U.S. patent application number 10/875356 was filed with the patent office on 2004-11-25 for indole derivatives and their use for the treatment of osteoporosis amongst other applications.
Invention is credited to Farina, Carlo, Gagliardi, Stefania, Novella, Pietro.
Application Number | 20040235930 10/875356 |
Document ID | / |
Family ID | 10855696 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235930 |
Kind Code |
A1 |
Farina, Carlo ; et
al. |
November 25, 2004 |
Indole derivatives and their use for the treatment of osteoporosis
amongst other applications
Abstract
A compound of formula (I) or a salt thereof, or a solvate
thereof, wherein: R.sub.1 and R.sub.2 each independently represents
C.sub.1-6alkoxy or halo; R.sub.3 and R.sub.4 each independently
represents hydrogen, C.sub.1-6alkoxy, arylC.sub.1-6alkoxy, hydroxy,
carboxyC.sub.1-6alkoxy, hydroxyC.sub.1-6alkoxy,
dihydroxyC.sub.1-6alkoxy, mono- and
di-(C.sub.1-6alkyl)aminoC.sub.1-6alkoxy or aminoC.sub.1-6alkoxy,
and R.sub.5 represents --NR.sub.sR.sub.t wherein R.sub.s and
R.sub.t each independently represent hydrogen, unsubstituted or
substituted C.sub.1-6alkyl, or unsubstituted or substituted
heterocyclyl, a process for the preparation of such a compound, a
pharmaceutical composition containing such a compound and the use
of the compound or composition in medicine. Particular indole
compounds are selective for mammalian osteoclasts, acting to
selectively inhibit their bone resorbing activity. These compounds
are therefore considered to be particularly useful for the
treatment and/or prophylaxis of diseases associated with loss of
bone mass, such as osteoporosis and related osteopenic diseases,
Paget's disease, hyperparathyroidism and related diseases. These
compounds are also considered to possess antitumour activity,
antiviral activity (for example against Semliki Forest, Vesicular
Stomatitis, Newcastle Disease, Influenza A and B, HIV viruses),
antiulcer activity (for example the compounds may be useful for the
treatment of chronic gastritis and peptic ulcer induced by
Helicobacter pylori), immunosuppressant activity, antilipidemic
activity, antiatherosclerotic activity and to be useful for the
treatment of AIDS and Alzheimer's disease. Furthermore, these
compounds are also considered useful in inhibiting angiogenesis,
i.e., the formation of new blood vessels which is observed in
various types of pathological conditions (angiogenic diseases) such
as rheumatoid arthritis, diabetic retinopathy, psoriasis and solid
tumours.
Inventors: |
Farina, Carlo; (Milan,
IT) ; Gagliardi, Stefania; (Milan, IT) ;
Novella, Pietro; (Milan, IT) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Family ID: |
10855696 |
Appl. No.: |
10/875356 |
Filed: |
June 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10875356 |
Jun 24, 2004 |
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10018443 |
Dec 12, 2001 |
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6787550 |
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10018443 |
Dec 12, 2001 |
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PCT/EP00/05672 |
Jun 16, 2000 |
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Current U.S.
Class: |
514/414 ;
514/415 |
Current CPC
Class: |
A61P 27/02 20180101;
A61P 17/06 20180101; C07D 403/12 20130101; A61P 1/00 20180101; A61P
35/02 20180101; A61P 13/12 20180101; A61P 31/04 20180101; A61P 3/06
20180101; A61P 19/08 20180101; A61P 1/04 20180101; A61P 19/10
20180101; C07D 209/18 20130101; C07D 401/12 20130101; A61P 29/00
20180101; A61P 17/00 20180101; A61P 19/02 20180101; A61P 37/06
20180101; A61P 43/00 20180101; A61P 31/18 20180101; A61P 25/28
20180101; A61P 31/16 20180101; A61P 35/00 20180101; A61P 11/00
20180101; A61P 9/10 20180101; A61P 31/12 20180101 |
Class at
Publication: |
514/414 ;
514/415 |
International
Class: |
A61K 031/405 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 1999 |
GB |
9914371.1 |
Claims
1. A method for the treatment of tumors, in a human or non-human
animal, which comprises administering an effective, non-toxic,
amount of a compound of formula (I) 61wherein; R.sub.1 and R.sub.2
each independently represents C.sub.1-6alkoxy or halo; R.sub.3 and
R.sub.4 each independently represents hydrogen, C.sub.1-6alkoxy,
arylC.sub.1-6alkoxy, hydroxy, carboxyC.sub.1-6alkoxy,
hydroxyC.sub.1-6alkoxy, dihydroxyC.sub.1-6alkoxy, mono- and
di-(C.sub.1-6alkyl)aminoC.sub.1-6alkoxy or aminoC.sub.1-6alkoxy
and; R.sub.5 represents --NR.sub.sR.sub.t wherein R.sub.s and
R.sub.t each independently represent hydrogen, unsubstituted or
substituted C.sub.1-6alkyl, or unsubstituted or substituted
heterocyclyl; or a salt thereof or a solvate thereof, to a human or
non-human mammal in need thereof.
2. A method according to claim 1 wherein said tumor is selected
from leukemia and solid tumors.
3. A method according to claim 2, wherein said solid tumor is
selected from renal cancer, melanoma, colon cancer, and lung
cancer.
4. A method according to claim 1, wherein R.sub.4 is hydrogen or
methoxy.
5. A method according to claim 1, wherein R.sub.s or R.sub.t
represent unsubstituted or substituted C.sub.1-6alkyl, or
unsubstituted or substituted heterocyclyl.
6. A method according to claim 1, wherein R.sub.s or R.sub.t
represent 3-[4-(3-methoxyphenyl)piperazin-1-yl]propyl or
3-[4-(2-pyrimidinyl)pipera- zin-1-yl]propyl.
7. A method according to claim 1 wherein R.sub.s or R.sub.t
represent an unsubstituted or substituted piperidinyl group.
8. A method according to claim 1, wherein R.sub.s or R.sub.t
represent a 1,2,2,6,6-pentamethylpiperidin-4-yl group or a
2,2,6,6-tetramethylpiperid- in-4-yl group.
9. A method according to claim 1, wherein R.sub.t is hydrogen.
10. A method according to claim 1 wherein R.sub.s is
3-[4-(3-methoxyphenyl)piperazin-1-yl]propyl or
3-[4-(2-pyrimidinyl)pipera- zin-1-yl]propyl, and R.sub.t is
hydrogen or a salt thereof or a solvate thereof.
11. A method according to claim 1 wherein R.sub.s is 3-pyridyl or
3-(6-methoxy)pyridyl, and R.sub.t is hydrogen or a salt thereof or
a solvate thereof.
12. A method according to claim 1 wherein R.sub.s is
2,2,6,6-tetramethylpiperidin-4-yl, R.sub.t is hydrogen, R.sub.3 is
3-ethoxy, and R.sub.1 is 5-chloro or 5-methoxy or a salt thereof or
a solvate thereof.
13. A method according to claim 1 wherein R.sub.s is
2,2,6,6-tetramethylpiperidin-4-yl, R.sub.t is hydrogen, R.sub.3 is
2-methoxy, 3-methoxy, 3-ethoxy, 3-propoxy, 3-benzyloxy,
3-(2-carboxyethoxy), 3-(2-hydroxyethoxy), 3-(2,3-dihydroxypropoxy),
3-(2-dimethylaminoethoxy) or 3-(3-aminopropoxy) and 3-hydroxy and
R.sub.4 is 5-methoxy or hydrogen or a salt thereof or a solvate
thereof.
14. A method according to claim 1 wherein R.sub.s is
1,2,2,6,6-pentamethylpiperidin-4-yl, R.sub.t is hydrogen, R.sub.3
is 2-methoxy or 3-ethoxy, and R.sub.4 is 5-methoxy or hydrogen or a
salt thereof or a solvate thereof.
15. A method according to claim 1 wherein; R.sub.s is
1-benzylpiperidin-4-yl, 1-(4-ethoxycarbonyl)butylpiperydin-4-yl,
1-(4-hydroxycarbonyl)butylpiperydin-4-yl and R.sub.t is hydrogen or
a salt thereof or a solvate thereof.
16. A method according to claim 1, wherein said compound of formula
(I) is selected from the group consisting of:
4-(5,6-dichloro-1H-indol-2-yl)-3-e-
thoxy-N-(2,2,6,6-tetramethylpiperidin-4-yl)-benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-3-benzyloxy-N-(2,2,6,6-tetramethylpiperidi-
n-4-yl)-benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-3-hydroxy-N-(2,2,6,6-tet-
ramethylpiperidin-4-yl)-benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-3-propox-
y-N-(2,2,6,6-tetramethylpiperidin-4-yl)benzamide;
4-(5,6-dichloro-1H-indol-
-2-yl)-N-[3-[4-(3-methoxyphenyl)piperazin-1-yl]propyl]-3-methoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-[3-[4-(2-pyrimidyl)piperazin-1-yl]propyl-
]-3-methoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetramethy-
lpiperidin-4-yl)-3-methoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(1,2-
,2,6,6-pentamethylpiperidin-4-yl)-3-ethoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(3-pyridyl)-3-ethoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(3-(6-methoxypyridyl))-3-ethoxybenzamide-
;
4-(5,6-dichloro-1H-indol-2-yl)-N-(1-benzylpiperidin-4-yl)-3-ethoxybenzam-
ide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)--
2,5-dimethoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(1,2,2,6,6-pentam-
ethylpiperidin-4-yl)-2,5-dimethoxybenzamide;
4-(5-methoxy-6-chloro-1H-indo-
l-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)-3-ethoxybenzamide;
4-(5,6-dichloro-1H-indol-2-yl)-3-methoxy-N-((1-ethoxycarbonylpentyl)piper-
idin-4-yl)-benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-3-methoxy-N-((carboxy-
butyl)piperidin-4-yl)benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(2,2,6,6--
tetramethylpiperidin-4-yl)-3-(hydroxycarbonylmethoxy)benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)-3-(2-
-hydroxy-ethoxy)benzamide;
4-(5,6-dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
ethylpiperidin-4-yl)-3-(3-amino-propoxy)benzamide;
4-(5,6-dichloro-1H-indo-
l-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)-3-(2-dimethylamino-ethoxy)be-
nzamide, and;
4-(5,6-dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetramethylpiperid-
in-4-yl)-3-(2,3-hydroxy-propoxy)benzamide.
Description
[0001] This invention relates to certain novel compounds, to a
process for preparing such compounds, to pharmaceutical
compositions containing such compounds and to the use of such
compounds and compositions in medicine.
[0002] Diseases associated with loss of bone mass are known to be
caused by over activity of osteoclast cells. It is also known that
certain compounds, usually related to bafilomycin, are useful for
treating such diseases. For example, International Application
Publication Number WO 91/06296 (Aktiebolaget Astra) discloses
certain bafilomycin macrolides for the treatment of bone affecting
diseases.
[0003] However, bafilomycin derivatives are not selective for
osteoclasts in humans. The use of these compounds is therefore
associated with unacceptable toxicity due to generalised blockade
of other essential v-ATPases. Indeed, to date there is no known
treatment which is selective for the human osteoclasts.
[0004] The search for a successful treatment for diseases
associated with loss of bone mass in humans is further complicated
in that the nature of the therapeutic target for the selective
inhibition of the osteoclasts is controversial. Thus Baron et al
(International Application Publication Number WO 93/01280) indicate
that a specific vacuolar ATPase (v-ATPase) has been identified in
osteoclasts as a potential therapeutic target. However, the Baron
work was carried out in chickens and Hall et al (Bone and Mineral
27, 159-166, (1994)), in a study relating to mammals, conclude that
in contrast to avian osteoclast v-ATPase, mammalian osteoclast
v-ATPase is pharmacologically similar to the v-ATPase in other
cells and, therefore, it is unlikely to be a good therapeutic
target.
[0005] It has now surprisingly been found that particular indole
compounds are selective for mammalian osteoclasts, acting to
selectively inhibit their bone resorbing activity. These compounds
are therefore considered to be particularly useful for the
treatment and/or prophylaxis of diseases associated with loss of
bone mass, such as osteoporosis and related osteopenic diseases,
Paget's disease, hyperparathyroidism and related diseases. These
compounds are also considered to possess antitumour activity,
antiviral activity (for example against Semliki Forest, Vesicular
Stomatitis, Newcastle Disease, Influenza A and B, HIV viruses),
antiulcer activity (for example the compounds may be useful for the
treatment of chronic gastritis and peptic ulcer induced by
Helicobacter pylori), immunosupressant activity, antilipidemic
activity, antiatherosclerotic activity and to be useful for the
treatment of AIDS and Alzheimer's disease. Furthermore, these
compounds are also considered useful in inhibiting angiogenesis
i.e. the formation of new blood vessels which is observed in
various types of pathological conditions (angiogenic diseases) such
as rheumatoid arthritis, diabetic retinopathy, psoriasis and solid
tumours.
[0006] Accordingly, the invention provides a compound of formula
(I) 1
[0007] or a salt thereof, or a solvate thereof,
[0008] wherein;
[0009] R.sub.1 and R.sub.2 each independently represents
C.sub.1-6alkoxy or halo;
[0010] R.sub.3 and R.sub.4 each independently represents hydrogen,
C.sub.1-6alkoxy, arylC.sub.1-6alkoxy, hydroxy,
carboxyC.sub.1-6alkoxy, hydroxyC.sub.1-6alkoxy,
dihydroxyC.sub.1-6alkoxy, mono- and
di-(C.sub.1-6alkyl)aminoC.sub.1-6alkoxy or aminoC.sub.1-6alkoxy,
and;
[0011] R.sub.5 represents --NR.sub.sR.sub.t wherein R.sub.s and
R.sub.t each independently represent hydrogen, unsubstituted or
substituted C.sub.1-6alkyl, or unsubstituted or substituted
heterocyclyl.
[0012] Suitably, R.sub.1 and R.sub.2 each independently represents
methoxy or chloro.
[0013] Suitable positions for substitution for R.sub.1 or R.sub.2
are the 4, 5, 6 or 7 position.
[0014] Favourably R.sub.1 or R.sub.2 are at the 5 or 6
position.
[0015] Preferably, R.sub.1 is 5-chloro and R.sub.2 is 6-chloro.
[0016] Suitably, R.sub.3 is hydroxy, methoxy, ethoxy, propoxy,
benzyloxy, carboxyethoxy, hydroxyethoxy, dihydroxypropoxy,
dimethylaminoethoxy or aminopropoxy.
[0017] Suitably, R.sub.3 is located ortho or meta to the
--COR.sub.5 moiety.
[0018] Suitably, R.sub.4 is hydrogen or methoxy.
[0019] Suitably, R.sub.4 is located meta to the --COR.sub.5
moiety.
[0020] Suitably, R.sub.s or R.sub.t represent unsubstituted or
substituted C.sub.1-6alkyl, or unsubstituted or substituted
heterocyclyl.
[0021] When R.sub.s or R.sub.t represent unsubstituted or
substituted C.sub.1-6alkyl, suitable C.sub.1-6alkyl groups are
ethyl, propyl and butyl.
[0022] When R.sub.s or R.sub.t represent substituted alkyl,
favoured groups are 3-[4-(3-methoxyphenyl)piperazin-1-yl]propyl and
3-[4-(2-pyrimidinyl)piperazin-1-yl]propyl.
[0023] Suitably, R.sub.s or R.sub.t represent an unsubstituted or
substituted piperidinyl group.
[0024] Favourably, R.sub.s or R.sub.t represent an unsubstituted or
substituted 4-piperidinyl group.
[0025] When R.sub.s or R.sub.t represent a substituted piperidinyl
group, suitable substituents include C.sub.1-6alkyl, fused
C.sub.3-8cycloalkyl, arylC.sub.1-6alkyl, hydroxyC.sub.1-6alkyl,
polyhydroxyC.sub.1-6alkyl, C.sub.1-6alkoxycarbonylC.sub.1-6alkyl,
carboxyC.sub.1-6alkyl, and aminoC.sub.1-6alkyl.
[0026] Favoured substituents for piperidinyl groups are
C.sub.1-6alkyl groups, especially methyl groups.
[0027] When R.sub.s or R.sub.t represent a substituted piperidinyl
group, it is preferred that the substituents are attached to one or
both of the carbon atoms alpha to the nitrogen atom.
[0028] Examples of substituted piperidinyl groups are
1,2,2,6,6-pentamethylpiperidin-4-yl and
2,2,6,6-tetramethylpiperidin-4-yl groups.
[0029] Favourably, R.sub.t is hydrogen.
[0030] There is a subgroup of compounds falling wholly within
formula (I), being of formula (I') 2
[0031] wherein;
[0032] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are as
defined in formula (I), with the proviso that formula (I') does not
include;
[0033]
4-(5,6-dichloro-1H-indol-2-yl)-N-(1,2,2,6,6-pentamethylpiperidin-4--
yl)benzamide;
[0034]
4-(5,6-dichloro-1H-indol-2-yl)-3-methoxy-N-(1,2,2,6,6-pentamethylpi-
peridin-4-yl)benzamide;
[0035]
4-(5,6-dichloro-1H-indol-2-yl)-2-methoxy-N-(1,2,2,6,6-pentamethylpi-
peridin-4-yl)benzamide;
[0036]
4-(5,6-dichloro-1H-indol-2-yl)-3-methoxy-N-(3-diethylaminopropyl)be-
nzamide;
[0037]
4-(5,6-dichloro-1H-indol-2-yl)-2-methoxy-N-(3-diethylaminopropyl)be-
nzamide;
[0038]
4-(5,6-dichloro-1H-indol-2-yl)-3-methoxy-N-[3-[4-(3-chlorophenyl)pi-
perazinyl]propyl]benzamide;
[0039]
4-(5,6-dichloro-1H-indol-2-yl)-2-methoxy-N-[3-[4-(3-chlorophenyl)pi-
perazinyl]propyl]benzamide;
[0040]
4-(5,6-dichloro-1H-indol-2-yl)-3-methoxy-N-(2,2,6,6-tetramethylpipe-
ridin-4-yl)-N-methylbenzamide, or;
[0041]
4-(5,6-dichloro-1H-indol-2-yl)-2-methoxy-N-(2,2,6,6-tetramethylpipe-
ridin-4-yl)-N-methylbenzamide.
[0042] It is considered that compounds of formula (I') are novel.
Accordingly, the present invention provides a compound of formula
(I') or a salt thereof or a solvate thereof.
[0043] There is a subgroup of compounds falling wholly within
formula (I) of formula (IA) 3
[0044] wherein;
[0045] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are as
defined in formula (I), R.sub.s is
3-[4-(3-methoxyphenyl)piperazin-1-yl]propyl or
3-[4-(2-pyrimidinyl)piperazin-1-yl]propyl, and R.sub.t is hydrogen.
It is considered that compounds of formula (IA) are novel.
[0046] Accordingly, the present invention provides a compound of
formula (IA) or a salt thereof or a solvate thereof.
[0047] There is a subgroup of compounds falling wholly within
formula (I) of formula (IB) 4
[0048] wherein;
[0049] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are as
defined in formula (I), R.sub.s is 3-pyridyl or
3-(6-methoxy)pyridyl, and R.sub.t is hydrogen. It is considered
that compounds of formula (IB) are novel.
[0050] Accordingly, the present invention provides a compound of
formula (IB) or a salt thereof or a solvate thereof.
[0051] There is a subgroup of compounds falling wholly within
formula (I) of formula (IC) 5
[0052] wherein;
[0053] R.sub.2, R.sub.4, and R.sub.5 are as defined in formula (I),
R.sub.s is 2,2,6,6-tetramethylpiperidin-4-yl, R.sub.t is hydrogen,
R.sub.3 is 3-ethoxy, and R.sub.1 is 5-chloro or 5-methoxy. It is
considered that compounds of formula (IC) are novel.
[0054] Accordingly, the present invention provides a compound of
formula (IC) or a salt thereof or a solvate thereof.
[0055] There is a subgroup of compounds falling wholly within
formula (I) of formula (ID) 6
[0056] wherein;
[0057] R.sub.1, R.sub.2, R.sub.4, and R.sub.5 are as defined in
formula (I), R.sub.s is 2,2,6,6-tetramethylpiperidin-4-yl, R.sub.t
is hydrogen, R.sub.3 is 2-methoxy, 3-methoxy, 3-ethoxy, 3-propoxy,
3-benzyloxy, 3-(2-carboxyethoxy), 3-(2-hydroxyethoxy),
3-(2,3-dihydroxypropoxy), 3-(2-dimethylaminoethoxy) or
3-(3-aminopropoxy) and 3-hydroxy and R.sub.4 is 5-methoxy or
hydrogen. It is considered that compounds of formula (ID) are
novel.
[0058] Accordingly, the present invention provides a compound of
formula (ID) or a salt thereof or a solvate thereof.
[0059] There is a subgroup of compound falling wholly within
formula (I) of formula (IE) 7
[0060] wherein;
[0061] R.sub.1, R.sub.2, R.sub.4, and R.sub.5 are as defined in
formula (I), R.sub.s is 1,2,2,6,6-pentamethylpiperidin-4-yl,
R.sub.t is hydrogen, and R.sub.3 is 2-methoxy or 3-ethoxy, and
R.sub.4 is 5-methoxy or hydrogen. It is considered that compounds
of formula (IE) are novel.
[0062] Accordingly, the present invention provides a compound of
formula (IE) or a salt thereof or a solvate thereof.
[0063] There is a subgroup of compounds falling wholly within
formula (I) of formula (IF) 8
[0064] wherein;
[0065] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are as
defined in formula (I), R.sub.s is 1-benzylpiperidin-4-yl,
1-(4-ethoxycarbonyl)butyl- piperydin-4-yl,
1-(4-hydroxycarbonyl)butylpiperydin-4-yl and R.sub.t is hydrogen.
It is considered that compounds of formula (IF) are novel.
[0066] Accordingly, the present invention provides a compound of
formula (IF) or a salt thereof or a solvate thereof.
[0067] As used herein, the term "alkyl" includes straight or
branched chain alkyl groups having from 1 to 12, suitably 1 to 6,
preferably 1 to 4, carbon atoms, such as methyl, ethyl, n- and
iso-propyl and n-iso-, tert-butyl and pentyl groups, and also
includes such alkyl groups when forming part of other groups such
as alkoxy or alkanoyl groups.
[0068] Suitable substituents for any alkyl groups include
heterocyclyl groups, for example piperazinyl.
[0069] As used herein, the term "aryl" includes phenyl and
naphthyl, especially phenyl.
[0070] Suitable optional substituents for any aryl group include up
to 5 substituents, suitably up to 3 substituents, selected from
alkyl, alkoxy, thioalkyl, hydroxy, halo, aryl, heterocyclyl,
trifluoromethyl, alkylcarbonyl, cyano, nitro, or a group
--NR.sub.uR.sub.v wherein R.sub.u and R.sub.v each independently
represent hydrogen, alkyl or alkylcarbonyl.
[0071] Suitable arylalkyl groups include phenylethyl and benzyl
groups, especially benzyl. Preferably, substituted aralkyl groups
are substituted in the aryl moiety.
[0072] As used herein, the terms "heterocyclic" and "heterocyclyl"
include saturated or unsaturated single or fused ring heterocyclic
groups, each ring having 4 to 11 ring atoms, especially 5 to 8,
preferably 5, 6 or 7 which ring atoms include 1, 2 or 3 heteroatoms
selected from O, S, or N. Examples of such groups include
piperidyl, pyridyl, piperazinyl, and pyrimidinyl.
[0073] Suitable optional substituents for any heterocyclyl group
includes those mentioned herein with respect to the aryl group.
[0074] As used herein, the term "halogen" or "halo" includes
fluoro, chloro, bromo and iodo, suitably fluoro and chloro,
favourably chloro.
[0075] When used herein "acyl" includes alkyl carbonyl.
[0076] Certain of the compounds of formula (I) may contain chiral
atoms and/or multiple bonds and may therefore exist as
stereoisomers. The invention extends to all stereoisomeric forms of
the compounds of formula (I) including geometric isomers,
diastereoisomers, enantiomers and mixtures thereof, including
racemic modifications. Stereoisomers may be separated or resolved
by conventional methods or any given isomer may be obtained by
conventional stereospecific or asymmetric syntheses.
[0077] Suitable salts are pharmaceutically acceptable salts.
[0078] Suitable pharmaceutically acceptable salts include acid
addition salts and salts of carboxy groups.
[0079] Suitable pharmaceutically acceptable acid addition salts
include salts with inorganic acids such, for example, as
hydrochloric acid, hydrobromic acid, orthophosphoric acid or
sulphuric acid, or with organic acids such, for example as
methanesulphonic acid, toluenesulphonic acid, acetic acid,
propionic acid, lactic acid, citric acid, fumaric acid, malic acid,
succinic acid, salicylic acid, maleic acid, glycerophosphoric acid
or acetylsalicylic acid.
[0080] Suitable pharmaceutically acceptable salts of carboxy groups
include metal salts, such as for example aluminium, alkali metal
salts such as sodium or potassium and lithium, alkaline earth metal
salts such as calcium or magnesium and ammonium or substituted
ammonium salts for example those with C.sub.1-6alkylamines such as
triethylamine, hydroxyC.sub.1-6alkylamines such as
2-hydroxyethylamine, bis(2-hydroxyethyl)amine or
tri(2-hydroxyethyl)amine, cycloalkylamines such as
dicyclohexylamine, or with procaine, 1,4-dibenzylpiperidine,
N-benzyl-b-phenethylamine, dehydroabietylamine,
N,N'-bisdehydroabietylami- ne, glucamine, N-methylglucamine, or
bases of the pyridine type such as pyridine, collidine, or
quinoline.
[0081] Suitable solvates of the compounds of the formula (I) are
pharmaceutically acceptable solvates, such as hydrates.
[0082] The salts and/or solvates of the compounds of the formula
(I) which are not pharmaceutically acceptable may be useful as
intermediates in the preparation of pharmaceutically acceptable
salts and/or solvates of compounds of formula (I) or the compounds
of the formula (I) themselves, and as such form another aspect of
the present invention.
[0083] A compound of formula (I) may be prepared by amidation of a
suitable carboxylic acid with a suitable amine. Accordingly, the
present invention also provides a process for the preparation of a
compound of formula (I) or a salt thereof or a solvate thereof,
which process comprises the amidation of a compound of formula (II)
9
[0084] wherein;
[0085] R.sub.1', R.sub.2', R.sub.3', and R.sub.4' each respectively
represent R.sub.1, R.sub.2, R.sub.3, and R.sub.4 as defined in
relation to formula (I) or a protected form thereof, with a
compound of formula (III)
HNR.sub.s'R.sub.t' (III)
[0086] wherein;
[0087] R.sub.s' and R.sub.t' each represent R.sub.s and R.sub.t as
defined in relation to formula (I) or a protected form thereof and
thereafter, as necessary, carrying out one or more of the following
steps:
[0088] (i) converting one compound of formula (I) into another
compound of formula (I);
[0089] (ii) removing any protecting group;
[0090] (iii) preparing a salt or a solvate of the compound so
formed.
[0091] Suitable amidation methods include treating the compound of
formula (II) with a compound of formula (III).
[0092] The reaction between the compounds of formula (II) and (III)
may be carried out under the appropriate conventional amidation
conditions, for example in an aprotic solvent such as
dimethylformamide, acetonitrile and tetrahydrofuran, at any
temperature providing a suitable rate of formation of the required
product, conveniently at ambient temperature; preferably the
amidation reaction is carried out in the presence of a peptide
coupling reagent such as 1-hydroxybenzotriazole (HOBT), and/or
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(WSC).
[0093] A compound of formula (II) may be prepared by cyclising a
compound of formula (IV) 10
[0094] wherein;
[0095] R.sub.1', R.sub.2', R.sub.3', and R.sub.4' are as defined in
relation to formula (II) and R.sub.p represents a protected
carboxyl group or a group convertible into a carboxyl group and
thereafter, as required, converting the group R.sub.p into a
carboxyl group.
[0096] Suitably, the cyclisation reaction is carried out under
reductive cyclisation conditions, for example by using powdered
iron/acetic acid mixtures or an alkali metal hydrogensulphite, such
as sodium hydrogensulphite, in any suitable solvent such as
tetrahydrofuran, ethanol, methanol, or water or mixtures thereof,
at any temperature providing a suitable rate of formation of the
required product, such as an elevated temperature, conveniently at
the reflux temperature of the solvent.
[0097] When Rp is a protected carboxyl group, suitable groups
include lower alkoxy carbonyl groups, for example methoxy or ethoxy
carbonyl groups, which may be removed by conventional hydrolysis
methods, for example by use of basic hydrolysis using ethanolic
potassium hydroxide.
[0098] When Rp is a group convertible into a carboxyl group,
suitable groups include cyano group. Such groups may be converted
into carboxyl groups using conventional methods for example when
R.sub.p is a cyano group it may be converted into a carboxyl group
by hydrolysis using conventional methods, for example by use of
basic hydrolysis using potassium hydroxide solution in ethanol at
reflux.
[0099] A preferred value of Rp is a cyano group.
[0100] A compound of formula (IV) is prepared by reacting a
compound of formula (V) 11
[0101] wherein;
[0102] R.sub.1' and R.sub.2' are as defined in relation to formula
(II) with a compound of formula (VI) 12
[0103] wherein;
[0104] R.sub.3, R.sub.4, and Rp are as defined in relation to
formula (IV) and L.sub.1 represents a leaving group, such as a
halogen group, for example a chloro group.
[0105] The reaction between the compounds of formula (V) and (VI)
may be carried out in an inert hydrocarbon solvent, such as
cyclohexane, at any temperature providing a suitable rate of
formation of the required product, preferably at an elevated
temperature, such as the reflux temperature of the solvent and in
presence of a base, preferably a tertiary amine such as
triethylamine.
[0106] The reaction between the compounds of formulae (V) and (VI)
proceeds via an intermediate which is not usually isolated and
which provides the required compound of formula (IV) on heating in
situ. In an alternative aspect, the intermediate is isolated
thereby providing an alternative preparation of the compound of
formula (IV) wherein the compound of formula (VII) 13
[0107] wherein;
[0108] R.sub.1', R.sub.2', R.sub.3', and R.sub.4' are as defined in
relation to formula (II) and Rp is as defined in relation to
formula (IV), is heated to provide the compound of formula (IV) as
hereinbefore defined.
[0109] The conversion of compound (VII) into the compound of
formula (IV) is conveniently carried out in a polar solvent
mixture, such as dioxane and water, usually at the reflux
temperature of the solvent mixture in conditions analogous to those
described in J. Het. Chem. 11, 219-221, (1974).
[0110] The compounds of formula (V) are known compounds or they are
prepared using methods analogous to those used to prepare known
compounds, such as those disclosed by Meervein et al Ann. Chem.
641, 1 (1961) and Org. Synth. Collective VII, 34-41.
[0111] The compounds of formula (VI) are known, are commercially
available, or they are prepared using methods analogous to those
used to prepare known compounds, such as those described in J.
March, Advanced Organic Chemistry, 3rd Edition (1985), Wiley
Interscience.
[0112] The compounds of formula (III) are known or they are
prepared using methods analogous to those used to prepare known
compounds, such as those described in J. March, Advanced Organic
Chemistry, 3rd Edition (1985), Wiley Interscience.
[0113] Amines of general formula HNRs'Rt' may be prepared using the
methods known in the art for the preparation of amines, for example
as taught in Houben-Weil, Methoden der Organischen Chemie, Vol.
XI/1 (1957) and Vol. E16d/2 (1992), Georg Thieme Verlag,
Stuttgart.
[0114] Alternatively a compound of formula (II) may be prepared by
cyclising a compound of formula (VIII) 14
[0115] wherein;
[0116] R.sub.1', R.sub.2', R.sub.3', and R.sub.4' are as defined in
relation to formula (II) and R.sub.p' represents a protected
carboxyl group or a group convertible into a carboxyl group and
thereafter, as required, converting the group R.sub.p' into a
carboxyl group.
[0117] Suitably, the cyclisation reaction is carried out using
Suzuki reaction conditions, using a palladium catalyst, such as
bis(acetonitrile)palladium (II) chloride, in presence of an organic
base, such as triethylamine, in any suitable solvent such as
tetrahydrofuran thereof at any temperature providing a suitable
rate of formation of the required product, preferably at an
elevated temperature, such as the reflux temperature of the
solvent.
[0118] When R.sub.p' is a protected carboxyl group, suitable
protecting groups include alkyoxy carbonyl groups, for example
benzyloxy carbonyl, which may be removed by conventional hydrolysis
methods, for example by use of basic hydrolysis using ethanolic
potassium hydroxide.
[0119] A preferred value of R.sub.p' is a benzyloxycarbonyl
group.
[0120] A compound of formula (VIII) is prepared by reacting a
compound of formula (IX) 15
[0121] wherein;
[0122] R.sub.1' and R.sub.2' are as defined in relation to formula
(II) with a compound of formula (X) 16
[0123] wherein;
[0124] R.sub.3', R.sub.4' are as defined in relation to formula
(II) and Rp' is as defined in fromula (VIII).
[0125] The reaction between the compounds of formula (IX) and (X)
may be carried out in an aprotic solvent, such as tetrahydrofuran,
at any temperature providing a suitable rate of formation of the
required product, preferably from 0-25.degree. C., in presence of a
palladium catalyst, preferably bis(triphenylphosphine)palladium
(II) chloride, and a copper salt, preferably copper (I) iodide.
[0126] The compounds of formula (IX) are known compounds or they
are prepared using methods analogous to those used to prepare known
compounds, such as those disclosed by Yu M. S. et al Tetrahedron
Letters, 39, 9347, (1998).
[0127] The compounds of formula (X) are prepared by reacting a
compound of formula (XI) 17
[0128] wherein;
[0129] R.sub.3', R.sub.4' are as defined in relation to formula
(II) and Rp' is as defined in formula (VIII) with a compound of
formula (XII)
.ident.--SiMe (XII)
[0130] The reaction between the compounds of formula (XI) and (XII)
may be carried out in an aprotic solvent, such as tetrahydrofuran,
at any temperature providing a suitable rate of formation of the
required product, preferably from 0-25.degree. C., in presence of a
palladium catalyst, preferably bis(triphenylphosphine)palladium
(II) chloride, and a copper salt, preferably copper (I) iodide and
in presence of a base, preferably a tertiary amine such as
triethylamine.
[0131] The reaction between the compounds of formulae (XI) and
(XII) proceeds via an intermediate which, if desired, is not
isolated, and which provides the required compound of formula (X)
by removing the protecting silyl group with n-tetrabutylammonium
fluoride. In an alternative aspect, the intermediate is isolated
thereby providing an alternative preparation of the compound of
formula (X) wherein the compound of formula (XIII) 18
[0132] wherein;
[0133] R.sub.3', and R.sub.4' are as defined in relation to formula
(II) and Rp' is as defined in relation to formula (VIII) is heated
to provide the compound of formula (X) as hereinbefore defined.
[0134] The compounds of formula (XI) are known compounds or they
are prepared using methods analogous to those used to prepare known
compounds, such as those disclosed by Buehler, C. A. et al., J. Am.
Chem. Soc. 68, 574 (1946).
[0135] The compounds of formula (XII) are known, are commercially
available, or they are prepared using methods analogous to those
used to prepare known compounds, such as those described in J.
March, Advanced Organic Chemistry, 3rd Edition (1985), Wiley
Interscience.
[0136] A compound of formula (I) or a salt thereof or a solvate
thereof may be isolated from the above mentioned processes
according to standard chemical procedures.
[0137] The preparation of salts and/or solvates of the compounds of
formula (I) may be performed using the appropriate conventional
procedure.
[0138] If required mixtures of isomers of the compounds of the
invention may be separated into individual stereoisomers by
conventional means. For example enantiomers may be resolved by the
use of an optically active acid as a resolving agent. Suitable
optically active acids which may be used as resolving agents are
described in "Topics in Stereochemistry", Vol. 6, Wiley
Interscience, 1971, Allinger, N. L. and Eliel, W. L. Eds.
[0139] Alternatively, any enantiomer of a compound of the invention
may be obtained by stereospecific synthesis using optically pure
starting materials of known configuration.
[0140] The absolute configuration of compounds may be determined by
conventional methods such as X-ray crystallographic techniques.
[0141] The protection of any reactive group may be carried out at
any appropriate stage in the aforementioned processes. Suitable
protecting groups include those used conventionally in the art for
the particular group being protected. Protecting groups may be
prepared and removed using the appropriate conventional procedure,
for example hydroxy groups, including diols, may be protected as
the silylated derivatives by treatment with an appropriate
silylating agent such as
di-tert-butylsilylbis(trifluoromethanesulphonate). The silyl group
may then be removed using conventional procedures such as treatment
with hydrogen fluoride, preferably in the form of a pyridine
complex and optionally in the presence of alumina, or by treatment
with acetyl chloride in methanol. Alternatively benzyloxy groups
may be used to protect phenolic groups, the benzyloxy group may be
removed using catalytic hydrogenolysis using such catalysts as
palladium (II) chloride or 10% palladium on carbon.
[0142] Amino groups may be protected using any conventional
protecting group, for example tert-butyl esters of carbamic acid
may be formed by treating the amino group with
di-tert-butyldicarbonate, the amino group being regenerated by
hydrolysing the ester under acidic conditions, using for example
hydrogen chloride in aqueous ethanol or trifluoroacetic acid in
methylene dichloride. An amino group may be protected as a benzyl
derivative, prepared from the appropriate amine and a benzyl halide
under basic conditions, the benzyl group being removed by catalytic
hydrogenolysis, using for example a palladium on carbon
catalyst.
[0143] Indole NH groups and the like may be protected using any
conventional group, for example benzenesulphonyl, methylsulphonyl,
tosyl, formyl, acetyl (all of which are removable by treatment with
alkaline reagents), benzyl (removable either with sodium in liquid
ammonia or with AlCl.sub.3 in toluene), allyl (removable by
treatment with rhodium (III) chloride under acidic conditions),
benzyloxycarbonyl (removable either by catalytic hydrogenation or
by alkaline treatment), trifluoroacetyl (removable by either
alkaline or acidic treatment), t-butyldimethylsilyl (removable by
treatment with tetrabutylammonium fluoride),
2-(trimethylsilyl)ethoxymethyl (SEM) (removable by treatment with
tetrabutylammonium fluoride in the presence of ethylendiamine),
methoxymethyl (MOM) or methoxyethyl (MEM) groups (removed by mild
acidic treatment).
[0144] Carboxyl groups may be protected as alkyl esters, for
example methyl esters, which esters may be prepared and removed
using conventional procedures, one convenient method for converting
carbomethoxy to carboxyl is to use aqueous lithium hydroxide.
[0145] A leaving group is any group that will, under the reaction
conditions, cleave from the starting material, thus promoting
reaction at a specified site. Suitable examples of such groups
unless otherwise specified are halogen groups, mesyloxy,
p-nitrobenzensulphonyloxy and tosyloxy groups.
[0146] The salts, esters, amides and solvates of the compounds
mentioned herein may as required be produced by methods
conventional in the art. For example, acid addition salts may be
prepared by treating a compound of formula (I) with the appropriate
acid.
[0147] Esters of carboxylic acids may be prepared by conventional
esterification procedures, for example alkyl esters may be prepared
by treating the required carboxylic acid with the appropriate
alkanol, generally under acidic conditions.
[0148] Amides may be prepared using conventional amidation
procedures, for example amides of formula CONR.sub.s'R.sub.t' may
be prepared by treating the relevant carboxylic acid with an amine
of formula HNR.sub.s'R.sub.t', wherein R.sub.s' and R.sub.t' are as
defined above. Alternatively, a C.sub.1-6 alkyl ester such as a
methyl ester of the acid may be treated with an amine of the above
defined formula HNR.sub.s'R.sub.t' to provide the required amide,
optionally in presence of trimethylalluminium following the
procedure described in Tetrahedron Lett. 48, 4171-4173, (1977).
[0149] As mentioned above the compounds of the invention are
indicated as having useful therapeutic properties.
[0150] Of particular interest is the osteoporosis associated with
the peri and post menopausal conditions. Also encompassed are the
treatment and prophylaxis of Paget's disease, hypercalcemia
associated with bone neoplasms and all the types of osteoporotic
diseases as classified below according to their etiology:
[0151] Primary Osteoporosis
[0152] Involutional
[0153] Type I or postmenopausal
[0154] Type II or senile
[0155] Juvenile
[0156] Idiopathic in young adults
[0157] Secondary Osteoporosis
[0158] Endocrine abnormality
[0159] Hyperthyroidism
[0160] Hypogonadism
[0161] Ovarian agenesis or Turner's syndrome
[0162] Hyperadrenocorticism or Cushing's syndrome
[0163] Hyperparathyroidism
[0164] Bone marrow abnormalities
[0165] Multiple myeloma and related disorders
[0166] Systemic mastocytosis
[0167] Disseminated carcinoma
[0168] Gaucher's disease
[0169] Connective tissue abnormalities
[0170] Osteogenesis imperfecta
[0171] Homocystinuria
[0172] Ehlers-Danlos syndrome
[0173] Marfan's syndrome
[0174] Menke's syndrome
[0175] Miscellaneous causes
[0176] Immobilisation or weightlessness
[0177] Sudeck's atrophy
[0178] Chronic obstructive pulmonary disease
[0179] Chronic alcoholism
[0180] Chronic heparin administration
[0181] Chronic ingestion of anticonvulsant drugs
[0182] In addition the invention encompasses the treatment of
tumours, especially those related to renal cancer, melanoma, colon
cancer, lung cancer and leukemia, viral conditions (for example
those involving Semliki Forest virus, Vesicular Stomatitis virus,
Newcastle Disease virus, Influenza A and B viruses, HIV virus),
ulcers (for example chronic gastritis and peptic ulcer induced by
Helicobacter pylori), for use as immunosupressant agents in
autoimmune diseases and transplantation, antilipidemic agents for
the treatment and/or prevention of hypercholesterolemic and
atherosclerotic diseases and to be useful for the treatment of AIDS
and Alzheimer's disease. These compounds are also considered useful
in treating angiogenic diseases, i.e. those pathological conditions
which are dependent on angiogenesis, such as rheumatoid arthritis,
diabetic retinopathy, psoriasis and solid tumours.
[0183] The present invention therefore provides a method for the
treatment and/or prophylaxis of diseases associated with over
activity of osteoclasts in mammals which method comprises the
administration of an effective non-toxic amount of a compound of
formula (I), or a pharmaceutically acceptable salt thereof, or a
pharmaceutically acceptable solvate thereof.
[0184] In a further aspect, the present invention provides a method
for the treatment of osteoporosis and related osteopenic diseases
in a human or non-human mammal, which comprises administering an
effective, non-toxic, amount of a compound of formula (I) or a
pharmaceutically acceptable salt thereof or a pharmaceutically
acceptable solvate thereof, to a human or non-human mammal in need
thereof.
[0185] In a further aspect, the present invention also provides a
method for the treatment of tumours, especially those related to
renal cancer, melanoma, colon cancer, lung cancer and leukemia,
viral conditions (for example those involving Semliki Forest,
Vesicular Stomatitis, Newcastle Disease, Influenza A and B, HIV
viruses), ulcers (for example chronic gastritis and peptic ulcer
induced by Helicobacter pylori), autoimmune diseases and
transplantation, for the treatment and/or prevention of
hypercholesterolemic and atherosclerotic diseases, ADS and
Alzheimer's disease, angiogenic diseases, such as rheumatoid
arthritis, diabetic retinopathy, psoriasis and solid tumours, in a
human or non-human mammal, which comprises administering an
effective, non-toxic, amount of a compound of formula (I) or a
pharmaceutically acceptable salt thereof or a pharmaceutically
acceptable solvate thereof, to a human or non-human mammal in need
thereof.
[0186] In a still further aspect, the present invention a compound
of formula (I) or a pharmaceutically acceptable salt thereof, or a
pharmaceutically acceptable solvate thereof, for use as an active
therapeutic substance.
[0187] In a further aspect, the present invention provides a
compound of formula (I) or a pharmaceutically acceptable salt
thereof or a pharmaceutically acceptable solvate thereof for use in
the treatment or prophylaxis of diseases associated with over
activity of osteoclasts in mammals.
[0188] In a further aspect, the present invention provides a
compound of formula (I) or a pharmaceutically acceptable salt
thereof or a pharmaceutically acceptable solvate thereof, for use
in the treatment of or prophylaxis of osteoporosis and related
osteopenic diseases.
[0189] In a further aspect, the present invention provides a
compound of formula (I) or a pharmaceutically acceptable salt
thereof or a pharmaceutically acceptable solvate thereof for use in
the treatment of tumours, especially those related to renal cancer,
melanoma, colon cancer, lung cancer and leukemia, viral conditions
(for example those involving Semliki Forest, Vesicular Stomatitis,
Newcastle Disease, Influenza A and B, HIV viruses), ulcers (for
example chronic gastritis and peptic ulcer induced by Helicobacter
pylori), autoimmune diseases and transplantation, for the treatment
and/or prevention of hypercholesterolemic and atherosclerotic
diseases, AIDS and Alzheimer's disease, angiogenic diseases, such
as rheumatoid arthritis, diabetic retinopathy, psoriasis and solid
tumours, in a human or non-human mammal.
[0190] A compound of formula (I), or a pharmaceutically acceptable
salt thereof or a pharmaceutically acceptable solvate thereof, may
be administered per se or, preferably, as a pharmaceutical
composition also comprising a pharmaceutically acceptable
carrier.
[0191] Accordingly, the present invention also provides a
pharmaceutical composition comprising a compound of formula (I) or
a pharmaceutically acceptable salt thereof or a pharmaceutically
acceptable solvate thereof and a pharmaceutically acceptable
carrier therefor.
[0192] Active compounds or a pharmaceutically acceptable salt
thereof or a pharmaceutically acceptable solvate thereof are
normally administered in unit dosage form.
[0193] An amount effective to treat the disorders hereinbefore
described depends upon such factors as the efficacy of the active
compounds, the particular nature of the pharmaceutically acceptable
salt or pharmaceutically acceptable solvate chosen, the nature and
severity of the disorders being treated and the weight of the
mammal. However, a unit dose will normally contain 0.01 to 50 mg,
for example 1 to 25 mg, of the compound of the invention. Unit
doses will normally be administered once or more than once a day,
for example 1, 2, 3, 4, 5 or 6 times a day, more usually 1 to 3 or
2 to 4 times a day such that the total daily dose is normally in
the range, for a 70 kg adult of 0.01 to 250 mg, more usually 1 to
100 mg, for example 5 to 70 mg, that is in the range of
approximately 0.0001 to 3.5 mg/kg/day, more usually 0.01 to 1.5
mg/kg/day, for example 0.05 to 0.7 mg/kg/day.
[0194] In such treatments the active compound may be administered
by any suitable route, e.g. by the oral, parenteral or topical
routes. For such use, the compound will normally be employed in the
form of a pharmaceutical composition in association with a human or
veterinary pharmaceutical carrier, diluent and/or excipient,
although the exact form of the composition will naturally depend on
the mode of administration.
[0195] Compositions are prepared by admixture and are suitably
adapted for oral, parenteral or topical administration, and as such
may be in the form of tablets, capsules, oral liquid preparations,
powders, granules, lozenges, pastilles, reconstitutable powders,
injectable and infusable solutions or suspensions, suppositories
and transdermal devices. Orally administrable compositions are
preferred, in particular shaped oral compositions, since they are
more convenient for general use.
[0196] Tablets and capsules for oral administration are usually
presented in a unit dose, and contain conventional excipients such
as binding agents, fillers, diluents, tabletting agents,
lubricants, disintegrants, colourants, flavourings, and wetting
agents. The tablets may be coated according to well known methods
in the art.
[0197] Suitable fillers for use include cellulose, mannitol,
lactose and other similar agents.
[0198] Suitable disintegrants include starch, polyvinylpyriolidone
and starch derivatives such as sodium starch glycollate. Suitable
lubricants include, for example, magnesium stearate. Suitable
pharmaceutically acceptable wetting agents include sodium lauryl
sulphate.
[0199] These solid oral compositions may be prepared by
conventional methods of blending, filling, tabletting or the like.
Repeated blending operations may be used to distribute the active
agent throughout those compositions employing large quantities of
fillers. Such operations are, of course, conventional in the
art.
[0200] Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups, or
elixirs, or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents, for example sorbitol, syrup, methyl cellulose, gelatin,
hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate
gel or hydrogenated edible fats, emulsifying agents, for example
lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles
(which may include edible oils), for example, almond oil,
fractionated coconut oil, oily esters such as esters of glycerine,
propylene glycol, or ethyl alcohol; preservatives, for example
methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired
conventional flavouring or colouring agents.
[0201] For parenteral administration, fluid unit dose forms are
prepared containing a compound of the present invention and a
sterile vehicle. The compound, depending on the vehicle and the
concentration, can be either suspended or dissolved. Parenteral
solutions are normally prepared by dissolving the compound in a
vehicle and filter sterilising before filling into a suitable vial
or ampoule and sealing. Advantageously, adjuvants such as a local
anaesthetic, preservatives and buffering agents are also dissolved
in the vehicle. To enhance the stability, the composition can be
frozen after filling into the vial and the water removed under
vacuum.
[0202] Parenteral suspensions are prepared in substantially the
same manner except that the compound is suspended in the vehicle
instead of being dissolved and sterilised by exposure to ethylene
oxide before suspending in the sterile vehicle. Advantageously, a
surfactant or wetting agent is included in the composition to
facilitate uniform distribution of the active compound.
[0203] For topical administration, the composition may be in the
form of a transdermal ointment or patch for systemic delivery of
the active compound and may be prepared in a conventional manner,
for example, as described in the standard textbooks such as
`Dermatological Formulations`--B. W. Barry (Drugs and the
Pharmaceutical Sciences--Dekker) or Harrys Cosmeticology (Leonard
Hill Books).
[0204] Accordingly, the present invention provides the use of a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, or a pharmaceutically acceptable solvate thereof, for the
manufacture of a medicament for the treatment and/or prophylaxis of
diseases associated with over activity of osteoclasts in
mammals.
[0205] In a further aspect, the present invention provides the use
of a compound of formula (I), or a pharmaceutically acceptable salt
thereof, or a pharmaceutically acceptable solvate thereof, for the
manufacture of a medicament for the treatment and/or prophylaxis of
osteoporosis and related osteopenic diseases.
[0206] In a still further aspect, the present invention provides
the use of a compound of formula (I), or a pharmaceutically
acceptable salt thereof, or a pharmaceutically acceptable solvate
thereof, for the manufacture of a medicament for the treatment of
tumours, especially those related to renal cancer, melanoma, colon
cancer, lung cancer and leukemia., viral conditions (for example
those involving Semliki Forest, Vesicular Stomatitis, Newcastle
Disease, Influenza A and B, HIV viruses), ulcers (for example
chronic gastritis and peptic ulcer induced by Helicobacter pylori),
autoimmune diseases and transplantation, for the treatment and/or
prevention of hypercholesterolemic and atherosclerotic diseases,
AIDS and Alzheimer's disease, angiogenic diseases, such as
rheumatoid arthritis, diabetic retinopathy, psoriasis and solid
tumours.
[0207] No unacceptable toxicological effects are expected with
compounds of the invention when administered in accordance with the
invention. As is common practice, the compositions will usually be
accompanied by written or printed directions for use in the medical
treatment concerned.
[0208] The following, descriptions, examples and pharmacological
methods illustrate the invention but do not limit it in any
way.
DESCRIPTIONS AND EXAMPLES
[0209] Description 1:
trans-4,5-Dichloro-2-nitro-.beta.-dimethylaminostyre- ne 19
[0210] A solution of 10.3 g (50 mmol) of
4,5-dichloro-2-nitrotoluene (Helv. Chim. Acta 1936, 19, 434-439) in
a mixture of 11.9 g (100 mmol) N,N-dimethylformamide dimethylacetal
in DMF (25 ml) was heated at 100.degree. C. for 16 h. The dark
reaction mixture was concentrated in vacuo, the residue diluted
with methylene chloride and washed twice with water. The organic
solution was dried over MgSO.sub.4, then concentrated in vacuo
affording 12.6 g (48 mmol, yield 96.5%) of the crude title compound
as dark red crystals.
[0211] Description 2: 2-Methoxy-4-cyanobenzoyl chloride 20
[0212] 2-Methoxy-4-cyanobenzoic acid (Tetrahedron Letters, 1986,
27(49), 5997-6000) (1 g, 5.6 mmol) was dissolved in
CH.sub.2Cl.sub.2 (20 ml). Oxalyl chloride (1.5 ml, 8.2 mmol) was
rapidly introduced into the solution and a drop of DMF was added. A
vigorous reaction took place with the abundant evolution of gaseous
products. The solution was stirred for 1 h then allowed to stand
over night. Solvent was removed using a rotary evaporator to leave
1.1 g of an off white solid (5.6 mmol, yield 99%) that was used
without further purification.
[0213] Description 3:
3-Methoxy-4-[2-[(4,5-dichloro-2-nitro)phenyl]-1-oxo--
ethyl]-benzonitrile 21
[0214] 2-Methoxy-4-cyanobenzoyl chloride (1.1 g, 5.6 mmol),
prepared as in Description 3, was added portionwise to a stirred
solution of trans-4,5-dichloro-2-nitro-.beta.-dimethylaminostyrene
(1.47 g, 5.6 mmol) and triethylamine (1.5 ml, 10 mmol) in
cyclohexane (20 ml). The solution was then refluxed for 16 h. The
reaction was cooled and all the volatile products removed using a
rotary evaporator. A dark residue was obtained which was then
dissolved in CH.sub.2Cl.sub.2 (40 ml) and washed once with 10%
Na.sub.2CO.sub.3 solution (20 ml). The organic layer was then dried
with anhydrous Na.sub.2SO.sub.4, filtered and the solvent removed
using a rotary evaporator. Dark brown to black powder (2.42 g) was
obtained that was dissolved in as little ethyl acetate as possible
and hexane was added to this solution to precipitate light brown
powder (1.72 g, mp=167-170.degree. C.) that was used without
further purification in the next step.
[0215] This crude intermediate (1.2 g) was dissolved in 1,4-dioxane
(20 ml) and water (10 ml) was added. The solution was refluxed for
48 h, filtered while still hot and then chilled in an ice water
bath. Yellow to brown crystals were collected on a Buchner funnel
obtaining 0.60 g (1.6 mmol, yield 30%) of the title compound,
mp=171-174.degree. C.
[0216] .sup.1H NMR (CDCl.sub.3) .delta.=8.27 (s, 1H); 7.81 (d,1H);
7.49 (s, 1H); 7.35 (dd, 1H); 7.28 (d, 1H); 4.61 (s, 2H); 4.00 (s,
3H).
[0217] Description 4:
3-Methoxy-4-(5,6-dichloro-1H-indol-2-yl)-benzonitril- e 22
[0218]
3-Methoxy-4-[2-(4,5-dichloro-2-nitro)phenyl-1-oxo-ethyl]-benzonitri-
le (0.4 g, 1.0 mmol) was dissolved in EtOH (10 ml) and AcOH (10
ml). The solution was brought gently to reflux and iron powder (0.5
g, 9 mmol) was added in small portions over the period of an hour.
The solution was refluxed for 12 h after which the solvents were
removed using a rotary evaporator. The residue was extracted
several times with THF. After removal of the solvent, crude
3-methoxy-4-(5,6-dichloro-1H-indol-2-yl)-be- nzonitrile (0.35 g,
1.0 mmol, yield 100%) was obtained that was used in the next step
without further purification. mp=241-244.degree. C.
[0219] .sup.1H NMR (DMSO-d.sub.6) .delta.=11.60 (s br, 1H); 7.98
(d, 1H); 7.85 (s, 1H); 7.67 (s, 1H); 7.65 (d, 1H); 7.55 (dd, 1H);
7.14 (s, 1H); 4.00 (s, 3H).
[0220] Description 5:
3-Methoxy-4-(5,6-dichloro-1H-indol-2-yl)-benzoic acid 23
[0221] 3-Methoxy-4-(5,6-dichloro-1H-indol-2-yl)-benzonitrile (0.35
g, 1.0 mmol) was suspended in 30% NaOH (20 ml) and 95% EtOH (20
ml). The mixture was refluxed for 12 h and then allowed to cool to
room temperature. The suspension was concentrated to about half
volume using a rotary evaporator and then filtered on a Buchner
funnel obtaining a tan to yellow coloured powder. This was stirred
for 2 hour in 10% HCl. The solution was then filter to yield 0.256
g (0.76 mmol, yield 69%) of the crude title compound that was
purified by chromatography to yield 150 mg of pure title compound,
mp>270.degree. C.
[0222] .sup.1H NMR (DMSO-d.sub.6) .delta.=11.60 (broad s, 1H); 7.92
(d, 1H); 7.83 (s, 1H); 7.66 (m, 3H); 7.10 (s, 1H); 4.02 (s,
3H).
[0223] Description 6: 2-Ethoxy-4-aminobenzoic acid 24
[0224] A suspension of methyl 2-ethoxy-4-acetamidobenzoate (50 g,
211 mmol) in aqueous solution of NaOH (15% W/W, 200 ml) was gently
refluxed for 16 hours. The resulting pale brown solution was
allowed to cool to room temperature and then further cooled in an
ice water bath. Concentrated HCl (37% w/w) was added until the
solution reached a pH of 6. The solid precipitated from the
solution was filtered under vacuum, dried at 50.degree. C. to give
38.3 g of the title compound (yield 100%).
[0225] Description 7: 2-Ethoxy-4-cyanobenzoic acid 25
[0226] In a 1 l reactor equipped with a sealed mechanical stirrer,
CuCN (12 g, 134 mmol) were suspended in 100 ml of distilled water.
NaCN (18.3 g, 373 mmol) was added with vigorous stirring and the
internal temperature was kept below 40.degree. C. until all the
CuCN went into solution. The suspension of 2-ethoxy-4-aminobenzoic
acid (20 g, 110 mmol) in water (200 ml) and concentrated HCl (33
ml) was stirred and cooled in an ice bath. When the temperature
reached 5.degree. C., a solution of NaNO.sub.2 (9.7 g, 140 mmol) in
water (30 ml) was added dropwise at such a rate as to maintain the
temperature below 5.degree. C. When all the NaNO.sub.2 was added,
the solution was slowly introduced through an ice cooled dropping
funnel into the reactor containing the NaCN/CuCN solution. A
reaction took place with the vigorous formation of N.sub.2. A few
drops of octanol were added to keep the foaming under control.
Stirring was continued for 4 h. The resulting suspension was then
extracted with ethyl acetate (3.times.100 ml) and the organic phase
dried over MgSO.sub.4 and evaporated under vacuum obtaining 15 g of
the title compound (yield 71.1%) as a light brown powder,
mp=170-172.degree. C.
[0227] Description 8:
3-Ethoxy-4-(5,6-dichloro-1H-indol-2-yl)-benzoic acid 26
[0228] The title compound was prepared starting from
2-ethoxy-4-cyanobenzoic acid, prepared as in Description 7,
following the procedure of Description 1-5. The title compound was
prepared with an overall yield of 18%, based on the
2-ethoxy-4-cyanobenzoic acid.
[0229] .sup.1H NMR (DMSO-d.sub.6) .delta.=11.63 (s br, 1H); 7.89
(d, 1H); 7.83 (s, 1H); 7.65 (s, 1H); 7.64 (d, 1H); 7.63 (s, 1H);
7.13 (s br, 1H); 4.27 (q, 2H); 1.48 (t, 3H).
[0230] Description 9: Dimethyl 2,5-dimethoxyterephthalate 27
[0231] A suspension of 2,5-dihydroxyterephthalic acid (5 g, 25
mmol), K.sub.2CO.sub.3 (10 g, 72 mmol) and dimethyl sulphate (11
ml, 116 mmol) in acetone (100 ml) was stirred and refluxed for 24
h. The mixture was filtered while still hot and the solvent was
evaporated to about half of the original volume. On cooling white
needles precipitated and were filtered and dried obtaining 4.6 g of
the title compound (yield 73%), mp=141-143.degree. C.
[0232] Description 10: 2,5-Dimethoxyterephthalic acid monomethyl
ester 28
[0233] A suspension of dimethyl 2,5-dimethoxyterephthalate (4 g,
15.7 mmol), prepared as in Description 9, in methanolic KOH (0.86 g
of KOH in 100 ml MeOH) was refluxed for 3 h. The solution was
cooled and the solvent removed under vacuum. The residue was
treated with dilute HCl and the solid filtered off. The crude
mixture was purified by column chromatogrphy using 1:1 ethyl
acetate/hexane as a solvent obtaining 1.72 g of the title compound
(yield 44.7%), mp=123-124.degree. C.
[0234] Description 11:
2,5-Dimethoxy-4-(5,6-dichloro-1H-indol-2-yl)-benzoi- c acid 29
[0235] The title compound was prepared starting from
2,5-dimethoxyterephthalic acid monomethyl ester, prepared as in
Description 10, following the procedure of Description 1-5. The
title compound was prepared with an overall yield of 46%, based on
2,5-dimethoxyterephthalic acid monomethyl ester
[0236] .sup.1H NMR (DMSO-d.sub.6) .delta.=11.38 (s br, 1H); 7.79
(s, 1H); 7.68 (s, 1H); 7.51 (s, 1H); 7.43 (s, 1H); 7.11 (d, 1H);
3.92 (s, 3H); 3.91 (s, 3H).
[0237] Description 12: 4-Bromo-3-hydroxybenzoic acid 30
[0238] This acid was prepared in similar way to that described by
Buehler et al (Buehler, C. A., Harris, J. O., Shacklett, C and
Block, B. P.; J. Am. Chem. Soc., 68, 574-577 (1946)). To a stirred
suspension of 3-hydroxybenzoic acid (50.0 g, 0.362 mol) in acetic
acid (495 ml), at RT under argon, was added a solution of bromine
(57.97 g, 0.3627 mol) in acetic acid (192 ml) over 2 h. During the
addition the internal temperature rose from 18.0 to 22.0.degree. C.
The mixture was stirred for 21 h and then concentrated under
vacuum, where approximately (500 ml) of distillate was collected.
The resulting concentrated solution was stored at 4.degree. C. for
2 h. The resulting white solid was filtered off and washed with
cold water (100 ml). This solid was disolved in a minimum volume of
boiling water (220 ml), filtered and allowed to cool to room
temperature. The resulting solid was removed by filtration, washed
with cold water and dried in a vaccum oven at 58.degree. C. to give
the title compound (12.50 g, yield 15.9%), mp=231-232.degree. C.,
(lit mp 225-226.degree. C.).sup.1.
[0239] .sup.1H NMR (400.13 MHz, DMSO-d.sub.6): 13.00 (1H, bs,
CO.sub.2H), 10.64 (1H, bs, OH), 7.60 (1H, d, H-5, J.sub.5-6 8.0
Hz), 7.52 (1H, d, H-2, J.sub.2-6 2.0 Hz), 7.29 (1H, dd, H-6,
J.sub.2-6 2.0 Hz, J.sub.5-6 8.0 Hz)
[0240] Description 13: 3-Benzyloxy-4-bromobenzoic acid, benzyl
ester 31
[0241] To a stirred suspension of NaH (10.47 g, 60% by wt, 0.2616
mol) in THF (1.40 L), at 13.1.degree. C. under argon, was added a
solution of 4-bromo-3-hydroxybenzoic acid (28.11 g, 0.130 mol) in
THF (400 ml) over 1.25 h maintaining the internal temperature in
the range 4-15.degree. C. After 1.5 h a solution of benzyl bromide
(44.3 g, 0.2591 mol) in THF (70 ml) was added over 0.5 h. To the
resulting suspension was added DMF (500 ml) and the mixture allowed
to warm to room temperature. After a further 15 h extra DMF was
added (1.5 L) to the suspension. After approximately 30 minutes the
reaction mixture was essentially clear and TLC analysis (25%
EtOAc/hexane) indicated that effectively all of the starting
material had been consumed. The reaction mixture was quenched with
dilute ammonium chloride (100 ml) and concentrated in vacuo. To the
concentrate was added ethyl acetate (1.0 L) and dilute ammonium
chloride (1.0 L) and the fractions separated. The aqueous fraction
was back extracted with ethyl acetate (1.times.450 ml and
1.times.300 ml). The total organic fraction was dried over sodium
sulphate, filtered and concentrated in vacuo. The resulting solid
was crystallised from ethanol:water (9:1; vol;vol, 200 ml), heated
at reflux, to which was added ethanol (65 ml) until all the
material had dissolved. Water was then added dropwise (17 ml),
followed by extra ethanol (8.0 ml). The resulting solution was
allowed to slowly cool to ambient temperature. The resulting solid
was filtered off and dried (44.54 g). This solid was then dissolved
in hot ethanol (227 ml) and water (5.0 ml) added and then allowed
to slowly cool to ambient temperature. The resulting crystals were
filtered off, washed with cold ethanol (25 ml) and dried in a
vacuum oven at 50.degree. C. to give the title compound (36.79 g,
yield 71.5%). mp=80.5-81.5.degree. C.
[0242] .sup.1H NMR (400.13 MHz, CDCl.sub.3): 7.67-7.28 (13H,
complex m, aromatics), 5.35 (2H, s, CH.sub.2Ph), 5.20 (2H, s,
CH.sub.2Ph).
[0243] Description 14: 3-Benzyloxy-4-trimethylsilanylethynyl
benzoic acid, benzyl ester 32
[0244] 3-Benzyloxy-4-bromobenzoic acid, benzyl ester (33.00 g,
83.07 mmol) was placed in a three necked flask and the atmosphere
was replaced, under vacuum, with argon using a Firestone valve. The
solid was dissolved in THF (215 ml), with stirring, and then
triethylamine (396 ml) was added. The mixture was cooled in an
ice/water bath and the solution degassed five times as described
above. Copper (I) iodide (127 mg, 0.66 mmol) and
bis(triphenylphosphine)palladium (II) chloride (933 mg, 1.33 mmol)
were quickly added and the solution degassed twice more.
Trimethylsilylacetylene (17.6 ml, 124.6 mmol) was then added
dropwise, by syringe, over ten minutes. The cooling bath was then
removed and the solution allowed to slowly warm to ambient
temperature. After 21 h TLC analysis (25% EtOAc/hexane) indicated
that essentially all the starting material had been consumed. The
solvents were removed by evaporation in vacuo and ethyl acetate
(500 ml) and dilute brine (300 ml) were added. The organic fraction
was separated and the aqueous phase was back extracted with ethyl
acetate (1.times.250 ml and 1.times.100 ml). The total organic
fraction was dried over sodium sulphate, filtered and evaporated to
give a crude brown solid (36.2 g). The solid was dissolved in hot
ethyl acetate (80 ml) where upon the title compound crystallised.
This solid was removed by filtration, washed with ethyl acetate and
dried in a vacuum oven at 40.degree. C. to give pure title compound
(13.75 g, yield 39%), mp=110.5-111.5.degree. C.
[0245] .sup.1H NMR (400.13M Hz, CDCl.sub.3): 7.66-7.28 (13H,
complex m, aromatics), 5.36 (2H, s, CH.sub.2Ph), 5.19 (2H, s,
CH.sub.2Ph), 0.26 (9H, s, Si(Me.sub.3).sub.3).
[0246] The filtrate was loaded on to a silica gel cartridge (400 g,
Biotage 75M) which was then eluted with 50% ethyl acetate/hexane,
the product fractions were pooled and concentrated in vacuo to give
title compound (21.1 g, yield 60%) as a slightly impure light brown
solid, which was used without further purification.
[0247] Description 15: 3-Benzyloxy-4-ethynylbenzoic acid, benzyl
ester 33
[0248] To a stirred solution of
3-benzyloxy-4-trimethylsilanylethynylbenzo- ic acid, benzyl ester
(13.5 g, 32.56 mmol) in THF (182 ml), under argon at -55.degree.
C., was added a solution of tetra-n-butylammonium fluoride (33.9
ml, 1.0M in THF) dropwise over 7 minutes. After a further 3 minutes
a sample was removed and TLC analysis (25% EtOAc/hexane) indicated
that all the starting material had been consumed. The reaction was
quenched by the addition of a solution of hydrochloric acid (0.4M,
100 ml) in dilute ammonium chloride. Then ethyl acetate (250 ml)
was added and the fractions separated. The aqueous fraction was
back extracted with ethyl acetate (1.times.200 ml and 1.times.50
ml). The total organic fraction was dried over sodium sulphate,
filtered and evaporated to give a brown oil (14.3 g). The oil was
taken up in toluene (40 ml) and loaded on to a silica gel cartridge
(400 g, Biotage 75M). The cartridge was then eluted as follows:
hexane (400 ml), hexane:toluene (1:1, vol:vol, 5.0 L) and finally
hexane:toluene (1:1, vol:vol, 2.3 L) containing 4% diethyl ether.
The pure fractions were pooled and evaporated in vacuo to give the
title compound (7.86 g, yield 70.5%) as a white solid.
[0249] .sup.1H NMR (400.13 MHz, CDCl.sub.3): 7.67-7.27 (13H,
complex m, aromatics), 5.34 (2H, s, CH.sub.2Ph), 5.22 (2H, s,
CH.sub.2Ph), 3.44 (1H, s, acetylenic).
[0250] Description 16: 4,5-Dichloro-2-iodoaniline 34
[0251] To a stirred solution of 3,4-dichloroaniline (1.944 g, 12.0
mmol) in acetic acid (40 ml) and under argon at ambient temperature
was added a solution of iodine monochloride (2.96 g, 18.23 mmol) in
acetic acid (25 ml plus 5 ml wash) dropwise over 22 minutes. After
stirring for 1.5 h a solid had precipitated which was filtered off
and washed with 5% sodium thiosulphate (100 ml). The solid was
dissolved in ethyl acetate (100 ml) and washed with saturated
sodium carbonate (100 ml) and water (100 ml). The resulting solid
was dried over sodium sulphate, filtered and evaporated to give a
dark coloured solid (2.69 g). The crude solid was dissolved in
EtOAc/hexane (1:1, vol:vol, 6.0 ml) and loaded on a to a silica
cartridge (90 g, Biotage) which was eluted as follows: hexane (600
ml), 4% EtOAc in hexane (1 L) and 5% EtOAc in hexane (1 L). The
pure fractions were concentrated in vacuo to give the title
compound as an off white solid (0.912 g, yield 26.4%).
[0252] .sup.1H NMR (400.13 MHz, CDCl.sub.3): 7.66 (1H, s, H-3),
6.81 (1H, s, H-6), 4.14 (2H, bs, --NH.sub.2). MS (AP.sup.+): m/z
288.0 and 290.1 (MH.sup.+).
[0253] Description 17:
4-(2-Amino-4,5-dichloro-phenylethynyl)-3-benzyloxyb- enzoic acid,
benzyl ester 35
[0254] 3-Benzyloxy-4-ethynylbenzoic acid, benzyl ester (820 mg,
2.395 mmol) and 4,5-dichloro-2-iodoaniline (710 mg, 2.467 mmol)
were placed in a three necked flask and the atmosphere was
replaced, under vacuum, with argon using a Firestone valve. The
solids were then dissolved in THF (13.0 ml), with stirring, and
then triethylamine (20 ml) was added. The mixture was cooled in an
ice/water bath and the solution degassed four times as described
above. Copper (I) iodide (4 mg, 0.02 mmol) and
bis(triphenylphosphine)palladium (II) chloride (27 mg, 0.04 mmol)
were quickly added and the solution degassed twice more. The
cooling bath was then removed and the solution allowed to slowly
warm to ambient temperature. After stirring for 6 h at ambient
temperature, TLC analysis (25% EtOAc/hexane) indicated that all the
starting 3-benzyloxy-4-ethynylbenzoic acid, benzyl ester had been
consumed. The solvents were removed by evaporation in vacuo and
ethyl acetate (50 ml) and dilute sodium hydrogen carbonate (25 ml)
were added. The organic fraction was separated and the aqueous
phase was back extracted with ethyl acetate (1.times.25 ml). The
total organic fraction was dried over sodium sulphate, filtered and
evaporated to give a pale yellow solid (1.28 g). This solid was
dissolved in chloroform (50 ml), silica gel added (Merck 9385, 3.25
g) and the mixture concentrated in vacuo. The crude material,
preloaded on silica, was purified by chromatography (Biotage 40 g).
The cartridge was eluted with chloroform:hexane (70:30), the pure
fractions were pooled and concentrated in vacuo to give the title
compound (1.113 g, yield 92.5%) as a pale yellow solid. A sample
was recrystallised from toluene:chloroform (45:55),
mp=152.5-153.5.degree. C.
[0255] .sup.1H NMR (400.13 MHz, CDCl.sub.3): 7.71 (2H, m,
aromatic), 7.57-7.20 (12H, complex m, aromatics), 6.63 (1H, s,
aromatic), 5.38 (2H, s, CH.sub.2Ph), 5.15 (2H, s, CH.sub.2Ph), 3.94
(2H, s, --NH.sub.2). MS (AP.sup.+): m/z 502.2 and 502.4
(MH.sup.+).
[0256] Description 18:
3-Benzyloxy-4-(5,6-dicholoro-1H-indol-2-yl)benzoic acid, benzyl
ester 36
[0257] 4-(2-Amino-4,5-dichloro-phenylethynyl)-3-benzyloxybenzoic
acid, benzyl ester (28.32 g, 56.38 mmol) was dissolved in warm
acetonitrile (1.50 L), with stirring under argon. The mixture was
cooled in an ice/water bath and the solution degassed on low
vacuum, using a Firestone valve, with the atmosphere being relaced
with argon. This procedure was repeated five times. The stirred
mixture was warmed to 66.degree. C. and
bis(acetonitrile)palladium(II) chloride (1.666 g, 5.638 mmol)
quickly added and then warmed to 75.degree. C. After 1.5 h a sample
was removed and analysed by TLC (25% EtOAc/hexane), which indicated
that all the starting material had been consumed. The reaction was
allowed to slowly cool to ambient temperature, whereupon the title
compound crystallised out of solution. The solid was removed by
filtration, washed with cold acetonitrile (125 ml) and dried in a
vacuum oven to give the title compound (21.375 g, yield 75.5%) as a
white solid, mp=168-169.degree. C.
[0258] .sup.1H NMR (400.13 MHz, CDCl.sub.3): 9.79 (1H, bs, --NH),
7.90-7.76 (3H, complex m, aromatics), 7.68 (1H, s, aromatic),
7.55-7.32 (10H, complex m, aromatics), 7.22 (1H, s, aromatic), 6.91
(1H, m, aromatic), 5.39 (2H, s, CH.sub.2Ph), 5.29 (2H, s,
CH.sub.2Ph). MS (AP.sup.+); m/z 502.2 and 502.4 (MH.sup.+).
[0259] Description 19:
3-Benzyloxy-4-(5,6-dicholoro-1H-indol-2-yl)benzoic acid. 37
[0260] A solution of
3-benzyloxy-4-(5,6-dicholoro-1H-indol-2-yl)benzoic acid, benzyl
ester. (0.2 g, 0.398 mmol) in EtOH (10 ml) and THF (5 ml) with NaOH
(5.3 mg, 1.33 mmol) was refluxed for 3 h. After cooling the solvent
was removed under vacuum and the residue treated with 37% HCl. The
precipitate was filtered, washed with water and dried under vacuum
to give 0.14 g (yield 85%) of the title compound as a yellow
powder, mp=>250.degree. C.
Example 1
4-(5,6-Dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethylpiperidin-4--
yl)-benzamide
[0261] 38
[0262] To a suspension of
3-ethoxy-4-(5,6-dichloro-1H-indol-2-yl)-benzoic acid (200 mg, 0.57
mmol), prepared as in Description 8, in CH.sub.3CN (14 ml) and THF
(6 ml), WSC(N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride) (104 mg, 55 mmol) and 1-hydroxybenzotriazole (77 mg,
0.57 mmol) were added and the reaction was refluxed for 3 h.
[0263] 4-Amino-2,2,6,6-tetramethylpiperidine (108 mg, 0.7 mmol)
were introduced into the reaction mixture and refluxing continued
for another 2 h. The reaction was cooled and the solvent was
removed under vacuum. The residue was treated with 30 ml of 10%
NaOH solution and then filtered. The resulting solid was washed
with water, dried and purified by chromatography on silica gel to
yield 154 mg of the title compound as light yellow powder (yield
55%), mp=253-255.degree. C.
[0264] .sup.1H NMR (DMSO-d.sub.6) o=11.56 (s br, 1H); 8.28 (d br,
1H); 7.85 (d, 1H); 7.82 (s, 1H) 7.64 (s, 1H); 7.59 (s, 1H); 7.57
(d, 1H); 7.11 (s, 1H); 4.40-4.21 (m, 1H); 4.30 (q, 2H); 1.80 (d br,
2H); 1.50 (t, 3H); 1.30 (m, 2H); 1.28 (s, 6H); 1.15 (s, 6H).
[0265] ESI POS; AQA; solvent: MeOH/spray 3 kV/skimmer: 20 V/probe
135.degree. C.: m/z 488 (MH.sup.+)
Example 2
4-(5,6-Dichloro-1H-indol-2-yl)-3-benzyloxy-N-(2,2,6,6-tetramethylpiperidin-
-4-yl)-benzamide
[0266] 39
[0267] To a solution of
3-benzyloxy-4-(5,6-dichloro-1H-indol-2-yl)benzoic acid (0.14 g,
0.34 mmol), prepared as in Description 19, in THF (14 ml),
WSC(N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride)
(0.078 g, 0.408 mmol) and 1-hydroxybenzotriazole (0.0551 g, 0.408
mmol) was refluxed for 6 h. A solution of
4-amino-2,2,6,6-tetramethylpiperidine (0.064 g, 0.408 mmol) in THF
(2 ml) was added dropwise and refluxed for additional 2 h. The
solvent was removed under vacuum and the residue suspended in
water. The solid was filtrated and dried under vacuum. The solid
was triturated with CH.sub.3CN (5 ml) to give 0.094 g (yield 45%)
of the title compound as a white powder, mp=220.degree. C.
[0268] .sup.1H-NMR (DMSO-d.sub.6) .delta.=11.64 (s br, 1H); 8.24 (d
br, 1H); 7.87 (d, 1H); 7.76 (s, 1H); 7.68 (d, 1H); 7.62 (s, 1H);
7.59 (dd, 1H); 7.52 (d, 2H); 7.42 (dd, 2H); 7.34 (dd, 1H); 7.06 (d,
1H); 5.40 (s, 2H); 4.38-4.23 (m, 1H); 1.76 (dd, 2H); 1.25 (dd, 2H);
1.23 (s, 6H); 1.12 (s, 6H).
[0269] ESI POS; AQA; solvent: MeOH/spray 3 kV/skimmer: 20 V/probe
135.degree. C.: m/z 550 (MH.sup.+)
Example 3
4-(5,6-Dichloro-1H-indol-2-yl)-3-hydroxy-N-(2,2,6,6-tetramethylpiperidin-4-
-yl)-benzamide
[0270] 40
[0271] A mixture of
4-(5,6-dichloro-1H-indol-2-yl)-3-benzyloxy-N-(2,2,6,6--
tetramethylpiperidin-4-yl)-benzamide (0.08 g, 0.145 mmol), prepared
as described in Example 2, in 37% HCl (5 ml) and EtOH (5 ml) was
refluxed for 3 h. After cooling the solvent was removed under
vacuum. The resulting solid was purified by column chromatography
over silica gel (CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 86:10:0.6) to
yield, after trituration with CH.sub.3CN, 0.02 g (yield 39%) of the
title compound as a white solid, mp>270.degree. C.
[0272] .sup.1H-NMR (DMSO-d.sub.6) .delta.=11.44 (s br, 1H); 8.16 (d
br, 1H); 7.80 (d, 1H); 7.78 (s, 1H); 7.69 (s, 1H); 7.48 (d, 1H);
7.39 (dd, 1H); 7.08 (s,1H);4.364.20 (m, 1H); 1.73 (dd, 2H); 1.23
(dd, 2H); 1.23 (s,6H); 1.11 (s, 6H).
[0273] ESI POS; AQA; solvent: MeOH/spray 3 kV/skimmer: 20 VI probe
135.degree. C.: m/z 460 (MH.sup.+)
Example 4
4-(5,6-Dichloro-1H-indol-2-yl)-3-propoxy-N-(2,2,6,6-tetramethylpiperidin-4-
-yl)benzamide
[0274] 41
[0275] A mixture of
4-(5,6-dichloro-1H-indol-2-yl)-3-hydroxy-N-(2,2,6,6-te-
tramethylpiperidin-4-yl)-benzamide (0.05 g, 0.109 mmol), prepared
as described in Example 3, K.sub.2CO.sub.3 (0.045 g, 0.33 mmol) and
2-bromo-propane (0.046 mg, 0.33 mmol) in aceton (5 ml) was refluxed
for 8 h. After cooling the mixture was filtered and the organic
phase was removed under vacuum. the residue was chromatographed
over silica gel with CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 32%
(86:10:0.6). The compound obtained was triturated with iPr.sub.2O
and filtered to obtain 0.017 mg (yield 37%) of the title compound
as a white powder, mp>250.degree. C.
[0276] .sup.1H-NMR (DMSO-d.sub.6) .delta.=11.55 (s br, 1H); 8.19 (d
br, 1H); 7.84 (d, 1H); 7.83 (s, 1H); 7.62 (s, 1H); 7.58 (s, 1H)7.56
(dd, 1H); 7.10 (d, 1H); 4.37-4.24 (m, 1H); 4.18 (t, 2H); 1.96-1.84
(m, 2H); 1.73 (dd, 2H); 1.20 (dd, 2H); 1.20 (s, 6H); 1.08 (s, 6H);
1.05 (t, 3H).
[0277] ESI POS; AQA; solvent: MeOH/spray 3 kV/skimmer: 20 V/probe
135.degree. C.: m/z 502 (MH.sup.+).
[0278] The Example compounds listed in Table 1 were prepared
according to the procedure of Example 1:
1TABLE 1 42 Ex. Name R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 MS MP
.degree. C. N.M.R. 5
4-(5,6-Dichloro-1H-indol-2-yl)-N-[3-{4-(3-meth-
oxyphenyl)piperazin-1-yl]pro- pyl}-3-methoxybenzamide 5-Cl 6-Cl
3-OMe --H 43 A) ESI POS; TSQ 700; solvent: MeOH/spray 4.5
kV/skimmer: 60 V/ capillary 220.degree. C.: m/z 474 (MH.sup.+) B)
CID Offset = -56 V: m/z 567; 375; 318. 201-205 .sup.1H-NMR
#(DMSO-d.sub.6) .delta. =11.53(s br, 1H); 8.55(t br, 1H); 7.88(d,
1H); 7.81(s, 1H); 7.66s, 1H); 7.60(d, 1H); 7.57(dd, 1H); 7.10 (dd,
1H); 7.04(m, 1H); 6.51(dd, 1H); 6.43(dd, 1H); 6.35dd, 1H); 4.00(s,
3H); 3.70(s, 3H); 3.30 (m, 2H); 3.13(m, 4H); 2.58(m, 4H); 2.41(t,
2H); 1.80-1.72(m, 2H). 6
4-(5,6-Dichloro-1H-indol-2-yl)-N-[3-[4-(2-py-
rimidyl)piperazin-1-yl]pr- opyl]-3-meth- oxybenzamide 5-Cl 6-Cl
3-OMe --H 44 A) ESI POS; TSQ 700; solvent: MeOH/spray 4.5
kV/skimmer: 60 V/ capillary 220.degree. C.: m/z 539 (MH.sup.+) B)
CID Offset = -56 V: m/z 539; 575; 318. 234-237 .sup.1H-NMR
(DMSO-d.sub.6 at #343 K).delta.=11.30(s br, 1H); 8.32(d, 2H);
8.31(t br, 1H); 7.85(d, 1H); 7.79 (s, 1H); 7.68(s, 1H); 7.60s br,
1H); 7.56(d, 1H); 7.01(s br, 1H); 6.59(t, 1H); 4.02(s, 3H): 3.77
(m, 4H); 3.40(dt, 2H); 2.50-2.40 (m, 6H); 1.87-1.73(m, 2H). 7
4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
methylpiperidin-4-yl)-3-meth- oxybenzamide 5-Cl 6-Cl 3-OMe --H 45
A) ESI POS; TSQ 700; solvent: MeOH/spray 4.5 kV/skimmer: 60 V/
capillary 220.degree. C.: m/z 474 (MH.sup.+) B) CID Offset = -56 V:
m/z 474; 457; 401; 318; 290; 123; 58. 151-156
.sup.1H-NMR(DMSO-d.sub.6) #.delta. =11.30(s br, 1H); 7.95(d br,
1H); 7.82(d, 1H); 7.77(s, 1H); 7.68(s, 1H); 7.61(d, 1H); 7.56(dd,
1H); 7.01 (d, 1H); 4.45-4.30(m, 1H); 4.01 (s, 3H); 1.80(m, 2H);
1.30(m, 2H); 1.30(s, 6H); 1.15(s, 6H). 8
4-(5,6-Dichloro-1H-indol-2-yl)-N-(1,2,2,6,6-pen-
tamethylpiperidin-4-yl)-3-eth- oxybenzamide 5-Cl 6-Cl 3-OEt --H 46
ESI POS; AQA; solvent: MeOH/spray 3 kV/ skimmer: 20 V/probe
135.degree. C.: m/z 502(MH.sup.+) >250
.sup.1H-NMR(DMSO-d.sub.6).delta. =11.55(s br, 1H); 8.22(d br, 1H);
7.85(d, 1H); 7.82(s, 1H); 7.63(s, #1H); 7.58(s, 1H); 7.57(d, 1H);
7.10(s br, 1H); 4.29(q, 2H); 4.29-4.14(m, 1H); 2.22(s, 3H);
1.75(dd, 2H); 1.50(t, 2H); 1.49(t, 3H); 1.12(s, 6H); 1.08(s, 6H). 9
4-(5,6-Dichloro-1H-indol-2-yl)-N-(3-py- ridyl)-3-ethoxybenzamide
5-Cl 6-Cl 3-OEt --H 47 El; TSQ 700; 180.degree. C.; 70 V; 200 uA:
m/z 425(M+.); 332; 304; 212. 237-239
.sup.1H-NMR(DMSO-d.sub.6).delta. =11.60(s br, 1H); 10.49(s, 1H);
8.95(d, 1H); 8.33(dd, 1H); 8.20(ddd, 1H); 7.95(d, 1H); 7.84(s, 1H);
7.72(d, 1H); 7.70(s, 1H); #7.66 (s, 1H); 7.41(dd, 1H); 7.18(d, 1H);
4.35(q, 2H); 1.50(t, 3H). 10 4-(5,6-Dichloro-1H-indol-2-yl)-N-(3--
(6-meth- oxypyridyl))-3-ethoxybenzamide 5-Cl 6-Cl 3-OEt --H 48 El;
TSQ 700; 180.degree. C.; 70 V; 200 uA: m/z 455(M+.); 332; 304.
258-260 .sup.1H-NMR (DMSOd.sub.6).delta. = 11.60(s br, 1H);
10.30(s, 1H); 8.52(d, 1H); 8.05dd, 1H); 7.94(d, 1H); 7.83(s, 1H);
7.70(d, 1H); 7.69(s, 1H); 7.65(s, 1H); #7.15(s, 1H); 6.86(d, 1H);
4.34(q, 2H); 3.87(s, 3H); 1.50(t, 3H). 11
4-(5,6-Dichloro-1H-indol-2-yl)-N-(1-benzy- l-
piperidin-4-yl)-3-eth- oxybenzamidehydrochloride 5-Cl 6-Cl 3-OEt
--H 49 El; TSQ 700; 180.degree. C.; 70 V; 200 uA: m/z 521(M+.);
350; 332; 212; 173; 132; 91; 82. 295
.sup.1H-NMR(DMSO-d.sub.6).delta. =11.65(s br, 1H); 8.30(d br, 1H);
7.86(d, 1H); 7.81(s, 1H); 7.65(s, 1H); 7.58 #(s, 1H); 7.56(d, 1H);
7.36-7.21 (m, 5H); 7.10(s, 1H); 4.29 (q, 2H); 3.89-3.72(s, 1H);
3.49(s, 2H); 2.83(d br, 2H); 2.02(dd br, 2H); 1.80(m, 2H); 1.61(dq,
2H); 1.49(t, 3H). 12
4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
methylpiperidin-4-yl)-2,- 5-di- methoxybenzamide 5-Cl 6-Cl 2-OMe
5-OMe 50 A) El; TSQ 700; 400 mA; 70 V: m/z 567(MH.sup.+) B) ESI
POS; AQA; solvent: MeOH/spray 3 kV/skimmer: 20 V/ probe 135.degree.
C.: m/z 504 (MH.sup.+) 148-152 .sup.1H-NMR (DMSO-d.sub.6).delta. #=
11.60(s br, 1H); 7.95(d br, 1H); 7.82(s, 1H); 7.69(s, 1H); 7.52(s,
2H); 7.13(s br, 1H); 4.40-4.21(m, 1H); 4.00(s, 3H); 3.92(s, 3H);
1.78 (dd, 2H); 1.20(s, 6H); 1.12(dd, 2H); 1.06(s, 6H). 13
4-(5,6-Dichloro-1H-in- dol-2-yl)-N-(1,2,2,6,6-penta-
methylpiperidin-4-yl)-2,5-di- methoxybenzamide 5-Cl 6-Cl 2-OMe
5-OMe 51 A) ESI POS; TSQ 700; solvent: MeOH/spray 4.5 kV/skimmer:
60 V/ capillary 220.degree. C.: m/z 518 (MH+) B) ESI DAU +518
(Collision gas: Argon): m/z 518; 487; 431; 365; 348; #123 143-147
.sup.1NMR(DMSO-d.sub.6).del- ta. =11.58(s br, 1H); 7.95(d br, 1H);
7.81(s, 1H); 7.68(s, 1H); 7.51(s, 2H); 7.13(s br, 1H); 4.28-4.10(m,
1H); 3.95(s, 3H); 3.90(s, 3H); 2.20(s, 3H); 1.78(dd, 2H); 1.39 (dd,
2H); 1.10(s, 6H); 1.06(s, 6H). 14 4-(5-Methoxy-6-chloro-1H-in-
dol-2-yl)-N-(2,2,6,6-tetra- methylpiperidin-4-yl)-3-eth-
oxybenzamide 5-OMe 6-Cl 3-OEt --H 52 ESI POS; AQA; solvent:
MeOH/spray 3 kV/ skimmer: 20 V/probe 135.degree. C.: m/z 484(MH+).
231-232 .sup.1H-NMR(DMSO-d.sub.6).delta. =11.18(d br, 1H); 8.18(d,
1H); 7.83(d, 1H); 7.56(s, 1H); #7.55(d, 1H); 7.46(d, 1H); 7.24(s,
1H); 7.07(d, 1H); 4.38-4.25(m, 1H); 4.27(q, 2H); 3.85(s, 3H);
1.73(dd, 2H); 1.50 (t, 3H); 1.20(dd, 2H); 1.20(s, 6H); 1.08(s, 6H).
15 4-(4,5-Dichloro-1H-indol-2-yl)-3-meth- oxy-N-((1-eth-
oxycarbonylbutyl)piperidin-4-yl)ben- zamide 5-Cl 6-Cl 3-OMe --H 53
ESI POS; AQA; solvent: MeOH/spray 3 kV/ skimmer: 20 V/probe
135.degree. C.: m/z 546(MH+). 188-190
.sup.1H-NMR(DMSO-d.sub.6).delta. =11.53(s br, 1H); 8.27(d, 1H);
7.87(d, 1H); 7.81(s, #1H) (s, 1H); 7.59(s, 1H); 7.57(d, 1H);
7.06(d, 1H); 4.06(q, 2H); 4.02(s, 3H); 3.86-3.70 (m, 1H); 2.86(d
br, 2H); 2.33-2.25(m, 4H); 1.94(dd, 2H); 1.81(dd, 2H); 1.66-1.38(m,
6H); 1.19(t, 3H). 16 4-(5,6-Dichloro-1H-indo- l-2-yl)-3-meth-
oxy-N-((carboxylbutyl)piperidin-4-yl)benzmiade 5-Cl 6-Cl 3-OMe --H
54 ESI POS; AQA; solvent: MeOH/spray 3 dV/ skimmer: 20 V/probe
135.degree. C: m/z 518(MH+) >250 .sup.1H-NMR
(DMSO-d.sub.6).delta.(343- K) = 11.34 (s br, 1H); 8.17(d br, 1H);
7.86(d, 1H); 7.77(s, 1H); 7.67(s, 1H); 7.60(d1H); #7.58 (dd, 1H);
7.03(d, 1H); 4.03(s, 3H); 4.03-3.88(m, 1H); 3.17(m, 4H);
2.77-2.50(m, 4H); 1.97(m, 2H); 1.88-1.75(m, 2H); 1.68-1.52(m,
4H).
[0279] The Example compounds listed in Table 2 were prepared
according to the procedure of Example 4:
2TABLE 2 55 Ex. Name R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 MP
.degree. C. N.M.R. 17
4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
methylpiperidin-4-yl)-3-(hy- droxycarbonylmethoxy)ben- zamide 5-Cl
6-Cl 3-OCH.sub.2COOH --H 56 260 .sup.1H-NMR(DMSO-d.sub.6) = 14.50(s
br, 1H); 8.60(s br, 1H); 8.50(d, 1H); 7.88(d, 1H); 7.77(s, 1H);
7.60(s, 1H); 7.54(s, 1H); 7.50(d, 1H); 7.00(s, 1H); 4.57(s, 2H);
4.45 (m, 1H); 2.00(d, 2H); #1.68(dd, 2H); 1.40(s, 6H); 1.38(s, 6H).
18 4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
methylpiperidin-4-yl)-3-- (2-hy- droxy-ethoxy)benzamide 5-Cl 6-Cl
3-OCH.sub.2CH.sub.2OH --H 57 270-272 .sup.1H-NMR(DMSO-d.sub.6) =
11.65(s, 1H); 8.22(d, 1H); 7.88(d, 1H); 7.80(s, 1H); 7.60-7.52(m,
3H); 7.15(s, 1H); 5.30(m, 1H); 4.35-4.20(m, 3H); 3.90(m, 2H);
1.70(d, 2H); 1.20(s, 6H); #1.12(m, 2H); 1.07(s, 6H). 19
4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,- 2,6,6-tetra-
methylpiperidin-4-yl)-3-(3-amino- propoxy)benzamide 5-Cl 6-Cl
3-O(CH.sub.2).sub.3NH.sub.2 --H 58 194 .sup.1H-NMR(DMSO-d.sub.6) =
11.70(s, 1H); 8.23(d, 1H); 7.80-7.75(m, 2H); 7.65-7.50(m, 3H);
7.05(s, 1H); 4.35-4.20(m, 3H); 3.35(m, 2H); 2.00(m, 2H);
1.75-1.60(m, 2H); 1.20(m, 2H); #1.18(s, 6H); 1.05(s, 6H). 20
4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
methylpiperidin-4-yl)-3-- (2-di- methylamino-ethoxy)benzamide 5-Cl
6-Cl 3-O(CH.sub.2).sub.2N(Me).sub- .2 --H 59 263-265
.sup.1H-NMR(DMSO-d.sub.6) = 12.25(s, 1H); 8.21(d br, 1H); 7.85(m,
1H); 7.80(s, 1H); 7.60(s, 1H); 7.56-7.49(m, 3H); 7.09(s, 1H);
4.40-4.25(m, 3H); 2.78(m, 2H); #2.35(s, 6H); 1.71(m, 2H); 1.19(s,
6H); 1.13(m, 2H); 1.06(s, 6H). 21
4-(5,6-Dichloro-1H-indol-2-yl)-N-(2,2,6,6-tetra-
methylpiperidin-4-yl)-3-- (2,3-di- hydroxy-propoxy)benzamide 5-Cl
6-Cl 3-OCH.sub.2CH(OH)CH.sub.2OH --H 60 252
.sup.1H-NMR(DMSO-d.sub.6) = 11.75(s br, 1H); 8.34(s br, 1H);
7.90(d, 1H); 7.80(s, 1H); 7.65(s, 1H); 7.60(s, 1H); 7.56(d, 1H);
7.15(s, 1H); 5.50(d, 1H); 5.04(t, 1H); #4.40-4.24(m, 2H);
4.20-4.00(m, 2H); 1.90-1.75(m, 2H); 1.40-1.10(m, 14H).
[0280] Biological Assays
[0281] Background. It is known that, upon attachment to bone, an
electrogenic H.sup.+-adenosine triphosphatase (ATPase) is polarised
to the osteoclast-bone interface. The pump transports massive
quantities of protons into the resorption microenvironment to
effect mobilisation of the bone mineral and to create the acidic pH
required by collagenases to degrade the bone matrix.
[0282] The vacuolar nature of the osteoclast proton pump was
originally recognised by Blair [H. C. Blair at al., Science, 245,
855 (1989)] and than confirmed by Bekker [P. J. Bekker et al., J.
Bone Min. Res., 5, 569 (1990)] and Vnnen [H. K. Vnnen et al., J.
Cell. Biol., 111, 1305 (1990)]. Evidence was based upon
preparations of ruffled membrane fragments from avian osteoclasts
(obtained from the medullar bone of calcium-starved egg-laying
hens). The resulting membrane vesicles acidify in response to ATP,
which is easily assessed by measuring the fluorescence quench of
acridine orange, a weak base which accumulates into acidic
compartments.
[0283] The biochemical pattern indicated that the osteoclast proton
pump belonged to the vacuolar-like ATPases since proton transport
was inhibited by N-ethylmaleimide (NEM), a sulphydryl reagent, and
by bafilomycin A.sub.1, a selective inhibitor of vacuolar
H.sup.+-ATPases [J. E. Bowman et al., Proc. Natl. Acad. Sci. USA,
85, 7972 (1988)], whilst it was not inhibited by ouabain, an
inhibitor of Na.sup.+/K.sup.+-ATPases; sodium orthovanadate, an
inhibitor of P-ATPases, or by omeprazole or SCH 28080, both of
which are inhibitors of gastric H.sup.+/K.sup.+-ATPase [J. P.
Mattsson et al., Acta Physiol. Scand., 146, 253 (1992)].
[0284] It is known that specific inhibitors of vacuolar ATPases,
such as bafilomycin A.sub.1, are able to inhibit bone resorption in
osteoclast cultures [K Sundquist et al., Biochem. Biophys. Res.
Commun. 168, 309-313 (1990)]
[0285] Inhibition of Proton Transport and v-ATPase Activity in
Membrane Vesicles. Preparation of crude bone microsomes from
calcium-starved egg-laying hens. Vesicles were prepared from
medullar bone obtained from tibiae and femurs of egg-laying hens
which were calcium-starved for at least 15 days. Briefly, bone
fragments were scraped with a 24 scalpel blade, suspended in 40 ml
of isolation medium (0.2 M sucrose, 50 mM KCl, 10 mM Hepes, 1 mM
EGTA, 2 mM dithiotheitrol, pH 7.4) and filtered through a 100 .mu.m
pore size nylon mesh. The whole procedure was performed at
4.degree. C. After homogenisation in a potter (20 strokes) in 40 ml
of isolation medium an initial centrifugation
(6,500.times.g.sub.max.times.2- 0 min) was performed to remove
mitochondria and lysosomes. The supernatant was centrifuged at
100,000.times.g.sub.max for 1 hr and the pellet was collected in 1
ml of isolation medium, divided into 200 .mu.l aliquots,
immediately frozen in liquid nitrogen and stored at -80.degree. C.
The protein content was determined using a Biorad colourimetric kit
according to Bradford [M. Bradford, Anal. Biochem., 72, 248
(1976)]. For the proton transport assay, 5-10 .mu.l of membranes
were used.
[0286] Purification of osteoclast membranes. 1 ml of crude
microsomal vesicles prepared above were applied (about 0.2 ml per
tube) on the top of a sucrose step-gradient consisting of 3.5 ml of
15%, 30% and 45% (w/w) sucrose in isolation medium and centrifuged
at 280,000 g.sub.max for 2 h (SW 41 Ti rotor). After centrifugation
the 30-45% sucrose interfaces were collected, diluted approx.
20-fold in isolation medium and pelletted at 100,000 g.sub.max for
1 hour (SW 28 rotor). The pellet was then resuspended in 1 ml of
isolation medium, aliquoted and frozen in liquid N.sub.2 and stored
at -80.degree. C. until used.
[0287] Human kidney membranes were obtained from the cortex of a
human kidney, frozen immediately after surgery, according to the
method reported in the literature for bovine kidney (S. Gluck, J.
Biol. Chem., 265, 21957 (1990)).
[0288] Preparation of human osteoclast microsomal vesicles.
Osteoclast-like giant cells isolated from osteoclastoma tumor were
homogenized with a glass-teflon homogenizer (1000 rpm.times.20
strokes), and the material was centrifuged at 6000.times.gmax for
20 minutes. The resulting pellet was then spun at 100000.times.gmax
for 60 minutes to pellet the microsomal fraction. Resuspended in 1
ml of isolation medium pH 7.4, frozen by liquid nitrogen immersion
and stored at -80.degree. C. until used.
[0289] Proton transport in membrane vesicles was assessed,
semi-quantitatively, by measuring the initial slope of fluorescence
quench of acridine orange (excitation 490 nm; emission 530 nm)
after addition of 5-20 .mu.l of membrane vesicles in 1 ml of buffer
containing 0.2 M sucrose, 50 mM KCl, 10 mM Hepes pH 7.4, 1 mM
ATP.Na2, 1 mM CDTA, 5 .mu.M valinomycin and 4 .mu.M acridine
orange. The reaction was started by addition of 5 mM MgSO4. Results
were expressed as the percent of the mean of two controls.
[0290] Inhibition of bafilomycin-sensitive ATPase activity was
assessed in purified membrane vesicles by measuring the release of
inorganic phosphate (Pi) during 30 min of incubation at 37.degree.
C. in a 96-well plate either in the presence or in the absence of
bafilomycin A1. The reaction medium contained 1 mM ATP, 10 mM
HEPES-Tris pH 8, 50 mM KCl, 5 uM valinomycin, 5 uM nigericin, 1 mM
CDTA-Tris, 100 uM ammonium molybdate, 0.2 M sucrose and membranes
(20 ug protein/ml). The reaction was initiated by MgSO.sub.4 (8-arm
pipette) and stopped, after 30 min, by addition of 4 volumes of the
malachite green reagent (96-arm pipette) prepared according to Chan
[Anal. Biochem. 157, 375 (1986)]. Absorbance at 650 nm was measured
after 2 min using a microplate reader. Results are expressed as
nmol (Pi).times.mg protein.sup.-1.times.min.sup.-1 and, for each
experiment, represent the mean.+-.sem of triplicates.
[0291] Pharmacological Data
[0292] Compounds described in the present invention are able to
inhibit bafilomycin-sensitive ATPase of chicken osteoclast in a
range from 50 nM to 2 .mu.M and of human osteoclast in a range from
30 nM to 5 .mu.M.
* * * * *