U.S. patent application number 16/036949 was filed with the patent office on 2019-08-29 for substituted chromenes for treatment of fibrosis or non-alcoholic steatohepatitis.
This patent application is currently assigned to OSTEONEUROGEN INC.. The applicant listed for this patent is OSTEONEUROGEN INC.. Invention is credited to Han Soo KIM, Ik Hwan KIM, Jun Hwan KIM, Ho Sup YOON, Byung Soo YOUN.
Application Number | 20190263789 16/036949 |
Document ID | / |
Family ID | 62913875 |
Filed Date | 2019-08-29 |
![](/patent/app/20190263789/US20190263789A1-20190829-C00001.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00002.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00003.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00004.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00005.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00006.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00007.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00008.png)
![](/patent/app/20190263789/US20190263789A1-20190829-C00009.png)
![](/patent/app/20190263789/US20190263789A1-20190829-D00001.png)
![](/patent/app/20190263789/US20190263789A1-20190829-D00002.png)
View All Diagrams
United States Patent
Application |
20190263789 |
Kind Code |
A1 |
YOUN; Byung Soo ; et
al. |
August 29, 2019 |
SUBSTITUTED CHROMENES FOR TREATMENT OF FIBROSIS OR NON-ALCOHOLIC
STEATOHEPATITIS
Abstract
The present invention relates to a novel compound and a
composition for the prevention, alleviation or treatment of
fibrosis or non-alcoholic steatohepatitis, which contains the
compound as an active ingredient, and more particularly to a novel
compound of Formula 1, which has an excellent effect on the
prevention, alleviation or treatment of fibrosis, and to a
composition for the prevention, alleviation or treatment of
fibrosis or non-alcoholic steatohepatitis, which contains the
compound as an active ingredient. The novel compound can regulate
the activation of EMT (epithelial-mesenchymal transition) by
effectively regulating the expression of snail and vimentin which
are regulators of EMT, and thus can effectively prevent, alleviate
or treat fibrosis. Furthermore, the novel compound has very good
pharmacokinetics. In addition, the novel compound of the present
invention can effectively inhibit fibrosis of liver cells, and thus
can also effectively alleviate or treat non-alcoholic
steatohepatitis.
Inventors: |
YOUN; Byung Soo; (Seoul,
KR) ; KIM; Jun Hwan; (Incheon, KR) ; KIM; Han
Soo; (Seoul, KR) ; YOON; Ho Sup; (Nanyang,
SG) ; KIM; Ik Hwan; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OSTEONEUROGEN INC. |
Seoul |
|
KR |
|
|
Assignee: |
OSTEONEUROGEN INC.
Seoul
KR
|
Family ID: |
62913875 |
Appl. No.: |
16/036949 |
Filed: |
July 17, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23V 2002/00 20130101;
C07D 405/12 20130101; A61P 1/16 20180101; A23L 33/10 20160801 |
International
Class: |
C07D 405/12 20060101
C07D405/12; A23L 33/10 20060101 A23L033/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2018 |
KR |
10-2018-0023415 |
Claims
1-10. (canceled)
11. A compound represented by the Formula 1: ##STR00008## or a
pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
C.sub.1-5 alkyl, C.sub.5-6 cycloalkyl, C.sub.5-6 heterocycloalkyl,
C.sub.6-12 aryl, or a C.sub.5-6 heteroaryl, where the C.sub.5-6
heterocycloalkyl and C.sub.5-6 heteroaryl each independently
contain at least one heteroatom selected from the group consisting
of O and N; R.sub.2 is hydrogen, ethyl, acetyl, acetoxy, carboxy,
benzoyloxy or 3,4,5-trihydroxybenzoyloxy; R.sub.3 is hydrogen,
hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy; R.sub.4
is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or
benzoyloxy; and R.sub.5 is hydrogen, hydroxy, methyl, methoxy,
acetoxy, carboxy or benzoyloxy.
12. The compound according to claim 11, or a pharmaceutically
acceptable salt thereof, wherein R.sub.1 is methyl, ethyl,
cyclopentyl, cyclohexyl, phenyl or benzyl.
13. The compound according to claim 11, or a pharmaceutically
acceptable salt thereof, wherein: R.sub.1 is methyl; R.sub.2 is
hydrogen; R.sub.3 is hydrogen, hydroxy or methoxy; R.sub.4 is
hydroxy or methoxy; and R.sub.5 is hydrogen, hydroxy or
methoxy.
14. The compound according to claim 11, wherein the compound is
selected from the group consisting of Formula 2, Formula 3, Formula
4 and Formula 5: ##STR00009## or a pharmaceutically acceptable salt
thereof.
15. A pharmaceutical composition containing pharmaceutically
acceptable carrier and a compound according to claim 11, or a
pharmaceutically acceptable salt thereof, as an active
ingredient.
16. A method for the treatment of fibrosis in a subject, comprising
administering to the subject in need thereof a therapeutically
effective amount of the pharmaceutical composition according to
claim 15.
17. The method of claim 16, wherein the fibrosis is selected from
the group consisting of idiopathic pulmonary fibrosis,
myelofibrosis, liver fibrosis, and kidney fibrosis.
18. A food composition containing a food-acceptable carrier and a
compound according to claim 11, or a pharmaceutically acceptable
salt thereof, as an active ingredient.
19. A method for the treatment of fibrosis in a subject, comprising
administering to the subject in need thereof a therapeutically
effective amount of the food composition according to claim 18.
20. The method of claim 19, wherein the fibrosis is selected from
the group consisting of idiopathic pulmonary fibrosis,
myelofibrosis, liver fibrosis, and kidney fibrosis.
21. A method for the treatment of non-alcoholic steatohepatitis in
a subject, comprising administering to the subject in need thereof
a therapeutically effective amount of the pharmaceutical
composition according to claim 15.
22. A method for the treatment of non-alcoholic steatohepatitis in
a subject, comprising administering to the subject in need thereof
a therapeutically effective amount of the food composition
according to claim 18.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] present invention relates to a novel compound and a
composition for the prevention, alleviation or treatment of
fibrosis or non-alcoholic steatohepatitis, which contains the
compound as an active ingredient, and more particularly to a novel
compound of Formula 1, which has an excellent effect on the
prevention, alleviation or treatment of fibrosis, and to a
composition for the prevention, alleviation or treatment of
fibrosis or non-alcoholic steatohepatitis, which contains the
compound as an active ingredient.
Description of the Prior Art
[0002] Fibrosis is a disease in which excess fibrous connective
tissue is formed in an organ or tissue in a reparative or reactive
process. This fibrous connective tissue is as opposed to formation
of normal fibrous tissue. When excess fibrous connective tissue is
formed in an organ or tissue, the tissue becomes hard and the
inflow of body fluids is reduced, so that its original function in
vivo cannot be sufficiently performed. Fibrosis is known to be
caused by injury, inflammation, burns, radiation, chemotherapy,
lymphedema or the like. Problems associated with fibrosis vary
depending on the location at which fibrous connective tissue is
formed, and the liver, secretory organs, lungs and the like are
mainly damaged by fibrosis. Typical examples of fibrosis include
idiopathic pulmonary fibrosis (IPF), myelofibrosis, liver fibrosis
and kidney fibrosis.
[0003] Currently known therapeutic agents against fibrosis include
Pirfenidone (a therapeutic agent against idiopathic pulmonary
fibrosis), Nintedanib (a therapeutic agent against idiopathic
pulmonary fibrosis), Ruxolitinib (a therapeutic agent against
myelofibrosis) and the like. However, there is a need to develop
new therapeutic agents which are more effective, safe for the human
body and easy to formulate.
[0004] Accordingly, the present inventor has conducted various
studies related to fibrosis in order to a new therapeutic agent
against fibrosis, and particularly paid attention on
epithelial-mesenchymal transition (EMT) (hereinafter referred to as
"EMT").
[0005] EMT refers to a phenomenon in which normal epithelial cells
are genetically reprogrammed into mesenchymal cells whose
morphology is likely to change, due to changes in the cytoskeleton
in the intermediate stage while normal cells transform into tumor
cells. Therefore, thinking that inhibition of EMT-related protein
expression may inhibit tumor metastasis and proliferation, and thus
many researchers have conducted studies related to EMT in order to
develop tumor therapeutic agents. Several hundred regulators of EMT
are known, including Twist, Snail, Slug, E-cadherin, vimentin,
collagen11a1 and the like.
[0006] As described above, studies on EMT and regulators of the EMT
have been conducted mostly for cancer or tumors. However, the
present inventor has focused on the relationship between EMT and
fibrosis based on some existing research results, and have expected
that fibrosis can be prevented and treated if EMT can be
regulated.
[0007] Accordingly, focusing on the relationship between EMT and
fibrosis, the present inventor has conducted studies to develop a
substance capable of effectively preventing, alleviating or
treating fibrosis, and as a result, has found that a compound
represented by Formula 1 as described in the specification exhibits
an excellent effect on the prevention, alleviation or treatment of
fibrosis by effectively regulating EMT and that non-alcoholic
steatohepatitis can also be effectively alleviated or treated due
to this effect, thereby completing the present invention.
SUMMARY OF THE INVENTION
[0008] Therefore, it is a main object of the present invention to
provide a novel compound having an excellent effect on the
prevention, alleviation or treatment of fibrosis.
[0009] Another object of the present invention is to provide a
composition for the prevention, alleviation or treatment of
fibrosis, which contains the compound as an active ingredient.
[0010] Still another object of the present invention is to provide
a composition for the alleviation or treatment of non-alcoholic
steatohepatitis, which contains the compound as an active
ingredient.
[0011] In accordance with one aspect, the present invention
provides a compound represented by the following Formula 1 or a
pharmaceutically acceptable salt thereof:
##STR00001##
wherein R.sub.1 is a substituted or unsubstituted C.sub.1-5 linear
or branched alkyl, a C.sub.5-6 cycloalkyl, a C.sub.5-6 cycloalkyl
containing at least one heteroatom selected from among O and N, a
substituted or unsubstituted C.sub.6-12 aryl, or a C.sub.5-6
heteroaryl containing at least one heteroatom selected from among O
and N; R.sub.2 is hydrogen, ethyl, acetyl, acetoxy, carboxy,
benzoyloxy or 3,4,5-trihydroxybenzoyloxy; and R.sub.3 to R.sub.5
are each independently hydrogen, hydroxyl, methyl, methoxy,
acetoxy, carboxy or benzoyloxy.
[0012] In accordance with another aspect, the present invention
provides a pharmaceutical composition for the prevention or
treatment of fibrosis, which contains the compound or a
pharmaceutically acceptable salt thereof as an active
ingredient.
[0013] In accordance with still another aspect, the present
invention provides a food composition for the prevention or
alleviation of fibrosis, which contains the compound or a
pharmaceutically acceptable salt thereof as an active
ingredient.
[0014] In accordance with still another aspect, the present
invention provides a pharmaceutical composition for treatment of
non-alcoholic steatohepatitis (NASH), which contains the compound
or a pharmaceutically acceptable salt thereof as an active
ingredient.
[0015] In accordance with yet another aspect, the present invention
provides a food composition for alleviation of non-alcoholic
steatohepatitis, which contains the compound or a pharmaceutically
acceptable salt thereof as an active ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows a compound of the present invention.
[0017] FIGS. 2 and 3 show a process of synthesizing a compound of
the present invention according to one example of the present
invention.
[0018] FIG. 4 shows the results of LCMS-NMR analysis of
7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one produced in a
process of synthesizing a compound of the present invention
according to one example of the present invention.
[0019] FIG. 5 shows the results of LCMS-NMR analysis of
7-(benzyloxy)-5-methoxy-2-phenyl-4H-chromen-4-one produced in a
process of synthesizing a compound of the present invention
according to one example of the present invention.
[0020] FIG. 6 shows the results of LCMS-NMR analysis of
1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethan-1-one produced in
a process of synthesizing a compound of the present invention
according to one example of the present invention.
[0021] FIG. 7 shows the results of LCMS-NMR analysis of
1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethane-1-one
produced in a process of synthesizing a compound of the present
invention according to one example of the present invention.
[0022] FIG. 8 shows the results of LCMS-NMR analysis of
1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethan-1-one produced
in a process of synthesizing a compound of the present invention
according to one example of the present invention.
[0023] FIG. 9 shows the results of LCMS-NMR analysis of
(E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxypheny-
l)prop-2-en-1-one produced in a process of synthesizing a compound
of the present invention according to one example of the present
invention.
[0024] FIG. 10 shows the results of LCMS-NMR analysis of
5-(benzyloxy)-2-(3,4-dimethoxyphenyl)-6,7-dimethoxy-4H-chromen-4-one
produced in a process of synthesizing a compound of the present
invention according to one example of the present invention.
[0025] FIG. 11 shows the results of LCMS-NMR analysis of
2-(3,4-dimethoxyphenyl)-7-hydroxy-5,6-dimethoxy-4H-chromen-4-one
produced in a process of synthesizing a compound of the present
invention according to one example of the present invention.
[0026] FIG. 12 shows the results of LCMS-NMR analysis of
2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one
produced in a process of synthesizing a compound of the present
invention according to one example of the present invention.
[0027] FIG. 13 shows the results of LCMS-NMR analysis of
7-(2-bromoethoxy)-2-(3,4-dimethoxyphenyl-5-hydroxy-6-methoxy-4H-chromen-4-
-one produced in a process of synthesizing a compound of the
present invention according to one example of the present
invention.
[0028] FIG. 14 shows the results of LCMS-NMR analysis of
4-(2-((2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4-oxo-4H-chromen-7-yl)-
oxy)ethyl)piperazin-2-one (a compound of Formula 2) produced in a
process of synthesizing a compound of the present invention
according to one example of the present invention.
[0029] FIG. 15 shows the inhibitory effect of a compound of the
present invention against fibrosis of ONGHEPA1 cells which are
mesenchymal stem cells (MSCs) derived from hepatic stellate cells
(HSCs).
[0030] FIG. 16 shows the inhibitory effect of a compound of the
present invention against expression of alpha-smooth muscle actin
(.alpha.-SMA) which is a representative marker of EMT
(epithelial-mesenchymal transition) in fibrosis of ONGHEPA1
cells.
[0031] FIG. 17 shows the inhibitory effect of a compound of the
present invention against fibrosis of diseased human lung
fibroblasts (DHLFs) isolated from lung fibrosis patients.
[0032] FIG. 18 shows the inhibitory effect of a compound of the
present invention against fibrosis of the A549 cell line which is a
human lung adenocarcinoma cell line.
[0033] FIG. 19 shows the inhibitory effect of a compound of the
present invention against expression of Snail and Vimentin which
are representative markers of EMT in the A549 cell line.
[0034] FIG. 20 shows the results of evaluating the metabolic
stability of a compound of the present invention for liver
microsomes.
[0035] FIG. 21 shows the results of evaluating the pharmacokinetics
of a compound of the present invention after oral
administration.
[0036] FIGS. 22 to 24 show the results of examining the effects of
comparative compounds for a compound of the present invention on
cell fibrosis.
[0037] FIG. 25 shows the results of comparing the pharmacokinetics
of a compound of the present invention after formulation.
[0038] FIG. 26 is a graph showing the change in body weight of
fibrotic animal models by administration of a compound of the
present invention or a control compound (pirfenidone). Sham, a
normal animal model; Vehicle Control, a non-compound-treated
fibrotic animal model orally administered only with 30% HPCD
(hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk PO (30%
HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk.
[0039] FIG. 27 is a graph showing the change in right lung weight
of fibrotic animal models by administration of a compound of the
present invention or a control compound (pirfenidone). Sham, a
normal animal model; Vehicle Control, a non-compound-treated
fibrotic animal model orally administered only with 30% HPCD
(hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk PO (30%
HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk.
[0040] FIG. 28 is a graph showing the change in hydroxyproline
amount in right lung of fibrotic animal models by administration of
a compound of the present invention or a control compound
(pirfenidone). Sham, a normal animal model; Vehicle Control, a
non-compound-treated fibrotic animal model orally administered only
with 30% HPCD (hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk
PO (30% HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk.
[0041] FIG. 29 is a graph showing the change in collagen amount in
right lung of fibrotic animal models by administration of a
compound of the present invention or a control compound
(pirfenidone). Sham, a normal animal model; Vehicle Control, a
non-compound-treated fibrotic animal model orally administered only
with 30% HPCD (hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk
PO (30% HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk.
[0042] FIG. 30 shows the results of performing histopathology
collagen morphometry of the left lung tissues of fibrotic animal
models administered with a compound of the present invention or a
control compound (pirfenidone). Sham, a normal animal model;
Vehicle Control, a non-compound-treated fibrotic animal model
orally administered only with 30% HPCD
(hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk PO (30%
HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk.
[0043] FIG. 31 shows the results of examining the concentration of
a compound present in the plasma of fibrotic animal models
administered with a compound of the present invention or a control
compound (pirfenidone). Pirfenidone 100mpk PO (30% HPCD), a
fibrotic animal model orally administered with Pirfenidone,
dissolved in 30% HPCD, at 100 mpk; Compound 1 10-100mpk PO,
fibrotic animal models orally administered with a compound of the
present invention, dissolved in 30% HPCD, at 10-100 mpk.
[0044] FIG. 32 shows the results of examining the concentration of
a compound present in the right lungs of fibrotic animal models
administered with a compound of the present invention or a control
compound (pirfenidone). Pirfenidone 100mpk PO (30% HPCD), a
fibrotic animal model orally administered with Pirfenidone,
dissolved in 30% HPCD, at 100 mpk; Compound 1 10-100mpk PO,
fibrotic animal models orally administered with a compound of the
present invention, dissolved in 30% HPCD, at 10-100 mpk.
[0045] FIGS. 33 to 38 show the results of H&E and Sirius Red
staining of the left lung tissues of experimental animal models.
Sham, a normal animal model; Vehicle Control, a
non-compound-treated fibrotic animal model orally administered only
with 30% HPCD (hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk
PO (30% HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk; *, Alveolar space; black arrow, Alveolar septa; #, Bronchiole;
white arrow head, area with prominent fibrosis; white arrow, area
invaded by inflammatory cells.
[0046] FIGS. 39 to 41 shows the results of H&E staining of the
liver tissues of experimental animal models. Sham, a normal animal
model; Vehicle Control, a non-compound-treated fibrotic animal
model orally administered only with 30% HPCD
(hydroxypropyl-beta-cyclodextrin); Pirfenidone 100mpk PO (30%
HPCD), a fibrotic animal model orally administered with
Pirfenidone, dissolved in 30% HPCD, at 100 mpk; Compound 1
10-100mpk PO, fibrotic animal models orally administered with a
compound of the present invention, dissolved in 30% HPCD, at 10-100
mpk; CV, Central vein; black arrow, area of necrosis developed.
[0047] FIG. 42 shows the results of H&E staining of the liver
tissues of non-alcoholic steatohepatitis animal models administered
with a compound of the present invention or a control compound
(telmisartan or OCA), and also shows the morphology of the livers.
Vehicle Control, a non-compound-treated non-alcoholic
steatohepatitis animal model orally administered only with 0.5%
CMC+1% Tween80 aqueous solution; Compound 1 50-100mpk, a
non-alcoholic steatohepatitis animal model orally administered with
a compound of the present invention, dissolved in 0.5% CMC+1%
Tween80 aqueous solution, at 50-100 mpk; Telmisartan 30mpk, a
non-alcoholic steatohepatitis animal model orally administered with
Telmisartan, dissolved in 0.5% CMC+1% Tween80 aqueous solution, at
30 mpk; OCA 50mpk, a non-alcoholic steatohepatitis animal model
orally administered with OCA, dissolved in 0.5% CMC+1% Tween80
aqueous solution, at 30 mpk.
[0048] FIG. 43 shows the results of statistically processing the
NAFLD (nonalcoholic fatty liver disease) activity score by t-test
on the basis of the degrees of inflammation, fibrosis, liver cell
ballooning and adipose accumulation in the liver tissues of
non-alcoholic steatohepatitis animal models administered with a
compound of the present invention or a control compound
(telmisartan or OCA). Vehicle Control, a non-compound-treated
non-alcoholic steatohepatitis animal model orally administered only
with 0.5% CMC+1% Tween80 aqueous solution; Compound low-high,
non-alcoholic steatohepatitis animal models orally administered
with a compound of the present invention, dissolved in 0.5% CMC+1%
Tween80 aqueous solution, at 5-100 mpk; Telmisartan, a
non-alcoholic steatohepatitis animal models orally administered
with Telmisartan, dissolved in 0.5% CMC+1% Tween80 aqueous
solution, at 30 mpk; OCA, a non-alcoholic steatohepatitis animal
models orally administered with OCA, dissolved in 0.5% CMC+1%
Tween80 aqueous solution, at 30 mpk.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention provides a novel compound represented
by the following Formula 1 or a pharmaceutically acceptable salt
thereof:
##STR00002##
wherein R.sub.1 is a substituted or unsubstituted C.sub.1-5 linear
or branched alkyl, a C.sub.5-6 cycloalkyl, a C.sub.5-6 cycloalkyl
containing at least one heteroatom selected from among O and N, a
substituted or unsubstituted C.sub.6-12 aryl, or a C.sub.5-6
heteroaryl containing at least one heteroatom selected from among O
and N; R.sub.2 is hydrogen, ethyl, acetyl, acetoxy, carboxy,
benzoyloxy or 3,4,5-trihydroxybenzoyloxy; and R.sub.3 to R.sub.5
are each independently hydrogen, hydroxyl, methyl, methoxy,
acetoxy, carboxy or benzoyloxy.
[0050] The novel compound or pharmaceutically acceptable salt
thereof according to the present invention can prevent, alleviate
or treat fibrosis.
[0051] The compound or pharmaceutically acceptable salt thereof
according to the present invention can inhibit cell fibrosis by
inhibiting expression of important factors such as .alpha.-SMA
(alpha-smooth muscle actin), Snail and Vimentin, which are involved
in EMT (epithelial-mesenchymal transition).
[0052] Due to this effect, the compound or pharmaceutically
acceptable salt thereof according to the present invention can
prevent, alleviate or treat fibrosis which is a disease in which
cells in an organ or tissue become fibrous by any cause.
[0053] Furthermore, nonalcoholic steatohepatitis (NASH) is also a
disease in which fibrosis of liver cells occurs. Thus, the novel
compound or pharmaceutically acceptable salt thereof according to
the present invention can alleviate or treat this nonalcoholic
steatohepatitis.
[0054] In particular, the compound or pharmaceutically acceptable
salt thereof according to the present invention can strongly
inhibit the growth and fibrosis of cells already programmed to
become fibrous and can restore these cells into normal cells. This
effect means that a state in which fibrosis has progressed can be
restored into a normal state, supporting that the compound or
pharmaceutically acceptable salt thereof according to the present
invention can exhibit a strong effect on the treatment of
fibrosis.
[0055] The compound or pharmaceutically acceptable salt thereof
according to the present invention is neither easily degraded by
liver microsomes nor transferred to other materials, and thus can
exhibit a long-lasting effect while maintaining its original
structure in vivo. Furthermore, it is advantageous for formulation
because the solubility thereof in phosphate buffer which is
generally used is not low. Moreover, it is safe for the human body
because the inhibitory activity thereof against CYP450 is low. In
addition, it has a very high administration easiness because it can
be absorbed rapidly in vivo even after oral administration.
[0056] For the fibrosis inhibitory effect, in vivo stability,
safety for the human body and the like as described above, R.sub.1
in the compound or pharmaceutically acceptable salt thereof
according to the present invention is preferably methyl, ethyl,
cyclopentyl, cyclohexyl, phenyl or benzyl. More preferably, R.sub.1
is methyl. In addition, preferably, R.sub.2 is hydrogen, R.sub.4 is
hydroxy or methoxy, and R.sub.3 and R.sub.5 are each independently
hydrogen, hydroxy or methoxy. More preferably, the compound or
pharmaceutically acceptable salt thereof according to the present
invention is a compound represented by any one of the following
Formulas 2 to 5 or a pharmaceutically acceptable salt thereof:
##STR00003##
[0057] The compound of the present invention may be prepared by the
method shown in the following Reaction Schemes 1 and 2:
##STR00004##
##STR00005##
wherein R.sub.1 to R.sub.5 correspond to R.sub.1 to R.sub.5 of
Formula 1.
[0058] Based on the above-described effect, the present invention
provides a pharmaceutical composition for the prevention or
treatment of fibrosis, which contains the compound or a
pharmaceutically acceptable salt thereof as an active
ingredient.
[0059] In this regard, the fibrosis is preferably any one selected
from the group consisting of idiopathic pulmonary fibrosis,
myelofibrosis, liver fibrosis and kidney fibrosis.
[0060] The present invention also provides a food composition for
the prevention or alleviation of fibrosis, which contains the
compound or a pharmaceutically acceptable salt thereof as an active
ingredient.
[0061] In this regard, the fibrosis is preferably any one selected
from the group consisting of idiopathic pulmonary fibrosis,
myelofibrosis, liver fibrosis and kidney fibrosis.
[0062] The present invention also provides a pharmaceutical
composition for treatment of non-alcoholic steatohepatitis (NASH),
which contains the compound or a pharmaceutically acceptable salt
thereof as an active ingredient.
[0063] The present invention also provides a food composition for
alleviation of non-alcoholic steatohepatitis (NASH), which contains
the compound or a pharmaceutically acceptable salt thereof as an
active ingredient.
[0064] The pharmaceutical composition of the present invention may
be a composition containing the compound or pharmaceutically
acceptable salt thereof according to the present invention alone or
in combination with a pharmaceutically acceptable carrier.
[0065] It is believed that the pharmaceutical composition of the
present invention may contain the compound or pharmaceutically
acceptable salt thereof according to the present invention in an
amount of 0.0001 to 100 wt % based on the total weight of the
composition.
[0066] It is believed that the pharmaceutical composition of the
present invention may be administered orally or parenterally in
clinical practice. For parenteral administration, the
pharmaceutical composition may be administered by intraperitoneal
injection, intrarectal injection, subcutaneous injection,
intravenous injection, intramuscular injection, intrauterine dural
injection, intracerebrovascular injection or intrathoracic
injection, and may be used as a general drug formulation.
[0067] The pharmaceutical composition of the present invention may
be used alone or in combination with surgery, radiotherapy,
hormonal therapy, chemotherapy and other methods employing
biological reaction regulators.
[0068] The daily dose of the compound or pharmaceutically
acceptable salt thereof contained in the pharmaceutical composition
of the present invention may be about 0.0001 g to 100 mg/kg/day,
preferably 0.001 g to 10 mg/kg/day, on the basis of in the
composition, and may be administrated once or several times a day,
with the range thereof being variable depending on patient's
weight, age, sex, health condition, diet, administration time,
administration mode, excretion rate, and severity of the
disease.
[0069] For oral or parenteral administration in clinical practice,
the pharmaceutical composition may be formulated in various forms
by using diluents or excipients, including fillers, extenders,
binders, wetting agents, disintegrants, surfactants and the like,
which are generally used.
[0070] In particular, the compound of the present invention can be
absorbed rapidly in vivo even after oral administration. In view of
bioavailability and in vivo stability, the compound of the present
invention is preferably formulated using NMP
(N-methyl-2-pyrrolidone), PEG400, SOLUTOL HS and water or
formulated using HPCD (hydroxypropyl-beta-cyclodextrin). In this
regard, the ratio of NMP:PEG400:SOLUTOL HS:water is preferably
5-15:10-30:10-30:40-60 (v/v), more preferably
8-12:15-25:15-25:45-55 (v/v). Where the compound of the present
invention is formulated using HPCD, a method of dissolving the
compound in 10-50% (w/v) HPCD aqueous solution is preferably used.
More preferably, 20-40% (w/v) HPCD aqueous solution is used.
[0071] When the compound of the present invention, formulated with
HPCD as described above, is orally administered for the purpose of
preventing, alleviating or treating fibrosis, may preferably be
administered at a dose of 1 to 50 mg/kg/day, more preferably 5 to
20 mg/kg/day, for mice, and may preferably be administered at a
dose of 0.08 to 4 mg/kg/day, more preferably 0.4 to 1.6 mg/kg/day,
for humans.
[0072] In addition, the compound of the present invention may also
be formulated using CMC (carboxymethyl cellulose) and Tween80 such
that the concentration of CMC is 0.1 to 1% (w/v) and the
concentration of Tween80 is 0.1 to 2%. When the compound of the
present invention, formulated with CMC and Tween80 as described
above, is orally administered for the purpose of preventing,
alleviating or treating non-alcoholic steatohepatitis, it may
preferably be administered at a dose of 1 to 70 mg/kg/day, more
preferably 5 to 60 mg/kg/day, for rats, and may preferably be
administered at a dose of 0.16 to 11.4 mg/kg/day, more preferably
0.8 to 9.7 mg/kg/day, for humans.
[0073] The pharmaceutical composition of the present invention may
contain, in addition to the compound or pharmaceutically acceptable
salt thereof according to the present invention, at least one
active ingredient showing the same or similar function.
[0074] The food composition of the present invention may be a
composition containing the compound or pharmaceutically acceptable
salt thereof according to the present invention alone or in
combination with a food-acceptable carrier. In this case, the
content of the compound or pharmaceutically acceptable salt thereof
according to the present invention may be suitably controlled
according to a conventional method based on the content thereof in
the pharmaceutical composition and the dose thereof. It is believed
that the food composition of the present invention may be in the
form of processed meat products, fish meat products, Tofu, Muk
(jellied food), porridge, noodles such as ramen noodles, seasonings
such as soy sauce, soybean paste, red pepper paste, mixed soybean
paste or the like, sauces, confectionery, dairy products such as
fermented milk, cheese or the like, pickled foods such as kimchi,
pickled vegetables or the like, or drinks such as fruit drinks,
vegetable drinks, soy milk, fermented drinks or the like. In
addition, the food-acceptable carrier may also be the
pharmaceutically acceptable carrier as described above.
[0075] Hereinafter, the present invention will be described in more
detail with reference to examples and experimental examples. It is
to be understood, however, that these examples and experimental
examples are for illustrative purposes only and are not intended to
limit the scope of the present invention.
Example 1: Synthesis of Compound of the Present Invention
1-1: Synthesis of 7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one
(Precursor Compound 2) (Step-1)
[0076] This step is a step of performing step-1 shown in FIG. 2,
and the detailed description thereof is as follows.
[0077] To a stirred suspension of
5,7-dihydroxy-2-phenyl-4H-chromen-4-one (precursor compound 1) (75
g; 0.294 mol; 1 equiv) in acetone (700 mL) was added potassium
carbonate (121.8 g; 0.442 mol; 3.0 equiv) and benzyl bromide (75.5
g; 0.442 mol; 1.5 equiv) in drops at 0.degree. C. The reaction
mixture was warmed to room temperature and then heated at
60.degree. C. for 5 h. The reaction completion was confirmed by TLC
(8:2/PE:EtOAc; R.sub.f.about.0.5). The mixture was allowed to cool
to room temperature and K.sub.2CO.sub.3 was removed by filtration
and the cake was washed with DCM several times until there was no
product was intact. The combined filtrate was concentrated to
dryness and the resulted solid was slurred with diethyl ether (200
mL), filtered and dried under suction to afford
7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (precursor
compound 2) as yellow solid (yield: 90.0 g; 88.6%).
[0078] The obtained
7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (precursor
compound 2) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 4.
[0079] LCMS: Mass found; (345.0; M+1).
[0080] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0081] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0082] Rt (min): 3.46; Area %--97.97.
[0083] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 12.83 (s, 1H),
8.10-8.12 (m, 2H), 7.62-7.65 (m, 3H), 7.51-7.62 (m, 2H), 7.43-7.50
(m, 2H), 7.38-7.41 (m, 3H), 7.06 (s, 1H), 6.93 (s, 1H), 6.51 (d,
J=2.40 Hz, 1H), 5.27 (s, 2H).
1-2. Synthesis of 7-(benzyloxy)-5-methoxy-2-phenyl-4H-chromen-4-one
(Precursor Compound 3) (Step-2)
[0084] This step is a step of performing step-2 shown in FIG. 2,
and the detailed description thereof is as follows.
[0085] To a stirred suspension of
7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (precursor
compound 2) (90 g; 0.261 mol; 1 equiv) in acetone (900 mL) was
added solid. KOH (43.9 g; 0.784 mol; 3 equiv) at room temperature.
The reaction mixture was warmed to 60.degree. C. and added dimethyl
sulfate (37.1 mL; 0.392 mol; 1.5 equiv) dropwise at 60.degree. C.
The reaction mixture was stirred at 60.degree. C. for 5 h. The
reaction completion was determined by TLC (1:1/PE:EtOAc;
R.sub.f.about.0.2). The mixture was allowed to cool to room
temperature and acidified with 10% aq. HCl solution until pH was
adjusted to 2. The resulted precipitate was collected by filtration
and washed with water, dried under suction for 12 h to afford
7-(benzyloxy)-5-methoxy-2-phenyl-4H-chromen-4-one (precursor
compound 3) as yellow solid (yield: 90 g; 96%).
[0086] The obtained
7-(benzyloxy)-5-methoxy-2-phenyl-4H-chromen-4-one (precursor
compound 3) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 5.
[0087] LCMS: Mass found; (359.0; M+1).
[0088] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode; Column: Zorbax extended C18 (50.times.4.6 mm, 5
.mu.m).
[0089] Rt (min): 2.94; Area %--97.85.
[0090] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 8.04-8.06 (m,
2H), 7.46-7.58 (m, 5H), 7.38-7.44 (m, 3H), 7.00 (d, J=2.00 Hz, 1H),
6.80 (d, J=1.60 Hz, 1H), 6.62 (d, J=2.00 Hz, 1H), 5.27 (s, 2H),
3.83 (s, 3H).
1-3: Synthesis of
1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethan-1-one (Precursor
Compound 4) (Step-3)
[0091] This step is a step of performing step-3 shown in FIG. 2,
and the detailed description thereof is as follows.
[0092] To a stirred suspension of
7-(benzyloxy)-5-methoxy-2-phenyl-4H-chromen-4-one (precursor
compound 3) (90 g; 0.251 mol; 1 equiv) in aqueous sodium hydroxide
solution (50%; 686 mL; 8.79 mol; 35 equiv) was added pyridine
(417.1 mL; 5.02 mol; 20 equiv) at room temperature. The dark brown
mixture was vigorously stirred and treated with diethylene glycol
(475 mL, 5.02 mol, 20 equiv) in drops. The mixture was heated to
100.degree. C. and stirred for 2 h. The reaction completion was
confirmed by TLC (1:1/PE:EtOAc; R.sub.f.about.0.5). The mixture was
cooled to 0.degree. C. and the pH was adjusted to 1 with 12N
aqueous hydrochloric acid solution. The aqueous portion was
extracted with ethyl acetate (2.times.500 mL). The combined organic
phase was washed with saturated aqueous sodium bicarbonate
solution, water and brine solution. Dried with sodium sulfate, and
the solvent was removed under reduced pressure. The resulted
residue was re dissolved in diethyl ether (700 mL) and the
insoluble dark particles were removed by filtration. The filtrate
was concentrated in vacuum to afford
1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethan-1-one (precursor
compound 4) as pale yellow solid (yield: 70 g; 97%).
[0093] The obtained
1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethan-1-one (precursor
compound 4) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 6.
[0094] LCMS: Mass found; (273.0; M+1).
[0095] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0096] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0097] Rt (min): 3.15; Area %--93.79.
[0098] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 13.77 (s, 1H),
7.35-7.47 (m, 5H), 6.18-6.21 (d, 2H), 5.18 (s, 2H), 3.86 (s, 3H),
2.51 (s, 3H).
1-4: Synthesis of
1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethan-1-one
(Precursor Compound 5) (Step-4)
[0099] This step is a step of performing step-4 shown in FIG. 2,
and the detailed description thereof is as follows.
[0100] To a stirred suspension of
1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethan-1-one (precursor
compound 4) (30 g; 0.110 mol; 1 equiv) in aq. tetraethyl ammonium
hydroxide solution (35%; 632 mL; 1.43 mol; 13 equiv) was added
pyridine (69.4 mL; 0.0.836 mol; 7.6 equiv) in drops. The reaction
mixture became clear dark solution. In a separate flask, potassium
persulfate (50.49 g; 0.187 mol; 1.7 equiv) in water (1 L) was taken
and to this solution, the above reaction mixture was added in drops
at room temperature and continued to stir for another 24 h. After
confirming the consumption of starting material by TLC, conc. HCl
was added to the reaction mixture to adjust the pH to 1-2 at
0.degree. C. The resulted brownish gummy residue was passed through
filtration and the aq. filtrate was further washed with diethyl
ether (1.times.100 mL).
[0101] The separated aqueous layer was treated with sodium sulfite
(11.09 g; 0.0.088 mol; 0.8 equiv), con HCl (110 mL) and benzene
(220 mL). This reaction mixture was heated to 95.degree. C. for 1
h. The reaction completion was determined by TLC (8:2/PE:EtOAc;
R.sub.f 0.3). The reaction mixture was cooled to 0.degree. C. and
extracted with ethyl acetate (2.times.300 mL). The combined organic
phase was washed with brine solution, dried with sodium sulfate and
the solvent was removed under reduced pressure. The residue was
purified by column chromatography over silica gel (60-120 mesh)
eluting with ethyl acetate (10-12%) in pet ether as an eluent to
afford 1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethan-1-one
(precursor compound 5) as yellow solid (yield: 10 g; 31%).
[0102] The obtained
1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethan-1-one
(precursor compound 5) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 7.
[0103] LCMS: Mass found; (289.0; M+1).
[0104] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0105] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0106] Rt (min): 2.68; Area %--91.01.
[0107] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 12.76 (s, 1H),
8.37 (s, 1H), 7.41-7.51 (m, 2H), 7.31-7.38 (m, 3H), 6.38 (s, 1H),
5.21 (s, 2H), 3.84 (s, 3H), 2.51 (s, 3H).
1-5: Synthesis of
1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethan-1-one
(Precursor Compound 6) (Step-5)
[0108] This step is a step of performing step-5 shown in FIG. 2,
and the detailed description thereof is as follows.
[0109] To a stirred suspension of
1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethan-1-one
(precursor compound 5) (28 g; 0.097 mol; 1 equiv) in acetone (300
mL) was added K.sub.2CO.sub.3 (20 g; 0.145 mol; 1.5 equiv) at RT.
The reaction mixture was warmed to 60.degree. C. and then added
dimethyl sulfate (18.2 mL; 0.145 mol; 2 equiv) dropwise at
60.degree. C. The reaction mixture was stirred at 60.degree. C. for
5 h. After confirming the reaction completion by TLC, the mixture
was allowed to cool to room temperature and K.sub.2CO.sub.3 was
removed by filtration and the cake was washed with DCM several
times until there was no product remain. The combined filtrate was
concentrated to dryness and the resulted residue was purified by
column chromatography over silica gel (60-120 mesh) eluting with
ethyl acetate (8-10%) in pet ether as an eluent to afford
1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethan-1-one
(precursor compound 6) as white solid (yield: 25 g; 85%).
[0110] The obtained
1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethan-1-one
(precursor compound 6) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 8.
[0111] LCMS: Mass found; (303.0; M+1).
[0112] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0113] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0114] Rt (min): 3.11; Area %--99.85.
[0115] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 12.95 (s, 1H),
7.36-7.48 (m, 5H), 6.45 (s, 1H), 5.20 (s, 2H), 3.96 (s, 3H), 3.92
(s, 3H), 2.51 (s, 3H).
1-6: Synthesis of
(E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxypheny-
l)prop-2-en-1-one (Precursor Compound 7) (Step-6)
[0116] This step is a step of performing step-6 shown in FIG. 2,
and the detailed description thereof is as follows.
[0117] To a stirred suspension of
1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethan-1-one
(precursor compound 6) (25 g; 0.082 mol; 1 equiv) and
3,4-dimethoxybenzaldehyde (16.4 g; 0.099 mol; 1.2 equiv) in ethanol
(200 mL) was added solution of KOH (46 g; 0.0.82 mol; 1 equiv) in
water at room temperature. The reaction mixture was stirred at room
temperature for 24 h. TLC (6:4/PE:EtOAc; R.sub.f 0.4) confirmed
that 70-75% product formation and the unreacted starting materials
were intact after 24 h. The mixture was concentrated in vacuum and
the residue was partitioned between aq. sodium bisulfate and DCM.
The separated organic phase was washed with water, brine solution
and dried with sodium sulfate. Concentrated under vacuum and the
resulted residue was slurred with diethyl ether (100 mL), filtered
and dried under suction to obtain
(E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxypheny-
l)prop-2-en-1-one (precursor compound 7) as yellow solid (yield:
23.0 g; 39%).
[0118] The obtained
(E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxypheny-
l)prop-2-en-1-one (precursor compound 7) was analyzed by LCMS-NMR
under the following conditions, and the results are shown in FIG.
9.
[0119] LCMS: Mass found; (450.9; M+1).
[0120] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0121] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0122] Rt (min): 3.34; Area %--98.16.
[0123] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 11.96 (s, 1H),
7.49-7.57 (m, 3H), 7.49-7.57 (m, 4H), 7.29-7.39 (m, 2H), 7.03 (d,
J=8.40 Hz, 1H), 6.47 (s, 1H), 5.19 (s, 2H), 3.85 (s, 3H), 3.83 (s,
3H), 3.77 (s, 3H), 3.74 (s, 3H).
1-7: Synthesis of
5-(benzyloxy)-2-(3,4-dimethoxyphenyl)-6,7-dimethoxy-4H-chromen-4-one
(Precursor Compound 8) (Step-7)
[0124] This step is a step of performing step-7 shown in FIG. 3,
and the detailed description thereof is as follows.
[0125] To a stirred suspension of
(E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxypheny-
l)prop-2-en-1-one (precursor compound 7) (21 g; 0.0466 mol; 1
equiv) in isoamyl alcohol (300 mL) was added selenium dioxide (21
g; 0.466 mol; 10 equiv) at room temperature. The mixture was heated
to 140.degree. C. and stirred for 7 h. After confirming the
reaction completion by TLC (4:6/PE:EtOAc; R.sub.f.about.0.2), the
mixture was allowed to cool to room temperature and the resulted
dark particles were removed by filtration on celite pad. The pad
was washed with DCM and the filtrate was concentrated under vacuum.
The resulted residue was diluted with DCM (500 mL), washed with aq.
NaHCO.sub.3 solution, water and brine. Dried with sodium sulfate
and the solvent was removed under reduced pressure The residue was
purified by column chromatography over silica gel (60-120 mesh)
eluting with ethyl acetate (50-60%) in pet ether as an eluent to
afford
5-(benzyloxy)-2-(3,4-dimethoxyphenyl)-6,7-dimethoxy-4H-chromen-4-o-
ne (precursor compound 8) as yellow solid (yield: 16 g; 76%).
[0126] The obtained
5-(benzyloxy)-2-(3,4-dimethoxyphenyl)-6,7-dimethoxy-4H-chromen-4-one
(precursor compound 8) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 10.
[0127] LCMS: Mass found; (449.0; M+1).
[0128] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0129] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0130] Rt (min): 2.83; Area %--98.62.
[0131] 1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 7.54 (d, J=8.40 Hz,
1H), 7.45 (t, J=7.60 Hz, 2H), 7.37 (d, J=8.40 Hz, 2H), 7.13 (d,
J=8.80 Hz, 2H), 6.82 (s, 1H), 5.30 (s, 2H), 3.89 (s, 3H), 3.85 (s,
3H), 3.82 (s, 3H), 3.77 (s, 3H).
1-8: Synthesis of
2-(3,4-dimethoxyphenyl)-7-hydroxy-5,6-dimethoxy-4H-chromen-4-one
(Precursor Compound 9) (Step-8)
[0132] This step is a step of performing step-8 shown in FIG. 3,
and the detailed description thereof is as follows.
[0133] To a stirred solution of
5-(benzyloxy)-2-(3,4-dimethoxyphenyl)-6,7-dimethoxy-4H-chromen-4-one
(precursor compound 8) (19 g; 0.0424 mol; 1 equiv) in chloroform
(200 mL) was added 10% Pd/C (3.8 g) and the mixture was
hydrogenated under ballon atmosphere at room temperature for 6-7 h.
After the reaction was completed by TLC, the catalyst was filtered
through celite pad. The pad was washed with 20% MeOH in DCM and the
combined filtrate was concentrated in vacuum to afford dark brown
solid. The solid was triturated with ethyl acetate (80 mL),
filtered and dried under suction to obtain
2-(3,4-dimethoxyphenyl)-7-hydroxy-5,6-dimethoxy-4H-chromen-4-on- e
(precursor compound 9) as yellow solid (yield: 11.0 g; 72%).
[0134] The obtained
2-(3,4-dimethoxyphenyl)-7-hydroxy-5,6-dimethoxy-4H-chromen-4-one
(precursor compound 9) was analyzed by LCMS-NMR under the following
conditions, and the results are shown in FIG. 11.
[0135] LCMS: Mass found; (359.0; M+1).
[0136] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0137] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0138] Rt (min): 2.07; Area %--99.63.
[0139] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 10.69 (s, 1H),
7.61 (d, J=8.40 Hz, 1H), 7.51 (s, 1H), 7.11 (d, J=8.40 Hz, 1H),
6.90 (s, 1H), 6.74 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.80 (s,
3H), 3.77 (s, 3H).
1-9: Synthesis of
2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one
(Precursor Compound 10) (Step-9)
[0140] This step is a step of performing step-9 shown in FIG. 3,
and the detailed description thereof is as follows.
[0141] To a stirred solution of
2-(3,4-dimethoxyphenyl)-7-hydroxy-5,6-dimethoxy-4H-chromen-4-one
(precursor compound 9) (11 g; 0.0307 mol; 1 equiv) in acetonitrile
(100 mL) was added AlCl.sub.3 (20.3 g; 0.153 mol; 5 equiv) in
portions at room temperature and then the mixture was refluxed at
90.degree. C. for 2 h. The reaction completion was determined by
TLC and the solvent was evaporated to dryness. The resulted residue
was treated with aq. HCl (10%; 200 mL) and chloroform (200 mL) and
refluxed until the reaction mixture became clear. After the
reaction was completed by TLC (7:3/PE:EtOAc; R.sub.f0.4), the
reaction mixture was cooled to room temperature and the organic
layer was separated. The aq. layer was again extracted with DCM
(1.times.100 mL) and the combined organic layer was washed with
water, brine solution. Dried over sodium sulfate and concentrated.
The residue was purified by column chromatography over silica gel
(60-120 mesh) eluting with DCM as an eluent to afford
2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one
(precursor compound 10) as a yellow solid (yield: 7.0 g; 66%).
[0142] The obtained
2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one
(precursor compound 10) was analyzed by LCMS-NMR under the
following conditions, and the results are shown in FIG. 12.
[0143] LCMS: Mass found; (344.9; M+1).
[0144] Method: A--0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate: 1.5
mL/min; +ve mode.
[0145] Column: Zorbax extended C18 (50.times.4.6 mm, 5 .mu.m).
[0146] Rt (min): 2.44; Area %--98.32.
[0147] HPLC: 97.64%.
[0148] Mobile Phase: A: 0.1% TFA in WATER, B: ACN; Flow Rate: 1.0
mL/min.
[0149] Column: Atlatis dC-18 (4.6.times.250) mm; 5 u; Rt (min):
12.68; Area %--97.64.
[0150] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 13.05 (s, 1H),
10.73 (s, 1H), 7.68-7.71 (m, 1H), 7.58 (d, J=2.04 Hz, 1H), 7.14 (d,
J=8.64 Hz, 1H), 6.99 (s, 1H), 6.65 (s, 1H), 3.89 (s, 3H), 3.86 (s,
3H), 3.76 (s, 3H).
[0151] .sup.13C-NMR (100 MHz, DMSO-d6): .delta. 182.6, 163.8,
157.8, 153.1, 152.8, 152.5, 149.4, 131.8, 123.4, 120.4, 112.1,
109.9, 104.5, 103.8, 94.8, 60.4, 56.3, 56.2.
1-10: Synthesis of
7-(2-bromoethoxy)-2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4H-chromen--
4-one (Precursor Compound 11) (Step-10)
[0152] This step is a step of performing step-10 shown in FIG. 3,
and the detailed description thereof is as follows.
[0153] To a stirred mixture of
2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one
(precursor compound 10) (10 g, 29 mmol, 1 equiv.) and potassium
carbonate (12.04 g, 87.1 mmol, 3.0 equiv.) in THF (400 mL, 40 vol.)
were added 1,2-dibromo ethane (27.28 g, 145.2 mol, 5 equiv.) and
TBAI (1.05 g, 0.0029 mol, 0.1 equiv.) and the resulting mixture was
refluxed at 60.degree. C. for 16 h. After confirming the completion
of reaction by TLC analysis, THF was removed under reduced pressure
to afford a solid. The crude product was washed with excess of THF
and methanol to obtain
7-(2-bromoethoxy)-2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4H-chromen--
4-one (precursor compound 11) as a solid (yield: 8 g; 61%). The
product was used for the next step without any further
purification.
[0154] The obtained
7-(2-bromoethoxy)-2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4H-chromen--
4-one (precursor compound 11) was analyzed by LCMS-NMR under the
following conditions, and the results are shown in FIG. 13.
[0155] LCMS: Mass found; (451.0; M+1).
[0156] Mobile phase: A: 0.1% HCOOH in H.sub.2O, B: ACN; Flow Rate:
1.5 mL/min; +ve mode.
[0157] Column: Atlantis dC18 (50.times.4.6 mm, 5 .mu.m).
[0158] Rt (min): 3.14; Area %--80.41.
[0159] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. 12.91 (s, 1H),
7.73 (dd, J=5.6, 2.0 Hz, 1H), 7.60 (d, J=2.0 Hz, 1H), 7.15 (d,
J=8.4 Hz, 1H), 7.06 (s, 1H), 7.02 (s, 1H), 4.51 (t, J=5.2 Hz, 2H),
3.91 (s, 3H), 3.85 (s, 3H), 3.79 (s, 3H).
1-11: Synthesis of
4-(2-((2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4-oxo-4H-chromen-7-yl)-
oxy)ethyl) piperazin-2-one (Compound 1) (Step-11)
[0160] This step is a step of performing step-11 shown in FIG. 3,
and the detailed description thereof is as follows.
[0161] A solution of
7-(2-bromoethoxy)-3-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4H-chromen--
4-one (precursor compound 11) (8 g, 17.7 mmol, 1.0 equiv.) and
piperazine-2-one (5.33 g, 53.3 mmol, 3.0 equiv.) in acetonitrile
(80 mL) was refluxed under nitrogen atmosphere for 12 h. After
confirming the completion of the reaction by TLC, acetonitrile was
removed under reduced pressure. The resultant gum was triturated
with DCM and pet-ether to get a solid, which was washed
subsequently with methanol, DCM and THF and dried well to afford
4-(2-((2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4-oxo-4H-chromen-7-yl)-
oxy)ethyl)piperazin-2-one (compound 1; Formula 2) (yield: 3.2 g;
41%).
[0162] The obtained
4-(2-((2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-4-oxo-4H-chromen-7-yl)-
oxy)ethyl)piperazin-2-one (compound 1) was analyzed by LCMS-NMR
under the following conditions, and the results are shown in FIG.
14.
[0163] LCMS: Mass found (471.2; M+1).
[0164] Mobile Phase: A: 0.1% Formic Acid in H.sub.2O; B: CAN.
[0165] Column: Atlantis dC18 (50.times.4.6 mm, 5 .mu.m).
[0166] Rt (min): 1.515; Area %--95.488.
[0167] HPLC: 97.03%.
[0168] Mobile Phase: A: 0.1% TFA in H.sub.2O; B: Acetonitrile.
[0169] Column: Atlantis dC18 (50.times.4.6 mm, 5 .mu.m).
[0170] Rt (min): 9.74; Area %--97.03.
[0171] .sup.1H NMR: (400 MHz, DMSO-d.sub.6): .delta. 12.89 (s, 1H),
7.75 (br s. 1H), 7.70 (dd, J=8.0, 1.6 Hz, 1H), 7.58 (s, 1H), 7.13
(d, J=8.5 Hz, 1H), 7.02 (d, J=10.4 Hz, 2H), 4.28 (t, J=5.2 Hz, 2H),
3.89 (s, 3H), 3.86 (s, 3H), 3.75 (s, 3H), 3.17 (br s, 2H), 3.11 (s,
2H), 2.87 (t, J=5.2 Hz, 2H), 2.73 (t, J=5.2 Hz, 2H).
[0172] .sup.13C-NMR (100 MHz, CDCl.sub.3): 182.4, 169.2, 163.9,
157.5, 153.1, 152.9, 152.2, 149.1, 132.7, 123.4, 119.9, 111.0,
108.6, 106.1, 104.1, 91.4, 67.1, 60.7, 57.0, 56.0, 55.9, 55.5,
49.4, 41.1.
Experimental Example 1: Examination of the Effect of Compound of
the Present Invention Against Fibrosis
1-1: Experiment on Effect Using Mesenchymal Stem Cells
[0173] In order to examine the effect of a compound of the present
invention against fibrosis, ONGHEPA1 cells (KCTC13086BP), which are
mesenchymal stem cells (MSCs) derived from rat hepatic stellate
cells (HSCs) and capable of proliferating indefinitely, were first
used. Fibrosis of the ONGHEPA1 cells can be induced by a simple
method of treating the cells with TGF-.beta. (transforming growth
factor beta) or PDGF (platelet-derived growth factor).
[0174] The ONGHEPA1 cells were seeded into medium, cultured for 24
hours, and then treated with TGF-.beta. (5 ng/ml) to induce cell
fibrosis. Alternatively, the cells were co-treated with TGF-.beta.
and the compound of the present invention (50 .mu.M in DMSO) and
cultured for 24 hours. Next, the degree of fibrosis of the cells,
that is, the degree of differentiation into myofibroblasts, was
examined with a phase contrast microscope (200.times.).
[0175] In this regard, the detailed experimental method followed
the paper of Kim et al. (Han-Soo Kim, Jun-Hwan Kim, Ji Yong Lee,
Young-Min Yoon, Ik-Hwan Kim, Ho-Sup Yoon, Byung-Soo Youn. Small
molecule-mediated reprogramming of epithelial-mesenchymal
transition thereby blocking fibrosis. bioRxiv preprint first posted
online Feb. 16, 2017; doi: http://dx.doi.org/10.1101/106591).
[0176] As a result, as shown in FIG. 15, the control group treated
with TGF-.beta. differentiated into elongated myofibroblasts, and
thus showed a typical fibrosis symptom, whereas the test group
treated with TGF-.beta. plus the compound of the present invention
showed no fibrosis symptom such that it did not differ from the
normal control group.
[0177] In addition, in order to examine whether the above-described
effect would be attributable to the effect against
epithelial-mesenchymal transition (EMT) (hereinafter referred to as
"EMT"), the expression of .alpha.-SMA (alpha-smooth muscle actin),
which is a representative marker of EMT, in the cells of each test
group, was examined by immunofluorescent staining and nuclear
staining (DAPI staining).
[0178] As a result, as shown in FIG. 16, the expression of
.alpha.-SMA in the control group treated with TGF-.beta. was
significantly high, whereas the expression of .alpha.-SMA in the
test group treated with TGF-.beta. plus the compound of the present
invention was inhibited such that it did not differ from that in
the normal control group.
[0179] These results support that the compound of the present
invention strongly inhibits the differentiation of cells into
myofibroblasts by affecting the EMT of the cells, and thus can
effectively prevent or treat fibrosis, particularly fibrosis or
non-alcoholic steatohepatitis (NASH) which occurs in the liver.
1-2: Experiment on Effect Using Fibroblasts Isolated from Lung
Fibrosis Patients
[0180] Diseased human lung fibroblasts (DHLF) (Lonza, Swiss)
isolated from lung fibrosis patients were seeded into medium,
cultured for 24 hours, and then treated with TGF-.beta. (5 ng/ml)
to induce fibrosis. Alternatively, the fibroblasts were co-treated
with TGF-.beta. and the compound of the present invention (50 .mu.M
in DMSO) and cultured for 24, 48 or 72 hours. Next, the degree of
differentiation into myofibroblasts was examined with a phase
contrast microscope (200.times.).
[0181] As a result, as shown in FIG. 17, the untreated group and
the control group treated with TGF-.beta. differentiated into
elongated myofibroblasts, and thus showed a typical fibrosis
symptom, whereas the test group treated with TGF-.beta. plus the
compound of the present invention showed little or no fibrosis
symptom, and the growth of the cells was also inhibited.
[0182] These results support that the compound of the present
invention strongly inhibits the growth and fibrosis of fibroblasts
already programmed to become fibrous and restores the fibroblasts
to normal cells, indicating that it can effectively prevent or
treat fibrosis, particularly fibrosis such as idiopathic pulmonary
fibrosis which occurs in lungs.
1-3: Experiment on Effect Using Lung Adenocarcinoma Cell Line
[0183] The A549 cell line, which is a lung adenocarcinoma cell line
used as a lung EMT-related study model, was seeded into medium,
cultured for 24 hours, and then treated with TGF-.beta. (5 ng/ml).
Alternatively, the A549 cell line was co-treated with TGF-.beta.
and the compound of the present invention (25 or 50 .mu.M in DMSO)
and cultured for 24 or 48 hours. Next, the cells were examined with
a phase contrast microscope (200.times.).
[0184] As a result, as shown in FIG. 18, the control group treated
with TGF-.beta. differentiated into myofibroblasts, whereas
myofibroblast differentiation of the test group treated with
TGF-.beta. plus the compound of the present invention was
inhibited.
[0185] In addition, in order to examine whether the effect as
described above would be attributable to the effect against EMT,
the expression patterns of Snail and Vimentin, which are
representative markers of EMT, in the cells of each test group,
were analyzed by real-time PCR.
[0186] As a result, as shown in FIG. 19, the expression of these
markers in the control group treated with TGF-.beta. significantly
increased, whereas the expression of these markers in the test
group treated with TGF-.beta. plus the compound of the present
invention was significantly inhibited.
[0187] These results support that the compound of the present
invention strongly inhibits the differentiation of cells into
myofibroblasts by affecting the EMT of the cells, and thus can
effectively prevent or treat fibrosis, particularly fibrosis such
as idiopathic pulmonary fibrosis which occurs in lungs.
Experimental Example 2: Examination of Pharmacokinetics of Compound
of the Present Invention
2-1: Metabolic Stability
[0188] The compound of the present invention was incubated with rat
liver microsomes and NADPH, and then the rate of elimination of the
compound was measured, thereby determining the intrinsic clearance
value (CLint value) of the compound. As controls, verapamil and
atenolol were used.
[0189] As a result, it was shown that the compound of the present
invention was metabolically stable such that it could be comparable
with atenolol (see Table 1 below and FIG. 20).
TABLE-US-00001 TABLE 1 Rat liver microsomes CL int Compound t1/2
(.mu.l/min/ Clearance name (min) mg protein) value Compound 1 71.43
19.40 Moderate Verapamil 6.37 217.68 High Atenolol 9415.85 0.15
Low
2-2: Solubility in Buffer
[0190] The compound of the present invention was added to phosphate
buffer (pH 7.4) at a concentration of 1 mg/ml and stirred. After 16
hours, the sample was filtered through a filter, and the
supernatant was analyzed by HPLC-UV, thereby determining the
content of compound dissolved. As controls, caffeine and diethyl
stilbesterol were used.
[0191] As a result, it was shown that the solubility of the
compound of the present invention in the buffer was lower than that
of caffeine, but higher than that of diethyl stilbesterol (see
Table 2 below).
TABLE-US-00002 TABLE 2 Compound name Solubility (.mu.g/ml) Caffeine
972.68 Diethyl stilbesterol 4.50 Compound 1 9.59
2-3: Whether to Inhibit CYP450
[0192] Whether the compound of the present invention inhibits
CYP3A4, CYP2D6 and CYP2C9 was examined. As controls, ketoconazole,
quinidine and sulfaphenazole were used.
[0193] As a result, it was shown that the compound of the present
invention had slight inhibitory activity against CYP2C9, but this
inhibitory activity was lower than that of the control
sulfaphenazole, and the compound of the present invention had
little or very low inhibitory activity against CYP3A4 and CYP2D6
(see Table 3 below).
TABLE-US-00003 TABLE 3 3A4- 3A4- midazolam testosterone 2D6 2C9
Compound IC50 IC50 IC50 IC50 name (.mu.M) (.mu.M) (.mu.M) (.mu.M)
Compound 1 >50 49.72 >50 5.54 Ketoconazole 0.027 0.023 na na
Quinidine na na 0.047 na Sulfaphenazole na na na 0.40 * na: not
activity
2-4: Pharmacokinetics after Oral Administration
[0194] To examine the pharmacokinetics of the compound after oral
administration, rat models were used.
[0195] Male SD rats (weighed 250 to 300 g) were divided into
several groups, each consisting of 3 rats, and the compound of the
present invention was orally administered thereto at 200 mpk, after
which the amount of the compound present in the plasma was measured
with the passage of time. For oral administration, the compound was
formulated with 0.5% CMC and about 1% Tween-80 [Tween80: 0.5% CMC
aqueous solution=1:99 (v/v)] and used.
[0196] As a result, it was shown that the compound of the present
invention could be absorbed rapidly in vivo after oral
administration (see Table 4 below and FIG. 21).
TABLE-US-00004 TABLE 4 Compound 1 PK parameters (200 mpk, po) Cmax
(ng/ml) 260.8 .+-. 41.4 Tmax (h) 1 .+-. 0 AUC (inf) (h*ng/ml) 426.5
.+-. 67.7 AUC (0-24) (h*ng/ml) 420.1 .+-. 69.3 AUC_% Extrap (obs)
1.6 .+-. 0.7 MRT (inf) (h) 1.7 .+-. 0.2 t1/2 (h) 1.5 .+-. 0.4 *Mean
.+-. SD (n = 3 rats/group)
Comparative Experimental Example 1: Examination of the Effect of
Comparative Examples Against Fibrosis
[0197] The effects of the following comparative compounds against
fibrosis were examined in the same manner as described in
Experimental Example 1-1 above.
[0198] As a result, as shown in FIGS. 22 to 24, unlike the compound
of the present invention, the comparative compounds failed to
induce cell death by cytotoxicity and to block the progression of
cell fibrosis.
##STR00006## ##STR00007##
Experimental Example 3: Pharmacokinetics of Formulations
[0199] For effective formulation of the compound of the present
invention, the pharmacokinetics of various formulations after oral
administration were examined according to the same method as
described in Experimental Example 2-4 above.
[0200] As a result, as shown in Table 5 below, when the compound
was formulated using Tween80: 0.5% CMC aqueous solution (1:99 v/v)
(Formulation Example 2), the bioavailability of the compound was as
low as 1.4%, and when the compound was formulated using
NMP:ethanol:PEG200:normal saline (5:10:30:55 v/v) (Formulation
Example 4), the bioavailability of the compound was about 3%. On
the other hand, it was shown that when the compound was formulated
using NMP:PEG400:SOLUTOL HS:water (10:20:20:50 v/v) (Formulation
Example 3), the bioavailability of the compound was greatly
improved to about 9%, and when the compound was formulated using
30% HPCD aqueous solution (Formulation Example 5), the
bioavailability of the compound was also greatly improved to about
7%.
TABLE-US-00005 TABLE 5 PK parameters Compound 1 Compound 1 Compound
1 Compound 1 Compound 1 Compound 1 (1 mpk, i.v) (10 mpk, po) (200
mpk, po) (10 mpk, po) (10 mpk, po) (10 mpk, po) Formulation
Formulation Formulation Formulation Formulation Formulation Example
1 Example 2 Example 2 Example 3 Example 4 Example 5 C.sub.o (ng/ml)
506.4 .+-. 129.7 -- -- -- -- -- Cmax (ng/ml) -- 22.3 .+-. 8.9 260.8
.+-. 41.4 62.2 .+-. 37.5 46.7 .+-. 8.8 393.3 .+-. 299.3 Tmax (h) --
0.4 .+-. 0.1 1 .+-. 0 0.25 .+-. 0.1 0.25 .+-. 0.0 1.3 .+-. 0.6
AUC.sub.(inf) (h*ng/ml) 149.3 .+-. 3.0 426.5 .+-. 67.7 155.2 .+-.
7.5 46.4 .+-. 14.1 1075.9 .+-. 326.9 AUC.sub.(0-t) (h*ng/ml) 146.6
.+-. 2.2 20.4 .+-. 6.2 420.1 .+-. 69.3 126.2 .+-. 19.1 42.5 .+-.
16.0 1000.5 .+-. 313.5 AUC_% Extrap (obs) 1.8 .+-. 0.5 -- 1.6 .+-.
0.7 -- -- 7.0 .+-. 5.2 Vd (LAT) 3.6 .+-. 0.25 -- -- -- -- -- CLp
(L/hr/kg) 6.7 .+-. 0.4 -- -- -- -- -- MRT(inf) (h) 0.4 .+-. 0.0 --
1.7 .+-. 0.2 -- -- 3.2 .+-. 0.7 t.sub.1/2 (h) 0.38 .+-. 0.03 -- 1.5
.+-. 0.4 -- -- 1.9 .+-. 0.6 Bioavailability (%) -- 1.4 1.4 9 3 7 *
Formulation Example 1 Use of 100% DMSO as i.v. vehicle *
Formulation Example 2 Use of Tween80: 0.5% CMC aqueous solution (1:
99 v/v) * Formulation Example 3 Use of NMP: PEG400: SOLUTOL HS:
water (10: 20: 20: 50 v/v) * Formulation Example 4 NMP: Use of
ethanol: PEG200: normal saline (5: 10: 30: 55 v/v) * Formulation
Example 5 Use of 30% (w/v) HPCD (hydroxypropyl-beta-cyclodextrin)
aqueous solution.
[0201] In addition, as shown in FIG. 25, the compound in
Formulation Example 2 was absorbed rapidly in vivo after oral
administration and was maintained in the plasma for a long period
of time without being eliminated, compared to the compound in
Formulation Example 3, indicating that it could exhibit a
long-lasting effect.
[0202] In addition, formulation was attempted by other methods, but
in most cases, there was a problem in that the compound was not
completely dissolved and the solution became turbid. Due to this
problem, these methods were excluded from additional
pharmacokinetic studies.
Experimental Example 4: Examination of the Effect of Compound of
the Present Invention Against Fibrosis in Animal Models
[0203] 5-week-old male C57BL/6 mice (weighed 18.2 to 20.5 g)
(KOATECH, Korea) were used as experimental animals and divided into
several groups, each consisting of 5 animals.
[0204] The experimental animals were housed in a housing box having
a size of 369 L.times.156 W.times.132H (mm) (EU, USA, UK GL
compliance) made of a polysulfone material in a SPF (Specific
Pathogen Free) and BSL (Bio Safety Level) 2 grade facility. The
number of animals in each housing box was 2 to 3 during the period
of quarantine and acclimatization and was also 2 to 3 during the
experimental period, and the housing conditions were set to a
temperature of 22.+-.2.degree. C., a relative humidity of
50.0.+-.15.0%, a ventilation cycle of 10 to 20 times/hr, a
light-dark cycle (a photoperiod) of 12 hr/day (07:00 to 19:00), and
an illumination intensity of 150 to 300 Lux.
[0205] Pulmonary fibrosis was induced by injecting a bleomycin
solution directly into the lungs via the trachea according to the
intratracheal instillation (IT) method of Kremer, Laxer and Berkman
et al. Specifically, C57BL/6J mice were anesthetized through
inhalation with 70% N.sub.2O and 30% O.sub.2 gas and 1.5%
isoflurane, and the skin of the anterior neck thereof was excised
and the organs under the muscle thereof were exposed and then
carefully excised using ophthalmic surgical scissors. 50 .mu.L of a
solution of bleomycin in distilled water was injected directly into
the lungs all at once via the excised organ by use of a 1 mL
syringe fitted with a 19-gauge injection needle having a blunt tip.
Immediately after the injection, the excised skin of the anterior
neck was sutured and the mice were allowed to recover from the
anesthetic, and then housed in a general housing cage. The
administration of bleomycin was performed using a visual
instillobot, and bleomycin-HCl 40 .mu.g/50 .mu.L was administered
once and a pulmonary fibrosis induction period of 12 days was
set.
[0206] The compound of the present invention was used after
dissolution in 30% HPCD aqueous solution (Formulation Example 5),
and the dose for each individual was calculated based on the recent
body weight thereof. 12 Days after the administration of bleomycin,
the compound of the present invention was administered once a day
(5 times a week) for 2 weeks. As a control, pirfenidone which is a
therapeutic agent for idiopathic pulmonary fibrosis was
administered in the same manner.
[0207] Changes in the body weight for 0-15 days after
administration of the compound of the present invention or
pirfenidone were examined, and the results are shown in FIG.
26.
[0208] On 15 days after administration of the compound, the mice
were sacrificed, and then the right lung of each mouse was
dissected and the weight thereof was measured. As a result, it was
shown that as pulmonary fibrosis was induced by administration of
bleomycin, the weight of the right lung increased, and the compound
of the present invention significantly inhibited this increase in
the right lung weight (see FIG. 27). In particular, it is noted
that the inhibitory effect of the compound in the group
administered at 10 mpk was the highest compared to that in the
group administered at 50 or 100 mpk and was also higher than that
in the group administered with pirfenidone 100 mpk.
[0209] The amounts of hydroxyproline (HP) and collagen in the
dissected right lung were measured using a known method. As a
result, it was shown that as pulmonary fibrosis was induced by
administration of bleomycin, the amounts of hydroxyproline (HP) and
collagen in the dissected right lung increased, and the compound of
the present invention significantly inhibited this increase (see
FIGS. 28 and 29). The results of measurement of hydroxyproline
indicated that the inhibitory effect of the compound of the present
invention was highest in the group administered at 100 mpk, and the
results of measurement of collagen indicated that the inhibitory
effect of the compound of the present invention was highest in the
group administered at 100 mpk, like the results of measurement of
the right lung weight.
[0210] Histopathology collagen morphometry of the dissected left
lung tissue was performed using a known method (calculating the
area of collagen in an optical image obtained by Sirius red
staining of the lung tissue sample). As a result, it was shown that
as pulmonary fibrosis was induced by administration of bleomycin,
an increased amount of collagen was observed, and the compound of
the present invention significantly inhibited this increase (see
Table 6 below and FIG. 30).
TABLE-US-00006 TABLE 6 T-test T-test Mean SEM Protection P value
Significance Collagen Collagen Collagen Collagen Collagen Group
Histopathology % % % % % 1 Sham 0.6 0.1 2 Vehicle control 6.0 0.7
0.0000 *** 3 Pirfenidone 100 mpk 4.1 0.7 34 0.0653 NS PO (30% HPCD)
4 Compound 1 10 mpk PO 2.8 0.4 59 0.0005 *** 5 Compound 1 50 mpk PO
2.4 0.6 66 0.0006 *** 6 Compound 1 100 mpk 2.8 0.7 59 0.0041 ** PO
NS: not significant
[0211] The amounts of the compound present in the plasma and lung
of each of the sacrificed mice (finally administered with each
compound at 1 hour before sacrifice) were measured. As a result, it
was shown that in the group administered with pirfenidone, the
concentration of the compound in the plasma and lung was high,
whereas in the group administered with the compound of the present
invention, the concentration of the compound was relatively very
low (see FIGS. 31 and 32). This suggests that although the compound
of the present invention was present in the plasma and lung at very
lower concentrations than pirfenidone, it exhibited the excellent
fibrosis inhibitory effect as described above, supporting the
superiority of the compound of the present invention. If it is
attempted to formulate the compound of the present invention in
various manners, it is expected that the compound of the present
invention can effectively treat fibrosis even at lower
concentrations.
[0212] The pathological conditions of the left lung tissue
dissected using a known method were examined by H&E and Sirius
Red staining. As a result, it was shown that as pulmonary fibrosis
was induced by administration of bleomycin, the densities of cells
and proteins in the lung tissue increased, and tissue fibrosis and
inflammatory cell invasion were observed, but the compound of the
present invention significantly inhibited such fibrosis symptoms
(see FIGS. 33 to 38). In particular, it is noted that the
inhibitory effect of the compound in the group administered at 10
mpk was the highest compared to that in the groups administered at
other concentrations.
[0213] In order to examine the effect of each treatment in this
Experiment on the liver, the liver of each of the sacrificed mice
was dissected, and the pathological conditions of the liver tissue
were examined by H&E staining. As a result, it shown that
hepatotoxic symptoms such as liver tissue invasion appeared due to
administration of bleomycin, and the group administered with
pirfenidone and the groups administered with the compound of the
present invention at 50 and 100 mpk also showed hepatotoxic
symptoms due to bleomycin, but the group administered with the
compound of the present invention at 10 mpk showed the same
condition as that of normal liver tissue, even though it was
treated with bleomycin (see FIGS. 39 to 41). This suggests that
when the compound of the present invention is administered at 10
mpk, it can exhibit a preventive, alleviating or therapeutic effect
against hepatotoxicity.
Experimental Example 5: Examination of the Effect of Compound of
the Present Invention Against Non-Alcoholic Steatohepatitis in
Animal Models
[0214] STAM mice (SMC Laboratories, Inc., Japan), which are
non-alcoholic steatohepatitis animal models, were used as
experimental animals. These animal models were administered with
streptozotocin immediately after birth, and fed with high-fat-diet
after 3 weeks. Then, in the mouse model, non-alcoholic
steatohepatitis occurred at 6 to 9 weeks, and liver fibrosis,
cirrhosis and liver cancer occurred within 9 to 12 weeks.
[0215] Over 6 to 9 weeks after administration of streptozotocin,
the compound of the present invention, dissolved in a mixture
solution of Tween80 and 0.5% CMC aqueous solution (1:99 (v/v))
(Formulation Example 2), was orally administered to the animal
models once a day (five times a week). As controls, telmisartan and
OCA (obeticholic acid) were administered in the same manner.
[0216] At 8 weeks after administration of streptozotocin (a time
point after 3-week administration of the compound), the mice were
sacrificed, the liver of each mouse was dissected, the invasion of
inflammatory cells was observed by Sirius Red staining of the liver
tissue, and fibrosis of the liver tissue was observed by Sirius Red
staining (see FIG. 42). In addition, the degrees of inflammation,
fibrosis, liver cell ballooning and adipose accumulation observed
were measured, data integration was performed, and the NAFLD
(nonalcoholic fatty liver disease) activity score was statistically
processed by t-test. As a result, it was shown that statistically
significant anti-NASH effects appeared in the group administered
with the compound of the present invention at 50 mpk and in the
group administered with telmisartan at 30 mpk (see FIG. 43).
[0217] As described above, the novel compound of the present
invention can regulate the activation of EMT
(epithelial-mesenchymal transition) by effectively regulating the
expression of snail and vimentin which are regulators of EMT, and
thus can effectively prevent, alleviate or treat fibrosis.
Furthermore, the novel compound of the present invention has very
good pharmacokinetics so that it can be absorbed rapidly in vivo
even after oral administration, can exhibit stable effects in vivo
and can be safely used without significant side effects. In
addition, the novel compound of the present invention can
effectively inhibit fibrosis of liver cells, and thus can also
effectively alleviate or treat non-alcoholic steatohepatitis.
* * * * *
References