Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
  • C07K5/126—Tetrapeptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/52—Cyclic peptides containing at least one abnormal peptide link with only normal peptide links in the ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to antagonists of ⁇ 4 ⁇ 7 integrin, and more particularly to cyclic peptide antagonists.
• Integrins are transmembrane receptors that are the bridges for cell-cell and cell-extracellular matrix (ECM) interactions. When triggered, integrins trigger chemical pathways to the interior (signal transduction), such as the chemical composition and mechanical status of the ECM. Integrins are obligate heterodimers, having two different chains: the ⁇ (alpha) and ⁇ (beta) subunits.
• the ⁇ 4 ⁇ 7 integrin is expressed on lymphocytes and is responsible for T-cell homing into gut- associated lymphoid tissues through its binding to mucosal addressin cell adhesion molecule (MAdCAM), which is present on high endothelial venules of mucosal lymphoid organs.
• MAdCAM mucosal addressin cell adhesion molecule
• Inhibitors of specific integrin-iigand interactions have been shown effective as anti-inflammatory agents for the treatment of various autoimmune diseases.
• monoclonal antibodies displaying high binding affinity for ⁇ 4 ⁇ 7 have displayed therapeutic benefits for gastrointestinal auto- inflammatory/autoimmune diseases, such as Crohn's disease, and ulcerative colitis.
• a multimer comprising a plurality of compounds covalently linked together, the compounds independently being of formula (I):
• R 1 is H; lower alkyl; aryl; heteroaryl; alkenyl; or heterocycie; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;
• R 2 and R 3 are each independently an amino acid chain of a proteinogenic or a non- proteinogenic alpha-amino acid, provided that R 2 and R 3 may be covalently linked to each other to form a ring;
• R 4 and R 6 are each independently H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycie; acids of the formula -C(O)OH; esters of the formula -C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula -C(O)NR**R***, wherein R** and R*** are independently selected from H, alkyl and aryl; -CH 2 C(O)R, wherein R is selected from -OH, lower alkyl, aryl, - lowera!kyl-aryl, or -NRaRb, where Ra and Rb are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; or -C(O)
• R 6 is H, lower alkyl, benzyl, alkenyl, lower alkyloxy; aryl; heteroaryl; heterocycle; -C(O)R****, wherein R**** is independently selected from alkyl, aryl, heteroaryl, amino, aminoalkyl, aminoaryl, aminoheteroaryl, alkoxy, aryioxy, heteroaryioxy; -CH 2 C(O)R; or -C(O)Rc; all of which are optionally substituted at one or more substitutabie positions with one or more suitable substituents, or along with R 7 or R 6 , a cyclic side chain of a proteinogenic or a non-proteinogenic amino acid having, the N-terminus thereof being the N-R 6 , wherein the proteinogenic or a non-proteinogenic amino acid can be substituted with a suitable substituent;
• X 1 is Leucine or tert-butyl-Ala
• X 2 is Asp
• X 3 is any amino acid listed under column X 3 of Table 1 B.
• a pharmaceutical composition comprising the multimer described herein along with the pharmaceutically acceptable carrier.
• the pharmaceutical composition may be formulated for any one of oral delivery, topical delivery and parenteral delivery.
• a method of treating inflammation or an autoimmune disease in a patient comprising administering to the patient a therapeutically effective amount of the multimer described herein.
• the inflammation or an autoimmune disease is gastrointestinal.
• a method for treating a condition in a patient associated with a biological function of an ⁇ 4 ⁇ 7 integrin the method comprising administering to the patient a therapeutically effective amount of the multimer described herein.
• a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the multimer described herein, wherein the disease or condition is a local or systemic infection of a virus or retrovirus-
• a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the multimer described herein, wherein the hepatitis A, B or C, hepatic encephalopathy, non-alcoholic steatohepatitis, cirrhosis, variceal bleeding, hemochromatosis, Wilson disease, tyrosinemia, alpha- 1 -antitrypsin deficiency, glycogen storage disease, hepatocellular carcinoma, liver cancer, primary biliary cholangitis, primary sclerosing cholangitis, primary biliary sclerosis, biliary tract disease, autoimmune hepatitis, or graft-versus-host disease.
• Figure 1 shows representative compounds of the present application, namely from the following classes, 18-membered ring, 21-membered ring, 21-membered ring (non-canonical, i.e. having a delta amino acid), 22-membered ring, and 24-membered ring.
• Figure 2 shows a representative 18-membered ring compound along with variations made at certain positions with corresponding ⁇ 4 ⁇ 7 integrin ELISA IC50 binding values associated with those variations.
• Figure 3 shows a representative 21-membered ring compound along with variations made at certain positions with corresponding ⁇ 4 ⁇ 7 integrin ELISA IC50 binding values associated with those variations.
• Figure 4 shows a representative 21-membered ring (non-canonical, i.e. having a delta amino acid) compound along with variations made at certain positions with corresponding ⁇ 4 ⁇ 7 integrin ELISA IC50 binding values associated with those variations.
• Figure 5 shows a representative 22-membered ring compound along with variations made at certain positions with corresponding ⁇ 4 ⁇ 7 integrin ELISA IC50 binding values associated with those variations.
• Figure 6A and 6B show representative NMR data for a muitimeric molecule, Compound No. 390, with 1 H- and 1 H- 1 H TOCSY NMR spectra recorded at 25 °C
• Figure 7 shows the binding to ⁇ 4 ⁇ 7 integrin measured as a MADCAM-1 competition assay in human whole blood for: a) representative monomeric Compound 456 (ET4062) and muitimeric Compound No.s 534 (ET4113) and 535 (ET4110), derived from Compound 456, and; b) representative monomeric Compound 340 (ET2451) and muitimeric Compound No.s 390 (ET3755) and 517 (ET3764), derived from Compound 340.
• Figure 8 Shows the detection of ⁇ 4 ⁇ 7+ Th memory cells trafficking in the mesenteric lymph nodes in mice suffering from DSS-induced colitis treated for 4 days with Compound No. 517 (ET3764) or vehicle.
• Figure 9 shows the ⁇ 4 ⁇ 7+ Th memory lymphocyte content in mesenteric lymph nodes taken from mice exposed to DSS irritant and treated for 4 days with various concentrations of Compound No. 517 (ET3764) or control (SMEDDS vehicle)
• Figure 10 shows the receptor occupancy of representative multimeric compounds on ⁇ 4 ⁇ 7- positive T helper memory cells as measured in a MADCAM-1 competition assay in human whole blood.
• Figure 11 shows the receptor occupancy of representative naceilin dimers on ⁇ 4 ⁇ 7-negative Th memory cells as measured in a VCAM-1 competition assay in human whole blood.
• Table 1 shows compounds exhibiting ⁇ 4 ⁇ 7 integrin affinity, selectivity and/or activity; and specifically with respect to these compounds: (A) the structure of the linker portion; (B) the structure of the peptide portion; and (C) and (C') the affinity, selectivity and activity values.
• R2 is H and R3 is CH3, the carbon atom bearing R2 and R3 has S-configuration.
• R2 is CH3 and R3 is H, the carbon atom bearing R2 and R3 has R-configuration.
• R2 is H and R3 is CH2-S-Ph, the carbon atom bearing R2 and R3 has S-configuration.
• R4 is H and R5 is C(O)-NH-tert-Butyl, the carbon atom bearing R4 and R5 has S- configuration.
• R4 is C(O)-NH-tert-Butyl and R5 is H, the carbon atom bearing R4 and R5 has R- configuration.
• R6 and R7 are both Pro, the R6 and R7 substituents are covalently bound and form the pyrrolidine ring of Pro.
• R6 and R8 are both dPro, the R6 and R8 substituents are covalently bound and form the pyrrolidine ring of dPro. If R6 and R7 are both [(4S)-fluoro-Pro], the R6 and R7 substituents are covalently bound and form the pyrrolidine ring of [(4S)-fluoro-Pro].
• R7 is Nva and R8 is H, the carbon atom bearing R7 and R8 has S-configuration.
• R6 and R7 are both Hyp, the R6 and R7 substituents are covalently bound and form the pyrrolidine ring of Hyp.
• Table 1X is a correspondence table linking the compounds described herein with the synthesis protocols outlined in the methods and materials.
• Table 2 shows multimeric compounds exhibiting ⁇ 4 ⁇ 7 integrin affinity, selectivity and/or activity; and specifically with respect to these compounds: (A) the structure of the linker portion; (B) the structure of the peptide portion; and (C) the affinity, selectivity and activity values.
• R6 and R7 are both Pro, the R6 and R7 substituents are covalently bound and form the pyrrolidine ring of Pro. If R6 and R7 are both Hyp, the R6 and R7 substituents are covalently bound and form the pyrrolidine ring of Hyp.
• Table 2X is a correspondence table linking the multimers described herein with the synthesis protocols outlined in the methods and materials, m/z is (M + 2H/2) and additional information regarding the linker.
• a multimer comprising a plurality of compounds covalently linked together, the compounds independently being of formula (i):
• R 1 is H; lower alkyl; aryl; heteroaryl; alkenyl; or heterocycle; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;
• R 2 and R 3 are each independently an amino acid chain of a proteinogenic or a non- proteinogenic alpha-amino acid, provided that R 2 and R 3 may be covalently linked to each other to form a ring;
• R 4 and R E are each independently H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; acids of the formula -C(O)OH; esters of the formula -C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula -C(O)NR**R***, wherein R** and R*** are independently selected from H, alkyl and aryl; -CH 2 C(O)R, wherein R is selected from -OH, lower alkyl, aryl, - loweralkyl-aryl, or -NRaRb, where Ra and Rb are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; or -C(O)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl- aryl; or -lower alkyl-ORd, wherein Rd is a suitable protecting group or OH group; all of
• R B is H, lower alkyl, benzyl, alkenyl, lower alkyloxy; aryl; heteroaryl; heterocycle; -C(O)R****, wherein R**** is independently selected from alkyl, aryl, heteroaryl, amino, aminoalkyl, aminoaryl, aminoheteroaryl, alkoxy, aryloxy, heteroaryloxy; -CH 2 C(O)R; or -C(O)Rc; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents, or along with R 7 or R 6 , a cyclic side chain of a proteinogenic or a non-proteinogenic amino acid having, the N-terminus thereof being the N-R 6 , wherein the proteinogenic or a non-proteinogenic amino acid can be substituted with a suitable substituent;
• X 1 is Leucine or tert-butyl-Ala
• X 2 is Asp
• X 3 is any amino acid listed under column X 3 of Table 1B.
• Multimerized, specifically dimerized, versions of certain compounds described herein exhibited affinity, selectivity and activity, summarized in Tables 2A, 2B and 2C.
• amino acid refers to molecules containing an amine group, a carboxylic acid group and a side chain that varies.
• Amino acid is meant to include not only the twenty amino acids commonly found in proteins but also non-standard amino acids and unnatural amino acid derivatives known to those of skill in the art, and therefore includes, but is not limited to, alpha, beta and gamma amino acids
• Peptides are polymers of at least two amino acids and may include standard, non-standard, and unnatural amino acids.
• a peptide is a polymer of two or more amino acids.
• suitable substituent as used in the context of the present invention is meant to include independently H; hydroxyl; cyano; alkyl, such as lower alky!, such as methyl, ethyl, propyl, n-butyl, t-butyl, hexyl and the like; alkoxy, such as lower alkoxy such as methoxy, ethoxy, and the like; aryloxy, such as phenoxy and the like; vinyl; alkenyl, such as hexenyl and the like; alkynyl; formyl; haloaikyl, such as lower haloaikyi which includes CF 3 , CCI 3 and the like; halide; aryl, such as phenyl and napthyl; heteroaryl, such as thienyl and furanyl and the like; amide such as C(O)NR a R b, where R a and R b are independently selected from lower alkyl, aryl
• a suitable substituent as used in the context of the present invention is meant to denote a substituent that does not interfere with the formation of the desired product by the processes of the present invention.
• the term "lower alkyl” as used herein either alone or in combination with another substituent means acyclic, straight or branched chain alkyl substituent containing from one to six carbons and includes for example, methyl, ethyl, 1- methylethyl, 1-methylpropyl, 2-methyIpropyl, and the like, A similar use of the term is to be understood for "lower alkoxy”, “lower thioalkyl”, “lower alkenyl” and the like in respect of the number of carbon atoms.
• lower alkoxy as used herein includes methoxy, ethoxy, i-butoxy.
• alkyl encompasses lower alkyl, and also includes alkyl groups having more than six carbon atoms, such as, for example, acyclic, straight or branched chain alkyl substituents having seven to ten carbon atoms.
• aryl as used herein, either alone or in combination with another substituent, means an aromatic monocyclic system or an aromatic polycyclic system.
• aryl includes a phenyl or a napthyl ring, and may also include larger aromatic polycyclic systems, such as fluorescent (eg. anthracene) or radioactive labels and their derivatives.
• heteroaryl as used herein, either alone or in combination with another substituent means a 5, 6, or 7-membered unsaturated heterocyc!e containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur and which form an aromatic system.
• heteroaryl also includes a polycyclic aromatic system comprising a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur.
• cycloalkyl as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent that includes for example, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
• cycloalkyl-alkyl- as used herein means an alkyl radical to which a cycloalkyl radical is directly linked; and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopenty!methyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl and 2-cyclohexylethyl.
• alkyl or “lower alkyl” terms is to be understood for aryl-alkyl-, aryl-loweralkyl- (eg. benzyl), -lower alkyl-alkenyl (eg.
• aryl-alkyl- means an alky! radical, to which an aryl is bonded.
• aryl-alkyl- include, but are not limited to, benzyl (phenylmethyl), 1-phenylethyl, 2-phenylethyl and phenylpropyl.
• heterocycle either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a three- to seven-mem bered saturated or unsaturated (including aromatic) cyclic compound containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur.
• heterocycles include, but are not limited to, aziridine, epoxide, azetidine, pyrrolidine, tetrahydro- furan, thiazolidine, pyrrole, thiophene, hydantoin, diazepine, imidazole, isoxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine, homopiperazine, 1 ,4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide or pyrimidine, and the like.
• alkenyl as used herein, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a double bond.
• examples of such radicals include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl.
• alkynyf as used herein is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a triple bond.
• examples of such radicals include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.
• alkoxy as used herein, either alone or in combination with another radical, means the radical -0-(C 1-n )alkyl wherein alkyl is as defined above containing 1 or more carbon atoms, and includes for example methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Where n is 1 to 6, the term “lower alkoxy” applies, as noted above, whereas the term “alkoxy” encompasses "lower alkoxy” as well as alkoxy groups where n is greater than 6 (for example, n - 7 to 10).
• aryloxy as used herein alone or in combination with another radical means -O-aryl, wherein aryl is defined as noted above.
• a protecting group or protective group is a substituent introduced into a molecule to obtain chemoselectivity in a subsequent chemical reaction.
• Many protecting groups are known in the art and a skilled person would understand the kinds of protecting groups that would be incorporated and could be used in connection with the methods described herein.
• protecting group based peptide synthesis typically solid phase peptide synthesis, the desired peptide is prepared by the step-wise addition of amino acid moieties to a building peptide chain.
• the two most widely used protocols, in solid-phase synthesis employ tert-butyloxycarbonyl (Boc) or 9- fluorenylmethoxycarbonyl (Fmoc) as amino protecting groups.
• Amino protecting groups generally protect an amino group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used amino protecting groups are disclosed in Greene, T. W. et al., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons (1999).
• Amino protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o- nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl, 4-chIorobenzoyl, 4 ⁇ bromobenzoyl, 4- nitrobenzoyl, and the like; sulfonyl groups such as benzenesu!fonyl, p-toluenesulfonyl and the like; alkoxy- or aryloxy-carbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p
• Amine protecting groups also include cyclic amino protecting groups such as phthaloyl and dithiosuccinimidyl, which incorporate the amino nitrogen into a heterocycle.
• amino protecting groups include formyl, acetyl, benzoyl,, pivaloyl, t- butylacetyl, phenylsulfonyl, Alloc, Teoc, benzyl, Fmoc, Boc and Cbz. It is well within the skill of the ordinary artisan to select and use the appropriate amino protecting group for the synthetic task at hand.
• R 1 is H.
• R 2 or R 3 is covalently linked to R 1 to form proline having NR 1 as the N- terminus.
• R 2 and R 3 are not both H. In some embodiments, R 2 and R 3 are each independently selected from the group consisting of amino acid chains of a proteinogenic or a non-proteinogenic alpha-amino acids.
• R 2 and R 3 are H and CH 3 respectively or vice versa.
• R 2 or R 3 is -CH2-S-R s , wherein R s is selected from lower alkyl; lower amino alkyl; aryl; heteroaryl; alkenyl; or heterocycle; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; preferably R 5 is phenyl or phenyl substituted with lower alkyl, halogen; or lower amino alkyl.
• R 4 and R 5 are not both H.
• R** and R*** are not both H.
• R 4 and R 5 are each independently H, or C(O)-NHR', wherein R f is H or a lower alkyl.
• R t is tert-butyl or H.
• R 6 is H.
• R 6 and either R 8 or R 9 form a ring resulting in a proline residue having N- R 6 as its N-terminus.
• n is 1.
• X 1 is Leu.
• X 2 is Asp.
• X 3 is Thr. In some embodiments, X 3 is Val.
• X 3 is He.
• X y and X z are each independently a proteinogenic or non-proteinogenic alpha-amino acid.
• X z is a proteinogenic or non-proteinogenic beta-amino acid. In some embodiments, X z is betaHomoLys or MethylbetaHomoLys. In some embodiments, X y and X z are each a primary amino acid.
• X y and X z are each any amino acid listed under column X y and column X z respectively of Table 1 B.
• the compound is any one of compounds 1-389 and 456 or the multimer is any one of compounds 390-397 and 457-538.
• the mu!timer is a dimer, trimer, tetramer, or pentamer.
• the monomer compounds are linked by a linker. In some embodiments, the compounds are linked together at a carbon, nitrogen, oxygen, sulphur or other atom associated with R 2 , R 3 , R 4 , R 5 , R 6 , R 7 /R 8 , X z , or X y .
• linkers may be used to multimerize the macrocycles described herein, including esters, amides, amines or mixed amides/amines. Additional linkages include, but are not limited to, ethers, thioethers, thioesters, disulphides, sulfoxides, sulfones, sulfonamides, sulfamates, sulfamides, carbamates, ureas, carbonates, phosphodiesters, phosphonamides, phosphoramidates, heterocycles such as triazoles from azide-alkyne cycloaddition ("Click" chemistry).
• monomeric macrocycles can be covalently attached to linkers via carbon-carbon single bond linkages, carbon-carbon double bond linkages or carbon-carbon triple bond linkages.
• monomeric macrocycles can be covalently attached directly to a second, third or fourth monomeric macrocycle via any of the above linkages; in this case no formal linker moiety is present.
• the multimer is a homo-multimer.
• the multimer is a hetero-multimer.
• pharmaceutically acceptable salts of the compounds described herein there is provided pharmaceutically acceptable salts of the compounds described herein.
• pharmaceutically acceptable salt represents salts or zwitterionic forms of the compounds of the present invention which are water or oil-soluble or dispersible, which are suitable for treatment of diseases without undue toxicity, irritation, and allergic response; which are commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use.
• the salts can be prepared during the final isolation and purification of the compounds or separately by treatment of an amino group with a suitable acid.
• Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate
• amino groups in the compounds of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyi, and diamyi sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
• acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
• any of the peptide compounds described herein are salt forms, e.g., acetate salts.
• a pharmaceutical composition comprising the multimer described herein along with the pharmaceutically acceptable carrier.
• the pharmaceutical composition may be formulated for any one of oral delivery, topical delivery and parenteral delivery.
• pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
• a method of treating inflammation or an autoimmune disease in a patient comprising administering to the patient a therapeutically effective amount of the multimer described herein.
• the inflammation or an autoimmune disease is gastrointestinal.
• a method for treating a condition in a patient associated with a biological function of an ⁇ 4 ⁇ 7 integrin comprising administering to the patient a therapeutically effective amount of the multimer described herein.
• the condition or disease is Inflammatory Bowel Disease (IBD), ulcerative colitis, Crohn's disease, Celiac disease (nontropical Sprue), enteropathy associated with seronegative arthropathies, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, radiotherapy, chemotherapy, pouchitis resulting after proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, primary sclerosing cholangitis, human immunodeficiency virus (HIV) infection in the
• the condition is pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma or graft versus host disease.
• a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the multimer described herein, wherein the disease or condition is a local or systemic infection of a virus or retrovirus.
• the a virus or retrovirus is echovirus 1 and 8, echovirus 9/Barty Strain, human papilloma viruses, hantaviruses, rotaviruses, adenoviruses, foot and mouth disease virus, coxsackievirus A9, human parechovirus 1 or human immunodeficiency virus type 1.
• a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the multimer described herein, wherein the hepatitis A, B or C, hepatic encephalopathy, non-alcoholic steatohepatitis, cirrhosis, variceal bleeding, hemochromatosis, Wilson disease, tyrosinemia, alpha-1 -antitrypsin deficiency, glycogen storage disease, hepatocellular carcinoma, liver cancer, primary biliary cholangitis, primary sclerosing cholangitis, primary biliary sclerosis, biliary tract disease, autoimmune hepatitis, or graft-versus-host disease.
• the multimer inhibits binding of ⁇ 4 ⁇ 7 integrin to MAdCAM.
• the compound selectively inhibits binding of ⁇ 4 ⁇ 7 integrin to MAdCAM.
• the patient is preferably a human.
• inhibition As used herein, “inhibition,” “treatment,” “treating,” and “ameliorating” are used interchangeably and refer to, e.g., stasis of symptoms, prolongation of survival, partial or full amelioration of symptoms, and partial or full eradication of a condition, disease or disorder in a subject, e.g., a rnammal-
• prevent or “prevention” includes (i) preventing or inhibiting the disease, injury, or condition from occurring in a subject, e.g.., a mammal, in particular, when such subject is predisposed to the condition but has not yet been diagnosed as having it; or (ii) reducing the likelihood that the disease, injury, or condition will occur in the subject.
• therapeutically effective amount refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual, A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.
• the compound is administered by a form of administration selected from the group consisting of oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, nebulization, sublingual, buccal, parenteral, rectal, vaginal, and topical.
• the compound is administered as an initial does followed by one or more subsequent doses and the minimum interval between any two doses is a period of less than 1 day, and wherein each of the doses comprises an effective amount of the compound.
• the effective amount of the compound is the amount sufficient to achieve at least one of the following selected from the group consisting of: a) about 50% or greater saturation of MAdCAM binding sites on ⁇ 4 ⁇ 7 integrin molecules; b) about 50% or greater inhibition of ⁇ 4 ⁇ 7 integrin expression on the cell surface; and c) about 50% or greater saturation of MAdCAM binding sites on ⁇ 4 ⁇ 7 molecules and about 50% or greater inhibition of ⁇ 4 ⁇ 7 integrin expression on the cell surface, wherein i) the saturation is maintained for a period consistent with a dosing frequency of no more than twice daily; ii) the inhibition is maintained for a period consistent with a dosing frequency of no more than twice daily; or iii) the saturation and the inhibition are each maintained for a period consistent with a
• the compound is administered at an interval selected from the group consisting of around the clock, hourly, every four hours, once daily, twice daily, three times daily, four times daily, every other day, weekly, bi-weekly, and monthly.
• Protocol A General nacellin synthesis 1.
• Fmoc amino acid (1.1 eq. with respect to resin) was dissolved in CH 2 CI 2 (10 mL/g of resin). If the amino acid did not dissolve completely, DMF was added slowly dropwise until a homogeneous mixture persisted upon stirring/sonication. The 2-chlorotrityl resin was allowed to swell in CH 2 CI 2 (5 mL/g of resin) for 15 minutes. The CH 2 CI 2 was then drained and the Fmoc amino acid solution was added to the vessel containing the 2-CI Trt resin. DIPEA was added (2 eq. with respect to the amino acid) and the vessel was agitated for five minutes. Another 2 eq.
• Nosyl protection The deprotected resin was stirred in CH 2 CI 2 (5 mL/mmol of resin) and DIPEA (6.5 eq.). A solution of Nosyl chloride (4.0 eq.) was added slowly, dropwise, over 30 minutes, to avoid a rapid exothermic reaction. After the addition was complete, stirring was continued at room temperature for three hours. The resulting nosyl-protected resin was filtered and washed with CH 2 CI 2 , MeOH, CH Z CI 2 , and THF.
• N-Methylation To a suspension of resin in THF (10 mL/mmol of resin) was added a solution of triphenylphosphine (5 eq.) in THF (2 M) and MeOH (10 eq.). The stirring suspension was cooled in an ice bath. A solution of DIAD (5 eq.) in THF (1 M) was added dropwise, via addition funnel. After addition was complete the bath was removed and the reaction was stirred at room temperature for an additional 90 minutes. The resin was filtered, washed with THF (x4), CH 2 CI 2 (x3), and THF (x2). 5.
• Nosyl-deprotection To a suspension of resin in NMP (10 mL/mmol of resin) was added 2- mercaptoethanol (10.1 eq.) and DBU (5.0 eq.). The solution became a dark green colour. After five minutes, the resin was filtered, washed with DMF until washes ran colourless. This procedure was repeated a second time, and the resin was then washed a final time with CH 2 CI 2 ,
• Fmoc protection To a suspension of resin in CH 2 CI 2 (7 mL/mmol of resin) was added a solution of Fmoc-CI (4 eq.) in CH 2 C1 2 (7 mL), and DIPEA (6.1 eq.). The suspension was stirred at room temperature for four hours then filtered and washed with CH 2 CI 2 (x2), MeOH (x2), CH 2 CI 2 (x2), and Et 2 O (x2).
• Protocol C Reductive amination 1.
• Fmoc Weinreb amide formation a mixture of Fmoc amino acid (1 mmol), ⁇ , ⁇ - dimethylhydroxylamine-HCl (1.2 eq.), and HCTU (1.2 eq.) in CH 2 Cl 2 (6.5 mL), was cooled to 0 °C. DIPEA (3 eq.) was then slowly added to the stirring mixture. The cooling bath was removed and the reaction was stirred at room temperature for 16 h. A 10% solution of HCI (4 mL) was added resulting in the formation of a precipitate, which was removed through filtration. The filtrate was washed with 10% HCI (3 x 4 mL) and brine (2 x 4 mL). The organic phase was then dried over Na 2 SO 4 . The solvent was removed under reduced pressure to give crude Fmoc Weinreb amide, which was used in the next reaction without purification.
• Reductive amination on-resin the linear peptide on-resin was placed in a solid-phase peptide synthesis reaction vessel and diluted with DMF (22 mL/g of resin). The Fmoc aldehyde (4.0 eq.) was added and the reaction was left to shake overnight The solution was then drained and the resin was washed with CH 2 Cl 2 (x3) and DMF (x3). The resin was then diluted with a mixture of MeOH/CH 2 CI 2 (22 mL/g of resin, 1:3 ratio) and NaBH 4 (7 eq.) was subsequently added.
• Protocol D Fragment-based macrocyclization a) In a two-dram vial, 0.1 mmol of the linear peptide and DEPBT (1.5 eq.) were dissolved in 5 mL of freshly distilled THF (0.02 M). DIPEA (3 eq.) was then added and the reaction mixture was left to stir overnight at room temperature (16 h). Tetraalkylammonium carbonate resin (Biotage®, 6 eq.) was then added to the reaction mixture and stirring was continued for an additional 24 h. The reaction was then filtered through a solid-phase extraction vessel and rinsed with CH 2 CI 2 (2 mL). The filtrate and washes were combined and the solvent was removed under reduced pressure.
• Protocol E Aziridine aldehyde-based macrocvclization
• Protocol F Nucleophilic ring-opening of acyl aziridine, post macrocvclization a) Thioacetic acid/thiobenzoic acid: the corresponding thio acid (4 eq.) was added to the crude reaction mixture. Reaction progress was monitored by LC-MS, and was generally complete after 1-2 hours.
• Protocol G Suzuki coupling, post macrocvclization a
• an iodo-Phe-containing macrocycle (0,1 mmol), Na 2 C0 3 (2 eq.), substituted boronic acid (1.1 eq.) and 4 mL of water: acetonitrile (1. ⁇ ratio) were combined in a microwave vial.
• the mixture was treated with N 2 gas flow for 10 minutes.
• silicon based Pd-catalyst (Siliacat-DPP Pd heterogenous catalyst, 0.05 eq.) was added.
• the reaction vial was sealed and placed in the microwave for 10 minutes at 120 'C (reaction time and temperature were increased to 30 min.
• Suzuki couplings with macrocycles that were prepared using 3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-bromobenzoic acid were conducted as follows: A mixture of crude macrocyclic Compound 340 that had been orthogonally protected as the ⁇ -ierf-butyl ester of the Asp residue and the ferf-butyl ether of the Thr residue (200 mg, 0.22 mmol) and 4-(4-Boc-piperazino) phenylboronic acid pinacol ester (171 mg, 0.44 mmol) were dissolved in a 1,2-dimethoxyethane (5.4 mL) and Ethanol (1.2 mL
• Protocol H General Ulmann coupling, post macrocvclization
• the peptide macrocycle (0.018 mmol) was placed in a 2-dram via! containing 2 mL of dry CH 2 C1 2 .
• Cu(OAc) 2 (1 eq.), benzene boronic acid (2 eq.) and 4 A (oven- dried) molecular sieves were then added to the vial followed by DIPEA (4 eq.).
• DIPEA 4 eq.
• the contents of the vial were stirred at room temperature overnight.
• the reaction progress was assessed by LCMS. Once the reaction was deemed complete, the mixture was filtered through a Celite plug and the solvent was removed under reduced pressure.
• Protocol I General global deprotection and cleavage Deprotection of the side chain protecting groups was achieved by dissolving the peptides in 2 mL of a cleavage cocktail consisting of TFA:H 2 O:TlS (95:2.5:2.5) for two hours (for sensitive peptides the mixture of TFA:H 2 O:TIS (95:2.5:2.5) may be substituted for a mixture of TFA:CH 2 Cl 2 (50:50)). Subsequently, the cleavage mixture was evaporated under reduced pressure and the peptides were precipitated twice from chilled diethyl ether/hexanes (or ted- butyl methyl ether).
• Protocol J General cleavage of reductivelv-labile protecting groups a) Pd/C and formic acid debenzylation: the benzyl protected macrocycle (0.35 mmol) was dissolved in MeOH (8 mL) with 10% formic acid, 10 % wt. Pd/C (Sigma-Aldrich, 37 mg, 0.1 Eq) and heated to 55 °C for 1h to 4h. Once the reaction was deemed complete, the mixture was filtered through a Celite plug, washed with MeOH and the solvent was removed under reduced pressure.
• Raney Ni desulfurization/debenzylation Raney Ni slurry (1-2 mL) was added directly to the cyclization reaction mixture and stirred vigorously overnight. The vial was then centrifuged and the liquid was transferred using a pipette to a tared vial. MeOH was added to the vial containing Raney Ni. The vial was then sonicated, vortexed, and centrifuged. Again, the liquid was transferred to a tared vial. This process was repeated with EtOAc and then a final time with MeOH. The combined washes were then removed under reduced pressure and the residue dried under vacuum. Protocol K: Amidation of side chain, post macrocvclization
• the macrocycle (4 ⁇ ) was dissolved in DMSO (200 ⁇ 1_). DIPEA (5 eq.) was then added. In a separate vial, 5 mg of fluorescent dye as the NHS ester was dissolved in 200 ⁇ L of DMSO. The macrocycle solution was then added to the solution of the fluorescent label. The reaction mixture was stirred overnight. Reaction progress was checked by LC-MS in the morning and then the solvent was removed by lyophilizatton.
• Protocol N Linker synthesis for multimerization a) Preparation of Acyl chloride linkers: Di-, tri- or tetra-carboxylic acids (1 eq.) and CH 2 CI 2 (0.114 M concentration) were added to a two-dram vial. SOCl 2 (15 eq. per carboxylic acid) was then added and the reaction mixture was left to stir for four hours at room temperature (some substrates required heating at 70 °C overnight for full solution and/or conversion). The solvent was removed via N 2 flow. The residue was dissolved in 3 mL of dry CH 2 CI 2 which was then removed under N 2 flow. This process was performed two additional times in an attempt to remove any free HCl from the sample.
• MD005067-2) was treated with ⁇ /s-Benzotriazole-activated Diphenic acid (1 eq.) in CH 3 CN ,(0.011 M) containing DIPEA (10 eq.).
• the reaction mixture was stirred for 16h (monitored by LC-MS)
• the solvent was removed by rotoevaporation and the crude material was submitted to reverse-phase silica chromatography (Biotage) to obtain the purified di-terf- butyl ester intermediate. Removal of the tert-butyl ester groups was effected by Protocol I.
• the diacid linker was isolated as a crude and used as such in muitimerization reactions without further manipulation.
• the solvent was removed by rotoevaporation and the crude material was submitted to reverse-phase silica chromatography (Biotage) to obtain the purified di-ie/f- butyl ester intermediate. Removal of the tert-butyl ester groups was effected by Protocot I. The diacid linker was isoiated as a crude and used as such in muitimerization reactions without further manipulation.
• Protocol O Nacellin Muitimerization a) Muitimerization of amine-containing monomeric macrocydes using bis- or tris-acyl chloride- activated linkers: The corresponding acyl chloride (0.35 mmol, 1.0 eq.), freshly prepared and under Argon atmosphere, was dissolved in anhydrous CH 2 Cl 2 (5 mL; note that iarger scale reactions required more-concentrated solution to produce higher-yielding dimerizations). Monomeric macrocycle (2, 3 or 4 eq.
• reaction mixture was stirred for 16h (monitored by LC-MS). Nal (8.5 mg, 0.05708 mmol, 2 eq) was then added and the reaction mixture was heated at 50°C for 2h. The solvent was removed in vacuo and the crude material was submitted to reverse-phase silica chromatography (Biotage) to obtain the purified product.
• a 96-well Microlon plate (Greiner, 655001) was coated with 100 ⁇ per well of a solution of 1ng/ml recombinant integrin ⁇ 4 ⁇ 7 (R&D Systems, 5397-A3-050) in carbonate buffer (50 mM, pH 9.6). The plate was incubated at 4°C overnight. The solution was removed and 250 ⁇ blocking buffer (50 mM Tris, 150 mM NaCI, 1 mM MnCl 2 , 1 % BSA, 0,05% Tween) was added per well. The plate was then incubated for 1 hour at room temperature.
• the plate was washed three times with wash buffer (50 mM Tris, 100 mM NaCI, 1 mM MnCI 2 , 0,05% Tween).
• wash buffer 50 mM Tris, 100 mM NaCI, 1 mM MnCI 2 , 0,05% Tween.
• 50 ⁇ of compound diluted in assay buffer was added by transfer from a compound serial dilution plate.
• 50 ⁇ recombinant MAdCAM-Fc (R&D systems, 6056-MC-050) at a concentration of 0.1 ug/ml in assay buffer 50 mM Tris, 150 mM NaCI, 1 mM MnCI 2 , 0,1 % BSA, 0,05% Tween
• the plate was incubated at room temperature with shaking (300 rpm) for 2 hours to reach binding equilibrium.
• the plate was washed three times in wash buffer and 100 ⁇ anti-human IgG Fc specific-HRP (Abeam, Ab97225) diluted at 1 :2000 in assay buffer was added to each well.
• the plate was incubated at room temperature for 1 hour under agitation.
• the plate was then washed three times and 100 ⁇ of 1 ,3',5,5'- Tetramethylbenxidie (TMB, KPl 5120-0083) was then added to each well.
• TMB 1 ,3',5,5'- Tetramethylbenxidie
• a 96-well Microlon plate (Greiner, 655001) was coated with 100 ⁇ per well of a solution of 0.5 ⁇ g/ml recombinant integrin ⁇ 4 ⁇ 1 (R&D Systems, 5397-A3-050) in carbonate buffer (50 mM, pH 9,6). The plate was incubated at 4°C overnight. The solution was removed and 250 ⁇ blocking buffer (50 mM Tris, 150 mM NaCI, 1 mM MnCI 2 , 1 % BSA, 0,05% Tween) was added per well. The plate was then incubated for 1 hour at room temperature.
• the plate was washed three times with wash buffer (50 mM Tris, 100 mM NaCI, 1 mM MnCI 2 , 0,05% Tween).
• wash buffer 50 mM Tris, 100 mM NaCI, 1 mM MnCI 2 , 0,05% Tween.
• 50 ⁇ of compound diluted in assay buffer was added by transfer from a compound serial dilution plate.
• 50 ⁇ recombinant VCAM-Fc (R&D systems, 862-VC-100) at a concentration of 0.1 ⁇ g/ml in assay buffer 50 mM Tris, 150 mM NaCI, 1 mM MnCI 2 , 0,1 % BSA, 0,05% Tween was added to each well.
• the plate was incubated at room temperature with shaking (300 rpm) for 2 hours to reach binding equilibrium- Then the plate was washed three times in wash buffer and 100 ⁇ anti-human IgG Fc specific-HRP (Abeam, Ab97225) diluted at 1:2000 in assay buffer was added to each well. The plate was incubated at room temperature for 1 hour under agitation. The plate was then washed three times and 100 ⁇ of 1.3'.5.5'-Tetramethylbenxidie (TMB, (TMB, KPL 5120-0083) was then added to each well. The reaction was stopped after 2 minute-incubation by adding 50 ⁇ of 1 M H 2 SO 4 and optical absorbance was read at 450 nM.
• TMB 1.3'.5.5'-Tetramethylbenxidie
• RPMI8866 human cells (Sigma #95041316) were cultured in RPMI 1640 medium (HyClone SH30027.1) supplemented with 10% FBS (Seradigm) and 1% Penicillin-Streptomycin.
• a 96-well plate (Costar, 3603) was coated with 100 ml/well of human recombinant MAdCAM-1 Fc Chimera (R&D Systems, 6056-MC-050) solution at 0.25 ⁇ g/ml in coating buffer (50mM sodium carbonate, pH 9.6).
• RPMI8866 cells were resuspended at 10 million cells/ml in PBS containing 5 mM calcein and incubated at 37 °C for 30 min in a 50 ml tube. PBS was added to fill the tube, cells were spun down and resuspended in RPMI 1540 medium to 2 million/ml.
• RAMOS human cells (ATCC CRL-1596) were cultured in RPMI 1640 medium (HyClone SH30027.1) supplemented with 10% FBS (Seradigm) and 1% Penicillin-Streptomycin.
• a 96- well plate (Costar, 3603) was coated with 100 ml/well of recombinant human VCAM-1 Fc Chimera (R&D systems, 862-VC-100) solution at 0.25 ⁇ g/ml in coating buffer (50mM sodium carbonate, pH 9.6). The plate was incubated overnight at 4°C and washed twice with 150 ⁇ per well wash buffer (0.05% Tween 20 in PBS), blocked with 250 ⁇ per well blocking buffer (1% non-fat dry milk in PBS), for 1 hour at room temperature.
• RAMOS cells were resuspended at 10 million cells/ml in PBS containing 5 mM calcein and incubated at 37 °C for 30 min in a 50 ml tube. PBS was added to fill the tube, cells were spun down and resuspended in RPM1 1640 medium to 2 million/ml. Compounds were diluted by serial dilution in binding buffer (1.5 mM CaCI 2l 0.5 mM MnCI 2 .50 mM Tris-HCI, pH 7.5) to a final volume of 50 ⁇ per well at 2x concentration.
• binding buffer 1.5 mM CaCI 2l 0.5 mM MnCI 2 .50 mM Tris-HCI, pH 7.5
• the plate was washed once with 300 ⁇ of PBS, 50 ⁇ of compound and 50 ⁇ of cells (100,000 cells) were transferred to each well and the plate was incubated in the dark at 37°C, 5% C0 2 for 45 min to allow cell adhesion.
• the plate was emptied by inverting and blotting on paper towels and washed manually twice with PBS. After last wash, 100 pL of PBS was added to wells and the fluorescence was read (Ex485/Em 5 i_) using a plate reader (Tecan Infinite 1000). To calculate the dose response, the fluorescence value of control wells not containing cells was subtracted from each test well.
• Receptor occupancy in primary cells was determined by measuring the amount of biotinylated human recombinant MAdCAM-1-FC or human recombinant VCAM-1-Fc bound to selected cell populations using flow cytometry.
• Human recombinant MAdCAM-1-FC or human recombinant VCAM-1-FC were biotinylated using commercially available reagents and protocol (Pierce).
• Cells were washed with 1 mL stain buffer-FBS (BD Biosciences) and resuspended in 100 ⁇ stain Buffer-FBS (BD Biosciences) containing 4mM MnCl 2 .
• Biotinylated-rhMAdCAM-1 was applied at a saturating concentration of 1200 ng/mL to compete with test article binding and incubated at room temperature for 1 hour.
• CD45 F1TC BioLegend 200 ug/ml
• CD29 APC Cy7 BioLegend 100 ug/mt
• Integrin beta7 PE BioLegend concentration 50 pg/rnL
• CD49d V421 BioLegend 50 Mg/mL
• CD3 V510 BioLegend 30 Mg/mL
• CD4 PECy7 BioLegend 100 pg/mL
• CD45RO PerCP BioLegend 200 pg/mL
• the cells were then washed with stain-buffer-FBS and resuspended in 150 microL stain buffer-FBS for acquisition on the flow cytometer (BD FACSCantoTM flow cytometer and BDFACSDivaTM software).
• FACS data was acquire by electronic gating on the basis of forward versus side scatter, The cytometer was set to collect 20,000 events in each tube. Cell population were determined using the following markers, CD45+, CD3+, CD4+,CD45RO+, CD49d+ , integrin b7, biotinylated ligands.
• Compound receptor occupancy was defined as the decrease in the number of integrin ⁇ 7 + or integrin ⁇ 7 - ⁇ cells binding biotinylated rhMAdCAM-1 or rhVCAM-1, respectively.
• Receptor occupancy was calculated with the following equation: 100-((% ligand-positive cells with compound/% ligand-positive cells DMSO)*100)
• the animal care facility employed is accredited by the Canadian Council on Animal Care (CCAC). This study was approved by a certified Animal Care Committee and complied with CACC standards and regulations governing the use of animals for research. The animals were housed under standardized environmental conditions. . A standard certified commercial rodent diet was provided ad libitum. Tap water was provided ad libitum at all times.
• CCAC Canadian Council on Animal Care
• DSA Dextran sulfate sodium
• DAI Body weight and disease activity index
• 1- blood in stool negative Hemoccult, positive Hemoccult, blood traces in stool visible, rectal bleeding
• 2- stool consistency normal, soft but still formed, very soft, diarrhea
• 3- body weight loss From Day 6 to day 9, Compound No. 517 (ET03764) or the vehicle were administered orally daily at 5 mL/kg.
• Multimeric compounds were generally more potent in cellular assays.
• Multimeric compounds were generally more potent in their ability to inhibit cell adhesion than their constituent monomers.
• Compound No. 340 (ET2451) and Compound No. 456 (ET4062) had IC50 of 175 and 199 nM respectively in the RPMI8866 cell adhesion assays (Table 1C and 1C).
• Multimeric compounds with over 10-fold greater potency than their constituent monomeric compounds were generated .
• Compound No. 517 (ET3764) a homodimer of Compound No. 340 (ET2451)
• Compound multimers generated from monomeric Compound 456 (ET4062) also showed higher binding affinity (Table 2C).
• Dimeric Compound No.s 534 (ET4113) and 535 (ET4110) demonstrated IC50 of 38 and 76 nM respectively while the corresponding parent monomeric Compound No. 456 (ET4062) only reached 15% receptor occupancy at the maximum concentration of 1000 nM.
• the dimeric Compound No.s 517 (ET3764) and 390 (ET3755) competed with a saturating amount of MAdCAM, with EC50 of 38 and 90 nM respectively within the same study.
• the corresponding monomeric Compound 340 (ET2451) reached 50% receptor occupancy at low concentrations but no concentration- response curve couid be obtained. This could be the result of non-specific binding of the monomeric compound to the cell.
• Multimeric compounds showed enhanced selectivity for integrin ⁇ 4 ⁇ 7 ⁇ er integrin ⁇ 4 ⁇ 1.
• Multimeric compounds had generally higher selectivity for integrin ⁇ 4 ⁇ 7 over integrin ⁇ 4 ⁇ 1 than their monomeric constituents.
• monomeric Compound No.s 340 (ET2451) and 456 (ET4062) showed 16- and 45-fold selectivity, respectively, when comparing ⁇ 4 ⁇ 7 versus ⁇ 4 ⁇ 1 cell adhesion assays.
• DSS-treated mice Dextran Sodium Sulfate (DSS) induces chronic colitis in experimental animals when given orally in drinking water for five days followed by no DSS in drinking water. Chronic inflammation is associated with the infiltration of leucocytes from the blood to intestinal tissues.
• DSS Dextran Sodium Sulfate
• mice were exposed for 5 days to dextran sulfate in their drinking water. On days 6 to 9, compounds or vehicle were administered orally daily. Mesenteric lymph nodes were collected 4 hours following the last dose and assessed.
• Compound No. 517 (ET3764) reduced the detection of integrin helper memory lymphocytes in the mesenteric lymph nodes (MLN).
• Compound No. 517 administered at a dose of 80 mg/kg, reduced the number of ⁇ 4 ⁇ 7+ positive lymphocytes by 60%.

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