WO2015036927A1

WO2015036927A1 - Immunomodulating peptidomimetic derivatives

Info

Publication number: WO2015036927A1
Application number: PCT/IB2014/064380
Authority: WO
Inventors: Pottayil Govindan Nair Sasikumar; Muralidhara Ramachandra; Seetharamaiah Setty Sudarshan Naremaddepalli
Original assignee: Aurigene Discovery Technologies Limited
Priority date: 2013-09-10
Filing date: 2014-09-10
Publication date: 2015-03-19

Abstract

The present invention relates to immunomodulating peptidomimetic derivatives as therapeutic agents capable of inhibiting the programmed cell death 1 (PD1) signalling pathway. The invention also refers to derivatives of the therapeutic agents. The invention also encompasses the use of the said therapeutic agents and derivatives for treatment of disorders via immunopotentiation comprising inhibition of immunosuppressive signal induced due to PD-1, PD-L1, or PD-L2 and therapies using them.

Description

IMMUNOMODULATING PEPTIDOMIMETIC DERIVATIVES

This application claims the benefit of Indian provisional application number 4044/CHE/2013, filed on September 10, 2013 and Indian provisional application number 4045/CHE/2013, filed on September 10, 2013; which hereby incorporated by reference. TECHNICAL FIELD

The present invention relates to immunomodulating peptidomimetic derivatives. The invention also relates to pharmaceutical compositions comprising the said peptidomimetic compounds and their derivatives as therapeutic agents.

BACKGROUND OF THE INVENTION

Programmed cell death- 1 (PD-1) is a member of the CD28 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. PD-1 and its ligands are broadly expressed and exert a wider range of immunoregulatory roles in T cells activation and tolerance compared with other CD28 members. PD-1 and its ligands are involved in attenuating infectious immunity and tumor immunity, and facilitating chronic infection and tumor progression. The biological significance of PD- 1 and its ligand suggests the therapeutic potential of manipulation of PD- 1 pathway against various human diseases (Hyun-Tak Jin, et al., Curr Top Microbiol Immunol. (2011); 350:17-37).

T-cell activation and dysfunction relies on direct and modulated receptors. Based on their functional outcome, co-signaling molecules can be divided as co-stimulators and co-inhibitors, which positively and negatively control the priming, growth, differentiation and functional maturation of a T-cell response (Li Shi, et al., Journal of Hematology & Oncology 2013, 6:74).

Therapeutic antibodies that block the programmed cell death protein-1 (PD-1) immune checkpoint pathway prevent T-cell down regulation and promote immune responses against cancer. Several PD-1 pathway inhibitors have shown robust activity in various phases of clinical trials (RD Harvey, Clinical Pharmacology & Therapeutics (2014); 96 2, 214-223).

Programmed death-1 (PD-1) is a co-receptor that is expressed predominantly by T cells. The binding of PD-1 to its ligands, PD-L1 or PD-L2, is vital for the physiological regulation of the immune system. A major functional role of the PD-1 signaling pathway is the inhibition of self-reactive T cells, which serve to protect against autoimmune diseases. Elimination of the PD-1 pathway can therefore result in the breakdown of immune tolerance that can ultimately lead to the development of pathogenic autoimmunity. Conversely, tumor cells can at times co-opt the PD-1 pathway to escape from immunosurveillance mechanisms. Therefore, blockade of the PD-1 pathway has become an attractive target in cancer therapy. Current approaches include six agents that are either PD-1 and PD-L1 targeted neutralizing antibodies or fusion proteins. More than forty clinical trials are underway to better define the role of PD-1 blockade in variety of tumor types. (Ariel Pedoeem et al., Clinical Immunology (2014), 153(1), 145-152).

International applications WO 01/14557, WO 02/079499, WO 2002/086083, WO 03/042402, WO 2004/004771 , WO 2004/056875, WO2006121168, WO2008156712, WO2010077634, WO2011066389, WO2014055897, WO2014059173, WO2014100079 and US patent US08735553 report PD-1 or PD-L1 inhibitory antibodies or fusion proteins.

Further, International applications, WO2011161699, WO2012/168944, WO2013144704 and WO2013132317report peptides or peptidomimetic compounds which are capable of suppressing and/or inhibiting the programmed cell death 1 (PD1) signaling pathway.

Still there is a need for more potent, better and/or selective immune modulators of PD-1 pathway. The present invention provides peptidomimetic compounds which are capable of suppressing and/or inhibiting the programmed cell death 1 (PD1) signalling pathway.

SUMMARY OF INVENTION

In accordance with the present invention, provided are peptidomimetic compounds or a pharmaceutically acceptable salt or a stereoisomer thereof, which are capable of suppressing and/or inhibiting the programmed cell death 1 (PD1) signalling pathway.

In one aspect, the present invention provides a peptidomimetic compounds of formula (I):

^Rl ^R2

P— [Aaal]_x— N— N— [Aaa2]_y— C(O)— [Aaa3]_z— Q (I)

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, x, y and z are integers, each independently selected from 1 to 2; each of [Aaal], [Aaa2] and [Aaa3] represents x, y and z independently selected amino acid residues, with a proviso that [Aaal] is not selected from Ser, Thr, Ala or Leu if

(i) [Aaa2] is selected from Asp, Asn, Glu or Gin;

(ii) [Aaa3] is selected from Ser, Asp, Ala, He, Phe, Trp, Glu or Thr; and

(iii) each of x, y and z is 1 ;

wherein, [Aaal], [Aaa2] and [Aaa3] are optionally substituted by hydroxyl; Ri and R₂ are independently hydrogen or alkyl;

P represents free N-terminus, alkylated N-terminus or acylated N-terminus of [Aaal];

Q represents free C-terminus, amidated C-terminus or esterified C-terminus of [Aaa3]; and

the bond between [Aaal] and -NRi- and/or between [Aaa2] and -NR₂- is a peptide bond or a reduced peptide bond.

In a further aspect of the present invention, it relates to the pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or a stereoisomer and processes for preparing thereof.

In yet another aspect of the present invention, it provides use of peptidomimetic compounds of formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof, which are capable of suppressing and/or inhibiting the programmed cell death 1 (PD1) signaling pathway.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptidomimetic compounds as therapeutic agents useful for treatment of disorders via immunopotentiation comprising inhibition of immunosuppressive signal induced due to PD-1, PD-L1 , or PD-L2 and therapies using them.

Each embodiment is provided by way of explanation of the invention, and not by way of limitation of the invention. In fact, it will be apparent to those skilled in the art that various modification and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment. Thus it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present invention are disclosed in, or are obvious from, the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not to be construed as limiting the broader aspects of the present invention.

In one embodiment, the present invention relates to compounds of formula (I)

^Rl ^R2

I I

P— [Aaal]_x— N— N— [Aaa2]_y— C(O)— [Aaa3]_z— Q (I)

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, x, y and z are integers, each independently selected from 1 to 2;

each of [Aaal], [Aaa2] and [Aaa3] represents x, y and z independently selected amino acid residues, with a proviso that [Aaal] is not selected from Ser, Thr, Ala or Leu if

(i) [Aaa2] is selected from Asp, Asn, Glu or Gin;

(ii) [Aaa3] is selected from Ser, Asp, Ala, He, Phe, Trp, Glu or Thr; and

(iii) each of x, y and z is 1 ;

In yet another embodiment, the present invention provides compounds of formula (IA)

H

-[Aaal]_x— N— N— [Aaa2]_y— C(O)— [Aaa3]_z— Q

H

(IA)

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, x, y, z, [Aaal], [Aaa2], [Aaa3], P and Q are same as defined in compound of formula (I). In yet another embodiment, the present invention provides peptidomimetic compounds of formula (IB):

or a stereoisomer or a pharmaceutically acceptable salt thereof;

wherein;

each of Ri ' , R₂' and R3 independently represents side chain of an amino acid with the proviso that Ri ' is not a side chain of Ser, Thr, Ala or Leu if R₂' is a side chain of Asp, Asn, Glu or Gin and R₃ is a side chain of Ser, Asp, Ala, He, Phe, Trp, Glu or Thr;

R4 is -OR or -NH₂;

R is hydrogen, alkyl or arylalkyl;

R₅ and R₆ are independently selected from hydrogen, alkyl or acyl;

each of R7 and R7' independently represents hydrogen or alkyl;

both R_a and R_a' represent hydrogen; or together represent an oxo (=0) group; both R_b and R_b' represent hydrogen; or together represent an oxo (=0) group. In yet another embodiment, the present invention provides compounds of the formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, [Aaal ] is selected from the group consisting of: Ser, Ala, Cys, Asp, Phe, Pro, Asn and Lys.

In yet another embodiment, the present invention provides compounds of the formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, [Aaa2] is selected from the group consisting of: Ala, Asn, Phe, Lys, Thr and Phe;

In yet another embodiment, the present invention provides compounds of the formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, [Aaa3] is selected from the group consisting of: Thr, Ala, Phe, Ser, Lys, Gly, val and Asn.

The embodiment below are illustrative of the present invention and are not intended to limit the claims to the specific embodiments exemplified.

According to one embodiment, specifically provided are compounds of the formula (I), in which Ri is hydrogen. According to another embodiment, specifically provided are compounds of the formula (I), in which R₂ is hydrogen.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which P is free N-terminus.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which Q is free C-terminus.

According to yet another embodiment, specifically provided are compounds of the formula (I), in which x, y and z are independently are 1.

According to yet another embodiment, specifically provided are compounds of the formula (I), in which x, y and z do not simultaneously have a value of 1.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which x is 2, y is 1 and z is 1.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which x is 1, y is 1 and z is 2.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which [Aaal] is selected from the group of: Ser, Ala, Asp, Phe and Lys.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which [Aaa2] is selected from the group of: Ala and Asn.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA), in which [Aaa3] selected from the group of: Thr, Ala, Lys and Gly.

According to yet another embodiment, specifically provided are compounds of the formula (I) and (IA) in which one, more or all amino acid/s is/are D amino acid/s.

In an embodiment, specific compounds of formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof; without any limitation are enumerated in table 1. Table 1

pharmaceutical administration. In one embodiment, the present invention provides a pharmaceutical composition comprising the compound as disclosed, and a pharmaceutically acceptable carrier or a diluent.

In another embodiment, the said pharmaceutical composition further comprising at least one of an anticancer agent, chemotherapy agent, or antiproliferative compound.

In one embodiment, use of compounds as disclosed in the present invention for use as a medicament.

In another embodiment, use of compounds as disclosed in the present invention in the manufacture of a medicament for the treatment of cancer or infectious disease.

In another embodiment, use of the compounds as disclosed in the present invention in the manufacture of a medicament for the treatment bone cancer, cancer of the head or neck, pancreatic cancer, skin cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

In another embodiment, the present invention provides the compounds as disclosed in the present invention for use in the treatment of cancer.

In another embodiment, the present invention provides the compounds as disclosed in the present invention for use in the treatment of infectious disease.

In one embodiment, the present invention provides the compounds as disclosed in the present invention for use as a medicament for the treatment of bacterial infectious disease, a viral infectious disease or a fungal infectious disease. In one embodiment, the present invention provides a method of treatment of cancer, wherein the method comprises administration of an effective amount of the compound of the present invention to the subject in need thereof.

In another embodiment the present invention provides a method of modulating an immune response mediated by PD-1 signaling pathway in a subject, comprising administering to the subject therapeutically effective amount of the compound of the present invention such that the immune response in the subject is modulated.

In yet another embodiment the present invention provides a method of inhibiting growth of tumour cells and/or metastasis in a subject, comprising administering to the subject a therapeutically effective amount of compound of the present invention capable of inhibiting the programmed cell death 1 (PD1) signaling pathway.

The said tumour cells include cancer such as but not limited to bone cancer, cancer of the head or neck, pancreatic cancer, skin cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

In yet another further embodiment the present invention provides a method of treating an infectious disease in a subject comprising administering to the subject a therapeutically effective amount of the compound of the present inventioncapable of inhibiting the programmed cell death 1 (PD1) signaling pathway such that the subject is treated for the infectious disease. Still yet another embodiment of the present invention provides a method of treating bacterial, viral and fungal infections in a subject comprising administering to the subject a therapeutically effective amount of the compound of the present invention capable of inhibiting the programmed cell death 1 (PD1) signalling pathway such that the subject is treated for the bacterial, viral and fungal infections.

The infectious disease includes but not limited to HIV, Influenza, Herpes, Giardia, Malaria, Leishmania, the pathogenic infection by the virus Hepatitis (A, B, & C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus, pathogenic infection by the bacteria chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, E. coli, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria, pathogenic infection by the fungi Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum, and pathogenic infection by the parasites Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, Nippostrongylus brasiliensis.

The compounds of the present invention may be used as single drugs or as a pharmaceutical composition in which the compound is mixed with various pharmacologically acceptable materials.

The pharmaceutical composition is usually administered by oral or inhalation routes, but can be administered by parenteral administration route. In the practice of this invention, compositions can be administered, for example, by orally, intravenous infusion, topically, intraperitoneally, intravesically or intrathecally. Examples of the parenteral administration includes but not limited to intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Oral administration, parenteral administration, subcutaneous administration and intravenous administration are the preferred methods of administration.

The dosage of the compounds of the present invention varies depending on age, weight, symptom, therapeutic efficacy, dosing regimen and/or treatment time. Generally, they may be administered by oral or inhalation routes, in an amount of 1 mg to 100 mg per time, from once a couple of days, once 3 days, once 2 days, once a day to a couple of times a day, in the case of an adult, or continuously administered by oral or inhalation routes from 1 to 24 hours a day. Since the dosage is affected by various conditions, an amount less than the above dosage may sometimes work well enough, or higher dosage may be required in some cases.

The compounds of the present invention may be administered in combination with other drugs for (1) complementation and/or enhancement of prevention and/or therapeutic efficacy of the preventive and/or therapeutic drug of the present invention, (2) dynamics, absorption improvement, dosage reduction of the preventive and/or therapeutic drug of the present invention, and/or (3) reduction of the side effects of the preventive and/or therapeutic drug of the present invention.

A concomitant medicine comprising the compounds of the present invention and other drug may be administered as a combination preparation in which both components are contained in a single formulation, or administered as separate formulations. The administration by separate formulations includes simultaneous administration and administration with some time intervals. In the case of the administration with some time intervals, the compound of the present invention can be administered first, followed by another drug or another drug can be administered first, followed by the compound of the present invention. The administration method of the respective drugs may be the same or different.

The dosage of the other drug can be properly selected, based on a dosage that has been clinically used. The compounding ratio of the compound of the present invention and the other drug can be properly selected according to age and weight of a subject to be administered, administration method, administration time, disorder to be treated, symptom and combination thereof. For example, the other drug may be used in an amount of 0.01 to 100 parts by mass, based on 1 part by mass of the compound of the present invention. The other drug may be a combination of two or more kind of arbitrary drugs in a proper proportion. The other drug that complements and/or enhances the preventive and/or therapeutic efficacy of the compound of the present invention includes not only those that have already been discovered, but those that will be discovered in future, based on the above mechanism.

Diseases on which this concomitant use exerts a preventive and/or therapeutic effect are not particularly limited. The concomitant medicine can be used for any diseases, as long as it complements and/or enhances the preventive and/or therapeutic efficacy of the compound of the present invention.

The compound(s) of the present invention can be used with an existing chemotherapeutic concomitantly or in a mixture form. Examples of the chemotherapeutic include an alkylation agent, nitrosourea agent, antimetabolite, anticancer antibiotics, vegetable-origin alkaloid, topoisomerase inhibitor, hormone drug, hormone antagonist, aromatase inhibitor, P-glycoprotein inhibitor, platinum complex derivative, other immunotherapeutic drugs and other anticancer drugs. Further, it can be used with a cancer treatment adjunct, such as a leucopenia (neutropenia) treatment drug, thrombocytopenia treatment drug, antiemetic and cancer pain intervention drug, concomitantly or in a mixture form.

In one embodiment, the compound(s) of the present invention can be used with other immunomodulators and/or a potentiating agent concomitantly or in a mixture form. Examples of the immunomodulator include various cytokines, vaccines and adjuvants. Examples of these cytokines, vaccines and adjuvants that stimulates immune responses include but not limited to GM-CSF, M-CSF, G-CSF, interferon-a, β, or γ, IL-1 , IL-2, IL- 3 , IL-12, Poly (I:C) and C_PG.

In another embodiment, the potentiating agents includes cyclophosphamide and analogs of cyclophosphamide, anti-TGF and Imatinib (Gleevac), a mitosis inhibitor, such as paclitaxel, Sunitinib (Sutent) or other antiangiogenic agents, an aromatase inhibitor, such as letrozole, an A2a adenosine receptor (A2AR) antagonist, an angiogenesis inhibitor, anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

As used herein, the term "acyl" refers to RC(O)-, wherein R is alkyl as defined below. Examples of acyl group include, but are not limited to -C(0)CH₃, -C(0)CH₂CH_3j -C(0)(CH₂)₂CH₃, -C(0)(CH₂)₃CH₃, -C(0)(CH₂)₄CH₃ or -C(0)(CH₂)₅CH₃.

As used herein the term "alkyl" refers to a hydrocarbon chain radical that includes solely carbon and hydrogen atoms in the backbone, containing no unsaturation, having from one to twenty carbon atoms (i.e., Ci-2o alkyl) or one to ten carbon atoms (i.e. , Ci-io alkyl) or one to five carbon atoms (i.e., C₁-5 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., including but not limited to methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, isopentyl or neopentyl. Unless set forth or recited to the contrary, all alkyl groups described or claimed herein may be straight chain or branched, substituted or unsubstituted.

As used herein, the term "amidated C-terminus" refers to that the C-terminal of the amino acid in amide form.

As used herein, the term "amide form" refers to primary, secondary and/or tertiary amides and may be represented by the formula -C(0)NR_xR_y, wherein each of R_x and R_y independently represents hydrogen or alkyl.

As used herein, the term "amino" refers to -NH₂ group. Unless set forth or recited to the contrary, all amino groups described or claimed herein may be substituted or unsubstituted.

As used herein, the term "amino acid" refers to amino acids having L or D stereochemistry at the alpha carbon.

As used herein, the term 'compound(s)' refers to the compounds disclosed in the present invention.

As used herein, the term "comprise" or "comprising" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

As used herein the term "ester" refers to (Q-Ce) linear or branched alkyl, (C4- Cio)aryl, (C4-Cio)heteroaryl or arylalkyl esters. As used herein the term "esterified C-terminus" refers to that the C-terminal of the amino acid in ester form.

As used herein the term "free C-terminus' refers to that the C-terminal of the amino acid in -CO₂H form.

As used herein, the term "hydroxyl" refers to -OH group.

As used herein, the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

As used herein, the term "optionally substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: alkyl, alkoxy, acyl, halo, and hydroxyl. It is understood that the substituent may be further substituted.

"Pharmaceutically acceptable salt" is taken to mean an active ingredient, which comprises a compound of the formula (I) in the form of one of its salts, in particular if this salt form imparts improved pharmacokinetic properties on the active ingredient compared with the free form of the active ingredient or any other salt form of the active ingredient used earlier. The pharmaceutically acceptable salt form of the active ingredient can also provide this active ingredient for the first time with a desired pharmacokinetic property which it did not have earlier and can even have a positive influence on the pharmacodynamics of this active ingredient with respect to its therapeutic efficacy in the body.

"Pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.

The term "stereoisomer" refers to any enantiomers, diastereoisomers, or geometrical isomers of the compounds of formula (I), wherever they are chiral or when they bear one or more double bond. When the compounds of the formula (I) and related formulae are chiral, they can exist in racemic or in optically active form. Since the pharmaceutical activity of the racemates or stereoisomers of the compounds according to the invention may differ, it may be desirable to use the enantiomers. In these cases, the end product or even the intermediates can be separated into enantiomeric compounds by chemical or physical measures known to the person skilled in the art or even employed as such in the synthesis. In the case of racemic amines, diastereomers are formed from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids such as the R and S forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (for example N-benzoylproline or N- benzenesulfonylproline), or the various optically active camphorsulfonic acids. Also advantageous is chromatographic enantiomer resolution with the aid of an optically active resolving agent (for example dinitrobenzoylphenylglycine, cellulose triacetate or other derivatives of carbohydrates or chirally derivatised methacrylate polymers immobilised on silica gel).

The term "subject" includes mammals (especially humans) and other animals, such as domestic animals (e.g., household pets including cats and dogs) and non- domestic animals (such as wildlife).

"Therapeutically effective amount" or "efficient amount" means sufficient amount of the compound(s) of the present invention that (i) treats or prevents the particular disease, disorder or syndrome (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, disorder or syndrome or (iii) prevents or delays the onset of one or more symptoms of the particular disease, disorder or syndrome described herein. In the case of cancer, the therapeutically effective amount of the drug may decrease the number of cancer cells; decrease the cancer size; inhibit (i.e., slow to some extent and alternatively stop) cancer cell infiltration into peripheral organs; suppress (i.e., slow to some extent and alternatively stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. In the case of infectious disease states, the therapeutic effective amount is an amount sufficient to decrease or alleviate an infectious diseases, the symptoms of an infections caused by bacterial, viral and fungal.

Naturally-occurring amino acids are identified throughout the specification by the conventional three-letter abbreviations indicated in the below table 2.

Table 2 (Amino acid codes)

Glutamic acid Glu Serine Ser

Glutamine Gin Threonine Thr

Glycine Gly Valine Val

The abbreviations used in the entire specification may be summarized hereinbelow with their particular meaning.

°C (degree Celsius); δ (delta); % (percentage); brine (NaCl solution); CH₂CI₂/DCM (Dichloromethane); BnBr (Benzyl bromide); br s (Broad singlet); Boc (Tert-butyloxycarbonyl); Bzl (Benzyloxy-carbonyl); CS₂CO₃ (Caesium carbonate); d (Doublet); DIPEA (Ν,Ν-Diisopropylethylamine); DMF (Dimethyl formamide); DMSO (Dimethyl sulphoxide); DMSO-d₆ (Deuterated DMSO); EDC.HC1/EDCI (1 -(3 -Dimethyl aminopropyl)-3-carbodiimide hydrochloride); Et₂NH (Diethylamine); Fmoc (9- Fluorenylmethyloxycarbonyl); g or gr (gram); H or ¾ (Hydrogen); ¾0 (Water); HOBt/HOBT (1 -Hydroxy benzotriazole); HCl (Hydrochloric acid); h or hr (Hours); Hz (Hertz); HPLC (High-performance liquid chromatography); K₂CO₃ (Potassium carbonate); LCMS (Liquid chromatography mass spectroscopy); MeOH (Methanol); mmol (Millimoles); M (Molar); μΐ (Microlitre); mL (Millilitre); mg (Milligram); m (Multiplet); MHz (Megahertz); MS (ES) (Mass spectroscopy-electro spray); rnin/min (Minutes); Na (Sodium); NaHC0₃ (Sodium bicarbonate); NH₂NH₂.H₂0 (Hydrazine hydrate); NMM (N- methyl morpholine); Na₂S0₄ (Sodium sulphate); N₂ (Nitrogen); NMR (Nuclear magnetic resonance spectroscopy); Pd/C (Palladium on carbon); PD-L1 (Programmed death-ligand 1); PD-L2 (Programmed cell death 1 ligand 2); prep- HPLC/preparative HPLC (Preparative High-performance liquid chromatography); S (Singlet); 'Bu/tBu (Tertiary butyl); TEA/Et₃N (Triethylamine); TLC (Thin Layer Chromatography); THF (Tetrahydrofuran); TIPS (Triisopropylsilane); TFA/CF3COOH (Trifluoroacetic acid); t (Triplet); t_{R = (}Retention time); TPP (Triphenylphosphine); Trt (Trityl or Triphenylmethyl), etc.

EXPERIMENTAL

An embodiment of the present invention provides the preparation of compounds of formula (I) according to the procedures of the following examples, using appropriate materials. Those skilled in the art will understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. Moreover, by utilizing the procedures described in detail, one of ordinary skill in the art can prepare additional compounds of the present invention. The starting materials are generally available from commercial sources such as Sigma-Aldrich, USA or Germany; Chem-Impex USA; G.L. Biochem, China and Spectrochem, India.

Purification and characterization of compounds

Analytical HPLC method: Analytical HPLC was performed using on ZIC HILIC 200A° column (4.6 mm x 250 mm, 5 μπι), Flow rate: 1.0 mL / min. The elution conditions used are: Buffer A: 5 mmol ammonium acetate, Buffer B: Acetonitrile, Equilibration of the column with 90 % buffer B and elution by a gradient of 90 % to 40 % buffer B during 30 min.

Preparative HPLC Method: The crude material was purified by preparative HPLC using ZIC HILIC 200A° column (21.2 mm x 150 mm, 5 μπι). The elution conditions used are Eluent: A: 5 mmol ammonium acetate B: Acetonitrile, Flow rate: 20 mL / min. The compound was eluted by gradient elution 0-2 min = 90 % buffer B, 2-20 min = 90- 40 % buffer B with a flow rate of 20 mL/min.

Example 1: Synthesis of compound 1

Step la:

Potassium carbonate (3.9 g, 28 mmol) and methyl iodide (3.26 g, 22mmol) were added to a solution of la (5.0 g, 19.15 mmol) in DMF (50 mL) and stirred at room temperature for 3 h. The completeness of the reaction was confirmed by TLC analysis. DMF was evaporated from the reaction mass and partitioned between water and ethyl acetate. Organic layer was washed with water, brine, dried over Na₂S0₄ and evaporated under reduced pressure to yield crude compound. The resulting crude compound was dissolved in methanol (50 mL), 99% hydrazine hydrate (12.5 mL) was added to the crude compound solution and stirred at room temperature for 6 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mixture was evaporated under reduced pressure to yield 5 g of compound lb (Yield: 96.15%), LCMS: 276.3 (M+H)⁺. Step lb:

1c 1d

DIPEA (4.3 g, 33.5 mmol) was added slowly to a stirred solution of lc (3 g, 13.4 mmol), HOBt (2.2 g, 16.1 mmol) and EDC.HCl (3.1 g, 16.1 mmol) in DCM (50 mL) at room temperature. To the above reaction mixture lb (3.7 g, 13.4 mmol) was added slowly and stirred at room temperature for 12 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mixture was then quenched with ice, precipitate was filtered and the solid re-crystallized with CH₂C1₂ to yield 6 g of Id, (Yield: 93 %), LCMS: 481.4 (M+H)⁺.

Step lc:

To a solution of Id (1.5 g) in methanol (20 mL) under inert atmosphere 10% Pd/C (0.5 g) was added and stirred for 1 h under H₂ atmosphere. The completion of the reaction was confirmed by TLC analysis. The Pd/C catalyst was removed by filtration through a Celite® pad, and washed with 50 mL of methanol. The combined organic filtrate on evaporation under reduced pressure to yield 1 g of le (Yield: 93%), LCMS: 347.2 (M+H)⁺.

Step Id:

1e 1g

The urea linkage was carried out using coupling of le (1 g, 2.8 mmol) in THF (20 mL) at room temperature, with If (1.24 g, 2.8 mmol). The coupling was initiated by the addition of TEA (0.42 g, 4.2 mmol) and the resultant mixture was stirred at room temperature. After the completion of 20 h, THF was evaporated from the reaction mass, and partitioned between water and ethyl acetate. Organic layer was washed with water, brine, dried over Na₂SC>4 and evaporated under reduced pressure to yield crude compound which was further washed with diethyl ether to yield 1.4 g of lg. LCMS: 638.2 (M+H)⁺. Step le:

ig in To a solution of lg (1.4 g) in methanol (10 mL) under inert atmosphere, 10% Pd-C (0.4 g) was added and stirred at room temperature for 1 h under H₂ atmosphere. The completion of the reaction was confirmed by TLC analysis. The Pd-C catalyst was then removed by filtration through a Celite® pad, which was then washed with 40 mL of methanol. The combined organic filtrate was evaporated under reduced pressure to obtain the desired 1.1 g of lh. LCMS: 548.1 (M+H)⁺

Step If:

1 h 1

To a solution of lh (1 g) in CH₂CI₂ (10 mL), trifluoroacetic acid (10 mL) and catalytic amount of triisopropylsilane were added and stirred for 3 h at room temperature. The resulting solution was concentrated in vacuum to yield 0.2 g of compound 1 (Yield:

32.6%), LCMS: 336.2 (M+H)⁺ The crude solid material was purified as preparative

HPLC method described under experimental conditions (yield: 30 mg, HPLC: t_R= 13.9 min).

To a solution of Fmoc-Thr(tBu)-OH (15.0 g, 37.7 mmol) in 100.0 mL of DMF, Cs₂C0₃ (14.8 g, 45.2 mmol) was added and the resutling mixture was cooled to 0 °C. To the reaction mixture benzyl bromide (7.74 g, 345.2 mmol) was added and the solution was stirred for 30 min at ice cold temperature followed by at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure and diluted with ethyl acetate (150 mL). The organic layer was washed with water (2 x 100 mL), brine (1 x 100 mL) and dried over Na₂S0₄. The filtered solution was concentrated and purified by silica gel column chromatogrophy (Eluent: 0-30% ethyl acetate in Hexane) to yeild 18.5 g of li as a white solid. LCMS: 433.1 (M-0'BU+H)⁺, 397.2 (M-OBzl)⁺.

Fmoc group on li was deprotected by adding diethylamine (40.0 mL) to li (10.0 g, 20.5 mmol) in CH₂CI₂ (40.0 mL). After an hour the resulting solution was concentrated in vacuum and the thick-residue was purified by column chromatography over neutral alumina (Eluent: 0-50% ethyl acetate in hexane then 0-5% methanol in chloroform) to yield 3.9 g ⁺.

To a stirred solution of lj (1.6 g, 6.0 mmol) in CH₂C1₂ (30 mL), TEA (1.2 g, 12.0 mmol) was added followed by the addition of 4-nitrophenyl chloroformate (1.3 g, 6.6 mmol) in CH₂CI₂ (10 mL). The resultant mixture was stirred at room temperature for 12 h. The completion of the reaction was confirmed by TLC analysis. After completion of reaction, the reaction mixture was diluted with CH₂CI₂ (50 mL) and washed with 1.0 M of sodium bisulphate (50 mL x 2) and 1.0 M sodium carbonate (50 mL x 2), dried over Na₂S0₄ and evaporated under reduced pressure to yield crude compound If, which was further purified by silica gel column chromatography (eluent: 0-20% ethyl acetate in Hexane) to yield 0.8 g of If. ^lU NMR (DMSO-d₆, 300 MHz): δ 8.23-8.27 (m, 2H), 7. ,27 '-7.39 (d, 7H), 5.99-6.01 (m, 1H), 5.10-5.25 (m, 2H), 4.28-4.31 (m, 2H), 1.28-1.30 (d, 3H), 1.12 (s, 9H).

Example 2: Synthesis of co

The compound 2 was synthesised by similar procedure as depicted in Example 1 using Boc-L-Ala-OH instead of Boc-L-Ser(tBu)-OH (compound la) and L-Ala-OtBu instead of L-Thr(tBu)-OBzl (compound lj) to yield 1.5 g of crude compound 2. The crude solid material (0.5 g) was purified as preparative HPLC method described under experimental conditions (yield: 0.17 g, analytical HPLC: t_R = 15.2 min), LCMS: 290.5 (M+H)⁺.

Example 3: Synthesis of

The compound 3 was synthesised by similar procedure as depicted in Example 1 using Boc-L-Cys(tBu)-OH instead of Boc-L-Ser(tBu)-OH (compound l a) and Cbz-Asn-OH instead of Cbz-Ala-OH (compound lc). Yield: 2 g (crude); LCMS: 434.4 (M+H₂0)⁺. Example 4: Synthesis of compound 4

The compound 4 was synthesised by similar procedure as depicted in Example 1 using Boc-L-Phe-OH instead of Boc-L-Ser(tBu)-OH (compound l a) and Cbz-Asn-OH instead of Cbz-Ala-OH (compound lc). The crude solid material (0.06 g) was purified as preparative HPLC method described under experimental conditions. LCMS: 439.2 (M+H)⁺, HPLC: t_R = 8.63 min.

Example 5: Synthesis of compound 5

The compound 5 was synthesised by similar procedure as depicted in Example 1 using Boc-L-Asp(OtBu)-OH instead of Boc-L-Ser(tBu)-OH (compound l a). The crude solid material (0.35 g) was purified as preparative HPLC method described under experimental conditions. LCMS: 364.1 (M+H)⁺, HPLC: t_R = 12.19 min.

Example 6: Synthesis of compound 6

The compound 6 was synthesised by similar procedure as depicted in Example 1 using Boc-L-Phe-OH instead of Boc-L-Ser(tBu)-OH (compound l a), Cbz-Phe-OH instead of Cbz-Ala-OH (compound lc) and H-Phe-OtBu instead of L-Thr(tBu)-OBzl (compound lj). The crude solid material (0.18 g) was purified as preparative HPLC method described under experimental conditions. LCMS: 518.5 (M+H)⁺, HPLC: t_R = 4.8 min. Example 7: Synthesis of

The compound 7 was synthesised by similar procedure as depicted in Example 1 using Boc-L-Hyp-OMe instead of Boc-L-Ser(tBu)-OH and Fmoc-Asn-OH instead of Cbz-Ala- OH (compound lc). The crude solid material (0.6 g) was purified as preparative HPLC method described under experimental conditions. LCMS: 405.2 (M+H)⁺, HPLC: t_R = 15.15 min.

Example 8: Synthesis of compound 8

The compound 8 was synthesised by similar procedure as depicted in Example 1 using Boc-Lys(Boc)-OH instead of Boc-L-Ser(tBu)-OH (compound la) and Fmoc-Asn-OH instead of Cbz-Ala-OH (compound lc). The crude solid material (0.15 g) was purified as preparative HPLC method described under experimental conditions. LCMS: 420.5 (M+H)⁺, HPLC: t_R = 20.15 min.

Example 9: Synthesis of compound 9

The compound 9 was synthesised using by procedure as depicted in Example 1 using Fmoc-Lys(Boc)-OH instead of Cbz-L-Ala-OH (compound lc). The crude solid material (1 g) was purified as preparative HPLC method described under experimental conditions LCMS: 393.2 (M+H)⁺, HPLC: t_R = 9.95 min.

Example 10: Synthesis of compound 10

The compound 10 was synthesised using similar procedure as depicted in Example 1 using Fmoc-Asn(Trt)-OH instead of Cbz-L-Ala-OH (compound lc) and H-Lys(Boc)- OtBu instead of L-Thr(tBu)-OBzl (compound lj). The crude solid material (1 g) was purified as preparative HPLC method described under experimental conditions. LCMS: 406.2 (M+H)⁺, HPLC: t_R = 16.91 min.

Example 11: Synthesis of compound 11

Step 11a:

Potassium carbonate (7.0 g, 50.8 mmol) and methyl iodide (2.5 g, 18.6 mmol) were added to a solution of compound 11a (5.0 g, 16.9 mmol) in DMF (35 mL) and stirred at room temperature for 3 h. The completeness of the reaction was confirmed by TLC analysis. DMF was evaporated from the reaction mass, and partitioned between water and ethyl acetate. Organic layer was washed with water, brine, dried over Na₂S0₄ and evaporated under reduced pressure get the desired 4.4 g of compound lib (Yield: 84.6%). LCMS: 310.1 (M+H)⁺ _.

Step lib:

To a solution of compound lib (4.4 g) in methanol (25 mL) under inert atmosphere 10% Pd/C (0.8 g) was added and stirred for 10 h under ¾ atmosphere. The completion of the reaction was confirmed by TLC analysis. The Pd/C catalyst was then removed by filtration through a Celite® bed and washed with 50 mL of methanol. The combined organic filtrate on evaporation under reduced pressure to yield 2.0 g of compound 11c (Yield: 80%). LCMS: 176.1 (M+H)⁺.

Step 11c:

DIPEA (2.2 g, 17.1 mmol) was added slowly to a stirred solution of 11c (1.1 g, 5.7 mmol), HOBt (0.9 g, 6.8 mmol) and EDC.HC1 (1.3 g, 6.8 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature for 5 min. To the above reaction mixture compound lid (1.0 g, 5.7 mmol) was added slowly and stirred at room temperature for 12 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mixture was then quenched with ice and the precipitate was filtered. The solid re-crystallized with CH₂CI₂ to yield 1.1 g of compound lie, (Yield: 57.8%). LCMS: 347.4 (M+H)⁺.

Step lid:

To a solution of compound lie (1.1 g, 3.17 mmol) in methanol (10 mL), 99% hydrazine hydrate (1.2 mL) was added and stirred at room temperature for 6 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mixture was evaporated under reduced pressure to yield lg of compound llf (Yield: 91.15%), LCMS: 347.4 (M+H)⁺.

The compound 11 was synthesised by similar procedure as depicted in Example 1 using compound llf in place compound lb (Example 1 , step la) and Cbz-Asn-OH in place of Cbz-Ala-OH to yield 0.25 g crude material of the title compound 11, LCMS: 450.2 (M+H)⁺.

Example 12: Synthesis of compound 12

Step 12a:

12a 12b

Potassium carbonate (4.4 g, 32.2 mmol) and methyl iodide (2.5 g, 17.7 mmol) were added to a solution of compound 12a (5.0 g, 16.1 mmol) in DMF and stirred at room temperature for 2 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mixture was partitioned between water and ethyl acetate. Organic layer was washed with water, brine, dried over Na₂SC>4 and evaporated under reduced pressure to get 5.0 g of compound 12b, LCMS: 326.5 (M+H)⁺

Step 12b:

12b 12c

Hydrazine hydrate (6.9 g, 138 mmol) was added to a solution of compound 12b (4.5 g, 13.8 mmol) in methanol and stirred at room temperature for 2 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mixture was evaporated under reduced pressure; the residue obtained was partitioned between water and ethyl acetate. Organic layer was washed with water, brine, dried over Na₂S0₄ and evaporated under reduced pressure yielded 3.2 g of compound 12c. LCMS: 326.2 (M+H)⁺ _.

Step 12c:

12c 12d 12e DIPEA (3.2 g, 25.3 mmol) was added slowly to a stirred solution of 12d (2.7 g, 9.2 mmol), HOBt (1.36 g, 10.1 mmol) and EDC.HC1 (1.93 g, 10.1 mmol) in DCM and stirred at room temperature. To the above reaction mixture 12c (3.0 g, 9.2 mmol) was added slowly and stirred at room temperature for 1 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mass was partitioned between ethyl acetate and water. Organic layer was washed with citric acid, NaHCOs_, brine, and dried over Na₂S0₄ and evaporated under reduced pressure to give crude compound. The crude was washed with diethyl ether and dried to yield 4.52 g of compound 12e; LCMS: 619.4(M+Na) ⁺.

Fmoc group was deprotected by the addition of diethylamine (13.5 mL) to a solution of compound 12e (4.5 g, 7.54 mmol) in CH₂CI₂ at -10 °C. The reaction was stirred at room temperature for 3 h. Progress of reaction was monitored by TLC. After completion, the resulting solution was concentrated in vacuum to get a thick gummy residue. The crude compound was purified by silica gel (60-120mesh) column chromatography (eluent: 5% methanol in DCM) to afford 2.2 g of compound 12f, LCMS: 375.5 (M+H)⁺, 397.4 (M+Na)⁺.

Step 12e:

The urea linkage was carried out using coupling of 12f (1.5 g, 4.0 mmol) in DMF at room temperature, with 12g (1.72 g, 4.0 mmol). The coupling was initiated by the addition of TEA (0.81 g, 8.0 mmol) and the resultant mixture was stirred at room temperature for 30 min. Progress of reaction was monitored by TLC. After completion, the reaction mass was partitioned between ethyl acetate and water. Organic layer was washed with citric acid, brine, dried over Na₂S0₄ and evaporated under reduced pressure to give crude compound. The resulting solid was washed with ether and dried to afford 1.1 g of Compound 12h.

Step 12f:

To a solution of compound 12h (1.1 g, 1.65 mmol) in methanol, was added 10% Pd-C (0.15 g) under inert atmosphere. The mixture was stirred for 1 h under H₂ atmosphere at room temperature. The completion of the reaction was confirmed by TLC analysis. The Pd-C catalyst was then removed by filtration through a Celite® pad and washed with methanol (2 x 50 mL). The combined organic filtrate on evaporation under reduced pressure resulted in the isolation of 0.7 g compound 12i, LCMS: 576.3 (M+H)⁺, 598.4 (M+H)⁺

Step 12g:

To a stirred solution of 12j (10 g, 16.7 mmol) in DMF, potassium carbonate (4.62 g, 33.6 mmol) and BnBr (3.2 g, 18.5 mmol) were added and continued reaction at room temperature for 2 h. The progress of reaction was monitored by TLC. After completion, the reaction mass was partitioned between ethyl acetate and water. Organic layer was washed with water, brine, and dried over Na₂SC>4 and evaporated under reduced pressure. The resulting solid was washed with n-hexane and dried to yield 10.0 g of compound 12k.

Step 12h:

12k Fmoc group was deprotected by the addition of diethylamine (30 mL) to a solution of compound 12k (10 g, 14.5 mmol) in CH₂CI₂ at -10 °C. The reaction was stirred at room temperature for 3 h. Progress of reaction was monitored by TLC. After completion, the resulting solution was concentrated in vacuum to get a thick gummy residue. The crude compound was purified by silica gel (60-120mesh) column chromatography (eluent: 5% methanol in DCM) to afford 6.0 g of compound 121.

Step 12i:

DIPEA (4.8 g, 3.8 mmol) was added slowly to a stirred solution of 12i (0.83 g, 1.5 mmol), HOBt (0.22 g, 1.65 mmol) and EDC.HCI (0.32 g, 1.65 mmol) in DCM and stirred at room temperature. To the above reaction mixture 121 (0.47 g, 1.5 mmol) was added slowly and stirred at room temperature for 1 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mass was partitioned between ethyl acetate and water. Organic layer was washed with citric acid, NaHC0_3, brine, and dried over Na₂S0₄ and evaporated under reduced pressure to give crude compound. The crude was washed with diethyl ether and dried to yield 1.0 g of compound 12m.

Step 12j:

To a solution of compound 12m (1.0 g, 0.98 mmol) in methanol, was added 10% Pd-C (0.1 g) under inert atmosphere. The mixture was stirred for 1 h under ¾ atmosphere at room temperature. The completion of the reaction was confirmed by TLC analysis. The Pd-C catalyst was then removed by filtration through a Celite® pad and washed with methanol (2 x 50 mL). The combined organic filtrate on evaporation under reduced pressure resulted in the isolation of 0.77 g compound 12n, LCMS: 932.7 (M+H)⁺, 954.8 (M+Na)⁺.

Step 12k:

To a solution of compound 12n (0.77 g, 0.77 mmol) in CH₂CI₂, trifluoroacetic acid (2.5 mL) and catalytic amount of triisopropylsilane were added and stirred at room temperature for 2 h. The resulting solution was concentrated under nitrogen and the solid material was further washed with diethyl ether, dried under vacuum to afford 0.5 g of crude compound. Crude compound was further purified by preparative HPLC method described under experimental conditions to yield pure compound 12, LCMS: 478.2. (M+H)⁺.

Example 13: Synthesis of compound 13

Ste

1 3a 1 3b

1 3c

Compound 13b (2.5 g, 5.9 mmol), TEA (1.2 g, 11.8 mmol) dissolved in DMF were added drop wise to a solution of 13a (1.5 g, 5.9 mmol) at 0 °C for urea bond formation and the mixture was stirred at room temperature for 30 min. The progress of the reaction was monitored by TLC analysis. After completion, the reaction mass was partitioned between ethyl acetate and water. Organic layer was washed with water, brine, and dried over Na₂S0₄ and evaporated under reduced pressure to give crude compound. The crude was further crystallized from hexane to yield 1.2 g of compound 13c.

Step 13b:

13c ^3d

To a solution of compound 13c (1.2 g, 2.35 mmol) in methanol (20 mL), was added 10% Pd-C (0.15 g) under inert atmosphere. The mixture was stirred for 1 h under ¾ atmosphere at room temperature. The completion of the reaction was confirmed by TLC analysis. The Pd-C catalyst was then removed by filtration through a Celite® pad and washed with methanol (2 x 75 mL). The combined organic filtrate on evaporation under reduced pressure resulted in the isolation of 0.7 g compound 13d, LCMS: 419.4 (M+H)⁺. Step 13c:

Compound 13e was synthesised by similar procedure as depicted for compound 1 If in step 1 Id of Example 11 using Boc-Ser(OtBu)-OH instead of Boc-Ala-OH (compound l id) and Fmoc-Asn(Trt)-OH instead of Cbz-Ser(OtBu)-OH (compound 11a). Yield: 2.5 g; LCMS: 654.3 (M+Na)⁺.

DIPEA (0.35 g, 2.8 mmol) was slowly added to a stirred solution of compound 13d (0.46 g, 1.1 mmol), compound 13e (0.7 g, 1.1 mmol), HOBt (0.16 g, 1.2 mmol) and EDC.HCI (0.23 g, 1.2 mmol) in DCM at 0°C. The reaction mixture was stirred at room temperature for 1 h. The completeness of the reaction was confirmed by TLC analysis. The reaction mass was partitioned between ethyl acetate and water. Organic layer was washed with citric acid, NaHCC>3, brine, and dried over Na₂SC¼ and evaporated under reduced pressure to give crude compound. The crude was washed with diethyl ether and dried to afford 0.82 g of compound 13f. LCMS: 1032.9 (M+H)⁺.

Step 13d:

To a solution of compound 13f (0.8 g, 0.77 mmol) in CH₂CI₂, trifluoroacetic acid (1 mL) and catalytic amount of triisopropylsilane were added and stirred at room temperature for 2 h. The resulting solution was concentrated under nitrogen and the solid material was further washed with diethyl ether, dried under vacuum to afford 0.5 g of crude compound. Crude compound was further purified by preparative HPLC method described under experimental conditions to yield pure compound 13, LCMS: 466.3 (M+H)⁺, HPLC: t_R = 16.41 min.

Example 14: Synthesis of compound 14

The compound 14 was synthesised by similar procedure as depicted in Example 12 using Boc-Phe-OH instead of Boc-Asp(OtBu)-OH (compound 12d); Fmoc-Asn-OH instead of Fmoc-Ala-OH (compound 12a) and H-Gly-OtBu instead of H-Asn(Trt)-OBzl (compound 12 1). The crude solid material was purified as preparative HPLC method described under experimental conditions. LCMS: 496.4 (M+H)⁺, HPLC: t_R = 11.6 min.

The compounds shown in below table 3, which can be prepared by following similar procedure as described above with suitable modification known to the one ordinary skilled in the art are also included in the scope of the present application.

Table 3

Rescue of mouse splenocvte proliferation in the presence of recombinant PD- L1/PD-L2:

Recombinant mouse PD-Ll (rm-PDL-1, cat no: 1019-B7-100; R&D Systems) were used as the source of PD-Ll.

Requirement:

Mouse splenocytes harvested from 6-8 weeks old C57 BL6 mice; RPMI 1640 (GIBCO, Cat # 11875); DMEM with high glucose (GIBCO, Cat # D6429); Fetal Bovine Serum [Hyclone, Cat # SH30071.03]; Penicillin (10000unit/ml)-Streptomycin(10,000μg/ml) Liquid (GIBCO, Cat # 15140-122); MEM Sodium Pyruvate solution lOOmM (lOOx), Liquid (GIBCO, Cat # 11360); Nonessential amino acid (GIBCO, Cat # 11140); L- Glutamine (GIBCO, Cat # 25030); Anti-CD3 antibody (eBiosciences - 16-0032); Anti- CD28 antibody (eBiosciences - 16-0281); ACK lysis buffer (lmL) (GIBCO, Cat # - A10492); Histopaque (density- 1.083 gm/mL) (SIGMA 10831); Trypan blue solution (SIGMA-T8154); 2 mL Norm Ject Luer Lock syringe- (Sigma 2014-12); 40 μΜ nylon cell strainer (BD FALCON 35230); Hemacytometer (Bright line-SIGMA Z359629); FACS Buffer (PBS/0.1% BSA): Phosphate Buffered Saline (PBS) pH 7.2 (HiMedia TS1006) with 0.1 % Bovine Serum Albumin (BSA) (SIGMA A7050) and sodium azide (SIGMA 08591); 5 mM stock solution of CFSE: CFSE stock solution was prepared by diluting lyophilized CFSE with 180 of Dimethyl sulfoxide (DMSO C₂H₆SO, SIGMA-D-5879) and aliquoted in to tubes for further use. Working concentrations were titrated from 10 μΜ to 1 μΜ. (eBioscience-650850-85); 0.05% Trypsin and 0.02% EDTA (SIGMA 59417C); 96-well format ELISA plates (Corning CLS3390); BD FACS caliber (E6016); Recombinant mouse B7-H1/PDL1 Fc Chimera, (rm-PD-Ll cat no: 1019-B7-100).

Protocol

Splenocyte preparation and culturing:

Splenocytes harvested in a 50 mL falcon tube by mashing mouse spleen in a 40 μπι cell strainer were further treated with 1 mL ACK lysis buffer for 5 min at room temperature. After washing with 9 mL of RPMI complete media, cells were re-suspended in 3 mL of lxPBS in a 15 mL tube. 3 mL of Histopaque was added carefully to the bottom of the tube without disturbing overlaying splenocyte suspension. After centrifuging at 800xg for 20 min at room temperature, the opaque layer of splenocytes was collected carefully without disturbing / mixing the layers. Splenocytes were washed twice with cold lxPBS followed by total cell counting using Trypan Blue exclusion method and used further for cell based assays.

Splenocytes were cultured in RPMI complete media (RPMI + 10% fetal bovine serum + 1 mM sodium pyruvate + 10,000units/ml penicillin and 10,000μg/ml streptomycin) and maintained in a CO₂ incubator with 5% C(¾ at 37°C.

CFSE Proliferation assay:

CFSE is a dye that passively diffuses into cells and binds to intracellular proteins. lxlO⁶ cells/mL of harvested splenocytes were treated with 5 μΜ of CFSE in pre-warmed lxPBS/0.1% BSA solution for 10 min at 37°C. Excess CFSE was quenched using 5 volumes of ice-cold culture media to the cells and incubated on ice for 5 min. CFSE labelled splenocytes were further given three washes with ice cold complete RPMI media. CFSE labelled lxlO⁵ splenocytes added to wells containing either MDA-MB231 cells (lxlO⁵ cells cultured in high glucose DMEM medium) or recombinant human PDL- 1 (100 ng/mL) and test compounds. Splenocytes were stimulated with anti-mouse CD3 and anti- mouse CD28 antibody (1 μg/mL each), and the culture was further incubated for 72 h at 37 °C with 5% CO₂. Cells were harvested and washed thrice with ice cold FACS buffer and % proliferation was analyzed by flow cytometry with 488 nm excitation and 521 nm emission filters.

Data compilation, processing and inference:

Percent splenocyte proliferation was analyzed using cell quest FACS program and percent rescue of splenocyte proliferation by compound was estimated after deduction of % background proliferation value and normalising to % stimulated splenocyte proliferation (positive control) as 100%.

Stimulated splenocytes: Splenocytes + anti-CD3/CD28 stimulation

Background proliferation: Splenocytes + anti-CD3/CD28 + PD-L1

Compound proliferation: Splenocytes + anti-CD3/CD28 + PD-L1 + Compound

Compound effect is examined by adding required concentration of compound to anti- CD3/CD28 stimulated splenocytes in presence of ligand (PDL-1)

Table 4

Claims

We claim:

1. A compound of formula (I)

^Ri ^R2

P— [Aaal]_x— N— N— [Aaa2]_y— C(O)— [Aaa3]_z— Q (I)

(i) [Aaa2] is selected from Asp, Asn, Glu or Gin;

(ii) [Aaa3] is selected from Ser, Asp, Ala, He, Phe, Trp, Glu or Thr; and

(iii) each of x, y and z is 1 ;

wherein, [Aaal], [Aaa2] and [Aaa3] are optionally substituted by hydroxyl;

Ri and R₂ are independently hydrogen or alkyl;

The compound according to claim 1 , wherein the compound of formula (I) is compound of formula (IA):

H

P— [Aaal]_x— N— N— [Aaa2]_y— C(O)— [Aaa3]_z— Q

H

(IA)

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, x, y, z, [Aaal], [Aaa2], [Aaa3], P and Q are same as defined in claiml.

The compound according to any one of claims 1 to 2, wherein x, y and z are independently 1.

The compound according to any one of claims 1 to 2, wherein x, y and z simultaneously do not have a value of 1.

5. The compound according to any one of claims 1 , 2 or 4, wherein x is 1 or 2, y is 1 and z is 1 or 2.

6. The compound according to claim 1 , or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein [Aaal] is selected from the group consisting of: Ser, Ala, Cys, Asp, Phe, Pro, Asn and Lys.

7. The compound according to claim 1 , or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein [Aaa2] is selected from the group consisting of: Ala, Asn, Phe, Lys, Thr and Phe.

8. The compound according to claim 1 , or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein [Aaa3] is selected from the group consisting of: Thr, Ala, Phe, Ser, Lys, Gly, val and Asn.

9. The compound according to any one of claims 1 to 8, wherein P is free N-terminus and Q is free C-terminus.

10. A compound is selected from the group consisting of

or a pharmaceutically acceptable sa t or a stereoisomer thereof.

11. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt or a stereoisomer thereof, and a pharmaceutically acceptable carrier or excipient.

12. The pharmaceutical composition according to claim 11, further comprising at least one of an anticancer agent, chemotherapy agent, or antiproliferative compound.

13. Use of a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt or a stereoisomer thereof, as a medicament.

14. Use of a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt or a stereoisomer thereof, in the manufacture of a medicament for the treatment of cancer or infectious disease.

15. The use according to claim 14, wherein the cancer is selected from bone cancer, cancer of the head or neck, pancreatic cancer, skin cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

16. The use according to claim 14, wherein the infectious disease is a bacterial infectious disease, a viral infectious disease or a fungal infectious disease.

17. A method of modulating an immune response mediated by PD-1 signaling pathway in a subject, comprising administering to the subject therapeutically effective amount of a compound according to any one of claims 1 to 10.

18. A method of inhibiting growth of tumour cells and/or metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 10.

19. The method of claim 18, wherein the tumour cells are of a cancer selected from the group consisting of breast cancer, colon cancer, lung cancer, melanoma, prostate cancer and renal cancer.

20. The method of claim 18, wherein the tumour cells are of a cancer selected from the list consisting of bone cancer, cancer of the head or neck, pancreatic cancer, skin cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

A method of treating an infectious disease in a subject comprising administering to the subject a therapeutically effective amount of compound according to any one of claims 1 to 10.

A method of treating bacterial, viral and fungal infections in a subject comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 10.

A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of compound according to any one of claims 1 to 10.