U.S. patent application number 14/989954 was filed with the patent office on 2016-04-28 for cyclic peptidomimetic compounds as immunomodulators.
This patent application is currently assigned to Aurigene Discovery Technologies Limited. The applicant listed for this patent is Seetharamaiah Setty Sudarshan Naremaddepalli, Muralidhara Ramachandra, Pottayil Govindan Nair Sasikumar. Invention is credited to Seetharamaiah Setty Sudarshan Naremaddepalli, Muralidhara Ramachandra, Pottayil Govindan Nair Sasikumar.
Application Number | 20160113901 14/989954 |
Document ID | / |
Family ID | 51688370 |
Filed Date | 2016-04-28 |
United States Patent
Application |
20160113901 |
Kind Code |
A1 |
Sasikumar; Pottayil Govindan Nair ;
et al. |
April 28, 2016 |
Cyclic Peptidomimetic Compounds as Immunomodulators
Abstract
The present invention relates to cyclic peptidomimetic compounds
as therapeutic agents capable of inhibiting the programmed cell
death 1 (PD1) signalling pathway. The invention also relates 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.
Inventors: |
Sasikumar; Pottayil Govindan
Nair; (Bangalore, IN) ; Ramachandra; Muralidhara;
(Bangalore, IN) ; Naremaddepalli; Seetharamaiah Setty
Sudarshan; (Bangalore, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sasikumar; Pottayil Govindan Nair
Ramachandra; Muralidhara
Naremaddepalli; Seetharamaiah Setty Sudarshan |
Bangalore
Bangalore
Bangalore |
|
IN
IN
IN |
|
|
Assignee: |
Aurigene Discovery Technologies
Limited
Bangalore
IN
|
Family ID: |
51688370 |
Appl. No.: |
14/989954 |
Filed: |
January 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14478806 |
Sep 5, 2014 |
9233940 |
|
|
14989954 |
|
|
|
|
Current U.S.
Class: |
424/278.1 |
Current CPC
Class: |
Y02A 50/401 20180101;
Y02A 50/411 20180101; Y02A 50/467 20180101; A61P 31/10 20180101;
Y02A 50/463 20180101; C07K 7/64 20130101; Y02A 50/465 20180101;
Y02A 50/473 20180101; A61P 31/00 20180101; A61K 31/38 20130101;
A61P 35/00 20180101; A61P 35/02 20180101; Y02A 50/30 20180101; Y02A
50/469 20180101; Y02A 50/479 20180101; A61P 37/02 20180101; Y02A
50/385 20180101; A61P 31/12 20180101; Y02A 50/481 20180101; Y02A
50/478 20180101; A61K 38/00 20130101; Y02A 50/406 20180101; A61P
31/04 20180101; C07D 259/00 20130101; A61K 31/395 20130101; C07D
273/00 20130101; Y02A 50/414 20180101; Y02A 50/471 20180101; Y02A
50/409 20180101 |
International
Class: |
A61K 31/395 20060101
A61K031/395 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 6, 2013 |
IN |
4010/CHE/2013 |
Claims
1. A method of modulating an immune response mediated by PD-1
signaling pathway in a subject, comprising administering to the
subject a therapeutically effective amount of a compound of formula
(I): ##STR00047## or a pharmaceutically acceptable salt or a
stereoisomer thereof; wherein, R.sub.1 is a side chain of amino
acid Ala, Ser, Thr or Leu; R.sub.2 is a side chain of amino acid
Asp, Glu, Gln or Asn; [Aaa] is an amino acid residue Ser, Asp, Ala,
Ile, Phe, Trp, Lys, Glu or Thr; R.sub.3 is hydrogen or alkyl; each
of R.sub.4 and R.sub.4' independently are hydrogen or alkyl; both
R.sub.a and R.sub.a' are hydrogen; or together are an oxo (.dbd.O)
group; both R.sub.b and R.sub.b' are hydrogen; or together are an
oxo (.dbd.O) group; L is ##STR00048## X is CH.sub.2, O or S;
R.sub.5 is hydrogen or alkyl; m is an integer from 1 to 3; and n is
an integer from 2 to 20.
2. The method according to claim 1, wherein the compound of formula
(I) is a compound of formula (IA): ##STR00049## or a
pharmaceutically acceptable salt or a stereoisomer thereof;
wherein, R.sub.1 is a side chain of amino acid Ala, Ser, Thr or
Leu; R.sub.2 is a side chain of amino acid Asp, Glu, Gln or Asn;
[Aaa] is an amino acid residue selected from Ser, Asp, Ala, Ile,
Phe, Trp, Lys, Glu or Thr; R.sub.3 is hydrogen or alkyl; each of
R.sub.4 and R.sub.4' independently are hydrogen or alkyl; both
R.sub.a and R.sub.a' are hydrogen; or together are an oxo (.dbd.O)
group; and both R.sub.b and R.sub.b' are hydrogen; or together are
an oxo (.dbd.O) group.
3. The method according to claim 1, wherein the compound of formula
(I) is a compound of formula (IB): ##STR00050## or a
pharmaceutically acceptable salt or a stereoisomer thereof;
wherein, R.sub.1 is a side chain of amino acid Ala, Ser, Thr or
Leu; R.sub.2 is a side chain of amino acid Asp, Glu, Gln or Asn;
and [Aaa] is an amino acid residue selected from Ser, Asp, Ala,
Ile, Phe, Trp, Lys, Glu or Thr.
4. The method according to claim 1, wherein the compound of formula
(I) is a compound of formula (IC): ##STR00051## or a
pharmaceutically acceptable salt or a stereoisomer thereof;
wherein, R.sub.1 is a side chain of amino acid Ala, Ser, Thr or
Leu; R.sub.2 is a side chain of amino acid Asp, Glu, Gln or Asn;
and [Aaa] is an amino acid residue selected from Ser, Asp, Ala,
Ile, Phe, Trp, Lys, Glu or Thr.
5. The method according to claim 1, wherein the compound of formula
(I) is a compound in which R.sub.1 is a side chain of amino acid
Ser or Thr; R.sub.2 is aside chain of amino acid Asp, Asn or Glu;
[Aaa] is an amino acid residue Ser or Thr; R.sub.3, R.sub.4 and
R.sub.4' independently are hydrogen; both R.sub.a and R.sub.a'
together represent an oxo (.dbd.O) group; both R.sub.b and R.sub.b'
together represent an oxo (.dbd.O) group; L is
--C(O)--(CH.sub.2).sub.m--(X--CH.sub.2--CH.sub.2).sub.n--NH--; X is
CH.sub.2 or 0; m is an integer from 1 to 3; and n is an integer
from 2 to 20; or a pharmaceutically acceptable salt or a
stereoisomer thereof.
6. The method according to claim 1, wherein the compound of formula
(I) is a compound in which R.sub.4 is C.sub.1-5 alkyl; or R.sub.4'
is C.sub.1-5 alkyl.
7. The method according to claim 1, wherein the compound of formula
(I) has a structure selected from the group consisting of
TABLE-US-00006 Compound No. Structure 1 ##STR00052## 2 ##STR00053##
3 ##STR00054## 4 ##STR00055## 5 ##STR00056## 6 ##STR00057## 7
##STR00058## 8 ##STR00059## 9 ##STR00060## 10 ##STR00061## 11
##STR00062## 12 ##STR00063## 13 ##STR00064## 14 ##STR00065## 15
##STR00066## 16 ##STR00067## 17 ##STR00068## 18 ##STR00069## 19
##STR00070## 20 ##STR00071## 21 ##STR00072## 22 ##STR00073## 23
##STR00074## 24 ##STR00075## and 25 ##STR00076##
or a pharmaceutically acceptable salt or a stereoisomer
thereof.
8. 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 of formula (I):
##STR00077## or a pharmaceutically acceptable salt or a
stereoisomer thereof; wherein, R.sub.1 is a side chain of amino
acid Ala, Ser, Thr or Leu; R.sub.2 is a side chain of amino acid
Asp, Glu, Gln or Asn; [Aaa] is an amino acid residue selected from
Ser, Asp, Ala, Ile, Phe, Trp, Lys, Glu or Thr; R.sub.3 is hydrogen
or alkyl; each of R.sub.4 and R.sub.4' independently are hydrogen
or alkyl; both R.sub.a and R.sub.a' are hydrogen; or together are
an oxo (.dbd.O) group; both R.sub.b and R.sub.b' are hydrogen; or
together are an oxo (.dbd.O) group; L is ##STR00078## X is
CH.sub.2, O or S; R.sub.5 is hydrogen or alkyl; m is an integer
from 1 to 3; and n is an integer from 2 to 20.
9. The method of claim 8, wherein the tumour cells are from a
cancer selected from the group consisting of breast cancer, colon
cancer, lung cancer, melanoma, prostate cancer and renal
cancer.
10. The method of claim 8, wherein the tumour cells are from a
cancer selected from the group consisting of bone cancer, cancer of
the head or neck, pancreatic cancer, skin cancer, cutaneous or
intraocular malignant endometrium, carcinoma of the cervix,
carcinoma of the vagina, carcinoma of the vulva, Hogkin'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
reter, 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.
11. A method of treating an infectious disease in a subject
comprising administering to the subject a therapeutically effective
amount of a compound of formula (I): ##STR00079## or a
pharmaceutically acceptable salt or a stereoisomer thereof;
wherein, R.sub.1 is a side chain of amino acid Ala, Ser, Thr or
Leu; R.sub.2 is a side chain of amino acid Asp, Glu, Gln or Asn;
[Aaa] is an amino acid residue selected from Ser, Asp, Ala, Ile,
Phe, Trp, Lys, Glu or Thr; R.sub.3 is hydrogen or alkyl; each of
R.sub.4 and R.sub.4' independently are hydrogen or alkyl; both
R.sub.a and R.sub.a' are hydrogen; or together are an oxo (.dbd.O)
group; both R.sub.b and R.sub.b' are hydrogen; or together are an
oxo (.dbd.O) group; L is ##STR00080## X is CH.sub.2, O or S;
R.sub.5 is hydrogen or alkyl; m is an integer from 1 to 3; and n is
an integer from 2 to 20.
12. The method according to claim 11, wherein the infectious
disease is a bacterial infectious disease, a viral infectious
disease or a fungal infectious disease.
13. A method for treating a cancer or infectious disease,
comprising administering to the subject a therapeutically effective
amount of a pharmaceutical composition comprising at least one of
the compounds according to claim 7 and a pharmaceutically
acceptable carrier or excipient.
14. The method according to claim 13, wherein the cancer is breast
cancer, colon cancer, lung cancer, melanoma, prostate cancer, or
renal cancer.
15. The method according to claim 13, wherein the infectious
disease is a bacterial infectious disease, a viral infectious
disease or a fungal infectious disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional under 35 U.S.C. .sctn.120
of pending nonprovisional application U.S. Ser. No. 14/478,806,
filed Sep. 5, 2014, which claims the benefit of priority under 35
U.S.C. .sctn.119(a) of Indian provisional application number
4010/CHE/2013, filed on Sep. 6, 2013, now abandoned, both of which
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field of the Invention
[0003] The present invention relates to cyclic peptidomimetic
compounds therapeutically useful as immune modulators. The
invention also relates to pharmaceutical compositions comprising
said cyclic peptidomimetic compounds as therapeutic agents.
[0004] 2. Description of the Related Art
[0005] 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 (Ariel Pedoeem et al., Curr
Top Microbiol Immunol. (2011); 350:17-37).
[0006] 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).
[0007] 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 on-going clinical trials (RD Harvey, Clinical
Pharmacology & Therapeutics (2014); 96 2, 214-223).
[0008] 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 (Hyun-Tak Jin et al.,
Clinical Immunology (Amsterdam, Netherlands) (2014), 153(1),
145-152).
[0009] 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 U.S. Ser.
No. 08/735,553 report PD-1 or PD-L1 inhibitory antibodies or fusion
proteins.
[0010] Further, International applications, WO2011161699,
WO2012/168944, WO2013144704 and WO2013132317 report peptides or
peptidomimetic compounds which are capable of suppressing and/or
inhibiting the programmed cell death 1 (PD1) signaling pathway.
[0011] Still there is a need for more potent, better and/or
selective immune modulators of PD-1 pathway. The present invention
provides cyclic peptidomimetic compounds which are capable of
suppressing and/or inhibiting the programmed cell death 1 (PD1)
signaling pathway.
SUMMARY OF THE INVENTION
[0012] In accordance with the present invention, cyclic
peptidomimetic compounds or a stereoisomer thereof or a
pharmaceutically acceptable salt thereof or pharmaceutical
compositions thereof are provided which suppress and/or inhibit the
programmed cell death 1 (PD1) signalling pathway.
[0013] In one aspect, the present invention provides cyclic
peptidomimetic compounds of formula (I):
##STR00001##
or a pharmaceutically acceptable salt or a stereoisomer thereof;
wherein,
[0014] R.sub.1 is a side chain of amino acid Ala, Ser, Thr or
Leu;
[0015] R.sub.2 is a side chain of amino acid Asp, Glu, Gln or
Asn;
[0016] [Aaa] is an amino acid residue selected from Ser, Asp, Ala,
Ile, Phe, Trp, Lys, Glu or Thr;
[0017] R.sub.3 is hydrogen or alkyl;
[0018] each of R.sub.4 and R.sub.4' independently are hydrogen or
alkyl;
[0019] both R.sub.a and R.sub.a' are hydrogen; or together are an
oxo (.dbd.O) group;
[0020] both R.sub.b and R.sub.b' are hydrogen; or together are an
oxo (.dbd.O) group;
[0021] L is
##STR00002##
[0022] X is CH.sub.2, 0 or S;
[0023] R.sub.5 is hydrogen or alkyl;
[0024] m is an integer from 1 to 3; and
[0025] n is an integer from 2 to 20.
[0026] In a further aspect of the present invention, there is
provided a pharmaceutical composition comprising a compound of
formula (I) or a pharmaceutically acceptable salt or a stereoisomer
and processes for preparing thereof.
[0027] In yet another aspect of the present invention, there is
provided methods for suppressing and/or inhibiting the programmed
cell death 1 (PD1) signaling pathway in a subject by administering
cyclic peptidomimetic compounds of formula (I) or a
pharmaceutically acceptable salt or a stereoisomer thereof or
pharmaceutical compositions thereof.
[0028] In yet another aspect of the present invention, there is
provided methods for inhibiting growth of tumour cells and/or
metastasis in a subject by administering cyclic peptidomimetic
compounds of formula (I) or a pharmaceutically acceptable salt or a
stereoisomer thereof or pharmaceutical compositions thereof.
[0029] In yet another aspect of the present invention, there is
provided methods for treating an infectious disease or a bacterial,
viral and fungal infections in a subject by administering cyclic
peptidomimetic compounds of formula (I) or a pharmaceutically
acceptable salt or a stereoisomer thereof or pharmaceutical
compositions thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions and certain embodiments of the
invention briefly summarized above are illustrated in the appended
drawings. These drawings form a part of the specification. It is to
be noted, however, that the appended drawings illustrate preferred
embodiments of the invention and therefore are not to be considered
limiting in their scope.
[0031] FIGS. 1A-1C depict the chemical synthetic scheme for
Compound 1.
[0032] FIGS. 2A-2B depict the chemical synthetic scheme for
Compound 2.
[0033] FIGS. 3A-3C depict the chemical synthetic scheme for
Compound 3.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention provides cyclic 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.
[0035] 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.
[0036] In one embodiment, the present invention relates to
compounds of formula (I)
##STR00003##
or a stereoisomer or a pharmaceutically acceptable salt thereof;
wherein;
[0037] R.sub.1 is side chain of amino acid Ala, Ser, Thr or
Leu;
[0038] R.sub.2 is a side chain of amino acid Asp, Glu, Gln or
Asn;
[0039] [Aaa] is an amino acid residue Ser, Asp, Ala, Ile, Phe, Trp,
Lys, Glu, or Thr;
[0040] R.sub.3 is hydrogen or alkyl;
[0041] each of R.sub.4 and R.sub.4' independently are hydrogen or
alkyl;
[0042] both R.sub.a and R.sub.a' are hydrogen; or together are an
oxo (.dbd.O) group;
[0043] both R.sub.b and R.sub.b' are hydrogen; or together are an
oxo (.dbd.O) group;
[0044] L is
##STR00004##
[0045] X is CH.sub.2, O or S;
[0046] R.sub.5 is hydrogen or alkyl;
[0047] m is an integer from 1 to 3; and
[0048] n is an integer from 2 to 20.
[0049] In a particular embodiment of the compounds of formula (I),
the invention comprises a particular series of compounds of formula
(IA):
##STR00005##
wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.4', R.sub.a,
R.sub.a', R.sub.b, R.sub.b.sup.' and [Aaa] are same as defined in
formula (I).
[0050] In a particular embodiment of the compounds of formula (I),
the invention comprises a particular series of compounds of formula
(IB):
##STR00006##
wherein, R.sub.1, R.sub.2 and [Aaa] are same as defined in formula
(I).
[0051] In yet another embodiment of the compounds of formula (I),
the invention comprises a particular series of compounds of formula
(IC):
##STR00007##
wherein, R.sub.1, R.sub.2 and [Aaa] are same as defined in formula
(I).
[0052] In yet another embodiment, the present invention provides
compounds of formula (I), wherein,
[0053] R.sub.1 is a side chain of amino acid Ser or Thr;
[0054] R.sub.2 is a side chain of amino acid of Asp, Asn or
Glu;
[0055] [Aaa] is an amino acid residue selected from Ser or Thr;
[0056] R.sub.3, R.sub.4 and R.sub.4' independently are
hydrogen;
[0057] both R.sub.a and R.sub.a' together are an oxo (.dbd.O)
group;
[0058] both R.sub.b and R.sub.b' together are an oxo (.dbd.O)
group;
[0059] L is
--C(O)--(CH.sub.2).sub.m--(X--CH.sub.2--CH.sub.2).sub.n--NH--;
[0060] X is CH.sub.2 or O;
[0061] m is an integer from 1 to 3; and
[0062] n is an integer from 2 to 20;
or a pharmaceutically acceptable salt or a stereoisomer
thereof.
[0063] The embodiments below are illustrative of the present
invention and are not intended to limit the claims to the specific
embodiments exemplified.
[0064] In one embodiment, specifically provided are compounds of
the formula (I), in which L is
##STR00008##
wherein X, n and R.sub.5 are the same as defined in formula
(I).
[0065] In another embodiment, specifically provided are compounds
of the formula (I), in which L is
##STR00009##
wherein m, n and R.sub.5 are the same as defined in formula
(I).
[0066] In yet another embodiment, specifically provided are the
compounds of the formula (I), in which L is
--C(O)--(CH.sub.2).sub.m--(X--CH.sub.2--CH.sub.2).sub.n--NH--;
wherein m, n and X are the same as defined in formula (I).
[0067] In yet another embodiment, specifically provided are
compounds of the formula (I), in which L is
##STR00010##
wherein m, X and R.sub.5 are the same as defined in formula
(I).
[0068] In yet another embodiment, specifically provided are
compounds of the formula (I), in which L is
--C(O)--CH.sub.2--(OCH.sub.2CH.sub.2).sub.2--NH--.
[0069] In another embodiment, specifically provided are compounds
of the formula (I), (IA), (IB) and (IC), in which R.sub.1 is side
chain of Ser.
[0070] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA), (IB) and (IC) in which R.sub.1
is side chain of Thr.
[0071] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA) and (IB), in which R.sub.2 is
side chain of Asp.
[0072] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA), (IB) and (IC) in which R.sub.2
is side chain of Asn.
[0073] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA), (IB) and (IC) in which R.sub.2
is side chain of Glu.
[0074] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA), (IB) and (IC) in which [Aaa] is
Ser.
[0075] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA), (IB) and (IC) in which [Aaa] is
Thr.
[0076] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA) and (IB) in which [Aaa] is
Asp.
[0077] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA) and (IB) in which [Aaa] is Lys
or Ile.
[0078] In yet another embodiment, specifically provided are
compounds of the formula (I), (IA) and (IB), in which one, more or
all amino acid/s is/are D amino acid/s.
[0079] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which R.sub.4' is
C.sub.1-5alkyl such as methyl.
[0080] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which R.sub.4 is
C.sub.1-5 alkyl such as methyl.
[0081] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which both R.sub.a and
R.sub.a' are hydrogen.
[0082] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which both R.sub.b and
R.sub.b' are hydrogen.
[0083] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which both R.sub.a and
R.sub.a' together represent an oxo (.dbd.O) group.
[0084] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which both R.sub.b and
R.sub.b' together represent an oxo (.dbd.O) group.
[0085] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which both R.sub.4 and
R.sub.4' are hydrogen.
[0086] In yet another embodiment, specifically provided are
compounds of the formula (I) and (IA), in which R.sub.3 is
hydrogen.
[0087] In an embodiment, specific compounds of formula (I) without
any limitation are enumerated in Table (1):
TABLE-US-00001 TABLE 1 Compound No. Structure 1 ##STR00011## 2
##STR00012## 3 ##STR00013## 4 ##STR00014## 5 ##STR00015## 6
##STR00016## 7 ##STR00017## 8 ##STR00018## 9 ##STR00019## 10
##STR00020## 11 ##STR00021## 12 ##STR00022## 13 ##STR00023## 14
##STR00024##
[0088] or a pharmaceutically acceptable salt or a stereoisomer
thereof thereof.
[0089] In one embodiment, the present invention provides a
pharmaceutical composition comprising the compound as disclosed,
and a pharmaceutically acceptable carrier or diluent.
[0090] In another embodiment, the pharmaceutical composition
comprises at least one additional pharmaceutical agent wherein the
additional pharmaceutical agent is an anticancer agent,
chemotherapy agent, or antiproliferative compound.
[0091] The compounds as disclosed in the present invention are
formulated for pharmaceutical administration.
[0092] In one embodiment, the present invention provides use of the
compounds as disclosed in the present invention for the preparation
of a medicament for the treatment of cancer.
[0093] In one embodiment, the present invention provides use of the
compounds as disclosed in the present invention for the preparation
of a medicament for the treatment of infectious diseases or
bacterial, viral and fungal infections.
[0094] 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 or
of a pharmaceutical composition thereof to the subject in need
thereof.
[0095] In one embodiment, the present invention provides a method
for inhibiting growth of tumour cells and/or metastasis by
administering an effective amount of the compound of the present
invention or of a pharmaceutical composition thereof to the subject
in need thereof.
[0096] Representative tumour cells include cancer such as but not
limited to melanoma, renal cancer, prostate cancer, breast cancer,
colon cancer and lung cancer, bone cancer, pancreatic cancer, skin
cancer, cancer of the head or neck, cutaneous or intraocular
malignant melanoma, uterine cancer, ovarian cancer, rectal cancer,
cancer of the anal region, stomach cancer, testicular 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.
[0097] Still yet another embodiment of the present invention
provides a method of treatment of infection via immunopotentiation
caused by inhibition of immunosuppressive signal induced by PD-1,
PD-L1, or PD-L2, wherein the method comprises administration of an
effective amount of the compound of the present invention or of a
pharmaceutical composition thereof to the subject in need
thereof.
[0098] 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,
Naegleria fowleri, Acanthamoeba sp., Giardia lambia,
Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax,
Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania
donovani, Toxoplasma gondi, Nippostrongylus brasiliensis.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] The compound 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.
[0107] 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-.alpha., .beta., or .gamma., IL-1, IL-2, IL-3, IL-12,
Poly (I:C) and C.sub.pG.
[0108] In another embodiment, the potentiating agents includes
cyclophosphamide and analogs of cyclophosphamide, anti-TGF.beta.
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.
[0109] 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.
[0110] 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., C.sub.1-20 alkyl) or one to ten carbon atoms
(i.e., C.sub.1-10 alkyl) or one to five carbon atoms (i.e.,
C.sub.1-5 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 acid" refers to amino acids having L or D stereochemistry at
the alpha carbon.
[0111] As used herein, the term `compound(s)` refers to the
compounds disclosed in the present invention.
[0112] 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.
[0113] As used herein, the term "including" as well as other forms,
such as "include", "includes," and "included," is not limiting.
[0114] "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.
[0115] "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.
[0116] The term "stereoisomer" refers to any enantiomers,
diastereomers, 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).
[0117] 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).
[0118] "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.
[0119] Naturally-occurring amino acids are identified throughout
the specification by the conventional three-letter abbreviations
indicated in the below table 2:
TABLE-US-00002 TABLE 2 Name 3-letter code Alanine Ala Asparagine
Asn Aspartic acid Asp Glutamic acid Glu Glutamine Gln Isoleucine
Ile Leucine Leu Lysine Lys Phenylalanine Phe Serine Ser Threonine
Thr Tryptophan Trp
[0120] The abbreviations used in the entire specification may be
summarized herein below with their particular meaning.
[0121] .degree. C. (degree Celsius); .delta. (delta); %
(percentage); brine (NaCl solution); bs or brs (Broad singlet); Bzl
(Benzyl); Cbz (Carboxybenzyl); Cbz-Cl (Benzyl chloroformate);
CH.sub.2Cl.sub.2/DCM (Dichloromethane); Cs.sub.2CO.sub.3 (Cesium
carbonate); DMF (Dimethyl formamide); DMSO (Dimethyl sulfoxide);
DIPEA/DIEA (N,N-Diisopropyl ethylamine); DMSO-d.sub.6 (Deuterated
DMSO); d (Doublet); EtOAc (Ethyl acetate); Et.sub.2NH
(Diethylamine); Fmoc (Fluorenylmethyloxycarbonyl); Fmoc-Cl
(Fluorenylmethyloxycarbonyl chloride) g or gr (gram); H or H.sub.2
(Hydrogen); H.sub.2O (Water); HOBt/HOBT (1-Hydroxy benzotriazole);
HCl (Hydrochloric acid); h or hr (Hours); HATU
(2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium
hexafluoro phosphate methanaminium); Hz (Hertz); HPLC
(High-performance liquid chromatography); LCMS (Liquid
chromatography mass spectroscopy); MeOH/CH.sub.3OH (Methanol); mmol
(Millimoles); M (Molar); .mu.l/.mu.L (Microlitre); mL (Millilitre);
mg (Milligram); min (minutes); m (Multiplet); mm (Millimeter); MHz
(Megahertz); MS (ES) (Mass spectroscopy-electro spray); min
(Minutes); Na (Sodium); NaOBu.sup.t (Sodium tert-butoxide);
NH.sub.2NH.sub.2.H.sub.2O (Hydrazine hydrate); Na.sub.2SO.sub.4
(Sodium sulphate); N.sub.2 (Nitrogen); NMR (Nuclear magnetic
resonance spectroscopy); NaHCO.sub.3 (Sodium bicarbonate);
Pd-C(Palladiun on carbon); 10% Pd/C (10% palladium activated
carbon); Pd(OH).sub.2 (palladium hydroxide); PD-L1 (Programmed
death-ligand 1); PD-L2 (Programmed cell death 1 ligand 2); prep
HPLC/prep-HPLC (Preparative High-performance liquid
chromatography); PyBOP
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate); RT/rt (room temperature); S (Singlet);
.sup.tBu/tBu (Tertiary butyl) TEA/Et.sub.3N (Triethyl amine); TFA
(Trifluoroaceticacid); TLC (Thin Layer Chromatography); THF
(Tetrahydrofuran); TFA/CF.sub.3COOH (Trifluoro acetic acid); t
(Triplet); t.sub.R=(Retention time), etc.
[0122] 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.
[0123] 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
[0124] Analytical HPLC method: Analytical HPLC was performed using
on ZIC HILIC 200A.degree. column (4.6 mm.times.250 mm, 5 .mu.m),
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.
[0125] Preparative HPLC Method A: The crude material was purified
by preparative HPLC using ZIC HILIC 200A.degree. column (21.2
mm.times.150 mm, 5 .mu.m). The elution conditions used are Eluent:
A: 5 mmol ammonium acetate B: Acetonitrile, Flow rate: 18 mL/min.
The compound was eluted by gradient elution 0-3 min=90% buffer B,
3-20 min=90-40% buffer B with a flow rate of 20 mL/min.
[0126] Preparative HPLC Method B: Prep HPLC was performed using on
ZIC HILIC 200A.degree. column (10 mm.times.250 mm, 5 .mu.m), Flow
rate: 5.0 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 20 min. LCMS was performed on AP1 2000 LC/MS/MS
triple quad (Applied biosystems) 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.
Example 1
Synthesis of Compound 1
[0127] FIGS. 1A-1C illustrate Steps 1a to 1c.
[0128] Step 1a: Sodium hydroxide (12.2 g, 305 mmol) and Cbz-Cl
(12.5 g, 73 mmol) were added to a solution of compound 1a (10.0 g,
61 mmol) in water (100 mL) and stirred at room temperature for 3 h.
The completeness of the reaction was confirmed by TLC analysis. The
reaction mass was partitioned between citric acid solution and
ethyl acetate. Organic layer was washed with water, brine, dried
over Na.sub.2SO.sub.4 and evaporated under reduced pressure to
yield 11 g of compound 1b (Yield: 61.1%). LCMS: 298.0
(M+H).sup.+.
[0129] Step 1b: DIPEA (3.5 g, 26.8 mmol) was added slowly to a
stirred solution of compound 1b (4.0 g, 13.4 mmol) and HATU (5.6 g,
14.7 mmol) in DMF (50 mL) and was allowed to stir at room
temperature for 5 more min. To the above reaction mixture
L-Ser(.sup.tBu)-OMe.HCl (3.5 g, 20.1 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
quenched with ice, precipitated solid was filtered and
re-crystallized with CH.sub.2Cl.sub.2 to yield 6 g of compound 1c,
LCMS: 454.8 (M+H).sup.+.
[0130] Step 1c: 99% Hydrazine hydrate solution (10 mL) was added
slowly to a stirred solution of compound 1c (6 g) in methanol (50
mL) and stirred at room temperature for 2 h. The completion of the
reaction was confirmed by TLC. The reaction mixture on evaporation
under reduced pressure yielded 5.8 g of compound 1d. LCMS: 455.0
(M+H).sup.+.
[0131] Step 1d: DIPEA (3.3 g, 25.4 mmol) was added slowly to a
stirred solution of compound 1d (5.8 g, 12.7 mmol), HATU (5.8 g,
15.2 mmol) in DMF (50 mL) and was allowed to stir at room
temperature for 5 min. Fmoc-L-Asn-OH (4.9 g, 14.0 mmol) was further
added to reaction mixture 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, precipitated solid was
filtered and re-crystallized with CH.sub.2Cl.sub.2 to yield 5.9 g
of compound 1e, LCMS: 791.0 (M+H).sup.+.
[0132] Step 1e: Fmoc group of compound 1e [(5.9 g in
CH.sub.2Cl.sub.2 (60 mL)] was deprotected using diethylamine (60
mL) and the completion of the reaction was confirmed by TLC
analysis. The reaction mixture on evaporation under reduced
pressure yielded 1.6 g of compound 1f. LCMS: 568.8 (M+H).sup.+.
[0133] Step 1f: Compound 1l (1.3 g, 3.0 mmol) and compound 1f (1.60
g, 2.8 mmol) was dissolved in THF (10 mL) and stirred at room
temperature. Coupling was initiated by the addition of
triethylamine (0.57 g, 5.6 mmol) to the above reaction mixture and
the reaction was allowed to stir for 12 h at room temperature. The
completeness of the reaction was confirmed by TLC analysis. Organic
layer was washed with NaHCO.sub.3, citric acid solution, brine,
dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure
to yield 0.45 g of compound1g.
[0134] Step 1g: Cbz group and benzyl ester deprotection was carried
out on compound 1g (0.45 g) in methanol using palladium hydroxide
(0.5 g) for 1 h at room temperature. The completeness of the
reaction was confirmed by TLC analysis. Palladium hydroxide was
removed by Celite.RTM. bed filtration and the filtrate was
evaporated under reduced pressure to yield 0.34 g of compound 1h.
LCMS: 636.0 (M+H).sup.+.
[0135] Step 1h: Cyclization of compound 1h (0.1 g, 0.15 mmol) was
carried out using HOBT (0.06 g, 0.47 mmol) and PyBOP (0.24 g, 0.47
mmol) in THF (50 mL). The reaction was initiated by slow addition
of DIPEA (0.06 g, 0.47 mmol) and further stirred at room
temperature for 12 h. The reaction mixture was evaporated and
washed with diethyl ether to yield 0.05 g of compound1i. LCMS:
617.9 (M+H).sup.+.
[0136] Step 1i: The acid sensitive protecting group on compound1i
[(0.08 g) in CH.sub.2Cl.sub.2 (0.9 mL)] was removed using TFA (0.9
mL). The reaction mixture was stirred at room temperature for 4 h,
followed by evaporation and washing with diethyl ether yielded 0.05
g of crude compound 1. The crude solid material was purified using
preparative HPLC method-A described under experimental conditions
(yield: 9 mg). LCMS: 506.4 (M+H).sup.+; HPLC: t.sub.R=12.3 min.
[0137] Synthesis of compound 1l
(NO.sub.2--C.sub.6H.sub.4--OCO-Thr(.sup.tBu)-OBzl,): To a solution
of Fmoc-Thr(.sup.tBu)-OH (15.0 g, 37.7 mmol) in 100.0 mL of DMF,
Cs.sub.2CO.sub.3 (14.8 g, 45.2 mmol) was added and the resulting
mixture was cooled to 0.degree. C. To the cooled 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 room
temperature for 12 h. The reaction mixture was further concentrated
under reduced pressure and diluted with ethyl acetate (150 mL). The
organic layer was washed with water (2.times.100 mL), brine
(1.times.100 mL) and dried over Na.sub.2SO.sub.4. The filtered
solution was concentrated and purified by silica gel column
chromatogrophy (Eluent: 0-30% ethyl acetate in Hexane) to yield
18.5 g of intermediate 1j as a white solid. LCMS: 433.1
(M-O.sup.tBu+H).sup.+. Fmoc group on compound 1j (10.0 g, 20.5
mmol) in CH.sub.2Cl.sub.2 (40.0 mL) was deprotected by stirring it
with diethylamine (40.0 mL) for 1 h at room temperature. 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 3.5 g of intermediate 1k (Yield: 70%). LCMS: 266.5
(M+H).sup.+. TEA (1.2 g, 12.0 mmol) was added to a stirred solution
of intermediate 1k (1.6 g, 6.0 mmol) in CH.sub.2Cl.sub.2 (30 mL).
Solution of 4-nitrophenyl chloroformate (1.3 g, 6.6 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added to the above stirred solution
and the reaction was continued for 12 h at room temperature. The
completion of the reaction was confirmed by TLC analysis After
completion of reaction, the reaction mixture was diluted with
CH.sub.2Cl.sub.2 (50 mL) and washed with 1.0 M of sodium bisulphate
(50 mL.times.2) and 1.0 M sodium carbonate (50 mL.times.2), dried
over Na.sub.2SO.sub.4 and evaporated under reduced pressure to
yield crude compound 1l, which was further purified by silica gel
column chromatography (eluent: 0-20% ethyl acetate in hexane) to
yield 0.8 g of compound 1l. .sup.1H NMR (DMSO-d.sub.6, 300 MHz):
.delta. 1.04 (s, 9H), 1.16 (d, 3H), 4.11 (m, 1H), 5.11 (m, 3H),
6.91 (d, 2H), 7.40 (m, 5H), 8.10 (d, 2H), 8.26 (brs, 1H).
Example 2
Synthesis of Compound 2
[0138] FIGS. 2A-2B illustrates Steps 2a and 2h.
[0139] Step 2a: DIPEA (2.71 g, 21 mmol) was added slowly to a
stirred solution of compound 1b (3.12 g, 10.5 mmol), HATU (4.41 g,
11.6 mmol) in DMF (30 mL) and was allowed to stir at room
temperature for 5 min. To the above reaction mixture
D-Thr(.sup.tBu)-OMe.HCl (2.0 g, 10.5 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
re-crystallized with CH.sub.2Cl.sub.2 to yield 4.2 g of compound
2a. LCMS: 491.2 (M+Na).sup.+.
[0140] Step 2b: 99% Hydrazine hydrate solution (5 mL) was added
slowly to a stirred solution of compound 2a (4.2 g) in methanol (40
mL) and the completion of the reaction was confirmed by TLC
analysis. The reaction mixture on evaporation under reduced
pressure yielded 4.2 g of compound 2b (Yield: 90%). LCMS: 469.4
(M+H).sup.+.
[0141] Step 2c: DIPEA (2.7 mL, 20.9 mmol) was added slowly to a
stirred solution of compound 2b (4.9 g, 10.5 mmol), HATU (4.8 g,
12.5 mmol) in DMF (50 mL). To the above stirred solution,
Fmoc-D-Asn(Trt)-OH (6.2 g, 10.5 mmol) was added and further stirred
at room temperature for 12 h. The completeness of the reaction was
confirmed by TLC analysis. The reaction mixture was diluted with
EtOAc (30 mL) and washed with 1.0 M sodium carbonate (20
mL.times.2), 10% citric acid (20 mL.times.2), water (20
mL.times.2), dried over Na.sub.2SO.sub.4 and evaporated under
reduced pressure to yield 10 g of crude intermediate 2c and was
further purified by silica gel column chromatography (eluent: 0-5%
MeOH in EtOAc) to yield 5 g of compound 2c. LCMS: 1047.7
(M+H).sup.+.
[0142] Step 2d: Fmoc deprotection of compound2c [(3.2 g) in
CH.sub.2Cl.sub.2 (10 mL)] was carried out using diethylamine (10
mL). The completion of the reaction was confirmed by TLC analysis.
The resulting solution on evaporation under reduced pressure
yielded 1.2 g of compound 2d.
[0143] Step 2e: Triethylamine (0.32 g, 3.2 mmol) was added slowly
to initiate the coupling of compound 2d (1.3 g, 1.6 mmol) and
compound 2h (0.79 g, 1.9 mmol) in THF (20 mL). The resulting
solution was further allowed to stir for 12 h at room temperature
and completeness of the reaction was confirmed by TLC analysis.
Organic layer was washed with NaHCO.sub.3, citric acid solution,
brine, dried over Na.sub.2SO.sub.4 and evaporated under reduced
pressure to yield 1.3 g of compound 2e.
[0144] Step 2f: Cbz group and benzyl ester deprotection was carried
out on compound 2e (1.3 g) in methanol using palladium hydroxide
(1.0 g) for 1 h at room temperature. The completeness of the
reaction was confirmed by TLC analysis. Palladium hydroxide was
removed by Celite.RTM. bed filtration and the filtrate was
evaporated under reduced pressure to yield 0.45 g of compound 2f.
LCMS: 878.4 (M+H).sup.+.
[0145] Step 2g: DIPEA (0.2 g, 1.5 mmol) was added slowly to a
stirred solution of compound 2f (0.45 g, 0.51 mmol), HOBT (0.21 g,
1.53 mmol) and PyBOP (0.8 g, 1.53 mmol) in THF (200 mL). The
reaction mixture was further stirred at room temperature for 12 h.
The completeness of the reaction was confirmed by TLC analysis. The
reaction mixture was evaporated and washed with diethyl ether to
yield 0.41 g of intermediate 2g. LCMS: 860.7 (M+H).sup.+.
[0146] Step 2h: To a solution of compound 2g (0.4 g) in
CH.sub.2Cl.sub.2 (5 mL), trifluoroacetic acid (5 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 crude compound 2. The crude solid material was
purified (yield: 10 mg,) using preparative HPLC method-B described
under experimental conditions. LCMS: 506.6 (M+H).sup.+; HPLC:
t.sub.R=12.4 min.
Synthesis of compound 2h
(NO.sub.2--C.sub.6H.sub.4--OCO-D-Ser(.sup.tBu)-OBzl)
[0147] The compound was synthesised using similar procedure as
exemplified in (example 1, compound 1l) using
Fmoc-D-Ser(.sup.tBu)-OH instead of Fmoc-Thr(.sup.tBu)-OH to yield 1
g crude material of 2h.
Example 3
Synthesis of Compound 3
[0148] FIGS. 3A-3C illustrates Steps 3a and 3k.
[0149] Step 3a: To a stirred solution of compound 1a (5.0 g, 30.6
mmol) in 1,4-Dioxane (50 mL), Sodium carbonate (8.12 g, 76.5 mmol,
dissolved in 10 mL water) and (Boc).sub.2O (9.98 mL, 45.7 mmol)
were added and stirred at room temperature for 12 h. The progress
of reaction was monitored by TLC. The reaction mass was partitioned
between diethyl ether and water. Then aqueous layer was made acidic
(pH=3) by 3N HCl solution and was extracted with DCM (2.times.200
mL). Organic layer was washed with water, brine, dried over
Na.sub.2SO.sub.4 and evaporated under reduced pressure to yield 50
g of pure 3a (Yield: 62.1%). LCMS: 263.0 (M+H).sup.+.
[0150] Step 3b: DIPEA (6.5 mL, 37.8 mmol) was added slowly to a
stirred solution of compound 3b (5 g, 12.6 mmol), compound 3c (2.72
g, 15 mmol), HOBt (2.55 g, 18.9 mmol) and EDC.HCl (3.62 g, 18.9
mmol) in DMF (75 mL) at 0.degree. C. The reaction mixture was
further stirred at room temperature for 12 h. The progress of the
reaction was confirmed by TLC analysis. The reaction mass was
partitioned between ethyl acetate and water. Organic layer was
washed with water, brine, dried over Na.sub.2SO.sub.4 and
evaporated under reduced pressure to yield crude. The crude
compound was purified by silica gel (60-120 mesh) column
chromatography using hexane/ethyl acetate (40:60) as elute to yield
5.2 g of compound 3d, (Yield: 71.0%). LCMS: 560.6 (M+H).sup.+.
[0151] Step 3c: To a stirred solution of compound 3d (5 g, 8.9
mmol) in dry DCM, diethylamine (50 mL) was added dropwise at
-10.degree. C. and stirred for 1 h at room temperature. After
completion of reaction, the mixture was evaporated under reduced
pressure to give crude compound. The crude was purified with (1:1)
n-pentane/diethyl ether wash and dried under high vacuum to yield
3.5 g of compound 3e.LCMS: 338.58 (M+H).sup.+.
[0152] Step 3d: NMM (1.4 mL, 14.0 mmol) was added slowly to a
stirred solution of compound 3a (3 g, 11.3 mmol), compound 3e (4.3
g, 12.9 mmol) and HATU (6.5 g, 17.1 mmol) in DMF (75 mL) at
0.degree. C. The reaction mixture was further stirred for 6 h at
room temperature. 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, dried over
Na.sub.2SO.sub.4 and evaporated under reduced pressure to give
crude compound. The crude was purified by silica gel column
chromatography (Eluent: 50% hexane in ethyl acetate) to yield 4.8 g
of compound 3f, LCMS: 583.7 (M+H).sup.+.
[0153] Step 3e: To a stirred solution of compound 3f (4.5 g, 7.7
mmol) in MeOH, Pd/C (2.0 g) was added slowly and stirred under
H.sub.2 atmosphere for 4 h at room temperature. Progress of
reaction was monitored by TLC. After completion, the reaction mass
was filtered through Celite.RTM. and washed with MeOH (2.times.150
mL). The resulting filtrate was evaporated under reduced pressure
to yield 3 g of compound 3g LCMS: 448.6 (M+H).sup.+.
[0154] Step 3f: To a stirred solution of Cbz-D-Asn-OH (1.78 g, 6.7
mmol) and compound 3g (3.0 g, 6.8 mmol) in DMF (75 mL), DCC (4.12
g, 20.3 mmol) and HOBT (1.8 g, 13.5 mmol) were added slowly at
0.degree. C. The reaction mixture was stirred for 48 h at room
temperature. 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, dried over
Na.sub.2SO.sub.4 and evaporated under reduced pressure to yield
crude. The crude compound was purified by silica gel (60-120 mesh)
column chromatography (Eluent: 4% MeOH in DCM) to yield 2.5 g of
Compound 3h, LCMS: 697.50 (M+H).sup.+.
[0155] Step 3g: To a stirred solution of compound 3h (2.5 g, 3.6
mmol) in MeOH (50 mL), Pd/C (1.2 g) was added and stirred under
H.sub.2 atmosphere for 4 h at room temperature. Progress of
reaction was monitored by TLC. After completion, the reaction mass
was filtered through Celite.RTM. and washed with MeOH (2.times.150
mL). The resulting filtrate was evaporated under reduced pressure
to yield 1.5 g of compound 3i, LCMS: 563.6 (M+H).sup.+.
[0156] Step 3h: Compound 3i (1.3 g, 2.3 mmol), TEA (0.43 mL, 3.5
mmol) in DMF (25 mL) and was added dropwise slowly to a solution of
3j(1.1 g, 2.6 mmol) at -10.degree. C. The mixture was further
stirred at room temperature for 2h. Progress of reaction was
monitored by TLC. After completion, the reaction mass was
partitioned between ethyl acetate and water. Organic layer was
washed with NaHCO.sub.3, citric acid solution, brine, then organic
layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced
pressure to give crude. The crude was purified by silica gel
(60-120 mesh) column chromatography (hexane/ethyl acetate (10:90)
as elute) to yield 1.2 g of compound 3k. LCMS: 840.6
(M+H).sup.+.
[0157] Step 3i: To a solution of compound 3k (1.0 g, 1.2 mmol) in
CH.sub.2Cl.sub.2 (10 mL), trifluoroacetic acid (10 mL) and
catalytic amount of triisopropylsilane were added and stirred for 3
h at room temperature to remove the acid sensitive protecting
groups. The resulting solution was concentrated in vacuum and
washed with diethyl ether to afford 1.0 g of crude compound 31,
LCMS: 628.65 (M+H).sup.+.
[0158] Step 3j: To a stirred solution of compound 31 (1.0 g, 1.5
mmol) in THF, PyBOP (2.4 g, 4.7 mmol), HOBT (0.6, 4.7 mmol) and
DIPEA (0.8 mL, 4.7 mmol) were added slowly and stirred for 12 h at
room temperature. The reaction mass was partitioned between water
and ethyl acetate. Organic layer was washed with water, brine,
dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure
to give crude. Crude compound was washed with diethyl ether to
yield 0.8 g of compound 3m, LCMS: 610.5 (M+H).sup.+.
[0159] Step 3k: To a stirred solution of compound 3m (0.8 g, 1.3
mmol) in MeOH (30 mL), Pd(OH).sub.2 (0.4 g) was added and stirred
under H.sub.2 atmosphere for 4 h at room temperature. Progress of
reaction was monitored by TLC. After completion, the reaction mass
was filtered through Celite.RTM. and washed with MeOH (2.times.150
mL). The resulting filtrate was evaporated under reduced pressure
to yield 0.7 g of compound3. LCMS: 520.5 (M+H).sup.+; HPLC:
t.sub.R=9.9 min.
[0160] Synthesis of Intermediate 3c: To a stirred solution of
compound 3n (4 g, 88.3 mmol) and compound 3o (10.2 mL, 71.2 mmol)
in DCM (150 mL), TEA (14.4 mL, 105 mmol) was added dropwise at
-78.degree. C. The reaction mixture was allowed to attain room
temperature and stirred for 12 h. Progress of reaction was
monitored by TLC. After completion, the reaction mass was
partitioned between water and DCM. Organic layer was washed with
water, brine, dried over Na.sub.2SO.sub.4 and evaporated under
reduced pressure to yield crude compound and was purified by silica
gel (60-120 mesh) column chromatography (Eluent: 80% ethyl acetate
in hexane) to yield 10 g of Compound 3c, LCMS: 181.18
(M+H).sup.+.
[0161] Synthesis of 3j
(NO.sub.2--C.sub.6H.sub.4-OCO-D-Ser(Bzl)-O.sup.tBu): The compound
was synthesised using similar procedure as exemplified in (example
1, compound 1k) using Fmoc-D-Ser(Bzl)-O.sup.tBu instead of
Fmoc-Thr(.sup.tBu)-OH to yield 1.5 g of compound 3j.
Example 4
Synthesis of Compound 4
##STR00025##
[0163] The compound was synthesised using similar procedure as
depicted in Example 2 (compound 2) using 7-aminoheptanoic acid
instead compound 1a to yield0.3 g crude material of the title
compound. The crude solid material was purified using preparative
HPLC described under experimental conditions. LCMS: 488.2
(M+H).sup.+; HPLC: t.sub.R=11.9 min.
[0164] The compounds in Table 3 below were prepared based on the
synthetic procedures described above.
TABLE-US-00003 TABLE 3 Compound LCMS HPLC No. Structure (M +
H).sup.+ (t.sub.R in min.) 5 ##STR00026## 518.2 9.7 6 ##STR00027##
534.2 12.4 7 ##STR00028## 532.9 -- 8 ##STR00029## 520.2 9.03 9
##STR00030## 492.1 12.9 10 ##STR00031## 520.2 11.23 11 ##STR00032##
507.2 11.96 12 ##STR00033## 493.2 8.35 13 ##STR00034## 521.3 12.2
14 ##STR00035## 535.4 11.2
[0165] The compounds shown in below table 4, 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-US-00004 TABLE 4 Compound No. Structure 15 ##STR00036## 16
##STR00037## 17 ##STR00038## 18 ##STR00039## 19 ##STR00040## 20
##STR00041## 21 ##STR00042## 22 ##STR00043## 23 ##STR00044## 24
##STR00045## 25 ##STR00046## and
Example 5
Rescue of Mouse Splenocyte Proliferation in the Presence of
Recombinant PD-L1
[0166] Recombinant mouse PD-L1 (rm-PDL-1, cat no: 1019-B7-100;
R&D Systems) were used as the source of PD-L1.
Requirement:
[0167] 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
(10000 unit/ml)-Streptomycin(10,000 .mu.g/ml) Liquid (GIBCO, Cat
#15140-122); MEM Sodium Pyruvate solution 100 mM (100.times.),
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 (1 mL) (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 .mu.m 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 .mu.L of Dimethyl
sulfoxide (DMSO C.sub.2H.sub.6SO, SIGMA-D-5879) and aliquoted in to
tubes for further use. Working concentrations were titrated from 10
.mu.m to 1 .mu.m. (eBioscience-650850-85);
[0168] 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-L1 cat no:
1019-B7-100).
Protocol
Splenocyte Preparation and Culturing:
[0169] Splenocytes harvested in a 50 mL falcon tube by mashing
mouse spleen in a 40 .mu.m cell strainer were further treated with
1 ml ACK lysis buffer for 5 mins at room temperature. After washing
with 9 mL of RPMI complete media, cells were re-suspended in 3 ml
of 1.times.PBS 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 800.times.g 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 1.times.PBS followed by
total cell counting using Trypan Blue exclusion method and used
further for cell based assays.
[0170] Splenocytes were cultured in RPMI complete media (RPMI+10%
fetal bovine serum+1 mM sodium pyruvate+10,000 units/mL penicillin
and 10,000 .mu.g/mL streptomycin) and maintained in a CO.sub.2
incubator with 5% CO.sub.2 at 37.degree. C.
CFSE Proliferation Assay:
[0171] CFSE is a dye that passively diffuses into cells and binds
to intracellular proteins. 1.times.10.sup.6 cells/ml of harvested
splenocytes were treated with 5 .mu.m of CFSE in pre-warmed
1.times.PBS/0.1% BSA solution for 10 mins at 37.degree. 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 1.times.10.sup.5 splenocytes added to wells
containing either MDA-MB231 cells (1.times.10.sup.5 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 .mu.g/mL each), and the culture was
further incubated for 72 h at 37.degree. C. with 5% CO.sub.2. Cells
were harvested and washed thrice with ice cold FACS buffer and %
proliferation was analysed by flow cytometry with 488 nm excitation
and 521 nm emission filters.
Data Compilation, Processing and Inference:
[0172] Percent splenocyte proliferation was analysed 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%.
Background proliferation: Splenocytes+anti-CD3/CD28+PD-L1
Stimulated splenocytes: Splenocytes+anti-CD3/CD28 stimulation
Compound proliferation: Splenocytes+anti-CD3/CD28+PD-L1+Compound
Compound effect is examined by adding required conc. of compound to
anti-CD3/CD28 stimulated splenocytes in presence of ligand (PDL-1)
Table 5 shows the rescue of mouse splenocyte proliferation
inhibited by recombinant mouse PDL1 using CFSE based assay:
TABLE-US-00005 TABLE 5 Percent rescue of splenocyte Compound
proliferation @ 100 nM compound No. concentration 1 95 2 94 3 58 4
49 5 53 8 68 9 73 10 89 12 91
* * * * *