AU758886B2 - Cell adhesion inhibitors - Google Patents

Description

1

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT a S 0 .000

S

.5.5 Name of Applicant: Actual Inventors: Address for Service: BIOGEN, INC.

Ko-Chung LIN, Steven P. ADAMS, Alfredo C. CASTRO, Craig N.

ZIMMERMAN, Julio Hernan CUERVO, Wen-Cherng LEE, Charles E.

HAMMOND, Mary Beth CARTER, Ronald G. ALMQUIST and Carol Lee

ENSINGER

CULLEN CO., Patent Trade Mark Attorneys, 239 George Street, Brisbane, Qld. 4000, Australia.

.06 9 Invention Title: CELL ADHESION INHIBITORS The following statement is a full description of this invention, including the best method of performing it known to us: la CELL ADHESION INHIBITORS TECHNICAL FIELD OF THE INVENTION The present invention relates to novel compounds that are useful for inhibition and prevention .of cell adhesion and cell adhesion-mediated pathologies.

This invention also relates to pharmaceutical formulations comprising these compounds and methods of using them for inhibition and prevention of cell adhesion 10 and cell adhesion-mediated pathologies. The compounds and pharmaceutical compositions of this invention can be used as therapeutic or prophylactic agents. They are particularly well-suited for treatment of many inflammatory and autoimmune diseases.

15 BACKGROUND OF THE INVENTION Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. As such, cell adhesion constitutes one of the fundamental mechanisms underlying numerous biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hemopoietic cells out of blood vessels and to the site of injury. As such, cell 2 adhesion plays a role in pathologies such as inflammation and immune reactions in mammals.

Investigations into the molecular basis for cell adhesion have revealed that various cell-surface macromolecules collectively known as cell adhesion molecules or receptors mediate cell-cell and cellmatrix interactions. For example, proteins of the superfamily called "integrins" are key mediators in adhesive interactions between hematopoietic cells and 10 their microenvironment Hemler, "VLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.", Ann. Rev. Immunol., 8, p. 365 (1990)).

Integrins are non-covalent heterodimeric complexes consisting of two subunits called a and B. There are at 15 least 12 different a subunits (al-a6, a-L, a-M, a-X, a- IIB, a-V and a-E) and at least 9 different B (81-89) subunits. Based on the type of its a and 8 subunit components, each integrin molecule is categorized into a subfamily.

a481 integrin, also known as very late antigen- *4 or CD49d/CD29, is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell-matrix adhesive interactions (M.E.

Hemler, Ann. Rev. Immunol., 8, p. 365 (1990)). It serves as a receptor for the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-i as well as to the extracellular matrix protein fibronectin (Ruegg et al., J. Cell Biol., 177, p. 179 (1991); Wayner et al., J. Cell Biol., 105, p.

1873 (1987); Kramer et al., J. Biol. Chem., 264, p. 4684 (1989); Gehlsen et al. Science, 24, p. 1228 (1988)).

Anti-VLA4 monoclonal antibodies have been shown 3 to inhibit VLA4-dependent adhesive interactions both in vitro and in jvi (Ferguson et al. Proc. Natl. Acad.

SIL, 88, p. 8072 (1991); Ferguson et al., J. immunol., 150, p. 1172 (1993)). Results of in vivo experiments suggest that this inhibition of VLA-4-dependent cell adhesion may prevent or inhibit several inflammatory and autoimmune pathologies L. Lobb et al., "The Pathophysiologic Role of a4 Integrins In Vivo", J. Cin.

Invest., 94, pp. 1722-28 (1994)).

10 In order to identify the minimum active amino acid sequence necessary to bind VLA-4, Komoriya et al.

("The Minimal Essential Sequence for a Major Cell Type- Specific Adhesion Site (CS1) Within the Alternatively Spliced Type III Connecting Segment Domain of Fibronectin 15 Is Leucine-Aspartic Acid-Valine", J. Biol. Chem., 266 pp. 15075-79 (1991)) synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin. They identified an Eamino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr

(SEQ

ID NO: as well as two smaller overlapping S" pentapeptides, Glu-Ile-Leu-Asp-Val [SEQ ID NO: 2i and Leu-Asp-Val-Pro-Ser [SEQ ID NO: that possessed inhibitory activity against FN-dependent cell adhesion.

These results suggested the tripeptide Leu-Asp-Val as a minimum sequence for cell-adhesion activity. It was later shown that Leu-Asp-Val binds only to lymphocytes that express an activated form of VLA-4, thus bringing into question the utility of such a peptide in vivo (E.A.

Wayner et al., "Activation-Dependent Recognition by Hematopoietic Cells of the LDV Sequence in the V Region of Fibronectin", J. Cell. Biol., 116(2), pp. 489-497 4 (1992)). However, certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo A. Ferguson et al., "Two Integrin Binding Peptides Abrogate T-cell-Mediated Immune Responses In Vivo", Proc.

Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991); and S. M.

Wahl et al., "Synthetic Fibronectin Peptides Suppress Arthritis in Rats' by Interrupting Leukocyte Adhesion and Recruitment", J. Clin. Invest., 94, pp. 665-62 (1994)].

A cyclic pentapeptide, Arg-Cys-Asp-TPro-Cys (wherein TPro denotes 4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to FN has also been described Nowlin et al, "A Novel Cyclic Pentapeptide Inhibits a4l1 and a5pl Integrin-mediated Cell Adhesion", J.

Biol. Chem., 268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862). This peptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a commmon motif in the recognition site for several extracellular-matrix proteins.

Despite these advances, there remains a need for 20 small, specific inhibitors of VLA-4-dependent cell adhesion. Ideally, such inhibitors would be semi-peptidic or non-peptidic so that they may be orally administered.

Such compounds would provide useful agents for treatment, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 binding. International patent applications WO 94/15958 and WO 92/00995 describe cyclic peptide and peptidomimetic 5 compounds with cell adhesion modulating activity.

International patent applications WO 93/08823 and WO 92/08464 describe guanidinyl-, urea- and thioureacontaining cell adhesion modulating compounds. United States Patent 5,260,277 describes guanidinyl cell adhesion modulation compounds.

SUMMARY OF THE INVENTION The present invention solves this problem by providing novel semi-peptidic compounds that inhibit the 1 0 binding of ligands to VLA-4. These compounds are useful for inhibition, prevention and suppression of VLA-4mediated cell adhesion and pathologies associated with that adhesion, such as inflammation and immune reactions.

The compounds of this invention may be used alone or in combination with other therapeutic or prophylactic agents to inhibit, prevent or suppress cell adhesion. This invention also provides pharmaceutical formulations containing these VLA-4-mediated cell adhesion inhibitors and methods of using the compounds and compositions of the invention for inhibition of cell adhesion.

According to one embodiment of this invention, these novel compounds, compositions and methods are advantageously used to treat inflammatory and immune diseases. The present invention also provides methods for preparing the compounds of this invention and intermediates useful in those methods.

DETAILED DESCRIPTION OF THE INVENTION The following abbreviations are used in the description: -6 "esignation Reagent or Frament Ac acetyl Boc benzyl Boc tert-butoxycarbonyl Bu butyl Cbz carbobenzyloxy Cy cyclohexyl CyM cyclohexylmethyl DIPEA diisopropylethylamine EDC 1- (3-diethylaminopropyl) -3ethylcarbodiimide HOBT l-hydroxybenzotriazole hydrate -amyl isoamy.

i-Pn isopentyl i-Pr isopropyl Me methyl 2-MPUBA 4- 2 -methylphenyl) urea) 2-MPUPAphenylmethylamino 2-MUPA4- 2 -methylphenyl) urea) phenylacetyl NMP N-methylpyrrolidinone NMM N-methylmorpholine Ph phenyl PUPA 4- -phenylurea)phenylacetyl Su succinimidyl 1, 1,3, 3 -tetramethyluronium tetra fluoroborate TEA tr ethylamine 30 TEA trifluoroacetic acid :TEAM r2.s (hydroxy)methylai4nomethane Definitions As used herein, the term "alkyl", alone or in combination, refers to a straight-chain or branched-chain alkyl radical contai.ning from 1 to 10, preferably from I to 6 and more preferably from 1 to 4, carbon atoms.

Examples of such radicals include, Out are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, Jsobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and the-like.

7 The term "alkenyl", alone or in combination, refers to a straight-chain or branched-chain alkenyl radical containing from 2 to 10, preferably from 2 to 6 and more preferably from 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Zbutenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl and the like.

The term "alkynyl", alone or in combination, 10 refers to a straight-chain or branched-chain alkynyl radical containing from 2 to 10, preferably from 2 to E and more preferably from 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethynvl (acetylenyl), propynyl, propargyl, butynyl, hexynyl, 15 decynyl and the like.

The term "cycloalkyl", alone or in combination, refers to a cyclic alkyl radical containing from 3-1C, preferably from 3-8 and more preferably from 3-6, carbon atoms and may be optionally aryl-fused. Examples of such radicals include, but are not limited tc, cyclopropyi, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term "cycloalkenyl", alone or in combination, refers to a cyclic carbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms and one cr more double bonds. Examples of such cycloalkenyl radicals include, but.are not limited to, cyclopentenyl, cyclohexenyl, cyclopentadienyl and the like.

The term "aryl" refers to a carbocyclic aromatic group selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, and anthracenyl; or a heterocyclio aromatic group selected from the group consisting of furyl, thienyl, 8pyridyl, pyrrolyl, oxazolyly, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1 2 ,3-triazoly., 1,3,4thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5triaziriyl, 1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzofb~furanyl, 2,3dihydrobenzofuranyl, benzo thiophenyl, lH-indazolyi, benzirnidazolyl, benzthiazolyl, purinyl, 4 H-quinolizinyl, quinolinyl, isoquinolinyl, cinnoJlinyl, phthalazinyl, quiriazolinyl, quinoxalinyl, 1,8-naphthyridinyl, *pteridinyl, carbazolyl, acridinyl, hnzyl phenothiazinyl, phenoxaziny-l, pyrazolo triazinyl and the like.

"Aryl", "cycloalkyP' and "cycloalkenyl" groups, as defined in this application may independently contain up to th.ree substituents which are independently selected from the group consisting of halogen, hydroxy21, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl', cyano, carboxy, carboalkoxy, Ar'-substituted alkyl., Ar'-substituted alkenyl or alkynyl, 1,2dioxvme:hylene, l,2-dioxyethylnakx, leoyo *.:alkynoxv, Ar'-substituted alkoxy, Ar'-substituted alkenoxv or alkynoxy, alkylamino, alkenylamino or alkynylamino, Ar'-substituted alkylamino, Ar'-substitutedj alkenylamino or alkynylamino, Ar'-substituted carbonyioxy, alkylcarbonyloxy, aliphatic or aromatic acyl, A-'-substituted acyl, Ar'-substituted alkylcarbonyloxy, Ar'-substituted carbonylamino, Ar'substituted amino, Ar'-substituted oxy, Ar'-substituted carbonvi, alkylcarbonylamino, Ar'-substituted alkylcarbonvlamino, alkoxy-carbonylamino, Ar' -substituted alkoxycarbonyl-amino, Ar' -oxycarbonylamino, 9alkylsulfonylamino, mono- or bis- (Arl-sulfonyl) amino, Ar' -substituted alkyl-sulfonylamino, morpholinocarbonylamino, thiomorpholinocarbonylamino,

N-

alkyl guanidino, N-Ar' guanidino, N-N-(Ar',alkyl) guanidino, N,N- (Ar' ,Ar' )guanidino, N,N-dialkyl guanidino, N,N,N-trialkyl guanidino, N-alkyl urea, N,N-dialkyl urea, N-Ar' urea, N,N-(Ar',alkyl) urea, N,N-(Ar') 2 urea, aralkyloxycarbony1-substituted alkyl, aralkylaminocarbonyl, thioaryloxy and the like; 10 wherein is defined similarly to aryl, but contains up to three substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, trifluoronethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, 1, 2 -dioxymethylene, 1, 2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylamino or alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl, alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl or N,N-dialkyl urea.

The term "aralkyl", alone or in combination, refers to an aryl substituted alkyl radical, wherein the term "alkyl" and "aryl" are as defined above. Examples of suitable aralkyl radicals include, but are not limited to, phenylmethyl, phenethyl, phenylhexyl, diphenylmethyl, pyridylmethyl, tetrazolylmethyl, furylmethyl, imidazolylmethyl, indolylmethyl, thienylpropyl and the like.

The term "alkoxy", alone or in combination, refers to an alkyl ether radical, wherein the term "alkyl" is as defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy and the like.

10 The term "alkenoxy", alone or in combination, refers to a radical of formula alkenyl-O-, wherein the term "alkenyl" is as defined above provided that the radical is not an enol ether. Examples of suitable alkenoxy radicals include, but are not limited to, allyloxy, E- and Z- 3 -methyl-2-propenoxy and the like.

The term "alkynyloxy", alone or in combination, refers to a radical of formula alkynyl-O-, wherein the term "alkynyl" is as defined above provided that the 1 0 radical is not an ynol ether. Examples of suitable alkynoxy radicals include, but are not limited to, propargyloxy, 2 -butynyloxy and the like.

The term "thioalkoxy" refers to a thioether radical of formula alkyl-S-, wherein alkyl is as defined above.

The term "alkylaminc", alone or in combination, refers to a mono- or di-alkyl-substituted amino radical a radical of formula alkyl-NH- or (alkyl)2-N-, wherein the term "alkyl" is as defined above. Examples of suitable alkylamino radicals include, but are nct limited to, methylamino, ethylamino, propylamino, isopropylamino, t-butylamino, N,N-diethylamir.c and the like.

The term "alkenylamino", alone or in combination, refers to a radical of formula alkenyl-NHor (alkenyl) 2 wherein the term "alkenyl" is as def:-.e above, provided that the radical is not an enamine.

A.

example of such alkenyiamino radicals is the allylaminc radical.

The term "alkynylamino", alone or in combination, refers to a radical of formula alkynyl-NHor (alkynyl) 2 wherein the term "alkynyl" is as defined 11 above, provided that the radical is not an ynamine. An example of such alkynylamino radicals is the propargyl amino radical.

The term "aryloxy", alone or in combination, refers to a radical of formula aryl-O-, wherein aryl is as defined above. Examples of aryloxy radicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxy and the like.

The term "arylamino", alone or in combination, refers to a radical of formula aryl-NH-, wherein aryl is as defined above. Examples of arylamino radicals include, but are not limited to, phenylamino (anilido), naphthylamino, 3- and 4-pyridylamino and the like.

The term "biaryl", alone or in combination, 15 refers to a radical of formula aryl-aryl-, wherein the term "aryl" is as defined above.

The term "thioaryl", alone or in combination, refers to a radical of formula aryl-S-, wherein the term "aryl" is as defined above. An example of a thioaryl radical is the thiophenyl radical.

The term "aryl-fused cycioalkyl", alone or in combination, refers to a cycioalkyl radical which shares two adjacent atoms with an aryl radical, wherein the terms "cycloalkyl" and "aryl" are as defined above. An example of an aryl-fused cycloalkyl radical is the benzofused cycloburyl radical.

The term "aliphatic acyl", alone or in combination, refers to radicals of formula alkyl-CO-, alkenyl-CO- and alkynyl-CO-derived from an alkane-, alkene- or alkyncarboxylic acid, wherein the terms "alkyl", "alkenyl" and "alkynyl" are as defined above.

Examples of such aliphatic acyl radicals include, but are 12 not limited to, acetyl, propionyl, butyryl, valeryl, 4methylvaleryl, acryloyl, crotyl, propiolyl, methylpropiolyl and the like.

The terms "aromatic acyl" or "aroyl", alone or in combination, refers to a radical of formula aryl-CO-, wherein the term "aryl" is as defined above. Examples of suitable aromatic acyl radicals include, but are not limited to, benzoyl, 4-halobenzoyl, 4 -carboxybenzoyl, o naphthoyl, pyridylcarbonyl and the like.

1 0 The term "heterocycloyl", alone or in combination, refers to radicals of formula heterocycle- CO-, wherein the term "heterocycle" is as defined below.

Examples of suitable heterocycloyl radicals include but are not limited to, tetrahydrofuranylcarbonyl, 15 piperidinylcarbonyl, tetrahydrothiophenecarbonyl and the like.

The terms "morpholinocarbonyl" and "thiomorpholinocarbonyl", alone or in combination with other terms, refer to an N-carbonyiated morpholino and an N-carbonylated thiomorpholino radical, respectively.

The term "alkylcarbonylamino", alone or in combinazion, refers to a radical cf formula alkyl-CONH, wherein the term "alkyl" is as defined above.

The term "alkoxycarbonylamino", alone or in combination, refers to a radical of formula alkyl-OCONH-, wherein the term "alkyl" is as defined above.

The term "alkylsulfonylamino", alone or in combination, refers to a radical of formula alkyl-SO 2

NH-,

wherein the term "alkyl" is as defined above.

The term "arylsulfonylamino", alone or in combination, refers to a radical of formula aryl-SO 2

NH-,

wherein the term "aryl" is as defined above.

13 The term "IN-alkylurea"l, alone or in combination, refers to a radical of formula alkyl-NH-Co- NH-, wherein the term "alkyl" is as defined above.

The term "IN-arylureal", alone or in combination, refers to a radical of formula aryl-NH-CO-NH-, wherein the term "aryl" is as defined above.

The term "halogen" means fluorine, chlorine, bromine and iodine.

*The terms "heterocycle" and "heterocyclic 10 ring", alone or in combination, refer to a non-aromatic 3- to lO-membered ring containing at least one endocyclic N, 0 or S atom. The heterocycle may optionally be aryl- *fused. The heterocycle may also be optionally substituted with one to three substituents which are independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, aralkyl, alkenyl, alkynyl, aryl, cyano, carboxy, carboalkoxy, Ar'substituted alkyl, Ar'-substituted alkenyl or alkynyl, l, 2 -dioxymethyle'ne, l, 2 -dioxyethylene, alkoxy, alkenoxy :or alkynoxy, Ar'-substituted alkoxy, Ar'-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino or alkynylamino, Ar'-substituted alkylamino, Ar'-substituted alkenylamino or alkynylamino, Ar'-substituted carbonyloxy, alkylcarbonyloxy, aliphatic or aromatic acyl, Ar'-substituted acyl, Mr'-substituted alkylcarbonyloxy, Ar'-substituted carbonylamino, Ar' substituted amino, Ar'-substituted oxy, Ar'-substituted carbonyl, alkylcarbonylamino, Ar' Substituted alkylcarbonylamino, alkoxy-carbonylamino, Ar '-substituted alkoxycarbonyl-amino, Ar' -oxycarbonylamino, alkylsulfonylamino, mono- or bis- (Ar'-sulfonyl) amino, 14 Ar'-substjtuted alkyl-sulfonylamino, morphol inocarbonyl amino, thiomorpholinocarbonylamino,

N-

alkyl guanidino, N-Ar' guanidino, N-N-(Ar',alkyl) guanidino, N,N-(Ar',Ar')guanidino, N,N-dialkyl guanidino, N,N,N-trialkyl guanidino, N-alkyl urea, N,N-dialkyl urea, N-Ar' urea, N,N-(Ar',alkyl) urea, N,N-(Ar') 2 urea, aralkoxycarbonyl-substituted alkyl, carboxyalkyl, oxo, arylsulfonyl and aralkylaminocarbonyl.

The term "leaving group" generally refers to groups readily displaceable by a nucleophile, such as an amine, and alcohol or a thiol nucleophile. Such leaving groups are well known and include carboxylates,

N-

hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides), triflates, tosylates, mesylates, alkoxy, 15 thioalkoxy and the like.

The term "hydrophobic group" refers tc a aroup which is resistant to uniting witrtt or absorbing -water.

:Examples of such hydrophobic groups include, bu,: are no-: limited to, methyl, ethyl, propy, butyl, pentyl, hexy-, phenyl, benzyl, naphthyl, N-benzylimidazolyl, mehlhieh. and the like.

0.The term "acidic functional group" reffers group which has an acidic hydrogen within it. Exarrnc_-es of such groups include, but-are not limited to, carboxylic acid, tetrazole, imidazole, hydroxyl, mercapto, hydroxylaminocarbonyl, sulfonic acid, sulf-1n1z acid, phosphoric acid and phosphonic acid.

The terms "activated derivative of a s-_ic4abv protected a-amino acid" and "activated substitutedphenylacetic acid derivative" refer to derivatives of carboxylic acids wherein the -OH group is replaced by a superior leaving group. Examples of activated acid 15 derivatives include, but are not limited to, the corresponding acyl halides acid fluoride, acid chloride and acid bromide), corresponding activated esters nitrophenyl ester, the ester of 1hydroxybenzotriazole, HOBT, or the ester of hydroxysuccinimide, HOSu), and other conventional derivatives within the skill of the art.

The terms "protected" or "protecting group" refer to a suitable chemical group which may be attached 1 0 to a functional group of a molecule, then removed at a later stage to reveal the intact functional group and molecule. Examples of suitable protecting groups for various functional groups are described in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 15 2d. Ed., John Wiley and Sons (1991); L. Fieser and M.

Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); L. Paquette, ed.

Encyclopedia of Reaaents for Organic Synthesis, John Wiley and Sons (1995).

The compounds of this invention contain one or more asymmetric carbon atoms and thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be of the R or S configuration. Although the specific compounds exemplified in this application may be depicted in a particular stereochemical configuration, compounds having either the opposite stereochemistry at any given chiral center or mixtures thereof are envisioned as part of the invention. Although amino acids and amino acid side chains may be depicted in a particular configuration, 16 both natural and unnatural forms are envisioned as part of the invention.

In view of the above definitions, other chemical terms used throughout this application can be easily understood by those of skill in the art. Terms may be used alone or in any combination thereof. The preferred and more preferred chain lengths of the radicals apply to all such combinations.

This invention provides compounds which are 1 0 capable of inhibiting VLA-4-mediated cell adhesion by inhibiting the binding of ligands to that receptor.

These compounds are represented by formula 3) -X (I) and phar-aceutically acceptable derivatives thereof; wherein: Z is selected from the group consisting of alkyl; aliphatic acyl optionally substituted with Nalkyl- cr N-arylamido; aroyl; heterocycloyl; alkyl- or arylsulfonyl; aralkylcarbonyl optionally substituted with aryl; heterocycloalkylcarbonyl; alkoxycarbonyl; araikylcxycarbonyl; cycloalkylcarbonyl optionally fused with aryl; heterocycloalkoxycarbonyl; alkylaminocarbonyl; arylaminc carbonyl and aralkylaminocarbonyl optionally substitu:ed with bis(alkylsulfonyl)amino, alkoxycarbonylamino or alkenyl; alkylsulfonyl; aralkylsulfonyl; arylsulfonyl; cycloalkylsulfonyl optionally fused with aryl; heterocyclylsulfonyl; heterocyzlylalkylsulfonyl; aralkoxycarbonyl; aryloxycarbonyl; cycloalkyloxycarbonyl; 17 heterocyclyloxycarbonyl; heterocyclylalkoxycarbonyl; mono- or di-alkylaminocarbonyl 'optionally substituted with aryl; (alkyl) (aralkyl)aminocarbonyl; mono- or diaralkylaminocarbonyl; mono- or di-arylaminocarbonyl; (aryl) (alkyl)aminocarbony.; mono- or dicycloalkylaminocarbonyl; heterocycl1yl amino carbonyl; heterocyclylal kylaminocarbonyl; (alkyl) (heterocyclyl) aminocarbonyl; (alkyl) (heterocyclylalkyl) aminocarbonyl; (aralkyl) (heterocyclyl)aminocarbonyl; (aralkyl) (heterocyclylalkyl) aminocarbonyl; alkenoyl optionally substituted with aryl; alkenylsulfonyl o p i n a l s u s i u e i h a y l y o l o t o a l opinalsubstituted with aryl; alkynyl optionally substituted with aryl; cycloalkenylcarbonyl; cycloalkenylsulfonyl; cycloalkylalkanoyl; cycloalkylalkylsulfonyl; arylaroyl, biarylsulfonyl; alkoxysulfonyl; aralkoxysulfonyl; alkylaminosulfonyl; aryloxysulfonyl; arylaminosulfonyl; N-arylureasubstituted alkanoyl; N-arylurea-substituted alkylsulfonyl; cycloalkenyl-substituted carbonyl; ***cycloalkenyl-substituted sulfonyl; alkenoxycarbonyl optionally substituted with aryl; alkenoxysulfonyl optionally substituted with aryl; alkynoxycarbonyl optionally substituted with aryl; alkynoxysulfonyl optionally substituted with aryl; alkenyl- or alkynylaminocarbonyl optionally substituted with aryl; alkenylor alkynyl-aminosulfonyl optionally substituted with aryl; acylamino-substituted alkanoyl; acylantinosubstituted alkylsulfonyl; alinocarbonyl -subs ti tuted alkanoyl; carbamoyl- substituted alkanoyl; carbamoylsubstituted alkylsulfonyl; heterocyclylalkanoyl; 18 heterocyclylaminosulfonyi; carboxyalkyl-substituted.

aralkoyl; carboxyalkyl-substituted aralkylsulfonyl; oxocarbocyclyl-fused. aroyl; oxocarbocyclyl-fused arylsulfonyl; heterocyclylalkanoyl; N' ,N'-alkyl, aryihydrazinocarbonyl; aryloxy-substituted alkanoyl. and heterocyclylalkylsulfonyl.

Y' is 2

Y

2 is -N(Ri)-C(R 2 :each

Y

3 is represented by the formula C (A3) -C each R: is independently selected from the group consisting of hydrogen, alkyl, and aralkyl; alkenyl; alkynyl; cycloalkyl; cycloalkenyl; cycloalkylalkyl; aryl; aminoalkyl; mono- or di-alkyl1-substituted amninoalkyl; mono- or di-aralkyl-substituted aminoalkyl; hydroxyalkyl; alkoxyalkyl; mercaptoalkyl; thioalkoxyalkyl A: is selected from the group consisting of amino acid side chains and corresponding protected derivatives; cycloalkyl'; and aikyl optionally subsrtituted with amino, acylamino, amino-substituted acylamino, :alkoxycarbonylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl', aralkoxycarbonyl, aminocarbonyl, alkylamainocarbonyl, dialkylarninocarbonyl, (alkyl) (aralkyl)aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl;, hydroxyl, carboxyalkylaminoc-arbonyl, hydroxylaminocarbonyl7, mercapto, thioalkoxy or heterocycle; A 2 is selected: from the group consisting of acidic functional groups and alkyl optionally substituted with an acidic functional group, protected acidic functional group or aryl; 19 each A 3 is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; aryl; cycloalkyl; and alkyl optionally substituted with amino, acylamino, aminosubstituted acylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylarninocarbonyl, (alkyl) (aralkyl) aminocarbonyl, aralkyJlaminocarbonyl, diaralkylaminocarbonyl, hydroxyl, carboxyalkylaminocarbonyl, hydroxylaminocarbonyl, mercapto, thioalkoxy or heterocycle; .or R: and any A are taken together with. the atoms to which they are attached form a 3- to 6 -membered ring heterocycle; each RI is independently selected from the group consisting of hydrogen and alkyl; n is an integer from 0 to 8; and is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; alkylaminc.

optionally substituted with'hydroxy, aminocarbonyl, N- :alkylaminocarbonyl, carboxy or alkoxycarbonyl; dialkylamino; cycloalkylamino; dicycloalkylamino; cycloalkylalkyaamino; (alkyl) (aryl)amino; aralkylaminc optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle; and (mono- or bis-carboxyl-ic -acid) -substituted alkylamine; he terocycl ylamino;, heterocyclyl-substituted alkylamino and wherein the compound of formula I is expressly not N' -carboxymethyl-N- (phenylacetylLleucy..L.asparty>.Lphenylalanyl-L-prolyl)piperazine when Z~phenylacetyl y 2

Y

3 n=2, and X=4carboxymethylpiperazinyl) and expressly not phenylacetyl- 20 L-leucyl-L-aspartyl-L-phenylalanyl-D-proline amide when Z=phenylacetyl,

Y

2 Y=F/p, n=2, and X=NH 2 A "pharmaceutically acceptable derivative" denotes any pharmaceutically acceptable salt, ester, salt of such ester, amide or salt of such amide of a compound of this invention. The invention also includes any other compound which, upon administration to a patient, is capable of providing (directly or indirectly) a compound of this invention a prodrug). The invention also 10 includes metabolites or residues of a compound of this invention characterized by the ability to inhibit, prevent cr suppress cell adhesion and cell adhesionmediated pathologies.

In a preferred embodiment of this invention,

A:

is selected from the group consisting of cycloalkyl; heterocyclic ring (when A: and R: are taken together); and alkyl optionally substituted with amino, acylamino, amino-substituted acylamino, aryl, carboxy, cycloalkyl, hydroxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylar.inocarbonyl, (alkyli (aralkyl)aminocarbonyl, aralkylarinocarbonyl, diaralkylaminocarbonyl, alkoxycarbonylamino, mercapto, thioalkoxy or heterocycle.

More preferably, A- is selected from the group consisting of aminocarbonylethyl, benzyl, n-butyl, isobutyl, carboxyethyl, cyclohexyl, 1-hydroxyethyi, hydroxymethyl, mercaptomethyl, 1-methylpropyl, methylthioethyl, n-propyl, isopropyl, methoxycarbonylaminobutyl, 6 -aminohexanoylaminobutyl and (when A: and R: are taken together) azetidine, aziridine, pyrrolidine, .and piperidine.

21 Even more preferably, A' is selected from the group consisting of benzyl, n-butyl, isobutyl, methylthioethyl, cyclohexyl, 1-methylpropyl, n-propyl and isopropyl. An alternate preferred A' is (when A' and R' are taken together) pyrrolidine.

In an alternate preferred embodiment, A 2 is selected from the group consisting of alkyl optionally substituted with amino, aminocarbonyl, aryl, alkoxycarbonyl, aralkyloxycarbonyl, 10 hydroxylaminocarbonyl, carboxy, NH-containing heterocycle, hydroxy, or mercapto; aralkyl optionally substituted with amino, aminocarbonyl, carboxy, NHcontaining heterocycle, hydroxy, or mercapto; and heterocyclic ring (when A 2 and R' are taken together) More preferably, A 2 is selected from the group consisting of carboxymethyl, 2-carboxyethyl, 1carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl, imidazolylmethyl, N-Bn-imidazolylmethyl, phenyl, carbomethoxymethyl, carbobenzyloxymethyl, and (when A' and RI are taken together) azetidine, aziridine, pyrrolidine, and piperidine.

Even more preferably, A 2 is selected from the group consisting of carboxymethyl, 2-carboxyethyl, 1carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl and imidazolylmethyl.

According to another preferred embodiment, A' is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; cycloalkyl; and alkyl optionally substituted with aryl, cycloalkyl, carboxy, hydroxylaminocarbonyl, alkoxy, aralkyloxy, mercapto, N-containing heterocycle, carboxyalkylaminocarbonyl or amino-substituted acylamino.

22 More preferably, A 3 is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; cyclohexyl; and alkyl optionally substituted with phenyl, cyclohexyl, carboxy, hydroxylaminocarbonyl, methoxy, benzyloxy, mercapto, N-benzylimidazoayl, biotinyl, tetrazolyl, valinyl-N-carbonyl or 6 -aminohexanoylamino.

According to another preferred embodiment, each Y3 is independently selected from the group consisting of amino acids and corresponding protected derivatives.

According to another preferred embodiment, is leucinyl

R

2 A*=i-Bu) Y- is aspartyl R- A K=carboxymethyl); n=2; and Y is valinylprolinyl A3=i-Pr) R' with A-=proline).

In another preferred embodiment, X is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; mono- and dialkylamino optionally *substituted with hydroxy, aminocarbonyl,

N-

alkylaminocarbonyl, carboxy or al'koxycarbonyl; di-Lal kyl amino; cycloalkylaminc; cycloalkylalkylarmano; :dicycloalkylamino; (alkyl) (aryl)amino; aralkylam-no opt ionally substituted with carboxy; diaralkylaminc; arylamino; N-containing heterocycle; bis-carboxylic acidsubstituted alkylainine and (mono- or biscarboxymethylaminocarbonyl-substituted-N-containin heterocycle.

More preferably, X is selected from the group consisting of amino, methylamino, isopropylamino, isobutylamino, n-butylamino, t-butylamino, isoamylamino, isopentylamino, hexylamino, cyclohexylamino, cyc lohexylmethyl amino, methylphenylamnino, phenylmethylamino, pheny'Lamino, 4- 23 methoxyphenylmethyl amino, dimethylamino, di isopropyl amino, diisobutylamino, hydroxy, methoxy, nbutoxy, t-butoxy, benzyloxy, 2 -pipe ridinecarboxyl ic acid, N'I- (a,caxI-bi s-carboxymethyl) 2 -piper idinecarboxamide, N' cabxmty--ierdncroaie 1-hydroxymethy1-2methyipropylamino, 1-N' -methylamido-l-methylethylamino, 3, 3 -dimethylbutyl amino, J-N'-methylamidobutylamino,

I-

amido-2-methylbutylamino, 1-carbomethoxy-2- .methylbutylamino, l-N'-methylamido-2-methylbutylamino, 1- 10 carboxy-l-phenylmethylamino, lorpholjino, piperidinyl,

N-

phenylpiperazinyl, pipecolinyl, and piperazinyl.

According to another preferred embodiment, Z is *selected from the group consisting of aliphatic acyl, aroyl, aralkylcarbonyl, heterocycloyl, alkoxycarbonyl, aralkyloxycarbonyl and heterocycloalkylcarbonyl. More preferably, Z is a (N-Ar'-urea)-para-substituted aralkylcarbonyl group and even more preferably, Z is a (N-Ar' -urea) -para--substituted phenylmethylcarbonyl group or (N-Ar'-urea) -para-substituted pyridylmethylcarbonyl group. Even more preferably, Z is a (N-orthosubstituted-Ar'urea) -para-substituted phenylmethylcarbonyl group or (N-meta-substjtuted- Ar'urea) -para-substituted phenylmethylcarbonyl group.

Examples of some specific preferred compounds of this invention are provided in Table 1.

24

(I)

wherein Y' is 2) WA) -C (0) y 2 is -N (R 2 (A 2 each y 3 is represented by the formula 2 (A 3 C 10 For A: and A 3 a single letter code refers to the side chain of the corresponding amino acid designated by that letter. A capital letter A) 4ndicates the L-amino acid while a small letter a) indicates the D-amino acid. Both capital and small letters L(l)) 15 indicates a mixture.

Unless expressly noted to the contrary; compounds in this table have RI and R 2 as hydrogen.

Cmpd Z A' A 2

(A

3

X

20 1 3-methoxy-4-(N'-phenyl L D V/P

OH

-urea)phenylacetyl 2 3 -methoxy-4-(NW-phenyl M D V/P OHI ~urea)phenylacetyl_____ :3 6 -methoxy-5-(N'-(2-methyIphenyi) L I

L

25 a.

a. a a 9 a.

a a a a.

a a.

a a *a a a a S. a a a.

a a.

26 Crnpd 38 3 4 -hydroNphenyl)propionyl L D VIP 39 3 -yrxpev)propionyl L D

VIP

Boc L D V/P 41 2 -quinolinecarbonyl L D

V/P

42 phenylacetyi L 0 V/pipecoliny 43 phenylacetyl L D V/n-butyl 0 0 000 0 0: 44 46 47 2 -quinolinecarbonyl 4 -methoxyphenylacetyl 3 -(4-hydroxyphenyl)propionylb-enzyiaminocarbonyl

L

(N-Me)-L (N-Me)-L

D

D

D

48 49 50 51 52 53 54 P-tolylaminocarbonyl phenylacetyl phenylacetyl phenylacetyl 2-quinolmnecarbonyl phenylacetyl phenylacetyl phenylacetyi 2-quinolinecarbonyl 2-quinolinecarbonyl 1 ,2,3,4-tetrahydro-2.

quinolinecarbonyl

L

n-prpy

L

L

L

L

L

L

L

L

L

D

D

D

D

D

V/n-butyl

V

V

V

V

V

V

fl-propyl n-propyl 2-butyl 2-butyl 2-butyl 2 butyl 2-butyl 2-butyl _(O-Me)-T

T

T n x

OH

OMe OMe OMe

OH

OH

OH

NHMe NHMe NHMe NHMe NHMe NHNap NHMe NHMe

NH,

OMe NHMe OMe NHMe NHMe NHM e ~4Ht-Bu lorpholino 59 61 2-quinolinecarbonyl 2-quinolinecarbonyl 2 -quinolinecarbonyl

L

L

L

62 1 Boc L D T I ~JHt-Bu 27 Cmpd Z A' A 2 (A 3 x 63 2-N-Boc-amino-1 .2,3.4-tetrahydro- L D V OH 2-naphthoyl_____ 64 3-phenylpropionyl L D V OH 3-(4-hydroxyphenyl)-2-bis- L D V OH (methylsutfonyl)aminopropionyl 66 3-(4-hydraxyphenyl)-2-N-Boc- L D V OH aminopropionyl_____ 67 2-amino-i,2,3,4-tetrahydro-2- L D V OH na phthoylTFA salt 68 Boc D V

OH

69 3-isoquinolinecarbonyl L D V OH 3-isoguinolinecarbonyl D V-

OH

71 1 ,2,3,4-tetrahydro-3- D V

OH

isoquinolinecarbonyl 72 naphthoyl L D V OH 73 1 ,2.3,4-tetrahydro-2-naphthoyl L I Dv

OH

74 naphthoyl 0 V-

OH

1 .2,3,4-tetrahydro-2-naphthoyl D V

OH

76 5-phenylpentanoyl 0 V-

OH

77 2-pyridinecarbonyl L D V OH 78 2-pyridinecarbonyl D V -OH 79 3-tetrahydrofurancarbonyl L D V OH 2-tetrahydrofurancarbonyl L D V OH 81 3-isoguinolinecarbonyl F D V OH 82 3-isoguinolinecarbonyl A R'Me) D V OH 83 3-isoguinolinecarbonyl pyclohexyl D V OH 84 1 ,2.3,4-tetrahydro-3- cyclohexyl D V OH isoquinolinecarbonyl 3-isoquinolinecarbonyl cyctohexyl- D V OH meth 86 1,2,3,4-tetrahydro-3- cyclohexyl- D V OH isopuinolinecarbonyl lmethd 28 Cmpd Z A' A 2 (A)x 87 3 -isoquinolinecarbonyl D F -OH 88 l,2,3,4-tetrahydro-3- D L -OH isoguinolinecarbony 89 3 -isoguinolinecarbonyl D L

OH

.1.2.3.4-tetrahydro-3- L D L OH "is~unolinecarbonyl 91 3 -isoquinolinecarbonyl L D L OH 92 1.2,3,4-tetrahydro-3- L D F OH isoguinolinecarbonyl 93 3 -isoguinolinecarbonyl L D F OH 94 2-guinolinecarbonyt L D V OH 3.3-diphenylpropionyl L D v OH 96 1 .2.3,4-tetrahydro.3- A D V OH isoquinolinecarbonyl 97 3 -isoguinoline arbonyl A D v OH 98 5-phenylpentanoyl L I D V -OH 99 indole-2-carbonyl L D V OH 100 3 -(4-hydroxy)ehenylpropionyl L D -NHi-Bu 101 benzoyl L D NHi-Bu 102 5-phenylpentanoyl L D NHi-amvl 103 3-(4-hydroxy)phenylpropionyl I L D NK-amyI 104 6-phenylhexanoyl L D v OH 105 benzoyl L D V OH 106 5-phenylpentanoyl L I D NHi-Bu 107 N-phenylsuccinamoyl L D V OH 108 N-4-fluorophenyisuccinamoyl L D V OH 109 N-methyl-N-phenylsuccinamoyl L D V OH 110 1.2,3.4-tetrahydro-2- L D NHi-amyl quinoilnecarbonyl__________ 11N-phenylsuccinamoyl L D NHi-Bu 112 3-phenylpropyl (N-Mel-L 10-Me)-D -V 01e 29 Cmpd Z A' A 2 (A)x 113 benzoyt (N-Me)-L D v

OH

114 1,2.3,4-tetrahydro-2- L D V NHHex guinolinecarbonyl__________ 115 1 2,3,4-tetrahydro-2- L D V 4-phenyl- Iguinolinecarbonyl _____piperidine 116 3 4 -hydroxy)phenylpropionyl L D NHHex 117 3 4 -hydroxy)phenylpropionyl L D 118 3 4 -h droxy)phenylpropionyl L _-NBu) 119 1 3 -h droXY)phenylpropiony; L D V NHHex 10

S

S

S 15 S

S

S

K'1 1quinolinecarbonyi j 21 2-quinolinecarbonyl L D v 122 1 .2,3,4-tetrahydro-2. L D

V

guinolinecarbonyl 123 2 -quinolinecarbonyl L D

V

124 1 ,2,3,4-tetrahydro-2. L D v Iguinolinecarbonyl__________ 125 2-unlncroy L D D 126 2 -pyridinecarbonyl 127 2-quinolinecarbonyl

L

128 4-methoxybenzoyl (N-Me)-L 129 phenylacetyl y

D

D

V-

V

V

V

V

NMePh NMePh NH-4-fluorophenyl NH-4-fluorophenyl_ NHPh NHPh NHMe 4-phenylpiperazinyl NHMe NH Me NHMe NHMe NHMe P 0 131 phenylacetyl R

D

132 phenylacetyl N D 133 2-N-Boc-amino-1 2 ,3,4-tetrahydro- D

V

_____2-naphthovl 134 12-N-phenylacetylamino-i Z 3,4.t D t V NHMe tetrahydro-2-naphthoyl

JI

135I Boc D I PI G 1 OH 30 *5

S

S

*5 Cmpd Z A' x 136 phenylacetyl D P G OH 137 phenylacetyl L D -N-f bis- (carboxy)methylJpipecolinamido 138 phenylacetyl L D P N H-fbis- (carboxy)- Smethyl] 139 phenytacetyl L D -N- [carboxym ethyl]pipecolinamide 140 3-phenylpropionyl (N-Me)-L D V OMe 141 4-hydroxyphenylacetyl (N-Me)-L D V OMe 142 2-quinolinecarbonyl (N-Me)-L D V -OMe 143 4-±phenylbutyryl (N-Me)-L D V OMe 144 4-(N'-2-hydroxy- L D V/P OH phenylurea)phenylacety 145 PUPA L D V/P OH 146 4-(N'-2-hydroxy- M D V/P OH phenylurea)phenylacetyl 147 3-methoxy-4-(N'. L D VIP NH, phenylurea)phenylacetyl______ 148 2-MPUPA L D VIP NH, 149 Boc D V P OH 150 5-phenylpentanoyl D V P OH 151 2-allyi-4-phenylbutyryl V p

OH

152 acetyl F L DN OH 153 benzoyl F L D/V OH 154 1 ,2,3.4-tetrahydro-3- L D V OMe gun -iearbonyl 155 4ohenylbutyrvl L D

OH

31 Cmpd Z A' A 2

(A

2 x 156 3 -isoquinolinecarbonyl L D V -OMe 157 3-isoguinolinecarbonyt L D NHi-Bu 158 2-quinolinecarbonyl L D V -Ot-Bu 159 2 -quinolinecarbonyl L v

OH

160 2 -quinolinecarbonyl L 0 D OH 161 4-phenylbutyryl L D -NHi-Bu 162 3-phenylpropionyl L D NHi-Bu 163 benzoyl G L D NHi-Bu 164 2-guinolinecarbonyl L D V NHMe 165 __4-methoxybenzoyl L D NHi-Bu 166 4-phenylbutyryl L D V OMe 167 Boc L D vim OMe 168 2-quinolinecarbonyl L D V/M OMe 169 N-n-butylaminocarbonyl D V- OMe 170 2-quinolinecarbonyl L D T OMe 171 N-t-butylaminocarbonyl L D NHi-Bu 172 benzoyl G D V OMe 173 benzoyl G (0-Me)-D V OMe 174 2-quinolinecarbonyl L D NH(1 -hydraxymethyl-2methyl- _propyf) 175 2-quinolinecarbonyt L D V morpholino 176 4-methoxyphenylacetyl L D T OMe -177 4-methoxyphenyisulfonyl L D T OMe 178 2-quinolinecarbonyl L D V NH, -179 2-quinolinecarbonyl (N-Me)-L 0 v NHMe 180 phenylacetyl (N-Me)-L D V NHMe 1181 phenvlacetvi L D v NHMe 32

S

S.

Cmpd Z A' A 2

X

182 3-phenylpropionyl (N-Me)-L D V NHMe 183 Phenvlacety,1 M D V NHMe 184 3-phenylpropionyl (N-Me)-L D V NHMe 185 2-quinolinecarbonyt L D A (R 2 =Me) NHMe 186 2-guinolinecarbonyl L D vim OH 187 phenylaminocarbonyl L D V NHMe 188 4-hydroxyphenylacetyl (N-Me)-L D V N HMe 189 phenylsulfonyl L D V NHMe 190 phenylacetyl L D (0-Me)-T OMe 191 phenylacetyl L D T OMe 192 phenylacetyl L -D (O-Bn)-T OMe 193 phenylacetyl L 0 (O-Ac)-T OMe 194 phenylacetyl V D V NHMe 195 -2-quinolinecarbonyl L D T On-Bu 196 phenylacetyl L D V On-Bu 197 2-quinotinecarbonyl L D T NH(4methoxy- 198 2-quinolinecarbonyl L D NH(3,3dimethyl-nbutyl) 199 PUPA D V/P NH- 200 PUPA L d V/P NH, 201 PUPA L D viP NH, 202 2-MPUPA D V/p OH aminohexa noyI -KI 203 PUPA L D V OH 204 PUPA L D V NHMe 205 PUPA L D V NHi Bu 206 2-MPUPA L D VIP OH 33 Cmpd Z A' A 2

(A

3 x 207 2-MPUPA L D phenyi OH 208 PUPA L D V/P NH, 209* PUPA I D VIP NH, 210 PUPA L d VIP NH, 211 PUPA L 0 VIP NH, 212 PUPA Id V/p NH, 213 PUPA L D NHBn 214 PUPA L D morpholino 215 PUPA L D NHi-Pr 216 PUPA L D NHCy 217 PUPA L D NHi-Bu 218 PUPA L D piperidinyl 219 2-MPUPA M D D NH, 220 2-MPUPA M D L NH, 221 2-MPUPA M D V NH, 222 2-MPUPA M D I NH.

223 2-MPUPA M D E NH, 224 2-MPUPA M D T NH-.

-225 2-MPUPA M D M NH-.

226 2-MPUPA M D. n NH.

227 2-MPUPA M D e NH,.

228 2-MPUPA M D -W NH, 229 2-MPUPA M D -s NH, 230 2-MPUPA L D D NH, 231 2-MPUPA L D L NH, 232 2-MPUPA L D v NH, 233 2-MPUPA L D I NH, 234 2-MPUPA L D E N H, 34 I 1 7 ICmpd [235 2-M Z A' A 2 (A 3 PUPA I L II 236 237 238 239 240 I 2-MPUPA L I D j M 2-MPUPA In 2-MPUPA 2-MPUPA L D e 24VIPUPA t I I rn 2-PP L D 241 242 243 244 245 246 247 248 249 250 251 252 253 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA p

P

P

p

P

P

P

P

P

P

P

T

T

D

D

D

D

D

D

D

D

D

D

D

D

D

D

L

V

E

T

M

n e w s

D

L

V

E

T

M

n e x

NH

NH

NH,

NH,

NH

NH

NH,

NH

NH,

NH,

NH,

NH,

NH,

NH,

NH,

NH,

NH.

NH,

NH,

-NH,

NH,

NH,

NH,

NH,

NH,

254 2-MPUPA F 4 255 2-PJPA 256 2-MPUPA T

D

2-PUPA 258 259 260 I Li I 2-MPUPA 2-MPUPA 2-MPUPA

T

T

T

D

D I I I T 1 4- 261 2-MPUPA I vv NH, 262 2-MPUPA I s I I NH 263 2-MPUPA NH, I. L LI 35 a.

a a a.

a C. a a.

a a a a a.

Cmpd Z A' A' (A 3 x 264 2-MPUPA E D L NH, 265 2-MPUPA E D V NH 266 2-MPUPA E D INH, 267 2-MPUPA E D E NH 268 2-MPUPA E D T NH 269 2-MPUPA E D M NH, 270 2-MPUPA E D n NH-.

271 2-MPUPA E D e NH- 272 2-MPUPA E D w NH- 273 2-MPUPA E D s NH, 274 2-MPUPA C D v NH.

275 2-MPUPA S D D NH, 276 2-MPUPA s D L NH- 277 2-MPUPA S D v NH, 278 2-MPUPA s D I NH, 279 2-MPUPA S D E NH, 280 2-MPUPA S D T NH.

281 2-MPUPA S D M NH.

282 2-MPUPA s D n NH- 283 2-MPUPA S D e NH- 284 2-MPUPA S D w NH.

285 2-MPUPA S D s NH- 286 2-MPUPA ID D N H, 287 2-MPUPA D L NH, 288 2-MPUPA D 0 V NH, 289 2-MPUPA ID I NH, 290 2-MPUPA D E NH, 291 2,MPUPA D T -NH, 292 2-MPUPA ID M NH., 36 Cmpdl z A' A 2 4(A~)n 293 2-MPUPA 2-MPUP LV n 294 2-MPUPA ID e 295 2-MPUPA ID w 296 2-MPUPA ID s 297 2-MPUPA Q D D 298 2-MPUPA Q D L 299 2-.MPUPA_ Q D v 4.

4 4*4* 4O @4 4 300 301 2-MPUPA 2-MPUPA 0

D

302 303 304 305 306 307 308 309 310 311 312

Q

2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2-MPUPA 2..MPUPA 2-MPUPA 2-MP UPA 2-MPUPA 0

Q

Q

L

D

D

D

D

D

D

E

E

E

T

M

n e

D

S

x

NH,

NH

NH

NH,

NH

NH

NH,

NH,

NH,

NH,

NH,

NH,

NH,

NH,

NH

S.

S

4*44 4

*SSS

4 5

S

.4 5 4s *4

E

D

I/

2-MPUPA L

FE'

P

___LI

E

2-MPUPAp 3141 2..MPUPA I T T E 1 315 316 317 318 2-MPUPA 4 -(N'-2-pyridylurea)phenylacetyI 3 -methoxy-4-(N'-(2-methylphenyl)urea)phenviacetvi

M

L

L

D

D

D

D

V

D

V/P

NH_

NH,

NH,

NH,

NH,

OIH

NH,

morpholino I I .L t

PUPA

L

I

I

319 320

PUPA

PUPA

L

NHi-Pr Klwtl I v I 37 C.

C

*C

C.

Cmpd Z A' A 2

(A

3

X

321 PUPA L D V NHBn 322 PUPA L D V piperidinyl 323 PUPA L D V NHi-Bu 324 PUPA L D VIP N HCy 325 PUPA L D V/P piperidinyl 326 PUPA L D V/P NHBn 327 PUPA L D V/P NHi-Pr 328 PUPA L D V/P NHi-Bu 329 2-MPUPA L D V morpholino 330 N-3-(4-hydroxyphenyl) pipecolyl I D NHi-Bu 331 N-3-(4-hydroxyphenyl)..propionyl P D NHi-Bu 332 3-isoquinolinecarbonyl L

OH

methyl-2butyroyl)-N 333 4-methylpentanoyl D NHC M 334 Cbz -CH-C v OMe (N of CH.CHA(C

A')-D

335 3 -(4-hydroxyphenyI)propionyl -CH..CH: V OMe (N of A-5 CHKCH,-(C 336 4 2 -fluorophenylurea)phenylacetyI L D V/P OH 337 2-MPUPA L D V/P/S OH 338 2-MPUPA L D V/P/SIT OH 339 2-MPUPA V L P/D OH 340 2-MPUPA v I p/d OH 341 2-MPUPA L P V/D OH 342 2-MPUPA P D -OH 343 hydrogen pv d/I 2-MPUBA 344 hydrogen v d I 2-MPUBA 345 2-MPUPA EL D V OH WO 97/03094 38 Cmpd Z A' A 2

(A.X

346 4-(N-(6-methyl-2- L D V/P OH pyddypurea)phenylacetyl 347 L D V/P OH fluorophenylurea)phenylacety 348 4-phenylbutyroyl (N-Me)-L I D V NHMe 349 phenylacetyl S D V NHMe 350 phenylacelyl K D V NHMe 351 phenylacetyl L D A (R 2 =Me) NHMe 352 phenylacetyl L D (O-Bn)-S NHMe -353 2-quinolinecarbonyl L D (O-Bn)-S NHMe 354 Bcc L D T NHBu 355 8cc L D V/P OH -356 2-quinolinecarbonyl L D -V/P OH 357 4-(N'-2-pyridylurea)phenylacetyI L D VIP NH- -358 2-MPUPA L D*THAM V/P OT-HAM 359 2-MPUPA L D*Na VIP ONa 360 2-MPUPA Het' 361 2-MPUPA IHet' 362 2-MPUPA L~l Het 2 363 2-MPUPA L~l Het 3 364 2-MPUPA Het -Het- 365 2-MPUPA L(l) Het' 366 fluorenylmethoxycarbonyl L D V OH 367 3-methoxyphenylacetyl L 0 V OH 368 3- 3-methylindol l)propionyl L D V OH 369 2-phenyl-3-methyl-pyrazol-4- L D V OH ____ylcarbonyl 370 6-methylbenzpyrimidon-2- L 0 OH- ____Vlcarbonyl 39 Cmpd Z A' A 2 (A 3

X

371 4-oxo-4,5,6,7- L D V OH tetrahydrobenzo[blfuran-3 ytcarbanyl 372 L D V OH (phenvlacetylenyI))pyridinecarbonyI 373 3 -(2-phenytthio)-pyndinecarbonyI L D V OH 374 -4-propylbenzoyl L D V OH 375 4 2 -(3-pyridinyl))thiazolecarbonyI L I D V -OH 376 4 -(2-(4-pyndinyl))thiazolecarbonyI L D V OH 37 7 5-(2-(3-pyridinyl))thiophenesulfonyI L D V OH 378 5-C 2 -(l-pyrrolyl))pvridinecarbonyI L D V OH 379 N,N-(4-trfluoromethylpyridin-2. L D V OH y)methylhydrazinocarbonyl 380 2 7quinoxalinylaminocarbonyl L D V OH 381 N-(4-trfifuoromethylpyridin-2 L D V OH yI)piperazinocarbonyl 382 L D V OH trifluoromethyl)phenylsulfanyl)- 11,2 .3,4-tetrahydrothiophenesulfonyI 383 l-(4-chlorophenylmethyl)pyrroidin. L D V OH I 2-on-4-ylcarbonyl 384 1-(2-furanylmethy)Ipyrrolidin-2-on L D V OH 4-ylcarbonyl_____ 385 2-(l -pyrrolyl)benzoyl L 0 V OH 386 6-chlorochroman-3-ylcarbonyl L D v OH 387 2,3-dihydrobenzofuran-5-ylcarbonyI L D V OH 388 4,6-dimethylpyrazolo[1 ,5-cjtriazin-3- L D V OH yicarbonyl 389 3,4-benzocyclohexanoyl L D V OH 390 nrbornylacety L D V OH 391 1 ,2,3,4-tetrahydro-9- L D V OH I acddinylcarbonyl 392 5,6,7,8- L D V OH tetrahvdronaphthylaminocarbong 40 0* 0 00 0 Cmpd Z A' A 2

(A

3 393 3-(2-(4-methylthiophenoxy))- L D v OH pyridinecarbonyt 394 2-(6-methoxynaphth-2y)propionyI L D V -OH 395 (2-naphthyioxy)acetyl L D v -OH 396 3-.quinuclidinylaminocarbonyl L D V OH 397 L D v OH tetra hydroisoguinoline)carbonyl 398 adamantan-2-ylcarbonyl L D V OH 399 (2-pyddyl)acetyl L D V OH 400 6 -methylcyclohexen-2-ylcarbonyl L D V OH 401 _(3-guinolinyl)acetyl L D V OH 402 4 2 -butyl)phenylaminocarbony L D v OH 403 1 ,4-dihydro-I -ethyl-7-methyl-4-oxo- L D v OH 1 .8-naphthyrldin-3-ylcarbonyt 404 (2-thienyl)acetyl L D v OH 405 4-(2-propyl)benzoy L D v OH 406 3.4-methylenedoxybenzoyl L D V OH 407 2 -(S-(2-pyridy))thiophenecarbonyl__ L D V OH 408 N-iminodibenzylcarbonyl L D v OH 409 2-MPUPA P D INHMe 410 2-MPUPA P D I OMe 411 2-MPUPA P D I OH 412 2-MPUPA -CH,CH,- D I OH (N of 413 2-MPUPA PE W Ne 414 2-MPUPA P E I NMe 415 2-MPUPA P E I OH 416 2-MPUPA P E -OH 41 where Het', He t 2 He t 3 Het', and Het 5 in Table I are defined below: 0 9

S

C0 2

H

Heti IaOMe 2

H

Het3

KN

0- C0 2

H

'N Het2 :N 0 OMe OMe C0 2

H

Na Het4 0 OMe KNO Hets The more preferred compounds of formula are selected from the group consisting of compound numbers 1, 2, 4, 144, 145, 146, 147, 148, 206, 315, 316, 317, 337, 338, 345, 346, 347, 357, 358 and 359 as identified in Table 1. Even more preferred compounds of formula

(I)

42 are selected from the group consisting of compound numbers 1, 206, 316, 358 and 359 as identified in Table 1. The most preferred compounds of formula are selected from the group consisting of compound numbers 358 and 359 as identified in Table 1.

Other compounds of this invention are compounds of formula

II:

K-

(II)

and pharmaceutically acceptable derivatives thereof, 10 wherein; K is selected from the group consisting of hydrogen, alkyl, aliphatic acyl, aroyl, aralkylcarbonyl, heterocycloyl, sulfonyl, aralkylcarbonyl, heterocycloalkylcarbonyl, alkoxycarbonyl, 15 aralkyloxycarbonyl, heterocycloalkoxycarbonyl, alkylaminocarbonyl and aralkylaminocarbonyl; J is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; alkylamino optionally substituted with hydroxy, aminocarbonyl,

N-

20 alkylaminocarbonyl, carboxy or alkoxycarbonyl; dialkylamino; cycloalkylamino; dicycloalkylamino; (alkyl)(aryl)amino; aralkylamino optionally substituted with carboxy; diaralkylamino; arylamino; and (mono- or bis-carboxylic acid)-substituted alkylamine; and each

Y

2 y 3 A, A 3

R

2 and n is independently as defined in formula I above.

Compounds of this invention may be synthesized using any conventional technique. Preferably, these compounds are chemically synthesized from readily 43 available starting materials, such as a-amino acids and their functional equivalents. Modular and convergent methods for the synthesis of these compounds are also preferred. In a convergent approach, for example, large sections of the final product are brought together in the last stages of the synthesis, rather than by incremental addition of small pieces to a growing molecular chain.

According to one embodiment, compounds of the present invention may be synthesized in the following 1 0 manner. A protected amino acid or functional equivalent is coupled to an appropriate activated ester moiety. The coupled product, if suitably functionalized, may be further reacted with yet another activated ester moiety.

This material can be further manipulated to give the desired compounds of the invention. At each step of the above sequence, the ester can be hydrolyzed to the corresponding acid to give another compound of the invention. This acid may also be converted to a corresponding acid derivative by standard methods.

Alternatively, the activated ester moieties mentioned above can be attached together first, then the resulting compound can be attached to additional amino acids or their functional group equivalents. At this point the final manipulations and/or necessary deprotection steps can be performed.

In another embodiment, under suitable conditions the desired functionalities can be incorporated (protected or unprotected) in one of the activated ester moieties. That ester is then coupled with an amino acid derivative or a moiety consisting of an amino acid.derivative previously coupled to an activated ester. The resulting product can then be 44 subjected to any deprotection steps, if necessary, to give compounds of the invention.

Alternatively, the compounds of this invention may be synthesized using solid support techniques. The core amino acid or their functional equivalent groups are assembled using standard reiterative coupling methodology on a resin. When the desired core is complete, the resulting fragment can be coupled with an activated ester moiety and/or the tethered end of the fragment may be 1 0 further derivatized to give the desired product.

Appropiate protection/deprotection methods may be used at any point during the synthetic sequence.

The compounds of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system blood, lymphatic system, central nervous system), increase oral availability, increase 20 solubility to allow administration by injection, alter metabolism and alter rate of excretion. Examples of these modifications include, but are not limited to, esterification with polyethylene glycols, derivatization with pivolates or fatty acid substituents, conversion to carbamates, hydroxylation of aromatic rings, and heteroatom-substitution in aromatic rings.

As used throughout this application, the term "patient" refers to mammals, including humans. And the term "cell" refers to mammalian cells, including human cells.

Once synthesized, the activities and VLA-4 specificities of the compounds according to this 45 invention may be determined using in vitro and in vivo assays.

For example, the cell adhesion inhibitory activity of these compounds may be measured by determining the concentration of inhibitor required to block the binding of VLA-4-expressing cells to fibronectin- or CS1-coated plates. In this assay microtiter wells are coated with either fibronectin (containing the CS-1 sequence) or CS-1. If CS-1 is used, it must be conjugated to a carrier protein, such as bovine serum albumin, in order to bind to the wells.

Once the wells are coated, varying concentrations of the t. est compound are then added together with appropriately labelled, VLA-4-expressing cells. Alternatively, the test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells.

The cells are allowed to incubate in the wells for at least 30 minutes. Following incubation, the wells are emptied and washed. Inhibition cf binding is measured by quantitating the fluorescence or radioactivity bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound.

VLA-4-expressing cells that may be utilized in this assay include Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral blood lymophocytes (PBLs). The cells used in this assay may be fluorescently or radioactively labelled.

A direct binding assay may also be employed to quantitate the inhibitory activity of the compounds of this invention. In this assay, a VCAM-IgG fusion protein containing the first two immunoglobulin domains of VCAM 46 (D1D2) attached above the hinge region of an IgG1 molecule ("VCAM 2D-IgG"), is conjugated to a marker enzyme, such as alkaline phosphatase The synthesis of this VCAM-IgG fusion is described in PCT publication WO 90/13300, the disclosure of which is herein incorporated by reference. The conjugation of that fusion to a marker enzyme is achieved by crosslinking methods well-known in the art.

The VCAM-IgG enzyme conjugate is then placed in the wells of a multi-well filtration plate, such as that contained in the Millipore Multiscreen Assay System (Millipore Corp., Bedford, MA). Varying concentrations cf the test inhibitory compound are then added to the wells followed by addition of VLA-4-expressing cells.

The cells, compound and VCAM-IgG enzyme conjugate are ixed together and allowed to incubate at room temperature.

Following incubation, the wells are vacuum drained, leaving behind the cells and any bound VCAM.

20 Quantitation of bound VCAM is determined by adding an appropriate colorimetric substrate for the enzyme .:conjugated to VCAM-IgG and determining the amount of reaction product. Decreased reaction product indicates increased binding inhibitory activity.

In order to assess the VLA-4 inhibitory specificity of the compounds of this invention, assays for other major groups of integrins, 82 and 83, as well as other 81 integrins, such as VLA-5, VLA-6 and o487 are performed. These assays may be similar to the adhesion inhibition and direct binding assays described above, substituting the appropriate integrin-expressing cell and corresponding ligand. For example, 47 polymorphonuclear cells (PMNs) express B2 integrins on their surface and bind to ICAM. B3 integrins are involved in platelet aggregation and inhibition may be measured in a standard platelet aggregation assay. binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds to laminin. a4B7 is a recently discovered homologue of VLA-4, which also binds fibronectin and VCAM. Specificity with respect to a47 is determined in a binding assay that utilizes the above-described

VCAM-

1 0 IgG-enzyme marker conjugate and a cell line that expresses a4B7, but not VLA-4, such as RPMI-8866 cells.

Once VLA-4-specific inhibitors are identified, they may be further characterized in in vivo assays. One such assay tests the inhibition of contact hypersensitivity in an animal, such as described by P.L.

Chisholm et al., "Monoclonal Antibodies to the Integrin a -4 Subunit Inhibit the Murine Contact Hypersensitivity Response", Eur. J. Immunol., 23, pp. 682-688 (1993) and n "Current Protocols in Immunology", J. E. Coligan, et 20 al., Eds., John Wiley Sons, New York, 1, pp. 4.2.1- 4.2.5 (1991), the disclosures of which is herein incorporated by reference. In this assay, the skin of the animal is sensitized by exposure to an irritant, such as dinitrofluorobenzene, followed by light physical irritation, such as scratching the skin lightly with a sharp edge. Following a recovery period, the animals are re-sensitized following the same procedure. Several days after sensitization, one ear of the animal is exposed to the chemical irritant, while the other ear is treated with a non-irritant control solution. Shortly after treating the'ears, the animals are given various doses of the VLA-4 inhibitor by subcutaneous injection. In vivo 48 inhibition of cell adhesion-associated inflammation is assessed by measuring the ear swelling response of the animal in the treated versus untreated ear. Swelling is measured using calipers or other suitable instrument to measure ear thickness. In this manner, one may identify those inhibitors of this invention which are best suited for inhibiting inflammation.

Another in vivo assay that may be employed to test the inhibitors of this invention is the sheep asthma 10 assay. This assay is performed essentially as described in W. M. Abraham et al., "a-Integrins Mediate Antigeninduced Late Bronchial Responses and Prolonged Airway Hyperresponsiveness in Sheep", J. Clin. Invest., 93, pp.

776-87 (1994), the disclosure of which is herein incorporated by reference. This assay measures inhibition of Ascaris antigen-induced late phase airway responses and airway hyperresponsiveness in asthmatic sheep.

The compounds of the present invention may be 20 used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids and bases.

Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2 -hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2 -naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, 49 tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-Dglucamine, tris(hydroxymethyl)methylamine and salts with amino acids such as arginine, lysine, and so forth.

Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such S 10 as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.

9. The compounds of the present invention may be formulated into pharmaceutical compositions that may be administered orally, parenterally, by inhalation spray, 20 topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions of this invention comprise any of the compounds of the present invention, or pharmaceutically acceptable derivatives thereof, together with any pharmaceutically acceptable carrier. The term "carrier" as used herein includes acceptable adjuvants and vehicles. Pharmaceutically acceptable carriers that may be used in the 50 pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium 10 trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

According to this invention, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable 20 dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.

In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and.its glyceride derivatives are useful in the preparation of injectables, as do natural 51 pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.

In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch.

:o Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

f Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable nonirritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal 52 tract. Suitable topical formulations are readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.

Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in 1 one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical .compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited 20 to, mineral oil, sorbitan monostearate, polysorbate cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspension in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

Thepharmaceutical compositions of this invention may also be administered by nasal aerosol or 53 inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

The amount of active ingredient that may be 10 combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the aspecific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular 0000.0 20 disease being treated. The amount of active ingredient may also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is coadministered.

The dosage and dose rate of the compounds of this invention effective to prevent, suppress or inhibit cell adhesion will depend on a variety of factors, such as the nature of the inhibitor, the size of the patient, the goal of the treatment, the nature of the pathology to be treated, the specific pharmaceutical composition used, and the judgment of the treating physician. Dosage levels of between about 0.001 and about 100 mg/kg body weight per day, preferably between about 0.1 and about 54 mg/kg body weight per day of the active ingredient compound are useful.

According to another embodiment compositions containing a compound of this invention may also comprise an additional agent selected from the group consisting of corticosteroids, bronchodilators, antiasthmatics (mast cell stabilizers), antiinflammatories, antirheumatics, immunosuppressants, antimetabolites, immunonodulators, antipsoriatics and antidiabetics. Specific compounds 1. 0 within each of these classes may be selected from any of those listed under the appropriate group headings in ."Comprehensive Medicinal Chemistry", Pergamon Press, Oxford, England, pp. 970-986 (1990), the disclosure of which is herein incorporated by reference. Also included within this group are compounds such as theophylline, sulfasalazine and aminosalicylates (antiinflammatories); cyclosporin, FK-506, and rapamycin (immunosuppressants); cyclophosphamide and methotrexate (antimetabolites) and interferons (immunomodulators).

S 20 According to other embodiments, the invention provides methods for preventing, inhibiting or Ssuppressing cell adhesion-associated inflammation and cell adhesion-associated immune or autoimmune responses.

VLA4-associated cell adhesion plays a central role in a variety of inflammation, immune and autoimmune diseases.

Thus, inhibition of cell adhesion by the compounds of this invention may be utilized in methods of treating or preventing inflammatory, immune and autoimmune diseases.

Preferably the diseases to be treated with the methods of this invention are selected from asthma, arthritis, psoriasis, transplantation rejection, multiple sclerosis, diabetes and inflammatory bowel disease.

55 These methods may employ the compounds of this invention in a monotherapy or in combination with an anti-inflammatory or immunosuppressive agent. Such combination therapies include administration of the agents in a single dosage form or in multiple dosage forms administered at the same time or at different times.

In order that this invention may be more fully understood, the following examples are set forth. These 10 examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

EXAPLE

S

GENERAL PROCEDURES FOR AMIDE BOND FORMATION IN SOLUTION: 15 PROCEDURE A: coupling with EDC/HOBt A solution of carboxylic acid (1.2 eq.) in DMF at 0

O

C was treated with HOBT (1.8 eq.) and EDC (1.4 eq.).

The mixture was stirred at 0°C for 1 to 2 h and then the free amine (1.0 eq., neutralized with TEA or DIPEA) was added. After stirring at RT for more than 3 h, the reaction mixture was diluted with ethyl acetate, washed with water 5% aqueous citric acid sat. NaHCO, and brine dried (Na,SO, or MgSO 4 and concentrated in vacuo.

56 PROCEDURE B coupling using activated ester (Nhydroxysuccinate or chloride) A solution of free amine (1-1.

2 eq.,neutralized with TEA or DIPEA) in CH 2 Cl 2 was treated with activated ester or acyl halide (1 eq.) at 0 OC or RT. After stirring at RT for over 1 h, the reaction mixture was washed with 5% aqueous citric acid sat. NaHCO- (2X), and brine dried (Na 2 SO4 or MgSO 4 and concentrated in vacuo.

1 0 GENERAL PROCEDURE FOR UREA FORMATION IN SOLUTION: PROCEDURE C: formation of urea with isocyanate and amine.

A solution of amine (1 eq.) and TEA (1 eq.) in

CH

2 C12 was treated with an isocyanate (1 eq.) and was S 15 stirred at RT for over 0.5 h. After concentration in vacuo, the product was either used as is or purified by chromatography.

GENERAL PROCEDURES FOR DEPROTECTION IN SOLUTION: PROCEDURE D removal of BOC with TFA S. 20 A solution of tBuOC(OINH-R (where R is alkyl optionally substituted with any number of suitable functional groups) in CH,C1- at 0 °C was treated with trifluoroacetic acid. The reaction was allowed to war.

to RT and stirred for 1 to 2 h. After concentration in vacuo the resulting amine/TFA salt was stored and neutralized with TEA or DIPEA prior to use.

PROCEDURE E removal of BOC with HC1 57 A solution of tBuOC(O)NH-R (where R is alkyl optionally substituted with any number of suitable functional groups) in dioxane at 0 °C was treated with 4N HC1 in dioxane. The reaction was allowed to warm to RT and stirred for 1 to 2 h. After concentration in vacuo the resulting amine/HCl salt was stored and neutralized with TEA or DIPEA prior to use.

PROCEDURE F: hydrogenation A mixture of starting material and 10% Pd/C in 10 methanol, water, ethyl acetate, and/or DMF was vigorously stirred under hydrogen (40 to 50 psi) for more than 2 h at RT. The resulting mixture was filtered through a plug :of Celite and the filtrate concentrated in vacuo.

GENERAL PROCEDURES FOR AMIDE BOND FORMATION ON SOLID

SUPPORT:

SPROCEDURE G: coupling with DCC/HOBt A mixture of resin (see below for preparation of resirn MCB1), tBuOC(O)NH-AA,-CO:H (where AA is an amino acid or functional equivalent) or R.-CO..H (10 HOBt (10 DCC (10 eq) and N-methylmorpholine (3 eq) in NMP was shaken for over 0.5 h at RT. The resin was then washed with NMP (2X) and CHC1. (3X).

PROCEDURE H: displacement from resin with amine A mixture of resin and amine (xs) in DMF was shaken for 6 h at RT. The resin was then washed with methanol (3X) and the combined solutions concentrated in vacuo.

58 GENERAL PROCEDURES FOR DEPROTECTION ON SOLID SUPPORT: PROCEDURE I: removal of BOC with TFA/CHC1 2 A mixture of resin and 50% TFA/CH 2 C1 2 was shaken for over 0.5 h at RT. The resin was then washed with

CH

2 C1 2 (2X) isopropanol (lX) and CH 2 C1 2 (3X).

PRCEUR HF with scavengers The protected product was treated with HF at to 0 0 c for over 1.5 h in the presence of anisole or :thioanisole as scavenger. The HF was removed with a stream of N 2 at 0 0

C.

SYNTHESIS OF COMM2~ON INTERMEDIATES Succinimidyl 3 -Iaoquimolinecprbpxylpte (i0n-OSu) A solution of 3 -isoquinolinecarboxyljc acid 15 (1.2 eq.) in DMF at 0 0 C was treated with EDC 4 eq.).

The mixture was stirred at 0 OC for 1 to 2 h and then N- *hydroxysuccinimide (1.0 eq.) was added. After stirring at RT for more than 3 h, the reaction mixture was poured into 602: sat. NaHCO, and the product filtered: 'H NMR (CDCl, 300 MHz, ppm) 9.35 1H), 8. 67 1H) 8.09 (mn, 1H), 7. 96 (mn, 1F) 7.82 (in, 2H-) 2. 94 4H).

Succininidvi 2 -Ounlie rboXvlpt n OnO-m A solution of 2-quinoline carboxylic acid (1.2 eq.) in DMF at 0 0 C was treated with EDC (1.4 eq.) The mixture was stirred at 0 0 C for 1 to 2 h and then Nhydroxysuccinimide (1.0 eq.) was added. After stirring at RT for more than 3 h, the reaction mixture was poured into sat. NaHCO3 and the product filtered: 1H NNR (CDCl 3 300 MHz, ppm) 8.35 1H), 8.27 1H) 8.19 (d, 59 IH), 7.87 1H), 7.80 1H), 7.68 Cm, 1H) 2. 91 (s, 4H).

Methyl 4 -1socvanatophenvlacetpte fKCH: A well-stirred cold solution of methyl paminophenyl acetate (9.8 g, 59.4 mmol) in CH 2 Cl 2 (200 mL) and TEA (25 niL, 18 g, 178.2 nimol) was treated with COCi 2 (96 mL of 1.9 M solution in toluene) over 1 h. The reaction mixture was stirred at 0 OC for an additional 1 h. The reaction mixture was concentrated and 3:1 10 ether/pet ether (125mL) was added. The mixture was filtered and the filtrate concentrated to give KC1 as a '-brown liquid. The crude product was purified by distillation (118-120 0 C/1.Onun) to afford pure KC1 g, 75%) as a colorless liquid: !H NMR (CDCl 3 300 M4Hz, ppm) 7.20 J 8.4 Hz), 7.02 J3 8.4 Hz), 3.69 (s, 3H), 3. 48 2H).

4 -PhenvluraidophePnvlaPctic acid: 4 -Phenylureidophenylacetic acid was prepared using procedure C with 4 -amino-phenylacetic acid and 20 phenyl isocyanate: 1 H NMR CCDSOCD,, 300 MHz, ppm) 8.72- 8.64 (mn, 2H), 7.44 2H), 7.36 2H), 7.28 2H), 7.16 Cd, 2H), 6.96 1W), 3.52 2H) m/z 272.

4 -o-Tolvlureidolhenvlacetic acrid: 4 -o-Tolylureidophenylacetic acid was prepared using procedure C with 4-amino-phenylacetic acid and otolyl isocyanate: 'H NMR (CDSOCD 3 300 MHz,. ppm) 8.97 Cs, 1H) 7.88 Cs, 1H), 7.83 Cd, 1H) 7.38 Cd, 2H) 7.17- 60 7.09 (in, 4H), 6.92 1H), 3.48 2H), 2.23 3H); m/z 285.

4-( 2 -FluoroDhenvl)ureidohenvlacetic acid: 4-( 2 -Fluorophenyl)ureidophenylacetic acid was prepared using procedure C with 2-fluoroaniline and KC1: 'H NMR (CD 3

SOCD

3 300 MHz, ppm) 9.00 1H),8.51 2.4 Hz, 1H), 8.14 (dd, 8.3 Hz, 1.5 Hz, 1H), 7.37 8.5 Hz, 2H), 7.07-7.25 4H), 6.99 IX), 3.48 2H).

4- 2 -Hdroxvvhenvlureido)Dhe vacetic acid: 4 2 -Hydroxyphenylureido)phenylacetic acid was prepared using procedure C with 2 -hydroxyaniline and KC1: 'H NMR (CDISOCD 3 300 MHz, ppm) 9.90 1H),9.25 1H), 8.12 8.02 (bd, 1H), 7.37 2H), 7.13 2H), 6.70-6.97 3H), 3.48 2H).

N-Succjnimidl 4 2 3 -methvlpovridlureidp)henvlcetate Prepared in three steps as follows: Procedure C with 2 -amino-3-methylpyridine and KC1 to give methyl 4 2 3 -methylpyridyiureido)phenylacetate.

A solution of methyl methylpyridylureido)phenylacetate (1 eq.) in methanol was treated with 1 N NaOH (2 The reaction was stirred for 16 h, then acidified carefully with 1 N HCl to pH 7 then with acetic acid to pH 3. The product was filtered and washed with methanol then ether to give 4- 3 -methylpyridylureido)phenylacetic acid: 'H NMR (CD4SOCD, 300 MHz, ppm) 11.97 1H), 8.64 (brs, 1H), 8.31 1H), 7.69 1H), 7.62 8.4 Hz, 2H), 7.33 61 8.4 Hz, 2H), 7.09 (mn, 1H), 3.62 2Hi), 2.38 (s, 3H); in/z 286.

A solution of mnethylpyridylureido)phenylacetic acid (1

N-

hydroxysuccinimide (1.2 and EDC (1.2 eq.) in DMF was made basic (pH 10) with TEA. After stirring at RT for over 12 h, the reaction was poured into 60% sat. MaHCO 3 and the product filtered: 'H NNR (CDSOCD 3 300 MHz, ppm) 12.04 1H), 8.84 1H), 8. 31 1H), 7.72 (mn, 3H) 10 7.42 (mn, 2H), 7.10 m, 1H), 4.18 2H), 2.98 4H), 2.38 m/z 383.

N-Succinimidvl 4- 2 -ovridvlure ido) rhenvlpacetate: Prepared in three steps as follows: Procedure C with 2-arninopyridine and KCI to 15 give methyl 4 2 -pyridylureido)phenylacetate: -H NMR *4*.13 30 ~,pm 8.20 2H), 7.62-7.51 (m 3H), 7.33 2H), 7.01 2H), 6.89-6.85 (mn, 1H), 3.70 (s, .4 4 3H), 3.59 2H).

A solution of methyl 4-(2pyridylureido)phenylacetate (5.7 g, 20.0 innol) in 9 *methanol %'20 mL) was treated with 1 N NaOH (40 mL) .The reaction was stirred for 16 h, then acidified carefully with 1 N HCl to pH 7 then with acetic acid to pH 3. The product was filtered and washed with methanol then ether to give 4 2 -pyridyl)ureidophenylacetic acid (4.7 g, 87%) as a white powder: 1H NMR (CD.,SOCD 3 300 MHz, ppm) 10.62 (bs, 1H), 9.53 (bs, 1H), 8.39 1H), 7.82 (t, 111), 7.63-7.55 (mn, 1H), 7.33-7.27 2H), 7.14-7.08 (in, 1H), 3.62 3H).

A solution of 4 2 -pyridyliureidophenylacetic acid (1 N-hydroxysuccininide (1.2 eq.) and EDC (1.2 62 eq.) in DMF was made basic (pH 10) with TEA. After stirring at RT for over 12 h, the reaction was poured into 60% sat. NaHCO 3 and the product filtered: 'H NNR

(CD

3

SOCD

3 300 MHz, ppm) 10.08 1H), 9.57 lIH), 8.39 (mn, 1H), 7.86 (in, lH), 7.62 (mn, 3H), 7.38 2H), 7.12 (mn, 1H), 4.15 2H), 2.91 4H); in/z 369.

3 -Methoxvy4ohnvureidophenviacetic acid: Prepared in six steps front 3 -methoxy-4-nitrobenzoic acid :as follows: 10 A mixture of 3 -methoxy-4-nitrobenzoic acid (2.01 g, 10.2 nunol) and thionyl chloride (2.3 mL, 31.5 nunol) was stirred at 80-90 0 C for 1.5 h. The reaction was concentrated and the residue diluted with ether. The organic solution was washed with sat. aq. NaHCO, (2 X) 15 then sat. aq. NaCI, dried (MgSO,) and concentrated to afford 3 -iethoxy-4-nitrobenzoyl chloride (1.92 g, 87-z) as a white solid: !H NNR (CDCl3, 300 MHz, ppm) 7.95-7.70 (mn, :3H) 4 .0 6 3H).

A cold (0 OC) solution of TMSCHN 2 (2 M in hexane, 1.5 inL, 3.0 inmol) and triethylamine (420 pL, mmnd) was treated with a solution of 3 -methoxy-4- S. nitrobenzoyl chloride (0.52 g, 2.4 nunol) in acetonitrile inL). The reaction was stirred at 0 OC for 24 h and then concentrated. The residue was slurried with sat.

aq. NaHCO3 and the mixture extracted with ether (3X) The combined ether washes were washed with water, then sat.

aq. NaCl, dried (MgSO 4 and concentrated to afford diazo- 3 -miethoxy-4-nitroacetophenone (0.53 g, 100% as a yellow foam: 'H NMR (CDCl 3 300 MHz, ppm) 7.88 10 Hz, 63 11) 7.61 1H) 7.27 10 Hz, 1H) 5.97 lH) 4.02 3H).

A refluxing solution of &-diazo-3-methoxy-4nitroacetophenone (7.95 g, 35.9 mmol) in t-BuOH (100 mL) was treated with a filtered solution of silver benzoate (2.50 g, 10.9 nimol) in triethylamine (15 mL) dropwise over 1 h. After refluxing for 45 rnin,-decolorizing carbon was added and the hot mixture filtered through a pad of Celite. The filtrate was concentrated and the residue diluted with ethyl acetate. The organic solution a was washed with 5% aq. NaHCO 3 (2 X) H:O, 5% aq. citric acid, H 2 01 then sat. aq. NaCi, dried (MgS0 4 and concentrated to afford t-butyl 3-methoxy-4eq egnitrophenylacetate (8.92 g, 93%) as a brown oil: IH NM~R (CDCl 3 300 M4Hz, ppm) 7.83 8. 3 Hz, 1H) 7.03 1H) 6.93 8.3 Hz, 11H), 3.97 3H), 3.58 2H), 1.45 sees(s, 9H).

A mixture of t-butyl 3-methoxy-4nitrophenylacetate (0.144 g, 0.539 nimol) and 10% Pd on carbon (0.155 g) in ethyl acetate (8 mL) and methanol (2 mL) was stirred under H 2 (40-60 psi) for 2 h. The mixture :was filtered through Celite and the filtrate concentrated to afford t-butyl 4 -amino- 3 -methoxypheriylacetate (0.123 g, 96%) as a light yellow oil: 1H NNR (CDC1 3 300 MHz, ppm) 6.70 3H), 4.04 (bs, 2H), 3.84 3H), 3.42 (s, 2H), 1.43 9H).

Procedure C with t-butyl 4-amino-3methoxyphenylacetate and phenyl isocyanate gave t-butyl 3 -methoxy-4-phenylureidophenylacetate: IH NMR (CDCl 3 300 MHz, ppm) 8.00 (d,11 Hz, 1H) 7.65-6.94 Cm, 7H), 6.80 (d, Hz, 1H), 6.74 1H), 3.68 3H), 3.45 2H), 1.44 9H).

64 A solution of t-butyl 3-methoxy-4phenylureidophenylacetate 108 g, 0. 303 mmol) in trifluoroacetic acid (5.0 mL) was stirred for 30 min.

The reaction was concentrated and the residue coevaporated with methylene chloride (2X) then ether to afford 3 -methoxy-4-phenylureidophenylacetic acid (0.090 g, 99%) as a white foam: 'H NMR (CDSOCD 3 300 MHz, ppm) 9.28 Cs,1H), 8.18 1H), 8.02 7.5 Hz, 1H), 7.58- 7.15 (in, 5H) 6. 91 (bin, 2H) 6.77 7. 5 Hz, 1H) 3.85 3H), 3.49 2H).

PJ-Succlinid-L- 3-ehx--h-vlxeoeyaeae A solution of 3-methoxy-4phenylureidophenylacetic acid (1 eq. in DMF at 0 0 C was treated with EDC (1.1 The mixture was stirred at 0 CC for 1 to 2 h and then N-hydroxysuccinimide 1 eq. was added. After stirring at RT for more than 3 h, the reaction mixture was poured into 60 sat. NaHCO, and the N-succinimidyl 3 -methoxy- 4 phenylureidophenyl acetate filtered.

20 N-SUccinimidyl 2-ehx-3- .:tolylurei do) yridvilacetate: Prepared in six steps from 2 6 -dichloro-3-nitropyridine as follows: A slurry of 2 6 -dichloro-3-nitropyrijdine (92%, 9.9 g, 47 mmol) and K 2 C0 3 powder 5 g, 47 mmol) in methanol (100 mL) was stirred for a week at RT. The reaction was filtered and concentrated. The residue was partitioned in ethyl acetate and 60% sat. aq. NaHCO 3 The organic solution was washed with 60% sat. aq. NaHCO, (2X),

H

2 0, then sat. aq. NaCi, dried (MgSO 4 and concentrated to 65 afford 2-hoo6mtoy--irprdn and 2-chloro-6methoxy-3-nitropyridjne (8.9 g, 100%) as a light Yellow solid: -H NMR (CDCl 3 300 MHz, ppm) 8.31 8.3 Hiz, 1H) 8.28 8.9 Hz, lH), 7.10 8.3 Hz, 1H), 6.82 8.9 Hz, IH), 4.15 3H) 4.06 3H) A mixture of 2 -chloro- 6-methoxy 5ni tropyri din and 2 clr-6mtoy ntoyidn (8.9 g, 47 inmol), t-butyl methyl malonate (10 mL, 60 inmol), and NaH 3.1 g, 120 Inmol) in THF (250 mL) was stirred at RT for 24 10 h. The reaction was concentrated and the residue treated with trifluoroacetic acid (200 mL) for 2 h. The reaction was concentrated and the product separated by flash chromatography (silica gel, 95:5 hexane-ethyl acetate) to afford methyl 6-2mtoy3ntoprdlctt (3.3 g, 62%) as a yellow oil: 'H NMR (CDCl 3 300 MHz, ppm) 8.27 Cd, 8. 0 Hz, 1H) 7. 04 8. 0 Hz, 1H), 4.09 3H-) 3. 2H) 3.75 3H) A mixture of methyl 6 -(2-methoxy-3- .nitro) pyr-4dylacetate (0.047 g, 0.21 xmnol) and 10% Pd on 20 carbon (0.063 g) in ethyl acetate (2 mL) and ethanol (1 niL) was szirred under H, (40-50 psi) for 6 h. The mixture was filtered through Celite and the filtrate concentrated* :to afford methyl -m hx-3 mn) yiy c at (0.041 g, 100%) as a light yellow oil: TN NMR (CDC1 3 300 MHz, ppm) 6.82 7.6 Hz, iN), 6.65 7.6 Hz, 1H), 3.94 3H) 3.70 Cs, 3H) 3.65 Cs, 2H) Procedure C with methyl 6 (2-methoxy-3amino) pyridyl acetate and o-tolyl isocyanate to give methyl 6- 2 -methoxy-3-o-tolylureido) pyridylacetate: 1H NMR 300 MHz, ppm) 8.33 7.9 Hz, lH), 7.51 Cd, 7. 8 Hz, IH) 7..41 1H) 7. 17 (in, 2H) 7. 08 Cm, 2H) 66 6.77 7.9 Hz, 1H), 3.81 3H), 3.71 3H), 3.67 2H) 2.20 3H) A solution of methyl 6 -(2-methoxy-3-otolylureido)pyridylacetate (0.023 g, 0.070 mmol) in methanol (1.0 mL) was treated with 2 M LiOH (90 pL, 0.18 mmol) The reaction was stirred for 18 h, diluted with

H

2 0 0 niL) and washed with ether (2X) .The aqueous solution was then acidified with 5% aq. citric acid. The product was filtered and washed with H 2 0 then ether to give 6-(-ehx-3 -oyued)prdlc i acid (0.014 g, 64%) as a white solid: IH NMR (CD_,OD, 300 MHz, ppm) 8.50-8.25 (in, 3H1), 7.60 (bd, 111), 7.28-7.00 (mn, 3H1), 4.01 3H), 3.69 2H1), 2.30 3H); MS, m/z 316.

A solution of 6 -(2-methoxy--3-otolylureido)pyridylacetic acid (1.61 g, 5.10 mmol) in DMF at C 0 C was treated with EDC 00 g, 5~.2 mmol). The mixture was stirred at 0 'C for 1 to 2 h and then Nhydroxysuccinmide (0.60 g, 5.2 inmol) was added. After stirring at RT for more than 3 h, the reaction mixture was noured into 60% sat. NaHCO, and the N-succinimidyl 6- 2 -rethoxy-3-o-tolylureido)pyridylacetate filtered.

H-L: 'OBn)

V-NHCH

3 H-LD(OBn)V-NHCH, was prepared by sequentially usi.-g procedure B with BOC-Val-Ogu and methylamine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, Procedure B with BOC-Leu-OSu, then procedure

D.

H-Lr 'O0n)V-OCH 3 H-LL2,OBn)V-C11 3 was prepared by sequentially using procedure B with BOC-Asp(OBn)-OSu ana 11-Val-OMe, 67 procedure D, procedure B with BOC-Leu-OSu, then procedure

D.

H-LD tOBri)V-O~n: H-LD(OBfl)V-OBn was prepared by sequentially using procedure B with BOG-ASP (OBn)-OSu and H-Val-OBn, procedure D, procedure B with BOC-Leu-OSu, then procedure

D.

H-LD (OBn) VP-OBn:.

H-LD(OBn)VP-OBn was prepared by sequentially 10 using procedure B with BOC-Val-Ogu and ?H-Pro-OBn, procedure D, procedure B with BOC-Asp (OBn) -OSu, procedure D, procedure B with BOC-Leu-OSu, then procedure

D.

1--LD (O~n)VP-OMe: H-LD(OBn)VP-OMe was prepared by sequentially using procedure A with BOC-Va-OH and H-Pro-O'e, procedure D, procedure B with BOC-Aso(OBn)-OSu, procedure D, procedure B with BOC-Leu-OSu, then procedure

D.

HLVOHwas prprdby sequentiallyusn procedure B with BOC-Val-OSu and H-Prc-OBn, procedure

D,

procedure B with BOC-Asp (OBn) -OSu, procedure D, procedure B with BOC-Leu-OSu, procedure F, then procedure

D.

H-MD (O~n)VP-O)Rn: H-MD(OBn)VP-OBn was prepared by sequentially using procedure B with BOC-Val-OSu and H-Pro-OBn, procedure D, procedure B with BOC-Asp OBn) -OSu, procedure D, procedure B with BOC-Met-OSu, then procedure

D.

68 H-LD VP-NH 2 H-LD(OBn)

VP-NH

2 was prepared by sequentially using procedure B with BOC-Val-OSu and H-Pro--NH 2 procedure D, procedure B with BOC-Asp(OBn)-osu, procedure D, procedure B with BOC-Leu-OSu, then procedure

D.

Resin (MBCI): Modified resin MBC1 (0.437 mruol/g) was synthesized according to the literature procedure (see: Richter, et al., Tetrahedron Lett. 35, p. 5547 (1994) ).MBC1 was treated with 50 TFA/CH.Cl- and triethylsilane for 2 h at RT then washed with CHCl: (2X), isopropanol (lX) and CHCl- (3X) before use.

MBC2 was prepared by sequentially using procedure G with BOC-Asp(OBn)-OH, procedure 1, procedure G with BOC-Leu-OH, procedure 1, then procedure G with 4phenylureidophenylacetic acid.

MBC3: MBC3 was prepared by sequentially using 20 procedure G with BOC-Val-OH, procedure 1, procedure

G

with BOC-Asp(OBn)-OH, procedure I, procedure G with BOC- Leu-OH, procedure 1, then procedure G with 4phenylureidophenylacetic acid.

MBC4 was prepared by sequentially using procedure G with BOC-Pro-OH, procedure I, procedure

G

with BOC-Val-OH, procedure I, procedure G with BOC- Asp(OBn)-OH, procedure I, procedure G with BOC-Leu-OH, 69 procedure I, then procedure G with 4phenylureidophenylacetic acid.

Example 2 Compound 77: Compound 77 was prepared by using procedure A with picolinic acid and H-LD(OBn)V-OBn then procedure

F.

Purification by HPLC gave the title compound: m/z 451.

.:Example 3 Compound 64: Compound 64 was prepared by using procedure A with hydrocinnamic acid and H-LD(OBn)V-OBn then procedure

F.

SPurification by HPLC gave the title compound: m/z 478.

Example 4 Compound 155: Compound 155 was prepared by using procedure B with S" chloro 4 -phenylbutyrate and H-LD(OBn)V-OBn then procedure F. Purification by HPLC gave the title compound: m/z 492.

Example Compound 157: Compound 157 was prepared by using procedure B with BOC- Asp(OBn)-OSu and isobutylamine, procedure D, procedure

B

with BOC-Leu-OSu, procedure D, procedure B with iQn-OSu, then procedure F. Purification by HPLC gave the title compound: m/z 457.

70 Compound 164: Compound 164 was prepared by using procedure B with Qn- OSu and H-LD(OBn)V-NHCH 3 then procedure F. Purification by HPLC gave the title compound: m/z 514.

Example 7 Compound 174: Compound 174 was prepared by using procedure B with BOC- Asp(OBn)-OSu and valinol, procedure D, procedure B with 10 BOC-Leu-OSu, procedure D, procedure B with Qn-OSu, then procedure F. Purification by HPLC gave the title compound: m/z 487.

Example 8 Compound 177: Compound 177 was prepared by using procedure B with BOC- AspLOBn)-OSu and H-Thr-OCH,, procedure D, procedure B with BOC-Leu-OSu, procedure D, procedure B with 4methoxybenzenesulfonyl chloride, then procedure

F.

Purification by HPLC gave the title compound: m/z 532.

Example 9 Compound 180: Compound 180 was prepared by using procedure B with BCC- Val-CSu and methylamine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-N- MeLeu-OSu, procedure D, procedure B with phenylacetyl chloride, then procedure F. Purification by HPLC gave the title compound: m/z 491.

71 Example Compound 189: Compound 189 was prepared by using procedure B with BOC- Val-OSu and methylamine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-Leu- OSu, procedure D, procedure B with phenylsulfonyl chloride, then procedure F. Purification by HPLC gave the title compound: m/z 499.

Example 11 10 Compound 345: Compound 345 was prepared by using procedure A with 4-otolylureidophenylacetic acid and H-LD(OBn)V-OBn then e° procedure F. Purification by HPLC gave the title compound: m/z 606.

Example 12 Compound 206: Compound 206 was prepared by using procedure A with 4-otolylureidophenylacetic acid and H-LD(OBn)VP-OBn then procedure F. Purification by HPLC gave the title 20 compound: m/z 709.

Example 13 Compound 144: Compoound 144 was prepared by using procedure A with 4- 2 -hydroxyphenylureido)phenylacetic acid and H-LD(OBn)VP- OBn then procedure F. Purification by HPLC gave the title compound: m/z 711, 24.6 min (gradient 8).

72 Compound 145: Compound 145 was prepared by using procedure A with 4phenylureidophenylacetic acid and H-LD(OBn)VP-OBn then procedure F. Purification by HPLC gave the title compound: m/z 695, 26.8 min (gradient 8).

Example Compound 146: Compound 146 was prepared by using procedure A with 4-(2- 10 hydroxyphenylureido)phenylacetic acid and H-MD(OBn)VP-OBn then procedure F. Purification by HPLC gave the title compound: m/z 729, 22.4 min (gradient 8).

Example 16 Compound 1: Compound 1 was prepared by using procedure A with 3methoxy- 4 -phenylureidophenylacetic acid and H-LD(OBn)VP- OBn then procedure F. Purification by HPLC gave the title compound: m/z 725, 28.5 min (gradient 8).

Example 17 Compound 2: Compound 2 was prepared by using procedure A with 3methoxy-4-phenylureidophenylacetic acid and H-MD(OBn)VP- OBn then procedure F. Purification by HPLC gave the title compound: m/z 743, 27.0 min (gradient 8).

73 Compound 32.5: Compound 315 was prepared by using procedure A with 4-otolylureidophenylacetic acid and H-MD(OBfl)VP-QBn then procedure F. Purification by HPLC gave the title compound: mlz 727.

Compound 346: *Compound 346 was prepared by using procedure B with N- Hydroxysuccinimidyl 4 (3-rnethylpyridylureido)phenylacetate and H-LDVP-QH. Purification by H-ILC gave Compound 346: m/z 710.

Exml Compound 316:.

Compound -316 was prepared by using procedure B with N- 01*0hydroxysuccinyimidyl 4- 2 -pyridylureido) phenylacetate anc-J 5:.H-LDVP--OH. Purification by HPLC gave the title compound: .*00 m/z 696.

00 Examnie 21 Compound 4: Compound 4 was prepared by using procedure B with Nhydroxysuccinimidyl 6- (2-methoxy-3-otolylureicoc) pyridyl acetate and H-LDVP-OH. Purification~ by HPLC gave the title compound: mhz '740, 30.7 min (gradient 8).

74 Example 22 Compound 147: compound 147 was prepared by using procedure B with Nhydroxysuccinimidyl 3-methoxy-4 -phenylureidophenylacetate and H-LD (OBn) VP-NH 2 then procedure F. Purification by HPLC gave the title compound: m/z 724, 26.-7 min (gradient 8).

Compound 148: 10 Compound 148 was prepared by using procedure A with 4-otojlylureidophenylacetic acid and H-LD(OBfl)VP-NH- then procedure F. Purification by HPLC gave the title compound: m/z 708, 26.0 min (gradient 8).

Examiple 24 Compound 317: Compound 31'7 was prepared by using procedure B with Nhydroxysuccinimidyl 6- (2-methoxy-3-otolyiurei do) pyridyi1acetate and H-LD(OBn)VP-NH. then procedure F. Purification by HPLC gave the title compound: m/z 739, 28.0 min 1gradient 8).

Example Compound 336: Compound 336 was prepared by using procedure A with 4- (2fluorophenyl)ureidophenylacetic acid and H-LD (OBn)VP-OBn then p~rocedure F. Purification by HPLC gave the title compound: m/z 713.

75 Compound 32: Compound 32 was prepared by using procedure B with iQn- OSu and H-LD(OBn)VP-OBn then procedure F. Purification by HPLC gave the title compound: m/z 598, 24.7 min (gradient 8).

Example 27 Compound 34: Compound 34 was prepared by using procedure B with 10 phenylacetyl chloride and H-LD(OBn VP-OBn then procedure F. Purification by HPLC gave the title compound: m/z 561, 23.7 min (gradient 8).

Example 28 Compound 39: 15 Compound 39 was prepared by using procedure A with hydroxyphenyl)propionic acid and H-LD(OBn)VP-OMe then procedure F. Purification by HPLC gave Compound 39: m/z 591, 21.5 min (gradient 8).

*o

S

Example 29 20 Compound 42: Crude compound 42 was prepared by sequentially using procedure A with BOC-Val-OH and H-homoPro-OBn, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-Leu-OSu, procedure D, procedure B with phenyl acetyl chloride then procedure

F.

Purification by HPLC gave the title compound: m/z 575, 26.4 min (gradient 8).

76 Compound 52: Compound 52 was prepared by sequentially using procedure A with BOC-norVal-OH and methylamine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-Leu-OSu, procedure D, procedure B with Qn-OSu then procedure F. Purification by HPLC gave the title compound: m/z 518, 30.2 min (gradient 8).

Example 31 10 Compound 46: Compound 46 was prepared by sequentially using procedure A with BOC-Val-OH and methylamine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure A with BOC-N-MeLeu-OH, procedure D, procedure A with 3-(4- 15 hydroxyphenyl)propionic acid then procedure F.

Purification by HPLC gave the title compound: m/z 521, 18.7 min (gradient 8).

Example 32 Compound 61: 20 Compound 61 was prepared by sequentially using procedure B with BOC-Thr-OSu and morpholine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-Leu-OSu, procedure D, procedure B with Qn-OSu then procedure F. Purification by HPLC gave the title compound: m/z 572, 24.0 min (gradient 8).

77 Example 33 Compound 213: Compound 213 was prepared by using procedure H with MBC2 and benzyamine then procedure J: m/z 588.

Example 34 Compound 214: Compound 214 was prepared by using procedure H with MBC2 and morpholine then procedure J: m/z 568.

Example 1 0 Compound 215: :Compound 215 was prepared by using procedure H with MBC2 and isopropylamine then procedure J: m/z 540.

Example 36 Compound 216: 15 Compound 216 was prepared by using procedure H with MBC2 and cyclohexylamine then procedure J: m/z 580.

SExample 37 Compound 217: Compound 21- was prepared by using procedure H with MBC: and isobutylamine then procedure J: m/z 554.

Example 38 Compound 218: Compound 218 was prepared by using procedure H with MBC2 and piperdine then procedure J: m/z 566.

78 Compound 318: Compound 318 was prepared by using procedure H with MBC3 and morpholine then procedure J: m/z 667.

Example Compound 319: Compound 319 was prepared by using procedure H with MBC3 and isopropylamine then procedure J: m/z 640.

Example 41 10 Compound 320: Compound 320 was prepared by using procedure H with MBC3 and cyclohexylamine then procedure J: m/z 679.

Example 42 Compound 321: 15 Compound 321 was prepared by using procedure H with MBC3 and benzylamine then procedure J: m/z 687.

Example 43 Compound 322: Compound 322 was prepared by using procedure H with MBC3 and piperidine then procedure J: m/z 665.

Example 44 Compound 323: Compound 323 was prepared by using procedure H with MBC3 and isobutylamine then procedure J: m/z 653.

79 Compound 324: Compound 324 was prepared by using procedure H with MBC4 and cyclohexylamine then procedure J: m/z 777.

Example 46 Compound 325: Compound 325 was prepared by using procedure H with MBC4 and piperdine then procedure J: m/z 763.

Example 47 1 0 Compound 326: Compound 326 was prepared by using procedure H with MBC4 and benzylamine then procedure J: m/z 785.

Example 48 Compound 327: 15 Compound 327 was prepared by using procedure H with MBC4 and isopropylamine then procedure J: m/z 736.

Example 49 Compound 328: Compound 328 was prepared by using procedure H with MBC4 and isobutylamine then procedure J: m/z 750.

Example Compound 363 A. A mixture of o-tolylureidophenylacetic acid (3.53 g, 12.4 mmol), H-Leu-OtBu-HCl (2.78 g, 12.4 mmol), TBTU (3.98 g, 12.4,immol), and iPr 2 NEt (4.32 mL, 24.8 mmmol) in DMF (25 mL) was stirred overnight at RT. The product was precipitated by addition of H 2 0 (10 mL). The solids were collected by filtration on a medium frit, washing with 2:1 DMF/H 2 0 (35 mL), H 2 0 (25 mL), and Et 2 O (2 x 25 mL), and dried on the filter (4.18 g, 74%) All of this product was suspended in CH 2 C1 2 (16 mL) and treated with TFA (16 mL) and stirred at RT 2 hr. The reaction was concentrated to a syrup which was evaporated from CH 2 Cl..

(2 x 20 mL). The residue was triturated with Et 2 O at RT for 2 hr. The solids were collected by filtration on a medium frit, washing with EtO (50 mL), and dried on the filter (3.40 g, MS (FAB) 398.

B. A mixture of DCC (0.206 g, 1.0 inmol) and HOBT (0.135 g, 1.0 mmiol) in EtOAc (6 mL) was stirred at RT 20 min until homogeneous. Fmoc-Asp-OtBu (0.411 g, 1.0 xnmol), piperonylamine (0.12 mL, 1.0 iniol), and Nmethylmorpholine (0.22 mU, 2.0 iniol) were added. After stirring overnight, the reaction was filtered to remove solids and the cake washed with fresh EtOAc (10 mU) The filtrate was washed with H 2 0 5% citric acid 20 NaHCO 3 (1x) and brine and dried (MgSO 4 Flash column chromatography on SiO 2 eluting with 100% CHClI to 2% MeOH/CHCl 3 provided 0.54 g (100%) of pure product as a solid: mp =128-130*C; TLC(2% MeOH/CHCl 3 R,=0.10; MS (FAB) 545; 'H NMR (CDC13, 300 MHz, ppm) 7.75-7.72 (mn, 2H), 7.59-7.56 (mn, 2H), 7.40-7.34 mn, 7.30-7.25 (in, 2H), 6.71-6.66 m i, 3H), 6.13-6.10 (mn, 2H), 5.84 2H), 4.46 (mn, 1H), 4.38-4.16 (mn, 5H), 2.86 (dd, IN, J=4.7, 15.6 Hz), 2.72 (dd, 1H, J=4.16, 15.6 Hz), 1.45 9H).

C. The product from Example 5OB (0.2 g, 0.46 mniol), piperidine (0.45 mU, 4.6 inmol), and CH 2 C1 2 (0.45 mU) were 81 stirred at RT for 90 min. The reaction was evaporated to a solid residue. Flash column chromatography on Si0 2 using a MeOH/EtOAc gradient provided product (0.138 g, 93%) as a colorless oil: MS (FAB) 323; MeOH/EtOAc) Rf=0.15; 'H NMR (CDCl 3 300 MHz, ppm) 7.63 (br s, 1H), 6.75-6.68 (in, 3H), 5.90 2H), 4.34 (dd, 1H, J-5.7, 14.7 Hz), 4.28 (dd, 1H, J=5.7, 14.7 Hz), 3.65 (dd, 1H, J=3.4, 9.3 Hz), 2.62 (dd, 1H, J=3-4, 15.7 Hz), 2.38 (dd, 1H, J=9.3, 15.7 Hz) 1. 74 2H) 1. 42 9H-).

10 D The product from Example 50C (2.55 g, 7.91 mmol) and Eschenimoser's salt (1.61 g, 8.70 minol) were refluxed in .MeCN (80 mL) under an inert atmosphere for 42 hr. The reaction was cooled to RT and evaporated to dryness. The residue was diluted with 5% NaHC0 3 and extracted with 15 EtOAc The combined organic extracts were washed with 5% NaHCO 3 (Ix) H 2 0 (lx) and brine (lx) and dried (MgSo 4 The crude product was dissolved in Et 2 O (250 inL) andpasedthrough a short pad of SiO 2 eluting with Et,O followed by EtOAc. The slightly impure product thus 20 obtained was further purified by trituration with ice .cold Et 2 O (30 mL) and collected by filtration to give a white solid (0.904 g, mp=121-123 0 C; TLC MeOH/CHCl 3 R,=0.59; 1H NMR (CDC1 3 300 MHz, ppm) 6.75-6.66 (in, 3H), 5.92 28), 4.66 (A of AB, 1H, J=14.7 Hz), 4.23 (B of AB, lH, J=14.7 Hz), 4.15 (ABq, 2H, J=11.9 Hz), 3.68 (dd, dd, 1H, J=5.2, 10.9 Hz), 2.72 (dd, 1H, J=5.2, 17.3 Hz), 2.41 (dd, 1H, J=10.9, 17.3 Hz), 1.45 9H); C,H,N for C:-H 22

N

2 0 5 theory- C:61.07, H:6.63, N:8.38, found- C:60.80, H:6.59, N:8.22.

82 E. The product from Example 50D (0.50 g, 1.5 mmoJ4, the product from Example 50A (0.596 g, 1.5 mmcl), and EDC (0.314 g, 1.64 mmol) were stirred in NMP (3 mL) at RT for 48 hr. The reaction was poured into EtOAc (60 luL), washed with IIO (8 x 6 mL) brine (Ix) and dried (MgSO 4 Flash column chromatography on SiO 2 eluting with 100% CHCl 1 to 30% EtOAc/CHC1 3 provided product 94 g, 88%) as a pale yellow oil: MS (FAB) 714; TLC (10% MeOH/CHCI 3 R,=0.40; IH NMR (CDCl 3 300 MHz, ppm) consistent with 10 structure and indicative of diastereorners.

F. The product from Example 50E (0.94 g, 1.32 mmcl) was *stirred in TFA (10 mL) at RT for 3 hr. The reaction was concentrated to dryness and the residue evaporated from

CH,

2 C1- (3 x 10 ml). The crude product was triturated with Et 2 O at RT, collected by filtration and dried on the filter (0.733g, 84%) MS (FAB) 658 680 (M+Na) TLC HOAc/EtOAc) R=O.lS; NMR (d'-DMSO, 300 MHz, ppm) consistent with structure and indicative of diastereomers.

Compound 364: A. Il- the same manner as described in Example 5OB, Fmoc- Asp-OtBu (8.23 g, 20.0 mmcl) was reacted with H-Gly- OBn*H0.l (4.03 g, 20.0 mmol) Flash column chromatography on Sic:'D, using an EtOAc/hexane gradient provided product (9.8 g, 88%) as a waxy solid: MS (FAB) 559; TLC MeOH/CHCl 1 Rf=0.71; 'H1 NMR (CDC1 3 300 Mliz, ppm) 7.73 2H, J=7.5 Hz) 7.59 2i.f J=*7.4 7.40-7.26 (mn, 9H) 6.44 (br s, 1H) 83 6.09 1H, J=8.3 Hz), 5.13 2H), 4.52-4.49 (in, 1H), 4.41-4.29 (in, 2H), 4.21 1H, J=7.1 Hz), 4.04 2H, J=5.2 Hz), 2.95 (dd, 1H, J=4.6, 15.7 Hz), 2.79 (dd, 1H, J=4.3, 15.7 Hz), 1.46 9H).

B. The product of Example 51A (9.8 g, 17.54 inmol) was deprotected in the manner described in Example Filtration through a pad of SiO. with 100% EtOAc followed by 5% MeOH/CHCl 3 provided product (41.24 g, 72%) as an oil: MS (FAB) 337; TLC MeOH/EtOAc) R.=0.15; 41 NMR (CDC1 3 300 MHz, ppm) 8.00 1H, J=5.4 Hz;, 7.30-7.21 (in, 5.07 2H), 3.98 (AB of ABX, 2H, J=5.4, 18.1 Hz), 3.60 (dd, 1H, J=3.4, 9.2 Hz) 2.60 (dd, IH, J=3.4, 5.4 Hz) 2.38 (dd, 1H, J=9.2, 15.4 Hz), 1.79 (br s, 2H), 1.36 (s, 9H).

C. The product of Example 51B (4.24 g, 12.60 mmcl) was *:cyclized in the manner described in Example SOD. Flash column chromatography using an Et0Ac/CHC1., gradient provided product as a syrup (1.4 g, MS (FAB) 349; TLC (1:1 EtOAc/CHCl 3 Rf=0.53; NMR (CDCI,, 300 MHz, ppm) 20 7.35-7.25 (in, 5H) 5. 11 2H) 4.21 (A of AB, 1H, J=17.5 Hz), 3.95 (B of AB, IH, J=17.5 Hz), 3.71 (dd, 1Hi, J=5.1, 11.2 Hz), 2.68 (dd, 1H, J=5.1, 17.2 Hz), 2.36 (dd, 1H, J=11.2, 17.2 Hz), 1.43 9H).

D. The product of Example 5iC (1.40 g, 4.02 mmcl) was coupled with the product of Example 50A using the procedure of Example 50E. Flash column chromatography using a CHCl3/EtOAc gradient provided product as a brittle, pale yellow foam (2.21 g, MS (FAB) 728; TLC (1:1 CHCl 3 /EtOAc) Rf=0.28; 'H NMR (CDC1 3 300 MHz, ppm) 84 consistent with structure and indicative of diastereomers.

E. The product of Example 51D (0.15 g, 0.21 mmol) was deprotected and purified as described in Example The product was obtained as an off-white solid (0.127 g, MS (FAB) 672 695 TLC (9:1:0.1 CHC1,/MeOH/AcOH) Rf=0.54; 'H NMR (d[-DMSO, 300 MHz, ppm) consistent with structure and indicative of diastereomers.

xml Compound 365 A. The product from Example 51E (0.-100 g, 0. 15 inmol; 4methoxybenzylamine (20 gL, 0.15 mmol), and TBTU (0.0482 g, 0.15 mmol) in NMP (0.3 mL) were treated with iPr.NEt (78 P1iL, 0.45 mxnol) After stirring overnight at R1T, the *reaction was di luted with EtOAc (10 mL) washed wit- H. C x 2 mL) 5 citric acid (2 x 2 mL) NaHCO, (2 x 2 mL) and brine (1 x 2 mL) and dried (MgSO 4 Filtration .through a short pad of SiC:, eluting with MeCH/CHCOI.

followed by 4'7 MeOH/C HC1 4 provided product as a foam~ (0.087 g, MS (FAB) 792; TLC (9:1 C-HClJ.MeOH) R=0. 41; :H NMR (CDCI>, 300 MHz, ppm, consist-ent wit.h structure and indicative of diastereomers.

B. A suspension of the product of Example 52A (0.087 g, 0.11 numol) and Degussa type E101 NE/W 10 Pd/c (0.017 g) in MeCH (10 mL) was hydrogenated under 25 psi H. for 18 hr. The reaction was filtered through Celite, rinsing with MeOl. The filtrate was evaporated to dryness. The 85 residue was triturated with Et20 and the resultant beige solids collected by filtration (36.1 mg, MS (FAB) 701 723 'H NMR (d 6 -DMSO, 300 MHz, ppm) consistent with structure and indicative of diastereomers.

Example 53 Inhibition of VLA4-Dependent Adhesion to BSA-Csl This assay was used to assess the potency of VLA4-directed inhibitory compounds of this invention.

10 1. Coniugation of CS1 to BSA *o We dissolved BSA-SMCC (Pierce Chemical, Rockford, IL; Catalog 77115) in H20 at a concentration of 10 mg/mL. [SEQ ID NO:4]: Cys-Tyr-Asp-Glu-Leu-Pro-Gln- Leu-Val-Thr-Leu-Pro-His-Pro-Asn-Leu-His-Gly-Pro-Glu-Ile- Leu-Asp-Val-Pro-Ser-Thr ("Cys-Tyr-CS1 peptide"), which we synthesized by conventional solid phase chemistry and purified by HPLC, was dissolved in 10mM HEPES pH 5, 50 mM Na C l and 0.1 mM EDTA also at a concentration of 10 mg/mL.

We then mixed 500 UL of BSA-SMCC, 250 pL of Cys-Tyr-CSl peptide and 75 pL of 1 mM HEPES pH 7.5 and allowed the conjugation reaction to proceed for 30 minutes. We stopped the reaction by adding 1 UL of betamercaptoethanol. Samples were analyzed for cross-linking by SDS-PAGE. This reaction produced multiple molecules of the Cys-Tyr-CS1 peptide conjugate to each BSA molecule.

2. Preparaion of Plates for Adhesion Assay We coated the wells of a Linbro titertek polystyrene 96-well flat bottom plate (Flow Laboratories, Maclean, VA; catalog #76-231-05) with 100 pL of the 86 above-described BSA-CS1 solution diluted to 1 Pg/mL in 0.05 M NaHCO 3 (15mM NaHCO 3 35mM Na 2 C0 3 pH 9.2. Some wells were not coated with CS1 in order to assess nonspecific cell binding (NSB). The plate was then incubated overnight at 4"C.

Following this incubation, the contents of the wells were removed by inverting and blotting the plate.

All of the wells were then blocked with 100 pL of li BSA in PBS, 0.02% NaN 3 for a minimum of one hour at room 10 temperature.

3. Preparation of Fluorescently Labelled Ramos Cells Ramos cells are grown, maintained and labelled in RPMI 1640 culture medium containing 1% BSA. Just oo prior to running the assay, we added 2',7'-bis-(2- S 15 carboxyethyl)-5 (and carboxyfluorescein acetoxymethyl ester ("BCECF-AM"; Molecular Probes Inc., Eugene, Oregon; catalog #B-1150) to a final concentration of 2uM to a culture of Ramos cells (4 x 10" cells/mL). We incubated the cells for 20 minutes at 37 0

C.

S" 20 Following labelling, the cells were washed twice in assay buffer (24 mM TRIS, 137 mM NaC1, 2.7 mM KC1, pH 7.4, containing 0.1% BSA and 2mM glucose) to remove any cations originating from the culture medium.

The cells were then resuspended in assay buffer to 4 x 10 cells/mL and 2mM MnCl 2 was added to upregulate VLA4 on the surface of the cells.

4. Runnina the Assay Immediately prior to running the assay, we removed the BSA blocking solution from the 96-well plates and washed the wells with 100 uL of assay buffer. We then added to each well 25 uL of test cell adhesion 87 inhibitory compound at 2x the final concentration and pL of the labelled Ramos cells. Final concentrations were selected across a range of anticipated usually between 0.01 nM 10 pM. Each concentration of compound was tested in triplicate. The compound and cells are allowed to incubate for 30 minutes at room temperature.

We then emptied the contents of the plate and washed the wells 4 times with assay buffer. Using a 10 light microscope, we examined the NSB wells. If more than a few cells are bound to those wells, we washed the plate once more to remove the excess non-specifically bound cells.

Binding of the Ramos cells to the CS1 peptide- 15 coated wells was measured by adding 100 pL of assay buffer to each well and quantitating fluorescence in a Millipore Cytofluor 2300 System plate reader set at 485 nm excitation and 530 nm emission. Binding was expressed as an IC50 the concentration of inhibitor at which of control binding occurs. Percent binding is calculated by the formula: [(FTB Fus) x 100 binding, where FTB is total fluorescence bound to CSl-containing wells without added inhibitor; F. is fluorescence bound in wells lacking CS1; and F, is fluorescence bound in wells containing an inhibitor of this invention.

Other compounds according to this invention were similarly assayed. The IC50 range for each of these compounds is indicated in the table below: 88 CpIC,,-- C mp 1c Cmpd J2d

#~I

I A 30 C 59 B 88 C 2 A 31 C 60 C 89 C 3 A 32 C 61 a 90 c 4 A 33 C 62 C 91 C C 34 B 63 IC 92 C 6 C 35 B 64 C -93 C 7 C 36 C 65 C 94 C 8 C 37 C 66 C 95 C 9 -C 38 C 67 C 96 C C 39 C 68 C 97 C 11 C 40 C 69 C 98 C 12 C 41 B 70 C 99 C 13 B 42 8 71 C 100 C 14 C 43 a 72 C 101o C C 44 B 73 C 102 C 16 C 45 C 74 C 103 C 17 C 46 C 75 C 104 C 18 B 47 C 76 C 105 C 19 C 48 C 77 C 106 C 20 C 49 C 78 C 107 C 21 C 50 C 79 C 108 C 22 C 51 C 80 C 109 C 23 C 52 C 81 C 110 C 24 -C 53 C 82 C ill C C 54 C 83 C 112 C 26 C 55 C 84 C 113 C 27 C 56 B 85 C 114 C 28 C 57 B 86 C 115 C 89 i Ii I Cmpd 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 Ic I Cmpd# IC. I

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

c

C

C

C

C

C

C

c

C

C

.9 m a 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160, 161 162 163 164 165 166 167 168 169

A

A

A

C

C

C

C

C

C

C

B

B

C

C

C

C

C

C

B

C

C

B

C

175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 Cmpd

IC

B

C

C

C

C

B

C

C

C

C

C

C

C

C

C

nd

C

C

B

C

B

Bn

B

B

A

Cmpd 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231

B

A

B

A

B

B

B

B

B

B

B

B

B

A

A

A

A

A

A

A

A

A

A

A

A

A

141 1 C 1 17m t 141 c 1 142 143 144 145

C

C

A

A

171 172 173 174 0

C

C

C

p 200 201 202 ini I I -1 90 Cmpd# IC Cmpd IC, Cm Cmpd I 233 A 262 A 291 A 320 B 234 A 263 A 292 A 321 B 235 A 264 A 293 A 322 A 236 A 265 A 294 A 323 B 237 A 266 A 295 A 324 B 238 A 267 A 296 A 325 B 239 A 268 A 297 A 326 A 240 A 269 A 298 A 327 A 241 A 270 A 299 A 328 A 242 A 271 A 300 A 329 A 243 A 272 A 301 A 330 C 244 A 273 A 302 A 331 C 245 A 274 A 303 A 332 C 246 A 275 A 304 A 333 C 247 A 276 A 305 A 334 C 248 A 277 A 306 A 335 C 249 A 278 A 307 A 336 A 250 A 279 A 308 A 337 A 251 A 280 A 309 A 338 A 252 A 281 A 310 A 339 C 253 A 282 A 311 A 340 C 254 A 283 A 312 A 341 C 255 A 284 A 313 A 342 C 256 A 285 A 314 A 343 C 257 A 286 A 315 A 344 C 258 A 287 A 316 A 345 A 259 A 288 A 317 A 346 A 260 A 289 A 318 A 347 A 9A1 1 A Q A siQ R 4R a..

C. C C C 91 1 1 3 i I CmPd 49 C 366 C 383 nd 401 350 C 367 C 384 nd 402 351 C 368 C 385 nd 403 352 C 369 C 386 -C 404 353 C 370 C 387 C 405 354 C 371 B 388 nd 406 355 nd 372 C 389 nd 407 356 nd 373 C 390 C 08

IC

nd nd

C

C

C

C

C

C

B

b 357 358 nd 374

C

391

C

409 1 A 37 n t_41 14 1 1 A 360 C 377 ndd 394 C j412

B

361 C 1378 End 395 C 1413 362 1c 379 nd 396 n Ed 41 B

I

380 'Q j. II 415 14A I

B

I I ZZ nuI f' 38 nd 4nn I nlu _r A1 I Table abbreviations: A -<5Onm; B 50nm-l0pm: C ->1O0im nd -not determined. All compounds tested in this table demonstrated an 1 mM.

zu Example Direct Binding Of VLA4-Presentino Cer To rLM TCN We next examined the of the compounds of this invention to inhibit VCAM/VLA t4 binding, utiliZ~r-c a VCAM-.IgG-alkaline phosphatase corrugate. To carry ou: this assay, we used the Mil~lipore Muitiscreen Assay System (Millipore Corp., Bedford, MA) to wash the cells efficiently.

1. Prenaration of AM-JG-AP corigates The construction of VCAM 2D-IgG expression vectors, transfection of CH0 cells with those constructs 92 and purification of the resulting expression product is described in PCT publication WO 90/13300, the disclosure of which is herein incorporated by reference.

1.2 ml of purified VCAM 2D-IgG (5 mg/ml in mM HEPES, pH 7.5) was reacted with 44 pl of Traut's reagent (2-iminothiolane, 20 mg/ml in water; Pierce Chemical, Rockford, IL.) at room temperature for minutes. The sameple was desalted on a 15 ml Sephadex Gcolumn equilibrated with 100 mM NaC1, 10 mM MES, pH 5.0. One ml fractions were collected and absorbance at 280 nm was determined. The two peak fractions were pooled.

One ml of calf intestinal alkaline phosphatase (19 mg/ml; Pierce Chemical, Rockford, IL) was reacted 15 with 100 pl of sulfo-SMCC (30 mg/ml in water) and 100 il 1 M HEPES, pH 7.5 for 35 minutes at room temperature.

The reaction mix was desalted on a 12 ml Sephadex column equilibrated with 150 mM NaCI, 10 mM HEPES, pH 6.0. One ml fractions were collected and absorbance at 280 nm was determined. The two peak fractions were pooled and stored on ice.

The alkaline phosphatase-SMCC and VCAM 2D-IgGiminothilane adducts were cross-linked at a molar ratio of 2:lin Tris-HCL, pH 7.5 by incubation at room temperature for 30 minutes. Extent of cross-linking was determined by SDS-PAGE. The cross-linked products were stabilized by the addition of 2 mM MgCl: and 0.25 mM ZnCl and stored at 4°C.

2. Binding Assay We first blocked a 96-well filtration plate for by adding 275 pL of PBS containing 0.1% Tween 20 and 2% BSA ("blocking buffer") to each well and incubating for 1 93 hour at room temperature. The plate was then placed onto a vacuum manifold and the blocking buffer was drained through the bottom of the filtration wells into a waste collection tray. Then we washed the wells three times with 200-250 uL of Tris-buffered saline, containing 0.1% BSA, 2 mM glucose and 1 mM HEPES, pH 7.5 ("assay buffer") to wash out any remaining blocking buffer. We then drained the plates and blotted them on paper towels to remove buffer on the underside of the plate.

10 We then prepared a stock solution of VCAM-IgG- AP (4 pg/mL in assay buffer) and filtered it through a 0.2 p low protein binding syringe filter (Gelman Sciences, Ann Arbor, MI 4454). This solution was then diluted 1:10 in assay buffer and 25 uL was added to every well of the washed plate.

e diluted the cell adhesion inhibitor being tested to 2x final concentration in assay buffer and S*added 25 pL of each dilution to triplicate wells in the plate. Final concentrations used ranged from 0.01 nM 10 UM. Control wells for total binding and non-specific binding received 25 pL of assay buffer, instead of inhibitor. Total binding wells contained cells and VCAM- IgG-AP in assay buffer. Non-specific binding wells contained only VCAM-IgG-AP in assay buffer.

Jurkat cells were washed once in assay buffer to remove growth medium and resuspended at 8 x 10 6 /mL in assay buffer containing 2 mM MnCl 2 We added 50 ul of Jurkat cells to every well, except the non-specific binding wells, which received 50 pL of assay buffer to maintain a final assay volume of 100 pL per well. We gently mixed the contents of the wells by tapping the 94 sides of the plate. The plate was then allowed to incubate undisturbed for 60 minutes at room temperature.

At the end of the 60 minute incubation, we placed the plate on the vacuum manifold to drain the wells. We carefully added 100 pL of assay buffer containing ImM MnCl 2 (wash buffer) to each well so as not to disturb the cells on the bottom. The wash buffer was removed by vacuum and the plate was washed again with 150 pL of wash buffer. After draining the wash buffer again, 10 the underside of the plate was blotted on paper towels.

Next, we prepared a 10 mg/mL solution of 4nitrophenylphosphate in 0.1 M glycine, 1 mM ZnC12, pH 10.5 (substrate buffer) and added 100 pL immediately added to each well. The plate was incubated for minutes at room temperature to allow the colorimetric reaction to proceed. We stopped the reaction by adding 100 1l of 3 N NaOH to each well.

The contents of the 96-well filtration plate was then transferred directly into a 96-well flat bottom plate using the vacuum manifold. The plate was read at a wavelength of 405 nm to determine the amount of VCAM conjugate bound to the cells. Percent binding is calculated by the formula: [(AT ANs) (AI x 100 binding, where is the absorbance at 405 nm of CSl-containing wells without added inhibitor; is the absorbance at 405 n. in wells lacking CS1; and A, is absorbance at 405 nm in wells containing an inhibitor of this invention We assayed other compounds of this invention in the same assay. The IC50 values are comparable to those derived from the CS1 binding assay described in the previcus example, although certain compounds demonstrated 95 up to 10-fold greater binding in this assay than in the previous assay.

Example Inhibition Of Mouse Contact Hypersensitivity We anesthetized 20-g female Balb/c mice (Jackson Laboratories, Bar Harbor, ME) with sodium pentobarbital (90 mg/kg, A 3 cm 2 patch of abdominal skin was then exposed by closely shaving the fur. The skin was then scrubbed with 70% ethanol, 10 followed by application of 25 pL of 0.5% DNFB in 4:1 v/v acetone:olive oil onto the bare abdominal skin. We then lightly scratched the skin with the applying pipet tip to Sencourage mild inflammation. Twenty four hours after the initial sensitization we again sensitized the mouse with 25 UL of 0.5% DNFB at same abdominal skin location, again followed by light scratching with the pipet tip. The second sensitization was performed while restraining the unanesthetized mouse.

n Day 5 (120 hours after the initial sensitization), we anesthetized the mice with 90:10 mg/kg ketamine:xylazine, i.p. and applied a sub-irritant dose of 10 pL of 0.2% DNFB to the dorsal surface of the left ear. The right ear received a similar application of the 4:1 v/v acetone:olive oil vehicle.

Four hours after challenging the immune response, we administered various concentrations of the inhibitors of this invention to the mice in 100 pL sodium phosphate buffer, pH 8.8, and 3% v/v DMSO by subcutaneous injection. Less soluble inhibitors occasionally required up to 30% DMSO addition the highest concentrations tested. Groups of 8 mice were used for each treatment tested. Positive (PS2 anti-mouse VLA-4 96 antibody, 8 mg/kg, and negative control (phosphate-buffered physiological saline, PBS, 100 uL DMSO in PBS, 100 uL groups were routinely tested for comparison as part of the assay of test compounds.

Twenty four hours after challenge mice were again anesthetized with ketamine:xylazine and the ear thickness of both ears measured with an engineer's micrometer to an accuracy of 10 4 inches. The ear -i 10 swelling response for each mouse was the difference between its control- and DNFB-challenged ear thickness Typical uninhibited ear swelling responses were 65-75 x 10 in. Inhibition of the ear swelling response was judged by comparison of treated groups with their negative control group. Percent inhibition was calculated as: (mean negative control group (mean test group ear ear swellin) swe .n x :00 20 I mean negative control group ear swelling

L

Statistical significance of the difference among treatment groups was evaluated using one-way analysis of variance followed by computation of the Tukey-Kramer Honestly Significant Difference (JMP, SAS Institute using p<0.05.

The inhibitors of this invention cause a statistically significant reduction in the ear swelling response of DNFB-treated mice as compared to uninhibited control animals.

Examrle i56 Inhibition Of Ascaris Antigen-Induced Late 97 Phase Airway Sensitivity In Alleraic Shee Sheep which had previously been shown to develop both early and late bronchial responses to Ascaris suum antigen were used in this study. The protocol used for the experiment was that described in W.

M. Abraham et al., J. Clin. Invest., 93, pp. 776-87 (1994), except that the VLA-4 inhibitors of this invention were administered to the animals was dissolved in 3-4 ml of 50% aqueous ethanol and delivered by aerosol 10 spray.

The results showed that all of the VLA-4 inhibitors of this invention inhibited the airway responses associated with administration of Ascaris suum antigen.

While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other compounds and methods which utilize the compounds of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims .:appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.

98 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Biogen, Inc. (except US) (BI STREET: 14 Cambridge Center CITY: Cambridge STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 02142 TELEPHONE: 617-679-2200 TELEFAX: 617-679-2838 (A NAME: Ko-Chung Lin (US only) (E STREET: 253 Lincoln Street (C CITY: Lexington (r STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 02173 (A NAME: Steven P. Adams (US only) (E STREET: 12 Berkley Lane (C CITY: Andover STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 01810 NAME: Alfredc C. Castro :'JS only! *STREET: 31 Gienwood Avenue (C CITY: Woburn STATE: Massachusetts (E COUNTRY: United States of America POSTAL CODE (ZIP): 01801 S(A NAME: Craig N. Zimmerman 'US only) STREET: 134 Highland Avenue #6 ,C CITY: Somerville STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 02143 (A NAME: Julio Hernan Cuervo ,US only) STREET: 16 Elmer Street #303 (C CITY: Cambridge STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 02138 (A NAME: Wen-Cherng Lee (US only) (E STREET: 192 Spring Street (C CITY: Lexington (E STATE: Massachusetts (E COUNTRY: United States of America 99 POSTAL CODE (ZIP): 02173 NAME: Charles E. Hammond (US only) STREET: 4 Chester Avenue CITY: Burlington STATE: Massachusetts COUNTRY: United States of America POSTAL CODE (ZIP): 01803 NAME: Mary Beth Carter (US only) STREET: 106 Sycamore Street CITY: Belmont STATE: Massachusetts COUNTRY: United States of America POSTAL CODE (ZIP): 02178 NAME: Ronald G. Almquist ci JS nly STREET: 50 Solomon Pierce Roa:: CITY: Lexington STATE: Massachusetts COUNTRY: United States of America POSTAL CODE (ZIP): 02173 NAME: Carol Lee Ensinger (US only; STREET: 732 Princeton Blvd. Apt CITY: Lowell STATE: Massachusetts COUNTRY: United States cf America POSTAL CODE (ZIP): 01851 (ii) TITLE OF INVENTION: CELL ADHESIOX INHIBITORS iii) NUMBER OF SEQUENCES: 4 S(iv) COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/498,237 FILING DATE: 11-JUL-1995 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 100 (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Glu Ile Leu Asp Val Pro Ser Thr 1 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (i.ii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Glu Ile Leu Asp Val 1 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) Leu 1 SEQUENCE DESCRIPTION: SEQ ID NO: 3: Asp Val Pro Ser INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 101 (i)MOLECULE TYPE: peptide, (i)HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Cys Tyr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu 1 5 10 His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

Claims (27)

1. A cell adhesion inhibitory compound of formula Z-(yl)(y 2 3 (D) and pharmaceutically acceptable derivatives thereof; wherein: Z is selected from the group consisting of alkylsulfonyl; aralkylsulfonyl; arylsulfonyl; cycloalkylsulfonyl optionally fused with aryl; heterocyclylsulfonyl; heterocyclylalkylsulfonyl; alkenylsulfonyl optionally substituted with aryl; ailcynylsulfonyl optionally substituted with aryl; cycloalkenylsulfonyl; cycloallcylalkylsulfonyl; biarylsulfonyl; alkoxysulfonyl; aralkoxysulfonyl; alkylaminosulfonyl; aryloxysulfonyl; arylaninosulfonyl; N-arylurea-substituted alkylsulfonyl; cycloalkenyl-substituted sulfonyl; alkenoxysulfonyl optionally substituted with aryl; alkynoxysulfonyl optionally substituted with aryl; alkenyl or alkynyl-aminosulfonyl optionally substituted with aryl; acylainino-substituted alkylsulfonyl; carbamoyl-substituted alkylsulfonyl; heterocyclylaminosulfonyl; carboxyalkyl-substituted aralkylsulfonyl; oxocarbocyclyl-fused arylsulfonyl; and aryloxy-substituted heterocyclylalkylsulfonyl; Y' is Y' is each Y 3 is represented by the formula 2 )(A 3 each R' is independently selected from the group consisting of hydrogen, alkyl, and aralkyl; alkenyl; alkynyl; cycloalkyl; cycloalkenyl; cycloalkylalkyl; aryl; aminoalkyl; mono- or di-alkyl-substituted aniinoalkyl; mono- or di-aralkyl-substituted aminoalcyl; hydroxyalkyl; alkoxyalcyl; mercaptoalkyl; thioalkoxyalkyl A' is selected from the group consisting of am-ino acid side chains and corresponding protected derivatives; cycloalkyl; and alkyl. optionally substituted with 103 amnino, acylainino, amino-substituted acylamino, alkoxycarbonylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocaiibonyl, dialkylaminocarbonyl, (allyl)(aralkyl)aminocarbonyl, aralkylalninocarbonyl, diarallcylaminocarbonyl, hydroxyl, carboxyalkaminocarbonyl, hydroxylaminocarbonyl, mercapto, thioalkoxy or heterocycle; A 2 is selected from the group consisting of acidic functional groups and alkyl optionally substituted with an acidic functional group, protected acidic functional group or aryl; each A 3 is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; aryl; cycloallcyl; and alkyl optionally substituted with amino, acylamino, amino-substituted acylamino, aryl, cycloallcyl, carboxy, alkaxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, diallcylaminocarbonyi, alkcyl)(arallcyl)amiiiocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, hydroxyl, carboxyalkylaminocarbonyl, hydroxylaminocarbonyl, mercapto, thioalkoxy or heterocycle; or R' and any A are taken together with the atoms to which they are attached form a 3-to 6 -membered ring heterocycle; each R 2 is independently selected from the group consisting of hydrogen and alkyl; n is an integer from 0 to 8; and X is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; allcylaniino optionally substituted with hydroxy, amninocarbonyl, N- alkylaniinocarbonyl, carboxy or alkoxycarbonyl; dialkylamnino; cycloalkylanino; dicycloalkylamjino; cycloallcylalkylanino; (N-alkyl)(N-aryl)amino; aralkylamino optionally substituted with carboxy; diaralkylamino; arylamnino; heterocycle; and (mono- or bis-carboxylic acid)-substituted alkylamine; heterocyclylamino; heterocyclyl-substituted. alkylamino.

2. The cell adhesion inhibitory compound according to claim 1, wherein: Z is selected from the group consisting of alkylsulfonyl; arylsulfonyl; arailcylsulfonyl; heterocyclylsulfonyl; heterocyclylalkylsulfonyl; cycloallcylsulfonyl; and cycloalkylakulonyl; 104 each R' is independently selected from the group consisting of hydrogen, alkyl, and aralkyl; and X is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; alkylamino optionally substituted with hydroxy, aminocarbonyl, N- alkylaminocarbonyl, carboxy or alkoxycarbonyl; dialcylaxnino; cycloalkylamino; dicycloalkylarnino; cycloalkylalkylamino; (alkyl)(aryl)amino; aralkylamnino optionally substituted with carboxy; diaralkylamnino; arylamino; heterocycle; and (mono- or bis- carboxylic acid)-substituted alkylamine.

3. The cell adhesion inhibitory compound according to claim 1, wherein A' is selected from the group consisting of cycloalkyl; heterocyclic ring (when A' and R' are taken together); and alkyl optionally substituted with amino, acylamino, amino-substituted acylamino, aryl, carboxy, cycloalkyl, hydroxy, alkoxy, aralkyloxy, alkoxycarbonyl, arailkoxycarbonyl, aniinocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, ,(tukyl)(aralkyl)aminocarhonyl, aralkylamninocarbonyl, diaralkylaminocarbonyl, alkoxycarbonylanino, mercapto, thioalkoxy or heterocycle.

4. The cell adhesion inhibitory compound according to claim 3, wherein A' 'is selected from the group consisting of aminocarbonylethyl, benzyl, n-butyl, isobutyl, carboxyethyl, cyclohexyl, 1 -hydroxyethyl, hydroxymethyl, mercaptomethyl, 1- methyipropyl, methyltbioethyl, n-propyl, isopropyl, methoxycarbonylaminobutyl, 6- aminohexanloylaminobutyl and (when Aland R' are taken together) azetidine, aziridine, pyrrolidine, and piperidine. The cell adhesion inhibitory compound according to claim 4, wherein A' is selected from the group consisting of benzyl, n-butyl, isobutyl, methylthioethyl, cyclohexyl, 1 -methyipropyl, n-propyl and isopropyl.

6. The cell adhesion inhibitory compound according to claim 3, wherein A' 'is (when A' and R' are take n together) an optionally bridged heterocyclic ring. 105

7. The cell adhesion inhibitory compound according to claim 6, wherein the heterocyclic ring is azetidine, aziridine, pyrrolidine, and piperidine.

8. The cell adhesion inhibitory compound according to claim 7, wherein the heterocyclic ring is pyrrolidine.

9. The cell adhesion inhibitory compound according to claim 1, wherein A 2 is selected from the group consisting of alkyl optionally substituted with amino, aminocarbonyl, aryl, alkoxycarbonyl, aralkyloxycarbonyl, hydroxylaminocarbonyl, carboxy, NH-containing heterocycle, hydroxy, or mercapto; aralkyl optionally substituted with amino, aminocarbonyl, carboxy, NH-containing heterocycle, hydroxy, or mercapto; and heterocyclic ring (when A 2 and R' are taken together). The cell adhesion inhibitory compound according to claim 9 wherein A is selected from the group consisting of carboxymethyl, 2-carboxyethyl, 1-carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl, imidazolylmethyl, N-Bn- imidazolylmethyl, phenyl, carbomethoxymethyl, carbobenzyloxymethyl, and (when A 2 and R are taken together) azetidine, aziridine, pyrrolidine and piperidine.

11. The cell adhesion inhibitory compound according to claim 10 wherein A 2 is Sselected from the group consisting of carboxymethyl, 2-carboxyethyl, 1-carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl and imidazolylmethyl.

12. The cell adhesion inhibitory compound according to claim 1, wherein A 3 is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; cycloalkyl; and alkyl optionally substituted with aryl, cycloalkyl, carboxy, hydroxylaminocarbonyl, alkoxy, aralkyloxy, mercapto, N-containing heterocycle, carboxyalkylaminocarbonyl or amino-substituted acylamino.

13. The cell adhesion inhibitory compound according to claim 12, wherein A is independently selected from the group consisting of amino acid side chains and 106 corresponding protected derivatives; cyclohexyl; and alkyl optionally substituted with phenyl, cyclohexyl, carboxy, hydroxylaminocarbonyl, methoxy, benzyloxy, mercapto, N- benzylimidazolyl, biotinyl, tetrazolyl, valinyl-N-carbonyl or 6 -aminohexanoylanuno.

14. The cell adhesion inhibitory compound according to claim 1, wherein each y 3 is independently selected from the group consisting of amino acids and corresponding protected derivatives. The cell adhesion inhibitory compound according to claim 1, wherein: n is 2; Y' is leucinyl; Y2 is aspartyl; and Y3 is valinylprolinyl.

16. The cell adhesion inhibitory compound according to claim 1, wherein X is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; mono- and diallcylamino optionally substituted with hydroxy, aminocarbonyl, N- alklaninocarbonyl.- carboxy or alkoxycarbonyl; dialkylamino; cycloalkylamino; cycloalkylalkylamino; dicycloalkylamino; (N-alkyl)(N-aryl)amnino; aralkylamnino optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle containing at least .one endocyclic N atom; and bis-carboxylic. acid'substituted alkylamine.

17. The cell adhesion inhibitory compound according to claim 1, wherein X is .:selected from the group consisting of amino, methylamino, isopropylamino, isobutylamnino, n-butylamino, t-butylamino, isoamylamino, isopentylamnino, hexylamino, cyclohexylamino, cyclohexylmethylaxnjno, methyiphenylamino, phenylmethylamino, phenylamino, 4- methoxyphenylmethylamnijo, dimethylamino, diisopropylaniino, diisobutylamino, hydroxy, methoxy, n-butoxy, t-butoxy, benzyloxy, 2-piperidinecarboxylic acid, N'-(a,ax'-bis- carboxyethyl)-2-piperidinecarboxamide, N'-caboxymethyl-2..piperidinecarboxanride, 1- hydroxymethy-2-methylpropylanmjno, 1-N'-methylamido. 1-methylethylamino, 3,3- 107 dimethylbutylamino, I -N'-methylamidobutylamino, l-axnido-2-methylbutylaxnino, 1- carbomethoxy-2-methiylbutylamino, 1 -N'-methylamido-2-methylbutylamino, 1 -carboxy- I1- phenylmethylarnino, morpholino, N-phenylpiperazinyl, pipecolinyl, and piperazinyl.

18. The cell adhesion inhibitory compound according to claim 1, wherein Z is selected from the group consisting of arylsulfonyl, aralkylsulfonyl, heterocyclylsulfonyl, aryloxysulfonyl, arailkyloxysulfonyl, and heterocyclylallcylsulfonyl.

19. The cell adhesion inhibitory compound according to claim 18, wherein Z is arylsulfonyl. :20. The cell adhesion inhibitory compound according to claim 19, wherein Z is a halo-substituted arylsulfonyl.

21. The cell adhesion inhibitory compound according to claim 18, wherein Z is a (N-Ar'-urea)-substituted aralkylsulfonyl group.

22. The cell adhesion inhibitory compound according to claim 21, wherein Z is a (N-Ar'-urea)-substituted phenylmethylsulfonyl group.'

23. The cell adhesion inhibitory compound according to claim 22, wherein Z is a (N-Ar'-urea)-para-substituted phenylmethylsulfonyl group. :24. The cell adhesion inhibitory compound according to claim 1, wherein A' is selected from the group consisting of cycloalkyl; optionally bridged heterocyclic ring (when A' and R1 are taken together); and alkyl optionally substituted with amino, acylamino, amino-substituted acylamino, aryl, carboxy, cycloalkyl, hydroxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aiuinocarbonyl, alcylamninocarbonyl, dialkylaminocarbonyl, (al kyl)(aralkyl)aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, alkoxycarbonylamino, mercapto, tbioalkoxy or heterocycle; A 2 is 108 selected from the group consisting of alkyl optionally substituted with amino, aininocarbonyl, aryl, alkoxycarbonyl, arailcyloxycarbonyl, hydroxylaminocarbonyl, carboxy, Nil-containing heterocycle, hydroxy, or mercapto; aralkyl. optionally substituted with amino, aminocarbonyl, carboxy, NI--containing heterocycle, hydroxy, or mercapto; and heterocyclic ring (when A 2 and R' are taken together); A 3 is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; cyclohexyl; and alkyl optionally substituted with phenyl, cyclohexyl, carboxy, hydroxylaminocarbonyl, methoxy, benzyloxy, mercapto, N-benzylimidazolyl, biotinyl, tetrazolyl, valinyl-N-carbonyl or 6 -aminohexanoylamino; X is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; mono-and dialkylamino optionally substituted with hydroxy, aminocarbonyl, N-allcylaminocarbonyl, carboxy or alkoxycarbonyl; dialkylamino; cycloalkylamino; cycloalkylalcylamino; dicycloalkylamino; *N-alkyl)(N-aryl)amino; aralkylamnino optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle containing at least one endocyclic N atom; and bis-carboxylic acid substituted alkylamine; and Z is selected from the group consisting of arylsulfonyl, aralkylsulfonyl, heterocyclylsulfonyl, aryloxysulfonyl, aralkyloxysulfonyl, and heterocyolylalkylsulfonlyl. The cell adhesion inhibitory compound according to claim 24, wherein A' is selected from the group consisting of aminocarbonylethyl, benzyl, n-butyl, isobutyl, 0 carboxyethyl, cyclohexyl. 1 -hydroxyethyl, hydroxymethyl, mercaptomethyl, 1 006 methyipropyl, methylthioethyl, n-propyl, isopropyl, methoxycarbonylaminobutyl, 6- 0 :..0aminohexanoylaminobutyl and (when Aland R' are taken together) azetidine, aziridine, pyrrolidine, and piperidine; A 2 is selected from the group consisting of carboxymethyl, 2- :carboxyethyl, I-carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercapto methyl, inidazolylmethyl, N-Bn-imidazolylmethyl, phenyl, carbomethoxymethyl, carbobenzyloxymethyl, and (when A 2 and R' are taken together) azetidine, aziridine, pyrrolidine and piperidine; X is selected from the group consisting of amino, methylamino, isopropylamino, isobutylarnino, n-butylamino, t-butylaminu, isoamylamino. isopentylamino hexylamino, cyclohexylamino, cyclohexylniethylamino, methyiphenylamino, phenylniethylamino, phenylamino, 4 -methoxyphenylmethylaniino, dimethylaniino, 109 diisopropylamino, diisobutylamjno, hydroxy, methoxy, n-butoxy, t-butoxy, benzYloxy, 2- piperidinecarboxylic acid; N'(~'bscroyty)2pprdncroaie N'- carboxymethyl-2piperidilecarboxmie 1 -hydroxymethyl-2-methylpropylmno, 1 -Nv- methylamido-i1-methylethylainino, 3,3 -dimethylbutylamino, 1-N' -methylamidobutylamino, l-amido-2-methylbutylamino, I -carbomethoxy-2-.methylbutylamino, I -N'-methylainido-2 methylbutylamino, l-carboxy- I1-phenylmethylamino, morpholino, N- phenylpiperazinyl, pipecolinyl, and piperazinyl; and Z is a (N-Ar'-urea)-substituted aralkylsulfonyl group.

26. The cell adhesion inhibitory compound according to claim 25, wherein A' is selected from the group consisting of benzyl, n-butyl, isobutyl, methylthioethyl, cyclohexyl, 1 -methylpropyl, n-propyl, isopropyl and (when A 2 and R' are taken together) azetidine, aziridine, pyrrolidine, and piperidine; A is selected from the group consisting of carboxymethyl, 2-carboxyethyl, l-carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl and imidazolylmethyl; and Z is a (N-Ar'-urea)-substituted *..,phenylmethylsulfonyl group.

27. A pharmaceutical composition comprising a compound according to any one of claims 1 to 26 in an amount effective for prevention, inhibition or suppression of cell adhesion and a phanmaceutically acceptable carrier.

28. The pharmaceutical composition according to claim 27, further comprising an agent selected from the group consisting of corticosteroids, bronchodilators, antiastbmatics, *~*antiiflamnmatories, antirhieumatics, inimunosuppressants, antimetabolites, S* immunomodulators, antipsoriatics and antidiabetics.

29. A method of preventing, inhibiting or suppressing cell adhesion in a mammal comprising the step of administering to said mammral the pharmaceutical composition according to claim 27 or 28. 3 0. The method according to claim 29, wherein said method is used for preventing, 110 inhibiting or suppressing inflammation.

31. The method according to claim 30, wherein said inflammation is cell-adhesion associated inflammation.

32. The method according to claim 29, wherein said method is used for preventing, inhibiting or suppressing an immune or autoimmune response.

33. The method according to claim 32, wherein said immune or autoimmune response is cell-adhesion associated immune or autoimmune response.

34. The method according to claim 29, wherein said method is used to treat or prevent a disease selected from the group consisting of asthma, arthritis, psoriasis, transplantation rejection, multiple sclerosis, diabetes and inflammatory bowel disease. *o DATED this 20 th day of December 2000 BIOGEN, INC. By their Patent Attorneys :...CULLEN CO. Sr *:eT R 1 ~tr- OP