4 ⁇ , integrin receptors on one cell engage multiple VCAM-1 ligands on another cell, and together provide a strong and stable adhesive bond.
• small molecule inhibitors of ⁇ 4 ⁇ ⁇ integrin must achieve a high degree of receptor occupancy for disruption of a significant number of these adhesive interactions.
• inhibitory compounds exhibit a very steep titration curve, since inhibition begins with 85-90% receptor occupancy and is complete when 95-100% of the receptors are occupied. With such a narrow dynamic range there is considerable assay to assay variation in cell-based adhesion studies.
• A is alkylene or alkenylene.
• This invention provides novel compounds of Formula I
• R 1 is alkyl, aryl. heteroaryl. aralkyl or heteroaralkyl:
• R 2 is H, alkyl. aryl, heteroaryl, aralkyl, or heteroaralkyl; or R' and R 2 may be taken together to form a saturated or unsaturated heterocycloalkyl;
• R 3 is H, alkyl. aryl. heteroaryl, aralkyl, or heteroaralkyl;
• A is aryl or heteroaryl; and
• x, y and z are independently 0, 1, 2, 3, or a pharmaceutical salt thereof.
• R 1 is alkyl of 1 to 10 carbon atoms, aralkyl of 7 to 1 1 carbon atoms, or heteroaralkyl of 7 to 1 1 members having 1 to 3 heteroatoms. In still more preferred embodiments of the present invention R 1 is straight chain alkyl of 4 to 8 carbon atoms, benzyl, benzhydryl, phenethyl, pyridylmethyl or pyridylethyl.
• R 2 is preferably hydrogen, alkyl of 1 to 10 carbon atoms or aralkyl of 7 to 1 1 carbon atoms. More preferably R 2 is hydrogen, alkyl of 1 to 6 carbon atoms, benzyl or naphfhylmethyl. Alternatively, when R 1 and R 2 are taken together, they preferably form a substituted heterocycloalkyl of 5 to 7 members having 1 to 3 heteroatoms selected from N, O and S.
• A is preferably substituted or unsubstituted aryl.
• the substituent is preferably selected from -NR 4 COR 5 , -OCONR 6 R 7 or -O(CHdonating)mNR 6 R 7 wherein R 4 is hydrogen or alkyl of 1 to 3 carbon atoms, R " is substituted or unsubstituted aryl, heteroaryl or heterocycloalkyl, R 6 and R 7 are independently, hydrogen or alkyl of 1 to 3 carbon atoms, or R 6 and R 7 , taken together may form a substituted heterocycloalkyl, and m is an integer from 1 to 6. In some embodiments of the present invention it is preferred that x and y are 0 and z is 1.
• R 3 is preferably hydrogen in some aspects of the invention.
• preferred substituents are alkyl of 1 to 3 carbon atoms, aryl, -COR * or -COOR 9 wherein R 8 is alkyl of 1 to 3 carbon atoms, aryl of 5 or 6 carbon atoms or aralkyl of 6 or 7 carbon atoms, and R 9 is hydrogen, alkyl of 1 to 3 carbon atoms, aryl of 5 or 6 carbon atoms or aralkyl of 6 or 7 carbon atoms.
• R 1 is alkyl, aralkyl or heteroaralkyl
• A is phenyl
• x and y are 0, and z is 1.
• More preferred compounds of the present invention are the following compounds:
• Alkyl' as used herein means a branched or straight chain having from 1 to
• alkyl groups include methyl, ethyl, propyl. isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl.
• Alkyl may be substituted and unsubstituted.
• Aryl' as used herein means mono or bicyclic aromatic ring having from 5 to
• Monocyclic rings preferably have 5 or 6 members and bicyclic rings preferably have 8, 9 or 10 membered ring structures.
• Exemplary aryl groups include phenyl and naphthyl.
• Aryl may be substituted or unsubstituted.
• Aralkyl as used herein means an aryl-alkyl group in which the aryl and alkyl group are previously defined. Exemplary aralkyl groups include benzyl and phenethyl. The aralkyl may be substituted or unsubstituted.
• Halogen is chlorine, fluorine, iodine or bromine.
• Heteroaryl whether used alone or as part of a group such as “heteroaralkyl' " means 5 to 10 membered mono or bicyclic aromatic ring having from 1 to 3 heteroatoms selected from N. O and S.
• Exemplary heteroaryls include pyridyl, pyrazinyl, pyridazinyl. pyrimidinyl, indolyl, imidazolyl, pyrazolyl and pyrrolyl.
• Preferred heteroaryl groups include lH-indoly-3-yl, pyridin-3-yl, pyridin-4-yl, and 1H- imidazol-4-yl.
• the heteroaryl may be substituted or unsubstituted.
• Hetcraralkyl means an heteroaryl-alkyl group in which the heteroaryl and alkyl are as previously described.
• exemplary heteroaralkyls include pyridylmethyl, pyridylethyl. thienylethyl, thienylmethyl. indolylmethyl. and furylmethyl.
• the heteraralkyl may be substituted or unsubstituted.
• Heterocycloalkyl refers to a monocycloalkyl having from 5 to 10 members including one or more heteroatoms selected from N, O or S.
• the heterocycloalkyl may be saturated or unsatured and may be substituted or unsubstituted.
• Suitable substituents are unsubstituted and include, but are not limited to, alkyl of 1 to 3 carbon atoms, halogen, -CN, -NO 2 , perhaloalkyl of 1 to 3 carbon atoms, aryl. aralkyl, -NR COR 5 , -CO 2 R 4 , -OR 4 , -OCONR 6 R 7 or -O(CH 2 )mNR 6 R 7 wherein R 4 is hydrogen, alkyl of 1 to 3 carbon atoms, or aralkyl of 7-10 carbon atoms, R 5 is aryl.
• R 6 and R 7 are independently, hydrogen or alkyl of 1 to 3 carbon atoms, or R 6 and R 7 , taken together may form a heterocycloalkyl, and m is an integer from 1 to 6.
• Carbon number refers to the number of carbons in the carbon backbone and does not include carbon atoms occurring in substituents thereof.
• Pharmaceutically acceptable salts are the acid addition salts which can be formed from a compound of the above general formula and a pharmaceutically acceptable acid such as phosphoric, sulfuric. hydrochloric, hydrobromic, citric, maleic, succinic, fumaric, acetic, lactic, nitric, sulfonic, p-toluene sulfonic, methane sulfonic acid, and the like.
• a pharmaceutically acceptable acid such as phosphoric, sulfuric. hydrochloric, hydrobromic, citric, maleic, succinic, fumaric, acetic, lactic, nitric, sulfonic, p-toluene sulfonic, methane sulfonic acid, and the like.
• the compounds of this invention contain a chiral center, providing for various seteroisomeric forms of the compounds such as racemic mixtures as well as the individual optical isomers.
• the individual isomers can be prepared directly or by asymmetric or stercospecific synthesis or by conventional separation of optical isomers from the racemic mixture.
• Novel compounds of Formula I are prepared by the sequential addition of appropriate amine nucleophilcs to 3.4-diethoxy-3-cyclobutene- 1.2-dione in alcoholic solvent, followed by hydrolysis of the precursor carboxylic acid ester to the parent acid by treatment with aqueous base as shown in the following reaction schemes.
• Example 8 2-(3.4-Dioxo-2-r(pyridin-3-ylmethyl)-aminol-cvclobut-l-enylamino)-3-phenyl-pro pionic acid methyl ester
• the lithium salt of the title compound was obtained in 66% yield as a light yellow solid.
• the modified procedure requires removing the volatiles in vacuo from the reaction mixture following completion of ester hydrolysis (usually 3 hours at room temperature), followed by partitioning the reaction mixture between EtOAc and water. The aqueous phase is then lyophilized to afford the lithium salt as an amorphous powder.
• Example 36 N-r2-rMethyl(2-phenylethyl)aminol-3.4-dioxo-l -cyclobuten- l-yl]-4-r(4- pyridinylcarbonyl)aminol-L-phenylalanine methyl ester
• IR (KBr, cm “1 ) 3375 (br), 2900, 1800, 1660, 1575, 1530, 1410, 1325.
• Example 47 N-12-(Methyl-pyridin-3-ylmethyl-amino)-3.4-dioxo-cvclobut-l-enyl]-4-r(pyridine-3- carbonyl)-aminol-L-phenylalanine methyl ester
• IR (KBr, cm “1 ) 3410, 2920, 1810, 1730, 1580, 1530, 1410, 1220.
• VLA-4 binding activity of exemplary compounds was measured by measuring the inhibition of the interaction of soluble VCAM-1 with Jurkat cells (ATCC #TIB-153) which express high levels of ⁇ 4 ⁇ , integrin (VLA-4) using a modification of the fluorescence activated cell sorter (FACS) assay described by Yednock, et al., J. Biol. Chem., 1995, 270:28740.
• FACS fluorescence activated cell sorter
• Jurkat cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin and glutamine as described by Yednock, supra.
• Recombinant soluble VCAM-1 (rsVCAM-1) was produced in a baculovirus expression system as a chimeric fusion protein containing the seven immunoglobulin domains of VCAM-1 on the N-terminus and the human IgG, heavy chain constant region on the C-terminus as described by Yednock, supra. Supernatant containing approximately lOug/ml rs VCAM-1 was collected after 72 hours and used in the assay without purification.
• Jurkat cells (approximately 10 7 cells/ml) were treated with 1.5mM MnCl 2 and
• Cells were washed twice and resuspended in 100 ⁇ l of a 1 : 100 dilution of FITC-conjugated goat anti-human IgG to detect the human Ig-VCAM-1 construct diluted in assay media containing 2.5% mouse serum to block potential cross- reactivity with cell surface bound 15/7. Cells were incubated on ice for 30 minutes in the dark. Cells were washed twice and analyzed with a standard FACS analysis as described in Yednock, et al., supra, on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
• compounds of the present invention exhibit high affinity for VLA-4, and can effectively inhibit the interaction of VLA-4 with VCAM.
• the compounds are useful for the treatment of inflammatory and autoimmune diseases including, but not limited to multiple sclerosis, meningitis, asthma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel syndrome, rheumatoid arthritis, tumor metastasis, tissue transplantation, and myocardial ischemia.
• the compounds of the present invention can be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases.
• These salts include, but are not limited to, salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and salts with organic acids such as acetic acid, oxalic acid, succinic acid, and maleic acid.
• Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium.
• the compounds of the present invention can also be used in the form of esters at the C-terminus; carbamates, amides and the like at the N-terminus or other conventional "pro-drug" forms which, when administered, convert to the active moiety in vivo.
• Compounds of the present invention may be administered in combination with one or more pharmaceutically acceptable carriers, for example, solvents, diluents and the like.
• Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils.
• Adjuvents customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA. These compounds may be administered orally as well as by intravenous, intramuscular, or subcutaneous routes.
• formulations may contain, for example, from about 0.05 to 5% of suspending agent, syrups containing, for example, from about 10 to 50% of sugar, or elixirs containing, for example, from about 20 to 50% ethanol, and the like.
• formulation may be, for example, sterile injectable solutions or suspensions containing from about 0.05 to 5% suspending agent in an isotonic medium.
• Such pharmaceutical preparations may contain, for example, from about 25 to about 90% by weight of active ingredient in combination with a carrier, and more preferably between about 5% and 60% by weight of active ingredient.
• compositions from the standpoint of ease of preparation and administration are solid compositions, particularly tablets and hard-filled or liquid-filled capsules. Oral administration of the compounds is preferred.
• the dosage requirements can be determined by one skilled in the art and will vary with the particular composition employed, the route of administration, the severity of the symptoms presented and the particular subject being treated.

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