U.S. patent application number 09/747563 was filed with the patent office on 2002-04-11 for pyrazole compounds.
Invention is credited to Chafeev, Mikhail, Costello, Penelope C., Daynard, Timothy Scott, Leung, Danny, Sanghera, Jasbinder, Wang, Shisen, Yan, Jun, Zhang, Zaihui.
Application Number | 20020042501 09/747563 |
Document ID | / |
Family ID | 27067766 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020042501 |
Kind Code |
A1 |
Zhang, Zaihui ; et
al. |
April 11, 2002 |
Pyrazole compounds
Abstract
Pharmaceutical compositions and compounds are provided. The
compounds of the invention have anti-proliferative activity, and
may promote apoptosis in cells lacking normal regulation of cell
cycle and death. In one embodiment of the invention, formulations
of the compounds in combination with a physiologically acceptable
carrier are provided. The pharmaceutical formulations are useful in
the treatment of hyperproliferative disorders, which disorders
include tumor growth, lymphoproliferative diseases, angiogenesis.
The compounds of the invention are substituted pyrazoles and
pyrazolines.
Inventors: |
Zhang, Zaihui; (Richmond,
CA) ; Yan, Jun; (Coquitlam, CA) ; Leung,
Danny; (Coquitlam, CA) ; Costello, Penelope C.;
(Vancouver, CA) ; Sanghera, Jasbinder; (Richmond,
CA) ; Daynard, Timothy Scott; (Vancouver, CA)
; Wang, Shisen; (Coquitlam, CA) ; Chafeev,
Mikhail; (Vancouver, CA) |
Correspondence
Address: |
Pamela J. Sherwood
BOZICEVIC, FIELD & FRANCIS LLP
Suite 200
200 Middlefield Road
Menlo Park
CA
94025
US
|
Family ID: |
27067766 |
Appl. No.: |
09/747563 |
Filed: |
December 22, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09747563 |
Dec 22, 2000 |
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09544908 |
Apr 7, 2000 |
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6214813 |
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Current U.S.
Class: |
534/769 |
Current CPC
Class: |
A61K 31/4152 20130101;
A61K 31/454 20130101; A61K 31/4155 20130101; C07D 403/14 20130101;
C07D 409/14 20130101; A61K 31/53 20130101; C07D 409/12 20130101;
A61K 31/4178 20130101; C07D 405/12 20130101; C07D 231/52 20130101;
C07D 231/46 20130101; C07D 403/12 20130101; C07D 401/12 20130101;
A61K 31/427 20130101; C07D 401/14 20130101; C07D 413/12 20130101;
C07D 231/38 20130101; C07D 417/12 20130101; A61P 35/00 20180101;
A61K 31/415 20130101; A61K 31/5377 20130101 |
Class at
Publication: |
534/769 |
International
Class: |
C09B 029/46 |
Claims
What is claimed is:
1. A composition comprising a compound of formula (1) 90and
stereoisomers, polymorphs, solvates, and pharmaceutically
acceptable salts thereof, and a pharmaceutically acceptable
carrier, diluent or excipient, where R.sup.1 is selected from
alkyl, aryl and heteroaryl, wherein each of alkyl, aryl and
heteroaryl may be substituted with one or more groups selected from
C.sub.1-C.sub.20alkyl, C.sub.6-C.sub.10aryl, heteroalkyl and
heteroaryl; R.sup.2 is selected from H and direct bond; R.sup.3 and
R.sup.4 are selected from --NH.sub.2 and NHC(.dbd.O)R.sup.5;
R.sup.5 is selected from R.sup.6, R.sup.7, and R.sup.8, where
R.sup.6 is selected from alkyl, heteroalkyl, aryl and heteroaryl;
R.sup.7 is selected from (R.sup.6).sub.k-alkylene,
(R.sup.6).sub.k-heteroalkylene, (R.sup.6).sub.k-arylene and
(R.sup.6).sub.k-heteroarylene; R.sup.8 is selected from
(R.sup.7).sub.k-alkylene, (R.sup.7).sub.k-heteroalkylene,
(R.sup.7).sub.k-arylene, and (R.sup.7).sub.k-heteroarylene; and k
is selected from 0, 1, 2, 3, 4 and 5; and n is selected from 0, 1,
2, 3, 4 or 5.
2. The composition of claim 1 wherein R.sup.1 is
C.sub.1-C.sub.20alkyl.
3. The composition of claim 2 wherein R.sup.1 is
C.sub.1-C.sub.6alkyl and each of R.sup.3 and R.sup.4 are
--NH.sub.2.
4. The composition of claim 1 wherein R.sup.1 is aryl.
5. The composition of claim 4 wherein R.sup.1 is aryl selected from
phenyl and naphthyl, the phenyl and napthyl substituted with at
least one heteroalkyl selected from alkoxy, carboxy and halide.
6. The composition of claim 5 wherein each of R.sup.3 and R.sup.4
are --NH.sub.2.
7. The composition of claim 1 wherein R.sup.5 is selected from
carbocyclic and heterocyclic groups.
8. The composition of claim 7 wherein the carbocyclic and
heterocyclic groups contain 5-12 ring atoms.
9. The composition of claim 1 wherein R.sup.5 is a carbocyclic
group selected from monocyclic and fused ring groups.
10. The composition of claim 1 wherein R.sup.5 is a heterocyclic
group containing from 1-3 heteroatoms selected from nitrogen and
sulfur.
11. The composition of claim 1 wherein R.sup.5 is selected from
R.sup.6 and R.sup.7, where R.sup.6 is selected from alkyl,
heteroalkyl, aryl and heteroaryl; R.sup.7 is selected from
(R.sup.6).sub.k-alkylene, (R.sup.6).sub.k-heteroalkylene,
(R.sup.6).sub.k-arylene and (R.sup.6).sub.k-heteroarylene; and k is
selected from 0, 1, 2, 3,4 and 5.
12. The composition of claim 1 wherein R.sup.5 is selected from
4-fluorophenyl, 3-ethylphenyl, 3-methoxyphenyl, 3-chlorophenyl,
3-fluorophenyl, 3-fluoro-4-methoxyphenyl, naphthalen-2-yl,
4-trifluoromethylphenyl, 3-phenoxyphenyl, biphenyl-2-yl,
2-Bromophenyl, 3-Bromophenyl, 4-Bromophenyl, 4-phenoxyphenyl,
4-iodophenyl, 4-bromonaphthalen-1-yl, o-tolyl, 2,6-difluorophenyl,
3,4-difluorophenyl, benzo[1,3]dioxol-5-yl, 4-methylsulfanylphenyl,
2,3-dihydrobenzo[1,4]dioxi- n-6-yl, 3-chloro-4-methoxyphenyl,
3,4-dichlorophenyl, 3,5-dichlorophenyl, 2-isopropylphenyl,
3,4-dimethoxyphenyl, 3-trifluoromethylphenyl,
3-methoxy-5-trifluoromethylphenyl, 2-chlorophenyl, 3-iodophenyl,
9-ethyl-9H-carbazol-3-yl, 2-benzenesulfonylphenyl, phenyl,
pyridin-3-yl, 6-methoxybenzothiazol-2-yl, benzotiazol-2-yl,
pyrazol-3-yl, pyridin-4-yl, 2,3,4,5,6-pentafluorophenyl, 3-[1
H]-1,2,4-triazolo, 3,5-difluorophenyl, and
2,3,4-trifluorophenyl.
13. The composition of claim 12 wherein each of R.sup.3 and R.sup.4
are --NH.sub.2.
14. The composition of claim 13 wherein R.sup.1 is
C.sub.1-C.sub.20alkyl.
15. The composition of claim 13 wherein R.sup.1 is aryl.
16. The composition of claim 13 wherein R.sup.1 is heteroaryl.
17. The composition of claim 1 wherein the compound of formula (1)
is selected from
(3,5-diamino-4-phenylazo-pyrazol-1-yl)phenylmethanone;
4-(3,5-diamino-4-phenylazopyrazol-1-yl)benzoic acid;
3,5-diamino-1-phenyl-4-phenylazopyrazole;
3,5-diamino-1-(4-Bromophenyl)-4- -phenylazopyrazole;
3,5-diamino-1-(4-fluorophenyl)-4-phenylazopyrazole; and
3,5-diamino-1-methyl-4-phenylazopyrazole.
18. A composition comprising a compound of formula (2) 91and
stereoisomers, solvates, and pharmaceutically acceptable salts
thereof, and a pharmaceutically acceptable carrier, diluent or
excipient, where R.sup.1 and R.sup.2 are selected from H and direct
bond; R.sup.3 and R.sup.4 are selected from --NH.sub.2,
NHC(.dbd.O)R.sup.5 and .dbd.O; and Ar is an aryl group.
19. A composition of claim 18 wherein Ar is phenyl having one or
more substituents selected from alkyl, aryl, heteroalkyl and
heteroaryl.
20. A composition of claim 19 wherein the substituents are selected
from benzenesulfonyl, bromide, carbonylethoxy, carbonylmethoxy,
chloride, dioxolyl, dioxinyl, ethyl, fluoride, hydroxyl, iodide,
iso-propyl, methoxy, methyl, methylthio, phenoxy, phenyl, propyl,
and trifluoromethyl.
21. A composition of claim 20 wherein the compound is selected from
3,5-diamino-4-[(4-fluorophenyl)hydrazono]pyrazole;
3-[N'-(3,5-diaminopyrazol-4-ylidene)hydrazino]phenol;
3,5-diamino-4-[(3-ethylphenyl)hydrazono]pyrazol;
3,5-diamino-4-[(3-methox- yphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-chlorophenyl)hydrazono]pyraz- ole;
3,5-diamino-4-[(3-fluorophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-fluoro-4-methoxyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(4-trifluoromethylphenyl)hydrazono]pyrazole;
4-[(3-phenoxyphenyl)hydrazono]pyrazole;
4-[N'-(3,5-diaminopyrazol-4-ylide- ne)hydrazino]benzoic acid ethyl
ester; 3,5-diamino-4-[(biphenyl-2-yl)hydra- zono]pyrazole;
3,5-diamino-4-[(2-Bromophenyl)hydrazono]pyrazol;
3,5-diamino-4-[(3-Bromophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(4-Bromo- phenyl)hydrazono]pyrazole;
3,5-diamino-4-[(4-phenoxyphenyl)hydrazono]pyraz- ole;
3,5-diamino-4-[(4-iodophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(o-tolyl)hydrazono]pyrazole;
3,5-diamino-4-[(2,6-difluorop- henyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,4-difluorophenyl)hydrazono]pyr- azole;
3,5-diamino-4-[(benzo[1,3]dioxol-5-yl)hydrazono]pyrazole;
3,5-diamino-4-[(4-methylsulfanylphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(2,3-dihydrobenzo[1,4]dioxin-6-yl)-hydrazono]pyrazole;
3,5-diamino-4-[(3-chloro-4-methoxyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,4-dichlorophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,5-dichlorophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(2-isopropylphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,4-dimethoxyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-trifluoromethylphenyl)hydrazono]pyrazole;
3-[N'-(3,5-diaminopyrazol-4-ylidene)hydrazino]benzoic acid ethyl
ester;
3,5-diamino-4-[(3-methoxy-5-trifluoromethylphenyl)-hydrazono]pyrazole;
3,5-diamino-4-[(2-chloro phenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-iodophenyl)hydrazono]pyrazole; and
3,5-diamino-4-[(2-benzenesulfonylphenyl)hydrazono]pyrazole.
22. A composition of claim 18 wherein Ar is naphthyl optionally
having one or more substituents selected from alkyl, aryl,
heteroalkyl and heteroaryl.
23. A composition of claim 22 wherein the substituents are selected
from benzenesulfonyl, bromide, carbonylethoxy, carbonylmethoxy,
chloride, dioxolyl, dioxinyl, ethyl, fluoride, hydroxyl, iodide,
iso-propyl, methoxy, methyl, methylthio, phenoxy, phenyl, propyl,
and trifluoromethyl.
24. A composition of claim 23 wherein the compound is selected from
3,5-diamino-4-[(naphthalen-2-yl)hydrazono]pyrazole; and
3,5-diamino-4-[(4-bromonaphthalen-1-yl)hydrazono]pyrazole.
25. A composition comprising a compound of formula (1) 92and
stereoisomers, solvates, and pharmaceutically acceptable salts
thereof, and a pharmaceutically acceptable carrier, diluent or
excipient, where R.sup.1 and R.sup.2 are selected from H and direct
bond; R.sup.3 and R.sup.4 are selected from --NH.sub.2 and
NHC(.dbd.O)R.sup.5; R.sup.5 is selected from heteroaryl and
substituted heteroaryl, where a substituent on substituted
heteroaryl is selected from alkyl, heteroalkyl, aryl and
heteroaryl.
26. A composition according to claim 25 wherein the compound is
selected from:
3,5-diamino-4-[(9-ethyl-9H-carbazol-3-yl)hydrazono]pyrazole;
3,5-diamino-4-[(pyridin-3-yl)hydrazono]pyrazole;
3,5-diamino-4-[(6-methox- ybenzothiazol-2-yl)hydrazono]pyrazole;
and 3,5-diamino-4-[(benzothiazol-2-- yl )hydrazono]pyrazole.
Description
[0001] This application in a continuation-in-part of U.S. patent
application Ser. No. 09/544,908, filed Apr. 7, 2000.
BACKGROUND OF THE INVENTION
[0002] It has become increasingly clear in recent years that cell
death is as important to the health of a multicellular organism as
cell division: where proliferation exists, so must a means of
regulating its cellular progeny. By repeated cell division and
differentiation throughout development or tissue repair, surplus or
even harmful cells are generated, and they must be removed or
killed. In adults, senescent cells are removed and replaced by
newly generated cells to maintain homeostasis.
[0003] The delicate interplay between growth and cell death in an
organism is mirrored in the complex molecular balance that
determines whether an individual cell undergoes division; arrests
in the cell cycle; or commits to programmed cell death. Signal
transduction is the term describing the process of conversion of
extracellular signals, such as hormones, growth factors,
neurotransmitters, cytokines, and others, to a specific
intracellular response such as gene expression, cell division, or
apoptosis. This process begins at the cell membrane where an
external stimulus initiates a cascade of enzymatic reactions inside
the cell that typically include phosphorylation of proteins as
mediators of downstream processes which most often end in an event
in the cell nucleus. The checks and balances of these signal
transduction pathways can be thought of as overlapping networks of
interacting molecules that control "go-no go" control points. Since
almost all known diseases exhibit dysfunctional aspects in these
networks, there has been a great deal of enthusiasm for research
that provides targets and therapeutic agents based on signal
transduction components linked to disease.
[0004] Dysregulation of cell proliferation, or a lack of
appropriate cell death, has wide ranging clinical implications. A
number of diseases associated with such dysregulation involve
hyperproliferation, inflammation, tissue remodelling and repair.
Familiar indications in this category include cancers, restenosis,
neointimal hyperplasia, angiogenesis, endometriosis,
lymphoproliferative disorders, graft-rejection, polyposis, loss of
neural function in the case of tissue remodelling, and the like.
Such cells may lose the normal regulatory control of cell division,
and may also fail to undergo appropriate cell death.
[0005] In one example, epithelial cells, endothelial cells, muscle
cells, and others undergo apoptosis when they lose contact with
extracellular matrix, or bind through an inappropriate integrin.
This phenomenon, which has been termed "anoikis" (the Greek word
for "homelessness"), prevents shed epithelial cells from colonizing
elsewhere, thus protecting against neoplasia, endometriosis, and
the like. It is also an important mechanism in the initial
cavitation step of embryonic development, in mammary gland
involution, and has been exploited to prevent tumor angiogenesis.
Epithelial cells may become resistant to anoikis through
overactivation of integrin signaling. Anoikis resistance can also
arise from the loss of apoptotic signaling, for example, by
overexpression of Bcl-2 or inhibition of caspase activity.
[0006] An aspect of hyperproliferation that is often linked to
tumor growth is angiogenesis. The growth of new blood vessels is
essential for the later stages of solid tumor growth. Angiogenesis
is caused by the migration and proliferation of the endothelial
cells that form blood vessels.
[0007] In another example, a major group of systemic autoimmune
diseases is associated with abnormal lymphoproliferation, as a
result of defects in the termination of lymphocyte activation and
growth. Often such diseases are associated with inflammation, for
example with rheumatoid arthritis, insulin dependent diabetes
mellitus, multiple sclerosis, systemic lupus erythematosus, and the
like. Recent progress has been made in understanding the causes and
consequences of these abnormalities. At the molecular level,
multiple defects may occur, which result in a failure to set up a
functional apoptotic machinery.
[0008] The development of compounds that inhibit hyperproliferative
diseases, particularly where undesirable cells are selectively
targeted, is of great medical and commercial interest.
[0009] Relevant literature:
[0010] The regulation of integrin linked kinase by
phosphatidylinositol (3,4,5) trisphosphate is described by
Delcommenne et al. (1998) Proc Natl Acad Sci 95:11211-6. Activated
nitrites in heterocyclic synthesis are discussed in Kandeel et al.
(1985) J. Chem. Soc. Perkin. Trans 1499.
SUMMARY OF THE INVENTION
[0011] Pharmaceutical compositions and compounds are provided. The
compounds of the invention are substituted pyrazoles and
pyrazolines. In one embodiment of the invention, formulations of
the compounds in combination with a physiologically acceptable
carrier are provided. The pharmaceutical formulations are useful in
the treatment of disorders associated with hyperproliferation and
tissue remodelling or repair. The compounds are also active in the
inhibition of specific protein kinases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph illustrating the anti-tumor activity of
KP-15792 in a murine model using Lewis Lung Carcinoma cells.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0013] The present invention provides novel compounds, compositions
and methods as set forth within this specification. In general, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs, unless clearly indicated otherwise. For
clarification, listed below are definitions for certain terms used
herein to describe the present invention. These definitions apply
to the terms as they are used throughout this specification, unless
otherwise clearly indicated.
Definition of Terms
[0014] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. For example, "a compound" refers to one or more of such
compounds, while "the enzyme" includes a particular enzyme as well
as other family members and equivalents thereof as known to those
skilled in the art.
[0015] "Alkyl" is a monovalent, saturated or unsaturated, straight,
branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon
group. In various embodiments, the alkyl group has 1-20 carbon
atoms, i.e., is a C1-C20 (or C.sub.1-C.sub.20) group, or is a
C1-C18 group, a C1-C12 group, a C1-C6 group, or a C1-C4 group.
Independently, in various embodiments, the alkyl group: has zero
branches (i.e., is a straight chain), one branch, two branches, or
more than two branches; is saturated; is unsaturated (where an
unsaturated alkyl group may have one double bond, two double bonds,
more than two double bonds, and/or one triple bond, two triple
bonds, or more than three triple bonds); is, or includes, a cyclic
structure; is acyclic. Exemplary alkyl groups include C.sub.1alkyl
(i.e., --CH.sub.3 (methyl)), C.sub.2alkyl (i.e., --CH.sub.2CH.sub.3
(ethyl), --CH.dbd.CH.sub.2 (ethenyl) and --C.dbd.CH (ethynyl)) and
C.sub.3alkyl (i.e., --CH.sub.2CH.sub.2CH.sub.3 (n-propyl),
--CH(CH.sub.3).sub.2 (i-propyl), --CH.dbd.CH--CH.sub.3
(1-propenyl), --C.dbd.-C--CH.sub.3 (1-propynyl),
--CH.sub.2--CH.dbd.CH.sub.2 (2-propenyl), --CH.sub.2--C.dbd.-CH
(2-propynyl), --C(CH.sub.3).dbd.CH.sub.2 (1-methylethenyl), and
--CH(CH.sub.2).sub.2 (cyclopropyl)).
[0016] "Ar" indicates a carbocyclic aryl group selected from
phenyl, substituted phenyl, naphthyl, and substituted naphthyl.
Suitable substituents on a phenyl or naphthyl ring include
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, carboxyl,
carbonyl(C.sub.1-C.sub.6)alkoxy, halogen, hydroxyl, nitro,
--SO.sub.3H, and amino.
[0017] "Aryl" is a monovalent, aromatic, hydrocarbon, ring system.
The ring system may be monocyclic or fused polycyclic (e.g.,
bicyclic, tricyclic, etc.). In various embodiments, the monocyclic
aryl ring is C5-C10, or C5-C7, or C5-C6, where these carbon numbers
refer to the number of carbon atoms that form the ring system. A C6
ring system, i.e., a phenyl ring, is a preferred aryl group. In
various embodiments, the polycyclic ring is a bicyclic aryl group,
where preferred bicyclic aryl groups are C8-C12, or C9-C10. A
naphthyl ring, which has 10 carbon atoms, is a preferred polycyclic
aryl group.
[0018] "Arylene" is a polyvalent, aromatic hydrocarbon, ring
system. The ring system may be monocyclic or fused polycyclic
(e.g., bicyclic, tricyclic, etc.). In various embodiments, the
monocyclic arylene group is C5-C10, or C5-C7, or C5-C6, where these
carbon numbers refer to the number of carbon atoms that form the
ring system. A C6 ring system, i.e., a phenylene ring, is a
preferred aryl group. In various embodiments, the polycyclic ring
is a bicyclic arylene group, where preferred bicyclic arylene
groups are C8-C12, or C9-C10. A naphthylene ring, which has 10
carbon atoms, is a preferred polycyclic aryl group. The arylene
group may be divalent, i.e., it has two open sites that each bond
to another group; or trivalent, i.e., it has three open sites that
each bond to another group; or it may have more than three open
sites.
[0019] "Carbocycle" refers to a ring formed exclusively from
carbon, which may be saturated or unsaturated, including aromatic.
The ring may be monocyclic (e.g., cyclohexyl, phenyl), bicyclic
(e.g., norbornyl), polycyclic (e.g., adamantyl) or contain a fused
ring system (e.g., decalinyl, naphthyl). In one embodiment, the
ring is monocyclic and formed from 5, 6 or 7 carbons. In one
embodiment, the ring is bicyclic and formed from 7, 8 or 9 carbons.
In one embodiment, the ring is polycyclic and formed from 9, 10 or
11 carbons. In one embodiment, the ring includes a fused ring
system and is formed from 8-12 carbons. Thus, in one embodiment,
the carbocycle is formed from 5-12 ring carbons.
[0020] "Heteroalkyl" is an alkyl group (as defined herein) wherein
at least one of the carbon atoms is replaced with a heteroatom.
Preferred heteroatoms are nitrogen, oxygen, sulfur, and halogen. A
heteroatom may, but typically does not, have the same number of
valence sites as carbon. Accordingly, when a carbon is replaced
with a heteroatom, the number of hydrogens bonded to the heteroatom
may need to be increased or decreased to match the number of
valence sites of the heteroatom. For instance, if carbon (valence
of four) is replaced with nitrogen (valence of three), then one of
the hydrogens formerly attached to the replaced carbon must be
deleted. Likewise, if carbon is replaced with halogen (valence of
one), then three (i.e., all) of the hydrogens formerly bonded to
the replaced carbon must be deleted. As another example,
trifluoromethyl is a heteroalkyl group wherein the three methyl
groups of a t-butyl group are replaced by fluorine.
[0021] "Heteroalkylene" is an alkylene group (as defined herein)
wherein at least one of the carbon atoms is replaced with a
heteroatom. Preferred heteroatoms are nitrogen, oxygen, sulfur, and
halogen. A heteroatom may, but typically does not, have the same
number of valence sites as carbon. Accordingly, when a carbon is
replaced with a heteroatom, the number of hydrogens bonded to the
heteroatom may need to be increased or decreased to match the
number of valence sites of the heteroatom, as explained elsewhere
herein.
[0022] "Heteroaryl" is a monovalent aromatic ring system containing
carbon and at least one heteroatom in the ring. The heteroaryl
group may, in various embodiments, have one heteroatom, or 1-2
heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms in the ring.
Heteroaryl rings may be monocyclic or polycyclic, where the
polycyclic ring may contained fused, spiro or bridged ring
junctions. In one embodiment, the heteroaryl is selected from
monocyclic and bicyclic. Monocyclic heteroaryl rings may contain
from about 5 to about 10 member atoms (carbon and heteroatoms),
preferably from 5-7, and most preferably from 5-6 member atoms in
the ring. Bicyclic heteroaryl rings may contain from about 8-12
member atoms, or 9-10 member atoms in the ring. The heteroaryl ring
may be unsubstituted or substituted. In one embodiment, the
heteroaryl ring is unsubstituted. In another embodiment, the
heteroaryl ring is substituted. Exemplary heteroaryl groups include
benzofuran, benzothiophene, furan, imidazole, indole, isothiazole,
oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine,
pyrimidine, pyrrole, quinoline, thiazole and thiophene.
[0023] "Heteroarylene" is a polyvalent aromatic ring system
containing carbon and at least one heteroatom in the ring. In other
words, a heteroarylene group is a heteroaryl group that has more
than one open site for bonding to other groups. The heteroarylene
group may, in various embodiments, have one heteroatom, or 1-2
heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms in the ring.
Heteroarylene rings may be monocyclic or polycyclic, where the
polycyclic ring may contained fused, spiro or bridged ring
junctions. In one embodiment, the heteroaryl is selected from
monocyclic and bicyclic. Monocyclic heteroarylene rings may contain
from about 5 to about 10 member atoms (carbon and heteroatoms),
preferably from 5-7, and most preferably from 5-6 member atoms in
the ring. Bicyclic heteroarylene rings may contain from about 8-12
member atoms, or 9-10 member atoms in the ring.
[0024] "Heteroatom" is a halogen, nitrogen, oxygen, silicon or
sulfur atom. Groups containing more than one heteroatom may contain
different heteroatoms.
[0025] "Heterocycle" refers to a ring containing at least one
carbon and at least one heteroatom. The ring may be monocyclic
(e.g., morpholinyl, pyridyl), bicyclic (e.g., bicyclo[2.2.2]octyl
with a nitrogen at one bridgehead position), polycyclic, or contain
a fused ring system. In one embodiment, the ring is monocyclic and
formed from 5, 6 or 7 atoms. In one embodiment, the ring is
bicyclic and formed from 7, 8 or 9 atoms. In one embodiment, the
ring is polycyclic and formed from 9, 10 or 11 atoms. In one
embodiment, the ring includes a fused ring system and is formed
from 8-12 atoms. Thus, in one embodiment, the heterocycle is formed
from 5-12 ring atoms. In one embodiment, the heteroatom is selected
from oxygen, nitrogen and sulfur. In one embodiment, the
heterocycle contains 1, 2 or 3 heteroatoms.
[0026] As used herein, and unless otherwise specified, the terms
carbocyclic and heterocyclic encompass both substituted and
unsubstituted carbocyclic and heterocyclic groups. In one
embodiment, the substitution present on a carbocyclic or
heterocyclic group is selected from alkyl, heteroalkyl, aryl, and
heteroaryl, preferably alkyl and heteroalkyl. In one embodiment,
the alkyl and heteroalkyl substitution present on a carbocyclic or
heterocyclic group is selected from C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, halogen, nitro, hydroxyl, cyano, sulfonic
acid (i.e., --SO.sub.3H), carboxylic acid, carboxylate ester (i.e.,
--CO.sub.2R where R is, e.g., C.sub.1-C.sub.10alkyl), amino,
alkylamino, dialkylamino, acyl (i.e., R--C(.dbd.O)--), and
acylamino (i.e., R--C(.dbd.O)--NH-- where R is, e.g.,
C.sub.1-C.sub.10alkyl). For example, and unless otherwise
specified, the terms cyclohexyl and phenyl refer to both
substituted and unsubstituted cyclohexyl and phenyl.
[0027] "Pharmaceutically acceptable salt" and "salts thereof" in
the compounds of the present invention refers to acid addition
salts and base addition salts.
[0028] Acid addition salts refer to those salts formed from
compounds of the present invention and inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and/or organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like.
[0029] Base addition salts refer to those salts formed from
compounds of the present invention and inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Suitable
salts include the ammonium, potassium, sodium, calcium and
magnesium salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, 2-diethylaminoethanol, trimethamine,
dicyclohexylamine, lysine, arginine, histidine, caffeine,
procaines, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, methylglucamine, theobromine, purines, piperazine,
piperidine, N-ethylpiperidine, and the like.
Compounds
[0030] In one aspect the present invention provides compounds of
formula (1), as set forth below.
[0031] In another aspect the present invention provides
compositions comprising a compound of formula (1) 1
[0032] and stereoisomers, solvates, and pharmaceutically acceptable
salts thereof, and a pharmaceutically acceptable carrier, diluent
or excipient, where
[0033] R.sup.1 and R.sup.2 are selected from direct bond, H, and
alkyl;
[0034] R.sup.3 and R.sup.4 are selected from --NH.sub.2,
NHC(.dbd.O)R.sup.5, and .dbd.O;
[0035] R.sup.5 is selected from R.sup.6, R.sup.7, and R.sup.8,
where R.sup.6 is selected from alkyl, heteroalkyl, aryl and
heteroaryl; R.sup.7 is selected from (R.sup.6).sub.k-alkylene,
(R.sup.6).sub.k-heteroalkylene- , (R.sup.6).sub.k-arylene and
(R.sup.6).sub.k-heteroarylene; R.sup.8 is selected from
(R.sup.7).sub.k-alkylene, (R.sup.7).sub.k-heteroalkylene,
(R.sup.7).sub.k-arylene, and (R.sup.7).sub.k-heteroarylene; and k
is selected from 0, 1, 2, 3, 4 and 5; and
[0036] n is selected from 0, 1, 2, 3, 4 or 5.
[0037] In another aspect, the present invention provides compounds
of formula (1) and compositions comprising compounds of formula (1)
as drawn above, wherein R.sup.1 is selected from alkyl, aryl and
heteroaryl, wherein each of alkyl, aryl and heteroaryl may be
substituted with one or more groups selected from
C.sub.1-C.sub.20alkyl, C.sub.6-C.sub.10aryl, heteroalkyl and
heteroaryl; R.sup.2 is selected from H and direct bond; R.sup.1 and
R.sup.1 are selected from --NH.sub.2 and NHC(.dbd.O)R.sup.5;
R.sup.5 is selected from R.sup.6, R.sup.7, and R.sup.8, where
R.sup.6 is selected from alkyl, heteroalkyl, aryl and heteroaryl;
RF is selected from (R.sup.6).sub.k-3alkylene,
(R.sup.6).sub.k-heteroalkylene, (R.sup.6).sub.k-arylene and
(R.sup.6).sub.k-heteroarylene; R.sup.8 is selected from
(R.sup.7).sub.k-alkylene, (R.sup.7).sub.k-heteroalkylene,
(R.sup.7).sub.k-arylene, and (R.sup.7).sub.k-heteroarylene; and k
is selected from 0, 1, 2, 3, 4 and 5; and n is selected from 0, 1,
2, 3, 4 or 5.
[0038] In various embodiments, R.sup.1 is C.sub.1-C.sub.20alkyl; or
R.sup.1 is C.sub.1-C.sub.6alkyl and each of R.sup.3 and R.sup.4 are
--NH.sub.2; or R.sup.1 is aryl; or R.sup.1 is aryl selected from
phenyl and naphthyl, the phenyl and napthyl substituted with at
least one heteroalkyl selected from alkoxy, carboxy and halide;
and/or each of R.sup.3 and R.sup.4 are --NH.sub.2; and/or R.sup.5
is selected from carbocyclic and heterocyclic groups, wherein the
carbocyclic and heterocyclic groups may optionally contain 5-12
ring atoms. In other embodiments, wherein R.sup.5 is a carbocyclic
group selected from monocyclic and fused ring groups, or R.sup.5 is
a heterocyclic group containing from 1-3 nitrogens; or R.sup.5 is
selected from R.sup.6 and R.sup.7, where R.sup.6 is selected from
alkyl, heteroalkyl, aryl and heteroaryl; R.sup.7 is selected from
(R.sup.6).sub.k-alkylene, (R.sup.6).sub.k-heteroalkylene,
(R.sup.6).sub.k-arylene and (R.sup.6).sub.k-heteroarylene; and k is
selected from 0, 1, 2, 3, 4 and 5.
[0039] In another embodiment, R.sup.5 is selected from
4-fluorophenyl, 3-ethylphenyl, 3-methoxyphenyl, 3-chlorophenyl,
3-fluorophenyl, 3-fluoro-4-methoxyphenyl, naphthalen-2-yl,
4-trifluoromethylphenyl, 3-phenoxyphenyl, biphenyl-2-yl,
2-Bromophenyl, 3-Bromophenyl, 4-Bromophenyl, 4-phenoxyphenyl,
4-iodophenyl, 4-bromonaphthalen-1-yl, o-tolyl, 2,6-difluorophenyl,
3,4-difluorophenyl, benzo[1,3]dioxol-5-yl, 4-methylsulfanylphenyl,
2,3-dihydrobenzo[1,4]dioxin-6-yl, 3-chloro-4-methoxyphenyl,
3,4-dichlorophenyl, 3,5-dichlorophenyl, 2-isopropylphenyl,
3,4-dimethoxyphenyl, 3-trifluoromethylphenyl,
3-methoxy-5-trifluoromethylphenyl, 2-chlorophenyl, 3-iodophenyl,
9-ethyl-9H-carbazol-3-yl, 2-benzenesulfonylphenyl, phenyl,
pyridin-3-yl, 6-methoxybenzothiazol-2-yl, benzotiazol-2-yl,
pyrazol-3-yl, pyridin-4-yl, 2,3,4,5,6-pentafluorophenyl, 3-[1
H]-1,2,4-triazolo, 3,5-difluorophenyl, and 2,3,4-trifluorophenyl,
where each of R.sup.3 and R.sup.4 may be --NH.sub.2; and where
R.sup.1 may be C.sub.1-C.sub.20alkyl, and/or aryl; and/or
heteroaryl.
[0040] In one aspect, the present invention provides a compound of
formula (1) selected from
(3,5-diamino-4-phenylazo-pyrazol-1-yl)phenylmethanone;
4-(3,5-diamino-4-phenylazopyrazol-1-yl)benzoic acid;
3,5-diamino-1-phenyl-4-phenylazopyrazole;
3,5-diamino-1-(4-Bromophenyl)-4- -phenylazopyrazole;
3,5-diamino-1-(4-fluorophenyl)-4-phenylazopyrazole; and
3,5-diamino-1-methyl-4-phenylazopyrazole.
[0041] In another aspect, the present invention provides compounds
of formula (2), and compositions comprising a compound of formula
(2) 2
[0042] and stereoisomers, solvates, and pharmaceutically acceptable
salts thereof, and a pharmaceutically acceptable carrier, diluent
or excipient, where R.sup.1 and R.sup.2 are selected from H and
direct bond; R.sup.3 and R.sup.4 are selected from --NH.sub.2,
NHC(.dbd.O)R.sup.5 and .dbd.O; and Ar is an aryl group. In one
aspect, Ar is phenyl having one or more substituents selected from
alkyl, aryl, heteroalkyl and heteroaryl, where optionally the
substituents are selected from benzenesulfonyl, bromide,
carbonylethoxy, carbonylmethoxy, chloride, dioxolyl, dioxinyl,
ethyl, fluoride, hydroxyl, iodide, isopropyl, methoxy, methyl,
methylthio, phenoxy, phenyl, propyl, and trifluoromethyl. Specific
compounds of the invention are
3,5-diamino-4-[(4-fluorophenyl)hydrazono]pyrazole;
3-[N'-(3,5-diaminopyrazol-4-ylidene)hydrazino]-phenol;
3,5-diamino-4-[(3-ethylphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-metho- xyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-chlorophenyl)hydrazono]-pyr- azole;
3,5-diamino-4-[(3-fluorophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-fluoro-4-methoxyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(4-trifluoromethylphenyl)hydrazono]pyrazole;
4-[(3-phenoxyphenyl)hydrazono]pyrazole;
4-[N'-(3,5-diaminopyrazol-4-ylide- ne)hydrazino]benzoic acid ethyl
ester; 3,5-diamino-4-[(biphenyl-2-yl)hydra- zono]pyrazole;
3,5-diamino-4-[(2-Bromophenyl)hydrazono]-pyrazole;
3,5-diamino-4-[(3-Bromophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(4-Bromo- phenyl)hydrazono]pyrazole;
3,5-diamino-4-[(4-phenoxyphenyl)hydrazono]-pyra- zole;
3,5-diamino-4-[(4-iodophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(o-tolyl)hydrazono]pyrazole;
3,5-diamino-4-[(2,6-difluorop- henyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,4-difluorophenyl)hydrazono]pyr- azole;
3,5-diamino-4-[(benzo[1,3]dioxol-5-yl)hydrazono]pyrazole;
3,5-diamino-4-[(4-methylsulfanylphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(2,3-dihydrobenzo[1,4]dioxin-6-yl)-hydrazono]pyrazole;
3,5-diamino-4-[(3-chloro-4-methoxyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,4-dichlorophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,5-dichlorophenyl)hydrazono]-pyrazole;
3,5-diamino-4-[(2-isopropylphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3,4-dimethoxyphenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-trifluoromethylphenyl)hydrazono]pyrazole;
3-[N'-(3,5-diaminopyrazol4-ylidene)hydrazino]benzoic acid ethyl
ester;
3,5-diamino-4-[(3-methoxy-5-trifluoromethylphenyl)-hydrazono]pyrazole;
3,5-diamino-4-[(2-chlorophenyl)hydrazono]pyrazole;
3,5-diamino-4-[(3-iodophenyl)hydrazono]pyrazole; and
3,5-diamino-4-[(2-benzenesulfonylphenyl)hydrazono]-pyrazole.
[0043] In another aspect, the present invention provides compounds
of formula (2) wherein Ar is naphthyl optionally having one or more
substituents selected from alkyl, aryl, heteroalkyl and heteroaryl,
where suitable substituents are benzenesulfonyl, bromide,
carbonylethoxy, carbonylmethoxy, chloride, dioxolyl, dioxinyl,
ethyl, fluoride, hydroxyl, iodide, iso-propyl, methoxy, methyl,
methylthio, phenoxy, phenyl, propyl, and trifluoromethyl; and
specific compounds of the invention are:
3,5-diamino-4-[(naphthalen-2-yl)hydrazono]pyrazole; and
3,5-diamino-4-[(4-bromonaphthalen-1-yl)hydrazono]pyrazole.
[0044] In compounds of formula (1), the structural moiety 3
[0045] is used to represent a family of tautomeric structures. In
part, the particular tautomeric structure(s) encompassed by formula
(1) depend on the selected of R.sup.3 and R.sup.4. When R.sup.3 and
R.sup.4 are each --NH.sub.2, then R.sup.1 and R.sup.2 are --H
and/or direct bonds, as shown in the Scheme below. 4
[0046] In one embodiment, R.sup.5 is selected from carbocyclic and
heterocyclic groups, where the carbocyclic and heterocyclic groups
preferably contain from 5 to 12 ring atoms. In one embodiment,
R.sup.5 is a carbocyclic group. In one embodiment, R.sup.5 is a
heterocyclic group.
[0047] In one embodiment, R.sup.5 is selected from the carbocyclic
groups phenyl and naphthyl. As noted previously, a carbocyclic
group may be substituted or unsubstituted. Accordingly, in this
embodiment, the phenyl or naphthyl group may be substituted with
one or more of, for example, alkyl, alkoxy, hydroxyl, sulfonic
acid, carboxylic acid, halogen, amino and acetylamino.
[0048] In one embodiment, R.sup.5 is selected from a heterocyclic
group of the formula 5
[0049] where p is selected from 1, 2 and 3. As used herein, the
moiety 6
[0050] represents a six-membered ring that optionally contains
unsaturation and necessarily includes 1, 2 or 3 ring nitrogens.
Examples include 7 8
[0051] In one embodiment, R.sup.3 and R.sup.4 are each amino
(--NH.sub.2) groups. In another embodiment, one of R.sup.3 and
R.sup.4 is an amino group while the other of R.sup.3 and R.sup.4 is
a carbonyl (.dbd.O) group. In one embodiment, both of R.sup.3 and
R.sup.4 are carbonyl groups. In one embodiment, n is 0. In another
embodiment, n is selected from 0, 1 and 2. In another embodiment, n
is selected from 1, 2, 3 and 4.
[0052] In one embodiment, the compounds and/or compositions and/or
methods of the present invention exclude a compound of formula (1)
wherein R.sup.1=H, and/or R.sup.2=H, and/or R.sup.3=amino, and/or
R.sup.4=amino, and/or n=0, and/or R.sup.5=4-methoxyphenyl.
Synthetic Methods
[0053] Compounds as set forth in compositions and methods of the
present invention may be prepared by methods disclosed in the
literature, and/or as summarized in Scheme 1. 9
[0054] Thus, a primary amine of the formula H.sub.2N--R.sup.9
(where R.sup.9 has been selected to represent
--(CH.sub.2).sub.n--R.sup.5 of formula (1)) is diazotised by
treatment sodium nitrite and hydrochloric acid. The intermediate
diazo compound (enclosed by a box in Scheme 1) will, in the
presence of base (e.g., sodium acetate as shown in Scheme 1) react
with compounds containing an active methylene group, i.e., a
compound including a methylene group (--CH.sub.2--) flanked by
electron withdrawing groups such as cyano (--CN) and/or ester
(--COOR), to provide an azo compound. This azo compound may be
reacted with a hydrazine derivative to provide compounds of the
present invention.
[0055] In Scheme 1, R.sup.1 and R.sup.2 are each preferably
hydrogen. However, either or both of R.sup.1 and R.sup.2 may be an
alkyl group.
Pharmaceutical Formulations
[0056] The compounds of this invention can be incorporated into a
variety of formulations for therapeutic administration. More
particularly, the compounds of the present invention can be
formulated into pharmaceutical compositions by combination with
appropriate pharmaceutically acceptable carriers or diluents, and
may be formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants, gels,
microspheres, and aerosols. As such, administration of the
compounds can be achieved in various ways, including oral, buccal,
rectal, parenteral, intraperitoneal, intradermal, transdermal,
intracheal, etc., administration. The active agent may be systemic
after administration or may be localized by the use of regional
administration, intramural administration, or use of an implant
that acts to retain the active dose at the site of
implantation.
[0057] In pharmaceutical dosage forms, the compounds may be
administered in the form of their pharmaceutically acceptable
salts. They may also be used in appropriate association with other
pharmaceutically active compounds. The following methods and
excipients are merely exemplary and are in no way limiting.
[0058] For oral preparations, the compounds can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0059] The compounds can be formulated into preparations for
injections by dissolving, suspending or emulsifying them in an
aqueous or nonaqueous solvent, such as vegetable or other similar
oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0060] The compounds can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0061] Furthermore, the compounds can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0062] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more compounds of the present invention.
Similarly, unit dosage forms for injection or intravenous
administration may comprise the compound of the present invention
in a composition as a solution in sterile water, normal saline or
another pharmaceutically acceptable carrier.
[0063] Implants for sustained release formulations are well known
in the art. Implants are formulated as microspheres, slabs, etc.
with biodegradable or non-biodegradable polymers. For example,
polymers of lactic acid and/or glycolic acid form an erodible
polymer that is well tolerated by the host. The implant containing
the inhibitory compounds is placed in proximity to the site of the
tumor, so that the local concentration of active agent is increased
relative to the rest of the body.
[0064] The term "unit dosage form", as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0065] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0066] The combined use of the provided inhibitory compounds and
other cytotoxic agents has the advantages that the required dosages
for the individual drugs is lower, and the effect of the different
drugs complementary. Depending on the patient and condition being
treated and on the administration route, the subject inhibitory
compounds may be administered in dosages of 0.1 .mu.g to 10 mg/kg
body weight per day. The range is broad, since in general the
efficacy of a therapeutic effect for different mammals varies
widely with doses typically being 20, 30 or even 40 times smaller
(per unit body weight) in man than in the rat. Similarly the mode
of administration can have a large effect on dosage. Thus for
example oral dosages in the rat may be ten times the injection
dose. Higher doses may be used for localized routes of
delivery.
[0067] A typical dosage may be a solution suitable for intravenous
administration; a tablet taken from two to six times daily, or one
time-release capsule or tablet taken once a day and containing a
proportionally higher content of active ingredient, etc. The
time-release effect may be obtained by capsule materials that
dissolve at different pH values, by capsules that release slowly by
osmotic pressure, or by any other known means of controlled
release.
[0068] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Some of the specific compounds are more potent than others.
Preferred dosages for-a given compound are readily determinable by
those of skill in the art by a variety of means. A preferred means
is to measure the physiological potency of a given compound.
[0069] For use in the subject methods, the subject compounds may be
formulated with other pharmaceutically active agents, particularly
other anti-metastatic, anti-tumor or anti-angiogenic agents.
Angiostatic compounds of interest include angiostatin, endostatin,
carboxy terminal peptides of collagen alpha (XV), etc. Cytotoxic
and cytostatic agents of interest include adriamycin, alkeran,
Ara-C, BICNU, busulfan, CNNU, cisplatinum, cytoxan, daunorubicin,
DTIC, 5-FU, hydrea, ifosfamide, methotrexate, mithramycin,
mitomycin, mitoxantrone, nitrogen mustard, velban, vincristine,
vinblastine, VP-16, carboplatinum, fludarabine, gemcitabine,
idarubicin, irinotecan, leustatin, navelbine, taxol, taxotere,
topotecan, etc.
Methods of Use
[0070] The subject compounds are administered to a subject having a
hyperproliferative disorders, e.g. to inhibit tumor growth, to
inhibit angiogenesis, to decrease inflammation associated with a
lymphoproliferative disorder, to inhibit graft rejection, or
neurological damage due to tissue repair, etc. The present
compounds are useful for prophylactic or therapeutic purposes. As
used herein, the term "treating" is used to refer to both
prevention of disease, and treatment of pre-existing conditions.
The prevention of proliferation is accomplished by administration
of the subject compounds prior to development of overt disease,
e.g. to prevent the regrowth of tumors, prevent metastatic growth,
diminish restenosis associated with cardiovascular surgery, etc.
Alternatively the compounds are used to treat ongoing disease, by
stabilizing or improving the clinical symptoms of the patient.
[0071] The host, or patient, may be from any mammalian species,
e.g. primate sp., particularly humans; rodents, including mice,
rats and hamsters; rabbits; equines, bovines, canines, felines;
etc. Animal models are of interest for experimental investigations,
providing a model for treatment of human disease.
[0072] The susceptibility of a particular cell to treatment with
the subject compounds may be determined by in vitro testing.
Typically a culture of the cell is combined with a subject compound
at varying concentrations for a period of time sufficient to allow
the active agents to induce cell death or inhibit migration,
usually between about one h and one week. For in vitro testing,
cultured cells from a biopsy sample may be used. The viable cells
left after treatment are then counted.
[0073] The dose will vary depending on the specific compound
utilized, specific disorder, patient status, etc. Typically a
therapeutic dose will be sufficient to substantially decrease the
undesirable cell population in the targeted tissue, while
maintaining patient viability. Treatment will generally be
continued until there is a substantial reduction, e.g. at least
about 50%, decrease in the cell burden, and may be continued until
there are essentially none of the undesirable cells detected in the
body.
[0074] The compounds also find use in the specific inhibition of
signaling pathway mediated by protein kinases. Protein kinases are
involved in signaling pathways for such important cellular
activities as responses to extracellular signals and cell cycle
checkpoints. Inhibition of specific protein kinases provides a
means of intervening in these signaling pathways, for example to
block the effect of an extracellular signal, to release a cell from
cell cycle checkpoint, etc. Defects in the activity of protein
kinases are associated with a variety of pathological or clinical
conditions, where there is a defect in signaling mediated by
protein kinases. Such conditions include those associated with
defects in cell cycle regulation or in response to extracellular
signals, e.g. hyperglycemia and diabetes Type I and Type II,
immunological disorders, e.g. autoimmune and immunodeficiency
diseases; hyperproliferative disorders, which may include
psoriasis, arthritis, inflammation, angiogenesis, endometriosis,
scarring, cancer, etc.
[0075] The compounds of the present invention are active in
inhibiting purified kinase proteins, i.e. there is a decrease in
the phosphorylation of a specific substrate in the presence of the
compound. A protein kinase of particular interest in integrin
linked kinase (ILK). ILK is a serine threonine kinase. The DNA and
predicted amino acid sequence may be accessed at Genbank, no.
U40282, or as published in Hannigan et al. (1996) Nature 379:91-96.
ILK regulates integrin extracellular activity (ECM interactions)
from inside the cell via its direct interaction with the integrin
subunit. Interfering with ILK activity allows the specific
targeting of integrin function, while leaving other essential
signaling pathways intact. Increased levels of cellular ILK
activity short circuits the normal requirement for adhesion to
extracellular membrane in regulating cell growth. Thus, inhibiting
ILK activity may inhibit anchorage-independent cell growth.
[0076] It is also known that many cell types undergo apoptosis if
the appropriate contacts with extracellular matrix proteins are not
maintained (anoikis). The induction of apoptosis by the subject
compounds in such cells predicts an association with the ILK
signaling pathway.
[0077] The compounds of the present invention bind to protein
kinases at a high affinity, and find use as affinity reagents for
the isolation and/or purification of such kinases. Affinity
chromatography is used as a method of separating and purifying
protein kinases and phosphatases using the biochemical affinity of
the enzyme for inhibitors that act on it. The compounds are coupled
to a matrix or gel. Preferably a microsphere or matrix is used as
the support. Such supports are known in the art and commercially
available. The inhibitor coupled support is used to separate an
enzyme that binds to the inhibitor from a complex mixture, e.g. a
cell lysate, that may optionally be partially purified. The sample
mixture is contacted with the inhibitor coupled support under
conditions that minimize non-specific binding. Methods known in the
art include columns, gels, capillaries, etc. The unbound compounds
are washed free of the resin, and the bound proteins are then
eluted in a suitable buffer.
[0078] The compounds of the invention may also be useful as
reagents for studying signal transduction or any of the clinical
disorders listed throughout this application.
Hyper-Proliferative Disorders of Interest
[0079] There are many disorders associated with a dysregulation of
cellular proliferation. The conditions of interest include, but are
not limited to, the following conditions.
[0080] The subject methods are applied to the treatment of a
variety of conditions where there is proliferation and/or migration
of smooth muscle cells, and/or inflammatory cells into the intimal
layer of a vessel, resulting in restricted blood flow through that
vessel, i.e. neointimal occlusive lesions. Occlusive vascular
conditions of interest include atherosclerosis, graft coronary
vascular disease after transplantation, vein graft stenosis,
peri-anastomatic prosthetic graft stenosis, restenosis after
angioplasty or stent placement, and the like.
[0081] Diseases where there is hyperproliferation and tissue
remodelling or repair of reproductive tissue, e.g. uterine,
testicular and ovarian carcinomas, endometriosis, squamous and
glandular epithelial carcinomas of the cervix, etc. are reduced in
cell number by administration of the subject compounds.
[0082] Tumor cells are characterized by uncontrolled growth,
invasion to surrounding tissues, and metastatic spread to distant
sites. Growth and expansion requires an ability not only to
proliferate, but also to down-modulate cell death (apoptosis) and
activate angiogenesis to produce a tumor neovasculature.
Angiogenesis may be inhibited by affecting the cellular ability to
interact with the extracellular environment and to migrate, which
is an integrin-specific function, or by regulating apoptosis of the
endothelial cells. Integrins function in cell-to-cell and
cell-to-extracellular matrix (ECM) adhesive interactions and
transduce signals from the ECM to the cell interior and vice versa.
Since these properties implicate integrin involvement in cell
migration, invasion, intra- and extra-vasation, and platelet
interaction, a role for integrins in tumor growth and metastasis is
obvious.
[0083] Tumors of interest for treatment include carcinomas, e.g.
colon, duodenal, prostate, breast, melanoma, ductal, hepatic,
pancreatic, renal, endometrial, stomach, dysplastic oral mucosa,
polyposis, invasive oral cancer, non-small cell lung carcinoma,
transitional and squamous cell urinary carcinoma etc.; neurological
malignancies, e.g. neuroblastoma, gliomas, etc.; hematological
malignancies, e.g. childhood acute leukaemia, non-Hodgkin's
lymphomas, chronic lymphocytic leukaemia, malignant cutaneous
T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma,
lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia,
bullous pemphigoid, discoid lupus erythematosus, lichen planus,
etc.; and the like.
[0084] Some cancers of particular interest include breast cancers,
which are primarily adenocarcinoma subtypes. Ductal carcinoma in
situ is the most common type of noninvasive breast cancer. In DCIS,
the malignant cells have not metastasized through the walls of the
ducts into the fatty tissue of the breast. Infiltrating (or
invasive) ductal carcinoma (IDC) has metastasized through the wall
of the duct and invaded the fatty tissue of the breast.
Infiltrating (or invasive) lobular carcinoma (ILC) is similar to
IDC, in that it has the potential metastasize elsewhere in the
body. About 10% to 15% of invasive breast cancers are invasive
lobular carcinomas.
[0085] Also of interest is non-small cell lung carcinoma. Non-small
cell lung cancer (NSCLC) is made up of three general subtypes of
lung cancer. Epidermoid carcinoma (also called squamous cell
carcinoma) usually starts in one of the larger bronchial tubes and
grows relatively slowly. The size of these tumors can range from
very small to quite large. Adenocarcinoma starts growing near the
outside surface of the lung and may vary in both size and growth
rate. Some slowly growing adenocarcinomas are described as alveolar
cell cancer. Large cell carcinoma starts near the surface of the
lung, grows rapidly, and the growth is usually fairly large when
diagnosed. Other less common forms of lung cancer are carcinoid,
cylindroma, mucoepidermoid, and malignant mesothelioma.
[0086] Melanoma is a malignant tumor of melanocytes. Although most
melanomas arise in the skin, they also may arise from mucosal
surfaces or at other sites to which neural crest cells migrate.
Melanoma occurs predominantly in adults, and more than half of the
cases arise in apparently normal areas of the skin. Prognosis is
affected by clinical and histological factors and by anatomic
location of the lesion. Thickness and/or level of invasion of the
melanoma, mitotic index, tumor infiltrating lymphocytes, and
ulceration or bleeding at the primary site affect the prognosis.
Clinical staging is based on whether the tumor has spread to
regional lymph nodes or distant sites. For disease clinically
confined to the primary site, the greater the thickness and depth
of local invasion of the melanoma, the higher the chance of lymph
node metastases and the worse the prognosis. Melanoma can spread by
local extension (through lymphatics) and/or by hematogenous routes
to distant sites. Any organ may be involved by metastases, but
lungs and liver are common sites.
[0087] Other hyperproliferative diseases of interest relate to
epidermal hyperproliferation, tissue remodelling and repair. For
example, the chronic skin inflammation of psoriasis is associated
with hyperplastic epidermal keratinocytes as well as infiltrating
mononuclear cells, including CD4+ memory T cells, neutrophils and
macrophages.
[0088] The proliferation of immune cells is associated with a
number of autoimmune and lymphoproliferative disorders. Diseases of
interest include multiple sclerosis, rheumatoid arthritis and
insulin dependent diabetes mellitus. Evidence suggests that
abnormalities in apoptosis play a part in the pathogenesis of
systemic lupus erythematosus (SLE). Other lymphoproliferative
conditions the inherited disorder of lymphocyte apoptosis, which is
an autoimmune lymphoproliferative syndrome, as well as a number of
leukemias and lymphomas. Symptoms of allergies to environmental and
food agents, as well as inflammatory bowel disease, may also be
alleviated by the compounds of the invention.
[0089] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
EXAMPLES
Example 1
In Vitro Screen
[0090] Compounds were screened using a series of disease related
kinase targets, such as integrin linked kinase-1. Synthesized
libraries of compounds are tested against each of the targets to
find compounds that inhibit one of the targets preferentially. The
desired in vitro potency of the inhibitor is such that the compound
is useful as a therapeutic agent, i.e. in the nanomolar or
micromolar range.
[0091] Inhibition of the targets is measured by scintillation
counting; the incorporation of radioactive phosphate onto a
specific substrate which is immobilized onto a filter paper at the
end of the assay. To provide meaningful measurements of inhibition,
the assays are performed both in the absence and presence of
specific and known inhibitors, and the amount of incorporated
radioactivity is compared to provide a baseline measurement.
[0092] The baseline activity is the amount of radioactivity
incorporated in the absence of inhibitor. The amount of
radioactivity incorporated in the presence of an inhibitor is
called the `sample activity`, and the % inhibition is expressed by
the following formula:
% inhibition=100-(sample activity/baseline activity*100)
[0093] and is usually expressed in conjunction with the compound
concentration. By using a range of inhibitor concentrations, the
IC.sub.50 of an inhibitor is estimated (i.e. the concentration at
which enzymatic activity is reduced by 50%). The IC.sub.50 of
various compounds against a particular target can be compared,
where a lower IC.sub.50 indicates a more potent compound.
[0094] Materials and Methods
[0095] Inhibition Assay:
[0096] Compounds listed in Table 1 were lyophilized and stored at
-20.degree. C. Stock solutions were made by weighing out the
compounds and dissolving them in dimethyl sulfoxide (DMSO) to a
standard concentration, usually 20 mM, and stored at -20.degree. C.
The compounds were diluted to a starting intermediate concentration
of 250 .mu.M in 1% DMSO, then serially diluted across a row of a 96
well plate using serial 2 fold dilution steps. Diluted 100% DMSO
was used as a negative control.
[0097] 5 .mu.l of each compound dilution were robotically pipetted
to Costar serocluster plates maintaining the same plate format. All
assays consisted of the following volumes:
[0098] 5 .mu.l diluted compound
[0099] 10 .mu.l enzyme preparation
[0100] 5 .mu.l substrate
[0101] 5 .mu.l assay ATP
[0102] and were then incubated 15 min at room temperature.
[0103] From each reaction, 10 .mu.l of reaction mix was spotted
onto Millipore Multiscreen-PH opaque plates and washed 2.times.10
min in 1% phosphoric acid. The plates were dried for at 40.degree.
C. for 30 min, then the substrate phosphate complexes were
quantitated by scintillation counting. These Millipore plates are
in a 96 well format with immobilized P81 phosphocellulose
membranes. Both the phosphorylated and non-phosphorylated form of
the substrate bind to the membrane while ATP (unincorporated
phosphate) is removed in the subsequent wash steps. Results are
shown in Table 1 below.
[0104] Integrin Linked Kinase:
[0105] The target integrin linked kinase is a full-length
recombinant protein expressed in sF9 insect cells by baculovirus
infection. The ILK1 substrate is CKRRRLASLR-amide.
[0106] Recombinant ILK protein was expressed using cultured insect
cells and a baculovirus expression system. Standard techniques for
DNA manipulation were used to produce recombinant DNA molecules and
baculoviruses (Sambrook. J., Fritsch, E. F. and Maniatis, T. 1989.
Molecular cloning, a laboratory manual. Second edition. Cold Spring
Harbor Laboratory Press. NY; Crossen, R. and Gruenwald, S. 1998.
Baculovirus expression Vector System Manual. 5.sup.th Edition.
Pharmingen, San Diego, Calif.) but the isolation of active ILK
required some ingenuity.
[0107] The ILK open reading frame (Hannigan et al., supra.),
excluding the 5' and 3' untranslated regions, was inserted into the
baculovirus transfer vector pAcG2T (Pharmingen) to produce a GST
fusion protein under the control of the strong AcNPV polyhedrin
promoter. A large scale plasmid preparation of the resulting
transfer construct was made using a Qiagen Plasmid Midi Kit. This
ILK transfer construct was then co-transfected with BaculoGold DNA
(Pharmingen) into Sf9 insect cells (Invitrogen) and a high titre
preparation of GST-ILK recombinant baculovirus was produced by
amplification in Sf9 cells. Liter scale expression of GST-ILK
recombinant protein was done in 1000 ml spinner flasks (Bellco) by
infection of Hi5 insect cells (Invitrogen) grown in Ex-Cell 400
Serum Free Media (JRH Biosciences) at a multiplicity of infection
of approximately 5. The cells were harvested three days after
infection and lysed in Hypotonic Lysis Buffer (HLB; 10 mM
imidazole, 5 mM EDTA, 0.1% .beta.-mercaptoethanol, 10 ug/ml PMSF, 1
mM benzamidine) by sonication. The lysate was centrifuged at 10,000
g for 20 min and the supernatant was discarded. The pellet was
washed twice in HLB and then washed twice in High Salt Buffer
("HSB"; 500 mM NaCl, 10 mM imidazole, 5 mM EDTA, 0.1%
.beta.-mercaptoethanol, 10 ug/ml PMSF, 1 mM benzamidine). The
pellet was then resuspended in DNAse-ATP Buffer ("DAB"; 10 mM
MgCl.sub.2, 1 mM MnCl.sub.2, .beta.-methyl aspartic acid, 2 mM NaF,
0.55 mg/ml ATP, lug/ml DNAse 1, 1% NP-40, 10 mM imidazole, 5 mM
EDTA, 0.1% .beta.-mercaptoethanol, 10 ug/ml PMSF, 1 mM benzamidine)
and stirred for 30 min at room temperature, and then centrifuged at
10,000.times.g for 20 min. The pellet was resuspended in High Salt
Detergent buffer ("HDB"; 1% NP-40, 1% Triton X-100, 500 mM NaCl, 10
mM imidazole, 5 mM EDTA, 0.1% .beta.-mercaptoethanol, 10 ug/ml
PMSF, 1 mM benzamidine), stirred for 30 min at room temperature,
and then centrufuged at 10,000 g for 20 min. The pellet was then
washed once in each of HDB, HSB, and HLB, centrifuging at 10,000 g
each time. Finally, the pellet was resuspended in HLB.
[0108] The recombinant ILK expressed in insect cells with a
baculovirus system was solubilized by treating the insoluble ILK
protein with DNAse I and detergents. This produced an ILK protein
preparation in the form of a microparticle suspension. This
preparation had a high specific activity and was amenable to
automated kinase assays.
1TABLE 1 Activity of Analogs of KP-15792 Codes Chemical Name
Structure MW IC.sub.50 (.mu.M) KP-15792 3,5-diamine-4-(p-
methoxyphenyl)hydrazono- pyrazole 10 232.24 1 KP-23194
3,5-diamino-4- phenylhydrazonopyrazole 11 202.21 0.6 KP-23195
3,5-diamino-4-(p-methylphenyl) hydrazonopyrazole 12 216.24 5.3
KP-23197 3,5-diamino-4-(3-hydroxy-4-
methoxyphenyl)hydrazonopyrazole 13 248.24 4 KP-23198
3-amino-4-phenylazo-2-pyrazolin-5- one 14 203.20 17.1 KP-23199
3-amino-4-(p-methylphenylazo)-2- pyrazolin-5-one 15 217.23 >20
KP-23200 3-amino-4-(p-methoxyphenylazo)-2- pyrazolin-5-one 16
233.22 >20 KP-23201 3-amino-4-(3-hydroxy-4-
methyoxyphenylazo)-2-pyrazolin-- 5-one 17 249.22 18 KP-23202
4-phenylhydrazonopyrazolin-3,5- -dione 18 204.18 >20 KP-23203
4-(p-methylphenyl)hydrazo- nopyrazolin- 3,5-dione 19 218.21 >20
KP-23204 4-(p- methoxyphenyl)hydrazonopyrazolin- 3,5-dione 20
234.21 >20 KP-23205 4-(3-hydroxy-4-
methyoxyphenyl)hydrazonopyrazolin- 3,5-dione 21 250.21 >20
KP-27288 4-[N'-(3,5-diaminopyrazole-4-
ylidene)hydrazino]benzenesulfonic acid 22 282.27 >20 KP-27289
3,5-diamino-4- morpholinylhydrazonopyrazole 23 211.22 9.6 KP-27290
3,5-diamino-4-(2- morpholinylethyl)hydrazonopyrazole 24 239.28 8.2
KP-27291 3,5-diamino-4-(2- imidazolyl)hydrazonopyrazole 25 192.18
28 KP-27292 3,5-diamino-4-(3-pyrazolyl) hydrazonopyrazole 26 192.18
8 KP-27293 3,5-diamino-4-(2- thiazolyl)hydrazonopyrazole 27 209.22
0.9 KP-27386 3,5-diamino-4-(4- piperidinylmethyl)hydrazonopyrazole
28 223.28 13 KP-27387 3,5-diamino-4-(3-
[1,2,4]triazinyl)hydrazonopyrazole 29 205.18 >20 KP-27294
3,5-diamino-4-(1-naphthyl) hydrazonopyrazole 30 252.27 0.6 KP-27295
4-[N'-(3,5-Diaminopyrazol-4- ylidene)hydrazino]naphtha- lene-1-
sulfonic acid 31 332.33 19.3 KP-27388 4-[N'-(3,5-diaminopyrazol-4-
ylidene)hydrazino]benzoic acid 32 246.22 13 KP-27389
3,5-diamino-4-(p- hydroxyphenyl)hydrazonopyrazol- e 33 218.21
>20 KP-27390 3,5-diamino-4-(p- chlorophenyl)hydrazonopyrazole 34
236.66 1.2 KP-27391 3,5-diamino-4-(p-(n-
propyl)phenyl)hydrazonopyrazole 35 258.32 4.6 KP-27392
3,5-diamino-4-(p- acetoaminophenyl)hydrazonopyrazole 36 274.3 5
KP-27393 3,5-diamino-4-(2- hydroxynaphthyl)hydrazonopyrazole 37
268.27 3
Example 4
Synthesis of 3,5-Diamino-4-(p-methoxyphenyl)hydrazonopyrazole
[0109] Unless otherwise stated, chemical reactants and reagents
were obtained from standard chemical supply houses, such as Aldrich
(Milwaukee, WI; www.aldrich.sial.com); and Lancaster Synthesis,
Inc. (Windham, N.H.; www.lancaster.co.uk).
[0110] To a flask containing p-anisidine (5.46 g, 44.3 mmol) and
concentrated HCl solution (11 mL) in 75 mL of water, cooled in an
ice water bath, was added sodium nitrite solution (4.57 g, 66.3
mmol). The resulting mixture was then added to a solution of
malononitrile (4.79 g, 72.6 mmol) in a mixture of MeOH (12 mL) and
water (25 mL). A large quantity of yellow solid quickly
precipitated. The mixture was stirred for about 30 minutes at room
temperature. The solid was collected and purified by
recrystallization in hot EtOH. The product (6.17 g, 70%) was
obtained as a yellow solid.
[0111] To a suspension of the yellow solid (2.00 g) prepared above
in 10 mL of EtOH was added hydrazine hydrate (2.0 mL). This mixture
was refluxed for about 3 h. The yellow solid was collected and
purified by recrystallization in hot EtOH. The product was isolated
as yellow cotton like solid (1.50 g, 65%). m.p.: 263-265.degree. C.
.sup.1H NMR (ppm, in DMSO-d.sub.6): 10.73 (s, br, 1 H), 7.69 (m, 2
H), 6.99 (m, 2 H), 6.00 (s, br, 4 H), 3.81 (s, 3 H). .sup.13C NMR
(ppm, in DMSO-d.sub.6): 158.4, 147.6, 121.7, 114.0, 113.4, 99.9,
55.3. FTIR (cm.sup.-1, KBr pellet): 3401, 3301, 3187, 1603, 562,
1498, 1248,1033, 828. Mass spectrometry (m/e, EI): 232 (M.sup.+,
100%). Elemental analysis for C.sub.10H.sub.12N.sub.60
(obtained/calcd.): C 52.28/51.72, H 5.18/5.21, N 35.88/36.19.
Example 5
Additional Syntheses According To Procedure of Example 4.
[0112] The following compounds were synthesized using
malononitrile, following essentially the same procedure as
described above in Example 4:
[0113] 3,5-diamino-4-phenylhydrazonopyrazole;
[0114] 3,5-diamino-4-(p-methylphenyl)hydrazonopyrazole;
[0115]
3,5-diamino-4-(3-hydroxy-4-methoxyphenyl)hydrazonopyrazole;
[0116]
4-[N'-(3,5-Diaminopyrazole-4-ylidene)hydrazino]benzenesulfonic
acid;
[0117] 3,5-diamino-4-morpholinylhydrazonopyrazole;
[0118] 3,5-diamino-4-(2-morpholinylethyl)hydrazonopyrazole;
[0119] 3,5-diamino-4-(2-imidazolyl)hydrazonopyrazole;
[0120] 3,5-diamino-4-(3-pyrazoly)hydrazonopyrazole;
[0121] 3,5-diamino-4-(2-thiazolyl)hydrazonopyrazole;
[0122] 3,5-diamino-4-(2-naphthyl)hydrazonopyrazole;
[0123] 3,5-diamino-4-(l
-(3-sulfonyl)naphthalyl)hydrazonopyrazole;
[0124] 3,5-diamino-4-(4-piperidinylmethyl)hydrazonopyrazole;
[0125] 3,5-diamino-4-(3-[1,2,4]triazinyl)hydrazonopyrazole;
[0126]
4-[N'-(3,5-diaminopyrazol-4-ylidene)hydrazino]naphthalene-1-sulfoni-
c acid;
[0127] 4-[N'-(3,5-diaminopyrazol-4-ylidene)hydrazino]benzoic
acid;
[0128] 3,5-diamino-4-(p-hydroxyphenyl)hydrazonopyrazole;
[0129] 3,5-diamino-4-(p-chlorophenyl)hydrazonopyrazole;
[0130] 3,5-diamino-4-(p-(n-propyl)phenyl)hydrazonopyrazole;
[0131] 3,5-diamino-4-(p-acetoaminophenyl)hydrazonopyrazole; and
[0132] 3,5-diamino-4-(2-hydroxynaphthyl)hydrazonopyrazole.
Example 6
Additional Syntheses According To Procedure of Example 4, using
Ethyl Cyanoacetate
[0133] The following compounds were synthesized using ethyl
cyanoacetate instead of malononitrile, but otherwise following
essentially the same procedure as described above in Example 4:
[0134] 3-amino-4-phenylazo-2-pyrazolin-5-one;
[0135] 3-amino-4-(p-methylphenylazo)-2-pyrazolin-5-one;
[0136] 3-amino-4-(p-methoxyphenylazo)-2-pyrazolin-5-one; and
[0137]
3-amino-4-(3-hydroxy-4-methyoxyphenylazo)-2-pyrazolin-5-one.
Example 7
Additional Syntheses According To Procedure of Example 4, using
Diethyl Malonate
[0138] The following compounds were synthesized using diethyl
malonate instead of malononitrile, but otherwise following
essentially the same procedure as described above in Example 4:
[0139] 4-phenylhydrazonopyrazolin-3,5-dione;
[0140] 4-(p-methylphenyl)hydrazonopyrazolin-3,5-dione;
[0141] 4-(p-methoxyphenyl)hydrazonopyrazolin-3,5-dione; and
[0142]
4-(3-hydroxy-4-methyoxyphenyl)hydrazonopyrazolin-3,5-dione.
EXAMPLE 8
Synthesis of 3,5-Diamino-4-[(4-Fluorophenyl)Hydrazono]Pyrazole
[0143] 38
[0144] 4-Fluoroaniline (95 .mu.L, 1.0 mmol) was weighed into a 25
mL test tube. Deionized water (1-2 mL) was added to the test tube
and the suspension was cooled to below 5.degree. C. in an ice bath.
Concentrated HCl (250 .mu.L, 3.0 mmol) was added dropwise to the
mixture. If the solution remained inhomogeneous, DMF was added
until all the solids had dissolved (0-2 mL). An aqueous sodium
nitrite solution (290 .mu.L of a 5.25 M solution, 1.5 mmol) was
added dropwise to this mixture and allowed to stir for
approximately 5 minutes. The resulting clear pale yellow solution
was then added dropwise to a second 25 mL test tube containing 1.4
mL of an ice cold aqueous solution which was 1.82 M (2.3 mmol) in
sodium acetate trihydrate and 1.09 M (1.5 mmol) in malononitrile. A
precipitate formed immediately. The reaction solution was stirred
for 1-2 hrs while warming to room temperature. The solution was
then filtered and the precipitate was washed twice with 5 mL of
deionized water. The product was dried overnight under vacuum to
yield 169 mg (90%) of the desired malononitrile derivative as a
yellow solid. A portion of this solid (94 mg, 0.5 mmol) was weighed
into a 25 mL test tube. Anhydrous ethanol (1.5 mL) was added and
the slurry was heated to 75.degree. C. Once the solid had
dissolved, hydrazine hydrate (1 mmol) was added dropwise via
micropipette. A precipitate usually formed within 10 minutes. The
reaction was monitored for the disappearance of the starting
material by TLC, as well as, for the appearance of a more polar
spot due to the product. Once the reaction was complete, the
solution was allowed to cool to room temperature. The solid was
isolated by filtration, washed with ethanol, and dried to yield 17
mg (15%) of the title compound as a mustard coloured solid.
EXAMPLE 9
Synthesis of
3-[N'-(3,5-Diaminopyrazol-4-Ylidene)Hydrazino]Phenol
[0145] 39
[0146] 3-[N'-(3,5-Diaminopyrazol-4-ylidene)hydrazino]phenol was
prepared using 93 mg (0.5 mmol) of
2-[(3-hydroxyphenyl)hydrazono]malononitrile, which was derived from
3-aminophenol (109 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. After heating for 4
hrs, a small amount of solid had formed. The solid was filtered off
and the filtrate was concentrated to a gummy black solid. This
material was dissolved in ethyl acetate and a small amount of gummy
solid was precipitated from the solution by the addition of
hexanes. The solid was removed by filtration and the filtrate was
again concentrated. The resulting solid was purified by flash
chromatography eluting with methylene chloride/methanol (7:1) to
yield 45 mg (33%) of the title compound as a black solid.
EXAMPLE 10
Synthesis of 3,5-Diamino-4-[(3-Ethylphenyl)Hydrazono]Pyrazole
[0147] 40
[0148] 3,5,-Diamino-4-[(3-ethylphenyl)hydrazono]pyrazole was
prepared using 99 mg (0.5 mmol) of
2-[(3-ethylphenyl)hydrazono]malononitrile, which was derived from
3-ethylaniline (124 .mu.L, 1.0 mmol) and malononitrile (1.5 mmol)
as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 10 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, precipitated from an ethyl acetate solution by the
addition of hexanes, and dried to yield 12 mg (10%) of the title
compound as a yellow solid.
EXAMPLE 11
Synthesis of 3,5-Diamino-4-[(3-Methoxyphenyl)Hydrazono]Pyrazole
[0149] 41
[0150] The title compound was prepared using 100 mg (0.5 mmol) of
2-[(3-methoxyphenyl)-hydrazono]malononitrile, which was derived
from m-anisidine (112 .mu.L, 1.0 mmol) and malononitrile (1.5 mmol)
as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 10 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, recrystallized from ethanol, and dried to yield 25 mg
(22%) of the title compound as a brownish orange solid.
EXAMPLE 12
Synthesis of 3,5-Diamino-4-[(3-Chlorophenyl)Hydrazono]Pyrazole
[0151] 42
[0152] This compound was prepared using 102 mg (0.5 mmol) of
2-[(3-chlorophenyl)-hydrazono]malononitrile, which was derived from
3-chloroaniline (106 .mu.L, 1.0 mmol) and malononitrile (1.5 mmol)
as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 5 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, precipitated from an ethyl acetate solution by the
addition of hexanes, and dried to yield 17 mg (14%) of the title
compound as a yellow solid. .sup.1H NMR (ppm, DMSO-d.sub.6): 5.98
(br, s, 2H), 6.38 (br, s, 2H), 7.18 (d, 1H), 7.40 (t, 1H), 7.60 (d,
1H), 7.69 (s, 1H), 10.78 (s, 1 H).
EXAMPLE 13
Synthesis of 3,5-Diamino-4-[(3-Fluorophenyl)Hydrazono]Pyrazole
[0153] 43
[0154] This compound was prepared using 94 mg (0.5 mmol) of
2-[(3-fluorophenyl)-hydrazono]malononitrile, which was derived from
3-fluoroaniline (96 .mu.L, 1.0 mmol) and malononitrile (1.5 mmol)
as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 5 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, washed with ethanol, and dried to yield 41 mg (37%) of
the title compound as a yellow solid. .sup.1H NMR (ppm,
DMSO-d.sub.6): 6.2 (br s, 4H), 7.0 (t, 1H), 7.35-7.62 (m, 3H),
10.80 (s, 1H).
EXAMPLE 14
Synthesis of
3,5-Diamino-4-[(3-Fluoro-4-Methoxyphenyl)Hydrazono]Pyrazole
[0155] 44
[0156] This compound was prepared using 109 mg (0.5 mmol) of
2-[(3-fluoro-4-methoxyphenyl)hydrazono]malononitrile, which was
derived from 3-fluoro-p-anisidine (141 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Precipitate formed in the reaction tube immediately after
the addition of hydrazine hydrate. The resulting solid was isolated
by filtration, washed with ethanol, and dried to yield 85 mg (68%)
of the title compound as a mustard coloured solid.
EXAMPLE 15
Synthesis of 3,5-Diamino-4-[(Naphthalen-2-Yl)Hydrazono]Pyrazole
[0157] 45
[0158] The title compound was prepared using 110 mg (0.5 mmol) of
2-[(naphthalen-2-yl)hydrazono]malononitrile, which was derived from
2-aminonaphthalene (143 mg, 1.0 mmol) and malononitrile (1.5 mmol)
as described in Example 8, and hydrazine hydrate. The hydrazine
hydrate was added to the solution at a temperature of 75.degree. C.
despite the fact that the starting material had not fully
dissolved. The solution cleared briefly and then a precipitate
formed. The resulting solid was isolated by filtration, washed with
ethanol, and dried to yield 86 mg (67%) of the title compound as a
tan coloured solid.
EXAMPLE 16
Synthesis
3,5-Diamino-4-[(4-Trifluoromethylphenyl)Hydrazono]Pyrazole
[0159] 46
[0160] This compound was prepared using 119 mg (0.5 mmol) of
2-[(4-trifluoromethylphenyl)-hydrazono]malononitrile, which was
derived from 4-(trifluoromethyl)aniline (126 .mu.L, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. No precipitate had formed after heating at 75.degree. C.
for 1 hr, however, analysis of the reaction solution by TLC
indicated that no starting material remained. The solution was
allowed to cool to room temperature and the solvent was evaporated.
The residue was dissolved in ethyl acetate and then precipitated by
the addition of hexanes. The resulting solid was isolated by
filtration and dried to yield 67 mg (50%) of the title compound as
a greenish brown solid. .sup.1H NMR (ppm, DMSO-d.sub.6): 6.03 (br
s, 2H), 6.48 (br s, 2H), 7.63 (d, 2H), 7.80 (d, 2H), 10.80 (br s, 1
H).
EXAMPLE 17
Sysnthesis of 4-[(3-Phenoxyphenyl)Hydrazono]Pyrazole
[0161] 47
[0162] The title compound was prepared using 131 mg (0.5 mmol) of
2-[(3-phenoxyphenyl)-hydrazono]malononitrile, which was derived
from 3-phenoxyaniline (185 mg, 1.0 mmol) and malononitrile (1.5
mmol) as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 5 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, recrystallized from ethanol, and dried to yield 87 mg
(59%) of the title compound as a mustard coloured solid.
EXAMPLE 18
Synthesis of 4-[N'-(3,5-Diaminopyrazol-4-Ylidene)Hydrazino]Benzoic
Acid
[0163] 48
[0164] This compound was prepared using 121 mg (0.5 mmol) of
4-(N'-dicyanomethylene-hydrazino)benzoic acid ethyl ester, which
was derived from ethyl 4-aminobenzoate (165 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. The hydrazine hydrate was added to the solution at a
temperature of 75.degree. C. The solution cleared briefly and then
a precipitate formed. The resulting solid was isolated by
filtration, washed with ethanol, and dried to yield 45 mg (33%) of
the title compound as a yellow solid.
EXAMPLE 19
Synthesis of 3,5-Diamino-4-[(Biphenyl-2-Yl)Hydrazono]Pyrazole
[0165] 49
[0166] This compound was prepared using 123 mg (0.5 mmol) of
2-[(biphenyl-2-yl)hydrazono]-malononitrile, which was derived from
2-aminobiphenyl (169 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. Precipitate formed
in the reaction tube immediately after the addition of the
hydrazine hydrate then the solution cleared. Very little solid
remained after heating the reaction at 75.degree. C. for 1 hr,
however, analysis of the reaction solution by TLC indicated that no
starting material remained. The solution was allowed to cool to
room temperature and the solvent was evaporated. The residue was
dissolved in ethyl acetate and then precipitated by the addition of
hexanes. The resulting solid was isolated by filtration and dried
to yield 85 mg (61%) of the title compound as an orange solid.
[0167] EXAMPLE 20
Synthesis of 3,5-Diamino-4-[(2-Bromophenyl)Hydrazono]Pyrazole
[0168] 50
[0169] This compound was prepared using 125 mg (0.5 mmol) of
2-[(2-Bromophenyl)-hydrazono]malononitrile, which was derived from
2-bromoaniline (172 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. Very little solid
had formed after heating the reaction at 75.degree. C. for 1 hr,
however, analysis of the reaction solution by TLC indicated that no
starting material remained. The solution was allowed to cool to
room temperature and the solvent was evaporated. The residue was
dissolved in ethyl acetate and then precipitated by the addition of
hexanes. The resulting solid was isolated by filtration and dried
to yield 102 mg (73%) of the title compound as an orange solid.
EXAMPLE 21
Synthesis of 3,5-Diamino-4-[(3-Bromophenyl)Hydrazono]Pyrazole
[0170] 51
[0171] This compound was prepared using 125 mg (0.5 mmol) of
2-[(3-Bromophenyl)-hydrazono]malononitrile, which was derived from
3-bromoaniline (172 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. The hydrazine
hydrate was added to the solution at a temperature of 75.degree. C.
despite the fact that the starting material had not fully
dissolved. The solution cleared briefly and then a precipitate
formed. The resulting solid was isolated by filtration, washed with
ethanol, and dried to yield 93 mg (66%) of the title compound as an
orange solid. .sup.1H NMR (ppm, DMSO-d.sub.6): 6.2 (br s, 4H),
7.21-7.32 (m, 2H), 7.50-7.62 (m, 1H), 7.90 (s, 1H), 10.71 (s,
1H).
EXAMPLE 22
Synthesis of 3,5-Diamino-4-[(4-Bromophenyl)Hydrazono]Pyrazole
[0172] 52
[0173] This compound was prepared using 125 mg (0.5 mmol) of
2-[(4-Bromophenyl)-hydrazono]malononitrile, which was derived from
4-bromoaniline (172 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. The hydrazine
hydrate was added to the solution at a temperature of 75.degree. C.
despite the fact that the starting material had not fully
dissolved. The solution cleared briefly and then a precipitate
formed. The resulting solid was isolated by filtration, washed with
ethanol, and dried to yield 109 mg (78%) of the title compound as a
yellow solid. .sup.1H NMR (ppm, DMSO-d.sub.6): 6.15 (br s, 4H),
7.52 (d, 2H), 7.61 (d, 2H), 10.71 (s, 1H).
EXAMPLE 23
Synthesis of 3,5-Diamino-4-[(4-Phenoxyphenyl)Hydrazono]Pyrazole
[0174] 53
[0175] This compound was prepared using 131 mg (0.5 mmol) of
2-[(4-phenoxyphenyl)-hydrazono]malononitrile, which was derived
from 4-phenoxyaniline (185 mg, 1.0 mmol) and malononitrile (1.5
mmol) as described in Example 8, and hydrazine hydrate. The
hydrazine hydrate was added to the solution at a temperature of
75.degree. C. despite the fact that the starting material had not
fully dissolved. The solution cleared briefly and then a
precipitate formed. The resulting solid was isolated by filtration,
washed with ethanol, and dried to yield 90 mg (61%) of the title
compound as an orange solid.
EXAMPLE 24
Synthesis of 3,5-Diamino-4-[(4-Iodophenyl)Hydrazono]Pyrazole
[0176] 54
[0177] This compound was prepared using 148 mg (0.5 mmol) of
2-[(4-iodophenyl)hydrazono]-malononitrile, which was derived from
4-iodoaniline (219 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. The hydrazine
hydrate was added to the solution at a temperature of 75.degree. C.
despite the fact that the starting material had not fully
dissolved. The solution cleared briefly and then a precipitate
formed. The resulting solid was isolated by filtration, washed with
ethanol, and dried to yield 114 mg (70%) of the title compound as a
yellow solid.
EXAMPLE 25
Synthesis of
3,5-Diamino-4-[(4-Bromonaphthalen-1-Yl)Hydrazono]Pyrazole
[0178] 55
[0179] This compound was prepared using 149 mg (0.5 mmol) of
2-[(4-bomonaphthalen-1-yl)-hydrazono]malononitrile, which was
derived from 1-amino-4-bromonaphthalene (222 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. The hydrazine hydrate was added to the solution at a
temperature of 75.degree. C. despite the fact that the starting
material had not fully dissolved. The solution cleared. Very little
solid had formed after heating the reaction at 75.degree. C. for 1
hr, however, analysis of the reaction solution by TLC indicated
that no starting material remained. The solution was allowed to
cool to room temperature and the solvent was evaporated. The
residue was dissolved in methanol and then precipitated by the
addition of water. The resulting solid was isolated by filtration
and dried to yield 42 mg (26%) of the title compound as a brown
solid.
EXAMPLE 26
Synthesis of 3,5-Diamino-4-[(O-Tolyl)Hydrazono]Pyrazole
[0180] 56
[0181] This compound was prepared using 92 mg (0.5 mmol) of
2-(o-tolylhydrazono)-malononitrile, which was derived from
4-toluidine (107 .mu.L, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. Very little solid
had formed after heating the reaction at 75.degree. C. for 1 hr,
however, analysis of the reaction solution by TLC indicated that no
starting material remained. The solution was allowed to cool to
room temperature and the solvent was evaporated. The residue was
dissolved in ethyl acetate and then precipitated by the addition of
hexanes. The resulting solid was isolated by filtration and dried
to yield 43 mg (40%) of the title compound as a yellow solid.
EXAMPLE 27
Synthesis of
3,5-Diamino-4-[(2,6-Difluorophenyl)Hydrazono]Pyrazole
[0182] 57
[0183] This compound was prepared using 103 mg (0.5 mmol) of
2-[(2,6-difluorophenyl)-hydrazono]malononitrile, which was derived
from 2,6-difluoroaniline (108 .mu.L, 1.0 mmol) and malononitrile
(1.5 mmol) as described in Example 8, and hydrazine hydrate.
Precipitate formed in the reaction tube approximately 10 min after
the addition of hydrazine hydrate. The resulting solid was isolated
by filtration, washed with ethanol, and dried to yield 44 mg (37%)
of the title compound as an orange solid.
EXAMPLE 28
Synthesis of
3,5-Diamino-4-[(3,4-Difluorophenyl)Hydrazono]Pyrazole
[0184] 58
[0185] This compound was prepared using 103 mg (0.5 mmol) of
2-[(3,4-difluorophenyl)-hydrazono]malononitrile, which was derived
from 3,4-difluoroaniline (99 .mu.L, 1.0 mmol) and malononitrile
(1.5 mmol) as described in Example 8, and hydrazine hydrate.
Precipitate formed in the reaction tube approximately 5 min after
the addition of hydrazine hydrate. The resulting solid was isolated
by filtration, washed with ethanol, and dried to yield 45 mg (38%)
of the title compound as a yellow solid. .sup.1H NMR (ppm,
DMSO-d.sub.6): 6.18 (br s, 4H), 7.28-7.55 (m, 2H), 7.70-7.82 (m,
1H), 10.80 (br s, 1H).
EXAMPLE 29
Synthesis of
3,5-Diamino-4-[(Benzo[1,3]Dioxol-5-Yl)Hydrazono]Pyrazole
[0186] 59
[0187] This compound was prepared using 107 mg (0.5 mmol) of
2-(benzo[1,3]dioxol-5-yl-hydrazono)malononitrile, which was derived
from 3,4-methylenedioxyaniline (137 mg, 1.0 mmol) and malononitrile
(1.5 mmol) as described in Example 8, and hydrazine hydrate.
Precipitate formed in the reaction tube approximately 10 min after
the addition of hydrazine hydrate. The resulting black solid was
isolated by filtration, dissolved in acetone, and hexanes was added
to precipitate a small amount of black solid. The solid was removed
by filtration and the filtrate was concentrated to yield 1.0 mg (1%
yield) of the title compound as a black solid. .sup.1H NMR (200
MHz, d.sup.6 DMSO) .delta.: 6.0 (brs, 6H), 6.92 (d, 1H), 7.18 (d,
1H), 7.38 (s, 1H), 10.60 (br s, 1H).
EXAMPLE 30
Synthesis of
3,5-Diamino-4-[(4-Methylsulfanylphenyl)Hydrazono]Pyrazole
[0188] 60
[0189] This compound was prepared using 108 mg (0.5 mmol) of
2-[(4-methylsulfanylphenyl)-hydrazono]malononitrile, which was
derived from 4-methylthioaniline (117 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Precipitate formed in the reaction tube immediately after
the addition of hydrazine hydrate. The resulting solid was isolated
by filtration, washed with ethanol, and dried to yield 95 mg (77%)
of the title compound as an orange solid.
EXAMPLE 31
Synthesis of
3,5-Diamino-4-[(2,3-Dihydrobenzo[1,4]Dioxin-6-Yl)-Hydrazono]P-
yrazole
[0190] 61
[0191] This compound was prepared using 114 mg (0.5 mmol) of
2-[(2,3-dihydro-benzo[1,4]dioxin-6-yl)hydrazono]malononitrile,
which was derived from 1,4-benzodiozan-6-amine (151 mg, 1.0 mmol)
and malononitrile (1.5 mmol) as described in Example 8, and
hydrazine hydrate. The hydrazine hydrate was added to the solution
at a temperature of 75.degree. C. despite the fact that the
starting material had not fully dissolved. The solution cleared.
Very little solid had formed after heating the reaction at
75.degree. C. for 1 hr, however, analysis of the reaction solution
by TLC indicated that no starting material remained. The solution
was allowed to cool to room temperature and the solvent was
evaporated. The residue was dissolved in ethyl acetate and then
precipitated by the addition of hexanes. The resulting solid was
isolated by filtration and dried to yield 35 mg (27%) of the title
compound as a tan coloured solid.
EXAMPLE 32
Synthesis of
3,5-Diamino-4-[(3-Chloro-4-Methoxyphenyl)Hydrazono]Pyrazole
[0192] 62
[0193] This compound was prepared using 117 mg (0.5 mmol) of
2-[(3-chloro-4-methoxy-phenyl)hydrazono]malononitrile, which was
derived from 3-chloro-4-anisidine (157 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Precipitate formed in the reaction tube immediately after
the addition of hydrazine hydrate. The resulting solid was isolated
by filtration, washed with ethanol, and dried to yield 93 mg (70%)
of the title compound as a yellow solid.
EXAMPLE 33
Synthesis of
3,5-Diamino-4-[(3,4-Dichlorophenyl)Hydrazono]Pyrazole
[0194] 63
[0195] This compound was prepared using 120 mg (0.5 mmol) of
2-[(3,4-dichlorophenyl)-hydrazono]malononitrile, which was derived
from 3,4-dichloroaniline (162 mg, 1.0 mmol) and malononitrile (1.5
mmol) as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube immediately after the addition of
hydrazine hydrate. The resulting solid was isolated by filtration,
precipitated from an ethyl acetate solution by the addition of
hexanes, and dried to yield 53 mg (39%) of the title compound as a
yellow solid. .sup.1H NMR (ppm, DMSO-d.sub.6): 6.30 (br, s, 4H),
7.55-7.79 (m, 2H), 7.95 (s, 1 H), 10.80 (s, 1 H).
EXAMPLE 34
Synthesis of
3,5-Diamino-4-[(3,5-Dichlorophenyl)Hydrazono]Pyrazole
[0196] 64
[0197] This compound was prepared using 120 mg (0.5 mmol) of
2-[(3,5-dichlorophenyl)-hydrazono]malononitrile, which was derived
from 3,5-dichloroaniline (162 mg, 1.0 mmol) and malononitrile (1.5
mmol) as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 5 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, precipitated from an ethyl acetate solution by the
addition of hexanes, and dried to yield 25 mg (18%) of the title
compound as a yellow solid.
EXAMPLE 35
Synthesis of
3,5-Diamino-4-[(2-Isopropylphenyl)Hydrazono]Pyrazole
[0198] 65
[0199] This compound was prepared using 106 mg (0.5 mmol) of
2-[(2-isopropylphenyl)-hydrazono]malononitrile, which was derived
from 2-isopropylaniline (142 .mu.L, 1.0 mmol) and malononitrile
(1.5 mmol) as described in Example 8, and hydrazine hydrate.
Precipitate formed in the reaction tube approximately 5 min after
the addition of hydrazine hydrate. The resulting solid was isolated
by filtration, washed with ethanol, and dried to yield 90 mg (73%)
of the title compound as a greenish yellow solid.
EXAMPLE 36
Synthesis of
3,5-Diamino-4-[(3,4-Dimethoxyphenyl)Hydrazono]Pyrazole
[0200] 66
[0201] This compound was prepared using 115 mg (0.5 mmol) of
2-[(3,4-dimethoxyphenyl)-hydrazono]malononitrile, which was derived
from 4-aminoveratrole (153 mg, 1.0 mmol) and malononitrile (1.5
mmol) as described in Example 8, and hydrazine hydrate. The
hydrazine hydrate was added to the solution at a temperature of
75.degree. C. despite the fact that the starting material had not
fully dissolved. The solution cleared. Very little solid had formed
after heating the reaction at 75.degree. C. for 1 hr, however,
analysis of the reaction solution by TLC indicated that no starting
material remained. The solution was allowed to cool to room
temperature and the solvent was evaporated. The residue was
dissolved in ethyl acetate and then precipitated by the addition of
hexanes. The resulting solid was isolated by filtration and dried
to yield 46 mg (35%) of the title compound as a mustard coloured
solid.
EXAMPLE 37
Synthesis of
3,5-Diamino-4-[(3-Trifluoromethylphenyl)Hydrazono]Pyrazole
[0202] 67
[0203] This compound was prepared using 119 mg (0.5 mmol) of
2-[(3-trifluoromethylphenyl)-hydrazono]malononitrile, which was
derived from 3-(trifluoromethyl)aniline (125 .mu.L, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Precipitate formed in the reaction tube approximately 10
min after the addition of hydrazine hydrate. The resulting solid
was isolated by filtration, washed with ethanol, and dried to yield
43 mg (31%) of the title compound as a yellow solid.
EXAMPLE 38
Synthesis of 3-[N'-(3,5-Diaminopyrazol-4-Ylidene)Hydrazino]benzoic
Acid Ethyl Ester
[0204] 68
[0205] The title compound was prepared using 121 mg (0.5 mmol) of
3-(N'-dicyanomethylene-hydrazino)benzoic acid ethyl ester, which
was derived from 3-aminobenzoate (149 .mu.L, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Precipitate formed in the reaction tube approximately 10
min after the addition of hydrazine hydrate. The resulting solid
was isolated by filtration, washed with ethanol, and dried to yield
58 mg (42%) of the title compound as a light brown solid.
EXAMPLE 39
Synthesis of
3,5-Diamino-4-[(3-Methoxy-5-Trifluoromethylphenyl)Hydrazono]P-
yrazole
[0206] 69
[0207] This compound was prepared using 134 mg (0.5 mmol) of
2-[(3-methoxy-5-trifluoromethylphenyl)hydrazono]malononitrile,
which was derived from 3-methoxy-5-trifluoromethylaniline (191 mg,
1.0 mmol) and malononitrile (1.5 mmol) as described in Example 8,
and hydrazine hydrate. Very little solid had formed after heating
the reaction at 75.degree. C. for 1 hr, however, analysis of the
reaction solution by TLC indicated that no starting material
remained. The solution was allowed to cool to room temperature and
the solvent was evaporated. The residue was dissolved in ethyl
acetate and then precipitated by the addition of hexanes. The
resulting solid was isolated by filtration and dried to yield 10 mg
(7%) of the title compound as a yellow solid.
EXAMPLE 40
Synthesis of 3,5-Diamino-4-[(2-Chlorophenyl)Hydrazono]Pyrazole
[0208] 70
[0209] This compound was prepared using 102 mg (0.5 mmol) of
2-[(2-chlorophenyl)-hydrazono]malononitrile, which was derived from
2-chloroaniline (105 .mu.L, 1.0 mmol) and malononitrile (1.5 mmol)
as described in Example 8, and hydrazine hydrate. Precipitate
formed in the reaction tube approximately 10 min after the addition
of hydrazine hydrate. The resulting solid was isolated by
filtration, washed with ethanol, and dried to yield 34 mg (29%) of
the title compound as a yellow solid.
EXAMPLE 41
Synthesis of 3,5-Diamino-4-[(3-Iodophenyl)Hydrazono]Pyrazole
[0210] 71
[0211] This compound was prepared using 148 mg (0.5 mmol) of
2-[(3-iodophenyl)hydrazono]-malononitrile, which was derived from
3-iodoaniline (219 mg, 1.0 mmol) and malononitrile (1.5 mmol) as
described in Example 8, and hydrazine hydrate. The hydrazine
hydrate was added to the solution at a temperature of 75.degree. C.
despite the fact that the starting material had not fully
dissolved. The solution cleared briefly and then a precipitate
formed. The resulting solid was isolated by filtration, washed with
ethanol, and dried to yield 122 mg (74%) of the title compound as a
mustard coloured solid.
EXAMPLE 42
Synthesis of
3,5-Diamino-4-[(9-Ethyl-9H-Carbazol-3-Yl)Hydrazono]Pyrazole
[0212] 72
[0213] This compound was prepared using 143 mg (0.5 mmol) of
2-[(9-ethyl-9H-carbazol-3-yl)-hydrazono]malononitrile, which was
derived from 3-amino-9-ethylcarbazole (210 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Solids had not formed after heating the reaction at
75.degree. C. for 1 hr, however, analysis of the reaction solution
by TLC indicated that no starting material remained. The solution
was allowed to cool to room temperature and the solvent was
evaporated. The residue was dissolved in ethyl acetate and then
precipitated by the addition of hexanes. The resulting solid was
isolated by filtration and dried to yield 46 mg (29%) of the title
compound as a black solid.
EXAMPLE 43
Synthesis of
3,5-Diamino-4-[(2-Benzenesulfonylphenyl)Hydrazono]Pyrazole
[0214] 73
[0215] This compound was prepared using 94 mg (0.5 mmol) of
2-[(2-benzenesulfonylphenyl)-hydrazono]malononitrile, which was
derived from 2-(phenylsulfonyl)aniline (233 mg, 1.0 mmol) and
malononitrile (1.5 mmol) as described in Example 8, and hydrazine
hydrate. Precipitate formed in the reaction tube approximately 20
min after the addition of hydrazine hydrate. The resulting solid
was isolated by filtration, washed with ethanol, and dried to yield
68 mg (20%) of the title compound as an orange coloured solid.
EXAMPLE 44
Synthesis of 3,5-Diamino-1-Phenyl-4-PhenylazoPyrazole
[0216] 74
[0217] This compound was prepared using 200 mg (1.2 mmol) of
2-(phenylhydrazono)-malononitrile, which was derived from aniline
(10 mL, 107 mmol) and malononitrile (161 mmol) as described in
Example 8, and phenylhydrazine (767 mg, 7.1 mmol). Solids had not
formed after heating the reaction at 75.degree. C. for 3 hrs,
however, analysis of the reaction solution by TLC indicated that no
starting material remained. The solution was allowed to cool to
room temperature and the solvent was evaporated. The residue was
dissolved in ethyl acetate and then precipitated by the addition of
hexanes. The resulting solid was isolated by filtration and dried
to yield 77 mg (23%) of the title compound as an orange coloured
solid.
EXAMPLE 45
Synthesis of
(3,5-Diamino-4-Phenylazo-Pyrazol-1-Yl)Phenylmethanone
[0218] 75
[0219] This compound was prepared using 85 mg (0.5 mmol) of
2-(phenylhydrazono)-malononitrile, which was derived from aniline
(10 mL, 107 mmol) and malononitrile (161 mmol) as described in
Example 8, and benzoic hydrazide (68 mg, 0.5 mmol). Solids had not
formed after heating the reaction at 75.degree. C. for 3 hrs,
however, analysis of the reaction solution by TLC indicated that no
starting material remained. The solution was allowed to cool to
room temperature and the solvent was evaporated. The residue was
dissolved in methanol and then precipitated by the addition of
water. The resulting solid was isolated by filtration and dried to
yield 20 mg (13%) of the title compound as an orange coloured
solid.
EXAMPLE 46
Synthesis of 3,5-Diamino-1-(4-Bromophenyl)-4-PhenylazoPyrazole
[0220] 76
[0221] This compound was prepared using 85 mg (0.5 mmol) of
2-(phenylhydrazono) malononitrile, which was derived from aniline
(10 mL, 107 mmol) and malononitrile (161 mmol) as described in
Example 8, and 4-bromophenylhydrazine hydrochloride (112 mg, 0.5
mmol) with the addition of 0.5 mL of 5% sodium hydroxide solution.
Solids had not formed after heating the reaction at 75.degree. C.
for 3 hrs, however, analysis of the reaction solution by TLC
indicated that no starting material remained. The solution was
allowed to cool to room temperature and the solvent was evaporated.
The residue was dissolved in methanol and then precipitated by the
addition of water. The resulting solid was isolated by filtration
and dried to yield 49 mg (27%) of the title compound as a brown
solid.
EXAMPLE 47
Synthesis of 4-(3,5-Diamino-4-Phenylazopyrazol-1-Yl)Benzoic
Acid
[0222] 77
[0223] This compound was prepared using 85 mg (0.5 mmol) of
2-(phenylhydrazono)-malononitrile, which was derived from aniline
(10 mL, 107 mmol) and malononitrile (161 mmol) as described in
Example 8, and 4-hydrazinobenzoic acid (76 mg, 0.5 mmol). After
reacting for 4 hrs, the reaction remained as a slurry; however,
analysis of the reaction solution by TLC indicated that no starting
material remained. The resulting solid was isolated by filtration,
washed with ethanol, and dried to yield 22 mg (14%) of the title
compound as a brown solid.
EXAMPLE 48
Synthesis of 3,5-Diamino-1-(4-Fluorophenyl)-4-PhenylazoPyrazole
[0224] 78
[0225] This compound was prepared using 85 mg (0.5 mmol) of
2-(phenylhydrazono)-malononitrile, which was derived from aniline
(10 mL, 107 mmol) and malononitrile (161 mmol) as described in
Example 8, and 4-fluorophenylhydrazine hydrochloride (81 mg, 0.5
mmol) with the addition of 0.5 mL of 5% sodium hydroxide solution.
After reacting for 4 hrs, very little solid had formed; however,
analysis of the reaction solution by TLC indicated that no starting
material remained. The resulting solid was removed by filtration
and the solvent was evaporated from the filtrate to yield 29 mg
(20%) of the title compound as a brown solid.
EXAMPLE 49
Synthesis of 3,5-Diamino-4-[(Pyridin-3-Yl)Hydrazono]Pyrazole
[0226] 79
[0227] This compound was prepared using
2-[(pyridin-3-yl)hydrazono]malonon- itrile (342 mg, 2 mmole), which
was derived from 3-aminopyridine (940 mg, 10 mmole)) and
malononitrile (858 mg, 13 mmol) as described in Example 8, and
hydrazine hydrate (110 mg, 2.2 mmole) in ethanol. Solids had not
formed after heating the reaction at 80.degree. C. for 40 minutes,
however, analysis of the reaction solution by TLC indicated that no
starting material remained. The solution was allowed to cool to
room temperature and the solvent was evaporated. The product was
obtained after upon re-crystallization from ethanol as a yellow
solid (150 mg). .sup.1H NMR (ppm, DMSO-d.sub.6): 6.18 (br., s, 4H),
7.20 (dd, 1H), 8.00 (dd, 1H), 8.38 (d, 1H), 8.85 (s, 1H), 10.77
(br., s, 1H).
EXAMPLE 50
Synthesis of
5-Amino-4-l(3-Fluorophenyl)Hydrazonol-2,4-Dihydropyrazole-3-O-
ne
[0228] 80
[0229] This compound was prepared using
2-[(3-fluorophenyl)hydrazono]malon- onitrile (145 mg, 0.5 mmole),
which was derived from 3-fluoroaniline (111 mg, 1 mmole)) and
cyclohexyl cyanoacetate (217 mg, 1.3 mmole) as described in Example
8, and hydrazine hydrate (25 mg, 0.5 mmole) in ethanol. Solids had
not formed after heating the reaction at 80.degree. C. for 40
minutes, however, analysis of the reaction solution by TLC
indicated that no starting material remained. The solution was
allowed to cool to room temperature and concentrated. The product
was obtained after filtration as a yellow solid (73 mg). .sup.1H
NMR (ppm, DMSO-d.sub.6): 5.95 (br., s, 2H), 6.90 (m, 1H), 7.20-7.65
(m, 3H), 10.56 (br., s, 1H), 12.77 (br., s, 1H).
EXAMPLE 51
Synthesis of
3,5-Diamino-4-[(6-Methoxybenzothiazol-2-Yl)Hydrazono]Pyrazole
[0230] 81
[0231] This compound was prepared using
2-[(6-methoxybenzothiazol-2-yl)hyd- razono]-malononitrile (200 mg),
which was derived from 2-amino-6-methoxybensothiazole (1.17 g) and
malononitrile (0.82 g) as described in Example 8, and hydrazine
hydrate (0.2 mL) in ethanol. Solids had not formed after heating
the reaction at 40.degree. C. for 2 hrs. The solution was allowed
to cool to room temperature and concentrated. The product was
obtained after column chromatography purification (80 mg, 35%).
EXAMPLE 52
Synthesis of
3,5-Diamino-4-[(Benzothiazol-2-Yl)Hydrazono]Pyrazole
[0232] 82
[0233] This compound was prepared using
2-[(6-benzothiazol-2-yl)hydrazono]- malononitrile (80 mg), which
was derived from 2-aminobensothiazole (925 mg) and malononitrile
(0.65 g) as described in Example 8, and hydrazine hydrate (0.1 mL)
in ethanol. Solids had not formed after heating the reaction at
60.degree. C. for 3 hrs. The solution was allowed to cool to room
temperature and concentrated. The product was obtained after thin
layer chromatography purification (47 mg, 50%).
EXAMPLE 53
Synthesis of 3,5-Diamino-4-[(Pyrazol-3-Yl)Hydrazono]Pyrazole
[0234] 83
[0235] This compound was prepared using 3-aminopyrazole (0.5 g),
malononitrile (1.8 g), and hydrazine hydrate (0.3 g) as described
in Example 1. The product was obtained after column chromatography
purification (157 mg, 14%).
EXAMPLE 54
Synthesis of 3,5-Diamino-4-[(Pyridin-4-Yl)Hydrazono]Pyrazole
[0236] 84
[0237] 4-Aminopyridine (0.36 g) was dissolved in a mixture of 2 ml
of H.sub.3PO.sub.4 (85%) and 1 ml of HNO.sub.3 (68%). The solution
was cooled at -5.degree. C. and then NaNO.sub.2 (0.28 g) solution
was added. After being stirred at 0.degree. C. for 1 hr., the
mixture was added dropwise into a solution of malononitrile (0.5
g), acetic acid (2.4 g), KOAc (6.3 g) and Na.sub.2CO.sub.3 (5.6 g).
The resulting mixture was kept stirring at 0.degree. C. for 1 hr.,
and 100 mL of water was added. The solid obtained after being
filtered and dried was redissolved in 5 mL of EtOH and hydrazine
hydrate (0.5 g) was added at 40.degree. C. After one hour of
reaction, the solid precipitated upon cooling to 0.degree. C. was
collected by filtration and the pure product was obtained after
re-crystallization from EtOH (278, mg, 36%).
EXAMPLE 55
Synthesis of
3,5-Diamino-4-[(2,3,4,5,6-Pentafluorophenyl)Hydrazono]Pyrazol-
e
[0238] 85
[0239] Pentafluoroaniline (1.0 g) dissolved in 12 mL of
CH.sub.3COOH was added into a solution of NaNO.sub.2 (0.41 g) in
concentrated H.sub.2SO.sub.4 at 5.degree. C. The reaction mixture
was kept stirring at 5.degree. C. for 1 hr and then slowly added
into a solution of malononitrile (1.0 g) mixed with 37 g of NaOAc
in 50 mL of H.sub.20 at 5-10.degree. C. The reaction mixture was
extracted with EtOAc (3.times.150 mL) ah hour later. The combined
organic phase was washed with brine, dried with anhydrous
MgSO.sub.4 and then evaporated. The residue was dissolved in 5 ml
of anhydrous EtOH and 0.2 g of N.sub.2H.sub.4 was added to it at
40.degree. C. After being stirred at 70.degree. C. for 2 hrs., the
solvents were removed and the residue was purified by column
yielding 87 mg of the product (5.4%).
EXAMPLE 56
Synthesis of 3,5-Diamino-4-[(31,2,4-Triazolo)Hydrazono]Pyrazole
[0240] 86
[0241] This compound was prepared using the same method as
described in Example 1. 3-Amino-1,2,4-triazole (0.88 g),
malononitrile (1.0 g), and hydrazine hydrate (0.5 ml) yielded 34 mg
of the product (6%).
EXAMPLE 57
Synthesis of
3,5-Diamino-4-[(3,5-Difluorophenyl)Hydrazono]Pyrazole
[0242] 87
[0243] This compound was prepared using the same method as
described in Example 1. 3,5-Difluoroaniline (0.31 g), malononitrile
(0.4 g) and hydrazine hydtrate (0.2 g) yielded 0.201 g of the
product (35%).
EXAMPLE 58
Synthesis of
3,5-Diamino-4-[(2,3,4-Trifluorophenyl)Hydrazono]Pyrazole
[0244] 88
[0245] This compound was prepared using the method as described in
Example 1. 2,3,4-Trifluoroaniline (0.36 g), malononitrile (0.4 g)
and hydrazine hydrate (0.2 g) yielded 0.337 g of the product
(54%).
EXAMPLE 59
Synthesis of 3,5-Diamino-1-Methyl-4-PhenylazoPyrazole
[0246] 89
[0247] This compound was prepared using the method as described in
Example 1 using 2-phenylhydrazonomalononitrile (425 mg) and
methylhydrazine sulfate (720 mg). The product was purified by
column chromatography and afforded a yellow solid.
* * * * *
References