MXPA01005968A - 3,4-diamino-3-cyclobutene-1,2-dione derivatives which inhibit leukocyte adhesion mediated by vla-4 - Google Patents

Abstract

Compounds of formula (I) which inhibit leukocyte adhesion mediated by interaction of the a4b1 integrin (VLA-4) with its counterreceptor VCAM-1, and their use for the treatment of inflammatory and autoimmune diseases.

Description

DERIVATIVES OF 3,4-DIAMINO-3-CICLOBUTENO-1, 2-DIONA WHICH INHIBIT THE ADHESION OF LEUKOCYTES MEDIATED BY INTEGRINE ALFA4, BETAI (VIA-4) Field of the Invention This invention relates to novel N-substituted 3, 4-diamino-3-cyclobutene-1, 2-dione derivatives which inhibit leukocyte adhesion mediated by the integrin a4ß interaction. (VLA-4) with its VCAM-1 counter-receptor, and its use for the treatment of inflammatory and autoimmune diseases.

Background of the Invention VLA-4 (also referred to as integrin a, jβ and CD49d / CD29), first identified by He ler and Takada (Hemler and Takada, European Patent Applicati on, Publi cation Do not . 330, 506, published August 30, 1989) is a member of the ßi integrin family of cell surface receptors, each of which comprises two subunits, one chain and one ßi chain. There are at least nine ßi integrins, all share the same ßi chain and each has a different chain. These nine receptors all bind to a different complement of the various matrix molecules of cells such as fibronectin, laminin and collagen. The VLA-4, for example, REF: 129769 ßi integrins in that it also binds to different molecules of the matrix that are expressed by endothelial cells and other cells. These different molecules of the matrix include VCAM-1, which is expressed in the endothelial cells of the human umbilical vein, activated by the cytosine in a culture. Epitopes other than VLA-4 are responsible for fibronectin and VCAM-1 binds to the activities and it has been shown that each activity is independently inhibited (Elices, et al., Cell, 60: 577-584 (1990)) . Intercellular adhesion mediated by VLA-4 and other cell surface receptors is associated with a large number of inflammatory responses. At the site of a lesion or other inflammatory stimulus, activated vascular endothelial cells express molecules that are adhesive to leukocytes. The mechanics of leukocyte adhesion to endothelial cells involves, in part, the recognition and binding of cell surface receptors in leukocytes to the corresponding cell surface molecules in endothelial cells. Once they are joined, the leukocytes migrate through the wall of the blood vessels to enter the injured site and release the chemical mediators to fight the infection. For reviews of adhesion receptors of the immune system, see, for example, Springer (Springer, Na ture, 346: 425-434 (1990)) and Osborn (Osborn, Cell, 62: 3-6 (1990)). Inflammatory disorders of the brain, such as multiple sclerosis (MS) and meningitis, are examples of central nervous system disorders in which the endothelium / leukocyte adhesion mechanism results in destruction for the other tissue healthy brain Large numbers of leukocytes migrate through the blood-brain barrier (BBB) in subjects with these inflammatory diseases. Leukocytes release toxic mediators that cause extensive tissue damage resulting in impaired nerve conduction and paralysis. In other organ systems, tissue damage also occurs through an adhesion mechanism resulting in the migration or activation of leukocytes. For example, it has been shown that the initial attack after myocardial ischemia to the heart tissue can be further complicated by the entry of leukocytes into the injured tissue causing an even further attack (Vedder, et al., Surgery, 106: 509 (1989)). Other inflammatory conditions mediated by an adhesion mechanism include asthma (Pretolani, et al., J. Exp. Med., 180: 795 (1994); collaborators, Clin. Invest., 93: 776 (1994): Mulligan et al., Immunology, 150: 2407 (1993)), Alzheimer's disease, atherosclerosis (Cybulsky et al., Science, 251: 788 (1991)).; Li and collaborators, Atheroscler. Thromb., .13: 197 (1993)), dementia due to AIDS (Sasseville et al., Am. J. Path., 144: 27 (1994)), diabetes (Yang et al., Proc. Nat. Acad. Science ( USA), 90: 10494 (1993), Burkly et al., Diabetes, 43: 526 (1994), Baron et al., J. Clin. Invest., 93 ^: 1700 (1994)), inflammatory bowel disease (Hamann et al. collaborators, Immunology, 152: 3238 (1994)), multiple sclerosis (Yednock et al., Nature, 356: 63 (1992); Baron et al., J. Exp. Med., 177: 57 (1993)), rheumatoid arthritis ( van Dinther-Janssen et al., Annals, Rheumatic Dis., 52: 672 (1993), Elices et al., J. Clin. Invest.,: 93: 405 (1994), Postigo et al., J. Clin. Invest., 89: 1445 (1991)), tissue transplantation (Paul et al., Transpl. Proceed., 25: 813 (1993), and tumor metastasis (Okahara et al., Can. Res., 5_4: 3233 (1994); Paavonen and collaborators, Int. J. Can., 5: 8: 298 (1994); Schadendorf et al., J. Path 170: 429 (199 3)). Because the importance of VLA-4 in inflammatory and autoimmune conditions, it is desirable to test the presence of VLA-4 in biological samples and for the compounds which inhibit cell adhesion. Individually, each receptor / ligand interaction is rapidly reversible; however, during the process of cell adhesion, multiple integrin a4β? in one cell they are coupled to multiple ligands of VCAM-1 in another cell, and together they provide a strong and stable adhesive bond. In order to prevent cell adhesion, the small molecule inhibitors of integrin a4β? they must achieve a high degree of receptor occupancy for the disruption of a significant number of these adhesive interactions. In addition, due to the multivalency of the adhesive interaction, the inhibitory compounds exhibit a very steep titration curve, since the inhibition starts with 85-90% occupancy of the receptors and is completed when 95-100% of the receptors are occupied . With this reduced dynamic range there is a considerable trial-to-trial variation in cell-based adhesion studies. An assay is desired which can detect the presence of an individual VCAM-1 molecule with an individual receptor and thus prevent a variation of test to assay. The N-substituted 3, 4-diamino-3-cyclobutene-l, 2-dione derivatives have been taught. The Japanese patent JP05229999 A2 930907 describes cyclobutene diones which are symmetrically disubstituted with α-amino acids. U.S. Patent No. 5,168,103 issued December 1, 1992 and assigned to American Home Products, discloses cyclobutenedione derivatives having the formula (2) where A is alkylene or alkenylene. It is taught that these compounds are useful as N-methyl-D-aspartate antagonists.

Description of the invention This invention provides new compounds of Formula I wherein R1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; R 2 is H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R1 and R2 can be taken together to form a saturated or unsaturated heterocycloalkyl; R3 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. In some preferred embodiments of the present invention R1 is alkyl of 1 to 10 carbon atoms, aralkyl of 7 to 11 carbon atoms, or heteroaralkyl of 7 to 11 members having 1 to 3 heteroatoms. In still more preferred embodiments of the present invention R1 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 11 carbon atoms. More preferably R2 is hydrogen, alkyl of 1 to 6 carbon atoms, benzyl or naphthylmethyl.

Alternatively, when R1 and R2 are taken together, they preferably form a 5- to 7-membered substituted heterocycloalkyl having 1 to 3 heieroatoms selected from N, 0 and S. A is preferably substituted or unsubstituted aryl. When A is substituted, the substituent is preferably selected from -NRCOR5, -OCONR6R7 or -0 (CH2) mNR6R7 wherein R4 is hydrogen or alkyl of 1 to 3 carbon atoms, R5 is unsubstituted or substituted aryl, heteroaryl or heterocycloalkyl, R6 and R7 are independently, hydrogen or alkyl of 1 to 3 carbon atoms, or R6 and R7, taken together can form a substituted heterocycloalkyl, and is an integer from 1 to 6. In some embodiments of the present invention it is preferred that xyy be 0 and z is 1. R 'is preferably hydrogen in some aspects of the invention. For purposes of defining the preferred substituted heterocycloalkyl, preferred substituents are alkyl of 1 to 3 carbon atoms, aryl, -COR8 or -COOR9 wherein R8 is alkyl of 1 to 3 carbon atoms, aryl of 5 or 6 carbon atoms or aralkyl of 6 or 7 carbon atoms, and R9 is hydrogen, alkyl of 1 to 3 carbon atoms, aryl of 5 or 6 carbon atoms or aralkyl of 6 or 7 carbon atoms. In some embodiments of the present invention R 1 is alkyl, aralkyl or heteroaralkyl, A is phenyl, x and y are 0 and z is 1. The most preferred compounds of the present invention are the following compounds: [2- (Benzylamino) -3, 4- dioxo-cyclobut-l-enyl] -L-phenylalanine; [2- (Benzhydrylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine; 2-. { 2- [2- (lH-Indol-3-yl) -ethylamino] -3,4-dioxo-cyclobut-1-enylamino} -L-phenylalanine; . { 3, 4-Dioxo-2- [(pyridin-3-ylmethyl) -amino] -cyclobut-1-enyl} -L-phenylalanine; [2- (Benzyl-hexyl-amino) -3, -dioxo-cyclobut-l-enyl] -L-phenylalanine; (2-Dibenzylamino-3, 4-dioxo-cyclobut-l-enylamino) -L-phenylslanine; (S) -2- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3-phenyl-propionic acid; (S) -2- [2- (hexyl-naphthalen-2-ylmethyl-amino) -3,4-dioxo-cyclobut-1-enylamino) -3-phenyl-propionic acid; Acid (S) -2-. { 2- [(4-Dimethylamino-benzyl) -hexyl-amino] -3,4-dioxo-cyclobut-1-enylamino} -3-phenyl-propionic; N- [3, -DIOXO-2- (4-phenyl-piperazin-1-yl) -cyclobut-1-en-1-yl] -L-phenylalanine; (S) -2- [2- (4-Acetyl-piperazin-1-yl) -3,4-dioxo-cyclobut-1-enylamino] -3-phenyl-propionic acid; (S) -3- (4-Benzylamino-phenyl) -2- (2-dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -propionic acid; (S) -3- (1-Benzyl-1H-imidazol-4-yl) -2- (2-dihexylamino-3, 4-dioxo-cyclobut-1-enylamino) -propionic acid; N- (2-Dihexylamino-3,4-dioxo-cyclobut-1-ethylamino) -O- (3-dimethylamino-propyl) -L-tyrosine; N- [2- [Methyl [2- (4-pyridinyl) ethyl] amino] -3,4-dioxo-1-cyclobuten-l-yl] -4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine; N- [2- [Methyl (2-phenylethyl) amino] -3,4-dioxo-l-cyclobuten-1-yl] -4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine; N- [2- (Dihexylamino) -3,4-dioxo-l-cyclobuten-1-yl] -4- [(4-p; -ridinylcarbonyl) -amino] -L-phenylalanine; N- [2- (Methyl-pyridin-3-ylmethylamino) -3,4-dioxo-cyclobut-1-enyl] -4- [(pyridine-4-carbonyl) -amino] -L-phenylalanine; N- [2- (Dihexylamino) -3,4-dioxo-l-cyclobuten-l-yl] -4- [(3-pyridinylcarbonyl) -amino] -L-phenylalanine; N- [2- [Methyl [2- (4-pyridinyl) ethyl] amino] -3,4-dioxo-1-cyclobuten-l-yl] -4- [(3-pyridinylcarbonyl) amino] -L-phenylalanine; N- [2- (Methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo-cyclo-b-1-enyl] -4- [(pyridine-3-carbonyl) -amino] -L-phenylalanine; N-. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-1-enyl} -L-phenylalanine; Methyl ester of N- [2- (dihexylamino) -3,4-dioxo-1-cyclobuten-1-yl] -. { 4- [4- (N-carboxy-benzoyl) piperidinylcarbonyl] amino} -L-phenylalanine. (2S) -3- (4-Dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-phenethyl-amino) -3,4-dioxo-cyclobut-1-enylamino] -propionic acid; (2S) -2- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3- (4-dimethyl-carbamoyloxy-phenyl) -propionic acid; (2S) -3- (4-Dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo-cyclobut-1-enylamide] -propionic acid; Acid (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2-. { 2- [Methyl- (2-pyridin-4-yl-ethyl) amino] -3,4-dioxo-cyclobut-1-enylamino} -propionic; (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl] -2- [2- (methyl-phenylethyl-amino) -3,4-dioxo-cyclobut-1-enylamino] -propionic acid; (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl) -2- acid. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3, -dioxo-cyclobat-1-enylamino] -propionic; and (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl) -2- acid. { 2-dihexylamino] -3,4-dioxo-cyclobut-1-enylamino} -propionic; or a pharmaceutical salt thereof.

"Alkyl" as used herein means a branched or straight chain having from 1 to 10 carbon atoms and more preferably from 1 to 8 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl. The alkyl may be substituted or unsubstituted.

"Aryl" as used herein means a mono or bicyclic aromatic ring having from 5 to 12 carbon atoms. The monocyclic rings preferably have 5 or 6 members and the bicyclic rings preferably have ring structures of 8, 9 or 10 members. Exemplary aryl groups include phenyl and naphthyl. The aryl can be substituted or unsubstituted.

"Aralkyl" as used herein means an aryl-alkyl group in which the aryl and alkyle group is previously defined. The aralkyl groups Exemplary include benzyl and phenethyl. Aralkyl can 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 a 5- or 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-indol-3-yl, pyridin-3-yl, pyridin-4-yl, and lH-imidazol-4-yl. Heteroaryl can be substituted or unsubstituted.

"Heteroaralkyl" means a heteroaryl-alkyl group in which the heteroaryl and alkyl are as previously described. Exemplary heteroaralkyls include pyridylmethyl, pyridylethyl, thienylethyl, thienylmethyl, indolylmethyl and furylmethyl. The heteroaralkyl can be substituted or unsubstituted.

"Heterocycloalkyl" refers to a monocycloalkyl having from 5 to 10 members that includes one or more heteroatoms selected from N, O or S. heterocycloalkyl can be saturated or unsaturated and can be substituted or unsubstituted. Suitable substituents, unless otherwise indicated, are unsubstituted and include, but are not limited to, alkyl of 1 to 3 carbon atoms, halogen, -CN, -N02, perhaloalkyl of 1 to 3 carbon atoms, aryl, aralkyl, -NRCOR5 -C02R4, -OR4, -OCONR6R7 or -0 (CH2) mNR6R7 wherein R4 is hydrogen, alkyl of 1 to 3 carbon atoms or aralkyl of 7-10 carbon atoms, R6 is aryl, heteroaryl or heterocycloalkyl, R6 and R7 are independently, hydrogen or alkyl of 1 to 3 carbon atoms, or R6 and R7, taken together, can form a heterocycloalkyl and m is a integer from 1 to 6. The carbon number refers to the number of carbons in the carbon backbone and does not include carbon atoms that occur in the carbon substituents. Where the terms are used in combination, the definition for each individual part of the combination applies unless otherwise defined. The pharmaceutically acceptable salts are the acid addition salts which can be formed of 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, sulphonic, p-toluene sulfonic, methane sulfonic and the like. The compounds of this invention contain a chiral center, which provides various stereoisomeric 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 stereospecific synthesis or by conventional separation of optical isomers from the racemic mixture. The new compounds of Formula I are prepared by the sequential addition of appropriate amine nucleophiles to 3,4-diethoxy-3-cyclobutene-1,2-dione in an alcohol solvent, followed by hydrolysis of the carboxylic acid ester precursor. to the acid of origin by treatment with an aqueous base as shown in the following reaction schemes.

Reaction Scheme 1 EtO 'Et In the case where the product of Method 1 contains X = OH, the corresponding carbamates are prepared as in Scheme 2.

Reaction Scheme 2 Method 5 The carbamate products are then further processed by means of Method 2 above. The method for preparing the compounds of Formula I as described above is exemplified in the following specific examples. These examples are illustrative and does not mean that they are limiting to this description in any way. Other methods for preparing the compounds of the present invention may be apparent to those skilled in the art. The reactants and reagents used are either commercially available or can be prepared according to standard procedures in the literature.

Example 1 (Method 1) [2- (Benzylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester To a stirred solution of L-phenylalanine methyl ester HCl (2.0 mmol, 431 mg) in pure ethanol (20 mL) was added triethylamine (2.2 mmol, 202 mg, 278 μL) and the resulting solution was stirred at room temperature for 15 minutes. Subsequently, pure 3, 4-diethoxy-3-cyclobutene-1,2-dione (2 mmol, 340 mg, 296 μL) was added dropwise and the resulting solution was stirred at room temperature overnight, during which a solid white color precipitated from the solution. The volatiles were removed in vacuo and the residue was taken up in EtOAc and partitioned between EtOAc and water. The organics were dried (Na2SO4) and purified by flash chromatography (Si02: 1) 20% EtOAc / hexane: 2) 30% EtOAc / hexane: 3) 40% EtOAc / hexane) to give the title compound as a colorless oil (551 mg, 85%).

X H NMR (DMSO-de, 300 MHz) d 9.1 (broad d, 1 H), 7.24 (m, 6 H), 4.88 (m, 1 H), 4.55 (m, 3 H), 4.16 (m, 2 H), 3.23 (dd, 1H), 2.96 (m, 1H), 1.30 (m, 2H).

Example 2 (Method 2) [2- (Benzylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester To a stirred solution of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester (0.33 mmol, 100 mg) in pure ethanol (3 mL) was added pure benzylamine dropwise. (0.36 mmole, 39 mg, 40 μL) at room temperature. The resulting solution was stirred at room temperature overnight, during which a white solid was precipitated from the solution. The volatile substances were removed in vacuo and the residue Absorbed in EtOAc and partitioned between EtOAc and water. The organics were dried (Na 2 SO), concentrated in vacuo and purified by flash chromatography (SiO 2: EtOAc / hexane) to give the title compound as a white solid (113 mg, 94%).

NMR? (DMSO-de, 400 MHz) d 7.85 (s broad, 1H), 7.66 (s broad, 1 H), 7.37 (m, 2H), 7.26 (m, 6H), 7.13 (m, 2H), 5.1 (m , 1H), 4.68 (m, 2H), 3.68 (s, 3H), 3.16 (dd, 1H, 1H, J = 13.9, 5.4 Hz), 3.03 (m, 1H).

Example 3 (Method 3) [2- (Benzylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine To a stirred solution of [2- (benzylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester (0.16 mmol, 60 mg) in THF (5 mL) was added aqueous LiOH (1.0 K, 0.16 mmol, 160 μL) and the resulting solution was stirred at room temperature for 3 hours. The Volatile substances were removed in vacuo and the residue was partitioned between 0.1M acetic acid and EtOAc. The organics were dried (Na2SO4) and concentrated in vacuo to give the title compound as a white solid, m.p. = 215-216 ° (33 mg, 59%).

XH NMR (DMSO-de, 400 MHz) d 13.1 (broad s, 1H), 7.89 (broad s, 1H), 7.59 (broad s, 1H), 7.37 (m, 2H), 7.26 (m, 6H), 7.14 (m, 2H), 4.91 (m, 1H), 4.68 (m, 2H), 3.17 (m, 1H), 3.01 (m, 1H). MS (El, m / e (%)) 350 (17, M +), 259 (16), 91 (100).

Example 4 (Method 2) [2- (Benzhydrylamino) -3,4-dioxo-cyclobut-1-enyl] -L-phenylalanine methyl ester Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester and diphenyl-methylamine in 88% yield .

X H NMR (DMSO-de, 400 MHz) d 8.38 (broad s, 1H), 7.68 (broad s, 1H), 7.39 (m, 4H), 7.26 (m, 9H), 7.12 (m, 2H), 6.33 (m m, 1H), 5.01 (m, 1H), 3.69 (s, 3H), 3.16 (dd, 1H, J = 13.4, 5.6 Hz), 3.05 (dd, 1H, J = 13.4, 5.6 Hz).

Example 5 (Method 3) [2- (benzhydrylamino) -3, -dioxo-cyclobut-l-enyl] -L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 76% yield as a white solid, m.p. = 187-188 ° C.

XH NMR (DMSO-de, 400 MHz) d 13.2 (broad s, 1H), 8.41 (broad s, 1H), 7.62 (broad s, 1 H), 7.38 (m, 4H), 7.30 (m, 2H), 7.24 (m, 7H), 7.13 (d, 2H, J = 7.02 Hz), 6.34 (m, 1H), 4. 90 (M, 1H), 3.16 (dd, 1H, J = 13.7, 4.9 Hz), 3.05 (m, 1 HOUR) . MS ((+) FAB m / e (%)) 449 (14, (M + Na) +), 427 (45, (M + H) +), 217 (331, 167 (100). , cm "1) 3200, 1790, 1730, 1640, 1575, 1530, 1440, 710.

Analysis Calculated for C26H22N204 • 0.25 H20: C, 72.45; H, 5.26: N, 6.50. Found: C, 72.69; H, 5.22: N, 6.67.

Example 6 (Method 2) 2- methyl ester. { 2- [2- (lH-indol-3-yl) -ethylamino] -3, -dioxo-cyclobut-1-enylamino} -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester and tryptamine in 86% yield.

XH NMR (DMSO-de, 400 MHz) d 10.88 (s, 1H), 7.68 (broad s, 1H), 7.57 (d, 2H, J = 7.8 Hz), 7.33 (d, 1H, J = 7.8 Hz), 7.23 (m, 3H), 7.12 (m, 3H), 7.07, (dt, 1H, J = 7.03, 1.1 Hz), 6.96 (dt, 1H, J = 7.03, 1.1 Hz), 5.0 (m, 1H), 3.79 (m, 2H), 3.67 (s, 3H), 3.13 (m, 1H), 3.02 (m, 1H), 2.91 (m, 2H).

Example 7 (Method 3) 2-. { 2- [2-- (lH-Indol-3-yl) -ethylamino] -3,4-dioxo-cyclobut-1-enylamino} -L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 61% yield as a white solid.

NMR Hl (DMSO-de, 400 MHz) d 13.3 (broad s, 1H), 10.8 (s, 1H), 7.58 (d, 2H, J = 7.8 Hz), 7.32 (d, 1H, J = 8.1 Hz), 7.10 - 7.30 (m, 6H), 7.06 (, 1H), 6.97 (m, 1H), 4.90 (m, 1H), 3.79 (m, 2H), 3.38 (q, 1H, J = 7.0 Hz), 3.14 ( dd, 1H, J = 13.9, 4.7 Hz), 3.01 (dd, 1H, J = 13.9, 4.7 Hz), 2.92 (m, 2H). MS (El, m / e (%)) 403 (4, M +), 385 (16), 294 (60), 143 (100).

Analysis Calculated for C23H2? N304 • 0.4 H20; C, 67.27: H, 5.35: N, 10.23. Found: C, 67.58; H, 5.82; N, 9.78.

Example 8 (Method 2) 2- Methyl ester. { 3, 4-dioxo-2- [(pyridin-3-ylmethyl) -amino] -cyclobut-1-enylamino} -3-phenyl-propionic Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine and 3-pyridylmethylamine in 81% yield , pf = 191-192 ° C.

XH NMR (DMSO-de, 400 MHz) d 8.51 (d, 2H, J = 5.1 Hz), 7.87 (s broad, 1H), 7.68 (broad s, 2H), 7.40 (dd, 1H, J = 4.7, 7. 6 Hz), 7.24 (m, 3H), 7.13 (d, 2H, J = 7.2 Hz), 5.01 (broad s, 1H), 4.72 (d, 2H, 5.7 Hz), 3.68 (s, 3H), 3.17 ( dd, 1H, J = 5.2, 13.7 Hz), 3.03 (m, 1H). MS (El, m / e (%)) 365 (6, M +), 337 (7), 274 (15), 242 (40), 214 (18), 186 (13), 146 (44), 44 (100). IR (KBr, cm "1) 3175, 2960, 1800, 1745, 1650, 1570, 1480, 1430, 1310, 1280.

Analysis Calculated for C29H? 9N5? 4; C, 65 74; H, 5 24; N, eleven . fifty . Found: C, 65.22; H, 5.15; N, 11.27.

Example 9 (Method 3). { 3, 4-DIOXO-2- [(pyridin-3-ylmethyl) -amino] -cyclobut-1-enyl) L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 9% yield as a white solid, m.p. = 259-261 ° C. 2 H NMR (DMSO-de, 300 MHz) d 13.25 (broad s, 1H), 8.53 (broad d, 2H), 7.93 (broad s, 1H), 7.69 (broad d, 2H), 7.41 (m, 1H), 7.20 (m, 5H), 4.91 (m, 1H), 4.73 (m, 2H), 3.18 (dd, 1H), 3.03 (m, 1H). MS ((+) FAB, m / e (%)) 352 (10, (M + H) +), 232 (17), 179 (23), 157 (100).

Example 10 (Method 2) [2- (Benzyl-hexyl-amino) -3,4-dioxo-cyclobut-1-enyl] -L-phenylalanine methyl ester Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobutyl-l-enyl] -L-phenylalanine and benzyl (hexyl) -amine methyl ester at 57 % yield as a light yellow oil which was immediately taken to the subsequent reaction.

Example 11 (Method 3) [2- (Benzyl-hexyl-amino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 41% yield as a yellow foam, m.p. = 61-65 ° C.

NMR? H (DMSO-de, 400 MHz) d 13.1 (s, 1H), 7.79 (s broad, 1H), 7.35 (dd, 3H, J = 10.6, 6.9 Hz), 7.21 (m, 6H), 5.14, (m, 1H), 4.67 (broad s, 2H), 3.38 (broad m, 2H), 3.26 ( dd, 2H, J = 14, 4.0 Hz), 2.98 (dd, 1H, J = 14.1, 11.2 Hz), 1. 38 (broad s, 2H), 1.15 (m, 6H), 0.82 (t, 3H, J - 6.8 Hz). MS ((+) FAB, m / e (%)) 457 (76, (M + Na) +), 435 (100, (M + H) +), 389 (13), 192 (35). IR (KBr, cm "1) 3290, 2940, 1800, 1740, 1675, 1570, 1520, 700.

Analysis Calculated for C26H30N2O4 • 0.25 H20; C, 71.13; H, 7. 00; N, 6.38. Found: C, 71.31; H, 7.00; N, 6.20.

Example 12 (Method 2) Methyl ester of (2-dibenzylamino-3,4-dioxo-cyclobut-l-enylamino) -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine and dibenzylamine in 44% yield as a solid light yellow color which was immediately taken to the subsequent reaction.

Example 13 (Method 3) (2-Dibenzylamino-3, 4-dioxo-cyclobut-1-enylamino) -L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 79% yield as yellow solid ur.

NMR? Ti (DMSO-de, 400 MHz) d 13.13 (s, 1H), 8.01 (d, 1H, J = 9. 0 Hz), 7.35 (m, 6H), 7.20 (m, 8H), 5.19 (m, 1H), 4.55 (s broad, 4H), 3.26 (dd, 2H, J = 3.9, 14.0 Hz), 2.97 (m , 1 HOUR) .

MS (El, m / e (%)) 440 (20, M +), 349 (16), 91 (100). IR (KBr, cm "1) 3450-3250 (broad), 2925, 1800, 1740, 1680, 1570, 1520, 1445, 1265, 700. Analysis Calculated for C 7H24N204; C, 73. 62; H, 5. 49; N, 6. 36. Found: C, 72.48; H, 5.41; N, 6.01.

Example 14 (Method 2) (S) -2- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3-phenyl-propionic acid methyl ester Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine and dihexylamine in 62% yield as a solid light yellow color which was immediately taken to the subsequent reaction.

Example 15 (Method 3) (S) -2- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3-phenyl-propionic acid Following the procedure of Method 3 above, the title compound was obtained in 86% yield as a colorless oil.

XH NMR (DMSO-d6, 400 MHz) d 13.1 (broad s, 1H), 7.58 (d, 1H, J = 9.2 Hz), 7.2 (m, 5H), 5.09 (m, 1H), 3.44 (s broad, 4H), 3.24 (dd, 1H, J = 4.0, 13.8 Hz), 3.0 (dd, 1H, J = 11.3, 14.1 Hz), 1.43 (broad s, 4H), 1.02 (m, 12H), 0.84 (t, 6H, J = 7.0 Hz). MS (El, m / e (%) 428 (100, M +), 372 (36), 337 (55), 224 (30).

Example 16 (Method 2) (S) -2- [2- (hexyl-naphthalen-2-ylmethyl-amino) -3,4-dioxo-cyclobut-l-enylamino] -3-phenyl-propionic acid methyl ester Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine methyl ester and (2-naphthalenyl-methyl) hexylamine in 63% of performance as a colorless oil which was immediately brought to the subsequent reaction.

Example 17 (Method 3) (S) -2- [2- (hexyl-naphthalen-2-ylmethyl-amino) -3,4-dioxo-cyclobut-1-enylamino] -3-phenyl-propionic acid Following the procedure of Method 3 above, the title compound was obtained in 80% yield as a light yellow solid, m.p. = 62-70 ° C.

NMR? (DMSO-de, 400 MHz) d 13.15 (broad s, 1H), 7.91 (m, 2H), 7.80 (s, 1H), 7.52 (m, 2H), 7.29 (m, 2H) 7.21 (m, 6H) , 5.17 (m, 1H), 4.84 (broad s, 2H), 3.24 (dd, 2H, J = 3.7, 14.3 Hz), 2.99 (m, 1H), 1.48-1.2 (m, 3H), 1.13 (s, 6H), 0.78 (t, 3H, J = 6.7 Hz). MS (El, m / e (%)) 484 (5, M +), 439 (4), 219 (28), 44 (100).

Example 18 (Method 2) Methyl ester of (S) -2- acid. { 2- [(4-dimethylamino-benzyl) -hexyl-amino] -3, -dioxo-cyclobut-1-enylamino} -3-phenyl-propionic Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine and (4-dimethyl-aminobenzyl) hexylamine in 59% yield as a colorless oil which was immediately taken to the subsequent reaction.

Example 19 (Method 3) Acid (S) -2-. { 2- [(4-Dimethylamino-benzyl) -hexyl-amino] -3,4-dioxo-cyclobut-1-enylamino} -3-phenyl-propionic Following the procedure of Method 3 above, the title compound was obtained in 97% yield as a white solid, m.p. = 77-80 ° C.

XH NMR (DMSO-de, 400 MHz) d 13.07 (s, 1H), 7.78 (broad s, 1H), 7.23 (m, 5H), 7.02 (broad s, 2H), 6.65 (d, 2H, J = 7.9) Hz), 5.16 (broad s, 1H), 4.49 (broad s, 2H), 3.26 (dd, 1H, J = 3.7, 13.8 Hz), 3.0 (m, 1H), 2.87 (s, 6H), 1.48 (s) broad, 3H), 1.15 (m, 7H), 0.82 (t, 3H, J = 6.8 Hz). MS ((+) FAB, m / e (%)) 500 (100, [M + Na] +), 478 (34, [M + H] +), 455 (17), 357 (32).

Example 20 (Method 2) N- [3,4-dioxo-2- (4-phenyl-piperazin-1-yl) -cyclobut-1-en-1-yl-L-phenylalanine methyl ester Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine and 1-phenyl-piperazine in 63% of yield as a white solid which immediately led to the subsequent reaction.

Example 21 (Method 3) N- [3, 4-DIOXO-2- (4-phenyl-piperazin-1-yl) -cyclobut-1-en-1-yl] -L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 65% yield as a white solid, m.p. = 165-167 ° C.

XH NMR (DMSO-de, 400 MHz) d 13.1 (broad s, 1H), 8.01 (d, 1H, J = 9.0 Hz) 7.29-7.16 (m, 7H), 6.98 (d, 2H, J = 8.1 Hz) , 6.82 (t, 1H, J = 7.2Hz), 5.08 (m, 1H), 3.77 (broad s, 4H), 3.24 (dd, 2H, J = 4.0, 14. OHz), 3.19 (t, 3H, J = 5.0 Hz), 2.98 (dd, 1H, J = 11.0, 13.8 Hz). MS (El, m / e (%)) 405 (48, M +), 361 (6), 304 (5), 44 (100).

Example 22 (Method 2) (S) -2- [2- (4-Acetyl-piperazin-1-yl) -3,4-dioxo-cyclobut-1-enylamino] -3-phenyl-propionic acid methyl ester Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3, -dioxo-cyclobut-1-enyl] -L-phenylalanine and 1-acetylpiperazine in 77% yield as a white solid which immediately led to the subsequent reaction.

Example 23 (Method 3) (S) -2- [2- (4-Acetyl-piperazin-1-yl) -3,4-dioxo-cyclobut-1-enylamino] -3-phenyl-propionic acid Following the procedure of Method 3 above, the title compound was obtained in 39% yield as a white solid, m.p. = 155-158 ° C.

NMR? E (DMSO-de, 400 MHz) d 13.1 (broad s, 1H), 7.96 (d, 1H, J = 9.2 Hz) 7.24 (m, 5H), 5.07 (m, 1H), 3.66 (s broad, 2H), 3.57 (broad s, 3H), 3.50 (d, 3H, J = 4.2 Hz), 3.23 (dd, 1H, J = 4.2, 13.8 Hz), 2.97 (dd, 1H, J = 11.0, 13.8 Hz) 2.03 (s, 3H). MS (El, m / e (%)) 371 (21, M +). 270 (10).

Example 24 (Method 1) [2-Ethoxy-3,4-dioxo-cyclobutyl-l-enyl] -L- (4-benzoylamino) phenylalanine methyl ester Following the procedure of Method 1 above, the title compound was obtained from methyl ester hydrochloride of L- (4-benzoylamino) phenylalanine and 3,4-diethoxy-3-cyclobutene-1,2-dione in 64% of performance.

X H NMR (DMSO-de, 300 MHz) d 10.2 (s, 1H), 9.1 (broad dd, 1H), 7.93 (dd, 2H), 7.69 (d, 2H), 7.53 (m, 3H), 7.2 (d , 2H), 4.59 (m, 3H), 3.7 (s, 3H), 3.21 (dd, 1H), 2.93 (broad m, 1H), 1.31 (m, 3H).

Example 25 (Method 2) (S) -3- (4-Benzoylamino-phenyl) -2- (2-dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -propionic acid methyl ester Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L- (4-benzoylamino) phenylalanine methyl ester and dihexylamine in 70% yield as a white solid which immediately led to the subsequent reaction.

Example 26 (Method 3) (S) -3- (4-Benzoylamino-phenyl) -2- (2-dihexylamino-3, -dioxy-cyclobut-1-enylamino) -propionic acid Following the procedure of Method 3 above, the title compound was obtained in 61% yield as a white solid, m.p. = 95-100 ° C.

X H NMR (DMSO-de, 400 MHz) d 13.1 (broad s, 1H), 10.17 (s, 1H), 7.91 (m, 2H), 7.68 (d, 2H, J = 8.6 Hz), 7.6 - 7.48 (m , 4H), 7.19 (d, 2H, J = 8.6 Hz), 5.09 (m, 1H), 3.45 (broad m, 4H), 3.22 (dd, 1H, J = 3.8, 13.9 Hz), 2.98 (dd) , 1H, J = 11.2, 13.8 Hz), 1.43 (broad s, 4H), 1.20 (s, 12H), 0.80 (t, 6H, J = 6.7 Hz). MS ((+) FAB, m / e (%)) 570 (51, [M + Na] +), 548 (25, [M + H] +), 210 (10), 105 (100).

Analysis Calculated for C32H4? N30 • 0.4 H20; C, 69.26; H, 7.59; N, 7.57. Found: C, 69.14; H, 7.55; N, 7.52.

Example 27 (Method 1) [2-Ethoxy-3,4-dioxo-cyclobutyl-l-enyl] -L- (im-benzyl) histidine methyl ester Following the procedure of Method 1 above, the title compound was obtained from methyl ester hydrochloride of L- (im-benzyl) histidine and 3,4-diethoxy-3-cyclobutene-1,2-dione in 58% of performance.

X H NMR (DMSO-de, 300 MHz) d 9.0 (broad dd, 1H), 7.67 (s, 1H), 7.31 (m, 1H), 7.16 (d, 2H), 6.91 (broad s, 1H), 5.12 ( s, 2H), 4.6 (m, 3H), 3.62 (d, 3H), 3.05 (dd, 1H), 2.9 (m, 1H), 1.3 (m, 3 H).

Example 28 (Method 2) Methyl ester of (S) -3- (l-benzyl-lH-imidazol-4-yl) -2- (2-dihexylamino-3,4-dioxo-cyclobut-l-enylamino) - propionic Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-1-enyl] -L- (1J-benzyl) histidine and dihexylamine in 94% yield as a white solid which was immediately taken to the subsequent reaction.

Example 29 (Method 3) (S) -3- (1-Benzyl-1H-imidazol-4-yl) -2- (2-dihexylamino-3, 4-di-3-oxo-cyclobut-1-enylamino) -propionic acid Following the procedure of Method 3 above, the title compound was obtained in 45% yield as a white solid, m.p. = 75-80 ° C.

XH NMR (DMSO-de, 400 MHz) d 7.87 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H), 7.29 (m, 3H), 7.16 (dd, J = 6.4, 1.8 Hz, 2H) , 6.92 (s, 1HI, 5.13 (s, 2H), 5.04 (q, J = 8.3, 5.8 Hz, 1H), 3.55 (broad, 4H), 3.01 (m, 2H), 1.49 (broad s, 4H), 1.21 (s, 13H), 0.82 (s, 6H). MS ((+) FAB, m / e (%)) 509 (100, [M + H] +), 185 (30), 172 (40).

Analyzes Calculated for C29H4oN404 • 0. 5 H20; C, 67.28; H, 7 98; M, 10 82 Found: C, 67.53; H, 8.10; N, 10.47.

Example 30 (Method 1) Methyl ester [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -0- (3-dimethylaminopropyl) -L-tyrosine Following the procedure of Method 1 above, the title compound was obtained from 0- (3-dimethylaminopropyl) -L-tyrosine methyl ester hydrochloride and 3,4-diethoxy-3-cyclobutene-1,2-dione hydrochloride in 71% yield X H NMR (DMSO-de, 300 MHz) d 9.1 (broad d, 1H), 7.12 (d, 2H), 6.82 (d, 2H), 4.89 (m, 1H), 4.6 (m, 2H), 3.93 (t , 2H), 3.69 (s, 3H), 3.1 (m, 1H), 2.88 (m, 1H), 2.31 (t, 2H), 2.12 (s, 6H), 1.8 (m, 2H), 1.3 (m, 3H).

Example 31 (Method 2) N- (2-Dihexylamino-3, 4-dioxo-cyclobut-1-enylamino) -0- (3-dimethylamino-propyl) -L-tyrosine methyl ester Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -0- (3-dimethylaminopropyl) -L-tyrosine methyl ester and dihexylamine in 23% yield as a white solid which was immediately brought into the subsequent reaction.

Example 32 (Method 3 (modified)) N- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -0- (3-dimethylamino-propyl) -L-tyrosine Following a modification procedure of Method 3 above, the lithium salt of the title compound was obtained in 66% yield as a light yellow solid. The modified procedure requires removing volatile substances in vacuo from the reaction mixture following completion of the ester hydrolysis (usually 3 hours at room temperature), followed by division of the reaction mixture between EtOAc and water. The aqueous phase was then lyophilized to give the lithium salt as an amorphous powder. 1N-NMR (DMSO-de, 400 MHz) d 7.08 (d, 1H, J = 6.6 Hz), 6.95 (d, 2H, J = 8.6 Hz), 6.68 (d, 2H, J = 8.6 Hz), 4.28 (m , 1H), 3.87. (t, 2H, J = 6.4 Hz), 3.3 (broad s, 4H), 3.05 (d, 2H, J == 5.1 Hz) 2.31 (t, 2H, J = 7.1 Hz), 2.11 (s, 6H), 1.78 (t, 2H, J = 6.9 Hz), 1.43 (broad s, 4H), 1.19 (broad m, 12H), 0.82 (t, 6H, J = 7.0 Hz).

MS ((+) FAB, m / e (%)) 536 (100, [M + Li] +), 530 (50, [M + H] +). IR (KB.r, cm "1) 3400, 2960, 2930, 2880, 1800, 1575, 1520, 1240.

Example 33 (Method 1) [2-Ethoxy-3,4-dioxo-cyclobut-l-enyl] -4- [(4-pi? Idinylcarbonyl) amino] -L-phenylalanine methyl ester Following the procedure of Method 1 above, the title compound was obtained from hydrochloride of methyl 4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine and 3, 4-diethoxy-3-cyclobutene-l, 2 -Diona in 71% of performance.

X H NMR (DMSO-de, 300 MHz) d 10.47 (s, 1H), 9.12 (broad dd, 1H), 8..78 (dd, 2H), 7.83 (d, 2H), 7.69 (d, 2H), 7.23 (d, 2H), 4.9 (broad m, 1H), 4.59 (m, 2H), 3.7 (s, 3H), 3.22 (dd, 1H), 2.94 (m, 1H), 1.31 (m, 3H).

Example 34 (Method 2) N- [2- [Methyl [2- (4-pyridinyl) ethoxy] ammonium] -3,4-dicxo-l-cyclobuten-l-yl] -4- [4- pyridinylcarbonyl) amino] -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from ester methyl [2-ethoxy-3, 4-dioxo-cyclobut-l-enyl] -4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine and methyl- [2- (4-pyridinyl) ethyl] amine in 41% yield as a yellow foam, which was immediately brought to the subsequent reaction.

Example 35 (Method 3 (modified)) N- [2- [Methyl] -2- (4-pyridinyl) ethyl] amino] -3, -dioxo-l-cyclobuten-1-yl] -4- [(4- pyridinylcarbonyl) amino] -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 73% yield as a yellow solid.

XH NMR (DMSO-d6, 400 MHz) d 10.45 (s, 1H), 8.74 (dd, J = 1.5, 4.6 Hz, 2H), 8.40 (d, J = 4.8 Hz, 2H), 7.81 (d, J = 6.1 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.40 (broad m, 1H), 7. 20 (d, J = 5.3 Hz, 2H), 7.10 (d, J = 8.3 Hz, 2H), 4.40 (m, 1H), 3.70 (broad s, 2H), 3.10 (m, 6H), 2.81 (m, 2H). MS ((+) , m / e (%)) 506 (100, [M + Li] +), 500 (50, [M + H] +). IR (KBr, cm "1) 3400, 1575, 1530, 1410, 1320.

Analysis Calculated for C27H24N505Li • 3.5 H20; C, 56.98; H, . 49; N ,. 12.31. Found: C, 56.96; H, 5.34; N, 11.82.

Example 36 (Method 2) N- [2- [Methyl (2-phenylethyl) amino] -3,4-dioxo-1-cyclobuten-l-yl] -4- [(4-pyridinylcarbonyl) amino] -methyl ester L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from methyl ester [2-ethoxy-3, 4-dioxo-cyclobut-l-enyl] -4- [(4-pyridin: .lcarbonil) amino ] -L-phenylalanine and methyl- (2-phenethyl) amine in 93% yield as a colorless foam which was immediately taken to the subsequent reaction.

Example 37 (Method 3 (modified)) N- [2- [Methyl (2-phenylethyl) amino] -3,4-dioxo-l-cyclobuten-l-yl] -4- [i 4-pyridinecarbonyl) amino] - L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 90% yield as a white solid.

NMR lE (DMSO-de, 400 MHz) d 10.45 (s, 1H), 8.73 (m, 2H), 7.80 (d, J = 5.7 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 7.32 -7.08 (m, 8H), 4.38 (d, J = 4.6 Hz, 1H), 3.67 (broad s, 2H), 3.08 (m, 5H), 2.79 (m, 2H). MS ((+) FAB, m / e (%)) 521 (100, [M + Na] +), 505 (85, [M + Li] +], 499 (60, [M + H] +). IR (KBr, cm "1) 3400, 1810, 1660, 1580, 1530, 1410, 1330.

Example 38 (Method 2) N- [2- [Diexylamino] -3,4-dioxo-l-cyclobuten-1-yl] -4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine methyl ester Following the procedure of Method 2 above, the title compound was obtained from methyl ester [2-ethoxy-3, 4-dioxo-cyclobut-l-enyl] -4- [(4-pyridinylcarbonyl) amino] -L phenylalanine and dihexylamine in 87% yield as a light yellow foam which was immediately taken to the subsequent reaction.

Example 39 (Method 3 (modified)) N- [2- (Dihexylamino) -3,4-dioxo-l-cyclobuten-l-yl] -4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 92% yield as a white solid.

XH NMR (DMSO-de, 400 MHz) d 10.46 (s, 1H), 9.75 (dd, J = 4.4, 1.8 Hz, 2H), 7.83 (dd, J = 4.4, 1.8 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.11 (d, J = 6.2 Hz, 1H), 7.05 (d, 8.6 Hz, 2H), 4.33 (q, J = 5.5 Hz, 1H), 3.65 (s broad, 2H), 3.25 (s) broad, 2H), 3.11 (d, J 5.7 Hz, 3H), 1.43 (broad s, 4H), 1.15 (wide i, 11 Hz), 0.78 (s, 6H). MS ((+) FAB, m / e (%)) 555 (100, [M + Li] +), 549 (97, [M + H] +). IR (KBr, cm "1) 3330, 2910, 1800, 1660, 1580, 1520, 1410, 1300.

Analysis Calculated for C3? H39N405Li • 2 H20; C, 62.99, H, 7.33; N, 9.48. Found: C, 62.65; H, 7.23; N, 9.31.

Example 40 (Method 2) N- [2- (Methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo-cyclobut-1-enyl] -4- [(pyridine-4-carbonyl) -methyl ester amino] -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -4- [(4-pyridinylcarbonyl) amino] methyl ester] -L-phenylalanine and methyl- (3-pyridinylmethyl) amine in 88% yield as an incoloration foam which was immediately taken to the subsequent reaction.

Example 41 (Method 3 (modified)) N- [2- (Methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo-cyclobut-l-enyl] -4- [(pyridine-4-carbonyl) - amino] -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 85% yield as a yellow solid.

X H NMR (DMSO-de, 400 MHz) d 10.47 (s, 1H), 8.77 (dd, J = 4.4, 1.8 Hz, 2H), 8.50 (m, 2H), 7.84 (m, 2H), 7.70 (broad m , 1H), 7.58 (d, J = 8.6 Hz, 3H), 7.39 (dd, J = 7.6, 4.9 Hz, 1H), 7.11 (d, J = 8.3 Hz, 2H), 4.7 (d, J = 15.2 Hz , 1H), 4.68 (broad m, 1H), 4.54 (broad s, 1H), 3.18 (dd, J 13.6, 4.3 Hz, 1H), 3.01 (s, 3H), 2.94 (dd, J = 13.6, 8.3 Hz , 1 HOUR) . MS ((+), (-) ESI, m / e (%)) 486 (82, [M + H] +), 484 (58, [MH] "). IR (KBr, cm" 1) 3400, 1800, 1620, 1580, 1530, 1410, 1325.

Example 42 (Method 1) [2-Ethoxy-3,4-dioxo-cyclobut-l-enyl] -4- [(3-pi? Idynylcarbonyl) amino] -L-phenylalanine methyl ester Following the procedure of Method 1 above, the title compound was obtained from 4- [(3-pyridinylcarbonyl) amino] -L-phenylalanine methyl ester hydrochloride and 3,4-diethoxy-3-cyclobutene-1,2-methyl ester. -Diona in 74% of performance.

X H NMR (DMSO-de, 300 MHz) d 10.4 (s, 1H), 9.1 (broad dd, 1H), 9.08 (d, 1H), 8.75 (dd, 1H), 8.27 (dt, 1H), 7.68 (d , 2H), 7.56 (dd, 1H), 7.23 (d, 2H), 4.9 (m, 1H), 4.58 (m, 2H), 3.68 (s, 3H), 3.23 (dd, 1H), 2.96 ( m, 1H), 1.32 (m, 3H).

Example 43 (Method 2) N- [2- (Dihexylamino) -3,4-dioxo-l-cyclobuten-1-yl] -4- [(3-pyridinylcarbonyl) amino] -L-phenylalanine methyl ester Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -4- [(3-pyridinylcarbonyl) amino] -L methyl ester. -phenylalanine and dihexylamine in 95% of: performance as a yellow foam which was immediately taken to the subsequent reaction.

Example 44 (Method 3 (modified)) N- [2- (Dihexylamino) -3,4-dioxo-l-cyclobuten-l-yl] -4- [(3-pyridinylcarbonyl) amino] -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 53% yield as a white solid.

X H NMR (DMSO-de, 400 MHz) d 10.38 (s, 1H), 9.06 (dd, J = 2.4, 1.8 Hz, 1H), 8.72 (dd, J = 4.7, 1.7 Hz, 1H), 8.25 (dt, J = 2.0 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.54 (m, 1H), 7.09 Id, J = 6.4 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H), 4.45 (m, 1H), 3.28 (broad s, 4H), 3.11 (dd, J = 13.6, 4.8 Hz, 1H), 2.99 (dd, J = 13.8, 7.2 Hz, 1H), 1.40 (s broad, 4H) , 1.16 (s broad, 12H), 0.74 (s, 6H). MS ((+) FAB, m / e (%)) 555 (43, [M + Li] +), 549 (100, [M + H] +).

IR (KBr, cm "1) 3375 (broad), 2900, 1800, 1660, 1575, 1530, 1410, 1315.

Example 45 (Method 2) N- [2- [Methyl [2- (4-pyridinyl) ethyl] amino] -3,4-dioxo-l-cyclobuten-l-yl] -4- [(3- pyridinylcarbonyl) amino] -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -4- [(3-pyridinylcarbonyl) amino] -L methyl ester. phenylalanine and methyl- [2- (4-pyridinyl) ethyl] amine in 54% yield as a yellow foam which was immediately taken to the subsequent reaction.

Example 6 (Method 3 (modified)) N- [2- [Methyl [2- (4-pyridinyl) ethyl] amino] -3,4-dioxo-l-cyclobuten-1-yl] -4- [(3- pyridinylcarbonyl) amino] -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 81% yield as a pale yellow solid.

NMR t (DMSO-de, 400 MHz) d 10.39 (s, 1H), 9.04 (d, J = 1.8 Hz, 1H), 8.71 (dd, J = 4.8, 1.8 Hz, 1H), 8.39 (d, J = 4.6 Hz, 2H), 8.24 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.52 (m, 1H), 7.43 (broad s, 1H), 7.19 (d, J = 5.3 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 4.42 (d, J = 4.4 Hz, 1H), 3.70 (broad s, 2H), 3.10 (m, 5H), 2.81 (, 2H) ). MS ((+) ESI, m / e (%)) 506 (25, [M + Li] +), 500 (100, [M + H] +). IR (KBr, cm "1) 3400 (broad), 1810, 1660, 1580, 1535, 1410, 1320.

Analysis Calculated for C27H24N505Li • 2. 5 H20; C, 58 90; H, 5 49; N, 12 72 Found: C, 58.68; H, 5.25; N, 12.46.

Example 47 (Method 2) N- [2- (Methyl-pyridin-3-ylmethyl] -amino) -3,4-dioxo-cyclobut-l-enyl] -4- [(pyridine-3-carbonyl) methyl ester] -amino] -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -4- [(3-pyridylcarbonyl) amino] methyl ester] -L-phenylalanine and methyl- (3-pyridyl-methyl) amine in 66% yield as a colorless foam which was immediately brought to the subsequent reaction.

Example 48 (Method 3 (modified)) N- [2- (Kethyl-pyridin-3-ylmethylamino) -3,4-dioxo-cyclobut-l-enyl] -4- [(pyridine-3-carbonyl) - amino] -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 73% yield as a white solid.

X H NMR (DMSO-de, 400 MHz) d 10.4 (s, 1H), 9.08 (d, J = 2.2 Hz, 1H), 8.73 (dd, J = 4.8, 1.8 Hz, 1H), 8.5 (m, 2H) 8.28 (dt, J = 2.0 Hz, 1H), 7.72 (broad m, 1H), 7.57 (m, 4H), 7.39 (dd, J = 7.7, 4.8 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H ), 4.76 id, J = 14.9 Hz, 1H), 4.66 (broad m, 1H), 4.55 (broad s, 1H), 3.18 (dd, J = 13.6, 4.3 Hz, 1H), 3.01 (s, 3H), 2.94 (dd, J = 13.8, 8.2 Hz, 1H). MS ((-), (+) ESI, m / e (%)) 486 (18, [M + H] +), 484 (100, [MH] ") IR (KBr, crn'1) 3275 (broad ), 1800, 1660, 1580, 1530, 1410, 1315.

Analysis Calculated for C2eH22N505Li • 2.8 H20; C, 57.62; H, . 13; N, 12.92. Found: C, 57.56; H, 4.74; N, 12.73.

Example 49 (Method 2) N- methyl ester. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dι-3-cyclobut-1-enyl} -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine and methyl- [2- (4- pyridinyl) ethyl] amine in 55% yield as a clear oil which was immediately brought to the subsequent reaction.

Example 50 (Method 3 (modified)) N-. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-1-enyl} -L-phenylalanine Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 87% yield as a white solid.

XH NMR (DMSO-de, 400 MHz) d 8.4 (d, J = 5.7 Hz, 2H), 7.42 (broad, 1H), 7.2 (d, J = 5.7 Hz, 2H), 7.11 (m, 5H), 4.39 (d, J "4.6 Hz, .1H), 3.68 (broad s, 2H), 3.18 (m, 5H), 2.78 (m, 2H). MS ((+) FAB, m / e (%)) 402 (45, [M + Na] +), 380 (100, [M + H] +). IR (KBr, cm "1) 3375, 1800, 1580, 1530, 1410.

Analysis Calculated for C2? H20N3O4Li • 1. 5 H20; C, 61 twenty-one; H, 5 63; N, 10 twenty . Found: C, 61.00; H, 5.44; N, 10.05.

Example 51 (Method 1) [2-Ethoxy-3,4-dioxo-cyclobut-l-enyl] - methyl ester. { 4- [4- (N-carboxybenzoyl) piperidinylcarbonyl] amino} -L-phenylalanine Following the procedure of Method 1 above, the title compound was obtained from (4- [4- (N-carboxybenzoyl) piperidinylcarbonyl] amino] -L-phenylalanine methyl ester hydrochloride and 3,4-diethoxy- 3-cyclobutene-l, 2-dione in 37% yield.

Example 52 (Method 2) Methyl ester of N- [2- (dihexylamino) -3,4-dioxo-l-cyclobuten-1-yl] -. { 4- [4- (N-carboxybenzoyl) piperidinylcarbonyl] amino} -L-phenylalanine Following the procedure of Method 2 above, the title compound was obtained from [2-ethoxy-3,4-dioxo-cyclobutyl-l-enyl] - methyl ester. { 4- [4- (N-carboxybenzoyl) piperidinyl-carbonyl] -amino} -L-phenylalanine and dihexylamine in 50% yield as a clear oil which was immediately taken to the subsequent reaction.

Example 53 (Method 3) Methyl ester of N- [2- (dihexylamino) -3,4-dioxo-l-cyclobuten-1-yl] -. { 4- [4- (N-carboxybenzoyl) -piperidinylcarbonyl] amino} -L-phenylalanine Following the procedure of Method 3 above, the title compound was obtained in 75% yield as a light yellow solid, m.p. = 75-80 ° C.

NMR? H (DMSO-de, 400 MHz) d 13.01 (broad s, 1H), 9.83 (s, 1H), 7.57 (d, 1H, J = 9.0 Hz), 7.47 (d, 2H, J = 8.3 Hz) , 7.33 (m, 4H), 7.12 (d, 2H, J = 8.6 Hz), 5.07 (s, 2H), 5.03 (m, 1H), 4.04 (d, 2H, J = 13.2 Hz), 3.5 (broad m , 4H), 3.28 (broad s, under H20, 1H), 3.16 (dd, 1H, J = 3.8 Hz), 2.93 (dd, 1H, J = 11.0 Hz), 2.85 (broad m, 2H), 1.76 (m , 2H), 1.48 (m, 6H), 1.20 (broad s, 13H), 0.82 (t, 6H, J = 6.7 Hz). MS ([M + H] +, m / e (%)) 689 (30 ), 555 (25), 186 (65), 91 (100), IR (KBr, cm "1) 3320, 2930, 1810, 1675, 1580, 1520, 1235.

Analysis Calculated for C39H52N 0, C, 68. 00; H, 7 61; N, 8 13 Found: C, 67.60; H, 7.79; N, 7.95.

Example 54 (Method 1) [2-Ethoxy-3,4-dioxo-cyclobut-l-enyl] -L-tyrosine methyl ester Following the procedure of Method 1 above, the title compound was obtained from L-tyrosine methyl ester hydrochloride and 3,4-diethoxy-3-cyclobutene-1,2-dione in 95% yield.

Example 55 (Method 4) (2S) -3- (4-Dimethylcarbamoyloxy-phenyl) -2-ethoxy-3,4-dioxo-cyclobut-1-enylamino] -propionic acid methyl ester To a solution of [2-ethoxy-3, 4-dioxo-cyclobut-1-enyl] -L-tyrosine methyl ester (1.6 mmol, 500 mg) in pyridine (15 mL) was added pure dimethylcarbamyl chloride (4.7 mmol. , 505 mg, 433 μL) dropwise and the resulting solution was heated at 40 ° C for 18 hours. The volatiles were removed in vacuo and the residue was partitioned between EtOAc and IN HCl. The organic substances are washed with additional IN HCl, water, and brine and dried (Na 2 SO 4). Purification by flash chromatography (SiO2, 60% EtOAc / hexane) gave the title compound as a yellow foam (282 mg, 45% yield).

Example 56 (Method 2) (2S) -3- (4-Dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-phenethyl-amino) -3,4-dioxo-cyclobut-1-enylamino] - methyl ester - propionic Following the procedure of Method 2 above, the title compound was obtained from (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2-ethoxy-3,4-dioxo-cyclobut-1-enylamino acid methyl ester ] -propionic and methyl- (2-phene-yl) amine in 75% yield as a colorless foam which was immediately taken to the subsequent reaction.

NMR? (DMSO-de, 400 MHz) d 7.85 (d, 1H, J = 9.0 Hz), 7.22 (m, 8H), 7.01 (d, 2H, J = 8.6 Hz), 5.12 (m, 1H), 3.72 (m broad, 1H), 3.68 (s, 3H), 3.21 (dd, 1H, J = 4.5 Hz), 3.09 (s, 3H) ,. 2.99 (s, 4H), 2.88 (s, 3H), 2.81 (m, 2H).

Example 57 (Method 3 (modified)) (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-phenethyl-amino) -3,4-dioxo-cyclobut-1-enylamino] acid - propionic Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 82% yield as a white solid.

NMR (DMSO-de, 400 MHz) d 7.33 (broad m, 1H), 7.26 (m, 5H), 7.1 (d, 2H, J = 8.6 Hz), 6.88 (d, 2H, J = 8.6 Hz), 4.36 (d, 1H, J = 4.8 Hz), 3.65 (broad m, 2H), 3.12 (dd, 2H, J = 5.1 Hz), 3.07 (s, 3H), 2.98 (s, 3H), 2.86 (s, 3H) ), 2.79 (m, 2H). MS ((+) FAB, m / e (%)) 488 (55, [M + Na] +), 472 (60, [M + Li] +), 466 (100, [M + H] +). IR (KBr, cm "1) 3410, 2910, 1810, 1725, 1580, 1530, 1410, 1210.

Analysis Calculated for C25H26 3? 6Li • 1. 5 H20; C, 60 24; H, 5 86; N, 8 43 Found: C, 60.40; H, 5.65; N, 8.27.

Example 58 (Method 2) (2S) -2- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3- (4-dimethylcarbamoyl-oxy-phenyl) -propionic acid methyl ester Following the method of Method 2 above, the title compound was obtained from (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2-ethoxy-3, -dioxo-cyclobut-1-enylamino] - methyl ester - propionic and dihexylamine in 40% yield as a yellow oil which was immediately taken to the subsequent reaction.

Example 59 (Method 3) (2S) -2- (2-Dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3- (4-dimethylcarbamoyl-oxy-phenyl) -propionic acid Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 70% yield as a light yellow solid.

XH NMR (DMSO-de, 400 MHz) d 7.11 (d, 1H, J = 6.4 Hz), 7.045 (m, 2H), 6.87 (m, 2H), 4.28 (m, 1H), 3.5 (broad m, 4H ), 3.12 (d, 2H, J = 5.3 Hz), 2.99 (s, 3H), 2.87 (s, 3H), 1.43 (broad m, 4H), 1.18 (broad m, 12H), 0.82 (t, 6H, J = 6.9 Hz). MS ((+) ESI, m / e (%)) 533 (30, (M + NH 4 +) +), 516 (100, (M + H) +). IR (KBr, cm "1) 3400, 2910, 1800, 1730, 1580, 1520, 1380, 1220.

Analysis Calculated for C28H4oN306Li • 1.25 H20; C, 61.77; H, 7.87; N, 7.72 Found: C, 61.67; H, 7.42; N, 7.45.

Example 60 (Method 2) (2S) -3- (4-Dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo-cyclobut-1-methyl ester) -enylamino] -propionic Following the procedure of Method 2 above, the title compound was obtained from ester (2S) -3- (4-Dimethylcarbamoyloxy-phenyl) -2-ethoxy-3, -dioxo-cyclobut-1-enylamino) -propionic acid and methyl- (3-pyridinylmethyl) amine methylate in 82% yield as a colorless foam which was immediately taken to the subsequent reaction.

Example 61 (Method 3 (modified)) (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-pyridin-3-ylmethyl-amino) -3, -dioxo-cyclobutyl- acid- enylamino] -propionic Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 88% yield as a light yellow solid.

XH NMR (DMSO-d6, 400 MHz) 8.50 (m, 2H), 7.67 (broad m, 1H), 7.61 (d, 1H, J = 7.7 Hz), 7.38 (dd, 1H, J = 4.7 Hz), 7.12 (d, 2H, J = 8.6 Hz), 6.88 (d, 2H, J = 8.3 Hz), 4.0 (m, 2H), 4. 51 (broad s, 1H), 3.18 (dd, 1H, J = 4.3 Hz), 3.02 (s, 3H), 2.99 (s, 3H), 2.94 (m, 1H), 2.88 (s, 3 H). MS ((+) ESI, m / e (%)) 459 (19, [M + Li] +), 453 (100, [M + H] +). IR (KBr, cm "1) 3410, 2920, 1810, 1730, 1580, 1530, 1410, 1220.

Analysis Calculated for C23H23N406Li • 1.5 H20; • C, 56.90; H, 5.40; N, 11.54. Found: C, 56.63; H, 5.17; N, 11.41.

Example 62 (Method 2) Methyl ester of (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2- acid. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobu-1-enylamino} -propionic Following the procedure of Method 2 above, the title compound was obtained from (2S) -3- [4-dimethylcarbamoyloxy-phenyl] -2-ethoxy-3,4-dioxo-cyclobut-1-enylamino acid methyl ester ] -propionic and methyl- [2- (4-pyridinyl) ethyl] amine in 77% yield as a colorless foam which was immediately taken to the subsequent reaction.

Example 63 (Method 3 (modified)) (2S) -3- [4-dimethylcarbamoyloxy-phenyl] -2- acid. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-1-enylamino} -propionic Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 88% yield as a yellow solid.

XH NMR (DMSO-de, 400 MHz) d 8.41 (d, 2H, J = 5.5 Hz), 7.41 (m, 1H), 7.21 (d, 2H, J = 5.7 Hz), 7.09 (d, 2H, J = 8.6 Hz), 6.88 (d, 2H, J = 8.6 Hz), 4.37 (m, 1H), 3.70 (broad m, 2H), 3.09 (m, 5H), 2.98 (s, 3H), 2.86 (s, 3H ), 2.80 (m ampl Lo, 2H). MS ((+) ESI, m / e (%)) 473 (20, [M + Li] +), 467 (100, [M + H] +).

IR (KBr, cm "1) 3410, 2930, 1800, 1770, 1580, 1530, 1410, 1210, 116.0.

Calculated Analysis for C24H25N406Li • 2.0 H20; C, 56.69; H, 5.75 / N, 11.02.

Found: C, 56.82; H, 5.43; N, 10.89.

Example 64 (Method 5) (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl] -2-ethoxy-3, -dioxo-cyclobat-1-enylamino] -propionic acid methyl ester To a solution of phosgene (1.9M solution in toluene, 7.8 mmol, 4.1 mL) in CH2C12 (80 mL) at 0 ° C was added a solution of methyl ester of [2-ethoxy-3,4-dioxo-cyclobut-1] -enyl] -L-tyrosine (7.8 mmol, 2.5 g) and pyridine (8.0 mmol, 633 mg; 647 μL) in CH2C12 (10 mL) dropwise for 15 minutes. The resulting solution was stirred at 0 ° C for 30 minutes and one solution was N-methylpiperazine (11.7 mmol, 1.2 g, 1.3 mL) and pyridine (11.7 mmol, 929 mg, 950 μL) in CH2C12 (10 mL) then added. Drop exhausts for 30 minutes. The resulting solution was warmed to room temperature and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was partitioned between EtOAc and water. The organic substances were dried (Na 2 SO 4), concentrated and purified by flash chromatography (SiO 2, Et 3 N 5% / EtOAc) to give the title compound as a colorless foam (1.3 g, 37%).

Example 65 (Method 2) (2S) -3- [4- (4-Methylpiperazinyl) carbamo] -lucy-phenyl] -2- [2- (methyl-phenethyl-amino) -3,4-dioxo-cyclobutyl methyl ester -1-enylamino] -propionic Following the procedure of Method 2 above, the title compound was obtained from (2S) -3- [4- (4-methylpiperazinyl) carbamoyloxy-phenyl] -2-ethoxy-3, 4-dioxo- cyclobut-1-enylamino] -propionic and methyl- (2-phenethyl) amine in 95% yield as a colorless foam which was immediately brought to the subsequent reaction.

Example 66 (Method 3) (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl] -2- [2- (methyl-phenethyl-amino) -3,4-dioxo-cyclobut-1-enylamino acid ] -propionic Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 90% yield as a white solid.

XH NMR (DMSO-de, 400 MHz) d 7.32-7.16 (m, 6H), 7.09 (d, 2H, "J = 8.6 Hz), 6.90 (d, 2H, J = 8.6 Hz), 4.32 (m, 1H ), 3.65 (broad m, 2H), 3.51 (broad s, 2H), 3.38 (broad s, 2H), 3.08 (ir., 5H), 2.79 (broad m, 2H), 2.31 (m, 4H), 2.19 (s, 3H) MS (FAE, m / e (%)) 543 (35, (M + Na) +), 527 (40, (M + Li)) 521 (100). IR (KBr, cm " 1) 3410, 2930, 1810, 1725, 1580, 1530, 1410, 1210.

Analysis Calculated for C28H9N406Li • 2.0 H20; C, 59.78; H, 6.27; N, 9.96. Found: C, 60.04; H, 6.07; N, 9.77.

Example 67 (Method 2) (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl) -2- methyl ester. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-1-enylamino} -propionic Following the procedure of Method 2 above, the title compound was obtained from ester methyl (2S) -3- [4- (4-methylpiperazinyl)] carbamoyloxy-phenyl) -2-ethoxy-3,4-dioxo-cyclobut-1-enylamino] -propionic acid and methyl- [2- (4- pyridinyl) ethyl] amine in 62% yield as a yellow foam which was immediately brought to the subsequent reaction.

Example 68 (Method 3 (modified)) (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl) -2- acid. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-1-enylamino} -propionic Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 80% yield as an off-white solid.

XH NMR (DMSO-d6, 400 MHz) d 8.41 (d, 2H, J = 5.9 Hz), 7.39 (m, 1H), 7.21 (d, 2H, J = 5.7 Hz), 7.09 (d, 2H, J = 8.6 Hz), 6.90 (d, 2H, J = 8.6 Hz), 4.38 (m, 1H), 3.69 (broad m, 2H), 3.51 (broad s, 2H), 3.35 (broad s, 4H, low peak H20 ), 3.09 (m, 4H), 2.82 (m, 2H), 2.30 (m, 4H), 2.19 (s, 3H). MS ((+) FAB, m / e (%)) 534 (100, [M + 2 Li] +), 528 (50, [M + Li] +). IR (KBr, cm "1) 3400 (broad), 2920, 1810, 1720, 1580, 1530, 1410.

Example 69 (Method 2) Methyl ester of (2S) -3- [4- (4-methylpiperazinyl) carbamoyloxy-phenyl) -2- acid. { 2-dihexylamino] -3,4-dioxo-cyclobut-1-enylamino} -propionic Following the procedure of Method 2 above, the title compound was obtained from (2S) -3- [4- (4-methylpiperazinyl)] carbamoyloxy-phenyl) -2-ethoxy-3,4-dioxoic acid methyl ester -cyclobut-1-enylamino] -propionic and dihexylamine in 85% yield as a colorless foam which was immediately taken to the subsequent reaction.

Example 70 (Method 3 (modified)) (2S) -3- [4- (4-Methylpiperazinyl) carbamoyloxy-phenyl) -3- acid. { 2-dihexylamino] -3,4-dioxo-cyclobut-1-enylamino} -propionic Following the procedure of Method 3 (modified) above, the title compound was obtained as its corresponding lithium salt in 84% yield as a white solid.

NMR XH (DMSO-de, 400 MHz) d 7.11 (d, 1H, J = 6.4 Hz), 7.05 (m, 2H), 6.88 (m, 2H), 4.29 (, 1H), 3.65 (broad m, 2H), 3. 55 (s broad, 3H), 3.34 (broad s, 4H, low peak H20), 3.12 (d, 2H, J = 5.3 Hz), 2.31 (m, 4H), 2.19 (s, 3H), 1. 43 (broad m, 4H), 1.18 (broad m, 11H), 0.82 (t, 6H, J - 6.9 Hz), EM ((+) FAB, m / e (%)) 577 (100, [M + Li ] +), 531 (20), 186 (45), 127 (80). IR (KBr, cpf1) 3410, 2920, 1810, 1580, 1520, 1410.

Analysis. Calculated for C3? H45N406Li • 1.5 H20; C, 61.68; H, 8.02; N, 9.28. Found: C, 61.43; H, 7.78; N, 9.04.

The above compounds were tested for the binding activity of VLA-4 using the following monovalent FACS assay. The IC50 value for a compound reflects 50% occupancy of the receivers. The assay can accurately measure the activity of the compounds with an IC 50 value ranging from 0.5 nM to 1 mM.

FACS Monovalent Assay for the Link of Integrina a4ß? / VCAM-l The binding activity of VLA-4 of the exemplary compounds was measured by measuring the inhibition of soluble VCAM-1 interaction with Jurkat cells (ATCC # TIB-153) which express high levels of a4β integrin. (VLA-4) using a modification of the fluorescence activated cell sorter assay (FACS) described by Yednock et al., J. Biol. Chem., 1995, 270: 28740. VCAM-1 interacts with the cell surface in a form dependent on integrin a4β ?. Jurkat cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin and glutamine as described by Yednock, supra.

Soluble, recombinant VCAM-1 (VCAM-lsr) was produced in a baculovirus expression system as a chimeric fusion protein containing the seven immunoglobulin domains of VCAM-1 at the N-terminus and the constant region of the heavy chain of human IgGi in the termination C as described by Yednock, supra. The supernatant containing approximately 10 ug / ml of VCAM-lsr was collected after 72 hours and used in the assay without purification. Jurkat cells (approximately 10 7 cells / ml) were treated with 1.5 mM MnCl 2 and 5 μg / ml 15/7 for 30 minutes on ice to activate ßi integrin. Mn + 2 activates the receptor to increase the binding of the ligand, and 15/7 is a monoclonal antibody that recognizes an activated / occupied conformation by ligands of the integrin a4β? and closes the molecule within this conformation to stabilize because of that the interaction of VCAM-1 / integrin a4ß ?. Yednock et al., supra. Antibodies similar to 15/7 have been prepared and can be used in this assay. For example, see, Luque et al., 1996, J. Biol. Chem., 271: 11067. The aliquots of 25 μl of cells were incubated for 30 minutes at room temperature with the compounds using a normal 5-dot serial dilution. 15 μl of baculovirus supernatant was added containing VCAMsr-Fc to the cells and incubated for 30 minutes on ice as described in Yednock et al., supra. The cells were washed twice and resuspended in 10 μl of a 1: 100 dilution of goat antihuman IgG conjugated with FITC to detect the human Ig-VCAM-1 construct diluted in the test media containing mouse serum 2.5 % to block cross-reactivity, potential with 15/7 bound to the cell surface. The cells were incubated on ice for 30 minutes in the dark. The cells were washed twice and analyzed with normal FACS analysis as described in Yednock, et al., Supra in the FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The data is shown in Table 1.

Table 1 In this manner, the 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, metastasis. tumor, 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 aleic 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 in the N-terminus or other conventional "pro-drug" forms which, when administered, are converted to the active portion in vivo.

The compounds of the present invention can 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, nonionic surfactants and edible oils such as corn, peanut and sesame oils. Adjuvants commonly employed in the preparation of pharmaceutical compositions such as flavoring agents, coloring agents, preservatives and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA, can advantageously be included. These compounds can be administered orally as well as by intravenous, intramuscular or subcutaneous routes. When administered orally in forms such as tablets, capsules, dispersible powders, granules or suspensions, the formulations may contain, for example, from about 0.05% to 5% suspending agent, syrups containing, for example, about 10%. at 50% sugar, or elixirs containing, for example, from about 20 to 50% ethanol and the like. When the administration is parenterally, the formulation may be, for example, injectable, sterile or suspensions containing from about 0.05 to 5% of 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. Preferred pharmaceutical compositions from the point of view of the preparation and administration case are solid compositions, particularly tablets and hard capsules filled or liquid filled. Oral administration of the compounds is preferred. The dosage requirements can be determined by a person of skill 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.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (39)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A compound of the formula: characterized in that R1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; R 2 is H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R1 and R2 can be taken together to form a saturated or unsaturated heterocyclic ring; R3 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.
2. 2. The compound according to claim 1, characterized in that A is phenyl, R1 is alkyl, R2 and R3 are H, x and y are 0 and z is 1.
3. 3. The compound according to claim 2, characterized in that A is substituted phenyl. .
4. The compound according to claim 1, characterized in that A is phenyl, R1 is heteroaralkyl, R2 and R3 are H, xyy are 0 and z is 1.
5. The compound according to claim 1, characterized in that A is substituted aryl, and the substituent is OCONR6R7.
6. 6. A compound according to claim 1, characterized in that it is [2- (benzylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine; or a pharmaceutical salt thereof.
7. 7. A compound according to claim 1, characterized in that it is [2- (benzhydrylamino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine; or a pharmaceutical salt thereof.
8. 8. A compound according to claim 1, characterized in that it is 2-. { 2- [2- (lH-indol-3-yl) -ethylamino] -3,4-dioxo-cyclobut-1-enylamino} -L-phenylalanine; or a pharmaceutical salt thereof.
9. 9. A compound according to claim 1, characterized in that it is. { 3, 4-dioxo-2- [(pyridin-3-ylmethyl) -amino] -cyclobut-1-enyl} -L-phenylalanine; or a pharmaceutical salt thereof.
10. 10. A compound according to claim 1, characterized in that it is [2- (benzyl-hexyl-amino) -3,4-dioxo-cyclobut-l-enyl] -L-phenylalanine; or a pharmaceutical salt thereof.
11. 11. A compound according to claim 1, characterized in that it is (2-dibenzylamino-3,4-dioxo-cyclobut-1-enylamino) -L-phenylalanine; or a pharmaceutical salt thereof.
12. 12. A compound according to claim 1, characterized in that it is (S) -2- (2-dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -3-phenyl-propionic acid; or a pharmaceutical salt thereof.
13. 13. A compound according to claim 1, characterized in that it is (S) -2- [2- (hexyl-naphthalen-2-ylmethyl-amino) -3,4-dioxo-cyclobut-l-enylamino) -3 acid. phenylpropionic; or a pharmaceutical salt thereof.
14. 14. A compound according to claim 1, characterized in that it is (S) -2- acid. { 2- [(4-dim3-tilamino-benzyl) -hexyl-amino] -3, -dioxo-cyclobut-1-enylamino} -3-phenyl-propionic; or a pharmaceutical salt thereof.
15. 15. A compound according to claim 1, characterized in that it is N- [3, 4-dioxo-2- (4-phenyl-piperazin-1-yl) -cyclobut-1-en-1-yl] -L-phenylalanine; or a pharmaceutical salt thereof.
16. 16. A compound according to claim 1, characterized in that it is (S) -2- [2- (4-acetyl-piperazin-1-yl) -3,4-dioxo-cyclobut-1-enylamino] -3 acid. phenylpropionic; or a pharmaceutical salt thereof.
17. 17. A compound according to claim 1, characterized in that it is (S) -3- (4-benzylamino-phenyl) -2- (2-dihexylamino-3,4-dioxo-cyclobut-1-enylamino) -propionic acid.; or a pharmaceutical salt thereof.
18. 18. A compound according to claim 1, characterized in that it is (S) -3- (l-benzyl-lH-imidazol-4-yl) -2- (2-dihexylamino-3,4-dioxo-cyclobutyl) acid. -1-enylamino) -propionic; or a pharmaceutical salt thereof.
19. 19. A compound according to claim 1, characterized in that it is N- (2-dihexylamino-3,4-dioxo-cyclobut-l-enylamino) -O- (3-dimethylamino-propyl) -L-tyrosine; or a pharmaceutical salt thereof.
20. 20. A compound according to claim 1, characterized in that it is N- [2- [methyl [2- (4-pyridinyl) ethyl] amino] -3,4-dioxo-l-cyclobuten-1-yl] -4 - [(4-pyridyrylcarbonyl) amino] -L-phenylalanine; or a pharmaceutical salt thereof.
21. 21. A compound according to claim 1, characterized in that it is N- [2- [methyl (2-phenylethyl) amino] -3,4-dioxo-l-cyclobuten-l-yl] -4- [(4-pyridinylcarbonyl) amino] -L-phenylalanine; or a pharmaceutical salt thereof.
22. 22. A compound according to claim 1, characterized in that it is N- [2- (dihexylamino) -3,4-dioxo-l-cyclobuten-l-yl] -4- [(4-pyridinylcarbonyl) -amino] - L-phenylalanine; or a pharmaceutical salt thereof.
23. 23. A compound according to claim 1, characterized in that it is N- [2- (methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo-cyclobut-l-enyl] -4- [(pyridine- 4-carbonyl) -amino] -L-phenylalanine; or a pharmaceutical salt thereof.
24. 24. A compound according to claim 1, characterized in that it is N- [2- (dihexylamino) -3,4-dioxo-l-cyclobuten-l-yl] -4- [(3-pyridinylcarbonyl) amino] -L phenylalanine; or a pharmaceutical salt thereof.
25. 25. A compound according to claim 1, characterized in that it is N- [2- [methyl [2- (4-pyridinyl) ethyl] amino] -3,4-dioxo-l-cyclobuten-1-yl] -4 - [(3-pyridinylcarbonyl) amino] -L-phenylalanine; or a pharmaceutical salt thereof.
26. 26. A compound according to claim 1, characterized in that it is N- [2- (methyl-pyridyl-3-ylmethyl-amino) -3,4-dioxo-cyclobut-l-enyl] -4- [(pyridin-3 -carbonyl) -amino] -L-phenylalanine; or a pharmaceutical salt thereof.
27. 27. A compound according to claim 1, characterized in that it is N-. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-l-enyl} -L-phenylalanine; or a pharmaceutical salt thereof.
28. 28, A compound according to claim 1, characterized in that it is N- [2- (dihexylamino) -3,4-dioxo-l-cyclobuten-1-yl] - methyl ester. { 4- [4- (N-carboxybenzoyl) -piperidinylcarbonyl] amino} -L-phenylalanine; or a pharmaceutical salt thereof.
29. 29. A compound according to claim 1, characterized in that it is (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-phenethyl-amino) -3,4-dioxo-cyclobut- 1-enylamino] -propionic; or a pharmaceutical salt thereof.
30. 30. A compound according to claim 1, characterized in that it is (2S) -2- (2-dihexy.Lamino-3,4-dioxo-cyclobut-1-enylamino) -3- (4-dimethyl-carbamoyloxy). phenyl) -propionic; or a pharmaceutical salt thereof.
31. 31. A compound according to claim 1, characterized in that it is (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2- [2- (methyl-pyridin-3-ylmethyl-amino) -3,4-dioxo- cyclobut-1-enylamino] -propionic; or a pharmaceutical salt thereof.
32. 32. A compound according to claim 1, characterized in that it is (2S) -3- (4-dimethylcarbamoyloxy-phenyl) -2- acid. { 2- [Methyl- (2-pyridin-4-yl-ethyl) amino] -3,4-dioxo-cyclobut-1-enylamino} -propionic; or a pharmaceutical salt thereof.
33. 33. A compound according to claim 1, characterized in that it is (2S) -3- [4- (4-methylpiperazinyl) carbamoyloxy-phenyl] -2- [2- (methyl-phenylethyl-amino) -3,4 acid. -dioxo-cyclobut-1-enylamino] -propionic; or a pharmaceutical salt thereof.
34. 34. A compound according to claim 1, characterized in that it is (2S) -3- [4- (4-methylpiperazinyl) carbamoyloxy-phenyl) -2- acid. { 2- [Methyl- (2-pyridin-4-yl-ethyl) -amino] -3,4-dioxo-cyclobut-1-enylamino] -propionic acid; or a pharmaceutical salt thereof.
35. 35. A compound according to claim 1, characterized in that it is (2S) -3- [4- (4-methylpiperazinyl) carbamoyloxy-phenyl) -2- acid. { 2-dihexylamino] -3,4-dioxo-cyclobut-1-enylamino} -propionic; or a pharmaceutical salt thereof.
36. 36. The use of a compound of the formula: wherein R1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; R 2 is H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R1 and R2 can be taken together to form a saturated or unsaturated heterocyclic ring; R3 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, characterized in that it is for the preparation of a medicament for inhibiting the adhesion of leukocytes in a patient suffering from a condition associated with leukocyte adhesion.
37. 37. The use of a compound of the formula: wherein R1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; R 2 is H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R1 and R2 can be taken together to form a saturated or unsaturated heterocyclic ring; R3 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, characterized in that it is for the preparation of a medicament for treating a patient suffering from inflammatory diseases.
38. 38. The use according to claim 36, characterized in that the inflammatory disease is selected from the group consisting of multiple sclerosis, meningitis, asthma, Alzheimer's disease, atherosclerosis, dementia due to AIDS, diabetes, inflammatory bowel disease, rheumatoid arthritis, tumor metastasis, transp.Lante of tissue and myocardial ischemia.
39. 39. A pharmaceutical composition, characterized in that it comprises a compound of the formula: wherein R1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; R 2 is H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R1 and R2 can be taken together to form a saturated or unsaturated heterocyclic ring; R3 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. DERIVATIVES OF 3,4-DIAMINO-3-CICLOBUTENO-1, 2-DIONA WHICH INHIBIT THE ADHESION OF LEUKOCYTES MEDIATED BY INTEGRINE ALFA4, BETAI (VLA-4) SUMMARY OF THE INVENTION The compounds of the formula (I) which inhibit the adhesion of leukocytes mediated by the interaction of the integrin a4β? (VLA-4) with its counter-receptor VCAM-1, and its use for the treatment of inflammatory and autoimmune diseases.