U.S. patent application number 11/200456 was filed with the patent office on 2006-03-23 for nitrogenous heterocyclic compounds.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Michio Ichimura, Shinichi Ide, Kenji Matsuno, Yuji Nomoto, Shoji Oda, Anjali Pandey, Junko Sasaki, Robert M. Scarborough, Eiji Tsukuda.
Application Number | 20060063770 11/200456 |
Document ID | / |
Family ID | 22847506 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060063770 |
Kind Code |
A1 |
Pandey; Anjali ; et
al. |
March 23, 2006 |
Nitrogenous heterocyclic compounds
Abstract
The present invention relates to nitrogen-containing
heterocyclic compounds and pharmaceutically acceptable salts
thereof which have inhibitory activity on the phosphorylation of
kinases, which inhibits the activity of such kinases. The invention
is also related to a method of inhibiting kinases and treating
disease states in a mammal by inhibiting the phosphorylation of
kinases. In a particular aspect the present invention provides
nitrogen-containing heterocyclic compounds and pharmaceutically
acceptable salts thereof which inhibit phosphorylation of a PDGF
receptor to hinder abnormal cell growth and cell wandering, and a
method for preventing or treating cell-proliferative diseases such
as arteriosclerosis, vascular reobstruction, cancer and
glomerulosclerosis.
Inventors: |
Pandey; Anjali; (Fremont,
CA) ; Scarborough; Robert M.; (Half Moon Bay, CA)
; Matsuno; Kenji; (Tokyo, JP) ; Ichimura;
Michio; (Tokyo, JP) ; Nomoto; Yuji; (Shizuoka,
JP) ; Ide; Shinichi; (Tokyo, JP) ; Tsukuda;
Eiji; (Tokyo, JP) ; Sasaki; Junko; (Akita,
JP) ; Oda; Shoji; (Tokyo, JP) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
Kyowa Hakko Kogyo Co., Ltd.
Tokyo
|
Family ID: |
22847506 |
Appl. No.: |
11/200456 |
Filed: |
August 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10344737 |
Mar 18, 2004 |
6956039 |
|
|
PCT/US01/41751 |
Aug 17, 2001 |
|
|
|
11200456 |
Aug 8, 2005 |
|
|
|
60226089 |
Aug 18, 2000 |
|
|
|
Current U.S.
Class: |
514/252.17 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 239/94 20130101; C07D 403/12 20130101; C07D 401/12
20130101 |
Class at
Publication: |
514/252.17 |
International
Class: |
A61K 31/517 20060101
A61K031/517 |
Claims
1. A method of inhibiting phosphorylation of PDGF receptor in a
patient comprising the step of administering a compound selected
from the group consisting of: ##STR36## ##STR37## wherein when the
compound is of Formula I(a) or I(b), R.sup.1 is a member selected
from the group consisting of: CN, O-methyl, --O-ethyl, --O-propyl,
--O-isopropyl, --O-butyl, --O-t-butyl, --O-isoamyl, 1-naphthyloxy,
2-naphthyloxy, 4-indolyloxy, 5-indolyloxy, 5-isoquinolyloxy, and
position isomers and homologs thereof; and when the compound is of
Formula I(c), I(d), I(e), I(f), I(g) or I(h), R.sup.1 is a member
selected from the group consisting of: CN, --O--C.sub.1-8alkyl that
is straight or branched chained, --O-phenyl, -naphthyloxy,
-indolyloxy and -isoquinolinyloxy; n is 1, 2 or 3; R.sup.3 is a
member selected from the group consisting of: --OH, --O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, --NH.sub.2, --N(--CH.sub.3).sub.2,
--NH(--CH.sub.2-phenyl), --NH(-Phenyl), --CN ##STR38## and all
pharmaceutically acceptable isomers, salts, hydrates, solvates and
prodrug derivatives thereof to the patient.
2. A method for inhibiting abnormal cell growth and cell wandering
in a patient and thereby preventing or treating a
cell-proliferative disease, comprising the step of administering a
compound selected from the group consisting of: ##STR39## ##STR40##
wherein when the compound is of Formula I(a) or I(b), R.sup.1 is a
member selected from the group consisting of: CN, O-methyl,
--O-ethyl, --O-propyl, --O-isopropyl, --O-butyl, --O-t-butyl,
--O-isoamyl, 1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy,
5-indolyloxy, 5-isoquinolyloxy, and position isomers and homo logs
thereof; and when the compound is of Formula I(c), I(d), I(e),
I(f), I(g) or I(h), R.sup.1 is a member selected from the group
consisting of: CN, --O--C.sub.1-8alkyl that is straight or branched
chained, --O-phenyl, -naphthyloxy, -indolyloxy and
-isoquinolinyloxy; n is 1, 2 or 3; R.sup.3 is a member selected
from the group consisting of: --OH, --O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, --NH.sub.2, --N(--CH.sub.3).sub.2,
--NH(--CH.sub.2-phenyl), --NH(-phenyl), --CN ##STR41## and all
pharmaceutically acceptable isomers, salts, hydrates, solvates and
prodrug derivatives thereof to the patient.
3. A method according to claim 2, wherein said cell-proliferative
disease is selected from the group consisting of arteriosclerosis,
vascular re-obstruction, restenosis, cancer and glomerulosclerosis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
10/344,737, filed Mar. 18, 2004, which is a national stage of
PCT/US01/41751, filed Aug. 17, 2001, which is an application
claiming the benefit under 35 USC 119(e) of 60/226,089, filed Aug.
18, 2000, the content of which is incorporated herein by
reference
TECHNICAL FIELD
[0002] The present invention relates to nitrogen-containing
heterocyclic compounds and pharmaceutically acceptable salts
thereof which have inhibitory activity on the phosphorylation of
kinases, which inhibits the activity of such kinases. The invention
is also related to a method of inhibiting kinases and treating
disease states in a mammal by inhibiting the phosphorylation of
kinases.
BACKGROUND ART
[0003] PDGF (platelet-derived growth factor) is known to act as an
aggravating factor for cell-proliferative diseases such as
arteriosclerosis, vascular reobstruction after percutaneous
coronary angioplasty and bypass operation, cancer,
glomerulonephritis, glomerulosclerosis, psoriasis and articular
rheumatism [Cell, 46, 155-169 (1986); Science, 253, 1129-1132
(1991); Nippon Rinsho (Japanese J. of Clinical Medicine), 50,
3038-3045 (1992); Nephrol Dial Transplant, 10, 787-795 (1995);
Kidney International, 43 (Suppl. 39), 86-89 (1993); Journal of
Rheumatology, 21, 1507-1511 (1994); Scandinavian Journal of
Immunology, 27, 285-294 (1988), etc.].
[0004] As for quinazoline derivatives which are useful as drugs,
N,N-dimethyl-4-(6,7-dimethoxy-4-quinazolinyl)-1-piperazine
carboxamide is described as a bronchodilator in South African
Patent No. 67 06512 (1968). Dimethoxyquinazoline derivatives are
described as inhibitors of phosphorylation of epidermal growth
factor (EGF) receptor in Japanese Published Unexamined Patent
Application No. 208911/93 and WO 96/09294. Quinoline derivatives
having benzodiazepin receptor agonist activity are described in
Pharmacology Biochemistry and Behavior, 53, 87-97 (1996) and
European Journal of Medicinal Chemistry, 31, 417-425 (1996), and
quinoline derivatives which are useful as anti-parasite agents are
described in Indian Journal of Chemistry, 26B, 550-555 (1987).
[0005] Inhibitors of phosphorylation of PDGF receptor so far known
include bismono- and bicyclic aryl compounds and heteroaryl
compounds (WO 92/20642), quinoxaline derivatives [Cancer Research,
54, 6106 (1994)], pyrimidine derivatives (Japanese Published
Unexamined Patent Application No. 87834/94) and dimethoxyquinoline
derivatives [Abstracts of the 16th Annual Meeting of the
Pharmaceutical Society of Japan (Kanazawa) (1996), 2, p. 275,
29(C2) 15-2].
DISCLOSURE OF THE INVENTION
[0006] An object of the present invention is to provide
nitrogen-containing heterocyclic compounds and pharmaceutically
acceptable salts thereof which have inhibitory activity on the
phosphorylation of kinases, which inhibits the activity of the
kinases. Particularly, important kinase inhibition according to the
invention is of receptor tyrosine kinases including
platelet-derived growth factor (PDGF) receptor, Flt3, CSF-1R,
epidermal growth factor receptor (EGRF), fibroblast growth factor
(FGF), vascular endothelial growth factor receptor (VEGFR) and
others. Another class of kinase inhibition according to the
invention is inhibitory activity nonreceptor tyrosine kinases
including src and abl, and the like. A third class of kinase
inhibition according to the invention is inhibitory activity toward
serine/threonine kinases, including such kinases as MAPK, MEK and
cyclin dependent kinases (CDKs) that mediate cell prolifetation,
AKT and CDK such that mediate cell survival and NIK that regulate
inflammatory responses. Inhibition of such kinases can be used to
treat diseases involving cell survival, proliferation and
migration, including cardiovascular disease, such as
arteriosclerosis and vascular reobstruction, cancer,
glomerulosclerosis fibrotic diseases and inflammation, as well as
the general treatment of cell-proliferative diseases.
[0007] In a preferred embodiment, the present invention provides
compounds and pharmaceutically acceptable salts thereof which
inhibit or prevent inhibition of phosphorylation of at least one
PDGF receptor by at least one tyrosine kinase. Such PDGF receptor
kinase inhibition can hinder abnormal cell growth and cell
wandering, and thus such compounds are useful for the prevention or
treatment of cell-proliferative diseases such as arteriosclerosis,
vascular reobstruction, cancer and glomerulosclerosis.
[0008] The present invention relates to nitrogen-containing
heterocyclic compounds represented by formula I as follows:
##STR1## wherein [0009] R.sup.1 is a member selected from the group
consisting of:
[0010] --CN, --O--C.sub.1-8 alkyl that is a straight or branched
chain, --O-phenyl, --O-naphthyl, --O-indolyl and --O-isoquinolinyl;
[0011] R.sup.2 and R.sup.4 are each independently a member selected
from the group consisting of:
[0012] hydrogen, --O(--CH.sub.2)--CH.sub.3,
--O--CH.sub.2--CH.dbd.CH.sub.2, --O--CH.sub.2--C.ident.CH and
--O(--CH.sub.2).sub.n--R.sup.3; providing that one of the R.sup.2
and R.sup.4 groups is hydrogen and the remaining R.sup.2 or R.sup.4
group is other than hydrogen; [0013] n is 1, 2 or 3; [0014] R.sup.3
is a member selected from the group consisting of: [0015] --OH,
--O--CH.sub.3, --O--CH.sub.2--CH.sub.3, --NH.sub.2,
--N(--CH.sub.3).sub.2, --NH(--CH.sub.2-phenyl), --NH(-phenyl), --CN
##STR2##
[0016] and all pharmaceutically acceptable isomers, salts,
hydrates, solvates and prodrug derivatives thereof.
[0017] Particularly preferred compounds according to formula above
are such compounds wherein R.sup.1 is a member selected from the
group consisting of CN, --O-methyl, --O-ethyl, --O-propyl,
--O-isopropyl, --O-butyl, --O-t-butyl, --O-isoamyl, 1-naphthyloxy,
2-naphthyloxy, 4-indolyloxy, 5-indolyloxy, 5-isoquinolyloxy, and
position isomers and homologs thereof, and all pharmaceutically
acceptable isomers, salts, hydrates, solvates and prodrug
derivatives of such compounds.
[0018] The pharmaceutically acceptable salts of the compounds
according to formula (I) include pharmaceutically acceptable acid
addition salts, metal salts, ammonium salts, organic amine addition
salts, amino acid addition salts, etc. Examples of the
pharmaceutically acceptable acid addition salts of the compounds of
formula (I) are inorganic acid addition salts such as
hydrochloride, sulfate and phosphate, and organic acid addition
salts such as acetate, maleate, fumarate, tartrate, citrate and
methanesulfonate. Examples of the pharmaceutically acceptable metal
salts are alkali metal salts such as sodium salt and potassium
salt, alkaline earth metal salts such as magnesium salt and calcium
salt, aluminum salt and zinc salt. Examples of the pharmaceutically
acceptable ammonium salts are ammonium salt and tetramethyl
ammonium salt. Examples of the pharmaceutically acceptable organic
amine addition salts include heterocyclic amine salts such as
morpholine and piperidine salts. Examples of the pharmaceutically
acceptable amino acid addition salts are salts with lysine, glycine
and phenylalanine.
[0019] In a preferred embodiment the invention provides compounds
according to formula I(a) and formula I(b) as follows: ##STR3##
wherein [0020] R.sup.1 is a member selected from the group
consisting of:
[0021] --CN, --O--C.sub.1-8 alkyl that is a straight or branched
chain, --O-phenyl, --O-naphthyl, --O-indolyl and
--O-isoquinolinyl;
[0022] and all pharmaceutically acceptable isomers, salts,
hydrates, solvates and prodrug derivatives thereof.
[0023] In another preferred embodiment the invention provides
compounds according to formula (Ic) and formula (Id) as follows:
##STR4## wherein [0024] R.sup.1 is a member selected from the group
consisting of:
[0025] --CN, --O--C.sub.1-8 alkyl that is a straight or branched
chain, --O-phenyl, --O-naphthyl, --O-indolyl and
--O-isoquinolinyl;
[0026] and all pharmaceutically acceptable isomers, salts,
hydrates, solvates and prodrug derivatives thereof.
[0027] In still another preferred embodiment the invention provides
compounds according to formula I(e) and formula I(f) as follows:
##STR5## wherein [0028] R.sup.1 is a member selected from the group
consisting of:
[0029] --CN, --O--C.sub.1-8 alkyl that is a straight or branched
chain, --O-phenyl, --O-naphthyl, --O-indolyl and
--O-isoquinolinyl;
[0030] and all pharmaceutically acceptable isomers, salts,
hydrates, solvates and prodrug derivatives thereof.
[0031] In yet another preferred embodiment the invention provides
compounds according to formula I(g) and formula I(h) as follows:
##STR6## wherein [0032] R.sup.1 is a member selected from the group
consisting of:
[0033] --CN, --O--C.sub.1-8 alkyl that is a straight or branched
chain, --O-phenyl, --O-naphthyl, --O-indolyl and --O-isoquinolinyl;
[0034] R.sup.3 is a member selected from the group consisting of:
[0035] --OH, --O--CH.sub.3, --O--CH.sub.2--CH.sub.3, --NH.sub.2,
--N(--CH.sub.3).sub.2, --NH(--CH.sub.2-phenyl), --NH(-phenyl), --CN
##STR7## and all pharmaceutically acceptable isomers, salts,
hydrates, solvates and prodrug derivatives thereof.
[0036] The pharmaceutically acceptable salts of the compounds
according to formula (I) include pharmaceutically acceptable acid
addition salts, metal salts, ammonium salts, organic amine addition
salts, amino acid addition salts, etc.
[0037] The present invention is not limited by the above listed
compounds. Analogs of the bicyclic compounds are contemplated.
[0038] Further, an especially preferred embodiment of the present
invention is a compound selected from the group consisting of:
N-[4-(methylethoxy)phenyl]{4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]pi-
perazinyl}carboxamide
[0039] ##STR8##
N-(4-cyanophenyl){4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl-
}carboxamide
[0040] ##STR9##
N-(4-cyanophenyl){4-[7-(2-pyrrolidinylethoxy)quinazolin-4-yl]piperazinyl}c-
arboxamide
[0041] ##STR10##
N-[4-(methylethoxy)phenyl]{4-[7-(2-pyrrolidinylethoxy)quinazolin-4-yl]pipe-
razinyl}carboxamide
[0042] ##STR11##
N-[4-(methylethoxy)phenyl]{4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperaz-
inyl}carboxamide
[0043] ##STR12##
{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phe-
nyl]carboxamide
[0044] ##STR13##
N-(4-cyanophenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carbox-
amide
[0045] ##STR14##
N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carb-
oxamide
[0046] ##STR15##
{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)car-
boxamide
[0047] ##STR16##
N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperaziny-
l}carboxamide
[0048] ##STR17##
N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piper-
azinyl}carboxamide
[0049] ##STR18##
N-[4-(methylethoxy)phenyl]{4-[7-(3-piperidylpropoxy)
quinazolin-4-yl]piperazinyl)carboxamide
[0050] ##STR19##
{4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)ca-
rboxamide
[0051] ##STR20##
N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazin-
yl}carboxamide
[0052] ##STR21##
N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]pipe-
razinyl}carboxamide
[0053] ##STR22##
N-(4-cyanophenyl){4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl}car-
boxamide
[0054] ##STR23##
N-[4-(methylethoxy)phenyl]{4-[7-(3-morpholin-4-ylpropoxy)
quinazolin-4-yl]piperazinyl}carboxamide
[0055] ##STR24##
N-(4-cyanophenyl){4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperaziny-
l}carboxamide
[0056] ##STR25##
N-[4-(methylethoxy)phenyl]{4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]pip-
erazinyl}carboxamide
[0057] ##STR26##
N-(4-cyanophenyl){4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]piperazinyl}-
carboxamide
[0058] ##STR27##
N-(4-cyanophenyl){4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}carbo-
xamide
[0059] ##STR28##
{4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)ph-
enyl]carboxamide
[0060] ##STR29## and all pharmaceutically acceptable isomers,
salts, hydrates, solvates and prodrug derivatives thereof.
[0061] The compounds may be prepared using methods and procedures
as generally described in WO 98/14431 published Sep. 12, 1998,
which is incorporated herein by reference. Starting materials may
be made or obtained as described therein as well.
[0062] Leaving groups such as halogen, lower alkoxy, lower
alkylthio, lower alkylsulfonyloxy, arylsulfonyloxy, etc, may be
utilized when necessary except for the reaction point, followed by
deprotection. Suitable amino protective groups are, for example,
those described in T. W. Greene, Protective Groups in Organic
Synthesis, John Wiley & Sons Inc. (1981), etc., such as
ethoxycarbonyl, t-butoxycarbonyl, acetyl and benzyl. The protective
groups can be introduced and eliminated according to conventional
methods used in organic synthetic chemistry [e.g., T. W. Greene,
Protective Groups in Organic Synthesis, John Wiley & Sons Inc.
(1981)].
[0063] In such processes, if the defined groups change under the
conditions of the working method or are not appropriate for
carrying out the method, the desired compound can be obtained by
using the methods for introducing and eliminating protective groups
which are conventionally used in organic synthetic chemistry [e.g.,
T. W. Greene, Protective Groups in Organic Synthesis, John Wiley
& Sons Inc. (1981)], etc. Conversion of functional groups
contained in the substituents can be carried out by known methods
[e.g., R. C. Larock, Comprehensive Organic Transformations (1989)]
in addition to the above-described processes, and some of the
active compounds of formula I may be utilized as intermediates for
further synthesizing novel derivatives according to formula I.
[0064] The intermediates and the desired compounds in the processes
described above can be isolated and purified by purification
methods conventionally used in organic synthetic chemistry, for
example, neutralization, filtration, extraction, washing, drying,
concentration, recrystallization, and various kinds of
chromatography. The intermediates may be subjected to the
subsequent reaction without purification.
[0065] There may be tautomers for some formula I, and the present
invention covers all possible isomers including tautomers and
mixtures thereof. Where chiral carbons lend themselves to two
different enantiomers, both enantiomers are contemplated as well as
procedures for separating the two enantiomers.
[0066] In the case where a salt of a compound of formula I is
desired and the compound is produced in the form of the desired
salt, it can be subjected to purification as such. In the case
where a compound of formula I is produced in the free state and its
salt is desired, the compound of formula I is dissolved or
suspended in a suitable organic solvent, followed by addition of an
acid or a base to form a salt.
[0067] The following non-limiting reaction Schemes I, II and III
illustrate preferred embodiments of the invention with respect to
making compounds according to the invention. ##STR30##
[0068] Scheme I describes the synthesis
7-(2-methoxyethoxy)-4-piperazinylquinazoline a key intermediate
that will be utilized in the synthesis of various targets as
described in Scheme II. The 2-amino-4-fluorobenzoic acid is
esterified followed by cyclization with formamide at elevated
temperature to afford 7-fluoro-4-quinazolinone. The 7-fluoro group
is displaced with several alkoxides generated by treating with NaH
in DMF at 100.degree. C. The 7-alkoxy-4-quinazolinone is
intermediate is converted to 7-alkoxy-4-chloroquinazoline with
thionyl chloride. The key intermediate I was obtained by treating
7-alkoxysubstituted-4-Cl-quinazoline with piperazine in an
appropriate solvent, such as isopropanol, acetonitrile, or THF at
room or reflux temperature for 1-6 h in presence of base
triethylamine or pyridine.
[0069] This illustrated Scheme II provides the synthesis of various
substituted urea from the intermediate obtained in Scheme I, or by
other procedures. The reaction of intermediate I with various
isocyanates afforded the final urea compounds. In cases where the
isocyanates are not commercially available, the piperazine
intermediate is treated with phosgene to give carbamoyl chloride
intermediate followed by reaction with various substituted
anilines. The piperazine intermediate can also be treated with
p-nitrophenyl chloroformate to afford nitrophenyl carbamate
intermediate that can be treated with various anilines to afford
the desired ureas.
[0070] If the urea compound has a terminal NH.sub.2 group (or one
or more of the hydrogen atoms on this amino group is replaced by a
displaceable substituent), then this compound may be utilized an
intermediate compound with which to produce a urea compound
terminated with a --NH-phenyl-R.sup.1 groups. Alternatively, if the
a different R.sup.1 group is desired on the phenyl group, a
replaceable para position leaving group phenyl substituent may be
displaced after coupling to provide the particular R.sup.1
substituent as described for formula I, above. ##STR31##
[0071] Scheme III describes the synthesis of
6-(2-methoxyethoxy)-4-piperazinylquinazoline as a key intermediate
that will be utilized in the synthesis of various positional isomer
targets. ##STR32##
[0072] Such procedures for producing the claimed compounds are
merely an illustration of a preferred aspect of the invention.
Other procedures and adaptations will be apparent to one of
ordinary skill in the art upon views these reaction schemes and the
structures of the compounds according to the invention. Such
procedures are deemed to be within the scope of the present
invention.
[0073] Also, the compounds of formula I and pharmaceutically
acceptable salts thereof may exist in the form of adducts with
water (hydrates) or various solvents, which are also within the
scope of the present invention.
[0074] The following non-limiting examples are provided to better
illustrate the present invention.
EXAMPLE 1
[0075] The intermediate
4-piperazinyl-7-(2-piperidylethoxy)quinazoline was prepared using
the procedures as generally described in Scheme 1 as follows:
##STR33##
[0076] Step A: To the ethanol solution (15 mL) of
2-amino-4-fluorobenzoic acid (840 mg, 5.41 mmmol) was added thionyl
chloride (1.18 mL, 16.23 mmol) and the resulting suspension was
refluxed overnight. The solvent was evaporated to the residue added
EtOAc, washed with 10% NaOH solution, dried, filtered and
evaporated to afford desired ethyl ester as a solid 981 mg, 82%).
MS (ES) 184(M+H)
[0077] Step B: To the formamide (6 mL) solution of
ethyl-2-amino-4-fluorobenzoate (811 mg, 4.43 mmol) added ammonium
formate (0.450 g, 7.14 mmol) and the reaction mixture was heated at
140.degree. C. overnight. After cooling added water and ethyl
acetate. The layers were separated, the EtOAc layer was dried,
filtered and evaporated to give desired quinazolinone (1 g,
quantitative). MS (ES) 165 (M+H)
[0078] Step C: To the DMF solution (3 mL) of 1-piperidineethanol
(0.689 mL, 5.18 mmol) at 0.degree. C. added sodium hydride (0.518
g, 12.95 mmol) and the mixture was stirred for 30 min. To this cold
solution added DMF solution (3 mL) of intermediate quinazolinone
(0.284 g, 1.73 mmol, from Step B) and the mixture was heated at
75.degree. C. overnight. The solvent was evaporated and the residue
purified by RP-HPLC to afford desired product as a creamy solid
(0.459 g, 97%)
[0079] Step D: A mixture of 7-(2-piperidylethoxy)-4-quinazolinone
(0.459 g, 1.68 mmol), from Step C) and POCl.sub.3 (5 mL) was heated
at 75.degree. C. overnight. After cooling excess POCl.sub.3 was
removed by evaporation and the residue azeotroped with toluene to
afford intermediate, 4-chloro-7-(2-piperidylethoxy)quinazoline (600
mg, 95%)
[0080] Step E: To the isopropanol solution (10 mL) of
4-chloroquinazoline (0.615 g, 2.11 mmol, from Step D) added
piperazine (0.727 g, 8.44 mmol) and heated the reaction for 4 h at
100.degree. C. The solvent was evaporated and the residue purified
by RP-HPLC to afford 4-piperazinyl-7-(2-piperidylethoxy)quinazoline
as a white solid (0.630 g, 87%).
EXAMPLE 2
[0081] Preparation of N-(4-cyanophenyl)
{4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}-carboxamide
##STR34##
[0082] To DMF solution (2 mL) of
4-piperazinyl-7-(2-piperidylethoxy)quinazoline (from Example 1,
Step E, 0.287 g, 0.84 mmol) added DMF solution (2 mL) of
4-cyanophenylisocyanate (0.181 g, 1.26 mmol) and the reaction was
stirred at room temperature overnight. The solvent was evaporated
and residue purified by RP-HPLC to afford desired product
N-(4-cyanophenyl)
{4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide as
a white solid (200 mg, 50%). MS (ES) 487(M+H)
EXAMPLES 3-4
[0083] N-(4-cyanophenyl)
{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-carboxamide was
prepared using the procedures as described above in Example 1 and 2
except that 2-methoxyethanol was used instead of
1-piperidineethanol as an alkoxide anion, to provide the title
compound. ##STR35##
[0084] The pharmacological activities of the compounds of the
present invention are obtained by following the test example
procedures as follows, for example.
Biological Test Assay Type 1
Inhibitory Effect on Compounds on Autophosphorylation of Platelet
Derived Growth Factor .beta.-PDGF Receptor
(1) HR5 Phosphorylation Assay
[0085] The HR5 cell line is a cell line of CHO cells engineered to
overexpress human .beta.-PDGFR, which cell line is available from
the ATCC. The expression level of .beta.-PDGFR in HR5 cells is
around 5.times.10.sup.4 receptor per cell. For the phosphorylation
assay according to the invention, HR5 cells were grown to
confluency in 96-well microtiter plates under standard tissue
culture conditions, followed by serum-starvation for 16 hours.
Quiescent cells were incubated at 37.degree. C. without or with
increasing concentrations of the test compound (0.01-30 uM) for 30
minutes followed by the addition of 8 nM PDGF BB for 10 minutes.
Cells were lysed in 100 mM Tris, pH7.5, 750 mM NaCl, 0.5% Triton
X-100, 10 mM sodium pyrophosphate, 50 mM NaF, 10 ug/ml aprotinin,
10 ug/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium
vanadate, and the lysate was cleared by centrifugation at
15,000.times.g for 5 minutes. Clarified lysates were transferred
into a second microtiter plate in which the wells were previously
coated with 500 ng/well of 1B5B11 anti-.beta.-PDGFR mAb, and then
incubated for two hours at room temperature. After washing three
times with binding buffer (0.3% gelatin, 25 mM Hepes pH 7.5, 100 mM
NaCl, 0.01% Tween-20), 250 ng/ml of rabbit polyclonal
anti-phosphotyrosine antibody (Transduction Laboratory) was added
and plates were incubated at 37.degree. C. for 60 minutes.
Subsequently, each well was washed three times with binding buffer
and incubated with 1 ug/ml of horse radish peroxidase-conjugated
anti-rabbit antibody (Boehringer Mannheim) at 37.degree. C. for 60
minutes. Wells were washed prior to adding ABTS (Sigma), and the
rate of substrate formation was monitored at 650 nm. The assay
results are reported as IC.sub.50 (expressed as the concentration
of a compound according to the invention that inhibits the PDGF
receptor phosphorylation by 50%) as compared to control cells that
are not exposed to a compound according to the invention.
[0086] Examples of such IC.sub.50 test results in the HR5 assay for
compounds according to the invention are set forth below in Table
1.
(2) MG63 Phosphorylation Assay
[0087] The MG63 cell line is a human osteosarcoma tumor cell line
available from the ATCC. This assay is for measuring endogenous
.beta.-PDGFR phosphorylation in MG63 cells. The assay conditions
are the same as those described at for HR5 cell, except that
PDGF-BB stimulation is provided in the presence or absence of 45%
human plasma. The HR5 assay results are reported as an IC.sub.50
(expressed as the concentration of a compound according to the
invention that inhibits the PDGF receptor phosphorylation by 50%)
as compared to control cells that are not exposed to a compound
according to the invention.
[0088] Examples of such IC.sub.50 test results in the MG63 assay
for compounds according to the invention are set forth below in
Table 1.
[0089] The assay results for Compound Examples 1 and 2 are set
forth in Table 1 below. TABLE-US-00001 TABLE 1 MG63 w/human plasma
HR5 Example Compound IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) Example 1
0.103 0.150 Example 2 3.56 2.27
Biological Test Assay Type 2 Growth Inhibition Against Smooth
Muscle Cells
[0090] Vascular smooth muscle cells are isolated from a pig aorta
by explanation and used for the test. The cells are put into wells
of a 96-well plate (8000 cells/well) and cultured in Dulbeccois
modified Eagle's medium (DMEM; Nissui Pharmaceutical Co., Ltd.)
containing 10% fetal bovine serum (FBS; Hyclone) for 4 days. Then,
the cells are further cultured in DMEM containing 0.1% FBS for 3
days, and are synchronized at the cell growth stationary phase.
[0091] To each well is added DMEM containing 0.1% FBS and a test
sample at a varied concentration, and the cell growth is brought
about by PDGF-BB (SIGMA, final concentration: 20 ng/ml). After
culturing for 3 days, the cell growth is measured using a cell
growth assay kit (Boehringer Mannheim) according to the XTT method
[J. Immunol. Methods, 142, 257-265 (1991)], and the cell growth
score is calculated by the following equation. Cell growth
score=100.times.{1-(M-PO)/(P100-PO)}wherein P100=absorbance by XTT
reagent when stimulated by PDGF-BB; PO=absorbance by XTT reagent
when not stimulated by PDGF-BB, and M=absorbance by XTT reagent
after addition of a sample when stimulated by PDGF-BB.
[0092] The test result is expressed as the concentration of a test
compound which inhibits the cell growth by 50% (IC.sub.50).
Biological Test Assay Type 3
Inhibitory Effect on Hypertrophy of Vascular Intima
[0093] Male SD rats (weight: 375-445 g, Charles River, golden
standard) are anesthetized with sodium pentobarbital (50 mg/kg,
i.p.), and then the neck of each animal is incised by the median
incision, followed by retrograde insertion of a balloon catheter
(2F, Edwards Laboratories) into the left external carotid. After
the above treatment is repeated seven times, the catheter is pulled
out, the left external carotid is ligated, and the wound is
sutured. A test compound is suspended in a 0.5% solution of Tween
80 in an aqueous solution of sodium chloride to a concentration of
20 mg/ml in the case of intraperitoneal administration and in a
0.5% solution of methyl cellulose 400 to a concentration of 6 mg/ml
in the case of oral administration. The suspension is administered
once a day in the case of intraperitoneal administration and once
or twice a day in the case of oral administration for a period of
15 days starting on the day before the balloon injury. On the 14th
day after the balloon injury, the animal is killed and its left
carotid is extirpated. The tissues are fixed with formalin, wrapped
in paraffin and sliced, followed by Elastica Wangeeson staining.
The area of the cross section of the vascular tissues (intima and
media) is measured with an image analyzer (Luzex F, NIRECO) and the
intima/media area ratio (I/M) is regarded as the degree of
hypertrophy of the vascular intima.
[0094] From the results obtained, it is apparent when the
hypertrophy of vascular intima is significantly inhibited by
administration of the compounds of the present invention.
Biological Test Assay Type 4
Evaluation by the Use of a Rat Adjuvant Arthritis Model
[0095] Dead cells of Mycobacterium bacterium (Difco Laboratories
Inc.) are disrupted in agate mortar and suspended in liquid
paraffin to the final concentration of 6.6 mg/ml, followed by
sterilization with high pressure steam. Then, 100 ml of the
suspension is subcutaneously injected into the right hind foot pad
of each animal of groups of female 8-weeks-old Lewis rats (Charles
River Japan) (6 animals/group) to induce adjuvant arthritis. A test
compound is suspended in a 0.5% solution of methyl cellulose to the
final concentration of 3 mg/ml, and from just before the induction
of arthritis, the suspension is orally administered in an amount of
100 ml/100 g of the body weight once a day, 5 days a week. To a
control group is administered a 0.5% solution of methyl cellulose.
A normal group is given no adjuvant treatment or test compound
administration. The administration of the test compound is
continued till the 18th day after the adjuvant treatment. On the
17th day, the number of leukocytes in peripheral blood are counted,
and on the 18th day, all the blood is collected, followed by
dissection.
[0096] The change in body weight with the passage of time, the
change of edema in hind foot with the passage of time, the weight
of spleen and thymus, the number of leukocytes in peripheral blood,
the hydroxyproline content of urine, the glucosaminoglycan content
of urine, the SH concentration in serum, the concentration of
nitrogen monoxide in serum and the concentration of mucoprotein in
serum are measured and evaluated. The volume of each of both hind
feet are measured using a rat's hind foot edema measurement device
(TK-101, Unicom). The number of leukocytes in peripheral blood are
counted using an automatic multichannel blood cell counter (Sysmex
K-2000, Toa Iyo Denshi Co., Ltd.). The hydroxyproline content of
urine is measured according to the method described in Ikeda, et
al., Annual Report of Tokyo Metropolitan Research Laboratories P.
H., , 36, 277 (1985), and the glucosaminoglycan content is measured
according to the method described in Moriyama, et al., Hinyo Kiyo,
40, 565 (1994) and Klompmakers, et al., Analytical Biochemistry,
153, 80 (1986). The SH concentration in serum is measured according
to the method described in Miesel, et al., Inflammation, 17, 595
(1993), and the concentration of nitrogen monoxide is measured
according to the method of Tracey, et al., Journal of Pharmacology
& Experimental Therapeutics, 272, 1011 (1995). The
concentration of mucoprotein is measured using Aspro GP Kit (Otsuka
Pharmaceutical Co., Ltd.). The percentage inhibition for each
indication is calculated according to the following equation. %
Inhibition={(Control group-Compound-administered group)/(Control
group-Normal group)}.times.100.
[0097] From the results obtain from such assays, it is apparent
when the compound according to the invention inhibits the
occurrence of adjuvant arthritis.
Biological Test Assay Type 5
Activity on a Mesangial Proliferative Glomerulonephritis Model
[0098] Anti-rat Thy-1.1 monoclonal antibody OX-7 (Sedaren) is
administered to male Wister-Kyoto rats (Charles River Japan, 160 g,
6 animals/group) in an amount of 1.0 mg/kg by intravenous
administration through the tail vein. A test compound is suspended
in a 0.5% solution of methylcellulose and the resulting suspension
is administered to each of the rats twice a day for a period of 7
days starting on the day before the administration of OX-7. On the
7th day after the OX-7 administration, when mesangial cell growth
and extracellular matrix hypertrophy become prominent, the left
kidney of each rat is extirpated, fixed with 20% buffered formalin
for 6 hours and wrapped in paraffin, followed by slicing. The
obtained pieces are subjected to immune tissue staining using
antibody PC10 (DAKO) against an intranuclear antigen of
proliferative cells. After comparative staining with Methyl Green
staining solution using diaminobenzidine as a color developer, the
paraffin pieces are enclosed. Half of the glomeruli in a kidney
piece are observed and the number of the cells in one glomerulus
which are positive to the intranuclear antigen of proliferative
cells are calculated. The test for the significance of difference
is carried out by the Wilcoxon test.
[0099] From such results, it is apparent when the compounds
according to the present invention show alleviating activity on
mesangial proliferative glomerulonephritis.
[0100] The compounds of formula (I) and pharmaceutically acceptable
salts thereof can be administered as such, but it is usually
preferred to administer them in the form of pharmaceutical
compositions, which are used for animals and human beings.
[0101] It is preferred to employ the administration route which is
the most effective for the treatment. For example, administration
is made orally or non-orally by intrarectal, intraoral,
subcutaneous, intramuscular or intravenous administration.
[0102] Examples of the forms for administration are capsules,
tablets, granules, powders, syrups, emulsions, suppositories and
injections.
[0103] Liquid compositions such as emulsions and syrups which are
appropriate for oral administration can be prepared using water,
sugars such as sucrose, sorbitol and fructose, glycols such as
polyethylene glycol and propylene glycol, oils such as sesame oil,
olive oil and soybean oil, preservatives such as benzoates, flavors
such as strawberry flavor and peppermint, etc.
[0104] Capsules, tablets, powders and granules can be prepared
using excipients such as lactose, glucose, sucrose and mannitol,
disintegrating agents such as starch and sodium alginate,
lubricants such as magnesium stearate and talc, binders such as
polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants
such as fatty acid esters, plasticizers such as glycerin, etc.
[0105] Compositions suitable for non-oral administration preferably
comprise a sterilized aqueous preparation containing an active
compound which is isotonic to the recipient's blood. For example,
injections are prepared using a carrier which comprises a salt
solution, a glucose solution, or a mixture of a salt solution and a
glucose solution.
[0106] Compositions for topical application are prepared by
dissolving or suspending an active compound in one or more kinds of
solvents such as mineral oil, petroleum and polyhydric alcohol, or
other bases used for topical drugs.
[0107] Compositions for intestinal administration are prepared
using ordinary carriers such as cacao fat, hydrogenated fat and
hydrogenated fat carboxylic acid, and are provided as
suppositories.
[0108] The compositions for non-oral administration may
additionally be formulated to contain one or more kinds of
additives selected from glycols, oils, flavors, preservatives
(including antioxidants), excipients, disintegrating agents,
lubricants, binders, surfactants and plasticizers which are used
for the preparation of compositions for oral administration.
[0109] The effective dose and the administration schedule for each
of the compounds of formula (I) or a pharmaceutically acceptable
salt thereof will vary depending on the administration route, the
patient's age and body weight, and the type or degree of the
diseases to be treated. However, it is generally appropriate to
administer a compound of formula (I) or a pharmaceutically
acceptable salt thereof in a dose of 0.01-1000 mg/adult/day,
preferably 5-500 mg/adult/day, in one to several parts.
[0110] All the compounds of the present invention can be
immediately applied to the treatment of kinase-dependent diseases
of mammals as kinase inhibitors, specifically, those relating to
tyrosine kinase. Specifically preferred are the compounds which
have IC50 within the range of 10 nM-10 .mu.M. Even more preferred
are compounds which have IC50 within the range of 10 .mu.M to -1
.mu.M. Most preferred are compounds which have an IC50 value which
is smaller than 1 .mu.M.
[0111] Specific compounds of the present invention which have an
activity to specifically inhibit one of the three types of protein
kinase (for example, kinase which phosphorylates tyrosine, kinase
which phosphorylates tyrosine and threonine, and kinase which
phosphorylates threonine) can be selected. Tyrosine
kinase-dependent diseases include hyperproliferative malfunction
which is caused or maintained by abnormal tyrosine kinase activity.
Examples thereof include psoriasis, pulmonary fibrosis,
glomerulonephritis, cancer, atherosclerosis and anti-angiopoiesis
(for example, tumor growth and diabetic retinopathy). Current
knowledge of the relationship between other classes of kinase and
specific diseases is insufficient. However, compounds having
specific PTK-inhibiting activity have a useful treatment effect.
Other classes of kinase have also been recognized in the same
manner. Quercetin, genistein and staurosporin, which are all
PTK-inhibitors, inhibit many kinds of protein kinase in addition to
tyrosine kinase. However, as a result of their lack of the
specificity, their cytotoxicity is high. Therefore, a PTK-inhibitor
(or an inhibitor of other classes of kinase) which is apt to bring
about undesirable side effects because of the lack of selectivity
can be identified by the use of an ordinary test to measure
cytotoxicity.
[0112] The present invention provides nitrogen-containing
heterocyclic compounds and pharmaceutically acceptable salts
thereof which inhibit phosphorylation of PDGF receptor to hinder
abnormal cell growth and cell wandering and thus are useful for the
prevention or treatment of cell-proliferative diseases such as
arteriosclerosis, vascular reobstruction, cancer and
glomerulosclerosis.
[0113] Although the present invention has been described in some
detail by way of illustration for purposes of clarity of
understanding, it will be apparent to those of ordinary skill in
the art that various modifications and equivalents can be made
without departing from the spirit and scope of the invention. It
should be understood that the foregoing discussion and examples
merely present a detailed description of certain preferred
embodiments. All the patents, journal articles and other documents
discussed or cited above are herein incorporated by reference in
their entirety.
* * * * *