U.S. patent number RE37,650 [Application Number 09/496,399] was granted by the patent office on 2002-04-09 for aryl and heteroaryl quinazoline compounds which inhibit csf-1r receptor tyrosine kinase.
This patent grant is currently assigned to Aventis Pharmacetical Products, Inc.. Invention is credited to Chin-Yi Jenny Hsu, Susan E. Johnson, Martin P. Maguire, Michael R. Myers, Paul E. Persons, Alfred P. Spada, Asher Zilberstein.
United States Patent |
RE37,650 |
Myers , et al. |
April 9, 2002 |
Aryl and heteroaryl quinazoline compounds which inhibit CSF-1R
receptor tyrosine kinase
Abstract
This invention relates to the treatment of intimation in a
patient suffering from such disorder. More specifically, the
invention relates to mono- and/or bicyclic aryl or heteroaryl
quinazoline compounds in the treatment of inflammation.
Inventors: |
Myers; Michael R. (Reading,
PA), Spada; Alfred P. (Lansdale, PA), Maguire; Martin
P. (Mont Clare, PA), Persons; Paul E. (King of Prussia,
PA), Zilberstein; Asher (Broomall, PA), Hsu; Chin-Yi
Jenny (West Chester, PA), Johnson; Susan E. (Upper
Darby, PA) |
Assignee: |
Aventis Pharmacetical Products,
Inc. (Bridgewater, NJ)
|
Family
ID: |
46276642 |
Appl.
No.: |
09/496,399 |
Filed: |
February 2, 2000 |
PCT
Filed: |
December 08, 1994 |
PCT No.: |
PCT/US94/14180 |
371
Date: |
June 04, 1996 |
102(e)
Date: |
June 04, 1996 |
PCT
Pub. No.: |
WO95/15758 |
PCT
Pub. Date: |
June 15, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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229886 |
Apr 19, 1994 |
5710158 |
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166199 |
Dec 10, 1993 |
5480883 |
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988515 |
Dec 10, 1992 |
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698420 |
May 10, 1991 |
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988515 |
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PCTUS9203736 |
May 6, 1992 |
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Reissue of: |
652444 |
Jun 4, 1996 |
05714493 |
Feb 3, 1998 |
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Current U.S.
Class: |
514/262.1;
514/230.5; 514/248; 514/249; 514/252.02; 514/252.16; 514/263.3;
514/264.1; 514/266.1 |
Current CPC
Class: |
A61K
31/517 (20130101); A61K 31/5377 (20130101); C07C
43/2055 (20130101); C07C 43/225 (20130101); C07C
43/23 (20130101); C07D 213/30 (20130101); C07D
213/64 (20130101); C07D 215/14 (20130101); C07D
215/18 (20130101); C07D 215/20 (20130101); C07D
215/233 (20130101); C07D 215/50 (20130101); C07D
239/74 (20130101); C07D 239/88 (20130101); C07D
239/91 (20130101); C07D 239/93 (20130101); C07D
239/94 (20130101); C07D 241/42 (20130101); C07D
241/52 (20130101); C07D 265/22 (20130101); C07D
277/64 (20130101); C07D 401/04 (20130101); C07D
403/04 (20130101); C07D 403/12 (20130101); C07D
405/04 (20130101); C07D 405/12 (20130101); C07D
409/04 (20130101); C07D 471/04 (20130101) |
Current International
Class: |
A61K
31/5375 (20060101); A61K 31/5377 (20060101); C07C
43/00 (20060101); C07D 405/00 (20060101); C07C
43/225 (20060101); C07D 241/00 (20060101); C07D
215/00 (20060101); C07D 241/42 (20060101); C07D
241/52 (20060101); C07D 239/88 (20060101); C07D
403/04 (20060101); C07D 239/91 (20060101); C07D
239/74 (20060101); C07D 239/93 (20060101); C07D
239/94 (20060101); C07C 43/205 (20060101); C07D
405/12 (20060101); C07D 403/00 (20060101); C07D
409/00 (20060101); C07C 43/23 (20060101); C07D
403/12 (20060101); C07D 239/00 (20060101); C07D
405/04 (20060101); C07D 265/00 (20060101); C07D
277/64 (20060101); C07D 215/50 (20060101); C07D
409/04 (20060101); C07D 215/18 (20060101); C07D
265/22 (20060101); C07D 215/14 (20060101); C07D
215/233 (20060101); C07D 215/20 (20060101); C07D
213/00 (20060101); C07D 213/30 (20060101); C07D
213/64 (20060101); C07D 277/00 (20060101); C07D
471/00 (20060101); C07D 401/00 (20060101); C07D
401/04 (20060101); C07D 471/04 (20060101); A61K
031/517 (); A61K 031/536 () |
Field of
Search: |
;514/230.5,248,249,252.02,252.16,258,259,266 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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9331010 |
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Jul 1993 |
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AU |
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0 520 722 |
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Dec 1992 |
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EP |
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0 566 266 |
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Oct 1993 |
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EP |
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0 635 498 |
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Jan 1995 |
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EP |
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1543560 |
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Apr 1979 |
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GB |
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WO92/20642 |
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Nov 1992 |
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WO |
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WO95/23141 |
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Aug 1995 |
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WO |
|
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704(1988). .
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|
Primary Examiner: Raymond; Richard L.
Attorney, Agent or Firm: Synnestvedt & Lechner LLP
Parent Case Text
This application is a .Iadd.reissue application of U.S. Ser. No.
08/652,444, filed Jun. 4, 1996, now U.S. Pat. No. 5,714,493, which
is a Section 371 application of PCT International Application Ser.
No. PCT/US94/14180, filed Dec. 8, 1994, and a
.Iaddend.continuation-in-part application of U.S. Ser. No.
08/229,886, filed Apr. 19, 1994, .Iadd.now U.S. Pat. No. 5,710,158,
.Iaddend.which is a continuation-in-part of Ser. No. 08/166,199,
filed Dec. 10, 1993, now U.S. Pat. No. 5,480,883, which is a
continuation-in-part of Ser. No. 07/988,515, filed Dec. 10, 1992,
now abandoned which is a continuation-in-part application of U.S.
Ser. No. 07/698,420, filed May 10, 1991 and a continuation-in-part
application of PCT International Application Ser. No.
PCT/US92/03736, filed May 6, 1992, which has entered the U.S.
National Stage as Ser. No. 08/146,072, filed Nov. 8, 1993.Iadd.,
now U.S. Pat. No. 5,409,930.Iaddend..
Claims
We claim:
1. A method for the treatment of inflammation in a patient
suffering from such disorder comprising administering to said
patient an effective amount of the compound of formula:
##STR24##
wherein
Ar is a substituted or unsubstituted benzene, pyrrole, thiophene,
furan, thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine,
2(1H)-pyridone, 4(1H)-pyridone, pyrazine, pyrimidine, pyridazine,
isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetralin,
naphthyridine, benzofuran, benzothiophene, indole,
2,3-dihydroindole, 1H-indazole, indoline, benzopyrazole,
1,3-benzodioxole, benzoxazole, purine, coumarin, chromone,
quinoline, tetrahydroquinoline, isoquinoline, benzimidazole,
quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine,
pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine, 1
H-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,
1(2H)-isoquinoline, 1,4-binzisoxazine, benzothiazole, quinoxaline,
quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide,
quinazoline-N-oxide, benzoxazine, phthalazine, or cinnoline;
X is a bond, O, S, SO, SO.sub.2, OCH.sub.2, C--C, C.ident.C, C--S,
SCH.sub.2, NH, NHCH.sub.2, NR.sub.4 or NR.sub.4 CH.sub.2 ;
R is independently, located at any appropriate position of Ar,
hydrogen, alkyl, alkenyl, phenyl, aralkyl, aralkenyl, hydroxy,
hydroxyalkyl, alkoxy, alkoxyalkyl, aralkoxy, aryloxy, acyloxy,
halo, haloalkyl, nitro, cyano, amino, mono- and di-alkylamino,
acylamino, carboxy, carboxyalkyl, carbalkoxy, carbaralkoxy,
carbalkoxyalkyl, carbalkoxyalkenyl, aminoalkoxy, amido,
mono-alkylamido, di-alkylamido, N, N-cycloalkylamido, sulfonyl,
mono-alkyl sulfonyl, di-alkyl sulfonyl, sulfamoyl, mono-alkyl
sulfamoyl, di-alkyl sulfamoyl, halophenyl or benzoyl;
R.sub.4 is alkyl or benzyl;
R.sub.5 is hydrogen, alkyl, alkylthio, cycloalkyl, hydroxy, alkoxy,
aralkoxy, aryl, halo, haloalkyl, carboxy or carbalkoxy; and
R.sub.6 and R.sub.7 are alkoxy or aralkoxy; or
a pharmaceutically acceptable salt thereof,
provided that: when R.sub.5 is hydrogen, R.sub.6 and R.sub.7 are
methoxy, and X is a bond, then Ar is other than R substitute phenyl
wherein R is hydrogen of (mono- or di-)methoxy; or when R.sub.5 is
hydrogen, R.sub.6 and R.sub.7 are alkoxy, X is NHCH.sub.2 or
NR.sub.4, and R.sub.4 is hydrogen, then Ar is other than R
substituted Ar is phenyl wherein R is hydrogen; or when R.sub.5 is
hydrogen or methoxy, R.sub.6 and R.sub.7 are methoxy, and X is
NHCH.sub.2, then Ar is other than R substituted pyridinyl wherein R
is hydrogen, mono-alkyl or mono-hydroxy, or Ar is other than R
substituted indol-3-yl wherein R is hydrogen..Iadd.
2. The method according to claim 1 wherein said compound is
selected from the group consisting of:
4-(3,4,5-trimethoxyphenylamino)-6,7-dimethoxyquinazoline
4-(naphthalen-2-ylethynyl)-6,7-dimethoxyquinazoline,
4-(4-hydroxyphenyl)-6,7-dimethoxyquinazoline,
4-(naphthalen-1-yl)-6,7-dimethoxyquinazoline,
4-(naphthalen-2-yl)-6,7-dimethoxyquinazoline,
4-(phenylacetylenyl-6,7-dimethoxyquinazoline,
4-(fluoro-4-methoxyphenyl)-6,7-dimethoxyquinazoline,
4-(3-phenylphenyl)-6,7-dimethoxyquinazoline,
4-(2-phenylethylenyl)-6,7-dimethoxyquinazoline,
4-(2-methoxypyridin-5-yl)-6,7-dimethoxyquinazoline,
4-(1-benzyl-indol-3-yl)-6,7-dimethoxyquinazoline,
4-(indol-3-yl)-6,7-dimethoxyquinazoline,
4-(1-methylindol-3-yl)-6,7-dimethoxyquinazoline,
4-(1-methylsulphonylindol-3-yl)-6,7-dimethoxyquinazoline,
4-(N-methyl-3,4,5-trimethoxyanilino)-6,7-dimethoxyquinazoline,
(.+-.)-4-(2-methyl-1,2,3,4-tetrahydroquinolin-1-yl)-6,7-dimethoxyquinazolin
e,
4-(1,2,3,4-tetrahydroquinolin-1-yl)-6,7-dimethoxyquinazoline,
4-(N-methyl-4-methoxyanilino)-6,7-dimethoxyquinazoline,
4-(N-methyl-4-chloroanilino)-6,7-dimethoxyquinazoline,
4-(2,3-dihydroindol-1-yl)-6,7-dimethoxyquinazoline,
4-(N-methyl-3-trifluoromethylanilino)-6,7-dimethoxyquinazoline,
4-(N-methyl-3-chloroanilino)-6,7-dimethoxyquinazoline-4-yl),
and
4-(naphthalen-1-ylethynyl)-6,7-dimethoxyquinazoline; or a
pharmaceutically acceptable salt thereof..Iaddend..Iadd.
3. The method according to claim 1 wherein said compound is
selected from the group consisting of:
4-(indazol-5-ylamino)-6,7-dimethoxyquinazoline,
4-(N-methylanilino)-6,7-dimethoxyquinazoline,
4-(N-benzylanilino)-6,7-dimethoxyquinazoline,
4-(N-ethyl-3-chloroanilino)-6,7-dimethoxyquinazoline,
4-(N-methyl-4-methylanilino)-6,7-dimethoxyquinazoline,
4-(4-methoxybenzylamino)-6,7-dimethoxyquinazoline,
4-(3,5-dimethoxybenzylamino)-6,7-dimethoxyquinazoline,
4-(3,4,5-trimethoxyphenoxy)-6,7-dimethoxyquinazoline,
4-(3-methoxythiophenoxy)-6,7-dimethoxyquinazoline,
4-[N-(5-indanyl)amino]-6,7-dimethoxyquinazoline,
4-(3-chlorothiophenoxy)-6,7-dimethoxyquinazoline,
4-(3-aminopyrazolyl)-6,7-dimethoxyquinazoline,
4-(1,4-benzodioxan-6-ylamino)-6,7-dimethoxyquinazoline,
4-(.alpha.-naphthylamino)-6,7-dimethoxyquinazoline,
4-(.beta.-naphthylamino)-6,7-dimethoxyquinazoline,
4-(cyclohexylamino)-6,7-dimethoxyquinazoline,
4-(N-methylanilino)-6,7-dimethoxyquinazoline, and
4-(3-chlorophenoxy)-6,7-dimethoxyquinazoline; or a pharmaceutically
acceptable salt thereof..Iaddend..Iadd.
4. The method according to claim 1 wherein said compound is
4-(indol-3-yl)-6,7-dimethoxyquinazoline..Iaddend..Iadd.
5. The method according to claim 1 wherein said compound is
4-(.alpha.-naphthylamino)-6,7-dimethoxyquinazoline..Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the modulation and/or inhibition of cell
signaling, cell proliferation, the control of abnormal cell growth
and cell inflammatory response. More specifically, this invention
relates to the use of mono- and/or bicyclic aryl or heteroaryl
quinazoline compounds which exhibit selective inhibition of
differentiation, proliferation or mediator release by effectively
inhibiting CSF-1R tyrosine kinase activity.
Normal cell growth is believed to be triggered by the exposure of
the cellular substrate to one or more growth factors, examples of
which are insulin, epidermal growth factor (EGF) and
platelet-derived growth factor (PDGF). Receptors for such growth
factor are imbedded in and penetrate through the cellular membrane.
The initiation of cellular reproduction is believed to occur when a
growth factor binds to the corresponding receptor on the external
surface of the cellular membrane. This growth factor-receptor
binding alters the chemical characteristics of that portion of the
receptor which exists within the cell and which function as an
enzyme to catalyze phosphorylation of either an intercellular
substrate or the receptor itself, the latter being referred to as
autophosphorylation. Examples of such phosphorylating enzymes
include tyrosine kinases, which catalyze phosphorylation of
tyrosine amino acid residues of substrate proteins.
Many disease states are characterized by the uncontrolled growth of
cells. These disease states involve a variety of cell types and
include disorders such as cancer, leukemia, psoriasis, inflammatory
diseases, bone diseases, atherosclerosis and restenosis occurring
subsequent to angioplastic procedures. The inhibition of tyrosine
kinases is believed to have utility in the control of deregulated
cellular proliferation, i.e., cellular proliferative disorders.
Initiation of autophosphorylation, i.e., phosphorylation of the
growth factor receptor itself, and of the phosphorylation of a host
of intracellular substrates are some of the biochemical events
which are involved in mediator release and cell proliferation.
REPORTED DEVELOPMENTS
Inhibitors of p56.sup.Ick tyrosine kinase have been reported in the
literature by Bolen, J. B. et al. FASEB J. 1992, 3403., Mustelin,
T. et al. TIBS 1993,215.; Eichmann, K. Angew. Chem. Int. Ed. Eng.
1993, 54.; and Klausner, R. D. Samelson, L. E. Cell 1991 , 875.
These include compounds that are potent but nonselective
inhibitors, such as staurosporine, which is competitive with ATP or
compounds that are very weak tyrosine kinase inhibitors, but are
somewhat selective, such as the flavonoid quercetin.
A series of dihydroxy-isoquinolines have been been reported by
Burke, T. R. et al. (Biorg. & Med. Chem. Lett. 1992, 1771;J.
Med. Chem. 1993 3010 and J. Med. Chem. 1993,3015) that have potent
p56.sup.Ick inhibiting activity. Potential therapeutic uses for
selective inhibitors of p56.sup.Ick include the treatment of
autoimmune diseases such as rheumatoid arthritis or transplant
rejection.
p56.sup.Ick, which is a non-receptor tyrosine kinase, has been
shown to be important in intracellular signaling in T-cells. It is
assumed that inhibitors of p56.sup.Ick kinase activity perturb the
activation of T-cells and therefore a selective inhibitor could
prove useful in the treatment of T-cell mediated conditions such as
organ rejection, rheumatoid arthritis or other auto-immune
diseases.
SUMMARY OF THE INVENTION
The present invention described compounds which are inhibitors of
the colony stimulating factor-1 receptor tyrosine kinase, CSF-1R,
activity and have activity in a p56.sup.Ick cell-free assay. These
compounds do not appear to have any significant serine/threonine
kinase inhibitory activity and in addition, compounds within the
scope of this invention do not demonstrate significant PDGF-R
activity in a cell-free assay. Compounds of this invention are also
weak inhibitors of PDGF-induced mitogenesis which may suggest that
these compounds inhibit other src-like tyrosine kinases involved in
the signal transduction pathway.
Compounds within the scope of this invention are inhibitors of the
colony stimulating factor-1 receptor tyrosine kinase, CSF-1R,
activity. A selective inhibitor of the tyrosine kinase activity of
this receptor, which is closely related to the platelet-derived
growth factor receptor (PDGF-R), has never been reported. Compounds
of this invention are selective inhibitors of CSF-1R tyrosine
kinase activity and are useful for elucidating the importance of
CSF-1 and CSF-1 receptor signaling in bone remodeling and
hematopoeisis. In addition compounds inhibiting growth
factor-induced CSF and/or Ick signalling are described herein.
In accordance with the present invention, there is provided
pharmaceutical compositions for inhibiting abnormal cell
proliferation and/or differentiation or mediator release in a
patient suffering from a disorder characterized by such
proliferation activity, comprising the administration to a patient
a tyrosine kinase composition which effectively inhibits CSF-1R
tyrosine kinase activity in a CSF-1R inhibiting effective amount of
a mono- aryl or hetero-aryl quinazoline compound exhibiting
inhibition of differentiation, proliferation or mediator release
activity wherein each aryl group is a ring system containing 0-4
hetero atoms, said compound being optionally substituted or
polysubstituted.
Another aspect of the present invention relates to a method of
inhibiting abnormal cell proliferation and/or differentiation or
mediator release comprising, in admixture with a pharmaceutically
acceptable carrier, a pharmaceutically effective amount of a
compound of the aforementioned type. Another aspect of this
invention comprises compounds useful in the practice of the present
method.
With respect to the aspects of this invention, the compounds
described by Formula I below constitute a class of the
aforementioned mono- and bicicyclic aryl or heteroaryl quinazoline
compounds for use in the practice of the present invention:
##STR1##
wherein
Ar is a substituted or unsubstituted mono- or bi-cyclic aryl or
heteroaryl ring system of about 5 to about 12 atoms and where each
monocyclic ring may contain 0 to about 3 hetero atoms, and each
bicyclic ring may contain 0 to about 4 hetero atoms selected from
N, O and S provided said hetero atoms are not vicinal oxygen and/or
sulfur atoms and where the substituents may be located at any
appropriate position of the ring system and are described by
R.;
X is a bond, O, S, SO, SO.sub.2, OCH.sub.2, C--C, C.ident.C, C--S,
SCH.sub.2, NH, NHCH.sub.2, NR.sub.4 and NR.sub.4 CH.sub.2 ;
R independently includes hydrogen, alkyl, alkenyl, phenyl, aralkyl,
aralkenyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, aralkoxy,
aryloxy, acyloxy, halo, haloalkyl, nitro, cyano, amino, mono- and
di-alkylamino, acylamino, carboxy, carboxyalkyl, carbalkoxy,
carbaralkoxy, carbalkoxyalkyl, carbalkoxyalkenyl, aminoalkoxy,
amido, mono- and di-alkylamido and N,N-cycloalkylamido, alkylthio,
alkylsulfinyl, sulfonyl, mono- and di-alkyl sulfonyl, sulfamoyl,
mono- and di- alkyl sulfamoyl, halophenyl of benzoyl; and R and R
together may also form a ketone group.
R.sub.4 is alkyl, --CH.sub.2 --CH.sub.2 -- or --CH.sub.2 --CH.sub.2
--CH.sub.2 --; and
R.sub.5, R.sub.6 and R.sub.7 are independently hydrogen, alkyl,
alkylthio, cycloalkyl, hydroxy, alkoxy, aralkoxy, aryl, halo,
haloalkyl, carboxy or carbalkoxy; or
a pharmaceutically acceptable salt thereof.
Preferred Ar monocyclic aryl or heteroaryl rings include
substituted or unsubstituted benzene, pyrrole, thiophene, furan,
thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine,
2(1H)-pyridone, 4(1H)-pyridone, pyrazine, pyrimidine, pyridazine,
isothiazole, isoxazole, oxazole and tetrazole.
Preferred Ar bicyclic aryl or heteroaryl rings include substituted
and unsubstituted naphthalene, tetralin, naphthyridine, benzofuran,
benzothiophene, indole, 2,3-dihydroindole, 1H-indazole, indoline,
benzopyrazole, 1,3-benzodioxole, benzoxazole, purine, coumarin,
chromone, quinoline, tetrahydroquinoline, isoquinoline,
benzimidazole, quinazoline, pyrido[2,3-b]pyrazine, pyrido
[3,4-b]pyrazine, pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine,
1H- pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,
1(2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline,
quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide,
quinazoline-N-oxide, benzoxazine, phthalazine, or cinnoline.
More preferred Ar rings include substituted and unsubstituted
benzene, pyridine, thiophene, naphthalene, quinoline, indole and
1H-pyrazole[3,4-d]-pyrimidine.
Preferred R substituents include hydrogen, alkyl, alkenyl, hydroxy,
alkoxy, halo, haloalkyl, amino, mono-and di-alkylamino, acylamino,
carboxy, carbalkoxy, amido, mono- and di-alkylamido,
N,N-cycloalkylamido, alkylthio, alkylsulfinyl, alkylsulfonyl or
sulfamoyl, alkyl, alkenyl, phenyl, aralkyl, aralkenyl, and R may
also form a keto group.
As employed above and through this disclosure, the following terms,
unless otherwise indicated, shall be understood to have the
following meanings:
"Monocyclic aryl of heteroaryl" means a carbocylcic or heterocyclic
aromatic ring. Preferred rings include phenyl, thienyl, pyridyl,
2(1H)-pyridonyl, 4(1H)-pyridonyl, furyl, pyrimidinyl, imidazolyl,
thiazolyl, oxazolyl and tetrazolyl.
"Bicyclic aryl or heteroaryl" means a bicyclic ring system composed
of two fused carbocyclic and/or heterocyclic aromatic rings.
Preferred rings include naphthyl, indolyl, benzothienyl,
benzofuranyl, quinolinyl, chromonyl, 1(2H)-isoquinolonyl,
isoquinolinyl, benzimidazolyl, benzothiazolyl, quinoxalinyl,
naphthyridinyl, cinnolinyl, phthalazinyl, pyrido[2,3-b]pyrazinyl,
pyrido[3,4-b]pyrazinyl, pyrido[3,2-c]pyridazinyl,
pyrido[3,4-b]-pyridinyl, pteridinyl, and quinazolinyl.
"Alkyl" means a saturated aliphatic hydrocarbon, either branched-
or straight-chained. Preferred alkyl is "lower-alkyl" having about
1 to about 6 carbon atoms. Examples of alkyl include methyl, ethyl,
n-propyl, isopropyl, butyl, secbutyl, t-butyl, amyl and hexyl.
"Cycloalkyl" means a cyclic aliphatic group comprising from about
three to about seven carbon atoms. Preferred cycloalkyl groups
include cyclopropyl, cyclobutyl, clclohexyl and cycloheptyl.
"Alkoxy" refers to an alkyl-O-group. Preferred alkoxy groups
include methoxy, ethoxy, propoxy and butoxy.
"Aryloxy" refers to an aryl-O-group. The preferred aryloxy group is
phenoxy.
"Aralkyl" means an alkyl group substituted by an aryl radical. The
preferred aralkyl groups are benzyl or phenethyl.
The preferred aralkoxy groups are benzyloxy and phenethoxy.
The preferred acyloxy groups are acetoxy and benzyloxy;
"Halo" means halogen. Preferred halogens include chloride, bromide
and fluoride.
The preferred haloalkyl groups are mono-, di- and
trifluoromethyl.
The more preferred compounds of this invention include those
compounds of Formula I where
Ar is phenyl or naphthyl;
R is hydrogen, alkyl, alkoxy hydroxy, halo or trifluoromethyl.
X is a bond, NH or NR.sub.4 ; and
R.sub.5, R.sub.6 and R.sub.7 are independently hydrogen or
alkoxy.
The most preferred compounds are those described where
Ar is phenyl;
X is NH or NMe; and
R.sub.5, R.sub.6 and R.sub.7 are independently hydrogen or
methoxy.
It is intended that N-oxides of the above described
aminoquinazolines are encompassed within the scope of this
invention.
Special embodiments of this invention inhibiting the growth factor
or tyrosine kinase include the following:
A. Compounds of Formula I where:
X is a bond, NR.sub.4, S or O, the inhibiting cell proliferation
and/or differentiation or mediator release is especially
characterized by CSF-1 activity.
B. Compounds of Formula I where:
X is a bond, NH, S or O, the inhibiting cell proliferation and/or
differentiation or mediator release is especially characterized by
Ick/EGF activity.
C. Compounds of Formula I where:
X is a bond and Ar is phenyl, indolyl, pyrrolyl, thienyl, pyridyl,
naphthyl, a bicyclic aryl, a bicyclic heteroaryl or substituted
phenyl, indoly, pyrrolyl, thienyl, pyridyl, naphthyl, bicyclic
aryl, bicyclic heteroaryl, the inhibiting cell proliferation and/or
differentiation or mediator release is especially characterized by
Ick activity.
D. Compounds of Formula I where:
X is NH, R.sub.6 and R.sub.7 are alkoxy and Ar is phenyl having at
least one substituent in the 3, 4 and / or 5 positions of hydroxy
or alkoxy, the inhibiting cell proliferation and/or differentiation
or mediator release is especially characterized by Ick
activity.
The compounds of this invention may be useful in the form of the
free base, in the form of salts and as a hydrate. All forms are
within the scope of the invention. Acid addition salts may be
formed and are simply a more convenient form for use; and in
practice, use of the salt form inherently amounts to use of the
base form. The acids which can be used to prepare the acid addition
salts include preferably those which produce, when combined with
the free base, pharmaceutically acceptable salts, that is, salts
whose anions are non-toxic to the animal organism in pharmaceutical
doses of the salts, so that the beneficial properties inherent in
the free base are not vitiated by side effects ascribable to the
anions. Although pharmaceutically acceptable salts of said basic
compound are preferred, all acid addition salts are useful as
sources of the free base form even if the particular salt per se is
desired only as an intermediate product as, for example, when the
salt is formed only for purposes of purification and
identification, or when it is used as an intermediate in preparing
a pharmaceutically acceptable salt by ion exchange procedures.
Pharmaceutically acceptable salts within the scope Of the invention
include those derived from the following acids: mineral acids such
as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic
acid; and organic acids such as acetic acid, citric acid, lactic
acid, tartaric acid, malonic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclohexylsulfamic acid, quinic acid, and the like.
The corresponding acid addition salts comprise the following:
hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate,
lactate, tartrate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toleuenesulfonate, cyclohexylsulfamate and
quinate, respectively.
The acid addition salts of the compounds of this invention are
prepared either by dissolving the free base in aqueous or
aqueous-alcohol solution or other suitable solvents containing the
appropriate acid and isolating the salt by evaporating the
solution, or by reacting the free base and acid in an organic
solvent, in which case the salt separates directly or can be
obtained by concentration of the solution.
The compounds of this invention may be prepared by employing
procedures known in the literature starting from known compounds or
readily prepared intermediates. Exemplary general procedures
follow.
In general the compounds useful for the method of inhibiting cell
proliferation and/or differentiation or mediator release may be
prepared by the coupling reaction of a palladium catalyzed aryl or
heteroarylstannane with an aryl or heteroarylhalide or triflate.
##STR2##
where A is halogen or triflate and B is trialkylstannane and R is
as previously described.
The 4-haloquinazoline starting materials are prepared in the
classical way using anthranilic acid derivatives and formamide at
reflux to provide the intermediate quinazolinones. Subsequent
treatment with POCl.sub.3 at about 110.degree. C. for about two
hours provides the chloroquinazolines. The final products are
prepared via a condensation with the appropriate aniline derivative
in a polar solvent such as ethanol. In the case of the phenoxy or
thiophenoxy derivatives, the metal salt (preferably Na) is prepared
and refluxed for several hours with the appropriate haloquinazoline
in a solvent such as THF. ##STR3##
The aryl and heteroarylstannanes may be prepared from the
corresponding halide (preferably bromide or iodide) by conversion
to the aryllithium by reaction with t-butyllithium at decreased
temperatures, preferably about -78.degree. C. followed by reaction
with a halotrialkylstannane. ##STR4##
Of course these products may also be prepared in the reverse manner
using the aryl or heteroarylhalides with the the corresponding
stannane. ##STR5##
The quinazoline stannanes intermediates may be prepared by the
action in trimethyltin sodium on aryl halides as described in Chem.
Pharm. Bull. 1982, 30, 1731-1737:
The preparation of the compounds useful in this invention are
described in Applicants' copending applications U.S. Ser. No.
08/166,199, filed Dec. 10, 1993 and U.S. Ser. No. 08/229,886, filed
Apr. 19, 1994 of which this application claims priority. U.S. Ser.
No. 08/166,199 and U.S. Ser. No. 08/229,886 are hereby incorporated
herein by reference.
Further, the following examples are representative of the processes
used to synthesis the compounds of this invention.
The below examples and those described in U.S. Ser. No. 08/166,199
may be followed to prepare any of the desired compounds of this
invention. A representative list of compounds which may be prepared
is shown below.
EXAMPLE 1
4-(3-chlorophenoxy)-6,7-dimethoxyquinazoline
THF (5 ml) and NaH (60% disp in oil, approx. 28 mg) is added to a
dry flask maintained under inert atmosphere at room temperature.
3-Chlorophenol (0.09 g) is added as a soln. in THF (1 mL) and
stirring is continued until the solution became clear.
4-Chloro-6,7-dimethoxyquinazoline is added all at once (as the
solid) and stirring was maintained overnight at RT. The solution is
partitioned between CH.sub.2 Cl.sub.2 and 5% NaOH. The organic
layer is washed with brine, dried (Na.sub.2 SO.sub.4) and
concentrated. Flash column chromatography (40% EtOAc/Hex) provided
the pure compound. An analytical sample is obtained by
recrystallization from EtOAc/Hex to provide
4-(3-chlorophenoxy)-6,7-dimethoxyquinazoline (0.05 g), white
needles, m.p. 152.degree.-153.degree. C.
EXAMPLE 2
4-(1-methylsulphonylindol-3-yl)-6,7-dimethoxyquinazoline
Step A
N-methylsulfonyl-3-trimethylstannylindole
A solution of 5 g (15.57 mmol) of N-methylsulfonyl-3-iodoindole
(5.1 g; 15.57 mmol) of hexamethylditin and 0.89 g (0.78 mmol) of Pd
(PPh3).sub.4 in 75 mL of dry toluene is flushed thoroughly with
nitrogen and heated to 90.degree. C. for 4 hours. The mixture is
then evaporated and chromatographed on silica gel (eluting with
hexane and then with 10% ethyl acetate/hexane to give
N-methylsulfonyl-3-trimethylstannylindole which is used directly in
the next step.
Step B
4-(1-methylsulphonylindol-3-yl)-6,7-dimethoxyquinazoline
A solution of 1.33 g (4.01 mmol) of
N-methylsulfonyl-3-trimethyl-stannylindole, 750 mg (3.34 mmol) of
4-chloro-6,7-dimethoxyquinazoline and 0.19 g (5mol % 0.16 mmol) of
Pd (PPh.sub.3).sub.4 in 10 ml of dry dimethylformamide is flushed
thoroughly with nitrogen and heated to 90.degree. C. for 12 hours.
The reaction mixture is diluted with methylene chloride washed with
10% ammonium hydroxide and stirred vigorously and then washed with
water and the combined organics are washed with brine (75 mL),
dried (MgSO.sub.4) and evaporated to dryness. Recrystallization
from ethyl acetate yields
4-(1-methylsulphonylindol-3-yl)-6,7-dimethoxyquinazoline
(m.p.>220.degree. C.).
The above examples may be followed to prepare any of the desired
compounds of this invention. A representative list of compounds
which may be prepared are shown below.
6,7-dimethoxy-4-naphthalen-2-ylethynylquinazoline, m.p.
158.degree.-161.degree. C.
4-(4-hydroxyphenyl)-6,7-dimethoxyquinazolinehydrochloride,
m.p.>270.degree. C. (dec)
4-(naphthalen-1-yl)-6,7-dimethoxyquinazoline, m.p.
144.degree.-147.degree. C.
4-(naphthalen-2-yl)-6,7-dimethoxyquinazoline, m.p.
115.degree.-118.degree. C.
4-phenylacetylenyl-6,7-dimethoxyquinazoline, m.p.
146.degree.-148.degree. C.
4-(3-fluoro-4-methoxyphenyl)-6,7-dimethoxyquinazoline, m.p.
207.degree.-210.degree. C.
4-(3-phenylphenyl)-6,7-dimethoxyquinazoline, m.p.
160.degree.-163.degree. C.
4-(2-phenylethylenyl)-6,7-dimethoxyquinazoline, m.p.
168.degree.-169.degree. C.
4-(2-methoxypyridin-5-yl)-6,7-dimethoxyquinazoline, m.p.
175.degree.-176.degree. C.
4-(1-benzylindol-3-yl)-6,7-dimethoxyquinazoline, m.p.
148.degree.-150.degree. C.
4-(indol-3-yl)-6,7-dimethoxyquinazoline, m.p. >240.degree. C.
(dec)
4-(1-methylindol-3-yl)-6,7-dimethoxyquinazoline hydrochloride, m.p.
>230.degree. C. (dec)
4-(1-methylsulphonylindol-3-yl)-6,7-dimethoxyquinazoline, m.p.
>220.degree. C. (dec)
4-(4-phenylpiperidin-1-yl)-6,7-dimethoxyquinazoline, m.p.
150.degree.-151.degree. C.
4-[4-(3-chlorophenyl)piperazin-1-yl]-6,7-dimethoxyquinazoline, m.p.
155.degree.-156.degree. C.
4-(N-methyl-3,4,5-trimethoxyanilino)-6,7-dimethoxyquinazoline, m.p.
149.degree.-151.degree. C.
(+-)-4-(2-methyl-1,2,3,4-tetrahydroquinolin-1-yl)-6,7-dimethoxyquinazoline
hydrochloride, m.p. 198.degree.-201.degree. C. (dec)
4-(1,2,3,4-tetrahydroquinolin-1-yl)-6,7-dimethoxyquinazoline
hydrochloride, m.p. 195.degree.-197.degree. C. (dec)
4-(N-methyl-4-methoxyanilino)-6,7-dimethoxyquinazoline
hydrochloride, m.p. 202.degree.-205.degree. C.
4-(N-methyl-4-chloroanilino)-6,7-dimethoxyquinazoline
hydrochloride, m.p. 220.degree.-222.degree. C.
4-(2,3-dihydroindol-1-yl)-6,7-dimethoxyquinazoline hydrochloride,
m.p. 226.degree.-229.degree. C. (dec)
N-(6,7-dimethoxyquinazolin-4-yl)-N-methyl-N-(3-trifluoromethyphenyl)amine
hydrochloride, m.p. 240.degree.-243.degree. C.
N-(3-chlorophenyl)-N-(6,7-dimethoxyquinazolin-4-yl)-N-methylamine
hydrochloride, m.p. 235.degree.-237.degree. C.
N-(3-chlorophenyl)-N-(quinazolin-4-yl)-N-methyl-amine
hydrochloride, m.p. 233.degree.-235.degree. C.
6,7-dimethoxy-4-naphthalen-1-yl-ethynylquinazoline, m.p.
175.degree.-177.degree. C.
4-(thien-3-yl)-6,7-dimethoxyquinazoline, m.p.
148.5.degree.-151.5.degree. C.
4-benzyl-6,7-dimethoxyquinazoline, m.p. 122.5.degree.-125.degree.
C. (6,7-dimethoxyquinazolin-4-yl)-5-indazolylamine hydrochloride,
m.p. 261.degree.-263.degree. C. (dec)
N-(6,7-dimethoxyquinazolin-4-yl)-N-phenyl-N-ethylamine
hydrochloride, m.p. 227.degree.-230.degree. C. (dec)
N-benzyl-N-(6,7-dimethoxyquinazolin-4-yl)-N-phenylamine
hydrochloride, m.p. 269.degree.-271.degree. C.
N-(6-chloroquinazolin-4-yl)-N-methyl-N-phenylamine, m.p.
106.degree.-108.degree. C.
N-(3-chloro-phenyl)-N-6,7-dimethoxyquinazolin-4-yl)-N-ethylamine
hydrochloride, m.p. 261.degree.-263.degree. C.
N-(6,7-dimethoxyquinazolin-4-yl)-N-methyl-N-p-tolylamine
hydrochloride, m.p. 230.degree.-234.degree. C. (dec)
N-benzyl-N-(6,7-dimethoxyquinazolin-4-yl)amine, m.p.
220.degree.-225.degree. C.
N-(4-methoxybenzyl)-N-(6,7-dimethoxyquinazolin-4-yl) amine, m.p.
194.degree.-198.degree. C.
N-(3,5-dimethoxybenzyl)-N-(6,7-dimethoxyquinazolin-4-yl)amine
hydrochloride, m.p. 265.degree.-269.degree. C.
4-(3,4,5-trimethoxyphenoxy)-6,7-dimethoxyquinazoline, m.p.
228.degree.-232.degree. C.
N-(quinazolin-4-yl)-N-phenyl-N-methylamine hydrochloride, m.p.
242.degree.-246.degree. C. (dec)
N-(6,7-dimethoxyquinazolin-4-yl)-N-(4-morpholin-4-ylphenyl)amine
hydrochloride, m.p. 231.degree.-235.degree. C. (dec)
4-(3-methoxythiophenoxy)-6,7-dimethoxyquinazoline, m.p.
139.5.degree.-141.5.degree. C.
4-[N-(5-indanyl)amino]-6,7-dimethoxyquinazoline hydrochloride, m.p.
244.degree.-246.degree. C. (dec)
4-(3-chlorothiophenoxy)-6,7-dimethoxyquinazoline, m.p.
152.degree.-153.5.degree. C.
4-(3-aminopyrazolyl)-6,7-dimethoxyquinazoline hydrochloride, m.p.
262.degree.-264.degree. C. (dec)
4-(1,4-benzodioxan-6-ylamino)-6,7-dimethoxyquinazoline
hydrochloride, m.p. 267.degree.-269.degree. C. (dec)
6,7-dimethoxy-4-(.alpha.-naphthylamino)quinazoline hydrochloride,
m.p. >250.degree. C.
6,7-dimethoxy-4-(.beta.-naphthylamino)quinazoline hydrochloride,
m.p. >250.degree. C.
.[.4-(cyclohexylanilino)-6,7-dimethoxyquinazoline.].
.Iadd.4-(cyclohexylamino)-6,7-dimethoxyquinazoline.Iaddend., m.p.
239.degree.-244.degree. C.
4-(3,4,5-trimethoxyanilino)-6,7-dimethoxyquinazoline hydrochloride,
m.p. 260.degree.-265.degree. C.
6,7-dimethoxy-4-(N-methylanilino)quinazoline hydrochloride, m.p.
>230.degree. C.
4-(3-chlorophenoxy)-6,7-dimethoxyquinazoline, m.p.
152.degree.-153.degree. C.
6,7-dimethoxy-4-(1-naphthylthio)-quinazoline, m. p.
174.5-176.5.degree. C.
6,7-dimethoxy-4-(2-naphthylthio)-quinazoline, m. p.
178.degree.-179.degree. C.
6,7-dimethoxy-4-(1-naphthyloxy)-quinazoline, m.p.
214.degree.-215.5.degree. C.
6,7-dimethoxy-4-(2- naphthyloxy)-quinazoline, m.p.
169.degree.-170.degree. C.
N-(6,7-dimethoxy-quinolazolin-4-yl)-N-(naphth-2-yl)-N-ethylamine
hydrochloride, m.p. 236.degree.-239.degree. C. (dec)
6,7-dimethoxy-4-(naphthalene-2-sulfinyl)quinazoline, m.p.
182.5.degree.-185.degree. C.
6,7-dimethoxy-4-(naphthalene-2-sulfonyl)quinazoline
4-(3-chloroanilino)-6,7-dimethylquinazoline hydrochloride, m.p.
271.degree.-274.degree. C.
4-(3,5-dimethylanilino-6,7-dimethylquinazoline hydrochloride, m.p.
>275.degree. C.
4-(N-methyl-4-methylanilino)-6,7-dimethylquinazoline hydrochloride,
m.p. 235.degree.-238.degree. C.
6,7-dimethyl-4-(1-naphthylamino)quinazoline hydrochloride, m.p.
244.degree.-247.degree. C.
6,7-dimethyl-4-(7-trifluoromethyl-3,4-dihydro-2H-quinolin-1-yl)quinazoline
hydrochloride, m.p. 240.degree. C.
4-(N-methyl-3-methylanilino)-6,7-dimethylquinazoline hydrochloride,
m.p. 205.degree.-207.degree. C.
4-(3-chlorophenylthio)-6,7-dimethylquinazoline hydrochloride, m.p.
197.degree.-202.degree. C.
4-(1-naphthylthio)-6,7-dimethylquinazoline hydrochloride, m.p.
204.degree.-209.degree. C.
4-(3,4-dimethoxyphenylthio)quinazoline, m.p.
115.degree.-117.degree. C.
PREPARATION OF PHARMACEUTICAL COMPOSITIONS AND PHARMACOLOGICAL TEST
SECTION
Compounds within the scope of this invention exhibit significant
activity as protein tyrosine kinase inhibitors and possess
therapeutic value as cellular antiproliferative agents for the
treatment of certain conditions including psoriasis,
atherosclerosis and restenosis injuries. Further, specific
inhibitors of CSF-1R tyrosine kinase activity are useful for
elucidating the importance of CSF-1 and CSF-1 receptor signaling in
bone remodeling and hematopoeisis. Compounds within the scope of
the present invention exhibit the modulation and/or inhibition of
cell signaling, cell proliferation, cell inflammatory response, the
control of abnormal cell growth and can be used in preventing or
delaying the occurrence or reoccurrence of such conditions or
otherwise treating the condition.
To determine the effectiveness of compounds of this invention, the
pharmacological tests described below, which are accepted in the
art and recognized to correlate with pharmacological activity in
mammals, are utilized. Compounds within the scope of this invention
have been subjected to these various tests, and the results
obtained are believed to correlate to useful cellular
differentiation mediator activity. The below described tests are
useful in determining the inhibition of the colony stimulating
factor-1 receptor tyrosine kinase (CSF-1R) activity. The ability to
inhibit the p561.sup.Ick tyrosine kinase activity of compounds
disclosed herein is described. The results of these tests are
believed to provide sufficient information to persons skilled in
the pharmacological and medicinal chemistry arts to determine the
parameters for using the studied compounds in one or more of the
therapies described herein.
EGF-Receptor Purification
EGF-receptor purification is based on the procedure of Yarden and
Schlessinger. A431 cells are grown in 80 cm.sup.2 bottles to
confluency (2.times.10.sup.7 cells per bottle). The cells are
washed twice with PBS and harvested with PBS containing 11.0 mmol
EDTA (1 hour at 37.degree. C., and centrifuged at 600 g for 10
minutes. The cells are solubilized in 1 ml per 2.times.10.sup.7
cells of cold solubilization buffer (50 mmol Hepes buffer, pH 7.6,
1% Triton X-100, 150 mmol NaCl, 5 mmol EGTA, 1 mmol PMSF, 50
.mu.g/ml aprotinin, 25 mmol benzomidine, 5 .mu.g/ml leupigptic, and
10 .mu.g/ml soybean trypsin inhibitor) for 20 minutes at 4.degree.
C. After centrifugation at 100,000 g for 30 minutes, the
supernatant is loaded onto a WGA-agarose column (100 .mu.l of
packed resin per 2.times.10.sup.7 cells) and shaken for 2 hours at
4.degree. C. The unabsorbed material is removed and the resin
washed twice with HTN buffer (50 mmol Hepes, pH 7.6, 0.1% Triton
X-100, 150 mmol NaCl), twice with HTN buffer containing 1M NaCl,
and twice with HTNG buffer (50 mmol Hepes, pH 7.6, 0.1% Triton
X-100, 150 mmol NaCl, and 10% glycerol). The EGF receptor is eluted
batchwise with HTNG buffer containing 0.5M N-acetyI-D-glucosamine
(200 .mu.l per 2.times.10.sup.7 cells). The eluted material is
stored in aliquots at -70.degree. C. and diluted before use with
TMTNG buffer (50 mmol Tris-Mes buffer, pH 7.6, 0.1% Triton X-100,
150 mmol NaCl, 10% glycerol).
ATP and EGF Dependence of Autophosphorylation
WGA-purified EGF receptor from A431 cells (0.5 .mu.g/assay is
activated with EGF (0.85 .mu.M) for 20 minutes at 4.degree. C. The
assay is performed at 15.degree. C. and initiated by addition of
Mg(Ac).sub.2 (60 mmol), Tris-Mes buffer, pH 7.6 (50 mmol), [.sup.32
P]ATP (carrier free, 5 .mu.Ci/assay), and increasing concentrations
of nonradioactive ATP. The assay is terminated after 10-sec by
addition of SDS sample buffer. The samples are run on a 6% SDS
polyacrylamide gel. The gel is dried and autoradiographed as
described above. The relevant radioactive bands are cut and counted
in the Cerenkov mode. The K.sub.m for ATP determined in this
fashion is found to be 7.2 .mu.M. With use of the 10-sec assay
protocol, the EGF concentration dependence of EGF-RK
autophosphorylation is determined.
Inhibition of EGF-R Autophosphorylation
A431 cells are grown to confluence on human fibronectin coated
tissue culture dishes. After washing 2 times with ice-cold PBS,
cells are lysed by the addition of 500 .mu.l/dish of lysis buffer
(50 mmol Hepes, pH 7.5, 150 mmol NaCl, 1.5 mmol MgCl.sub.2, 1 mmol
EGTA, 10% glycerol, 1% triton X-100, 1 mmol PMSF, 1 mg/ml
aprotinin, 1 mg/ml leupeptin) and incubating 5 minutes at 4.degree.
C. After EGF stimulation (500 .mu.g/ml 10 minutes at 37.degree. C.)
immunoprecipitation is performed with anti EGF-R (Ab 108) and the
autophosphorylation reaction (50 .mu.l aliquots, 3 .mu.Ci
[.gamma.-.sup.32 P]ATP) sample is carried out in the presence of 2
or 10 .mu.M of compound of the present invention, for 2 minutes at
4.degree. C. The reaction is stopped by adding hot electrophoresis
sample buffer. SDA-PAGE analysis (7.5% els) is followed by
autoradiography and the reaction is quantitated by densitometry
scanning of the x-ray films.
Cell Culture
Cells termed HER 14 and K721A (=DK) are prepared by transfecting
NIH3T3 cells (clone 2.2) (From C. Fryling, NCI, NIH), which lack
endogenous EGF-receptors, with cDNA constructs of wild-type
EGF-receptor or mutant EGF-receptor lacking tyrosine kinase
activity (in which Lys 721 at the ATP-binding site is replace by an
Ala residue, respectively). All cells are grown in DMEM with 10%
calf serum (Hyclone, Logan, Utah).
Further tests which show the effectiveness and selectivity of
compounds of this invention to inhibit dell proliferation and/or
differentiation of mediator release are as follows.
CSF-1R Cell-free Autophosphorylation Assay
For a regular 28 tube assay (14 samples per 15 well gel):
In 2 ml eppendorf tube: 140 mg protein A sepharose (5
mg/sample)
Swell in 20 mM Hepes pH 7.5 and wash 2x in Hepes
Add 280 .lambda..alpha.-CSF-1R
20 min RT shaking
Wash 3 .times. in HNTG pH 7.5: 20 mM Hepes 150 mM NaCl 0.1% triton
X-100 10% glycerol In 15 ml tube: 2.8 ml lysate 20 mM Hepes lysis
buffer: 1.5 mM MgCl.sub.2 150 mM NaCl 1 mM EGTA 10% glycerol 1%
triton X-100
Protease inhibitors added fresh:
PMSF: 8 mg/ml=2500x in 100% ETCH, store frozen, add 100 .lambda./10
ml lysis buffer
Aprotinin: 10 mg/ml=250x in H.sub.2 O, store frozen, add 40
.lambda./10 ml lysis buffer
Leupeptin: 1mg/ml=250 x in H20, store frozen, add 40 .mu./10 ml
lysis buffer
Add washed beads to stimulated lysate and incubate 90 min 4.degree.
C. on rotator or shaking
prepare 28 compound tubes:
make 40 mM solutions of compounds in 100% DMSO
make serial dilutions in 50 mM Tris pH 7.5+10 mM MnCl.sub.2
aliquot 10.lambda. compound solution into each 1 ml eppendorf
reaction tube waiting on ice, control blanks get 10% buffer
Wash beads 1x HNTG, 2x 10 nM Tris pH 7.5
Add 10.lambda. ATP solution: 312.lambda. 50 mM Tris pH 7.5 + 10 mM
MnCl.sub.2 2.7.lambda. cold ATP (stock of 10 mM in 5 mM) Tris = 20
.mu.M final) 351.sup.32 P-ATP (10.mu. Ci/sample)
Vortex, incubate 10 min on ice
Add 45.lambda. 2x SDS-sample buffer, heat 95.degree. C. 6 min
7.5% SDS-PAGE, fix, dry, expose (usually 4 hrs)
Ick Kinase: Immunoprecipitated from Jurkat lysate
A. Jurkat cells (human T-cell leukemia, ATCC clone #E6-1) are grown
in suspension in RPMI 1640 medium with 10% fetal calf serum, 100
U/ml penicillin/streptomycin, and 2 mM L-glutamine in a 37.degree.
C. incubator at 5% CO.sub.2.
B. Cells are grown to 1-1.5.times.10.sup.6 cells/ml media, pelleted
by centrifugation, and lysed in lysis buffer at 10.sup.8 cells/ml
buffer (50 mM tris (pH 8), 150 nM NaCl, 5 mM EDTA, 10% glycerol,
and 1% NP-40, to which fresh protease and phosphatase inhibitors
are added as described above for A431 lysate). Lysates stored at
-70.degree. C.
C. Immunoprecipitation: 3-4 mg Protein-A sepharose/sample washed 2x
20 mM Hepes (pH 7.5). 1 ul .alpha.-Ick antibody (prepared as
polyclonals in rabbits using a peptide antigen corresponding to the
N-terminal region of human Ick) per sample added to the Protein-A
and shaken 20 min at room temperature. After washing 3X HNTG,
lysate from 2.times.10.sup.6 cells is added to each sample, rotated
2 hr at 4.degree. C., then washed 3x HNTG (2nd wash containing 0.5N
NaCl). If all samples contain identical concentrations of the
enzyme, then the immuno-precipitation can be done in bulk and
alloquoted to appropriate numbers of tubes prior to assay
set-up.
D. Compounds screening in the cell-free Ick kinase assay: Compounds
(40 mM stocks in DMSO) are initially screened at concentrations of
10 and 100 uM in samples containing Ick immuno-precipitated from
2.times.10.sup.6 cells, 5 uM cdc2 (a p34.sup.cdc 2-derived
synthetic peptide (N6-20) prepared by R. Howk, RPR).sup.7, 5 mM
MnCl.sub.2, 5 uM ATP and 30 uCi g.sup.32 P-ATP (6000 Ci/mmol, NEN)
in 20 mM hepes (pH 7.5) for 5 min at 30.degree. C. Samples are
analyzed by 5-15% SDS-PAGE and autoradiography as described for
EGFR kinase assays.
E. Intact cell activation/inhibition
studies:.about.5.times.10.sup.7 cells per sample in 1 ml media are
activated with either 10 ug a-CD3 (clone Cris 7, Biodesign) for 1
min at 37.degree. C. or 20 ng PMA and 10 ug PHA for 20 min at
37.degree. C. in the presence and absence of compound (added
earlier so that the total time of compound incubation is 30 min).
Incubations are terminated by centrifugation and lysis (as
described). Samples are analyzed by immunoprecipitation (aPY (100
ul/10.sup.8 cells), a-PLC (100 ul/10.sup.8 cells), or a-zeta (20
ul/10.sup.8 cells)), followed by SDS-PAGE and western blotting onto
nitrocellulose and immunoblotting using RC20 recombinant aPY-HRP
Transduction Labs) and ECL (Amersham).
cAMP-dependent Protein Kinase (PKA) Assay
Selectivity assay for compounds is performed as follows. Each
sample contains 0.4 pmolar units PKA (from rabbit muscle, Sigma), 1
uM cAMP, 50 uM Tris-HCL (pH7), 10 mM MgAc, 50 ug BSA, 16 uM
Kemptide substrate (specific cAMP kinase phosphate acceptor whose
sequence corresponds to the pig liver pyruvate kinase
phosphorylation site), 16 uM ATP, 16 uCi .sup.32 P-ATP (6000
Ci/mmol, NEN), +/- compound and dH.sub.2 O to a final volume of 200
ul. Reactions proceed for 5 min. at 30.degree. C., and are
terminated by the addition of 100 ul 375 mM H.sub.3 PO.sub.4. 50 ul
each sample spotted onto Whatman P81 phosphocellulose filters,
which are washed 3X (15 min.) in 75 mM H.sub.3 PO.sub.4, followed
by an acetone rinse and dry (Cerenkov) counting.
In view of the results of the above test, compounds of the present
invention can be shown to be selective.
The following tables show examples of representative compounds of
this invention and their test results as determined by the above
inhibition of CSR-1R and Ick procedures.
Structure ##STR6## Ick activity IC.sub.50 (.mu.M) CSF-R activity
IC.sub.50 (.mu.M) ##STR7## >10 6 ##STR8## 100 7 ##STR9## >50
0.18 ##STR10## >50 0.5 ##STR11## >50 4.0 ##STR12## 10 >100
##STR13## 0.5 >100 ##STR14## 50 >50 ##STR15## 10 >50
##STR16## 2.5 >50 ##STR17## 10 >20 ##STR18## 20 .gtoreq.20
##STR19## 1 >50 ##STR20## 2.5 3 ##STR21## 5 1 ##STR22## 5 >50
##STR23## 2.9 15
The results obtained by the above experimental methods evidence the
useful CSF-1R receptor protein tyrosine kinase inhibition of
properties of compounds within the scope of the present invention
and possess therapeutic value as cellular antiproliferative agents.
The above pharmacological test results may be used to determine the
dosage and mode of administration for the particular therapy
sought.
The compounds of the present invention can be administered to a
mammalian host in a variety of forms adapted to the chosen route of
administration, i.e., orally, or parenterally. Parenteral
administration in this respect includes administration by the
following routes: intravenous, intramuscular, subcutaneous,
intraocular, intrasynovial, transepithelial including transdermal,
ophthalmic, sublingual and buccal; topically including ophthalmic,
dermal, ocular, rectal and nasal inhalation via insufflation and
aerosol and rectal systemic.
The active compound may be orally administered, for example, with
an inert diluent or with an assimilable edible carrier, or it may
be enclosed in hard or soft shell gelatin capsules, or it may be
compressed into tablets, or it may be incorporated directly with
the food of the diet. For oral therapeutic administration, the
active compound may be incorporated wit excipient and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 0.1% of
active compound. The percentage of the compositions and
preparations may, of course, be varied and may conveniently be
between about 2 to about 6% of the weight of the unit. The amount
of active compound in such therapeutically useful compositions is
such that a suitable dosage will be obtained. Preferred
compositions or preparations according to the present invention are
prepared so that an oral dosage unit form contains between about 1
and 1000 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain
the following: A binder such as gum tragacanth, acacia, corn starch
or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin may be added
or a flavoring agent such as peppermint, oil of wintergreen, or
cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or orange flavor.
Of course, any material used in preparing any dosage unit form
should be pharmaceutically pure and substantially non-toxic in the
amounts employed. In addition, the active compound may be
incorporated into sustained-release preparations and
formulations.
The active compound may also be administered parenterally or
intraperitoneally. Solutions of the active compound as a free base
or pharmacologically acceptable salt can be prepared in water
suitably mixed with a surfactant such as hydroxypropylcellulose.
Dispersion can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the for must be sterile and must be fluid
to the extent that easy syringability exists. It may be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
use of agents delaying absorption, for example, aluminum
monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the
active compound in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredient into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred method
of preparation are vacuum drying and the freeze drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof.
The therapeutic compounds of this invention may be administered to
a mammal alone or in combination with pharmaceutically acceptable
carriers, as noted above, the proportion of which is determined by
the solubility and chemical nature of the compound, chosen route of
administration and standard pharmaceutical practice.
The dosage of the present therapeutic agents which will be most
suitable for prophylaxis or treatment will vary with the form of
administration, the particular compound chosen and the
physiological characteristics of the particular patient under
treatment. Generally, small dosages will be used initially and if
necessary, will be increased by small increments until the optimum
effect under the circumstances is reached. The therapeutic human
dosage, based on physiological studies using rats, will generally
be from about 0.01 mg to about 100 mg/kg of body weight per day or
from about 0.4 mg to about 10 g or higher although it may be
administered in several different dosage units from once to several
times a day. Oral administration requires higher dosages.
* * * * *