WO2015044900A1 - Therapeutic immunomodulating compounds - Google Patents

Abstract

The present invention relates to therapeutic immunomodulating compounds capable of inhibiting the programmed cell death 1 (PD1) signalling pathway. The invention also encompasses the use of the said therapeutic compounds and their 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

THERAPEUTIC IMMUNOMODULATING COMPOUNDS

This application claims the benefit of Indian provisional application number 4404/CHE/2013, filed on September 27, 2013; which hereby incorporated by reference. TECHNICAL FIELD

The present invention relates to therapeutic immunomodulating compounds useful for treatment of disorders via immuno-potentiation comprising inhibition of immunosuppressive signal induced due to PD-1, PD-L1 , or PD-L2 and therapies using them.

The invention also relates to pharmaceutical compositions comprising the said therapeutic immunomodulating compounds thereof.

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, WO2006/121168, WO2008/156712, WO2010/077634, WO2011/066389, WO2014/055897, WO2014/059173, WO2014/100079 and US patent US08735553 report PD-1 or PD-L1 inhibitory antibodies or fusion proteins.

Further, International applications, WO2011/161699, WO2012/168944, WO2013/144704 and WO2013/132317 report peptides or peptidomimetic compounds which are capable of suppressing and/or inhibiting the programmed cell death 1 (PDl) signaling pathway.

In view of the current ongoing research and disclosures as discussed above, it would thus be desirable to explore further therapeutically usefulness of immunomodulatory compounds as peptides or modified peptides.

The present invention therefore provides therapeutically useful immunomodulating compounds.

SUMMARY OF INVENTION

The present invention relates to therapeutic immunomodulating compounds and its derivatives or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof; which are capable of modulating the PDl signalling pathway.

In one aspect, the present invention provides therapeutic immunomodulating compound of formula (I):

(I)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein,

X is selected from Lys, Glu or Ser;

Y is selected from Glu, Gin or Lys;

Li is -CO-(CH₂)_n-NH-, -NH-(CH₂)_n-CO-, an amide bond between X and Y or absent;

'n' is selected from 1 to 5;

Z is (Aaa3)_q-L₂;

L₂ is -NH-(CH₂)_n-CO- or absent;

Aaal, Aaa2 and Aaa3 each represent independently an amino acid residue;

m and p are independently selected from 0 to 29;

q is selected from 1 to 29; and

R represents -CONH₂ of a C-terminal carboxylic acid moiety or is absent;

provided that in formula (I),

(i) at least one of the peptide bonds (-CO-NH-) is a reduced peptide bond (- CH₂-NH-);

or

(ii) at least one of the amino acids is N-alkylated;

or

(iii) combination of (i) and (ii).

It should be understood that the formula (I) structurally encompasses all stereoisomers, enantiomers and diastereomers, and pharmaceutically acceptable salts that may be contemplated from the chemical structure of the genera described herein.

In another aspect of the present invention, it provides compounds of formula (I) therapeutically useful in the treatment or prevention of disease or disorder, where there is an advantage in modulation of the PD1 signalling pathway.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides therapeutic immunomodulating compounds 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 modifications 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 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, f formula (I):

(I)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein,

X is selected from Lys, Glu or Ser;

Y is selected from Glu, Gin or Lys;

Li is -CO-(CH₂)_n-NH-, -NH-(CH₂)_n-CO-, an amide bond between X and Y or absent;

'n' is selected from 1 to 5;

-NH-(CH₂)_n-CO- or absent;

Aaal, Aaa2 and Aaa3 each represent independently an amino acid residue;

m and p are independently selected from 0 to 29;

q is selected from 1 to 29; and

R represents -CONH₂ of a C-terminal carboxylic acid moiety or is absent;

provided that in formula (I), (i) at least one of the peptide bonds (-CO-NH-) is a reduced peptide bond (- CH₂-NH-);

or

(ii) at least one of the amino acids is N-alkylated;

or

(iii) combination of (i) and (ii).

In yet another embodiment, the compound of formula (I), wherein the compound of formula (I) is a compound of formula (IA):

(IA)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa2, Aaa3, m, p, q and R are same as defined in formula (I). In yet another further embodiment, the compound of formula (I), wherein the compound of formula (I) is a compound of formula (IB):

(IB)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa3, m, q and R are same as defined in formula (I).

In yet another further embodiment, the compound of formula (I), wherein the compound of formula (I) is a compound of formula (IC):

(IC)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaa2, Aaa3, p, q and R are same as defined in formula (I).

In yet another further embodiment, the compound of formula (I), wherein the compound of formula (I) is a compound of formula (ID):

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa2, m, p and R are same as defined in formula (I).

In yet another further embodiment, the compound of formula (I), wherein the compound of formula (I) is a compound of formula (IE):

(Aaa1)_m x z Y (Aaa2)_p— R

(IE)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa2, X, Y, Z, m, p and R are same as defined in formula (I).

In yet another further embodiment, the compound of formula (I), wherein m and p are independently selected from 0 to 4.

In yet another further embodiment, the compound of formula (I), wherein m is 1 and p is 2. In yet another further embodiment, the compound of formula (I), wherein Li is an amide bond between X and Y.

In yet another further embodiment, the compound of formula (I), wherein R represents -CONH2 of a C-terminal carboxylic acid moiety.

The embodiments 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 m is 0 or 1.

According to another embodiment, specifically provided are compounds of the formula (I), in which Aaal is Ser.

According to yet another embodiment, specifically provided are compounds of the formula (I), in which X is Glu.

According to yet another embodiment, specifically provided are compounds of the formula (I), in which Z is (Aaa3)_q.

According to the preceding embodiment, specifically provided are compounds of the formula (I), in which Aaa3 is selected from Thr and Ser.

According to another embodiment, specifically provided are compounds of the formula (I), in which q is 2.

According to yet another embodiment, specifically provided are compounds of the formula (I), in which Y is Lys.

According to yet another embodiment, specifically provided are compounds of the formula (I), in which p is 0, 1 or 2.

According to one embodiment, specifically provided are compounds of the formula (I), in which Aaa2 is selected from Ser and Phe.

According to another embodiment, specifically provided are compounds of the formula (IA), in which m is 0 orl.

According to one embodiment, specifically provided are compounds of the formula (IA), in which Aaal is Ser.

According to the preceding embodiment, specifically provided are compounds of the formula (IA), in which q is 2.

According to yet another embodiment, specifically provided are compounds of the formula (IA), in which Aaa3 is selected from Thr and Ser. According to yet another embodiment, specifically provided are compounds of the formula (IA), in which p is 0, 1 or 2.

According to yet another embodiment, specifically provided are compounds of the formula (IA), in which Aaa2 is selected from Ser and Phe.

According to yet another embodiment, specifically provided are compounds of the formula (IA), in which R represents -CONH2 of a C-terminal carboxylic acid moiety.

According to yet another embodiment, specifically provided are compounds of the formula (IA), in which R is absent.

According to one embodiment, specifically provided are compounds of the formula (IE), in which m is 1.

According to another embodiment, specifically provided are compounds of the formula (IE), in which Aaal is Ser.

According to yet another embodiment, specifically provided are compounds of the formula (IE), in which X is Glu.

According to yet another embodiment, specifically provided are compounds of the formula (IE), in which Z is (Aaa3)_q.

According to the preceding embodiment, specifically provided are compounds of the formula (IE), in which Aaa3 is selected from Thr and Ser.

According to the preceding embodiment, specifically provided are compounds of the formula (IE), in which q is 2.

According to one embodiment, specifically provided are compounds of the formula (IE), in which Y is Lys.

According to yet another embodiment, specifically provided are compounds of the formula (IE), in which p is 2.

According to yet another embodiment, specifically provided are compounds of the formula (IE), in which Aaa2 is selected from Ser and Phe.

According to yet another embodiment, specifically provided are compounds of the formula (IE), in which R represents -CONH2 of a C-terminal carboxylic acid moiety.

According to yet another embodiment, specifically provided are compounds of the formula (IE), in which R is absent.

According to yet another embodiment, specifically provided are compounds of the formula (I), (IA), (IB), (IC), (ID) and (IE), in which at least one of the peptide bonds, (-CO-NH-) is a reduced peptide bond (-CH2-NH-) or at least one of the amino acids is N- alkylated.

According to yet another embodiment, specifically provided are compounds of the formula (I), (IA), (IB), (IC), (ID) and (IE), in which at least one of the peptide bonds, (-CO-NH-) is a reduced peptide bond (-CH2-NH-) and at least one of the amino acids is N-alkylated.

According to the preceding embodiment, specifically provided are compounds of the formula (I), (IA), (IB), (IC), (ID) and (IE), in which the N-alkylated is preferably N- methylated.

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

In an embodiment, specific compounds of formula (I) without any limitation are enumerated in Table 1.

Table 1

as a ree ase or a p armaceut ca sa t o a pept e er vat ve o ormu a I or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof.

The present invention provides immunosuppression modulating peptides capable of modulating the PD1 signalling pathway.

The present invention further provides modifications, derivatives of the peptides and pharmaceutical compositions comprising the peptides for treatment of cancer or infection via immunopotentiation caused by inhibition of immunosuppressive signal induced by PD-1, PD-L1 or PD-L2 and therapies using them, immunopotentiative substrates included as the active ingredients.

In the compounds of the present invention one or more of the amino acids of the peptide may be D-amino acid(s) with a view to provide improved stability in vivo.

The invention includes compounds as described above, formulated for pharmaceutical administration, typically by combination with a pharmaceutically acceptable carrier or diluent.

The invention includes compounds as described above for use in a method of medical treatment, e.g. in the treatment of cancer, treatment of bacterial, viral and fungal infections.

The invention further includes a method of screening compounds for ability to block interaction between PD-1 and a PD-1 ligand, comprising contacting candidate compounds of the kind described above with PD-1 or a PD-1 ligand binding portion of PD-1 and with a PD-1 ligand or a PD-1 binding portion of a PD-1 ligand, and measuring the extent of PD-l/PD-1 ligand binding. In addition, compounds of the invention may be combined with carrier molecules such as dendrimers, e.g. PAMAM dendrimers, liposomes, micro-particles and nanoparticles such as polycyanoacrylate nanoparticles.

In one embodiment the present invention provides the compounds having the ability to inhibit the programmed cell death 1 (PD1) signalling pathway and being capable of reducing PD-L1 or PD-L2 binding to PD-1 and resulting immunosuppressive signalling by PD- 1.

Further embodiment of the present invention relates to the compounds as disclosed in the present invention, wherein one or more of the amino acids of the peptide moiety the compounds is substituted with a D- amino acid.

The compounds as disclosed in the present invention are formulated for 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 comprises at least one of an anticancer agent, chemotherapy agent or antiproliferative compound.

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

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

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

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 invention capable 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.

Particularly, since the compound of the present invention exhibits an effect of stimulating or proliferating lymphoid cells, the concomitant use is able to reduce a dosage of chemotherapeutics commonly used or an irradiation dosage in radio therapy. This results in suppression of side effects that accompany with chemotherapy and radio therapy.

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.

The concomitant use of the compound of the present invention and a cancer antigen is able to give an additive or synergetic enhancement effect. Examples of the cancer antigen include HLA-Al and HLA-A2 derived peptides derived from MAGE-1 or MAGE-3 of malignant melanoma, MART-1 and gplOO, HER2/neu peptide of breast cancer and ovarian cancer, MUC-1 peptide of adenocarcinoma and NY-ESO-1 of metastatic cancer.

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 "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-₂0 alkyl) or one to ten carbon atoms

(i.e., Ci-₁0 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 "amino" refers to -Nth 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.

The term 'compound(s)' as used herein comprises peptides and modified peptides as 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 "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

The term "N-alkylated" refers to an amino acid residue in which the nitrogen of the amino acid residue is alkylated. For example, N-alkylated amino acid residues have the formula: R_aNH-CHR_e-C(0)-. R_a is an alkyl group as defined above and R_e represents side chain of the amino acid.

The term 'peptide' is used herein to designate a sequence of natural amino acids bonded in said sequence by peptide bonds.

The term "peptide bond" as used herein refers to the chemical bond between carbon and nitrogen in the bivalent group CONH that unites amino acid residues in a peptide.

Modifications of the peptides discussed hereinafter and wherever relevant may include replacements of some of the L-amino acids by D-amino acids, bonding of amino acids at other than alpha amino groups, including at side chain amino or carboxylic groups.

Modifications of the peptides discussed hereinafter and wherever relevant further comprise modified peptide bond between two amino acids, wherein the typical peptide bond (-CONH-) between two amino acids is replaced with reduced amide bond (-

CH₂NH-).

"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" refers to 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 by the conventional three-letter abbreviations indicated in the below Table 2.

Table 2 (Amino acid codes)

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

°C (degree Celsius); % (percentage); Boc (Tertiary-Butyloxycarbonyl Group); brine (NaCl solution); CH₂C1₂/DCM (Dichloromethane); DBU (1 ,8- Diazabicyclo[5.4.0]undec-7-ene); DIC (Ν,Ν'-Diisopropylcarbodiimide); DIPEA (N,N- Diisopropylethylamine); DMF (Dimethyl formamide); DMSO (Dimethyl sulphoxide); DMSO-d₆ (Deuterated DMSO); ECF (Ethyl chloroformate); EtOH (Ethanol); equiv (Equivalents); Fmoc (Fluorenylmethyloxycarbonyl group); g or gr (gram); H or ¾ (Hydrogen) ; HATU ( 1 - [Bis(dimethylamino)methylene]- 1 H- 1 ,2,3-triazolo [4,5 - b]pyridinium 3-oxid hexafluorophosphate) ¾0 (Water); HO AT (l-Hydroxy-7- azabenzotriazole); HOBt (1-Hydroxy benzotriazole); h or hr (Hours); Hz (Hertz); HPLC (High-performance liquid chromatography); LCMS (Liquid chromatography mass spectroscopy); MeOH (Methanol); mmol (Millimoles); M (Molar); μΐ (Microlitre); mL (Millilitre); mg (Milligram); MS (ES) (Mass spectroscopy-electro spray); min. (Minutes); Na (Sodium); NaHCOs (Sodium bicarbonate); NMM (N-methyl morpholine); Na2S04 (Sodium sulphate); PD-L1 (Programmed death-ligand 1); PD-L2 (Programmed cell death 1 ligand 2); P2O5 (Phosphorus pentoxide); preparative HPLC (Preparative High-performance liquid chromatography); PyBOP (Benzotriazol-1 - yloxy)tripyrrolidinophosphonium hexafluorophosphate); 'Bu/tBu (tertiary butyl); TLC (Thin Layer Chromatography); THF (Tetrahydrofuran); TIPS (Triisopropylsilane); TFA (Trifluoroacetic acid); etc.

EXPERIMENTAL

Different immunomodulatory compounds of the present invention were prepared via solid phase peptide synthesis, which was carried out manually using either a custom made glass reactor with a frit or polyethylene vessel equipped with a polypropylene filter.

All the compounds described herein, including compounds of general formula (I),

(IA), (IB), (IC), (ID) and (IE) and specific examples are prepared using techniques known to one skilled in the art through the procedure depicted below or by other methods. Furthermore, in the following procedures, where specific acids, bases, reagents, coupling agents, solvents, etc. are mentioned, it is understood that other suitable acids, bases, reagents, coupling agents etc. may be used and are included within the scope of the present invention. The compounds obtained by using the below procedures may be of insufficient purity. These compounds can be purified by using any of the methods for purification of organic compounds known to persons skilled in the art, for example, crystallization or silica gel or alumina column chromatography using different solvents in suitable ratios. All possible stereoisomers are envisioned within the scope of this invention.

Purification and characterization of compounds

Analytical HPLC method: Analytical HPLC was performed using on Aeris Peptide column (4.6 mm x 250 mm, 3.6 μπι), Flow rate: 1.0 mL / min. The elution conditions used are: Buffer A: 0.1 % TFA, Buffer B: Acetonitrile, Equilibration of the column with

2% buffer B and elution by a gradient of 0-2 min = 2% buffer B, 2-15 min = 2-70% buffer B, 15-20 min = 70-95% buffer B with a flow rate of 1 mL/min. Preparative HPLC Method: The crude material was purified by preparative HPLC using Kromasil C18 column (21.2 mm x 150 mm, 10 μπι). The elution conditions used are Eluent: A: 0.1% TFA, B: Acetonitrile, Flow rate: 15 mL / min. The compound was eluted by gradient elution 0-2 min = 2% buffer B, 2-5 min=2% buffer B, 5-25 min = 15- 35% buffer B with a flow rate of 15 mL/min.

General procedures

I. Removal of Allyloxycarbonyl (Alloc /OA11) group of side chain of Lysine and Glutamic acid

After the completion of the linear protected peptide sequence, the Alloc- protecting group from lysine and allyl protecting group from glutamic acid were removed from the peptidyl resin by treating with tetrakistriphenylphosphinepalladium (0) (5 equiv) and phenylsilane (10 equiv) in a solution of chloroform N-methylpyrrolidine (95/5 v/v) for 6 h under argon. The resin (2 g) was washed with a solution of 10% NMP in chloroform (6 x 35 mL); 1 % DIPEA in DMF (6 x 35 mL); DCM (6 x 35 mL); DMF (6 x 35 mL); and finally with DMF (3 x 35 mL each); The deprotection and resulting free amino group was confirmed by Kaiser test.

II. Lactam bridge/Cyclisation

A solution of HOBt/DIC (5 equiv and 7 equiv respectively relative to resin loading) in DMF was added to the resin and coupling was carried out overnight. After 18 h the resin was filtered and washed with DMF/DCM/DMF (6 x 35 mL). Kaiser test was carried out and in case of slight blue colouration, the peptidyl resin was capped with acetylating mixture (pyridine/DCM/acetic anhydride: 8:8: 1). After 30 min, the resin was filtered and washed with DMF/DCM/DMF (6 x 35 mL each)

III. Lactam bridge/Cyclisation/coupling

PyBOP (2 equiv), HOBt (2 equiv), DIPEA (2 equiv) was added to the OA11 and Alloc deprotected resin (1.0 g) in DMF and reaction was allowed to rotate at room temperature for 12 h. The resulting resin was washed with DMF/DCM/DMF (6 x 35 mL). Coupling was confirmed by negative Kaiser test.

IV. Cleavage of protected peptide from CTC resin:

The CTC resin was de -protected by treating it with 30% of HFIP in DCM for 2 h to generate the peptide as protected peptide. After completion of deprotection, the resin was filtered and washed with DCM. The filtrate containing side chain protected peptide was evaporated under nitrogen. Chilled cold ether was added to the concentrated solution to precipitate the peptide as white solid. The solid was centrifuged and thoroughly washed with diethyl ether and lyophilised.

V. Cleavage of peptidyl resin and global deprotection

The peptidyl resin was washed with MeOH (6 x 35 mL) and solvent ether (3 x 35 mL) and dried under vacuum. The cleavage of the peptides from the solid support is achieved by treating the peptidyl resin with cleavage cocktail (90% TFA / 5% TIPS / 5% water) at room temperature for 3.0 h. Cleavage mixture was collected by filtration and the resin was washed with TFA and DCM. The excess TFA and DCM was concentrated to small volume under nitrogen and DCM was added to the residue and evaporated under nitrogen. The process was repeated 3-4 times to remove most of the volatile impurities. The residue was cooled to 0 °C and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged and the supernatant ether was removed and fresh ether was added to the peptide and re-centrifuged. The residue was dissolved in Millipore water and lyophilized to obtain the crude peptide.

Characterization of peptide

LCMS was performed on API 2000 LC/MS/MS triple quad (Applied bio systems) with Agilent 1100 series HPLC with G1315 B DAD, using Mercury MS column or using Agilent LC/MSD VL single quad with Agilent 1100 series HPLC with G1315 B DAD, using Mercury MS column or using Shimadzu LCMS 2020 single quad with Prominence UFLC system with SPD-20 A DAD

For further illustration of methods of preparing the compounds of the present invention, the following examples are disclosed below.

Example 1: S

Desiccated Rink Amide MBHA-Amide resin (100-200 mesh, 0.50 mmol/g, 2 g) was placed in a polyethylene vessel equipped with a polypropylene filter. Resin was swelled in DCM (35 mL) for 30 min and DMF (35 mL) for 30 min. The Fmoc group of the Rink Amide MBHA-Amide was deprotected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min (35 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deportation was positive. The C-terminal amino acid, Fmoc- Phe-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (1.1 mL, 7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. After the first amino acid attachment, the unreacted amino group, if any, in the resin is capped, using acetic anhydride/pyridine/DCM (1 :8:8) for 10 and 20 minutes to avoid any deletion of the sequence. After capping, resin is washed with DCM (6 x 35 mL), DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). The Fmoc group on the C-terminal amino acid attached peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (35 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive. Fmoc-Ser(tBu)-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (1.1 mL,7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. The Fmoc group of the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (35 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive. Next amino acid in the peptide sequence Fmoc-Lys(Alloc)-OH (2.2 g, 5 equiv) in dry

DMF was added to the deprotected resin and coupling was initiated with DIC (1.1 mL, 7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. Next amino acid in the peptide sequence Fmoc-Ser(tBu)-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (1.1 mL, 7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. Next amino acid in the peptide sequence Fmoc- Thr(tBu)-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (1.1 mL, 7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. On completion of threonine coupling Fmoc- group on the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (20 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deportation was positive. Next amino acid Fmoc-Glu(OAll)-OH (2.1 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (1.1 mL, 7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. The Fmoc group of the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (20 mL). The resin was washed with DMF (6 x 15 mL), DCM (6 x 15 mL) and DMF (6 x 15 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive. Next amino acid Boc-N-methyl-Ser(tBu)-OH (1.5 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with

DIC (1.1 mL, 7 equiv) and HOBt (0.94 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. After the completion of the linear protected peptide sequence, the Alloc- protecting group from Lys (Alloc) and allyl protecting group from glutamic acid was removed as mentioned in the general procedure I using tetrakistriphenylphosphinepalladium (0) (5.7 g, 5 equiv) and phenylsilane (1.2 mL, 10 equiv). The lactam bridge was carried out as mentioned in the general procedure II using HOBt (0.7 g, 5 equiv)/DIC (0.8 mL, 7 equiv) method. The peptidyl resin was cleaved as mentioned in procedure V for cleavage to yield 450 mg crude peptide. The compound was further purified by reverse phase preparative HPLC using the method as detailed in experimental conditions. LCMS: 780.0 [M+H]⁺.

Example 2: Synt

Synthesis was carried out as explained in procedure for compound 1 , using Rink Amide MBHA-Amide resin (100-200 mesh, 0.50 mmol/g, 1 g). The C-terminal amino acid was coupled as Fmoc-Phe-OH (0.970 g, 5 equiv) using DIC (0.5 mL, 7 equiv) and HOBt (0.47 g, 5 equiv) in DMF (20 mL). The remaining amino acids; Fmoc-Ser(tBu)-OH (0.960 g, 5 equiv), Fmoc-Lys(Alloc)-OH (1.1 g, 5 equiv), Fmoc-Ser(tBu)-OH (1.9 g, 5 equiv), Fmoc-N-methyl Thr(tBu)-OH (1.0 g, 5 equiv), Fmoc-Glu(OAll)-OH (1.0 g, 5 equiv), and Boc-Ser(tBu)-OH (0.7 g, 5 equiv) were coupled sequentially by following analogous procedure as mentioned in Example 1. After the completion of the linear protected peptide sequence, the Alloc protecting group from lysine and allyl protecting group from glutamic acid was removed as mentioned in the general procedure I using tetrakistriphenylphosphinepalladium (0) (2.85 g, 5 equiv) and phenylsilane (0.6 mL, 10 equiv). The lactam bridge was carried out as mentioned in the general procedure II using HOBt (0.67 g, 5 equiv)/DIC (0.9 mL, 7 equiv) method. The peptidyl resin was cleaved as mentioned in procedure V for cleavage to yield 330 mg crude peptide. The compound was further purified by reverse phase preparative HPLC using the method as detailed in experimental conditions. LCMS: 780.1 [M+H]⁺.

Compounds 28 to 35 were synthesised using similar procedure as depicted in Example 2.

Table 3

Exa

The C-terminal amino acid, Fmoc-Phe-OH (0.69 g, 1.5 equiv) in dry DCM (20 mL) was added to the pre swelled chlorotrityl chloride resin (CTC) (1.0 g, 1.2 mmol/g) in dry

DCM (distilled over P₂0₅) for an hour, followed by the addition of DIPEA (0.98 mL, 4.8 equiv) and allowed to rotate for overnight in a Robbins scientific rotator at room temperature. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). After the first amino acid attachment, the resin was capped, using 2% of DIPEA in an equal amount of DCM/MeOH (20 mL) mixture for 5 and 15 minutes to avoid any deletion of the sequence. After capping, resin is washed with DCM (6 x 35 mL), DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). The Fmoc-group on the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (35 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc- deprotection was positive. Fmoc-Ser(tBu)-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (0.77 mL, 5 equiv) and HOBt (0.675 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. The Fmoc group of the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (35 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). A solution of o-NBS-Cl (0.886 g, 4 equiv), collidine (1.3 mL, 10 equiv) in NMP (25 mL) was added to the Fmoc- deprotected H-Ser(tBu)-Phe dipeptide attached to CTC resin and the reaction was shaken for 15 min at room temperature. The o-NBS protected dipeptidyl resin was washed with NMP (6 times) and was confirmed by negative Kaiser test. N-methylation on o-NBS protected peptide was carried out by adding initially a solution of DBU (0.45 mL, 3 equiv) in NMP (10 mL) followed by dimethyl sulfate (0.94 mL, 10 equiv) in NMP (10 mL) and in each step the reaction was rotated for 3-5 min at room temperature. The resin was filtered and washed with NMP and the N-methylation process was repeated for one more cycle of DBU and dimethyl sulfate addition. Finally the resin was washed with NMP (6 times) and the deprotection of o-NBS from the peptidyl resin was carried out by adding a solution of mercaptoethanol (0.7 mL, 10 equiv), DBU (0.75 mL, 5 equiv) in NMP (20 mL) at room temperature for 5 min. The deprotection procedure was repeated once again and the resin was washed with NMP (6 times). The deprotection was confirmed by positive Kaiser test. Next amino acid in the peptide sequence, Fmoc- Lys(Alloc)-OH (2.2 g, 2 equiv, ) in dry DMF was added to the deprotected resin and coupling was initiated with HATU (0.76 g, 2 equiv), HOAt (0.27 g, 2 equiv) and DIPEA (0.5 mL, 3 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. Next amino acid in the peptide sequence Fmoc-Ser(tBu)-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (0.77 mL, 5 equiv) and HOBt (0.675 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. Next amino acid in the peptide sequence Fmoc-Thr(tBu)-OH (1.9 g, 5 equiv) in dry DMF was added to the deprotected resin and coupling was initiated with DIC (0.77 mL, 5 equiv) and HOBt (0.675 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. On completion of threonine coupling Fmoc group on the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (20 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deportation was positive. N-methylation on threonine was carried out using a solution of o-NBS-Cl (0.886 g, 4 equiv) and collidine (1.3 mL, 10 equiv) in NMP for 15 min at room temperature. The o-NBS protected peptidyl resin was washed with NMP (6 times) and was confirmed by negative Kaiser test. N-methylation on o-NBS protected peptide was carried out by adding initially a solution of DBU (0.45 mL, 3 equiv) in NMP followed by dimethyl sulfate (0.94 mL, 10 equiv) in NMP and in each step the reaction was rotated for 3-5min at room temperature. The resin was filtered and washed with NMP and the N-methylation process was repeated for one more cycle of DBU and dimethyl sulfate addition. Finally the resin was washed with NMP (6 times) and the deprotection of o-NBS from the peptidyl resin was carried out by adding a solution of mercaptoethanol (0.7 mL, 10 equiv), DBU (0.75 mL, 5 equiv) in NMP at room temperature for 5 min. The deprotection procedure was repeated once again and the resin was washed with NMP (6 times). The deprotection was confirmed by positive Kaiser test. Peptide chain is further elongated by the addition of subsequent amino acid Fmoc-Glu(OAll)-OH (1.6 g, 2 equiv) in dry DMF to the Fmoc- deprotected peptidyl resin. Coupling was initiated with HATU (0.76 g, 2 equiv), HOAT (0.27 g, 2 equiv) and DIPEA (0.5 mL, 3 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. The Fmoc group of the peptidyl resin was de- protected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (20 mL). The resin was washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive. N-terminal amino acid was attached to the peptidyl resin using Fmoc- Ser(tBu)-OH (1.9 g, 5 equiv) and coupling was initiated with DIC (0.77 mL, 5 equiv) and HOBt (0.67 g, 5 equiv) in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF (6 x 35 mL), DCM (6 x 35 mL) and DMF (6 x 35 mL). Kaiser test on peptide resin aliquot upon completion of coupling was negative. On completion the target peptide sequence, Fmoc group on the peptidyl resin was deprotected by treating it twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (20 mL). The resin was washed with DMF (6 x 15 mL), DCM (6 x 15 mL) and DMF (6 x 15 mL). Kaiser test on peptide resin aliquot upon completion of Fmoc-deportation was positive. N-methylation on o-NBS protected peptide was carried out by adding initially a solution of DBU (0.45 mL, 3 equiv) in NMP followed by dimethyl sulfate (0.94 mL, 10 equiv) in NMP and in each step the reaction was rotated for 3-5min at room temperature. The resin was filtered and washed with NMP and the N- methylation process was repeated for one more cycle of DBU and dimethyl sulfate addition. Finally the resin was washed with NMP (6 times) and the deprotection of o- NBS from the peptidyl resin was carried out by adding a solution of mercaptoethanol (0.7 mL, 10 equiv), DBU (0.75 mL, 5 equiv) in NMP at room temperature for 5 min. The deprotection procedure was repeated once again and the resin was washed with NMP (6 times). The deprotection was confirmed by positive Kaiser test. After the completion of the linear protected peptide sequence, the Alloc- protecting group from Lys( Alloc) and allyl protecting group from glutamic acid was removed as mentioned in the general procedure I using tetrakistriphenylphosphinepalladium (0) (1.1 g, 1 equiv) and phenylsilane (1.25 mL, 10 equiv). The lactam bridge was carried out as mentioned in the general procedure III using PyBOP (1.56 g, 3 equiv), HOBt (0.40 g, 3 equiv) and DIPEA (0.5 mL, 3 equiv) method. The peptidyl resin was cleaved as mentioned in procedure V to yield 280 mg crude compound The compound was further purified by reverse phase preparative HPLC using the method as detailed in experimental conditions. LCMS: 809.5 [M+H]⁺.

Compounds, 6 and 8 were synthesised using similar procedure as depicted in Example 3 whereas compounds 3, 5, 7 and 9 were synthesised as linear peptide by excluding the lactam bridging step using similar procedure as depicted in Example 3.

Table 4

E

Step 10a:

To a stirred solution of Fmoc-Thr(tBu)-OH 10a (10 g, 1.0 equiv) in THF (100 mL), NMM (2.8 g, 1.1 equiv) and ECF (2.9 g, 1.1 equiv) were added at -20 °C and reaction was continued for 30 min. NaBFU (1.9 g, 2.0 equiv) dissolved in minimum amount of water was added to above reaction mixture and stirred at 0 °C for 15 min and at room temperature for 2 h. Progress of the reaction was monitored by TLC. After completion of the reaction THF from reaction mass was evaporated and crude product was diluted with EtOAc (50 mL). The organic layer was washed with water (25 mL x 2), 10% NaHCCte (25 mL x 2), 10% citric acid sol (25 mL x 2), brine solution (25 mL x 2), dried over anhydrous Na₂SC¼ and concentrated under reduced pressure. The crude compound was further purified by silica gel column chromatography using (30% of EtOAc in hexane) to yield 9.0 g of compound 10b, LCMS: 384.0 [M+H]⁺.

Step 10b:

10b 10c

Dess-Martin periodinane (7.7 g, 1.5 equiv) was added to a stirred solution of compound 10b (4.5 g, 1.0 equiv) in dry DCM (50 mL) at 0 °C and the resulting mixture was further stirred for 3 h at room temperature. Reaction mass was concentrated and the crude compound obtained was purified by silica gel column chromatography using EtOAc and ra-hexane (1 :9 ratio) to yield 3.8 g compound 10c, LCMS: 382.2 [M+H]⁺.

Step 10c:

To a stirred solution of compound 10c (2.7 g, 1.0 equiv) in EtOH at ice cold temperature, H-Phe-OBzl (2.0 g, 1.0 equiv), acetic acid (0.2 mL) were added. After 30 min of stirring NaBtU (0.57 g, 2.0 equiv) was added to the reaction mass and reaction was allowed to continue for 3 h at room temeperature. The completion of the reaction was monitored by TLC. After completion, reaction was quenched with water and diluted with EtOAc. The organic layer was separated and concentrated under reduced pressure to yield crude compound. The crude compound was purified by silica gel column chromatography using 20% of EtOAc in «-hexane to yield 1.1 g of compound lOd, LCMS: 617.3 [M+H]⁺.

Step lOd:

(Boc)₂0 (1.06 g, 3 equiv), NMM (0.2 g, 1.2 equiv) and DMAP (0.02 g, 0.1 equiv) was added to a solution of compound lOd (1.0 g, 1.0 equiv) in DCM at room temperature and stirred for 12 h. The completion of the reaction was monitored by TLC. After completion, reaction mass was diluted with DCM. The organic layer was washed with water (25 mL x 2), 10% citric acid sol (25 mL x 2), brine solution (25 mL x 2), dried over anhydrous Na2S04 and concentrated under reduced pressure to get crude compound. The crude compound obtained was purified by silica gel column chromatography using 15% EtOAc in «-hexane to yield 1.0 g of compound lOe, LCMS: 717.1 [M+H]⁺.

Step lOe:

Pd/C (0.1 g) was added under nitrogen atmosphere to a solution of benzyl ester protected compound lOe (1.0 g, 1.0 equiv) in HPLC grade EtOAc (20 mL) and AcOH (0.1 mL). The resulting reaction mass was stirred under hydrogen pressure in autoclave for 2 h. After completion of the reaction (monitored by TLC), Pd/C from the reaction mass was filtered through celite bed and washed the bed with MeOH (50 mL x 2) and EtOAc (50 mL x 2). The combined organic layer was concentrated under reduced pressure to yield 0.6 g of crude compound lOf, LCMS: 627.1 [M+H]⁺.

Step lOf:

The compound 10 was synthesised using similar procedure as depicted in

Example 2 using Rink Amide MB HA- Amide resin. The C-terminal amino acid was coupled as Fmoc-Phe-OH. The remaining amino acids; Fmoc-Ser(tBu)-OH, Fmoc- Lys(Boc)-OH, Fmoc-Thr(tBu)-^[CH₂NH]-Ser(tBu)-OH (lOf), Fmoc-Glu(OtBu)-OH, and Boc-Ser(tBu)-OH were coupled sequentially by following analogous procedure as mentioned in example 2. After the completion of the linear protected peptide sequence, resin was cleaved as mentioned in procedure example 1 for cleavage to yield 350 mg crude peptide. LCMS: 770.5 [M+H]⁺.

Compounds 11, 12 and 13 were synthesised using similar procedure as depicted in Example 4.

Table 5

Example 5: Synthesis of compound 14

The compound 14 was synthesised using similar procedure as depicted in Example 2 using Rink Amide MB HA- Amide resin. The C-terminal amino acid was coupled as Fmoc-Phe-OH. The remaining amino acids; Fmoc-Ser(tBu)-OH, Fmoc- Lys(Alloc)-OH, Fmoc-Thr(tBu)^[CH₂NH]-Ser(tBu)-OH (lOf), Fmoc-Glu(OAll)-OH, and Boc-Ser(tBu)-OH were coupled sequentially by following analogous procedure as mentioned in example 2. The lactam bridge was carried out as mentioned in the general procedure III and peptidyl resin was cleaved as mentioned in example 1 for cleavage to yield 450 mg crude peptide. LCMS: 752.1 [M+H]⁺.

Compounds 15-17 were synthesised using similar procedure as depicted in Example 5.

Table 6

Example 6: Synthesis of compound 18

Compound Fmoc-Ser(tBu)-^[CH2NH]-Phe-OtBu (18a) was synthesised using similar procedure as depicted for compound lOd in example 4.

Step 18a:

To compound 18a (1.0 g, 1.7 mmol) in CH2CI2 (5 mL) diethylamine (5 mL) was added and the reaction mixture was stirred at room, temperature for 1 h. The resulting solution was concentrated in vacuum to get gummy residue. The crude compound was purified by neutral alumina column chromatography (Eluent: 0-50% ethyl acetate in hexane then 0- 5% methanol in chloroform) to get 0.4 g of 18b. LCMS: 351.1 [M+H]⁺.

Step 18b:

The compound 18 was synthesised using similar procedure as depicted in Example 3. The C-terminal amino acid of Fmoc-Lys(Alloc)-OH was coupled to CTC resin (100-200 mesh, 1.2 mmol/g, 1 g). The remaining amino acids, Fmoc-Ser(tBu)-OH, Fmoc- Thr(tBu)-OH, Fmoc-Glu(OAll)-OH, and Boc-Ser(tBu)-OH were coupled sequentially by following analogous procedure as mentioned in example 3. After completion of the linear sequence, the Alloc and OA11 protecting groups from lysine and glutamic acid were removed as mentioned in the example 1 using tetrakis(triphenylphosphine) palladium(O) and phenylsilane. Lactam bridiging of free γ-COOH group of glutamic acid and ε-ΝΗ₂ of lysine in the peptidyl resin was carried using PyBOP/HOBt method as mentioned in general procedure-Ill. After completion of the cyclization protected cyclic peptide from the CTC resin was cleaved as mentioned in the general procedure IV using 30% of HFIP. The C-terminal acid free protected peptide was coupled to dipeptide H- Ser(tBu)-^[CH₂NH]-Phe-OtBu (18b) using PyBOP, HOBt and DIPEA as mentioned in the general procedure-Ill. Finally the protected peptide was cleaved as mentioned in the example 1 and the crude peptide was purified by preparative HPLC purification to yield pure compound 18.

Compound 21 was synthesised using similar procedure as depicted in Example 6. Linear compounds 19 and 20 were synthesised using similar procedure excluding the lactam bridging step as depicted in Example 6.

Table 7

S

Compound Fmoc-Ser(tBu)-^[CH2NH]-Phe-NH2 (23b) was synthesised using similar procedure as depicted for compound lOd in Example 4. Step 23b: XOtBu XOtBu

H ° Et,NH/DCM H °

NH,

H i

23b r„ 23c ^{^}Ph

Fmoc group of compound 23b (0.8 g, 1.0 equiv) was deprotected as mentioned for the synthesis of compound 18b in example 6. The crude compound was purified by neutral alumina column chromatography (Eluent: 2% of CHCb in MeOH) to get 0.35 g of compound 23c. LCMS: 294.1 [M+H]⁺.

The compound 23 was synthesised using similar procedure as depicted in Example 6 (compound 18) using compound 23c in place of compound 18b to yield compound 23.

Compound 25 was synthesised using similar procedure as depicted in Example 7. Linear compounds 22 and 24 were synthesised using similar procedure excluding the lactam bridging step as depicted in Example 7.

Table 8

Example 8: S

Step 26a:

Compound Fmoc-Lys(Alloc)-^[CH2NH]-Ser(tBu)-Phe-NH2 (26b) was synthesised using similar procedure by coupling of Fmoc-Lys(Alloc)-H to H-Ser(tBu)-Phe-NH2 as depicted for compound lOd in example 4.

Step 26b:

Fmoc- group of compound 26b (1.5 g, 3.4 mmol) was deprotected as mentioned for the synthesis of compound 18b in example 6. The crude compound was purified by neutral alumina column chromatography (Fluent: 5% of CHCb in MeOH) to get 1.0 g of 26c. LCMS: 537.1 [Μ+Η]⁺·

Step 26c:

Synthesis of compound 26 was carried out as explained in procedure for example 3. The C-terminal of Fmoc-Ser(tBu)-OH was coupled to CTC resin (100-200 mesh, 1.2 mmol/g, 1 g) as described in example 3. The remaining amino acids, Fmoc-NMe-Thr(tBu)-OH, Fmoc-Glu(OAll)-OH, and Boc-Ser(tBu)-OH were coupled sequentially by following analogous procedure as mentioned in example 3. After completion of the sequence protected peptide from the CTC resin was cleaved as mentioned in the general procedure IV using 30% of HFIP. C-terminal acid of protected peptide was coupled to H- Lys(Alloc)-^[CH₂NH]-Ser(tBu)-Phe-NH₂ (26c) using PyBOP/HOBt method as mentioned in general procedure III. After completion of the linear sequence, the Alloc and OA11 protecting groups from Lys( Alloc), Glu(OAll) of peptidyl resin were removed as mentioned in the example 1 using tetrakis(triphenylphosphine)palladium(0) and phenylsilane. Lactam bridging of free γ-COOH group of glutamic acid and ε-ΝΗ2 of lysine in the peptidyl resin was carried using PyBOP, HOBt and DIPEA as mentioned in example 3. Finally the protected peptide was cleaved as mentioned in the general procedure-V to yield crude compound 26. The compound was further purified by reverse phase preparative HPLC using the method as detailed in experimental conditions. LCMS: 766.2 [M+H]⁺.

Compound 27 was synthesised using similar procedure as depicted in Example 7.

Table 9

The compounds shown in below Table 10 , 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 10

Rescue of mouse splenocvte proliferation in the presence of recombinant PD

L1/PD-L2:

Recombinant mouse PD-L1 (rm-PDL-1, cat no: 1019-B7-100; R&D Systems) were used as the source of PD-L1.

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% CO2 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-Ll

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

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

Table 11

Claims

We Claim:

1 . A compound of Formula (I):

(I)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein,

X is selected from Lys, Glu or Ser;

Y is selected from Glu, Gin or Lys;

Li is -CO-(CH₂)n-NH-, -NH-(CH₂)_n-CO-, an amide bond between X and Y or absent;

'n' is selected from 1 to 5;

L₂ is -NH-(CH₂)n-CO- or absent;

Aaal, Aaa2 and Aaa3 each represent independently an amino acid residue;

m and p are independently selected from 0 to 29;

q is selected from 1 to 29; and

R represents -CONH2 of a C-terminal carboxylic acid moiety or is absent;

provided that in formula (I),

(i) at least one of the peptide bonds (-CO-NH-) is a reduced peptide bond (- or

(ii) at least one of the amino acids is N-alkylated;

or

(iii) combination of (i) and (ii).

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

(IA)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa2, Aaa3, m, p, q and R are same as defined in claim 1.

3 . The compound according to claim 1, wherein the compound of formula (I) is a compound of formula (IB):

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa3, m, q and R are same as defined in claim 1.

4 . The compound according to claim 1, wherein the compound of formula (I) is a compound of formula (IC):

(IC) or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaa2, Aaa3, p, q and R are same as defined in claim 1.

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

(ID)

or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof;

wherein, Aaal , Aaa2, m, p and R are same as defined in claim 1.

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

(Aaa1 )_m x z Y (Aaa2)_p— R

(IE)

or a pharmaceutical salt of a peptide derivative of formula (I), or a stereoisomer of a peptide derivative of formula (I) or pharmaceutical salt thereof;

wherein, Aaal , Aaa2, X, Y, Z, m, p and R are same as defined in claim 1.

The compound according to any one of claims 1, 2, 5 or 6, wherein m and p are independently selected from 0 to 4.

The compound according to any one of claims 1 , 2, 5, 6 or 7, wherein m is 1 and p is 2.

The compound according to claim 1 , wherein Li is an amide bond between X and Y.

The compound according to any one of claims 1 to 6, wherein R represents - CONH2 of a C-terminal carboxylic acid moiety.

A compound according to claim 1 is selected from the group consisting of:

as a free base or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof.

A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 11 or a pharmaceutical salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof; and a pharmaceutically acceptable carrier or excipient.

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

A compound according to any one of claims 1 to 11, or a pharmaceutical acceptable salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof, for use as a medicament.

A compound according to any one of claims 1 to 11, or a pharmaceutical acceptable salt of a peptide derivative of formula (I) or a stereoisomer of a peptide derivative of formula (I) or a pharmaceutical salt thereof, for use as a medicament for the treatment of cancer or infectious disease.

The compound for use according to claim 15, 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.

The compound for use according to claim 15, wherein the infectious disease is a bacterial infectious disease, a viral infectious disease or a fungal infectious disease.

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 11.

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 11.

The method of claim 19, 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.

The method of claim 19, 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 11.

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 11.