U.S. patent application number 13/962523 was filed with the patent office on 2013-12-05 for substituted indolyl alkyl amino derivatives as novel inhibitors of histone deacetylase.
This patent application is currently assigned to Janssen Pharmaceutica N.V.. The applicant listed for this patent is Partick Rene Angibaud, Janine Arts, Isabelle Noelle Constance Pilatte, Bruno Roux, Peter Ten Holte, Kristof Van Emelen, Marc Gustaaf Celine VERDONCK. Invention is credited to Partick Rene Angibaud, Janine Arts, Isabelle Noelle Constance Pilatte, Bruno Roux, Peter Ten Holte, Kristof Van Emelen, Marc Gustaaf Celine VERDONCK.
Application Number | 20130324568 13/962523 |
Document ID | / |
Family ID | 34979048 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130324568 |
Kind Code |
A1 |
VERDONCK; Marc Gustaaf Celine ;
et al. |
December 5, 2013 |
SUBSTITUTED INDOLYL ALKYL AMINO DERIVATIVES AS NOVEL INHIBITORS OF
HISTONE DEACETYLASE
Abstract
This invention comprises the novel compounds of formula (I)
##STR00001## wherein R.sup.1, R.sup.2, R.sup.3, X and Y have
defined meanings, having histone deacetylase inhibiting enzymatic
activity; their preparation, compositions containing them and their
use as a medicine.
Inventors: |
VERDONCK; Marc Gustaaf Celine;
(Gierle, BE) ; Angibaud; Partick Rene;
(Fontaine-Bellenger, FR) ; Roux; Bruno; (Saint
Leger du Bourg-Denis, FR) ; Pilatte; Isabelle Noelle
Constance; (Louviers, FR) ; Ten Holte; Peter;
(Beerse, BE) ; Arts; Janine; (Breda, NL) ;
Van Emelen; Kristof; (Sint-Niklaas, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VERDONCK; Marc Gustaaf Celine
Angibaud; Partick Rene
Roux; Bruno
Pilatte; Isabelle Noelle Constance
Ten Holte; Peter
Arts; Janine
Van Emelen; Kristof |
Gierle
Fontaine-Bellenger
Saint Leger du Bourg-Denis
Louviers
Beerse
Breda
Sint-Niklaas |
|
BE
FR
FR
FR
BE
NL
BE |
|
|
Assignee: |
Janssen Pharmaceutica N.V.
Beerse
BE
|
Family ID: |
34979048 |
Appl. No.: |
13/962523 |
Filed: |
August 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13027793 |
Feb 15, 2011 |
8524728 |
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13962523 |
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11572563 |
Jan 23, 2007 |
8193205 |
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PCT/EP2005/053612 |
Jul 25, 2005 |
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13027793 |
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60592182 |
Jul 29, 2004 |
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Current U.S.
Class: |
514/275 ;
514/318 |
Current CPC
Class: |
A61K 31/4545 20130101;
C07D 491/056 20130101; A61P 35/00 20180101; A61K 31/454 20130101;
C07D 401/14 20130101; A61K 31/506 20130101; A61P 35/02 20180101;
C07D 405/14 20130101; C07D 409/14 20130101; A61P 17/06 20180101;
A61P 35/04 20180101; C07D 491/04 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/275 ;
514/318 |
International
Class: |
C07D 401/14 20060101
C07D401/14; C07D 409/14 20060101 C07D409/14; C07D 491/056 20060101
C07D491/056; C07D 405/14 20060101 C07D405/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 2004 |
EP |
04077172.7 |
Claims
1-12. (canceled)
13. A method of treating a disorder related to the abnormal growth
of cells or the abnormal growth of transformed cells, in a
mammalian subject in need of inhibition of such abnormal cell
growth, comprising administering to the subject a therapeutically
effective amount of the compound of formula (I), ##STR00338## the
N-oxide forms, the pharmaceutically acceptable addition salts and
the stereo-chemically isomeric forms thereof, wherein each n is an
integer with value 0, 1 or 2 and when n is 0 then a direct bond is
intended; each m is an integer with value 1 or 2; each X is
independently N or CH; each Y is independently O, S, or NR.sup.4;
wherein each R.sup.4 is hydrogen, C.sub.1-6alkyl,
C.sub.1-6alkyloxyC.sub.1-6alkyl, C.sub.3-6cycloalkyl,
C.sub.3-6cycloalkylmethyl, phenylC.sub.1-6alkyl,
--C(.dbd.O)--CHR.sup.5R.sup.6 or
--S(.dbd.O).sub.2--N(CH.sub.3).sub.2; wherein each R.sup.5 and
R.sup.6 is independently hydrogen, amino, C.sub.1-6alkyl or
aminoC.sub.1-6alkyl; and when Y is NR.sup.4 and R.sup.2 is on the
7-position of the indolyl then R.sup.2 and R.sup.4 together can
form the bivalent radical --(CH.sub.2).sub.2-- (a-1), or
--(CH.sub.2).sub.3-- (a-2); R.sup.1 is hydrogen, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkyl, C.sub.1-6alkylsulfonyl,
C.sub.1-6alkylcarbonyl or mono- or di(C.sub.1-6
alkyl)aminosulfonyl; R.sup.2 is hydrogen, hydroxy, amino, halo,
C.sub.1-6alkyl, cyano, C.sub.2-6alkenyl, polyhaloC.sub.1-6alkyl,
nitro, phenyl, C.sub.1-6alkylcarbonyl, hydroxycarbonyl,
C.sub.1-6alkylcarbonylamino, C.sub.1-6alkyloxy, or mono- or
di(C.sub.1-6alkyl)amino; R.sup.3 is hydrogen, C.sub.1-6alkyl, or
C.sub.1-6alkyloxy; and when R.sup.2 and R.sup.3 are on adjacent
carbon atoms, they can form the bivalent radical
--O--CH.sub.2--O--.
14. The method of claim 13 in which the abnormal growth of cells
comprises cell growth independent of normal regulatory mechanisms,
said mechanisms including the direct inhibition of tumour growth by
growth arrest, terminal differentiation or apoptosis of cancer
cells, and the indirect inhibition of tumour growth by inhibition
of the neovascularization of tumours.
15. The method of claim 14 in which the abnormal growth of cells is
c caused by histone deacetylase (HDAC) activity.
16. A method of inhibiting abnormal cellular proliferative
conditions caused by histone deacetylase (HDAC) activity, in a
human subject in need of inhibition of histone deacetylase (HDAC)
activity, comprising administering to the subject a therapeutically
effective amount of the compound of formula (I), ##STR00339## the
N-oxide forms, the pharmaceutically acceptable addition salts and
the stereo-chemically isomeric forms thereof, wherein each n is an
integer with value 0, 1 or 2 and when n is 0 then a direct bond is
intended; each m is an integer with value 1 or 2; each X is
independently N or CH; each Y is independently O, S, or NR.sup.4;
wherein each R.sup.4 is hydrogen, C.sub.1-6alkyl,
C.sub.1-6alkyloxyC.sub.1-6alkyl, C.sub.3-6cycloalkyl,
C.sub.3-6cycloalkylmethyl, phenylC.sub.1-6alkyl,
--C(.dbd.O)--CHR.sup.5R.sup.6 or
--S(.dbd.O).sub.2--N(CH.sub.3).sub.2; wherein each R.sup.5 and
R.sup.6 is independently hydrogen, amino, C.sub.1-6alkyl or
aminoC.sub.1-6alkyl; and when Y is NR.sup.4 and R.sup.2 is on the
7-position of the indolyl then R.sup.2 and R.sup.4 together can
form the bivalent radical --(CH.sub.2).sub.2-- (a-1), or
--(CH.sub.2).sub.3-- (a-2); R.sup.1 is hydrogen, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkyl, C.sub.1-6alkylsulfonyl,
C.sub.1-6alkylcarbonyl or mono- or di(C.sub.1-6alkyl)aminosulfonyl;
R.sup.2 is hydrogen, hydroxy, amino, halo, C.sub.1-6alkyl, cyano,
C.sub.2-6alkenyl, polyhaloC.sub.1-6alkyl, nitro, phenyl,
C.sub.1-6alkylcarbonyl, hydroxycarbonyl,
C.sub.1-6alkylcarbonylamino, C.sub.1-6alkyloxy, or mono- or
di(C.sub.1-6alkyl)amino; R.sup.3 is hydrogen, C.sub.1-6alkyl, or
C.sub.1-6alkyloxy; and when R.sup.2 and R.sup.3 are on adjacent
carbon atoms, they can form the bivalent radical
--O--CH.sub.2--O--.
17. A method of inhibiting tumor growth in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of the compound of formula (I), ##STR00340## the
N-oxide forms, the pharmaceutically acceptable addition salts and
the stereo-chemically isomeric forms thereof, wherein each n is an
integer with value 0, 1 or 2 and when n is 0 then a direct bond is
intended; each m is an integer with value 1 or 2; each X is
independently N or CH; each Y is independently O, S, or NR.sup.4;
wherein each R.sup.4 is hydrogen, C.sub.1-6alkyl,
C.sub.1-6alkyloxyC.sub.1-6alkyl, C.sub.3-6cycloalkyl,
C.sub.3-6cycloalkylmethyl, phenylC.sub.1-6alkyl,
--C(.dbd.O)--CHR.sup.5R.sup.6 or
--S(.dbd.O).sub.2--N(CH.sub.3).sub.2; wherein each R.sup.5 and
R.sup.6 is independently hydrogen, amino, C.sub.1-6alkyl or
aminoC.sub.1-6alkyl; and when Y is NR.sup.4 and R.sup.2 is on the
7-position of the indolyl then R.sup.2 and R.sup.4 together can
form the bivalent radical --(CH.sub.2).sub.2-- (a-1), or
--(CH.sub.2).sub.3-- (a-2); R.sup.1 is hydrogen, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkyl, C.sub.1-6alkylsulfonyl,
C.sub.1-6alkylcarbonyl or mono- or di(C.sub.1-6alkyl)aminosulfonyl;
R.sup.2 is hydrogen, hydroxy, amino, halo, C.sub.1-6alkyl, cyano,
C.sub.2-6alkenyl, polyhaloC.sub.1-6alkyl, nitro, phenyl,
C.sub.1-6alkylcarbonyl, hydroxycarbonyl,
C.sub.1-6alkylcarbonylamino, C.sub.1-6alkyloxy, or mono- or
di(C.sub.1-6alkyl)amino; R.sup.3 is hydrogen, C.sub.1-6alkyl, or
C.sub.1-6alkyloxy; and when R.sup.2 and R.sup.3 are on adjacent
carbon atoms, they can form the bivalent radical
--O--CH.sub.2--O--.
18. The method of claim 17 in which the tumor comprises lung
cancer, adenocarcinoma lung cancer, non-small cell lung cancer,
pancreatic cancer, exocrine pancreatic carcinoma, colon cancers,
colon adenocarcinoma and colon adenoma, prostate cancer, advanced
prostate cancer, hematopoietic tumours of lymphoid lineage, acute
lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, myeloid
leukemias, acute myelogenous leukemia (AML), thyroid follicular
cancer, myelodysplastic syndrome (MDS), tumours of mesenchymal
origin, fibrosarcomas, rhabdomyosarcomas, melanomas,
teratocarcinomas, neuroblastomas, gliomas, benign tumours of the
skin, keratoacanthomas, breast carcinoma, advanced breast cancer,
kidney carcinoma, bladder carcinoma and epidermal carcinoma.
Description
[0001] This invention concerns compounds having histone deacetylase
(HDAC) inhibiting enzymatic activity. It further relates to
processes for their preparation, to compositions comprising them,
as well as their use, both in vitro and in vivo, to inhibit HDAC
and as a medicine, for instance as a medicine to inhibit
proliferative conditions, such as cancer and psoriasis.
[0002] Nuclear histones are known as integral and dynamic
components of the machinery responsible for regulating gene
transcription and other DNA-templated processes such as
replication, repair, recombination, and chromosome segregation.
They are the subject of post-translational modifications including
acetylation, phosphorylation, methylation, ubiquitination, and
ADP-ribosylation.
[0003] Histone deacetylase(s), herein referred to as "HDACs", are
enzymes that catalyze the removal of the acetyl modification on
lysine residues of proteins, including the core nucleosomal
histones H2A, H2B, H3 and H4. Together with histone
acetyltransferase(s), herein referred to as "HATs", HDACs regulate
the level of acetylation of the histones. The balance of
acetylation of nucleosomal histones plays an important role in
transcription of many genes. Hypoacetylation of histones is
associated with condensed chromatin structure resulting in the
repression of gene transcription, whereas acetylated histones are
associated with a more open chromatin structure and activation of
transcription.
[0004] Eleven structurally related HDACs have been described and
fall into two classes. Class I HDACs consist of HDAC 1, 2, 3, 8 and
11 whereas class II HDACs consist of HDAC 4, 5, 6, 7, 9 and 10.
Members of a third class of HDACs are structurally unrelated to the
class I and class II HDACs. Class I/II HDACs operate by
zinc-dependent mechanisms, whereas class III HDACs are
NAD-dependent.
[0005] In addition to histones, other proteins have also been the
substrate for acetylation, in particular transcriptionfactors such
as p53, GATA-1 and E2F; nuclear receptors such as the
glucocorticoid receptor, the thyroid receptors, the estrogen
receptors; and cell-cycle regulating proteins such as pRb.
Acetylation of proteins has been linked with protein stabilization,
such as p53 stabilization, recruitment of cofactors and increased
DNA binding. p53 is a tumour suppressor that can induce cell cycle
arrest or apoptosis in response to a variety of stress signals,
such as DNA damage. The main target for p53-induced cell cycle
arrest seems to be the p21 gene. Next to its activation by p53, p21
has been identified by virtue of its association with
cyclin/cyclin-dependent kinase complexes resulting in cell cycle
arrest at both G1 and G2 phases, its up-regulation during
senescence, and its interaction with the proliferating cell nuclear
antigen.
[0006] The study of inhibitors of HDACs indicates that they play an
important role in cell cycle arrest, cellular differentiation,
apoptosis and reversal of transformed phenotypes.
[0007] The inhibitor Trichostatin A (TSA), for example, causes cell
cycle arrest at both G1 and G2 phases, reverts the transformed
phenotype of different cell lines, and induces differentiation of
Friend leukemia cells and others. TSA (and suberoylanilide
hydroxamic acid SAHA) have been reported to inhibit cell growth,
induce terminal differentiation, and prevent the formation of
tumours in mice (Finnin et al., Nature, 401: 188-193, 1999).
[0008] Trichostatin A has also been reported to be useful in the
treatment of fibrosis, e.g. liver fibrosis and liver chirrhosis.
(Geerts et al., European Patent Application EP 0 827 742, published
11 Mar., 1998).
[0009] The pharmacophore for HDAC inhibitors consists of a
metal-binding domain, which interacts with the zinc-containing
active site of HDACs, a linker domain, and a surface recognition
domain or capping region, which interacts with residues on the rim
of the active site.
[0010] Inhibitors of HDACs have also been reported to induce p21
gene expression. The transcriptional activation of the p21 gene by
these inhibitors is promoted by chromatin remodelling, following
acetylation of histones H3 and H4 in the p21 promotor region. This
activation of p21 occurs in a p53-independent fashion and thus HDAC
inhibitors are operative in cells with mutated p53 genes, a
hallmark of numerous tumours.
[0011] In addition HDAC inhibitors can have indirect activities
such as augmentation of the host immune response and inhibition of
tumor angiogenesis and thus can suppress the growth of primary
tumors and impede metastasis (Mai et al., Medicinal Research
Reviews, 25: 261-309).
[0012] In view of the above, HDAC inhibitors can have great
potential in the treatment of cell proliferative diseases or
conditions, including tumours with mutated p53 genes.
[0013] Patent application EP1472216 published on Aug. 14, 2003
discloses bicyclic hydroxamates as inhibitors of histone
deacetylase.
[0014] Patent applications EP1485099, EP1485348, EP1485353,
EP1485354, EP1485364, EP1485365, EP1485370, EP1485378 published on
18 Sep., 2003, amongst others, disclose substituted
piperazinylpyrimidinylhydroxamic acids as inhibitors of histone
deacetylase furthermore EP1485365 discloses R306465.
[0015] Patent application EP1492534 published on 9 Oct., 2003,
discloses carbamic acid compounds comprising a piperazine linkage,
as HDAC inhibitors.
[0016] Patent application EP1495002 published on 23 Oct., 2003,
disclose substituted piperazinyl phenyl benzamide compounds, as
histone deacetylase inhibitors.
[0017] Patent application WO04/009536 published on 29 Jan., 2004,
discloses derivatives containing an alkyl linker between the aryl
group and the hydroxamate, as histone deacetylase inhibitors.
[0018] Patent application EP1525199 published on 12 Feb., 2004,
discloses (hetero)arylalkenyl substituted bicyclic hydroxamates, as
histone deacetylase inhibitors.
[0019] Patent application WO04/063146 published on 29 Jul. 2004,
discloses derivatives of N-hydroxy-benzamide derivatives with
anti-inflammatory and antitumour activity.
[0020] Patent application WO04/063169 published on 29 Jul. 2004,
discloses substituted aryl hydroxamate derivatives as histone
deacetylase inhibitors.
[0021] Patent application WO04/072047 published on 26 Aug. 2004,
discloses indoles, benzimidazoles and naphhimidazoles as histone
deacetylase inhibitors.
[0022] Patent application WO04/082638 published on 30 Sep. 2004,
discloses hydroxamates linked to non-aromatic heterocyclic ring
systems as histone deacetylase inhibitors.
[0023] Patent application WO04/092115 published on 28 Oct. 2004,
discloses hydroxamate derivatives as histone deacetylase
inhibitors.
[0024] Patent application WO05/028447 published on 31 Mar. 2005,
discloses benzimidazoles as histone deacetylase inhibitors.
[0025] Patent applications WO05/030704 and WO05/030705 published on
7 Apr. 2005, discloses benzamides as histone deacetylase
inhibitors.
[0026] Patent application WO05/040101 published on 6 May 2005,
disloses acylurea connected and sulfonylurea connected hydroxamates
as histone deacetylase inhibitors.
[0027] Patent application WO05/040161 also published on 6 May 2005,
discloses biaryl linked hydroxamates as histone deacetylase
inhibitors.
[0028] The compounds of the present invention differ from the prior
art in structure, in their pharmacological activity and/or
pharmacological potency.
[0029] The problem to be solved is to provide histone deacetylase
inhibitors with high enzymatic and cellular activity that have
increased bioavailability and/or in vivo potency.
[0030] The novel compounds of the present invention solve the
above-described problem.
[0031] The compounds of the present invention show excellent
histone deacetylase inhibiting enzymatic and cellular activity.
They have a high capacity to activiate the p21 gene, both at the
cellular and the in vivo level. They have a desirable
pharmacokinetic profile and low affinity for the P450 enzymes,
which reduces the risk of adverse drug-drug interaction allowing
also for a wider safety margin.
[0032] Advantageous features of the present compounds are metabolic
stability, solubility and/or p21 induction capacity. More in
particular the compounds of the present invention have increased
half-lives in rat hepatocytes, have an increased
solubility/stability in aqueous solutions and/or have enhanced in
vivo p21 promoter inducing capacities.
[0033] This invention concerns compounds of formula (I)
##STR00002##
the N-oxide forms, the pharmaceutically acceptable addition salts
and the stereo-chemically isomeric forms thereof, wherein [0034]
each n is an integer with value 0, 1 or 2 and when n is 0 then a
direct bond is intended; [0035] each m is an integer with value 1
or 2; [0036] each X is independently N or CH; [0037] each Y is
independently O, S, or NR.sup.4; wherein [0038] each R.sup.4 is
hydrogen, C.sub.1-6alkyl, C.sub.1-6alkyloxyC.sub.1-6alkyl,
C.sub.3-6cycloalkyl, C.sub.3-6cycloalkylmethyl,
phenylC.sub.1-6alkyl, --C(.dbd.O)--CHR.sup.5R.sup.6 or
--S(.dbd.O).sub.2--N(CH.sub.3).sub.2; wherein [0039] each R.sup.5
and R.sup.6 is independently hydrogen, amino, C.sub.1-6alkyl or
aminoC.sub.1-6alkyl; and [0040] when Y is NR.sup.4 and R.sup.2 is
on the 7-position of the indolyl then R.sup.2 and R.sup.4 together
can form the bivalent radical
[0040] --(CH.sub.2).sub.2-- (a-1), or
--(CH.sub.2).sub.3-- (a-2); [0041] R.sup.1 is hydrogen,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl, C.sub.1-6alkylsulfonyl,
C.sub.1-6alkylcarbonyl or mono- or di(C.sub.1-6alkyl)aminosulfonyl;
[0042] R.sup.2 is hydrogen, hydroxy, amino, halo, C.sub.1-6alkyl,
cyano, C.sub.2-6alkenyl, polyhaloC.sub.1-6alkyl, nitro, phenyl,
C.sub.1-6alkylcarbonyl, hydroxycarbonyl,
C.sub.1-6alkylcarbonylamino, C.sub.1-6alkyloxy, or mono- or
di(C.sub.1-6alkyl)amino; [0043] R.sup.3 is hydrogen,
C.sub.1-6alkyl, or C.sub.1-6alkyloxy; and [0044] when R.sup.2 and
R.sup.3 are on adjacent carbon atoms, they can form the bivalent
radical --O--CH.sub.2--O--.
[0045] Lines drawn into the bicyclic ring systems from substituents
indicate that the bonds may be attached to any of the suitable ring
atoms of the bicyclic ring system.
[0046] The term "histone deacetylase inhibitor" or "inhibitor of
histone deacetylase" is used to identify a compound, which is
capable of interacting with a histone deacetylase and inhibiting
its activity, more particularly its enzymatic activity. Inhibiting
histone deacetylase enzymatic activity means reducing the ability
of a histone deacetylase to remove an acetyl group from a histone.
Preferably, such inhibition is specific, i.e. the histone
deacetylase inhibitor reduces the ability of a histone deacetylase
to remove an acetyl group from a histone at a concentration that is
lower than the concentration of the inhibitor that is required to
produce some other, unrelated biological effect.
[0047] As used in the foregoing definitions and hereinafter, halo
is generic to fluoro, chloro, bromo and iodo; C.sub.1-2alkyl
straight chain saturated hydrocarbon radicals having 1 or 2 carbon
atoms such as, e.g. methyl or ethyl; C.sub.1-6alkyl defines
C.sub.1-2alkyl and straight and branched chain saturated
hydrocarbon radicals having from 3 to 6 carbon atoms such as, e.g.
propyl, butyl, 1-methylethyl, 2-methylpropyl, pentyl,
2-methyl-butyl, hexyl, 2-methylpentyl and the like; and
polyhaloC.sub.1-6alkyl defines C.sub.1-6alkyl containing three
identical or different halo substituents for example
trifluoromethyl; C.sub.2-6alkenyl defines straight and branched
chain hydrocarbon radicals containing one double bond and having
from 2 to 6 carbon atoms such as, for example, ethenyl, 2-propenyl,
3-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, and the
like; C.sub.3-6cycloalkyl includes cyclic hydrocarbon groups having
from 3 to 6 carbons, such as cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, and the like.
[0048] Pharmaceutically acceptable addition salts encompass
pharmaceutically acceptable acid addition salts and
pharmaceutically acceptable base addition salts. The
pharmaceutically acceptable acid addition salts as mentioned
hereinabove are meant to comprise the therapeutically active
non-toxic acid addition salt forms, which the compounds of formula
(I) are able to form. The compounds of formula (I) which have basic
properties can be converted in their pharmaceutically acceptable
acid addition salts by treating said base form with an appropriate
acid. Appropriate acids comprise, for example, inorganic acids such
as hydrohalic acids, e.g. hydrochloric or hydrobromic acid;
sulfuric; nitric; phosphoric and the like acids; or organic acids
such as, for example, acetic, trifluoroacetic, propanoic,
hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e.
butanedioic acid), maleic, fumaric, malic, tartaric, citric,
methanesulfonic, ethanesulfonic, benzenesulfonic,
p-toluenesulfonic, cyclamic, salicylic, p-amino-salicylic, pamoic
and the like acids.
[0049] The compounds of formula (I) which have acidic properties
may be converted in their pharmaceutically acceptable base addition
salts by treating said acid form with a suitable organic or
inorganic base. Appropriate base salt forms comprise, for example,
the ammonium salts, the alkali and earth alkaline metal salts, e.g.
the lithium, sodium, potassium, magnesium, calcium salts and the
like, salts with organic bases, e.g. the benzathine,
N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids
such as, for example, arginine, lysine and the like.
[0050] The term "acid or base addition salts" also comprises the
hydrates and the solvent addition forms, which the compounds of
formula (I) are able to form. Examples of such forms are e.g.
hydrates, alcoholates and the like.
[0051] The term "stereochemically isomeric forms of compounds of
formula (I)", as used herein, defines all possible compounds made
up of the same atoms bonded by the same sequence of bonds but
having different three-dimensional structures, which are not
interchangeable, which the compounds of formula (I) may possess.
Unless otherwise mentioned or indicated, the chemical designation
of a compound encompasses the mixture of all possible
stereochemically isomeric forms, which said compound may possess.
Said mixture may contain all diastereomers and/or enantiomers of
the basic molecular structure of said compound. All
stereochemically isomeric forms of the compounds of formula (I)
both in pure form or in admixture with each other are intended to
be embraced within the scope of the present invention.
[0052] The N-oxide forms of the compounds of formula (I) are meant
to comprise those compounds of formula (I) wherein one or several
nitrogen atoms are oxidized to the so-called N-oxide, particularly
those N-oxides wherein one or more of the piperidine-, piperazine
or pyridazinyl-nitrogens are N-oxidized.
[0053] Some of the compounds of formula (I) may also exist in their
tautomeric forms. Such forms although not explicitly indicated in
the above formula are intended to be included within the scope of
the present invention.
[0054] Whenever used hereinafter, the term "compounds of formula
(I)" is meant to include also the pharmaceutically acceptable
addition salts and all stereoisomeric forms.
[0055] As used herein, the terms "histone deacetylase" and "HDAC"
are intended to refer to any one of a family of enzymes that remove
acetyl groups from the .epsilon.-amino groups of lysine residues at
the N-terminus of a histone. Unless otherwise indicated by context,
the term "histone" is meant to refer to any histone protein,
including H1, H2A, H2B, H3, H4, and H5, from any species. Human
HDAC proteins or gene products, include, but are not limited to,
HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8,
HDAC-9 HDAC-10 and HDAC-11. The histone deacetylase can also be
derived from a protozoal or fungal source.
[0056] A first group of interesting compounds consists of those
compounds of formula (I) wherein one or more of the following
restrictions apply: [0057] a) each R.sup.4 is hydrogen,
C.sub.1-6alkyl, C.sub.3-6cycloalkyl, C.sub.3-6cycloalkylmethyl,
--C(.dbd.O)--CHR.sup.5R.sup.6 or
--S(.dbd.O).sub.2--N(CH.sub.3).sub.2; [0058] b) R.sup.1 is
hydrogen, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl,
C.sub.1-6alkylsulfonyl, or mono- or
di(C.sub.1-6alkyl)aminosulfonyl; or [0059] c) R.sup.3 is
hydrogen.
[0060] A second group of interesting compounds consists of those
compounds of formula (I) wherein one or more of the following
restrictions apply:
a) each n is an integer with value 0 or 1; b) each X is
independently N; c) each R.sup.4 is hydrogen or C.sub.1-6alkyl; d)
R.sup.1 is hydrogen, C.sub.1-6alkyl or hydroxyC.sub.1-6alkyl; or e)
R.sup.2 is hydrogen, halo, C.sub.1-6alkyl or C.sub.1-6alkyloxy.
[0061] A third group of interesting compounds consists of those
compounds of formula (I) wherein one or more of the following
restrictions apply: [0062] a) each n is an integer with value 0 or
1; [0063] b) each R.sup.4 is hydrogen, C.sub.1-6alkyl,
C.sub.1-6alkyloxyC.sub.1-6alkyl, C.sub.3-6cycloalkyl or
phenylC.sub.1-6alkyl; [0064] c) R.sup.1 is hydrogen,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl, C.sub.1-6alkylcarbonyl or
C.sub.1-6alkylsulfonyl; or [0065] d) R.sup.2 is hydrogen, halo,
C.sub.1-6alkyl, cyano, nitro or C.sub.1-6alkyloxy.
[0066] A fourth group of interesting compounds consists of those
compounds of formula (I) wherein one or more of the following
restrictions apply: [0067] a) each n is an integer with value 0 or
1; [0068] b) each m is an integer with value 1; [0069] c) each
R.sup.4 is hydrogen, C.sub.1-6alkyloxyC.sub.1-6alkyl,
C.sub.3-6cycloalkyl or phenylC.sub.1-6alkyl; [0070] c) R.sup.1 is
hydrogen, hydroxyC.sub.1-6alkyl, C.sub.1-6alkylcarbonyl or
C.sub.1-6alkylsulfonyl; [0071] d) R.sup.2 is hydrogen, halo, cyano,
nitro or C.sub.1-6alkyloxy; or [0072] e) R.sup.3 is
C.sub.1-6alkyloxy; or [0073] f) when R.sup.2 and R.sup.3 are on
adjacent carbon atoms, they can form the bivalent radical
--O--CH.sub.2--O--.
[0074] A fifth group of interesting compounds consists of those
compounds of formula (I) wherein one or more of the following
restrictions apply:
a) each n is an integer with value 1; b) each m is an integer with
value 1; c) each X is independently N; d) each Y is independently
NR.sup.4; e) each R.sup.4 is C.sub.1-6alkyl; f) R.sup.1 is
hydrogen; g) R.sup.2 is hydrogen or halo; or h) R.sup.3 is
hydrogen.
[0075] A group of preferred compounds consists of those compounds
of formula (I) wherein each n is an integer with value 0 or 1; each
R.sup.4 is hydrogen, C.sub.1-6alkyl,
C.sub.1-6alkyloxyC.sub.1-6alkyl, C.sub.3-6cycloalkyl or
phenylC.sub.1-6alkyl; R.sup.1 is hydrogen, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkyl, C.sub.1-6alkylcarbonyl or
C.sub.1-6alkylsulfonyl; and R.sup.2 is hydrogen, halo,
C.sub.1-6alkyl, cyano, nitro, polyhaloC.sub.1-6alkyl or
C.sub.1-6alkyloxy.
[0076] A group of more preferred compounds consists of those
compounds of formula (I) wherein each n is an integer with value 1;
each m is an integer with value 1; each X is independently N; each
Y is independently NR.sup.4; each R.sup.4 is C.sub.1-6alkyl;
R.sup.1 is hydrogen; R.sup.2 is hydrogen or halo; and R.sup.3 is
hydrogen. [0077] The most preferred compounds are compound No. 1a,
compound No. 30, compound No. 39 and compound No. 50
##STR00003##
[0078] The compounds of formula (I) and (II), their
pharmaceutically acceptable salts and N-oxides and stereochemically
isomeric forms thereof may be prepared in conventional manner. The
starting materials and some of the intermediates are known
compounds and are commercially available or may be prepared
according to conventional reaction procedures as generally known in
the art or as described in patent applications EP1485099,
EP1485348, EP1485353, EP1485354, EP1485364, EP1485365, EP1485370,
and EP1485378. Some preparation methods will be described
hereinafter in more detail. Other methods for obtaining final
compounds of formula (I) are described in the examples.
[0079] a) Hydroxamic acids of formula (I) may be prepared by
reacting an intermediate of formula (II), wherein Q is
tetrahydropyranyloxyaminocarbonyl, herein referred to as
intermediates of formula (II-a), with an appropriate acid, such as
for example, trifluoro acetic acid. Said reaction is performed in
an appropriate solvent, such as, for example, methanol or
dichloromethane.
##STR00004##
[0080] b) Alternatively, hydroxamic acids of formula (I) may be
prepared by reacting an intermediate of formula (II), wherein Q is
C.sub.1-2alkyloxycarbonyl, herein referred to as intermediates of
formula (II-c), with hydroxylamine, in the presence of a base such
as for example sodium hydroxyde. Said reaction is performed in an
appropriate solvent, such as, for example, methanol.
##STR00005##
[0081] The compounds of formula (I) may also be converted into each
other via art-known reactions or functional group transformations.
A number of such transformations are already described hereinabove.
Other examples are hydrolysis of carboxylic esters to the
corresponding carboxylic acid or alcohol; hydrolysis of amides to
the corresponding carboxylic acids or amines; hydrolysis of
nitriles to the corresponding amides; amino groups on imidazole or
phenyl may be replaced by a hydrogen by art-known diazotation
reactions and subsequent replacement of the diazo-group by
hydrogen; alcohols may be converted into esters and ethers; primary
amines may be converted into secondary or tertiary amines; double
bonds may be hydrogenated to the corresponding single bond; an iodo
radical on a phenyl group may be converted in to an ester group by
carbon monoxide insertion in the presence of a suitable palladium
catalyst.
[0082] The present invention also concerns intermediates of formula
(II)
##STR00006##
the N-oxide forms, the pharmaceutically acceptable addition salts
and the stereo-chemically isomeric forms thereof, wherein [0083]
each n is an integer with value 0, 1 or 2 and when n is 0 then a
direct bond is intended; [0084] each m is an integer with value 1
or 2; [0085] each X is independently N or CH; [0086] each Y is
independently O, S, or NR.sup.4; wherein [0087] each R.sup.4 is
hydrogen, C.sub.1-6alkyl, C.sub.1-6alkyloxyC.sub.1-6alkyl,
C.sub.3-6cycloalkyl, C.sub.3-6cycloalkylmethyl,
phenylC.sub.1-6alkyl, --C(.dbd.O)--CHR.sup.5R.sup.6 or
--S(.dbd.O).sub.2--N(CH.sub.3).sub.2; wherein [0088] each R.sup.5
and R.sup.6 is independently hydrogen, amino, C.sub.1-6alkyl or
aminoC.sub.1-6alkyl; and [0089] when Y is NR.sup.4 and R.sup.2 is
on the 7-position of the indolyl then R.sup.2 and R.sup.4 together
can form the bivalent radical
[0089] --(CH.sub.2).sub.2-- (a-1), or
--(CH.sub.2).sub.3-- (a-2); [0090] R.sup.1 is hydrogen,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl, C.sub.1-6alkylsulfonyl,
C.sub.1-6alkylcarbonyl or mono- or di(C.sub.1-6alkyl)aminosulfonyl;
[0091] R.sup.2 is hydrogen, hydroxy, amino, halo, C.sub.1-6alkyl,
cyano, C.sub.2-6alkenyl, polyhaloC.sub.1-6alkyl, nitro, phenyl,
C.sub.1-6alkylcarbonyl, hydroxycarbonyl,
C.sub.1-6alkylcarbonylamino, C.sub.1-6alkyloxy, or mono- or
di(C.sub.1-6alkyl)amino; [0092] R.sup.3 is hydrogen,
C.sub.1-6alkyl, or C.sub.1-6alkyloxy; [0093] when R.sup.2 and
R.sup.3 are on adjacent carbon atoms, they can form the bivalent
radical --O--CH.sub.2--O--; and [0094] Q is
C.sub.1-2alkyloxycarbonyl, hydroxycarbonyl or
tetrahydropyranyloxyaminocarbonyl.
[0095] Groups of interesting, preferred, more preferred and most
preferred compounds can be defined for the compounds of formula
(II), in accordance with the groups defined for the compounds of
formula (I).
[0096] a) Intermediates of formula (II-a) may be prepared by
reacting an intermediate of formula (III) with an intermediate of
formula (II), wherein Q is hydroxycarbonyl, herein referred to as
intermediates of formula (II-b), in the presence of appropriate
reagents such as
N'-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,
monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). The
reaction may be performed in the presence of a base such as
triethylamine, in a suitable solvent, such as, a mixture of
dichloromethane and tetrahydrofuran.
##STR00007##
[0097] b) Intermediates of formula (II-a) may also be prepared by
reacting an intermediate of formula (VI) with the appropriate
carboxaldehyde of formula (V), wherein t is an integer with value 0
or 1, and when t is 0 then a direct bond is intended, in the
presence of an appropriate reagent, such as a sodium borohydride,
in a suitable solvent such as dichloroethane or methanol.
##STR00008##
[0098] c) Intermediates of formula (II-b) may be prepared by
reacting an intermediate of formula (II), wherein Q is methyl- or
ethyloxycarbonyl (C.sub.1-2alkyl), herein referred to as
intermediates of formula (II-c), with an appropriate acidic
solution, e.g. hydrochloric acid, or basic solution, e.g. hydrogen
bromide or sodiumhydroxide, in a suitable solvent e.g. an alcohol,
such as ethanol or propanol.
##STR00009##
[0099] d) The intermediates of formula (II-c) may be prepared by
reacting the carboxylic acid ethyl ester of formula (IV) with the
appropriate carboxaldehyde of formula (V), in the presence of an
appropriate reagent, such as a sodium borohydride e.g. sodium
tetrahydroborate, in a suitable solvent such as an alcohol e.g.
methanol.
##STR00010##
[0100] e) In an identical way, the intermediates of formula (II-c)
may be prepared by reacting an intermediate of formula (XIV) with
the appropriate intermediate of formula (XV), in the presence of an
appropriate reagent, such as a sodium borohydride e.g. sodium
tetrahydroborate, in a suitable solvent such as an alcohol e.g.
methanol.
##STR00011##
[0101] f) Intermediates of formula (II-c) may also be prepared by
reacting an intermediate of formula (X) with an intermediate of
formula (XI) wherein W is an appropriate leaving group such as, for
example, halo, e.g. fluoro, chloro, bromo or iodo, or a sulfonyloxy
radical such as methylsulfonyloxy,
[0102] 4-methylphenylsulfonyloxy and the like. The reaction can be
performed in a reaction-inert solvent such as, for example, an
alcohol, e.g. methanol, ethanol,
[0103] 2-methoxy-ethanol, propanol, butanol and the like; an ether,
e.g. 4,4-dioxane, 1,1'-oxybispropane and the like; a ketone, e.g.
4-methyl-2-pentanone; or
[0104] N,N-dimethylformamide, nitrobenzene, acetonitrile and the
like. The addition of an appropriate base such as, for example, an
alkali or earth alkaline metal carbonate or hydrogen carbonate,
e.g. triethylamine or sodium carbonate, may be utilized to pick up
the acid which is liberated during the course of the reaction. A
small amount of an appropriate metal iodide, e.g., sodium or
potassium iodide may be added to promote the reaction. Stirring may
enhance the rate of the reaction. The reaction may conveniently be
carried out at a temperature ranging between room temperature and
the reflux temperature of the reaction mixture and, if desired, the
reaction may be carried out at an increased pressure.
##STR00012##
[0105] g) In an identical way, intermediates of formula (II-c) may
be prepared by reacting an intermediate of formula (XII) with an
intermediate of formula (XIII), wherein W is an appropriate leaving
group as described above.
##STR00013##
[0106] The intermediates of formula (VI) may be prepared by
reacting the intermediate of formula (VII) with piperidine in a
suitable solvent e.g. dichloromethane.
##STR00014##
[0107] The intermediates of formula (VII) may be prepared by
reacting an intermediate of formula (VIII) with an intermediate of
formula (III), in the presence of appropriate reagents such as
N'-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,
monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). The
reaction may be performed in the presence of a base such as
triethylamine, in a suitable solvent, such as, a mixture of
dichloromethane and tetrahydrofuran.
##STR00015##
[0108] Intermediates of formula (VIII) may be prepared by reacting
an intermediate of formula (IX) with an intermediate of formula
(IV), wherein R.sup.1 is hydrogen, herein referred to as
intermediates of formula (IV-a), in the presence of sodium
hydroxide, in a suitable solvent, such as tetrahydrofuran, followed
by neutralization with hydrochloric acid and addition of sodium
carbonate.
##STR00016##
[0109] The compounds of formula (I) and some of the intermediates
may have at least one stereogenic centre in their structure. This
stereogenic centre may be present in an R or an S
configuration.
[0110] The compounds of formula (I) as prepared in the hereinabove
described processes are generally racemic mixtures of enantiomers,
which can be separated from one another following art-known
resolution procedures. The racemic compounds of formula (I) may be
converted into the corresponding diastereomeric salt forms by
reaction with a suitable chiral acid. Said diastereomeric salt
forms are subsequently separated, for example, by selective or
fractional crystallization and the enantiomers are liberated there
from by alkali. An alternative manner of separating the
enantiomeric forms of the compounds of formula (I) involves liquid
chromatography using a chiral stationary phase. Said pure
stereochemically isomeric forms may also be derived from the
corresponding pure stereochemically isomeric forms of the
appropriate starting materials, provided that the reaction occurs
stereospecifically. Preferably if a specific stereoisomer is
desired, said compound would be synthesized by stereospecific
methods of preparation. These methods will advantageously employ
enantiomerically pure starting materials.
[0111] The compounds of formula (I), the pharmaceutically
acceptable acid addition salts and stereoisomeric forms thereof
have valuable pharmacological properties in that they have a
histone deacetylase (HDAC) inhibitory effect.
[0112] This invention provides a method for inhibiting the abnormal
growth of cells, including transformed cells, by administering an
effective amount of a compound of the invention. Abnormal growth of
cells refers to cell growth independent of normal regulatory
mechanisms (e.g. loss of contact inhibition). This includes the
inhibition of tumour growth both directly by causing growth arrest,
terminal differentiation and/or apoptosis of cancer cells, and
indirectly, by inhibiting neovascularization of tumours.
[0113] This invention also provides a method for inhibiting tumour
growth by administering an effective amount of a compound of the
present invention, to a subject, e.g. a mammal (and more
particularly a human) in need of such treatment. In particular,
this invention provides a method for inhibiting the growth of
tumours by the administration of an effective amount of the
compounds of the present invention. Examples of tumours which may
be inhibited, but are not limited to, lung cancer (e.g.
adenocarcinoma and including non-small cell lung cancer),
pancreatic cancers (e.g. pancreatic carcinoma such as, for example
exocrine pancreatic carcinoma), colon cancers (e.g. colorectal
carcinomas, such as, for example, colon adenocarcinoma and colon
adenoma), prostate cancer including the advanced disease,
hematopoietic tumours of lymphoid lineage (e.g. acute lymphocytic
leukemia, B-cell lymphoma, Burkitt's lymphoma), myeloid leukemias
(for example, acute myelogenous leukemia (AML)), thyroid follicular
cancer, myelodysplastic syndrome (MDS), tumours of mesenchymal
origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanomas,
teratocarcinomas, neuroblastomas, gliomas, benign tumour of the
skin (e.g. keratoacanthomas), breast carcinoma (e.g. advanced
breast cancer), kidney carcinoma, ovary carcinoma, bladder
carcinoma and epidermal carcinoma.
[0114] The compound according to the invention may be used for
other therapeutic purposes, for example: [0115] a) the
sensitisation of tumours to radiotherapy by administering the
compound according to the invention before, during or after
irradiation of the tumour for treating cancer; [0116] b) treating
arthropathies and osteopathological conditions such as rheumatoid
arthritis, osteoarthritis, juvenile arthritis, gout, polyarthritis,
psoriatic arthritis, ankylosing spondylitis and systemic lupus
erythematosus; [0117] c) inhibiting smooth muscle cell
proliferation including vascular proliferative disorders,
atherosclerosis and restenosis; [0118] d) treating inflammatory
conditions and dermal conditions such as ulcerative colitis,
Crohn's disease, allergic rhinitis, graft vs. host disease,
conjunctivitis, asthma, ARDS, Behcets disease, transplant
rejection, uticaria, allergic dermatitis, alopecia greata,
scleroderma, exanthema, eczema, dermatomyositis, acne, diabetes,
systemic lupus erythematosis, Kawasaki's disease, multiple
sclerosis, emphysema, cystic fibrosis and chronic bronchitis;
[0119] e) treating endometriosis, uterine fibroids, dysfunctional
uterine bleeding and endometrial hyperplasia; [0120] f) treating
ocular vascularisation including vasculopathy affecting retinal and
choroidal vessels; [0121] g) treating a cardiac dysfunction; [0122]
h) inhibiting immunosuppressive conditions such as the treatment of
HIV infections; [0123] i) treating renal dysfunction; [0124] j)
suppressing endocrine disorders; [0125] k) inhibiting dysfunction
of gluconeogenesis; [0126] l) treating a neuropathology for example
Parkinson's disease or a neuropathology that results in a cognitive
disorder, for example, Alzheimer's disease or polyglutamine related
neuronal diseases; [0127] m) treating psychiatric disorders for
example schizophrenia, bipolar disorder, depression, anxiety and
psychosis; [0128] n) inhibiting a neuromuscular pathology, for
example, amylotrophic lateral sclerosis; [0129] o) treating spinal
muscular atrophy; [0130] p) treating other pathologic conditions
amenable to treatment by potentiating expression of a gene; [0131]
q) enhancing gene therapy; [0132] r) inhibiting adipogenesis;
[0133] s) treating parasitosis such as malaria.
[0134] Hence, the present invention discloses the compounds of
formula (I) for use as a medicine as well as the use of these
compounds of formula (I) for the manufacture of a medicament for
treating one or more of the above mentioned conditions.
[0135] The compounds of formula (I), the pharmaceutically
acceptable acid addition salts and stereoisomeric forms thereof can
have valuable diagnostic properties in that they can be used for
detecting or identifying a HDAC in a biological sample comprising
detecting or measuring the formation of a complex between a
labelled compound and a HDAC.
[0136] The detecting or identifying methods can use compounds that
are labelled with labelling agents such as radioisotopes, enzymes,
fluorescent substances, luminous substances, etc. Examples of the
radioisotopes include .sup.125I, .sup.131I, .sup.3H and .sup.14C.
Enzymes are usually made detectable by conjugation of an
appropriate substrate which, in turn catalyses a detectable
reaction. Examples thereof include, for example,
beta-galactosidase, beta-glucosidase, alkaline phosphatase,
peroxidase and malate dehydrogenase, preferably horseradish
peroxidase. The luminous substances include, for example, luminol,
luminol derivatives, luciferin, aequorin and luciferase.
[0137] Biological samples can be defined as body tissue or body
fluids. Examples of body fluids are cerebrospinal fluid, blood,
plasma, serum, urine, sputum, saliva and the like.
[0138] In view of their useful pharmacological properties, the
subject compounds may be formulated into various pharmaceutical
forms for administration purposes.
[0139] To prepare the pharmaceutical compositions of this
invention, an effective amount of a particular compound, in base or
acid addition salt form, as the active ingredient is combined in
intimate admixture with a pharmaceutically acceptable carrier,
which carrier may take a wide variety of forms depending on the
form of preparation desired for administration. These
pharmaceutical compositions are desirably in unitary dosage form
suitable, preferably, for administration orally, rectally,
percutaneously, or by parenteral injection. For example, in
preparing the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed, such as, for example, water,
glycols, oils, alcohols and the like in the case of oral liquid
preparations such as suspensions, syrups, elixirs and solutions; or
solid carriers such as starches, sugars, kaolin, lubricants,
binders, disintegrating agents and the like in the case of powders,
pills, capsules and tablets.
[0140] Because of their ease in administration, tablets and
capsules represent the most advantageous oral dosage unit form, in
which case solid pharmaceutical carriers are obviously employed.
For parenteral compositions, the carrier will usually comprise
sterile water, at least in large part, though other ingredients, to
aid solubility for example, may be included. Injectable solutions,
for example, may be prepared in which the carrier comprises saline
solution, glucose solution or a mixture of saline and glucose
solution. Injectable suspensions may also be prepared in which case
appropriate liquid carriers, suspending agents and the like may be
employed. In the compositions suitable for percutaneous
administration, the carrier optionally comprises a penetration
enhancing agent and/or a suitable wetting agent, optionally
combined with suitable additives of any nature in minor
proportions, which additives do not cause a significant deleterious
effect to the skin. Said additives may facilitate the
administration to the skin and/or may be helpful for preparing the
desired compositions. These compositions may be administered in
various ways, e.g., as a transdermal patch, as a spot-on or as an
ointment.
[0141] It is especially advantageous to formulate the
aforementioned pharmaceutical compositions in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form
as used in the specification and claims herein refers to physically
discrete units suitable as unitary dosages, each unit containing a
predetermined quantity of active ingredient, calculated to produce
the desired therapeutic effect, in association with the required
pharmaceutical carrier. Examples of such dosage unit forms are
tablets (including scored or coated tablets), capsules, pills,
powder packets, wafers, injectable solutions or suspensions,
teaspoonfuls, tablespoonfuls and the like, and segregated multiples
thereof.
[0142] Those skilled in the art could easily determine the
effective amount from the test results presented hereinafter. In
general it is contemplated that a therapeutically effective amount
would be from 0.005 mg/kg to 100 mg/kg body weight, and in
particular from 0.005 mg/kg to 10 mg/kg body weight. It may be
appropriate to administer the required dose as two, three, four or
more sub-doses at appropriate intervals throughout the day. Said
sub-doses may be formulated as unit dosage forms, for example,
containing 0.5 to 500 mg, and in particular 10 mg to 500 mg of
active ingredient per unit dosage form.
[0143] As another aspect of the present invention a combination of
a HDAC-inhibitor with another anticancer agent is envisaged,
especially for use as a medicine, more specifically in the
treatment of cancer or related diseases.
[0144] For the treatment of the above conditions, the compounds of
the invention may be advantageously employed in combination with
one or more other medicinal agents, more particularly, with other
anti-cancer agents. Examples of anti-cancer agents are: [0145]
platinum coordination compounds for example cisplatin, carboplatin
or oxalyplatin; [0146] taxane compounds for example paclitaxel or
docetaxel; [0147] topoisomerase I inhibitors such as camptothecin
compounds for example irinotecan or topotecan; [0148] topoisomerase
II inhibitors such as anti-tumour podophyllotoxin derivatives for
example etoposide or teniposide; [0149] anti-tumour vinca alkaloids
for example vinblastine, vincristine or vinorelbine; [0150]
anti-tumour nucleoside derivatives for example 5-fluorouracil,
gemcitabine or capecitabine; [0151] alkylating agents such as
nitrogen mustard or nitrosourea for example cyclophosphamide,
chlorambucil, carmustine or lomustine; [0152] anti-tumour
anthracycline derivatives for example daunorubicin, doxorubicin,
idarubicin or mitoxantrone; [0153] HER2 antibodies for example
trastuzumab; [0154] estrogen receptor antagonists or selective
estrogen receptor modulators for example tamoxifen, toremifene,
droloxifene, faslodex or raloxifene; [0155] aromatase inhibitors
such as exemestane, anastrozole, letrazole and vorozole; [0156]
differentiating agents such as retinoids, vitamin D and retinoic
acid metabolism blocking agents (RAMBA) for example accutane;
[0157] DNA methyl transferase inhibitors for example azacytidine;
[0158] kinase inhibitors for example flavoperidol, imatinib
mesylate or gefitinib; [0159] farnesyltransferase inhibitors;
[0160] other HDAC inhibitors; [0161] inhibitors of the
ubiquitin-proteasome pathway for example Velcade; or [0162]
Yondelis.
[0163] The term "platinum coordination compound" is used herein to
denote any tumour cell growth inhibiting platinum coordination
compound which provides platinum in the form of an ion.
[0164] The term "taxane compounds" indicates a class of compounds
having the taxane ring system and related to or derived from
extracts from certain species of yew (Taxus) trees.
[0165] The term "topisomerase inhibitors" is used to indicate
enzymes that are capable of altering DNA topology in eukaryotic
cells. They are critical for important cellular functions and cell
proliferation. There are two classes of topoisomerases in
eukaryotic cells, namely type I and type II. Topoisomerase I is a
monomeric enzyme of approximately 100,000 molecular weight. The
enzyme binds to DNA and introduces a transient single-strand break,
unwinds the double helix (or allows it to unwind) and subsequently
reseals the break before dissociating from the DNA strand.
Topisomerase II has a similar mechanism of action which involves
the induction of DNA strand breaks or the formation of free
radicals.
[0166] The term "camptothecin compounds" is used to indicate
compounds that are related to or derived from the parent
camptothecin compound which is a water-insoluble alkaloid derived
from the Chinese tree Camptothecin acuminata and the Indian tree
Nothapodytes foetida.
[0167] The term "podophyllotoxin compounds" is used to indicate
compounds that are related to or derived from the parent
podophyllotoxin, which is extracted from the mandrake plant.
[0168] The term "anti-tumour vinca alkaloids" is used to indicate
compounds that are related to or derived from extracts of the
periwinkle plant (Vinca rosea).
[0169] The term "alkylating agents" encompass a diverse group of
chemicals that have the common feature that they have the capacity
to contribute, under physiological conditions, alkyl groups to
biologically vital macromolecules such as DNA. With most active
alkylating moieties are generated in vivo after complex degradative
reactions, some of which are enzymatic. The most important
pharmacological actions of the alkylating agents are those that
disturb the fundamental mechanisms concerned with cell
proliferation in particular DNA synthesis and cell division. The
capacity of alkylating agents to interfere with DNA function and
integrity in rapidly proliferating tissues provides the basis for
their therapeutic applications and for many of their toxic
properties.
[0170] The term "anti-tumour anthracycline derivatives" comprise
antibiotics obtained from the fungus Strep. peuticus var. caesius
and their derivatives, characterised by having a tetracycline ring
structure with an unusual sugar, daunosamine, attached by a
glycosidic linkage.
[0171] Amplification of the human epidermal growth factor receptor
2 protein (HER 2) in primary breast carcinomas has been shown to
correlate with a poor clinical prognosis for certain patients.
Trastuzumab is a highly purified recombinant DNA-derived humanized
monoclonal IgG1 kappa antibody that binds with high affiniity and
specificity to the extracellular domain of the HER2 receptor.
[0172] Many breast cancers have estrogen receptors and growth of
these tumours can be stimulated by estrogen. The terms "estrogen
receptor antagonists" and "selective estrogen receptor modulators"
are used to indicate competitive inhibitors of estradiol binding to
the estrogen receptor (ER). Selective estrogen receptor modulators,
when bound to the ER, induces a change in the three-dimensional
shape of the receptor, modulating its binding to the estrogen
responsive element (ERE) on DNA.
[0173] In postmenopausal women, the principal source of circulating
estrogen is from conversion of adrenal and ovarian androgens
(androstenedione and testosterone) to estrogens (estrone and
estradiol) by the aromatase enzyme in peripheral tissues. Estrogen
deprivation through aromatase inhibition or inactivation is an
effective and selective treatment for some postmenopausal patients
with hormone-dependent breast cancer.
[0174] The term "antiestrogen agent" is used herein to include not
only estrogen receptor antagonists and selective estrogen receptor
modulators but also aromatase inhibitors as discussed above.
[0175] The term "differentiating agents" encompass compounds that
can, in various ways, inhibit cell proliferation and induce
differentiation. Vitamin D and retinoids are known to play a major
role in regulating growth and differentiation of a wide variety of
normal and malignant cell types. Retinoic acid metabolism blocking
agents (RAMBA's) increase the levels of endogenous retinoic acids
by inhibiting the cytochrome P450-mediated catabolism of retinoic
acids.
[0176] DNA methylation changes are among the most common
abnormalities in human neoplasia. Hypermethylation within the
promotors of selected genes is usually associated with inactivation
of the involved genes. The term "DNA methyl transferase inhibitors"
is used to indicate compounds that act through pharmacological
inhibition of DNA methyl transferase and reactivation of tumour
suppressor gene expression.
[0177] The term "kinase inhibitors" comprises potent inhibitors of
kinases that are involved in cell cycle progression and programmed
cell death (apoptosis).
[0178] The term "farnesyltransferase inhibitors" is used to
indicate compounds that were designed to prevent farnesylation of
Ras and other intracellular proteins. They have been shown to have
effect on malignant cell proliferation and survival.
[0179] The term "other HDAC inhibitors" comprises but is not
limited to: [0180] carboxylates for example butyrate, cinnamic
acid, 4-phenylbutyrate or valproic acid; [0181] hydroxamic acids
for example suberoylanilide hydroxamic acid (SAHA), piperazine
containing SAHA analogues, biaryl hydroxamate A-161906 and its
carbozolylether-, tetrahydropyridine- and tetralone-analogues,
bicyclic aryl-N-hydroxycarboxamides, pyroxamide, CG-1521, PXD-101,
sulfonamide hydroxamic acid, LAQ-824, LBH-589, trichostatin A
(TSA), oxamflatin, scriptaid, scriptaid related tricyclic
molecules, m-carboxy cinnamic acid bishydroxamic acid (CBHA),
CBHA-like hydroxamic acids, trapoxin-hydroxamic acid analogue,
R306465 and related benzoyl- and heteroaryl-hydroxamic acids,
aminosuberates and malonyldiamides; [0182] cyclic tetrapeptides for
example trapoxin, apidicin, depsipeptide, spiruchostatin-related
compounds, RedFK-228, sulfhydryl-containing cyclic tetrapeptides
(SCOPs), hydroxamic acid containing cyclic tetrapeptides (CHAPs),
TAN-174s and azumamides; [0183] benzamides for example MS-275 or
CI-994, or [0184] depudecin.
[0185] The term "inhibitors of the ubiquitin-proteasome pathway" is
used to indentify compounds that inhibit the targeted destruction
of cellular proteins in the proteasome, including cell cycle
regulatory proteins.
[0186] For the treatment of cancer the compounds according to the
present invention may be administered to a patient as described
above, in conjunction with irradiation. Irradiation means ionising
radiation and in particular gamma radiation, especially that
emitted by linear accelerators or by radionuclides that are in
common use today. The irradiation of the tumour by radionuclides
can be external or internal.
[0187] The present invention also relates to a combination
according to the invention of an anti-cancer agent and a HDAC
inhibitor according to the invention.
[0188] The present invention also relates to a combination
according to the invention for use in medical therapy for example
for inhibiting the growth of tumour cells.
[0189] The present invention also relates to a combinations
according to the invention for inhibiting the growth of tumour
cells.
[0190] The present invention also relates to a method of inhibiting
the growth of tumour cells in a human subject which comprises
administering to the subject an effective amount of a combination
according to the invention.
[0191] This invention further provides a method for inhibiting the
abnormal growth of cells, including transformed cells, by
administering an effective amount of a combination according to the
invention.
[0192] The other medicinal agent and HDAC inhibitor may be
administered simultaneously (e.g. in separate or unitary
compositions) or sequentially in either order. In the latter case,
the two compounds will be administered within a period and in an
amount and manner that is sufficient to ensure that an advantageous
or synergistic effect is achieved. It will be appreciated that the
preferred method and order of administration and the respective
dosage amounts and regimes for each component of the combination
will depend on the particular other medicinal agent and HDAC
inhibitor being administered, their route of administration, the
particular tumour being treated and the particular host being
treated. The optimum method and order of administration and the
dosage amounts and regime can be readily determined by those
skilled in the art using conventional methods and in view of the
information set out herein.
[0193] The platinum coordination compound is advantageously
administered in a dosage of 1 to 500 mg per square meter
(mg/m.sup.2) of body surface area, for example 50 to 400
mg/m.sup.2, particularly for cisplatin in a dosage of about 75
mg/m.sup.2 and for carboplatin in about 300 mg/m.sup.2 per course
of treatment.
[0194] The taxane compound is advantageously administered in a
dosage of 50 to 400 mg per square meter (mg/m.sup.2) of body
surface area, for example 75 to 250 mg/m.sup.2, particularly for
paclitaxel in a dosage of about 175 to 250 mg/m.sup.2 and for
docetaxel in about 75 to 150 mg/m.sup.2 per course of
treatment.
[0195] The camptothecin compound is advantageously administered in
a dosage of 0.1 to 400 mg per square meter (mg/m.sup.2) of body
surface area, for example 1 to 300 mg/m.sup.2, particularly for
irinotecan in a dosage of about 100 to 350 mg/m.sup.2 and for
topotecan in about 1 to 2 mg/m.sup.2 per course of treatment.
[0196] The anti-tumour podophyllotoxin derivative is advantageously
administered in a dosage of 30 to 300 mg per square meter
(mg/m.sup.2) of body surface area, for example 50 to 250
mg/m.sup.2, particularly for etoposide in a dosage of about 35 to
100 mg/m.sup.2 and for teniposide in about 50 to 250 mg/m.sup.2 per
course of treatment.
[0197] The anti-tumour vinca alkaloid is advantageously
administered in a dosage of 2 to 30 mg per square meter
(mg/m.sup.2) of body surface area, particularly for vinblastine in
a dosage of about 3 to 12 mg/m.sup.2, for vincristine in a dosage
of about 1 to 2 mg/m.sup.2, and for vinorelbine in dosage of about
10 to 30 mg/m.sup.2 per course of treatment.
[0198] The anti-tumour nucleoside derivative is advantageously
administered in a dosage of 200 to 2500 mg per square meter
(mg/m.sup.2) of body surface area, for example 700 to 1500
mg/m.sup.2, particularly for 5-FU in a dosage of 200 to 500
mg/m.sup.2, for gemcitabine in a dosage of about 800 to 1200
mg/m.sup.2 and for capecitabine in about 1000 to 2500 mg/m.sup.2
per course of treatment.
[0199] The alkylating agents such as nitrogen mustard or
nitrosourea is advantageously administered in a dosage of 100 to
500 mg per square meter (mg/m.sup.2) of body surface area, for
example 120 to 200 mg/m.sup.2, particularly for cyclophosphamide in
a dosage of about 100 to 500 mg/m.sup.2, for chlorambucil in a
dosage of about 0.1 to 0.2 mg/kg, for carmustine in a dosage of
about 150 to 200 mg/m.sup.2, and for lomustine in a dosage of about
100 to 150 mg/m.sup.2 per course of treatment.
[0200] The anti-tumour anthracycline derivative is advantageously
administered in a dosage of 10 to 75 mg per square meter
(mg/m.sup.2) of body surface area, for example 15 to 60 mg/m.sup.2,
particularly for doxorubicin in a dosage of about 40 to 75
mg/m.sup.2, for daunorubicin in a dosage of about 25 to 45
mg/m.sup.2, and for idarubicin in a dosage of about 10 to 15
mg/m.sup.2 per course of treatment.
[0201] Trastuzumab is advantageously administered in a dosage of 1
to 5 mg per square meter (mg/m.sup.2) of body surface area,
particularly 2 to 4 mg/m.sup.2 per course of treatment.
[0202] The antiestrogen agent is advantageously administered in a
dosage of about 1 to 100 mg daily depending on the particular agent
and the condition being treated. Tamoxifen is advantageously
administered orally in a dosage of 5 to 50 mg, preferably 10 to 20
mg twice a day, continuing the therapy for sufficient time to
achieve and maintain a therapeutic effect. Toremifene is
advantageously administered orally in a dosage of about 60 mg once
a day, continuing the therapy for sufficient time to achieve and
maintain a therapeutic effect. Anastrozole is advantageously
administered orally in a dosage of about 1 mg once a day.
Droloxifene is advantageously administered orally in a dosage of
about 20-100 mg once a day. Raloxifene is advantageously
administered orally in a dosage of about 60 mg once a day.
Exemestane is advantageously administered orally in a dosage of
about 25 mg once a day.
[0203] These dosages may be administered for example once, twice or
more per course of treatment, which may be repeated for example
every 7, 14, 21 or 28 days.
[0204] In view of their useful pharmacological properties, the
components of the combinations according to the invention, i.e. the
other medicinal agent and the HDAC inhibitor may be formulated into
various pharmaceutical forms for administration purposes. The
components may be formulated separately in individual
pharmaceutical compositions or in a unitary pharmaceutical
composition containing both components.
[0205] The present invention therefore also relates to a
pharmaceutical composition comprising the other medicinal agent and
the HDAC inhibitor together with one or more pharmaceutical
carriers.
[0206] The present invention also relates to a combination
according to the invention in the form of a pharmaceutical
composition comprising an anti-cancer agent and a HDAC inhibitor
according to the invention together with one or more pharmaceutical
carriers.
[0207] The present invention further relates to the use of a
combination according to the invention in the manufacture of a
pharmaceutical composition for inhibiting the growth of tumour
cells.
[0208] The present invention further relates to a product
containing as first active ingredient a HDAC inhibitor according to
the invention and as second active ingredient an anticancer agent,
as a combined preparation for simultaneous, separate or sequential
use in the treatment of patients suffering from cancer.
EXPERIMENTAL PART
[0209] The following examples are provided for purposes of
illustration.
[0210] Hereinafter, "DCM" is defined as dichloromethane, "DIPE" is
defined as diisopropyl ether, "DMA" is defined as is defined as
N,N-dimethylacetamide, "DMSO" is defined as dimethylsulfoxide,
"EDC" is defined as
N'-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,
monohydrochloride, "EtOAc" is defined as ethyl acetate, "EtOH" is
defined as ethanol, "HOBt" is defined as
1-hydroxy-1H-benzotriazole, "MeOH" is defined as methanol, "TFA" is
defined as trifluoroacetic acid and "THF" is defined as
tetrahydrofuran.
A. Preparation of the Intermediate Compounds
Example A1
a) Preparation of Intermediate 1
##STR00017##
[0212] A mixture of
2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylic acid,
ethyl ester (0.0114 mol), 1-methyl-1H-indole-3-carboxaldehyde
(0.017 mol) and MgSO.sub.4 (0.5 g) in MeOH (80 ml) was stirred and
refluxed for 15 hours, then cooled to room temperature. Sodium
tetrahydroborate (0.018 mol) was added portionwise. The mixture was
stirred at room temperature 5 hours, poured out into water and
extracted with EtOAc. The organic layer was separated, dried
(MgSO.sub.4), filtered, and the solvent was evaporated. The residue
(6.6 g) was purified by column chromatography over silica gel
(15-40 .mu.m) (eluent: DCM/MeOH/NH.sub.4OH 94/6/0.5). The pure
fractions were collected and the solvent was evaporated, yielding
4.3 g (90%) of intermediate 1.
b) Preparation of Intermediate 2
##STR00018##
[0214] A mixture of intermediate 1 (0.0037 mol) and sodium
hydroxide (0.0074 mol) in EtOH (60 ml) was stirred at 50.degree. C.
for 15 hours, then cooled to room temperature and the solvent was
evaporated till dryness, yielding 1.5 g (100%) of intermediate
2.
c) Preparation of Intermediate 3
##STR00019##
[0216] EDC (0.0075 mol), then HOBt (0.0075 mol) then
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.015 mol) were added
at room temperature to a mixture of intermediate 2 (0.005 mol) in
DCM (100 ml) and THF (100 ml) under N.sub.2 flow. The mixture was
stirred at 40.degree. C. for 4 hours, poured out into ice water and
extracted with DCM. The organic layer was separated, dried
(MgSO.sub.4), filtered, and the solvent was evaporated. The residue
(4 g) was purified by column chromatography over silica gel (15-40
.mu.m) (eluent: DCM/MeOH/NH.sub.4OH 96/4/0.5). The pure fractions
were collected and the solvent was evaporated, yielding 1 g (42%)
of intermediate 3. A fraction (0.051 g) was crystallized from DIPE.
The precipitate was filtered off and dried, yielding 0.03 g of
intermediate 3, melting point 70.degree. C.
Example A2
a) Preparation of Intermediate 4
##STR00020##
[0218] A mixture of
2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylic acid,
ethyl ester (0.0072 mol) in THF (40 ml) and sodium hydroxide 1N (40
ml) was stirred at room temperature overnight. Hydrochloric acid 1N
(40 ml) was added. The mixture was stirred for 10 minutes. Sodium
carbonate (0.0216 mol) was added. The mixture was stirred for 10
minutes.
1-[[(9H-fluoren-9-ylmethoxy)carbonyl]oxy]-2,5-pyrrolidinedione
(0.0072 mol) was added portionwise. The mixture was stirred at room
temperature for 6 hours, then cooled to 0.degree. C. and acidified
with hydrochloric acid. The precipitate was filtered, washed with
diethyl ether and dried, yielding 4.1 g (100%) of intermediate
4.
b) Preparation of Intermediate 5
##STR00021##
[0220] Triethylamine (0.02 mol), EDC (0.0082 mol) and HOBt (0.0082
mol) were added at room temperature to a mixture of intermediate 4
(0.0068 mol) in DCM/THF (200 ml) under N.sub.2 flow. The mixture
was stirred at room temperature for 15 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0082 mol) was added.
The mixture was stirred at room temperature for 48 hours, poured
out into water and extracted with DCM. The organic layer was washed
with NaHCO.sub.3 10%, dried (MgSO.sub.4), filtered, and the solvent
was evaporated. The residue (4 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 98/2/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 3.4 g (89%) of
intermediate 5.
c) Preparation of Intermediate 6
##STR00022##
[0222] A mixture of intermediate 5 (0.0355 mol) and piperidine
(0.089 mol) in DCM (400 ml) was stirred at 35.degree. C. during 72
hours. The solvent was evaporated. The residue was purified by
column chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 80/20/2). The pure fractions were collected and
the solvent was evaporated, yielding 6.7 g (56%). Part of the
residue (0.79 g) was crystallized from diethyl ether. The
precipitate was filtered off and dried, yielding 0.62 g of
intermediate 6, melting point 129.degree. C.
d) Preparation of Intermediate 7
##STR00023##
[0224] A mixture of intermediate 6 (0.0009 mol) and
5-chloro-1H-indole-3-carboxaldehyde (0.0012 mol) in
1,2-dichloro-ethane (30 ml) was stirred at room temperature
overnight. Tris(acetato-.alpha.-O)hydro-borate(1-), sodium (0.0013
mol) was added portionwise. The mixture was stirred at room
temperature for 4 hours, poured out into water/NaOH 3N and
extracted with DCM. The organic layer was separated, dried
(MgSO.sub.4), filtered, and the solvent was evaporated. The residue
(0.5 g) was purified by column chromatography over silica gel
(15-40 .mu.m) (eluent DCM/MeOH/NH.sub.4OH 93/7/0.5). Two fractions
were collected and the solvent was evaporated, yielding 0.07 g
(16%) of intermediate 7.
Example A3
a) Preparation of Intermediate 8
##STR00024##
[0226] A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic
acid, ethyl ester (0.094 mol) in acetonitrile (40 ml) was added at
10.degree. C. to a suspension of 4-piperidinemethanol (0.086 mol)
and potassium carbonate (0.172 mol) in acetonitrile (200 ml) under
N.sub.2 flow. The mixture was brought to room temperature, then
stirred for 4 hours, poured out into water and extracted with
EtOAc. The organic layer was separated, dried (MgSO.sub.4),
filtered, and the solvent was evaporated. The residue (23 g) was
crystallized from acetonitrile/diethyl ether. The precipitate was
filtered off and dried, yielding 7.8 g (34%) of intermediate 8. The
mother layer was evaporated. The residue (17 g) was purified by
column chromatography over silica gel (20-45 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 97/3/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 4.6 g (20%) of
intermediate 8, melting point 129.degree. C.
b) Preparation of Intermediate 9
##STR00025##
[0228] Triethylamine (0.038 mol) then methanesulfonyl chloride
(0.025 mol) were added at 0.degree. C. to a solution of
intermediate 8 (0.0189 mol) in DCM (80 ml) under N.sub.2 flow. The
mixture was stirred at 0.degree. C. for 2 hours and poured out into
ice water. The organic layer was separated, dried (MgSO.sub.4),
filtered, and the solvent was evaporated, yielding 6.5 g (100%) of
intermediate 9.
c) Preparation of Intermediate 10
##STR00026##
[0230] A mixture of intermediate 9 (0.0189 mol),
N-methyl-1H-indole-3-ethanamine (0.0172 mol) and potassium
carbonate (0.0344 mol) in acetonitrile (180 ml) was stirred and
refluxed for 24 hours, poured out into water and extracted with
EtOAc. The organic layer was separated, dried (MgSO.sub.4),
filtered, and the solvent was evaporated. The residue (8.5 g) was
purified by column chromatography over silica gel (70-200 .mu.m)
(eluent: DCM/MeOH/NH.sub.4OH 98/2/0 to 97/3/0.1). The pure
fractions were collected and the solvent was evaporated, yielding
1.25 g (20%) of intermediate 10.
d) Preparation of Intermediate 11
##STR00027##
[0232] A mixture of intermediate 10 (0.003 mol) and sodium
hydroxide (0.006 mol) in EtOH (80 ml) was stirred and refluxed
overnight, then cooled to room temperature and the solvent was
evaporated till dryness, yielding 1.3 g (100%) of intermediate 11,
mp. >260.degree. C.
e) Preparation of Intermediate 12
##STR00028##
[0234] EDC (0.0045 mol) then HOBt (0.0045 mol) were added at room
temperature to a mixture of intermediate 11 (0.003 mol) in THF (100
ml) and DCM (100 ml) under N.sub.2 flow. The mixture was stirred at
room temperature for 15 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.012 mol) was added.
The mixture was stirred at room temperature for 72 hours, poured
out into water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered, and the solvent was
evaporated. The residue (3 g) was purified by column chromatography
over silica gel (15-40 .mu.m)(eluent: DCM/MeOH/NH.sub.4OH
94/6/0.1). The pure fractions were collected and the solvent was
evaporated. The residue (0.82 g) was taken up in diethyl ether. The
precipitate was filtered off and dried, yielding 0.78 g of
intermediate 12, melting point 154.degree. C.
Example A4
a) Preparation of Intermediate 13
##STR00029##
[0236] A mixture of
2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylic acid,
ethyl ester (0.0049 mol), 1H-indole-3-ethanol, methanesulfonate
(ester) (0.0054 mol) and potassium carbonate (0.01 mol) in
acetonitrile (20 ml) was stirred and refluxed overnight, then
cooled, poured out into ice water and extracted with DCM. The
organic layer was separated, dried (MgSO.sub.4), filtered, and the
solvent was evaporated. The residue (2.2 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 96/4/0.2). The pure fractions were collected
and the solvent was evaporated, yielding 0.442 g (22%) of
intermediate 13, melting point 238.degree. C.
b) Preparation of Intermediate 14
##STR00030##
[0238] A mixture of intermediate 13 (0.0025 mol),
(2-bromoethoxy)(1,1-dimethylethyl)dimethyl-silane (0.0034 mol) and
N-ethyl-N-(1-methylethyl)-2-propanamine (0.0038 mol) in DMSO (20
ml) was stirred at 50.degree. C. for 15 hours, then cooled to room
temperature, poured out into ice water and extracted with DCM. The
organic layer was separated, dried (MgSO.sub.4), filtered, and the
solvent was evaporated. The residue (1.7 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 98/2/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 0.76 g (54%) of
intermediate 14.
c) Preparation of Intermediate 15
##STR00031##
[0240] A mixture of intermediate 14 (0.0013 mol) and sodium
hydroxide (0.0027 mol) in EtOH (40 ml) was stirred at 80.degree. C.
overnight, then cooled to room temperature and the solvent was
evaporated, yielding 0.752 (100%) of intermediate 15.
d) Preparation of Intermediate 16
##STR00032##
[0242] EDC (0.002 mol) then HOBt (0.002 mol) were added at room
temperature to a mixture of intermediate 15 (0.0013 mol) in THF (80
ml) and DCM (80 ml) under N.sub.2 flow. The mixture was stirred at
room temperature for 15 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0068 mol) was added.
The mixture was stirred at room temperature for 72 hours, poured
out into water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered, and the solvent was
evaporated. The residue (1.3 g) was purified by column
chromatography over silica gel (15-40 .mu.m)(eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 0.38 g of intermediate
16.
e) Preparation of Intermediate 17
##STR00033##
[0244] A mixture of intermediate 16 (0.0011 mol) and
tetrabutylammonium fluoride (0.0032 mol) in THF (10 ml) was stirred
at room temperature for 72 hours, poured out into water and
extracted with EtOAc. The organic layer was washed with water,
dried (MgSO.sub.4), filtered and the solvent was evaporated,
yielding 0.5 g (88%) of intermediate 17.
Example A5
a) Preparation of Intermediate 45
##STR00034##
[0246] A solution of 2-chloro-5-pyrimidinecarboxylic acid, methyl
ester (0.058 mol) in DMA (80 ml) was added dropwise to a solution
of 4-piperidinemethanamine (0.116 mol) and N-ethyldiisopropylamine
(0.145 mol) in DMA (150 ml) under N.sub.2 flow. The mixture was
stirred at room temperature for 1 hour and 30 minutes, poured out
into ice water and extracted with EtOAc, then with DCM. The organic
layer was washed with water, dried (MgSO.sub.4), filtered and the
solvent was evaporated. The residue was crystallized from DIPE. The
precipitate was filtered off and dried, yielding 10 g (65%) of
intermediate 45.
b) Preparation of Intermediate 46
##STR00035##
[0248] A mixture of intermediate 45 (0.0024 mol),
1-methyl-5-nitro-1H-indole-3-carboxaldehyde (0.0036 mol) and
MgSO.sub.4 (0.25 g) in MeOH (80 ml) was stirred at 60.degree. C.
overnight, then cooled to room temperature. Sodium tetrahydroborate
(0.0041 mol) was added portionwise. The mixture was stirred at room
temperature for 18 hours, poured out into water and extracted with
EtOAc. The organic layer was separated, dried (MgSO.sub.4),
filtered and the solvent was evaporated. The residue (1.1 g) was
crystallized from acetonitrile. The precipitate was filtered off
and dried, yielding 0.9 g (86%) of intermediate 46, melting
point:150.degree. C.
c) Preparation of Intermediate 47
##STR00036##
[0250] A mixture of intermediate 46 (0.002 mol) and sodium
hydroxide (0.008 mol) in EtOH (60 ml) was stirred at 60.degree. C.
overnight, then cooled to room temperature and evaporated. The
residue was taken up in diethyl ether. The precipitate was filtered
off and dried, yielding 0.6 g (67%) of intermediate 47.
d) Preparation of Intermediate 48
##STR00037##
[0252] EDC (0.0019 mol) and HOBt (0.0019 mol) were added at room
temperature to a solution of intermediate 47 (0.0013 mol) and
triethylamine (0.0039 mol) in DCM/THF (50/50) (100 ml) under
N.sub.2 flow. The mixture was stirred at room temperature for 15
minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0026 mol)
was added. The mixture was stirred at room temperature for 72
hours, poured out into water and extracted with DCM. The organic
layer was separated, dried (MgSO.sub.4), filtered and the solvent
was evaporated. The residue (1 g) was purified by column
chromatography over kromasil (5 .mu.m) (eluent: DCM/MeOH/NH.sub.4OH
98/2/0.2 to 92/8/0.2). The pure fractions were collected and the
solvent was evaporated, yielding 0.101 g (15%) of intermediate
48.
Example A6
a) Preparation of Intermediate 49
##STR00038##
[0254] A solution of 2-chloro-5-pyrimidinecarboxylic acid, methyl
ester (0.033 mol) in DCM (80 ml) was added at room temperature to a
solution of 4-piperidinemethanol (0.066 mol) and
N-ethyldiisopropylamine (0.083 mol) in DCM (100 ml) under N.sub.2
flow. The mixture was stirred at room temperature for 3 hours and
30 minutes, poured out into ice water and extracted with DCM. The
organic layer was separated, dried (MgSO.sub.4), filtered and the
solvent was evaporated. The residue was taken up in pentane. The
precipitate was filtered off and dried, yielding 7.88 g (95%1 of
intermediate 49.
b) Preparation of Intermediate 50
##STR00039##
[0256] DMSO (0.058 mol) was added dropwise at -78.degree. C. to a
solution of ethanedioyl dichloride (0.0278 mol) in DCM (50 ml)
under N.sub.2 flow. The mixture was stirred for 15 minutes. A
solution of intermediate 49 (0.023 mol) in DCM (200 ml) was added
dropwise. The mixture was stirred at -78.degree. C. for 1 hour and
30 minutes. Triethylamine (0.118 mol) was added dropwise. The
mixture was stirred at -78.degree. C. for 1 hour, poured out into
water and extracted with DCM. The organic layer was separated,
dried (MgSO.sub.4), filtered and the solvent was evaporated. The
residue was crystallized from diethyl ether. The precipitate was
filtered off and dried, yielding 3.06 g (54%) of intermediate
50.
c) Preparation of Intermediate 51
##STR00040##
[0258] Intermediate 50 (0.0122 mol) was added at 5.degree. C. to a
solution of 1-methyl-1H-indole-3-ethanamine (0.0122 mol) in MeOH
(270 ml) under N.sub.2 flow. The mixture was stirred a few minutes.
Sodiumcyanoborohydride (0.0183 mol) and acetic acid (0.0183 mol)
were added. The mixture was stirred at room temperature for 48
hours, poured out into potassium carbonate 10% and extracted with
DCM. The organic layer was separated, dried (MgSO.sub.4), filtered
and the solvent was evaporated. The residue (4.9 g) was purified by
column chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 97/3/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 1.2 g (24%) of
intermediate 51.
d) Preparation of Intermediate 52
##STR00041##
[0260] A mixture of intermediate 51 (0.0009 mol) and sodium
hydroxide (0.0039 mol) in EtOH (60 ml) was stirred and refluxed for
15 hours, then evaporated till dryness, yielding intermediate 52.
This intermediate was used directly in the next reaction step.
e) Preparation of Intermediate 53
##STR00042##
[0262] HOBt (0.0019 mol) then EDC (0.0019 mol) were added at room
temperature to a solution of intermediate 52 (0.0009 mol) and
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0019 mol) in DCM/THF
(130 ml). The mixture was stirred at room temperature for 48 hours,
poured out into water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered and the solvent was
evaporated. The residue (0.93 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 97/3/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 0.155 g (33%) of
intermediate 53.
Example A7
a) Preparation of Intermediate 54
##STR00043##
[0264] A mixture of
2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylic acid,
ethyl ester (0.0038 mol), 1-ethyl-1H-indole-3-carboxaldehyde
(0.0049 mol) and Pd/C 10% (0.5 g) in MeOH (20 ml) containing 1 ml
of a 10% thiopene solution in EtOH, was hydrogenated at room
temperature for 24 hours under a 3 bar pressure, then filtered over
celite. The solvent was evaporated till dryness. The residue (1.8
g) was purified by column chromatography over silica gel (15-40
.mu.m) (eluent: DCM/MeOH/NH.sub.4OH 95/5/0.2 to 93/7/0.5). The pure
fractions were collected and the solvent was evaporated, yielding
0.7 g (44%) of intermediate 54.
b) Preparation of Intermediate 55
##STR00044##
[0266] Sodium hydride 60% (0.009 mol) was added at 0.degree. C. to
a solution of intermediate 54 (0.0045 mol) in THF (50 ml) under
N.sub.2 flow. The mixture was stirred at room temperature for 1
hour. A solution of iodo-ethane (0.0062 mol) in THF (10 ml) was
added dropwise. The mixture was stirred at room temperature
overnight, poured out into water and extracted with EtOAc. The
organic layer was separated, dried (MgSO.sub.4), filtered, and the
solvent was evaporated. The residue (0.6 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 0.16 g (8%) of
intermediate 55.
c) Preparation of Intermediate 56
##STR00045##
[0268] A mixture of intermediate 55 (0.0003 mol) and sodium
hydroxide (0.03 g) in EtOH (15 ml) was stirred at 80.degree. C. for
6 hours, then evaporated till dryness, yielding 0.16 g (100%) of
intermediate 56.
d) Preparation of Intermediate 57
##STR00046##
[0270] EDC (0.0005 mol) and HOBt (0.0005 mol) were added at room
temperature to a mixture of intermediate 56 (0.0003 mol) in DCM (20
ml) and THF (20 ml) under N.sub.2 flow. The mixture was stirred for
15 minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0007 mol)
was added. The mixture was stirred at room temperature for 72
hours, poured out into water and extracted with DCM. The organic
layer was separated, dried (MgSO.sub.4), filtered, and the solvent
was evaporated. The residue (0.3 g) was purified by column
chromatography over kromasil (5 .mu.m) (eluent: DCM/MeOH/NH.sub.4OH
93/7/0.35). The pure fractions were collected and the solvent was
evaporated, yielding 0.03 g (16%) of intermediate 57.
Example A8
a) Preparation of Intermediate 58
##STR00047##
[0272] Sodium hydride (0.011 mol) was added at 5.degree. C. to a
solution of intermediate 13 (0.0037 mol) in THF (30 ml) under
N.sub.2 flow. The mixture was stirred for 30 minutes. A solution of
iodomethane (0.0081 mol) in THF (10 ml) was added dropwise. The
mixture was stirred at 10.degree. C. for 2 hours, then brought to
room temperature for 1 hour and 30 minutes, poured out into ice
water and extracted with DCM. The organic layer was separated,
dried (MgSO.sub.4), filtered, and the solvent was evaporated. The
residue (1.7 g) was purified by column chromatography over kromasil
(15-40 .mu.m) (eluent: DCM/MeOH/NH.sub.3OH 98/2/0.1). Two fractions
were collected and the solvent was evaporated, yielding 0.265 g of
intermediate 58 and 0.57 g (17%) of intermediate 10.
b) Preparation of Intermediate 59
##STR00048##
[0274] A mixture of intermediate 58 (0.0006 mol) and sodium
hydroxide (0.0012 mol) in EtOH (30 ml) was stirred at 80.degree. C.
overnight, then cooled to room temperature and the solvent was
evaporated, yielding 0.26 g (100%) of intermediate 59.
c) Preparation of Intermediate 60
##STR00049##
[0276] EDC (0.0009 mol) and HOBt (0.0009 mol) were added at room
temperature to a solution of intermediate 59 (0.0006 mol) in THF
(30 ml) and DCM (30 ml) under N.sub.2 flow. The mixture was stirred
at room temperature for 15 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0012 mol) was added.
The mixture was stirred at room temperature for 24 hours, poured
out into water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered, and the solvent was
evaporated. The residue (0.6 g) was purified by column
chromatography over kromasil (5 am) (eluent: DCM/MeOH/NH.sub.4OH
99/1/0.05 and 94/6/0.3). The pure fractions were collected and the
solvent was evaporated, yielding 0.1 g (33%) of intermediate
60.
Example A9
a) Preparation of Intermediate 61
##STR00050##
[0278] DMSO (0.127 mol) was added at -78.degree. C. to a solution
of ethanedioyl dichloride (0.061 mol) in DCM (110 ml) under N.sub.2
flow. The mixture was stirred for 15 minutes. A solution of
intermediate 8 (0.051 mol) in DCM (200 ml) was added. The mixture
was stirred at -78.degree. C. for 1 hour and 30 minutes.
Triethylamine (0.26 mol) was added dropwise. The mixture was
stirred at -78.degree. C. for 15 minutes, then brought to room
temperature for 2 hours and 30 minutes. Water was added. The
mixture was extracted with DCM. The organic layer was separated,
dried (MgSO.sub.4), filtered, and the solvent was evaporated. The
residue (14 g) was purified by column chromatography over silica
gel (20-45 .mu.m) (eluent: cyclohexane/EtOAc 70/30). The pure
fractions were collected and the solvent was evaporated, yielding
7.6 g (57%) of intermediate 61.
b) Preparation of Intermediate 62
##STR00051##
[0280] Sodiumcyanoborohydride (0.049 mol) and acetic acid (0.034
ml) were added at room temperature to a solution of
5-chloro-1-methyl-1H-Indole-3-ethanamine (0.031 mol) and
intermediate 61 (0.034 mol) in MeOH (700 ml) under N.sub.2 flow.
The mixture was stirred at room temperature for 24 hours, poured
out into potassium carbonate 10% and extracted with DCM. The
organic layer was separated, dried (MgSO.sub.4), filtered and the
solvent was evaporated. The residue (14.8 g) was purified by column
chromatography over silica gel (20-45 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.2). The pure fractions were collected
and the solvent was evaporated, yielding 4.52 g (32%) of
intermediate 62.
c) Preparation of Intermediate 63
##STR00052##
[0282] Methanesulfonyl chloride (0.0049 mol) was added at 5.degree.
C. to a solution of intermediate 62 (0.004 mol) and triethylamine
(0.008 mol) in DCM (150 ml) under N.sub.2 flow. The mixture was
stirred at room temperature for 24 hours, poured out into ice water
and extracted with DCM. The organic layer was separated, dried
(MgSO.sub.4), filtered and the solvent was evaporated. The residue
(2.39 g) was taken up in DIPE. The precipitate was filtered off and
dried, yielding 1.78 g (84%) of intermediate 63, melting point
162.degree. C.
d) Preparation of Intermediate 64
##STR00053##
[0284] A mixture of intermediate 63 (0.0032 mol) and sodium
hydroxide (0.0128 mol) in EtOH (150 ml) was stirred and refluxed
for 5 hours, then cooled to room temperature and taken up in
diethyl ether. The precipitate was filtered off and dried, yielding
1.57 g (99%) of intermediate 64, melting point >260.degree.
C.
e) Preparation of Intermediate 65
##STR00054##
[0286] EDC (0.0064 mol) and HOBt (0.0064 mol) were added at room
temperature to a solution of intermediate 64 (0.0032 mol) in THF
(160 ml) and DCM (160 ml) under N.sub.2 flow. The mixture was
stirred at room temperature for 30 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0064 mol) was added.
The mixture was stirred at room temperature for 3 days, poured out
into water and extracted with DCM. The organic layer was separated,
dried (MgSO.sub.4), filtered and the solvent was evaporated. The
residue (2.77 g) was purified by column chromatography over silica
gel (15-40 .mu.m) (eluent: DCM/MeOH/NH.sub.4OH 97/3/0.1). The pure
fractions were collected and the solvent was evaporated. The
residue (0.385 g) was crystallized from CH.sub.3CN/diethyl ether.
The precipitate was filtered off and dried, yielding 0.084 g of
intermediate 65, melting point 179.degree. C.
Example A10
a) Preparation of Intermediate 66
##STR00055##
[0288] A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic
acid, ethyl ester (0.094 mol) in acetonitrile (240 ml) was added at
room temperature to a solution of 4-piperidinyl-carbamic acid,
1,1-dimethylethyl ester (0.078 mol) and potassium carbonate (0.156
mol) in acetonitrile (120 ml) under N.sub.2 flow. The mixture was
stirred at room temperature overnight, poured out into ice water
and extracted with EtOAc. The organic layer was washed with water,
dried (MgSO.sub.4), filtered, and the solvent was evaporated. The
residue was crystallized from diethyl ether. The precipitate was
filtered off and dried, yielding 14.4 g (53%) of intermediate 66,
melting point 160.degree. C.
b) Preparation of Intermediate 67
##STR00056##
[0290] TFA (20 ml) was added at room temperature to a solution of
intermediate 66 (0.0225 mol) in DCM (110 ml). The mixture was
stirred at room temperature for 15 hours, poured out into water and
basified with potassium carbonate. The mixture was extracted with
DCM. The organic layer was separated, dried (MgSO4), filtered, and
the solvent was evaporated, yielding 5.5 g (98%) of intermediate
67.
Example A11
a) Preparation of Intermediate 68
##STR00057##
[0292] Sodium hydride 60% in oil (0.0069 mol) was added at
0.degree. C. to a solution of 2-methyl-1H-indole-3-acetic acid,
ethyl ester (0.0046 mol) in THF (10 ml) under N.sub.2 flow. The
mixture was stirred at room temperature for 1 hour. Iodo-ethane
(0.006 mol) was added. The mixture was stirred at room temperature
for 18 hours and poured out into EtOAc and saturated NaCl. The
organic layer was separated, dried (MgSO.sub.4), filtered and the
solvent was evaporated till dryness. The residue (1.1 g) was
purified by column chromatography over silica gel (15-40 .mu.m)
(eluent: cyclohexane/EtOAc 80/20). The pure fractions were
collected and the solvent was evaporated, yielding 0.73 g (65%) of
intermediate 68.
b) Preparation of Intermediate 69
##STR00058##
[0294] A solution of diisobuytlaluminium hydride in toluene (0.0045
mol) was added dropwise at -78.degree. C. to a solution of
intermediate 68 (0.003 mol) in DCM (15 ml) and 1,2-dimethoxyethane
(15 ml) (molecular sieves: 3 angstrom) under N.sub.2 flow. The
mixture was stirred at -78.degree. C. for 3 hours, then quenched
with HC13N and extracted with DCM. The organic layer was washed
with water, dried (MgSO.sub.4), filtered and the solvent was
evaporated till dryness, yielding 0.7 g (>100%) of intermediate
69.
c) Preparation of Intermediate 70
##STR00059##
[0296] Titanium (IV) ethoxide (0.0023 mol) was added to a mixture
of intermediate 67 (0.0021 mol) and intermediate 69 (0.0021 mol) in
1,2-dichloro-ethane (25 ml). The mixture was stirred at room
temperature for 30 minutes.
Tris(acetato-.alpha.-O)hydro-borate(1-), sodium (0.0023 mol) was
added portionwise. The mixture was stirred at room temperature for
18 hours, then quenched with NaHCO.sub.3 and extracted with DCM.
The organic layer was separated, dried (MgSO.sub.4), filtered and
the solvent was evaporated till dryness. The residue (1.2 g) was
purified by column chromatography over silica gel (Sum) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.1). The pure fractions were collected
and the solvent was evaporated yielding 0.2 g (21%) of intermediate
70.
d) Preparation of Intermediate 71
##STR00060##
[0298] A mixture of intermediate 70 (0.0004 mol) and sodium
hydroxide (0.0009 mol) in EtOH (30 ml) was stirred at 60.degree. C.
overnight, then cooled to room temperature and evaporated, yielding
0.2 g (100%) of intermediate 71.
e) Preparation of Intermediate 72
##STR00061##
[0300] EDC (0.0007 mol) and HOBt (0.1 g) were added at room
temperature to a solution of intermediate 71 (0.0004 mol) and
triethylamine (0.0009 mol) in DCM/THF (40 ml) under N.sub.2 flow.
The mixture was stirred at room temperature for 15 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0009 mol) was added.
The mixture was stirred at room temperature for 72 hours, poured
out into water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered and the solvent was
evaporated. The residue (0.4 g) was purified by column
chromatography over kromasil (5 .mu.m)(eluent: DCM/MeOH/NH.sub.4OH
98/2/0.1 to 90/10/1). The pure fractions were collected and the
solvent was evaporated, yielding 0.087 g (37%) of intermediate
72.
Example A12
a) Preparation of Intermediate 73
##STR00062##
[0302] Intermediate 50 (0.0046 mol) was added at 5.degree. C. to a
solution of 6-methoxy-1-methyl-1H-Indole-3-ethanamine (0.0046 mol)
in MeOH (100 ml) under N.sub.2 flow. The mixture was stirred for 30
minutes. Sodiumcyanoborohydride (0.0068 mol) then acetic acid
(0.0046 mol) were added portionwise. The mixture was stirred at
room temperature for 48 hours, poured out into potassium carbonate
10% and extracted with DCM. The organic layer was separated, dried
(MgSO.sub.4), filtered, and the solvent was evaporated. The residue
(4 g) was purified by column chromatography over silica gel (15-40
.mu.m)) (eluent: DCM/MeOH/NH.sub.4OH 96/4/0.2). The pure fractions
were collected and the solvent was evaporated. A part (0.7 g) of
the residue (2.2 g) was crystallized from acetonitrile. The
precipitate was filtered off and dried, yielding 0.43 g (61%) of
intermediate 73, melting point 122.degree. C.
b) Preparation of Intermediate 74
##STR00063##
[0304] A mixture of intermediate 73 (0.0015 mol) and sodium
hydroxide (0.006 mol) in EtOH (90 ml) was stirred and refluxed for
8 hours, then evaporated till dryness, yielding intermediate
74.
c) Preparation of Intermediate 75
##STR00064##
[0306] HOBt (0.003 mol) then EDC (0.003 mol) were added at room
temperature to a solution of intermediate 74 (0.0015 mol) and
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.003 mol) in THF/DCM
(200 ml) under N.sub.2 flow. The mixture was stirred at room
temperature for 48 hours, poured out into water and extracted with
DCM. The organic layer was separated, dried (MgSO.sub.4), filtered,
and the solvent was evaporated. The residue (1.1 g) was purified by
column chromatography over silica gel (10 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.5). The pure fractions were collected
and the solvent was evaporated. The residue (0.24 g) was purified
by column chromatography over silica gel (10 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.5). The pure fractions were collected
and the solvent was evaporated, yielding 0.17 g (21%) of
intermediate 75.
Example A13
a) Preparation of Intermediate 76
##STR00065##
[0308] A mixture of 6-methoxy-1H-indole-3-ethanamine (0.053 mol)
and 1,3-isobenzofurandione (0.058 mol) in toluene (130 ml) was
stirred and refluxed for 48 hours, then filtered. The filtrate was
evaporated, yielding 5.4 g (32%) of intermediate 76.
b) Preparation of Intermediate 77
##STR00066##
[0310] A solution of intermediate 76 (0.017 mol) in DMF (19 ml) was
added dropwise at room temperature to a suspension of sodium
hydride (0.034 mol) in DMF (11 ml) under N.sub.2 flow. The mixture
was stirred at room temperature for 1 hour and 30 minutes.
1-iodo-propane (0.034 mol) was added. The mixture was stirred at
room temperature for 1 hour and 15 minutes. Saturated NaCl was
added. The mixture was extracted with EtOAc. The organic layer was
washed with water, dried (MgSO.sub.4), filtered, and the solvent
was evaporated, yielding 4.9 g of intermediate 77. This product was
used directly in the next reaction step.
c) Preparation of Intermediate 78
##STR00067##
[0312] A mixture of intermediate 77 (0.068 mol) and hydrazine,
monohydrate (0.068 mol) in EtOH (60 ml) was stirred and refluxed
for 1 hour, poured out into water and extracted with DCM. The
organic layer was separated, dried (MgSO.sub.4), filtered, and the
solvent was evaporated, yielding 3.53 g of intermediate 78. This
product was used directly in the next reaction step.
d) Preparation of Intermediate 79
##STR00068##
[0314] Sodiumcyanoborohydride (0.024 mol) and acetic acid (0.0167
mol) were added at room temperature to a solution of intermediate
61 (0.0167 mol) and intermediate 78 (0.015 mol) in MeOH (380 ml)
under N.sub.2 flow. The mixture was stirred for 30 minutes, poured
out into potassium carbonate 10% and extracted with DCM. The
organic layer was separated, dried (MgSO.sub.4), filtered, and the
solvent was evaporated. The residue (8.84 g) was purified by column
chromatography over silica gel (15-40 nm) (eluent:
DCM/MeOH/NH.sub.4OH 96/4/0.2). The pure fractions were collected
and the solvent was evaporated, yielding 2.85 g (40%) of
intermediate 79.
e) Preparation of intermediate 80
##STR00069##
[0316] A mixture of intermediate 79 (0.0028 mol), iodo-ethane
(0.0056 mol) and triethylamine (0.0085 mol) in DMF (60 ml) was
stirred at 50.degree. C. for 7 hours, poured out into ice water and
extracted with EtOAc. The organic layer was washed with water,
dried (MgSO.sub.4), filtered and the solvent was evaporated. The
residue (1.8 g) was purified by column chromatography over kromasil
(5 nm) (eluent: DCM/MeOH 100/0 to 95/5). The pure fractions were
collected and the solvent was evaporated, yielding 1.1 g (78%) of
intermediate 80.
f) Preparation of Intermediate 81
##STR00070##
[0318] A mixture of intermediate 80 (0.0022 mol) and sodium
hydroxide (0.0088 mol) in EtOH (100 ml) was stirred and refluxed
for 6 hours, then stirred at room temperature overnight and
evaporated till dryness. The residue was taken up in diethyl ether.
The precipitate was filtered off and dried, yielding 0.965 g (88%)
of intermediate 81, melting point >260.degree. C.
g) Preparation of Intermediate 82
##STR00071##
[0320] EDC (0.0038 mol) and HOBt (0.0038 mol) were added at room
temperature to a solution of intermediate 81 (0.0019 mol) in THF
(100 ml) and DCM (100 ml) under N.sub.2 flow. The mixture was
stirred at room temperature for 30 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0038 mol) was added.
The mixture was stirred at room temperature for 48 hours, poured
out into water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered and the solvent was
evaporated. The residue (1.2 g) was purified by column
chromatography over kromasil (5 .mu.m) (eluent: DCM/MeOH/NH.sub.4OH
99/1/0.05 to 93/7/0.35). The pure fractions were collected and the
solvent was evaporated, yielding 0.114 g of intermediate 82.
Example A14
a) Preparation of Intermediate 83
##STR00072##
[0322] A mixture of intermediate 62 (0.0034 mol) and sodium
hydroxide (0.0134 mol) in EtOH (150 ml) was stirred at 80.degree.
C. for 3 hours, then cooled to room temperature and evaporated till
dryness. The residue was taken up in diethyl ether. The precipitate
was filtered off and dried, yielding 1.18 g (78%) of intermediate
83, melting point >260.degree. C.
b) Preparation of Intermediate 84
##STR00073##
[0324] EDC (0.0052 mol) and HOBt (0.0052 mol) were added at room
temperature to a solution of intermediate 83 (0.0026 mol) in THF
(120 ml) and DCM (120 ml) under N.sub.2 flow. The mixture was
stirred at room temperature for 30 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0052 mol) was added.
The mixture was stirred at room temperature for 6 days, poured out
into ice water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered and the solvent was
evaporated. The residue (2 g) was purified by column chromatography
over silica gel (15-40 .mu.m) (eluent: DCM/MeOH/NH.sub.4OH
96/4/0.5). The pure fractions were collected and the solvent was
evaporated. The residue (0.75 g, 55%) was purified by column
chromatography over kromasil (10 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 96/4/0.5). The pure fractions were collected
and the solvent was evaporated, yielding 0.625 g of intermediate
84. This product was used directly in the next reaction step.
Example A15
Preparation of Intermediate 85
##STR00074##
[0326] 4-Piperidinemethanamine (0.65 mol) and potassium carbonate
(96 g) were stirred in acetonitrile (1000 ml) and then a solution
of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester
(0.37 mol) in acetonitrile (500 ml) was added dropwise at room
temperature over 1 hour. The reaction mixture was stirred overnight
at room temperature and the solvent was evaporated. The residue was
stirred in water and the mixture was extracted with DCM
(2.times.500 ml). The organic layer was separated, washed with
water, dried (MgSO.sub.4), filtered off and the solvent was
evaporated. The residue was purified by column chromatography over
silica gel (eluent 1: EtOAc/Hexane 1/1; eluent 2: MeOH+ small
amount of NH.sub.4OH). The product fractions were collected,
stirred with potassium carbonate slurry and the mixture was
extracted with DCM. The organic layer was separated, dried
(MgSO.sub.4), filtered off and the solvent was evaporated, yielding
31 g (32%) of intermediate 85.
Example A16
a) Preparation of Intermediate 86
##STR00075##
[0328] Sodium hydride (0.0095 mol) was added at 5.degree. C. to a
solution of 5-chloro-1H-indole-3-carboxaldehyde (0.0056 mol) in THF
(52 ml) under N.sub.2 flow. The mixture was stirred at 0.degree. C.
for 1 hour. 1-Iodo-propane (0.0067 mol) was added. The mixture was
stirred at room temperature for 2 days, poured out into water and
extracted with EtOAc. The organic layer was separated, dried
(MgSO.sub.4), filtered, and the solvent was evaporated, yielding
1.5 g of intermediate 86. This product was used directly in the
next reaction step.
b) Preparation of Intermediate 87
##STR00076##
[0330] Sodiumcyanoborohydride (0.0068 mol) and acetic acid (0.0046
mol) were added at room temperature to a solution of intermediate
85 (0.0042 mol) and intermediate 86 (0.0051 mol) in MeOH (120 ml)
under N.sub.2 flow. The mixture was stirred and refluxed for 2
days, then cooled to room temperature, poured out into potassium
carbonate 10% and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered, and the solvent was
evaporated. The residue (2.42 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.2). The pure fractions were collected
and the solvent was evaporated, yielding 1.2 g (60%) of
intermediate 87.
c) Preparation of Intermediate 88
##STR00077##
[0332] A mixture of intermediate 87 (0.0025 mol) and sodium
hydroxide (0.01 mol) in EtOH (120 ml) was stirred and refluxed for
4 hours, then evaporated till dryness. The residue was taken up in
diethyl ether. The precipitate was filtered off and dried, yielding
0.845 g (72%) of intermediate 88, melting point>260.degree.
C.
d) Preparation of Intermediate 89
##STR00078##
[0334] EDC (0.0036 mol) and HOBt (0.0036 mol) were added at room
temperature to a solution of intermediate 88 (0.0018 mol) in THF
(90 ml) and DCM (90 ml) under N.sub.2 flow. The mixture was stirred
for 30 minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0036
mol) was added. The mixture was stirred at room temperature for 3
days, poured out into ice water and extracted with DCM. The organic
layer was separated, dried (MgSO.sub.4), filtered, and the solvent
was evaporated. The residue (1.3 g) was purified by column
chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 92/8/0.5). The pure fractions were collected
and the solvent was evaporated, yielding 0.54 g (56%) of
intermediate 89.
Example A17
a) Preparation of Intermediate 90
##STR00079##
[0336] Methanesulfonyl chloride (0.004 mol) was added at 10.degree.
C. to a solution of 1,2-dimethyl-1H-indole-3-ethanol (0.0026 mol)
and triethyl amine (0.008 mol) in DCM (10 ml) under N.sub.2 flow.
The mixture was stirred at 10.degree. C. for 4 hours. The solvent
was evaporated till dryness, yielding intermediate 90. This product
was used directly in the next reaction step.
b) Preparation of Intermediate 91
##STR00080##
[0338] A mixture of intermediate 85 (0.0054 mol), intermediate 90
(0.0075 mol) and potassium carbonate (0.021 mol) in acetonitrile
(150 ml) was stirred and refluxed for 2 days, then cooled to room
temperature, poured out into ice water and extracted with EtOAc.
The organic layer was separated, dried (MgSO.sub.4), filtered, and
the solvent was evaporated. The residue (1.88 g) was purified by
column chromatography over silica gel (15-40 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 97/3/0.1). The pure fractions were collected
and the solvent was evaporated, yielding 0.15 g (7%) of
intermediate 91.
c) Preparation of intermediate 92
##STR00081##
[0340] A mixture of intermediate 91 (0.0003 mol) in sodium
hydroxide (0.0014 mol) and EtOH (20 ml) was stirred and refluxed
for one day, then cooled to room temperature and evaporated till
dryness. The residue was taken up in diethyl ether. The precipitate
was filtered off and dried, yielding 0.12 g (82%) of intermediate
92, melting point>260.degree. C.
d) Preparation of Intermediate 93
##STR00082##
[0342] EDC (0.0005 mol) and HOBt (0.0005 mol) were added at room
temperature to a solution of intermediate 92 (0.0002 mol) in THF
(15 ml) and DCM (15 ml). The mixture was stirred for 15 minutes.
O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0005 mol) was added.
The mixture was stirred at room temperature for 4 days, poured out
into ice water and extracted with DCM. The organic layer was
separated, dried (MgSO.sub.4), filtered, and the solvent was
evaporated. The residue (0.14 g) was purified by column
chromatography over silica gel (10 .mu.m) (eluent:
DCM/MeOH/NH.sub.4OH 95/5/0.3). The pure fractions were collected
and the solvent was evaporated, yielding 0.035 g (25%) of
intermediate 93.
[0343] Table F-1 lists the intermediates that were prepared
according to one of the above Examples.
TABLE-US-00001 TABLE F-1 (intermediates) ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##
##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114##
##STR00115## ##STR00116## ##STR00117## ##STR00118## ##STR00119##
##STR00120## ##STR00121## ##STR00122## ##STR00123## ##STR00124##
##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129##
##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134##
##STR00135## ##STR00136## ##STR00137## ##STR00138## ##STR00139##
##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144##
##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149##
##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154##
##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159##
##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164##
##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169##
Interm. 109; Ex. [A1]; mp. .degree.C. ##STR00170## ##STR00171##
##STR00172## ##STR00173## ##STR00174## ##STR00175## ##STR00176##
##STR00177## ##STR00178## ##STR00179## ##STR00180## ##STR00181##
##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186##
##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191##
##STR00192## ##STR00193## ##STR00194## ##STR00195## ##STR00196##
##STR00197## ##STR00198## ##STR00199## ##STR00200## ##STR00201##
##STR00202## ##STR00203## ##STR00204##
##STR00205## ##STR00206## ##STR00207## ##STR00208## ##STR00209##
##STR00210## ##STR00211## ##STR00212## ##STR00213## ##STR00214##
##STR00215## ##STR00216## ##STR00217## ##STR00218## ##STR00219##
##STR00220## ##STR00221## ##STR00222## ##STR00223## ##STR00224##
##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229##
##STR00230## ##STR00231## ##STR00232## ##STR00233## ##STR00234##
##STR00235## ##STR00236## ##STR00237## ##STR00238##
##STR00239##
B. Preparation of the Final Compounds
Example B1
Preparation of Compound 1a
##STR00240##
[0345] TFA (2 ml) was added to a mixture of intermediate 3 (0.0006
mol) in MeOH (40 ml). The mixture was stirred at room temperature
for 30 hours. The solvent was evaporated. The residue was
crystallized from EtOAc/diethyl ether. The precipitate was filtered
off and dried, yielding 0.31 g (86%) of compound 1a, melting point
130.degree. C. alternative synthesis for compound 1
Preparation of Compound 1b
##STR00241##
[0347] Hydroxylamine (50% in water, 7.5 ml) and then NaOH 1N (15
ml) were added at 10.degree. C. to a mixture of intermediate 1
(0.0098 mol) in MeOH (10 ml). The mixture was stirred at room
temperature for 24 hours. The mixture was acidified to pH5-6 by
adding a solution of HCl 1N. The precipitate was filtered off
washed with diethylether and dried. The residue was (4.5 g) was
purified by column chromatography over silica gel LiChroprep.RTM.
NH2 (25-40 .mu.m) (eluent: DCM/MeOH/H.sub.2O 90/10/1). The pure
fractions were collected and the solvent was evaporated, yielding
3.1 g (80%). The HCl salt was prepared on a fraction (0.5 g) in
EtOH and the precipitate was filtered off washed with diethylether
and dried yielding 0.43 g of compound 1b, melting point 220.degree.
C.
Example B2
Preparation of Compound 2
##STR00242##
[0349] TFA (0.5 ml) was added to a mixture of intermediate 7
(0.0001 mol) in MeOH (10 ml) and the mixture was stirred at room
temperature for 24 hours. The solvent was evaporated. The residue
was crystallized from acetonitrile/diethyl ether. The precipitate
was filtered off and dried, yielding 0.036 g (50%) of compound 2,
melting point 205.degree. C.
Example B3
Preparation of Compound 3
##STR00243##
[0351] TFA (5 ml) was added at room temperature to a mixture of
intermediate 12 (0.0014 mol) in MeOH (100 ml). The mixture was
stirred at room temperature for 48 hours. The solvent was
evaporated till dryness. The residue was crystallized from
EtOAc/diethyl ether. The precipitate was filtered off and dried,
yielding 0.545 g (74%) of compound 3, melting point 121.degree.
C.
Example B4
Preparation of Compound 4
##STR00244##
[0353] TFA (0.5 ml) was added to a mixture of intermediate 17
(0.0009 mol) in MeOH (80 ml). The mixture was stirred at room
temperature for 4 days. The solvent was evaporated. The residue was
crystallized from diethyl ether. The precipitate was filtered off
and dried, yielding 0.19 g (32%) of compound 4, melting point
103.degree. C.
Example B5
Preparation of Compound 18
##STR00245##
[0355] A mixture of intermediate 48 (0.0002 mol) in TFA (0.75 ml)
and MeOH (15 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated. The residue was crystallized from diethyl
ether. The precipitate was filtered off and dried, yielding 0.071 g
(59%) of compound 18.
Example B6
Preparation of Compound 19
##STR00246##
[0357] A mixture of intermediate 53 (0.0003 mol) in TFA (1 ml) and
MeOH (20 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated till dryness. The residue was crystallized
from MeOH/CH.sub.3CN/diethyl ether. The precipitate was filtered
off and dried, yielding 0.133 g (80%) of compound 19, melting point
174.degree. C.
Example B7
Preparation of Compound 20
##STR00247##
[0359] A mixture of intermediate 57 (0.00005 mol) in TFA (0.25 ml)
and MeOH (10 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated. The residue (0.04 g) was crystallized from
CH.sub.3CN/diethyl ether. The precipitate was filtered off and
dried. The residue (0.04 g) was purified by column chromatography
over silica gel LiChroprep.RTM. NH.sub.2 (25-40 .mu.m) (eluent:
DCM/MeOH/H.sub.2O 80/20/2). The pure fractions were collected and
the solvent was evaporated, yielding 0.02 g (80%) of compound 20,
melting point 90.degree. C.
Example B8
Preparation of Compound 21
##STR00248##
[0361] A mixture of intermediate 60 (0.0001 mol) in TFA (0.5 ml)
and MeOH (10 ml) was stirred at room temperature overnight. The
solvent was evaporated. The residue (0.15 g) was purified by column
chromatography over silica gel LiChroprep.RTM. NH.sub.2 (25-40
.mu.m) (eluent: DCM/MeOH/H.sub.2O 80/20/2). The pure fractions were
collected and the solvent was evaporated, yielding 0.064 g (78%) of
compound 21, melting point 83.degree. C.
Example B9
Preparation of Compound 22
##STR00249##
[0363] A mixture of intermediate 65 (0.002 mol) in TFA (6 ml) and
MeOH (120 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated till dryness. The residue was crystallized
from CH.sub.3CN/MeOH/diethyl ether. The precipitate was filtered
off and dried, yielding 0.9 g (87%) of compound 22, melting point
183.degree. C.
Example B10
Preparation of Compound 23
##STR00250##
[0365] A mixture of intermediate 72 (0.0001 mol) in TFA (0.5 ml)
and MeOH (10 ml) was stirred at room temperature for 48 hours. The
solvent was evaporated. The residue was crystallized from diethyl
ether. The precipitate was filtered off and dried, yielding 0.059 g
(59%) of compound 23, melting point 182.degree. C.
Example B11
Preparation of Compound 24
##STR00251##
[0367] A mixture of intermediate 75 (0.0003 mol) in TFA (1 ml) and
MeOH (20 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated till dryness. The residue was crystallized
from MeOH/CH.sub.3CN/diethyl ether. The precipitate was filtered
off and dried, yielding 0.147 g (78%) of compound 24, melting point
160.degree. C.
Example B12
Preparation of Compound 25
##STR00252##
[0369] A mixture of intermediate 82 (0.0009 mol) in TFA (2.5 ml)
and MeOH (56 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated till dryness. The residue was purified by
column chromatography over silica gel (25-40 .mu.m) (eluent:
DCM/MeOH/H.sub.2O 90/10/1). The pure fractions were collected and
the solvent was evaporated. The residue was crystallized from DIPE.
The precipitate was filtered off and dried, yielding 0.286 g (53%)
of compound 25, melting point 80.degree. C.
Example B13
Preparation of Compound 26
##STR00253##
[0371] A mixture of intermediate 84 (0.0012 mol) in TFA (3 ml) and
MeOH (60 ml) was stirred at room temperature for 24 hours. The
solvent was evaporated till dryness. The residue was crystallized
from DCM/MeOH. The precipitate was filtered off, washed with
diethyl ether and dried, yielding 0.322 g (50%) of compound 26,
melting point 188.degree. C.
Example B14
Preparation of Compound 27
##STR00254##
[0373] A mixture of intermediate 89 (0.001 mol) in TFA (2.5 ml) and
MeOH (50 ml) was stirred at room temperature for 24 hours, then
evaporated till dryness. The residue was crystallized from
MeOH/CH.sub.3CN/diethyl ether. The precipitate was filtered off,
washed with water and dried, yielding 0.33 g (55%) of compound 27,
melting point 171.degree. C.
Example B15
Preparation of Compound 28
##STR00255##
[0375] A mixture of intermediate 93 (0.00007 mol) in TFA (0.2 ml)
and MeOH (4 ml) was stirred at room temperature for 3 days, then
evaporated till dryness, yielding 0.041 g (100%) of compound 28,
melting point 80.degree. C.
[0376] Table F-2 lists the compounds that were prepared according
to one of the above Examples. The following abbreviations were used
in the tables: .C.sub.2HF.sub.3O.sub.2 stands for the
trifluoroacetate salt, mp. stands for melting point.
TABLE-US-00002 TABLE F-2 (final compounds) ##STR00256##
##STR00257## ##STR00258## ##STR00259## ##STR00260## ##STR00261##
##STR00262## ##STR00263## ##STR00264## ##STR00265## ##STR00266##
##STR00267## ##STR00268## ##STR00269## ##STR00270## ##STR00271##
##STR00272## ##STR00273## ##STR00274## ##STR00275## ##STR00276##
##STR00277## ##STR00278## ##STR00279## ##STR00280## ##STR00281##
##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286##
##STR00287## ##STR00288## ##STR00289## ##STR00290## ##STR00291##
##STR00292## ##STR00293## ##STR00294## ##STR00295## ##STR00296##
##STR00297## ##STR00298## ##STR00299## ##STR00300## ##STR00301##
##STR00302## ##STR00303## ##STR00304## ##STR00305## ##STR00306##
##STR00307## ##STR00308## ##STR00309## ##STR00310## ##STR00311##
##STR00312## ##STR00313## ##STR00314## ##STR00315## ##STR00316##
##STR00317## ##STR00318## ##STR00319## ##STR00320## ##STR00321##
##STR00322## ##STR00323## ##STR00324## ##STR00325## ##STR00326##
##STR00327## ##STR00328## ##STR00329## ##STR00330## ##STR00331##
##STR00332## ##STR00333## ##STR00334## ##STR00335## ##STR00336##
##STR00337##
C. Pharmacological Example
[0377] The in vitro assay for inhibition of histone deacetylase
(see example C.1) measures the inhibition of HDAC enzymatic
activity obtained with the compounds of formula (I).
[0378] Cellular activity of the compounds of formula (I) was
determined on A2780 tumour cells using a colorimetric assay for
cell toxicity or survival (Mosmann Tim, Journal of Immunological
Methods 65: 55-63, 1983)(see example C.2).
[0379] The solubility of a compound measures the ability of a
compound to stay in solution. In a first method the ability of a
compound to stay in aqueous solution upon dilution (see example
C.3.a) is measured. DMSO-stock solutions are diluted with a single
aqueous buffer solvent in 3 consecutive steps. For every dilution
turbidity is measured with a nephelometer.
[0380] In a second method the solubility of a compound at different
pH's can be measured with the use of a Chemiluminescent Nitrogen
Detector (see example C.3.b).
[0381] A drug's permeability expresses its ability to move from one
medium into or through another. Specifically its ability to move
through the intestinal membrane into the blood stream and/or from
the blood stream into the target. Permeability (see example C.4)
can be measured through the formation of a filter-immobilized
artificial membrane phospholipid bilayer. In the filter-immobilized
artificial membrane assay, a "sandwich" is formed with a 96-well
microtitre plate and a 96-well filter plate, such that each
composite well is divided into two chambers with a donor solution
at the bottom and an acceptor solution at the top, separated by a
125 .mu.m micro-filter disc (0.45 .mu.m pores), coated with 2%
(wt/v) dodecane solution of dioleoylphosphatidyl-choline, under
conditions that multi-lamellar bilayers form inside the filter
channels when the system contacts an aqueous buffer solution. The
permeability of compounds through this artificial membrane is
measured in cm/s. The purpose is to look for the permeation of the
drugs through a parallel artificial membrane at 2 different pH's:
4.0 and 7.4. Compound detection is done with UV-spectrometry at
optimal wavelength between 250 and 500 nm.
[0382] Metabolism of drugs means that a lipid-soluble xenobiotic or
endobiotic compound is enzymatically transformed into (a) polar,
water-soluble, and excretable metabolite(s). The major organ for
drug metabolism is the liver. The metabolic products are often less
active than the parent drug or inactive. However, some metabolites
may have enhanced activity or toxic effects. Thus drug metabolism
may include both "detoxication" and "toxication" processes. One of
the major enzyme systems that determine the organism's capability
of dealing with drugs and chemicals is represented by the
cytochrome P450 monooxygenases, which are NADPH dependent enzymes.
Metabolic stability of compounds can be determined in vitro with
the use of subcellular human tissue (see example C.5.a.). Here
metabolic stability of the compounds is expressed as % of drug
metabolised after 15 minutes incubation of these compounds with
microsomes. Quantitation of the compounds was determined by LC-MS
analysis. Metabolic stability of compounds can also be determined
by calculating the half live of compounds in rat hepatocyte cells
(see example C.5.b.).
[0383] It has been shown that a wide variety of anti-tumoral agents
activate the p21 protein, including DNA damaging agents and histone
deacetylase inhibitors. DNA damaging agents activate the p21 gene
through the tumour suppressor p53, while histone deacetylase
inhibitors transcriptionally activates the p21 gene via the
transcription factor Spl. Thus, DNA damaging agents activate the
p21 promoter through the p53 responsive element while histone
deacetylase inhibitors activate the p21 promoter through sp1 sites
(located at the -60 bp to +40 bp region relative to the TATA box)
both leading to increased expression of the p21 protein. When the
p21 promoter in a cells consists of a p21 1300 bp promoter fragment
that does not comprise the p53 responsive elements it is
accordingly non-responsive to DNA damaging agents. The capacity of
compounds to induce p21 can be evaluated in several ways. A first
method is to treat tumour cells with the compound of interest and
after lysis of the cells detects p21 induction with the p21 enzyme
linked immunosorbent assay (WAF1 ELISA of Oncogene). The p21 assay
is a "sandwich" enzyme immunoassay employing both mouse monoclonal
and rabbit polyclonal antibodies. A rabbit polyclonal antibody,
specific for the human p21 protein, has been immobilized onto the
surface of the plastic wells provided in the kit. Any p21 present
in the sample to be assayed will bind to the capture antibody. The
biotinylated detector monoclonal antibody also recognizes human p21
protein, and will bind to any p21, which has been retained by the
capture antibody. The detector antibody, in turn, is bound by
horseradish peroxidase-conjugated streptavidin. The horseradish
peroxidase catalyses the conversion of the chromogenic substrate
tetra-methylbenzidine from a colorless solution to a blue solution
(or yellow after the addition of stopping reagent), the intensity
of which is proportional to the amount of p21 protein bound to the
plate. The colored reaction product is quantified using a
spectrophotometer. Quantitation is achieved by the construction of
a standard curve using known concentrations of p21 (provided
lyophilised). This assay can measures p21 induction as the
consequence of DNA damage or as the consequence of histone
deacetylase inhibition (see example C.6.a.).
[0384] Another method tests the capacity of compounds to induce p21
as the consequence of HDAC inhibition at the cellular level. The
cells can be stably transfected with an expression vector
containing a p21 1300 bp promoter fragment that does not comprise
the p53 responsive elements and wherein an increase of a reporter
gene expression, compared to the control levels, identifies the
compound as having p21 induction capacity. The reporter gene is a
fluorescent protein and the expression of the reporter gene is
measured as the amount of fluorescent light emitted (see example
C.6.b.). The last method is an in vivo method wherein mice are used
for screening the pharmaceutical activity of a compound. The above
described stably transformed tumour cells can be administered to
mice in an amount sufficient to effect production of a tumour.
After the tumour cells had sufficient time to form a tumour, a
potentially active compound can be administered to the animals and
the effect of said compound on the tumour cells is evaluated by
measuring the expression of the reporter gene. Incubation with
pharmaceutical active compounds will result in an increase of
reporter gene expression compared to the control levels (see
example C.6.c.)
[0385] Specific HDAC inhibitors should not inhibit other enzymes
like the abundant CYP P450 proteins. The CYP P450 (E. coli
expressed) proteins 3A4, 2D6 en 2C9 convert their specific
substrates into a fluorescent molecule. The CYP3A4 protein converts
7-benzyloxy-trifluoromethyl coumarin (BFC) into
7-hydroxy-trifluoromethyl coumarin. The CYP2D6 protein converts
342-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin
(AMMC) into
3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin
hydrochloride and the CYP2C9 protein converts
7-Methoxy-4-trifluoromethyl coumarin (MFC) into
7-hydroxy-trifluoromethyl coumarin. Compounds inhibiting the
enzymatic reaction will result in a decrease of fluoresent signal
(see example C.7).
Example C.I
In Vitro Assay for Inhibition of Histone Deacetylase
Example C.I.a
In Vitro Assay with [.sup.3H]-Labelled Substrate
[0386] HeLa nuclear extracts (supplier: Biomol) were incubated at
60 .mu.g/ml with 75 .mu.M of substrate. As a substrate for
measuring HDAC activity a synthetic peptide, i.e. the amino acids
14-21 of histone H4, was used. The substrate is biotinylated at the
NH.sub.2-terminal part with a 6-aminohexanoic acid spacer, and is
protected at the COOH-terminal part by an amide group and
specifically [.sup.3H]acetylated at lysine 16. The substrate,
biotin-(6-aminohexanoic)Gly-Ala-([.sup.3H]-acetyl-Lys-Arg-His-Arg-Lys-Val-
-NH.sub.2), was added in a buffer containing 25 mM Hepes, 1 M
sucrose, 0.1 mg/ml BSA and 0.01% Triton X-100 at pH 7.4. After 30
min the deacetylation reaction was terminated by the addition of
HCl and acetic acid. (final concentration 0.035 mM and 3.8 mM
respectively). After stopping the reaction, the free
.sup.3H-acetate was extracted with ethylacetate. After mixing and
centrifugation, the radioactivity in an aliquot of the upper
(organic) phase was counted in a .beta.-counter.
[0387] For each experiment, controls (containing HeLa nuclear
extract and DMSO without compound), a blank incubation (containing
DMSO but no HeLa nuclear extract or compound) and samples
(containing compound dissolved in DMSO and HeLa nuclear extract)
were run in parallel. In first instance, compounds were tested at a
concentration of 10.sup.-5M. When the compounds showed activity at
10.sup.-5M, a concentration-response curve was made wherein the
compounds were tested at concentrations between 10.sup.-5M and
10.sup.-12M. In each test the blank value was substracted from both
the control and the sample values. The control sample represented
100% of substrate deactylation. For each sample the radioactivity
was expressed as a percentage of the mean value of the controls.
When appropriate IC.sub.50-values (concentration of the drug,
needed to reduce the amount of metabolites to 50% of the control)
were computed using probit analysis for graded data. Herein the
effects of test compounds are expressed as pIC.sub.50 (the negative
log value of the IC.sub.50-value) (see Table F-3).
Example C.I.b
In Vitro Assay with Fluorescent-labelled Substrate
[0388] The HDAC Fluorescent Activity Assay/Drug Discovery Kit of
Biomol (cat.No: AK-500-0001) was used. The HDAC Fluorescent
Activity Assay is based on the Fluor de Lys (Fluorogenic Histone
deAcetylase Lysyl) substrate and developer combination. The Fluor
de Lys substrate, comprises an acetylated lysine side chain.
Deacetylation of the substrate sensitizes the substrate so that, in
the second step, treatment with the Fluor de Lys developer produces
a fluorophore.
[0389] HeLa nuclear extracts (supplier: Biomol) were incubated at
60 .mu.g/ml with 75 .mu.M of substrate. The Fluor de Lys substrate
was added in a buffer containing 25 mM Tris, 137 mM NaCl, 2.7 mM
KCl and 1 mM MgCl.sub.2.6H.sub.2O at pH 7.4. After 30 min, 1 volume
of the developer was added. The fluorophore was excited with 355 nm
light and the emitted light (450 nm) was be detected on a
fluorometric plate reader. For each experiment, controls
(containing HeLa nuclear extract and buffer), a blank incubation
(containing buffer but no HeLa nuclear extract) and samples
(containing compound dissolved in DMSO and further diluted in
buffer and HeLa nuclear extract) were run in parallel. In first
instance, compounds were tested at a concentration of 10.sup.-5M.
When the compounds showed activity at 10.sup.-5M, a
concentration-response curve was made wherein the compounds were
tested at concentrations between 10.sup.-5M and 10.sup.-9M. All
sample were tested 4 times. In each test the blank value was
substracted from both the control and the sample values. The
control sample represented 100% of substrate deactylation. For each
sample the fluorescence was expressed as a percentage of the mean
value of the controls. When appropriate IC.sub.50-values
(concentration of the drug, needed to reduce the amount of
metabolites to 50% of the control) were computed using probit
analysis for graded data. Herein the effects of test compounds are
expressed as pIC.sub.50 (the negative log value of the
IC.sub.50-value) (see Table F-3).
Example C.2
Determination of Antiproliferative Activity on A2780 Cells
[0390] All compounds tested were dissolved in DMSO and further
dilutions were made in culture medium. Final DMSO concentrations
never exceeded 0.1% (v/v) in cell proliferation assays. Controls
contained A2780 cells and DMSO without compound and blanks
contained DMSO but no cells. MTT was dissolved at 5 mg/ml in PBS. A
glycine buffer comprised of 0.1 M glycine and 0.1 M NaCl buffered
to pH 10.5 with NaOH (1 N) was prepared (all reagents were from
Merck).
[0391] The human A2780 ovarian carcinoma cells (a kind gift from
Dr. T. C. Hamilton [Fox Chase Cancer Centre, Pennsylvania, USA])
were cultured in RPMI 1640 medium supplemented with 2 mM
L-glutamine, 50 .mu.g/ml gentamicin and 10% fetal calf serum. Cells
were routinely kept as monolayer cultures at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere. Cells were passaged once a week
using a trypsin/EDTA solution at a split ratio of 1:40. All media
and supplements were obtained from Life Technologies. Cells were
free of mycoplasma contamination as determined using the Gen-Probe
Mycoplasma Tissue Culture kit (supplier: BioMerieux).
[0392] Cells were seeded in NUNC.TM. 96-well culture plates
(Supplier: Life Technologies) and allowed to adhere to the plastic
overnight. Densities used for plating were 1500 cells per well in a
total volume of 200 .mu.l medium. After cell adhesion to the
plates, medium was changed and drugs and/or solvents were added to
a final volume of 200 .mu.l. Following four days of incubation,
medium was replaced by 200 .mu.l fresh medium and cell density and
viability was assessed using an MTT-based assay. To each well, 25
.mu.l MTT solution was added and the cells were further incubated
for 2 hours at 37.degree. C. The medium was then carefully
aspirated and the blue MTT-formazan product was solubilized by
addition of 25 .mu.l glycine buffer followed by 100 .mu.l of DMSO.
The microtest plates were shaken for 10 min on a microplate shaker
and the absorbance at 540 nm was measured using an Emax 96-well
spectrophotometer (Supplier: Sopachem). Within an experiment, the
results for each experimental condition are the mean of 3 replicate
wells. For initial screening purposes, compounds were tested at a
single fixed concentration of 10.sup.-6 M. For active compounds,
the experiments were repeated to establish full
concentration-response curves. For each experiment, controls
(containing no drug) and a blank incubation (containing no cells or
drugs) were run in parallel. The blank value was subtracted from
all control and sample values. For each sample, the mean value for
cell growth (in absorbance units) was expressed as a percentage of
the mean value for cell growth of the control. When appropriate,
IC.sub.50-values (concentration of the drug, needed to reduce cell
growth to 50% of the control) were computed using probit analysis
for graded data (Finney, D J., Probit Analyses, 2.sup.nd Ed.
Chapter 10, Graded Responses, Cambridge University Press, Cambridge
1962). Herein the effects of test compounds are expressed as
plC.sub.50 (the negative log value of the IC.sub.50-value)(see
Table F-3).
Example C.3
Solubility/Stability
C.3.1. Solubility in Aqueas Media
[0393] In the first dilution step, 10 .mu.l of a concentrated
stock-solution of the active compound, solubilized in DMSO (5 mM),
was added to 100 .mu.l phosphate citrate buffer pH 7.4 and mixed.
In the second dilution step, an aliquot (20 .mu.l) of the first
dilution step was further dispensed in 100 .mu.l phosphate citrate
buffer pH 7.4 and mixed. Finally, in the third dilution step, a
sample (20 .mu.l) of the second dilution step was further diluted
in 100 .mu.l phosphate citrate buffer pH 7.4 and mixed. All
dilutions were performed in 96-well plates. Immediately after the
last dilution step the turbidity of the three consecutive dilution
steps were measured with a nephelometer. Dilution was done in
triplicate for each compound to exclude occasional errors. Based on
the turbidity measurements a ranking is performed into 3 classes.
Compounds with high solubility obtained a score of 3 and for this
compounds the first dilution is clear. Compounds with medium
solubility obtained a score of 2. For these compounds the first
dilution is unclear and the second dilution is clear. Compounds
with low solubility obtained a score of 1 and for these compounds
both the first and the second dilution are unclear (see Table
F-3).
C.3.b. Solubility/Stability at Different pH's
[0394] The solubility of a compound, at different pH's, can also be
measured with the use of a chemiluminescent nitrogen detector. (see
Table F-3).
Example C.4
Parallel Artificial Membrane Permeability Analysis
[0395] The stock samples (aliquots of 10 .mu.l of a stock solution
of 5 mM in 100% DMSO) were diluted in a deep-well or Pre-mix plate
containing 2 ml of an aqueous buffer system pH 4 or pH 7.4
(PSR4System Solution Concentrate (pION)).
[0396] Before samples were added to the reference plate, 150 .mu.l
of buffer was added to wells and a blank UV-measurement was
performed. Thereafter the buffer was discarded and the plate was
used as reference plate. All measurements were done in UV-resistant
plates (supplier: Costar or Greiner).
[0397] After the blank measurement of the reference plate, 150
.mu.l of the diluted samples was added to the reference plate and
200 .mu.l of the diluted samples was added to donorplate 1. An
acceptor filter plate 1 (supplier: Millipore, type:MAIP N45) was
coated with 4 .mu.l of the artificial membrane-forming solution
(1,2-Dioleoyl-sn-Glycer-3-Phosphocholine in Dodecane containing
0.1% 2,6-Di-tert-butyl-4-methylphenol and placed on top of donor
plate 1 to form a "sandwich". Buffer (200 .mu.l) was dispensed into
the acceptor wells on the top. The sandwich was covered with a lid
and stored for 18 h at room temperature in the dark.
[0398] A blank measurement of acceptor plate 2 was performed
through the addition of 150 .mu.l of buffer to the wells, followed
by an UV-measurement. After the blank measurement of acceptor plate
2 the buffer was discarded and 150 .mu.l of acceptor solution was
transferred from the acceptor filter plate 1 to the acceptor plate
2. Then the acceptor filter plate 1 was removed form the sandwich.
After the blank measurement of donor plate 2 (see above), 150 .mu.l
of the donor solution was transferred from donor plate 1 to donor
plate 2. The UV spectra of the donor plate 2, acceptor plate 2 and
reference plate wells were scanned (with a SpectraMAX 190). All the
spectra were processed to calculate permeability with the PSR4p
Command Software. All compounds were measured in triplo.
Carbamazepine, griseofulvin, acycloguanisine, atenolol, furosemide,
and chlorothiazide were used as standards in each experiment.
Compounds were ranked in 3 categories as having a low permeability
(mean effect <0.5.times.10.sup.-6 cm/s; score 1), a medium
permeability (1.times.10.sup.-6 cm/s>mean
effect.gtoreq.0.5.times.10.sup.-6 cm/s; score 2) or a high
permeability (.gtoreq.1.times.10.sup.-6 cm/s; score 3).
Example C.5
Metabolic Stability
Example C.5.a
[0399] Sub-cellular tissue preparations were made according to
Gorrod et al. (Xenobiotica 5: 453-462, 1975) by centrifugal
separation after mechanical homogenization of tissue. Liver tissue
was rinsed in ice-cold 0.1 M Tris-HCl (pH 7.4) buffer to wash
excess blood. Tissue was then blotted dry, weighed and chopped
coarsely using surgical scissors. The tissue pieces were
homogenized in 3 volumes of ice-cold 0.1 M phosphate buffer (pH
7.4) using either a Potter-S (Braun, Italy) equipped with a Teflon
pestle or a Sorvall Omni-Mix homogeniser, for 7.times.10 sec. In
both cases, the vessel was kept in/on ice during the homogenization
process.
[0400] Tissue homogenates were centrifuged at 9000.times.g for 20
minutes at 4.degree. C. using a Sorvall centrifuge or Beckman
Ultracentrifuge. The resulting supernatant was stored at
-80.degree. C. and is designated `S9`.
[0401] The S9 fraction can be further centrifuged at
100.000.times.g for 60 minutes (4.degree. C.) using a Beckman
ultracentrifuge. The resulting supernatant was carefully aspirated,
aliquoted and designated `cytosol`. The pellet was re-suspended in
0.1 M phosphate buffer (pH 7.4) in a final volume of 1 ml per 0.5 g
original tissue weight and designated `microsomes`.
[0402] All sub-cellular fractions were aliquoted, immediately
frozen in liquid nitrogen and stored at -80.degree. C. until
use.
[0403] For the samples to be tested, the incubation mixture
contained PBS (0.1M), compound (5 .mu.M), microsomes (1 mg/ml) and
a NADPH-generating system (0.8 mM glucose-6-phosphate, 0.8 mM
magnesium chloride and 0.8 Units of glucose-6-phosphate
dehydrogenase). Control samples contained the same material but the
microsomes were replaced by heat inactivated (10 min at 95 degrees
Celsius) microsomes. Recovery of the compounds in the control
samples was always 100%.
[0404] The mixtures were preincubated for 5 min at 37 degrees
Celsius. The reaction was started at timepoint zero (t=0) by
addition of 0.8 mM NADP and the samples were incubated for 15 min
(t=15). The reaction was terminated by the addition of 2 volumes of
DMSO. Then the samples were centrifuged for 10 min at 900.times.g
and the supernatants were stored at room temperature for no longer
as 24 h before analysis. All incubations were performed in duplo.
Analysis of the supernatants was performed with LC-MS analysis.
Elution of the samples was performed on a Xterra MS C18
(50.times.4.6 mm, 5 .mu.m, Waters, US). An Alliance 2790 (Supplier:
Waters, US) HPLC system was used. Elution was with buffer A (25 mM
ammoniumacetate (pH 5.2) in H.sub.2O/acetonitrile (95/5)), solvent
B being acetonitrile and solvent C methanol at a flow rate of 2.4
ml/min. The gradient employed was increasing the organic phase
concentration from 0% over 50% B and 50% C in 5 min up to 100% B in
1 min in a linear fashion and organic phase concentration was kept
stationary for an additional 1.5 min. Total injection volume of the
samples was 25 .mu.l.
[0405] A Quattro (supplier: Micromass, Manchester, UK) triple
quadrupole mass spectrometer fitted with and ESI source was used as
detector. The source and the desolvation temperature were set at
120 and 350.degree. C. respectively and nitrogen was used as
nebuliser and drying gas. Data were acquired in positive scan mode
(single ion reaction). Cone voltage was set at 10 V and the dwell
time was 1 sec.
[0406] Metabolic stability was expressed as % metabolism of the
compound after 15 min of incubation in the presence of active
microsomes
( E ( act ) ) ( % metabolism = 100 % - ( ( Total Ion Current ( TIC
) of E ( act ) at t = 15 TIC of E ( act ) at t = 0 ) .times. 100 )
. ##EQU00001##
Compounds that had a percentage metabolism less than 20% were
defined as highly metabolic stable. Compound that had a metabolism
between 20 and 70% were defined as intermediately stable and
compounds that showed a percentage metabolism higher than 70 were
defined as low metabolic stable. Three reference compounds were
always included whenever a metabolic stability screening was
performed. Verapamil was included as a compound with low metabolic
stability (% metabolism=73%). Cisapride was included as a compound
with medium metabolic stability (% metabolism 45%) and propanol was
included as a compound with intermediate to high metabolic
stability (25% metabolism). These reference compounds were used to
validate the metabolic stability assay. C.5.b: Metabolic Stability
with Rat Hepatocytes Cell Culture.
[0407] Rat hepatocytes were isolated from male Sprague Dowley rats.
The compounds were dissolved to a 5 mM stock solution in 100% DMSO
and incubated at a final concentration of 5 .mu.M for 0, 15, 30, 60
and 120 min with rat hepatocyte cell cultures (0.5 million viable
cells/0.5 ml) using 24-well plates.
[0408] Samples were prepared for LC-MS by addition of two volumes
of DMSO. The samples were thoroughly shaken and subsequently
centrifuged at 900 g for 10 min (room temperature). All experiments
were performed in triplicate. Of the resulting supernatant 50 .mu.l
was analysed by LC-MS.
[0409] For LC-MS, elution of samples was performed on a Hypersil
BDS C18 column (50.times.4.6 mm, 5 .mu.m, Thermohypersil, UK). The
HPLC system comprised a Surveyor delivery system (Surveyor Inc.,
San Jose, US) equipped with a Surveyor autosampler device. Elution
was with buffer A (10 mM ammoniumacetate (pH 6.9) in
H.sub.2O/Acetonitrile (95:5)) and solvent B (acetonitrile) at a
flow rate of 1.2 ml/min. The gradient employed was 0.5 min solvent
A as start condition followed by increasing the organic phase
concentration from 0% B till 95% B over 2 min in a linear fashion.
This phase was kept stationary for a further 2 min and reduced
again to 0% B within 0.5 min.
[0410] Total injection volume of samples was 50 .mu.L. Column oven
temperature was kept at 40.degree. C. The LC flow was splitted for
MS detection and 0.1 ml let into the source. An triple quadrupol
mass spectrometer TSQ Quantum (Thermofinnigan, LaJolla, USA) mass
spectrometer fitted with an ESI source was used for detection.
Source voltage was set at 3800 volt, the capillary temperature at
300 oC. The mass spectrometer was operated in positive ion mode in
SIM adjusted to the mass of M+H with a scan width of 1 Da for
quantification purposes. Instrument control, data acquisition and
processing were performed using the Xcalibur software
(ThermoFinnigan, San Jose, Calif., U.S.A). The metabolic stability
of compounds in rat hepatocytes was expressed as in vitro
half-lives.
[0411] As reference, compound R306465 (WO03/76422) was used (in
vitro half-live: 8 min) Compound 1 and compound 5 were tested and
had an in vitro half-live of 81 min. and 60 min. respectively.
Example C.6
p21 Induction Capacity
Example C.6.a
p21 Enzyme Linked Immunosorbent Assay
[0412] The following protocol has been applied to determine the p21
protein expression level in human A2780 ovarian carcinoma cells.
The A2780 cells (20000 cells/180 .mu.l) were seeded in 96 microwell
plates in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50
.mu.g/ml gentamicin and 10% fetal calf serum. 24 hours before the
lysis of the cells, compounds were added at final concentrations of
10.sup.-5, 10.sup.-6, 10.sup.-7 and 10.sup.-8 M. All compounds
tested were dissolved in DMSO and further dilutions were made in
culture medium. 24 hours after the addition of the compound, the
supernatants were removed from the cells. Cells were washed with
200 .mu.l ice-cold PBS. The wells were aspirated and 30 .mu.l of
lysisbuffer (50 mM Tris.HCl (pH 7.6), 150 mM NaCl, 1% Nonidet p40
and 10% glycerol) was added. The plates were incubated overnight at
-70.degree. C.
[0413] The appropriate number of microtiter wells were removed from
the foil pouch and placed into an empty well holder. A working
solution (1.times.) of the Wash Buffer (20.times. plate wash
concentrate: 100 ml 20-fold concentrated solution of PBS and
surfactant. Contains 2% chloroacetamide) was prepared. The
lyophilised p21WAF standard was reconstituted with distilled
H.sub.2O and further diluted with sample diluent (provided in the
kit)
[0414] The samples were prepared by diluting them 1:4 in sample
diluent. The samples (100 .mu.l) and the p21WAF1 standards (100
.mu.l) were pipetted into the appropriate wells and incubated at
room temperature for 2 hours. The wells were washed 3 times with
1.times. wash buffer and then 100 .mu.l of detector antibody
reagent (a solution of biotinylated monoclonal p21WAF1 antibody)
was pipetted into each well. The wells were incubated at room
temperature for 1 hour and then washed three times with 1.times.
wash buffer. The 400.times. conjugate (peroxidase streptavidine
conjugate: 400-fold concentrated solution) was diluted and 100
.mu.l of the 1.times. solution was added to the wells. The wells
were incubated at room temperature for 30 min and then washed 3
times with 1.times. wash buffer and 1 time with distilled H.sub.2O.
Substrate solution (chromogenic substrate) (100 .mu.l) was added to
the wells and the wells were incubated for 30 minutes in the dark
at room temperature. Stop solution was added to each well in the
same order as the previously added substrate solution. The
absorbance in each well was measured using a spectrophotometric
plate reader at dual wavelengths of 450/595 nm. For each
experiment, controls (containing no drug) and a blank incubation
(containing no cells or drugs) were run in parallel. The blank
value was substracted from all control and sample values. For each
sample, the value for p21WAF1 induction (in absorbance units) was
expressed as the percentage of the value for p21WAF1 present in the
control. Percentage induction higher than 130% was defined as
significant induction. Nine compounds were tested and all showed
significant induction at 10.sup.-6M.
Example C.6.b
Cellular Method
[0415] A2780 cells (ATCC) were cultivated in RPMI 1640 medium
supplemented with 10% FCS, 2 mM L-glutamine and gentamycine at
37.degree. C. in a humidified incubator with 5% CO.sub.2. All cell
culture solutions are provided by Gibco-BRL (Gaithersburg, Md.).
Other materials are provided by Nunc.
[0416] Genomic DNA was extracted from proliferating A2780 cells and
used as template for nested PCR isolation of the p21 promoter. The
first amplification was performed for 20 cycles at an annealing
temperature of 55.degree. C. using the oligonucleotide pair
GAGGGCGCGGTGCTTGG and TGCCGCCGCTCTCTCACC with the genomic DNA as
template. The resulting 4.5 kb fragment containing the -4551 to +88
fragment relative to the TATA box was re-amplified with the
oligonucleotides TCGGGTACCGAGGGCGCGGTGCTTGG and
ATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycles with annealing at
88.degree. C. resulting in a 4.5 kb fragment and subsequently with
the oligonucleotide pair TCGGGTACCGGTAGATGGGAGCGGATAGACACATC and
ATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycles with annealing at
88.degree. C. resulting in a 1.3 kb fragment containing the -1300
to +88 fragment relative to the TATA box. The restriction sites
XhoI and KpnI present in the oligonucleotides (underlined sequence)
were used for subcloning.
[0417] The luciferase reporter was removed from the pGL3-basic and
replaced by the ZsGreen reporter (from the pZsGreenl-N1 plasmid) at
KpnI and XbaI restriction sites. pGL3-basic-ZsGreen-1300 was
constructed via insertion of the above mentioned 1.3 kb fragment of
the human p21 promoter region into pGL3-basic-ZsGreen at the XhoI
and KpnI sites. All restriction enzymes are provided by Boehringer
Manheim (Germany). A2780 cells were plated into a 6-well plate at a
density of 2.times.10.sup.5 cells, incubated for 24 hours, and
transfected with 2 ug of pGL3-basic-ZsGreen-1300 and 0.2 ug of
pSV2neo vector by using Lipofectamine 2000 (Invitrogen, Brussels,
Belgium) as described by manufacturer. The transfected cells were
selected for 10 days with G418 (Gibco-BRL, Gaithersburg, Md.) and
single cell suspensions were grown. After three weeks, single
clones were obtained.
[0418] The A2780 selected clones were expanded and seeded at 10000
cells per well into 96-well plates. 24 hours after seeding, the
cells were treated for an additional 24 hours with compounds
(affecting sp1 sites in the proximal p21 promoter region).
Subsequently, cells were fixed with 4% PFA for 30' and
counterstained with Hoechst dye. The p21 promoter activation
leading to ZsGreen production and thus fluorescence, was monitored
by the Ascent Fluoroskan (Thermo Labsystems, Brussels,
Belgium).
[0419] For each experiment, controls (containing no drug) and a
blank incubation (containing no cells or drugs) were run in
parallel. The blank value was substracted from all control and
sample values. For each sample, the value for p21 induction was
expressed as the percentage of the value for p21 present in the
control. Percentage induction higher than 130% was defined as
significant induction.
[0420] Seventy one compounds were tested and all showed significant
induction at 10.sup.-6M.
Example C.6.c
In Vivo Method
[0421] A selected clone was injected subcutaneous (10.sup.7
cells/200 .mu.l) into the flank of nude mice and a calliper
measurable tumour was obtained after 12 days. From day 12 on,
animals were dosed, orally or intraveinally, daily during 6 days
with solvent and 20-40 mpk compound (4-10 animals each). Tumours
were evaluated for fluorescence by the in-house developed Automated
Whole Body Imaging System (Fluorescent stereomicroscope type
Olympus.RTM. SZX12 equipped with a GFP filter and coupled to a CCD
camera type JAI.RTM. CV-M90 controlled by a software package based
on the IMAQ Vision Software from National Instruments.RTM.). As
reference, compound R306465 (WO03/76422) was used. Compounds were
ranked as inactive (no fluorescence measurable), weaker, identical
or better than R306465. Compound 1 was tested and was better than
R306465.
Example C.7
P450 Inhibiting Capacity
[0422] All compounds tested were dissolved in DMSO (5 mM) and a
further dilution to 5 10.sup.-4 M was made in acetonitrile. Further
dilutions were made in assay buffer (0.1M NaK phosphate buffer pH
7.4) and the final solvent concentration was never higher than
2%.
[0423] The assay for the CYP3A4 protein comprises per well 15 pmol
P450/mg protein (in 0.01M NaKphosphate buffer+1.15% KCl), an NADPH
generating system (3.3 mM Glucose-6-phosphate, 0.4 U/ml
Glucose-6-phosphate dehydrogenase, 1.3 mM NADP and 3.3 mM
MgCl.sub.2.6H.sub.2O in assay buffer) and compound in a total assay
volume of 100 .mu.l. After a 5 min pre-incubation at 37.degree. C.
the enzymatic reaction was started with the addition of 150 .mu.M
of the fluoresent probe substrate BFC in assay buffer. After an
incubation of 30 minutes at room temperature the reaction was
terminated after addition of 2 volumes of acetonitrile. Fluorescent
determinations were carried out at an excitation wavelength of 405
nm and an emission wavelength of 535 nm. Ketoconazole
(IC.sub.50-value=3.times.10.sup.-8M) was included as reference
compound in this experiment. The assay for the CYP2D6 protein
comprises per well 6 pmol P450/mg protein (in 0.01M NaKphosphate
buffer+1.15% KCl), an NADPH generating system (0.41 mM
Glucose-6-phosphate, 0.4 U/ml Glucose-6-phosphate dehydrogenase,
0.0082 mM NADP and 0.41 mM MgCl.sub.2.6H.sub.2O in assay buffer)
and compound in a total assay volume of 100 .mu.l. After a 5 min
pre-incubation at 37.degree. C. the enzymatic reaction was started
with the addition of 3 .mu.M of the fluoresent probe substrate AMMC
in assay buffer. After an incubation of 45 minutes at room
temperature the reaction was terminated after addition of 2 volumes
of acetonitrile. Fluorescent determinations were carried out at an
excitation wavelength of 405 nm and an emission wavelength of 460
nm. Quinidine (IC.sub.50-value <5.times.10.sup.-8M) was included
as reference compound in this experiment.
[0424] The assay for the CYP2C9 protein comprises per well 15 pmol
P450/mg protein (in 0.01M NaKphosphate buffer+1.15% KCl), an NADPH
generating system (3.3 mM Glucose-6-phosphate, 0.4 U/ml
Glucose-6-phosphate dehydrogenase, 1.3 mM NADP and 3.3 mM
MgCl.sub.2.6H.sub.2O in assay buffer) and compound in a total assay
volume of 100 .mu.l. After a 5 min pre-incubation at 37.degree. C.
the enzymatic reaction was started with the addition of 200 .mu.M
of the fluoresent probe substrate MFC in assay buffer. After an
incubation of 30 minutes at room temperature the reaction was
terminated after addition of 2 volumes of acetonitrile. Fluorescent
determinations were carried out at an excitation wavelength of 405
nm and an emission wavelength of 535 nm. Sulfaphenazole
(IC.sub.50-value=6.8.times.10.sup.-7M) was included as reference
compound in this experiment.
[0425] For initial screening purposes, compounds were tested at a
single fixed concentration of 1.times.10.sup.-5 M. For active
compounds, the experiments were repeated to establish full
concentration-response curves. For each experiment, controls
(containing no drug) and a blank incubation (containing no enzyme
or drugs) were run in parallel. All compounds were assayed in
quadruplicate. The blank value was subtracted from all control and
sample values. For each sample, the mean value of P450 activity of
the sample (in relative fluorescence units) was expressed as a
percentage of the mean value of P450 activity of the control.
Percentage inhibition was expressed as 100% minus the mean value of
P450 activity of the sample. When appropriate, IC.sub.50-values
(concentration of the drug, needed to reduce P450 activity to 50%
of the control) were calculated.
TABLE-US-00003 TABLE F-3 lists the results of the compounds that
were tested according to example C.1.a., C.1.b, C.2, C.3.a. and
C.3.b. Enzymatic Enzymatic Cellular Solubility activity activity
activity C.3.b. Compound pIC50 pIC50 pIC50 Solubility pH = 2.3
number C.1.a. C.1.b C.2 C.3.a. (mg/ml) 5 8.6 6.5 2.0 1a 8.8 9.2 8.2
3 1.4 6 8.2 6.3 7 8.5 6.1 3 8.5 6.7 4 8.7 7.0 3 17 7.8 6.8 8 8.3
7.1 2.4 9 8.3 7.1 10 8.1 7.5 3 3.7 16 8.6 7.1 3 11 8.2 7.5 3 2.8 2
8.4 7.5 3 15 8.5 7.5 14 8.4 7.4 1.7 13 6.0 7.5 3 12 8.2 7.1 3 35
8.2 6.7 68 >9.0 7.2 2.5 63 >9.0 7.5 67 8.3 7.5 1.9 76 7.8 6.7
72 7.9 6.8 80 7.8 7.1 34 8.4 7.5 33 10.0 7.5 1.6 32 8.0 8.4 1.5 21
8.5 7.6 1.9 49 8.7 7.5 3.3 53 10.0 8.0 20 9.5 8.1 2.7 64 8.9 7.8 62
9.5 8.0 28 8.1 7.1 61 8.9 7.6 60 8.7 6.9 3.1 59 9.0 7.7 58 10.0 5.8
19 8.0 7.1 24 7.6 6.5 1.6 57 10.2 7.6 2.8 56 9.2 7.6 55 10.1 7.8
2.0 54 10.2 7.9 52 10.3 8.1 51 9.8 8.0 50 8.6 7.6 2.4 48 >9.0
8.2 66 >9.0 7.4 1.7 73 7.9 7.1 47 8.8 8.0 4.0 74 8.2 7.1 46 9.0
8.0 31 8.2 7.4 75 7.3 7.1 27 >9.0 8.0 45 9.0 6.7 1.7 44 8.2 7.4
1.8 43 8.5 7.8 2.1 29 7.9 6.9 42 8.3 7.5 71 7.5 6.5 70 8.5 6.8 69
7.6 6.7 25 7.6 7.0 41 8.4 5.8 40 8.5 7.5 23 8.4 7.5 22 9.1 7.2 26
>9.0 7.5 65 >9.0 7.5 1.6 39 9.0 7.5 2.0 30 9.0 7.5 2.1 38
>9.0 7.5 37 8.6 7.5 36 8.7 8.6 2.5 79 8.4 8.1 2.7 18 8.3 8.0 81
8.9 7.5
D. Composition Example
Film-Coated Tablets
Preparation of Tablet Core
[0426] A mixture of 100 g of a compound of formula (I), 570 g
lactose and 200 g starch is mixed well and thereafter humidified
with a solution of 5 g sodium dodecyl sulphate and 10 g
polyvinyl-pyrrolidone in about 200 ml of water. The wet powder
mixture is sieved, dried and sieved again. Then there is added 100
g microcrystalline cellulose and 15 g hydrogenated vegetable oil.
The whole is mixed well and compressed into tablets, giving 10.000
tablets, each comprising 10 mg of a compound of formula (I).
Coating
[0427] To a solution of 10 g methyl cellulose in 75 ml of
denaturated ethanol there is added a solution of 5 g of ethyl
cellulose in 150 ml of dichloromethane. Then there are added 75 ml
of dichloromethane and 2.5 ml 1,2,3-propanetriol 10 g of
polyethylene glycol is molten and dissolved in 75 ml of
dichloromethane. The latter solution is added to the former and
then there are added 2.5 g of magnesium octadecanoate, 5 g of
polyvinyl-pyrrolidone and 30 ml of concentrated colour suspension
and the whole is homogenated. The tablet cores are coated with the
thus obtained mixture in a coating apparatus.
* * * * *