AU3644500A - Cell adhesion inhibitors - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT 0* *000 0000 0 0* *0 0 a 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.

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. Immuno!., 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 8 (81-19) 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-1 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 3to inhibit VLA4-dependent adhesive interactions both in vitro and in vivo (Ferguson et al. Proc. Natl. Acad.

Sci., 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 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 8amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr

(SEQ

S* ID NO: as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val [SEQ ID NO: 2! 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 (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 10 (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 a401 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.

S. 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 S: 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 S. 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- T) e i Znj~j=Reagent or Fragment Ac acetyl Boc benzyl Boc tert-butox ycarbonyl BU butyl Cbz carbobenzyloxy Cy ccoey Cym cyclohexylmty DIPEAdiisopropylethylamine EDC 1- (3-diethylaminopropyl) -3ethylcarbodiimide HOBT 1-hydrxybenzotriazole hydrate i-Pn isopenty! i-Pr isopropyl Me methyl *2-MPUBA 4- 2 -methylphenyl) urea) PUPAphenylmethylamino 2-PP 4- (2-methylphenyl) urea) phenylacetyl *NMP N-methylpyrrolidinone NMM N-methylmorpholine Ph phenyl PUPA 4- -Phenylurea)phenylacetyl 25 Su succinimiciyl TBTU 2- (l!--benzoriazo1-:-yi...

1, 1,3, 3 -retramethyluroni u tetra fluoroborate TEA triethylamine 30 TFA trifluoroacetic acid THAM tr-,s (hydroxy) methylairinomethane As used herein, the term "alkyl", alone or rin combination, refers to a straight-chain or branched-chain alkyl radical contalning from 1 to 10, preferably from I to 6 and more preferably from 1 to 4, carbon atoms.

Examples of such radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, i sobutyl, 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, S0 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, ethynyl (acetylenyl), propynyl, propargyl, butynyl, hexynyl, 15 decynyl and the like.

The term "cycloalkyl", alone or in combinatlio., 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, cyclopropyl, cyciobutyl, 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 or 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, -8apyridyl, pyrrolyl, oxazolyly, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, l,2,3-triazolyl, 1,3,4thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indoliny., benzo[bI furanyl, 2,3dihydrobenzofuranyl, benzo~b) thiophenyl, lH-indazolyl, benzirnidazolyl, benzthiazolyl, purinyl, 4 H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1, 8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenotniazinyl, phenoxaziny-l, pyrazolofi, S-clltriazinyl and the like.

"Aryl", "cycloalkyl" and "cycloalkenyl" groups, 15 as defined in this application may independently contain to three substi-tuents which are independently selected from the group consisting of halogen, hydroxyl1, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl', cvano, carboxy, carboalkoxy, Ar'-substituted alkyl, Ar'-substituted alkenyl or a2.kynyl, 11,2di oxvme thviene, l,2-dioxyethv'Lene, alkoxy, alkenoxy or .:alkynoxv, Ar'-substituted alkoxy, Ar' -subs tituted alkenoxy or alkynoxy, alkylamino, alkenylamino or alkynylaminw, Ar'-substituted alkylamino, Ar'- subs t ituteC: alkenylamino or alkynylamino, Ar'-substituted carbonyiLoxy, alkylcarbonyloxy, aliphatic or aromatiC acyl, Af I-substituted acyl, Ar'-substituted alkylcarbonyloxy, Ar'-substituted carbonylamino, Ar'substituted amino, Arl-substituted oxy, Ar'-substituted carbonvi, alkylcarbonylamino, Ar' substituted alkylcarbonylamino, a 1koxy-ca.rbonyl amino, Ar' -substituted alkoxycarbonyl-amino, Ar' -oxycarbonylamino, 9alkylsulfonylamino,, mono- or bis- (Ar'-sulfonyl) amino, Ar' -substituted alkyl-sul fonylamino, morphol inocarbonyl amino, thiomo rphol1inocarbonyl amino, Nalkyl guanidino, N-Ar' guanidino, N-N-(Ar',alkyl) guanidino, N,N-(Ar',Ar')guanjdino, 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, aralkyloxycarbonyl-substituted alkyl, araJlkylaminocarbonyl, 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, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, 1, 2 -dioxymethylene, 1, 2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylamino or alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl, al kyl carbonyl amino, al kox ycarbonyl amino, 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, thienyipropyl 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-0-, 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 15 above.

The term "alkylaminc", alone or in combinatron, refers to a mono- or di-alkyl-substituted amino radical a radical of formula alkyl-NH- or (alkyl) 2 wherein the term "alkyl" is as defined above. Examoles of suitable alkylamino radicals include, but are nct limited to, methylamino, ethylamino, propylamino, isopropylamino, t-butylamino, N,N-diethylamir. and t-: 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.nez 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-0-, 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, 1 0 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, 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 cycloalkyl", alone or in combination, refers to a cycloalkyl radical which shares two adjacent atoms with an aryl radical, wherein the terms "cycloalkyl" and "aryl" are as defired 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, methyipropiolyl 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, naphthoyl, pyridylcarbonyl and the like.

S. S 10 The term "heterocycloyl", alone or in combination, refers to radicals of formula heterocycle- CO-, wherein the term "heterocycle" is as defined below.

41 Examples f suitable heterocycloyl radicals include but are not Limited to, tetrahydrofuranylcarbonyl, pipe ri dinvi ca rbonyl, tetrahydrothiophenecarbonyl and the like.

The terms "morpholinocarbonyl" and *5"thiomorp'holinocarbonyl", alone or in combination with other terms, refer to an N-carbonyiated morpholino and an N-carbonvl'ated thiomorpholino radical, respectively.

S SThe term "al kylcarbonyl amino", alone or in combinazicn, refers to a radical cf formula alkyl-CONH, wherein the term "'alkyl"' is as defined above.

The term "al1koxycarbonyl1amino", alone or in combinaticn, refers to a radical cf formula alkyl-OCONH-, wherein term "alkyl" is as defined above.

The term "alkylsulfonylamino", alone or in combinaticn, refers to a radical of formula alkyl-SOgNH-, wherein the term "alkyl" is as def2ined 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", alone or in combination, refers to a radical of formula alkyl-NH-Co- NH-, wherein the term "alkyl" is as defined above.

The term "N-arylurea", 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 ring", alone or in combination, refer to a non-aromatic 3- to lO-menibered ring containing at least one endocyclic N, 0 or S atom. The heterocycl~e may optionally be aryl- *fused. The heterocycle may also be optionally substituted with one to three substituents which are 15 independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, aralkyl, :alkenyl, alkynyl, aryl, cyano, carboxy, carboalkoxy, Arla substituted alkyl, Ar'-substituted alkenyl or alkynyl, l, 2 -dioxymethylene, 1, 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, Ar'-substituted alkylcarbonyloxy, Ar'-substituted carboriylamino, Ar' substituted amino, Ar'-substituted oxy, Ar'-substituted carbonyl, al kylcarbonyl amino, Ar '-substituted alIkyl carbonyl.amino, al1koxy-carbonyl amino, Ar '-substituted al koxycarbonyl 7amino, Ar'I -oxyca rbonyl amino, alkyl sul fonyl amino, mono- or bis- (Ar'-sulfonyl) amino, 14 Ar' -substituted al kyl-sul fonylamino, morpholinocarbonyl amino, thiomorpholinocarbonylamino,

N-

alkyl guanidino, N-Ar' guanidino, N-N-(Ar',alkyl) guanidino, N,N-(Ar' ,Ar' )guanidino, N,N-ciialkyl 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, a ral koxycarbonyl -subs ti tuted alkyl, carboxyalky~., oxo, arylsulfonyl and aralkylaminocarbonyl.

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

N-

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

The term "hydrophobic group" refers to a group.

which is resistant to unitina wit"-. or absorbing water.

:Examples of such hydrophobic groups include, bu,: are nc: limited to, methyl, ethyl, propy, butyl, pentyl, hexy-, phenyl, benzyl, naphthyl, N-benzylimidazolyl, methylthioethyl and the like.

The term "acidic functional group" refers

L

group which has an acidic hydrogen within it. Examrn'es of such groups include, but are not limited to, carboxylic acid, tetrazole, imidazole, hydroxyl, Inercapto, hydroxylaminocarbonyl, sulfonic acid, su2-flnI acid, phosphoric acid and phosphonic acid.

The terms "activated derivative of a sia' protected a-amino acid" and "activated substitutedpheriylacetic 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, 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.

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

The compounds of this invention contain one or S* 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 co that receptor.

These ccmpounds are represented by formula 2 3

(I)

*e and pharmaceutically acceptable derivatives thereof; 15 wherein: Z is selected from the group consisting of alkyl; aliphatic acyl optionally substituted with Nalkyi- cr N-arylamido; aroyl; heterocycloyl; alkyi- or arylsulfonyl; aralkylcarbonyl optionally substituted with aryl; he:erocycloalkylcarbonyl; alkoxycarbonyl; araikylcxycarbonyl; cycloalkylcarbonyl optionally fused with aryl; heterocycloalkoxycarbonyl; alkylaminocarbonyl; arylaminz carbonyl and aralkylaminocarbonyl optionally substituted with bis(alkylsulfonyl)amino, alkoxycarbonylamino or alkenyl; alkylsulfonyl; aralkyls-lfonyl; arylsulfonyl; cycloalkylsulfonyl optionally fused with aryl; heterocyclylsulfonyl; heterocyclylalkylsulfonyl; 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)aminocarbonyl; mono- or dicycloalkylaminocarbonyl.; heterocyclylaminocarbonyl; heterocyclylalkylaminocarbonyl; (alkyl) (heterocyclyl) arinocarbonyl; (alkyl) (heterocyclylalkyl) aminocarbonyl; (aralkyl) (heterocyclyl)aminocarbonyl; (aralkyl) (heterocyclylalkyl) aminocarbonyl; alkenoyl optionally substituted with aryul; alkenylsulfonyl optionally substituted with aryl; alkynoyl optionally substituted with aryl; alkynylsulfonyl optionally 15 substituted with aryl; cycloalkenylcarbonyl; cycloalkenylsulfonyl; cycloalkylalkanoyl; cycloalkylalkylsulfony.; arylaroyl, biarylsulfonyl; alkoxysulfonyl; aralkoxysulfonyl; alkylaminosulfonyl; aryloxysulfonyl; arylaminosultonyl; N-arylureasubstituted alkanoyl; N-arylurea-substituted .alkylsulfonyl; cycloalkenyl-substituted carbonyl.; cyc loalkenyl-subs ti tuted sulfonyl; alkenoxycarbonyl optionally substituted with aryl.; alkenoxysulfonyl optionally substituted with aryl; alkynoxycarbonyl.

optionally substituted with aryl; alkynoxysulfonyl optionally substituted with aryl.; alkenyl- or alkynylamninocarbonyl optionally substituted with aryl; alkenylor alkynyl-aminosulfonyl optionally substituted with aryl; acylamino- subs t ituted alkanoyl; acylaminosubstituted alkylsulfonyl; aminocarbonyl-substituted.

alkanoyl; carbamoyl- substituted alkanoyl; carbamoylsubstituted alkylsulfonyl; heterocyclylalkanoyl; 18 heterocyclylaminosulfonyl; carboxyalky1-substituted aralkoyl; carboxyalkyl-substituted aralkylsulfonyl; oxocarbocyclyl-f used aroyl; oxocarbocycly).-f used arylsulfonyl; heterocyclylalkanoyl; N' ,N'-alkyl, aryihydrazinocarbonyl; aryloxy-substituted alkanoyl and heterocyclylalkylsulfonyl.

Y' is 2 Y' is -N (RI-C (R 2 each Y' is represented by the formula C (A 3 -C each R: is independently selected from the group consisting of hydrogen, alkyl, and aralkyl; alkenyl; alkynyl; cycloalkyl; cycloalkenyl; cycloalkylalkyl; aryl1; aminoalkyl; mono- or di-alkyj.-substituted aminoalkyl; 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 a-.ky1I optionally subsr-ituted with armino, acylamino, amino- subs ti tut ed acylamino, :alkoxycarbonylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl', aralkoxycarbonyl, alninocarbonyl, alkyianuinocarbony., dialkylaminocarbonyl, (alkyl) (aralkyl)aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbony--., hydroxyl, carboxyalkylaminoc-arbonyl, hydroxylaminocarbonyl', mercapto, thioalkoxy or heterocycle; A' 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, araJlkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl) (aralkyl) aminocarbonyl, 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; alkylaminc optionally substituted with hydroxy, aminocarbonyl,

N-

.*alkylaminocarbonyl, carboxy or alkoxycarbonyl; **dialkylanino; cycloal kyl amino; dicycloalkylamino; cycloalkylalkylamino; (alkyl) (aryl)amino; aralkylaminc optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle; and (mono- or bis-carboxylic acid) -substituted alkylamine; heterocyclylamino; heterocyclyl-substituted alkylamino and wherein the compound of formula I is expressly not N' -carboxymethyl-N- (phenylacetyl-L-leucyl-L-aspartyl-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

3 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 includes metabol1ites or residues of a compound of this invention. characterized by the ability to inhibit, aprevent 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 R1 are taken tCogether) and alkyl optionally substituted with amino, acylamin,, :amino-substituted acylamino, aryl, carboxy, cycloalkyl, hydroxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl1, a. *dial kyla:nocarbonyl, (alkyl I (aralkv!laminocarbony.l, aralkylamr'nocarbonyl, diaralkyiaminocarbonyl, a! koxycarbonyl amino, mercapto, thioalkoxy or heterocyc-L

E.

More preferably, A- is select-ed from the group consisting of aminocarbonyl ethyl, benzyl, n-butyl, isobutyl, carboxyethyi, cyclohexyl, 1-hydroxyethyi, hydroxymethyl, mercaptomethyl, 1-methylpropyl, methylthioethyl, n-propyl, isopropyl1, methoxycarbonylaminobutyl, E-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' 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 A2 and R' are taken together).

More preferably, A is selected from the group consisting of carboxymethyl, 2-carboxyethyl, 1carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl, imidazolylmethyl, N-Bn-imidazolylmethyl, phenyl, carbomethoxymethyl, carbobenzyloxymethyl, and (when Az and R' are taken together) azetidine, aziridine, pyrrolidine, and piperidine.

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

According to another preferred embodiment, 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, hydroxylaninocarbonyl, 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, benzy].oxy, mercapto, N-benzylimidazolyl, biotinyl, tetrazolyl, valinyl-N-carbonyl or 6 aminohex ano yl amino.

According to another preferred embodiment, each

Y

3 is independently selected from the group consisting of amino acids and corresponding protected derivatives.

According to another preferred embodiment, Y' is :*leucinyl

R

2 A=i-Bu); Y- is aspartyl

R:=H,

A-=carboxyrethyl); n=2; and Y' is valinylprolinyl

A

3 =i-Pr) (R 2 R' with A=proline) In another preferred embodiment, X is selected from the group consisting of alkoxy; aryloxy; aralkyloxy; hydroxyl; amino; mono- and djalkylaino optionally substituted with hydroxy, aminocarbony1,

N-

alkylaminocarbonyl, carboxy cr al'koxycarbonyl; dialkylamino; cycloalkylamino; cycloalkylalkylamino; :dicycloalkylamino; (alkyl) (aryl) amino; aralkylaml no optionally substituted with carboxy; diaralkylaminc; arylamino; N-containing heterocycle; bis-carboxylic acidsubstituted alkylamine and (mono- or biscabx)mtyaioabnl-usti tdNcnaiin heterocycle.

More preferably, X is selected from the group consisting of amino, methylarnino, isopropylamino, isobutylamino, n-butylamino, t-butylamino, isoamylamino, isopentylamino, hexylamino, cycl ohexyl amino, cyc lohexylmethyl amino, methyiphenyl amino, phenylmethylamino, phenylamino, 4- 23 methoxyphenylmethyl amino, dimethylamino, di isopropyl amino, dii sobutyl amino, hydroxy, methoxy, nbutoxy, t-butoxy, benzyloxy, 2 -piperidinecarboxylic acid, N I-(a a b -abxmty)-2-ie dncroaie N' carboxyme thyl -2 -piper idineca roxami de, 1-hydroxymethyl-2methyipropyl amino, l-N'-methylamido-l-methylethy1amino, 3, 3-dimethylbutyl amino, Il-N '-methyl amidobut yl amino, 1amido-2 -methylbut yl amino, 1-carbomethoxy-2- .methylbutyl amino, l-N'-methylamido-2-methylbutylamino, 1carboxy-l-phenylmethylamino, morpholino, piperidinyl,

N-

phenylpiperazinyl, pipecolinyl, and piperazinyl.

According to another preferred embodiment, Z is .selected from the group consisting of aliphatic acyl, aroyl, aralkylcarbonyl, heterocyc.oyl, alkoxycarbonyl, aralkyloxycarbonyl and heterocycloalkylcarbonyl. More preferably, Z is a (N-Ar'-urea)-para-substituted aralkylcarbonyl group and even more preferably, Z is a (N A -u e aa.u stit t d p e y m t v c r o y r u or(N-Ar-urea -para-substituted pnylmethyl carbon grou group. Even more preferably, Z is a (N-ortho- :substituted-Ar' urea) -para-substituted phenylmethylcarbonyl group or (N-meta-substituted- Ar'urea) -Para- substituted phenylmethylcarbonyl group.

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

24

C

C Table- i.

wherein Y' is 2

Y

2 is -N(R)-C(R 2

(A

2 each Y 3 is represented by the formula -N(R)-C(R 2

(A

3 C For A and A3, a single letter code refers to the side chain of the corresponding amino acid designated by that letter. A capital letter A) :ndicates the L-amino acid while a small letter a) 'ndicates the D-amino acid. Both capital and small letters L(l)) indicates a mixture.

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

II r Cmpd 1 z 3-methoxy-4-(N-phenyl urea)Dhenvlacetvl

A'

L

A 2 X

OH

V/P

I,

r v/ 2 3-methory-4-(-phenyl M D urea~peyaey 3 1 6-methoxy-5-(N'-(2-methylphenyl)- J L D Surea)-2-pfidlacetyl

I

v NH, 6-methoxy-5-(N,-(2-methylphenyl)urea)-2-pyridyfacetyl II, 0 I L I 6 3 isoguinolinecarbonyl 3-isoquinolinecarbonyl

L

L

E

hydroxylaminocarb- Oylmethyl V I OH 71 3-isoauinofinearhnnvI I f

I

onylmethyl v u 8 3 -isoguinolinecarbonyI L (N-Bn)-H V OH 9 3-isoquinolinecarbonyl L C V OH 3 -isoquinoinecarbonyl L tetrazol-5- V OH I-methvl 25 Cmpd Z A' A 2 x 11 3 -isoguinolinecarbonyl L D ____NH-CyM 12 3 -isoquinolinecarbonyl L D

-OH

13 3 -(4-hydroxyphenyl)propionyl L (R 2 Me) D V OMe 14 3 4 -hydroxyphenyl)propionyl L D NH-CyM 515 3 4 -hydroxyphenvl)propionyl L di NHi-Bu 16 3-(4-hydroxyphenyl)propiono I d -NHi-Bu 17 3-(4-hydroxvpiiteryjpropionyl D 181-44hyroypenlprpin)_ N-e)L D NOi-B 18L 3 4 -hydroxfyphenyl)propionyl (NML D v OMe 19 2 3 4 -hydroxyphenyl)propionyl L D NM V OMe 020 3 4 -hydroxyphenyl)propionyl L D OMe ethyl .22 3 -$4-hydroxvphenyl)propionyl L L(N-Me)-D V OMe 523 te rhydro-3-isoguinolinecarbonyl L D

OH

24 3-phenylpropionyl L D NH-CyM 1525 4-phenylbutyryl L D NH-CyM .se26 5-phenylpentanoyl L D NH-CvM 27 tetrahydro-3-isoguinolinecarbonvI L (N-Bn)-H V OH S.S28 acetyl (N-6n)-L D V OMe .29 acetyl (N-phen- D V OMe 30 3-phenylpropionyl (N-phen- D V OMe :ethyl)-L 31 tetra hydro-3-isoq uinolinecarbonyL L E v

OH

32 3 -isoquinolinecarbonyl L D VIP OH 33 tetrahydro-3-isoguinolinecarbonyI L D V/P

OH

34 phenylacetyl L D V/p

OH

35 phenylacetyl L D v/p OMe 6 3-phenyl propionyl I L D V/P OH 3+7 3-phenylpropionyl L D VIPOe 26 Cmpd 38 3 -(4-hYdroxyphenyI)propiony L J D VI 3-(4-hyd xyphenvil ro inn I x 0 om

OM

3-(-hvroynhpvflrnnnnu i vir Boc L D 41 2 -auinolinecarbonvl L rI~r -r 1dM 42 phenylacetyl L D /pipecoliny

OH-

43 phenylacetyl L I :Vnbu_ eI e e e le

S.

be S 4 es..

0 5S 0 *000 0@ OS

S

S

5555 5 *55e @4 0 S 000 S *5O@

S

5S5O 6* @0 S 0 00 0 S 0O 2-quinolinecarbonyl V/n-butyl I 4 45 4-methoxyphenviacetvi NHiM

NHM~

I

46 47 4-hyroxyphenvI)prorpionvi (N-Me)-L I -f Me- D2 bezvamnocrbonv7 L7~ iil:.

48 p-tolylaminocal on I L D V NHMe 49 phenylacetyl n-propyl D v NHMe phenylacetyl L D v -NHNap 51 phenylacetyl L D n-propyl -NHMe 52 2-quinolinecarbonyl L D n-prapyl NHMe 53 phenylacetyl L D 2-butyl NH, 54 phenylacetyl L D 2-butyl OMe phenylacetyI L D 2-butyl NHMe 56 2-quinolinecarbonyl L D 2-butyl OMe 57 2-quinolinecarbonyl L D 2-butyl NHMe 58 1,2.3,4-tetrahydro-2- L D 2-butyl NHMe quinolinecarbonyl_____ 59 2-quinolinecarbonyl L D O-e-T N~ 2-quinolinecarbonyl L D T NHt-Bu 61 2-quinolinecarbonyl L P D T morpholino 62 Boc L D T _NHt-Bu 27 Cmpd z A' A' 2A) x 63 2-N-Boc-amino-1 .2,3,44etrahydro- L D V OH 2-naphthoyl 64 3-phenylpropionyl L D V OH 3-(4-hydroxyphenyl)-2-bis- L D V OH (methylsulfanyl)aminopropionyl 86 3-(4-hydroxyphenyl)-2-N-Boc- L D V OH aminopropionyl 67 2-amino-i ,2,3,4-tetrahydro-2- L D V OH na phthoyl TFA salt 68 Boc D v OH 69 3-isoquinolinecarbonyl L D V OH 3-isaquinolinecarbonyl D V- OH 71 1 ,2,3,4-tetrahydro-3- D V- OH isoguinolinecarbonyl 72 naphthoyl L D I V OH 73 1 1,2,3,4-tetrahydro-2-naphthoyl L D v OH 74 naphthoyl D V- OH 1 .2,3,4-tetrahydro-2-naphthoyl D V- OH 76 5-phenylpentanoyl D V- OH 77 2-pyridinecarbonyl L D V OH 78 2-pyridinecarbonyl 0 V- OH 79 3-tetrahydrofurancarbonyt L D V OH 2-tetrahydrofurancarbonyl L D V OH 81 3-isoquinolinecarbonyl F D V OH 82 3-isoquinolinecarbonyl A (R2=Me) D V OH 83 3-isoguinolinecarbonyl cyclohexyl D V OH 84 1 2.3,4-tetrahydro-3- cyclohexyl D V OH isoguinolinecarbanyl_____ 3-4soquinolinecarbonyl cyclohexyl- D V OH methAl 88 1,2.3,4-tetrahydro-3- cyclohexyl- D OH isoauinolinecarbonyl methy 28 Cmpd Z A' A 2 3 87 3 -isoquinolinecarbonyl D F -OH 88 1,2,3,4-tetrahydro-3- D L -OH isoguinolinecarbonyl 89 3 -4soquinolinecarbonyl D L -OH 1.2.3.4-tetrahydro-3- L D L OH is~quinolinecarbonyl 91 1 3 -isoquinolinecarbonyl L D L OH 92 1 2.3,4-tetra hyd ro-3- L D F OH isoguinolinecarbonyl 93 3 -isoquinolinecarbonyl L D F OH 94 2-quinolinecarbonyl L D V OH 3.3-diphenylpropionyl L D v -OH 98 1,2.3,4-tetrahydro.3- A D V OH isoquinolinecarbonyl_____ 97 3-isoquinolinecarbonyl A D v OH 98 5-phenylpentanoyl L D v OH 99 indole-2-carbonyl L D V OH 100 3-(4-hydroxy)phenylpropionyl L D -NH,-Bu 101 benzoyl L D NHi-Bu 102 5-phenylpentanoyl L D NHi-amyl 103 3-(4-hydroxy)phenylpropionyl L D NHi-amyl 104 6-phenylhexanoyl L D V -OH 105 benzoyl L D V OH 106 5-phenylpentanoyl L D NHi-8u 107 N-phenylsuccinamoyl L D V OH 108 N-4-fluorophenylsuccinamoyl L D V OH 109 N-methyl-N-phenylsuccinamoyl L D V OH 110 1.2,3.4-tetrahydro-2- L D NHi-amyl quinollnecarbonyl_____ ill N-phenylsuccinamoyl L D NHi-Bu__ 1112 13-phenyloropyl (N-Me)-L (0-Me)-D V OMe 29 Cmpd Z A' A' (A 3) x 113 benzoyl (N-Me)-L DI 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-phenylguinolinecarbonyl ______ipeddine 116 3-(4-hydroxy) Dhenylpropionyl L D -NHHex 117 3-(4-hydroxy) phenyipropionyl L D N iBu) 118 3-(4-hydront) phenyipropionyl L D -NtiBu) 119 3-(4-hydroxy) phenylpropionyl L D V -NHHex 120 l.2.3,4-tetrahydro-2- L 0 V NMePh qinieca rbon 121 2-cguinolinecarbonyl L D V NMePh 122 1 ,2.3.4-tetrahydro-2- L D v NH-4-fluguinolinecarbony ophenyl 123 2-quinolinecarbonyl L D V NH-4-fluorophenVI 124 1 .2.3.4-tetrahydro-2- L D v NHPh g u i n o l i n e c a r b o n y l 1;25 2-quinolinecarbonyl L D V NHPh 126 2-pyridinecarbonyl (N-Me)-L D v NHMe 127 2-quinolinecarbanyl L D V 4-phenylpiperazinvi 128 4-methoxybenzoyl (N-Me)-L D V NHMe 12Vhnlaey D V NHMe 130 p V NHMe 131 phenylacetyl R D v NHMe 132 phenylacetyl N D V NHMe 133 2-N-Boc-amino-1 .2,3,4-tetrahydro- D V NHMe 2-naphthoyl 134 2-N-phenylacetylamino-1 D V NHMe .I tetrahydro-2-naphthoyl 135 1Boc D I P G OH 30

S

Cmpd z A' A 2

X

136 phenylacetyl D P -G OH 137 phenylacetyt L D -N-[bis- (carboxy)methytjpipecolin.

am id 138 phenylacetyl L D P NH-Ibis- (carboxy)- _______methyl 139 phenylacetyl L D -Nfcarboxcym ethyllpipecolinamide 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-hnluyy (N-Me)-L D V OMe 144 4-(N'-2-hydroxy- L D V/P OH Phenylurea)phenylacetyl 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-atlyI-4-phenylbutyryl V P -p OH 152 acetyl F L DN OH 153 benzoyl F L DNt OH 154 1,2,3.4-tetrahydro-3- L D V OMe isoguinolinecarbonyl 155 4-phenylbutyryl L D V O 31 Cmpd Z A' A 2

(A

3 x 156 3-isoguinolinecarbonyl L D V OMe 1157 1 3-isoguinolinecarbonyl L I D -NHi-Bu 158 2-quinolinecarbonyl L D V -Ot-Bu 159 2-quinofinecarbonyl L (0-Bn)-D V OH 160 2-quinolinecarbonyl L D D OH 161 4-phenylbutyryl L D -NHi-Bu 162 3-phenylprapionyl L D -NHi-Bu 163 benzoyl G L D NHi-Bu 164 2-guinolinecarbonyl L D V NHMe 165 4-methoxy enzoyl L D -NHi-Bu 166 4-phenyl butyryl L D V OMe 167 Boc L D V/M 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 -hy.

droxymethyl-2methylpropyl) 175 2-quinolinecarbonyl L D V morpho- 176 4-methoxyphenylacetyl L 0 T OMe 177 4-methoxyphenylsulfonyl L I D T OMe 178 2-quinolinecarbonyl L D V NH, 179 2-quinolinecarbonyl (N-Me)-L D V NHMe 180 phenylacetyl (Me -L 0 V NHMe 181 henvlacetyl L D V NHMe 32 a a. Cmpd Z A' A 2 x 182 3-phenylpropionyl (N-Me)-L D V NHMe 183 phenylacetyl M D I V NHMe 184 3-phenylpropionyl (N-Me)-L D V NHMe 185 2-quinolinecarbonyl 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 NHMe 189 phenylsulfonyl L D V NHMe 190 phenylacetyl L I D (0-Me)-T OMe 191 phenylacetyl L D T OMe 192 phenylacetyl L D (O-Bn)-T OMe 193 phenylacetyl L D (0-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-quinolinecarbonyl L D T NH(4methoxybenzyl) 198 2-quinolinecarbonyl L D NH(3,3.

dimethyl-n- ___butyl) 199 PUPA I D V/P NH- 200 PUPA L d V/P NH, 201 PUPA L D V/P NH, 202 2-MPUPA D V/P OH aminohexa 203 PUPA L D V -OH 204 PUPA L D V NHMe 205 PUPA L D I V NH* By 206 2-MPUPA L D V/P O 33 Cmpd z A' A 2 x 207 2-MPUPA L D phenyl OH 205 PUPA L I D V/P NH, 209 PUPA I D VIP NH, 210 PUPA L d VIP NH,_ 5211 -PUPA L 0 VIP NH, 212 PUPA I I d v/P NH, 213 PUPA L D NHBn 214 PUPA L D morpho- ___lino 215 PUPA L D NHi-Pr 216 PUPA L I 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 0 V NH, 222 2-MPUPA M 0 I NH.

223 2-MPUPA M D E NH, 224 2-MPUPA M D T NH_ 225 2-MPUPA M 0. 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 0 s NH, 230 2-MPUPA L 0 D _NH, 231 2-MPUPA L 0 L NH, 232 2-MPUPA L 0 DV NH, 233 2-MPUPA L D INH, 234 2 -MPUPA L D E N.

34 a. a.

a Cmpd Z A' A'

X

235 2-MPUPA L D T NH, 236 12-MPUPA L D M NH, 237 2-MPUPA L D n NH 238 2-MPUPA L D eN 239 2-MPUPA L D w NH, 240 2-MPUPA L D s NH, 241 2-MPUPA P D D NH, 242 2-MPUPA P D L NH, 243 2-MPUPA P D v NH, 244 2-MPUPA P D I NH, 245 2-MPUPA P D E NH, 246 2-MPUPA P D T NH, 247 2-MPUPA P D M NH, 248 2-MPUPA P D n NH, 249 2-MPUPA P D e NH, 250 2-MPUPA P D w NH, 251 2-MPUPA P D s NH- 252 2-MPUPA T D D NH, 253 2-MPUPA T D L NH-.

254 2-MPUPA T D v NH, 255 2-MPUPA T D I NH,.

256 2-MPUPA T D E NH, 257 2-MPUPA T D T NH, 258 2-MPUPA T D M NH, 259 2-MPUPA T D n NH, 260 2-MPUPA T D e NH, 261 2-MPUPA T D w NH2 262 *2-MPUPA T D s NH, 263 2-MPUPA E -D D NH a.

a a a.

a a. a a 35 C.

S.

S

*5

S

*5 S S *5 Cmpd Z A' A' (A 3 x 264 2-MPUPA E DL

NH,

265 2-MPUPA E I D V NH, 266 2-MPUPA E D I _NHz_ 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 NHW 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 I D e NH- 284 2-MPUPA S D w NH- 285 2-MPUPA S D s NH- 286 2-MPUPA D D NH, 287 2-MPUPA ID L NH, 288 2-MPUPA D 0V

NH,

289 2-MPUPA D I NH, 290 2-MPUPA I D E NH, 291 2-rMPUPA I D T NH, 292 2-MPUPA I D M -[NH, 36 a

S

0O*S

SS

S S S. S.

Cmpd Z A' A' M.

3 x 293 2-MPUPA ID nl NH, 294 24vIPUPA ID e NH, 295 2-MPUPA ID w N 296 2-MPUPA ID S NH, 297 2-MPUPA Q D D N 298 2-MPUPA a0 D L

NH,

299 2-MPUPA Q D V NH, 300 2-MPUPA 0 D I NH, 3011 2-MPUPA 0 D E -NH, 302 2-MPUPA a0 D T NH, 303 2-MPUPA 0 D M NH, 304 2-MPUPA 0 D n NH, 305 2-MPUPA 0 D e NH, 306 2-MPUPA a D w NH, 307 2-MPUPA a D s NH, 308 2-MPUPA M E D NH, 309 12-MPUPA M E V NH, 310 2-MPUPA L E D NH-.

311 2-.MPUPA L E v NH- 312 2-MPUPA P E D NH, 313 2-MPUPA P E V NH2 314 2-MPUPA T E D NH, 315 2-MPUPA M I D V/P OH 316 _4-(N'-2-pyridyurea)phenylacetI L D VIP OH 317 3 -methoxy-4-(N'-(2-methylpheny)- L D V/P NH, urea)phenylacetyl______ 318 PUPA L D V morpholino 319 -PUPA L D V NHi-Pr 320 PUPA L D V NH~ 0 37 Cmpd Z A' A 2 (Ai 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 V/P NHCy 325 PUPA L D V/P piperidinyl 326 PUPA L I 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 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 NHCyM 334 Cbz -CH-CH- V OMe (N of CH,CH.,-(C 335 3-(4-hydroxyphenyl)propionyl -CHCH: V OMe (N of CHCH,-(C of 336 4-(2-fluorophenylurea)phenylacetyI L D VIP OH 337 2-MPUPA L 0 V/PIS OH 338 2-MPUPA L D VIP/SIT OH 339 2-MPUPA. V L P/D OH 340 2-MPUPA v I pld OH 341 2-MPUPA L P V/D OH 342 2-MPUPA P D -OH 343 hydrogen 1)v d/I 2-MPUBA 344 hydrogen v d I 2-MPUBA 345 2-MPUPA L D V OH WO 97/03094 38 Cmpd Z A' A' (AX X 346 4-(N-(6-methyl-2- L D V/P OH pyridylourea)phenylacetyl_____ 347 L D V/P OH fluorophenylurea)phenylacetl_____ 348 4-phenylbutyroyl (N-Me)-L D V NHMe 349 phenylacetyl S D V NHMe 350 phenylacetyl 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 Boc L 0 T NHBu 355 Boc L D V/P OH 356 2-quinolinecarbonyl L D V/P OH 357 4-(N'-2-pyridyturea)phenylacetyI L D V/P NH- 358 2-MPUPA L D*THAM VIP OTHAM 359 2-MPUPA L ID*Na V/P ONa 360 2-MPUPA L~l Het'- 361 2-MPUPA I Het' 362 2-MPUPA L(I Het' 363 2-MPUPA L~l Het 3 364 -MPUA L He4 364 2-MPUPA Het' 366 fluorenylmethoxycarbonyl L D V OH 367 3-methoxyphenylacetyl L D V OH 368 3-(3-methylindolyl)propionyl L D V OH 369 2-phenyl-3-methyl-pyrazol-4- L D V OH 370 6-methylbenzpynimidon-2- L D V OH vi~ycarbonyl 39 a Cmpd Z A A' (A 3

X

371 4-oxo-4,5,6,7- L D V OH tetrahyd robe nzo [b~fura n-3- ~~~ylcarbonyl___ 372 L D V OH (phenylacetylenyl))pyr~dinecarbonyI 373 3 -(2-phenytthio)-pyridinecarbony L D V OH 374 4-propylbenzoyl L D0 V OH 375 4-(2-(3-pyridinyl))thiazolecarbony L D V OH 376 4-(2-(4-pyndinyl))thiazolecarbonyI L D v OH 377 5-2(-ydnl)hohnsloy L I D V OH 378 5-( 2 -(l-pyrrolyl))pyridinecarbonyI L D V OH 379 N,N-(4-trifluoromethyipyridin-2- L D v OH vl)methylhydrazinocarbonyl_____ 1 0 380 2-quinoxalinylaminocarbanyl L D V OH 381 N-(4-trifluoromethylpyridin-2- L D V OH yl)piperazinocarbonyl 382 L D V OH trifluoromethyi)phenylsulfonyl).

1,2,3,4-tetrahydrothiophenesufony 383 1-(4-chlorophenylmethyl)pyrrolidin- L D V OH I -2-on-4-ylcarbonyt 384 1-(2-furanylmethy)Ipyrrolidin-2-on- L D V OH 4-ylcarbonyl 1 5 385 2-(l-pyrrolyl)benzoyl L D V OH 386 16-chlorochroman-3-ylcarbonyl L D V OH 387 [2,3-dihydrobenzofuran-5-ylcarbonyl L D V OH 388 4,6-dimethylpyrazolo[1 .5-cjtriazin-3- L D V OH I ycarbonyl 389 3,4-benzocyctohexanoyl L D V OH 390 norbornylacetyl L D V OHu 391 1,2,3,4-tetrahydro-9- L D V OH acndinylcarbonyl 392 5.6.7,8- L 0V OH tetrahdronahthlaminocarbonv E 40 S. Cmpd

ZA

t A(A) x 393 3-(2-(4-methylthiophenoxy))- L D V OH pyridinecarbonyl 394 2-(6-methoxynaphth-2-y)propionyI L D V OH 395 (2-naphthyloxy)acetyl L D V OH 396 1 3-cluinuclidinylaminocarbonyl L D V OH 397 L D v OH Itetra hydroisog uinoline)carbonyl 398 adamantan-2-ylcarbonyl L D v OH 399 (2-pyridyl)acetyl L D V OH 400 6-methylcyclohexen-2-ylcarbonyt L D V OH 401 (3-guinolinyl)acetyl L D V OH 402 4-(2-butyl)phenylaminocarbonyl L D v OH 403 1 .4-dihydro-1 -ethyl-7-methyl-4.oxo- L D V OH 18-naphthyidin-3-ylca rbonyl 404 (2-thienyl)acetyl L 0 V OH 405 4-(2-propyl~benzoyt L D v OH 406 3,4-methylenedoxyberizoyl L D V OH 407 2-(5-(2-pyridyl))thiophenecarbonyI L D V OH 408 N-iminodibenzylcarbonyl L D v OH 409 2-MPUPA P D I NHMe 410 2-MPUPA P D I ome 411 2-MPUPA P D I OH 412 2-MPUPA

-CH

2 CHI,-. D IOH (N of 413 2-MPUPA P E -WNe 414 2-MPUPA P E W Ne 415 2-MPUPA P E

OH

416 2-MPUPA P E -OH

S.

S

S

S.

41 where Het', He t 2 He t 3 He t 4 and Het 5 in Table 1. are defined below: 9 C0 2

H

P02 N Het3 N0> C0 2

H

N Het2 OMe OMe C0 2

H

N Het4 0 OMe

H

KNO Hets

KC

2

H

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 (1) 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-

(I)

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, 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, 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.

20 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 10 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 yivo 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 CSl-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 test 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 20 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 IgGi 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 0 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 mixed 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 1B integrins, such as VLA-5, VLA-6 and a487 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 82 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 a487 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 in "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 as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, .OS. 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.

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 S. 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 Pli, 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.

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

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.

Alternatively, the pharmaceutical compositions 20 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 excipienz 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 1.0 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, preferabi-, 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 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 specific 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 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 10 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).

20 According to other embodiments, the invention provides methods for preventing, inhibiting or suppressing 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.

EXAMPLES

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

C

2 1 was treated with activated ester or acyl halide (1 eq.) at 0 "C 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 SO< or MgSO 4 and concentrated in vacuo.

1 0 GENERAL PROCEDURE FOR UREA FORMATION IN SOLUTION: PROQCEDUE 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 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 20 A solution of tBuOC(ONH-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 warr.

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 1 0 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 S: of Celite and the filtrate concentrated in vacuo.

GENERAL PROCEDURES FOR AMIDE BOND FORMATION ON SOLID

SUPPORT:

PROCEDURE GZ: coupling with DCC/HOBt A mixture of resin (see below for preparation of resin 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 CH,C1; (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 BOG with TFA/CH 2 C1 2 A mixture of resin and 50% TFA/CH 2 Cl 2 Was shaken for over 0.5 h at RT. The resin was then washed with

CH

2 C1 2 (2X) isopropanol. (1X) and CH 2 Cl 2 (3X) PROCEDUJRE__J: HF with scavengers The protected product was treated with HF at to 0 *C for over 1.5 h in the presence of anisole or thioanisole as scavenger. The HF was removed with a 10 stream of N 2 at0 C SYNTHESIS OF COMMON INTERMEDIATES Succinimidyl 3 -Isgquinoline-carboxvlpte (i~n-OSu) A solution of 3 -isoquinolinecarboxylic acid (1.2 eq.) in DMF at 0 0 C was treated with EDC 4 eq.).

The mixture was stirred at 0 0 C for 1 to 2 h and then Nhyciroxysuccinimide (1.0 eq.) was added. After stirring at rT for more than 3 h, the reaction mixture was poured into 60:Z sat. NaHCO, and the product filtered: 'H NMR *S 20 (CDC'L, 300 MHz, ppm) 9.35 1H) 8. 67 1H) 8.09 (in, 1H), '7.96 (mn, 7.82 (mn, 2H), 2.94 4H).

Succinirnidvi 2 -Ouinolinecarboxylate A solution of 2-quinoline carboxylic acid (1.2 eq.) in DMF at 0 0 C was treated with EDC 4 eq.) The mixture was stirred at 0 DC for 1 to 2 h and then Nhydroxysuccinimide (1.0 eq.) was added. After stirring at RT f or more than 3 h, the reaction mixture was poured into 60i sat. NaHCO 3 and the product filtered: 1H NNR (CDCl 3 300 MHz, ppm) 8.35 1H) 8.27 1H) 8.19 (d, 59 1H), 7. 87 1H) 7. 80 (in, IH)t 7 .68 (in, 1H) 2. 91(s 4H).

Methyl 4-I socvanatphanlpcatate (KCI A well-stirred cold solution of methyl paminophenylacetate (9.8 g, 59.4 immcl) in CH 2 Cl 2 (200 mL) and TEA (25 m.L, 18 g, 178.2 mnmol) was treated with COC1 2 (96 mL of 1.9 M solution in toluene) over 1 h. The reaction mixture was stirred at 0 'C for an additional 1 h. The reaction mixture was concentrated and 3:1 ether/pet ether (125mL) was added. The mixture was filtered and the filtrate concentrated to give KC1 as a Nbrown liquid. The crude product was purified by distillation (118-120 OC/l.mm) to afford pure KC1 g, 75%) as a colorless liquid: IH NMR (CDCl 3 300 MHz, ppm) 7. 20 J 8. 4 Hz) 7.02 J 8.4 Hz), 3.69 (s, 3H), 3.48 2H).

4 -Phenvluraidg]2henvlpcetic acid: 4 -Phenylureidophenylacetic acid was prepared using procedure C with 4-amino-phenylacetic acid and 20 phenyl isocyanate: 111 NMR (CDSOCD,, 300 MHz, ppm) 8.72- 8.64 2H), 7.44 211), 7.36 2H), 7.28 2H]), 7.16 2H), 6.96 Ct, 3.52 Cs, 2H); rn/z 272.

4 -o-Tolvlureidop~henvlpc-etic- arid: 4 -o-Tolylureidophenylacetic acid was prepared using procedure C with 4-amino-phenylacetic acid and otolyl isocyanate: 'H NMR (CDSOCD, 300 MHz,. ppm) 8.97 1H), 7.88 1H), 7.83 1H), 7.38 2H1), 7.17- 60 7.09 (in, 4H), 6.92 1H), 3.48 2H), 2.23 3H); m/z 285.

4- 2 -Fluororphenv1)ureidophenvlpcetic acid;.

4- 2 -Fluorophenyl)ureiciophenylacetic 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, 1H1), 8.14 (dd, 8. 3 Hz, 1. 5 Hz, lH) 7. 37 8.5 Hz, 2H) 7.07-7.25 (mn, 4H) 6.99 (mn, 1H) 3.48 2H-) 4 2 -Hydroxyphenylure i do) phenyl ace tic acid was prepared using procedure C with 2-hydroxyaniline and KC1: 'H NMR (CD.,S0CD 3 300 MHz, ppm) 9.90 IH),9.25 1H) 8.12 8.02 (bd, 1H), 7.37 2H), 7.13 2H), 6.70-6.97 (in, 3H), 3.48 2H).

N-SuCcinimidvl 4 2 3 -methVlovridhlureido) phenvI acetate: Prepared in three steps as follows: 9Procedure C with 2 -amino- 3-methylpyridi ne and KC1 to give methyl 4 2 -(3-inethylpyridylureido) phenylacetate.

A solution of methyl 4- (3inethylpyricylureido)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 NI4R

(CD.,SOCD

3 300 M4Hz, ppm.) 11.97 1H), 8.64 (brs, 1H), 8 .31 1H), *7..69 (mn, 1H), 7.62 8.4 Hz, 2H), 7.33 61 8.4 Hz, 2H), 7.09 (mn, 1H), 3.62 2H), 2.38 (s, 3H); inlz 286.

A solution of mnethylpyridyjlureido) phenylacetic acid (1 Nhydroxysuccinimide (1.2 eq.) 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. NaHCO 3 and the product filtered: NMR (CDSOCD 3 300 MHz, ppm) 12.04 111), 8.84 IH), 8.31 1H), 7.72 (mn, 3H), 10 7.42 (in, 2H) 7. 10 (n,1H) 4.18 2H), 2.98 4H), 2.38 3H); m/z 383.

~@N-Succinimidyl 4 (2-nyridylure ido) ohenvi acetate:- Prepared in three steps as follows: Procedure C with 2-aininopyridine and KC1 to ivemethyl 4 2 -pyridylureido)phenylacetate: H. NNR (CDCl 3 1, 300 MHz, ppm) 8.20 2H), 7.62-7.51 (in, 3H), 7.33 2H), 7.01 2H), 6.89-6.85 (in, 1H), 3.70 (s, :3H) 3. 59 2H).

A solution of methyl 4- (2pyridylureido)phenylacetate (5.7 9, 20.0 imuol) in 9 9*methanol %(20 mL) was treated with 1 N NaOil (40 inL) .The reaction was stirred for 16 h, then acidified carefully with 1 N HiCl 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: 1 H NMR (CDSOCD 3 1, 300 M4Hz, ppm) 10.62 (bs, 1H), 9.53 (bs, 1H), 8.39 1H), 7.82 (t, 1H), 7.63-7.55 (mn, 1H), 7.33-7.27 2H), 7.14-7.08 (m, IH) 3.62 Cs, 3H).

A solution of 4 2 -pyridyl)ureidophenylacetic acid (1 N-hydroxysuccinimide (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: IiiINM

(CD

3

SOCD

3 300 MHz, ppm) 10.08 1H), 9.57 111), 8.39 (mn, 1H), 7.86 (in, 1H) 7.62 (mn, 3H1), 7.38 211), 7.12 (mn, 1H), 4.15 211), 2.91 411); in/z 369.

3 -Methoxv-4-ophenvlureidophenvlacet-ic acid: Prepared in six steps from 3 -inethoxy-4-nitrobenzoic acid *as follows: a 10 A mixture of 3 -methoxy-4-nitrobenzoic acid (2.01 g, 10.2 inmol) and thionyl chloride (2.3 mL, 31.:) 99miol) was stirred at 80-90 OC for 1.5 h. The reaction was concentrated and the residue diluted with ether. The organic solution was washed with sat. aq. Na-CO, (2 X)

H.

2 0, then sat. aq. NaCl, dried (MgSO 4 and concentrated to afford 3 -iethoxy-4-nitrobenzoyl chloride (1.92 g, 87&) as a white solid: !H NMR (CDC13, 300 MHz, ppm) 7.95-7.70 (mn, 31-1), 4.06 3H).

9 A cold (0 OC) solution of TMSCHN 2 (2 M in hexane, 1.5 mL, 3.0 inmol) and triethylainine (420 pL, 9 mmcl) was treated with a solution of 3 -methoxy-4ni.trobenzoyl chloride (0.52 g, 2.4 inmol) in acetonitrie mL). The reaction was stirred at 0 *C for 24 h and then concentrated. The residue was slurried with sat.

aq. NaHC0 3 and the mixture extracted with ether The combined ether washes were washed with water, then sat.

aq-. NaCl, dried (MgSO 4 and concentrated to afford diazo- 3 -methoxy-4-nitroacetophenone (0.53 g, 100% as a yellow foam: 1 H NI4R (CDCl 3 300 MHz, ppm) 7.88 10 Hz, 63 1H) 7. 61 1H) 7.27 10 Hz, 1H) 5. 97 1H) 4.02 3H1).

A refluxing solution of &)-diazo-3-methoxy-4ritroacetophenone (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 Inmol) in triethylamine (15 niL) dropwise over 1 h. After refluxing for 45 min,-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 was washed with 5% aq. NaHCO 3 (2 H:O, 5% aq. citric acid, 1120, then sat. aq. NaCi, dried (MgSO 4 and concentrated to afford t-butyl 3-methoxy-4- ~.nitrophenylacetate (8.92 g, 93%) as a brown oil: IH NNR (CDCl 3 300 M4Hz, ppm) 7.83 (di, 8. 3 Hz, 1H) 7.03 1H), 6.93 8.3 Hz, 1H), 3.97 3H), 3.58 2H), 1.45 9H).

A mixture of t-butyl 3-methoxy-4nitrophenylacetate (0.144 g, 0.539 mmol) and 10% Pd on carbon (0.155 g) in ethyl acetate (8 mL) and methanol (2 niL) 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-methoxyphenylacetate (0.123 g, 96%) as a light yellow oil: IH NNR (CDCI 3 300 MHz, ppm) 6.70 (in, 3H1) 4.04 (bs, 2H) 3.84 3H) 3.42 (s, 2H1), 1. 43 9H).

Procedure C with t-butyl 4-amino-3methoxyphenylacetate and phenyl isocyanate gave t-butyl 3 -methoxy- 4-phenylureiciophenyl acetate: IH NMR (CDCl 3 300 MHz, ppm) 8.00 (d,11 Hz, 1H1) 7.65-6.94 (mn, 711), 6.80 (d, Hz, 1H), 6.74 1H), 3.68 3H), 3.45 2H), 1.44 9H1).

64 A solution of t-butyl 3-methoxy-4phenylureidophenylacetate (0.108 g, 0.303 minol) in trifluoroacetic acid (5.0 niL) 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: 1 H N?4R (CDSOCD 3 300 MHz, ppm) 9.28 1H), 8. 18 1H) 8.02 7. 5 Hz, 1H) 7.58- 7.15 (in, 5H), 6.91 (bin, 2H), 6.77 7.5 Hiz, 1H), 3.85 3H) 3. 49 2H).

N-SuIccinimidvl 3 -methox.- 4 -1henvl ure idophenvl acetate.

A solution of 3-xnethoxy-4phenylureidophenylacetic acid (1 eq.) in DNF at 0 0 C was treated with EDC 1 The mixture was stirred at 0 *C for 1 to 2 h and then N-hydroxysuccinimide (1.1 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 -me thox y- 4 phen yl ure idophenyl acetate filtered.

20 N-Succinimidvl 6- (2-nethoxv-3-o- .:tolylurei-do) pvridvlpceta-te: Prepared in six steps from 2 6 -dichloro-3-nitropyridine as follows: A slurry of 2 6 -dichloro-3-nitropyridine (92 i, 9.9 g, 47 minol) and K 2 C0 3 powder (6.5 g, 47 nimol) 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), H201 then sat. aq. NaCl, dried (MgSO 4 and concentrated to 65 afford 2 -chloro-6-methoxy-5-nitropyridine 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, 1H), 7.10 8.3 Hz, 1H), 6.82 8.9 Hz, IH), 4.15 3H), 4.06 3H).

A mixture of 2 and 2 -chloro- 6-methoxy- 3 ni tropyri dine (8.9 g, 47 mmol), t-butyl methyl malonate (10 mL, 60 mmol), and NaH 3.1 g, 120 mmol) 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 f lash chromatography (silica gel, 95:5 hexane-ethyl acetate) to afford methyl E-( 2 -methoxy-3-nitro)pyridylacetate (3.3 g, 62%) as a yellow oil: '11 NMR (CDCl 3 300 MHz, ppm) 8.27 8.0 Hz, 1H), 7.04 8.0 Hz, 1H), 4.09 3H), 3.85 2H), 3.75 3H).

A mixture of methyl 6- (2-methoxy-3- *~ito p *-Jky-4-ylacetate (0.047 g, 0.21 mmol) and 1%Pd on carbon (0.063 g) in ethyl acetate (2 mL) and ethanol (1 mL) was stirred under H-l(40-50 psi) for 6 h. The mixture was filtered through Celite and the filtrate concentrated 9..to afford methyl -m hx-3 mn) yiy c at (0.041 g, 100%) as a light yellow oil: "H NMR (CDCl 3 300 MHz, ppm) 6. 82 7. 6 Hz, iN) 6. 65 7. 6 Hz, 1H) 3. 94 3H) 3. 70 3H) 3. 65 2H) Procedure C with methyl 6- (2-methoxy-3amino) pyr idyl acetate and o-tolyl isocyanate to give methyl 6- 2 -nethoxy-3-o-tolylureido) pyridylacetate:

IH

NMR (CDCI., 300 MHz, ppm) 8.33 '7.9 Hz, 1H) 7. 51 (d, 7.8 Hz, 7H), 741 1H), 7.17 (mn, 2H), 7.08 (in, 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 inmol) in methanol (1.0 mL) was treated with 2 M LiOH (90 pL, 0.18 mmiol) The reaction was stirred for 18 h, diluted with

H

2 0 (5.0 mL.) and washed with ether The aqueous solution was then acidified with 5% aq. citric acid. The product was filtered and washed with H 2 0 then ether to 10. give 6 2 -methoxy-3-o-tolylureido)pyridylacetic acid (0.014 g, 640b) as a white solid: IH NNR (CDIOD, 300 MHz, ppm) 8.50-8.25 (mn, 3H), 7.60 (lxi, IH), 7.28-7.00 (mI, 3H), 3H), 3.69 2H), 2.30 3H); MS, zn/z 316.

A solution of 6 -(2-rnethoxy-3-otolylureido)pyridylacetic acid (1.61 g, 5.10 inrol) in DMP at C 0 C was treated with EDC (1.00 g, 5.2 mmol) .The mixture was stirred at 0 *C for 1 to 2 h and then Nhydroxysuccinimide (0.60 g, 5.2 mmol) was added. After stirring at RT for more than 3 h, the reaction mixture 20 was poured into 60% sat. NaHCO, and the N-succinimnidyl 6- 2 -rethoxy-3-o-tolylureido)pyridylacetate filtered.

H-L 'fOBn)

V-NHCH

3 H-LD(OBn)V-NHCH. was prepared by sequentially usir'z procedure B with BOC-Val-OSu and methylamine, 2$ procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-Leu-OSu, then procedure

D.

H-LT:'OBn)V-OCH: H-LrL"OBn)V-OCH 3 was prepared by sequentially using procedure B with BOC--Asp (OBn) -OSu and H-Val-cjMe, 67 procedure D, procedure B with BOC-Leu--OSu, then procedure

D.

H-LD fOBn)V-OBn: H-LD(OBn)V--OBn was prepared by sequentially using procedure B with BOC-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 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-Leu-OSu, then procedure D.

H-LD (OBn-)VP-OMe: H-LD(OBn)VP-OMe was prepared by sequentially using procedure A with BOC-Val-OH and H-Pro-OMe, :procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-Leu-OSu, then procedure D.

.:H-LDVP-OH was prepared by sequentially using procedure B with BOC-Val-OSu and H-Prco-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 IVP- Ofn: 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 (ogn VP-NH 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(OBI)-OSu, procedure D, procedure B with BOC-Leu-OSu, then procedure D.

Resin (MBCI): Modified resin MBCl (0.437 nunol/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.C1 and triethylsilane for 2 h at RT then washed with CHCl- (2X), isopropanol (lX) and CI-1C1- (3X) before use.

MBC2: M'BC2 was prepared by sequentially using procedure G with BQC-Asp(OBn)-OH, procedure 1, procedure G with BOC-Leu-OH, procedure I, then procedure G with 4- :phenylureidophenylacetic acid.

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

G

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

MBC4 was prepared by sequentially using procedure G with BOC-Pro-OH, procedure 1, procedure G with BOC-Val-OH, procedure I, procedure G with BOC- Asp(OBn)-OH, procedure I, procedure G with BOC-tLeu-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: 1 0 Compound 64 was prepared by using procedure A with hydrocinnamic acid and H-LD(OBn)V-OBn then procedure F.

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

~Example 4 Compound 155: Compound 155 was prepared by using procedure B with 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 witn 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 ComDound 177: Compound 177 was prepared by using procedure B with BOC- Asp(OBn)-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 BC3- Val-OSu and methylamine, procedure D, procedure B with BOC-Asp(OBn)-OSu, procedure D, procedure B with BOC-N- MeLe--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 procedure F. Purification by HPLC gave the title compound: m/z 606.

15 1 5 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 S 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 315: Compound 315 was prepared by using procedure A with 4-otolylureidophenylacetic acid and H-MD(OBn)VP-OBn then procedure F. Purification by HPLC gave the title compound: m/z 727.

Compound 346: .Compound 346 was prepared by using procedure B with N- Hydroxysuccinimidyl 4- (3-me thylpyr idylureido)phenylacetate and H--LDVP-OH. Purification by HPLIC gave Compound 346: m/z 710.

Compound 316: Compound 316 was prepared by using procedure B with Nhydroxysuccinyjimidyl 4- 2 -pyridylureido)phenylacetate and H-LDVP-OH. Purification by HPLC gave the title compound: m/z 696.

Example 21 Compound 4: Compound was prepared by using procedure B with Nhydroxysuczinimidyl 6- (2-methoxy-3-otolylureido)pyridylacetate and H--LDVP-OH. Purif ication: by HPLC gave the title compound: m/z 740, 30.7 min (gradient 8).

74 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).

Example 23 Compound 148: 1 0 Compound 148 was prepared by using procedure A with 4-otolylureidophenylacetic acid and H-LD(OBn)VP-NH then procedure F. Purification by HPLC gave the title compound: m/z 708, 26.0 min (gradient 8).

oo ~Example 24 Compound 317: Compound 317 was prepared by using procedure B with Nhydroxysuccinimidyl 6-(2-methoxy-3-otolyiureido)pyridylacetate and H-LD(OBn)VP-NH then procedure F. Purification by HPLC gave the title 20 compound: m/z 739, 28.0 min (gradient 8).

Example Compound 336: Compound 336 was prepared by using procedure A with 4-(2fluorophenyl)ureidophenylacetic acid and H-LD(OBn)VP-OBn then procedure 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 1 0 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).

0 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 se@* 591, 21.5 min (gradient 8).

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 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 10 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.

Example 37 Compound 21 7 Compound 21~ was prepared by using procedure H with MBCC and isobutylamine then procedure 3: 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 Example 39 Compound 318: Compound 318 was prepared by using procedure H with MBC3 and morpholine then procedure J: m/z 667.

ExamT~1e Compound 319: Compound 319 was prepared by using procedure H with MBC3 and isopropylamine then procedure J: rn/z 640.

10 Compound 320: Compound 320 was prepared by using and cyclohexylamine then procedure Example 42 Compound 321: 15 Compound 321 was prepared by using and benzylamine then procedure J: procedure H with MBC3 J: m/z 679.

procedure H with MBC3 m/z 687.

Example 43 Comnound 322: Compound 322 was prepared by using procedure H- with MBC3 and-" niperi4dine then procedure rn/z 665.

Exampip, 44 Compound 323: Compound 323 was'prepared by using procedure H with MBC3 and isobutylamine then procedure J: xn/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 .mmol), and iPr 2 NEt (4.32 mL, 24.8 mmmol) in DMF (25 mL) was stirred overnight at RT. The product was 80 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 mnL), H~20 (25 mL), and Et 2 O (2 x 25 rmL), and dried on the filter (4.18 g, All of this product was suspended in CH 2 Cl 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,c1.

(2 x 20 mL). The residue was triturated with Et 2 O (lO0mL) at RT for 2 hr. The solids were collected by filtration on a medium f rit, 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 nunol) and HOBT (0.135 g, 1.0 nunol) in EtOAc (6 mL) was stirred at RT 20 min until homogeneous. Fmoc-Asp-OtBu (0.411 g, 1.0 inmol), piperonylainine (0.12 inL, 1.0 inmol), and Nmethylinorpholine (0.22 ruL, 2.0 minol) were added. After stirring overnight, the reaction was filtered to remove solids and the cake washed with fresh EtOAc (10 inL) The filtrate was washed with H 2 0 5% citric acid (lx) 20 NaHCO 3 (lx) and brine (lx) 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 0 C; TLC(2% MeOH/CHCl 3 R,=0.10;

MS

(FAB) 545; 1 H NMR (CDC1 3 300 MHz, ppm) 7.75-7.72 (mn, 2H), 7.59-7.56 (mn, 2H), 7.40-7.34 mn, 2H), 7.30-7.25 (mn, 2H), 6.71-6.66 3H), 6.13-6.10 (in, 2H), 5.84 2H), 41.46 (in, 1H), 4.38-4.16 (mn, 5H), 2.86 (dd, IH, 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 (0.2k g, 0.46 inmol), piperidine (0.45 mL, 4.6 mmiol), and CH 2 C1 2 (0.45 inL) were 81 stirred at RT for 90 mmi. The reaction was evaporated to a solid residue. Flash column chromatography on Sio, using a MeOH/EtOAc gradient provided product (0.138 g, 93%) as a colorless oil: ms (FAB) 323; MeOH/EtOAc) Rf=Q.15; IH NMR (CDCl 3 300 MHz, ppm) 7.63 (br s, 1H), 6.75-6.68 (in, 3H), 5.90 2H), 4.34 (dd, IH, J-5.7, 14.7 Hz), 4.28 (dd, 1H, J=5.7, 14.7 Hz), 3.65 (dci, 1H, J=3.4, 9.3 Hz), 2.62 (dci, 1H, J=3.4, 15.7 Hz), 2.38 (cid, 1H, J=9.3, 15.7 Hz) 1. 74 2H) 1. 42 9H).

D. The product from Example 5OC (2.55 g, 7.91 nimol) and Eschenmoser's salt (1.61 g, 8.70 nunol) 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% NaHCO 3 and extracted with EtOAc The combined organic extracts were washed with 5% NaHCO 3 (lx) H 2 0 (lx) and brine (lx) and dried :(MgSO 4 The crude product was dissolved in Et 2 O (250 mL) and passed through a short pad of 5102, 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 inL) and collected by filtration to give a white solid (0.904 g, 34%) mp=121-123*C; TLC MeOH/CHCl 3 R,=0.59; 'H NMR (CDC13, 300 MHz, ppm) 6.75-6.66 (in, 3H), 5.92 2H), 4.66 (A of AB, 1H, J=14.7 Hz), 4.23 (B of AB, lH, J=14.7 Hz), 4.15 (ABq, 21H, J=11.9 Hz), 3.68 (dci, dci, 1H, J=5.2, 10.9 Hz) 2.72 (dci, 1H, J=5.2, 17.3 Hz), 2.41 (dci, 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 The product f rom Example 5OD 50 g, 1.-5 inmol) the product from Example 50A 596 g, 1. 5 inmol) 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 mL), washed with H 2 0 (8 x 6 mL) brine (lx) and dried (MgSO 4 Flash column chromatography on Si0 2 eluting with 100% CHC1-, to 30% EtOAc/CHCl 3 provided product 94 g, 88%) as a pale yellow oil: MS (FAB) 714; TLC (10% MeOH/CHC 3 R,=0.40; 'H NMR (CDCl 3 300 MHz, ppm) consistent with 10 structure and indicative of diastereomers.

F. The product from Example 50E (0.94 g, 1.32 mmol) was :stirred in TFA (10 mL) at RT for 3 hr. The reaction was concentrated to dryness and the residue evaporated from CH2l-(3x 10 Tecrude poutwastruaedwh 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,=0.15; 'H NNR (d'-DMSO, 300 MHz, ppm) consistent with structure and indicative of diastereoners.

ExaI~e 1 Compound 364: A. the same manner as described in Example 50B, Fmoc- Asp-OtBu (8.23 g, 20.0 inmol) was reacted with H-Gly- OBn*IICl (4.03 g, 20.0 inmol). Flash column chromatography on SiC:, using an EtOAc/hexane gradient provided product (9.8 g, 88%) as a waxy solid: MS (FAB) 559; TLC MeOH/CHC1 1 Rf=0.71; 1 H NMR (CDCl 3 300 MHz, ppm) 7.73 2H, J=7.5 Hz), 7.59 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 1H), 4.41-4.29 Cm, 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 mmol) was deprotected in the manner described in Example Filtration through a pad of SiO. with 100% EtOAc followed by 5% MeOH/CHC1 3 provided product ^31.24 g, 72%) as an oil: MS (FAB) 337; TLC MeOH/EtOAc) R.=0.15; IH NMR (CDCd 3 300 MHz, ppm) 8.00 Ct, 1H, J=5.4 Hz;, '7.30-7.21 (rn, 5.07 2H), 3.98 CAB of ABX, 2H, J=5.4, 18.1 Hz), 3.60 Cdd, 1H, J=3.4, 9.2 Hz), 2.60 (dcl, 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 mmol) was :cyclized in the manner described in Example SOD. Flash column chromatography using an EtOAc/CHC1., gradient provided product as a syrup (1.4 g, MS (FAB) 349; TLC (1:1 EtOAc/CHC1 3 Rf=0.53; NMR (CDCI,, 300 MHz, ppm) 20 7.35-7.25 Cm, 5H), 5.11 2H), 4.21 (A of AB, 18, J=17.5 Hz), 3.95 (B of A.B, lH, J=17.5 Hz), 3.71 Cdd, 1H, J=5.1, 11.2 Hz), 2.68 (dd, 18, J=5.1, 17.2 Hz), 2.36 (dd, 18, J=11.2, 17.2 Hz), 1.43 9H).

D. The product of Example 51C (1.40 g, 4.02 mmol) was coupled with the product of Example 50A using the procedure of Example 50E. Flash column chromatography using a CHCl 3 /EtOAc gradient provided product as a brittle, pale yellow foam (2.21 g, MS (FAR) 728; TLC (1:1 CHCl 3 /EtOAc) Rf=0.28; IH NMR CCDCl 3 300 MHz, ppm) 84 consistent with structure and indicative of diastereoners.

E. The product of Example 51D (0.15 g, 0.21 mmol) was deprotectei and purified as described in Example SOF.

The product was obtained as an off-white solid (0.127 g, MS (FAB) 672 695 (M+Na) TLC 1:0. 1 CHCl 3 /MeOH/AcOH) Rf=0.54; 41 NMR (c[-DMSQ, 300 MHz, ppm) consistent with structure and -indicative of diaastereomers.

Compound 365 A. The product from Example 51E (0.100 g, 0.15 mmol; 4methoxybenzylamine (20 uL, 0. 15 mxnol) and TBTU 0482 g, 0.15 mmnol) in 1NMP (0.3 m~L) were treated with iPr.-NEt (78 ul-, 0. 45 mrnol) .After stirring overnight at RTA, the reaction was di luted wi th EtOAc (10 rnL) washed wit- H.

x 2 niL) 5 citric acid (2 x 2 mL) NaHC., (2 x 2 rnL) and brine (1 x 2 niL) and dried (MgSO 4 Filtrataion :through a short pad of SiO:, eluting with MeOH/CHC1> followed by 4'r MeOH/C HCIl 4 provided product as a foan (0.087 g, MS (FAB) 792; TLC (9:1 C'HClJMeOH-) R,=0.41; HI NMR (CDC.L;, 300 MHz, ppm) consiscent wir' structure and indicative of ciastereomers.

B. A suspension of the product of Example 52A (0.087 g, 0.11 nunol) and Degussa type E101 NE/W l0 Pd/C (0.017 g) in MeOH (10 niL) was hydrogenated under 25 psi H. for 18 hr. The reaction was filtered through Celite, rinsing with MeOH. 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-CSI This assay was used to assess the potency of VLA4-directed inhibitory compounds of this invention.

10 1. Coniuaation of CS1 to BSA 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-GIn- Leu-Val-Thr-Leu-Pro-His-Pro-Asn-Leu-His-Gly-Pro-Glu-Ile- Leu-Asp-Val-?ro-Ser-Thr ("Cys-Tyr-CSl peptide"), which we synthesized by conventional solid phase chemistry and urified by HPLC, was dissolved in 10mM HEPES pH 5, 50 mM NaC1 and 0.1 mM EDTA also at a concentration of 10 mg/mL.

We then mixed 500 L of BSA-SMCC, 250 pL of Cys-Tyr-CSI 20 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 pL 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. Prepararion 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

CO

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 1% 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 prior to running the assay, we added 2',7'-bis-(2- 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 106 cells/mL). We incubated the cells for 20 minutes at 37 0

C.

20 Following labelling, the cells were washed twice in assay buffer (24 mM TRIS, 137 mM NaCI, 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. Running 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 UL 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 uL 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 FS) FN)]/(FT x 100 binding, where FTB is total fluorescence bound to CS1-containing wells without added inhibitor; as 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 a a. a.

Cmpd IC,, Cmpd IC,, Cmpd Cmpd _Ic.

1 A 30 C 59 B 88 c 2 A 31 C 60 C 89 -C 3 A 32 C 61 B 90 C 4 A 33 C 62 C 91 C C 34 B 63 C 92 c 6 C 35 B 64 C 93 C 7 C 36 C 65 C 94 C 8 C 37 C 65 c 95 C 9 C 38 C 67 C 96 C C 39 C 68 C97

C

11 C 40 C 69 C 98 C 12 C 41 B 70 C 99 C 13 B 42 B 71 C 100 C 14 C 43 B 72 C 101 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 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 112T 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 1 f _fl V I-1 1F;iC 89 3 U Cmpd# Cmpd Om d _aa p s Cmp I C= 117 C 146 A 175 B 204 B 118 C 147 A 176 C 205

B

119 C 148 A 177 C 206 A 120 121 122

C

C

C

150 151

C

C

179 180 I S S

OSS*

*SS S 123 124 125 126

C

152 153 C 181 182 I 18

C

B

C

C

208 209 210 211

B

A

B

B

B

B

C

154 Wc

C

211 U-ff v I ^Z13 127 128 129 130 131 132 133 134 135

I

C

C

C

156 157 158

B

B

C

185 186 187

C

C

C

214 215 216

B

B

B

B

C

C

C

C

C

C

C U I U

I

216 159 160 161 162

C

C

C

C

188 189 190 191

C

C

C

C

217 218 219 220

B

B

A

A

163 164

I

C

B

1 C 2201

A

192 193

B

C

221 222"

A

A

136 C 165 C 194 nd 223 A 137 C 166 C 195 C 224 A 138 C 167 C 196 C 225 A 139 C 168 B 197 B 226 A 140 C 169 C 198 C 227 A 141 C 170 C 199 B 228 A 142 C 171 C 200 B 229 A 143 C 172 C 201 B 230 A 144 A 173 C 202 B 231 A 14A A 174 R m _A )323 A 90 Cmpd IC Cmpd IC, Cmpd IC, 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 _61 A ?Qn A Q19 R B C 91

S

Cmp Ic. Cmpd lc" Cmpd lC", Cmpd

IC,

349 C 366 C 383 nd 401 fld 350 C 367 C 384 nd 402 nci 351 C 368 C 385 nd 403 C 352 C 369 C 386 C 404 C 5353 C 370 C 387 C 405 C 354 C 371 B 388 nd 40 6 C 355 nd 372 C 389 nd 407 -C 356 nd 373 C 390 C 408 C 357 nd 374 C 391 C 409 B1 358 A 375 B 392 nd 410 B 359 A 376 C 393 C 411 A 360 C 377 nd 394 C 1412 _B 361 C 378 nd 395 C 413 B 362 C 379 nd 396 n d 414 B 363 C 380 C 398 1 415 B 364 B 381 nd 399 41jj6_ B R nd Table abbreviatons: A -,c5Onm; 8B- 50nm-l Opm: C 10Ottm: nd not determined. All compounds tested in this table demonstrated an IC.1: 1 MM.

Example E Direct Bindina Of VLAZI-Presenting Ce''s 7o vcAM-IOG We next examined the ab'L2::y of the compounos of this invention to inhibit VCAiE/VLA4e binding, utiliZ17 2 z a VCAYM-IgG--alkaline phosphatase con -ugate. To carry this assay, we used the Millipore Mitiscreen Assay System (Millipore Corp., Bedford, MA) to wash the cells efficiently.

1. Preparation of VCAM-IgG-AP conjugates The construction of VCAM 2D-IgG expression vectors, transfection of CHO 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 1p 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 with 100 pl of sulfo-SMCC (30 mg/ml in water) and 100 pi 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 0

C.

2. Bindina 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 pL 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 pL was added to every well of the washed plate.

We diluted the cell adhesion inhibitor being tested to 2x final concentration in assay buffer and S. added 25 L of each dilution to triplicate wells in the plate. Final concentrations used ranged from 0.01 nM 10 pM. 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 106/mL in assay buffer containing 2 mM MnC1 2 We added 50 pl of Jurkat cells to every well, except the non-specific binding wells, which received 50 uL 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, S 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 ZnC1 2 pH '10.5 (substrate buffer) and added 100 pL immediately S* 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 pi 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 As) As)]/[(AT 3 x 100 binding, where A.s is the absorbance at 405 nm of CSl-containing wells without added inhibitor; is the absorbance at 405 rn. 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, 1 0 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 encourage mild inflammation. Twenty four hours after the initial sensitization we again sensitized the mouse with 15 25

U

L 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.

On 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 1 0

P

L 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 pL 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 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: r I (mean negative control group (mean test group ear I ear swelling) sweiino x 00 I mean negative control group ear swelling L

J

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.

Example 56 Inhibition Of Ascaris Antigen-Induced Late 97 Phase Airway Sensitivity In Alleraic Sheep 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 a'l of the VLA-4 inhibitors of this invention inhibited the airway responses associated with administration of Ascaris suum antigen.

15 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) STREET: 14 Cambridge Center (Cl CITY: Cambridge (E STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 02142 (G TELEPHONE: 617-679-2200 (H TELEFAX: 617-679-2838 (A NAME: Ko-Chung Lin (US only) (E STREET: 253 Lincoln Street (C CITY: Lexington STATE: Massachusetts (F COUNTRY: United States of America (F POSTAL CODE (ZIP): 02173 (A NAME: Steven P. Adams (US only) (B STREET: 12 Berkley Lane 9 (C CITY: Andover *9o* (2 STATE: Massachusetts (F COUNTRY: United States of Ameri-ca (F POSTAL CODE (ZIP) 01810 ,NAr,"E: Alfredc C. Castro US only! STREET: 31 Glenwood Avenue (C CITY: Woburn STATE: Massachusetts (7 COUNTRY: United States of America (F POSTAL CODE (ZIP): 01801 NA:E: Craig N. Zimmerman 'US only) (7 STREET: 134 Highland Avenue #6 :2 CITY: Somerville STATE: Massachusetts COUNTRY: United States of America (F POSTAL CODE (ZIP): 02143 (z NAME: Julio Hernan Cuervo 'US only) (B STREET: 16 Elmer Street #303 (C CITY: Cambridge C- STATE: Massachusetts (E COUNTRY: United States of America (F POSTAL CODE (ZIP): 02138 (A NAME: Wen-Cherng Lee (US only) (B STREET: 192 Spring Street (C CITY: Lexington (7 STATE: Massachusetts (7 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 ".eS cniy.

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 ADHESION INH:B3TORS diii) NUMBER OF SEQUENCES: 4 fiv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release t Version #1.30C EPO; (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 00/492,237 FILING DATE: 1l-JUL-1995 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 100 a a S4 a a.

(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 (iii) 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) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Leu Asp Val Pro Ser 1 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 101 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Cys Tyr Asp Giu Leu Pro Gin Leu Val Thr Leu Pro His Pro Asn Leu 1 5 10 His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

Claims (28)

1. A cell adhesion inhibitory compound of formula too 0: and pharmaceutically acceptable derivatives thereof; wherein: Z is selected from the croup consisting of aliphatic acyl optionally substituted with N- alkyl- or N-arylamido; aroyl; heterocycloyl; alkyl- or arylsulfonyl; aralkylcarbonyl optionally substituted with aryl1; heterocycloalkylcarbonyl; alkoxycarbonyl; aral*:-.lloxycarbonyl; cycloalkylcarbonyl optionally fused with ary:; heterocycloalkoxycarbonyA'; alkylamirilocarbonyl; aryi'amino carbonyl and a'ralkylamnocarbonyl optionally subs:f.tuted with bis (alkylsul fony.' amino, alkcxvcarbonylamino or alkenyl1; alkylsulfonyl; S aralkylsulfonyl; arylsulfonyl: cyzloal kylsulfonyl opticna1"y fused with aryl; heterc-cyclylsulfonyl; heter cyclylalkylsulfonyl; araikoxycarbonyl; ary-lcxvcarbonyl; cycloalkyloxycar*-cnyl; heterocyclyloxycarbonyl; heterocyzlylalkoxycarbonyl; mono- or di-alkylaminocarbonyl op':ionally substituted with aryl1; (alkyl) (aralkyl)aminocarbonyl; mono- or di- aralkviaminocarbonyl; mono- or di-arylaminocarbonyl; (ary.' (alkyl)aminocarbonyl; mono- or di- cycloalkylami.nocarbonyl; heterocyciylaminocarbonyl; heterocyclylal kylaminocarbonyl;. (alky.') (heterocyclyl) aminocarbony..; 103 (alkyl) (heterocyclylalkyl) aminocarbonyl; (aralkyl) (heterocyclyl) aminocarbonyl; (aralkyl) (heterocyclylalkyl) aminocarbonyl; alkenoyl optionally substituted with aryl; alkenylsulfonyl optionally substituted with aryl; alkynoyl optionally substituted with aryl; alkynylsulfonyl optionally substituted with aryl; cycloalkenylcar-bonyl; cycloalkenylsulfonyl; cycloalkylalkanoyl; cycloalkylalkylsulfonyl; arylaroyl, biarylsulfonyl; alkoxysulfonyl; aralkoxysulfony!; alkylaminosulfonyl; aryloxysulfonyl; arylaminosul fonyi'; N-arylurea- 0: substituted alkanoyl; N-arylurea-substituted alkylsulfonyl; cycloalkenyl-substituted carbonyl; cycloalkenyl-substituted sulfonyl; alkenoxycarbonyl optionally substituted with ary'l; alkenoxysulfonyl optionally substituted with aryl; alkynoxycarbonyl optionally substituted with aryl; alkynoxysulfonyl optionally substituted with aryl; alkenyl- or alkynyl- aminocarbonyl optionally substituted with aryl; alkenyl- or alkynyl-aminosulfonyl optionally substituted with aryl; acylamino- subs t.itutej alkanoyl; acylamnino- substituted alkylsulfonyl; aminocarbonyl-substituted alkanoyl; carbamoyl-substitutei alkanoyl; carbamoyl- substituted alkylsulfonyl: heterocycivialkanoyl; heterocyclylaminosulfonyl; carboxyalkyi-substituted aralkoyl; carboxyalkyl-substituted aralkylsulfonyl; oxocarbocyclyl-fused aroyl; oxocarbocyclyl-fused arylsulfonyl; heterocyclylalkanoyl; N' ,N'-alkyl, arylbydrazinocarbonyl; aryloxy-substituted alkanoyl and heterocyclylalkylsulfonyl. Y' is 2 V' is -N(R 1 104 each Y' is represented by the formula C (R 2 (A 3 each RI is independently selected from the group consisting of hydrogen, alkyl, and aralkyl; alkenyl; alkynyl; cycloalkyl; cycloalkenyl; cycloalkylalkyl; aryl; aminoalkyl; mono- or di-al kyl -subs ti tuted aminoalkyl; mono- or di-aralkyl-substizuted arninoalkyl; hydroxyalky'L; alkoxyalkyl; mercaptoalkyl; thioalkoxyalkyl A: is selected from the group consisting of amino acid side chains and corresponding protected derivatives; cycloalkyl; andi aikyl optionally substitutecd with amino, acylainino, amino-substituted aCylamino, *a I akoxycarbonyl amino, aryl1, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, ar-alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl) (aralkyl) aminocarbonv', aralkylaminocarbonyl1, diaral kvlaminocarbony., hv"tiroxyl, carboxyalkylaminocarbonyl, hy.,crox %I i.-noca rbonyi, mercapto, thioalkoxy or heT-er-o--cce; is selected 'from the group cons-,sting of :acidic functional groups and aiky. optionally substr:utei witih an acidic functional group, protected acidic functional group or aryl; each A' is independently selected fromr the grcuz consisting of amino acid side chains and corresponding protected derivatives; aryl-; cycloalkyl; and alkyl optionally substituted with amino, acylamino, amino- substituted acylamino, aryl, cycloalkyl, carboxy, aikoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbony.L, aminocarbonyl, al kylaminocarbonyi, dial kylaminocarbonyl, (alkyl) (aralkyl)aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, hydroxyl, 105 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' 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; alkylarnino optionally substituted with hydroxy, aminocarbonyl, N- alkylaminocarbonyl, carboxy or alkoxycarbonyl; and dialkylamino; cycloalkylamino; dicycloalkylamino; cycloalkylalkylamino; (alkyl) (aryl) amino; aralkylamino optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle; and (mono- or bis-carboxylic acid) -substituted alkylamine; heterocyclylamino; heterocyclyl -substituted alkylamino, and wherein the compound of formula I is expressly not N'- carboxymethyl-N- (phenylacetyl-L-leucyl-L-aspartyl-L- phenylalanyl-L-prolyl) piperazine when Z=phenylacetyl, Y 1 Y 2 y 3 n=2, and X=4- ~:carboxymethylpiperazinyl) and expressly not phenylacetyl- L-leucyl-L-aspartyl-L-phenylalanyl-D-proline amide when Z=phenylacetyl, Y 1 Y 2 y 3 n=2, and X=NH; with the proviso that Z is not benzoyl, phenylacetyl, pyridinylcarbonyl, pyridinylacetyl, anilinocarbonyl, 3- quinolinoyl, pyrazolylcarbonyl, tryptophyl, and 3,4- dihydroxylbenzoyl when y 2 is D, y 3 is V, I, F, P, W, Y or L and h is I; and when Y' is D and n is 0.

2. The cell adhesion inhibitory compound according to claim 1, wherein: Z is selected from the group consisting of alkyl; aliphatic acyl optionally substituted with N-alkyl- or N-arylamido; aroyl; heterocycloyl; alkyl- and arylsulfonyl; aralkylcarbonyl optionally substituted with 106 aryl; heterocycloalkylcarbonyl; alkoxycarbonyl; aralkyloxycarbonyl; cycloalkylcarbonyl optionally fused with aryl; heterocycloalkoxycarbonyl; alkylaminocarbonyl; arylaminocarbonyl and aralkylaminocarbonyl optionally substituted with bis-(alkylsulfonyl)amino, alkoxycarbonylamino or alkenyl; each RI 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- Salkylaminocarbonyl, carboxy or alkoxycarbonyl; dialkylamino; cycloalkylamino; dicycloalkylamino; cycloalkylalkylamino; (alkyl)(aryl)amino; aralkylamino optionally substituted with carboxy; diaralkylamino; 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, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl)(aralkyl)aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, alkoxycarbonylamino, mercapto, thioalkoxy or heterocycle. 107

4. The cell adhesion inhibitory compound according to claim 3, wherein A 1 is selected from the group consisting of aminocarbonylethyl, benzyl, n-butyl, isobutyl, carboxyethyl, cyclohexyl, 1-hydroxyethyl, hydroxymethyl, mercaptomethyl, 1-methylpropyl, methylthioethyl, n-propyl, isopropyl, methoxycarbonylaminobutyl,

6-aminohexanoylaminobutyl and (when A: and R' are taken together) azetidine, aziridine, Spyrrolidine, 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-methylpropyl, n-propyl and isopropyl. 6. The cell adhesion inhibitory compound according to claim 4, wherein A: is (when A' and R: are taken together) pyrrolidine.

7. 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)

8. The cell adhesion inhibitory compound according to claim 7 wherein A 2 is selected from the group 108 consisting of carboxymethyl, 2-carboxyethyl, 1- carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl, imidazolylmethyl, N-Bn-imidazolylmethyl, phenyl, carbomethoxymethyl, carbobenzyloxymethyl, and (when A 2 and R I are taken together) azetidine, aziridine, pyrrolidine and piperidine.

9. The cell adhesion inhibitory compound according to claim 8 wherein A 2 is selected from the group consisting of carboxymethyl, 2-carboxyethyl, 1- o carboxyethyl, hydroxylaminocarbonylmethyl, hydroxymethyl, mercaptomethyl and imidazolylmethyl. The cell adhesion inhibitory compound according to claim 1, wherein A- is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; cycloalkyl; and alkyl optionally substituted with aryi, cycloalkyl, carboxy, hydroxylaminocarbonyl, alkoxy, aralkyloxy, mercapto, N-containing heterocycle, carboxyalkylaminocarbonyl or amino-substituted acylaminc.

11. The cell adhesion inhibitory compound according to claim 10, wherein A' is independently selected from the group consisting of amino acid side chains and corresponding protected derivatives; cyclohexyl; and alkyi optionally substituted with pheny, cyclohexyl, carboxy, hydroxylaminocarbonyl, methoxy, benzyloxy, mercapto, N-benzylimidazolyl, biotinyl, tetrazolyl, valinyl-N-carbonyl or 6-aminohexanoylamino. 109

12. 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.

13. The cell adhesion inhibitory compound according to claim 1, wherein: n is 2; Y is leucinyl; Y. is aspartyl; and Y 3 is valinylprolinyl. S.

14. 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 dialkylamino optionally substituted wizh hydroxy, aminocarbonyl, N- alkylaminocarbonyl, carboxy or alkoxycarbonyl; dialkylamino; cycloalkylaminc; cycloalkylalkylamino; dicycloalkylamino; (alkyl)(aryl)amino; aralkylamino optionally substituted with carboxy; diaralkylamino; arylamino; N-containing heterocycle; bis-carboxylic acid- substituted alkylamine and (mono- or bis- carboxy)methylaminocarbonyl-substituted-N-containing heterocycle. The cell adhesion inhibitory compound according to claim 14, wherein X is selected from the group consisting of amino, methylamino, isopropylamino, isobutylamino, n-butylamino, t-butylamino, isoamyl, isopentylamino., hexylamino, cyclohexylamino, cyclohexylmethylamino, methylphenylamino, l10a phenylmethylamilo, phenyl amino, 4- me thoxyphenylmethYl aino, dimethylamino, diisopropylamino, di i obutyl amino, hydroxy, methoxy, n- butoxy, t-butoxy, benzyloxy, 2-piperidineCarboxylic acid, N -bis-carboxymethyl) -2-piperidinecarboxamide, N' carboxymethyl-2 -piperidinecarboxamide, 1 -hydroxymethyl -2- methyipropyl amino, 1-N' -methylamido--methylethylamino, 3 -dimethylbutyl am~ino, 1 -N'-methyl amidobutyl aino, 1- amino- 2 methylbutyl amino, 1 -carbomethoxy-2 methylbutylamino, 1 -N'I -methylamido- 2 -methylbutyl amino, 1- craboxy- 1-phenylmethylamino, morpholino, piperidinyl, N- phenylpiperazinyl, pipecolinyl, and piperazinyl.

16. The cell adhesion inhibitory compound according to claim 1, wherein Z is selected from the group consisting of aliphatic acyl, aroyl, aralkylcarbonyl, heterocycloyl, alkoxycarbonyl, aralkyloxycarbonyl and heterocycloalkylcarbolyl.

17. The cell adhesion inhibitory compound according to claim 16, wherein Z is a (N-Ar' -urea) -para- substituteclLaralkylcarbonyl group.

18. The cell adhesion inhibitory compound according to claim 17, wherein Z is a (N-Ar'-urea) -para- substituted phenylmethylcarbonyl group.

19. The cell adhesion inhibitory compound according to claim 2 selected from the group consisting of compound numbers 1 (L-Proline, 1-(IN-[IN-IN- 1 4- I (phenylamino] carbonyl amino] (3 -methoxyphenyl) I acetyl I -L 110Ob leucyl] -L-a-aspartyl] -L-valyl] 2 (L-Proline, 1- EN- [N- II phenylamino] carbonyl] amino] (3- methoxyphenyl) Iacetyl] -L-methionyl] -L-a-aspartyl] -L- valylI-), 4 (L-Valine, 1 N N 5 2 methyiphenyl) amino] carbonyl] amino] (6-methoxy-2-pyridyl)] acetyl] -L-leucyl] -L-a-aspartyl] 144 (L-Prolifle, 1-[N- EN- (2- hydroxy) phenylamino] carbonyll amino] phenyl] acetyl] -L- leucyl] -L-a-aspartyl] -L-valyl] 145 (L-Proline, 1- EN- [N [[Cphenylamino] carbonyl]I amino) phenyl]I acetyl]I -L leucyl] -L-a-aspartyl] -L-valyl] 146 (L-Proline, 1-E[N- [N- EN- hydroxy) phenylamino] carbonyl] amino] phenyl] acetyl] -L- methionyll -L-a-aspartyl] -L-valyl] 147 (L-Prolinamide, 1 EN- [4 [phenylamino] carbonyl]I amino] (3 methoxyphenyl) I acetyl] -L-leucyl] -L-a-aspartyl] -L-valylI 148 (L-Prolinamide, 1-E[N- methyiphenyl) amino] carbonyl] amino] phenyl] acetyl] -L- leucyl] -L-a-aspartyl] -L-valyl] 206 (L-Proline, 1- [N- methyiphenyl) amino] carbonyl] amino] phenyl] acetyl] -L- leucyl] -L-a-aspartyl] -L-valyl] 315 (L-Proline, 1- EN- methyiphenyl) amino] carbonyl] amino] phenyl] acetyl] -L- methionyl] -L-a-aspartyl] -L-valyl] 316 (L-Proline, 1- [N- [4 [(2-pyridylamino] carbonyl] amino] (phenyl) I acetyl]I L-leucyl] -L-a-aspartyl] -L-valyl] 317 (L-Prolinamide, 1- [[phenylaminol carbonyl] amino] (3- methoxyphenyl)]acetyl] -L-leucyl] -L-a-aspartyl] -L-valyl] 337 (L-Serine, 1-E(N-E[N- EN- [(2-methyl phenyl) amino] carbonyl] amino] phenyl] acetyl] -L-leucyl] -L-a- aspartyl] -L-valyl] -L-prolyl] 338 (L-threonine, 1-EN- EN- methyiphenyl) amino] carbonyl] amino] phenyl] acetyl] -L- leucyl] -L-a-aspartyl] -L-valyl] -L-prolyl] -L-seryl] 345 (L-Valine, 1- EN- methyiphenyl) amino) carbonyl] amino] phenyl] acetyl] -L- leucyl) -L-a-aspartyl] 346 (L-Proline, 1- 110c methoxy-2-pyridlyail carbonyl] amino] (phenyl) I acetyl) -L- leucyl] -L-a-aspartyl] -L-valyl] 347 (L-Proline, [N- fluorophenyl) amino] carbonyh] amino] phenyl] acetyl] -L- leucyl]-L-a-aspartyl]-L-valYll-), 357 (L-Prolinamide, 1- pyridyl) amino] carbonyl] amino] phenyl] acetyll leucyl) -L-a- aspartyl]-L-valyl]-), 358 (L-Proline, methyiphenyl) amino] carbonyl]I amino] phenyl]I acetyl]I -L leucyl]-L-a-aspartyl]-L-valyl]-, compound with 2 equivalents of 2-amino-2 (hydroxymethYl) 3-propanediol) and 359 (L-Proline, I- methyiphenyl) amino] carbonyl] amino] phenyl] acetyl] -L- leucyll -L-a-aspartyl] -L-valyh] disodium salt). WO 97/03094 PCT/US96/1570 111 The cell adhesion inhibitory compound according to claim 19 selected from the group consisting of compound numbers 1, 206, 316, 358 and 359.

21. The cell adhesion inhibitory compound according to claim 20 selected from the group consisting of compound numbers 358 and 359.

22. A pharmaceutical composition comprising a compound according to any one of claims 1 to 21 in an amount effective for prevention, inhibition or suppression of cell adhesion and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition according to claim 22, further comprising an agent selected from the group consisting of corticosteroids, bronchodilarors, antiasthmatics, antiinflammatories, antirheumatics, immunosuppressants, antimetabolites, immunomodulators, antipsoriatics and antidiabetics.

24. A method of preventing, inhibiting or suppressing cell adhesion in a mammal comprising the step of administering to said mammal the pharmaceutical composition according to claim 22 or 23. The method according to claim 24, wherein said method is used for preventing, inhibiting or suppressing inflammation. 112

26. The method according to claim 25, wherein said inflammation is cell-adhesion associated inflammation.

27. The method according to claim 24, wherein said method is used for preventing, inhibiting or suppressing an immune or autoimmune response.

28. The method according to claim 27, wherein said immune or autoimmune response is cell-adhesion associated immune or autoimmune response.

29. The method according to claim 24, 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. A cell adhesion inhibitory compound of 20 formula Z- (y 2 3 ),x and pharmaceutically acceptable derivatives thereof; wherein: Z is a (N-Ar-urea) -para-substituted aralkylcarbonyl group; Y' is -N(R -C(R 2 Y 2 is -N(R)-C(R 2 (A 2 )-C 0 each Y' is represented by the formula C(R 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 aminoalkyl; 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 113 derivatives; cycloalkyl; and alkyl optionally substituted with amino, acylamino, amino-substituted acylamino, alkoxycarbonylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl) (aralkyl) aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, hydroxyl, carboxyalkylaminocarbonyl, hydroxylaminocarbonyl, mercapto, thioalkoxy or heterocycle; A is selected from the group consisting of acidic functional groups and alkyl optionally substituted q 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; cycloalkyl; and alkyl optionally substituted with amino, acylamino, amino- substituted acylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl) (aralkyl) aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, hydroxyl, carboxyalkylaminocarbonyl, hydroxylaminocarbonyl, mercapto, thioalkoxy or heterocycle; or R 1 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; alkylamino optionally substituted with hydroxy, aminocarbonyl, N- alkylaminocarbonyl, carboxy or alkoxycarbonyl; dialkylamino; cycloalkylamino; dicycloalkylamino; cycloalkylalkylamino; (alkyl) (aryl) amino; aralkylamino optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle; and (mono- or bis-carboxylic 114 acid)-substituted alkylamine; heterocyclylamino; heterocyclyl-substituted alkylamino.

31. The cell adhesion inhibitory compound according to claim 30, wherein each Y' is independently selected from the group consisting of amino acids and corresponding protected derivatives.

32. The cell adhesion inhibitory compound 1. 0 according to claim 30, wherein: n is 2 o Y' is leucinyl; Y 2 is aspartyl; and y 3 is valinylprolinyl.

33. The cell adhesion inhibitory compound according to claim 30, wherein Z is a (N-Ar'-urea)-para- substituted phenylmethylcarbonyl group.

34. A cell adhesion inhibitory compound of formula Z- (y 2 (y 3 (I) and pharmaceutically acceptable derivatives thereof; wherein: Z is selected from the group consisting of alkyl; aliphatic acyl optionally substituted with N-alkyl- or N-arylamido; aroyl; heterocycloyl; alkyl- or arylsulfonyl; aralkylcarbonyl optionally substituted with aryl; heterocycloalkylcarbonyl; alkoxycarbonyl; aralkyloxycarbonyl; cycloalkylcarbonyl optionally fused with aryl; heterocycloalkoxycarbonyl; alkylaminocarbonyl; arylamino carbonyl and aralkylaminocarbonyl optionally substituted with bis(alkylsulfonyl)amino, alkoxycarbonylamino or alkenyl; alkylsulfonyl; aralkylsulfonyl; arylsulfonyl; cycloalkylsulfonyl optionally fused with aryl; heterocyclylsulfonyl; heterocyclylalkylsulfonyl; aralkoxycarbonyl; aryloxycarbonyl; cycloalkyloxycarbonyl; 115 heterocyclyloxycarbonyl; heterocyclylalkoxycarboiyl; mnono- or di-alkylaminocarbonyl optionally substituted with aryl; (alkyl) (aralkyl)aminocarbonyl; mono- or di- aralkylaminocarbonyl; mono- or di-arylaminocarbonyl; (aryl (alkyl)aminocarbonyl; mono- or di-cycloalkylaminocarbonyl; heterocyclylaminocarbonyl; heterocyclylalkylaminocarbonyl; (alkyl) (heterocyclyl) aminocarbonyl; (alkyl) (heterocyclylalkyl) aminocarbonyl; (aralkyl) (heterocyclyl) aminocarbonyl; (aralkyl) (heterocyclylalkyl)aminocarbonyl; alkenoyl optionally substituted with aryl; alkenylsulfonyl optionally substitutd with aryl; alkynoyl optionally substituted with aryl; alkynylsulfonyl optionally substituted with aryl; *cycloalkenylcarbonyl; cycloalkenylsulfonyl; cycloalkylalkanoyl; cycloalkylalkylsulfonyl; arylaroyl, C::biarylsulfonyl; alkoxysulfonyl; aralkoxysulfonyl; alkylaminosulfonyl; aryloxysulfonyl; arylaminosulfonyl; N- arylurea-substituted alkanoyl; N-arylurea- substituted .alkylsulfonyl; cycloalkenyl-substituted carbonyl; cycloalkenyl-substituted sulfonyl; alkenoxycarbonyl opinal susiue.ih ay; aknxsloy optionally substituted with aryl; alkenoxysulfonyl *optionally substituted with aryl; alkynoxycarbonyl optionally substituted with aryl; alkenyl- or alkynyl- aminocarbonyl optionally substituted with aryl; alkenyl- or alkynyl-aminosulfonyl optionally substituted with aryl; acylamino-substituted alkanoyl; acylamino- substituted alkylsulfonyl; aminocarbonyl-substituted alkanoyl; carbamoyl-substituted alkanoyl; carbomoyl -substituted alkylsulfonyl; heterocyclylalkanoyl; heterocycly].aminosulfonyl; carboxyalkyl -substituted aralkoyl; carboxyalkyl-substituted aralkylsulfonyl; oxocarbocyclyl-fused aroyl; oxocarbocyclyl -fused arylsulfonyl;. heterocyclylalkanoyl; N' -alkyl, arylhydrazinocarbonyl; aryloxy-substituted alkanoyl and heterocyclylalkylsulfonyl. Y" i s -N C(R 2 -C 116 0O S S S S. S S S. 55 S S *SS* S S S 5*SS *5 S SS. S S. S S S* Y 2 i s -N (R1) -C (R 2 WA) -C each y 3 is represented by the formula -N (R 1 C (R 2 (A 3 _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-alkyl-substituted aminoalkyl; mono- or di-aralkyl -substituted aminoalkyl; hydroxyalkyl; alkoxyalkyl; mercaptoalkyl; thicalkoxyalkyl 10 A' is selected from the group consisting of amino acid side chains and corresponding protected derivatives; cycloalkyl; and alkyl optionally substituted with amino, acylamino, amino-substituted acylamino,. alkoxycarbonylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl) (aralkyl) aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, hydroxyl, carboxyalkylaminocarbonyl, hydroxylaminocarbonyl, 20 mercapto, thioalkoxy or heterocycle; A2 is selected from the group consisting of acidic functional groups and alkyl. optionally substitutes with an acidic functional .group, protected acidic functional group or aryl; each A' 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, amino- substituted acylamino, aryl, cycloalkyl, carboxy, alkoxy, aralkyloxy, alkoxycarbonyl, aralkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, (alkyl) (aralkyl) aminocarbonyl, aralkylaminocarbonyl, diaralkylaminocarbonyl, hydroxyl carboxyalkylaminocarbonyl, hydroxylaminocarbonyl, mercapto, thioalkoxy or heterocycle; or R1 and any A are taken together with the atoms to which they are attached form a 3- to 6-membered 117 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; alkylamino optionally substituted with hydroxy, aminocarbonyl, N- alkylaminocarbonyl, carboxy or alkoxycarbonyl; and dialkylamino; cycloalkylamino; dicycloalkylamino; cycloalkylalkylamino; (alkyl) (aryl) amino; aralkylamino optionally substituted with carboxy; diaralkylamino; arylamino; heterocycle; and (mono- or bis-carboxylic acid) -substituted alkylamine; heterocyclylamino; heterocyclyl-substituted alkylamino, 15 and wherein the compound of formula I is expressly not N'- carboxymethyl-N-(phenylacetyl-L-leucyl-L-aspartyl-L- phenylalanyl-L-prolyl)piperazine when Z=phenylacetyl, Y 2 Y 3 n=2, and X=4- carboxymethylpiperazinyl) and expressly not phenylacetyl- 20 L-leucyl-L-aspartyl-L-phenylalanyl-D-proline amide Swhen Z=phenylacetyl, Y=L, Y 2 Y 3 n=2, and X=NH; with the proviso that X is not a cyclic ring containing or 6- atoms including a ring nitrogen atom forming an amide bond with the carboxyl group of Y 3 when Y 3 is V, I, F, P, W, Y, or L, and n is 1; and with the proviso that X is not an amido group substituted with a 6- or 7- membered ring or a fused 6,6- or 6,7- member lactam ring, when n is 0. DATED this 2 5 t h day of May 2000 BIOGEN, INC. By their Patent Attorneys CULLEN CO.