TW202325719A - Compounds - Google Patents

Abstract

Compounds of formula (I):

Description

compound

The present invention relates to novel compounds. The invention also relates to their preparation and their use in the treatment of various conditions mediated by α4β7 integrins, especially (but not exclusively) inflammatory conditions and/or autoimmune diseases such as inflammatory bowel disease.

Integrins are transmembrane receptors that bridge cell-cell and cell-extracellular matrix (ECM) interactions. It is involved in many cellular processes, including cell adhesion and migration, and regulation of gene expression.

Integrins are obligate heterodimers with two distinct chains: α (alpha) and β (beta) subunits. The α4β7 integrin is expressed on lymphocytes and is responsible for T-cell homing to gut-associated lymphoid tissues via binding to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) present on the high endothelial venules of mucosal lymphoid organs .

Inhibitors of specific integrin-ligand interactions have proven to be effective anti-inflammatory agents in the treatment of various autoimmune diseases. For example, monoclonal antibodies displaying high binding affinity for α4β7 have shown therapeutic benefit in autoinflammatory/autoimmune diseases of the gastrointestinal tract such as Crohn's disease and ulcerative colitis.

WO2022/002781 published after the earliest priority date of this application discloses a compound exhibiting α4β7 integrin antagonist activity. This compound is referred to as "Comparative Compound 1" in the Examples described herein.

Other compounds exhibiting α4β7 integrin antagonist activity are described in WO2018/085921. In particular, Compound 16 as described herein is referred to as "Comparative Compound 2" in the Examples described herein.

There is a need to develop improved α4β7 antagonists for the prevention or treatment of inflammatory conditions and/or autoimmune diseases.

In one aspect of the present invention, a compound of formula (I) is provided: or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention provides a pharmaceutical composition, which comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use as a medicament.

In another aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of an inflammatory condition and/or an autoimmune disease in a patient.

In another aspect, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for the manufacture of of drugs.

In another aspect, the present invention provides a method of treating an inflammatory condition and/or an autoimmune disease in a patient in need thereof, the method comprising administering to the patient a compound of formula (I) or a pharmaceutically acceptable salts or solvates.

Advantages and Surprising Findings The inventors have unexpectedly found that compounds of formula (I) as described herein, in particular Compound 1, are chemically more stable compared to the reference Compound 1, as described in more detail below. In particular, Compound 1 has been demonstrated to be more stable in aqueous and dry formulations compared to Control Compound 1. This finding was unexpected, as a single amino acid substitution would not have been predicted from the art to result in such a dramatic increase in stability.

The present inventors have also found that the compounds of formula (I) as described herein, in particular Compound 1, exhibit unexpectedly long half-lives when administered in vivo as compared to the control Compound 1 . This was unexpected given the small structural differences between the compounds.

The inventors have also surprisingly found that compounds of formula (I) as described herein, in particular compound 1, are chemically more stable compared to the control compound 2, as described in more detail below.

In one aspect of the present invention, there is provided a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or solvate thereof. In this specification, all references to compounds of formula (I) generally include references to their pharmaceutically acceptable salts and solvates.

In certain embodiments, according to the present invention there are provided pharmaceutically acceptable salts of the compounds described herein. As used herein, the term "pharmaceutically acceptable salt" denotes a salt or zwitterionic form of a compound of the present invention, which is water-soluble or oil-soluble or dispersible, suitable for the treatment of disease without undue toxicity, irritation and Hypersensitivity reaction; commensurate with a reasonable benefit/risk ratio and valid for its intended use. Salts can be prepared during the final isolation and purification of the compounds, either by treating the compounds with a suitable acid to protonate one or more nitrogen atoms, or with a suitable base to deprotonate one or more carboxylic acid groups. prepared separately. Salts also include zwitterionic forms of compounds wherein one or more amine groups are protonated and one or more carboxylic acid groups are deprotonated, but no counter-ion is present.

A representative acid addition salt is bicarbonate. One representative base addition salt is the ammonium salt.

Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of formula (I), including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof .

General Synthetic Methods Compounds of formula (I) can be synthesized using the methods generally shown in Scheme 1 . plan 1

In Scheme 1, the variables have the following meanings: PG ₁ is an amino protecting group; PG ₂ is a carboxyl protecting group; LG is a leaving group; X is a leaving group or a group convertible into a leaving group; _a is a C _1-6 alkyl group, or two groups R _a together _{form a} C _1-6 alkylene group, _{which is} fused with an aryl group as appropriate.

There is no particular restriction on the nature of each of the protecting groups PG ₁ and PG ₂ , as long as it fulfills the normal function of the protecting group, i.e. it can be attached to the relevant group, remaining attached to protect the group from its possible Any subsequent response to instability and can be removed when protection is no longer needed.

The protecting groups PG ₁ and PG ₂ are well known to those skilled in the art. Suitable examples are described in TW Greene and P. Wuts, "Protective Groups in Organic Synthesis", Wiley and Sons, 3rd edition, 1999.

Examples of amino protecting groups PG ₁ include the following: benzyloxycarbonyl (Cbz); p-methoxybenzylcarbonyl (Moz or MeOZ); tertiary butoxycarbonyl (BOC); 9-fenylmethoxy Carbonyl (Fmoc) group; Acetyl (Ac); Benzoyl (Bz); Benzyl (Bn); -Dimethoxybenzyl; p-methoxyphenyl (PMP); p-toluenesulfonyl (tosyl, Ts); Troc (trichloroethyl chloroformate); p-nitrobenzenesulfonyl (nosyl) and the o-nitrobenzenesulfonyl (Nps) group. Preferred _PG1 groups are BOC and Fmoc.

Examples of the carboxyl protecting group PG ₂ include esters, another part of _the ester group includes branched C _{3 -6} alkyl _, such as tertiary butyl; C _{3 -8} cycloalkyl, especially cyclohexyl; benzyl; 2,6 - esters of disubstituted phenols (eg 2,6-xylenol, 2,6-diisopropylphenol, 2,6-di-tertiary-butylphenol); silyl esters; and orthoesters. Preferred PG ₃ groups include branched C _{3 - 6} alkyl, C _{5 - 7} cycloalkyl and benzyl, especially tertiary butyl, cyclohexyl and benzyl.

Examples of leaving groups LG include halogens (especially chlorine, bromine or iodine) and sulfonate groups such as methanesulfonate, triflate and p-toluenesulfonate. Preferred is halogen, and especially bromine or iodine.

An example of a group X is a leaving group, as defined and exemplified above for the group LG. Examples of groups that can be converted into leaving groups include hydroxyl groups.

Compound (III) is formed from compound (II) by successive amide coupling reactions (a1) to (a4) with the relevant protected amino acids, followed by a final deprotection step (a5).

Each of steps (a1) to (a4) is performed by first removing the protecting group _PG1 from the amine terminus of the starting amino acid, followed by coupling the resulting amine with the carboxyl moiety of the next amino acid, usually in a suitable solvent in the presence of a conventional coupling agent, usually in the presence of a base. The carboxyl terminus of the compound of formula (II) and the intermediates involved in steps (a1 ) to (a4) must be protected during these amide coupling steps unless the solid phase synthesis methods mentioned below are used. Suitable carboxy protecting groups are as defined and exemplified above for the group _PG2 .

Methods for carrying out deprotection steps (a1) to (a5) and suitable deprotecting agents are well known to those skilled in the art. Suitable deprotecting agents are described in "Protective Groups in Organic Synthesis" mentioned above. For example, when PG ₁ is Fmoc, the deprotecting agent can be piperidine.

The coupling agent can be any reagent that facilitates the coupling of carboxylic acids and amines to produce amides. Examples of coupling agents are also well known to those skilled in the art. Typical coupling agents include carbodiimides such as diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC); ammonium and Reagents such as 1-[bis-(dimethylamino)methylonyl]-1 H -1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate ( HATU), 2-(1 H -benzotriazol-1-yl]-1,1,3,3-tetramethyl Hexafluorophosphate (HBTU) and O -(1 H -6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate (HCTU); and organophosphate reagents such as 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT). Couplers can also be used in combination with additives such as HOBT (1-Hydroxy-benzotriazole) or ethyl cyanohydroxyiminoacetate (Oxyma®). Oxyma and DIC are preferred.

The base is not particularly limited as long as it can act as a base and does not react with activated amino acids. Examples of suitable bases include tertiary amines including trimethylamine, triisopropylamine and diisopropylethylamine. Diisopropylethylamine is preferred.

The solvent is not particularly limited so long as it is inert to the reaction and capable of dissolving the reactants, at least to some extent. Examples of suitable solvents include: halogenated hydrocarbons such as dichloromethane (DCM); dimethylsulfoxide (DMSO); dimethylformamide (DMF); N-methylpyrrolidone (NMP) and mixtures thereof, Among them, DMF is preferred.

Advantageously, steps (a1) to (a5) are carried out on a solid phase. The support is usually an organic polymer, usually in the form of a resin, with functional groups capable of reacting with one end of the peptide chain to facilitate attachment of this end to the chain. Since the peptide remains covalently attached to the support throughout the synthesis, excess reagents and by-products can be removed by washing and filtration.

Typically, this involves reacting compound (II) with a suitable solid support such that the carboxyl terminus of compound (II) is bound to the solid support; step (a1) is performed by washing the solid support with a suitable deprotecting agent Deprotecting part to (a4); coupling part of steps (a1) to (a4) performed as described above; deprotecting step (a5) performed by washing the solid support with a suitable deprotecting agent ; and finally cleavage of the compound from the solid support to produce the compound of formula (III).

Examples of solid phase peptide synthesis and methods for performing them are well known to those skilled in the art, such as taught in "Solid Phase Synthesis: A Practical Guide", edited by S. Kates and F. Albericio, CRC Press, 2000.

Examples of organic polymers suitable for use in forming solid support resins include polystyrene, typically cross-linked polystyrene (obtained by copolymerization of styrene and divinylbenzene).

The functional group can be any group that allows attachment to an organic polymer while allowing the assembly of a partially protected peptide chain thereon, and can be cleaved without affecting the side chain protecting group. Examples of functional groups on which organic polymers can be functionalized include triarylmethyl halides, preferably trityl halides (especially trityl chloride); diaryl methyl halides, especially diphenylmethyl halides; halo; halobenzyl; and halomethyl. Polymers functionalized with trityl halides, especially 2-chlorotrityl-resins, are preferred because they allow the release of partially protected compounds of formula (III).

The initial association of compound (II) with a suitable solid support can be carried out by reacting compound (II) with a functionalized organic polymer providing a solid support in a suitable solvent.

For some resins, especially triarylmethyl halide resins, the reaction can be carried out in the presence of a base. The base must be soluble in the solvent. Examples of suitable bases include tertiary amines including trimethylamine, triisopropylamine and diisopropylethylamine, with diisopropylethylamine being preferred.

The solvent is not particularly limited so long as it is inert to the reaction, enables the organic polymer resin to swell therein, and dissolves the reactants, at least to some extent. Examples of suitable solvents include those solvents defined and exemplified above for steps (a1 ) to (a4) and mixtures thereof. DMF is preferred.

The final step of cleavage of the compound from the solid support to produce the compound of formula (III) can be carried out using a cleavage agent in a suitable solvent. The cleaving agent is typically an acid of sufficient strength and/or present in a concentration sufficient to cleave the compound of formula (III) from the solid support while leaving the protecting groups on the compound intact. Particularly preferred is 1,1,1,3,3,3-hexafluoroisopropanol (HFIP).

The solvent is not particularly limited so long as it is inert to the reaction and capable of dissolving the reactants, at least to some extent. Examples of suitable solvents include alcohols, such as methanol, ethanol, and isopropanol; halogenated hydrocarbons, such as dichloromethane (DCM), and mixtures thereof. DCM is preferred.

In step (b), compound (IV) is formed by cyclization of compound (III) in an intramolecular amide coupling reaction, usually in the presence of a conventional coupling agent, usually in the presence of a base, in a suitable solvent.

The coupling agent can be any reagent that facilitates the coupling of carboxylic acids and amines to produce amides. Examples of coupling agents include those defined and exemplified above for steps (al) to (a4). HATU is preferred.

The solvent is not particularly limited so long as it is inert to the reaction and capable of dissolving the reactants, at least to some extent. Examples of suitable solvents include those solvents defined and exemplified above for steps (a1 ) to (a4) and mixtures thereof. DMF is preferred.

The reaction is generally carried out under suitable conditions so that cyclization occurs and the intramolecular amide coupling required to produce the compound of formula (IV) predominates over any intermolecular reaction between two molecules of the compound of formula (III). Usually, the reaction is carried out in a dilution between 0.1 mM and 1000 mM, preferably 0.2 to 500 mM, more preferably 10 to 100 mM.

In step (c), compound (V) is formed by reacting compound (IV) with compound (VIII) in an aryl cross-coupling reaction, usually in the presence of a catalyst, usually in the presence of a base, in a suitable in solvent.

Compounds of formula (VIII) are commercially available. One such commercially available compound is 4-(4-(benzyloxycarbonyl)-piperyl)phenyl Acid pinacol esters in which PG ₁ is benzyloxycarbonyl and the two groups R _a together form 1,1,2,2-dimethylethylidene.

The catalyst can be any catalyst capable of catalyzing the reaction of an aryl halide or aryl sulfonate (e.g. mesylate or triflate) with an aryl Catalyst for the coupling of esters to form a carbon-carbon bond between two aryl groups. Examples of the catalyst include organic palladium reagents and organic nickel reagents, among which tetrakis(triphenylphosphine)palladium(0) is preferable.

The reaction is usually carried out in the presence of a base. Examples of suitable bases include alkali metal carbonates, such as sodium carbonate or potassium carbonate; alkali metal phosphates, such as sodium phosphate or potassium phosphate; and alkali metal alkoxides, such as sodium ethoxide or potassium ethoxide. Potassium phosphate is preferred.

The solvent is not particularly limited so long as it is inert to the reaction and capable of dissolving the reactants, at least to some extent. Examples of suitable solvents include water; alcohols, such as methanol, ethanol, and isopropanol; ethers, such as dioxane, 1,2-dimethoxyethane (DME); and mixtures thereof. A mixture of water and dioxane is preferred.

In step (d), compound (VI) is formed from compound (V) by removing the amino protecting group PG ₁ and the carboxyl protecting group PG ₂ . This is done in the presence of a suitable deprotecting reagent, usually in a suitable solvent.

Methods and suitable deprotecting reagents for carrying out deprotecting step (d) are well known to those skilled in the art. Suitable deprotecting reagents are described in "Protective Groups in Organic Synthesis" mentioned above. Examples of suitable deprotecting reagents include strong acids, with trifluoroacetic acid being preferred.

In step (e), compound (I) is formed by amide coupling reaction of compound (VI) and compound (IX), usually in a suitable solvent in the presence of a base.

To facilitate the reaction, a compound of formula (IX) wherein X is OH may first be converted into another compound of formula (IX) wherein X is a leaving group such as halo and especially wherein X is chloro. This is done by reacting a compound of formula (IX) wherein X is OH with a halogenating agent. Examples of suitable halogenating agents include acetyl chloride, thionyl chloride, phosphorus trichloride and phosphorus pentachloride, with acetyl chloride being preferred.

The base is not particularly limited as long as it can act as a base and does not react with the compound of formula (IX) in which X is a leaving group. Examples of suitable bases include those bases defined and exemplified above for steps (al) to (a4). Tertiary amines, especially diisopropylethylamine, are preferred.

Compounds of formula (VII) can be synthesized using the method generally shown in Scheme 2. Scenario 2

In scheme 2, LG has the same meaning as in scheme 1.

In step (a), compound (XII) is formed by reacting compound (X) with compound (XI), usually in the presence of an acid, in a suitable solvent. Methods and suitable conditions and reagents for carrying out reaction step (a) are well known to those skilled in the art. Compounds of formula (X) and (XI) are commercially available.

The acid is not particularly limited as long as it can function as an acid. Usually a strong acid is used. Examples of suitable acids include: inorganic acids, such as sulfuric acid and nitric acid; and organic acids, especially sulfonic acids, such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid. Sulfonic acids are preferred and trifluoromethanesulfonic acid is especially preferred.

The solvent is not particularly limited as long as it dissolves the reactants at least to some extent. In this case, it is especially preferred that the acid acts as a solvent. Sulfonic acids are preferred and trifluoromethanesulfonic acid is especially preferred.

In step (b), compound (XIII) is formed by reducing compound (XII) in a suitable solvent. Methods and suitable reducing agents for carrying out the reduction step (b) are well known to those skilled in the art.

The reducing agent can be any reagent capable of carrying out the reducing step. Examples of suitable reagents include alkali metal borohydrides, such as sodium borohydride and sodium tris(acetyloxy)borohydride, with sodium borohydride being preferred.

The solvent is not particularly limited so long as it is inert to the reaction and capable of dissolving the reactants, at least to some extent. Examples of suitable solvents include water; alcohols, such as methanol, ethanol, and isopropanol; ethers, such as diethyl ether and 1,2-dimethoxyethane (DME); and mixtures thereof. A mixture of water and isopropanol is preferred.

In step (c), amine (XIV) is formed by cleavage of amide (XIII), usually in the presence of an acid, in a suitable solvent. Methods and suitable acids for performing amide cleavage are well known to those skilled in the art.

The acid is not particularly limited as long as it can function as an acid. Usually a strong acid is used. Examples of suitable acids include: inorganic acids, such as hydrochloric acid, sulfuric acid, and nitric acid; and organic acids, especially sulfonic acids, such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid. Hydrohalic acids are preferred and hydrochloric acid is especially preferred.

The solvent is not particularly limited so long as it is inert to the reaction and capable of dissolving the reactants, at least to some extent. Examples of suitable solvents include water; alcohols, such as methanol, ethanol, and isopropanol; ethers, such as diethyl ether and 1,2-dimethoxyethane (DME); and mixtures thereof. A mixture of water and isopropanol is preferred.

In step (d), compounds of formula (VII) are formed by attaching the protecting group PG ₁ to the amine (XIV). Suitable protecting groups and suitable reagents for their introduction are well known to those skilled in the art and examples are described in "Protective Groups in Organic Synthesis" mentioned above. For example, when PG ₁ is Fmoc, a suitable reagent for introducing it may be N- ( 9H -oxene-9-ylmethoxycarbonyloxy)butanediimide.

Pharmaceutical composition In one aspect of the present invention, a pharmaceutical composition is provided, which comprises the compound of formula (I) as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be formulated for administration by either oral or parenteral routes. Examples of pharmaceutical carriers are well known to those skilled in the art.

Pharmaceutical compositions suitable for the delivery of compounds of the invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

Compounds of the invention can be administered orally. Oral administration can involve swallowing, allowing the compound to enter the gastrointestinal tract, and/or buccal, lingual, or sublingual administration, allowing the compound to enter the bloodstream directly from the mouth.

Formulations suitable for oral administration include solid, semi-solid and liquid systems such as lozenges; soft or hard capsules containing multiparticulate or nanoparticles; buccal tablets (including liquid fills); chewable tablets; gels ; rapidly dispersing dosage forms; films; oval suppositories; sprays; and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations can be used as fillers in soft or hard capsules (made, for example, of gelatin or hydroxypropylmethylcellulose), and generally comprise carriers such as water, ethanol, polyethylene glycol, propylene glycol, Methylcellulose or a suitable oil, and one or more emulsifying and/or suspending agents. Liquid formulations can also be prepared by reconstituting solids, eg, from sachets.

The compounds of the invention may also be used in rapidly dissolving, rapidly disintegrating dosage forms such as those described in Liang and Chen, Expert Opinion in Therapeutic Patents , H (6), 981-986, (2001 ).

For lozenge dosage forms, depending on the dosage, the drug may comprise from 1% to 80% by weight of the dosage form, more usually from 5% to 60% by weight of the dosage form. In addition to the drug, lozenges generally contain disintegrants. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose Vein, microcrystalline cellulose, hydroxypropyl cellulose substituted by lower carbon alkyl group, starch, pregelatinized starch and sodium alginate. Generally, the disintegrant will comprise 1% to 25% by weight of the dosage form, preferably 5% to 20% by weight.

Binders are often used to impart cohesive qualities to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycols, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose and hydroxypropylmethylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrate, and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline Cellulose, starch and calcium hydrogen phosphate dihydrate.

Tablets may also optionally contain surfactants, such as sodium lauryl sulfate and polysorbate 80; and glidants, such as silicon dioxide and talc. When present, the surfactant can comprise from 0.2% to 5% by weight of the tablet, and the glidant can comprise from 0.2% to 1% by weight of the tablet. Tablets generally also contain lubricating agents, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate and sodium lauryl sulfate. The lubricant generally constitutes 0.25% to 10% by weight of the lozenge, preferably 0.5% to 3% by weight.

Other possible ingredients include antioxidants, coloring agents, flavoring agents, preservatives and taste-masking agents.

An exemplary lozenge contains up to about 80% drug, about 10% to about 90% binder by weight, about 0% to about 85% diluent by weight, about 2% to about 10% disintegrant, and about 0.25 % by weight to about 10% by weight lubricant. Tablet blends may be compressed directly or by rollers to form tablets. Tablet blends or a portion of a blend may alternatively be wet granulated, dry granulated or melt granulated, melt agglomerated or extruded prior to tableting. The final formulation may comprise one or more layers, and may be coated or uncoated; it may even be encapsulated.

The formulation of tablets is discussed in H. Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (Marcel Dekker, New York, 1980).

Compounds of the invention may also be administered parenterally, that is, directly into the bloodstream, into a muscle or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous. Devices suitable for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are usually aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably buffered to a pH of 3 to 9), although in some applications parenteral formulations are more suitable for formulation As sterile non-aqueous solutions or in dry form to be combined with a suitable vehicle, such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example by lyophilization, is readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

Medical uses and methods of treatment In another aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use as a medicament.

In another aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of a condition in a patient related to the biological function of α4β7 integrin in the patient.

In another aspect, the present invention provides the use of the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a drug for treating a patient related to the biological function of the patient's α4β7 integrin. Medicines for the condition.

In one aspect of the present invention, there is provided a method for treating a condition in a patient related to the biological function of α4β7 integrin, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically effective amount thereof. acceptable salts or solvates.

In another aspect of the present invention, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use in treating inflammatory or autoimmune diseases in a patient. Preferably, the inflammatory or autoimmune disease is gastrointestinal.

In another aspect of the present invention, the present invention provides the use of the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for the manufacture of of drugs. Preferably, the inflammatory or autoimmune disease is gastrointestinal.

In one aspect of the present invention, there is provided a method of treating an inflammatory or autoimmune disease in a patient, comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof or solvates. Preferably, the inflammatory or autoimmune disease is gastrointestinal.

In some embodiments, the condition or disease is selected from the group consisting of inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease, microscopic colitis, collagenous colitis, Eosinocyte gastroenteritis, pouchitis after proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, cholangitis, pericholangitis, primary sclerosing cholangitis, gastrointestinal infection of human immunodeficiency virus (HIV ), graft-versus-host disease, and primary biliary sclerosis.

In preferred embodiments, the condition or disease is an inflammatory bowel disease, such as ulcerative colitis and/or Crohn's disease.

In another aspect of the present invention, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use in treating viral diseases in a patient. Preferably, the inflammatory or autoimmune disease is gastrointestinal.

In another aspect of the present invention, the present invention provides the use of the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for treating viral diseases in patients. Preferably, the inflammatory or autoimmune disease is gastrointestinal.

In one aspect of the present invention, there is provided a method of treating a viral disease in a patient, comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the disease or condition is a viral or retroviral localized or systemic infection. In one embodiment, the disease or condition is HIV/AIDS. In one embodiment, the disease or condition is HIV-1. In one embodiment, the disease or condition is HIV-2.

In some embodiments, the compound of formula (I) inhibits the binding of α4β7 integrin to MAdCAM. Preferably, the compound selectively inhibits the binding of α4β7 integrin to MAdCAM.

In any embodiment, the patient is preferably a human.

As used herein, the terms "disease", "disease" and "condition" are used interchangeably.

As used herein, "inhibit," "treatment," "treating," and "improvement" are used interchangeably and refer to, for example, arrest of symptoms, prolongation of survival, a portion of symptoms, or Complete improvement and partial or complete eradication of a condition, disease or disorder.

As used herein, "prevent" or "prevention" includes: (i) preventing or inhibiting the occurrence of a disease, injury or condition in an individual, such as a mammal, especially when such individual is susceptible to the condition but has not yet When diagnosed with the condition; or (ii) reducing the likelihood of the individual developing the disease, injury or condition.

As used herein, a "therapeutically effective amount" refers to an amount effective, at dosages and for a specified period of time, necessary to achieve the desired therapeutic result. A therapeutically effective amount of an agent can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the 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 agent are outweighed by the therapeutically beneficial effects.

In some embodiments, the compound is administered by an administration form selected from the group consisting of: oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporized, nebulized, lingual Lower, buccal, parenteral, rectal, vaginal, and topical. In certain embodiments, compounds are administered subcutaneously.

Administration Regardless of the route of administration chosen, the compound of the present invention and/or the pharmaceutical composition of the present invention can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. Actual dosage concentrations of the active ingredients in the pharmaceutical compositions of the invention may be varied so as to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being unacceptably toxic to the patient.

The selected dosage concentration will depend on a variety of factors, including the activity of the compound of the invention, or a pharmaceutically acceptable salt thereof; the route of administration; the time of administration; the rate of excretion or metabolism of the compound; the rate and extent of absorption; and the duration of treatment. ; other drugs, compounds and/or materials used in combination with the compound; age, sex, weight, condition, general health and previous medical history of the patient being treated; and similar factors well known in the medical art.

A physician or veterinarian generally skilled in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start doses of the compounds of the invention used in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such effective dosage will generally depend on the factors described herein.

In some embodiments, the compound is administered in an initial dose followed by one or more subsequent doses with a minimum interval between any two doses of a period of less than 1 day, and wherein each dose comprises an effective amount of compound.

In some embodiments, the effective amount of the compound is an amount sufficient to achieve at least one selected from the group consisting of: a) about 50% or greater saturation of the MAdCAM binding site on the α4β7 integrin molecule; b ) about 50% or greater inhibition of α4β7 integrin expression on the cell surface; and c) about 50% or greater saturation of MAdCAM binding sites on the α4β7 molecule and about 50% or greater saturation of α4β7 integrin expression on the cell surface % or greater inhibition, where i) saturation is maintained for a period consistent with dosing frequency not more than twice daily; ii) inhibition is maintained for a period consistent with dosing frequency not more than twice daily; or iii) Saturation and suppression are each maintained for a period of time consistent with a frequency of dosing not to exceed twice daily.

In some embodiments, the compound is administered at intervals selected from the group consisting of: around the clock, every hour, every four hours, once a day, twice a day, three times a day, four times a day, every other day, Weekly, biweekly and monthly.

In certain embodiments, the compound is administered once daily. In certain embodiments, the compound is administered every 2 days. In certain embodiments, the compound is administered every 3 days. In certain embodiments, the compound is administered every 4 days. In certain embodiments, the compound is administered every 5 days. In certain embodiments, the compound is administered every 6 days. In certain embodiments, the compound is administered weekly. In certain embodiments, the compound is administered every 2 weeks. In certain embodiments, the compound is administered every 3 weeks. In certain embodiments, the compound is administered every 4 weeks. In certain embodiments, the compound is administered monthly.

In certain embodiments, the compound is administered subcutaneously once daily. In certain embodiments, the compound is administered subcutaneously every 2 days. In certain embodiments, the compound is administered subcutaneously every 3 days. In certain embodiments, the compound is administered subcutaneously every 4 days. In certain embodiments, the compound is administered subcutaneously every 5 days. In certain embodiments, the compound is administered subcutaneously every 6 days. In certain embodiments, the compound is administered subcutaneously once a week. In certain embodiments, the compound is administered subcutaneously every 2 weeks. In certain embodiments, the compound is administered subcutaneously every 3 weeks. In certain embodiments, the compound is administered subcutaneously every 4 weeks. In certain embodiments, the compound is administered subcutaneously once a month.

In some embodiments, the compound is administered subcutaneously once daily at a dose of 1.4 to 5.6 μg/kg. In some embodiments, the compound is administered subcutaneously every 2 days at a dose of 2.8 to 12.9 μg/kg. In some embodiments, the compound is administered subcutaneously every 3 days at a dose of 4.5 to 24 μg/kg. In some embodiments, the compound is administered subcutaneously every 4 days at a dose of 6.1 to 40 μg/kg. In some embodiments, the compound is administered subcutaneously every 5 days at a dose of 8.1 to 59 μg/kg. In some embodiments, the compound is administered subcutaneously every 6 days at a dose of 10.3 to 89 μg/kg. In some embodiments, the compound is administered subcutaneously once a week at a dose of 12.7 to 142 μg/kg.

EXAMPLES The following examples illustrate the preparation of compounds of formula (I). Intermediates illustrate the preparation of suitable intermediates.

The following abbreviations are used: BSA Bovine Serum Albumin 2-CTC (2-Chlorophenyl) Diphenylmethyl Chloride (2-Chlorotrityl Chloride) DCM Dichloromethane DIC Diisopropylcarbodiimide DIPEA Di Isopropylethylamine DMF Dimethylformamide DMSO Dimethylidene Fmoc-OSu N- ( 9H -Oxime-9-ylmethoxycarbonyloxy)succinimideh, hr hours HATU 1- [Bis-(dimethylamino)methylonyl]-1 H -1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate HBTU [2-(1 H -Benzotriazol-1-yl]-1,1,3,3-tetramethyl Hexafluorophosphate HFIP 1,1,1,3,3,3-Hexafluoroisopropanol HOBT 1-Hydroxybenzotriazole HPLC HPLC IPA 2-propanol LCMS LC-MS MeCN Acetonitrile MeOH methanol min minutes Oxyma ethyl cyanohydroxyiminoacetate PBS phosphate-buffered saline TRIS ginseng (hydroxymethyl) aminomethane TFA trifluoroacetic acid THF tetrahydrofuran Reference compound 1 is compound 1 of WO2022/002781. Comparative compound 2 is compound 16 of WO2018/205008.

Synthesis of Intermediates Intermediates 1-4 Step (a) - Intermediate 1 In a 2.0 L round bottom flask equipped with a stir bar, 4-bromobenzoic acid (227 g, 1.13 mol, 1.0 eq.) was dissolved in trifluoromethanesulfonic acid (1.0 L) and cooled to 0° C. for 15 minutes with stirring. N- (Hydroxymethyl)phthalimide (200 g, 1.13 mol, 1.0 eq.) was added in portions over 15 minutes. The resulting mixture was stirred at room temperature for 18 hours. LCMS chromatogram of the crude reaction mixture confirmed the completion of the reaction as assessed by the disappearance of 4-bromobenzoic acid. The mixture was poured into an ice bath (approximately 2.5 L) with stirring. The resulting white solid was filtered and then washed with _H2O until the pH of the filtrate reached about 6, as assessed by wet pH paper. The resulting solid was placed under house vacuum to near dryness to afford crude intermediate 1.

Steps (b) and (c) - Intermediates 2 and 3 In a 12.0 L round bottom flask, suspend Intermediate 1 (1.13 mol, assuming 100% yield from previous step) in 7:1 isopropanol/ _H2O mixture (5.0 L) and stirred. Solid _NaBH4 (214 g, 5.65 mol, 5.0 eq.) was then added in small portions over 4 hours with stirring. The mixture was then stirred overnight at room temperature, over which time it slowly turned into a clear solution. LCMS analysis confirmed complete conversion of intermediate 1 to intermediate 2. The reaction was quenched by gradual addition of concentrated HCl to a constant pH of about 1 (monitored every 15 minutes with wet pH paper). The resulting cloudy mixture was allowed to warm to about 70°C for 5 hours to give a clear solution. LCMS analysis confirmed complete conversion of intermediate 2 to the hydrochloride salt of intermediate 3. The mixture was stirred overnight at room temperature and then the solvent was slowly removed under positive compressed air flow to afford crude intermediate 3.

Step (c) - Intermediate 4 In the same 12.0 L round bottom flask as in Step 2, Intermediate 3 (1.13 mol, assuming 100% yield from the previous step) was suspended in _H2O (5.0 L). The pH of the mixture was raised to about 8 using triethylamine as assessed by wet pH paper. A slurry of Fmoc-OSu (381 g, 1.13 mol, 1.0 eq.) in acetonitrile (500 mL) was added in one portion. The mixture was stirred at room temperature for about 65 hours and LCMS analysis confirmed complete consumption of intermediate 3. The mixture was diluted to ~11 L with _H2O , acidified to pH ~1 with concentrated HCl, assessed by wet pH paper, and then stirred for an additional 1.0 h. The resulting suspension was filtered, and the solid filter cake was washed with water (ca. 2 L). The solid filter cake was triturated with acetonitrile (ca. 3.5 L), filtered again and dried in house vacuum for 2 days to give Intermediate 4 (396.02 g, 0.876 mol, obtained from 4-bromobenzoic acid in 4 steps) as an off-white solid. The yield was 77%). This solid readily dissolved in DMF to a homogeneous solution.

Intermediate 5-9 Steps (a1) to (a6) - Intermediate 6 (a1) 2-CTC resin (1.5 g, 1.5 mmol/g, 2.25 mmol) was expanded with DCM (10 volumes) for 15 minutes and intermediate 5 (in WO2017/079821 Revealed in as compound 4A) (1.5 eq). DMF (10 volumes) containing DIPEA (4.0 eq.) was added to the resin in one portion. The reaction mass was gently agitated under nitrogen sparging at 25-28°C for 3 hours. The solvent was drained and the resin was washed with DMF (10 vol, 3 x 5 min).

The resin was capped in a mixture of 10% DIPEA + 20% MeOH + 70% DMF (mixture 10 volumes) for 30 minutes at 25-28°C. The suspension was gently agitated at 25-28°C for 30 minutes under nitrogen sparging. The solvent was drained and the resin was washed with DMF (3 x 10 vol, 3 x 5 min) and DCM (3 x 10 vol; 3 x 5 min).

Deprotection: 20% piperidine in DMF (10 volumes) was added to the 2-CTC resin and stirred gently at 25-28° C. for 15 minutes under nitrogen sparging. Drain the solvent. A second batch of 20% piperidine in DMF (10 volumes) was added to the 2-CTC resin with gentle agitation for 15 minutes at 25-28°C. Drain the solvent. The resin was washed with DMF (10 vol, 3 x 5 min) and DCM (10 vol; 3 x 5 min).

After deprotection, the resin was washed 5 times with DMF (10 volumes). (a2) Fmoc-L-Ile-OH (1.5 eq), (a3) Fmoc-L-Asp(tBu)-OH (1.5 eq), (a4) Fmoc-L-Leu-OH (1.5 eq) and (a5 ) 3-(Fmoc-aminomethyl)-4-bromobenzoic acid (Intermediate 4) (1.5 eq) followed by coupling in DMF (10 vol) for 2 hours using Oxyma (1.5 eq) and DIC (2 eq) each , and deprotected with 20 vol% piperidine in DMF for 30 minutes. The resin was washed with DCM (3 x 10 vol) and dried, yielding 2.3 g of peptidyl resin.

(a6) The peptidyl resin was treated with lysis mix (30 vol% HFIP/70 vol% DCM, 10 vol) for 30 minutes and then filtered. Repeat the process once. The combined filtrates were concentrated under reduced pressure to afford crude intermediate 6 (1.8 g). LCMS: 92%. Mass of desired compound: 882 (m+2H) observed at room temperature. HPLC: 86%

Step (b) - Intermediate 7 To a stirred solution of intermediate 6 (1.8 g, 2.048 mmol) in DMF (40 ml, 0.05 M) was added HATU (1.12 g, 3.07 mmol) followed by DIPEA (1.13 ml, 6.1 mmol) at 25 °C . The reaction mixture was stirred at 25°C for 3 hours. The progress of the reaction was monitored by LCMS analysis. After completion of the reaction, the reaction mass was diluted with ice/water (80 ml) and extracted with ethyl acetate (3×100 ml). The separated organic layer was washed again with brine (80 ml). The separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure at 35 °C to obtain Intermediate 7 (0.45 g) as an off-white solid.

LCMS: 58%, observed the desired compound mass 861 (m+H), HPLC: 45%

Step (c) - intermediate 8 at 25°C, to intermediate 7 (300 mg, 0.344 mmol), (4-(4-tertiary butoxycarbonyl)piperyl)-phenyl Acid pinacol ester (commercially available, see also CN110862380A, Step 1: Example 228c) (134 mg, 0.344 mmol) and K ₃ PO ₄ (218 mg, 1.032 mmol) in dioxane:H ₂ O (9:1 ) (9 mL, 30 V) to a stirred (degassed with nitrogen for 15 min) solution was added Pd(PPh ₃ ) ₄ (0.079 g, 0.0688 mmol). The reaction mixture was heated at 90°C for 8 hours. LCMS monitoring showed complete conversion of starting material. The mixture was diluted with water (20 ml), cooled to room temperature, filtered through a pad of Celite® and extracted with ethyl acetate (2 x 40 mL). The combined organic layers were dried over _Na2SO4 , filtered and concentrated under reduced pressure to afford crude intermediate 8 ( ₃₀₀ mg) as a light yellow solid. LCMS: 25%, mass of desired compound: 1046 (m+H).

Step (d) - Intermediate 9 Intermediate 8 (300 mg) was placed in a 25 ml round bottom flask and TFA:DCM (4.5 ml, 15 V) was added. The reaction mixture was stirred at 25°C for 2 hours. The progress of the reaction was monitored by LCMS analysis. After the reaction was complete, the solvent was removed in vacuo and washed with diethyl ether (3 x 10 ml) and dried to give crude product 300 mg (off-white solid). The crude material was submitted to preparative reverse phase HPLC, and after purification and lyophilization, the weight obtained was 65 mg. LCMS: 26%, mass desired compound 888.9 (m+H).

Example 1 2 , 2 '-(( S , S , S , S , S , S , 12 S , 8 S , 11 S , 14 S , 17 S , 18 S )-((([ 1 , 1 '- Benzene ] -2 , 2' - dicarbonyl ) bis ( piperone - 4,1 - diyl ) ) bis ( 4,1 - phenylene ) ) bis ( 14 - ( ( S ) -secondary butyl ) - 18- ( tertiary butylaminoformyl ) -8 - isobutyl - 17 - methyl - 2 , 6 , 9 , 12 , 15 - pentaoxy - 3 , 7 , 10 , 13 , 16 - penta Aza - 1 ( 2 , 1 ) -pyrrolidine - 5 ( 1 , 3 ) -phenylcycloctadecane - 56 , 11 - diyl )) diacetic acid Intermediate 9 (40 mg, 0.045 mmol) was placed in a 25 ml round bottom flask, and anhydrous DCM (4 ml, 10 V) was added at 0° C., followed by DIPEA (0.2 ml, 1.127 mmol), followed by addition at 0 °C. [1,1'-biphenyl]-2,2'-dicarbonyl dichloride (prepared according to US4818771; 6.2 mg, 0.0225 mmol) in DCM was added dropwise at °C. The reaction mixture was stirred at 25°C for 3 hours. The progress of the reaction was monitored by LCMS analysis. After completion of the reaction, the solvent was removed in vacuo and the residue was washed with diethyl ether (3 x 10 ml) and dried to give the crude product (50 mg) as an off-white solid. LCMS: 68%; mass of desired compound 992 (m/2+H).

The crude product was submitted to preparative HPLC and after purification and lyophilization 11 mg of compound was obtained with a purity of 97% (HPLC). The compound of Example 1 is also referred to as "compound 1" hereinafter.

Example 2 Testing of Compound 1 in Integrin α 4 β 7 -MAdCAM - 1 Cell Adhesion Assay Reagents and Materials : Recombinant human MAdCAM-1 (rhMAdCAM-1) was obtained from R&D systems (Catalog No. 6056-MC-050) .

The CellTrace™ CFSE Cell Proliferation Kit for Flow Cytometry was obtained from Thermo Fisher Scientific (Cat# C34554).

The peptides to be tested are aliquoted and reconstituted internally.

Coating Buffer (50 nM Sodium Carbonate, pH 9.6) (Merck Millipore, Cat# S2127).

Wash buffer (PBS without Mg ^{2 +} and Ca ^{2 +} + 0,05% Tween® 20).

Binding buffer (1.5 mM CaCl ₂ , 1 mM MnCl ₂ , 100 mM NaCl, 50 mM TRIS-HCl (pH 7.5)

Blocking buffer. The stock solution is PBS (without Mg ^{2 +} and Ca ^{2 +} ) + 10% BSA.

Bovine Serum Albumin (BSA) (Sigma, cat. no. 9430).

RPMI Cell Medium 1640 (1×) + Glutamax (Gibco Cat# 61870-010).

Fetal bovine serum (Thermo Fisher Scientific; Cat. No. A4766801 ).

Penicillin-Streptomycin (10,000 U/mL) (Thermo Fisher Scientific; Cat. No. 15140122).

Final Growth Medium: RPMI Cell Medium 1640 (1x) + Glutamax + 10% FBS + 1% Penicillin-Streptomycin.

Compound Dilution Trays (Thermo Fisher Scientific, Nunc, Cat. No. 249944).

Perkin Elmer TopSeal A plus was obtained from PerkinElmer (cat. no. 6050185).

Coolsink heating plate XT96F (Merck Millipore, catalog number BCS-536).

Envision 2105 Multimode Disc Reader (PerkinElmer, Cat. No. 2105-0010).

The F96 Maxisorp plate line from Nunc was obtained from Thermo Fisher Scientific (cat. no. 442404).

Cells: The human B-lymphocyte cell line RPMI 8866 was obtained from Merck via ECACC (European Collection of Authenticated Cell Cultures) (Catalog No. 95041316 and Lot No. 14G012).

Assay protocol : Day 0: RPMI 8866 cells were split at a concentration of 500.000 cells/ml.

Maxisorp plates were coated with 100 µL/well of coating buffer containing 1 µg/mL rhMAdCAM-1. Plates were sealed with TopSeal A plus and incubated overnight at 4°C.

Day 1: Plates were washed twice with 150 µL wash buffer and then blocked with 250 µL/well blocking buffer (1% BSA in PBS) for one hour at room temperature. CFSE Celltrace stock solution is prepared just before use by adding 18 µL DMSO to a vial of CellTrace™ Reagent and mixing well. Cells were pelleted by centrifugation and supernatant removed. Dilute the Cell trace DMSO stock solution in pre-warmed (37°C) PBS to a working concentration of 1 µM. Cells were gently resuspended in PBS dye solution to a concentration of 1 million cells/ml.

Cells were incubated at 37°C for 20 minutes in the dark. Five times the original staining volume of medium was added to the cells and incubated for 5 minutes. This step removes any free dye remaining in the solution.

Cells were pelleted by centrifugation and resuspended in fresh pre-warmed complete medium, and cells were counted. Cell suspensions were prepared at a concentration of 2 million cells/ml in binding buffer.

Compound 1 was reconstituted to a concentration of 10 mM in DMSO. Compound 1 was diluted to 2x final concentration in binding buffer via serial dilution in the compound dilution plate.

After one hour of blocking, the Maxisorp discs were washed three times with 250 µL of PBS (without Mg ^{2 +} and Ca ^{2 +} ). 50 µL of test compound was added to designated wells on the plate, and 50 µL of CFSE-labeled cells (100,000 cells/well) were added to those wells. Plates were incubated on pre-heated coolsink plates for 45 minutes at 37°C and 5% CO ₂ to allow cell attachment.

After incubation, unbound cells were washed away. Cell plates were emptied by inversion and liquid residue was removed by blotting on paper towels. Dishes were washed twice by adding 150 µL of PBS (without Mg ^{2 +} and Ca ^{2 +} ).

After the last wash, 100 µL of PBS (without Mg ^{2 +} and Ca ^{2 +} ) was added to each well and the fluorescence (Ex485/Em535) was read using a disc reader (Envision 2105 multimode disc reader). To calculate concentration effects, the fluorescence values of control wells without cells were subtracted from each test well.

Figure 1 shows the % inhibition of Compound 1 at different concentrations. _IC50 values were calculated using a 4 parameter non-linear fit of the data. The IC ₅₀ value of Compound 1 was found to be 3.5×10 ^{- 8} M.

Example 3 Pharmacokinetic Characterization of Compound 1 Sprague Dawley or Wistar rats (male, body weight approximately 250-350 g) were administered a single intravenous (bolus intravenous) injection of the peptide to be tested.

After intravenous administration of Compound 1 (dose 510 nmol/kg, dosing volume 2 mg/kg), at 10 minutes, 15 minutes, 40 minutes, 1 hour, 2 hours, 4 hours, 8 hours after administration 24 hours to draw blood samples. At each sampling time point, samples were drawn from rats by tail dissection. The administration vehicle is 20 mM phosphate buffer saline, 260 mM mannitol, pH 7.

Plasma samples were analyzed by liquid chromatography mass spectrometry (LC-MS/MS) after protein precipitation. Pharmacokinetic parameters were calculated using individual plasma concentrations using non-compartmental methods in Phoenix WinNonlin 8.3 or higher. The plasma terminal elimination half-life (T½) was determined as ln(2)/λz, where λz is the magnitude of the slope of the log-linear regression of the terminal log concentration versus time curve. AUC _inf is the area under the plasma concentration-time curve extrapolated to infinity (AUC _inf = AUC _last + C _last /λz, where C _last is the last observed plasma concentration). _C0 is the plasma concentration at the dosing time intercept and was back extrapolated by log-linear regression of the first two data points. Results for selected compounds are shown in Table 1. dose AUC _inf C ₀ T _1/2 compound nmol/kg (hr*nmol/L) (nmol/L) (hr) Compound 1 510 32200 90200 5.61 Table 1

Example 4 Chemical Stability of Compound 1 and Control Compound 1 In this example, the stability of Compound 1 was tested and compared with that of Control Compound 1 .

Materials and Methods The chemical stability of the compounds was tested in 20 mM phosphate pH 7, 20 mM phosphate pH 8 and 20 mM glycine pH 9 at 1 mg/mL in aqueous solution. Chemical stability was followed for two weeks at 40°C, and chemical stability was assessed using normalized endpoint purity.

HPLC prepared samples for determination of chemical purity were measured by reverse-phase HPLC at T=0, 1 and 2 weeks, using conventional HPLC equipment, such as Dionex® Ultimate 3000 system, when using a binary gradient, equipped with A column, such as a 1.5 x 150 mm Acquity UPLC peptide CSH column, with a suitable gradient of buffer A (0.3% trifluoroacetic acid in water) and buffer B (0.3% trifluoroacetic acid, 90% MeCN in water), Where the column temperature was 50°C and the gradient was from 20 to 80% MeCN over 19 minutes. Purity was determined by peak area.

Chemical Stability Stock Solution of Compound 1 and Reference Compound 1 The compound was carefully weighed and dissolved in MQ water to 2 mg/mL, and the pH was adjusted to neutral pH (pH 6-8). The stock solution was equilibrated at ambient temperature for 15 minutes, at which time no particles were visible. A 40 mM buffer stock solution was prepared for each pH condition tested.

Chemical Stability Analysis : Formulations for chemical testing were made by mixing peptide stocks and buffer stocks in a 1:1 ratio. This was done for each buffer/pH condition in Protein LoBind centrifuge tubes 2.0 mL (Eppendorf). These samples were placed in a thermostat set at 40°C for two weeks.

Measuring Chemical Stability : Chemical stability was assessed by taking a small volume and running it on a Dionex® Ultimate 3000 system. The resulting chromatograms were integrated and normalized to freshly lysed samples (T=0), yielding normalized purity data. Endpoint data after 2 weeks of storage at 40°C are reported in Table 2 below and shown in Figure 2. compound formulation Normalized purity after storage at 40°C for 2 weeks Control compound 1 20 mM Phosphate pH 7 95.6 Control compound 1 20 mM Phosphate pH 8 97.1 Control compound 1 20 mM Glycine pH 9 97.4 Compound 1 20 mM Phosphate pH 7 99.1 Compound 1 20 mM Phosphate pH 8 99.4 Compound 1 20 mM Glycine pH 9 99.7 Table 2

Example 5 Further Pharmacokinetic Characterization Method of Compound 1 Beagle dogs (male, body weight about 10-12 kg) were administered a single subcutaneous (sc) and intravenous (iv) injection of the peptide to be tested.

After subcutaneous and intravenous administration of selected compounds (at a dose of 200 µg/kg) in a volume of 0.1 ml/kg, at 0, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours post-dose Blood samples of control compound 1 were drawn at 1 hour, 48 hours, 72 hours and 144 hours, and at 0, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours after administration Blood samples of Compound 1 were drawn at 1 hour and 168 hours.

Blood was sampled via venflon on day 1 (excluding the 8 hour time point). Blood was sampled through the jugular vein using vacuum blood collection tubes and vacuum blood collection tube needles at the 8-hour sampling time and subsequent sampling time points. The administration vehicle is PBS buffer, pH 7.

Plasma samples were analyzed by liquid chromatography mass spectrometry (LC-MS/MS) after protein precipitation. Pharmacokinetic parameters were calculated using individual plasma concentrations using non-compartmental methods in Phoenix WinNonlin 8.3 or higher. The plasma terminal elimination half-life (T½) was determined as ln(2)/λz, where λz is the magnitude of the slope of the log-linear regression of the terminal log concentration versus time curve. AUC _inf is the area under the plasma concentration-time curve extrapolated to infinity (AUC _inf = AUC _last + C _last /λz, where C _last is the last observed plasma concentration). _C0 is back extrapolated by log-linear regression of the first two data points. Results for selected compounds are shown in Table 3. Compound number investment channel t _{½ Cmax} AUC _inf (hr) (nmol/L) (hr*nmol/L) Control compound 1 IV 23.5 625 3780 Control compound 1 SC 26.6 137 3060 Compound 1 IV 53.0 743 19300 Compound 1 SC 95.2 127 21800 table 3

As can be seen from Table 3, Compound 1 exhibited a significant t½ increase and higher AUC compared to the control Compound 1. This increase was unexpected considering the small structural differences between the compounds.

Example 6 Further Pharmacokinetic Characterization of Compound 1 Methods Beagle dogs (male, body weight approximately 10-12 kg) were administered a single subcutaneous (sc) injection of the peptide to be tested. After subcutaneous administration of selected compounds (doses of 10, 20, 50, 100, 200 and 400 µg/kg) in a volume of 0.1 ml/kg, at 0, 3, 6, 8, 12, 24, 48, Blood samples were drawn at 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 576, 744, 936, 1104 and 1272 hours.

Blood was sampled via venflon on day 1 (excluding the 8 hour time point). Blood was sampled through the jugular vein using vacuum blood collection tubes and vacuum blood collection tube needles at the 8-hour sampling time and subsequent sampling time points. The administration vehicle is PBS buffer, pH 7.

Plasma samples were analyzed by liquid chromatography mass spectrometry (LC-MS/MS) after protein precipitation. Pharmacokinetic parameters were calculated using individual plasma concentrations using non-compartmental methods in Phoenix WinNonlin 8.3 or higher. The plasma terminal elimination half-life (T½) was determined as ln(2)/λz, where λz is the magnitude of the slope of the log-linear regression of the terminal log concentration versus time curve. AUC _inf is the area under the plasma concentration-time curve extrapolated to infinity (AUC _inf = AUC _last + C _last /λz, where C _last is the last observed plasma concentration). _C0 is back extrapolated by log-linear regression of the first two data points. Results for selected compounds are shown in Table 4. Compound number investment channel dose t ½ _{Tmax Cmax} AUC _inf (µg/kg) (hr) (hr) (nmol/L) (hr*nmol/L) Compound 1 SC 10 340 20 18.1 2460 20 460 20 43.1 5680 50 340 20 102 11600 100 230 20 504 58600 200 230 20 417 72500 400 260 20 927 158000 Table 4

The data presented in Table 4 supplement the data in Table 3, showing that the t½ exhibited by Compound 1 is unexpectedly long following subcutaneous administration. This was unexpected considering its molecular structure and the relatively low t½ shown by the control Compound 1 in the results shown in Table 3.

Example 7 Test Compounds in Integrin α4β7 - MAdCAM Cell Adhesion Assay and Integrin α4β1 - VCAM - 1 Cell Adhesion Assay Recombinant human MAdCAM -1 (rhMAdCAM-1) line was obtained from R&D systems (cat. no. 6056 -MC-050).

Recombinant human VCAM-1 (rhVCAM-1) was obtained from R&D systems (catalogue number 862-VC-100).

The CellTrace™ CFSE Cell Proliferation Kit for Flow Cytometry was obtained from Thermo Fisher Scientific (Cat# C34554).

The peptides to be tested are aliquoted and reconstituted internally.

Coating Buffer (50 nM Sodium Carbonate, pH 9.6) (Merck Millipore, Cat# S2127).

Wash buffer (PBS without Mg ^{2 +} and Ca ^{2 +} + 0.05% Tween® 20).

DMEM Binding Buffer (Dulbecco Modified Eagle Medium, Phenol Red Free (DMEM) (Gibco Cat. No. 31053-028), ₁ mM MnCl (Sigma Aldrich, Cat. No. M1787-10x1ML) , 20 mM HEPES (pH 7.5) (Gibco Cat. No. 15630-056), 1 mM Sodium Pyruvate (Gibco Cat. No. 11360-039), 0.05% Casein (Sigma Aldrich, Cat. #C4765))

Blocking buffer: stock solution is PBS (without Mg ^{2 +} and Ca ^{2 +} ) + 10% BSA.

Bovine Serum Albumin (BSA) (Sigma, cat. no. 9430).

RPMI Cell Medium 1640 (1×) + Glutamax (Gibco Cat# 61870-010).

Fetal bovine serum (Thermo Fisher Scientific; Cat. No. A4766801 ).

Penicillin-Streptomycin (10,000 U/mL) (Thermo Fisher Scientific; Cat. No. 15140122).

Final Growth Medium: RPMI Cell Medium 1640 (1x) + Glutamax + 10% FBS + 1% Penicillin-Streptomycin.

Compound Dilution Trays (Thermo Fisher Scientific, Nunc, Cat. No. 249944).

Perkin Elmer TopSeal A plus was obtained from PerkinElmer (cat. no. 6050185).

Coolsink heating plate XT96F (Merck Millipore, catalog number BCS-536).

Envision 2105 Multimode Disc Reader (PerkinElmer, Cat. No. 2105-0010).

The F96 Maxisorp plate line from Nunc was obtained from Thermo Fisher Scientific (cat. no. 442404).

Cells: The human B-lymphocyte cell line RPMI 8866 was obtained from Merck via ECACC (European Collection of Authenticated Cell Cultures) (Catalog No. 95041316 and Lot No. 14G012). The human B lymphocyte cell line RAMOS was obtained from the New Zealand Pharma Culture Collection.

Assay protocol : Day 0: RPMI 8866 and RAMOS cells were split at a concentration of 500,000 cells/ml.

A Maxisorp plate was coated with 100 µL/well of coating buffer containing 1 µg/mL rhMAdCAM-1 and a second Maxisorp plate was coated with 100 µL/well of coating buffer containing 1 µg/mL rhVCAM-1 . Plates were sealed with TopSeal A plus and incubated overnight at 4°C.

Day 1: Plates were washed twice with 150 µL wash buffer and then blocked with 250 µL/well blocking buffer (1% BSA in PBS) for one hour at room temperature. CFSE Celltrace stock solution is prepared just before use by adding 18 µL DMSO to a vial of CellTrace™ Reagent and mixing well. Cells were pelleted by centrifugation and supernatant removed. Dilute the Cell trace DMSO stock solution in pre-warmed (37°C) PBS to a working concentration of 1 µM. Cells were gently resuspended in PBS dye solution to a concentration of 1 million cells/ml.

Cells were incubated at 37°C for 20 minutes in the dark. Five times the original staining volume of medium was added to the cells and incubated for 5 minutes. This step removes any free dye remaining in the solution.

Cells were pelleted by centrifugation and resuspended in fresh pre-warmed complete medium, and cells were counted. Cell suspensions were prepared at a concentration of 2 million cells/ml in DMEM binding buffer.

Compound 1 and control compounds 1 and 2 were reconstituted to a concentration of 10 mM in DMSO. In compound dilution plates, all compounds were diluted to 2x final concentration in DMEM binding buffer via serial dilution.

After one hour of blocking, the Maxisorp discs were washed three times with 250 µL of PBS (without Mg ^{2 +} and Ca ^{2 +} ). 50 µL of test compound was added to designated wells on the plate, and 50 µL of CFSE-labeled cells (100,000 cells/well) were added to those wells. CFSE-labeled RPMI 8866 cells were added to rhMAdCAM-1 coated dishes, and CFSE-labeled RAMOS cells were added to rhVCAM-1 coated dishes. Plates were incubated on pre-heated coolsink plates for 45 minutes at 37°C and 5% CO ₂ to allow cell attachment.

After incubation, unbound cells were washed away. Cell plates were emptied by inversion and liquid residue was removed by blotting on paper towels. Dishes were washed twice by adding 150 µL of PBS (without Mg ^{2 +} and Ca ^{2 +} ).

After the last wash, 100 µL of PBS (without Mg ^{2 +} and Ca ^{2 +} ) was added to each well and the fluorescence (Ex485/Em535) was read using a disc reader (Envision 2105 multimode disc reader). To calculate concentration effects, the fluorescence values of control wells without cells were subtracted from each test well. _IC50 values were calculated using a 4 parameter non-linear fit of the data.

Table 5 shows the IC ₅₀ values of compound 1 and control compounds on α4β7 and α4β1. Compound number α 4 β 7 RPMI8866/MAdCAM-1 adhesion α 4 β 1 RAMOS/VCAM-1 adhesion selectivity ratio IC ₅₀ (nM) IC ₅₀ (nM) Compound 1 7.2 115 16.0 Control compound 1 26.6 304 11.4 Control compound 2 7.5 122 16.3 table 5

Example 8 Testing Compound 1 in a Mouse Dextran Sodium Sulfate ( DSS ) Colitis Model The present study was conducted in a mouse model of dextran sodium sulfate (DSS)-induced ulcerative colitis (UC). DSS induced chronic colitis in experimental animals when given orally in drinking water for 6 days, followed by 6 days in the absence of DSS in drinking water. Chronic intestinal inflammation is associated with weight loss, diarrhea, blood in the stool, and infiltration of white blood cells from the blood into the intestinal tissue. Among these cells, T lymphocyte subsets such as Th1 and Th17 play an important role in the initiation and chronication of inflammation. The migration of T cells from blood to target tissues is a complex process controlled by molecularly distinct adhesion and signaling steps. Therefore, the adhesion mechanism of a4b7 and MAdCAM-1 may be closely involved in the migration of lymphocytes to intestinal inflammatory sites.

Evaluation of the therapeutic potential of Compound 1 was performed in a mouse model of dextran sodium sulfate (DSS)-induced ulcerative colitis (UC).

Animals Male C57BI/6 mice (Charles River, Germany), weighing 20-25 g on arrival, 8 weeks old, were used for this study. Upon arrival at the animal facility, a general health assessment was performed on all animals. A 5-day acclimatization period was allowed before the start of the study.

Animal Care Committee This study was performed in an animal facility accredited by the Association for Accreditation and Accreditation of Laboratory Animal Care (AALAC), CRO Selvita, Zagreb, Croatia. All animal-related research was carried out in accordance with 2010/63/EU and national legislation regulating the use of laboratory animals for scientific research and other purposes (Official Journal 55/13). The Institutional Committee for Animal Research Ethics (CARE-Zg) oversees that animal-related procedures do not compromise animal welfare.

Housing environment Animals were housed under standardized environmental conditions. Mice were housed in cages with a solid floor (TECNIPLAST cage, type III, polysulfonate material, surface 425 mm x 266 mm x 185 mm). Ten animals per cage were housed on irradiated laboratory animal corncob-free bedding (Scobis Due - Mucedola, Italy) and provided a cotton nest for nesting, a desirable paper shelter (Lillico Serving Biotechnology, UK) and Wooden Nibblers (Certified, Bio-Serv USA). Each cage was equipped with a manual water distribution system. Standard certified commercial rodent diets were provided ad libitum. Tap water was provided ad libitum. The diet and water are believed to be free of known contaminants that would interfere with the research objectives. Each cage has a corresponding group identification, indicating the treatment and identity of the animals kept in the cage. Mice from different treatment groups were not mixed. The animal room was maintained at a controlled temperature of 22 ± 2°C and a relative humidity of 55 ± 10%. A controlled lighting system ensures that animals have 12 hours of light and 12 hours of darkness per day. Maintain adequate ventilation with 15-20 air changes per hour.

Ulcerative colitis was induced in C57Bl / 6 mice by administering 1.5% (w/v) dextran sodium sulfate (DSS) salt in drinking water Reagent grade: MP Biomedicals, cat. no. 160110 (36-50 kDa ) induced experimental UC in mice for 6 days. On day 7, DSS drinking water was replaced with normal water until the end of the study on day 12. Group 1 (see Table 6) did not receive DSS in drinking water and served as a control for healthy colons. Group Number of animals / group DSS in drinking water (D0-D6) water only (D6-D12) Handling D-2-D11 Dosage concentration / timing and frequency of administration 1 5 water medium 10 mL/kg (D-2 and D-1 PO/TID) (D0-D11 PO/BID) 2 10 1.5% DSS medium 10 mL/kg (D-2 and D-1 PO/TID) (D0-D11 PO/BID) 3 10 Compound 1 10000 nmol/kg 10000 nmol/kg (20 mg/kg) (D-2 and D-1 PO/TID) (D0-D11 PO/BID) 4 10 Compound 1 1250 nmol/kg 1250 nmol/kg (2.5 mg/kg) (D-2 and D-1 PO/TID) (D0-D11 PO/BID) Table 6 PO = oral; TID = three times a day; BID = twice a day

The studied test article and vehicle were administered orally at 10 mL/kg. Dosing volumes were adjusted individually according to the body weight of each animal to achieve the target dose indicated for the individual group.

Preparation of Test Compounds and Vehicles test substance the solution medium 0.5% HPMC with 20 mM Phosphate pH 7.5 Compound 1 2 mg/mL or 0.25 mg/ml in 0.5% hydroxypropylmethylcellulose (HPMC) with 20 mM phosphate pH 7.5 Table 7

Vehicle was prepared once throughout the study and stored at +4°C.

Dosing solutions were prepared in the afternoon for the next day's dosing event, stored at +4°C and administered to animals at room temperature.

Vehicle and Compound 1 were administered TID on Days -2 and -1, and BID thereafter.

Disease Activity Index ( DAI ) Assessment To assess the severity of colitis, the Disease Activity Index (DAI) was calculated as a combined score of a) weight loss, b) stool firmness, and c) daily colorectal bleeding, with a maximum score of 12. Baseline DAI scores were determined on day -2.

Animals were weighed daily beginning on day -2 before the first DSS challenge, and % body weight change was calculated using the formula: [(body weight on day X - initial body weight)/initial body weight] x 100. Body weight change expressed as a single digit value (X%) was used to determine the weight loss score as described in Table 8.

Fecal samples were collected daily during animal handling. Place each fecal pellet into a 2 mL U-bottom Eppendorf tube filled with 1.0 mL of water and vortex at maximum speed for 3-5 seconds. For stool firmness, well-formed pellets are assigned 0 points, semi-formed pellets are assigned 1 point, pasty pellets (loose stool) are assigned 2 points, incomplete liquid stools (mild diarrhea) are assigned 3 points and liquid stools (severe diarrhea ) assigns 4 points.

A colorectal bleeding score was defined as 0 indicating the absence of blood in the stool or anal area, 2 indicating visible blood in the fecal particles and 4 indicating severe bleeding and/or blood around the anal area. Each score will be determined as shown in Table 8. Weight Loss (%) score stool firmness score colorectal bleeding score <1% 0 shaped and hard 0 does not exist 0 1-5% 1 molded and soft 1 5.1-10% 2 Loose stool 2 exist 2 10.1-15% 3 mild diarrhea (watery) 3 >15.1% 4 severe diarrhea 4 serious 4 Table 8 Disease Activity Index (DAI) of animals treated with DSS* *Table adapted from Current protocols in Pharmacology 72:5.58 (Bang, B. and Lichtenberger, LM 2016. Methods of inducing inflammatory bowel disease in mice. Curr. Protoc. Pharmacol. 72:5.58.1-5.58.42. doi: 10.1002/0471141755.ph0558s47.)

Colon weight and length measurements and colon sample collection for histopathology Colons, including the cecum, were removed after mice were euthanized at the end of the study. The length of the colon, excluding the length of the cecum, was measured by a ruler from the cecum to the anus. Next, the colon was flushed with cold PBS and weighed, and its length was measured. The length of the colon from the cecum to the anus was measured in centimeters (cm) with a ruler, excluding the length of the cecum. Colons were then flushed with cold PBS, weighed and cut into three sections. Segment 1 (proximal part) and segment 3 (distal part) were fixed in 10% formalin for histological analysis.

Colon Preparation and Histological Analysis Resected colons were divided into proximal and distal segments, dissected and embedded in paraffin whole (two pieces).

Histological analysis of PAS-stained slides was performed using Nishitani et al. ( Int Immunopharmacol . 2009 , 9 , 1444-51), as summarized in Table 9 below: severity of inflammation layer involved epithelial injury score degree 0 no inflammation no inflammation Epithelial intact No lesion 1 mild mucous membrane structural damage Dotted 2 Moderate Mucosa and submucosa erosion Multifocal 3 severe Transmural ulcer Diffuse Table 9

Results DAI scores were individually assessed based on the severity of three specific symptoms: blood in stool, stool firmness, and weight loss. Calculate the area under the curve (AUC) for each group (Figure 3). In the DSS-treated groups (DSS+vehicle; DSS+compound 1 10000 nmol/kg and DSS+compound 1 1250 nmol/kg), DAI score AUC increased after administration of DSS in drinking water. Compared with the vehicle control (no DSS) group, the AUC of the DAI score was significantly increased in the DSS vehicle group. Administration of the highest dose of Compound 1 (10000 nmol/kg) from day -2 resulted in a significant reduction in DAI score AUC compared to the DSS+vehicle group (Figure 3).

The key measure of disease severity in the DSS colitis model is weight loss, so weight gain is a good indicator of the therapeutic effect of a test compound. Weight change scores were included in the Disease Activity Index (DAI) score (see Table 8). The weight change score AUC of each group was calculated (Figure 4). In the DSS-treated groups (DSS+vehicle; DSS+compound 1 10000 nmol/kg and DSS+compound 1 1250 nmol/kg), body weight change score AUC increased after administration of DSS in drinking water. The body weight change score AUC was significantly increased in the DSS vehicle group compared to the vehicle control (no DSS) group. Compared with the DSS+vehicle group, a significant therapeutic effect was observed after administration of compound 1 (10000 nmol/kg) and compound 1 (1250 nmol/kg) from day -2, leading to a significant reduction in the weight change score AUC (Figure 4) .

Colon shortening is a key macroscopic change that correlates with disease severity in DSS colitis, and thus colon length is often studied by measuring colon length in centimeters (cm) as part of a termination procedure. The colon length (cm) of all animals in the experiment was measured (Figure 5). As shown in FIG. 5 , in the DSS-treated groups (DSS+vehicle; DSS+compound 1 10000 nmol/kg and DSS+compound 1 1250 nmol/kg), the colon length was shortened after administration of DSS in drinking water. Colon length was significantly reduced in the DSS+vehicle group compared to the vehicle control (no DSS) group. Administration of high dose of Compound 1 (10000 nmol/kg) and low dose of Compound 1 (1250 nmol/kg) from day -2 resulted in a significant increase in colon length compared to the DSS vehicle group (Figure 5).

Histological analysis of the colon is a means of assessing disease severity and inflammation in DSS-induced colitis. Colons of individual mice were histologically analyzed by using the Nishitani total score as described in Table 9. The Nishitani total score scored for the whole colon (proximal and distal colon) for each group is shown in FIG. 6 . In the DSS-treated groups (DSS+vehicle; DSS+compound 1 10000 nmol/kg and DSS+compound 1 1250 nmol/kg), the total colonic Nishitani score increased after administration of DSS in drinking water. The total Nishitani score was significantly increased in the DSS+vehicle group compared to the vehicle control (no DSS) group. Compared with the DSS+vehicle group, administration of high-dose Compound 1 (10000 nmol/kg) and low-dose Compound 1 (1250 nmol/kg) from day -2 resulted in a significant reduction in the total colonic Nishitani score (Fig. 6) .

Histological assessment of inflammation severity was performed as part of the Nishitani total score assessment of manifestations of DSS-induced colonic disease. Inflammation severity scores scored for the whole colon (proximal and distal colon) for each group are shown in FIG. 7 . In the DSS-treated groups (DSS+vehicle; DSS+compound 1 10000 nmol/kg and DSS+compound 1 1250 nmol/kg), histological inflammation severity scores increased after administration of DSS in drinking water. The total Nishitani score was significantly increased in the DSS+vehicle group compared to the vehicle control (no DSS) group. Compared with the DSS+vehicle group, administration of high dose of compound 1 (10000 nmol/kg) but not low dose of compound 1 (1250 nmol/kg) from day -2 resulted in a significant reduction in the severity score of the whole colonic inflammation (Fig. 7).

Together, these findings demonstrate the significant therapeutic effect of Compound 1 on multiple parameters of disease activity in a DSS-induced colitis mouse model, including improvement in Disease Activity Index (DAI) scores, reduction in colon shortening, reduction in histological damage of the colon and severity of inflammation degree.

Example 9 Chemical Stability of Compound 1 , Control Compound 1 and Control Compound 2 In this example, the stability of Compound 1 was tested and compared with the stability of Control Compound 1 and Control Compound 2.

Materials and Methods The chemical stability of the compounds was tested in 20 mM phosphate pH 7, 20 mM phosphate pH 8 and 20 mM glycine pH 9 at 1 mg/mL in aqueous solution. Chemical stability was followed for two weeks at 40°C, and chemical stability was assessed using normalized endpoint purity.

HPLC for chemical purity determination Reconstituted samples were reverse phase HPLC at T=0 and 40 days using conventional HPLC equipment, such as a Dionex® Ultimate 3000 system, for binary gradient applications, equipped with columns such as 1.5×150 mm Acquity UPLC peptide CSH column, and use a suitable gradient of buffer A (0.3% trifluoroacetic acid, aqueous solution) and buffer B (0.3% trifluoroacetic acid, 90% MeCN, aqueous solution) to measure, where The column temperature was 70°C and the gradient was from 20 to 80% MeCN over 49 minutes. Purity was determined by peak area.

Stock Solutions Compounds were carefully weighed out and dissolved in MQ water to 2 mg/mL, and the pH was adjusted to neutral pH (pH 6-8). The stock solution was equilibrated at ambient temperature for 15 minutes, at which point no visible particles were present. A 40 mM buffer stock solution was prepared for each pH condition tested.

Chemical Stability Analysis Formulations for chemical testing were made by mixing peptide stocks and buffer stocks in a 1:1 ratio. This was done for each buffer/pH condition in Protein LoBind tubes 2.0 mL (Eppendorf). These samples were placed in a thermostat set at 40°C for 40 days.

Measuring Chemical Stability Chemical stability was assessed by taking a small volume and running it on a Dionex® Ultimate 3000 system. The resulting chromatograms were integrated and normalized purity data were generated by normalizing to freshly dissolved samples (T=0). Endpoint data after 40 days of storage at 40°C are reported in Table 10 below and shown in Figure 8. compound formulation Normalized purity after storage at 40°C for 40 days Control compound 1 20 mM Phosphate pH 7 92 Control compound 1 20 mM Phosphate pH 8 93 Control compound 1 20 mM Glycine pH 9 92 Control compound 2 20 mM Phosphate pH 7 80 Control compound 2 20 mM Phosphate pH 8 82 Control compound 2 20 mM Glycine pH 9 77 Compound 1 20 mM Phosphate pH 7 99 Compound 1 20 mM Phosphate pH 8 99 Compound 1 20 mM Glycine pH 9 99 Table 10

Example 10 Chemical Stability of Compound 1 and Control Compound 1 under Dry Conditions In this example, the stability of Compound 1 was tested and compared to that of Control Compound 1 .

Materials and Methods The chemical stability of the compounds was tested under dry conditions. The chemical stability was followed for 3 months in a desiccator at 40°C, the relative humidity was set by a saturated aqueous solution of sodium chloride. Chemical stability was assessed using normalized endpoint purity.

HPLC for determination of chemical purity Prepared samples were reverse phase HPLC at T=0, 1, 2 and 3 months using conventional HPLC equipment, such as the Dionex® Ultimate 3000 system, for binary gradient applications, equipped with A column such as a 1.5 x 150 mm Acquity UPLC Peptide CSH column with an appropriate buffer A (0.3% TFA in water) and buffer B (0.3% TFA, 90% MeCN in water) gradient was measured with a column temperature of 50°C and a gradient from 20 to 80% MeCN over 19 minutes. Purity was determined by peak area.

Stock Solutions Compounds were carefully weighed out and dissolved in Milli-Q® Purified Water (MQ Water) to 2 mg/mL and pH adjusted as necessary to achieve dissolution, which in the case of Compound 1 was pH 2-3 and In the case of control compound 1 it was pH 6. The stock solution was equilibrated at ambient temperature for 15 minutes, at which point no visible particles were present.

Chemical Stability Analysis : Two dry formulations were generated for chemical stability testing. The first formulation was generated by diluting the stock solution of the compound to 1 mg/mL in MQ water without further pH adjustment, and the second formulation was produced by diluting the stock solution to 1 mg/mL with MQ water and using Adjust pH to 9 with NaOH/HCl to produce. These two formulations were aliquoted into Eppendorf tubes and a dry formulation was generated by lyophilization. For chemical stability assessment, the formulations were stored in a desiccator at 40°C with relative humidity set by saturated NaCl solution.

Measurement of Chemical Stability: Chemical stability was assessed by withdrawing one Eppendorf tube for each compound and formulation at each pull point. The dry formulation was dissolved using 20 mM phosphate pH 7.0 to a concentration of 1 mg/mL. Samples were then measured on a Dionex® Ultimate 3000 system. The resulting chromatograms were integrated and normalized purity data were generated by normalizing to the reconstituted dry formulation generated at T=0. Endpoint data after 3 months storage at 40°C are reported in Table 11 below and shown in Figure 9.

Compound 1 and control Compound 1 were tested for chemical stability in aqueous formulations (Example 9) and dry formulations (Example 10). The results show that compound 1 has greater stability in aqueous and dry formulations compared to control compound 1. compound formulation Normalized purity after storage at 40°C for three months Control compound 1 pH 6 86 Control compound 1 pH 9 87 Compound 1 pH 2-3 99 Compound 1 pH 9 99 Table 11

Example 11 Pharmacokinetic characterization of comparative compounds 1 and 2 Sprague Dawley or Wistar rats (male, body weight approximately 250-350 g) were administered a single intravenous (bolus intravenous) injection of the peptide to be tested. After intravenous administration of Compound 1 (dose 510 nmol/kg, dosing volume 2 mg/kg), at 10 minutes, 15 minutes, 40 minutes, 1 hour, 2 hours, 4 hours, 7 hours after administration , 24 hours, 30 hours and 48 hours to draw blood samples. At each sampling time point, samples were drawn from rats by tail dissection. The administration vehicle is 20 mM phosphate buffer saline, 260 mM mannitol, pH 7.

Plasma samples were analyzed by liquid chromatography mass spectrometry (LC-MS/MS) after protein precipitation. Pharmacokinetic parameters were calculated using individual plasma concentrations using non-compartmental methods in Phoenix WinNonlin 8.3 or higher. The plasma terminal elimination half-life (T½) was determined as ln(2)/λz, where λz is the magnitude of the slope of the log-linear regression of the terminal log concentration versus time curve. AUC _inf is the area under the plasma concentration-time curve extrapolated to infinity (AUC _inf = AUC _last + C _last /λz, where C _last is the last observed plasma concentration). _C0 is the plasma concentration at the dosing time intercept and was back extrapolated by log-linear regression of the first two data points.

Results for selected compounds are shown in Table 12. compound dose AUC _inf C ₀ T _1/2 nmol/kg (hr*nmol/L) (nmol/L) (hr) Control compound 1 511 4640 2900 2.39 Control compound 2 511 13500 4940 7.58 Table 12

All publications mentioned in the above specification are incorporated herein by reference. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and equivalents of the described modes for carrying out the invention which are obvious to those skilled in chemistry, pharmacy, or related fields are intended to be within the scope of the following claims.

Figure 1 shows the dose-response curve of Compound 1. 2 shows the normalized purity values of Compound 1 and Control Compound 1 after two weeks storage at 40° C., as determined by the method described in Example 4. FIG. Figure 3 shows a histogram of the area under the curve (AUC) disease activity index (DAI) scores expressed as mean ± SEM for each group for Compound 1 when tested in the DSS mouse colitis model according to Example 8. The dose concentration of Compound 1 is the p.o. dose administered per dosing. Statistical differences between groups were determined using one-way ANOVA followed by Dunnett's multiple comparisons test to compare groups to the DSS+vehicle group (black in histogram). *p < 0.05; ns = not significant. Figure 4 shows a histogram of the area under the curve (AUC) body weight change scores for Compound 1 expressed as mean ± SEM for each group when tested according to Example 8 in the DSS mouse colitis model. The dose concentration of Compound 1 is the p.o. dose administered per dosing. Statistical differences between groups were determined using one-way ANOVA followed by Dunnett's multiple comparison test comparing groups to the DSS+vehicle group (black in histogram). *p < 0.05; ns = not significant. Figure 5 shows the histogram colon length (cm) expressed as the mean ± SEM of each group for Compound 1 when tested according to Example 8 in the DSS mouse colitis model. The dose concentration of Compound 1 is the p.o. dose administered per dosing. Statistical differences between groups were determined using one-way ANOVA followed by Dunnett's multiple comparison test comparing groups to the DSS+vehicle group (black in histogram). *p < 0.05; ns = not significant. Figure 6 shows the histological analysis of the histogram of the whole colon by using the Nishitani total score of Compound 1 expressed as the median and range for each group when tested according to Example 8 in the DSS mouse colitis model. The dose concentration of Compound 1 is the p.o. dose administered per dosing. Statistical differences between groups were determined using one-way ANOVA followed by Dunnett's multiple comparison test comparing groups to the DSS+vehicle group (black in histogram). *p < 0.05; ns = not significant. For comparisons between the DSS+vehicle group and the vehicle (no DSS) control group, the Wilcoxon signed rank test was used; #=p<0.05. Figure 7 shows histological analysis of the histological analysis of histograms of whole colonic inflammation severity scores expressed as median and range for each group when tested in the DSS mouse colitis model according to Example 8. The dose concentration of Compound 1 is the p.o. dose administered per dosing. Statistical differences between groups were determined using a non-parametric Mann-Whitney test, comparing each group to the DSS+vehicle group (black in the histogram). *p < 0.05; ns = not significant. For comparisons between the DSS+vehicle group and the vehicle (no DSS) control group, the Wilcoxon signed rank test was used; #=p<0.05. 8 shows the normalized purity values of Compound 1 and Control Compounds 1 and 2 after storage at 40° C. for 40 days, as determined by the method described in Example 9. FIG. 9 shows the stability of Compound 1 compared to Control Compound 1 under dry conditions when tested according to the conditions described in Example 10.

Claims (11)

A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier. The compound of Claim 1 or a pharmaceutically acceptable salt or solvate thereof, which is used as a medicine. The compound of Claim 1 or a pharmaceutically acceptable salt or solvate thereof, which is used for treating inflammation or autoimmune diseases in patients. The compound according to claim 4 or a pharmaceutically acceptable salt or solvate thereof, wherein the inflammatory or autoimmune disease is a disease of the gastrointestinal tract. A compound as claimed in claim 1 or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of a condition or disease selected from the group consisting of: inflammatory bowel disease (IBD), ulcerative colitis, Crowe Crohn's disease, celiac disease, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, pouchitis after proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, cholangitis , pericholangitis, primary sclerosing cholangitis, gastrointestinal infection human immunodeficiency virus (HIV), graft versus host disease, primary biliary sclerosis. The compound according to claim 6 or a pharmaceutically acceptable salt or solvate thereof, wherein the condition is inflammatory bowel disease. The compound according to claim 7 or a pharmaceutically acceptable salt or solvate thereof, wherein the inflammatory bowel disease is ulcerative colitis. The compound according to claim 7 or a pharmaceutically acceptable salt or solvate thereof, wherein the inflammatory bowel disease is Crohn's disease. The compound of claim 1 or a pharmaceutically acceptable salt or solvate thereof is used for treating local or systemic infection of viruses or retroviruses. The compound according to claim 10 or a pharmaceutically acceptable salt or solvate thereof, wherein the virus is HIV.