U.S. patent application number 16/421867 was filed with the patent office on 2020-05-07 for compositions and methods of modulating 15-pgdh activity.
The applicant listed for this patent is CASE WESTERN RESERVE UNIVERSITY BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM UNIVERSITY OF KENTUCKY RESEARCH FOUNDATION. Invention is credited to Monika Antczak, KiBeom Bae, Amar Desai, Stanton Gerson, Sanford Markowitz, Bruce Posner, Joseph Ready, Hsin-Hsiung Tai, James K.V. Willson, Sung Yeun Yang, Youngyou Zhang.
Application Number | 20200140453 16/421867 |
Document ID | / |
Family ID | 49384006 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200140453 |
Kind Code |
A1 |
Markowitz; Sanford ; et
al. |
May 7, 2020 |
COMPOSITIONS AND METHODS OF MODULATING 15-PGDH ACTIVITY
Abstract
Compounds and methods of modulating 15-PGDH activity, modulating
tissue prostaglandin levels, treating disease, diseases disorders,
or conditions in which it is desired to modulate 15-PGDH activity
and/or prostaglandin levels include 15-PGDH inhibitors and 15-PGDH
activators described herein.
Inventors: |
Markowitz; Sanford; (Pepper
Pike, OH) ; Willson; James K.V.; (Dallas, TX)
; Posner; Bruce; (Richardson, OH) ; Ready;
Joseph; (Carrollton, TX) ; Zhang; Youngyou;
(Cleveland, OH) ; Tai; Hsin-Hsiung; (Lexington,
KY) ; Antczak; Monika; (Ft. Worth, TX) ;
Gerson; Stanton; (Hunting Valley, OH) ; Bae;
KiBeom; (Buk-gu, KR) ; Yang; Sung Yeun;
(Yeonje-Gu, KR) ; Desai; Amar; (Cleveland,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CASE WESTERN RESERVE UNIVERSITY
BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM
UNIVERSITY OF KENTUCKY RESEARCH FOUNDATION |
Cleveland
Austin
Lexingont |
OH
TX
KY |
US
US
US |
|
|
Family ID: |
49384006 |
Appl. No.: |
16/421867 |
Filed: |
May 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15359330 |
Nov 22, 2016 |
10301320 |
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16421867 |
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14395021 |
Oct 16, 2014 |
9790233 |
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PCT/US2013/036790 |
Apr 16, 2013 |
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15359330 |
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61624670 |
Apr 16, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 213/75 20130101;
A61P 7/00 20180101; A61P 7/02 20180101; C07D 209/08 20130101; C07D
277/46 20130101; C07D 495/04 20130101; A61P 1/00 20180101; C07D
215/08 20130101; A61P 29/00 20180101; A61K 31/519 20130101; C07D
237/20 20130101; A61P 19/00 20180101; A61P 43/00 20180101; C07C
2601/14 20170501; A61P 19/10 20180101; C07D 207/263 20130101; C07D
213/73 20130101; C07D 241/20 20130101; A61K 31/4365 20130101; A61P
19/08 20180101; A61P 7/06 20180101; C07D 213/76 20130101; A61P
37/06 20180101; A61P 1/16 20180101; C07D 213/71 20130101; C07D
295/185 20130101; C07D 405/12 20130101; A61P 1/04 20180101; C07D
239/42 20130101; A61P 17/02 20180101; C07D 275/03 20130101; C07C
311/21 20130101; C07D 261/14 20130101 |
International
Class: |
C07D 495/04 20060101
C07D495/04; C07C 311/21 20060101 C07C311/21; C07D 213/71 20060101
C07D213/71; C07D 213/73 20060101 C07D213/73; C07D 213/75 20060101
C07D213/75; C07D 213/76 20060101 C07D213/76; C07D 215/08 20060101
C07D215/08; C07D 237/20 20060101 C07D237/20; C07D 405/12 20060101
C07D405/12; C07D 239/42 20060101 C07D239/42; C07D 241/20 20060101
C07D241/20; C07D 261/14 20060101 C07D261/14; C07D 207/263 20060101
C07D207/263; C07D 275/03 20060101 C07D275/03; C07D 209/08 20060101
C07D209/08; C07D 277/46 20060101 C07D277/46; C07D 295/185 20060101
C07D295/185; A61K 31/4365 20060101 A61K031/4365; A61K 31/519
20060101 A61K031/519 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under Grant
No. R01CA127306, R01CA127306-03S1, AND 5P50CA150964, awarded by The
National Institutes of Health. The United States government may
have certain rights to the invention.
Claims
1-81. (canceled)
82: A method of treating oral and/or gastrointestinal diseases
associated with inflammation and/or ulcers in a subject in need
thereof, the method comprising: administering to the subject a
therapeutically effective amount of a 15-PGDH inhibitor.
83: The method of claim 82, wherein the ulcer comprises at least
one of a mucosal or submucosal ulcer.
84: The method of claim 82, wherein the gastrointestinal disease
comprises at least one of oral ulcers or gastrointestinal
ulcers.
85: The method of claim 82, wherein the gastrointestinal disease
comprises at least one of colitis, gastritis, or cryptitis.
86: The method of claim 82, wherein the gastrointestinal disease
comprises ulcerative colitis.
87: The method of claim 82, wherein the gastrointestinal disease
comprises inflammatory bowel disease.
88: The method of claim 82 wherein the 15-PGDH inhibitor is
administered at an amount effective to increase prostaglandin
levels in blood or tissue of the subject
89: The method of claim 82, wherein the 15-PGDH inhibitor is
administered to the subject at an amount effective to inhibit or
treat at least one of oral or gastrointestinal ulcer formation.
90: The method of claim 82, wherein the 15-PGDH inhibitor is
administered to the subject at an amount effective to inhibit or
treat at least one of oral or gastrointestinal inflammation.
91: The method of claim 82, wherein the 15-PGDH inhibitor comprises
a compound having formula (III): ##STR00264## wherein n is 1 or 2;
R.sub.1 is a C.sub.1-8 alkyl, which is linear, branched, or cyclic
and which is unsubstituted or substituted; R.sub.2 and R.sub.3 are
the same or different and are each selected from the group
consisting of a H, a lower alkyl group, (CH.sub.2).sub.n1OR'
(wherein n1=1, 2, or 3), CF.sub.3, CH.sub.2--CH.sub.2X,
O--CH.sub.2--CH.sub.2X, CH.sub.2--CH.sub.2--CH.sub.2X,
O--CH.sub.2--CH.sub.2X (wherein X=F, Cl, Br, or I), CN,
(C.dbd.O)--R', (C.dbd.O)N(R').sub.2, O(CO)R', COOR' (wherein R' is
H or a lower alkyl group); Z.sub.1 is S; X.sub.2 is N or C; R.sub.6
and R.sub.7 are optional and if present are the same or different
and are each selected from the group consisting of a H, F, Cl, Br,
I, a lower alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2, or
3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group); substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, and a substituted or
unsubstituted heterocyclyl; or a pharmaceutically acceptable salt
thereof.
92: A method of treating oral and/or gastrointestinal inflammation
and/or ulcers in a subject in need thereof, the method comprising:
administering to the subject a therapeutically effective amount of
a 15-PGDH inhibitor.
93: The method of claim 92, wherein the subject has at least one of
oral ulcers or gastrointestinal ulcers.
94: The method of claim 92, wherein the subject has at least one of
colitis, gastritis, or cryptitis.
95: The method of claim 92, wherein the subject has ulcerative
colitis.
96: The method of claim 92, wherein the subject has inflammatory
bowel disease.
97: The method of claim 92 wherein the 15-PGDH inhibitor is
administered at an amount effective to increase prostaglandin
levels in blood or tissue of the subject
98: The method of claim 92, wherein the 15-PGDH inhibitor is
administered to the subject at an amount effective to inhibit or
treat at least one of oral or gastrointestinal ulcer formation.
99: The method of claim 92, wherein the 15-PGDH inhibitor is
administered to the subject at an amount effective to inhibit or
treat at least one of oral or gastrointestinal inflammation.
100: The method of claim 92, wherein the 15-PGDH inhibitor
comprises a compound having formula (III): ##STR00265## wherein n
is 1 or 2; R.sub.1 is a C.sub.1-8 alkyl, which is linear, branched,
or cyclic and which is unsubstituted or substituted; R.sub.2 and
R.sub.3 are the same or different and are each selected from the
group consisting of a H, a lower alkyl group, (CH.sub.2).sub.n1OR'
(wherein n1=1, 2, or 3), CF.sub.3, CH.sub.2--CH.sub.2X,
O--CH.sub.2--CH.sub.2X, CH.sub.2--CH.sub.2--CH.sub.2X,
O--CH.sub.2--CH.sub.2X (wherein X=F, Cl, Br, or I), CN,
(C.dbd.O)--R', (C.dbd.O)N(R').sub.2, O(CO)R', COOR' (wherein R' is
H or a lower alkyl group); Z.sub.1 is S; X.sub.2 is N or C; R.sub.6
and R.sub.7 are optional and if present are the same or different
and are each selected from the group consisting of a H, F, Cl, Br,
I, a lower alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2, or
3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group); substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, and a substituted or
unsubstituted heterocyclyl; or a pharmaceutically acceptable salt
thereof.
Description
RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application No. 61/624,670, filed Apr. 16, 2012, the subject matter
of which is incorporated herein by reference in its entirety.
BACKGROUND
[0003] 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) represents
the key enzyme in the inactivation of a number of active
prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids
(HETEs) (e.g., by catalyzing oxidation of PGE.sub.2 to
15-keto-prostaglandin E2, 15k-PGE). The human enzyme is encoded by
the HPGD gene and consists of a homodimer with subunits of a size
of 29 kDa. The enzyme belongs to the evolutionarily conserved
superfamily of short-chain dehydrogenase/reductase enzymes (SDRs),
and according to the recently approved nomenclature for human
enzymes, it is named SDR36C1. Thus far, two forms of 15-PGDH have
been identified, NAD+-dependent type I 15-PGDH and the type II
NADP-dependent 15-PGDH, also known as carbonyl reductase 1 (CBR1,
SDR21C1). However, the preference of CBR1 for NADP and the high Km
values of CBR1 for most prostaglandin suggest that the majority of
the in vivo activity can be attributed to type I 15-PGDH.
[0004] Recent studies suggest that inhibitors of 15-PGDH and
activators of 15-PGDH could be therapeutically valuable. It has
been shown that there is an increase in the incidence of colon
tumors in 15-PGDH knockout mouse models. A more recent study
implicates increased 15-PGDH expression in the protection of
thrombin-mediated cell death. It is well known that 15-PGDH is
responsible for the inactivation of prostaglandin E2 (PGE.sub.2),
which is a downstream product of COX-2 metabolism. PGE.sub.2 has
been found to be neurotoxic both in vitro and in vivo; thus, COX-2
specific inhibitors, which decrease PGE.sub.2 release, exhibit
neuroprotective effects. PGE.sub.2 has also been shown to be
beneficial in a variety of biological processes, such as hair
density, dermal wound healing, and bone formation.
SUMMARY
[0005] Embodiments described herein relate to compounds and methods
of modulating 15-PGDH activities, modulating tissue prostaglandin
levels, and/or treating diseases, disorders, or conditions in which
it is desired to modulate 15-PGDH activity and/or prostaglandin
levels.
[0006] In some embodiments, a 15-PGDH inhibitor can be administered
to a tissue of a subject at an amount effective to increase
prostaglandin levels in the tissue. The 15-PGDH inhibitor can
include formula (I):
##STR00001## [0007] wherein n is 0-2; [0008] R.sub.1 is a C.sub.1-8
alkyl, which is linear, branched, or cyclic and which is
unsubstituted or substituted (e.g., R.sub.1 can be a C.sub.2-6
alkyl, C.sub.2-4 alkyl, or C.sub.4 alkyl, which is linear,
branched, or cyclic and which is unsubstituted or substituted);
[0009] R.sub.2 and R.sub.3 are the same or different and are each
selected from the group consisting of a H, a lower alkyl group,
(CH.sub.2).sub.n1OR' (wherein n1=1, 2, or 3), CF.sub.3,
CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', (C.dbd.O)N(R').sub.2, O(CO)R',
COOR' (wherein R' is H or a lower alkyl group); [0010] R.sub.4 and
R.sub.5 are the same or different and are each selected from the
group consisting of hydrogen, C.sub.1-C.sub.24 alkyl,
C.sub.2-C.sub.24 alkenyl, C.sub.2-C.sub.24 alkynyl,
C.sub.3-C.sub.20 aryl, C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24
aralkyl, halo, silyl, hydroxyl, sulfhydryl, C.sub.1-C.sub.24
alkoxy, C.sub.2-C.sub.24 alkenyloxy, C.sub.2-C.sub.24 alkynyloxy,
C.sub.5-C.sub.20 aryloxy, acyl (including C.sub.2-C.sub.24
alkylcarbonyl (--CO-alkyl) and C.sub.6-C.sub.20 arylcarbonyl
(--CO-aryl)), acyloxy (--O-acyl), C.sub.2-C.sub.24 alkoxycarbonyl
(--(CO)--O-alkyl), C.sub.6-C.sub.20 aryloxycarbonyl
(--(CO)--O-aryl), C.sub.2-C.sub.24 alkylcarbonato
(--O--(CO)--O-alkyl), C.sub.6-C.sub.20 arylcarbonato
(--O--(CO)--O-aryl), carboxy (--COOH), carboxylato (--COO--),
carbamoyl (--(CO)--NH.sub.2), C.sub.1-C.sub.24 alkyl-carbamoyl
(--(CO)--NH(C.sub.1-C.sub.24 alkyl)), arylcarbamoyl
(--(CO)--NH-aryl), thiocarbamoyl (--(CS)--NH.sub.2), carbamido
(--NH--(CO)--NH.sub.2), cyano (--CN), isocyano (--N.sup.+C.sup.-),
cyanato (--O--CN), isocyanato (--O--N.sup.+.dbd.C.sup.-),
isothiocyanato (--S--CN), azido (--N.dbd.N.sup.+.dbd.N.sup.-),
formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH.sub.2),
C.sub.1-C.sub.24 alkyl amino, C.sub.5-C.sub.20 aryl amino,
C.sub.2-C.sub.24 alkylamido (--NH--(CO)-alkyl), C.sub.6-C.sub.20
arylamido (--NH--(CO)-aryl), imino (--CR.dbd.NH where R is
hydrogen, C.sub.1-C.sub.24 alkyl, C.sub.5-C.sub.20 aryl,
C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24 aralkyl, etc.),
alkylimino (--CR.dbd.N(alkyl), where R=hydrogen, alkyl, aryl,
alkaryl, aralkyl, etc.), arylimino (--CR.dbd.N(aryl), where
R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (--NO.sub.2),
nitroso (--NO), sulfo (--SO.sub.2--OH), sulfonato
(--SO.sub.2--O--), C.sub.1-C.sub.24 alkylsulfanyl (--S-alkyl; also
termed "alkylthio"), arylsulfanyl (--S-aryl; also termed
"arylthio"), C.sub.1-C.sub.24 alkylsulfinyl (--(SO)-alkyl),
C.sub.5-C.sub.20 arylsulfinyl (--(SO)-aryl), C.sub.1-C.sub.24
alkylsulfonyl (--SO.sub.2-alkyl), C.sub.5-C.sub.20 arylsulfonyl
(--SO.sub.2-aryl), phosphono (--P(O)(OH).sub.2), phosphonato
(--P(O)(O.sup.-).sub.2), phosphinato (--P(O)(O.sup.-)), phospho
(--PO.sub.2), phosphino (--PH.sub.2), combinations thereof, and
wherein R.sub.4 and R.sub.5 may be linked to form a cyclic or
polycyclic ring, wherein the ring is a substituted or unsubstituted
aryl, a substituted or unsubstituted heteroaryl, a substituted or
unsubstituted cycloalkyl, and a substituted or unsubstituted
heterocyclyl; and pharmaceutically acceptable salts thereof.
[0011] In other embodiments, the 15-PGDH inhibitor can i) at 2.5
.mu.M concentration, stimulate a Vaco503 reporter cell line
expressing a 15-PGDH luciferase fusion construct to a luciferase
output level of greater than 70 (using a scale on which a value of
100 indicates a doubling of reporter output over baseline); ii) at
2.5 .mu.M concentration stimulate a V9m reporter cell line
expressing a 15-PGDH luciferase fusion construct to a luciferase
output level of greater than 75; iii) at 7.5 .mu.M concentration
stimulate a LS174T reporter cell line expressing a 15-PGDH
luciferase fusion construct to a luciferase output level of greater
than 70; iv) at 7.5 .mu.M concentration, does not activate a
negative control V9m cell line expressing TK-renilla luciferase
reporter to a level greater than 20; and v) inhibits the enzymatic
activity of recombinant 15-PGDH protein at an IC50 of less than 1
.mu.M.
[0012] In other embodiments, the 15-PGDH inhibitor can i) at 2.5
.mu.M concentration, stimulate a Vaco503 reporter cell line
expressing a 15-PGDH luciferase fusion construct to increase
luciferase output; ii) at 2.5 .mu.M concentration stimulate a V9m
reporter cell line expressing a 15-PGDH luciferase fusion construct
to increase luciferase output; iii) at 7.5 .mu.M concentration
stimulate a LS174T reporter cell line expressing a 15-PGDH
luciferase fusion construct to increase luciferase output; iv) at
7.5 .mu.M concentration, does not activate a negative control V9m
cell line expressing TK-renilla luciferase reporter to a luciferase
level greater than 20% above background; and v) inhibits the
enzymatic activity of recombinant 15-PGDH protein at an IC50 of
less than 1 .mu.M.
[0013] In other embodiments, the 15-PGDH inhibitor can inhibit the
enzymatic activity of recombinant 15-PGDH at an IC50 of less than 1
.mu.M, or preferably at an IC50 of less than 250 nM, or more
preferably at an IC50 of less than 50 nM, or more preferably at an
IC50 of less than 5 nM.
[0014] In still other embodiments, the 15-PGDH inhibitor can be
applied to skin of a subject to promote and/or stimulate
pigmentation of the skin and/or hair growth and/or inhibit hair
loss. The 15-PGDH inhibitor can also be administered to a subject
to promote wound healing, regenerate tissue, and/or treat at least
one of oral ulcers, ulcerative colitis, gastrointestinal ulcers,
inflammatory bowel disease, vascular insufficiency, colitis,
Raynaud's disease, Buerger's disease, diabetic neuropathy,
pulmonary artery hypertension, cardiovascular disease, diabetic
ulcers, renal disease, and erectile dysfunction. The 15-PGDH
inhibitor can further be administered to a subject in combination
with a prostanoid agonist for the purpose of enhancing the
therapeutic effect of the agonist in prostaglandin responsive
conditions.
[0015] In some embodiments, the 15-PGDH inhibitor can be
administered to tissue of a subject to increase tissue stem cells.
The 15-PGDH inhibitor can also be administered to a bone marrow
graft donor or a hematopoietic stem cell donor to increase the
fitness of a donor bone marrow graft or a donor hematopoietic stem
cell graft. The 15-PGDH inhibitor can be administered to bone
marrow of a subject to increase stem cells in the subject. The
15-PGDH inhibitor can further be administered to bone marrow of a
subject to increase the fitness of the marrow as a donor graft.
[0016] In other embodiments the 15-PGDH inhibitor can be
administered to a preparation of hematopoietic stem cells of a
subject to increase the fitness of the stem cell preparation as a
donor graft. The 15-PGDH inhibitor can also be administered to a
preparation of peripheral blood hematopoietic stem cells of a
subject to increase the fitness of the stem cell preparation as a
donor graft. The 15-PGDH inhibitor can further be administered to a
preparation of umbilical cord stem cells to increase the fitness of
the stem cell preparation as a donor graft.
[0017] In yet other embodiments, the 15-PGDH inhibitor can be
administered to a subject to mitigate bone marrow graft rejection,
to enhance bone marrow graft engraftment, and/or to enhance
engraftment of a hematopoietic stem cell graft, or an umbilical
cord stem cell graft.
[0018] In still other embodiment, the 15-PGDH inhibitor can be
administered to a subject or to a tissue graft of a subject to
mitigate graft rejection or to enhance graft engraftment.
[0019] In other embodiments, the 15-PGDH inhibitor can be
administered to a subject or to tissue of the subject to confer
resistance to toxic or lethal effects of exposure to radiation.
[0020] In other embodiments, the 15-PGDH inhibitor can be
administered to a subject for the treatment of osteoporosis, bone
fractures, or promoting healing after bone injury or joint
replacement.
[0021] In an alternative example, the 15-PGDH inhibitor can be
administered to a subject or to the liver of a subject to promote
liver regeneration following liver resection or following toxic
injury to the liver. In one instance, toxic injury to the liver may
be caused by overdose of acetaminophen or related hepatotoxic
compounds.
[0022] In still other embodiments of the application, a 15-PGDH
activator can be administered to a tissue of a subject at an amount
effective to increase 15-PGDH levels and decrease prostaglandin
levels in the tissue. The 15-PGDH activator can include formula
(IV):
##STR00002## [0023] wherein X.sub.3 and Y.sub.2 are independently C
or SO; [0024] U is OR'' (wherein R'' is H, a substituted or
unsubstituted alkyl group, or substituted or unsubstituted aryl
group) or
[0024] ##STR00003## [0025] R.sub.8, R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each selected from the group consisting of H, F,
Cl, Br, I, an alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2,
or 3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group), a substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted heterocyclyl, and R.sub.8 and R.sub.9 may be linked
to form a cyclic or polycyclic ring; and pharmaceutically
acceptable salts thereof.
[0026] In some embodiments, the activator can i) at 7.5 .mu.M
concentration, stimulate a Vaco503 reporter cell line expressing a
15-PGDH luciferase fusion construct to a luciferase output level of
greater than 50 (using a scale on which a value of 100 indicates a
doubling of reporter output over baseline); ii) at 7.5 .mu.M
concentration stimulate a V9m reporter cell line expressing a
15-PGDH luciferase fusion construct to a luciferase output level of
greater than 50; iii) at 7.5 .mu.M concentration stimulate a LS174T
reporter cell line expressing a 15-PGDH luciferase fusion construct
to a luciferase output level of greater than 50; iv) at 7.5 .mu.M
concentration, does not activate the negative control V9m cell line
expressing TK-renilla luciferase reporter to a level any greater
than 25; and v) against recombinant 15-PGDH protein the compound
shows an IC.sub.50 concentration for inhibiting 15-PGDH enzyme
activity of greater than or equal to 2.5 .mu.M.
[0027] In some embodiments, the activator can i) at 7.5 .mu.M
concentration, stimulate a Vaco503 reporter cell line expressing a
15-PGDH luciferase fusion construct to increase luciferase output;
ii) at 7.5 .mu.M concentration stimulate a V9m reporter cell line
expressing a 15-PGDH luciferase fusion construct to increase
luciferase output; iii) at 7.5 .mu.M concentration stimulate a
LS174T reporter cell line expressing a 15-PGDH luciferase fusion
construct to increase luciferase output; iv) at 7.5 .mu.M
concentration, does not activate the negative control V9m cell line
expressing TK-renilla luciferase reporter to a luciferase level any
greater than 25% above; and v) against recombinant 15-PGDH protein
the compound shows an IC.sub.50 concentration for inhibiting
15-PGDH enzyme activity of greater than or equal to 2.5 .mu.M.
[0028] In other embodiments, the 15-PGDH activator can be
administered to a subject to treat a neoplasia, such as a colon
neoplasia. The 15-PGDH activator can also be administered to a
subject to prevent neoplasia, such as a colon neoplasia. The
15-PGDH activator can also be administered to a subject to reduce
inflammation and/or pain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIGS. 1(A-C) illustrate graphs showing luciferase activity
of cells that express a 15-PGDH luciferase fusion construct created
by targeted gene knock-in of renilla luciferase into the last
coding exon of 15-PGDH treated with the compounds SW033291,
SW054384, and SW145753 at various concentrations. The activity is
demonstrated in three different colon cancer cell lines all
engineered to contain the 15-PGDH-luciferase fusion. These cell
lines are Vaco-9m (V9m), LS174T, Vaco503 (V503).
[0030] FIG. 2 illustrates western blots demonstrating the levels of
15-PGDH protein in cell lines V9M, LS174T, and V503 treated with
7.5 .mu.M of SW033291, SW054384, and SW145753 for 48 hours.
Untreated FET cells provide a positive control for 15-PGDH
expression.
[0031] FIG. 3 illustrates western blots demonstrating 15-PGDH
protein levels in colon cell lines treated with SW124531 (FET cells
treated with TGF-.beta. (10 ng/ml for 48 hours) are used as a
positive control for 15-PGDH expression in certain panels).
[0032] FIG. 4 illustrates western blots demonstrating the levels of
15-PGDH protein (wt-PGDH) expressed from a cDNA expression vector
in V400-S3-2-32 cells treated with 5 .mu.M SW124531, and protein
levels of a catalytically dead mutant 15-PGDH (mu-PGDH) also
expressed from a cDNA expression vector in V400-M3-2-72 cells
treated with SW124531.
[0033] FIGS. 5(A-C) illustrate 15-PGDH protein levels in V503 cells
treated with SW124531 as assayed by immuno-fluorescence (upper two
rows) and by western blot (lower panel).
[0034] FIGS. 6(A-F) illustrate graphs showing 15-PGDH mRNA levels
in colon cancer cell lines treated with SW033291.
[0035] FIGS. 7(A-C) illustrate graphs showing 15-PGDH mRNA levels
in colon cancer cell lines treated with SW033291.
[0036] FIGS. 8(A-C) illustrate graphs showing 15-PGDH mRNA levels
in colon cancer cell lines treated with SW054384 and SW145753.
[0037] FIGS. 9(A-I) illustrate graphs showing 15-PGDH mRNA levels
in colon cancer cell lines treated with 5 .mu.M SW124531.
[0038] FIGS. 10(A-C) illustrate graphs showing 15-PGDH activity in
cell lines treated with SW033291, SW054384, and SW145753. Activity
is measured as pmol PGE.sub.2/min/million cells.
[0039] FIGS. 11(A-D) illustrates a table and plots showing activity
of recombinant 15-PGDH protein (a 15-PGDH-GST fusion protein)
incubated with varying concentrations of the test compounds.
[0040] FIGS. 12(A-D) illustrate plots showing the activity of
recombinant 15-PGDH protein treated with SW033291 and SW054384,
with panels 12A and C measuring transfer of tritium from a
radiolabeled PGE2 substrate and panels 12 B and D measuring
generation of NADH by fluorescence.
[0041] FIG. 13 illustrates a table and plot showing 15-PGDH
activity measured by following transfer of tritium from a
radiolabeled PGE2 substrate in cells treated with SW124531 (upper
panel) and in recombinant 15-PGDH protein treated with SW124531
(lower panel).
[0042] FIGS. 14(A-B) illustrate melt curves and a table showing
different compound's ability to directly bind to recombinant
15-PGDH protein as measured by shifting the melting temperature of
the protein.
[0043] FIGS. 15(A-B) illustrate melt curves temperature of
catalytically inactive mutant 15-PGDH protein treated with the test
compounds.
[0044] FIGS. 16(A-B) illustrates graphs showing PGE.sub.2 levels
that are assayed in the medium of A549 cells that have been
stimulated by IL1-beta for 23 hours, with the test compounds.
[0045] FIG. 17 illustrates a graph showing the dose response effect
of SW033291 on PGE.sub.2 production from IL1-beta treated A549
cells.
[0046] FIGS. 18(A-B) illustrate graphs showing the in vivo
modulations by compounds (2.5 .mu.M) of PGDH activity as reflected
in PGE.sub.2 levels following addition of PGE.sub.2 into the medium
of Vaco-503 cells.
[0047] FIG. 19 illustrates images showing the activity of SW033291
in speeding the healing of a model wound consisting of a scratch in
a monolayer of HaCaT cells observed over 48 hours of treatment.
[0048] FIGS. 20(A-B) illustrate graphs showing the quantitation of
scratch width at 0 and 48 hours in the control, SW033291 (2.5
.mu.M) treated cells, and the TGF-beta (1 ng/ml) treated cells.
[0049] FIGS. 21(A-B) illustrate plots showing: (A) percent
inhibition of PGDH using titrations of 15-PGDH inhibitor SW033291
run at different 15-PGDH concentrations; and (B) the IC50 of
15-PGDH inhibitor SW033291 versus 15-PGDH concentration.
[0050] FIGS. 22(A-B) illustrate graphs showing: (A) 15-PGDH enzyme
inhibiting activity and (B) percent inhibition of activity due to
of SW033291 as measured before and after dialysis of the 15-PGDH
and SW033291 mixture.
[0051] FIGS. 23(A-B) illustrate a plot showing reaction rates and
relative reaction velocity of 15-PGDH at varying concentrations of
SW033291.
[0052] FIGS. 24(A-B) illustrate plots showing: (A) inhibition of
15-PGDH by SW033291 in the presence of PGE-2; and (B) IC50 of
SW033291 against 15-PGDH versus PGE2 concentration.
[0053] FIG. 25 illustrates a schematic diagram showing the
structure activity relationships of analogues of SW033291 versus
their IC50 against 15-PGDH.
[0054] FIG. 26 illustrates a schematic diagram showing additional
analogues of SW033291.
[0055] FIGS. 27(A-C) illustrate graphs showing luciferase activity
of colon cancer cell lines V503, LS174T, and V503 treated with 2.5
.mu.M and 7.5 .mu.M the compounds of FIG. 26.
[0056] FIG. 28 illustrates a graph showing percent inhibition of
15-PGDH activity by the compounds of FIG. 26.
[0057] FIGS. 29(A-B) illustrate plots showing the IC50 against
15-PGDH of SW033291 and SW0206980.
[0058] FIGS. 30(A-B) illustrate plots showing melting profiles of
SW0206890 and SW033291 binding to 15-PGDH.
[0059] FIGS. 31(A-C) illustrate plots showing percent inhibition of
15-PGDH activity by SW033291, SW206980, and SW206992.
[0060] FIGS. 32(A-C) illustrate graphs showing luciferase activity
of colon cancer cell lines V503, LS174T, and V503 treated with
various concentrations of SW033291.
[0061] FIG. 33(A-C) illustrate graphs showing luciferase activity
of colon cancer cell lines V503, LS174T, and V503 treated with
various concentrations of SW0206980.
[0062] FIG. 34(A-C) illustrate graphs showing luciferase activity
of colon cancer cell lines V503, LS174T, and V503 treated with
various concentrations of SW0206992.
[0063] FIGS. 35(A-B) illustrate plots showing melting profiles of
SW206992, SW0206890 and SW033291 binding to 15-PGDH.
[0064] FIGS. 36(A-B) illustrate plots showing melting profiles of
SW206992, SW0206890 and SW033291 binding to 15-PGDH.
[0065] FIGS. 37(A-C) illustrate graphs showing the effect of
SW206992, SW0206890 and SW033291 on the regulation of PGE-2 in A549
cells stimulated with IL1-Beta.
[0066] FIGS. 38(A-C) illustrate graphs showing the effect of
SW206992, SW0206890 and SW033291 on cell numbers in A549 cells
after stimulated with IL1-Beta.
[0067] FIG. 39 illustrates a schematic diagram of additional
analogues of SW033291.
[0068] FIGS. 40(A-C) illustrate graphs showing luciferase activity
of colon cancer cell lines V9M, LS174T, and V503 treated with 2.5
.mu.M and 7.5 .mu.M the compounds of FIG. 39.
[0069] FIG. 41 illustrates a graph showing percent inhibition of
15-PGDH activity by the compounds of FIG. 40.
[0070] FIGS. 42(A-D) illustrate a graph showing percent inhibition
of 15-PGDH activity by the compounds of FIG. 40.
[0071] FIG. 43 shows the dose response curve for induction of a
15-PGDH-luciferase fusion gene reporter in the V9m cell line
background of SW033291, SW208064, SW208065, SW208066, and
SW208067.
[0072] FIG. 44 illustrates titration curves of 15-PGDH inhibitor
compounds in an assay measuring effects on PGE2 levels in the
medium of A549 cells that have been stimulated with IL1-beta.
[0073] FIG. 45 is a plot showing weight change of FVB mice treated
with SW033291.
[0074] FIGS. 46(A-C) illustrate graphs showing: (A) total bone
marrow cellularity; (B) SKL population of wild type versus PGDH-/-
mice; and (C) average CFU counts in wild type versus PGDH-/- mice
(designated as either PGDH-/- or as PGDH).
[0075] FIG. 47 illustrates a graph showing CFU counts in wild type
bone marrow treated with SW033291 and PGE-2.
[0076] FIGS. 48(A-C) illustrate graphs showing: (A) bone marrow
cellularity of mice treated with SW033291; (B) SKL % in whole bone
marrow of mice treated with SW033291; and (C) CFU counts in mice
treated SW033291.
[0077] FIGS. 49(A-B) illustrate: (A) a schematic diagram following
CD45.2 antigen marked cells in lethally irradiated C57BL/6J mice
rescued with a bone marrow transplant from donor mice treated with
SW033291 or with vehicle; and (B) graphs showing chimerism, of
donor B-Cells, myeloid cells, and T-Cells after such treatment.
[0078] FIG. 50 illustrates a schematic diagram showing schema of a
study in which C57BL/61 mice are irradiated with 11 GY on day 0 and
followed by treatment with SW033291.
[0079] FIG. 51 illustrates a schematic diagram of a partial
hepatectomy.
[0080] FIGS. 52(A-D) illustrate photographs showing preoperative
and post-operative view of mouse liver.
[0081] FIGS. 53(A-D) illustrate photographs showing
post-hepatectomy views of the mouse liver (at left) and
regeneration of mouse liver on post-operative day 7 (at right).
[0082] FIGS. 54(A-B) illustrate micrographs of post-hepatectomy
mouse livers of mouse administered SW033291 and control vehicle,
with arrows designating mitotic figures.
[0083] FIG. 55 illustrates a graph showing mitosis in liver of
SW033291 treated mouse versus the control mouse.
[0084] FIG. 56 illustrates a graph showing the liver to body weight
ratios attained following partial hepatectomy in control versus
SW033291 treated C57Bl/6J mice.
[0085] FIG. 57 illustrates a graph showing the liver to body weight
ratios attained following partial hepatectomy in control versus
SW033291 twice daily treated C57Bl/6J mice.
[0086] FIG. 58 illustrates a graph reprising the liver to body
weight ratios attained following partial hepatectomy in control
versus SW033291 treated C57Bl/6J mice.
[0087] FIGS. 59(A-B) illustrate a graph and plot showing ALT levels
following partial hepatectomy in one mouse control versus one mouse
treated with SW033291.
[0088] FIG. 60 illustrates a graph showing serum bilirubin levels
following partial hepatectomy in a control mouse and a mouse
treated with SW033291.
[0089] FIG. 61 illustrates a graph showing the liver to body weight
ratios attained following partial hepatectomy in control versus
SW033291 treated FVB mice.
[0090] FIG. 62 illustrates a graph showing preoperative body
weights in control versus SW033291 treated FVB mice.
[0091] FIG. 63 illustrates a graph showing the weight of the
resected liver segment from mice treated with either SW033291 or
vehicle control and assayed for liver regeneration.
[0092] FIG. 64 illustrates a graph showing liver weights attained
post partial heptatectomy in SW033291 and control mice.
[0093] FIG. 65 illustrates a graph showing the liver to body weight
ratios obtained post partial hepatectomy in SW033291 treated and
control mice.
[0094] FIG. 66 illustrates a "box and whisker" plot comparing liver
to body weight ratio following partial hepatectomy of SW033291
treated and control FVB mice at post-operative day 4.
[0095] FIG. 67 illustrates a "box and whisker" plot comparing liver
to body weight ratio following partial hepatectomy of SW033291
treated and control FVB mice at post-operative day 7.
[0096] FIG. 68 illustrates a "box and whisker" plot comparing liver
to body weight ratio following partial hepatectomy of SW033291
treated and control FVB mice at post-operative day 4.
[0097] FIG. 69 illustrates photographs of S-phase cells following
partial hepatectomy on post-operative day 2 in livers of SW033291
treated and vehicle treated control mice.
[0098] FIG. 70 illustrates a photograph showing high powered
(40.times.) views of representative fields from the study of FIG.
69.
[0099] FIG. 71 illustrates a "box and whiskers" plot comparing
percent of BrdU positive cells in livers of SW033291 treated versus
vehicle control treated mice on post-operative day 2 following
partial hepatectomy.
[0100] FIG. 72 illustrates a graph showing the average changes from
baseline weight of the cohort of control versus SW033291 treated
mice all treated with 2% dextran sulfate sodium (DSS) in the
drinking water.
[0101] FIG. 73 illustrates a graph of the daily disease activity
index of the cohort of control versus SW033291 treated mice all
treated with 2% DSS in the drinking water.
[0102] FIG. 74 illustrates a graph showing the average changes from
baseline weight of the cohort of DSS treated mice receiving a
control vehicle versus SW033291.
[0103] FIGS. 75(A-B) illustrates: (A) a graph showing the number of
ulcers in a colon of DSS treated mice receiving a control vehicle
versus SW033291; and (B) photographs showing ulcers of DSS treated
mice receiving control (left) or SW033291 (right).
[0104] FIG. 76 illustrates a graph showing quantitation of ulcer
burden on day 15 of DSS treated mice.
[0105] FIGS. 77(A-B) illustrate photographs showing colonoscopic
findings and mouse endoscopic index of colitis severity (MEICs) for
a DSS treated mouse receiving a control vehicle or SW033291.
[0106] FIG. 78 illustrates a graph showing MEICS score of DSS
treated mice receiving a control vehicle or SW033291.
[0107] FIG. 79 illustrates photomicrographs of high powered fields
from the mid-colon on day 8 of the DSS protocol from control mice,
SW033291 treated mice (treatment) and 15-PGDH knockout mice (KO)
and a graph depicting sum of the average number of BrdU positive
cells per crypt in the distal plus middle colons of control (Cn),
SW033219 treated mice (Tx), and 15-PGDH knockout mice (KO) on day
1, day 8, and day 15 of the DSS treatment protocol.
[0108] FIG. 80 illustrates a graph showing colon length at day 22
of DSS treated mice receiving a control vehicle or SW033291.
[0109] FIG. 81 illustrates a schematic diagram of analogues of
SW054384.
[0110] FIGS. 82(A-C) illustrate graphs showing luciferase activity
of colon cancer cell lines V9M, LS174T, and V503 treated with 2.5
.mu.M and 7.5 .mu.M the compounds of FIG. 81.
[0111] FIG. 83 illustrates a graph showing percent inhibition of
15-PGDH activity by the compounds of FIG. 74.
[0112] FIGS. 84(A-C) illustrate: (A) a graph showing activity in
lowering PGE2 levels in media of A549 cells that are stimulated to
produce PEG2 by treatment using IL1-beta using compounds of FIG.
81; (B) a graph showing toxicity of A549 cells administered the
compounds of FIG. 81; and (C) photographs of A549 cells treated
with compounds of FIG. 81.
[0113] FIG. 85 illustrates a plot showing metabolic stability of
SW054384 by incubation with murine liver S9 microsomes.
[0114] FIG. 86 illustrates a plot showing metabolic stability of
SW0125991 by incubation with murine liver S9 microsomes
[0115] FIG. 87 illustrates a schematic diagram of analogues of
SW054384.
[0116] FIG. 88 illustrates a graph showing luciferase activity of
colon cancer cell V9m treated with 2.5 .mu.M and 7.5 .mu.M the
compounds of FIG. 87.
[0117] FIG. 89 illustrates a graph showing luciferase activity of
colon cancer LS174T cells treated with 2.5 .mu.M and 7.5 .mu.M the
compounds of FIG. 87.
[0118] FIG. 90 illustrates a graph showing luciferase activity of
colon cancer cell V503 treated with 2.5 .mu.M and 7.5 .mu.M the
compounds of FIG. 87.
[0119] FIG. 91 illustrates a graph showing percent inhibition of
15-PGDH activity by the compounds of FIG. 87.
[0120] FIG. 92 is a schematic illustration showing the structures
of 15-PGDH activators SW054384, SW125991, SW207997, SW207998, and
SW207999.
[0121] FIG. 93 illustrates a graph showing the activities of
SW054384, SW125991, SW207997, SW207998, SW207999 in lowering PGE2
levels in medium of A549 cells that have been treated with 2.5
.mu.M of each compound along with addition of 2.5 ng/ml
IL1-beta.
[0122] FIG. 94 illustrates titration curves of 15-PGDH activator
compounds in an assay measuring effects on PGE2 levels in the
medium of A549 cells that have been stimulated with IL1-beta.
[0123] FIG. 95 illustrates a photograph showing assessment of
toxicity of SW125991 by testing effect of increasing doses on
colony formation of A549 cells, Vaco9M (V9m) cells, LS174T cells,
and Vaco503 (V503) cells.
[0124] FIG. 96 illustrates a photograph showing assessment of
toxicity of SW207997 by testing effect of increasing doses on
colony formation of A549 cells, Vaco9M (V9m) cells, LS174T cells,
and Vaco503 (V503) cells.
[0125] FIG. 97 illustrates a photograph showing assessment of
toxicity of SW207998 by testing effect of increasing doses on
colony formation of A549 cells, Vaco9M (V9m) cells, LS174T cells,
and Vaco503 (V503) cells.
[0126] FIG. 98 illustrates a photograph showing assessment of
toxicity of SW207999 by testing effect of increasing doses on
colony formation of A549 cells, Vaco9M (V9m) cells, LS174T cells,
and Vaco503 (V503) cells.
DETAILED DESCRIPTION
[0127] For convenience, certain terms employed in the
specification, examples, and appended claims are collected here.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this application belongs.
[0128] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0129] The terms "comprise," "comprising," "include," "including,"
"have," and "having" are used in the inclusive, open sense, meaning
that additional elements may be included. The terms "such as",
"e.g.", as used herein are non-limiting and are for illustrative
purposes only. "Including" and "including but not limited to" are
used interchangeably.
[0130] The term "or" as used herein should be understood to mean
"and/or", unless the context clearly indicates otherwise.
[0131] It will be noted that the structure of some of the compounds
of the application include asymmetric (chiral) carbon atoms. It is
to be understood accordingly that the isomers arising from such
asymmetry are included herein, unless indicated otherwise. Such
isomers can be obtained in substantially pure form by classical
separation techniques and by stereochemically controlled synthesis.
The compounds of this application may exist in stereoisomeric form,
therefore can be produced as individual stereoisomers or as
mixtures.
[0132] The term "isomerism" means compounds that have identical
molecular formulae but that differ in the nature or the sequence of
bonding of their atoms or in the arrangement of their atoms in
space. Isomers that differ in the arrangement of their atoms in
space are termed "stereoisomers". Stereoisomers that are not mirror
images of one another are termed "diastereoisomers", and
stereoisomers that are non-superimposable mirror images are termed
"enantiomers", or sometimes optical isomers. A carbon atom bonded
to four nonidentical substituents is termed a "chiral center".
[0133] The term "chiral isomer" means a compound with at least one
chiral center. It has two enantiomeric forms of opposite chirality
and may exist either as an individual enantiomer or as a mixture of
enantiomers. A mixture containing equal amounts of individual
enantiomeric forms of opposite chirality is termed a "racemic
mixture". A compound that has more than one chiral center has 2n-1
enantiomeric pairs, where n is the number of chiral centers.
Compounds with more than one chiral center may exist as either an
individual diastereomer or as a mixture of diastereomers, termed a
"diastereomeric mixture". When one chiral center is present, a
stereoisomer may be characterized by the absolute configuration (R
or S) of that chiral center. Alternatively, when one or more chiral
centers are present, a stereoisomer may be characterized as (+) or
(-). Absolute configuration refers to the arrangement in space of
the substituents attached to the chiral center. The substituents
attached to the chiral center under consideration are ranked in
accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn
et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et
al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951
(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J.,
Chem. Educ. 1964, 41, 116).
[0134] The term "geometric Isomers" means the diastereomers that
owe their existence to hindered rotation about double bonds. These
configurations are differentiated in their names by the prefixes
cis and trans, or Z and E, which indicate that the groups are on
the same or opposite side of the double bond in the molecule
according to the Cahn-Ingold-Prelog rules. Further, the structures
and other compounds discussed in this application include all
atropic isomers thereof.
[0135] The term "atropic isomers" are a type of stereoisomer in
which the atoms of two isomers are arranged differently in space.
Atropic isomers owe their existence to a restricted rotation caused
by hindrance of rotation of large groups about a central bond. Such
atropic isomers typically exist as a mixture, however as a result
of recent advances in chromatography techniques, it has been
possible to separate mixtures of two atropic isomers in select
cases.
[0136] The terms "crystal polymorphs" or "polymorphs" or "crystal
forms" means crystal structures in which a compound (or salt or
solvate thereof) can crystallize in different crystal packing
arrangements, all of which have the same elemental composition.
Different crystal forms usually have different X-ray diffraction
patterns, infrared spectral, melting points, density hardness,
crystal shape, optical and electrical properties, stability and
solubility. Recrystallization solvent, rate of crystallization,
storage temperature, and other factors may cause one crystal form
to dominate. Crystal polymorphs of the compounds can be prepared by
crystallization under different conditions.
[0137] The term "derivative" refers to compounds that have a common
core structure, and are substituted with various groups as
described herein.
[0138] The term "bioisostere" refers to a compound resulting from
the exchange of an atom or of a group of atoms with another,
broadly similar, atom or group of atoms. The objective of a
bioisosteric replacement is to create a new compound with similar
biological properties to the parent compound. The bioisosteric
replacement may be physicochemically or topologically based.
Examples of carboxylic acid bioisosteres include acyl sulfonimides,
tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and
LaVoie, Chem. Rev. 96, 3147-3176 (1996).
[0139] The phrases "parenteral administration" and "administered
parenterally" are art-recognized terms, and include modes of
administration other than enteral and topical administration, such
as injections, and include, without limitation, intravenous,
intramuscular, intrapleural, intravascular, intrapericardial,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal and intrastemal injection and
infusion.
[0140] The term "treating" is art-recognized and includes
inhibiting a disease, disorder or condition in a subject, e.g.,
impeding its progress; and relieving the disease, disorder or
condition, e.g., causing regression of the disease, disorder and/or
condition. Treating the disease or condition includes ameliorating
at least one symptom of the particular disease or condition, even
if the underlying pathophysiology is not affected.
[0141] The term "preventing" is art-recognized and includes
stopping a disease, disorder or condition from occurring in a
subject, which may be predisposed to the disease, disorder and/or
condition but has not yet been diagnosed as having it. Preventing a
condition related to a disease includes stopping the condition from
occurring after the disease has been diagnosed but before the
condition has been diagnosed.
[0142] The term "pharmaceutical composition" refers to a
formulation containing the disclosed compounds in a form suitable
for administration to a subject. In a preferred embodiment, the
pharmaceutical composition is in bulk or in unit dosage form. The
unit dosage form is any of a variety of forms, including, for
example, a capsule, an IV bag, a tablet, a single pump on an
aerosol inhaler, or a vial. The quantity of active ingredient
(e.g., a formulation of the disclosed compound or salts thereof) in
a unit dose of composition is an effective amount and is varied
according to the particular treatment involved. One skilled in the
art will appreciate that it is sometimes necessary to make routine
variations to the dosage depending on the age and condition of the
patient. The dosage will also depend on the route of
administration. A variety of routes are contemplated, including
oral, pulmonary, rectal, parenteral, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, intranasal,
inhalational, and the like. Dosage forms for the topical or
transdermal administration of a compound described herein includes
powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, nebulized compounds, and inhalants. In a
preferred embodiment, the active compound is mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants that are required.
[0143] The term "flash dose" refers to compound formulations that
are rapidly dispersing dosage forms.
[0144] The term "immediate release" is defined as a release of
compound from a dosage form in a relatively brief period of time,
generally up to about 60 minutes. The term "modified release" is
defined to include delayed release, extended release, and pulsed
release. The term "pulsed release" is defined as a series of
releases of drug from a dosage form. The term "sustained release"
or "extended release" is defined as continuous release of a
compound from a dosage form over a prolonged period.
[0145] The phrase "pharmaceutically acceptable" is art-recognized.
In certain embodiments, the term includes compositions, polymers
and other materials and/or dosage forms which are, within the scope
of sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0146] The phrase "pharmaceutically acceptable carrier" is
art-recognized, and includes, for example, pharmaceutically
acceptable materials, compositions or vehicles, such as a liquid or
solid filler, diluent, excipient, solvent or encapsulating
material, involved in carrying or transporting any subject
composition from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in
the sense of being compatible with the other ingredients of a
subject composition and not injurious to the patient. In certain
embodiments, a pharmaceutically acceptable carrier is
non-pyrogenic. Some examples of materials which may serve as
pharmaceutically acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, sunflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0147] The compounds of the application are capable of further
forming salts. All of these forms are also contemplated herein.
[0148] "Pharmaceutically acceptable salt" of a compound means a
salt that is pharmaceutically acceptable and that possesses the
desired pharmacological activity of the parent compound. For
example, the salt can be an acid addition salt. One embodiment of
an acid addition salt is a hydrochloride salt. The pharmaceutically
acceptable salts can be synthesized from a parent compound that
contains a basic or acidic moiety by conventional chemical methods.
Generally, such salts can be prepared by reacting the free acid or
base forms of these compounds with a stoichiometric amount of the
appropriate base or acid in water or in an organic solvent, or in a
mixture of the two; generally, non-aqueous media like ether, ethyl
acetate, ethanol, isopropanol, or acetonitrile being preferred.
Lists of salts are found in Remington's Pharmaceutical Sciences,
18th ed. (Mack Publishing Company, 1990).
[0149] The compounds described herein can also be prepared as
esters, for example pharmaceutically acceptable esters. For
example, a carboxylic acid function group in a compound can be
converted to its corresponding ester, e.g., a methyl, ethyl, or
other ester. Also, an alcohol group in a compound can be converted
to its corresponding ester, e.g., an acetate, propionate, or other
ester.
[0150] The compounds described herein can also be prepared as
prodrugs, for example pharmaceutically acceptable prodrugs. The
terms "pro-drug" and "prodrug" are used interchangeably herein and
refer to any compound, which releases an active parent drug in
vivo. Since prodrugs are known to enhance numerous desirable
qualities of pharmaceuticals (e.g., solubility, bioavailability,
manufacturing, etc.) the compounds can be delivered in prodrug
form. Thus, the compounds described herein are intended to cover
prodrugs of the presently claimed compounds, methods of delivering
the same and compositions containing the same. "Prodrugs" are
intended to include any covalently bonded carriers that release an
active parent drug in vivo when such prodrug is administered to a
subject. Prodrugs are prepared by modifying functional groups
present in the compound in such a way that the modifications are
cleaved, either in routine manipulation or in vivo, to the parent
compound. Prodrugs include compounds wherein a hydroxy, amino,
sulfhydryl, carboxy, or carbonyl group is bonded to any group that
may be cleaved in vivo to form a free hydroxyl, free amino, free
sulfhydryl, free carboxy or free carbonyl group, respectively.
[0151] Examples of prodrugs include, but are not limited to, esters
(e.g., acetate, dialkylaminoacetates, formates, phosphates,
sulfates, and benzoate derivatives) and carbamates (e.g.,
N,N-dimethylaminocarbonyl) of hydroxy functional groups, ester
groups (e.g., ethyl esters, morpholinoethanol esters) of carboxyl
functional groups, N-acyl derivatives (e.g., N-acetyl)N-Mannich
bases, Schiff bases and enaminones of amino functional groups,
oximes, acetals, ketals and enol esters of ketone and aldehyde
functional groups in compounds of Formula I, and the like, See
Bundegaard, H. "Design of Prodrugs" p 1-92, Elesevier, New
York-Oxford (1985).
[0152] The term "protecting group" refers to a grouping of atoms
that when attached to a reactive group in a molecule masks, reduces
or prevents that reactivity. Examples of protecting groups can be
found in Green and Wuts, Protective Groups in Organic Chemistry,
(Wiley, 2.sup.nd ed. 1991); Harrison and Harrison et al.,
Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and
Sons, 1971-1996); and Kocienski, Protecting Groups, (Verlag,
3.sup.rd ed. 2003).
[0153] The term "amine protecting group" is intended to mean a
functional group that converts an amine, amide, or other
nitrogen-containing moiety into a different chemical group that is
substantially inert to the conditions of a particular chemical
reaction. Amine protecting groups are preferably removed easily and
selectively in good yield under conditions that do not affect other
functional groups of the molecule. Examples of amine protecting
groups include, but are not limited to, formyl, acetyl, benzyl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl
(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,
trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,
2-trimethylsilyl-ethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)
ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),
2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted
trityl groups, 9-fluorenylmethyloxycarbonyl (FMOC),
nitro-veratryloxycarbonyl (NVOC), and the like. Those of skill in
the art can identify other suitable amine protecting groups.
[0154] Representative hydroxy protecting groups include those where
the hydroxy group is either acylated or alkylated such as benzyl,
and trityl ethers as well as alkyl ethers, tetrahydropyranyl
ethers, trialkylsilyl ethers and allyl ethers.
[0155] Additionally, the salts of the compounds described herein,
can exist in either hydrated or unhydrated (the anhydrous) form or
as solvates with other solvent molecules. Nonlimiting examples of
hydrates include monohydrates, dihydrates, etc. Nonlimiting
examples of solvates include ethanol solvates, acetone solvates,
etc.
[0156] The term "solvates" means solvent addition forms that
contain either stoichiometric or non stoichiometric amounts of
solvent. Some compounds have a tendency to trap a fixed molar ratio
of solvent molecules in the crystalline solid state, thus forming a
solvate. If the solvent is water the solvate formed is a hydrate,
when the solvent is alcohol, the solvate formed is an alcoholate.
Hydrates are formed by the combination of one or more molecules of
water with one of the substances in which the water retains its
molecular state as H.sub.2O, such combination being able to form
one or more hydrate.
[0157] The compounds, salts and prodrugs described herein can exist
in several tautomeric forms, including the enol and imine form, and
the keto and enamine form and geometric isomers and mixtures
thereof. Tautomers exist as mixtures of a tautomeric set in
solution. In solid form, usually one tautomer predominates. Even
though one tautomer may be described, the present application
includes all tautomers of the present compounds. A tautomer is one
of two or more structural isomers that exist in equilibrium and are
readily converted from one isomeric form to another. This reaction
results in the formal migration of a hydrogen atom accompanied by a
switch of adjacent conjugated double bonds. In solutions where
tautomerization is possible, a chemical equilibrium of the
tautomers will be reached. The exact ratio of the tautomers depends
on several factors, including temperature, solvent, and pH. The
concept of tautomers that are interconvertible by tautomerizations
is called tautomerism.
[0158] Of the various types of tautomerism that are possible, two
are commonly observed. In keto-enol tautomerism a simultaneous
shift of electrons and a hydrogen atom occurs.
[0159] Tautomerizations can be catalyzed by: Base: 1.
deprotonation; 2. formation of a delocalized anion (e.g., an
enolate); 3. protonation at a different position of the anion;
Acid: 1. protonation; 2. formation of a delocalized cation; 3.
deprotonation at a different position adjacent to the cation.
[0160] The term "analogue" refers to a chemical compound that is
structurally similar to another but differs slightly in composition
(as in the replacement of one atom by an atom of a different
element or in the presence of a particular functional group, or the
replacement of one functional group by another functional group).
Thus, an analogue is a compound that is similar or comparable in
function and appearance, but not in structure or origin to the
reference compound.
[0161] A "patient," "subject," or "host" to be treated by the
subject method may mean either a human or non-human animal, such as
a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the
subject of the herein disclosed methods can be a human, non-human
primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig
or rodent. The term does not denote a particular age or sex. Thus,
adult and newborn subjects, as well as fetuses, whether male or
female, are intended to be covered. In one aspect, the subject is a
mammal. A patient refers to a subject afflicted with a disease or
disorder.
[0162] The terms "prophylactic" or "therapeutic" treatment is
art-recognized and includes administration to the host of one or
more of the subject compositions. If it is administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) then the treatment is
prophylactic, i.e., it protects the host against developing the
unwanted condition, whereas if it is administered after
manifestation of the unwanted condition, the treatment is
therapeutic (i.e., it is intended to diminish, ameliorate, or
stabilize the existing unwanted condition or side effects
thereof).
[0163] The terms "therapeutic agent", "drug", "medicament" and
"bioactive substance" are art-recognized and include molecules and
other agents that are biologically, physiologically, or
pharmacologically active substances that act locally or
systemically in a patient or subject to treat a disease or
condition. The terms include without limitation pharmaceutically
acceptable salts thereof and prodrugs. Such agents may be acidic,
basic, or salts; they may be neutral molecules, polar molecules, or
molecular complexes capable of hydrogen bonding; they may be
prodrugs in the form of ethers, esters, amides and the like that
are biologically activated when administered into a patient or
subject.
[0164] The phrase "therapeutically effective amount" or
"pharmaceutically effective amount" is an art-recognized term. In
certain embodiments, the term refers to an amount of a therapeutic
agent that produces some desired effect at a reasonable
benefit/risk ratio applicable to any medical treatment. In certain
embodiments, the term refers to that amount necessary or sufficient
to eliminate, reduce or maintain a target of a particular
therapeutic regimen. The effective amount may vary depending on
such factors as the disease or condition being treated, the
particular targeted constructs being administered, the size of the
subject or the severity of the disease or condition. One of
ordinary skill in the art may empirically determine the effective
amount of a particular compound without necessitating undue
experimentation. In certain embodiments, a therapeutically
effective amount of a therapeutic agent for in vivo use will likely
depend on a number of factors, including: the rate of release of an
agent from a polymer matrix, which will depend in part on the
chemical and physical characteristics of the polymer; the identity
of the agent; the mode and method of administration; and any other
materials incorporated in the polymer matrix in addition to the
agent.
[0165] The term "ED50" is art-recognized. In certain embodiments,
ED50 means the dose of a drug, which produces 50% of its maximum
response or effect, or alternatively, the dose, which produces a
pre-determined response in 50% of test subjects or preparations.
The term "LD50" is art-recognized. In certain embodiments, LD50
means the dose of a drug, which is lethal in 50% of test subjects.
The term "therapeutic index" is an art-recognized term, which
refers to the therapeutic index of a drug, defined as
LD50/ED50.
[0166] The terms "IC.sub.50," or "half maximal inhibitory
concentration" is intended to refer to the concentration of a
substance (e.g., a compound or a drug) that is required for 50%
inhibition of a biological process, or component of a process,
including a protein, subunit, organelle, ribonucleoprotein,
etc.
[0167] With respect to any chemical compounds, the present
application is intended to include all isotopes of atoms occurring
in the present compounds. Isotopes include those atoms having the
same atomic number but different mass numbers. By way of general
example and without limitation, isotopes of hydrogen include
tritium and deuterium, and isotopes of carbon include C-13 and
C-14.
[0168] When a bond to a substituent is shown to cross a bond
connecting two atoms in a ring, then such substituent can be bonded
to any atom in the ring. When a substituent is listed without
indicating the atom via which such substituent is bonded to the
rest of the compound of a given formula, then such substituent can
be bonded via any atom in such substituent. Combinations of
substituents and/or variables are permissible, but only if such
combinations result in stable compounds.
[0169] When an atom or a chemical moiety is followed by a
subscripted numeric range (e.g., C.sub.1-6), it is meant to
encompass each number within the range as well as all intermediate
ranges. For example, "C.sub.1-6 alkyl" is meant to include alkyl
groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5,
2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.
[0170] The term "alkyl" is intended to include both branched (e.g.,
isopropyl, tert-butyl, isobutyl), straight-chain e.g., methyl,
ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl),
and cycloalkyl (e.g., alicyclic) groups (e.g., cyclopropyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. Such aliphatic hydrocarbon groups have a specified number
of carbon atoms. For example, C.sub.1-6 alkyl is intended to
include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, and C.sub.6
alkyl groups. As used herein, "lower alkyl" refers to alkyl groups
having from 1 to 6 carbon atoms in the backbone of the carbon
chain. "Alkyl" further includes alkyl groups that have oxygen,
nitrogen, sulfur or phosphorous atoms replacing one or more
hydrocarbon backbone carbon atoms. In certain embodiments, a
straight chain or branched chain alkyl has six or fewer carbon
atoms in its backbone (e.g., C.sub.1-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain), for example four or fewer.
Likewise, certain cycloalkyls have from three to eight carbon atoms
in their ring structure, such as five or six carbons in the ring
structure.
[0171] The term "substituted alkyls" refers to alkyl moieties
having substituents replacing a hydrogen on one or more carbons of
the hydrocarbon backbone. Such substituents can include, for
example, alkyl, alkenyl, alkynyl, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Cycloalkyls can be further substituted, e.g., with the substituents
described above. An "alkylaryl" or an "aralkyl" moiety is an alkyl
substituted with an aryl (e.g., phenylmethyl (benzyl)). If not
otherwise indicated, the terms "alkyl" and "lower alkyl" include
linear, branched, cyclic, unsubstituted, substituted, and/or
heteroatom-containing alkyl or lower alkyl, respectively.
[0172] The term "alkenyl" refers to a linear, branched or cyclic
hydrocarbon group of 2 to about 24 carbon atoms containing at least
one double bond, such as ethenyl, n-propenyl, isopropenyl,
n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl,
eicosenyl, tetracosenyl, cyclopentenyl, cyclohexenyl, cyclooctenyl,
and the like. Generally, although again not necessarily, alkenyl
groups can contain 2 to about 18 carbon atoms, and more
particularly 2 to 12 carbon atoms. The term "lower alkenyl" refers
to an alkenyl group of 2 to 6 carbon atoms, and the specific term
"cycloalkenyl" intends a cyclic alkenyl group, preferably having 5
to 8 carbon atoms. The term "substituted alkenyl" refers to alkenyl
substituted with one or more substituent groups, and the terms
"heteroatom-containing alkenyl" and "heteroalkenyl" refer to
alkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which
at least one carbon atom is replaced with a heteroatom. If not
otherwise indicated, the terms "alkenyl" and "lower alkenyl"
include linear, branched, cyclic, unsubstituted, substituted,
and/or heteroatom-containing alkenyl and lower alkenyl,
respectively.
[0173] The term "alkynyl" refers to a linear or branched
hydrocarbon group of 2 to 24 carbon atoms containing at least one
triple bond, such as ethynyl, n-propynyl, and the like. Generally,
although again not necessarily, alkynyl groups can contain 2 to
about 18 carbon atoms, and more particularly can contain 2 to 12
carbon atoms. The term "lower alkynyl" intends an alkynyl group of
2 to 6 carbon atoms. The term "substituted alkynyl" refers to
alkynyl substituted with one or more substituent groups, and the
terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to
alkynyl in which at least one carbon atom is replaced with a
heteroatom. If not otherwise indicated, the terms "alkynyl" and
"lower alkynyl" include linear, branched, unsubstituted,
substituted, and/or heteroatom-containing alkynyl and lower
alkynyl, respectively.
[0174] The terms "alkyl", "alkenyl", and "alkynyl" are intended to
include moieties which are diradicals, i.e., having two points of
attachment. A nonlimiting example of such an alkyl moiety that is a
diradical is --CH.sub.2CH.sub.2--, i.e., a C.sub.2 alkyl group that
is covalently bonded via each terminal carbon atom to the remainder
of the molecule.
[0175] The term "alkoxy" refers to an alkyl group bound through a
single, terminal ether linkage; that is, an "alkoxy" group may be
represented as --O-alkyl where alkyl is as defined above. A "lower
alkoxy" group intends an alkoxy group containing 1 to 6 carbon
atoms, and includes, for example, methoxy, ethoxy, n-propoxy,
isopropoxy, t-butyloxy, etc. Preferred substituents identified as
"C.sub.1-C.sub.6 alkoxy" or "lower alkoxy" herein contain 1 to 3
carbon atoms, and particularly preferred such substituents contain
1 or 2 carbon atoms (i.e., methoxy and ethoxy).
[0176] The term "aryl" refers to an aromatic substituent containing
a single aromatic ring or multiple aromatic rings that are fused
together, directly linked, or indirectly linked (such that the
different aromatic rings are bound to a common group such as a
methylene or ethylene moiety). Aryl groups can contain 5 to 20
carbon atoms, and particularly preferred aryl groups can contain 5
to 14 carbon atoms. Examples of aryl groups include benzene,
phenyl, pyrrole, furan, thiophene, thiazole, isothiazole,
imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole,
pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
Furthermore, the term "aryl" includes multicyclic aryl groups,
e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,
benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,
methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,
benzofuran, purine, benzofuran, deazapurine, or indolizine. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles", "heterocycles," "heteroaryls"
or "heteroaromatics". The aromatic ring can be substituted at one
or more ring positions with such substituents as described above,
as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkylaminocarbonyl,
aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,
arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkylamino, dialkylamino,
arylamino, diaryl amino, and al kylaryl amino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can
also be fused or bridged with alicyclic or heterocyclic rings,
which are not aromatic so as to form a multicyclic system (e.g.,
tetralin, methylenedioxyphenyl). If not otherwise indicated, the
term "aryl" includes unsubstituted, substituted, and/or
heteroatom-containing aromatic substituents.
[0177] The term "alkaryl" refers to an aryl group with an alkyl
substituent, and the term "aralkyl" refers to an alkyl group with
an aryl substituent, wherein "aryl" and "alkyl" are as defined
above. Exemplary aralkyl groups contain 6 to 24 carbon atoms, and
particularly preferred aralkyl groups contain 6 to 16 carbon atoms.
Examples of aralkyl groups include, without limitation, benzyl,
2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl,
4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl,
4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for
example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl,
2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,
3-ethyl-cyclopenta-1,4-diene, and the like.
[0178] The terms "heterocyclyl" or "heterocyclic group" include
closed ring structures, e.g., 3- to 10-, or 4- to 7-membered rings,
which include one or more heteroatoms. "Heteroatom" includes atoms
of any element other than carbon or hydrogen. Examples of
heteroatoms include nitrogen, oxygen, sulfur and phosphorus.
[0179] Heterocyclyl groups can be saturated or unsaturated and
include pyrrolidine, oxolane, thiolane, piperidine, piperazine,
morpholine, lactones, lactams, such as azetidinones and
pyrrolidinones, sultams, and sultones. Heterocyclic groups such as
pyrrole and furan can have aromatic character. They include fused
ring structures, such as quinoline and isoquinoline. Other examples
of heterocyclic groups include pyridine and purine. The
heterocyclic ring can be substituted at one or more positions with
such substituents as described above, as for example, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic or
heteroaromatic moiety. Heterocyclic groups can also be substituted
at one or more constituent atoms with, for example, a lower alkyl,
a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower
alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, --CF.sub.3,
or --CN, or the like.
[0180] The term "halo" or "halogen" refers to fluoro, chloro,
bromo, and iodo. "Counterion" is used to represent a small,
negatively charged species such as fluoride, chloride, bromide,
iodide, hydroxide, acetate, and sulfate.
[0181] The terms "substituted" as in "substituted alkyl,"
"substituted aryl," and the like, as alluded to in some of the
aforementioned definitions, is meant that in the alkyl, aryl, or
other moiety, at least one hydrogen atom bound to a carbon (or
other) atom is replaced with one or more non-hydrogen substituents.
Examples of such substituents include, without limitation:
functional groups such as halo, hydroxyl, silyl, sulfhydryl,
C.sub.1-C.sub.24 alkoxy, C.sub.2-C.sub.24 alkenyloxy,
C.sub.2-C.sub.24 alkynyloxy, C.sub.5-C.sub.20 aryloxy, acyl
(including C.sub.2-C.sub.24 alkylcarbonyl (--CO-alkyl) and
C.sub.6-C.sub.20 arylcarbonyl (--CO-aryl)), acyloxy (--O-acyl),
C.sub.2-C.sub.24 alkoxycarbonyl (--(CO)--O-alkyl), C.sub.6-C.sub.20
aryloxycarbonyl (--(CO)--O-aryl), C.sub.2-C.sub.24 alkylcarbonato
(--O--(CO)--O-alkyl), C.sub.6-C.sub.20 arylcarbonato
(--O--(CO)--O-aryl), carboxy (--COOH), carboxylato (--COO--),
carbamoyl (--(CO)--NH.sub.2), mono-(C.sub.1-C.sub.24
alkyl)-substituted carbamoyl (--(CO)--NH(C.sub.1-C.sub.24 alkyl)),
di-(C.sub.1-C.sub.4 alkyl)-substituted carbamoyl
(--(CO)--N(C.sub.1-C.sub.24 alkyl).sub.2), mono-substituted
arylcarbamoyl (--(CO)--NH-aryl), thiocarbamoyl (--(CS)--NH.sub.2),
carbamido (--NH--(CO)--NH.sub.2), cyano (--CN), isocyano
(--N.sup.+C.sup.-), cyanato (--O--CN), isocyanato
(--ON.sup.+C.sup.-), isothiocyanato (--S--CN), azido
(--N.dbd.N.sup.+.dbd.N.sup.-), formyl (--(CO)--H), thioformyl
(--(CS)--H), amino (--NH.sub.2), mono- and di-(C.sub.1-C.sub.24
alkyl)-substituted amino, mono- and di-(C.sub.5-C.sub.20
aryl)-substituted amino, C.sub.2-C.sub.24 alkylamido
(--NH--(CO)-alkyl), C.sub.6-C.sub.20 arylamido (--NH--(CO)-aryl),
imino (--CR.dbd.NH where R=hydrogen, C.sub.1-C.sub.24 alkyl,
C.sub.5-C.sub.20 aryl, C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24
aralkyl, etc.), alkylimino (--CR.dbd.N(alkyl), where R=hydrogen,
alkyl, aryl, alkaryl, etc.), arylimino (--CR.dbd.N(aryl), where
R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (--NO.sub.2),
nitroso (--NO), sulfo (--SO.sub.2--OH), sulfonato
(--SO.sub.2--O.sup.-), C.sub.1-C.sub.24 alkylsulfanyl (--S-alkyl;
also termed "alkylthio"), arylsulfanyl (--S-aryl; also termed
"arylthio"), C.sub.1-C.sub.24 alkylsulfinyl (--(SO)-alkyl),
C.sub.5-C.sub.20 arylsulfinyl (--(SO)-aryl), C.sub.1-C.sub.24
alkylsulfonyl (--SO.sub.2-alkyl), C.sub.5-C.sub.20 arylsulfonyl
(--SO.sub.2-aryl), phosphono (--P(O)(OH).sub.2), phosphonato
(--P(O)(O.sup.-).sub.2), phosphinato (--P(O)(O.sup.-)), phospho
(--PO.sub.2), and phosphino (--PH.sub.2); and the hydrocarbyl
moieties C.sub.1-C.sub.24 alkyl, C.sub.2-C.sub.24 alkenyl,
C.sub.2-C.sub.24 alkynyl, C.sub.5-C.sub.20 aryl, C.sub.6-C.sub.24
alkaryl, and C.sub.6-C.sub.24 aralkyl.
[0182] In addition, the aforementioned functional groups may, if a
particular group permits, be further substituted with one or more
additional functional groups or with one or more hydrocarbyl
moieties such as those specifically enumerated above. Analogously,
the above-mentioned hydrocarbyl moieties may be further substituted
with one or more functional groups or additional hydrocarbyl
moieties such as those specifically enumerated.
[0183] When the term "substituted" appears prior to a list of
possible substituted groups, it is intended that the term apply to
every member of that group. For example, the phrase "substituted
alkyl, alkenyl, and aryl" is to be interpreted as "substituted
alkyl, substituted alkenyl, and substituted aryl." Analogously,
when the term "heteroatom-containing" appears prior to a list of
possible heteroatom-containing groups, it is intended that the term
apply to every member of that group. For example, the phrase
"heteroatom-containing alkyl, alkenyl, and aryl" is to be
interpreted as "heteroatom-containing alkyl, substituted alkenyl,
and substituted aryl.
[0184] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not. For example, the phrase "optionally
substituted" means that a non-hydrogen substituent may or may not
be present on a given atom, and, thus, the description includes
structures wherein a non-hydrogen substituent is present and
structures wherein a non-hydrogen substituent is not present.
[0185] The terms "stable compound" and "stable structure" are meant
to indicate a compound that is sufficiently robust to survive
isolation, and as appropriate, purification from a reaction
mixture, and formulation into an efficacious therapeutic agent.
[0186] The terms "free compound" is used herein to describe a
compound in the unbound state.
[0187] Throughout the description, where compositions are described
as having, including, or comprising, specific components, it is
contemplated that compositions also consist essentially of, or
consist of, the recited components. Similarly, where methods or
processes are described as having, including, or comprising
specific process steps, the processes also consist essentially of,
or consist of, the recited processing steps. Further, it should be
understood that the order of steps or order for performing certain
actions is immaterial so long as the compositions and methods
described herein remains operable. Moreover, two or more steps or
actions can be conducted simultaneously.
[0188] The term "small molecule" is an art-recognized term. In
certain embodiments, this term refers to a molecule, which has a
molecular weight of less than about 2000 amu, or less than about
1000 amu, and even less than about 500 amu.
[0189] All percentages and ratios used herein, unless otherwise
indicated, are by weight.
[0190] The term "neoplasm" refers to any abnormal mass of cells or
tissue as a result of neoplasia. The neoplasm may be benign,
potentially malignant (precancerous), or malignant (cancerous). An
adenoma is an example of a neoplasm.
[0191] The terms "adenoma", "colon adenoma" and "polyp" are used
herein to describe any precancerous neoplasm of the colon.
[0192] The term "colon" as used herein is intended to encompass the
right colon (including the cecum), the transverse colon, the left
colon and the rectum.
[0193] The terms "colorectal cancer" and "colon cancer" are used
interchangeably herein to refer to any cancerous neoplasia of the
colon (including the rectum, as defined above).
[0194] The terms "gene expression" or "protein expression" includes
any information pertaining to the amount of gene transcript or
protein present in a sample, as well as information about the rate
at which genes or proteins are produced or are accumulating or
being degraded (e.g., reporter gene data, data from nuclear runoff
experiments, pulse-chase data etc.). Certain kinds of data might be
viewed as relating to both gene and protein expression. For
example, protein levels in a cell are reflective of the level of
protein as well as the level of transcription, and such data is
intended to be included by the phrase "gene or protein expression
information". Such information may be given in the form of amounts
per cell, amounts relative to a control gene or protein, in
unitless measures, etc.; the term "information" is not to be
limited to any particular means of representation and is intended
to mean any representation that provides relevant information. The
term "expression levels" refers to a quantity reflected in or
derivable from the gene or protein expression data, whether the
data is directed to gene transcript accumulation or protein
accumulation or protein synthesis rates, etc.
[0195] The terms "healthy" and "normal" are used interchangeably
herein to refer to a subject or particular cell or tissue that is
devoid (at least to the limit of detection) of a disease
condition.
[0196] The term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term should also be understood to include analogues
of either RNA or DNA made from nucleotide analogues, and, as
applicable to the embodiment being described, single-stranded (such
as sense or antisense) and double-stranded polynucleotides. In some
embodiments, "nucleic acid" refers to inhibitory nucleic acids.
Some categories of inhibitory nucleic acid compounds include
antisense nucleic acids, RNAi constructs, and catalytic nucleic
acid constructs. Such categories of nucleic acids are well-known in
the art.
[0197] Embodiments described herein relate to compounds and methods
of modulating 15-PGDH activity, modulating tissue prostaglandin
levels, and/or treating diseases, disorders, or conditions in which
it is desired to modulate 15-PGDH activity and/or prostaglandin
levels. "Inhibitors," "activators," and "modulators" of 15-PGDH
expression or of 15-PGDH activity are used to refer to inhibitory,
activating, or modulating molecules, respectively, identified using
in vitro and in vivo assays for 15-PGDH expression or 15-PGDH
activity, e.g., ligands, agonists, antagonists, and their homologs
and mimetics. The term "modulator" includes inhibitors and
activators. Inhibitors are agents that, e.g., inhibit expression of
15-PGDH or bind to, partially or totally block stimulation,
decrease, prevent, delay activation, inactivate, desensitize, or
down regulate the activity of 15-PGDH, e.g., antagonists.
Activators are agents that, e.g., induce or activate the expression
of a 15-PGDH or bind to, stimulate, stabilize, increase, open,
activate, facilitate, or enhance activation, sensitize or up
regulate the activity of 15-PGDH, e.g., agonists. Modulators
include naturally occurring and synthetic ligands, small chemical
molecules, and the like.
[0198] 15-PGDH inhibitors described herein can provide a
pharmacologic method for elevating prostaglandin levels in tissue.
Known activities of prostaglandins include promoting hair growth,
promoting skin pigmentation, and promoting skin darkening or the
appearance of skin tanning. Known activities of prostaglandins also
include ameliorating pulmonary artery hypertension. 15-PGDH
inhibitors described herein may also be utilized to increase tissue
stem cell numbers for purposes that would include increasing
resistance to tissue damage by radiation, increasing resistance to
environmental exposures to radiation, increasing stem cell numbers
to increase fitness of bone marrow or other types of
transplantation (through either in vivo exposure to 15-PGDH
inhibitors described herein to increase stem cell numbers prior to
harvest of a transplanted tissue, or through ex vivo exposure of a
harvested tissue prior to transplant into a recipient host).
15-PGDH inhibitors described herein may also be utilized for
purposes that would include promoting liver regeneration, including
liver regeneration after liver resection, and liver regeneration
after toxic insults, which for example may be the toxic insult of
acetaminophen overdose. Prostaglandin signaling is also known to
promote wound healing, protect the stomach from ulceration, and
promote healing of ulcers of stomach and intestines. Additionally,
15-PGDH inhibitors described herein can promote activity of human
keratinocytes in "healing" scratches across cultures of
keratinocyte cells. Hence, 15-PGDH inhibitors described herein may
be utilized to also heal ulcers of other tissues, including, but
not limited to skin, and including but not limited to diabetic
ulcers. Further, 15-PGDH inhibitors described herein may be
utilized for the treatment of erectile dysfunction.
[0199] 15-PGDH activators described herein can increase levels of
15-PGDH protein in cells and in increase levels of 15-PGDH
enzymatic activity in cells. Increasing tissue levels of 15-PGDH
can decrease tissue levels of prostaglandins. Activities associated
with compounds that decrease tissue prostaglandins include
decreasing development of human tumors, particularly decreasing
development of human colon tumors. Accordingly, compounds that
increase tissue 15-PGDH activity can lower risk of development of
colon and other tumors. Compounds that increase 15-PDGH activity
can also be used to treat colon and other tumors. Compounds that
increase 15-PDGH may be used to treat or to prevent tumors when
given singly, or when given in combination with inhibitors of
cyclooxygenase-1 and/or cyclooxygenase-2 enzymes, or when given in
combination with other therapeutic agents.
[0200] 15-PGDH inhibitors and activators described herein can be
identified using assays in which putative modulator compounds are
applied to cells expressing 15-PGDH and then the functional effects
on 15-PGDH activity are determined. Samples or assays comprising
15-PGDH that are treated with a potential activator, inhibitor, or
modulator are compared to control samples without the inhibitor,
activator, or modulator to examine the extent of effect. Control
samples (untreated with modulators) are assigned a relative 15-PGDH
activity value of 100%. Inhibition of 15-PGDH is achieved when the
15-PGDH activity value relative to the control is about 80%,
optionally 50% or 25%, 10%, 5% or 1%. Activation of 15-PGDH is
achieved when the 15-PGDH activity or expression value relative to
the control is 105%, optionally 110%, optionally 125%, optionally
150%, optionally 200%, 300%, 400%, 500%, or 1000-3000% or more
higher.
[0201] Agents tested as modulators of 15-PGDH can be any small
chemical molecule or compound. Typically, test compounds will be
small chemical molecules, natural products, or peptides. The assays
are designed to screen large chemical libraries by automating the
assay steps and providing compounds from any convenient source to
assays, which are typically run in parallel (e.g., in microtiter
formats on microtiter plates in robotic assays). Modulators also
include agents designed to increase the level of 15-PGDH mRNA or
the level of translation from an mRNA.
[0202] In some embodiments, the modulator of 15-PGDH can be a
15-PGDH inhibitor that includes a compound having the following
formula (I):
##STR00004## [0203] wherein n is 0-2; [0204] R.sub.1 is a C.sub.1-8
alkyl, which is linear, branched, or cyclic and which is
unsubstituted or substituted (e.g., R.sub.1 can be C.sub.2-6 alkyl,
C.sub.2-4 alkyl, or C.sub.4 alkyl, which is linear, branched, or
cyclic and which is unsubstituted or substituted); [0205] R.sub.2
and R.sub.3 are the same or different and are each selected from
the group consisting of a H, a lower alkyl group,
(CH.sub.2).sub.n1OR' (wherein n1=1, 2, or 3), CF.sub.3,
CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', (C.dbd.O)N(R').sub.2, O(CO)R',
COOR' (wherein R' is H or a lower alkyl group); [0206] R.sub.4 and
R.sub.5 are the same or different and are each selected from the
group consisting of hydrogen, C.sub.1-C.sub.24 alkyl,
C.sub.2-C.sub.24 alkenyl, C.sub.2-C.sub.24 alkynyl,
C.sub.3-C.sub.20 aryl, C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24
aralkyl, halo, silyl, hydroxyl, sulfhydryl, C.sub.1-C.sub.24
alkoxy, C.sub.2-C.sub.24 alkenyloxy, C.sub.2-C.sub.24 alkynyloxy,
C.sub.5-C.sub.20 aryloxy, acyl (including C.sub.2-C.sub.24
alkylcarbonyl (--CO-alkyl) and C.sub.6-C.sub.20 arylcarbonyl
(--CO-aryl)), acyloxy (--O-acyl), C.sub.2-C.sub.24 alkoxycarbonyl
(--(CO)--O-alkyl), C.sub.6-C.sub.20 aryloxycarbonyl
(--(CO)--O-aryl), C.sub.2-C.sub.24 alkylcarbonato
(--O--(CO)--O-alkyl), C.sub.6-C.sub.20 arylcarbonato
(--O--(CO)--O-aryl), carboxy (--COOH), carboxylato (--COO--),
carbamoyl (--(CO)--NH.sub.2), C.sub.1-C.sub.24 alkyl-carbamoyl
(--(CO)--NH(C.sub.1-C.sub.24 alkyl)), arylcarbamoyl
(--(CO)--NH-aryl), thiocarbamoyl (--(CS)--NH.sub.2), carbamido
(--NH--(CO)--NH.sub.2), cyano (--CN), isocyano (--N.sup.+C.sup.-),
cyanato (--O--CN), isocyanato (--O--N.sup.+.dbd.C.sup.-),
isothiocyanato (--S--CN), azido (--N.dbd.N.sup.+.dbd.N.sup.-),
formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH.sub.2),
C.sub.1-C.sub.24 alkyl amino, C.sub.5-C.sub.20 aryl amino,
C.sub.2-C.sub.24 alkylamido (--NH--(CO)-alkyl), C.sub.6-C.sub.20
arylamido (--NH--(CO)-aryl), imino (--CR.dbd.NH where R is
hydrogen, C.sub.1-C.sub.24 alkyl, C.sub.5-C.sub.20 aryl,
C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24 aralkyl, etc.),
alkylimino (--CR.dbd.N(alkyl), where R=hydrogen, alkyl, aryl,
alkaryl, aralkyl, etc.), arylimino (--CR.dbd.N(aryl), where
R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (--NO.sub.2),
nitroso (--NO), sulfo (--SO.sub.2--OH), sulfonato
(--SO.sub.2--O--), C.sub.1-C.sub.24 alkylsulfanyl (--S-alkyl; also
termed "alkylthio"), arylsulfanyl (--S-aryl; also termed
"arylthio"), C.sub.1-C.sub.24 alkylsulfinyl (--(SO)-alkyl),
C.sub.5-C.sub.20 arylsulfinyl (--(SO)-aryl), C.sub.1-C.sub.24
alkylsulfonyl (--SO.sub.2-alkyl), C.sub.5-C.sub.20 arylsulfonyl
(--SO.sub.2-aryl), phosphono (--P(O)(OH).sub.2), phosphonato
(--P(O)(O.sup.-).sub.2), phosphinato (--P(O)(O.sup.-)), phospho
(--PO.sub.2), phosphino (--PH.sub.2), combinations thereof, and
wherein R.sub.4 and R.sub.5 may be linked to form a cyclic or
polycyclic ring, wherein the ring is a substituted or unsubstituted
aryl, a substituted or unsubstituted heteroaryl, a substituted or
unsubstituted cycloalkyl, and a substituted or unsubstituted
heterocyclyl; and pharmaceutically acceptable salts thereof.
[0207] In other embodiments, the 15-PGDH inhibitor can include a
compound having the following formula (II):
##STR00005## [0208] wherein n is 0-2; [0209] R.sub.1 is a C.sub.1-8
alkyl, which is linear, branched, or cyclic, and which is
unsubstituted or substituted; [0210] R.sub.2 and R.sub.3 are the
same or different and are each selected from the group consisting
of a H, a lower alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2,
or 3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', (C.dbd.O)N(R').sub.2, O(CO)R',
COOR' (wherein R' is H or a lower alkyl group); [0211] Z.sub.1 is
NR', O or S (wherein R' is H or a lower alkyl group); [0212]
X.sub.1 and Y.sub.1 are the same or different and are each selected
from the group consisting of hydrogen, C.sub.1-C.sub.24 alkyl,
C.sub.2-C.sub.24 alkenyl, C.sub.2-C.sub.24 alkynyl,
C.sub.3-C.sub.20 aryl, C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24
aralkyl, halo, silyl, hydroxyl, sulfhydryl, C.sub.1-C.sub.24
alkoxy, C.sub.2-C.sub.24 alkenyloxy, C.sub.2-C.sub.24 alkynyloxy,
C.sub.5-C.sub.20 aryloxy, acyl (including C.sub.2-C.sub.24
alkylcarbonyl (--CO-alkyl) and C.sub.6-C.sub.20 arylcarbonyl
(--CO-aryl)), acyloxy (--O-acyl), C.sub.2-C.sub.24 alkoxycarbonyl
(--(CO)--O-alkyl), C.sub.6-C.sub.20 aryloxycarbonyl
(--(CO)--O-aryl), C.sub.2-C.sub.24 alkylcarbonato
(--O--(CO)--O-alkyl), C.sub.6-C.sub.20 arylcarbonato
(--O--(CO)--O-aryl), carboxy (--COOH), carboxylato (--COO--),
carbamoyl (--(CO)--NH.sub.2), C.sub.1-C.sub.24 alkyl-carbamoyl
(--(CO)--NH(C.sub.1-C.sub.24 alkyl)), arylcarbamoyl
(--(CO)--NH-aryl), thiocarbamoyl (--(CS)--NH.sub.2), carbamido
(--NH--(CO)--NH.sub.2), cyano (--CN), isocyano (--N.sup.+C.sup.-),
cyanato (--O--CN), isocyanato (--O--N.sup.+.dbd.C.sup.-),
isothiocyanato (--S--CN), azido (--N.dbd.N.sup.+.dbd.N.sup.-),
formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH.sub.2),
C.sub.1-C.sub.24 alkyl amino, C.sub.5-C.sub.20 aryl amino,
C.sub.2-C.sub.24 alkylamido (--NH--(CO)-alkyl), C.sub.6-C.sub.20
arylamido (--NH--(CO)-aryl), imino (--CR.dbd.NH where R is
hydrogen, C.sub.1-C.sub.24 alkyl, C.sub.5-C.sub.20 aryl,
C.sub.6-C.sub.24 alkaryl, C.sub.6-C.sub.24 aralkyl, etc.),
alkylimino (--CR.dbd.N(alkyl), where R=hydrogen, alkyl, aryl,
alkaryl, aralkyl, etc.), arylimino (--CR.dbd.N(aryl), where
R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (--NO.sub.2),
nitroso (--NO), sulfo (--SO.sub.2--OH), sulfonato
(--SO.sub.2--O.sup.-), C.sub.1-C.sub.24 alkylsulfanyl (--S-alkyl;
also termed "alkylthio"), arylsulfanyl (--S-aryl; also termed
"arylthio"), C.sub.1-C.sub.24 alkylsulfinyl (--(SO)-alkyl),
C.sub.5-C.sub.20 arylsulfinyl (--(SO)-aryl), C.sub.1-C.sub.24
alkylsulfonyl (--SO.sub.2-alkyl), C.sub.5-C.sub.20 arylsulfonyl
(--SO.sub.2-aryl), phosphono (--P(O)(OH).sub.2), phosphonato
(--P(O)(O.sup.-).sub.2), phosphinato (--P(O)(O.sup.-)), phospho
(--PO.sub.2), phosphino (--PH.sub.2), combinations thereof, and
wherein X.sub.1 and Y.sub.1 may be linked to form a cyclic or
polycyclic ring, wherein the ring is a substituted or unsubstituted
aryl, a substituted or unsubstituted heteroaryl, a substituted or
unsubstituted cycloalkyl, and a substituted or unsubstituted
heterocyclyl; and pharmaceutically acceptable salts thereof.
[0213] Examples of 15-PGDH inhibitors having formulas (I) or (II)
include the following compounds:
##STR00006## ##STR00007## ##STR00008##
and pharmaceutically acceptable salts thereof.
[0214] In certain embodiments, the 15-PGDH inhibitor having formula
(I) or (II) can be selected that can ia) at 2.5 .mu.M
concentration, stimulate a Vaco503 reporter cell line expressing a
15-PGDH luciferase fusion construct to a luciferase output level of
greater than 70 (using a scale on which a value of 100 indicates a
doubling of reporter output over baseline); iia) at 2.5 .mu.M
concentration stimulate a V9m reporter cell line expressing a
15-PGDH luciferase fusion construct to a luciferase output level of
greater than 75; iiia) at 7.5 .mu.M concentration stimulate a
LS174T reporter cell line expressing a 15-PGDH luciferase fusion
construct to a luciferase output level of greater than 70; and iva)
at 7.5 .mu.M concentration, does not activate a negative control
V9m cell line expressing TK-renilla luciferase reporter to a level
greater than 20; and va) inhibits the enzymatic activity of
recombinant 15-PGDH protein at an IC50 of less than 1 .mu.M
[0215] In other embodiments, the 15-PGDH inhibitor can ib) at 2.5
.mu.M concentration, stimulate a Vaco503 reporter cell line
expressing a 15-PGDH luciferase fusion construct to increase
luciferase output; iib) at 2.5 .mu.M concentration stimulate a V9m
reporter cell line expressing a 15-PGDH luciferase fusion construct
to increase luciferase output; iiib) at 7.5 .mu.M concentration
stimulate a LS174T reporter cell line expressing a 15-PGDH
luciferase fusion construct to increase luciferase output; ivb) at
7.5 .mu.M concentration, does not activate a negative control V9m
cell line expressing TK-renilla luciferase reporter to a luciferase
level greater than 20% above background; and vb) inhibits the
enzymatic activity of recombinant 15-PGDH protein at an IC50 of
less than 1 .mu.M.
[0216] In other embodiments, the 15-PGDH inhibitor can inhibit the
enzymatic activity of recombinant 15-PGDH at: ic) an IC50 of less
than 1 uM, or preferably iic) at an IC50 of less than 250 nM, or
more preferably iiic) at an IC50 of less than 50 nM, or more
preferably iv) at an IC50 of less than 5 nM.
[0217] An example of a 15-PGDH inhibitor having formula (I) that
meets the above noted criteria (ia-va) includes a compound having
the formula (III): An example of a 15-PGDH inhibitor having formula
(I) that meets the above noted criteria (ib-vb) includes a compound
having the formula (III): An example of a 15-PGDH inhibitor having
formula (I) that meets the above noted criteria ic, and/or iic, and
or iiic, and or ivc, includes a compound having the formula (III):
In still other embodiments, the 15-PGDH inhibitor can include a
compound having the following formula (III):
##STR00009## [0218] wherein n is 0-2; [0219] R.sub.1 is a C.sub.1-8
alkyl, which is linear, branched, or cyclic and which is
unsubstituted or substituted; [0220] R.sub.2 and R.sub.3 are the
same or different and are each selected from the group consisting
of a H, a lower alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2,
or 3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', (C.dbd.O)N(R').sub.2, O(CO)R',
COOR' (wherein R' is H or a lower alkyl group); [0221] Z.sub.1 is
NR', O or S (wherein R' is H or a lower alkyl group); [0222]
X.sub.2 is N or C; [0223] R.sub.6 and R.sub.7 are optional and if
present are the same or different and are each selected from the
group consisting of a H, F, Cl, Br, I, a lower alkyl group,
(CH.sub.2).sub.n1OR' (wherein n1=1, 2, or 3), CF.sub.3,
CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group); substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, and a substituted or
unsubstituted heterocyclyl; and pharmaceutically acceptable salts
thereof.
[0224] 15-PGDH inhibitors having formula (III) can be synthesized
as shown:
##STR00010##
[0225] Any reaction solvent can be used in the above preparation
process as long as it is not involved in the reaction. For example,
the reaction solvent includes ethers such as diethyl ether,
tetrahydrofuran and dioxane; halogenized hydrocarbons, such as
dichloromethane and chloroform; amines such as pyridine, piperidine
and triethylamine; alkylketones, such as acetone, methylethylketone
and methylisobutyl; alcohols, such as methanol, ethanol and
propanol; non-protonic polar solvent, such as
N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile,
dimethylsulfoxide and hexamethyl phosphoric acid triamide. Among
non-reactive organic solvents that are ordinarily used in the
organic synthesis, preferable solvents are those from which water
generated in the reaction can be removed by a Dean-Stark trap. The
examples of such solvents include, but are not limited to benzene,
toluene, xylene and the like. The reaction product thus obtained
may be isolated and purified by condensation, extraction and the
like, which is ordinarily conducted in the field of the organic
synthesis, if desired, by silica gel column chromatography. The
individual enantiomers of PGDH inhibitors having the formula III
can be separated by a preparative HPLC using chromatography columns
containing chiral stationary phases.
[0226] Further, embodiments of this application include any
modifications for the preparation method of the 15-PGDH inhibitors
described above. In this connection, any intermediate product
obtainable from any step of the preparation method can be used as a
starting material in the other steps. Such starting material can be
formed in situ under certain reaction conditions. Reaction reagents
can also be used in the form of their salts or optical isomers.
[0227] Depending on the kinds of the substituents to be used in the
preparation of the 15-PGDH inhibitors, and the intermediate product
and the preparation method selected, novel 15-PGDH inhibitors can
be in the form of any possible isomers such as substantially pure
geometrical (cis or trans) isomers, optical isomers (enantiomers)
and racemates.
[0228] In some embodiments, a 15-PGDH inhibitor having formula
(III) can include a compound with the following formula:
##STR00011## [0229] and pharmaceutically acceptable salts
thereof.
[0230] Advantageously, the 15-PDGH inhibitor having formula (III)
was found to: i) inhibit recombinant 15-PGDH at 1 nM concentration;
ii) inhibit 15-PGDH in cell lines at 100 nM concentration, iii)
increase PGE.sub.2 production by cell lines; iv) is chemically
stable in aqueous solutions over broad pH range; v) is chemically
stable when incubated with hepatocyte extracts, vi) is chemically
stable when incubated with hepatocyte cell lines; vii) shows 253
minutes plasma half-life when injected IP into mice; and viii)
shows no immediate toxicity over 24 hours when injected IP into
mice at 0.6 .mu.mole/per mouse and at 1.2 .mu.mole/per mouse and
also no toxicity when injected IP into mice at 0.3 .mu.mole/per
mouse twice daily for 21 days.
[0231] In other embodiments, a 15-PGDH inhibitor having formula
(III) can include a compound with the following formula:
##STR00012## [0232] and pharmaceutically acceptable salts
thereof.
[0233] In still other embodiments, a 15-PGDH inhibitor having
formula (III) can include a compound with the following
formula:
##STR00013##
and pharmaceutically acceptable salts thereof.
[0234] In other embodiments, the 15-PDHG inhibitor can comprise a
(+) or (-) optical isomer of a 15-PGDH inhibitor having formula
(III). In still other embodiments, the 15-PDHG inhibitor can
comprise a mixture at least one of a (+) or (-) optical isomer of a
15-PGDH inhibitor having formula (III). For example, the 15-PGDH
inhibitor can comprise a mixture of: less than about 50% by weight
of the (-) optical isomer of a 15-PGDH inhibitor having formula
(III) and greater than about 50% by weight of the (+) optical
isomer of a 15-PGDH inhibitor having formula (III), less than about
25% by weight of the (-) optical isomer of a 15-PGDH inhibitor
having formula (III) and greater than about 75% by weight of the
(+) optical isomer of a 15-PGDH inhibitor having formula (III),
less than about 10% by weight of the (-) optical isomer of a
15-PGDH inhibitor having formula (III) and greater than about 90%
by weight of the (+) optical isomer of a 15-PGDH inhibitor having
formula (III), less than about 1% by weight of the (-) optical
isomer of a 15-PGDH inhibitor having formula (III) and greater than
about 99% by weight of the (+) optical isomer of a 15-PGDH
inhibitor having formula (III), greater than about 50% by weight of
the (-) optical isomer of a 15-PGDH inhibitor having formula (III)
and less than about 50% by weight of the (+) optical isomer of a
15-PGDH inhibitor having formula (III), greater than about 75% by
weight of the (-) optical isomer of a 15-PGDH inhibitor having
formula (III) and less than about 25% by weight of the (+) optical
isomer of a 15-PGDH inhibitor having formula (III), greater than
about 90% by weight of the (-) optical isomer of a 15-PGDH
inhibitor having formula (III) and less than about 10% by weight of
the (+) optical isomer of a 15-PGDH inhibitor having formula (III),
or greater than about 99% by weight of the (-) optical isomer of a
15-PGDH inhibitor having formula (III) and less than about 1% by
weight of the (+) optical isomer of a 15-PGDH inhibitor having
formula (III).
[0235] In a still further embodiment, the 15-PDGH inhibitor can
consist essentially of or consist of the (+) optical isomer of a
15-PGDH inhibitor having formula (III). In yet another embodiment,
the PDGH inhibitor can consist essentially of or consist of the (-)
optical isomer of a 15-PGDH inhibitor having formula (III).
[0236] The 15-PGDH inhibitors described herein can be used for the
prevention or the treatment of diseases that are associated with
15-PGDH and/or decreased prostaglandin levels and/or where it
desirable to increase prostaglandin levels in the subject. For
example, as discussed above, it is known that prostaglandins play
an important role in hair growth. Specifically, internal storage of
various types (A.sub.2, F.sub.2a, E.sub.2) of prostaglandins in the
various compartments of hair follicles or their adjacent skin
environments has been shown to be essential in maintaining and
increasing hair density (Colombe L et. al, 2007, Exp. Dermatol,
16(9), 762-9). It has been reported that 15-PGDH, which is involved
in the degradation of prostaglandins is present in the hair
follicle dermal papillae, inactivates prostaglandins, especially,
PGF.sub.2a and PGE.sub.2, to cause scalp damage and alopecia
(Michelet J F et. al., 2008, Exp. Dermatol, 17(10), 821-8). Thus,
the compounds described herein, which have a suppressive or
inhibitory activity against 15-PGDH that degrades prostaglandins,
can improve scalp damage, prevent alopecia and promote hair growth
and be used in a pharmaceutical composition for the prevention of
alopecia and the promotion of hair growth.
[0237] In other embodiments, the 15-PGDH inhibitors described
herein can be used in a pharmaceutical composition for promoting
and/or inducing and/or stimulating pigmentation of the skin and/or
skin appendages, and/or as an agent for preventing and/or limiting
depigmentation and/or whitening of the skin and/or skin appendages,
in particular as an agent for preventing and/or limiting
canities.
[0238] In still other embodiments, the 15-PGDH inhibitors described
herein can be used in a pharmaceutical composition for the
prevention or the treatment of cardiovascular disease and/or
diseases of vascular insufficiency, such as Raynaud's disease,
Buerger's disease, diabetic neuropathy, and pulmonary artery
hypertension. Prostaglandins including prostaglandin homologues
produced in the body have been known to maintain the proper action
of the blood vessel wall, especially to contribute to vasodilation
for blood flow, preventing platelet aggregation and modulating the
proliferation of smooth muscle that surrounds blood vessel walls
(Yan. Cheng et. al., 2006, J. Clin., Invest). In addition, the
inhibition of prostaglandins production or the loss of their
activity causes the degeneration of the endothelium in the blood
vessel walls, platelet aggregation and the dysfunction of cellular
mechanism in the smooth muscle. Among others, the production of
prostaglandins in blood vessels was shown to be decreased in
hypertension patients, including pulmonary artery hypertension.
[0239] In other embodiments, the 15-PGDH inhibitors described
herein can be used in a pharmaceutical composition for the
prevention or the treatment of oral and/or gastrointestinal
diseases, such as oral ulcers, gum disease, gastritis, colitis,
ulcerative colitis, and gastric ulcers. Gastritis and gastric
ulcer, representatives of the gastrointestinal diseases, are
defined as the conditions where gastrointestinal mucus membrane is
digested by gastric acid to form ulcer. In the stomach walls
generally consisting of mucosa, submucosa, muscle layer and serosa,
gastric ulcer even damages submucosa and muscle layer, while
gastritis damages mucosa only. Although the morbidity rates of
gastritis and gastric ulcer are relatively high, the causes thereof
have not been clarified yet. Until now, they are known to be caused
by an imbalance between aggressive factors and defensive factors,
that is, the increase in aggressive factors such as the increase in
gastric acid or pepsin secretion, or the decrease in defensive
factors such as structural or morphological deficit of the gastric
mucus membrane, the decrease in mucus and bicarbonate ion
secretion, the decrease in prostaglandin production, or the
like.
[0240] Currently available therapeutic agents for gastritis and
gastric ulcer comprise various drugs for strengthening the
defensive factors such as an antacid, which does not affect,
gastric acid secretion but neutralizes gastric acid that has been
already produced, an inhibitor of gastric acid secretion, a
promoter of prostaglandin secretion, and a coating agent for
stomach walls. Especially, prostaglandins are known to be essential
in maintaining the mechanism for protecting and defending gastric
mucus membrane (Wallace J L., 2008, Physiol Rev., 88(4), 1547-65,
S. J. Konturek et al., 2005, Journal of Physiology and
Pharmacology, 56(5)). In view of the above, since the 15-PGDH
inhibitors described herein show a suppressive or inhibitory
activity against 15-PGDH, which degrades prostaglandins that
protect gastric mucus membrane, they can be effective for the
prevention or the treatment of gastrointestinal diseases, inter
alia, gastritis and gastric ulcer.
[0241] In the kidney, prostaglandins modulate renal blood flow and
may serve to regulate urine formation by both renovascular and
tubular effects. In clinical studies, PGE.sub.1 has been used to
improve creatinine clearance in patients with chronic renal
disease, to prevent graft rejection and cyclosporine toxicity in
renal transplant patients, to reduce the urinary albumin excretion
rate and N-acetyl-beta-D-glucosaminidase levels in patients with
diabetic nephropathy (see Porter, Am., 1989, J. Cardiol., 64:
22E-26E). In addition, U.S. Pat. No. 5,807,895 discloses a method
of preventing renal dysfunction by intravenous administration of
prostaglandins such as PGE.sub.1, PGE.sub.2 and PGI.sub.2.
Furthermore, it has been reported that prostaglandins serve as
vasodilators in the kidney, and, thus, the inhibition of
prostaglandin production in the kidney results in renal dysfunction
(Hao. C M, 2008, Annu Rev Physiol, 70, 357.about.77).
[0242] Thus, the 15-PGDH inhibitors described herein, which have a
suppressive or inhibitory activity against 15-PGDH that degrades
prostaglandins, may be effective in the prevention or the treatment
of renal diseases that are associated with renal dysfunction.
[0243] The term "renal dysfunction" as used herein includes such
manifestations as follows: lower than normal creatinine clearance,
lower than normal free water clearance, higher than normal blood
urea, nitrogen, potassium and/or creatinine levels, altered
activity of kidney enzymes such as gamma glutamyl synthetase,
alanine phosphatidase, N-acetyl-beta-D-glucosaminidase, or
beta-w-microglobulin; and increase over normal levels of
macroalbuminuria.
[0244] Prostaglandins including PGE.sub.1, PGE.sub.2 and PGF.sub.2a
have also been shown to stimulate bone resorption and bone
formation to increase the volume and the strength of the bone (H.
Kawaguchi et. al., Clinical Orthop. Rel. Res., 313, 1995; J. Keller
et al., Eur. Jr. Exp. Musculoskeletal Res., 1, 1992, 8692).
Considering that 15-PGDH inhibits the activities of prostaglandins
as mentioned in the above, the inhibition of 15-PGDH activity may
lead to the promotion of bone resorption and bone formation that
are inhibited by 15-PGDH. Thus, the 15-PGDH inhibitors described
herein can be effective for the promotion of bone resorption and
bone formation by inhibiting 15-PGDH activity. 15-PGDH inhibitors
can also be used to increase bone density, treat osteoporosis,
promote healing of fractures, or promote healing after bone surgery
or joint replacement.
[0245] In yet other embodiments, the 15-PGDH inhibitors described
herein can effective for treating 15-PGDH expressing cancers.
Inhibition of 15-PGDH can inhibit the growth, proliferation, and
metastasis of 15-PGDH expressing cancers.
[0246] In still other embodiments, the 15-PGDH inhibitors described
herein can be effective for wound healing. Among various
prostaglandins, PGE.sub.2 is known to serve as a mediator for wound
healing. Therefore, when skin is injured by wounds or burns, the
inhibition of 15-PGDH activity can produce the treatment effect of
the wounds or the burns by PGE.sub.2.
[0247] Additionally, as discussed above, increased prostaglandin
levels have been shown to stimulate signaling through the Wnt
signaling pathway via increased beta-catenin mediated
transcriptional activity. Wnt signaling is known to be a key
pathway employed by tissue stem cells, and increasing PGE.sub.2
signaling has in model organisms been shown to increase numbers of
hematopoietic stem cells. Hence, 15-PGDH inhibitors described
herein may be utilized to increase tissue stem cell numbers for
purposes that would include increasing resistance to tissue damage
by radiation, increasing resistance to environmental exposures to
radiation, increasing stem cell numbers to increase fitness of bone
marrow or other types of transplantation (through either in vivo
exposure to 15-PGDH inhibitors described herein to increase stem
cell numbers prior to harvest of a transplanted tissue, or through
ex vivo exposure of a harvested tissue prior to transplant into a
recipient host, or through treatment of the recipient host either
before, during, or after receipt of the transplant).
[0248] In some embodiments, the 15-PGDH inhibitor can be
administered to a bone marrow graft donor or a hematopoietic stem
cell donor to increase the fitness of a donor bone marrow graft or
a donor hematopoietic stem cell graft.
[0249] In other embodiments, the 15-PGDH inhibitor can also be
administered to bone marrow of a subject to increase stem cells in
the subject or to increase the fitness of the marrow as a donor
graft.
[0250] In still other embodiments, the 15-PGDH inhibitor can be
administered to a preparation of hematopoietic stem cells,
peripheral blood hematopoietic stem cells, or umbilical cord stem
cells of the subject to increase the fitness of the stem cell
preparation as a donor graft or to decrease the number of units of
umbilical cord blood required for transplantation.
[0251] In yet other embodiments, the 15-PGDH inhibitor can be
administered to a subject to mitigate bone marrow graft rejection,
to enhance bone marrow graft engraftment, to enhance engraftment of
a hematopoietic stem cell graft, or an umbilical cord stem cell
graft, to enhance engraftment of a hematopoietic stem cell graft,
or an umbilical cord stem cell graft, and/or to decrease the number
of units of umbilical cord blood required for transplantation into
the subject. The administration can be, for example, following
treatment of the subject or the marrow of the subject with
radiation therapy, chemotherapy, or immunosuppressive therapy.
[0252] In other embodiments, the 15-PGDH inhibitor can be
administered to a recipient of a bone marrow transplant, of a
hematopoietic stem cell transplant, or of an umbilical cord stem
cell transplant, in order to decrease the administration of other
treatments or growth factors.
[0253] In further embodiments, the 15-PGDH inhibitor can be
administered to a subject or to a tissue graft of a subject to
mitigate graft rejection, to enhance graft engraftment, to enhance
graft engraftment following treatment of the subject or the marrow
of the subject with radiation therapy, chemotherapy, or
immunosuppressive therapy, to confer resistance to toxic or lethal
effects of exposure to radiation, confer resistance to the toxic
effect of Cytoxan, the toxic effect of fludarabine, the toxic
effect of chemotherapy, or the toxic effect of immunosuppressive
therapy, to decrease infection, and/or to decrease pulmonary
toxicity from radiation.
[0254] Additionally, in model organism PGE.sub.2 signaling
stimulates liver regeneration and increase survival after exposure
to hepatoxic agents, such as acetaminophen. Hence, 15-PGDH
inhibitors described herein may be utilized to increase liver
regeneration after liver resection, or to increase liver
regeneration and increase survival after exposures to hepatoxic
agents, including but not limited to acetaminophen and similar
compounds.
[0255] PGE1 analogues have also been used in the treatment of
erectile dysfunction. Accordingly, in some embodiments, 15-PGDH
inhibitors described herein can used either alone or combination
with a prostaglandin for the treatment of erectile dysfunction.
[0256] The 15-PGDH inhibitors described herein can be provided in a
pharmaceutical composition or cosmetic composition depending on the
pathological or cosmetic condition or disorder being treated. A
pharmaceutical composition containing the 15-PGDH inhibitors
described herein as an active ingredient may be manufactured by
mixing the derivative with a pharmaceutically acceptable carrier(s)
or an excipient(s) or diluting the 15-PGDH inhibitors with a
diluent(s) in accordance with conventional methods. The
pharmaceutical composition may further contain fillers,
anti-cohesives, lubricants, wetting agents, flavoring agents,
emulsifying agents, preservatives and the like. The pharmaceutical
composition may be formulated into a suitable formulation in
accordance with the methods known to those skilled in the art so
that it can provide an immediate, controlled or sustained release
of the 15-PGDH inhibitors after being administered into a
mammal.
[0257] In some embodiments, the pharmaceutical composition may be
formulated into a parenteral or oral dosage form. The solid dosage
form for oral administration may be manufactured by adding
excipient, if necessary, together with binder, disintegrants,
lubricants, coloring agents, and/or flavoring agents, to the
15-PGDH inhibitors and shaping the resulting mixture into the form
of tablets, sugar-coated pills, granules, powder or capsules. The
additives that can be added in the composition may be ordinary ones
in the art. For example, examples of the excipient include lactose,
sucrose, sodium chloride, glucose, starch, calcium carbonate,
kaolin, microcrystalline cellulose, silicate and the like.
Exemplary binders include water, ethanol, propanol, sweet syrup,
sucrose solution, starch solution, gelatin solution,
carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl
starch, methylcellulose, ethylcellulose, shellac, calcium
phosphonate and polypyrrolidone. Examples of the disintegrant
include dry starch, sodium arginate, agar powder, sodium
bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic
monoglyceride and lactose. Further, purified talc, stearates,
sodium borate, and polyethylene glycol may be used as a lubricant;
and sucrose, bitter orange peel, citric acid, tartaric acid, may be
used as a flavoring agent. In some embodiments, the pharmaceutical
composition can be made into aerosol formulations (e.g., they can
be nebulized) to be administered via inhalation.
[0258] The 15-PGDH inhibitors described herein may be combined with
flavoring agents, buffers, stabilizing agents, and the like and
incorporated into oral liquid dosage forms such as solutions,
syrups or elixirs in accordance with conventional methods. One
example of the buffers may be sodium citrate. Examples of the
stabilizing agents include tragacanth, acacia and gelatin.
[0259] In some embodiments, the 15-PGDH inhibitors described herein
may be incorporated into an injection dosage form, for example, for
a subcutaneous, intramuscular or intravenous route by adding
thereto pH adjusters, buffers, stabilizing agents, relaxants,
topical anesthetics. Examples of the pH adjusters and the buffers
include sodium citrate, sodium acetate and sodium phosphate.
Examples of the stabilizing agents include sodium pyrosulfite,
EDTA, thioglycolic acid and thiolactic acid. The topical
anesthetics may be procaine HCl, lidocaine HCl and the like. The
relaxants may be sodium chloride, glucose and the like.
[0260] In other embodiments, the 15-PGDH inhibitors described
herein may be incorporated into suppositories in accordance with
conventional methods by adding thereto pharmaceutically acceptable
carriers that are known in the art, for example, polyethylene
glycol, lanolin, cacao butter or fatty acid triglycerides, if
necessary, together with surfactants such as Tween.
[0261] The pharmaceutical composition may be formulated into
various dosage forms as discussed above and then administered
through various routes including an oral, inhalational,
transdermal, subcutaneous, intravenous or intramuscular route. The
dosage can be a pharmaceutically effective amount. The
pharmaceutically effective amount can be an amount of the 15-PGDH
inhibitor to treat or improve alopecia, cardiovascular disease,
gastrointestinal disease, wounds, and renal disease. The
pharmaceutically effective amount of the compound will be
appropriately determined depending on the kind and the severity of
the disease to be treated, age, sex, body weight and the physical
condition of the patients to be treated, administration route,
duration of therapy and the like. Generally, the effective amount
of the compound may be in the range of about 1 to 1,000 mg in the
oral administration, about 0.1 to 500 mg in the intravenous
administration, about 5 to 1,000 mg in the rectal administration.
Generally, the daily dosage for adults is in the range of about 0.1
to 5,000 mg, preferably about to 1,000 mg but cannot be determined
uniformly because it depends on age, sex, body weight and the
physical condition of the patients to be treated. The formulation
may be administered once a day or several times a day with a
divided dose.
[0262] Cosmetic compositions containing the 15-PGDH inhibitor can
include any substance or preparation intended to be brought into
contact with the various superficial parts of the human body
(epidermis, body hair and hair system, nails, lips and external
genital organs) or with the teeth or the buccal mucous membranes
for the purpose, exclusively or mainly, of cleansing them, of
giving them a fragrance, of modifying their appearance and/or of
correcting body odors and/or protecting them or of maintaining them
in good condition.
[0263] The cosmetic composition can comprise a cosmetically
acceptable medium that may be water or a mixture of water and at
least one solvent selected from among hydrophilic organic solvents,
lipophilic organic solvents, amphiphilic organic solvents, and
mixtures thereof.
[0264] For topical application, the cosmetic composition can be
administered in the form of aqueous, alcoholic, aqueous-alcoholic
or oily solutions or suspensions, or of a dispersion of the lotion
or serum type, of emulsions that have a liquid or semi-liquid
consistency or are pasty, obtained by dispersion of a fatty phase
in an aqueous phase (O/W) or vice versa (W/O) or multiple
emulsions, of a free or compacted powder to be used as it is or to
be incorporated into a physiologically acceptable medium, or else
of microcapsules or microparticles, or of vesicular dispersions of
ionic and/or nonionic type. It may thus be in the form of a salve,
a tincture, milks, a cream, an ointment, a powder, a patch, an
impregnated pad, a solution, an emulsion or a vesicular dispersion,
a lotion, aqueous or anhydrous gels, a spray, a suspension, a
shampoo, an aerosol or a foam. It may be anhydrous or aqueous. It
may also comprise solid preparations constituting soaps or
cleansing cakes.
[0265] The cosmetic compositions may in particular comprise a hair
care composition, and in particular a shampoo, a setting lotion, a
treating lotion, a styling cream or gel, restructuring lotions for
the hair, a mask, etc. The cosmetic compositions can be a cream, a
hair lotion, a shampoo or a conditioner. These can be used in
particular in treatments using an application that may or may not
be followed by rinsing, or else in the form of a shampoo. A
composition in the form of a foam, or else in the form of spray or
an aerosol, then comprising propellant under pressure, is also
intended. It can thus be in the form of a lotion, serum, milk,
cream, gel, salve, ointment, powder, balm, patch, impregnated pad,
cake or foam.
[0266] In particular, the compositions for application to the scalp
or the hair can be in the form of a hair care lotion, for example
for daily or twice-weekly application, of a shampoo or of a hair
conditioner, in particular for twice-weekly or weekly application,
of a liquid or solid soap for cleansing the scalp, for daily
application, of a hairstyle shaping product (lacquer, hair setting
product or styling gel), of a treatment mask, or of a foaming gel
or cream for cleansing the hair. These may also be in the form of a
hair dye or mascara to be applied with a brush or a comb.
[0267] Moreover, for topical application to the eyelashes or body
hair, the compositions may be in the form of a pigmented or
unpigmented mascara, to be applied with a brush to the eyelashes or
alternatively to beard or moustache hair. For a composition
administration by injection, the composition may be in the form of
an aqueous lotion or an oily suspension. For oral use, the
composition may be in the form of capsules, granules, oral syrups
or tablets. According to a particular embodiment, the composition
is in the form of a hair cream or hair lotion, a shampoo, a hair
conditioner or a mascara for the hair or for the eyelashes.
[0268] In a known manner, the cosmetic compositions may also
contain adjuvants that are normal in the cosmetics field, such as
hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic
additives, preservatives, antioxidants, solvents, fragrances,
fillers, UV-screening agents, odor absorbers and dyestuffs. The
amounts of these various adjuvants are those conventionally used in
the cosmetics field, and are for example from 0.1% to 20%, in
particular less than or equal to 10%, of the total weight of the
composition. According to their nature, these adjuvants can be
introduced into the fatty phase, into the aqueous phase and/or into
the lipid spherules.
[0269] In some embodiments, the 15-PGDH inhibitor can be
administered in a combinatorial therapy or combination therapy that
includes administration of a 15-PGDH inhibitor with one or more
additional active agents. The phrase "combinatorial therapy" or
"combination therapy" embraces the administration of the 15-PGDH
inhibitor, and one or more therapeutic agents as part of a specific
treatment regimen intended to provide beneficial effect from the
co-action of these therapeutic agents. Administration of these
therapeutic agents in combination typically is carried out over a
defined period (usually minutes, hours, days or weeks depending
upon the combination selected). "Combinatorial therapy" or
"combination therapy" is intended to embrace administration of
these therapeutic agents in a sequential manner, that is, wherein
each therapeutic agent is administered at a different time, as well
as administration of these therapeutic agents, or at least two of
the therapeutic agents, in a substantially simultaneous manner.
Substantially simultaneous administration can be accomplished, for
example by administering to the subject an individual dose having a
fixed ratio of each therapeutic agent or in multiple, individual
doses for each of the therapeutic agents. Sequential or
substantially simultaneous administration of each therapeutic agent
can be effected by any appropriate route including, but not limited
to, oral routes, intravenous routes, intramuscular routes, and
direct absorption through mucous membrane tissue. The therapeutic
agents can be administered by the same route or by different
routes. The sequence in which the therapeutic agents are
administered is not narrowly critical.
[0270] In some embodiments, the additional active agent can be
chosen in particular from lipoxygenase inhibitors as described in
EP 648488, the bradykinin inhibitors described in particular in EP
845700, prostaglandins and their derivatives, in particular those
described in WO 98/33497, WO 95/11003, JP 97-100091, JP 96-134242,
the agonists or antagonists of the receptors for prostaglandins,
and the nonprostanoic analogues of prostaglandins as described in
EP 1175891 and EP 1175890, WO 01/74307, WO 01/74313, WO 01/74314,
WO 01/74315 or WO 01/72268.
[0271] In other embodiments, the 15-PGDH inhibitors can be
administered in combination with active agents, such as
vasodilators, prostanoid agonists, antiandrogens, cyclosporins and
their analogues, antimicrobials, triterpenes, alone or as a
mixture. The vasodilators can include potassium channel agonists
including minoxidil and its derivatives, aminexil and the compounds
described in U.S. Pat. Nos. 3,382,247, 5,756,092, 5,772,990,
5,760,043, 5,466,694, 5,438,058, 4,973,474, chromakalin and
diazoxide. The antiandrogens can include 5.alpha.-reductase
inhibitors such as finasteride and the compounds described in U.S.
Pat. No. 5,516,779, cyprosterone acetate, azelaic acid, its salts
and its derivatives, and the compounds described in U.S. Pat. No.
5,480,913, flutamide and the compounds described in U.S. Pat. Nos.
5,411,981, 5,565,467 and 4,910,226. The antimicrobial compounds can
include selenium derivatives, ketoconazole, triclocarban,
triclosan, zinc pyrithione, itraconazole, asiatic acid, hinokitiol,
mipirocine, and the compounds described in EP 680745, clinycine
hydrochloride, benzoyl or benzyl peroxide and minocycline. The
anti-inflammatory agents can include inhibitors specific for Cox-2
such as for example NS-398 and DuP-697 (B. Batistini et al.,
DN&P 1994; 7(8):501-511) and/or inhibitors of lipoxygenases, in
particular 5-lipoxygenase, such as for example zileuton (F. J.
Alvarez & R. T. Slade, Pharmaceutical Res. 1992; 9(11):
1465-1473).
[0272] Other active compounds, which can be present in
pharmaceutical and/or cosmetic compositions can include aminexil
and its derivatives, 60-[(9Z,12Z)octadec-9,12-dienoyl]hexapyranose,
benzalkonium chloride, benzethonium chloride, phenol, oestradiol,
chlorpheniramine maleate, chlorophyllin derivatives, cholesterol,
cysteine, methionine, benzyl nicotinate, menthol, peppermint oil,
calcium panthotenate, panthenol, resorcinol, protein kinase C
inhibitors, prostaglandin H synthase 1 or COX-1 activators, or
COX-2 activators, glycosidase inhibitors, glycosaminoglycanase
inhibitors, pyroglutamic acid esters, hexosaccharidic or
acylhexosaccharidic acids, substituted ethylenearyls, N-acylated
amino acids, flavonoids, derivatives and analogues of ascomycin,
histamine antagonists, triterpenes, such as ursolic acid and the
compounds described in U.S. Pat. Nos. 5,529,769, 5,468,888,
5,631,282, saponins, proteoglycanase inhibitors, agonists and
antagonists of oestrogens, pseudopterins, cytokines and growth
factor promoters, IL-1 or IL-6 inhibitors, IL-10 promoters, TNF
inhibitors, vitamins, such as vitamin D, analogues of vitamin B12
and panthotenol, hydroxy acids, benzophenones, esterified fatty
acids, and hydantoin.
[0273] Pharmaceutical and/or cosmetic compositions including the
15-PGDH inhibitor described herein can additionally contain, for
example, at least one compound chosen from prostaglandins, in
particular prostaglandin PGE.sub.1, PGE.sub.2, their salts, their
esters, their analogues and their derivatives, in particular those
described in WO 98/33497, WO 95/11003, JP 97-100091, JP 96-134242,
in particular agonists of the prostaglandin receptors. It may in
particular contain at least one compound such as the agonists (in
acid form or in the form of a precursor, in particular in ester
form) of the prostaglandin F.sub.2.alpha. receptor, such as for
example latanoprost, fluprostenol, cloprostenol, bimatoprost,
unoprostone, the agonists (and their precursors, in particular the
esters such as travoprost) of the prostaglandin E.sub.2 receptors
such as 17-phenyl PGE.sub.2, viprostol, butaprost, misoprostol,
sulprostone, 16,16-dimethyl PGE.sub.2, 11-deoxy PGE.sub.1, 1-deoxy
PGE.sub.1, the agonists and their precursors, in particular esters,
of the prostacycline (IP) receptor such as cicaprost, iloprost,
isocarbacycline, beraprost, eprostenol, treprostinil, the agonists
and their precursors, in particular the esters, of the
prostaglandin D.sub.2 receptor such as BW245C
((4S)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidinehept-
-anoic acid), BW246C
((4R)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidinehept-
-anoic acid), the agonists and their precursors, in particular the
esters, of the receptor for the thromboxanes A2 (TP) such as I-BOP
([IS-[1a,2a(Z),
3b(1E,3S),4a]]-7-[3-[3-hydroxy-4-[4-(iodophenoxy)-1-butenyl]-7-oxabicyclo-
-[2.2.1]hept-2-yl]-5-heptenoic acid).
[0274] Advantageously, the composition can include at least one
15-PGDH inhibitor as defined above and at least one prostaglandin
or one prostaglandin derivative such as for example the
prostaglandins of series 2 including in particular PGF.sub.2.alpha.
and PGE.sub.2 in saline form or in the form of precursors, in
particular of the esters (example isopropyl esters), their
derivatives such as 16,16-dimethyl PGE.sub.2, 17-phenyl PGE.sub.2
and 16,16-dimethyl PGF.sub.2.alpha. 17-phenyl PGF.sub.2.alpha.,
prostaglandins of series 1 such as 11-deoxyprostaglandin E1,
1-deoxyprostaglandin E1 in saline or ester form, is their
analogues, in particular latanoprost, travoprost, fluprostenol,
unoprostone, bimatoprost, cloprostenol, viprostol, butaprost,
misoprostol, their salts or their esters.
[0275] In accordance with another aspect of the application, the
modulator of 15-PGDH can be a 15-PGDH activator that can promote or
stimulate the activity of 15-PGDH. In certain embodiments, the
15-PDGH activator can include a compound having the formula
(IV):
##STR00014## [0276] wherein X.sub.3 and Y.sub.2 are independently C
or SO; [0277] U is OR'' (wherein R'' is H, a substituted or
unsubstituted alkyl group, or substituted or unsubstituted aryl
group) or
[0277] ##STR00015## [0278] R.sub.8, R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each selected from the group consisting of H, F,
Cl, Br, I, an alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2,
or 3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group), a substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted heterocyclyl, and R.sub.8 and R.sub.9 may be linked
to form a cyclic or polycyclic ring; and pharmaceutically
acceptable salts thereof.
[0279] In other embodiments, the 15-PDGH activator can include a
compound having the formula (V):
##STR00016## [0280] wherein U is OR' (wherein R'' is H, a
substituted or unsubstituted alkyl group, or substituted or
unsubstituted aryl group) or
[0280] ##STR00017## [0281] R.sub.8, R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each selected from the group consisting of H, F,
Cl, Br, I, an alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2,
or 3), CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group), a substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted heterocyclyl, and R.sub.8 and R.sub.9 may be linked
to form a cyclic or polycyclic ring; and pharmaceutically
acceptable salts thereof.
[0282] In certain embodiments, a 15-PGDH activator having formula
(IV) or (V) can be selected that can: ia) at 7.5 .mu.M
concentration, stimulate a Vaco503 reporter cell line expressing a
15-PGDH luciferase fusion construct to a luciferase output level of
greater than 50 (using a scale on which a value of 100 indicates a
doubling of reporter output over baseline); iia) at 7.5 .mu.M
concentration stimulate a V9m reporter cell line expressing a
15-PGDH luciferase fusion construct to a luciferase output level of
greater than 50; iiia) at 7.5 .mu.M concentration stimulate a
LS174T reporter cell line expressing a 15-PGDH luciferase fusion
construct to a luciferase output level of greater than 50; iva) at
7.5 .mu.M concentration, does not activate the negative control V9m
cell line expressing TK-renilla luciferase reporter to a level any
greater than 25; and va) against recombinant 15-PGDH protein the
compound shows an IC50 concentration for inhibiting 15-PGDH enzyme
activity of greater than 2.5 .mu.M.
[0283] In certain embodiments, a 15-PGDH activator having formula
(IV) or (V) can be selected that can: ib) at 7.5 .mu.M
concentration, stimulate a Vaco503 reporter cell line expressing a
15-PGDH luciferase fusion construct to increase luciferase output;
iib) at 7.5 .mu.M concentration stimulate a V9m reporter cell line
expressing a 15-PGDH luciferase fusion construct to increase
luciferase output; iiib) at 7.5 .mu.M concentration stimulate a
LS174T reporter cell line expressing a 15-PGDH luciferase fusion
construct to increase luciferase output; ivb) at 7.5 .mu.M
concentration, does not activate the negative control V9m cell line
expressing TK-renilla luciferase reporter to a luciferase level any
greater than 25% above; and vb) against recombinant 15-PGDH protein
the compound shows an IC50 concentration for inhibiting 15-PGDH
enzyme activity of greater than or equal to 2.5 .mu.M.
[0284] In other embodiments, a 15-PGDH activator having formula
(IV) or (V) that meets the above noted criteria (ia-va) and/or that
meet the above noted criteria (ib-vb) includes a compound having
the formula (VI):
##STR00018## [0285] and pharmaceutically acceptable salts
thereof.
[0286] In other embodiments, the 15-PGDH activator can be an
analogue of a compound having the formula (VI). Such analogues can
have the following formula (VII):
##STR00019## [0287] wherein U is OR' (wherein R'' is H, a
substituted or unsubstituted alkyl group, or substituted or
unsubstituted aryl group) or
[0287] ##STR00020## [0288] R.sub.8 and R.sub.9 are each selected
from the group consisting of H, F, Cl, Br, I, an alkyl group,
(CH.sub.2).sub.n1OR' (wherein n1=1, 2, or 3), CF.sub.3,
CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group), a substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted heterocyclyl, and R.sub.8 and R.sub.9 may be linked
to form a cyclic or polycyclic ring; and pharmaceutically
acceptable salts thereof.
[0289] Examples of 15-PGDH activators having the formula (VII)
include:
##STR00021## ##STR00022##
and pharmaceutically acceptable salts thereof.
[0290] Other examples of compounds having formula (VII)
include:
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031##
##STR00032##
and pharmaceutically acceptable salts thereof.
[0291] In other embodiments, the 15-PGDH activator can be an
analogue of compound (VI) having the following formula (VIII):
##STR00033## [0292] wherein R.sub.10 is selected from the group
consisting of a substituted or unsubstituted aryl, a substituted or
unsubstituted cycloalkyl, and a substituted or unsubstituted
heterocyclyl; and pharmaceutically acceptable salts thereof.
[0293] Examples of 15-PGDH activators having the formula (VIII)
include:
##STR00034## ##STR00035##
and pharmaceutically acceptable salts thereof.
[0294] Still other examples of compounds having the formula (VIII)
include:
##STR00036##
and pharmaceutically acceptable salts thereof.
[0295] In still other embodiments, the 15-PGDH activator can be an
analogue of compound (VI) having the formula (IX):
##STR00037## [0296] wherein R.sub.11 is H, F, Cl, Br, I, a lower
alkyl group, (CH.sub.2).sub.n1OR' (wherein n1=1, 2, or 3),
CF.sub.3, CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X,
CH.sub.2--CH.sub.2--CH.sub.2X, O--CH.sub.2--CH.sub.2X (wherein X=F,
Cl, Br, or I), CN, (C.dbd.O)--R', N(R').sub.2, NO.sub.2,
(C.dbd.O)N(R').sub.2, O(CO)R', OR', SR', COOR' (wherein R' is H or
a lower alkyl group), a substituted or unsubstituted aryl, a
substituted or unsubstituted cycloalkyl, and a substituted or
unsubstituted heterocyclyl; and pharmaceutically acceptable salts
thereof.
[0297] Examples of 15-PGDH activators having the formula (IX)
include:
##STR00038##
and pharmaceutically acceptable salts thereof.
[0298] Still other examples of compounds having formula (IX)
include:
##STR00039## ##STR00040## ##STR00041## ##STR00042##
and pharmaceutically acceptable salts thereof.
[0299] In other embodiments, the 15-PGDH activator can be an
analogue of compound (IV) having the following formulas:
##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053##
and pharmaceutically acceptable salts thereof.
[0300] The 15-PGDH activators described herein can be used for the
prevention or the treatment of diseases that are associated with
decreased 15-PGDH levels and/or increased prostaglandin levels.
Increasing tissue levels of 15-PGDH should decrease tissue levels
of prostaglandins. Activities associated with compounds that
decrease tissue prostaglandins include decreasing development of
human tumors. For example, administration of 15-PGDH activators can
be used to treat patients with colon neoplasia, e.g., colon cancer
or colon adenoma, or to treat and prevent new disease in patients
with a history of colon neoplasia, or to reverse resistance to
NSAID therapy for neoplasia therapy or neoplasia preventive
therapy. Further, administration of 15-PGDH activators described
herein can be used to treat subjects having an NSAID-responsive
condition. In certain embodiments, 15-PGDH activators enhance
NSAID-responsiveness in subjects who are relatively unresponsive to
NSAID treatment.
[0301] The 15-PGDH activators described herein can be also be used
in a method of treating any NSAID-responsive condition. The
NSAID-responsive condition applies to a subject who is
NSAID-resistant or a subject who was determined to be resistant to
NSAID therapy. In the method, a therapeutically effective amount of
15-PGDH activators can be administered alone or in combination with
an effective an effective amount of 15-PGDH protein, cDNA, or an
active fragment thereof. The patient may be a subject at risk of
developing colon neoplasia (e.g., based on family history), or a
subject at risk of colon adenoma relapse, but is suspected of being
resistant to NSAID therapy. Further, the patient may be any subject
who is undergoing or about to undergo NSAID therapy for any
NSAID-responsive condition, but who experiences NSAID
resistance.
[0302] The 15-PGDH activators described herein can be provided in a
pharmaceutical composition that includes pharmaceutically
acceptable carrier. In some embodiments, the 15-PGDH activator can
be provided alone or in combination with other components (e.g., an
NSAID), can be made into aerosol formulations (i.e., they can be
"nebulized") to be administered via inhalation. The 15-PGDH
activator can also be provided alone or in combination with other
components in aqueous and non-aqueous solutions, isotonic sterile
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
Compositions including the 15-PGDH activator can be administered,
for example, orally, nasally, topically, intravenously,
intraperitoneally, or intrathecally. The formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampoules and vials. Solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. The modulators can also be administered as part of a
prepared food or drug.
[0303] The dose administered to a patient should be sufficient to
induce a beneficial response in the subject over time. The optimal
dose level for any patient will depend on a variety of factors
including the efficacy of the specific modulator employed, the age,
body weight, physical activity, and diet of the patient, on a
possible combination with other drugs, and on the severity of the
case of diabetes. It is recommended that the daily dosage of the
15-PGDH activator can be determined for each individual patient by
those skilled in the art. The size of the dose also will be
determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
compound in a particular subject.
[0304] In some embodiments, the 15-PGDH activator can be
administered in a combination therapy includes administration of a
single pharmaceutical dosage formulation that contains a 15-PGDH
activator and one or more additional active agents, as well as
administration of a 15-PGDH activator and each active agent in its
own separate pharmaceutical dosage formulation. For example, a
15-PGDH activator and celecoxib can be administered to the human
subject together in a single oral dosage composition, such as a
tablet or capsule, or each agent can be administered in separate
oral dosage formulations. In other embodiments, an NSAID, e.g.,
celecoxib or aspirin, may be administered with an effective amount
of the 15-PGDH activator. Where separate dosage formulations are
used, a 15-PGDH activator and one or more additional active agents
can be administered at essentially the same time (i.e.,
concurrently), or at separately staggered times (i.e.,
sequentially). Combination therapy is understood to include all
these regimens.
[0305] The invention is further illustrated by the following
example, which is not intended to limit the scope of the
claims.
Example 1
[0306] This Example describes the activities of four compounds with
respect to the enzyme 15-Prostaglandin Dehydrogenase (15-PGDH)
(encoded by the gene HPGD). The compounds are SW033291, SW054384,
SW124531, SW145753 and have the following formulas:
##STR00054##
[0307] FIG. 1 shows that SW033291, SW054384, and SW145753 all
increase luciferase activity of cells the express a 15-PGDH
luciferase fusion construct created by targeted gene knock-in of
renilla luciferase into the last coding exon of 15-PGDH. The
activity is demonstrated in three different colon cancer cell lines
all engineered to contain the 15-PGDH-luciferase fusion. These cell
lines are Vaco-9m (V9m), LS174T, Vaco503 (V503). SW054384 is in
general the best inducer, and shows maximum activity at 6.25 .mu.M.
Value of 1.0 on the Y-axis is the basal level of reporter activity
in cells treated with drug free DMSO vehicle.
[0308] FIG. 2 shows western blots demonstrating that SW033291,
SW054384, and SW145753 all increase levels of 15-PGDH protein in
cell lines V503, LS174T, and V503 treated with 7.5 .mu.M compound
for 48 hours. Untreated FET cells provide a positive control for
15-PGDH expression.
[0309] FIG. 3 shows western blot demonstrating SW124531 also
increases 15-PGDH protein levels in colon cell lines (FET cells
treated with TGF- (10 ng/ml) for 48 hours are used as a positive
control for 15-PGDH expression in certain panels).
[0310] FIG. 4 shows western blot demonstrating 5 .mu.M SW124531
increases levels of 15-PGDH protein (wt-PGDH) expressed from a cDNA
expression vector in V400-S3-2-32 cells, and also increases protein
levels of a catalytically dead mutant 15-PGDH (mu-PGDH) also
expressed from a cDNA expression vector in V400-M3-2-72 cells. As
these proteins are expressed from a heterologous CMV promoter, the
findings suggest that the compounds work directly on stabilizing
the 15-PGDH protein. The compounds show no effects on levels of a
related enzyme, 17-beta-estradiol-dehydrogenase.
[0311] FIG. 5 shows increase in 15-PGDH protein levels in V503
cells treated with SW124531 as assayed by immuno-fluorescence
(upper two rows) and by western blot (lower panel).
[0312] FIGS. 6-9 show that SW033291, SW054384, SW145753, and
SW124531 do not in general alter 15-PGDH mRNA levels in treated
colon cancer cell lines as assessed by real-time PCR. The only
exception is the slight increase in 15-PGDH mRNA in SW033291
treated V503 cells, which is less than the induction of 15-PGDH
protein as well as 15-PGDH-luciferase reporter levels seen in
SW033291 treated V503 cells. In these studies parental cell lines
(not containing the 15-PGDH-luciferase reporter) are employed.
[0313] FIG. 10 shows the effects of three compounds on total
15-PGDH activity in cell lines treated with the compounds. Cell
lines were treated with compounds at 7.5 .mu.M for 48 hours, and
then pelleted. Pellets were lysed and total 15-PGDH activity
measured and normalized to 1,000,000 input cells per pellet.
15-PGDH activity was assayed by measuring the transfer of tritium
from 15(S)-[15-3H] PGE.sub.2 to glutamate by coupling 15-PGDH with
glutamate dehydrogenase as described in (Chi X, Freeman B M, Tong
M, Zhao Y, Tai H H. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH)
is up-regulated by flurbiprofen and other non-steroidal
anti-inflammatory drugs in human colon cancer HT29 cells. Arch
Biochem Biophys. 2009; 487(2):139-45). Activity is measured as pmol
PGE.sub.2/min/million cells. As shown, SW033291 markedly inhibits
15-PGDH activity in all three of the cell lines tested. We conclude
that although SW033291 increases total 15-PGDH protein levels in
cells, it also inactivates 15-PGDH enzyme activity.
[0314] In contrast, 15-PGDH enzyme activity is increased in cells
treated with SW054384 and in cells treated with SW145753.
[0315] FIG. 11 shows the effect on activity of recombinant 15-PGDH
protein (a 15-PGDH-GST fusion protein) incubated with varying
concentrations of the test compounds, with 15-PGDH activity across
a range of compound concentrations recorded on the table and
displayed on the corresponding graphs. As shown, SW033291 is a
potent inhibitor of 15-PGDH activity, with an IC50 of <1.25 nM.
This contrasts with the IC50 of between 25 nM-62.5 nM measured for
the commercial 15-PGDH inhibitor available from Cayman Chemical
(Cayman catalogue item 10638, Cayman Chemical number 13695).
[0316] FIG. 11 also shows that at very high concentration SW054384
can inhibit recombinant 15-PGDH activity, with an IC50 of between 5
.mu.M-50 .mu.M. We conclude that SW054384 increases total 15-PGDH
level and activity in cells treated with 7.5 .mu.M compound, but
can inhibit in vitro recombinant 15-PGDH protein in vitro assays
using 5 .mu.M-50 .mu.M compound.
[0317] FIG. 11 also shows that SW145753 can inhibit activity of
recombinant 15-PGDH enzyme in an in vitro assay at an IC50 between
12-6.25 nM. This suggests the activity of SW145753 in increasing
versus in inhibiting 15-PGDH activity may be discordant in cells
versus in the in vitro assay (perhaps due to washout of drugs when
washing the cells), or may be concentration dependent.
[0318] FIG. 12 shows repeat testing of the effects of SW033291 and
SW054384 on activity of recombinant 15-PGDH protein tested in
vitro. Assays were done by measuring the transfer of tritium from
15(S)-[15-3H] PGE.sub.2 to glutamate (at 1 .mu.M PGE.sub.2
substrate) shown at left (panels A, C), or by direct fluorescence
monitoring of NADH generation by 15-PGDH (done at 20 .mu.M
PGE.sub.2 substrate) shown at right (panels B, D). SW033291 is
again confirmed as a highly potent 15-PGDH inhibitor with an IC50
of 0.7 nM as measured in the tritium assay and an IC50 of 1.6 nM as
measured in the fluorescence assay. The relative insensitivity of
the IC50 to substrate concentration suggests that SW033291 is a
non-competitive inhibitor of 15-PGDH.
[0319] SW054384 shows very weak inhibitory activity, with IC50s
that are 10,000 fold higher than that of SW033291 (8.4 .mu.M and 11
.mu.M in the tritium and fluorescent based assays respectively).
This is consistent with the activity of SW054384 being on balance
to increase 15-PGDH protein level and enzyme activity in cells.
[0320] FIG. 13 shows results of assays of 15-PGDH activity using
the tritium method in cells treated with SW124531 (upper panel) and
in recombinant 15-PGDH protein treated with SW124531 (lower panel).
SW124531 shows activity in increasing 15-PGDH activity in most cell
lines, though this activity is best in cell lines in which basal
15-PGDH activity is >10 units. SW124531 also inhibits activity
of 15-PGDH recombinant protein at an IC50 of 50 nM.
[0321] FIG. 14 shows assay of different compounds for ability to
directly bind to recombinant 15-PGDH protein as measured by
shifting the melting temperature of the protein. The melting of the
protein is followed by measurement of the fluorescence of SYPRO
Orange dye (Sigma # S5692) that increases as the dye binds to
hydrophobic residues exposed as the protein melt. The graph at
upper left shows the melt curves of 15-PGDH with all of the assays
done in the presence of the different compounds superimposed on
each other. The graph at upper right plots the negative derivative
of fluorescence versus temperature for each of the curves shown at
left, with the melting point measured as the temperature of the
negative peak (i.e., the point of most rapid change in the
fluorescence versus temperature plot). The results are shown in
tabular form on the table below. Lapatinib is used as a negative
control. There is no binding of any drug in the absence of enzyme
co-factor (either NAD or NADH). In the presence of either NAD or
NADH, SW033291 creates two peaks in the melting curve, with one of
these peaks displaced by 15 degrees Celsius, consistent with
SW033291 binding directly to 15-PGDH. SW124531 and SW145753 also
show evidence of direct binding to 15-PGDH. In this assay, SW054384
cannot be demonstrated to bind 15-PGDH. It is possible that
SW054384 does bind to 15-PGDH, but that the binding is weak and is
melted off at a temperature below the melting temperature of the
15-PGDH protein. Assays were done at both 10 .mu.M and 100 .mu.M
cofactor (testing both NAD and NADH), which compares well with the
published Km of NAD of 15.8 .mu.M.
[0322] FIG. 15 shows that none of the four compounds tested induce
a shift in the melting temperature of catalytically inactive mutant
15-PGDH protein. We interpret the induction of 15-PGDH mutant
protein by SW124531 as suggesting that SW124531 likely has weak
binding to mutant 15-PGDH that is able to stabilize protein at
37.degree. C., but with the drug melted off at a temperature below
50.degree. C., that is the melting temperature of the protein.
[0323] FIG. 16 shows the in vivo modulation by compounds of 15-PGDH
activity as reflected in PGE.sub.2 levels that are assayed in the
medium of A549 cells that have been stimulated by IL1-beta for 23
hours, with compound added for the last 5 hours (blue bars). The
increment in PGE.sub.2 level shows the clear inhibition of 15-PGDH
activity in the cells by addition of SW033291 (as well as SW145753,
SW124531, and a commercial 15-PGDH inhibitor from Cayman Chemical.
In an additional iteration (red bars)(2), SW054384 was added
commencing 24 hours before addition of IL1-beta, and then
maintained for the next 26 hours in the presence of IL1-beta. The
lower level of PGE.sub.2 produced supports that in these cells
SW054384 increased the 15-PGDH activity. Panel at left shows raw
data; whereas, panel at right shows data normalized for cell
numbers present at end of the experiment. PGE.sub.2 levels are
assayed by ELISA.
[0324] FIG. 17 shows the dose response of effect on SW033291 on
PGE.sub.2 production from IL1-beta treated A549 cells, as reflected
in PGE.sub.2 levels that are assayed in the medium of A549 cells
that have been stimulated by IL1-beta for 24 hours, with SW033291
added for the last 8 hours.
[0325] FIG. 18 shows the in vivo modulations by 2.5 .mu.M compounds
of 15-PGDH activity as reflected in PGE.sub.2 levels following
addition of PGE.sub.2 into the medium of Vaco-503 cells. In this
study cells are treated with compound for 24 hours after which
PGE.sub.2 is added into the medium. After an added 24 hours
PGE.sub.2 levels remaining in the medium are assayed by Elisa. Data
labeled "medium" is a control lane with PGE.sub.2 added to medium
alone, in the absence of cells. Data labeled DMSO is a control in
which cells are treated with DMSO only (the diluent for the
compounds). The difference between the "medium" and the "DMSO"
lanes represents the cell dependent degradation of PGE.sub.2 by
15-PGDH. Again demonstrated, is the near complete blockade of
15-PGDH activity by addition of 2.5 uM SW033291, as reflected by
the blockade in PGE.sub.2 degradation. Additionally demonstrated is
the stimulation of 15-PGDH activity by SW054384, as reflected by
the increased degradation of PGE.sub.2.
[0326] FIG. 19 shows the activity of 2.5 .mu.M SW033291 in speeding
the healing of a model wound consisting of a scratch in a monolayer
of HaCaT cells observed over 48 hours of treatment. TGF-beta serves
as the positive control in the assay.
[0327] FIG. 20 shows the quantitation of the width of the scratch
at 0 and 48 hours in the control, 2.5 .mu.M SW033291 treated cells,
and the TGF-beta (1 ng/ml) treated cells.
Example 2
Analysis of Analogues of Lead Compounds SW033291, a 15-PGDH
Inhibitor
[0328] This Example provides data on a group of structural
analogues of SW033291. Data provided includes level of induction of
a 15-PGDH-luciferase fusion gene reporter, recorded as % increased
luciferase activity over basal level, in three colon cancer cell
lines, V9m, V503, and LS174T, engineered to contain the reporter,
and treated with either 2.5 uM or 7.5 uM compound (i.e., Values are
recorded on a scale where 100 indicates of doubling of luciferase
activity over baseline level). Also recorded is the IC50 of each
compound for inhibiting enzymatic activity of recombinant 15-PGDH
in an in vitro assay.
TABLE-US-00001 TABLE 2 Enzyme V9M LS174T V503 V503 inhibi- V9M
reporter LS174T reporter reporter reporter tion reporter activity
reporter activity activity activity (IC.sub.50, activity (7.5
activity (7.5 (2.5 (7.5 Structures ID nM) (2.5 uM) uM) (2.5 uM) uM)
uM) uM) ##STR00055## SW033291 1.23 nM 98.24 93.16 123.46 106.73
126.32 99.34 ##STR00056## SW033291 isomer B 0.76 nM Note:
Structures shown are for illustrative purposes. We don't know which
structure corresponds to Isomer A or B. Residual activity of isomer
A may be due to small amounts of Isomer B present in the
preparation. ##STR00057## SW033291 isomer A 56.56 nM Note:
Structures shown are for illustrative purposes. We don't know which
structure corresponds to Isomer A or B. Residual activity of isomer
A may be due to small amounts of Isomer B present in the
preparation. ##STR00058## SW033292 1.51 ##STR00059## 413423 -5.65
-8.76 3.41 3.91 7.89 3.13 ##STR00060## 980653 8.83 11.96 5.76 10.99
-10.47 -15.21 ##STR00061## 405320 8.77 -15.45 9.95 -2.57 -7.73
-33.68 ##STR00062## SW208078 25 nM 36.16 34.90 72.71 40.01 87.73
83.77 ##STR00063## SW208079 125 nM 36.01 32.75 53.42 43.83 85.29
61.82 ##STR00064## SW033290 525 nM -34.33 -25.45 -0.24 -6.58 30.93
17.71 ##STR00065## SW208080 2.64 nM 102.08 98.65 117.81 116.64
103.70 127.19 ##STR00066## SW208081 18 nM 37.79 63.56 64.53 95.14
90.46 105.37 ##STR00067## SW206976 >7.5 uM 11.80 17.51 42.38
20.56 16.79 49.53 ##STR00068## SW206977 >7.5 uM 3.5028 0.45
34.88 29.35 33.00 37.62 ##STR00069## SW206978 >7.5 uM 7.5141
8.02 31.44 26.19 38.84 35.75 ##STR00070## SW206979 >7.5 uM
-12.59 -20.62 34.26 32.79 21.66 42.92 ##STR00071## SW206980 0.97 nM
99.37 92.71 117.24 92.15 129.57 108.51 ##STR00072## SW206992 1.411
nM 86.44 121.75 85.81 72.03 161.20 145.39 ##STR00073## SW208064
151.4 nM 82.58 50.11 126.18 96.23 126.18 96.23 ##STR00074##
SW208065 4.865 nM 120.19 118.92 73.50 87.74 73.50 87.74
##STR00075## SW208066 1.368 nM 122.72 111.63 123.89 93.74 123.89
93.74 ##STR00076## SW208067 2.395 nM 121.69 108.47 94.30 79.63
94.30 79.63 ##STR00077## SW208068 >7.5 uM 12.90 12.35 14.90
15.00 14.90 15.00 ##STR00078## SW208069 >7.5 uM -14.48 0.23
19.47 15.13 19.57 15.13 ##STR00079## SW208070 >7.5 uM 22.56
12.11 19.69 15.43 19.69 15.43 ##STR00080##
[0329] We first note that the 15-PGDH inhibitory activity of
SW033291 is at least 98% due to the activity of one of the two
optical isomers of this compound, designated isomer A and B. The
structural assignment of isomer A versus isomer B is not yet
established.
[0330] There is an important effect on the length of the carbon
side chain of SW033291 on the IC50 for inhibiting recombinant
15-PGDH in vitro. Compared to SW033291 (4 carbons): SW208080 (5
carbons) has IC50 1.5 times higher, SW208081 (6 carbons) has IC50
10 times higher, and SW208079 (1 carbon) has IC50 over 60-fold
higher, with marked loss of activity in inducing the cell line
reporters.
[0331] The sulfoxide group appears to be a critical substituent, as
inactive substitutions of the sulfoxide include the corresponding:
ketone (SW206976), amide (SW206977), ester (SW206978), and
carboxylic acid (SW206979). However, inhibitory activity is
observed for the sulfone analogs.
[0332] Deletion of the phenyl group on SW033291 (SW206980) lowers
the IC50 by half. SW206980 continues to be a highly active compound
in reporter induction when applied to reporter cell lines at 2.5 uM
concentration.
Example 3
[0333] The following Example describes the synthesis of SW033291
and analogues thereof as well as provides mass spectrometry NMR
confirmation of the structures.
##STR00081##
##STR00082##
[0334] SW033291
2-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
was prepared using procedure describe by Kalugin. To the solution
of
4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitrile
(0.53 mmol, 220 mg) in DMF (0.25 M)/EtOH (0.5 M) was added KOH
(0.32 mmol, 18 mg, 0.6 equiv., 0.1 M in water). The reaction
mixture was stirred at 35.degree. C. for 40 min. Once complete, the
reaction was diluted with EtOAc and washed with 10% aq. solution of
acidic acid, the organic phase was separated and aqueous layer was
extracted twice with EtOAc, dried over magnesium sulfate, filtered
and concentrated under reduced pressure to give 211 mg of SW033291
2-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
(96%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.67-7.60 (m, 1H),
7.57-7.35 (m, 7H), 7.10 (dd, J=5.0, 3.7 Hz, 1H), 4.54 (s, 2H), 3.26
(ddd, J=12.8, 9.1, 6.0 Hz, 1H), 3.09 (ddd, J=12.8, 9.1, 6.6 Hz,
1H), 1.83-1.61 (m, 2H), 1.53-1.38 (m, 2H), 0.93 (t, J=7.3 Hz, 3H).
ESI-MS (m/z): 413 [M+H].sup.+.
##STR00083##
[0335]
2-(((butylthio)methyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)nicotinon-
itrile. Acetic Acid (900 .mu.L) and hydrogen peroxide (0.57 mmol,
1.5 equiv., 30% solution in water) were added to the solution of
2-(((butylthio)methyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile
(0.38 mmol, 150 mg) in chloroform (900 .mu.L). The reaction mixture
was stirring at 32.degree. C. for 45 min. The reaction was then
diluted with EtOAc and washed with saturated NaHCO.sub.3 solution,
dried over magnesium sulfate, filtered and concentrated under
reduced pressure to give 153 mg of designed product (98%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.75 (dd, J=3.8, 1.1 Hz, 1H),
7.66-7.57 (m, 2H), 7.58-7.51 (m, 4H), 7.47 (s, 1H), 7.16 (dd,
J=5.0, 3.8 Hz, 1H), 4.74 (d, J=13.0 Hz, 1H), 4.41 (d, J=13.0 Hz,
1H), 2.97 (dt, J=13.0, 8.2 Hz, 1H), 2.81 (dt, J=12.9, 7.3 Hz, 1H),
1.94-1.76 (m, 2H), 1.53-1.38 (m, 2H), 0.94 (t, J=7.4 Hz, 3H).
ESI-MS (m/z): 413 [M+H].sup.+
##STR00084##
[0336]
2-(((butylthio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitri-
le. A mixture of
4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile
(0.34 mmol, 101 mg), butyl(chloromethyl)sulfane (0.34 mmol, 48 mg,
1.0 equiv.) and Et.sub.3N (0.51 mmol, 72 .mu.L, 1.5 equiv.) was
refluxed in dry CH.sub.3CN (350 .mu.L) for 20 min. The reaction
mixture was then diluted with EtOAc and water. The organic phase
was separated and aqueous layer was extracted twice with EtOAc. The
combined extractions were washed with saturated NaCl solution,
dried over magnesium sulfate, filtered and concentrated under
reduced pressure. The residue was purified by flash chromatography
to give 124 mg of designed product (92%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.70 (dd, J=3.8, 1.1 Hz, 1H), 7.64-7.56 (m,
1H), 7.55-7.47 (m, 5H), 7.40 (d, J=1.1 Hz, 1H), 7.14 (dd, J=5.0,
3.8 Hz, 1H), 4.53 (s, 2H), 2.74 (t, J=8.0 Hz, 2H), 1.72-1.57 (m,
2H), 1.49-1.34 (m, 2H), 0.90 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 397
[M+H].sup.+.
##STR00085##
[0337]
4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitr-
ile. To a solution of 3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one
(2.34 mmol, 500 mg) and cyanothioacetamide (7.0 mmol, 717 mg, 3.0
equiv.) in ethanol (7 mL), a few drops of piperidine were added.
The reaction was refluxed for 3 h. The solid that formed was
collected and recrystallized from acetic acid to give designed
product in 46% isolated yield.
[0338] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.17 (d, J=3.8
Hz, 1H), 7.96 (d, J=5.0 Hz, 1H), 7.74-7.62 (m, 2H), 7.54 (dd,
J=5.1, 2.0 Hz, 3H), 7.31-7.19 (m, 1H), 7.01 (s, 1H). ESI-MS (m/z):
295 [M+H].sup.+.
##STR00086##
##STR00087##
[0339] 3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one was prepared from
benzaldehyde and 1-(thiophen-2-yl)ethanone via aldol condensation
using procedure described by Azam showed in Scheme 2. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.88-7.80 (m, 2H), 7.67 (dd, J=4.9,
1.1 Hz, 1H), 7.66-7.59 (m, 2H), 7.47-7.34 (m, 4H), 7.18 (dd, J=5.0,
3.8 Hz, 1H). ESI-MS (m/z): 215 [M+H].sup.+.
##STR00088##
[0340] 3-phenyl-1-(thiazol-2-yl)prop-2-en-1-one was prepared from
via Wittig reaction using procedure described by Merino showed in
Scheme 2. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.06 (d,
J=3.0 Hz, 1H), 7.99 (s, 1H), 7.96 (s, 1H), 7.75-7.67 (m, 3H),
7.44-7.38 (m, 3H). ESI-MS (m/z): 216 [M+H].sup.+.
##STR00089##
[0341] SW208079-1-A
2-(methylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
was prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1 and 2.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.67-7.50 (m, 5H),
7.50-7.36 (m, 3H), 7.16-7.09 (m, 1H), 4.58 (s, 2H), 2.99 (s, 3H).
ESI-MS (m/z): 371 [M+H].sup.+.
##STR00090##
[0342] SW208080-1-A
2-(pentylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
was prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1 and 2.
.sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2) .delta. 7.98-7.36 (m, 8H),
7.33-6.85 (m, 1H), 4.47 (s, 2H), 3.28-3.15 (m, 1H), 3.09-2.99 (m,
1H), 1.81-1.59 (m, 2H), 1.50-1.25 (m, 4H), 0.88 (t, J=7.2 Hz, 3H).
ESI-MS (m/z): 427 [M+H].sup.+.
##STR00091##
[0343] SW208081-1-A
2-(hexylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
was prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1 and 2.
.sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2) .delta. 7.78-7.66 (m, 1H),
7.63-7.46 (m, 7H), 7.27-7.02 (m, 1H), 4.11 (s, 2H), 3.43-3.20 (m,
1H), 3.11 (ddd, J=13.8, 9.4, 6.4 Hz, 1H), 1.89-1.63 (m, 2H),
1.58-1.39 (m, 4H), 1.40-1.21 (m, 2H), 0.91 (d, J=6.8 Hz, 3H).
ESI-MS (m/z): 441 [M+H].sup.+.
##STR00092##
[0344] SW208066,
2-(butylsulfinyl)-4-phenyl-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine
was prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1 and 2.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.06 (s, 1H), 7.92 (d,
J=3.2 Hz, 1H), 7.65-7.39 (m, 6H), 4.63 (s, 2H), 3.28 (ddd, J=12.8,
9.0, 6.2 Hz, 1H), 3.11 (ddd, J=12.8, 9.0, 6.8 Hz, 1H), 1.85-1.63
(m, 2H), 1.56-1.42 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z):
414 [M+H].sup.+.
##STR00093##
[0345] SW206980,
2-(butylsulfinyl)-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine was
prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1 and 2.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.79 (d, J=8.5, 1H),
7.65-7.49 (m, 2H), 7.39 (dt, J=5.1, 0.7 Hz, 1H), 7.06 (dd, J=5.0,
3.7, Hz, 1H), 5.20 (s, 2H), 3.26 (ddd, J=12.8, 9.0, 6.2 Hz, 1H),
3.10 (ddd, J=12.8, 9.1, 6.6 Hz, 1H), 1.78-1.60 (m, 2H), 1.55-1.39
(m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 337 [M+H].sup.+
##STR00094##
[0346] SW206992,
2-(butylsulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine was
prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1 and 2.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.15 (d, J=8.5 Hz, 1H),
7.95 (d, J=8.5 Hz, 1H), 7.90 (d, J=3.2 Hz, 1H), 7.44 (d, J=3.2 Hz,
1H), 3.29 (ddd, J=12.7, 9.0, 6.2 Hz, 1H), 3.13 (ddd, J=12.8, 9.0,
6.7 Hz, 1H), 1.83-1.61 (m, 2H), 1.59-1.38 (m, 2H), 0.92 (t, J=7.3
Hz, 3H). ESI-MS (m/z): 338 [M+H].sup.+.
##STR00095##
[0347] SW208064, 2-(butylsulfinyl)thieno[2,3-b]pyridin-3-amine was
prepared by using synthetic procedures described for the
preparation of analog SW033291 and showed in Scheme 1. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.61 (dd, J=4.7, 1.6 Hz, 1H), 7.89
(dd, J=8.1, 1.6 Hz, 1H), 7.33 (dd, J=8.1, 4.6 Hz, 1H), 3.39-3.18
(m, 1H), 3.20-3.03 (m, 1H), 1.74 (p, J=7.6 Hz, 2H), 1.63-1.38 (m,
2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 255 [M+H].sup.+.
##STR00096##
[0348] SW208078-1-A
2-(butylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.
Acetic Acid (50 .mu.L) and hydrogen peroxide (0.036 mmol, 1.5
equiv., 30% solution in water) were added to the solution of
SW033291
2-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
(0.024 mmol, 10 mg) in chloroform (50 .mu.L). The reaction mixture
was stirring at 32.degree. C. for 4 h. The reaction was diluted
with EtOAc and washed with saturated NaHCO.sub.3 solution, dried
over magnesium sulfate, filtered and concentrated under reduced
pressure to give crude
2-(butylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine,
which was purified by flash chromatography in 8% isolated yield.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.71 (d, J=3.8 Hz, 1H),
7.64-7.54 (m, 3H), 7.53-7.42 (m, 4H), 7.15 (dd, J=5.0, 3.7 Hz, 1H),
5.09 (s, 2H), 3.38-3.02 (m, 2H), 1.92-1.67 (m, 2H), 1.52-1.28 (m,
2H), 0.92 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 429 [M+H].sup.+.
##STR00097##
[0349] SW033290-2-A
2-(methylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine
was prepared by using synthetic procedures described for the
preparation of analog SW208078-1-A and showed in Scheme 1 and 2.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.78-7.68 (m, 1H),
7.64-7.54 (m, 3H), 7.53-7.45 (m, 4H), 7.18-7.10 (m, 1H), 5.08 (s,
2H), 3.14 (s, 3H). ESI-MS (m/z): 387 [M+H].sup.+.
##STR00098##
[0350] SW208065,
6-(butylsulfinyl)-2,4-diphenylthieno[2,3-d]pyrimidin-5-amine. To
the solution of
4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitrile
(0.07 mmol, 30 mg) in DMF (0.25 M) was added KOH (0.035 mmol, 2 mg,
0.5 equiv., 0.1 M in water). The reaction mixture was stirred at
room temperature for 20 min. Once complete, the reaction was
diluted with EtOAc and washed with 5% aq. solution of acidic acid.
The organic phase was separated and aqueous layer was extracted
twice with EtOAc, dried over magnesium sulfate, filtered and
concentrated under reduced pressure to give crude product, which
was purified by flash chromatography in 70% isolated yield. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 8.73-8.37 (m, 2H), 7.78-7.68 (m,
2H), 7.66-7.55 (m, 3H), 7.53-7.40 (m, 3H), 4.83 (s, 2H), 3.30 (ddd,
J=12.7, 8.9, 6.3 Hz, 1H), 3.21-3.01 (m, 1H), 1.87-1.66 (m, 2H),
1.57-1.41 (m, 2H), 0.95 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 408
[M+H].sup.+
##STR00099##
[0351]
4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitri-
le. Acetic Acid (600 .mu.L) and hydrogen peroxide (0.37 mmol, 1.5
equiv., 30% solution in water) were added to the solution of
4-(((butylthio)methyl)thio)-2,6-diphenylpyrimidine-5-carbonitrile
(0.25 mmol, 98 mg) in chloroform (900 .mu.L). The reaction mixture
was stirring at 32.degree. C. for 45 min. Once complete, the
reaction was diluted with EtOAc and washed with saturated
NaHCO.sub.3 solution, dried over magnesium sulfate, filtered and
concentrated under reduced pressure to give 88 mg of designed
product (98%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.57 (dt,
J=7.7, 1.2 Hz, 2H), 8.28-8.05 (m, 2H), 7.80-7.40 (m, 6H), 4.82 (d,
J=13.2 Hz, 1H), 4.49 (d, J=13.3, 1H), 2.95 (dt, J=13.0, 8.1 Hz,
1H), 2.84 (dt, J=13.0, 7.3 Hz, 1H), 1.91-1.74 (m, 2H), 1.56-1.40
(m, 2H), 0.95 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 408 [M+H].sup.+
##STR00100##
[0352]
4-(((butylthio)methyl)thio)-2,6-diphenylpyrimidine-5-carbonitrile.
A mixture of
4,6-diphenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (0.35
mmol, 101 mg), butyl(chloromethyl)sulfane (0.35 mmol, 48 mg, 1.0
equiv.) and Et.sub.3N (0.87 mmol, 2.5 equiv.) was refluxed in dry
CH.sub.3CN (200 .mu.L) for 20 min. The reaction was diluted with
EtOAc and water. The organic phase was separated and aqueous layer
was extracted twice with EtOAc. The combined extractions were
washed with saturated NaCl solution, dried over magnesium sulfate,
filtered and concentrated under reduced pressure. The residue
obtained was then purified by flash chromatography to give 59 mg of
designed product (75%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.75-8.36 (m, 2H), 8.35-7.91 (m, 2H), 7.71-7.41 (m, 6H), 4.59 (s,
2H), 2.74 (t, J=7.5 Hz, 2H), 1.75-1.58 (m, 2H), 1.49-1.34 (m, 2H),
0.91 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 392 [M+H].sup.+.
##STR00101##
[0353] 4,6-diphenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile was
prepared according procedure described by Soto. A mixture of NaOiPr
(1.5 mmol, 1.0 equiv., prepared in situ from sodium and dry iPrOH),
benzothioamide (1.5 mmol, 205 mg, 1.0 equiv.) and
2-(ethoxy(phenyl)methylene)malononitrile (1.5 mmol, 297 mg, 1.5
equiv.) in iPrOH (75 mL) was stirred for 5 h at room temperature.
The reaction was then acidified with con. HCl and stirred
overnight, evaporated and obtained solid was recrystallized from
acetic acid to give 265 mg of
4,6-diphenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (61%).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.23-8.12 (m, 2H),
8.07-7.91 (m, 2H), 7.74-7.49 (m, 6H). ESI-MS (m/z): 290
[M+H].sup.+
##STR00102##
[0354] SW208067,
6-(butylsulfinyl)-4-phenyl-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-5-amin-
e was prepared by using synthetic procedures described for the
preparation of analog SW208065 and showed in Scheme 3. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.10 (dd, J=3.7, 1.3 Hz, 1H),
7.74-7.65 (m, 2H), 7.62-7.53 (m, 3H), 7.50 (dd, J=5.0, 1.2 Hz, 1H),
7.14 (dd, J=5.0, 3.7 Hz, 1H), 4.79 (s, 2H), 3.28 (ddd, J=12.8, 9.0,
6.2 Hz, 1H), 3.11 (ddd, J=12.8, 9.0, 6.7 Hz, 1H), 1.84-1.63 (m,
2H), 1.54-1.41 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 414
[M+H].sup.+.
##STR00103##
##STR00104##
[0355] SW206976-1,
1-(3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)pentan-1-o-
ne. To the solution of
2-((2-oxohexyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile
(0.13 mmol, 50 mg) in ethanol (500 .mu.L) was added KOH (0.13 mmol,
2 mg, 1.0 equiv.). The reaction mixture was stirred at 50.degree.
C. for 30 min. Once complete, the reaction was diluted with EtOAc
and washed with 10% aq. HCl. The organic phase was separated and
aqueous layer was extracted twice with EtOAc, dried over magnesium
sulfate, filtered and concentrated under reduced pressure to afford
designed product in 98% yield.
[0356] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.65 (dd, J=3.8,
1.1 Hz, 1H), 7.62-7.56 (m, 2H), 7.55-7.48 (m, 4H), 7.40 (s, 1H),
7.13 (dd, J=5.0, 3.8 Hz, 1H), 4.13 (s, 2H), 2.72 (t, J=7.4 Hz, 2H),
1.72-1.56 (m, 2H), 1.42-1.25 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).
ESI-MS (m/z): 393 [M+H].sup.+.
##STR00105##
[0357]
2-((2-oxohexyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile. A
mixture of
4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile
(0.068 mmol, 20 mg), Et.sub.3N (0.11 mmol, 15 .mu.L, 1.6 equiv.)
and 2-butyloxirane (0.11 mmol, 11 mg, 1.6 equiv.) in MeOH (500
.mu.L) was stirred at room temperature. When the reaction was
complete as judged by TLC, the reaction mixture was evaporated; the
crud product dissolved in DCM and DMP (0.10 mmol, 1.5 equiv.) was
added at 0.degree. C. The reaction mixture was stirred at room
temperature for 2 h and then was quenched by addition of 1:1
mixture of 20% Na.sub.2S.sub.2O.sub.3/NaHCO.sub.3 solution. The
organic layer was separated, dried over magnesium sulfate and the
solvent was removed under reduced pressure. The crude product was
purified by flash chromatography to afford designed product in 72%
yield. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.02 (s, 1H), 7.97
(d, J=3.1 Hz, 1H), 7.71-7.59 (m, 2H), 7.55 (d, J=3.2 Hz, 1H),
7.55-7.46 (m, 4H), 4.52 (s, 2H), 2.75 (t, J=7.8 Hz, 2H), 1.73-1.54
(m, 2H), 1.51-1.26 (m, 2H), 0.91 (t, J=7.3 Hz, 3H). ESI-MS (m/z):
393 [M+H].sup.+.
##STR00106##
[0358] SW206977,
3-amino-4-phenyl-N-propyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carbox-
amide. A mixture of
4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile
(0.12 mmol, 35 mg), 2-chloro-N-propylacetamide (0.12 mmol, 16 mg,
1.0 equiv.) and EtONa (0.19 mmol, 1.6 equiv.) in ethanol (1 mL) was
stirred at 50.degree. C. When the reaction was complete as judged
by TLC, the reaction was diluted with EtOAc and washed with 10% aq.
HCl. The organic phase was separated and aqueous layer was
extracted twice with EtOAc, dried over magnesium sulfate, filtered
and concentrated under reduced pressure to afford designed product
in 61% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.65 (d,
J=3.7, 1H), 7.58-7.49 (m, 3H), 7.49-7.38 (m, 4H), 7.10 (dd J=4.9,
3.7 Hz, 1H), 5.75 (s, 2H), 5.59-5.38 (m, 1H), 3.35 (td J=7.0, 5.9
Hz, 1H), 1.64-1.58 (m, 2H), 0.96 (t, J=7.4 Hz, 3H). ESI-MS (m/z):
394 [M+H].sup.+.
##STR00107##
[0359] SW206978, Ethyl
3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxylate.
A mixture of
4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile
(0.34 mmol, 100 mg), ethyl 2-chloroacetate (0.54 mmol, 1.6 equiv.)
and EtONa (0.54 mmol, 1.6 equiv.) in ethanol (1 mL) was stirred at
reflux. When the reaction was complete as judged by TLC, the
reaction was diluted with EtOAc and washed with 10% aq. HCl. The
organic phase was separated and aqueous layer was extracted twice
with EtOAc, dried over magnesium sulfate, filtered and concentrated
under reduced pressure to afford designed product in 79% yield.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.01 (d, J=3.7 Hz, 1H),
7.87-7.67 (m, 2H), 7.56 (d, J=6.5 Hz, 5H), 7.36-6.90 (m, 1H), 5.73
(s, 2H), 4.23 (q, J=7.1 Hz, 2H), 1.25 (t, J=7.0 Hz, 3H). ESI-MS
(m/z): 381[M+H].sup.+.
##STR00108##
[0360] SW206979,
3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxylic
acid. To a solution of
-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile
(0.34 mmol, 100 mg) and ethyl 2-chloroacetate (0.54 mmol, 1.6
equiv.) in ethanol (1 mL), Et.sub.3N (0.54 mmol, 1.6 equiv.) was
added. The reaction was refluxed for 20 min. The reaction was then
diluted with EtOAc and water. The organic phase was separated and
aqueous layer was extracted twice with EtOAc. The combined
extractions were washed with saturated NaCl solution, dried over
magnesium sulfate, filtered and concentrated under reduced pressure
to afford designed product. Ethyl
2-((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)acetate was
then dissolved in DMF and treated with 1M aq. NaOH at 50.degree. C.
to give SW206979,
3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carbo-
xylic acid in 63% yield. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 8.00 (dd, J=3.7, 1.1 Hz, 1H), 7.79-7.64 (m, 2H), 7.55 (dt,
J=7.6, 3.2 Hz, 5H), 7.16 (dd, J=5.0, 3.7 Hz, 1H), 5.72 (s, 2H).
ESI-MS (m/z): 353[M+H].sup.+.
##STR00109##
##STR00110##
[0361] SW208068, 2-(butylthio)pyridin-3-amine. To the solution of
butane-1-thiol (7.0 mmol, 628 mg, 1.1 equiv.) in THF (30 mL) was
added NaH (6.6 mmol, 158 mg, 1.05 equiv.) at 0.degree. C. After the
reaction mixture was stirred at room temperature for 30 min.
2-chloro-3-nitropyridine (6.33 mmol, 1.0 g) was portion wise added
and left with stirring at room temperature. for 2 h. Water was then
added to the reaction mixture, and the resulting mixture was
extracted with ethyl acetate. The organic layer was washed with a
saturated aqueous solution of sodium chloride, and dried over
sodium sulfate, filtered and concentrated under reduced pressure to
afford crude product. Because of difficulties with purification,
impure 2-(butylthio)-3-nitropyridine was directly used for the next
step. Nitropyridine (0.47 mmol, 100 mg) was dissolved in a mixed
solvent of acetic acid (3.3 ml) and conc. hydrochloric acid (130
.mu.L), and zinc (5.7 mmol, 370 mg) was added in small portions
while being cooled with ice. After the mixture was stirred for 30
minutes, the reaction mixture was filtered, and the filtrate was
neutralized with an aqueous solution of NaHCO.sub.3, and extracted
with DCM. The organic layer was washed with water and then with a
saturated aqueous solution of sodium chloride, and dried over
sodium sulfate. Subsequently, the solvent was evaporated to obtain
2-(butylthio)pyridin-3-amine as a pale yellow oil. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.94 (dd, J=4.1, 2.0 Hz, 1H), 7.05-6.51
(m, 2H), 3.84 (s, 2H), 3.51-2.95 (m, 2H), 1.72-1.60 (m, 2H),
1.56-1.36 (m, 2H), 0.91 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 183
[M+H].sup.+.
##STR00111##
[0362] SW208069, 2-(butylsulfinyl)-3-nitropyridine. To the solution
of butane-1-thiol (7.0 mmol, 628 mg, 1.1 equiv.) in THF (30 mL) was
added NaH (6.6 mmol, 158 mg, 1.05 equiv.) at 0.degree. C. After the
reaction mixture was stirred at room temperature for 30 min.
2-chloro-3-nitropyridine (6.33 mmol, 1.0 g) was portion wise added
and left with stirring at room temperature. for 2 h. Water was then
added to the reaction mixture, and the resulting mixture was
extracted with ethyl acetate. The organic layer was washed with a
saturated aqueous solution of sodium chloride, and dried over
magnesium sulfate, filtered and concentrated under reduced pressure
to afford crude product. Because of difficulties with purification,
impure 2-(butylthio)-3-nitropyridine was used directly for the next
step. Nitropyridine (0.47 mmol, 100 mg) was dissolved in a mixed
solvent of acetic acid (1.2 ml) and chloroform (1.2 mL), and
hydrogen peroxide (0.7 mmol, 1.5 equiv., 30% solution in water) was
added. After the mixture was stirred for 45 minutes, at 32.degree.
C., the reaction was diluted with EtOAc and washed with saturated
NaHCO.sub.3 solution, dried over magnesium sulfate, filtered and
concentrated under reduced pressure to
2-(butylsulfinyl)-3-nitropyridine.
[0363] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.14 (dd, J=4.6,
1.5 Hz, 1H), 8.54 (dd, J=8.2, 1.5 Hz, 1H), 7.67 (dd, J=8.2, 4.6 Hz,
1H), 3.18 (ddd, J=12.7, 9.3, 7.2 Hz, 1H), 3.00 (ddd, J=12.7, 9.1,
4.9 Hz, 1H), 2.17-1.92 (m, 1H), 1.91-1.70 (m, 1H), 1.68-1.35 (m,
2H), 0.96 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 229 [M+H].sup.+.
##STR00112##
[0364] SW208070, 2-(butylsulfinyl)pyridin-3-amine was prepared by
using synthetic procedure described for the preparation of analog
SW208069 and showed in Scheme 4. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.90 (dd, J=4.4, 1.4 Hz, 1H), 7.09 (dd, J=8.3, 4.4 Hz, 1H),
6.93 (dd, J=8.3, 1.4 Hz, 1H), 5.30 (s, 2H), 3.24 (ddd, J=13.0, 9.5,
5.4 Hz, 1H), 3.03 (ddd, J=13.0, 9.8, 6.3 Hz, 1H), 1.95-1.61 (m,
2H), 1.55-1.35 (m, 2H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 199
[M+H].sup.+.
Example 4
Analysis of the Mechanism of 15-PGDH Inhibition by SW033291 and
Related Compounds
[0365] The following Example provides data relating to the
mechanism of action by which SW033291 inhibits 15-PGDH.
[0366] Duplicate titrations of 15-PGDH Inhibitor (SW033291) were
run at 4 different concentrations of 15-PGDH (24 nM, 12 nM, 6 nM, 3
nM). Reactions contained the indicated concentration of enzyme, 250
.mu.M NAD(+), 25 .mu.M PGE-2, and were assembled and incubated at
room temperature for 3 minutes.
[0367] FIGS. 21 (A-B) show the shift in IC.sub.50 value with
changing enzyme concentration. The result is indicative of a
tight-binding mode of inhibition with dependency on enzyme:
inhibitor stoichiometry, rather than on the absolute concentration
of the inhibitor. In all cases, the IC.sub.50 values are less than
the enzyme concentration, indicating that nM drug is almost fully
bound by the enzyme.
[0368] FIGS. 22 (A-B) indicate that SW033291 behaves very much like
an irreversible inhibitor of 15-PGDH, and cannot be efficiently
dialyzed off the 15-PGDH protein.
[0369] The testing of whether SW033291 is a reversible inhibitor
proceeded by: [0370] (i) an 8 ul aliquot taken of 15-PGDH stock (8
mg/mL 15-PGDH in 500 .mu.L of 15-PGDH assay buffer)(4 nmol 15-PGDH,
4 .mu.M 15-PGDH), was incubated on ice with: (a) addition of 5
.mu.L of 100 mM NAD(+)+addition of 3.2 .mu.L of 2.5 mM SW033291
stock, then dialyzed versus 1 L buffer for 12 hours, followed by a
fresh 1 L of buffer for 12 more hours; or (b) addition of 5 .mu.L,
100 mM NAD(+)+addition of 3.2 .mu.L DMSO then dialyzed versus 1 L
buffer for 12 hours, followed by a fresh 1 L of buffer for 12 more
hours. [0371] (ii) Pre-dialysis, remove 1 .mu.L and dilute in 200
.mu.L assay Buffer (20 nM), then measure 15-PGDH activity. [0372]
(iii) Post-Dialysis, at 24 hrs, remove 1 .mu.L and dilute in 200
.mu.L assay Buffer (20 nM), then measure 15-PGDH activity.
[0373] Dialysis buffer is 50 mM Tris pH7.4, 40 mM NaCl, 0.1 mM DTT,
0.01% Tween-20.
[0374] Under the conditions of the assay SW033291 inhibited 91% of
15-PGDH pre-dialysis, and 85% of 15-PGDH activity
post-dialysis--that is dialysis did not reverse the inhibition of
15-PGDH. Control 15-PGDH protein that was dialyzed in the absence
of SW033291 remained fully active.
[0375] FIGS. 23(A-B) show (A) at upper right the reaction rates for
15-PGDH in the presence of a graded set of increasing
concentrations of SW033291. In the graph at upper right P is the
NADH concentration as a proxy for 15-keto-PGE2. (P+S) is the
starting PGE2 concentration of 20 .mu.M. The assay was carried out
in the presence of 10 nM recombinant 15-PGDH. In the graph at lower
left (B), Vo is the initial velocity of the reaction in the absence
of SW033291, and Vi is the initial velocity of the reaction in the
presence of the corresponding concentration of SW033291. The line
shows the curve generated by fitting the data to the Morrison
equation. The curve fitting yields a calculated a Ki.sup.App value
of 0.1015 nM. The dashed line intersects the X axis at 8.5 nM. This
represents the point at which [inhibitor]=[active enzyme] showing
that the enzyme preparation contains 85% active enzyme. In the
Morrison equation, Ki is the binding affinity of the inhibitor; [S]
is substrate concentration; and Km is the concentration of
substrate at which enzyme activity is at half maximal. Note that
IC50 is the functional strength of the inhibitor. Whereas the IC50
value for a compound may vary between experiments depending on
experimental conditions, the Ki is an absolute value. Ki is the
inhibition constant for a drug; the concentration of competing
ligand in a competition assay which would occupy 50% of the
receptors if no ligand were present.
[0376] FIGS. 24(A-B) show duplicate titrations of 15-PGDH Inhibitor
(SW033291) that were run at 6 different concentrations of PGE2
(1.25 uM-40 uM). In the graph at top, Y-axis is % inhibition of the
reaction by SW033291. The X-axis is the concentration of SW033291
in nM. Reactions contain 5.0 nM added 15-PGDH, 250 .mu.M NAD(+),
and indicated concentrations of PGE-2, were assembled and incubated
at room temperature for 60 minutes. The Km for PGE2 is
approximately 5 uM, and reactions run with PGE2 concentrations
below 5 uM go very slowly making it difficult to quantitate
inhibition by SW033291. However, in reactions with PGE2 at
concentrations of 5 uM-40 uM, the IC.sub.50 for SW033291 is
unaffected by the increasing PGE2 concentration, showing that the
inhibition is noncompetitive.
[0377] FIG. 25 shows the structure activity relationships of
analogues of SW033291 versus their IC50 against recombinant
15-PGDH. Assignments of structures to the two isomers, A and B, of
SW033291 are arbitrary, as the structure of the active isomer
(isomer B) has not been determined. The optical isomers of SW033291
were separated by preparative HPLC using a 10 mm.times.250 mm
Chiralcel ODH column, 5% isopropanol in hexanes, 1 mL/min. The `A`
isomer is the faster eluting isomer. The `B` isomer is the slower
eluting isomer.
[0378] The analogue family shows that SW033291, with a 4 carbon
side chain, is 2-fold more potent than SW208080 (5 carbon side
chain), 15-fold more potent than SW208081 (6 carbon side chain),
and 100-fold more potent than SW208079 (1 carbon side chain).
SW033291 is also 20-fold more potent than SW208078, the analogue
that converts the sulfoxide group to a sulfone.
[0379] FIG. 26 shows structures of additional SW033291 analogs that
convert the sulfoxide group to a ketone, an amide, an ester, or a
carboxylic acid. Also shown is structure SW206980 that deletes the
phenyl ring from SW033291.
[0380] FIGS. 27(A-C) show graphs that show the level of compound's
activity in inducing the 15-PGDH-luciferase fusion reporter in
three different test cell line backgrounds, V9m, LS174T, and V503.
Each compound was tested at two concentrations, 2.5 uM, and 7.5 uM.
Y-axis is luciferase activity.
[0381] At 2.5 .mu.M-7.5 .mu.M, SW206980, that deletes the phenyl
group of SW033291, shows activity comparable to SW033291 in all
three reporter lines.
[0382] Structures that have converted the sulfoxide group to a
ketone, amide, ester, or carboxylic acid show major loss of
activity in inducing the reporter.
[0383] FIG. 28 shows graphs that show the percent of 15-PGDH enzyme
activity that is inhibited at 2.5 uM and at 7.5 uM by each of the 5
test compounds. SW206980 that deletes the phenyl group of SW033291,
shows at these concentrations similar potency to SW03291 in
inhibiting 15-PGDH activity.
[0384] Structures that have converted the sulfoxide group to a
ketone, amide, ester, or carboxylic acid show major loss of
activity as 15-PGDH inhibitors.
[0385] FIGS. 29(A-B) show a titration curve that plots percent
inhibition of 15-PGDH enzyme activity at different concentrations
of SW033291 and SW0206980. Under identical assay conditions,
SW206980 shows a slightly lower IC50.
[0386] FIGS. 30(A-B) show that SW206980 binds directly to 15-PGDH
and markedly shifts its melting curve. Shown at left is the melt
curve of 15-PGDH as reflected by fluorescence of the hydrophobic
dye SYPRO Orange. Shown at right is the negative first derivative
of the melt curve.
[0387] Three conditions are plotted, that of 10 uM 15-PGDH, that of
10 uM 15-PGDH plus 10 uM SW206980, and that of 10 uM 15-PGDH plus
125 uM NADH plus 10 .mu.M SW206980. The melting temperature, as
reflected by the inflection point of the curve at right is shifted
by 20.degree. C., from 48-degrees up to 68-degrees, in the presence
of SW206980 and NADH, reflecting that SW206980 directly binds to
and markedly stabilizes the tertiary structure of 15-PGDH, in a
manner requiring the presence of the NADH cofactor.
[0388] FIGS. 31(A-C) show further analogues of SW033291 that build
on the previous finding that removal of the SW033291 phenyl ring
(SW206980) retained activity. The new analog (SW206992) adds a
nitrogen to the left-hand ring.
[0389] Table 3 provides a comparison of the properties of SW033291,
SW206980, and SW206992.
TABLE-US-00002 TABLE 3 Summary of three SW033291 analogs SW033291
SW206980 SW206992 IC.sub.50 1.59 nM 0.97 nM 1.411 nM Time to ~5
mins ~2 mins ~2 mins inhibition (10 nM) .DELTA. Tm (NADH)
19.degree. C. 15.5.degree. C. 19.degree. C. Concentration ~100 nM
>300 nM >1 um for Full Cell Line Reporter Induction
Hepatocyte Stable > T1/2 = stability couple hrs 80 mins Toxicity
>10 .mu.M >7.5 .mu.M >7.5 .mu.M
[0390] Time to inhibition refers to the time needed to inhibit the
generation of NADH by 15-PGDH from the moment with drug is added
into the reaction mix. Delta Tm refers to the shift in melting
temperature of recombinant 15-PGDH in the presence of drug (with
cofactor NADH also present). Concentration of Full Cell Line
Reporter Induction refers to the concentration of drug that needs
to be added to reporter cell line to achieve maximal induction of
the 15-PGDH-luciferase gene fusion reporter cassette, as measured
by luciferase assays. Hepatocyte stability refers to the half-life
of compound in the presence of hepatocytes in culture. Toxicity
refers to the concentration of compound needed to decrease cell
numbers in a cell culture assay.
[0391] FIGS. 32(A-C) show titration of induction by SW033291 of the
15-PGDH-luciferase gene fusion reporter in three different cell
line backgrounds. In general between 80-160 nM SW033291 exposure
for 24 hours is needed to induce maximal reporter induction.
[0392] FIGS. 33(A-C) show titration of induction by SW206980 of the
15-PGDH-luciferase gene fusion reporter in three different cell
line backgrounds. In general .gtoreq.300 nM SW206980 exposure for
24 hours is needed to induce maximal reporter induction.
[0393] FIGS. 34(A-C) show titration of induction by SW206992 of the
15-PGDH-luciferase gene fusion reporter in three different cell
line backgrounds. In general .gtoreq.1000 nM SW206992 exposure for
24 hours is needed to induce maximal reporter induction.
[0394] FIGS. 35(A-C) show the shift in the melt curve of
recombinant 15-PGDH protein (10 uM) by 20 uM SW033291, SW206980,
and SW206992 in the presence of the cofactor NAD (+)(100 uM).
Control melting temperature is 49 degrees centigrade. SW033291
shifts the melting temperature to 63 degrees. SW206980 shifts the
melting temperature to 61 degrees. SW206992 shifts the melting
temperature to 59 degrees. Thus all three compounds directly bind
to 15-PGDH and markedly increase the melting temperature of the
protein, with the order of the temperature shifts being
SW033291>SW206980>SW206992.
[0395] FIGS. 36(A-B) show the shift in the melt curve of
recombinant 15-PGDH protein (10 uM) by 20 uM SW033291, SW206980,
and SW206992 in the presence of the cofactor NADH (100 uM). Control
melting temperature is 55 degrees centigrade. SW033291 shifts the
melting temperature to 74 degrees. SW206980 shifts the melting
temperature to 70.5 degrees. SW206992 shifts the melting
temperature to 68.5 degrees. Thus all three compounds directly bind
to 15-PGDH and markedly increase the melting temperature of the
protein, with the order of the temperature shifts being
SW033291>SW206980>SW206992.
[0396] FIGS. 37(A-C) show a titration curve of 15-PGDH inhibitor
compounds in an assay measuring effect on PGE2 levels in the medium
of A549 cells that have been stimulated with IL1-beta. Highest PGE2
levels, 3000 pg/ml, are achieved with SW033291, with maximal effect
attained at 2.5 uM compound. Next highest PGE2 level, 2500 pg/ml
are achieved with SW206980, with maximal effect attained at 7.5 uM
compound. Lowest induction, to 2100 pg/ml PGE2 is achieved with
SW206992, with maximal effect attained at 2.5 uM. In these
reactions, A549 cells were maintained in F12K medium supplemented
with 10% fetal calf serum (FBS) and 50 .mu.g/mL gentamicin in a
humidified atmosphere containing 5% CO.sub.2 at 37.degree. C. Cells
were plated in 24-well plates (0.5 mL per well) at about 100,000
cells per well in duplicate and grown for 24 h before stimulation
with IL-1.beta. (1 ng/mL) overnight (16 h) to generate PGE2.
SW033291 and its analogs were added at the indicated
concentrations, and the incubation continued for 8 h. Medium was
collected, and the level of PGE2 was analyzed by enzyme
immunoassay. Data were analyzed from results of three independent
experiments.
[0397] FIGS. 38(A-C) show assays of cellular toxicity on A549 cells
at 24 hours of 15-PGDH inhibitors as assayed by CellTiter-Glo
measurement. No effect on CellTitre-Glo levels is seen by
concentrations of up to 10 uM of SW033291, SW206980, and
SW2206992.
[0398] FIG. 39 shows structures of 7 SW033291 analogues, SW208064,
SW208065, SW208066, SW208067, SW208068, SW208069, SW208070.
[0399] Table 4 provides tabular summary of the properties of 4
analogues, SW208064, SW208065, SW208066, SW208067, and in
particular lists the IC50 for each of these 4 compounds against 2.5
nM recombinant 15-PGDH.
TABLE-US-00003 TABLE 4 Summary of four SW033291 analogs from UTSW
set 8 SW033291 SW2068064 SW208065 SW208066 SW208067 IC.sub.50 1.23
nM 151.4 nM 4.865 nM 1.368 nM 2.395 nM Time to inhibition ~5 mins
(10 nM) .DELTA. Tm (NADH) 19.degree. C. 5.degree. C. 13.degree. C.
16.5.degree. C. 16.5.degree. C. Concentration for ~100 nM ~600 nM
~100 nM ~100 nM ~500 nM Full Cell Line Reporter Induction
Hepatocyte Stable > stability couple hrs Toxicity >10
.mu.M
[0400] Time to inhibition refers to the time needed to inhibit the
generation of NADH by 15-PGDH from the moment with drug is added
into the reaction mix. Delta Tm refers to the shift in melting
temperature of recombinant 15-PGDH in the presence of drug (with
cofactor NADH also present). Concentration of Full Cell Line
Reporter Induction refers to the concentration of drug that needs
to be added to reporter cell line to achieve maximal induction of
the 15-PGDH-luciferase gene fusion reporter cassette, as measured
by luciferase assays. Hepatocyte stability refers to the half-life
of compound in the presence of hepatocytes in culture. Toxicity
refers to the concentration of compound needed to decrease cell
numbers in a cell culture assay.
[0401] FIG. 40 provides graphical summary showing the activity of
each of the compounds in inducing a 15-PGDH-luciferase fusion gene
reporter introduced into three different colon cancer cell lines,
V9m, LS174T, and V503. Results are measured by assay of luciferase
activity after exposure of cells to compound at either 2.5 uM or
7.5 uM compounds concentration.
[0402] FIG. 41 provides graphical summary showing the activity of
each of the compounds in inhibiting the enzymatic activity of
recombinant 15-PGDH enzyme when compound is added at 2.5 uM and at
7.5 uM. 100% inhibition corresponds to complete inhibition of the
enzyme.
[0403] FIG. 42 shows measurement of IC for inhibiting 2.5 nM of
recombinant 15-PGDH when incubated across a range of concentrations
of SW208064, SW208065, SW208066, and SW208067. Y-axis of each graph
records percent inhibition of the 15-PGDH enzymatic activity. 100%
Inhibition corresponds to complete inhibition of the enzyme. X-axis
of each graph records the log of the inhibitor concentration
expressed in nM.
Example 5
Analysis of Toxicity of SW033291
[0404] Table 5 shows a summary of a group of 8-12 week old male FVB
mice in control or SW033291 treatment arms assessed for toxicity of
SW033291, with 6 mice in each arm of the study.
TABLE-US-00004 TABLE 5 Baseline Characteristics FVB male mice--8-12
weeks old Toxicity Study WT-Control WT-Treatment p-value Number 6 6
Sex M M Age (Days) 73.7.1 .+-. 4.7 73.2 .+-. 5.0 0.465 Weight (gm)
27.5 .+-. 2.4 26.8 .+-. 3.1 0.412
[0405] FIG. 43 shows the dose response curve for induction of a
15-PGDH-luciferase fusion gene reporter in the V9m cell line
background of SW033291, SW208064, SW208065, SW208066, and
SW208067.
[0406] FIG. 44 Shows titration curves of 15-PGDH inhibitor
compounds in an assay measuring effects on PGE2 levels in the
medium of A549 cells that have been stimulated with IL1-beta in the
same experimental design described for FIG. 37. At 100 nM
concentration of drug, the highest levels of PGE2 in the medium are
achieved by treating cells with SW208066 or with SW208067, after
which the next highest level of PGE2 in the medium is achieved by
treating cells with SW033291.
[0407] FIG. 45 shows the daily weights of a group of 8-12 week old
FVB mice treated with vehicle or with SW033291 IP at 5 mg/kg twice
daily for 21 days. SW033291 was administered in a vehicle of 10%
Ethanol, 5% Cremophor EL, 85% D5W at a concentration of 125 ug/200
ul. As shown, both vehicle and drug treated mice show equal weight
gain during the 21 day period, with no evidence for SW033291
reducing mouse weight. N=6 mice in both the SW033291 treated and
the vehicle treated arms.
Example 6
Analysis of Effect of SW033291 on Bone Marrow Function
[0408] This Example shows effects of SW033291 on bone marrow
function.
[0409] FIGS. 46(A-C) show analysis of bone marrow of wild-type mice
versus mice that are homozygous genetic knockouts for 15-PGDH
(PGDH-/- mice). Total bone marrow cellularity and percent of
Sca1+/c-Kit+ cells in lineage negative (SKL) cells are the same in
both sets of mice. However, bone marrow from 15-PGDH-/- mice shows
an approximately 50% increase in numbers of hematopoietic colonies
generated when marrow is plated into methylcelluose. Experimental
conditions are noted on the figure. 15-PGDH knockout mice are
denoted by label PGDH-/- and by label 15-PGDH.
[0410] FIG. 47 shows assay in which bone marrow is harvested from a
wild-type mouse, and incubated ex vivo on ice for 2 hours with
either SW033291 (0.5 uM), or 1 uM PGE2 or 1 uM 16,16-dimethyl PGE2
(dmPGE2). Treated marrow is again then plated into methylcellulose
for counting of hematopoietic colonies. SW033291 treated marrow
again shows an approximately 50% increase in the number of bone
marrow derived colonies generated. Under these conditions, a lesser
increase is seen in marrow treated with PGE2, and a slightly
greater increase is seen in marrow treated with dmPGE2.
[0411] FIGS. 48(A-C) show a study of C57BL/6J mice treated with IP
SW033291 administered in a vehicle of 10% Ethanol, 5% Cremophor EL,
85% D5W at a dose of 5 mg/kg or 20 mg/kg. Panel A shows mouse bone
marrow cellularity, white blood count (wbc), red blood count (rbc)
and platelets counts. Panel B shows percent of Sca1+/c-Kit+ cells
in lineage negative (SKL) cells are unchanged in SW033291 treated
mice. Panel C shows that marrow from SW033291 treated mice gives
rise to approximately 30% increase in numbers of hematopoietic
colonies generated when marrow is plated into methylcelluose.
Experimental conditions are noted on the figure.
[0412] FIGS. 49(A-B) show analysis of marrow from CD45.2 antigen
marked C57BL/6J mice that were treated with SW033291 5 mg/kg IP
daily for 3 doses in a vehicle of 10% Ethanol, 5% Cremophor EL, 85%
D5W or that were treated with vehicle alone. On day 3 mice were
sacrificed, marrow flushed and mixed at a 1:1 ratio with vehicle
treated CD45.1 marrow. 2 million whole BM cells were injected into
the tail vein of lethally irradiated CD45.1 mice and percent
chimerism measured via flow cytometry at weeks 8,