U.S. patent application number 12/781430 was filed with the patent office on 2010-09-02 for n-oxides of n-phenyl-2-pyramidine-amine derivatives.
Invention is credited to Klaus Olaf Bornsen, Peter End, Gerhard Gross, Paul William Manley, Ulrike Pfaar, Jurg Zimmermann.
Application Number | 20100222362 12/781430 |
Document ID | / |
Family ID | 9929594 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100222362 |
Kind Code |
A1 |
Bornsen; Klaus Olaf ; et
al. |
September 2, 2010 |
N-OXIDES OF N-PHENYL-2-PYRAMIDINE-AMINE DERIVATIVES
Abstract
The invention relates to N-phenyl-2-pyrimidine-amine derivatives
derivatives in which at least one nitrogen atom carries an oxygen
atom to form the corresponding N-oxides, to processes for the
preparation thereof, to pharmaceutical compositions comprising
those compounds, and to the use thereof in the preparation of
pharmaceutical compositions for the therapeutic treatment of
warm-blooded animals, including humans.
Inventors: |
Bornsen; Klaus Olaf;
(Staufen, DE) ; End; Peter; (Oberwill, CH)
; Gross; Gerhard; (Lorrach, DE) ; Pfaar;
Ulrike; (Rheinfelden, DE) ; Manley; Paul William;
(Arlesheim, CH) ; Zimmermann; Jurg; (Binningen,
CH) |
Correspondence
Address: |
NOVARTIS;CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 101/2
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
9929594 |
Appl. No.: |
12/781430 |
Filed: |
May 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12478082 |
Jun 4, 2009 |
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12781430 |
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10502291 |
Apr 29, 2005 |
7557105 |
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PCT/EP03/00613 |
Jan 22, 2003 |
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12478082 |
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Current U.S.
Class: |
514/253.01 ;
544/364 |
Current CPC
Class: |
A61P 17/06 20180101;
C07D 401/14 20130101; A61P 11/06 20180101; A61P 7/02 20180101; A61P
11/00 20180101; A61P 35/02 20180101; A61P 31/04 20180101; C07D
401/04 20130101; A61P 43/00 20180101; A61P 37/06 20180101; A61P
9/00 20180101; A61P 9/10 20180101; A61P 7/00 20180101; A61P 35/00
20180101 |
Class at
Publication: |
514/253.01 ;
544/364 |
International
Class: |
A61K 31/496 20060101
A61K031/496; C07D 401/14 20060101 C07D401/14; A61P 35/00 20060101
A61P035/00; A61P 17/06 20060101 A61P017/06; A61P 7/02 20060101
A61P007/02; A61P 9/10 20060101 A61P009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2002 |
GB |
0201508.9 |
Claims
1. A compound which is
4-[(methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-
-2-pyrinidinyl]-amino]-phenyl]-benzamide, or a pharmaceutically
acceptable salt thereof.
2. A pharmaceutical composition comprising a compound according to
claim 1 together with a pharmaceutically acceptable carrier.
3. A method of treating a warm-blooded animal suffering from a
proliferative disorder, which comprises administering to the
warm-blooded animal a dose effective against the disorder of a
compound according to claim 1.
Description
[0001] The invention relates to N-phenyl-2-pyrimidine-amine
derivatives in which at least one nitrogen atom carries an oxygen
atom to form the corresponding N-oxides, to processes for the
preparation thereof, to pharmaceutical compositions comprising
those compounds, and to the use thereof in the preparation of
pharmaceutical compositions for the therapeutic treatment of
warm-blooded animals, including humans.
[0002] The invention relates particularly to compounds of formula
I
##STR00001##
[0003] wherein
[0004] R.sub.1 is hydrogen or hydroxy,
[0005] R.sub.2 is hydrogen, lower alkyl or hydroxy-lower alkyl,
[0006] A is --NR.sub.5R.sub.6, --CR.sub.5R.sub.6 or
--OR.sub.5R.sub.6,
[0007] R.sub.5R.sub.6 together represent alkylene with four, five
or six carbon atoms, oxa-lower alkylene with one oxygen and three
or four carbon atoms, or aza-lower alkylene with one or two
nitrogen and two, three or four carbon atoms wherein the nitrogen
atom is unsubstituted or substituted by lower alkyl, hydroxy-lower
alkyl, or acetyl, and wherein lower alkylene in each case may be
partially or totally unsaturated and/or the carbon atoms of lower
alkylene may be substituted by lower alkyl, hydroxyl, lower alkoxy
or oxo when lower alkylene is not totally unsaturated, and wherein
at least one nitrogen atom carries an oxygen atom to form the
corresponding N-oxide or when no nitrogen atom carries an oxygen
atom, A is substituted by oxo on a ring carbon,
[0008] or a pharmaceutically acceptable salt of such a
compound.
[0009] Preferably A is substituted by oxo on a ring carbon.
[0010] Preferably A is pyrrolidino, piperidyl, piperidino,
piperazinyl, pyridyl, pyrrolidino, pyrrolidinyl, morpholino, lower
alkylpiperazino, N-methylpiperazino, 4-methyl-3-oxo-1-piperazinyl,
3-oxo-1-piperazinyl, 1H-imidazolyl, 1H-2-methylimidazolyl,
1H-4-methylimidazolyl or 1H-2,4-dimethylimidazolyl, cyclohexyl or
phenyl, optionally substituted by oxo on a ring carbon;
[0011] Most preferably "A" represents a piperazino group of the
following formula A'
##STR00002##
[0012] wherein
[0013] R.sub.3 represents, hydrogen, lower alkyl or acetyl.
[0014] Preference is given to compounds of formula I, wherein
[0015] R.sub.1 is hydrogen,
[0016] R.sub.2 is hydrogen, methyl or hydroxymethyl,
[0017] A is A', optionally substituted by oxo on a ring carbon,
[0018] R.sub.3 is methyl or hydrogen,
[0019] or salts of such compounds.
[0020] When "A" is substituted by oxo on a ring carbon, "A" is
preferably selected from lower alkyl-oxo-piperazino such as
4-methyl-3-oxo-1-piperazinyl or oxo-piperazino such as
3-oxo-1-piperazinyl, oxo-pyrrolidin, oxo-piperidino, oxo-piperidyl,
oxo-morpholino, oxo-cyclohexyl, succinimido or glutarimido.
[0021] The nitrogen atoms, which carry an oxygen atom to form the
corresponding N-oxides are preferably the ring nitrogen atoms
located on pyrimidine, pyrindinyl, "A" or piperazino group of the
formula A'.
[0022] By defining "R.sub.5R.sub.6 together", the applicant does
not include in the numbering the nitrogen, oxygen or carbon group
mentioned in NR.sub.5R.sub.6, CR.sub.5R.sub.6 or
OR.sub.5R.sub.6.
[0023] The prefix "lower" denotes a radical having up to and
including a maximum of 7, especially up to and including a maximum
of 4 carbon atoms, the radicals in question being either linear or
branched with single or multiple branching.
[0024] Lower alkyl is preferably alkyl with from and including 1 up
to and including 7, preferably from and including 1 to and
including 4, and is linear or branched; preferably, lower alkyl is
butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl,
such as n-propyl or isopropyl, ethyl or methyl. Preferably lower
alkyl is methyl, propyl or tert-butyl.
[0025] Hydroxy-lower alkyl, preferably hydroxymethyl,
2-hydroxyethyl or 2-hydroxy-2-propyl.
[0026] Lower alkoxy is especially methoxy, ethoxy, isopropyloxy, or
tert-butyloxy.
[0027] In a preferred aspect, the invention relates to compounds of
formula II
##STR00003##
[0028] wherein
[0029] R.sub.1 is hydrogen or hydroxy,
[0030] R.sub.2 is lower alkyl or hydroxy-lower alkyl,
[0031] R.sub.3 is hydrogen, methyl or acetyl, and
[0032] the stars indicate the nitrogen atoms which optionally carry
an oxygen atom to form the corresponding N-oxides,
[0033] with the proviso that at least one of the three nitrogen
atoms marked by a star carries an oxygen atom if R.sub.1 is
hydrogen, R.sub.2 is methyl and R.sub.3 is hydrogen or methyl, or
salts of such compounds.
[0034] Optionally, the nitrogen atoms of the 2-pyrimidine can also
carry one or two oxygen atoms to form the corresponding
N-oxides.
[0035] Preferably, a compound of formula II carries at least one
oxygen atom to form the corresponding N-oxide.
[0036] Optionally, the piperazinyl is substituted by oxo to from a
lower alkyl-oxo-piperazino such as 4-methyl-3-oxo-1-piperazinyl or
an oxo-piperazino such as 3-oxo-1-piperazinyl.
[0037] The term "lower" within the scope of compounds of formula II
denotes radicals having up to and including 7, preferably up to and
including 4 carbon atoms.
[0038] When R.sub.1 is hydroxy, the 3-pyridinyl moiety is
substituted by hydroxy at a ring carbon atom at position 2, 4, 5 or
6.
[0039] Lower alkyl R.sub.2 is preferably methyl.
[0040] Hydroxy-lower alkyl R.sub.2 is preferably hydroxymethyl.
[0041] Salts are especially the pharmaceutically acceptable salts
of compounds of formula I or II.
[0042] Such salts are formed, for example, as acid addition salts,
preferably with organic or inorganic acids, from compounds of
formula I or II with a basic nitrogen atom, especially the
pharmaceutically acceptable salts.
[0043] For isolation or purification purposes it is also possible
to use pharmaceutically unacceptable salts, for example picrates or
perchlorates. Only the pharmaceutically acceptable salts or free
compounds (if the occasion arises, in the form of pharmaceutical
compositions) attain therapeutic use, and these are therefore
preferred.
[0044] In view of the close relationship between the novel
compounds in free form and in the form of their salts, including
those salts that can be used as intermediates, for example in the
purification or identification of the novel compounds, hereinbefore
and hereinafter any reference to the free compounds is to be
understood as referring also to the corresponding salts, as
appropriate and expedient.
[0045] A compound of formula I or II possesses valuable
pharmacological properties and may, for example, be used as an
anti-tumour agent, as an agent to treat atherosclerosis, as an
agent to treat restenosis, as an anti-leukemic agent for the
prevention of transplantation-induced disorders, such as
obliterative bronchiolitis, and/or for preventing the invasion of
warm-blooded animal cells by certain bacteria, such as
Porphyromonas gingivalis.
[0046] The phosphorylation of proteins has long been known as an
essential step in the differentiation and division of cells.
Phosphorylation is catalysed by protein kinases subdivided into
serine/threonine and tyrosine kinases. The tyrosine kinases include
PDGF (Platelet-derived Growth Factor) receptor tyrosine kinase.
[0047] PDGF is a very commonly occurring growth factor, which plays
an important role both in normal growth and also in pathological
cell proliferation, such as is seen in carcinogenesis and in
diseases of the smooth-muscle cells of blood vessels, for example
in atherosclerosis and thrombosis.
[0048] The inhibition of PDGF-stimulated receptor tyrosine kinase
activity in vitro is measured in PDGF receptor immune complexes of
A431 cells, as described by E. Andrejauskas-Buchdunger and U.
Regenass in Cancer Research 52, 5353-5358 (1992). A compound of
formula I or II inhibits PDGF-dependent acellular receptor
phosphorylation. The inhibition of PDGF receptor tyrosine kinase is
measured in a microtitre ELISA assay (cf Trinks et al., J. Med.
Chem. 37, 1015-27 (1994).
[0049] The inhibition of PDGF receptor tyrosine kinase makes a
compound of formula I or II also suitable for the treatment of
tumour diseases, such as gliomas, sarcomas, prostate tumours, and
tumours of the colon, breast, and ovary.
[0050] A compound of formula I or II also inhibits cellular
processes involving the so-called stem-cell factor (SCF, also known
as the c-Kit ligand or steel factor), such as SCF receptor (Kit)
autophosphorylation and the SCF-stimulated activation of MAPK
kinase (mitogen-activated protein kinase).
[0051] In particular, a compound of formula I or II inhibits the
tyrosine kinase activity of c-Kit. This can be shown in a tyrosine
kinase inhibition assay using the cytoplasmatic kinase domain of
c-Kit. The assay is performed as follows: The baculovirus donor
vector pFbacG01 (GIBCO) is used to generate a recombinant
baculovirus that expresses the amino acid region amino acids
544-976 of the cytoplasmic kinase domains of human c-Kit. The
coding sequences for the cytoplasmic domain of c-Kit is amplified
by PCR from a human uterus c-DNA library (Clontech). The amplified
DNA fragment and the pFbacG01 vector are made compatible for
ligation by digestion with BamH1 and EcoRI. Ligation of these DNA
fragments results in the baculovirus donor plasmid c-Kit. The
production of the viruses, the expression of proteins in Sf9 cells
and the purification of the GST-fused proteins are performed as
follows:
[0052] Production of virus: Transfer vector (pFbacG01-c-Kit)
containing the c-Kit kinase domain is transfected into the DH10Bac
cell line (GIBCO) and the transfected cells are plated on selective
agar plates. Colonies without insertion of the fusion sequence into
the viral genome (carried by the bacteria) are blue. Single white
colonies are picked and viral DNA (bacmid) is isolated from the
bacteria by standard plasmid purification procedures. Sf9 cells or
Sf21 cells (American Type Culture Collection) are then transfected
in 25 cm.sup.2 flasks with the viral DNA using Cellfectin
reagent.
[0053] Determination of small scale protein expression in Sf9
cells: Virus containing media is collected from the transfected
cell culture and used for infection to increase its titre. Virus
containing media obtained after two rounds of infection is used for
large-scale protein expression. For large-scale protein expression
100 cm.sup.2 round tissue culture plates are seeded with
5.times.10.sup.7 cells/plate and infected with 1 mL of
virus-containing media (approx. 5 MOIs). After 3 days the cells are
scraped off the plate and centrifuged at 500 rpm for 5 min. Cell
pellets from 10-20, 100 cm.sup.2 plates, are resuspended in 50 mL
of ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1%
NP-40, 1 mM DTT, 1 mM PMSF). The cells are stirred on ice for 15
min and then centrifuged at 5000 rpms for 20 min.
[0054] Purification of GST-tagged protein: The centrifuged cell
lysate is loaded onto a 2 mL glutathione-sepharose column
(Pharmacia) and washed three times with 10 mL of 25 mM Tris-HCl, pH
7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCl. The GST-tagged protein is
eluted by 10 applications (1 mL each) of 25 mM Tris-HCl, pH 7.5, 10
mM reduced-glutathione, 100 mM NaCl, 1 mM DTT, 10% Glycerol and
stored at -70.degree. C. Kinase assay. Tyrosine protein kinase
assays with purified GST-c-Kit are carried out in a final volume of
30 .mu.l containing 200-1800 ng of enzyme protein (depending on the
specific activity), 20 mM Tris-HCl, pH 7.6, 3 mM MnCl.sub.2, 3 mM
MgCl.sub.2, 1 mM DTT, 10 .mu.M Na.sub.3VO.sub.4, 5 .mu.g/mL
poly(Glu,Tyr) 4:1, 1% DMSO, 1.0 .mu.M ATP and 0.1 .mu.Ci
[.gamma..sup.33 P] ATP. The activity is assayed in the presence or
absence of inhibitors, by measuring the incorporation of .sup.33P
from [.gamma..sup.33P] ATP into the poly(Glu,Tyr) 4:1 substrate.
The assay (30 .mu.L) is carried out in 96-well plates at ambient
temperature for 20 min under conditions described below and
terminated by the addition of 20 .mu.L of 125 mM EDTA.
Subsequently, 40 .mu.L it of the reaction mixture is transferred
onto Immobilon-PVDF membrane (Millipore, Bedford, Mass., USA)
previously soaked for 5 min with methanol, rinsed with water, then
soaked for 5 min with 0.5% H.sub.3PO.sub.4 and mounted on vacuum
manifold with disconnected vacuum source. After spotting all
samples, vacuum is connected and each well rinsed with 200 L 0.5%
H.sub.3PO.sub.4. Membranes are removed and washed 4.times. on a
shaker with 1.0% H.sub.3PO.sub.4 and once with ethanol. Membranes
are counted after drying at ambient temperature, mounting in
Packard TopCount 96-well frame, and addition of 10 .mu.L/well of
Microscint.TM. (Packard). IC.sub.50 values are calculated by linear
regression analysis of the percentage inhibition of each compound
in duplicate, at four concentrations (usually 0.01, 0.1, 1 and 10
.mu.M). One unit of protein kinase activity is defined as 1 nmole
of .sup.33P ATP transferred from [.gamma..sup.33P] ATP to the
substrate protein per minute per mg of protein at 37.degree. C.
[0055] A compound of formula I or II inhibits also the
autophosphorylation of SCF receptor (and c-Kit, a proto-oncogen).
Inhibition of the autophosphorylation of the SCF receptor can be
measured using e.g. MO7e cells, a human promegakaryocytic leukaemia
cell line which depends on SCF for proliferation. They are obtained
from Grover Bagby, Oregon Health Sciences University, USA. The
cells are cultivated in RPMI 1649 medium supplemented with 10 FBS
and 2.5 ng/ml GC-CMF. GM-SCF and SCF are commercially available.
Serum-deprived MO7e cells are prepared and incubated for 90 min at
37.degree. C. with the test substance before being stimulated with
recombinant SCF for 10 min at 37.degree. C. Identical quantities of
cell lysates are analysed by Western blot using antiphosphotyrosine
antibodies (Buchdunger et al., Proc. Natl. Acad. Sci (USA) 92,
2558-62 (1995)). The immunodecorated proteins are detected by means
of the ECL Western blotting system from Amersham (Amersham,
UK).
[0056] On the basis of the described properties, a compound of
formula I or II may be used not only as a tumour-inhibiting
substance, for example in small cell lung cancer, but also as an
agent to treat non-malignant proliferative disorders, such as
atherosclerosis, thrombosis, psoriasis, scleroderma, and fibrosis,
as well as for the protection of stem cells, for example to combat
the haemotoxic effect of chemotherapeutic agents, such as
5-fluoruracil, and in asthma. It may especially be used for the
treatment of diseases which respond to an inhibition of the PDGF
receptor kinase.
[0057] In addition, a compound of formula I or II prevents the
development of multidrug resistance in cancer therapy with other
chemotherapeutic agents or abolishes a pre-existing resistance to
other chemotherapeutic agents. Also regardless of the effect
described hereinbefore, a compound of formula I or II may be used
to advantage in combination with other antitumour agents, such as
especially other c-Kit inhibitors and inhibitors of Vascular
Endothelial Growth Factor (VEGF) receptor or c-Src activity.
[0058] Also Abl kinase, especially v-Abl kinase, is inhibited by a
compound of formula I or II. The inhibition of v-Abl tyrosine
kinase is determined by the methods of N. Lydon at al., Oncogene
Research 5, 161-173 (1990) and J. F. Geissler et al., Cancer
Research 52, 4492-8 (1992). In those methods
[Val.sup.5]-angiotensin II and [.gamma..sup.32P]-ATP are used as
substrates.
[0059] By analogy, a compound of formula I or II also inhibits
Bcr-Abl kinase (see Nature Medicine 2, 561-566 (1996)) and is thus
suitable for the treatment of Bcr-Abl-positive cancer and tumour
diseases, such as leukaemias (especially chronic myeloid leukaemia
and acute lymphoblastic leukaemia, where especially apoptotic
mechanisms of action are found), and also shows effects on the
subgroup of leukaemic stem cells as well as potential for the
purification of these cells in vitro after removal of said cells
(for example, bone marrow removal) and reimplantation of the cells
once they have been cleared of cancer cells (for example,
reimplantation of purified bone marrow cells).
[0060] Test for activity against c-Abl protein tyrosine kinase. The
test is conducted as a filter binding assay as follows: The
His-tagged kinase domain of c-Abl is cloned and expressed in the
baculovirus/Sf9 system as described by Bhat et al., J. Biol. Chem.
272, 16170-5 (1997). A protein of 37 kD (c-Abl kinase) is purified
by a two-step procedure over a cobalt metal chelate column followed
by an anion exchange column with a yield of 1-2 mg/L of Sf9 cells.
The purity of the c-Abl kinase is >90% as judged by SDS-PAGE
after Coomassie blue staining. The assay contains: c-Abl kinase (50
ng), 20 mM Tris.HCl, pH 7.5, 10 mM MgCl.sub.2, 10 .mu.M
Na.sub.3VO.sub.4, 1 mM DTT and 0.06 .mu.Ci/assay [.gamma..sup.33
P]-ATP (5 .mu.M ATP) using 30 .mu.g/mL poly-Ala,Glu,Lys,Tyr-6:2:5:1
(Poly-AEKY, Sigma P1152) in the presence of 1% DMSO, total volume
of 30 .mu.L. Reactions are terminated by adding 10 .mu.L of 250 mM
EDTA, and 30 .mu.L of the reaction mixture is transferred onto
Immobilon-PVDF membrane (Millipore, Bedford, Mass., USA) previously
soaked for 5 min with methanol, rinsed with water, then soaked for
5 min with 0.5% H.sub.3PO.sub.4 and mounted on vacuum manifold with
disconnected vacuum source. After spotting all samples, vacuum is
connected and each well rinsed with 200 .mu.L 0.5% H.sub.3PO.sub.4.
Membranes are removed and washed on a shaker with 0.5%
H.sub.3PO.sub.4 (4 times) and once with ethanol. Membranes are
counted after drying at ambient temperature, mounting in Packard
TopCount 96-well frame, and addition of 10 .mu.L/well of
Microscint.TM. (Packard).
[0061] Test for activity against Bcr-Abl. The murine myeloid
progenitor cell line 32Dcl3 transfected with the p210 Bcr-Abl
expression vector pGDp210Bcr/Abl (32D-bcr/abl) was obtained from J.
Griffin (Dana Faber Cancer Institute, Boston, Mass., USA). The
cells express the fusion Bcr-Abl protein with a constitutively
active Abl kinase and proliferate growth factor independent. The
cells are expanded in RPMI 1640 (AMIMED), 10% fetal calf serum, 2
mM glutamine (Gibco) ("complete medium"), and a working stock is
prepared by freezing aliquots of 2.times.10.sup.6 cells per vial in
freezing medium (95% FCS, 5% DMSO (SIGMA)). After thawing, the
cells are used during maximally 10-12 passages for the
experiments.
[0062] For cellular assays, compounds are dissolved in DMSO and
diluted with complete medium to yield a starting concentration of
10 .mu.M followed by preparation of serial 3-fold dilutions in
complete medium. 200,000 32D-Bcr/Abl cells in 50 .mu.L complete
medium are seeded per well in 96 well round bottom tissue culture
plates. 50 .mu.L per well of serial 3-fold dilutions of the test
compound are added to the cells in triplicates. Untreated cells are
used as control. The compound is incubated together with the cells
for 90 min at 37.degree. C., 5% CO.sub.2, followed by
centrifugation of the tissue culture plates at 1300 rpm (Beckman
GPR centrifuge) and removal of the supernatants by careful
aspiration taking care not to remove any of the pelleted cells. The
cell pellets are lysed by addition of 150 .mu.L lysis buffer (50 mM
Tris/HCl, pH 7.4, 150 mM sodium chloride, 5 mM EDTA, 1 mM EGTA, 1%
NP-40, 2 mM sodium ortho-vanadate, 1 mM PMSF, 50 .mu.g/mL aprotinin
and 80 pg/mL leupeptin) and either used immediately for the ELISA
or stored frozen in the plates at -20.degree. C. until usage.
[0063] Black ELISA plates (Packard HTRF-96 black plates) are
precoated over night at 4.degree. C. with 50 ng/well of the rabbit
polyclonal anti-abl-SH3 domain Ab 06-466 from Upstate in 50 .mu.L
PBS. After washing 3 times with 200 .mu.L/well PBS containing 0.05%
Tween20 (PBST) and 0.5% TopBlock (Juro), residual protein binding
sites are blocked with 200 .mu.L/well PBST, 3% TopBlock for 4 h at
room temperature followed by incubation with 50 .mu.L lysates of
untreated or compound-treated cells (20 .mu.g total protein per
well) for 3-4 h at 4.degree. C. After 3 washings, 50 .mu.L/well
anti-phosphotyrosine Ab PY20(AP) labeled with alkaline phosphatase
(Zymed) diluted to 0.2 .mu.g/mL in blocking buffer is added and
incubated over night (4.degree. C.). For all incubation steps the
plates are covered with plate sealers (Costar). Finally, the plates
are washed another three times with washing buffer and once with
deionized water before addition of 90 .mu.L/well of the
AP-substrate CDPStar RTU with Emerald II. The plates, now sealed
with Packard TopSeal.TM.-A plate sealers, are incubated for 45 min
at room temperature in the dark and luminescence is quantified by
measuring counts per second (CPS) with a Packard Top Count
Microplate Scintillation Counter (Top Count).
[0064] The difference between the ELISA-readout (CPS) obtained for
with the lysates of the untreated 32D-Bcr/Abl cells and the readout
for the assay-background (all components, but without cell lysate)
is calculated and taken as 100% reflecting the constitutively
phosphorylated Bcr-Abl protein present in these cells. The activity
of the compound on the Bcr-Abl kinase activity is expressed as
percent reduction of the Bcr-Abl phosphorylation. The values for
the IC.sub.50 and IC.sub.90 are determined from the dose response
curves by graphical extrapolation.
[0065] Test for activity against mutant Bcr-Abl: The activity of
compounds on the M351T mutant Bcr-Abl kinase activity is assessed
as described above, except that 32Dcl3 cells transfected with
mutant Bcr-Abl in place of p210 Bcr-Abl are utilised.
[0066] c-Raf-1 protein kinase assay: Recombinant c-Raf-1 protein is
obtained by triple infection of Sf21 cells with GST-c-Raf-1
recombinant baculovirus together with v-Src and v-Ras recombinant
baculoviruses that are required for active c-Raf-1 kinase
production (Williams et al., PNAS 1992; 89:2922-6). Active Ras
(v-Ras) is required to recruit c-Raf-1 to the cell membrane and
v-Src to phosphorylate c-Raf-1 to fully activate it. Cells are
seeded at 2.5.times.10.sup.7 cells per 150 mm dish and allowed to
attach to a 150 mm dish for 1 hr at RT. Media (SF90011 containing
10% FBS) is aspirated and recombinant baculovirus GST-c-Raf-1,
v-Ras and v-Src are added at MOI of 3.0, 2.5 and 2.5, respectively,
in a total volume of 4-5 mL. Cells are incubated for 1 hr at RT and
then 15 mL of medium is added. Infected cells are incubated for
48-72 hr at 27.degree. C. Infected Sf21 cells are scraped and
collected into a 50 mL tube and centrifuged for 10 min at 4.degree.
C. at 1100 g in a Sorvall centrifuge. The cell pellet is washed
once with ice cold PBS and lysed with 0.6 mL lysis buffer per
2.5.times.10.sup.7 cells. Complete lysis of cells is achieved after
10 min on ice with occasional pipetting. The cell lysates are
centrifuged for 10 min at 4.degree. C. at 14,500 g in a Sorvall
centrifuge with SS-34 rotor and the supernatant is transferred to a
fresh tube and stored at -80.degree. C. c-Raf-1 is purified from
cell lysates using 100 .mu.L of packed glutathione-sepharose 4B
beads equilibrated in ice cold PBS per 2.5.times.10.sup.7 cells.
GST-c-Raf-1 is allowed to bind to the beads at 4.degree. C. for 1
hr with rocking. Bound GST-c-Raf-1 with beads is transferred to a
column. The column is washed once with lysis buffer and twice with
ice cold Tris buffered saline. Ice cold elution buffer is added and
column flow is stopped to allow the free glutathione to disrupt the
interaction of GST-c-Raf-1 with glutathione sepharose beads.
Fractions (1 mL) are collected into pre-chilled tubes. Each tube
contains 10% glycerol (final concentration) to maintain kinase
activity during freeze thaw cycles. Purified fractions of
GST-c-Raf-1 kinase protein are stored at -80.degree. C.
[0067] I.kappa.B is used as substrate for the c-Raf-1 kinase.
I.kappa.B is expressed in bacteria as a His-tagged protein BL21.
LysS bacteria containing the I.kappa.B plasmid are grown to an
OD600 of 0.6 in LB medium, then induced to express the I.kappa.B
with IPTG (final concentration of 1 mM) for 3 hrs at 37.degree. C.
and then bacteria are lysed by sonication (microtip limit setting
for 3 times at 1 min each in sonication buffer [50 mM Tris pH 8.0,
1 mM DTT, 1 mM EDTA] and centrifuged at 10,000 g for 15 min. The
supernatant is mixed with ammonium sulfate to give a final
concentration of 30%. This mixture is rocked for 15 min at 4 C then
spun at 10,000 g for 15 min. The pellet is resuspended in binding
buffer (Novagen) containing 10 mM BSA. This solution is applied to
Ni-agarose (Novagen) and washed according to the Novagen manual.
I.kappa.B is eluted from the column using elution buffer (0.4 M
imidazole, 0.2 M NaCl, 8 mM Tris pH 7.9). Fractions containing
protein are dialysed in 50 mM Tris pH 8, 1 mM DTT.
[0068] The activity of c-Raf-1 protein kinase is assayed in the
presence or absence of inhibitors, by measuring the incorporation
of .sup.33P from [.gamma..sup.33P] ATP into I.kappa.B. The assay is
carried out in 96-well plates at ambient temperature for 60 min. It
contains (total volume of 30 .mu.L): c-Raf-1 kinase (400 ng), 25 mM
Tris.HCl, pH 7.5, 5 mM MgCl.sub.2, 5 mM MnCl.sub.2, 10 .mu.M
Na.sub.3VO.sub.4, 1 mM DTT and 0.3 .mu.Ci/assay [.gamma..sup.33
P]-ATP (10 .mu.M ATP) using 600 ng I.kappa.B in the presence of 1%
DMSO. Reactions are terminated by adding 10 .mu.L of 250 mM EDTA
and 30 .mu.L of the reaction mixture is transferred onto
Immobilon-PVDF membrane (Millipore, Bedford, Mass., USA) previously
soaked for 5 min with methanol, rinsed with water, then soaked for
5 min with 0.5% H.sub.3PO.sub.4 and mounted on vacuum manifold with
disconnected vacuum source. After spotting all samples, vacuum is
connected and each well rinsed with 200 .mu.L 0.5% H.sub.3PO.sub.4.
Membranes are removed and washed 4.times. on a shaker with 0.5%
H.sub.3PO.sub.4, once with ethanol. Membranes are counted after
drying at ambient temperature, mounting in Packard TopCount 96-well
frame, and addition of 10 .mu.L/well of Microscint.TM.
(Packard).
[0069] It has also been surprisingly discovered that our compounds
of formula I or II have an unexpected potential to serve as
hypoxia-selective products due to bio-reduction (deoxygenation) in
the cell especially in the tumours and in the brain.
Hypoxia-activated pro-drugs are especially useful in cancer therapy
since severe hypoxia occurs in solid tumour tissue or in the brain.
Hypoxic cells can be exploited for therapy by non-toxic,
hypoxia-activated pro-drugs. Thus because of the net reduction of
the N-oxide moiety, there is an higher uptake of compounds of
formula I or II in the tumours or in the brain and an accumulation
of the reduced form of the compounds of formula I or II in the
tumours or brain.
[0070] Another advantage of the compounds of formula I or II, is
superior effects to carrier-mediated efflux over compound A (by a
saturable system, probably P-gp). Consequences of this less
pronounced efflux is; [0071] a greater absorption [0072] higher
drug levels in the brain and [0073] higher drug levels in the
tumour.
[0074] This effect on P-gp and transport mechanism may be
demonstrated as follows:
[0075] Caco-2 cell monolayers grown on polyethylene terephthalate
(PET) filters (Falcon.TM.) for 21-25 days are used for transport
experiments. The flux of compounds across Caco-2 cell monolayers
grown on PET filters as well as across PET filters alone without
Caco-2 cells (for system validation) in the presence and absence of
the potent efflux pump inhibitors CsA and Verapamil, respectively,
are determined as follows: Prior to the transport experiment, the
culture medium in the acceptor compartment (0.2 ml for apical and
1.0 ml for basolateral sides) is replaced with acceptor solution
(HBSS, when relevant containing the inhibitor of interest)
preincubated at 37.degree. C. To start the experiment, the medium
in the donor compartment (0.35 ml for apical and 1.15 ml for
basolateral sides) is replaced with donor solution (compound in
HBSS, when relevant containing inhibitor of interest) preincubated
at 37.degree. C. Aliquots of 150 .mu.l are removed from the donor
and the acceptor side after about 1 and 120 minutes. Transport
experiments in both apical-to-basolateral and basolateral-to-apical
directions are performed in triplicate at 37.degree. C. in an
incubator without shaking.
[0076] Furthermore, the plasma protein binding of the compounds of
formula I or II is superior with regard to free fraction and/or
association with plasma proteins (e.g. albumin, .alpha.-1-acid
glycoprotein (AAG)), to that observed with compound A. A lower
extent of association to AAG results in less pronounced variability
of free fraction of N-oxides and also has an effect of free
fraction of compound A compounds. At the clinically relevant dose
of 400 mg daily dose (concentrations of 900-2600 ng/mL of compound
A), the free fraction of compound A ranges from 4 to 5%. Using
erythrocyte partitioning, compound A was mainly found associated
with albumin and alpha-1-acid glycoprotein (AAG). The fraction
associated with lipoproteins and gamma globulins was <5%. The
reduced plasma protein-binding of compounds of formula I or II is
shown by the following example.
[0077] The free (or unbound) fraction of compounds of formula I or
II is determined by the ultracentrifugation method which was used
also for compound A (see European patent application No. 1250140 or
International patent application WO 01/47507 filed on 22 Dec.
2000). Solutions of human serum albumin (40 .mu./L) and
.alpha.-1-acid glycoprotein (1 g/L) will be prepared in Soerensen
buffer pH 7.4 containing 0.9% NaCl (w/v). 30 .mu.L of the compounds
of formula I or II stock solutions are directly spiked into 3 mL of
the protein solutions to get the intended final concentrations of
300-5000 ng/mL compounds of formula I or II (ethanol final
concentration 0.5%, factor 1:200). After incubation for 30 min at
37.degree. C. under constant gentle agitation, the spiked protein
samples (n=3/4) are centrifuged at 200,000 g for at least 5 hours h
at 37.degree. C. (centrifuge with a fixed angle rotor) using
thick-walled polycarbonate centrifuge tubes. The spin is stopped
without braking. The concentration of compounds of formula I or II
are determined after incubation (before centrifugation) and after
centrifugation in the supernatant.
[0078] The pharmacokinetics of the compounds of formula I or II are
advantageous over compound A with regard to C.sub.max (highest
observed concentration in plasma in units mass/volume), half-live
(refers to the time after administration of the drug to observe a
diminution of one half of the measured pharmacological response; In
one aspect, the half-life is enhanced when the half-life is
increased by at least 50%) or AUC (plasma concentrations over time,
as defined by the Area Under the Curve (AUC) in units of
mass-time/volume) in plasma as a matter of transport mechanism
(e.g. P-gp). This advantageous pharmacokinetics is shown by giving
to animals (e.g. rats) a single dose of compounds of formula I or
II (one group of animals will be treated intravenously and one
group of animals will be treated per orally). Blood is taken at
selected time points (e.g. 0.083 min (iv: intra venous) and 0.25,
0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h after iv or po (per orally
dosing). Plasma is prepared immediately by centrifugation of blood.
Unchanged compounds of formula I or II and compound A are measured
in plasma using HPLC/UV detection or LC-MS.
[0079] Furthermore, the reduced plasma protein binding of the
compounds of formula I or II to plasma proteins can cause an
increase in the apparent volume of distribution due to higher
fraction of unbound drug (fu). Advantageously, the distribution of
the compounds of formula I or II into organs and tissues (including
the brain) is different from that of compound A. This can be shown
as follows.
[0080] Compounds of formula I or II and compound A in brain of mice
or rats upon dosing with non-radiolabelled N-oxide.
[0081] Animals (e.g. mice or rats) receive a single dose of
compounds of formula I or II (one group of animals is treated
intravenously and one group of animals is treated per orally).
Animals are sacrificed at selected time points (e.g. 0.083 min (iv)
and 1, 8, 24 and 48 h after iv or po dosing). Brain of treated
animals are taken, homogenates of brain are prepared and samples
are prepared (e.g. extraction of homogenate with organic solvent
such as methanol, acetonitrile or others) for analysis (HPLC/UV or
LC-MS) of compounds of formula I or II and compound A.
Concentrations of compounds of formula I or II and compound A in
brain and plasma is measured for determination of ratio
brain/plasma.
[0082] Distribution of radioactive substance(s) in mice or rats
upon dosing with radiolabelled N-oxide.
[0083] For the tissue distribution study, e.g. 10 mg/kg po of
radiolabelled N-oxide (e.g. [.sup.14C]-label; 100 .mu.Ci/kg b.w.)
is administered to animals. The uptake/distribution of radioactive
substance(s) throughout the body of the animal is investigated
using quantitative whole-body autoradioluminography (QWABL). The
animals arer sacrificed at selected time points and frozen in a
mixture of dry-ice and hexane at approx. -75.degree. C. Frozen
animals are embedded in a pre-cooled 2% aqueous gel of Na-CMC, at
approx. -75.degree. C.;. 40 .mu.m thick sections are obtained at
ca. -20.degree. C. in a CryoMacrocut cryomicrotome (Leica Instr.
GmbH, D-Nussloch). Dehydration of sections take place during 24-60
h at -23.degree. C. in the cryomicrotome. Sections exposure to BAS
III Imaging plates (Fuji Photo Film Co., Ltd., J-Tokyo) for 1 day
at room temperature in a lead shielding box to minimize the
increase of the background. The duration of exposure allows
detection of ca. 2 dpm/mg, i.e. the radioactivity concentration
corresponding to ca. 0.2-0.4% of the total radioactive dose if the
radioactivity was evenly distributed throughout the body. Scan is
performed in a Fuji BAS 2000 TR phosphor imager, immediately after
the end of the exposure, under controlled light conditions, at a
100 .mu.m scanning step with a 1024 gradation. Image analysis is
done as follows: The resulting photostimulated light data files are
corrected by subtracting the background, processed electronically
with the help of a MCID/M4 (3.0 Rev. 1.3) image analyzer (Imaging
Research, St. Catherines, Ontario, Canada) and automatically
converted into radioactivity concentrations using a 1st degree
polynomial calibration curve obtained from a radioactive blood
scale processed under similar conditions as the samples. Detection
(LD) and quantitation (QL) limits are determined by LD=mean of
background (n=10)+3 SD; QL=3 LD. The size of the measurement areas
are the same as that of each blood standard of the blood scale used
to set the calibration curve. Image files processed using the Adobe
Photoshop.RTM. software.
[0084] For distribution of total radioactive substance(s) Using
QWABL only half of the treated animal are used which makes possible
to use organs or tissues for determination of unchanged N-oxide
and/or compound A in selected samples using HPLC/UV or
HPLC-radioactivity and/or LC-MS.
[0085] Compounds of formula I or II have also less affinity to
CYP450s [1, 2], because of N-oxides which are more polar. These
enzymes are metabolizing most of the drugs on the market. Less
affinity translates to smaller drug/drug interaction potential.
Especially the blocking of a basic nitrogen like in a
piperazine/pyridine moiety reduce the affinity to CYP2D6 an enzyme
which binds substrates especially by ionic interaction with the
aspartate residue which requires a basic moiety like the nitrogen
in the piperazine ring system. A pool out of ten different human
liver microsomes are incubated with all cofactors necessary for
their metabolic activity (NADPH) with defined marker substrates for
the respective CYP450 isozyme specific activity. The potential
inhibitor is added with increasing concentrations and the metabolic
reactions are evaluated by the corresponding analytical method
(LC/MS, HPLC, Fluorescence). The conversion rate without inhibitor
is set to 100% and the inhibition rate is evaluated as the
concentration of inhibitor needed to suppress 50% of the conversion
(IC50). The following marker substrates are used:
TABLE-US-00001 Substrates CYP Preferred Acceptable 1A2
Ethoxyresorufin Caffeine (low turnover) Phenacetin Theophylline
(low turnover) Acetanilide (mostly applied in hepatocytes)
Methoxyresorufin 2A6 Coumarin 2C8 Paclitaxel (availability of
standards?) 2C9 S-Warfarin Tolbutamide Diclofenac (low turnover)
2C19 S-Mephenytoin (4-hydroxy metabolite) Omeprazole 2D6 Bufuralol
Metoprolol Dextromethorphan Debrisoquine Codeine (all with no
problems, but less commonly used) 2E1 Chlorzoxazone 4-Nitrophenol
Lauric Acid 3A4 Midazolam Nifedipine Testosterone (strongly
Felodipine recommended to use at Cyclosporin least two structurally
Terfenadine unrelated substrates) Erythromycin Simvastatin
[0086] In addition, a compound of formula I or II shows useful
effects in the treatment of disorders arising as a result of
transplantation, for example, allogenic transplantation, especially
tissue rejection, such as especially obliterative bronchiolitis
(OB), i.e. a chronic rejection of allogenic lung transplants. In
contrast to patients without OB, those with OB often show an
elevated PDGF concentration in bronchoalveolar lavage fluids. If a
compound of formula I or II is administered to rats with tracheal
allogenic transplants, for example in a dose of 50 mg/kg i.p., it
can be shown after removal of 10 transplants per group after 10 and
30 days for morphometric analysis of possible epithelial lesions
and occlusion of the airways, and investigation for
immunohistochemical pathways of action that, although a compound of
formula I or II has no significant effect on epithelial necrosis or
infiltration by inflammatory cells, it does markedly reduce
fibroproliferation and occlusion of the lumen compared with
controls. Synergistic effects with other immunomodulatory or
anti-inflammatory substances are possible, for example when used in
combination with cyclosporin A (CsA), rapamycin, or ascomycin, or
immunosuppressant analogues thereof, for example cyclosporin G,
FK-506 or comparable compounds; corticosteroids; cyclophosphamide;
azathioprine; methotrexate; brequinar; leflunomide; mizoribine;
mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualin;
immunsuppressant antibodies, especially monoclonal antibodies for
leucocyte receptors, for example MHC, CD2, CD3, CD4, CD7, CD25,
CD28, B7, CD45, CD58 or their ligands; or other immunomodulatory
compounds, such as CTLA4Ig. If CsA (1 mg/kg s.c.), for example, is
combined with a compound of formula I or II (50 mg/kg), synergism
may be observed.
[0087] A compound of formula I or II is also effective in diseases
associated with vascular smooth-muscle cell migration and
proliferation (where PDGF and PDGF receptor often also play a
role), such as restenosis and atherosclerosis. These effects and
the consequences thereof for the proliferation or migration of
vascular smooth-muscle cells in vitro and in vivo can be
demonstrated by administration of a compound of formula I or II and
also by investigating its effect on the thickening of the vascular
intima following mechanical injury in vivo.
[0088] A compound of formula I or II is used in 0.1N HCl or DMSO at
a concentration of 10 mM for in vitro studies. The stock solution
is further diluted with cell culture medium and used in
concentrations of 10 to 0.1 .mu.M for the experiments. For in vivo
administration, a compound of formula I or II is dissolved for
example in DMSO at a concentration of 200 mg/ml and then diluted
1:20 with 1% Tween in 0.9% saline solution. After sonication, a
clear solution is obtained. The stock solutions are prepared fresh
each day before administration. (The compound of formula I or II
may also be dissolved simply in deionised water for oral
administration or in 0.9% saline solution for parenteral
administration). Administration is carried out 24 hours before the
operation. A compound of formula I or II is administered to rats in
one dose of 50 mg/kg i.p. per day for the entire observation
period. Control rats are given the same formulation but without the
presence of a compound of formula I or II. Oral administration is
also possible.
[0089] Primary cultures of smooth-muscle aorta cells are isolated
from 9 to 11-day-old DA (AG-B4, RT1a) rat aorta using a
modification of the method described by Thyberg et al. (see
Differentiation 25, 156-67 (1983)). The aorta is opened by means of
a longitudinal incision and the endothelium carefully removed. The
adventitia and the tunica media are separated, and the tunica media
is digested with 0.1% collagenase and DNAse in phosphate-buffered
physiological saline for 30 min at 37.degree. C. The cells are
centrifuged, suspended in culture medium, and then allowed to grow
on plastic vials. The primary cells are used for the experiments
after passages 2 to 6. Subcultures are kept in DMEM (Dulbecco's
Modified Eagle's Medium), supplemented with 10% fetal calf serum, 2
mmol/ml glutamine, 100 mmol/ml streptomycin, and 100 IU/m1
penicillin. For identification purposes, the cells are left to grow
on glass slide covers and stained immunohistochemically using an
anti-.alpha.-actin antibody obtained from smooth-muscle cells (see
below).
[0090] The migration of smooth-muscle cells is quantified in vitro
using a Transwell cell culture insert (Costar, Cambridge, Mass.)
whose upper and lower compartments are separated by a polycarbonate
membrane of 8 .mu.l pore size. The cells (100 .mu.l at a
concentration of 1 million cells/ml) are exposed in the upper
compartment. After 2 hours, 60 ng/ml PDGF-BB or PDGF-AA (Upstate
Biotechnology Inc., Lake Placid, N.Y.) is added to the lower
compartment, supplemented with 0.5% fetal calf serum and 0.1%
bovine serum albumin, and the test compound of formula I or II is
added in concentrations of 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003
.mu.M. To measure fibronectin-dependent migration, the Transwell
chambers are covered with fibronectin at a concentration of 10
.mu.g/ml for 24 h at 4.degree. C. (human cellular fibronectin,
Upstate Biotechnology Inc.). After 24 hours' migration, the filters
are removed, fixed in methanol, and stained with Mayer's
haematoxylin and eosin. The migrated cells on the lower side of the
filter membrane are determined by counting the specified sectional
fields on the filters with the aid of a light microscope with a
magnification of 400.times.. The inhibition of migration is
quantified in terms of the percentage of cells versus with the
control. To exclude the possibility of a toxic effect, the
viability of the cells is tested by incorporation of 3H-thymidine
in DMEM, supplemented with 10% fetal calf serum. An inhibition of
migration induced by PDGF-AA and especially by PDGF-BB is observed
with a compound of formula I or II.
[0091] Experimental animals: the aorta and carotid artery of male
Wistar rats (purchased from the Laboratory Animal Centre of the
University of Helsinki, Finland) are denuded. The rats are
anaesthetised with 240 mg/kg chloral hydrate i.p. and Buprenorphine
(Temgesic, Reckitt & Coleman, Hull, UK) is administered for
perioperative and postoperative alleviation of pain. All animals
are given human care in keeping with the "Principles of Laboratory
Animal Care" and the "Guide for the Care and Use of Laboratory
Animals" of the NIH (NIH Publication 86-23, revised 1985). Rats
weighing 200-300 g were used for the denudation procedure. The left
common carotid artery is denuded of endothelium through the
intraluminal passage of a 2F embolectomy catheter (Baxter
Healthcare Corporation, Santa Ana, Calif., 27). To remove the
endothelium, the catheter is passed through the lumen three times,
inflated with 0.2 ml air. The external carotid is ligated after
removal of the catheter and the wound closed. The histological
changes are evaluated by reference to sections of mid-carotid 4
days after denudation. The thoracic aorta is denuded of endothelium
using a 2F Fogarty arterial embolectomy catheter. The catheter is
inserted into the thoracic aorta via the left iliac artery,
inflated with 0.2 ml air, and passed through the lumen five times
to remove the endothelium. The iliac artery is then ligated. Three
times (3, 7 and 14 days) are selected for evaluation of the
histological changes.
[0092] To quantify the proliferating cells, 3 different procedures
are used for labelling the cells with bromodeoxyuridine (BrdU)
after denudation of the rat carotid. In this model, the media cell
proliferation begins 24 h after denudation; cells in the intima
first appear after 72-96 hours. To quantify the proliferation of
smooth-muscle cells before the appearance of cells in the intima,
0.1 ml BrdU-labelling reagent (ZYMED, San Francisco, Calif.) is
administered i.v. during the postoperative period of 0 to 72 h
post-denudation (in total 0.1 ml 6 times). To quantify the
proliferation during the initial wave of migration, the rats were
given 3.times.0.1 ml BrdU-labelling reagent at 8-hour intervals
over a period of 72-96 hours after the operation. To quantify the
proliferation at the end of the initial wave of migration, a third
group of rats is given a pulsed dose of 0.3 ml BrdU three hours
before sacrifice.
[0093] Histological samples are fixed in 3% paraformaldehyde
solution for 4 h for embedding in paraffin. Morphological changes
are evaluated from paraffin sections stained with Mayer's
haematoxylin-eosin. The cell counts of different vessel sections
are calculated at a magnification of 400.times.. To identify cells
in culture and cells appearing in the neo-intima within four days
of the denudation injury, immunohistochemical staining of
acetone-fixed samples is carried out using an anti-.alpha.-actin
antibody obtained from smooth-muscle cells (Bio-Makor, Rehovot,
Israel). Primary smooth-muscle cells are identified on
acetone-fixed glass cover slides using the same staining method.
The sections are incubated with the primary antibody (dilution
1:2000), washed, and incubated consecutively with
peroxidase-conjugated rabbit-antimouse-Ig and goat-antirabbit-Ig,
followed by treatment with substrate solution with the chromogen
3-amino-9-ethylcarbazol and hydrogen peroxide. BrdU stains are
prepared from paraffin sections using a primary mouse antibody
(Bu20a, Dako, A/S, Denmark) and the Vectastain Elite ABC-Kit
(Vector Laboratories, Burliname, Calif.). The sections are
deparaffinised and treated by microwave at 500 W (2.times.5 min in
0.1M citrate buffer, pH 6), followed by treatment with 95%
formamide in 0.15 M trisodium citrate for 45 min at 70.degree. C.
Antibody dilutions are prepared according to the manufacturer's
specifications. The sections are counterstained with Mayer's
haematoxylin and eosin, and positive cells are counted separately
for the initima, media, and adventitia.
[0094] In the carotid of treated animals, a significant decrease is
found in the cell count for smooth-muscle cells. The adventitia and
the media showed a significant reduction in the cell count. As a
result of a compound of formula I or II, a slight decrease in the
absolute number of BrdU-labelled cells is seen in the intima,
media, and adventitia during the first two labelling periods (0-72
and 72-96 h), and after 93-96 h a decrease in the number of
labelled cells is seen in all compartments. Decreases in the number
of smooth-muscle cells are likewise found in the aorta-denuded
animals.
[0095] According to these findings, a compound of formula I or II
can thus inhibit the proliferation, and especially the migration,
of vascular smooth-muscle cells.
[0096] A compound of formula I or II is also capable of inhibiting
angiogenesis. This may be demonstrated as follows: a chamber
containing agar (0.8%) and heparin (2 U/ml) with or without growth
factor (VEGF 3 .mu.g/ml, PDGF 1 .mu.g/ml or bFGF 0.3 .mu.g/ml) is
implanted subcutaneously into normal mice (C57 BL/6). A compound of
formula I or II is administered orally in a dose showing good
anti-tumour activity in a nude mouse xenotransplant model. Dosing
is started one day before implantation of the chambers. The
chambers are removed after 5 days. The angiogenic efficacy is
quantified by measuring both the vascularised tissue which has
grown around the implant and the blood content of this tissue
(external blood). The blood is determined by measuring the
haemoglobin. Although the vessels do not grow into the agar, the
agar becomes intensely red if an antiangiogenic effect is present.
If a compound inhibits the increase in blood that is induced by the
growth factor, this is seen as an indication that the compound in
question is blocking the angiogenic effect of the growth factor
concerned. Inhibition of the weight but not the volume of blood
suggests an effect on the proliferation of fibroblasts. A
suppression of the control response suggests an inhibition of wound
healing. At an oral dose of 50 mg/kg once daily, a compound of
formula I or II inhibits the angiogenic effect of all three growth
factors (VEGF, PDFG, bFGF).
[0097] Interestingly, it was found that
4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-hydroxymethyl-3-[[4-(3-pyridinyl-
)-2-pyrimidinyl]-amino]-phenyl}benzamide,
4-[(4-methyl-4-oxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridiny-
l)-2-pyrimidinyl]-amino]-phenyl}-benzamide and
4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(1-oxido-3-pyridiny-
l)-2-pyrimidinyl]-amino]-phenyl]-benzamide represent metabolites of
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide (STI571 or imatinib, hereinafter
compound A) which can be found in the human body upon
administration of compound A. Compound A is described in EP 0 564
409 B1 and, in the form of the methane sulfonate salt, in WO
99/03854.
[0098] In addition to the before-mentioned metabolites, further
compound A metabolites were identified in monkeys such as
4-[(4-methylcarbonyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridiny-
l)-2-pyrimidinyl]-amino]-phenyl}benzamide,
4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-carboxy-3-[[4-(3-pyridinyl)-2-py-
rimidinyl]-amino]-phenyl}-benzamide,
4-carboxy-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-b-
enzamide,
4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(3-pyridin-
yl)-2-pyrimidinyl]-amino]-phenyl]-benzamide wherein the pyridinyl
moiety is substituted at a ring carbon atom by hydroxy, and
4-[(1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-
-amino]-phenyl]-benzamide wherein the pyridinyl moiety is
substituted at a ring carbon atom by hydroxy.
[0099] Preference is given to compounds of formula II, wherein
[0100] R.sub.1 is hydrogen,
[0101] R.sub.2 is methyl or hydroxymethyl,
[0102] R.sub.3 is methyl, and
[0103] the stars indicate the nitrogen atoms which optionally carry
an oxygen atom to form the corresponding N-oxides,
[0104] with the proviso that at least one of the three nitrogen
atoms marked by a star carries an oxygen atom if R.sub.2 is
methyl,
[0105] or salts of such compounds.
[0106] Special preference is further given to compounds of formula
II, wherein
[0107] R.sub.1 is hydrogen,
[0108] R.sub.2 is hydroxy-lower alkyl,
[0109] R.sub.3 is methyl, and
[0110] the stars indicate the nitrogen atoms which optionally carry
an oxygen atom to form the corresponding N-oxides,
[0111] or salts of such compounds.
[0112] Especially preferred are the compounds selected from
4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-hydroxymethyl-3-[[4-(3-pyridinyl-
)-2-pyrimidinyl]-amino]-phenyl}-benzamide,
4-[(4-methyl-4-oxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridiny-
l)-2-pyrimidinyl]-amino]-phenyl}-benzamide and
4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(1-oxido-3-pyridiny-
l)-2-pyrimidinyl]-amino]-phenyl]-benzamide, and pharmaceutically
acceptable salts of these compounds.
[0113] Very special preference is further given to a compound of
formula I or II mentioned in the Examples below, or a salt,
especially a pharmaceutically acceptable salt, thereof.
[0114] The compounds of formula I or II or salts thereof are
prepared in accordance with processes known per se, though not
previously described for the manufacture of the compounds of the
formula I or II, especially whereby
[0115] a) a compound of formula III
##STR00004##
[0116] wherein R.sub.1 and R.sub.2 have the meanings given under
formula I, is reacted with a compound of formula IV
##STR00005##
[0117] wherein A has the meanings given under formula I.
[0118] and a compound thus obtained is converted into a N-oxide of
formula I with a suitable oxidizing agent or if not converted into
a N-oxide, A has to be substituted by oxo on a ring carbon;
[0119] preferably,
[0120] a compound of formula Ill
##STR00006##
[0121] wherein R.sub.1 and R.sub.2 have the meanings given under
formula II and the star indicates a nitrogen atom which optionally
carries an oxygen atom, is reacted with a compound of formula
IV
##STR00007##
[0122] wherein R.sub.3 has the meanings given under formula II and
the stars indicate the nitrogen atoms which optionally carry an
oxygen atom;
[0123] and a compound thus obtained is optionally converted into a
N-oxide of formula II with a suitable oxidizing agent; or
[0124] b) a compound of formula V
##STR00008##
[0125] wherein R.sub.1 and R.sub.2 have the meanings given under
formula I, Hal is halo (e.g. --Cl, --Br, --F, --I), is reacted with
a compound of formula VI
AH (VI),
[0126] wherein A has the meanings given under formula I,
[0127] and a compound thus obtained is optionally converted into a
N-oxide of formula I with a suitable oxidizing agent;
[0128] preferably, a compound of formula V
##STR00009##
[0129] wherein R.sub.1 and R.sub.2 have the meanings given under
formula II, Hal is halo (e.g. --Cl, --Br, --F, --I) and the star
indicates a nitrogen atom which optionally carries an oxygen atom,
is reacted with a compound of formula VI
##STR00010##
[0130] wherein R.sub.3 has the meanings given under formula II and
the stars indicate the nitrogen atoms which optionally carry an
oxygen atom;
[0131] and a compound thus obtained is optionally converted into a
N-oxide of formula II with a suitable oxidizing agent;
[0132] whereby functional groups which are present in the starting
compounds of process a) or b) and are not intended to take part in
the reaction, are present in protected form if necessary, and
protecting groups that are present are cleaved, whereby the said
starting compounds may also exist in the form of salts provided
that a salt-forming group is present and a reaction in salt form is
possible;
[0133] and, if so desired, a compound of formula I or II obtained
by process a) or b) is converted into another compound of formula I
or II, an obtained free compound of formula I or II is converted
into a salt, an obtained salt of a compound of formula I or II is
converted into the free compound or another salt, and/or a mixture
of isomeric compounds of formula I or II is separated into the
individual isomers.
[0134] In a most preferred embodiment, compounds of formula I or II
are in a substantially pure form.
[0135] The term "substantially pure" is understood in the context
of the present invention to mean substantially free of biological
material such as found in the blood, especially less than 10%,
preferably less than 1%, and most preferably free of such
biological material.
[0136] Description of the Process Variants
[0137] A suitable oxidizing agent for converting a compound
obtained by process a) or b) into a N-oxide of formula I or II is
preferably hydrogen peroxide or a suitable peracid, for example a
suitable perbenzoic acid, such as especially m-chloro-perbenzoic
acid. The reaction is carried out in an inert solvent, for example
a halogenated hydrocarbon, such as dichloromethane, at temperatures
of approximately from -20.degree. C. to the boiling point of the
solvent in question, in general below +100.degree. C. If hydrogen
peroxide is used as the oxidizing agent, the reaction is preferably
carried out in water at about room temperature. The desired N-oxide
can then be purified using conventional methods such as e.g. column
chromatography or recrystallisation.
[0138] On the other hand, the N-oxides of formula I or II may be
prepared according to the process described in the preceding
paragraph by already oxidizing the starting materials used in the
synthesis of compounds of formula I or II.
[0139] Regarding Process a):
[0140] The reaction between a compound of formula III and a
compound of formula IV preferably takes place in a suitable inert
solvent, especially N,N-dimethylformamide, in the presence of
propylphosphonic anhydride (Fluka, Buchs, Switzerland) and a base
such as especially triethylamine, preferably at room
temperature.
[0141] Regarding Process b):
[0142] The reaction between a compound of formula V and a compound
of formula IV preferably takes place in a suitable inert solvent,
especially alcohols, e.g. lower alcohols such as especially
ethanol, at elevated temperature, preferably near the boiling
temperature of the solvent employed.
[0143] Halo present in a compound of formula V is e.g. fluoro,
chloro, bromo and iodo, preferably chloro.
[0144] Additional Process Steps
[0145] In the additional process steps, carried out as desired,
functional groups of the starting compounds which should not take
part in the reaction may be present in unprotected form or may be
protected for example by one or more protecting groups. The
protecting groups are then wholly or partly removed according `to
one of the known methods.
[0146] Protecting groups, and the manner in which they are
introduced and removed are described, for example, in "Protective
Groups in Organic Chemistry", Plenum Press, London, New York 1973,
and in "Methoden der organischen Chemie", Houben-Weyl, 4th edition,
Vol. 15/1, Georg-Thieme-Verlag, Stuttgart 1974 and in Theodora W.
Greene, "Protective Groups in Organic Synthesis", John Wiley &
Sons, New York 1981. A characteristic of protecting groups is that
they can be removed readily, i.e. without the occurrence of
undesired secondary reactions, for example by solvolysis,
reduction, photolysis or alternatively under physiological
conditions.
[0147] The end products of formula I or II may however also contain
substituents that can also be used as protecting groups in starting
materials for the preparation of other end products of formula I or
II. Thus, within the scope of this text, only a readily removable
group that is not a constituent of the particular desired end
product of formula I is designated a "protecting group", unless the
context indicates otherwise.
[0148] General Process Conditions
[0149] All process steps described here can be carried out under
known reaction conditions, preferably under those specifically
mentioned, in the absence of or usually in the presence of solvents
or diluents, preferably those that are inert to the reagents used
and able to dissolve them, in the absence or presence of catalysts,
condensing agents or neutralising agents, for example ion
exchangers, typically cation exchangers, for example in the
protonated (H.sup.+--) form, depending on the type of reaction
and/or reactants at reduced, normal, or elevated temperature, for
example in the range from -100.degree. C. to about 190.degree. C.,
preferably from about -80.degree. C. to about 150.degree. C., for
example at -80 to -60.degree. C., at RT, at -20 to 40.degree. C.,
at 0 to 100.degree. C. or at the boiling point of the solvent used,
under atmospheric pressure or in a closed vessel, if need be under
pressure, and/or in an inert, for example an argon or nitrogen,
atmosphere.
[0150] The invention relates also to those embodiments of the
process in which one starts from a compound obtainable at any stage
as an intermediate and carries out the missing steps, or breaks off
the process at any stage, or forms a starting material under the
reaction conditions, or uses said starting material in the form of
a reactive derivative or salt, or produces a compound obtainable by
means of the process according to the invention under those process
conditions, and further processes the said compound in situ. In the
preferred embodiment, one starts from those starting materials
which lead to the compounds described hereinabove as preferred.
[0151] In the preferred embodiment, a compound of formula I or II
is prepared according to the processes and process steps defined in
the Examples.
[0152] The compounds of formula I or II, including their salts, are
also obtainable in the form of hydrates, or their crystals can
include for example the solvent used for crystallisation (present
as solvates).
[0153] Starting Materials
[0154] New starting materials and/or intermediates, as well as
processes for the preparation thereof, are likewise the subject of
this invention. In the preferred embodiment, such starting
materials are used and reaction conditions so selected as to enable
the preferred compounds to be obtained.
[0155] The starting materials used in the above described process
are known, capable of being prepared according to known processes
(see also EP 0 564 409 B1), or commercially obtainable; in
particular, they can be prepared using processes as described in
the Examples.
[0156] In the preparation of starting materials, existing
functional groups which do not participate in the reaction should,
if necessary, be protected. Preferred protecting groups, their
introduction and their removal are described above or in the
Examples. In place of the respective starting materials and
transients, salts thereof may also be used for the reaction,
provided that salt-forming groups are present and the reaction with
a salt is also possible. Where the term starting materials is used
hereinbefore and hereinafter, the salts thereof are always
included, insofar as reasonable and possible.
[0157] A compound of formula III wherein R.sub.2 is lower alkyl and
the nitrogen atom marked by a star does not carry an oxygen atom as
a substituent can be prepared as described in EP 0 564 409 B1. Such
compounds may then be converted into the corresponding N-oxides
using a suitable oxidizing agent as described above under
"Description of the process variants".
[0158] A compound of formula III wherein R.sub.2 is hydroxy-lower
alkyl can be prepared analogously to Example 1 by starting with a
compound of the following formula VII:
##STR00011##
[0159] The remaining starting materials are known, capable of being
prepared according to known processes like those described in e.g.
EP 0 564 409 B1, or commercially available; or in particular, they
can be prepared using processes as described in the Examples.
Suitable N-phenyl -2-pyrimidine-amine derivatives to form the
corresponding N-oxides are also described in e.g. EP 0 564 409
B1.
[0160] The invention relates also to a process for the treatment of
warm-blooded animals, including humans, suffering from said
diseases, especially a tumour disease, wherein a quantity of a
compound of formula I or II which is effective against the disease
concerned, especially a quantity with antiproliferative and
especially tumour-inhibiting efficacy, is administered to
warm-blooded animals, including humans, in need of such treatment.
The invention relates moreover to the use of a compound of formula
I or II for the inhibition of the above-mentioned tyrosine kinases,
especially PDGF receptor kinase, v-Abl kinase, and/or c-Kit
receptor kinase, or for the preparation of pharmaceutical
compositions for use in treating warm-blooded animals, including
humans, especially for the treatment of tumours, such as gliomas,
ovarian tumours, prostate tumours, colon tumours, and tumours of
the lung, such as especially small cell lung carcinoma, and tumours
of the breast or other gynaecological tumours. Depending on
species, age, individual condition, mode of administration, and the
clinical picture in question, effective doses, for example daily
doses of about 1-2500 mg, preferably 1-1000 mg, especially 5-500
mg, are administered to warm-blooded animals, including humans, of
about 70 kg bodyweight.
[0161] Thus, in a further aspect, the present invention relates to
the use of N-phenyl -2-pyrimidine-amine derivatives in which at
least one nitrogen atom carries an oxygen atom to form the
corresponding N-oxides or a pharmaceutically acceptable salt of
such a compound for the preparation of a pharmaceutical composition
for the treatment of a proliferative disorder.
[0162] Preferably, the present invention relates to the use of a
compound of formula I or II or a pharmaceutically acceptable salt
of such a compound for the preparation of a pharmaceutical
composition for the treatment of a proliferative disorder.
[0163] Most preferably, the proliferative disorder is selected from
tumors or brain proliferative disorders.
[0164] The invention further provides a method of treating
warm-blooded animals, including humans, which comprises
administering to such a warm-blooded animal suffering from a
proliferative disorder, in a dose effective against said disorder,
a compound of formula I or II or a pharmaceutically acceptable salt
of such a compound.
[0165] In still another embodiment, the instant invention provides
a pharmaceutical composition comprising at least one N-phenyl
-2-pyrimidine-amine derivative in which at least one nitrogen atom
carries an oxygen atom to form the corresponding N-oxides or a
pharmaceutically acceptable salt thereof, together with a
pharmaceutically acceptable carrier.
[0166] Preferably, the instant invention provides a pharmaceutical
composition comprising a compound of formula I or II or a
pharmaceutically acceptable salt thereof, together with a
pharmaceutically acceptable carrier.
[0167] The compositions of the present invention may contain at
least one additional pharmaceutically active compound such as
4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin--
2-ylamino)phenyl]-benzamide.
[0168] Preferably a pharmaceutical composition for the treatment of
a proliferative disorder in warm-blooded animals, including humans,
comprising as an active ingredient a compound of formula I or II
according or a pharmaceutically acceptable salt of such a compound,
together with a pharmaceutically acceptable carrier.
[0169] Thus the invention relates also to pharmaceutical
compositions comprising as an active ingredient a compound of
formula I or II together with a pharmaceutically acceptable
carrier, especially for the prevention or treatment of one of the
said diseases, said pharmaceutical compositions being suitable for
e.g. topical, enteral, for example oral or rectal, or parenteral
administration. Especially tablets or gelatin capsules containing
the active substance together with diluents, for example lactose,
dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycerin,
and/or lubricants, for example silica, talc, stearic acid, or salts
thereof, typically magnesium or calcium stearate, and/or
polyethylene glycol, are used for oral administration. Tablets may
likewise contain binders, for example magnesium aluminium silicate,
starches, typically corn, wheat or rice starch, gelatin,
methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone, and, if so desired, disintegrants, for
example starches, agar, alginic acid, or a salt thereof, typically
sodium alginate, and/or effervescent mixtures, or adsorbents,
colouring agents, flavours, and sweetening agents. The
pharmacologically active compounds of the present invention may
further be used in the form of preparations for parenteral
administration or infusion solutions. Such solutions are preferably
isotonic aqueous solutions or suspensions, these possibly being
prepared before use, for example in the case of lyophilised
preparations containing the active substance either alone or
together with a carrier, for example mannitol. The pharmaceutical
substances may be sterilised and/or may contain excipients, for
example preservatives, stabilisers, wetting agents and/or
emulsifiers, solubilisers, salts for the regulation of osmotic
pressure, and/or buffers. The present pharmaceutical compositions
which, if so desired, may contain further pharmacologically active
substances, such as other c-Kit inhibitors or inhibitors of VEGF
receptor or c-Src activity, are prepared in a manner known r se,
for example by means of conventional mixing, granulating, coating,
dissolving or lyophilising processes, and contain from about 1% to
100%, especially from about 1% to about 20%, of the active
substance or substances.
EXAMPLES
[0170] The following Examples illustrate the invention but do not
limit the scope thereof in any way.
[0171] Abbreviations:
[0172] DMF N,N-Dimethylformamide
[0173] h hour(s)
[0174] min minute(s)
[0175] m.p. melting point
[0176] RT room temperature
[0177] THF tetrahydrofuran
Example 1
4-[(4-Methyl-1-piperazinyl)-methyl]-N-{4-hydroxymethyl-3-[[4-(3-pyridinyl)-
-2-pyrimidinyl]-amino]-phenyl}-benzamide
[0178] A solution of propylphosphonic anhydride in
N,N-dimethylformamide (Fluka, Buchs, Switzerland; 350 .mu.L of 50%,
0.6 mmol) is added in portions over 20 min to a stirred mixture of
N-(5-amino-2-hydroxymethyl-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine
(117 mg, 0.4 mmol), 4-[(4-methyl-1-piperazinyl)-methyl]-benzoic
acid dihydrochloride (123 mg, 0.4 mmol) and triethylamine (445
.mu.L, 3.2 mmol) in dry N,N-dimethylformamide (5 mL). The mixture
is stirred for 24 h at RT. The solvent is evaporated off under
reduced pressure and the residue is treated with saturated aqueous
sodium hydrogen carbonate solution (20 mL) and extracted with ethyl
acetate (2.times.20 mL). The combined extracts are washed with
saturated aqueous sodium chloride (15 mL), dried (MgSO.sub.4),
filtered and the solvent is evaporated off under reduced pressure
to yield the crude product which is purified by reverse phase high
pressure liquid chromatography (Nagel Polygoprep C.sub.18, 7 .mu.m,
300 .ANG.; Macherey-Nagel, Duren, Germany), eluent 0.1%
trifluoroacetic acid in water -0.1% trifluoroacetic acid in
acetonitrile. The fractions containing the pure product are
combined, basified with saturated aqueous sodium hydrogen carbonate
and evaporated to dryness under reduced pressure. The residue is
treated with saturated aqueous sodium hydrogen carbonate and
extracted with ethyl acetate (5.times.). The combined extracts are
washed with water, dried (MgSO.sub.4), filtered and the solvent is
evaporated off under reduced pressure to yield the product which is
recrystallised from methanol--ethyl acetate to give the title
compound as a pale-yellow crystalline solid, m.p. 196-198.degree.
C.
[0179] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.): 2.14 (s, 3H),
2.25-2.45 (m, 8H), 3.52 (s, 2H), 4.56 (s, 2H), 5.50 (br.s, 1H),
7.29 (d, J=8.3 Hz, 1H), 7.41 (dd, J=2.0, 8.3 Hz, 1H), 7.44 (d,
J=8.1 Hz, 2H), 7.50 (d, J=5.1 Hz, 1H), 7.52 (dd, J=3.3, 8.1 Hz,
1H), 7.93 (d, J=8.1 Hz, 2H), 8.56 (d, J=2.0 Hz, 1H), 8.57 (d, J=5.1
Hz, 1H), 8.59 (ddd, J=1. 4, 2.1, 8.1 Hz, 1H), 8.69 (dd, J=1.4, 3.3
Hz, 1H), 9.10 (s, 1H), 9.33 (d, J=2.1 Hz, 1H) and 10.22 (s,
1H).
Step 1.1: 2-Amino-4-nitrobenzenemethanol
[0180] A stirred solution of 2-amino-4-nitrobenzoic acid (Aldrich;
18.2 g, 100 mmol) in dry THF (500 mL) at 20.degree. C., is treated
with a solution of borane-THF complex (BH.sub.3.THF; Fluka; 100 mL
of 1.0 M), dropwise over 45 min to regulate the gas evolution. The
mixture is then heated at 65.degree. C. for 2 h. The stirred
mixture is then cooled to 0.degree. C., treated with water (20 mL)
and warmed to RT. Upon the cessation of gas evolution, hydrochloric
acid (20 mL of 12 M) is added and the mixture is then heated at
65.degree. C. for 30 min. The cooled mixture is then concentrated
to a volume of circa 150 mL by rotary evaporation under reduced
pressure to give a suspension. The suspension is filtered and the
precipitate is redissolved in ethyl acetate (500 mL) and washed
with saturated aqueous sodium hydrogen carbonate (2.times.150 mL).
The solution is dried (Na.sub.2SO.sub.4), filtered and the solvent
is evaporated off under reduced pressure to yield the crude product
which is purified by recrystallisation from ethyl acetate-hexane to
give the title compound as a yellow crystalline solid, m.p.
126-128.degree. C.
Step 1.2: 2-[(2-Propenyloxy)-methyl-5-nitrobenzenamine
[0181] A stirred solution of 2-amino-4-nitrobenzenemethanol (14.3
g, 85 mmol) in dry THF (350 mL) at 0.degree. C. under an argon
atmosphere, is treated dropwise over 35 min with a solution of
potassium tent-butylate in THF (Fluka; 85 mL of 1.0 M). The mixture
is stirred at 0.degree. C. for 15 min and then treated dropwise
over 50 min with a solution of allylbromide (7.9 mL, 94 mmol) in
dry THF (80 mL) at 0.degree. C. and then stirred at 20.degree. C.
for 90 min. The mixture is diluted with ethyl acetate (800 mL). The
resulting solution is washed with saturated aqueous ammonium
chloride (3.times.400 mL), dried (MgSO.sub.4), filtered and the
solvent is evaporated off under reduced pressure to yield the crude
product which is purified by column chromatography on silica gel,
eluent 50% ethyl acetate in hexane, to give the title compound as a
brown oil.
[0182] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.): 4.06 (d, J=5.3
Hz, 2H); 4.47 (s, 2H); 5.22 and 5.34 (dd, J=10.4, 17.3 Hz, 2H);
5.65 (br.s, 2H); 5.98 (m, J=5.3, 10.4, 17.3 Hz, 1H), 7.35 (d, J=8.3
Hz, 1H), 7.38 (dd, J=2.0, 8.3 Hz, 1H) and 7.52 (d, J=2.0 Hz,
1H).
Step 1.3: {2-[(2-Propenyloxy)-methyl]-5-nitrophenyl}-guanidine
[0183] Nitric acid (1.04 mL of 65%, 15 mmol) is added to a stirred
solution of 2-[(2-propenyloxy)-methyl]-5-nitrobenzenamine (3.15 g,
15 mmol) in ethanol (30 mL) at 20.degree. C. A solution of
cyanamide (0.95 g, 22.5 mmol) in water (1 mL) is then added
dropwise to the stirred mixture at 95.degree. C. over a period of
60 min. The mixture is heated at 95.degree. C. for 14 h, with
additional aliquots of cyanamide (total 2.2 g, 58 mmol) being added
throughout this period and with the acidity being periodically
adjusted to pH 3 by the addition of nitric acid (65%). The
resulting mixture is cooled to 0.degree. C., basified with aqueous
ammonia (5 mL of 25%), diluted with water (150 mL) and extracted
with ethyl acetate (3.times.100 mL). The combined extracts are
washed with saturated aqueous ammonium chloride (50 mL), dried
(MgSO.sub.4), filtered and the solvent is evaporated off under
reduced pressure to give the title compound as a brown oil, which
is used directly in the next step without further purification.
Step 1.4:
N-{2-[(2-Propenyloxy)-methyl-5-nitro-phenyl}-4-(3-pyridinyl)-2-p-
yrimidinamine
[0184] A stirred mixture of
{2-[(2-propenyloxy)-methyl]-5-nitro-phenyl}-guanidine (3.75 g, 15
mmol), 3-(dimethylamino)-1-(3-pyridinyl)-2-propen-1-one (2.60 g, 15
mmol) and ethyl diisopropylamine (2.6 mL, 15 mmol) in 1-butanol (50
mL) is heated at 120.degree. C. for 20 h. The solvent is then
evaporated off under reduced pressure to give a residue which is
dissolved in ethyl acetate (100 mL). The resulting mixture is
filtered (celite), washed with saturated aqueous sodium chloride
(50 mL), dried (MgSO.sub.4), filtered and the solvent is evaporated
off under reduced pressure to yield the crude product which is
purified by column chromatography on silica gel, eluent ethyl
acetate, and recrystallised from ethyl acetate-hexane to give the
title compound as a yellow crystalline solid, m.p. 213-215.degree.
C.
[0185] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.): 4.10 (d, J=5.3
Hz, 2H); 4.77 (s, 2H); 5.22 and 5.35 (dd, J=10.4, 17.3 Hz, 2H);
5.96 (m, J=5.3, 10.4, 17.3 Hz, 1H), 7.58 (dd, J=4.8, 7.9 Hz, 1H),
7.66 (d, J=8.4 Hz, 1H), 7.69 (d, J=5.2 Hz, 1H), 7.95 (ddd, J=1.2,
1.2, 7.9 Hz, 1H), 8.51 (dd, J=1.6, 8.4 Hz, 1H), 8.67 (d, J=5.2 Hz,
1H), 8.73 (dd, J=1.2, 4.8 Hz, 1H), 9.05 (d, J=1.2 Hz, 1H), 9.23
(br.s, 1H) and 9.35 (d, J=1.6 Hz, 1H).
Step 1.5:
N-(2-Hydroxymethyl-5-nitro-phenyl)-4-(3-pyridinyl)-2-pyrimidinam-
ine
[0186] Polymethylhydrosiloxane (860 mg),
tetrakis(triphenylphoshine)palladium (70 mg) and zinc chloride
(2.66 mL of 0.5 M in THF, 1.33 mmol) is added to a stirred solution
of
N-{2-[2-propenyloxy)-methyl]-5-nitro-phenyl}-4-(3-pyridinyl)-2-pyrimidina-
mine (2.60 g, 7.2 mmol) in dry THF (60 mL). The mixture is then
stirred under an argon atmosphere at 30.degree. C. for 30 h. The
solvent is then evaporated off under reduced pressure to give a
residue which is treated with saturated aqueous sodium chloride
solution (50 mL) and extracted with ethyl acetate (3.times.50 mL).
The combined extracts are dried (Na.sub.2SO4), filtered and the
solvent is evaporated off under reduced pressure to yield the crude
product which is recrystallised from THF to give the title compound
as a pale-yellow crystalline solid, m.p. 247-250.degree. C.
Step 1.6:
N-(5-Amino-2-hydroxymethyl-phenyl)-4-(3-pyridinyl)-2-pyrimidinam-
ine
[0187] A solution of
N-(2-hydroxymethyl-5-nitro-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine
(0.23 g, 0.71 mmol) in ethanol (230 mL) is hydrogenated at
atmospheric pressure over Raney nickel (0.2 g) at 25.degree. C. The
calculated amount of hydrogen is taken up in 13 h. The mixture is
then filtered and the solvent is evaporated off under reduced
pressure to yield the crude product which is purified by column
chromatography on silica gel, eluent 25% aqueous
ammonia-ethanol-dichloromethane (1:9:90), to give the title
compound as a yellow crystalline solid, m.p. 213-215.degree. C.
[0188] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.): 4.42 (d, J=5.1
Hz, 2H), 5.05 (br.s, 2H), 5.26 (t, J=5.1 Hz, 1H), 6.23 (dd, J=2.1,
8.0 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 7.35 (d, J=2.0 Hz, 1H), 7.45
(d, J=5.1 Hz, 1H), 7.56 (dd, J=4.7, 8.0 Hz, 1H), 8.47 (ddd, J=1.8,
1.8, 8.0 Hz, 1H), 8.53 (d, J=5.1 Hz, 1H), 8.70 (dd, J=1.4, 4.7 Hz,
1H), 8.88 (s, 1H) and 9.29 (d, J=2.4 Hz, 1H).
Example 2
4-[(4-Methyl-4-oxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl-
)-2-pyrimidinyl]-amino]-phenyl}-benzamide
[0189] 3-Chloroperoxybenzoic acid (Fluke, Buchs, Switzerland; 2.06
g of 55%, 4.27 mmol) is added to a stirred mixture of
4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyr-
imidinyl]-amino]-phenyl}-benzamide (prepared as described in EP 0
564 409 B1, Example 21; 2.00 g, 4.05 mmol) in dichloromethane (70
mL) at -20.degree. C. The resulting mixture is then stirred at RT
for 72 h. The solvent is then evaporated off under reduced pressure
to yield a mixture which is purified by column chromatography on
silica gel, eluent dichloromethane-methanol-water (70:30:5), to
give the title compound as a yellow crystalline solid, m.p.
154-158.degree. C.
Example 3
4-[(4-Methyl-1-piperazinyl)-methyl-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-
-2-pyrimidinyl]-amino]-phenyl]-benzamide
[0190] N-Methylpiperazine (99 mg, 1.0 mmol) is added to a stirred
suspension of
4-chloromethyl-N[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amin-
o]phenyl]-benzamide (220 mg, 0.49 mmol) in ethanol (5 mL). The
mixture is then stirred at 100.degree. C. for 15 h to give a
solution, which is then cooled to RT and treated with ethyl acetate
(200 mL). The resulting solution is washed with aqueous sodium
hydroxide (100 mL of 2M) and saturated aqueous sodium chloride
solution (100 mL), dried (Na.sub.2SO.sub.4), filtered and the
solvent is evaporated off under reduced pressure to yield the crude
product which is purified by column chromatography on silica gel,
eluent 25% aqueous ammonia-methanol-dichloromethane (0.5:10:90) to
give the title compound as a yellow crystalline solid, m.p.
232-235.degree. C.
Step 3.1:
N-[4-Methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]amino]-phe-
nyl]-benzamide
[0191] Utilising the procedure described in Example 2, but
employing
N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide
(prepared as described in EP 0 564 409 B1, Example 20) in place of
4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyr-
imidinyl]-amino]phenyl}benzamide, afforded the title compound which
is purified by column chromatography on silica gel, eluent 10%
methanol in dichloromethane, and recrystallised from ethanol to
give the title compound as a pale-yellow crystalline solid, m.p.
258-260.degree. C.
Step 3.2:
4-Methyl-N-3-[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl-1,3-benzened-
iamine
[0192] Hydrochloric acid (9 mL of 4M) is added to a suspension of
N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]phenyl]benza-
mide (0.43 g, 1.08 mmol) in n-propanol (9 mL) and the resulting
mixture is heated at 100.degree. C. for 34 h. The cooled mixture is
evaporated under reduced pressure to give an oil, which is
dissolved in water (10 mL), filtered and basified with aqueous
sodium hydroxide (4 M). The resulting precipitate is filtered,
washed with water and dried to yield the crude product, which is
recrystallised from ethanol to give the title compound as a yellow
crystalline solid, m.p. 104-106.degree. C.
Step 3.3:
4-Chloromethyl-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimid-
inyl]-amino]-phenyl]-benzamide
[0193] A solution 4-(chloromethyl)-benzoyl chloride (Fluka, Buchs,
Switzerland; 184 mg, 0.977 mmol) in dioxane (2 mL) is added
dropwise to a solution of
4-methyl-N-3-[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-1,3-benzenediamine
(275 mg, 0.937 mmol) in dioxane (5 mL) and the mixture is stirred
at 20.degree. C. for 75 min. A second portion of
4-(chloromethyl)-benzoyl chloride (60 mg, 0.317 mmol) dissolved in
dioxane (1 mL) is then added and the mixture is stirred for a
further 120 min. The resulting suspension is treated with ethyl
acetate (50 mL) to give a solution which is washed with aqueous
sodium hydroxide (2.times.50 mL of 2M). The ethyl acetate solution
is dried (Na.sub.2SO.sub.4), filtered and the solvent is evaporated
off under reduced pressure to yield the crude product which is
purified by column chromatography on silica gel, eluent 5% methanol
in dichloromethane to give the title compound as a yellow
crystalline solid, m.p. 224-226.degree. C.
Example 4
4-[(4-Methyl-1,4-dioxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyrid-
inyl)-2-pyrimidinyl-amino]-phenyl}-benzamide
[0194]
4-[(4-Methyl-1-piperazinyl)-methyl]-N-{4-methyl-34[4-(3-pyridinyl)--
2-pyrimidinyl]-amino]-phenyl}benzamide monomethanesulphonate (3.00
g, 5 mmol; prepared as described in WO 99/03854) is added to
aqueous hydrogen peroxide (30 mL of 3%) and the resulting solution
is stirred at 20.degree. C. for 160 h. The pH of the solution is
then adjusted to pH 14 with aqueous sodium hydroxide (4 M) and the
resulting suspension is stirred for 1.5 h. The crude product is
filtered off, washed with water, dried and purified by column
chromatography on silica gel, eluent 25% aqueous
ammonia-ethanol-dichloromethane (5:30:70), to give the title
compound as a yellow crystalline solid, m.p. 242-244.degree. C.
Example 5
4-[(4-methyl-1-piperazinyl)methyl]-N-[4-hydroxymethyl-3-[[4-(3-pyridinyl)--
2-pyrimidinyl]amino]phenyl]benzamide
[0195] A solution of propylphosphonic anhydride in
N,N-dimethylformamide (Fluka, Buchs, Switzerland; 350 .mu.L of 50%,
0.6 mmol) is added in portions over 20 minutes to a stirred mixture
of
N-[2-[5-amino-(2-hydroxy)methyl]phenyl]-4-(3-pyridinyl)-2-pyrimidinamine
as described in example 1, step 1.6 (117 mg, 0.4 mmol),
4-[(4-methyl-1-piperazinyl)methyl]benzoic acid, dihydrochloride
(123 mg, 0.4 mmol) and triethylamine (445 .mu.L, 3.2 mmol) in dry
N,N-dimethylformamide (5 mL). The mixture is stirred for 24 hours
at room temperature. The solvent is evaporated off under reduced
pressure and the residue is treated with saturated aqueous sodium
hydrogen carbonate solution (20 mL) and extracted with ethyl
acetate (2.times.20 mL). The combined extracts are washed with
saturated aqueous sodium chloride (15 mL), dried (MgSO.sub.4),
filtered and the solvent is evaporated off under reduced pressure
to yield the crude product which is purified by reverse phase high
pressure liquid chromatography (Nagel Polygoprep C.sub.18, 7 .mu.m,
300 .ANG.; Macherey-Nagel, Duren, Germany), eluent 0.1%
trifluoroacetic acid in water -0.1% trifluoroacetic acid in
acetonitrile. The fractions containing the pure product are
combined, basified with saturated aqueous sodium hydrogen carbonate
and evaporated to dryness under reduced pressure. The residue is
treated with saturated aqueous sodium hydrogen carbonate and
extracted with ethyl acetate (5.times.). The combined extracts are
washed with water, dried (MgSO.sub.4), filtered and the solvent is
evaporated off under reduced pressure to yield the product which is
recrystallised from methanol--ethyl acetate to give the title
compound as a pale-yellow crystalline solid, m.p. 196-198.degree.
C.
[0196] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.): 2.14 (s, 3H),
2.25-2.45 (m, 8H), 3.52 (s, 2H), 4.56 (s, 2H), 5.50 (br.s, 1H),
7.29 (d, J=8.3 Hz, 1H), 7.41 (dd, J=2.0, 8.3 Hz, 1H), 7.44 (d,
J=8.1 Hz, 2H), 7.50 (d, J=5.1 Hz, 1H), 7.52 (dd, J=3.3, 8.1 Hz,
1H), 7.93 (d, J=8.1 Hz, 2H), 8.56 (d, J=2.0 Hz, 1H), 8.57 (d, J=5.1
Hz, 1H), 8.59 (ddd, J=1.4, 2.1, 8.1 Hz, 1H), 8.69 (dd, J=1.4, 3.3
Hz, 1H), 9.10 (s, 1H), 9.33 (d, J=2.1 Hz, 1H) and 10.22 (s,
1H).
Example 6
4-[(3-Oxo-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidi-
nyl]amino]phenyl]benzamide
[0197] A solution of propylphosphonic anhydride in
N,N-dimethylformamide (Fluka, Buchs, Switzerland; 1.3 mL of 50%,
2.25 mmol) is added dropwise to a stirred mixture of
4-methyl-N-[4-(3-pyridinyl)-2-pyrimidinyl]-1,3-benzenediamine (416
mg, 1.5 mmol), 4-[(3-oxo-1-piperazinyl)methyl]benzoic acid (351 mg,
1.5 mmol) and triethylamine (1.7 mL, 12 mmol) in dry
N,N-dimethylformamide (4 mL). The mixture is stirred for 17 hours
at room temperature and then treated with saturated aqueous sodium
hydrogen carbonate solution (100 mL). The resulting precipitate is
filtered, washed with water, dried and recrystallised from methanol
to give the title compound as a cream crystalline solid.
[0198] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.) ppm 2.21 (s,
3H), 2.63 (t, J=5.49 Hz, 2H), 2,80 (s, 3H), 2.96 (s, 2H), 3.26 (t,
J=5.42 Hz, 2H), 3.60 (s, 2H), 7.19 (d, J=8.39 Hz, 1H), 7.42 (d,
J=5.19 Hz, 1H), 7.46 (m, 3H), 7.51 (dd, J=7.93, 4.73 Hz, 1H), 7.91
(d, J=8.09, 1H), 8.06 (s, 1H), 8.47 (m, 1H), 8.50 (d, J=5.04 Hz,
1H), 8.67 (dd, J=4.73, 1.53 Hz, 1H), 8.99 (s, 1H), 9.26 (d, J=1.98
Hz, 1H) and 10.18 (s, 1H).
4-[(3-Oxo-1-piperazinyl)methyl]benzoic acid
[0199] A mixture of 3-bromomethylbenzoic acid (4.30 g, 20 mmol),
piperazin-2-one (2.0 g, 20 mmol) and powdered potassium carbonate
(2.76 g, 20 mmol) in methanol (50 mL) is stirred for 17 hours at
room temperature. The resulting mixture is filtered and the solvent
is evaporated off under reduced pressure to give a residue which is
treated with hydrochloric acid (80 mL of 0.25 M) and stirred for 5
min. The precipitated product is filtered, washed with water, dried
and recrystallised from methanol to give the title compound as a
cream crystalline solid.
Example 7
4[[(4-Methyl-3-oxo-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-
-pyrimidinyl]amino]phenyl]benzamide
[0200] Utilising the procedure described for Example 6, but
employing 4-[(4-methyl-3-oxo-1-piperazinyl)methyl]benzoic acid in
place of 4-[(3-oxo-1-piperazinyl)methyl]benzoic acid afforded the
title compound as a yellow crystalline solid, m.p. 187-192.degree.
C.
[0201] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, .delta.) 2.21 (s, 3H),
2.55 (t, J=5.34 Hz, 2H), 2.91 (s, 2H), 3.15 (m, 2H), 3.61 (s, 2H),
7.19 (d, J=8.24 Hz, 1H), 7.42 (d, J=5.19 Hz, 1H), 7.46 (t, J=8.01
Hz, 1H), 7.48 (m, 2H), 7.51 (dd, J=7.86, 4.81 Hz, 1H), 7.77 (s,
1H), 7.91 (d, J=8.09 Hz, 2H), 8.07 (s, 1H), 8.47 (d, J=7.94 Hz,
1H), 8.50 (d, J=5.19 Hz, 1H), 8.67 (d, J=3.36 Hz, 1H), 8.99 (s,
1H), 9.27 (s, 1H) and 10.18 (s, 1H).
4-[(4-Methyl-3-oxo-1-piperazinyl)methyl]benzoic acid
[0202] Utilising the procedure described for
4-[(3-oxo-1-piperazinyl)methyl]benzoic acid, but employing
1-methylpiperazin-2-one in place of piperazin-2-one afforded the
title compound as cream crystalline solid.
Example 8
[0203] Tablets containing 100 mg of a compound of formula II, for
example one of the compounds of formula II described in the
Examples 1-4, are usually prepared in the following
composition:
TABLE-US-00002 Composition: Active ingredient 100 mg Crystalline
lactose 240 mg Avicel 80 mg PVPPXL 20 mg Aerosil 2 mg Magnesium
stearate 5 mg 447 mg
[0204] Preparation: The active substance is mixed with carrier
materials and compressed on a tableting machine (Korsch EKO, punch
diameter 10 mm).
[0205] Avicel is microcrystalline cellulose (FMC, Philadelphia,
USA).
[0206] PVPPXL is polyvinylpolypyrrolidone, cross-linked (BASF,
Germany).
[0207] Aerosil is silicon dioxide (Degussa, Germany).
Example 9
[0208] Capsules containing 100 mg of a compound of formula II, for
example one of the compounds of formula II described in the
Examples 1-4, are usually prepared in the following
composition:
TABLE-US-00003 Composition: Active ingredient 100 mg Avicel 200 mg
PVPPXL 15 mg Aerosil 2 mg Magnesium stearate 1.5 mg 318.5 mg
[0209] Preparation: The capsules are prepared by mixing the
components and filling the mixture into hard gelatin capsules, size
1.
* * * * *