U.S. patent application number 15/205853 was filed with the patent office on 2017-01-12 for pyrrolidine carboxamido derivatives and methods for preparing and using the same.
The applicant listed for this patent is Korea Research Institute of Chemical Technology, Research & Business Foundation Sungkyunkwan University. Invention is credited to Imran Ali, Chong Hak Chae, Gildon Choi, Moon Kook Jeon, Kwangho Lee, Youn Sook Lee, Seok Hee Park, Sang Dal Rhee.
Application Number | 20170008924 15/205853 |
Document ID | / |
Family ID | 57686085 |
Filed Date | 2017-01-12 |
United States Patent
Application |
20170008924 |
Kind Code |
A1 |
Lee; Kwangho ; et
al. |
January 12, 2017 |
PYRROLIDINE CARBOXAMIDO DERIVATIVES AND METHODS FOR PREPARING AND
USING THE SAME
Abstract
Pyrrolidine carboxamido derivatives, optical isomers thereof,
and salts thereof that are able to prevent, improve, and/or treat
inflammatory conditions, including inflammatory bowel disease, and
methods for preparing and using the same are provided.
Inventors: |
Lee; Kwangho; (Daejeon,
KR) ; Rhee; Sang Dal; (Daejeon, KR) ; Choi;
Gildon; (Daejeon, KR) ; Ali; Imran; (Daejeon,
KR) ; Chae; Chong Hak; (Daejeon, KR) ; Jeon;
Moon Kook; (Daejeon, KR) ; Park; Seok Hee;
(Suwon, KR) ; Lee; Youn Sook; (Suwon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Research & Business Foundation Sungkyunkwan University
Korea Research Institute of Chemical Technology |
Suwon
Daejeon |
|
KR
KR |
|
|
Family ID: |
57686085 |
Appl. No.: |
15/205853 |
Filed: |
July 8, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/1016 20130101;
C07K 5/1024 20130101; C07K 5/1008 20130101; C07K 5/0823 20130101;
A61P 1/00 20180101; C07D 403/06 20130101; C07D 207/04 20130101;
A61K 38/00 20130101; A61K 47/542 20170801 |
International
Class: |
C07K 5/117 20060101
C07K005/117; C07K 5/097 20060101 C07K005/097 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2015 |
KR |
10-2015-0097040 |
Claims
1. A compound represented by the following Formula 1, an optical
isomer thereof, or a pharmaceutically acceptable salt thereof.
##STR00041## wherein: n is 0, 1, or 2; A is -a.sup.1-, which is an
amino acid independently selected from the group consisting of
alanine, (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic
acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine
(Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile,
I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M),
phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S),
threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and
valine (Val, V), both terminal ends of the amino acid being coupled
to a carbonyl group or an amine group by an amide bond; and R.sup.1
is a straight chain or branched chain C.sub.1-36 alkyl, a straight
chain or branched chain C.sub.2-36 alkenyl including at least one
double bond, or a straight chain or branched chain C.sub.2-36
alkynyl including at least one triple bond.
2. The compound of claim 1, wherein: n is 0, 1, or 2; .sub.a.sup.1
is ##STR00042## both terminal ends of which is coupled to a
carbonyl group or amide group thereof by an amide bond; and R.sup.1
is a straight chain or branched chain C.sub.1-36 alkyl.
3. The compound of claim 1 selected from the group consisting of
the following compounds. ##STR00043## ##STR00044## ##STR00045##
##STR00046##
4. A method for preparing the compound of claim 1 represented by
Formula 1, which comprises, as represented by the following
Reaction Scheme 1: reacting a compound 2 with a compound 3 to
prepare a compound 4; hydrolyzing the compound 4 in the presence of
a base to prepare a compound 5; reacting the compound 5 with a
compound 6 to prepare a compound 7; hydrolyzing the compound 7 in
the presence of a base to prepare a compound 8; reacting the
compound 8 with a compound 9 to prepare a compound 10; hydrolyzing
the compound 10 in the presence of a base to prepare the compound
of claim 1. ##STR00047## ##STR00048## wherein A, R.sup.1 and n are
the same as defined in claim 1 and R.sup.2 is a straight chain or
branched chain C.sub.1-5 alkyl.
5. A composition for preventing, improving, or treating
inflammatory bowel disease, which comprises, as an active
component, the compound, the optical isomer, or the salt of claim
1.
6. The composition of claim 5, which inhibits formation of an
inflammatory signal transduction complex mediated by MyD88,
inhibits formation of an inflammatory signal transduction complex
mediated by Pellino-1, inhibits formation of an inflammatory signal
transduction complex mediated by Rip1, suppresses expression of at
least one protein selected from the group consisting of G-CSF,
IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta.,
IL-6, IL-9, MCP-1, MIP-3.alpha., IL12p40/70, MIG, TNF-.alpha., and
VCAM-1, or suppresses activity of NF-.kappa.B.
7. The composition of claim 5, wherein the inflammatory bowel
disease includes ulcerative colitis, Behcet's disease, and Crohn's
disease.
8. A method for preventing, improving, or treating inflammatory
bowel disease, which comprises administering to a subject in need a
composition containing, as an active component, the compound, the
optical isomer, or the salt of claim 1.
9. The method of claim 8, wherein the composition is administered
via oral, nasal, pulmonary, rectal, buccal, vaginal, ocular and
transdermal routes.
10. The method of claim 8, wherein the inflammatory bowel disease
includes ulcerative colitis, Behcet's disease, and Crohn's
disease.
11. The method of claim 8, wherein the composition, wherein the
composition inhibits formation of an inflammatory signal
transduction complex mediated by MyD88, inhibits formation of an
inflammatory signal transduction complex mediated by Pellino-1,
inhibits formation of an inflammatory signal transduction complex
mediated by Rip1, suppresses expression of at least one protein
selected from the group consisting of G-CSF, IL-2, SCF, VEGF,
CX3CL1, IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta., IL-6, IL-9, MCP-1,
MIP-3.alpha., ID 2p40/70, MIG, TNF-.alpha., and VCAM-1, or
suppresses activity of NF-.kappa.B.
12. A method for preventing, improving, or treating disease or
syndrome, which comprises administering to a subject in need a
composition containing, as an active component, the compound, the
optical isomer, or the salt of claim 1, wherein the disease or
syndrome involves formation of a Pellino-1 induced inflammatory
signal transduction complex containing MyD88, RIP1, or both.
13. The method of claim 12, wherein the disease or syndrome is
inflammatory bowel disease.
14. The method of claim 13, wherein the inflammatory bowel disease
includes ulcerative colitis, Behcet's disease, and Crohn's
disease.
15. The method of claim 12, wherein the disease or syndrome
includes multiple sclerosis, psoriasis, sepsis, geographic atrophy,
wet age-related macular disease, dry age-related macular disease,
diabetic retinopathy, infectious lung diseases, bacterial
pneumonia, viral pneumonia, diffuse large B-cell lymphoma, viral
infection, autoimmune disease, blood cancer including lymphoma, and
tumors in internal organs.
16. A composition for preventing, improving, or treating disease or
syndrome, which comprises, as an active component, the compound,
the optical isomer, or the salt of claim 1, wherein the disease or
syndrome includes geographic atrophy, wet age-related macular
disease, dry age-related macular disease, and diabetic
retinopathy.
17. The composition of claim 16, wherein the compound, the optimal
isomer, or the salt of claim 1 has a pharmaceutical effect on
retinal pigment epithelium cells.
18. The composition of claim 16, wherein the compound, the optimal
isomer, or the salt of claim 1 inhibits expression, in retinal
pigment epithelium cells, of at least one protein selected from the
group consisting of Nox-4, VEGF, VEGFR1, VEGFR2, Ang2, EPO and
EPOR.
19. The composition of claim 16, wherein the compound, the optimal
isomer, or the salt of claim 1 increases expression, in retinal
pigment epithelium cells, of Ang 1, Tie2, or both.
20. A composition for preventing, improving, or treating disease or
syndrome, which comprises, as an active component, the compound,
the optical isomer, or the salt of claim 1, wherein the disease or
syndrome includes sepsis and multiple sclerosis.
21. A composition for preventing, improving, or treating alopecia,
which comprises, as an active component, the compound, the optical
isomer, or the salt of claim 1, wherein the active component
inhibits expression of IL-6 in scalp and hair follicles.
22. A composition for preventing, improving, or treating disease or
syndrome, which comprises, as an active component, the compound,
the optical isomer, or the salt of claim 1, wherein the disease or
syndrome involves formation of a Pellino-1 induced inflammatory
signal transduction complex containing MyD88, RIP1, or both.
23. The composition of claim 22, wherein the disease or syndrome is
inflammatory bowel disease.
24. The composition of claim 23, wherein the inflammatory bowel
disease includes ulcerative colitis, Behcet's disease, and Crohn's
disease.
25. The composition of claim 22, wherein the disease or syndrome
includes multiple sclerosis, psoriasis, sepsis, geographic atrophy,
wet age-related macular disease, dry age-related macular disease,
diabetic retinopathy, infectious lung diseases, bacterial
pneumonia, viral pneumonia, diffuse large B-cell lymphoma, viral
infection, autoimmune disease, blood cancer including lymphoma, and
tumors in internal organs.
26. A method for suppressing expression of cytokines and/or
chemokines, which comprises administering to a subject in need a
composition containing, as an active component, the compound, the
optical isomer, or the salt of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to Korean
Application No. 10-2015-0097040 filed Jul. 8, 2015, which is
incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to pyrrolidine carboxamido
derivatives, optical isomers thereof, or pharmaceutically
acceptable salts thereof, and methods for preparing and using the
same.
BACKGROUND ART
[0003] Various compounds/compositions/methods including, but not
limited to, immunosuppressive drugs (e.g., infliximab),
aminosalicylic acids (e.g., sulfasalazine), and steroids have been
proposed as means for reducing cytokines and/or chemokines to
prevent and/or treat various diseases including, but not limited
to, inflammatory indications, cancers, and ophthalmic indications
(Expert opinion on emerging drugs (2015) 20(3):349-352; Cell.
(2010) March 19; 140(6): 883-899; Progress in Retinal and Eye
Research 37(2013) 68e89, which are incorporated herein by
reference). They are, however, unsatisfactory at least because they
are expensive, and/or involve side effects, and/or show low
therapeutic efficacy (P&T 41(2016), Jun no 6; Gut
56(2007):725-732; World J Gastroenterol (2005);11(16):2462-2466,
which are incorporated herein by reference). Therefore, there
remains a need for a new compound, composition, and/or a
method.
SUMMARY
[0004] The present invention is based on the discovery that certain
pyrrolidine carboxamido derivatives are able to suppress the
expression and activity of inflammatory cytokines (e.g., IL-6)
and/or chemokines and are able to remain at a sufficiently high
concentration in a target tissue/cell while being less exposed to
blood. The present invention is also based on the discovery that
certain pyrrolidine carboxamido derivatives are able to inhibit the
activity of NF-.kappa.B by stabilizing of I.kappa.B. The present
invention is further based on the discovery that certain
pyrrolidine carboxamido derivatives are able to disrupt the
formation of inflammatory signal transduction complex mediated by
myeloid differentiation primary response gene 88 (MyD88) and/or
receptor-interacting protein 1 (RIP1) that act in the downstream of
signaling pathway involving toll-like receptor 2/4 and
IL-1.beta..
[0005] In one aspect, the present invention provides compounds
represented by the following Formula 1, optical isomers thereof, or
pharmaceutically acceptable salts thereof.
##STR00001##
[0006] wherein: n is 0, 1, or 2; A is -a.sup.1-, which is an amino
acid independently selected from the group consisting of alanine,
(Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid
(Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine
(Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile,
I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M),
phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S),
threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and
valine (Val, V), both terminal ends of the amino acid being coupled
to a carbonyl group or an amine group by an amide bond; and R.sup.1
is a straight chain or branched chain C.sub.1-36 alkyl, a straight
chain or branched chain C.sub.2-36 alkenyl including at least one
double bond, or a straight chain or branched chain C.sub.2-36
alkynyl including at least one triple bond.
[0007] In another aspect, the present invention provides methods
for preparing the compounds, the optical isomers, and the
salts.
[0008] In still another aspect, the present invention provides
compositions for preventing, improving, and treating various
diseases (e.g., inflammatory indications, cancers, and ophthalmic
indications). The compositions each comprise, as an active
component, at least one of the compounds, at least one of the
optical isomers, or at least one of the salts.
[0009] In still yet another aspect, the present invention provides
methods for preventing, improving, or treating various diseases
(e.g., inflammatory indications, cancers, and ophthalmic
indications). The methods each comprise administering to a subject
in need a composition containing, as an active component, at least
one of the compounds, at least one of the optical isomers, or at
least one of the salts.
[0010] Compounds according to certain embodiments of the present
invention may inhibit decomposition of I.kappa.B in inflammation
signaling pathway mediated by MyD88 (myddosome complex) and/or RIP
1, thereby preventing NF-.kappa.B from being transported into
nucleus of a cell, resulting in suppression of expression of
cytokines and chemokines (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1,
IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta., IL-6, IL-9, MCP-1,
MIP-3.alpha., IL12p40/70, MIG, TNF-.alpha., and VCAM-1) and
preventing inflammation reaction that could otherwise be caused by
the expression thereof.
[0011] Other aspects and advantages of the present invention will
become apparent to the skilled in the art from a consideration of
the detailed description and the drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1A is an electrophoresis result showing that compounds
according to embodiments of the present invention suppress the
expression of IL-6.
[0013] FIG. 1B is a graph showing that compounds according to
embodiments of the present invention suppress the expression of
IL-6.
[0014] FIGS. 2A-2D are bar graphs that show that compounds
according to embodiments of the present invention suppress the
expression of cytokines and chemokines in a cell line RAW 264.7.
FIG. 2A is a bar graph that shows that the expression of GCSF,
IL-2, SCF, and VEGF was suppressed as statistically meaningful when
the cells were treated with the compounds of the present invention.
FIG. 2B is a bar graph that shows that the expression of CX3CL1,
IGFBP5, IGFBP6, IL-1 b, IL-6, and IL-9 was suppressed as
statistically meaningful when the cells were treated with the
compounds of the present invention. FIG. 2C is a bar graph that
shows that the expression of MCP-1, MIP-3a, IL12p40/70, MIG, TNF-a,
and VCAM-1 was suppressed as statistically meaningful when the
cells were treated with the compounds of the present invention.
FIG. 2D is a bar graph that shows that the expression of IL-1a was
suppressed as statistically meaningful when the cells were treated
with the compounds of the present invention.
[0015] FIG. 3 shows that compounds according to embodiments of the
present invention suppress the expression of IL-6 in a host in a
cell line RAW 264.7.
[0016] FIG. 4 shows that compounds according to embodiments of the
present invention inhibit the activity of NF-.kappa.B.
[0017] FIG. 5 shows that the compounds according to embodiments of
the present invention suppress NF-.kappa.B.
[0018] FIGS. 6A-6C are bar graphs that show that the compounds
according to embodiments of the present invention inhibit the
activity of NF-.kappa.B (FIG. 6A) while not affecting signal
transmission of TGF-.beta. (FIG. 6B) and BMP (FIG. 6C).
[0019] FIG. 7 is an immunoprecipitation result showing that the
compounds according to embodiments of the present invention inhibit
formation of inflammation signaling pathway protein complex
mediated by IRAK-1, MyD88, and/or RIP1 and showing that the
compounds according to embodiments of the present invention change
the concentration of I.kappa.B.
[0020] FIG. 8 is an immunoprecipitation result showing that the
compounds according to embodiments of the present invention can
disrupt formation of inflammation signaling pathway protein complex
mediated by IRAK-1, MyD88, and/or RIP1.
[0021] FIGS. 9A and 9B show that change in pretreatment
concentration of the compounds according to embodiments of the
present invention change concentration of I.kappa.B in RAW 264.7
macrophage cells (FIG. 9A) and BMDM cells (FIG. 9B).
[0022] FIG. 10 is a graph indicating disease activity index scores
in an animal model with DSS-induced chronic colitis according to
the dose of compounds according to embodiments of the present
invention in case of oral administration thereof.
[0023] FIG. 11A shows disease activity index scores representing
the ability of compounds according to embodiments of the present
invention to suppress acute colitis in an animal model with
DSS-induced acute colitis.
[0024] FIGS. 11B-11D shows the compounds according to embodiments
of the present invention affect the amount of expression of
chemokines (CCL2 (FIG. 11C), CCL20 (FIG. 11B), and CXCL1 (FIG.
11D)) in a mice model with DSS-induced chronic colitis.
[0025] FIG. 12 are images showing shapes of large intestinal villi
from a non-treated group.
[0026] FIG. 13 are images showing shapes of large intestinal villi
from a DDS-induced chronic colitis model group
[0027] FIG. 14 are images showing shapes of large intestine villi
from a group treated with a compound according to embodiments of
the present invention (compound 1.1).
[0028] FIG. 15 are images of large intestinal tissues obtained from
non-treated group, the DDS-induced chronic colitis model group, the
group treated with compound 1.1 (mpk) and the group treated with
sulfasalazine (500 mpk) as an anti-inflammatory drug for colitis
treatment.
[0029] FIG. 16 are images showing the morphology of large
intestinal mucous membranes obtained from the non-treated group,
the DDS-induced chronic colitis model group, the treated group with
compound 1.1 (100 mpk) and the group treated with sulfasalazine
(500 mpk) as an anti-inflammatory drug for colitis treatment.
[0030] FIG. 17 is a graph showing recovery level of large
intestinal wall in a non-treated group, a DDS-induced chronic
colitis model group, a group treated with compounds according to
embodiments of the present invention, and a group treated with
sulfasalazine.
[0031] FIG. 18 is a graph showing changes in blood concentration
over time of compounds according to embodiments of the present
invention via intravenous administration.
[0032] FIG. 19 is a graph showing changes in blood concentration
over time of compounds according to embodiments of the present
invention via oral administration.
[0033] FIG. 20A is a Western blot image confirming whether
compounds according to embodiments of the present invention inhibit
MAPK/ERK signaling pathway.
[0034] FIG. 20B is a diagram depicting signaling pathway of
toll-like receptors.
[0035] FIG. 21 is a Western blot image confirming whether compounds
according to embodiments of the present invention inhibit MAPK/ERK
signaling pathway.
[0036] FIG. 22A and FIG. 22B show the inhibition level of
NF-.kappa.B activation by compounds according to embodiments of the
present invention (FIG. 22A) and an IRAK1/4 inhibitor (FIG. 22B),
respectively.
[0037] FIGS. 23A and 23B are immunoblot images confirming whether
compounds according to embodiments of the present invention and an
IRK1/4 inhibitor change the concentration of I.kappa.B. FIG. 23A is
an immunoblot image showing cells pretreated with compound 1.1 (100
nM), IRAK1/4 inhibitor (25 .mu.M) and smaducin-6 (100 nM) and then
further treated with LPS (100 ng/ml). FIG. 23B is an immunoblot
image showing that RAK1/4 inhibitor also suppressed degradation of
I.kappa.B similar to compound 1.1.
[0038] FIG. 24A and FIG. 24B are an image and a graph,
respectively, comparing the ability of compounds according to
embodiments of the present invention to inhibit MAPK/ERK signaling
pathway and the ability of an IRAK1/4 inhibitor to inhibit MAPK/ERK
signaling pathway.
[0039] FIGS. 25A and 25B are Western blot images confirming whether
compounds according to embodiments of the present invention
suppress, in ARPE-19, expression of Nox-4, VEGF, VEGFR1, VEGFR2,
Ang-2, EPO, and EPOR and can increase the expression of Ang-1 and
Tie2. FIG. 25A is a Western blot image confirming that compounds
according to embodiments of the present invention suppress, in
ARPE-19, expression of Nox-4, VEGF, VEGFR1, and VEGFR2. FIG. 25B is
a Western blot image confirming that compounds according to
embodiments of the present invention suppress, in ARPE-19, Ang-2,
EPO, and EPOR, and can increase expression of Ang-1 and Tie2.
[0040] FIG. 25C is a qRT-PCR image confirming whether compounds
according to embodiments of the present invention suppress the
expression of VEGF in HRMEC.
[0041] FIG. 26 is an image showing that compounds according to
embodiments of the present invention suppress tube formation in
HRMEC.
[0042] FIGS. 27A and 27B are images showing that compounds
according to embodiments of the present invention suppress
activated oxygen increased in a mice model with STZ-induced type 1
diabetic retinopathy. FIG. 27A shows an image of the administration
schedule. FIG. 27B are images showing retina tissues stained with 5
.mu.M of dihydroethidium and measured active oxygen rates from each
group.
[0043] FIG. 28A is a graph showing that compounds according to
embodiments of the present invention have a therapeutic effect in
MOG-induced EAE mice.
[0044] FIG. 28B is a graph showing that compounds according to
embodiments of the present invention change the weight of
MOG-induced EAE mice.
[0045] FIG. 29 is a graph showing that compounds according to
embodiments of the present invention have a therapeutic effect in a
Cecal ligation and puncture (CLP) model.
DETAILED DESCRIPTION
[0046] 1. Definitions
[0047] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this disclosure belongs. The following
references, which are incorporated herein by reference, provide one
of skill with a general definition of many of the terms used in
this invention: The Cambridge Dictionary of Science and Technology
(Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger
et. al. (eds.), Springer Verlag (1991); and Hale & Marham, The
Harper Collins Dictionary of Biology (1991). As used herein, the
following terms have the meanings ascribed to them below, unless
specified otherwise.
[0048] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive.
[0049] Unless specifically stated or obvious from context, as used
herein, the terms "a", "an", and "the" are understood to be
singular or plural. Thus, for example, reference to "a compound"
includes mixtures of such compounds; reference to "a carrier"
includes mixtures of two or more carriers; and the like.
[0050] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%. 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein are modified by the term about.
[0051] The terms "active agent," "drug," and "pharmaceutical agent"
are used interchangeably herein to refer to a chemical material or
compound which, when administered to a subject (e.g., any animal
including a human or non-human animal) by any means and/or routes
induces a desired pharmacologic effect (e.g., such as a reduction
of inflammation).
[0052] The term "additive" as used herein may refer to any
additional components that may be added to the compositions
described herein. For example, additives may include excipients
(e.g., one or more excipients), antioxidants (e.g., one or more
antioxidants), stabilizers (e.g., one or more stabilizers),
preservatives (e.g., one or more preservatives), pH adjusting
and/or buffering agents (e.g., one or more pH adjusting and/or
buffering agents), tonicity adjusting agents (e.g., one or more
tonicity adjusting agents), thickening agents (e.g., one or more
thickening agents), suspending agents (e.g., one or more suspending
agents), binding agents (e.g., one or more binding agents),
viscosity-increasing agents (e.g., one or more viscosity-increasing
agents), and the like, provided that the additional components are
pharmaceutically acceptable for the particular condition to be
treated. The additives may also include processing agents and drug
delivery modifiers and enhancers, such as, for example, calcium
phosphate, magnesium stearate, talc, monosaccharides,
disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium
carboxymethyl cellulose, dextrose, hydroxypropyl-beta-cyclodextrin,
polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and
the like, as well as combinations of any two or more thereof. Other
suitable pharmaceutically acceptable excipients are described in
"Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey
(1991), and "Remington: The Science and Practice of Pharmacy,"
Lippincott Williams & Wilkins, Philadelphia, 20th edition
(2003) and 21st edition (2005), which are incorporated herein by
reference. The additives described herein may be used in any
suitable amounts.
[0053] As used herein, the term "administering" means oral
administration, administration as a suppository, topical contact,
intravenous, parenteral, intraperitoneal, intramuscular,
intralesional, intrathecal, intranasal or subcutaneous
administration, or the implantation of a slow-release device, e.g.,
a mini-osmotic pump, to a subject. Administration is by any route,
including parenteral and transmucosal (e.g., oral, nasal,
pulmonary, rectal, buccal, vaginal, ocular and transdermal
routes).
[0054] The terms "derivative" and "analog" are used herein
interchangeably, and refer to a compound that possesses the same
core as a parent compound, but differs from the parent compound in
bond order, in the absence or presence of one or more atoms and/or
groups of atoms, and combinations thereof. The derivative can
differ from the parent compound, for example, in one or more
substituents present on the core, which may include one or more
atoms, functional groups, or substructures. The derivative can also
differ from the parent compound in the bond order between atoms
within the core. In general, a derivative can be imagined to be
formed, at least theoretically, from the parent compound via
chemical and/or physical processes.
[0055] As used herein, "antioxidants" may refer to are man-made or
natural substances that may prevent or delay some types of cell
damage and/or oxidation. Antioxidants are found in many foods,
including fruits and vegetables. They are also available as dietary
supplements. Exemplary antioxidants may include: Meta-carotene,
Lutein, Lycopene, Selenium, Vitamin A, Vitamin C, and Vitamin E.
Other antioxidants known to one of skill in the art may also be
used. The antioxidants described herein may be used in any suitable
amount.
[0056] By "co-administer" it is meant that a compound or
composition described herein is administered at the same time, just
prior to, or just after the administration of additional therapies
or active agents or additives described herein. The compound or the
composition of the disclosure can be administered alone or can be
co-administered to a subject in need. Co-administration is meant to
include simultaneous or sequential administration of the compound
individually or in combination (more than one compound or agent).
The preparations can also be combined, when desired, with other
active substances.
[0057] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. Patent law and can mean " includes," "including," and the
like; "consisting essentially of" or "consists essentially"
likewise has the meaning ascribed in U.S. Patent law and the term
is open-ended, allowing for the presence of more than that which is
recited so long as basic or novel characteristics of that which is
recited is not changed by the presence of more than that which is
recited, but excludes prior art embodiments.
[0058] As used herein, "concurrent administration" includes
overlapping in duration at least in part. For example, when two
agents (e.g., any of the agents or class of agents described herein
that has bioactivity) are administered concurrently, their
administration occurs within a certain desired time. The agents'
administration may begin and end on the same day. The
administration of one agent can also precede the administration of
a second agent by day(s) as long as both agents are taken on the
same day at least once. Similarly, the administration of one agent
can extend beyond the administration of a second agent as long as
both agents are taken on the same day at least once. The active
agent(s) does not have to be taken at the same time each day to
include concurrent administration.
[0059] As used herein, an "effective amount" or "therapeutically
effective amount" is that amount sufficient to affect a desired
biological effect, such as beneficial results, including clinical
results. As such, an "effective amount" depends upon the context in
which it is being applied. An effective amount may vary according
to factors known in the art, such as the disease state, age, sex,
and weight of the individual being treated. Several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation. In
addition, the compounds, compositions, or formulations of this
disclosure can be administered as frequently as necessary to
achieve a therapeutic amount.
[0060] The term, "gel" as used herein may refer to a material which
is not a readily flowable liquid and not a solid, i.e., semi-solid.
Gels may be formed from naturally occurring or synthetic materials.
The gels can be non-ordered to slightly ordered showing some
birefringence, liquid crystal character. Gels may be administered
topically.
[0061] The term "inflammatory bowel disease" as used herein has its
usual medical meaning, and refers to a group of inflammatory
indications/conditions of a colon and small intestine. Exemplary
inflammatory bowel diseases may include, but are not limited to,
Crohn's disease, ulcerative colitis, Johne's disease, Behget's
syndrome, collagenous colitis, diversion colitis, indeterminate
colitis, infective colitis, ischaemic colitis, lymphocytic colitis,
and closely related diseases and disorders of the gastrointestinal
tract.
[0062] The term "inhibit," as used herein, means to prevent,
decrease, slow-down or arrest. In one embodiment, a compound,
composition, or formulation may be considered to inhibit the
viability of at least one protein (e.g., G-CSF, IL-2, SCF, VEGF,
CX3CL1, IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta., IL-6, IL-9, MCP-1,
MIP-3.alpha., IL12p40/70, MIG, TNF-.alpha., VCAM-1, and
NF-.kappa.B) when the amount or rate of the process or reaction
that takes place in the presence of the compound, composition, or
formulation is decreased by at least about 10% when compared to the
amount or rate in the absence of the compound, composition, or
formulation. In another embodiment, a compound, composition, or
formulation may be considered to inhibit a process or reaction when
the amount or rate of the process or reaction that takes place in
the presence of the compound, composition, or formulation is
decreased by at least about 20% when compared to the amount or rate
in the absence of the compound, composition, or formulation. In
other embodiments, a compound, composition, or formulation may be
considered to inhibit viability of one or more proteins (e.g.,
G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1.alpha.,
IL-1.beta., IL-6, IL-9, MCP-1, MIP-3.alpha., ID 2p40/70, MIG,
TNF-.alpha., VCAM-1, and NF-.kappa.B) when the amount or rate of
viability that takes place in the presence of the compound,
composition, or formulation is decreased by at least about 25%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 75% or
about 80% when compared to the amount or rate in the absence of the
compound, composition, or formulation. In still other embodiments,
a compound, composition, or formulation may be considered to
inhibit viability of one or more proteins, i.e. arresting its
development.
[0063] As used herein, "intermittent administration" includes the
administration of an active agent for a period of time (which can
be considered a "first period of administration"), followed by a
time during which the agent is not taken or is taken at a lower
maintenance dose (which can be considered "off-period") followed by
a period during which the agent is administered again (which can be
considered a "second period of administration"). Generally, during
the second phase of administration, the dosage level of the agent
will match that administered during the first period of
administration but can be increased or decreased as medically
necessary.
[0064] "Jelly" according to the current disclosure is a class of
gels, which are semisolid systems that consist of suspensions made
up either small inorganic particles or large organic molecules
interpenetrated by a liquid, in which the structural coherent
matrix contains a high portion of liquid, usually water.
[0065] "Liquid" as used herein is a dosage form consisting of a
composition in its liquid state. A liquid is pourable; it flows and
conforms to its container at room temperature. Liquids display
Newtonian or pseudoplastic flow behavior. In embodiments, a
"semi-liquid" as used herein may have properties of both a liquid
and another formulation (i.e., a suspension, an emulsion, a
solution, a cream, a gel, a jelly, and the like).
[0066] "Myeloid differentiation primary response gene 88" or
"MYD88" is a protein that, in humans, is encoded by the MYD88 gene.
MyD88 plays a central role in the innate and adaptive immune
response. This protein functions as an essential signal transducer
in the interleukin-1 and Toll-like receptor signaling pathways.
These pathways regulate that activation of numerous proinflammatory
genes. The encoded protein consists of an N-terminal death domain
and a C-terminal Toll-interleukin) receptor domain.
[0067] As used herein, the term "ointment" may refer to a highly
viscous liquid or semi-liquid formulation that may be used for
therapeutic treatment of a disease, syndrome, or condition (e.g.,
inflammatory bowel disease).
[0068] As used herein "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
The type of carrier can be selected based upon the intended route
of administration. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile topical solutions or
dispersion. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the composition
(e.g., Formula I as described herein, derivatives/analogues of
Formula I, or a pharmaceutically acceptable salt, solvent, hydrate,
or polymorph thereof), use thereof in the ophthalmic compositions
for the disclosure is contemplated.
[0069] "Pharmaceutical carriers" or "carriers" as used herein can
further include pharmaceutically acceptable carriers, excipients,
or stabilizers which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered
solution. Examples of physiologically acceptable carriers include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptide; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween.TM., polyethylene glycol (PEG), and
Pluronics.TM..
[0070] Additionally, "pharmaceutically acceptable" means approved
or approvable by a regulatory agency of the Federal or a state
government or the corresponding agency in countries other than the
United States, or that is listed in the U.S. Pharmacopoeia or other
generally recognized pharmacopoeia for use in animals, and more
particularly, in humans.
[0071] The terms, "pH agent" or "buffering agent" as used herein
may refer to compounds or buffers useful as pH regulators. These
include, but are not limited to, glycerol buffers, citrate buffers,
borate buffers, acetate buffers, gluconate buffers, phosphate
buffers, or citric acid-phosphate buffers may also be included. The
pH agent or buffering agent may be used in any suitable amount.
[0072] The term, "preservative" as described herein may refer to a
substance or chemical that prevents undesirable chemical changes of
the compound or compositions or formulas described herein. Suitable
preservatives may include, for example, benzalkonium chloride,
thimerosal, chlorobutanol, methyl paraben, propyl paraben,
phenylethyl alcohol, edetate disodium sorbic acid, Onamer M
Polyquat, cetyl bromide, cetyl pyridinium chloride, benzyl bromide,
EDTA, phenylmercury nitrate, phenylmercury acetate, thimerosal,
merthiolate, acetate and phenylmercury borate, polymyxin B
sulphate, methyl and propyl parabens, quaternary ammonium chloride,
sodium benzoate, sodium proprionate, and sodium perborate, and
other agents known to those skilled in the art, or a combination
thereof. The preservative may be used in any suitable amount.
[0073] The terms "prevent," "preventing," or "prevention," and
other grammatical equivalents as used herein, include to keep from
developing, occur, hinder or avert a disease or condition symptoms
as well as to decrease the occurrence of symptoms. The prevention
may be complete (i.e., no detectable symptoms) or partial, so that
fewer symptoms are observed than would likely occur absent
treatment. The terms further include a prophylactic benefit. For a
disease or condition to be prevented, the compositions may be
administered to a patient at risk of developing a particular
disease, or to a patient reporting one or more of the physiological
symptoms of a disease, even though a diagnosis of this disease may
not have been made.
[0074] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 as well as all intervening decimal values
between the aforementioned integers such as, for example, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges,
"nested sub-ranges" that extend from either end point of the range
are specifically contemplated. For example, a nested sub-range of
an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to
30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20,
and 50 to 10 in the other direction. Ranges can be expressed herein
as from "about" one particular value, and/or to "about" another
particular value. When such a range is expressed, another aspect
includes from the one particular value and/or to the other
particular value. Similarly, when values are expressed as
approximations, by use of the antecedent "about," it is understood
that the particular value forms another aspect. It is further
understood that the endpoints of each of the ranges are significant
both in relation to the other endpoint, and independently of the
other endpoint. It is also understood that there are a number of
values disclosed herein, and that each value is also herein
disclosed as "about" that particular value in addition to the value
itself. It is also understood that throughout the application, data
are provided in a number of different formats and that this data
represent endpoints and starting points and ranges for any
combination of the data points. For example, if a particular data
point "10" and a particular data point "15" are disclosed, it is
understood that greater than, greater than or equal to, less than,
less than or equal to, and equal to 10 and 15 are considered
disclosed as well as between 10 and 15. It is also understood that
each unit between two particular units are also disclosed. For
example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are
also disclosed.
[0075] "Receptor interacting protein" or "RIP1" as used herein
describes a protein kinase which is a crucial regulator of cell
survival and death. RIP1 and RIP2 also bear a C-terminal domain
belonging to the death domain superfamily, allowing recruitment to
large protein complexes initiating different signaling
pathways.
[0076] As used herein, "salts" or "salt form" or "pharmaceutically
accepted salts" may include base addition salts (formed with free
carboxyl or other anionic groups) which are derived from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium,
or ferric hydroxides, and such organic bases as isopropylamine,
triethylamine, 2-ethylamino-ethanol, histidine, procaine, and the
like. Such salts are formed as acid addition salts with any free
cationic groups and generally are formed with inorganic acids such
as, for example, hydrochloric, sulfuric, or phosphoric acids, or
organic acids such as acetic, citric, p-toluenesulfonic,
methanesulfonic acid, oxalic, tartaric, mandelic, and the like.
Salts of the disclosure may include amine salts formed by the
protonation of an amino group with inorganic acids such as
hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric
acid, phosphoric acid, and the like. Salts of the disclosure also
include amine salts formed by the protonation of an amino group
with suitable organic acids, such as p-toluenesulfonic acid, acetic
acid, and the like. Additional excipients which are contemplated
for use in the practice of the present disclosure are those
available to those of ordinary skill in the art, for example, those
found in the United States Pharmacopoeia Vol. XXII and National
Formulary Vol. XVII, U.S. Pharmacopoeia Convention, Inc.,
Rockville, Md. (1989), the relevant contents of which is
incorporated herein by reference.
[0077] The "semisolid gel" according to the current disclosure is a
semisolid. The semisolid formulation apparent viscosity may
increase with concentration.
[0078] As used herein, "sequential administration" includes that
the administration of two agents (e.g., the compounds or
compositions described herein) occurs separately on the same day or
do not occur on a same day (e.g., occurs on consecutive days).
[0079] "Solution" according to the current disclosure may be a
clear, homogeneous liquid dosage form that contains one or more
chemical substances dissolved in a solvent or mixture of mutually
miscible solvents. A solution is a liquid preparation that contains
one or more dissolved chemical substances in a suitable solvent or
mixture of mutually miscible solvents. Because molecules of a drug
substance in solution are uniformly dispersed, the use of solutions
as dosage forms generally provides assurance of uniform dosage upon
administration and good accuracy when the solution is diluted or
otherwise mixed.
[0080] The term "solvent," as used herein, refers to a liquid
solvent either aqueous or non-aqueous. The selection of the solvent
depends notably on the solubility of the composition on said
solvent and on the mode of administration. Aqueous solvent may
consist solely of water, or may consist of water plus one or more
miscible solvents, and may contain dissolved solutes such as
sugars, buffers, salts or other excipients. The more commonly used
non-aqueous solvents are the short-chain organic alcohols, such as,
methanol, ethanol, propanol, short-chain ketones, such as acetone,
and poly alcohols, such as glycerol. The solvent may be present in
any suitable amount
[0081] By "subject" or "patient" is meant either a human or
non-human animal, such as a mammal. "Subject" may include any
animal, including horses, dogs, cats, pigs, goats, rabbits,
hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep,
cattle, fish, and birds. A human subject may be referred to as a
patient.
[0082] "Suspension" as used herein is a liquid dosage form that
contains solid particles dispersed in a liquid vehicle.
[0083] As used herein, the term "syndrome" may refer to a group of
symptoms that consistently occur together or a condition
characterized by a set of associated symptoms. A syndrome (e.g.,
inflammatory bowel syndrome) may be a set of medical signs and
symptoms that are correlated with each other and often, are
correlated with a specific disease. A disease, on the other hand,
may be a health condition that has a clearly defined reason behind
it. A syndrome (from the Greek word meaning `run together`)
however, may produce a number of symptoms without an identifiable
cause. They may suggest the possibility of an underlying disease or
even the chances of developing a disease.
[0084] The terms "treat," "treating" or "treatment," and other
grammatical equivalents as used herein, include alleviating,
abating, ameliorating, or preventing a disease, condition (e.g.,
inflammatory bowel disease) or symptoms, preventing additional
symptoms, ameliorating or preventing the underlying metabolic
causes of symptoms, inhibiting the disease or condition, e.g.,
arresting the development of the disease or condition, relieving
the disease or condition, causing regression of the disease or
condition, relieving a condition caused by the disease or
condition, or stopping the symptoms of the disease or condition,
and are intended to include prophylaxis. The terms further include
achieving a therapeutic benefit and/or a prophylactic benefit. By
therapeutic benefit is meant eradication or amelioration of the
underlying disorder being treated. Also, a therapeutic benefit is
achieved with the eradication or amelioration of one or more of the
physiological symptoms associated with the underlying disorder such
that an improvement is observed in the patient, notwithstanding
that the patient may still be afflicted with the underlying
disorder.
[0085] As used herein, "viscosity" refers to a fluid's resistance
to flow. Viscosity agents may be used herein and include, for
example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl
cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose,
carboxymethyl cellulose, hydroxy propyl cellulose, other agents
known to those skilled in the art, or a combination thereof.
[0086] The term "weight percent" or "% (w/w)" refers to a
percentage of a component in a solution that is calculated on the
basis of weight for the component and the solvent. For example, a
1% (w/w) solution of a component would have 1 g of the component
dissolved in a 100 g of solvent. The term "volume percent" or "%
(v/v)" refers to a percentage of a component in a solution that is
calculated on the basis of volume for the component and the
solvent. For example, a 1% (v/v) solution of a component would have
1 ml of the component dissolved in a 100 ml of solvent. The term
"weight/volume percent" or "% (w/v)" refers to a percentage of a
component in a solution that is calculated on the basis of weight
for the component and on the basis of volume for the solvent. For
example, a 1.0% (w/v) solution of a component would have 1 g of the
component dissolved in a 100 ml of solvent 2. Compounds
[0087] As discussed above, one aspect of the present invention
provides a compound represented by the following Formula 1, an
optical isomer thereof, or a pharmaceutically acceptable salt
thereof.
##STR00002##
[0088] wherein: n is 0, 1, or 2; A is -a.sup.1-, which is an amino
acid independently selected from the group consisting of alanine,
(Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid
(Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine
(Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile,
I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M),
phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S),
threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and
valine (Val, V), both terminal ends of the amino acid being coupled
to a carbonyl group or an amine group by an amide bond; and R.sup.1
is a straight chain or branched chain C.sub.1-36 alkyl, a straight
chain or branched chain C.sub.2-36 alkenyl including at least one
double bond, or a straight chain or branched chain C.sub.2-36
alkynyl including at least one triple bond.
[0089] The term "compound of the present invention", and equivalent
expressions, are meant to embrace the compound of the Formula as
hereinbefore described, which expression includes the
pharmaceutically acceptable salts, and the solvates, e.g.,
hydrates, and the solvates of the pharmaceutically acceptable salts
where the context so permits.
[0090] In accordance with some embodiments of the invention,
a.sup.1 may be
##STR00003##
and R.sup.1 may be a straight chain or branched chain C.sub.1-36
alkyl.
[0091] Non-limiting examples of the compounds include the following
compounds:
##STR00004## ##STR00005## ##STR00006## ##STR00007##
[0092] Compounds according to embodiments of the present invention
are effective for preventing or treating various diseases including
inflammatory indications, cancers, and ophthalmic indications. More
particularly, the compounds are effective for suppressing the
expression of cytokines and/or chemokines (e.g., G-CSF, IL-2, SCF,
VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta., IL-6, IL-9,
MCP-1, MIP-3.alpha., ID 2p40/70, MIG, TNF-.alpha., and VCAM-1). The
compounds are also effective for inhibiting decomposition of
I.kappa.B in inflammation signaling pathway mediated by MyD88
(myddosome complex) and/or RIP 1, thereby preventing NF-.kappa.B
from being transported into nucleus of a cell. In addition,
effective concentration of the compounds in a targeted cell/tissue
remains for a sufficient time.
[0093] 3. Preparation Methods
[0094] Another aspect of the present invention provides a method
for preparing the compound represented by Formula 1. The method, as
illustrated by the Reaction Scheme 1 shown below, comprises:
reacting a compound 2 with a compound 3 to prepare a compound 4
(step 1); hydrolyzing the compound 4 in the presence of a base to
prepare a compound 5 (step 2); reacting the compound 5 with a
compound 6 to prepare a compound 7 (step 3); hydrolyzing the
compound 7 in the presence of a base to prepare a compound 8 (step
4); reacting the compound 8 with a compound 9 to prepare a compound
10 (step 5); hydrolyzing the compound 10 in the presence of a base
to prepare the compound of Formula I (step 6).
##STR00008## ##STR00009##
[0095] wherein A, R.sup.1 and n are the same as defined in claim 1
and R.sup.2 is a straight chain or branched chain C.sub.1-5
alkyl.
[0096] In some embodiments, in the step 1, the compound 2 may be
coupled with the compound 3 in the presence of
1-ethy-3-(3-dimethylaminopropyl)carbodiimide (EDCI),
hydroxybenzotriazole (HOBt), and a base. The base can be an organic
or inorganic base. Non-limiting examples of the organic base
include pyridine, triethylamine (TEA), N,N-diisopropylethlyamine
(DIPEA), and 1,8-diazabicyclo[5.4.0]unde-7-ene (DBU). Non-limiting
examples of the inorganic base include sodium hydroxide, sodium
carbonate, potassium carbonate, cesium carbonate, and sodium
hydride. These may be used stoichiometric or excess, alone or in
combination. Non-limiting examples of the solvent that can be used
to react the compound 2 with the compound 3 include an ether (e.g.,
tetrahydrofuran (THF), dioxane, ethyl ether and
1,2-dimethoxyethane), an alcohol (e.g., methanol, ethanol,
propanol, and butanol), dimethylformamide (DMF), dimethylsulfoxide
(DMSO), dichloromethane (DCM), dichloroethane, water, acetone,
benzenesulfonate, toluensulfonate, chlorobenzenesulfonate,
xylenesulfonate, ethylacetate, phenylacetate, phenylpropionate,
phenyl butyrate, citrate, lactate, hydroxybutyrate, glycolate,
maleate, tartrate, methansulfonate, propanesulfonate,
naphthalen-1-sulfonate, naphthalen-2-sulfonate, and mandelate. The
solvent can be used alone or in combination.
[0097] The base in the step 2 can be an organic or inorganic base.
Likewise, non-limiting examples of the organic base that can be
used in the step 2 include pyridine, triethylamine,
N,N-diisopropylethlyamine (DIPEA), and
1,8-diazabicyclo[5.4.0]unde-7-ene (DBU). Non-limiting examples of
the inorganic base include sodium hydroxide, sodium carbonate,
potassium carbonate, cesium carbonate, and sodium hydride. These
may be used stoichiometric or excess, alone or in combination.
Non-limiting examples of the solvent that can be used to react the
compound 4 with the compound 5 include an ether (e.g.,
tetrahydrofuran (THF), dioxane, ethyl ether and
1,2-dimethoxyethane), an alcohol (e.g., methanol, ethanol,
propanol, and butanol), dimethylformamide (DMF), dimethylsulfoxide
(DMSO), dichloromethane (DCM), dichloroethane, water, acetone,
benzenesulfonate, toluensulfonate, chlorobenzenesulfonate,
xylenesulfonate, ethylacetate, phenylacetate, phenylpropionate,
phenyl butyrate, citrate, lactate, hydroxybutyrate, glycolate,
mandelate, tartrate, methansulfonate, propanesulfonate,
naphthalen-1-sulfonate, naphthalen-2-sulfonate, and mandelate. The
solvent can be used alone or in combination.
[0098] The step 3 and the step 5 may be performed in the manner
identical or similar to the step 1. The step 4 and the step 6 may
be performed in the manner identical or similar to the step 2.
[0099] Preparation of Compound 2
[0100] Examples of the compound 2 represented by the following
Formula 2, which is the starting material of the Reaction Scheme 1,
may be prepared by, e.g., the Preparation
[0101] Method A described below.
##STR00010##
[0102] wherein n is 0, 1, or 2; A is -a.sup.1-, which is an amino
acid independently selected from the group consisting of alanine,
(Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid
(Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine
(Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile,
I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M),
phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S),
threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and
valine (Val, V), both terminal ends of the amino acid being coupled
to a carbonyl group or an amine group by an amide bond; and R.sup.1
is a straight chain or branched chain C.sub.1-36 alkyl, a straight
chain or branched chain C.sub.2-36 alkenyl including at least one
double bond, or a straight chain or branched chain C.sub.2-36
alkynyl including at least one triple bond
[0103] [Preparation Method A]
[0104] A compound represented by the Formula a shown below is
coupled with an amino acid selected from the group consisting of
alanine, (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic
acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine
(Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile,
I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M),
phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S),
threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and
valine (Val, V) in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
hydroxybenzotriazole, and a base to form an amide bond, thereby
preparing the compound 2.
##STR00011##
[0105] (R.sup.1 is same as defined in Formula 2). 4.
Compositions/Formulations
[0106] A still another aspect of the present invention provides a
composition for preventing, improving, and treating various
diseases (e.g., inflammatory indications, cancers, and ophthalmic
indications), which composition comprises, as an active component,
at least one of the compounds, at least one of the optical isomers,
or at least one of the salts.
[0107] Compositions in accordance with some embodiments may
suppress expression of cytokines and/or chemokines including G-CSF,
IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta.,
IL-6, IL-9, MCP-1, MIP-3.alpha., IL12p40/70, MIG, TNF-.alpha., and
VCAM-1. Compositions in accordance with other embodiments may
suppress activity of NF-.kappa.B. Compositions in accordance with
other embodiments may inhibit formation of an inflammatory signal
transduction complex mediated by MyD88. Compositions in accordance
with other embodiments may inhibit formation of an inflammatory
signal transduction complex mediated by RIP1. Compositions in
accordance with other embodiments may inhibit formation of an
inflammatory signal transduction complex mediated by Pellino-1.
[0108] In some embodiments, the present invention provides a
composition for preventing, improving, and treating inflammatory
bowel disease (including closely related disorders), which
comprises, as an active component, at least one of the compounds,
at least one of the optical isomers, or at least one of the salts.
The inflammatory bowel disease may include, but are not limited to,
ulcerative colitis, Behcet's disease, and Crohn's disease. The
composition may further comprise an additive.
[0109] In some embodiments, the present invention provides a
composition for preventing, improving, or treating multiple
sclerosis, psoriasis, sepsis, geographic atrophy, wet age-related
macular disease, dry age-related macular disease, diabetic
retinopathy, infectious lung diseases, bacterial pneumonia, viral
pneumonia, diffuse large B-cell lymphoma, viral infection,
autoimmune disease, blood cancer including lymphoma, and tumors in
internal organs, which comprises, as an active component, at least
one of the compounds, at least one of the optical isomers, or at
least one of the salts.
[0110] In some embodiments, the present invention provides a
composition for preventing, improving, or treating alopecia, which
comprises, as an active component, at least one of the compounds,
at least one of the optical isomers, or at least one of the salts,
wherein the active component inhibits expression of IL-6 in scalp
and hair follicles.
[0111] The present invention embraces formulations suitable for the
administration of the compounds described herein. The compounds
described herein can be in formulations (including pharmaceutical
compositions) with additives such as excipients (e.g., one or more
excipients), antioxidants (e.g., one or more antioxidants),
stabilizers (e.g., one or more stabilizers), preservatives (e.g.,
one or more preservatives), pH adjusting and/or buffering agents
(e.g., one or more pH adjusting and/or buffering agents), tonicity
adjusting agents (e.g., one or more tonicity adjusting agents),
thickening agents (e.g., one or more thickening agents), suspending
agents (e.g., one or more suspending agents), binding agents (e.g.,
one or more binding agents), viscosity-increasing agents (e.g., one
or more viscosity-increasing agents), and the like, provided that
the additional components are pharmaceutically acceptable for the
particular condition to be treated. In some embodiments, the
formulation may include combinations of two or more of the
additional components as described herein (e.g., 2, 3, 4, 5, 6, 7,
8, or more additional components). In some embodiments, the
additives include processing agents and drug delivery modifiers and
enhancers, such as, for example, calcium phosphate, magnesium
stearate, talc, monosaccharides, disaccharides, starch, gelatin,
cellulose, methyl cellulose, sodium carboxymethyl cellulose,
dextrose, hydroxypropyl-beta-cyclodextrin, polyvinylpyrrolidinone,
low melting waxes, ion exchange resins, and the like, as well as
combinations of any two or more thereof. Other suitable
pharmaceutically acceptable excipients are described in
"Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey
(1991), and "Remington: The Science and Practice of Pharmacy,"
Lippincott Williams & Wilkins, Philadelphia, 20th edition
(2003) and 21st edition (2005), which are incorporated herein by
reference.
[0112] Formulations of the pharmaceutical compositions appropriate
for administration by any medically acceptable means are included
in the invention. The pharmaceutical formulations may comprise a
pharmaceutically acceptable carrier appropriate to the means of
administration and a pharmaceutically acceptable compound
(composition). For example, formulations of the composition
described herein may be suitable for oral administration. They can
be formed in various forms including solutions, suspensions,
semi-liquids, semi-solids, gels, emulsions, ointments, tablets, and
creams. Tablet forms can include one or more of lactose, sucrose,
mannitol, sorbitol, calcium phosphates, corn starch, potato starch,
microcrystalline cellulose, gelatin, colloidal silicon dioxide,
talc, magnesium stearate, stearic acid, and other excipients,
colorants, fillers, binders, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers.
[0113] The compositions (formulations) may be administered via many
routes including, not limited to, oral, nasal, pulmonary, rectal,
buccal, vaginal, ocular, and transdermal routes. The mode,
frequency, and effective amount of administration of the
compositions (formulations) can be decided according to methods
known in the art and/or the methods described herein (e.g., oral
administration, 0.1-1,000 mg/day, once a day). For example, they
can be administered alone or in combination. For example, they can
be concurrently administered, co-administered, and/or
intermittently administered.
[0114] 5. Methods of Using Compounds, Compositions, or
Formulations
[0115] A further aspect of the present invention provides a method
for preventing, improving, or treating various diseases (e.g.,
inflammatory indications, cancers, and ophthalmic indications),
which comprises administering to a subject in need the composition
(or compound or formulation described herein).
[0116] In an embodiment, the present invention provides a method
for preventing, improving, or treating inflammatory bowel disease,
which comprises administering the composition (or compound or
formulation described herein) to a subject in need a composition
containing, as an active component, at least one of the compounds,
at least one of the optical isomers, or at least one of the
salts.
[0117] In another embodiment, the prevent invention provides a
method for preventing, improving, or treating disease or syndrome,
which method comprises administering to a subject in need a
composition containing, as an active component, at least one of the
compounds, at least one of the optical isomers, or at least one of
the salts. The disease or syndrome may involve formation of a
Pellino-1 induced inflammatory signal transduction complex
containing MyD88, RIP1, or both. The disease or syndrome may
include, but not limited to, multiple sclerosis, psoriasis, sepsis,
geographic atrophy, wet age-related macular disease, dry
age-related macular disease, diabetic retinopathy, infectious lung
diseases, bacterial pneumonia, viral pneumonia, diffuse large
B-cell lymphoma, viral infection, autoimmune disease, blood cancer
including lymphoma, and tumors in internal organs (e.g., liver,
lung, intestine, prostate, pancreas and the like).
[0118] In still another embodiment, the prevent invention provides
a method for preventing, improving, or treating geographic atrophy,
wet age-related macular disease, dry age-related macular disease,
or diabetic retinopathy, which method comprises administering to a
subject in need a composition containing, as an active component,
at least one of the compounds, at least one of the optical isomers,
or at least one of the salts. The compound(s), the optical
isomer(s), and the salt(s) may have a pharmaceutical effect on
retinal pigment epithelium cells. In retinal pigment epithelium
cells, they may inhibit expression of at least one protein selected
from the group consisting of Nox-4, VEGF, VEGFR1, VEGFR2, Ang2, EPO
and EPOR. In retinal pigment epithelium cells, they may increase
expression of Ang 1, Tie2, or both.
EXAMPLES
[0119] The present invention will be explained in more detail with
the following examples. The examples are presented solely for the
purpose of illustration of the present invention and the present
invention will not be limited to the examples.
Example 1
Preparation of Compounds
Example 1.1
(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl-
)pyrrolidine-2-carboxamido)acetamido)propanoic acid
(Pal-PPGY-OH)
##STR00012##
[0121] Step 1: Preparation of (S)-methyl
14(S)-1-balmitovlovrrolidine-2-carbonyl)pyroliddine-2-carboxylate
[0122] A mixture solution was made by mixing
(S)-1-palmitoylpyrrolidine-2-carboxylic acid (10.0 g, 28.3 mmol)
prepared in the step 2 of Example 1.2, EDCI (5.96 g, 31.1 mmol),
HOBt (4.20 g, 31.1 mmol), and triethylamine (11.8 mL, 84.9 mmol) in
dichloromethane. Proline methyl ester hydrochloride (5.15 g, 31.1
mmol) was added to the mixture solution. The resulting mixture was
agitated at room temperature overnight, concentrated under reduced
pressure, diluted with sodium bicarbonate aqueous solution, and
extracted with ethyl acetate three times. The whole organic layer
was washed with saline solution and washed with 1N HCl three times.
The resultant was washed with saline solution, dried with anhydrous
magnesium sulfate, and concentrated under reduced pressure to
obtain (S)-methyl
1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxlate
(11.4 g, yield 87%).
[0123] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 4.69-4.65 (m, 1H),
4.54-4.58 (m, 1H), 3.83-3.93 (m, 1H), 3.58-3.72 (m,5H), 3.45-3.53
(m, 1H), 1.89-2.31 (m, 10H), 1.60-1.64 (m, 2H), 1.25 (m, 24H), 0.88
(t, J=6.87 Hz, 3H).
[0124] MS (ESI), calcd for C.sub.27H.sub.48N.sub.2O.sub.4464.4,
found m/z465.2 (M+H.sup.+).
[0125] Step 2: Preparation of (S)-1-((S)-1-balmitovlovrrolidine
-2-carbonyl)pyrrolidine-2-carboxylic acid
[0126] (S)-methyl
1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxlate
(15.0 g, 32.3 mmol) prepared in the step 1 was mixed with
tetrahydrofuran. Sodium hydroxide (2.58 g, 64.6 mmol) aqueous
solution was added to the mixture solution. The resulting mixture
was agitated at room temperature overnight and concentrated. 1N HCl
was added to adjust the pH to 1.0. The aqueous layer thereof was
extracted with ethyl acetate three times. The whole organic layer
was dried with anhydrous magnesium sulfate and concentrated to
obtain
(S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic
acid (13.2 g, yield 91%) as white solid.
[0127] MS (ESI), calcd for C.sub.26H.sub.46N.sub.2O.sub.4450.3,
found m/z451.1 (M+H.sup.+).
[0128] Step 3: Preparation of ethyl
2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido-
)acetate
[0129]
(S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxyl-
ic acid (12 g, 26.6 mmol) prepared in the step 2 was mixed with
dichloromethane. Glycine ethyl ester hydrochloride (4.09 g, 29.3
mmol), EDCI (5.62 g, 29.3 mmol), HOBt (3.96 g, 29.3 mmol) and
triethyamine (11.1 mL, 79.8 mmol) were added to the mixture
solution. The resulting mixture was agitated at room temperature
overnight, concentrated under reduced pressure, diluted with sodium
carbonate aqueous solution, and extracted with ethyl acetate three
times. The whole organic layer was washed with saline solution and
washed with 1N HCl three times. The organic layer was washed with
saline solution, dried with anhydrous magnesium sulfate, and
concentrated to obtain ethyl
2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxyamid-
o)acetate (11.1 g, yield 78%).
[0130] MS (ESI), calcd for C.sub.30H.sub.53N.sub.3O.sub.5535.4,
found m/z536.5 (M+H.sup.+).
[0131] Step 4: Preparation of
2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido-
)acetic acid
[0132] Ethyl
2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxyamid-
o)acetate (12 g, 22.4 mmol) prepared in the step 3 was mixed with
tetrahydrofuran. Sodium hydroxide (1.79 g, 44.8 mmol) aqueous
solution was added to the mixture solution. The resulting mixture
was agitated at room temperature overnight and concentrated. 1N HCl
was added to adjust the pH to 1.0. The aqueous layer was extracted
with ethyl acetate three times. The whole organic layer was dried
with anhydrous magnesium sulfate and concentrated to obtain
2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido-
)acetic acid (9.5 g, yield 84%).
[0133] MS (ESI), calcd for C.sub.28H.sub.49N.sub.3O.sub.5507.4,
found m/z508.2 (M+H.sup.+).
[0134] Step 5: Preparation of (S)-methyl
3-(4-hydroxyphenyl)-2-(24(S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyr-
rolidine-2-carboxamido)acetamido)propanoate
[0135]
2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carbo-
xamido)acetic acid (10 g, 19.7 mmol) prepared in the step 4 was
mixed with dichloromethane. Tyrosine methyl ester (4.23 g, 21.7
mmol), EDCI (4.16 g, 21.7 mmol), HOBt (21.7 g, 21.7 mmol), and
triethylamine (8.19 mL, 59.1 mmol) were added to the mixture
solution. The resulting mixture was agitated at room temperature
overnight, concentrated under reduced pressure, diluted with sodium
bicarbonate aqueous solution, and extracted with ethyl acetate
three times. The whole organic layer was washed with saline
solution and washed with 1N HCl three times. The organic layer was
washed with saline solution, dried with anhydrous magnesium
sulfate, concentrated, and purified with MPLC to obtain (S)-methyl
3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)py-
rrolidine-2-carboxamido)acetamido)propanoate (8.4 g, yield
62%).
[0136] MS (ESI), calcd for C.sub.37H.sub.60N.sub.4O.sub.7684.4,
found m/z685.2 (M+H.sup.+).
[0137] Step 6: Preparation of
(S)-3-(4-hydroxyphenyl)-2-(24(S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl-
)pyrrolidine-2-carboxamido)acetamido)propanoic acid
[0138] (S)-methyl
3-(4-hydroxyphenyI)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)py-
rrolidine-2-carboxamido)acetamido)propanoate (4.9 g, 7.16 mmol)
prepared in the step 5 was mixed with tetrahydrofuran. Sodium
hydroxide (0.86 g, 21.5 mmol) aqueous solution was added to the
mixture solution. The resulting mixture was agitated at room
temperature overnight and concentrated. 1N HCl was added to adjust
the pH to 1.0. The aqueous layer was extracted with ethyl acetate
three times. The whole organic layer was dried with anhydrous
magnesium sulfate and concentrated to obtain
(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbony-
l)pyrrolidine-2-carboxamido)acetamido)propanoic acid (4.5 g, yield
93%).
[0139] .sup.1H-N MR (300 MHz, MeOD) .delta. 7.04 (d, J=8.31 Hz, 2H)
6.70 (d, J=8.37 Hz,2H), 4.38-4.68 (m,3H), 3.44-4.04 (m, 6H),
2.91-3.13 (m, 2H), 1.81-2.38 (m, 10H), 1.54-1.60 (m, 2H), 1.30 (m,
24H), 0.88 (t, J=6.63 Hz, 3H).
[0140] MS (ESI), calcd for C.sub.37H.sub.58N.sub.4O.sub.7670.4,
found m/z671.3 (M+H.sup.+).
Example 1.2
(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)ace-
tamido)propanoic acid (Pal-PGY-OH)
##STR00013##
[0142] Step 1: (S)-methyl 1-palmitoylpyrrolidine-2-carboxylate
[0143] Palmitic acid (7 g, 27.3 mmol), EDCI (5.78 g, 30.0 mmol),
HOBt (4.05 g, 30.0 mmol), and, triethylamine (11.4 mL, 81.9 mmol)
were mixed with dichloromethane. Proline methyl ester hydrochloride
(4.97 g, 30.0 mmol) was added to the mixture solution. The
resulting mixture was agitated at room temperature overnight,
concentrated under reduced pressure, diluted with sodium carbonate
aqueous solution, and extracted with ethyl acetate three times. The
whole organic layer was washed with saline solution and washed with
1N HCl three times. The organic layer was washed with saline
solution, dried with anhydrous magnesium sulfate, and concentrated
under reduced pressure to obtain (S)-methyl
1-palmitoylpyrrolidine-2-carboxylate (9.6 g, yield 96%) as viscous
liquid.
[0144] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 4.46-4.50 (m, 1H),
3.47-3.75 (m,5H), 1.90-2.36 (m, 6H), 1.59-1.69 (m, 2H), 1.25 (m,
24H), 0.88 (t, J=6.84 Hz, 3H)
[0145] MS (ESI), calcd for C.sub.22H.sub.41NO.sub.3367.3, found
m/z368 (M+H.sup.+).
[0146] Step 2: Preparation of
(S)-1-palmitoylpyrrolidine-2-carboxylic acid
[0147] (S)-methyl 1-palmitoylpyrrolidine-2-carboxylate (10.0 g,
27.2 mmol) prepared in the step 1 was mixed with tetrahydrofuran.
Sodium hydroxide (3.26 g, 81.6 mmol) aqueous solution was added to
the mixture solution. The resulting mixture was agitated at room
temperature overnight and concentrated. 1N HCl was added to adjust
the pH to 1.0. The aqueous layer was extracted with ethyl acetate
three times. The whole organic layer was dried with anhydrous
magnesium sulfate and concentrated to obtain
(S)-1-palmitoylpyrrolidine-2-carboxylic acid (8.6 g, yield 89%) as
white solid.
[0148] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 4.59-62 (m, 1H),
3.42-3.59 (m,2H), 2.46-2.53 (m, 1H), 2.33-2.38 (m, 2H), 1.93-2.01
(m, 3H), 1.62-1.69 (m, 2H), 1.25 (m, 24H), 0.88 (t, J=6.90 Hz,
3H)
[0149] MS (ESI), calcd for C.sub.21H.sub.39NO.sub.3353.3, found
m/z354.2 (M+H.sup.+).
[0150] Step 3: preparation of (S)-ethyl
2-(1-palmitoylpyrrolidine-2-carboxamido)acetate
[0151] (S)-1-palmitoylpyrrolidine-2-carboxylic acid (10 g, 28.3
mmol) prepared in the step 2, glycine ethyl ester hydrochloride
(4.34 g, 31.1 mmol), EDCI (5.76 g,31.1 mmol), HOBt (4.20 g,31.1
mmol), and triethyamine (1,5.7 mL, 113 mmol) were mixed with
dichloromethane. The resulting mixture was agitated at room
temperature overnight, concentrated under reduced pressure, diluted
with sodium carbonate aqueous solution, and extracted with ethyl
acetate three times. The whole organic layer was washed with saline
solution and washed with 1N HCl three times. The organic layer was
washed with saline solution, dried with anhydrous magnesium
sulfate, and concentrated to obtain (S)-ethyl
2-(1-palmitoylpyrrolidine-2-carboxamido)acetate (10.7 g, yield
86%).
[0152] MS (ESI), calcd for C.sub.29H.sub.46N.sub.2O.sub.4438.3,
found m/z439.1 (M+H.sup.+).
[0153] Step 4: Preparation of
(S)-2-(1-palmitoylpyrrolidine-2-carboxamido)acetic acid
[0154] (S)-ethyl 2-(1-palmitoylpyrrolidine-2-carboxamido)acetate
(12 g, 27.4 mmol) prepared in the step 3 was mixed with
tetrahydrofuran. Sodium hydroxide (2.20 g, 54.7 mmol) aqueous
solution was added to the mixture solution. The resulting mixture
was agitated at room temperature overnight and concentrated. 1N HCl
was added to adjust the pH to 1.0. The aqueous layer was extracted
with ethyl acetate three times. The whole organic layer was dried
with anhydrous magnesium sulfate and concentrated to obtain
(S)-2-(1-palmitoylpyrrolidine-2-carboxamido)acetic acid (10.2 g,
yield 91%) as white solid.
[0155] MS (ESI), calcd for C.sub.23H.sub.42N.sub.2O.sub.4410.3,
found m/z 411.3 (M+H.sup.+).
[0156] Step 5: Preparation of (S)-methyl
3-(4-hydroxyphenyl)-2-(24(S)-1-palmitoylpyrrolidine-2-carboxamido)acetami-
do)propanoate
[0157] (S)-2-(1-palmitoylpyrrolidine-2-carboxamido)acetic acid (7
g, 27.3 mmol) prepared in the step 4 was mixed with
dichloromethane. Tyrosine methyl ester (5.86 g, 30.0 mmol), EDCI
(5.78 g, 30.0 mmol), HOBt(4.05 g, 30.0 mmol), and triethylamine
(11.4 mL, 81.9 mmol) were added to the mixture solution. The
resulting mixture was agitated at room temperature overnight,
concentrated under reduced pressure, diluted with sodium
bicarbonate aqueous solution, and extracted with ethyl acetate
three times. The whole organic layer was washed with saline
solution and washed with 1N HCl three times. The organic layer was
washed with saline solution, dried with anhydrous magnesium
sulfate, concentrated, and purified with MPLC to obtain (S)-methyl
3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetam-
ido)propanoate (9.8 g, yield 61%).
[0158] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 7.25-7.50 (m, 3H),
6.93 (d, J=8.34 Hz, 2H) 6.70 (d, J=8.34 Hz,2H), 4.70-4.77 (m, 1H),
4.39-4.43 (m, 1H), 3.95-4.21 (m, 1H), 3.41-3.72 (m, 5H), 2.92-3.12
(m, 2H), 1.91-2.35 (m, 7H), 1.57-1.61 (m, 2H), 1.25 (m, 24H), 0.88
(t, J=6.87 Hz, 3H)
[0159] MS (ESI), calcd for C.sub.33H.sub.53N.sub.3O.sub.6587.4,
found m/z588.1 (M+H.sup.+).
[0160] Step 6: Preparation of
(S)-3-(4-hydroxyphenyl)-2-(24(S)-1-palmitoylpyrrolidine
-2-carboxamido)acetamido)propanoic acid
[0161] (S)-methyl
3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetam-
ido)propanoate (2.85 g, 4.85 mmol) prepared in the step 5 was mixed
with tetrahydrofuran. Sodium hydroxide (0.58 g, 14.6 mmol) aqueous
solution was added to the mixture solution. The resulting mixture
was agitated at room temperature overnight and concentrated. 1N HCl
was added to adjust the pH to 1.0. The aqueous layer was extracted
with ethyl acetate three times. The whole organic layer was dried
with anhydrous magnesium sulfate and concentrated to obtain
(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)ac-
etamido)propanoic acid (2.2 g, yield 79%) as white solid.
[0162] .sup.1H-NMR (300 MHz, MeOD) .delta. 7.03 (d, J=8.40 Hz,2H)
6.70 (d, J=8.40 Hz,2H), 4.58-4.61 (m,1H), 4.33-4.56 (m, 1H),
3.58-4.37 (m, 4H), 2.96-3.15 (m, 2H), 1.92-2.39 (m, 6H), 1.55-1.62
(m, 2H), 1.29 (m, 24H), 0.91 (t, J=6.87 Hz, 3H)
[0163] MS (ESI), calcd for C.sub.32H.sub.51N.sub.3O.sub.6573.4,
found m/z574.2(M+H.sup.+).
Example 1.3
Palmitoyl-L-alanyl-L-prolylglycyl-L-tyrosine (pal-APGY-OH)
[0164] The compound was prepared according to the following
Reaction Scheme 2.
##STR00014## ##STR00015##
[0165] The compound (1) (10 g, 46.5 mmol), the compound (2) (7.15
g, 51.2 mmol), EDCl.HCl (9.82 g, 51.2 mmol), HOBt (6.92 g, 51.2
mmol), and triethylamine (19.4 mL, 140 mmol) were mixed with
dichloromethane. The resulting mixture was agitated at room
temperature overnight, concentrated under reduced pressure, diluted
with sodium carbonate aqueous solution, and extracted with ethyl
acetate three times. The whole organic layer was washed with saline
solution and washed with 1N HCl three times. The organic layer was
washed with saline solution, dried with anhydrous magnesium
sulfate, and concentrated under reduced pressure to obtain the
compound (3) (yield 91%) as viscous liquid.
[0166] LC-MS (ESI): calcd for C.sub.14H.sub.24N.sub.2O.sub.5 300.2,
found m/z 301.2 (M+H.sup.+).
[0167] The compound (3) (12 g, 40 mmol) was mixed with
tetrahydrofuran. Sodium hydroxide (6.40 g, 160 mmol) aqueous
solution was added to the mixture solution. The resulting mixture
was agitated at room temperature overnight and concentrated. 1N HCl
was added to adjust the pH to 1.0. The aqueous layer was extracted
with ethyl acetate three times. The whole organic layer was dried
with anhydrous magnesium sulfate and concentrated to obtain the
compound (4) (yield 96%) as white solid.
[0168] LC-MS (ESI): calcd for C.sub.12H.sub.20N.sub.2O.sub.5 272.1,
found m/z 273.1 (M+H.sup.+).
[0169] The compound (4) (6.25 g, 23 mmol), the compound (5) (4.85
g, 25.3 mmol), EDCI.HCl (4.85 g, 25.3 mmol), HOBt (43.42 g, 25.3
mmol), and triethylamine (TEA, 12.8 mL, 96 mmol) were mixed with
dichloromethane. The resulting mixture was agitated at room
temperature overnight, concentrated under reduced pressure, diluted
with sodium carbonate aqueous solution, and extracted with ethyl
acetate three times. The whole organic layer was washed with
sodiumbicarbonate aqueous solution twice, washed with saline
solution, and washed with 1N HCl three times. The organic layer was
washed with saline solution, dried with anhydrous magnesium
sulfate, and concentrated to obtain the compound (6) (yield 87%) as
white solid.
[0170] LC-MS (ESI): calcd for C.sub.22H.sub.31 N.sub.3O.sub.7
449.2, found m/z 450.2 (M+H.sup.+).
[0171] The compound (6) (8 g, 17.8 mmol) was dissolved in ethyl
acetate. An excess amount of 4 N HCl in dioxane was added at room
temperature. The resulting mixture was agitated at room temperature
for 4 hours and concentrated under reduced pressure to obtain the
compound (7) as white solid.
[0172] LC-MS (ESI): calcd for C.sub.17H.sub.23N.sub.3O.sub.5 349.2,
found m/z 350.2 (M+H.sup.+).
[0173] The compound (7) (0.25 g, 0.65 mmol), the compound (8)
(Boc-alanine, 0.12 g, 0.65 mmol), EDCI.HCl (0.25 g, 1.30 mmol),
HOBt (0.18 g, 1.30 mmol), and triethylamine (0.36 mL, 2.60 mmol)
were mixed with dichlromethane. The resulting mixture was agitated
at room temperature overnight, concentrated under reduced pressure,
diluted with sodium carbonate aqueous solution, and extracted with
ethyl acetate three times. The whole organic layer was washed with
sodium bicarbonate aqueous solution twice, washed with saline
solution, and washed with 1N HCl three times. The organic layer was
washed with saline solution, dried with anhydrous magnesium
sulfate, concentrated, and purified by using MPLC
(dichloromethane/2-propanol) to obtain the compound (9) (yield
3%).
[0174] LC-MS (ESI): calcd for C.sub.25H.sub.36N.sub.4O.sub.8 520.3,
found m/z 520.7 (M+H.sup.+).
[0175] The compound (9) (0.11 g, 0.21 mmol) was dissolved in ethyl
acetate. An excess amount of 4 N HCl in dioxane was added at room
temperature and agitated at room temperature for 4 hours. The
resulting mixture was concentrated under reduced pressure to obtain
the compound (10) as white solid.
[0176] LC-MS (ESI): calcd for C.sub.20H.sub.28N.sub.4O.sub.6 420.2,
found m/z 420.6 (M+H.sup.+).
[0177] To a solution of palmitic acid (0.02 g, 0.08 mmol) in
dichloromethane was added compound (10) (0.04 g, 0.09 mmol),
EDCI.HCl (0.03 g, 0.16 mmol), HOBt (0.02 g, 0.16 mmol), and
triethylamine (0.04 mL, 0.32 mmol). The resultant was was agitated
at room temperature overnight, concentrated under reduced pressure,
diluted with sodium carbonate aqueous solution, and extracted with
ethyl acetate three times. The whole organic layer was washed with
saline solution, and washed with 1N HCl three times. The organic
layer was washed with saline solution, dried with anhydrous
magnesium sulfate, concentrated, and purified by using MPLC
(dichloromethane/2-propanol) to obtain the compound (11) (yield
58%).
[0178] LC-MS (ESI): calcd for C.sub.36H.sub.58N.sub.4O.sub.7 658.4,
found m/z 659.1 (M+H.sup.+)
[0179] The compound (11) (0.03 g, 0.05 mmol) was mixed with
tetrahydrofuran. Sodium hydroxide (0.008 g, 0.20 mmol) aqueous
solution was added. The resulting mixture was agitated at room
temperature overnight and concentrated. 1N HCl was added to adjust
the pH to 1.0. The aqueous layer was extracted with ethyl acetate
three times. The whole organic layer was dried with anhydrous
magnesium sulfate and concentrated to obtain the compound (12)
(yield 93%) as white solid.
[0180] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.05 (d, J=8.50
Hz, 2H), 6.70 (d, J=8.50 Hz, 2H), 4.59-4.65 (m, 2H), 4.39-4.41 (m,
1H), 3.96-3.99 (m, 1H), 3.84-3.89 (m, 1H), 3.65-3.76 (m, 2H),
3.10-3.14 (m, 1H), 2.97-3.01 (m, 1H), 2.20-2.24 (m, 3H), 2.09-2.14
(m, 1H), 1.96-2.03 (m, 2H), 1.58-1.60 (m, 3H), 1.31-1.36 (m, 30H),
0.92 (t, J=7.15 Hz, 3H). LC-MS (ESI): calcd for
C.sub.35H.sub.56N.sub.4O.sub.7 644.4, found m/z 644.6
(M+H.sup.+).
[0181] Compounds of Examples 1.4 through 1.15 were prepared in the
same manner as described in Example 1.3, using the compound (7) as
the starting material. NMR data of those compounds are shown
below.
##STR00016##
Example 1.4
[0182] Palmitoylglycyl-L-prolylglycyl-L-tyrosine (pal-GPGY-OH)
.sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.03 (d, J=8.50 Hz, 2H),
6.70 (d, J=8.50 Hz, 2H), 4.58-4.63 (m, 1H), 4.39-4.42 (m, 1H),
3.90-4.08 (m, 4H), 3.59-3.74 (m, 3H), 3.09-3.12 (m, 1H), 2.96-3.00
(m, 1H), 1.99-2.31 (m, 7H), 1.56-1.66 (m, 3H), 1.24-1.35 (m, 26H),
0.92 (t, J=7.05 Hz, 3H). LC-MS (ESI): calcd for
C.sub.34H.sub.54N.sub.4O.sub.7 630.4, found m/z 630.8
(M+H.sup.+).
##STR00017##
Example 1.5
[0183]
((9Z,12Z)-octadeca-9,12-dienoyl)glycyl-L-prolylglycyl-L-tyrosine
(linoleyl-GPGY-OH) .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.03
(d, J=8.50 Hz, 2H), 6.71 (d, J=8.50 Hz, 2H), 5.31-5.41 (m, 4H),
4.58-4.62 (m, 1H), 4.39-4.42 (m, 1H), 3.99-4.08 (m, 2H), 3.70-3.73
(m, 1H), 3.59-3.67 (m, 1H), 3.09-3.12 (m, 1H), 2.96-3.00 (m, 1H),
2.78-2.79 (m, 3H), 2.25-2.31 (m, 1H), 2.18-2.22 (m, 2H), 1.99-2.13
(m, 7H), 1.56-1.64 (m, 3H), 1.31-1.42 (m, 18H), 0.93 (t, J=7.10 Hz,
3H). LC-MS (ESI): calcd for C.sub.36H.sub.54N.sub.4O.sub.7 654.4,
found m/z 655 (M+H.sup.+).
##STR00018##
Example 1.6
Palmitoyl-L-phenylalanyl-L-prolylglycyl-L-tyrosine
(pal-FPGY-OH)
[0184] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.27-7.28 (m, 5H),
7.07 (d, J=8.45 Hz, 2H), 6.71 (d, J=8.30 Hz, 2H), 4.59-4.63 (m,
1H), 4.40-4.42 (m, 1H), 3.99-4.02 (m, 1H), 3.85-3.89 (m, 1H),
3.73-3.78 (m, 1H), 3.52-3.55 (m, 1H), 3.09-3.16 (m, 2H), 2.86-3.02
(m, 3H), 1.95-2.22 (m, 8H), 1.44-1.62 (m, 4H), 1.31 (m, 25H), 0.92
(t, J=7.10 Hz, 3H). LC-MS (ESI): calcd for
O.sub.41H.sub.60N.sub.4O.sub.7 720.4, found m/z 721.1
(M+H.sup.+).
##STR00019##
Example 1.7
Hexanoyl-L-prolyl-L-prolylglycyl-L-tyrosine (hexanoyl-PPGY-OH)
[0185] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.05 (d, J=8.50
Hz, 2H), 6.70 (d, J=8.50 Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m,
1H), 4.40-4.44 (m, 1H), 3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H),
3.73-3.76 (m, 1H), 3.51-3.68 (m, 3H), 3.08-3.12 (m, 1H), 2.97-3.02
(m, 1H), 2.31-2.41 (m, 2H), 2.20-2.29 (m, 2H), 1.92-2.12 (m, 5H),
(m, 8H), 1.57-1.66 (m, 3H), 1.31-1.38 (m, 5H), 0.93 (t, J=7.00 Hz,
3H). LC-MS (ESI): calcd for C.sub.27H.sub.38N.sub.4O.sub.7 530.3,
found m/Z 530.7 (M+H.sup.+).
##STR00020##
Example 1.8
Octanoyl-L-prolyl-L-prolylglycyl-L-tyrosine (octanoyl-PPGY-OH)
[0186] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.05 (d, J=8.50
Hz, 2H), 6.71 (d, J=8.50 Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m,
1H), 4.40-4.42 (m, 1H), 3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H),
3.73-3.77 (m, 1H), 3.51-3.68 (m, 3H), 3.08-3.12 (m, 1H), 2.97-3.02
(m, 1H), 2.18-2.34 (m, 4H), 1.92-2.12 (m, 5H), 1.57-1.61 (m, 2H),
1.32-1.35 (m, 10H), 0.92 (t, J=7.00 Hz, 3H). LC-MS (ESI): calcd for
C.sub.29H.sub.42N.sub.4O.sub.7 558.3, found m/z 558.5
(M+H.sup.+).
##STR00021##
Example 1.9
Decanoyl-L-prolyl-L-prolylglycyl-L-tyrosine (decanoyl-PPGY-OH)
[0187] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.03 (d, J=8.55
Hz, 2H), 6.71 (d, J=8.40 Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m,
1H), 4.40-4.42 (m, 1H), 3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H),
3.73-3.77 (m, 1H), 3.51-3.68 (m, 3H), 3.08-3.12 (m, 1H), 2.97-3.02
(m, 1H), 2.18-2.39 (m, 5H), 1.92-2.14 (m, 6H), 1.57-1.61 (m, 2H),
1.32-1.36 (m, 14H), 0.92 (t, J=7.15 Hz, 3H). LC-MS (ESI): calcd for
O.sub.31H.sub.46N.sub.4O.sub.7 586.3, found m/z 586.8
(M+H.sup.+).
##STR00022##
Example 1.10
Stearoyl-L-prolyl-L-prolylglycyl-L-tyrosine (stearoyl-PPGY-OH)
[0188] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.03 (d, J=8.55
Hz, 2H), 6.71 (d, J=8.25 Hz, 2H), 4.66-4.68 (m, 1H), 4.55-4.57 (m,
1H), 4.40-4.44 (m, 1H), 3.96-4.02 (m, 1H), 3.84-3.89 (m, 1H),
3.71-3.76 (m, 1H), 3.48-3.68 (m, 4H), 3.08-3.12 (m, 1H), 2.97-3.02
(m, 1H), 2.14-2.42 (m, 5H), 1.90-2.14 (m, 6H), 1.57-1.61 (m, 2H),
1.32-1.35 (m, 30H), 0.92 (t, J=7.15 Hz, 3H). LC-MS (ESI): calcd for
C.sub.39H.sub.62N.sub.4O.sub.7 698.5, found m/z 698.5
(M+H.sup.+).
##STR00023##
Example 1.11
Hex-5-enoyl-L-prolyl-L-prolylglycyl-L-tyrosine
(5-hexenoyl-PPGY-OH)
[0189] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.05 (d, J=8.55
Hz, 2H), 6.70 (d, J=8.25 Hz, 2H), 5.78-5.89 (m, 1H), 4.98-5.08 (m,
3H), 4.66-4.69 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.44 (m, 1H),
3.97-4.01 (m, 1H), 3.84-3.90 (m, 1H), 3.73-3.76 (m, 1H), 3.47-3.68
(m, 4H), 3.08-3.12 (m, 1H), 2.97-3.01 (m, 1H), 2.33-2.42 (m, 2H),
2.20-2.30 (m, 2H), 2.06-2.15 (m, 4H), 1.92-2.04 (m, 4H), 1.67-1.76
(m, 3H). LC-MS (ESI): calcd for C.sub.27H.sub.36N.sub.4O.sub.7
528.3, found m/z 529 (M+H.sup.+).
##STR00024##
Example 1.12
[0190] Oleoyl-L-prolyl-L-prolylglycyl-L-tyrosine (oleyl-PPGY-OH)
.sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.03 (d, J=8.50 Hz, 2H),
6.71 (d, J=8.50 Hz, 2H), 5.34-5.38 (m, 2H), 4.66-4.68 (m, 1H),
4.55-4.57 (m, 1H), 4.40-4.42 (m, 1H), 3.96-4.01 (m, 1H), 3.84-3.90
(m, 1H), 3.73-3.77 (m, 1H), 3.53-3.69 (m, 4H), 3.08-3.12 (m, 1H),
2.97-3.01 (m, 1H), 2.18-2.39 (m, 5H), 2.06-2.12 (m, 2H), 1.92-2.05
(m, 3H), 1.58-1.61 (m, 3H), 1.31-1.35 (m, 25H), 0.92 (t, J=7.0 Hz,
3H). LC-MS (ESI): calcd for C.sub.39H.sub.60N.sub.4O.sub.7 696.4,
found m/z 697.3 (M+H.sup.+).
##STR00025##
[0191] Example 1.13
[0192]
((9Z,12Z)-octadeca-9,12-dienoyl)-L-prolyl-L-prolylglycyl-L-tyrosine
(linoleyl-PPGY-OH) .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.05
(d, J=8.45 Hz, 2H), 6.71 (d, J=8.45 Hz, 2H), 5.32-5.41 (m, 4H),
4.66-4.68 (m, 1H), 4.55-4.57 (m, 1H), 4.40-4.43 (m, 1H), 3.96-4.01
(m, 1H), 3.84-3.89 (m, 1H), 3.73-3.77 (m, 1H), 3.53-3.68 (m, 4H),
3.08-3.12 (m, 1H), 2.97-3.01 (m, 1H), 2.80 (t, J=6.40 Hz, 2H),
2.19-2.39 (m, 5H), 1.93-2.13 (m, 10H), 1.59-1.61 (m, 3H), 1.31-1.40
(m, 15H), 0.92 (t, J=6.59 Hz, 3H). LC-MS (ESI): calcd for
C.sub.39H.sub.58N.sub.4O.sub.7 694.4, found m/z 695.2
(M+H.sup.+).
##STR00026##
Example 1.14
Palmitoyl-L-valyl-L-prolyl-L-prolylglycyl-L-tyrosine
(Pal-VPPGY-OH)
[0193] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.04 (d, J=8.35
Hz, 2H), 6.71 (d, J=8.35 Hz, 2H), 4.67-4.70 (m, 1H), 4.52-4.58 (m,
1H), 4.39-4.43 (m, 1H), 3.77-4.05 (m, 4H), 3.64-3.73 (m, 2H),
3.49-3.60 (m, 1H), 3.01-3.11 (m, 2H), 1.87-2.31 (m, 15H), 1.61-1.63
(m, 3H), 1.31 (m, 24H), 1.01 (d, J=6.58 Hz, 3H), 0.98 (d, J=6.58
Hz, 3H), 0.92 (t, J=6.96 Hz, 3H). LC-MS (ESI), calcd for
C.sub.42H.sub.67N.sub.5O.sub.8 769.5, found m/z 770.7
(M+H.sup.+).
##STR00027##
Example 1.15
Decanoyl-L-valyl-L-prolyl-L-prolylglycyl-L-tyrosine
(decanoyl-VPPGY-OH)
[0194] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 7.05 (d, J=8.45
Hz, 2H), 6.71 (d, J=8.45 Hz, 2H), 4.67-4.70 (m, 1H), 4.50-4.57 (m,
1H), 4.39-4.43 (m, 1H), 3.95-4.05 (m, 2H), 3.80-3.91 (m, 1H),
3.65-3.70 (m, 2H), 3.64-3.73 (m, 2H), 3.53-3.57 (m, 1H), 3.01-3.10
(m, 2H), 1.89-2.31 (m, 12H), 1.61-1.63 (m, 3H), 1.31 (m, 24H), 1.01
(d, J=6.68 Hz, 3H), 0.97 (d, J=6.55 Hz, 3H), 0.92 (t, J=7.05 Hz,
3H). LC-MS (ESI), calcd for C.sub.36H.sub.55N.sub.5O.sub.8 685.4,
found m/z 686.6 (M+H.sup.+).
[0195] Table 1 depicts the compounds according to Examples 1.1
through 1.15.
TABLE-US-00001 TABLE 1 Compounds according to Examples 1.1 through
1.15 Example No. (Compound No.) Chemical Formula 1.1 ##STR00028##
Pal-PPGY-OH 1.2 ##STR00029## Pal-PGY-OH 1.3 ##STR00030##
Pal-APGY-OH 1.4 ##STR00031## pal-GPGY-OH 1.5 linoleyl-GPGY-OH 1.6
##STR00032## pal-FPGY-OH 1.7 hexanoyl-PPGY-OH 1.8 octanoyl-PPGY-OH
1.9 ##STR00033## decanoyl-PPGY-OH 1.10 ##STR00034##
stearoyl-PPGY-OH 1.11 ##STR00035## 5-hexenoyl-PPGY-OH 1.12
##STR00036## oleyl-PPGY-OH 1.13 ##STR00037## linoleyl-PPGY-OH 1.14
##STR00038## Pal-VPPGY-OH 1.15 ##STR00039## decanoyl-VPPGY-OH
##STR00040## indicates data missing or illegible when filed
Example 2
Assays
Example 2.1
Suppression of Expression of IL-6
[0196] To evaluate suppression of expression of IL-6 by the
compounds of the present invention, the following experiment was
performed.
[0197] First, RAW 264.7 macrophage cells were purchased from
American Type Culture Collection (ATCC; Manassas, Va.), and were
culture at a temperature of 37.degree. C., under 5% CO.sub.2
atmosphere, using 10% Fetal Bovine Serum (FBS) and Dulbecco's
Modified Eagle's Medium (DMEM) containing 1%
penicillin/streptomycin.
[0198] The cultured RAW 264.7 macrophage cells were divided into a
6-well cell culture plate. After 24 hours, the cells were
pretreated with compound 1.1, compound 1.2, and smaducin-6,
respectively, in a concentration of 100 nM for 30 min, and further
treated with lipopolysaccharides (LPS) for two hours. These cells
were collected using TRIzol.RTM. and the RNA was extracted. From 2
.mu.g of the extracted RNA, cDNAs (complementary deoxyribonucleic
acids) were synthesized and reverse transcription polymerase chain
reactions (RT-PCR) and real-time polymerase chain reactions
(Real-Time PCR) were performed. Samples from the RT-PCR were
confirmed by electrophoresis on agarose gels and quantified using
densitometer. A glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
gene was used as a loading control for the interleukin-6 gene.
[0199] FIG. 1A shows images depicting suppression of expression of
interleukin-6 by compound 1.1 and compound 1.2, and FIG. 1B is a
graph showing the quantified suppression of expression of
interleukin-6 by the compound 1.1 and compound 1.2. As compared to
the untreated sample, when the cells treated with the compound
according to the present invention, the expression of
interleukin-6, which was induced by LPS treatment, was
suppressed.
[0200] In addition, RAW 264.7 macrophage cells cultured as
described above were also divided into a 6-well plate, after 24
hours, were pretreated with 100 nM of compound 1.1 for 30 minutes,
and further treated with LPS for two hours. The above cell culture
was collected and, using a cytokine array (Mouse cytokine array C3,
RayBiotech), changes in amounts of cytokine and chemokine were
quantified by a densitometer. The cytokines and chemokines detected
by the compound of the present invention included G-CSF, IL-2, SCF,
VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1.alpha., IL-1.beta., IL-6, IL-9,
MCP-1, MIP-3.alpha., ID 2p40/70, MIG, TNF-.alpha., and VCAM-1. As
compared to the untreated samples, when the cells were treated with
the compounds of the present invention, the expression of the above
cytokines and chemokines, which were induced by LPS treatment, were
suppressed as statistically meaningful (FIGS. 2A-2D).
[0201] RAW 264.7 macrophage cells were treated with varying
concentrations of compound 1.1 from 50 pM to 500 nM for 30 minutes,
and treated with100 ng/mL of LPS for two hours. The induced
expression of interleukin-6 was quantified as described above and
the results were presented in FIG. 3. In the RAW 264.7 macrophage
cells, the concentration of compound 1.1 where the expression of
interleukin-6 was suppressed to 50% or below (i.e. IC.sub.50) was
about 1.6 nM.
Example 2.2
Suppression of Activity of NF-.kappa.B
[0202] To evaluate whether NF-.kappa.B signal induced by LPS was
specifically suppressed by the compounds of the present invention,
the following experiments were performed.
[0203] Specifically, 5.times.NF-.kappa.B-Luc reporter plasmid was
transfected into RAW 264.7 cells using Effectene (Qiagen, USA). The
transfected cells were pretreated with 100 nM of compound 1.1 for
30 minutes, and further treated with LPS (100 ng/ml) for two hours,
and luciferase activity in the cells was measured (FIG. 4 depicts a
graph showing relative inhibition activities of compound 1.1 and
compound 1.2 to NF-.kappa.B activation). When the cells were
treated with compound 1.1 and compound 1.2 according to the present
invention, activation of NF-.kappa.B, which was induced by LPS, was
inhibited (FIG. 4).
[0204] In addition, 5.times.NF-.kappa.B-Luc reporter plasmid was
transfected into RAW 264.7 macrophage cells and treated with
compounds of the present invention, as described above. After 24
hours of the transfection, the cells were pretreated with DMSO
(control), compound 1.1, reference compound 1 (compound where
palmitic acid is removed from compound 1.1), and smaducin-6 were
treated at various concentrations of 100 pM, 1 nM, and 100 nM for
30 minutes, and then the cells were treated with 100 ng/mL of LPS
for two hours. Luciferase activity in the cells was measured and
the results are presented in FIG. 5.
[0205] As shown in FIG. 5, when the cells were pretreated with
compound 1.1 (similar to the cells pretreated with smaducin-6),
inhibition of the activity of NF-.kappa.B, which was induced by LPS
treatment, was increased as dose of compound 1.1 increased. In
contrast, when the cells were pre-treated with DMSO or reference
compound 1, the activity of the NF-.kappa.B was not inhibited in
dose dependent manner.
Example 2.3
Signaling Pathway Selectivity
[0206] To evaluate signaling pathway selectivity of compound 1.1
according to the present invention, the following experiments were
performed. The following experiments were performed to test whether
compound 1.1 specifically suppressed a signaling pathway induced by
an individual inducer.
[0207] Specifically, 5.times.NF-.kappa.B-Luc reporter plasmid,
SBE-Luc reporter plasmid, and BRE-Luc reporter plasmid were
individually transfected into Raw 264.7 macrophage cells. After 24
hours, the cells transfected with the NF-.kappa.B-Luc reporter
plasmid were treated with 100 ng/mL of LPS, the cells transfected
with the SBE-Luc reporter plasmid were treated with 5 ng/mL of
TFG-.beta.1, and the cells transfected with the BRE-Luc reporter
plasmid were treated with 100 ng/mL of BMP6 for two hours.
[0208] FIGS. 6A-6C is a graph showing the relative suppression
activities of compound 1.1 to different signaling pathways. As
shown in FIGS. 6A-6C, when the cells were treated with compound 1.1
according to the present invention, activation of NF-.kappa.B,
which was induced by LPS treatment, was inhibited. However,
activation of BRE, which was induced by the BMP6 treatment, or
activation of SBE, which was induced by the TFG-.beta.1 treatment,
was not inhibited by compound 1.1. Accordingly, compound 1.1
selectively inhibited activation of NF-.kappa.B signaling
pathway.
[0209] Particularly, recent studies have reported that, for
treating an inflammatory bowel disease, TFG-.beta. and BMP
signaling pathways may specifically relate to mucosal wound healing
and the like (Nature 449 (2007), 361-365, Am J Path, 162(2),
(2003), Nature lmmunol. 6, (2005), 507-514, J Cell Physiol. 196(2):
(2003); 258-64), and Nature Protocols, 8(3), (2013) 627-637, which
are incorporated herein by reference), and TFG-.beta. could be a
very important factor in control of the inflammatory condition
(dendritic cell conditioning) in intestines (J. Clin. Invest., 111
(2003), 1297-1308, Immunity, 10 (1999), 39-49, Eur. J. lmmunol., 36
(2006), 864-874, Immunity, 25 (2006), 319-329, Cell 118 (2004),
229-241, and J. lmmunol. 179 (2007), 2690-2694, which are
incorporated herein by reference). Accordingly, the compounds of
the present invention do not suppress activation of TFG-.beta. and
BMP signaling pathways which indicated that those compounds are
markedly effective for the treatment of inflammatory bowel
disease.
Example 2.4
Disruption of Formation of Signaling Complex and Degradation of
Inhibitor .kappa.B (I.kappa.B)
[0210] In order to test whether the compound of the present
invention can disrupt formation of inflammation signaling pathway
protein complex mediated by MyD88 and/or RIP1, e.g. Toll-like
receptors (TLRs) signaling pathway protein complex,
immunoprecipitation experiments were performed. At a same time, the
following experiments were performed to measure I.kappa.B
degradation by the compound of the present invention as measuring
changes of concentration of I.kappa.B.
[0211] Specifically, using antibodies corresponding to proteins
which relate to the formation of Toll-like receptors (TLRs)
signaling pathway protein complex (for example, IRAK1, TRAF6,
MyD88, RIP1 and Pellino-1), disruption of the formation of
inflammatory signaling pathway protein complexes was affirmed by
immunoprecipitation. As reference control, a relative amount of
expression of 13-actin from the total cell lysate was compared and
presented using Western blot.
[0212] RAW264.7 macrophage cells were individually retreated with
compound 1.1 and compound 1.2, and smaducin-6, and further treated
with LPS. These RAW264.7 macrophage cells were collected, lysed in
a lysis butter (PBS containing 0.5% Triton X-100, 20 mM HEPES (pH
7.4), 150 mM NaCl, 12.5mM .beta.-glycerol phosphate, 1.5 mM
MgCl.sub.2, 10 mM NaF, 2 mM DTT, 1 mM Na3O4V, 2 mM EGTA, and 1 mM
Protease Inhibitor(PMSF)), and centrifuged at 13000 rpm for 10
minutes. For immunoprecipitation assays, the supernatant was
incubated at 4.degree. C. for 12 hours, with protein-A agarose
beads and the antibodies corresponding to the above proteins, and
the beads were subsequently washed three times with the lysis
buffer. The immunoprecipitated substances were dissociated from the
beads with addition of 2.times. sample buffer, and were boiled. The
prepared samples were loaded on SDS-polyacrylamide gel (FIG.
7).
[0213] To measure changes in concentration of I.kappa.B, RAW264.7
macrophage cells were individually pre-treated with compound 1.1,
compound 1.2, and smaducin-6 at a concentration of 100 nM, and then
treated with LPS. The extracted cell lysate was used for
immunoblotting with I.kappa.B.alpha. antibody, and .beta.-actin was
used as reference (FIG. 7).
[0214] FIG. 7 shows immunoprecipitation images indicating that
compound 1.1 and compound 1.2 disrupted the inflammatory signaling
pathway protein complexes, which are mediated by MyD88 or RIP1 and
additionally indicating changes in the concentration of I.kappa.B
by these compounds. When the cells were treated with the compounds
according to the present invention, as compared to the control of
total cell lysate, the formation of inflammatory signaling pathway
protein complex (which is mediated by MyD88 or RIP1) was disrupted
(FIG. 7). Cells treated with the compound of the present invention
were stabilized by dephosphorylating I.kappa.B as compared to
expression of the reference of .beta.-actin.
[0215] When the cells were treated with the compounds according to
the present invention, as compared to the control of total cell
lysate, formation of the MyD88 protein complex and the RIP1 protein
complex, and activities thereof were substantially disrupted and
inhibited (FIG. 7). These results indicated that the compounds in
the present invention may be used for the treatment of diseases
related to Pellino-1. Furthermore, in addition to above described
inflammatory bowel diseases, the compounds of the present invention
may be effective for preventing or treating geographic atrophy,
wet- age-related macular disease (wet-AMD), dry-age-related macular
disease (dry-AMD), diabetic retinopathy, multiple sclerosis (MS),
lung inflammation, bacterial pneumonia, viral pneumonia, Diffuse
large B-cell lymphoma (DLBCL, GCB type or ABC type), and alopecia
(Journal of Clinical Investigation, 124(11), (2014), 4976-4988, J
Virology, 86(12), (2012), 6595-6604, Nature Medicine, 19(5),
(2013), 595-602, J. Immunol., 187 (2011), 1-14, J. Inv. Derm., 132
(2012), 43-49, Med. Inflamm., (2010), Article ID 928030, Hair The
Transplant, 4 (2014), 4:1, Exp. Derm., 17 (2007), 12-19, and DDT
Dis. Mech. 5 (2009), e163-171), which are incorporated herein by
reference).
[0216] Bone-marrow-derived macrophage (BMDM) cells were
individually pretreated with compound 1.1, reference compound 1,
and smaducin-6 at various concentrations of 100 pM, 1 nM, and 100
nM, and then further treated with 100 ng/mL of LPS. The extracted
cell lysates were used for immunoblot as described above, and the
results were shown in FIG. 8.
[0217] In case of cells pre-treated with compound 1.1 as compared
to cells pretreated with smaducin-6, formation of the inflammatory
signaling pathway complex (for example, Toll-like receptors (TLR)),
which was mediated by MyD88 and RIP1, was further disrupted in an
increasing dose dependent manner of compound 1.1 (FIG. 8).
Meanwhile, cells pretreated with DMSO or reference compound 1
formed the inflammatory signaling pathway complex, which is
mediated by MyD88 and RIP1 in a non-dose dependent manner.
[0218] Likewise, as compared to the control of total cell lysate,
when the cells were pre-treated with compound 1.1 of the present
invention, formation of the inflammatory signaling pathway complex,
which is mediated by MyD88 and RIP1, was substantially disrupted.
The experiments using BMDM cells with the compound of the present
invention indicated that the compound of the present invention is
effective for preventing and treating multi diseases relating to
MyD88, preventing and treating diseases relating to expression of
Pellino-1 such as viral infection (respiratory viral infection,
viral pneumonia), bacterial pneumonia, autoimmune disease, blood
cancer including lymphoma, tumors in various internal organs (e.g.,
liver, lung, intestine, prostate, pancreas and the like), and
preventing and treating multiple sclerosis (MS).
[0219] As depicted in FIGS. 9A and 9B, RAW 264.7 macrophage cells
(top FIG. 9A) and BMDM cells (FIG. 9B) were individually pretreated
with compound 1.1, reference compound 1, and smaducine-6 at various
concentrations of 100 pM, 1 nM, and 100nM and then further treated
with 100 ng/ml of LPS. The results were compared with expression of
reference of .beta.-actin. The expression of I.kappa.B was
increased in the cells as the dose of pretreated compound 1.1 and
smaducin-6 increased. Meanwhile, I.kappa.B was degraded in cells
pretreated with DMSO or reference compound 1 because it was shown
to be phosphorylated. Accordingly, compound 1.1 of the present
invention inhibited the degradation of I.kappa.B.
Example 2.5
Evaluating Correlation Between Dose and Disease Activity Index
[0220] To evaluate the disease activity index of the compounds of
the present invention in an animal model with chronic colitis
induced by dextran sulfate sodium (DSS), the following experiments
were performed.
[0221] Mice (7-8 weeks, female, C57BL/6) were fed 2% DSS polymer
(MW of about 50000 Da) in drinking water for 5-7 days, and colitis
was induced every 2 to 15 days. Compound 1.1 was then administered
orally to the mice, which had induced chronic colitis by DSS, in an
amount of 50 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg,
respectively, from the third day after feeding DDS, daily, for 11
days. Body weights, diarrhea and hemafecia of the mice were checked
daily and the disease activity indexes were measured (FIG. 10).
[0222] FIG. 10 is a graph indicating disease activity index scores
in the animal model with DSS-induced chronic colitis according to
the dose of orally administered compound 1.1 and compound 1.2. As
shown in FIG. 10, when compound 1.1 of the present invention was
administered at different doses from 50 mg/kg to 400 mg/kg, the
disease activity thereof was increased as the dose increased, and
the disease activity was saturated at a dose of 200 mg/kg.
Accordingly, the compound of the present invention
proportionally-increased activity in a dose dependent manner.
Example 2.6
Suppression of Activity of Bowel Disease
[0223] To evaluate the supersession activity of the compounds of
the present invention in an animal model with induced acute colitis
(which is induced by DSS), the following experiments were
performed.
[0224] Mice (7-8 weeks, female, C57BL/6) were fed 2% DSS polymer
(MW of about 50000 Da) in drinking water for 7-8 days, and colitis
was induced. Then, each mouse having acute colitis was administered
sulfasalazine in an amount of 500 mg/kg and compound 1.1 in an
amount of 100 mg/kg daily for 14 days. Body weights, diarrhea and
hemafecia of the mice were checked daily and disease activity
indexes (DAI) were measured (FIG. 11A).
[0225] The DAI was measured as follows:
[0226] 1) weight loss (0 point: no weight loss; 1 point: reduced
weight by 1-5%, 2 points: reduced weight by 6-10%, 3 points:
reduced weight by 11-20%; 4 points: reduced weight by 20% or
greater);
[0227] 2) diarrhea (0 point: normal bowel movement; 2 points: loose
bowel movement; 4 points: diarrhea); and
[0228] 3) hemafecia (0 point: normal stool; 2 points: mild blood in
stool; 4 points: heavy blood in stool).
[0229] FIG. 11A is a graph indicating disease activity index scores
presenting suppression activities of the administered compounds in
the animal model with DSS-induced acute colitis. As compared to
anti-inflammatory sulfasalazine, the compound of the present
invention had sufficient disease activity index score at reduced
dose, and thus, the compound of the present invention is more
effective for treating colitis (FIG. 11A).
[0230] Meanwhile, as shown in FIGS. 11B-11D, mice suffering from
chronic colitis induced by DSS were orally administered
sulfasalazine in an amount of 500 mg/kg and compound 1.1 in an
amount of 100 mg/kg daily for 10 days. After 10 days of
administration, large intestine tissues were obtained from the
mice, and expression of chemokines (CCL20, CCL2 and CX3CL1) in the
tissues was measured using real-time polymerase chain reaction
(Real-Time PCR) as described above. Compound 1.1 was an effective
chemokine blockers by inhibiting chemotaxis of pathogenic immune
cells into the inflamed tissues.
Example 2.7
Histological Analyses of Colon Villi
[0231] To confirm treatment effect of DDS-induced chronic colitis
by compound 1.1 of the present invention, large intestinal villi
from a non-treated group, a DDS-induced chronic colitis model group
and a group treated with compound 1.1 were photographed (FIGS.
12-16).
[0232] FIGS. 12-14 are images showing shapes of the large
intestinal villi from the non-treated group, the DDS-induced
chronic colitis model group and the group treated with compound
1.1. As shown in FIGS. 12-14, in comparison to the large intestinal
villi from the DSS-induced chronic colitis model, the villi from
the group treated with compound 1.1 were similar to the villi of
the non-treated group.
[0233] FIG. 15 shows photographic images of large intestinal
tissues obtained from non-treated group, the DDS-induced chronic
colitis model group, the group treated with compound 1.1 (100 mpk)
and the group treated with sulfasalazine (500 mpk) as an
anti-inflammatory drug for colitis treatment. FIG. 16 shows
photographic images of morphology of large intestinal mucous
membranes obtained from the non-treated group, the DDS-induced
chronic colitis model group, the treated group with compound 1.1
(100 mpk) and the treated group with sulfasalazine (500 mpk) as an
anti-inflammatory drug for colitis treatment, and the mucous
membranes were stained with Alcian blue. As shown in FIGS. 15-16,
when the DSS-induced chronic colitis model was treated with
compound 1.1, histological damages, which can be found in inflamed
tissues, were alleviated. In addition, in comparison to the
non-treated group and the group treated with sulfasalazine, the
group treated with compound 1.1 had greater recovery of the mucous
membranes by blocking inflamed cells into the tissue.
[0234] FIG. 17 is a graph showing recovery level of large
intestinal wall in the non-treated group, the DDS-induced chronic
colitis model group, the treated group with compound 1.1 (100 mpk)
and the treated group with sulfasalazine (500 mpk).
[0235] Specifically, as described in Nature Protocols, 8(3), (2013)
627-637, on the 8th day 8 of treatment in DDS-induced chronic
colitis models, FITC-Dextran was orally administered at dose of 44
mg per 100 g body weight. After four hours of administration, blood
in an amount of 300-400 .mu.L was collected from heart of the mice.
The FITC-Dextran released in blood stream was measured using a
spectrophotofluorometer.
[0236] FIG. 17 quantitatively shows that, by analyzing the
FITC-Dextran released in blood stream, tight junction in large
intestinal epithelial tissue caused by embolization of large
intestinal mucous membrane, or function at the large intestinal
wall were recovered as much as the current sulfasalazine treatment.
Accordingly, the compound of the present invention had a
significant effect on recovering the intestinal epithelial barrier
and tight junction functions in chronic colitis tissues.
Example 2.8
Plasma Concentration
[0237] To evaluate changes of the blood (blood stream)
concentration of intravenous and oral administration of compound
1.1 of the present invention, the following experiments were
performed.
[0238] Compound 1.1 was administered to a rat intravenously (I.V.,
5 mg/kg) or orally (50 mg/kg), and 24 hours after administration,
the concentration of compound 1.1 in plasma was measured (FIG. 18
and FIG. 19).
[0239] Changes in the concentration of the compound 1.1 when
intravenously administered at various time intervals were measured
(FIG. 18). The concentration was decreased to 1/100 of the initial
concentration thereof within 1-2 hours and within 8 hours from the
administration. Compound 1,1 was detected in blood at very low
concentration.
[0240] Changes in the blood concentration of compound 1.1 at
various time courses when orally administered were measured (FIG.
19). After 30 minutes from the oral administration, compound 1.1
was not detected in blood.
[0241] Table 2 shows pharmacokinetic parameters of compound
1.1.
TABLE-US-00002 TABLE 2 Pharmacokinetic parameters of compound 1.1
Profile 5 mg/kg (i.v.) 50 mg/kg (p.o.) In vivo Animal Male SD rat
Male SD rat PK Cmax (ng/mL) 6,017.3 .+-. 2,302.4 0 (Rat, single
Tmax (hr) 0.08 0 dose) AUClast 2,135.6 .+-. 848.1 0 (ng hr/mL) T1/2
(hr) -- 0 Vss (L/kg) 5.4 .+-. 1.0 -- CL (L/hr/kg) 2.7 .+-. 1.3
--
Example 2.9
Tissue Distribution
[0242] To evaluate tissue distribution of compound 1.1 according to
the present invention, the following experiments were
performed.
[0243] Compound 1.1 was orally administered to a rat at dose of 10
mg/kg, and after 2 hours and 8 hours, concentrations of compound
1.1 were measured in the small intestinal tissue, large intestinal
tissue, appendix tissue, retrimentum in small intestine and
retrimentum in large intestine. The results are shown in the
following Table 3.
TABLE-US-00003 TABLE 3 Concentration of compound 1.1 after oral
administration Concentration (ng/mL) Species 2 hours 8 hours small
intestinal tissue 75 .+-. 70.2 78.1 .+-. 59.9 large intestinal
tissue 30 .+-. 10 33.2 .+-. 28.8 appendix tissue 22.7 .+-. 2.56
48.2 .+-. 46 retrimentum in small 1118 .+-. 217 876 .+-. 900
intestine retrimentum in large ND 1278 .+-. 52.7 intestine ND: Not
Determined
[0244] Quantitation range: intestine 8-2000 ng/kg, retrimentum
30-1000 ng/mL
[0245] As shown in Table 3, compound 1.1 was distributed in
intestinal tissues such as small intestinal, large intestinal and
appendix tissues at 2 hours and 8 hours from administration in a
quantitative range, and further compound 1.1 was distributed in
internal tissues 8 hours after administration.
[0246] Accordingly, when orally administered, an effective
concentration of the compound of the present invention is
continuously maintained in intestinal tissues, even after 8 hours.
Because the compound of the present invention is a small molecule
drug, the compound can be readily taken up into the intestines such
that effective concentration thereof may be readily reached.
Accordingly, by oral administration, the compound described herein
may be used for efficiently treating inflammatory bowel
diseases.
Example 2.10
Inhibition of Activity of MAPK Signaling Pathway
[0247] To test whether compound 1.1 and compound 1.2 of the present
invention suppress MAPK/ERK signaling pathway, Western blot
analysis was performed (FIG. 20A).
[0248] RAW 246.7 macrophage cells were pretreated with compound
1.1, compound 1.2 and reference compound 1 at a concentration of
100 nM for 30 minutes and then further treated with LPS for 0 hour,
0.5 hour, 1 hour and 2 hours. Phosphorylation of MAPK signaling
pathway proteins (ERK1/2, JNK, p38) from the cell treated with
compound 1.1, and compound 1.2 were activated at 0.5 hour with the
same pattern as the case of reference compound 1, and after 1 hour
or 2 hours of treatment, phosphorylation of those proteins were
gradually decreased with the same pattern as the case with
reference compound 1. P-actin was used as reference control.
Changes of the concentration upon phosphorylation of each protein
were measured by immunoblotting using anti-phosphorylation
antibodies. Reference compound 1, compound 1,1 and compound 1.2 did
not inhibit phosphorylation of the MAPK signaling pathway
proteins.
[0249] FIG. 21 shows images determining the inhibition of
phosphorylation of MAPK signaling pathway proteins (ERK1/2, JNK,
p38). BMDM cells were pretreated with DMSO, reference compound 1,
compound 1.1 and smaducin-6 at various concentrations of 100 pM, 1
nM and 100 nM and then further treated with LPS 2 hours.
Thereafter, Western blot analysis was performed. Compound 1.1
according to the present invention was not related to suppressing
phosphorylation of MAPK/ERK signaling pathway proteins despite
increase of dose, which was similar to DMSO and reference compound
1.
Example 2.11
Comparison with IRAK1/4-Inhibitor
[0250] As described in Example 2.3, 5.times.NF-.kappa.B-Luc
reporter plasmid was transfected into RAW 246.7 macrophage cells.
After 24 hours, the cells transfected with NF-.kappa.B-Luc reporter
plasmid were pretreated with compound 1.1 (100 nM),
interleukin-1-receptor-associated-kinase-1/4 inhibitor (IRAK1/4)
inhibitor (25 .mu.M, CAS 509093-47-4), and smaducin-6 (100 nM) for
30 minutes, and then further treated with LPS (100 ng/ml) for 2
hours. Subsequently, luciferase activities in the cells were
measured.
[0251] FIGS. 22A and 22B show inhibition of NF-.kappa.B activation
by compound 1.1 (FIG. 22A) and IRAK1/4 inhibitor (FIG. 22B)
relatively. At a high IRAK1/4 inhibitor concentration (e.g. 25
.mu.M), NF-.kappa.B activation was inhibited, which was similar to
compound 1.1.
[0252] Changes in I.kappa.B concentration in the cell lysate of RAW
246.7 macrophage cells, which were pretreated with compound 1.1
(100 nM), IRAK1/4 inhibitor (25 .mu.M) and smaducin-6 (100 nM) and
then further treated with LPS (100 ng/ml), were measured (FIG.
23A). RAK1/4 inhibitor also suppressed degradation of I.kappa.B
similar to compound 1.1 (FIG. 23B).
[0253] FIGS. 24A and 24B shows a graph and images comparing
suppression of MAPK/ERK signaling pathway by compound 1.1 and
IRAK1/4 inhibitor. Specifically, RAW 246.7 macrophage cells were
pretreated with DMSO, compound 1.1 (100 nM), IRAK1/4 inhibitor (25
.mu.M), and smaducin-6 (100 nM) for 30 minutes, and then further
treated with LPS (100 ng/ml) for two hours. FIG. 24A specifically
shows immunoblotting results to evaluate whether phosphorylation of
MAPK signaling pathway proteins such as ERK, JNK, and p38 were
inhibited by the above treatment. .beta.-actin was used as
reference. Compound 1.1 and smaducin-6 neither suppressed MAPK
signaling pathway nor inhibited phosphorylation of the proteins. In
contrast, IRAK1/4 inhibitor suppressed at least one or more of MAPK
signaling pathways.
[0254] In addition, to check for an unexpected side effect (e.g.,
from the fact that IRAK1/4 inhibitor suppresses AP-1 transcription
signal in MAPK pathway induced with LPS, which is in contrary to
compound 1.1), the following experiments were performed.
[0255] RAW 246.7 macrophage cells were transfected with AP-1-Luc
reporter plasmid as described in Example 2.3. After 24 hours, the
transfected cells with AP-1-Luc reporter plasmid were pretreated
with compound 1.1 (100 nM), IRAK1/4 inhibitor (25 .mu.M), and
smaducin-6 (100 nM) for 30 minutes, and then further treated with
LPS (100 ng/ml) for 2 hours. Luciferase activity in the cells was
measured and results are presented in FIG. 24B. Compound 1.1 of the
present invention selectively inhibited activity of NF-.kappa.B
signaling pathway, but did not suppress MAPK signaling pathways
which is important to biological activity of cells. However,
IRAK1/4 inhibitor inhibited MAPK signaling pathway, which may
result in inhibiting AP-1 transcriptional factors, such that, when
applied in biological subject, unexpected side effects may
occur.
[0256] The results in Examples 2.1 to 2.11 above indicated that the
compounds of the present invention can (1) inhibit expression of
interleukin-6 and activity of NF-.kappa.B, which are induced with
LPS treatment; (2) disrupt inflammatory signaling pathways mediated
by MyD88 and RIP1; and (3) provide similar disease activity index
at less dose than the dose of sulfasalazine that is current
anti-inflammatory drug for colitis. In addition, when the compounds
in the present invention are orally administrated, concentration in
blood stream is low, whereas the effective concentration is
maintained in cells and/or tissues. In particular, in intestinal
tissues, the effective concentration thereof can be maintained even
after 8 hours of administration. Accordingly, the compounds of the
present invention are used for treating inflammatory disease in
intestinal tissues, and particularly, are effectively used for
preventing, alleviating and treating inflammatory bowel disease
such ulcerative colitis, Behcet's Disease, Crohn's disease and the
like.
Example 2.12
Effect on Retinal Pigment Epithelium Cells
[0257] Compound 1.1 influenced angiogenesis related factors or
suppression factors.
[0258] ARPE-19 cells were treated with 5.5 mM of glucose as
reference control. For experiment group, the ARPE-19 cells were
treated with 30 mM of glucose for 48 hours to induce high blood
glucose condition, and simultaneously treated with DMSO, compound
1.1 (10 nM), and compound 1.1 (50 nM). Changes in expression of
Nox-4, VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie-2, EPO, and EPOR
proteins were measured by Western blot analysis.
[0259] Ang1 and Tie-2, which are factors controlling hemorrhage by
reinforcing blood vessel, were increased according to increased
concentration of compound 1.1 (FIGS. 25A and 25B). However,
expression of Nox4 of producing factor of ROS (reactive oxygen
species), VEGF of inducing factor of new blood vessel, VEG FR1,2,
Ang2 of antagonizing factor for Ang1, and EPO and EPOR of factors
for diabetic retinopathy was reduced by treating with compound
1.1.
[0260] In addition, HRMEC cells were treated with 5.5 mM of glucose
for reference group. For experiment group, the HRMEC cells were
treated with 30 mM of glucose for 48 hours to induce high blood
glucose condition, and simultaneously treated with DMSO, compound
1.1 (10 nM), and compound 1.1 (50 nM). Changes in expression of
Nox-4, VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie-2, EPO, and EPOR
proteins were quantitatively measured by qRT-PCR (quantitative
RT-PCR). Expression of VEGF precursor as stimulating factor of new
blood vessel was reduced by treatment with compound 1.1 (FIG.
25C).
[0261] To evaluate the effect of compound 1.1 on tube formation
during angiogenesis, HRMEC cells (8.times.10.sup.3) were cultured
on Matrigel coated micro slides and treated with 20 ng/ml of VEGF
for 4 hours to induce the tube formation. Simultaneously, for
reference group, the cells were treated with DMSO, and for
experimental group, the cells were treated with 50 nM of compound
1.1 and 1 uM of calcein-AM. The cells were observed using
fluorescence microscope.
[0262] FIG. 26 shows images of tube formation in the cells using
fluorescence microscope, which can show that compound 1.1
suppressed tube formation. Indeed, in ARPE-19 cells of human
retinal pigment epithelium cells, compound 1.1 suppressed
expression of Nox-4, VEGF, VEGFR1, VEGFR2, Ang2, EPO, and EPOR
proteins according to a concentration gradient thereof, and
increased expression of Ang1 and Tie-2 was observed. As such,
compound 1.1 is effectively used for preventing, alleviating or
treating ophthalmic indications such as diabetic retinopathy. In
addition, by disrupting formation of signaling pathway complex of
Myd88, the compound 1.1 is effectively used for preventing,
alleviating or treating geographic atrophy, wet-age-related macular
disease (wet-AMD), dry-age-related macular disease (dry-AMD) and
the like (Cell. 149(4), (2012); 847-859).
Example 2.13
Effect on Diabetic Retinopathy
[0263] Streptozotocin (STZ) was administered at dose of 50mg/kg to
mice daily for 5 days, and a mouse model having induced diabetic
retinopathy was obtained. The experimental group of the mouse model
was injected with compound 1.1, in an amount of 0.2 .mu.g into one
eye of the mouse, from the 20.sup.th day to 24.sup.th days from the
administration, three times with two days of interval. After 50
days of administration, a DR sample that was not treated with
compound 1.1 and a DR sample treated with compound 1.1 were
collected.
[0264] The collected retina tissues from each mouse group as
described above were stained with 5 .mu.M of dihydroethidium and
active oxygen rates from each group were measured (FIG. 27B). The
DR sample injected with compound 1.1 showed reduced active oxygen
rate as compared to the non-treated DR sample. Accordingly,
compound 1.1 of the present invention is effectively used for
preventing, alleviating or treating ophthalmic indications such as
diabetic retinopathy from the biological experiments using
mice.
Example 2.14
Effect on Multiple Sclerosis
[0265] To confirm the effect of compound 1.1 on multiple sclerosis,
mice (10 weeks old, female) were sensitized with MOG35-55/CFU and
PTX and an experimental autoimmune encephalomyelitis mouse model
was obtained. Experimental methods were described in Oncotarget,
Vol. 7 (2016), No. 13, 15382-15393, which is incorporated herein by
reference.
[0266] As shown in FIG. 28A, compound 1.1 was hypodermically
injected at dose of 1 mg/kg and 40 mg/kg for 25 days once in every
other day. As reference group, 10000 unit of Interferon-beta was
hypodermically injected for 25 days, once every other day. Clinical
score were measured and presented. Consequently, compound 1.1
showed to be effective in the treatment for multiple sclerosis at
minimal dose of 1 mg/kg in comparison to the conventional drug
interferon-beta currently used for multiple sclerosis
treatment.
[0267] In addition, when changes in body weights were measured from
each experiment group, the group treated with compound 1.1 did not
have reduced weight as compared to the EAE disease model group
(FIG. 28B). In other words, compound 1.1, as compared to the
current primary drug for treating multiple sclerosis, e.g.
interferon-beta, exhibited the same or greater treatment effect and
safety, and thus, is effectively used for preventing, alleviating
or treating multiple sclerosis in experimental autoimmune
encephalomyelitis model.
Example 2.15
Effect on Septicemia
[0268] To confirm the effects of compound 1.1 on septicemia, 10
mice in each group (7-week old, male) were anesthetized, and
thereafter, the appendicies were exposed by incision of abdomen.
The lower portion of ileocecal valve of the exposed appendix was
tied and a single hole was made by using a 22 gage syringe needle.
The treated appendix was reinserted into the abdominal cavity, and
grafted using thread to obtain a septicemia-induced mice model
(cecal ligation and pucture model, CLP model). Experimental methods
were described in EMBO Mol Med. (2015) March 12; 7(5):577-92, which
is incorporated herein by reference.
[0269] Compound 1.1 was hypodermically injected to the treated mice
at dose of 1 mg/kg, after 2 hours from the CLP procedure, with 12
hour intervals, three times. As reference control, smaducin-6 was
hypodermically injected at dose of 12 mg/kg as same method used for
compound 1.1. Survival rates of the mice from each group were
measured (FIG. 29). Sham comparative group was not treated after
incision of abdomen and anastomosis, CLP+PBS comparative group was
treated with phosphate buffered saline (PBS) instead of drug
compound after cecal ligation and puncture model was prepared. As a
result, compound 1.1 (1 mg/kg) was effective for the treatment of
septicemia, which has not been previously treated with conventional
drugs at low dosages compared to high dosages of smadudin-6 (e.g.
12 mg/kg), with 60% of survival rate. In other words, compound 1.1
was effective on cecal ligation and puncture (CLP) model,
indicating that compound 1.1 is effectively used for preventing,
alleviating or treating septicemia.
[0270] <Formulation 1> Granules
TABLE-US-00004 Compound of Formula 1 2 g Lactose 1 g
[0271] The granules were prepared in accordance with the method
known in the art.
[0272] <Formulation 2> Tablets
TABLE-US-00005 Compound of Formula 1 100 mg Corn starch 100 mg
Lactose 100 mg Stearic magnesium 2 mg
[0273] The tablets were prepared in accordance with the method
known in the art.
[0274] <Formulation 3> Capsules
TABLE-US-00006 Compound of Formula 1 100 mg Corn starch 100 mg
Lactose 100 mg Stearic magnesium 2 mg
[0275] The capsules were prepared in accordance with the method
known in the art.
[0276] <Formulation 4> Injections
TABLE-US-00007 Compound of Formula 1 100 mg Mannitol 180 mg
Na.sub.2HPO.sub.4.cndot.2H.sub.2O 26 mg Distilled water 2974 mg
[0277] The injections were prepared in accordance with the method
known in the art.
REFERENCES
[0278] 1. Toxicology and Applied Pharmacology 279 (2014)
311-321
[0279] 2. International Immunopharmacology 23 (2014) 294-303
[0280] 3. Seminars in Immunology 26 (2014) 75-79
[0281] 4. Neurobiology of Aging 22 (2001) 863-871
[0282] 5. J. Immunology 175 (2005) 3463-3468
[0283] 6. Prot Natl Acad Sci. 86 (1989) 6367-6371; J. Immunol. 143
(1989) 3949-3955; Nature 332 (1988) 83-85
[0284] 7. FASEB J. 4 (1990) 2860-2867
[0285] 8. J. Immunol. 147 (1991) 2777-2786
[0286] 9. Cell 46(5) (1986) 705-16
[0287] 10. Annu Rev Immunol. 16 (1998) 225
[0288] 11. Nature 395 (1998) 297-300; Cell 93 (1998) 1231-1240
[0289] 12. Nature 449 (2007), 361-365
[0290] 13. American Journal of Pathology, 162(2), (2003)
[0291] 14. Nature Immunol. 6, (2005), 507-514
[0292] 15. J Cell Physiol. 196(2): (2003); 258-64)
[0293] 16. Nature Protocols, 8(3), (2013) 627-637.
[0294] 17. The Journal of Clinical Investigation, 124(11), (2014),
4976-4988.
[0295] 18. J. Virology, 86(12), (2012), 6595-6604.
[0296] 19. J. Clin. Invest., 111 (2003), 1297-1308.
[0297] 20. Immunity, 10 (1999), 39-49.
[0298] 21. Eur. J. Immunol., 36 (2006), 864-874.
[0299] 22. Immunity, 25 (2006), 319-329.
[0300] 23. Cell 118 (2004), 229-241.
[0301] 24. J. Immunol. 179 (2007), 2690-2694.
[0302] 25. Oncotarget, Vol. 7 (2016), No. 13,15382-15393.
[0303] 26. Cell. 149(4), (2012); 847-859.
[0304] 27. Nature Medicine, 19(5), (2013), 595-602.
[0305] 28. J. Immunol., 187 (2011), 1-14.
[0306] 29. J. Inv. Derm., 132 (2012), 43-49.
[0307] 30. Med. Inflamm., (2010), Article ID 928030
[0308] 31. Hair The Transplant, 4 (2014), 4:1
[0309] 32. Exp. Derm., 17 (2007), 12-19
[0310] 33. DDT Dis. Mech. 5 (2009), e163-171
[0311] 34. EMBO Mol Med. (2015) Mar 12; 7(5):577-92
[0312] 35. Expert opinion on emerging drugs (2015)
20(3):349-352
[0313] 36. Cell. (2010) March 19; 140(6): 883-899
[0314] 37. Progress in Retinal and Eye Research 37(2013) 68e89
[0315] 38. P&T 41(2016), Jun no 6
[0316] 39. Gut 56(2007):725-732.
[0317] 40. World J Gastroenterol (2005);11(16):2462-2466
[0318] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
* * * * *