U.S. patent application number 11/805763 was filed with the patent office on 2007-12-20 for non-peptide inhibition of t-lymphocyte activation and therapies related thereto.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Michael D. Cahalan, K. George Chandy, Stephan Grismer, Mark J. Miller, Heiko J. Rauer, Heike Wulff.
Application Number | 20070293554 11/805763 |
Document ID | / |
Family ID | 23903817 |
Filed Date | 2007-12-20 |
United States Patent
Application |
20070293554 |
Kind Code |
A1 |
Chandy; K. George ; et
al. |
December 20, 2007 |
Non-peptide inhibition of T-lymphocyte activation and therapies
related thereto
Abstract
Compounds, preparations and methods for immunosuppressive
treatment of autoimmune disorders, graft rejection and/or
graft/host disease. Therapeutically effective amounts of certain
substituted triarylmethane compounds, such as
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, are administered to
mammalian patients to selectively inhibit the calcium-activated
K.sup.+ channel (IKCa1) in lymphocytes, monocytes, macrophages,
platelets or endothelial cells without concomitant inhibition of
P450-dependent enzyme systems, resulting in reduction of antigen-,
cytokine-, or mitogen-induced calcium entry through store operated
calcium channels in these cells, suppression of cytokine production
by these cells, and inhibition of activation of these cells. Such
inhibition of the Ca.sup.++ activated K.sup.+ channel (IKCa1)
prevents the pre-Ca.sup.++ stage of cell activation and thus causes
immunosuppression and an anti-inflammatory response.
Inventors: |
Chandy; K. George; (Laguna
Beach, CA) ; Wulff; Heike; (Irvine, CA) ;
Cahalan; Michael D.; (Laguna Beach, CA) ; Grismer;
Stephan; (Blaustein, DE) ; Rauer; Heiko J.;
(Irvine, CA) ; Miller; Mark J.; (Brea,
CA) |
Correspondence
Address: |
STOUT, UXA, BUYAN & MULLINS, LLP
Suite #310
4 Venture
Irvine
CA
92618
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
23903817 |
Appl. No.: |
11/805763 |
Filed: |
May 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10402532 |
Mar 28, 2003 |
7235577 |
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11805763 |
May 25, 2007 |
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09479391 |
Jan 6, 2000 |
6803375 |
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10402532 |
Mar 28, 2003 |
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Current U.S.
Class: |
514/381 ;
514/406; 514/408; 514/730 |
Current CPC
Class: |
A61K 31/065 20130101;
A61K 31/415 20130101; A61K 31/40 20130101; A61K 31/425 20130101;
A61P 37/02 20180101; A61K 31/41 20130101; A61P 37/06 20180101; A61K
31/16 20130101; A61K 31/277 20130101 |
Class at
Publication: |
514/381 ;
514/406; 514/408; 514/730 |
International
Class: |
A61K 31/40 20060101
A61K031/40; A61K 31/065 20060101 A61K031/065; A61K 31/41 20060101
A61K031/41; A61K 31/415 20060101 A61K031/415; A61P 37/02 20060101
A61P037/02 |
Claims
1-18. (canceled)
19. A method for suppressing antigen- or cytokine- or
mitogen-stimulated calcium entry via store-operated calcium
channels in lymphocytes, monocytes, macrophages, platelets and
endothelial cells and/or cytokine production by these cells and/or
activation of these cells of a mammalian patient, without
concomitant cytochrome P450 inhibition, said method comprising the
step of administering to the patient a therapeutically effective
amount of a compound having the formula: ##STR8## wherein; X, Y and
Z are the same or different and are independently selected from
CH2, O, S, NR.sub.1, N.dbd.CH, CH.dbd.N and
R.sub.2--C.dbd.C--R.sub.3, where R.sub.2 and R.sub.3 are H or may
combine to form a saturated or unsaturated carbocyclic or
heterocyclic ring, optionally substituted with one or more R
groups; R.sub.1 is selected from H, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, acyl and aroyl, optionally substituted with
hydroxy, amino, substituted amino, cyano, alkoxy, halogen,
trihaloalkyl, nitro, thio, alkylthio, carboxy and alkoxycarbonyl
groups; R is selected from H, halogen, trihaloalkyl, hydroxy,
acyloxy, alkoxy, alkenyloxy, thio, alkylthio, nitro, cyano, ureido,
acyl, carboxy, alkoxycarbonyl, N--(R.sub.4)(R.sub.5) and saturated
or unsaturated, chiral or achiral, cyclic or acyclic, straight or
branched hydrocarbyl group with from 1 to 20 carbon atoms,
optionally substituted with hydroxy, halogen, trihaloalkyl,
alkylthio, alkoxy, carboxy, alkoxycarbonyl, oxoalkyl, cyano and
N--(R.sub.4)(R.sub.5) group, R.sub.4 and R.sub.5 are selected from
H, alkyl, alkenyl, alkynyl, cycloalkyl and acyl or R.sub.4 and
R.sub.5 may combine to form a ring, wherein a carbon may be
optionally substituted by a heteroatom selected from O, S or
N--R.sub.6, R.sub.6 is H, alkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyalkyl or carboxyalkyl, n is 0-5; m is 1 or 2; with the
proviso that when m is 1, Q is selected from OH, CN, carboxyalkyl,
N--(R.sub.7)(R.sub.8), where R.sub.7 and R.sub.8 are selected from
H, lower alkyl (1-4C), cycloalkyl, aryl, acyl, amido, or R.sub.7
and R.sub.8 may combine to form a saturated or unsaturated
heterocylic ring and optionally substituted with up to 3 additional
heteroatoms selected from N, O, and S; or --NH-heterocycle, where
the heterocycle is represented by thiazole, oxazole, ##STR9##
isoxazole, pyridine, pyrimidine, and purine and where U and V are
selected from H and O; and when m is 2, Q is a spacer of from 2-10
carbons either as a straight or branched, chiral or achiral, cyclic
or acyclic, saturated or unsaturated, hydrocarbon group such as
phenyl.
20. A method according to claim 19 wherein the compound is
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole.
21. A method according to claim 19 wherein the compound is
1-[(2-fluorophenyl)diphenylmethyl]-1H-pyrazole.
22. A method according to claim 19 wherein the compound is
1-[(4-chlorophenyl)diphenylmethyl]-1H-pyrazole.
23. A method according to claim 19 wherein the compound is
1-[(2-fluorophenyl)diphenylmethyl]-1H-pyrazole.
24. A method according to claim 19 wherein the compound is
1-[(2-chlorophenyl)diphenylmethyl]-1H-1,2,3,4-tetrazole.
25. A method according to claim 19 wherein the compound is
2-(2-chlorophenyl)-2,2-diphenylacetonitrile.
26. A method according to claim 19 wherein the compound is
2-(2-fluorophenyl)-2,2-diphenylacetonitrile.
27. A method according to claim 19 wherein the method is carried
out for the purpose of treating or preventing an autoimmune
disorder, transplant rejection or graft-versus-host disease in a
mammalian patient.
28. A method according to claim 19 wherein the method is carried
out for the purpose of causing immunomodulation.
29. A method according to claim 28 wherein the method is carried
out for the purpose of causing immunomodulation as a treatment for
an autoimmune disorder, to prevent transplant rejection or to treat
graft-versus-host disease.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to chemical compositions,
preparations and methods for medical treatment and more
particularly to the use of certain substituted triarylmethane
compounds for immunosuppressive treatment of autoimmune disorders
or inflammatory diseases, or the treatment or prevention of
transplant rejection or graft-versus-host disease in mammalian
patients.
BACKGROUND OF THE INVENTION
[0002] Organ transplantation has become routine in many parts of
the world. Transplants of liver, kidney, heart, lung and pancreas,
are now regularly performed as treatment for end-stage organ
disease. The outcomes of organ transplant procedures have
progressively improved with the development of refinements in
tissue typing, surgical techniques, and more effective
immunosuppressive treatments. However, rejection of transplanted
organs remains a major problem. T-lymphocytes play a central role
in the immune response and they are responsible, in large measure,
for the rejection of many transplanted organs. They are also
responsible for the so-called graft-versus host disease in which
transplanted bone marrow cells recognize and destroy MHC-mismatched
host tissues. Accordingly, drugs such as cyclosporin and FK506 that
suppress T-cell immunity are used to prevent transplant rejection
and graft-versus-host disease. Unfortunately, these T-cell
inhibiting drugs are toxic, with liver and renal toxicities
limiting their use.
[0003] Autoimmune diseases encompass a whole spectrum of clinical
disorders wherein a patient's immune system mistakenly attacks
self, targeting the cells, tissues, and organs of the patient's own
body. The following are some examples of autoimmune diseases,
categorized with respect to the target organ that is principally
affected by each such disease: TABLE-US-00001 Nervous System:
Multiple sclerosis Myasthenia gravis Autoimmune neuropathies such
as Guillain-Barre Autoimmune uveitis Blood: Autoimmune hemolytic
anemia Pernicious anemia Autoimmune thrombocytopenia Vascular:
Temporal arteritis Anti-phospholipid syndrome Vasculitides such as
Wegener's granulomatosis Behcet's disease Skin: Psoriasis
Dermatitis herpetiformis Pemphigus vulgaris Vitiligo
Gastrointestinal Tract: Crohn's Disease Ulcerative colitis Primary
biliary cirrhosis Autoimmune hepatitis Endocrine: Type 1 diabetes
mellitus Addison's Disease Grave's Disease Hashimoto's thyroiditis
Autoimmune oophoritis and orchitis Multiple Organs and/or
Musculoskeletal System: Rheumatoid arthritis Systemic lupus
erythematosus Scleroderma Polymyositis, dermatomyositis
Spondyloarthropathies such as ankylosing spondylitis Sjogren's
syndrome
[0004] Irrespective of the particular organ(s) affected,
T-lymphocytes are believed to contribute to the development of
autoimmune diseases. The currently available therapies for these
diseases are largely unsatisfactory and typically involve the use
of glucocorticoids (e.g. methylprednisolone, prednisone),
non-steroidal anti-inflammatory agents, gold salts, methotrexate,
antimalarials, and other immunosuppressants such as cyclosporin and
FK-506.
[0005] Thus, the search for additional immunosuppressive agents for
preventing transplant rejection and for the treatment of autoimmune
and inflammatory disorders occupies considerable attention in the
pharmaceutical industry. Since cytokines such as interferon-gamma
and tumor necrosis factor-alpha play a critical role in transplant
rejection and in the pathophysiology of autoimmune disorders, much
effort has been invested in the development of agents that suppress
their production, secretion and/or end-organ effect.
[0006] There is an excellent track record of treating nervous and
cardiovascular disorders with ion channel modulators - either
openers or blockers. Ion channel blockers as a general class,
represent the major therapeutic agents for treatment of stroke,
epilepsy and arrhythmias. Since ion channels play a major role in
the T-cell immune response, these channels may represent attractive
targets for pharmaceutical immunomodulation.
[0007] The early stages of T-cell activation may be conceptually
separated into pre-Ca.sup.++ and post-Ca.sup.++ events (Cahalan and
Chandy 1997, Curr. Opin. Biotechnol. 8: 749). Following engagement
of antigen with the T-cell antigen-receptor, activation of tyrosine
kinases and the generation of inositol 1,4,5-triphosphate leads to
the influx of Ca.sup.++ through store-operated calcium channels
(also known as Calcium-Release Activated Calcium or CRAC channels)
and the rise of cytoplasmic Ca.sup.2+ concentration (Cahalan and
Chandy 1997, Curr. Opin. Biotechnol. 8: 749; Kerschbaum and Cahalan
1999, Science 283: 836; Kerschbaum and Cahalan 1998; J. Gen.
Physiol. 111: 521). The rise in Ca.sup.++ activates the phosphatase
calcineurin, which then dephosphorylates a cytoplasmically
localized transcription factor (N-FAT) enabling it to accumulate in
the nucleus and bind to a promoter element of the interleukin-2
gene. Along with parallel events involving the activation of
protein kinase C and ras, gene transcription leads to lymphokine
secretion and to lymphocyte proliferation. Some genes require
long-lasting Ca.sup.++ signals while others require only a
transient rise of Ca.sup.++. Furthermore, Ca.sup.++ immobilization
of the T-cell at the site of antigen presentation helps to cement
the interaction between T-cell and the antigen-presenting cell and
thereby facilitate local signaling between the cells (Negulescu
1996, Immunity 4:421).
[0008] Ion channels underlie the Ca.sup.++ signal of T-lymphocytes.
Ca.sup.++ ions move across the plasma membrane through a channel
termed the store-operated Ca.sup.++ channel or the CRAC channel
which is activated by depletion of internal calcium stores like the
endoplasmic reticulum (Cahalan and Chandy 1997, Curr. Opin.
Biotechnol. 8: 749). Two distinct types of potassium channels
indirectly determine the driving force of calcium entry through the
store-operated Ca.sup.2+ channel (Cahalan and Chandy 1997, Curr.
Opin. Biotechnol. 8: 749). The first is the voltage-gated Kv1.3
channel (Cahalan 1985, J. Physiol. 385: 197; Grissmer 1990, Proc.
Natl. Acad. Sci. USA 87: 9411; Verheugen 1995, J. Gen. Physiol.
105: 765; Aiyar 1996, J. Biol. Chem. 271: 31013; Cahalan and Chandy
1997, Curr. Opin. Biotechnol. 8: 749) and the second is the
intermediate-conductance calcium-activated potassium channel, IKCa1
(Grissmer 1993, J. Gen. Physiol. 102: 601; Fanger 1999 J. Biol.
Chem. 274: 5746; Rauer 1999, J. Biol. Chem. 274: 21885) which is
also known as IK1 (VanDorpe 1998, J. Biol. Chem. 273: 21542), hSK4
(Joiner 1997, Proc. Natl. Acad. Sc. USA 94: 11013; Khanna 1999, J.
Biol. Chem. 274: 14838) and hKCa4 (Lodgson 1997, J. Biol. Chem.
272: 32723; Ghanshani 1998, Genomics 51: 160). When these potassium
channels open, the resulting efflux of K.sup.+ hyperpolarizes the
membrane, which in turn accentuates the entry of Ca.sup.++, which
is absolutely required for downstream activation events (Cahalan
and Chandy 1997, Curr. Opin. Biotechnol. 8: 749). Blockers of the
Kv1.3 and IKCa1 channels suppress human T-cell activation, when
applied independently, and produce greater suppression when applied
together (DeCoursey 1984, Nature 307: 465; Chandy J. Exp. Med. 160:
369; Koo 1997, J. Immunol. 158: 5120; Nguyen 1995, Mol. Pharmacol.
50: 1672; Hanson 1999, Br. J. Pharmacol. 126: 1707; Kalman 1998, J.
Biol. Chem. 278: 32697; Khanna 1999, J. Biol. Chem. 274: 14838;
Jensen 1999; Proc. Natl. Acad. Sc. USA 96: 10917). One mechanism
for the immunosuppression by K.sup.+ channel blockers is via
membrane depolarization, which reduces Ca.sup.++ entry through CRAC
channels in the T-cell membrane, which in turn leads to suppression
of calcium-dependent signaling events during human T-cell
activation (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8:
749; Koo 1999, Cell. Immunol. 197: 99).
[0009] Clotrimazole, a non-selective inhibitor of IKCa1, suppresses
mitogen-stimulated T-cell activation, especially of pre-activated
cells (Khanna 1999, J. Biol. Chem. 274: 14838; Jensen 1999, Proc.
Natl. Acad. Sci. USA 96: 10917). Clotrimazole and related
imidazoles, other than the compounds of this invention, have also
previously been described for use in treating rheumatoid arthritis,
an autoimmune disorder (Wojtulewski 1980, Ann. Rheum. Dis. 39: 469;
Wyburn-Mason 1976, U.S. Pat. No. 4,073,922; Wyburn-Mason 1987, U.S.
Pat. No. 183,941; Wyburn-Mason 1979, U.S. Pat. No. 4,218,449).
However, clotrimazole shows considerable toxicity with increasing
doses, toxicity being primarily associated with its potent
(nanomolar) inhibition of cytochrome P450 enzymes (Wojtulewski
1980, Ann. Rheum. Dis. 39: 469; Burgess 1972 Antimicrob. Agents
Chemother. 2: 423; Brugnara 1996, J. Clin Invest. 97: 1227). Thus,
there clearly is a need for newer analogs that block IKCa1 without
concomitant inhibition of cytochrome P450-dependent enzymes.
[0010] Other patents have described the use of clotrimazole,
related azole antimycotics (e.g., miconazole and econazole) and
related aromatic halides for the treatment of cancer (Halperin
1994, WO 96/08240; Halperin 1997 U.S. Pat. No. 5,633,274), but only
at micromolar concentrations (Benzaquen 1995, Nat. Med. 1: 534),
substantially greater than the concentrations required to block the
IKCa1 channel (.about.20-100 nM), suggesting that the mechanism of
suppression of proliferation might be unrelated to channel block.
Also at micromolar concentrations, clotrimazole, related azole
antimycotics (e.g., miconazole and econazole) and related aromatic
halides have been described for use in the treatment of
arteriosclerosis as a hyperproliferative disease (Halperin 1994, WO
94/189680 and U.S. Pat. No. 5,358,959), and for the treatment of
diseases characterized by neovascularization (Halperin 1996, U.S.
Pat. No. 5,512,591; Halperin 1997, U.S. Pat. No. 5,643,936 and U.S.
Pat. No. 5,591,763).
[0011] At least some of the triarylmethyl-1H-pyrazole compounds of
the present invention have also previously been described in PCT
International Publication WO 97/34599 entitled USE OF CLOTRIMAZOLE
AND RELATED COMPOUNDS IN THE TREATMENT OF DIARRHEA, as being
useable for the treatment of diarrhea, although they do not
constitute preferred embodiments of the inventions.
[0012] Also, PCT International Publication WO/97/34589 entitled
TRIARYL METHANE COMPOUNDS FOR SICKLE CELL DISEASE describes various
substituted triarylmethane compounds as effective treatments for
sickle cell disease due to their ability to inhibit ion flux
through the calcium activated potassium channel (Gardos channel) of
erythrocytes, which has now been shown to be encoded by the IKCa1
gene (VanDorpe 1998, J. Biol. Chem. 273: 21542). Clotrimazole, the
preferred compound in this invention is in phase II trials for the
treatment of sickle cell disease gene (VanDorpe 1998, J. Biol.
Chem. 273: 21542), but at higher doses causes toxic side effects
most likely due to its inhibition of cytochrome P450 enzymes. The
PCT International Publication WO/97/34589 also describes various
substituted triarylmethane compounds as effective treatments for
diseases characterized by unwanted or abnormal cell proliferation
(the examples cited being melanoma cells and fibroblast
proliferation), but at only micromolar concentrations (Benzaquen
1995, Nat. Med. 1: 534; Halperin 1997 U.S. Pat. No. 5,633,274; PCT
application WO/97/34589; PCT application WO/97/08240) which are
substantially higher than that required for block of the IKCa1
channel (half-block at 20-100 nM), suggesting that the mechanism of
suppression of cell proliferation might be unrelated to channel
block. Furthermore, since the three compounds used to support this
claim, clotrimazole, ketoconazole and miconazole (Benzaquen 1995,
Nat. Med. 1: 534) also inhibit cytochrome P450 enzymes at nanomolar
concentrations (Mason 1987, Steroids 50: 179; Morris 1992, FASEB J.
6: 752), the mechanism of suppression of abnormal proliferation may
be related to inhibition of these enzymes. Another possible
mechanism for suppression of proliferation stated in PCT
application WO/97/34589 is non-specific cytotoxicity. Therefore,
the claims in PCT application WO/97/34589 that suppression of
abnormal proliferation is due solely to alteration of transmembrane
ion fluxes cannot be substantiated.
[0013] WO 97/34589 does not describe or suggest that the
substituted triarylmethane compounds disclosed therein are capable
of selectively blocking the calcium activated potassium channels
encoded by the IKCa1 gene in resting and activated T-lymphocytes,
or that such compounds would, alone or in combination with other
inhibitors of T-cell signaling cascades, suppress antigen-,
cytokine- and/or mitogen-stimulated calcium-entry through
store-operated calcium channels, and/or cytokine production and/or
activation of human T-lymphocytes, without concomitant inhibition
of cytochrome P450 enzymes, leading to immunosuppressive activity
when administered to mammalian patients.
[0014] Given the shortcomings associated with the currently
available modes of therapy for autoimmune disorders, transplant
rejection and graft-versus-host disease, there remains a need for
the development of new immunosuppressive drugs that are capable of
selectively inhibiting the activation of lymphocytes with minimal
side effects, and without affecting the cytochrome P450 enzyme
system.
SUMMARY OF THE INVENTION
[0015] The present invention generally comprises pharmaceutical
preparations containing substituted triarylmethane compounds, as
listed in Appendix A, and methods for immunosuppressive treatment
of autoimmune disorders, graft rejection and/or graft-versus-host
disease by administering therapeutically effective amounts of such
compounds to mammalian patients.
[0016] In accordance with the invention, there is provided a method
for inhibiting antigen-, cytokine-, or mitogen-induced
calcium-entry through store-operated calcium channels, cytokine
production and cell activation in lymphocytes, monocytes,
macrophages and for treating autoimmune disorders, graft rejection
and/or graft-versus-host disease by administering to a mammalian
patient a therapeutically effective amount of at least one compound
having the general structural formula: ##STR1## [0017] Wherein,
[0018] X, Y and Z are same or different and are independently
selected from CH.sub.2, O, S, NR.sub.1, N.dbd.CH, CH.dbd.N and
R.sub.2--C.dbd.C--R.sub.3, where R.sub.2 and R.sub.3 are H or may
combine to form a saturated or unsaturated carbocyclic or
heterocyclic ring, optionally substituted with one or more R
groups; [0019] R.sub.1 is selected from H, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, acyl and aroyl, optionally substituted with
hydroxy, amino, substituted amino, cyano, alkoxy, halogen,
trihaloalkyl, nitro, thio, alkylthio, carboxy and alkoxycarbonyl
groups; [0020] R is selected from H, halogen, trihaloalkyl,
hydroxy, acyloxy, alkoxy, alkenyloxy, thio, alkylthio, nitro,
cyano, ureido, acyl, carboxy, alkoxycarbonyl, N--(R.sub.4)(R.sub.5)
and saturated or unsaturated, chiral or achiral, cyclic or acyclic,
straight or branched hydrocarbyl group with from 1 to 20 carbon
atoms, optionally substituted with hydroxy, halogen, trihaloalkyl,
alkylthio, alkoxy, carboxy, alkoxycarbonyl, oxoalkyl, cyano and
N--(R.sub.4)(R.sub.5) group, [0021] R.sub.4 and R.sub.5 are
selected from H, alkyl, alkenyl, alkynyl, cycloalkyl and acyl or
R.sub.4 and R.sub.5 may combine to form a ring, wherein a carbon
may be optionally substituted by a heteroatom selected from O, S or
N--R.sub.6, [0022] R.sub.6 is H, alkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyalkyl or carboxyalkyl, [0023] n is 0-5; m is 1
or 2; with the proviso that [0024] when m is 1, Q is selected from
OH, CN, carboxyalkyl, N--(R.sub.7)(R.sub.8), where R.sub.7 and
R.sub.8 are selected from H, lower alkyl (1-4C), cycloalkyl, aryl,
acyl, amido, or R.sub.7 and R.sub.8 may combine to form a saturated
or unsaturated heterocylic ring and optionally substituted with up
to 3 additional heteroatoms selected from N, O, and S; or [0025]
--NH-heterocycle, where the heterocycle is represented by thiazole,
oxazole, isoxazole, pyridine, pyrimidine, and purine and [0026]
where U and V are selected from H and O; and ##STR2## [0027] when m
is 2, Q is a spacer of from 2-10 carbons either as a straight or
branched hydrocarbon chain, or containing a hydrocarbon ring such
as phenyl. [0028] In the most preferred embodiment of this
invention, [0029] X, Y, and Z are R.sub.2--C.dbd.C--R.sub.3, where
R.sub.2 and R.sub.3 are H; [0030] R is selected from H and halogen,
preferably, F and Cl; [0031] m is 1; and [0032] Q is
--N--(R.sub.7)(R.sub.8), where R.sub.7 and R.sub.8 are selected
from H, acyl, amido, and R.sub.7 and R.sub.8 combine to form a
saturated or unsaturated heterocyclic ring, optionally substituted
with up to three heteroatoms selected from N, O, or S, for example,
pyrrolidine, piperidine, pyrazole, oxazole, isoxazole, tetrazole,
azepine, etc., which may be optionally substituted with a lower
alkyl or amino group.
[0033] Further in accordance with the invention, preferred
compounds of this invention having the general Formula I above, are
a group of triarylmethyl-1H-pyrazole compounds that have structural
Formula I-A below: ##STR3## [0034] Wherein: [0035] X, Y, and Z are
R.sub.2--C.dbd.C--R.sub.3, where R.sub.2 and R.sub.3 are H; [0036]
R is selected from H and halogen, preferably, F and Cl; Compounds
of Formula I-A have been determined to selectively inhibit the
intermediate-conductance calcium-activated potassium channel,
IKCa1, at low nanomolar concentrations, and exhibit 200-1500 fold
selectivity for this channel over other ion channels, and over
cytochrome P450-dependent enzymes.
[0037] Still further in accordance with the invention,
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (designated as T34
in Appendix A) and possibly other compounds of Formulas I and I-A
above, when administered to mammalian patients, inhibit (i.e.,
block or partially block) the intermediate conductance Ca.sup.++
activated K channel (IKCa1) expressed in resting and activated
lymphocytes, monocytes, macrophages, platelets and endothelial
cells. By inhibiting the IKCa1 channel in lymphocytes, monocytes,
macrophages, platelets and endothelial cells the present invention
prevents or deters Ca.sup.++ entry and, thus, disrupts the
signaling cascade that leads to cytokine production and cell
activation. It is by this mechanism, and possibly others, that the
compounds of Formulas I and I-A are useable to cause suppression of
immune and anti-inflammatory responses. Unlike clotrimazole, the
compounds of Formula I and I-A lack the imidazole moiety that is
believed to be responsible for inhibition of cytochrome
P450-dependent enzymes and, thus, the compounds of Formula II above
will inhibit or block the Ca.sup.++ activated K.sup.+ channel
(IKCa1) without also causing inhibition of cytochrome
P450-dependent enzymes. In this manner, the compounds of Formula I
and I-A can be administered in amounts that are effective to
inhibit (i.e., block or partially block) the Ca.sup.++ activated
K.sup.+ channel (IKCa1) without causing at least some of the toxic
side-effects associated with cytochrome P450 inhibition by
clotrimazole.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1a shows the block of an IKCa1 current (hIKCa1
expressed in COS-7 cells) by 100 nM T34.
[0039] FIG. 1b shows the Hill plot (nH=1.8) of the reduction of
slope conductance at -80 mV of IKCa1 currents in COS-7 cells
(n=15).
[0040] FIG. 1c is a graph showing the effect of 100 nM of T34 on
native IK.sub.Ca currents in a human T-lymphocyte after 4 days of
activation with 1 .mu.g/ml PHA. Currents were elicited by 200 ms
voltage ramps from -160 to 40 mV every 30 s with 1 .mu.M of free
Ca.sup.++ in the pipette solution.
[0041] FIG. 2 shows the hydrolysis of compound T34 and clotrimazole
at pH 7.4 and pH 5.0.
[0042] FIG. 3 shows the hydrolysis of compound T34 to T3 at
pH1.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0043] The following detailed description, and the examples
contained therein, are provided for the purpose of describing and
illustrating certain embodiments of the invention only and are not
intended to limit the scope of the invention in any way.
[0044] The present invention provides for the use of
therapeutically effective substituted triaryl methane compounds
that are more selective in inhibiting the said channel in nanomolar
concentrations and exhibiting no inhibitory effect on the
cytochrome P450-dependent enzyme systems at 50 times greater
concentrations. Because the imidazole moiety is responsible for
inhibition of cytochrome P-450-dependent enzymes, applicants have
synthesized compounds of Formula I and I-A above that do not
include the imidazole moiety, including instead other heterocyclic
groups. Applicants have also synthesized a range of
triaryl-methanols, amines, ureas, acetonitriles and related
compounds, as listed in Appendix A, by synthetic methodologies
outlined in Scheme 1 below. The triarylmethyl-1-H-pyrazoles of this
invention potently block IKCa1. Applicants have further discovered
that one particular compound of this invention having Structural
formula I-B below, exhibits .about.3-fold greater affinity for the
channel (K.sub.d=20 nM) than clotrimazole (K.sub.d=70 nM), and does
not inhibit cytochrome P450 3A4, the major xenobiotic metabolizing
enzyme in the human liver, even at a concentration of 10 .mu.M.
Four other compounds in this series (T39, T40, T46 and T84) are
more potent inhibitors of IKCa1 channels than clotrimazole (TABLE
8).
[0045] Furthermore, applicants have discovered that the ratio of
cytochrome P-450-dependent enzyme systems inhibition (EC.sub.50) to
IKCa1 inhibition (K.sub.d) needs to be >50-100 to achieve the
therapeutic effect for prevention of the diseases modulated by
IKCa1 channel without the aforementioned side effects evident in
clotrimazole and related imidazoles.
[0046] As a further test of selectivity, applicants have evaluated
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, one of the
compounds of this invention (designated as T-34 in Appendix A), on
other cloned and native ion channels, (Kv1.1-1.5, Kv3.1, Kv4.2,
Jurkat-SK.sub.Ca, BKCa, hSKM1-Na, CRAC and lymphocyte chloride
channels). All of these channels were blocked with K.sub.d
values.about.5 .mu.M. Thus, T34 was found to be a remarkably potent
and selective IKCa1 inhibitor. Because of its structural similarity
to clotrimazole and based on experimental data described in the
examples below, we expect that T34 (logP=4.0 versus 3.5 for
clotrimazole) will have a similar or slightly better
bioavailability than clotrimzole and, contrary to clotrimazole, no
side effects mediated by inhibition of cytochrome P450-dependant
enzymes.
[0047] The invention is particularly concerned with compounds for
effective treatments for auto-immune disorders, transplant
rejection, inflammatory disorders and graft-versus-host disease.
Accordingly, the present invention provides compositions and
methods suitable for treatment of said diseases, and further
provides therapy devoid of side effects associated with currently
available drugs on the market.
[0048] The present invention includes methods that are specifically
intended to suppress the immune system and reduce inflammation in a
mammalian patient who is in need of such treatment. Specifically,
the method of this invention is useful in treating and preventing
the resistance to transplantation rejection of organs or tissues
(such as heart, kidney, liver, lung, bone marrow, cornea, pancreas,
limb, nerves, medulla ossium, duodenum, small bowel, skin,
pancreatic islet etc. including xeno transplantation),
graft-versus-host diseases, autoimmune diseases such as rheumatoid
arthritis, systemic lupus erythematosis, Hashimoto's thyroiditis,
multiple sclerosis, myasthenia gravis, type I diabetes mellitus,
nephrotic syndrome, steroid-dependent and steroid-resistant
nephrosis, palmar-plantar pustolosis, allergic encephalomyelitis,
glomerulonephritis, Behcet's syndrome, ankylosing spondylitis,
polymyositis, fibromyositis, etc.
[0049] The compounds of this invention are also useful for treating
inflammatory, proliferative and hyperproliferative skin diseases
and cutaneous manifestations of immunologically mediated illnesses
such as psoriasis, psoriatic arthritis, atopic dermatitis, contact
dermatitis and other eczematous dermatitises, seborrhoic
dermatitis, Lichen planus, Pemphigus, bullous Pemphigus,
Epidermolysis bullosa, angiodemas, vasculilides, erythemas,
cutanous eosinophilias, acne, Alopecia areata, and
arteriosclerosis.
[0050] The compounds of the invention are further useful in the
treatment of respiratory diseases, for example sarcoidosis, fibroid
lung, idiopathic interstitial pneumonia, and reversible obstructive
airway diseases, including conditions such as asthma, including
bronchial asthma, allergic asthma, intrinsic asthma, extrinsic
asthma and dust asthma, bronchitis and the like. The compounds may
also be useful for the treating hepatic injury associated with
ischemia.
[0051] The compounds may also be indicated in certain eye diseases
such as keratoconjunctivitis, vernal conjunctivitis, keratitis,
uveitis, corneal leukoma, occular pemphigus, Mooren's ulcer,
Scleritis, Graves' ophtalmopathy, sympathetic ophthalmia and the
like.
[0052] The compounds are also useful treating inflammatory bowl
diseases (e.g. Crohn's disease), neurological diseases (including
Guillain-Barre syndrome, Meniere's disease, radiculopathy),
endocrine diseases (including hyperthyroidism and Basedow's
disease), hematological diseases (including pure red cell aplasia,
aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic
purpura, autoimmune hemolytic anemia, agranulocytosis and
anerythroplasia), bone diseases (including osteoporosis),
respiratory disease (including sarcoidosis, idiopathic interstitial
pneumonia), skin diseases (including dermatomyositis, leukoderma
vulgaris, ichthyosis vulgaris, photoallergic sensitivity and
cutanous T cell lymphoma), genitals (orchiitis, vulvitis),
circulatory diseases (including arteriosclerosis, polyarteritis
nodosa, vasculitis, Buerger's disease, and myocardosis), collagen
disorders (including scleroderma, aortitis syndrome, eosinophilic
fascitis, Wegener's granulomatosis, Sjogren's syndrome, periodontal
diseases), kidney diseases (including nephrotic syndrome,
hemolytic-uremic syndrome, Goodpasture's syndrome) and muscular
dystrophy. The compounds may also be useful for the treatment of
diseases including intestinal inflammations/allergies such as
Coeliac disease, proctitis, ulcerative colitis, eosinophilic
gastroenteritis, mastocytosis, Crohn's disease and ulcerative
colitis and food-related allergic diseases which have symptomatic
manifestations remote from the gastrointestinal tract, for example
migraine, rhinitis and eczema. Further, the invention can be used
for treating preventing or treating inflammation of mucosa or blood
vessels (such as leukotriene-mediated diseases), gastric ulcers,
vascular damage caused by ischemic diseases and thrombosis,
ischemic bowl diseases. Further, the invention will be useful for
treating multidrug resistance of tumor cells, (i.e. enhancing the
activity and/or sensitivity of chemotherapeutic agents).
[0053] The compounds may be useful for the treatment and prevention
of hepatic diseases such as immunogenic diseases (e.g. chronic
autoimmune liver diseases including autoimmune hepatitis, primary
biliary cirrhosis and sclerosing cholangitis), partial liver
resection, acute liver necrosis (e.g. necrosis caused by toxins,
viral hepatitis, shock or anoxia), B-virus hepatitis, nonA/nonB
hepatitis, cirrhosis. A. Compounds Useable in Accordance with this
Invention: ##STR4##
[0054] As stated in the above-set-forth summary of the invention,
the compounds of this invention are represented by Formula I below,
[0055] Wherein, [0056] X, Y and Z are same or different and are
independently selected from CH.sub.2, O, S, NR.sub.1, N.dbd.CH,
CH.dbd.N and R.sub.2--C.dbd.C--R.sub.3, where R.sub.2 and R.sub.3
are H or may combine to form a saturated or unsaturated carbocyclic
or heterocyclic ring, optionally substituted with one or more R
groups; [0057] R.sub.1 is selected from H, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, acyl and aroyl, optionally substituted with
hydroxy, amino, substituted amino, cyano, alkoxy, halogen,
trihaloalkyl, nitro, thio, alkylthio, carboxy and alkoxycarbonyl
groups; [0058] R is selected from H, halogen, trihaloalkyl,
hydroxy, acyloxy, alkoxy, alkenyloxy, thio, alkylthio, nitro,
cyano, ureido, acyl, carboxy, alkoxycarbonyl, N--(R.sub.4)(R.sub.5)
and saturated or unsaturated, chiral or achiral, cyclic or acyclic,
straight or branched hydrocarbyl group with from 1 to 20 carbon
atoms, optionally substituted with hydroxy, halogen, trihaloalkyl,
alkylthio, alkoxy, carboxy, alkoxycarbonyl, oxoalkyl, cyano and
N--(R.sub.4)(R.sub.5) group, [0059] R.sub.4 and R.sub.5 are
selected from H, alkyl, alkenyl, alkynyl, cycloalkyl and acyl or
R.sub.4 and R.sub.5 may combine to form a ring, wherein a carbon
may be optionally substituted by a heteroatom selected from O, S or
N--R.sub.6, [0060] R.sub.6 is H, alkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyalkyl or carboxyalkyl, [0061] n is 0-5; m is 1
or 2; with the proviso that [0062] when m is 1, Q is selected from
OH, CN, carboxyalkyl, N--(R.sub.7)(R.sub.8), where R.sub.7 and
R.sub.8 are selected from H, lower alkyl (1-4C), cycloalkyl, aryl,
acyl, amido, or R.sub.7 and R.sub.8 may combine to form a saturated
or unsaturated heterocylic ring and optionally substituted with up
to 3 additional heteroatoms selected from N, O, and S; or [0063]
--NH-heterocycle, where the heterocycle is represented by thiazole,
oxazole, isoxazole, pyridine, pyrimidine, and purine and [0064]
where U and V are selected from H and O; and ##STR5## [0065] when m
is 2, Q is a spacer of from 2-10 carbons either as a straight or
branched, chiral or achiral, cyclic or acyclic hydrocarbon group,
such as phenyl. [0066] In the most preferred embodiment of this
invention, [0067] X, Y, and Z are R.sub.2--C.dbd.C--R.sub.3, where
R.sub.2 and R.sub.3 are H; [0068] R is selected from H and halogen,
preferably, F and Cl; [0069] m is 1; and [0070] Q is
--N--(R.sub.7)(R.sub.8), where R.sub.7 and R.sub.8 are selected
from H, acyl, amido, and R.sub.7 and R.sub.8 combine to form a
saturated or unsaturated heterocyclic ring, optionally substituted
with up to three heteroatoms selected from N, O, or S, for example,
pyrrolidine, piperidine, pyrazole, oxazole, isoxazole, tetrazole,
azepine, etc., which may be optionally substituted with a lower
alkyl or amino group.
[0071] Some of the preferred compounds covered by Formula I
include, [0072] (2-chlorophenyl)diphenyl methanol (T3) [0073]
(2-thienyl)diphenyl methanol (T9) [0074]
N-[(2-chlorophenyl)diphenylmethyl]-urea (T33) [0075]
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrrole (T44) [0076]
N-[(2-chlorophenyl)diphenylmethyl]-N-(2-pyrimidyl)amine (T68)
[0077] (2-chlorophenyl)diphenylmethyl amine (T41) [0078]
N-(2-chlorophenyl)diphenylmethyl acetamide (T75) [0079]
2-(4-chlorophenyl)-2,2-diphenylacetonitrile (T26) [0080]
2-(2-chlorophenyl)-2,2-diphenylacetonitrile (T39) [0081]
2-[(2-chlorophenyl)(diphenyl)methyl]-1H-isoindole-1,3(2H)-dione
(T71) [0082] 1-[(2-chlorophenyl)diphenylmethyl]-1H-tetrazole
(T84).
[0083] In another preferred embodiment having the general Formula I
X, Y, and Z are each R.sub.2--C.dbd.C--R.sub.3 (where R.sub.2 and
R.sub.3 are H; [0084] R is selected from H and halogen, preferably,
F and Cl); m is 2; and [0085] Q is a spacer of from 2-10 carbons
either as a straight or branched hydrocarbon chain, or containing a
hydrocarbon ring such as phenyl. Some of the preferred compounds
covered by this embodiment include: [0086] N,N-1,2-ditritylamino
ethane (T21) [0087] 1,4-ditritylaminomethyl benzene (T23) [0088]
N,N-1,3-[(2-chlorophenyl)diphenylmethyl]amino propane (T49).
[0089] Further in accordance with the invention, preferred
compounds of this invention having the general Formula I above, are
a group of triarylmethyl-1H-pyrazole compounds that have structural
Formula I-A below: ##STR6## [0090] Wherein: [0091] X, Y, and Z are
R.sub.2--C.dbd.C--R.sub.3, where R.sub.2 and R.sub.3 are H; [0092]
R is selected from H and halogen, preferably, F and Cl;
[0093] Preferred compounds covered by Formula I-A include, [0094]
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) [0095]
1-[(2-fluorophenyl)diphenylmethyl]-1H-pyrazole (T46) [0096]
1-[(4-chlorophenyl)diphenylmethyl]-1H-pyrazole (T13) [0097]
1-[(2-fluorophenyl)diphenylmethyl]-1H-pyrazole (T28) B. Synthesis
of the Compounds:
[0098] The compounds of this invention may be prepared as outlined
in Scheme 1 and Example 1. The individual steps are described below
in the examples. The synthetic procedures described here are
exemplary and may be modified by those skilled in the art .
##STR7## C. Preferred Routes of Administration:
[0099] The compounds described herein, or pharmaceutically
acceptable salts or hydrates thereof, can be delivered to a patient
using a wide variety of routes or modes of administration. Suitable
routes of administration include, but are not limited to,
inhalation, transdermal, oral, rectal, transmucosal, intestinal and
parenteral administration, including intramuscular, subcutaneous
and intravenous injections.
[0100] The compounds described herein, or pharmaceutically
acceptable salts or hydrates thereof, may be administered singly or
in combination with other therapeutic agents, e.g. analgesics,
antibiotics, non-steroidal anti-inflammatory agents, steroids, and
other immunosuppressive drugs like cyclosporin A, rapamycin, FK506
or Kv1.3 selective blockers. At least one of the preferred
compounds, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole
designated as T34 in Appendix A, may be administered per se or in
the form of a pharmaceutical composition wherein the active
compound is in admixture with one or more physiologically
acceptable carriers, excipients or diluents. Pharmaceutical
compositions for use in accordance with the present invention may
be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active compounds
into preparations, which can be used pharmaceutically. Proper
formulation is dependent on the route of administration chosen. For
parenteral administration (bolus injection or continuous infusion),
the agents of the invention may be formulated in water-soluble form
in aqueous solutions, preferably in physiologically compatible
buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer. Additionally, suspensions of the
compounds may be prepared as oily injections with fatty oils,
synthetic fatty acid esters, or liposomes. The compounds may also
be formulated as a depot preparation. For oral administration, the
compounds can be formulated readily by combining the active
compound with pharmaceutically acceptable carriers well known in
the art. Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion for
patients to be treated. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol: cellulose preparations such as, for example maize starch,
wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
methyl cellulose, hydroxypropyl-methylcellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone.
C. EXAMPLES
[0101] The following examples serve to illustrate various aspects
of the invention and are not to be construed as limiting the
invention to those embodiments so exemplified.
Example 1
Synthesis of Triarylmethanols (General Method A)
[0102] 25 mmol of magnesium turnings and a catalytic amount of
iodine to initiate the reaction were stirred in 50 ml of anhydrous
diethyl ether. Then, a solution containing 25 mmol of the
appropriate aryl bromide in anhydrous diethyl ether (50 ml) was
slowly added allowing a gentle reflux. Once the addition was
complete the mixture was heated at reflux until all the magnesium
was consumed. Next, a solution of the required benzophenone (25
mmol) in anhydrous diethyl ether (50 ml) was slowly added. The
resulting mixture was heated at reflux for 5-12 h, then cooled to
0.degree. C. and poured into 100 ml of cold water. To dissolve the
precipitating magnesium hydroxide the mixture was acidified with
concentrated HCl. The organic phase was separated, and the aqueous
phase was extracted with diethyl ether. The combined organic phases
were washed with sodium bicarbonate solution (10%) and then dried
over sodium sulfate. Evaporation of the solvent gave the respective
triarylmethanol either as creamy solid or as an oil, which normally
was recrystallized from petroleum ether (40-60.degree. C.) several
times.
Example 2
Preparation of (2-Chlorophenyl)diphenyl methanol (Compound T3)
[0103] Following the procedure outlined in Example 1, 1.3 g (52
mmol) of magnesium turnings, 10.0 g (52 mmol) of
1-bromo-2-chlorobenzene and 9.4 g (52 mmol) benzophenone gave 9.81
g (64%) of (2-Chlorophenyl) diphenyl methanol (Compound T3), mp:
91.degree. C.
Example 3
[0104] Following the procedure outlined in Example 1, the following
triarylmethanols (Table 1) were prepared. TABLE-US-00002 TABLE 1
Designation on Melting Triarylmethanol Compound Appendix A Yield
Point (4-Chlorophenyl)diphenyl methanol T1 56% 82.degree. C.
(3-Chlorophenyl)diphenyl methanol T2 52% 53.degree. C.
Bis-(4-chlorophenyl)phenyl methanol T4 56% 86.degree. C.
Bis-(3-chlorophenyl)phenyl methanol T5 52% oil (2-Thienyl)diphenyl
methanol T9 64% 129.degree. C. (4-Fluorophenyl)diphenyl methanol
T12 58% 120.5.degree. C. (4-Fluorophenyl)(2-thienyl)phenyl T14 62%
75.degree. C. methanol Bis-(4-methoxyphenyl)phenyl T15 62% sticky
dark methanol red paste Tris-(4-methoxyphenyl) methanol T16 48%
75.degree. C. Di-(2-thienyl)phenyl methanol T35 54% 86.degree. C.
(2-Fluorophenyl)diphenyl methanol T36 69% 116.degree. C.
(2-Chlorophenyl)(2-thienyl)phenyl T43 58% 90.5.degree. C. methanol
Diphenyl(2-trifluoromethylphenyl) T54 57% 111.degree. C. methanol
Diphenyl(4-trifluoromethylphenyl) T55 68% oil methanol
Diphenyl(3-trifluoromethylphenyl) T56 62% 52.degree. C.
methanol
Example 4
Synthesis of Triaryl Chlorides (General Method B)
[0105] To a stirred suspension of 20 mmol of the corresponding
triarylmethanol in 100 ml of petroleum ether (40-60.degree. C.) was
added dropwise an excess of freshly distilled thionyl chloride. The
reaction mixture was stirred at room temperature for 30 min and
then heated under reflux for 1 h. Excess thionyl chloride was
removed by concentrating to dryness in vacuo. The residue was
suspended in 100 ml of petroleum ether and left in the refrigerator
overnight. The resulting crystals were filtered off and thoroughly
washed with petroleum ether. To avoid hydrolysis of these sensitive
triaryl chlorides, they were immediately used for further reactions
after being characterized by melting point and mass
spectrometry.
Example 5
Synthesis of (2-Chlorophenyl)diphenyl chloromethane
[0106] Following the procedure outlined in Example 4, 5.00 g (17.1
mmol) of (2-chlorophenyl)diphenyl methanol, designated as T-3 on
Appendix A, was treated with 2.5 ml thionyl chloride (34 mmol)
according to general method B to give 4.39 g (82%) of
(2-Chlorophenyl)diphenyl chloromethane, mp: 131.degree. C.
Example 6
Synthesis of Triarylmethylamines (General Method C)
[0107] To a solution of the appropriate triaryl chloride (5 mmol)
in anhydrous acetonitrile (100 ml) were added the desired amine or
urea (5 mmol) and triethylamine (5 mmol) as proton acceptor. The
resulting mixture was stirred and heated at reflux for 24 h.
Evaporation of the solvent afforded a creamy residue, which was
dissolved in 200 ml of methylene chloride. The mixture was washed
two times with 50 ml of water, dried over Na.sub.2SO.sub.4, and
concentrated in vacuo. The crude product was recrystallized from
petroleum ether (40-60.degree. C.)/methylene chloride.
Example 7
Preparation of 1-Tritylpyrrolidine (Compound T7)
[0108] 2.00 g (7.2 mmol) of trityl chloride was treated with 0.51 g
(7.2 mmol) pyrrolidine and 0.72 g (7.2 mmol) triethylamine
according to General Method C in Example 6 to give 1.86 g (82%) of
1-tritylpyrrolidine (T7), mp: 126.degree. C.
Example 8
[0109] Following the procedure in Example 6, the following
compounds (Table 2) were prepared. TABLE-US-00003 TABLE 2 Melting
Triarylmethylamines from an amine or urea Number Yield Point
1-Trityl-1H-pyrrole T10 79% 243.degree. C. N-trityl urea T24 58%
238.degree. C. N-[(4-Chlorophenyl)diphenylmethyl] urea T29 62%
228.degree. C. N-[(4-Fluorophenyl)diphenylmethyl] urea T31 66%
222.degree. C. N-[(2-Chlorophenyl)diphenylmethyl] urea T33 68%
243.degree. C. 1[(2-Chlorophenyl)diphenylmethyl]-1H-pyrrole T44 67%
184.degree. C. N-[(2-Fluorophenyl)diphenylmethyl] urea T45 66%
225.degree. C.
Example 9
Synthesis of Triarylmethylamines with a Heterocyclic Amine (General
Method D)
[0110] Especially with substituted pyrazoles and pyrimidines
General Method C tended to give unsatisfactory yields and oily,
dark byproducts, which where extremely difficult to remove even by
column chromatography. Therefore excessive amine was used as a
hydrogen acceptor instead of triethylamine. To a solution of the
required triaryl chloride (5 mmol) in anhydrous acetonitrile (100
ml) was added an excess of the required amine (10-20 mmol). After
stirring under reflux for 8 h the mixture was poured into cold
water (400 ml) and kept at 4.degree. C. for 2 h. The precipitate
formed was collected by vacuum filtration, thoroughly washed with
water to remove any of the remaining amine, and recrystallized from
ethanol.
Example 10
Preparation of 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole
(Compound T34)
[0111] 1.50 g (4.8 mmol) of 2-chlorotrityl chloride obtained under
Example 5 was reacted with 1.00 g (15 mmol) of pyrazole according
to general method D to give 1.26 g (76%) of
1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole, mp: 135.degree.
C.
Example 11
[0112] Following the procedure in Example 9, the following
compounds (Table 3) were prepared. TABLE-US-00004 TABLE 3
Designation on Melting Triarylmethylamines from Heterocyclic Amines
Appendix A Yield Point 1-Trityl-1H-pyrazole T11 82% 202.degree. C.
1-[(4-Chlorophenyl)diphenylmethyl]-1H-pyrazole T13 87% 133.degree.
C. 1-[Tris(4-methoxyphenyl)methyl]-1H-pyrazole T19 82% 158.degree.
C. 1-[(4-Fluorophenyl)diphenylmethyl]-1H-pyrazole T28 84%
145.degree. C. 1-[Diphenyl(2-thienyl)methyl]-1H-imidazole T37 78%
176.degree. C. 1-[Diphenyl(2-thienyl)methyl]-1H-pyrazole T38 83%
157.degree. C. 1-[(2-Fluorophenyl)diphenylmethyl]-1H-pyrazole T46
84% 192.degree. C. N-(1,3-thiazol-2yl)-N-tritylamine T57 79%
213.degree. C. 1-{Diphenyl[2-(trifluoromethyl)phenyl]methyl}-1H-
T58 46% 114.degree. C. pyrazole
1-{Diphenyl[2-(trifluoromethyl)phenyl]methyl}-3- T59 62%
107.degree. C. (trifluoromethyl)-1H-pyrazole
1-{Diphenyl[4-(trifluoromethyl)phenyl]methyl}-1H- T60 65%
135.degree. C. pyrazole
N-Diphenyl[4-(trifluoromethyl)phenyl]methyl-N- T61 58% 166.degree.
C. (1,3-thiazol-2-yl)amine
1-[(2-Chlorophenyl)diphenylmethyl]-3,5-dimethyl-1H- T62 68%
195.degree. C. pyrazole
1-[(2-Chlorophenyl)diphenylmethyl]-3-methyl-1H- T63 78% 118.degree.
C. pyrazole N-[(4-Chlorophenyl)diphenylmethyl]-N-(1,3- T64 62%
156.degree. C. thiazol-2yl)amine
1-[(2-Chlorophenyl)diphenylmethyl]-3- T65 64% 139.degree. C.
(trifluorormethyl)-1H-pyrazole
N-[(2-Chlorophenyl)diphenylmethyl]-N-(1,3- T66 72% 152.degree. C.
thiazol-2yl)amine N-[(2-Chlorophenyl)diphenylmethyl]-N-(4- T67 92%
115.degree. C. pyridyl)amine
N-[(2-Chlorophenyl)diphenylmethyl]-N-(2- T68 64% 162.degree. C.
pyrimidyl)amine N-[(2-Chlorophenyl)diphenylmethyl]-N-(2- T69 67%
115.degree. C. pyridyl)amine
N-[(4-Chlorophenyl)diphenylmethyl]-N-(4- T70 81% 214.degree. C.
pyridyl)amine 2-[(2-Chlorophenyl)diphenylmethyl]-1H-isoindole- T71
67% 168.degree. C. 1,3(2H)-dione
N-Diphenyl[2-(trifluoromethyl)phenyl]methyl-N- T72 65% 164.degree.
C. (1,3-thiazol-2yl)amine
N-Diphenyl[2-(trifluoromethyl)phenyl]methyl-N-(2- T73 78%
133.degree. C. pyrimidinyl)amine
N-[(2-Fluorophenyl)diphenylmethyl]-N-(1,3- T78 58% 169.degree. C.
thiazol-2yl)amine N-[(2-Chlorophenyl)diphenylmethyl]-N-(4-methyl-
T79 49% 168.degree. C. 1,3-thiazol-2yl)amine
N-{5-[(4-Nitrophenyl)sulfonyl]-1,3-thiazol-2yl}- T81 73%
135.degree. C. N[(2-chlorophenyl)(diphenyl)methyl]amine
1-[(2-Chlorophenyl)diphenylmethyl]-1H-1,2,3,4- T84 72% 129.degree.
C. tetrazole 1-[(2-Chlorophenyl)diphenylmethyl]-1H-1,3- T85 68%
168.degree. C. benzimidazole
Example 12
Preparation of N,N-1,2-Ditritylamino ethane (Compound T21)
[0113] 2.0 g (7.2 mmol) of trityl chloride, 0.21 g (3.6 mmol) of
1,2-diaminoethane and 0.72 g (7.2 mmol) of triethyl amine were
dissolved in methylene chloride and heated under reflux for 8 hours
as described under Example 6 (Ng 1995, Tetrahedron 51: 7883) to
yield 1.03 g (53%) of N,N-1,2-ditritylamino ethane, mp: 172.degree.
C.
Example 13
[0114] The procedure in Example 12 was followed to obtain the
following compounds (Table 4). TABLE-US-00005 TABLE 4 Melting
Bis-triarylmethyldiamines from Diamines Number Yield Point
N,N-1,3-Ditritylamino propane T22 58% 179.degree. C.
1,4-Ditritylaminomethyl benzene T23 64% 201.degree. C. N,N-1,2-[(2-
T48 62% 228.degree. C. Chlorophenyl)diphenylmethyl]amino ethane
N,N-1,3-[(2- T49 58% 198.degree. C.
Chlorophenyl)diphenylmethyl]amino propane
Example 14
Preparation of (2-chlorophenyl)diphenylmethyl amine (Compound
T41)
[0115] To a solution of 1.50 g (4.79 mmol) of
(2-Chlorophenyl)diphenyl chloromethane, obtained under Example 5,
in 100 ml of ethyl ether was added 100 ml of 25% ammonia solution
and the resulting mixture was vigorously stirred at room
temperature for 24 hours (Casadio 1973, J. Pharm. Sci. 62: 773).
The organic layer was separated and the aqueous layer was extracted
with ether. The combined organic phases were thoroughly washed with
water, dried over anhydrous sodium sulfate and evaporated to
dryness. The oily residue was crystallized from petroleum ether
(40-60.degree. C.) to give 1.10 g (78%) of the product, mp:
98.degree. C.
Example 15
[0116] Following the procedure set forth in Example 14, the
following three compounds were prepared (Table 5). TABLE-US-00006
TABLE 5 Melting Triarylmethylamines from Ammonia Number Yield Point
(4-Fluorophenyl)diphenylmethyl amine T42 81% 62.degree. C.
(2-Fluorophenyl)diphenylmethyl amine T47 79% 84.degree. C.
(2-Trifluoromethylphenyl)diphenylmethyl T82 62% 106.degree. C.
amine
Example 16
Preparation of N-(2-chlorophenyl)diphenylmethyl acetamide (T75)
[0117] 2.5 g (8.51 mmol) of (2-chlorophenyl)diphenylmethyl amine
obtained under Example 14 was acetylated with 30 ml of freshly
distilled acetic anhydride. The resulting mixture was stirred at
40.degree. C. for 4 hours, poured into 200 ml of cold water and
left in the refrigerator overnight. The precipitate was collected
by vacuum filtration and recrystallized from ethanol to yield 1.17
g (41%) of the product, mp: 181.degree. C.
Example 17
[0118] Following the procedure in Example 16 the following
N-triarylmethyl acetamides were prepared from the corresponding
amines obtained under Example 15 (Table 6). TABLE-US-00007 TABLE 6
N-Triarylmethylacetamides from corresponding Melting Amines Number
Yield Point N-(2-Fluorophenyl)diphenylmethyl acetamide T76 73%
215.degree. C. N-(2-Trifluoromethylphenyl)diphenylmethyl T83 83%
185.degree. C. acetamide
Example 18
Preparation of 2-(4-Chlorophenyl)-2,2-diphenylacetonitrile
(T26)
[0119] 2-(4-Chlorophenyl) 2,2-diphenylacetonitrile was synthesized
by carefully triturating 1.50 g (4.8 mmol) of 4-chlorotrityl
chloride with 1.00 g (11 mmol) of copper cyanide and the resulting
mixture was heated for 4 hours at 150.degree. C. without a solvent.
After cooling 50 ml of toluene was added, the mixture was filtered
and the filtrate was concentrated in vacuo. The resulting residue
was recrystallized from petroleum ether (40-60.degree. C.) to give
0.66 g (45%) of the triarylmethyl acetonitrile derivative.
Example 19
[0120] The following triarylmethyl acetonitriles were prepared by
the procedure outlined in Example 18 (Table 7) TABLE-US-00008 TABLE
7 Triarylmethylacetonitriles from corresponding Melting Chlorides
Number Yield Point 2-(4-Fluorophenyl) 2,2-diphenylacetonitrile T27
52% 76.degree. C. 2-(2-Chlorophenyl) 2,2-diphenylacetonitrile T39
52% 143.degree. C. 2-(2-Fluorophenyl) 2,2-diphenylacetonitrile T40
63% 144.degree. C.
[0121] Compounds T39 and T40 have been disclosed in Brugnara, PCT
Application WO 97/34589. Compounds T50 (4-pyridyl, diphenyl
methanol), T51 (2,2,2-Triphenyl propionic acid), T52
[(S)-(-)-.alpha.,.alpha.-diphenyl-2-pyrrolidine methanol] and T53
[(R)-(+)-.alpha.,.alpha.-diphenyl-2-pyrrolidine methanol] used in
the biological testing are commercially available from Aldrich
Chemical. Co., Milwaukee, Wis. 53201, USA.
[0122] The following example provides an exemplary, but not
limiting, formulation, for administering the compounds of the
invention to mammals. Any of the compounds described herein, or
pharmaceutically acceptable salts or hydrates thereof, may be
formulated as illustrated in the following example.
Example 20
Gelatin Capsules
[0123] Acid-resistant coated hard gelatin capsules are prepared
using the following ingredients: TABLE-US-00009 Compound T34 100
mg/capsule Starch dried 200 mg/capsule Magnesium stearate 10
mg/capsule
The above ingredients are mixed and filled into acid-resistant
coated hard gelatin capsules in 310 mg quantities.
Example 21
In Vitro Activity
[0124] The assays are generally applicable for demonstrating the in
vitro activity of compounds of General Formula (I).
A) Block of IKCa1
[0125] This example demonstrates the ability of the exemplary
compounds, to inhibit the cloned human IKCa1 channel. The cloning
of human IKCa1 has been previously reported (Fanger 1999, J. Biol.
Chem. 274: 5746). COS-7 cells, maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf
serum, 4 mM L-glutamine and 1 mM Na.sup.+ pyruvate, were
transiently transfected with hIKCa1.
[0126] Electrophysiological experiments were carried out in the
whole-cell configuration of the patch-clamp technique using an
EPC-9 amplifier (HEKA Elektronik, Lambrecht Germany) interfaced to
a computer running acquisition and analysis software (Pulse and
Pulsfit; HEKA Elektronik). Pipettes were pulled from soft glass
capillaries, coated with Sylgard (Dow-Corning, Midland, Mich.), and
fire polished to resistances of 2.0-4.5 M.OMEGA. measured in the
bath. COS-7 cells were trypsinized and plated on glass coverslips 3
h before measurement. For measurements of IK.sub.Ca currents an
internal pipette solution containing 145 mM K.sup.+ aspartate, 2 mM
MgCl.sub.2, 10 mM HEPES, 10 mM K.sub.2EGTA and 8.5 mM CaCl.sub.2
(rendering 1 .mu.M of free Ca.sup.++), adjusted to pH 7.2 with
NaOH, with an osmolarity of 290-310 mosM was used. To prevent
activation of the native chloride channels in COS-7 cells an
aspartate Ringer solution was used as an external solution (160 mM
Na.sup.+ aspartate, 4.5 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 5
mM 5 HEPES, adjusted to pH 7.4 with NaOH, with an osmolarity of
290-310 mosM). 200 ms voltage ramps from -120 mV to 40 mV every 10
s were used. The holding potential in all experiments was -80 mV.
Series resistance compensation was not employed. The reduction of
slope conductance at -80 mV was used to determine the K.sub.d-value
by fitting the values to the Hill equation with a Hill coefficient
of unity.
[0127] The results of this assay are provided in TABLE 8, below.
TABLE-US-00010 TABLE 8 K.sub.d values for block of IKCa1 stably
transfected into COS-7 cells Compound Designation in Appendix A
K.sub.d [nM] T1 550 T2 530 T3 520 (.+-.30) T4 No effect at 1 .mu.M
T5 No effect at 1 .mu.M T7 30 000 T8 No effect at 10 .mu.M T9 1500
T10 28 000 T11 2500 (.+-.400) T12 700 T13 90 (.+-.10) T14 800 T15
10000 T16 10000 T17 35 000 T18 40 000 T19 No effect at 1 .mu.M T20
No effect at 1 .mu.M T23 No effect at 1 .mu.M T24 8000 T26 750 T27
800 T28 200 T29 15000 T30 10000 T31 8000 T34 20 (.+-.3) T35 9000
T36 700 T37 1000 T38 1100 (.+-.100) T39 60 T40 60 T41 5000 T42 5000
T43 750 T45 1000 T46 40 (.+-.5) T47 2000 T48 30000 T49 32000 T54
700 T55 820 T56 650 T57 n.d. T58 5000 T59 25 000 (.+-.3) T60 1500
(.+-.0.3) T61 35 000 T62 12 000 T63 1100 (.+-.0.3) T64 20 000 T65
2000 (.+-.0.5) T66 15 000 T67 30 000 T68 900 T69 28 000 T70 11 000
T71 No effect at 10 .mu.M T72 25 000 T73 15 000 T74 600 T75 1200
T76 1200 T77 800 T78 12 000 T79 8000 T81 15 000 T82 2500 T83 1500
T84 45 (.+-.7) T85 No effect at 1 .mu.M T86 1500 (.+-.500)
B) Selectivity of Clotrimazole and
1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) for IKCa1 Over
Other Ion Channels
[0128] 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) was
judged to be the most potent compound of the exemplary compounds
covered by General Formula (I) and was further investigated for its
selectivity over a whole range of other ion channels. Clotrimazole,
the imidazole currently under clinical investigation for the
treatment of sickle cell disease and diarrhea was used as a
control. 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) was
also investigated for its ability to block native IKCa1 currents in
activated human T-lymphocytes and in the human colonic epithelial
cell line T84.
[0129] L929 cells stably expressing mKv1.1, rKv1.2, mKv1.3, and
mKv3.1 and MEL cells stably expressing hKv1.5 have been previously
described (Grissmer 1994, Mol. Pharmacol. 45: 1227). hKv1.4 and
rKv4.2 were stably expressed in LTK (HK1-7). Channel expression was
induced 8-12 h before the electrophysiological experiments by 4
.mu.M dexamethasone (Sigma). HEK-293 cells stably expressing the
skeletal muscle sodium channel hSkM1 (SCN4A) were generated in the
laboratory of Dr. F. Lehmann-Horn. HEK-293 cells stably expressing
hSIo.alpha. were obtained from Dr. Andrew Tinker (Center for
Clinical Pharmacology, University College London). L929, MEL, LTK
and HEK cells were maintained in DMEM containing 10%
heat-inactivated fetal calf serum (Summit Biotechnology, Fort
Collins, Colo.) and 250 .mu.g/ml G418 (Life Technologies, Inc.).
Rat basophilic leukemia (RBL) cells were maintained in Eagle's
minimum essential medium with 2 mM L-glutamine and 10% fetal calf
serum. CHO cell were maintained in F12K media (ATCC). Human
leukemic Jurkat E6-1 cells were cultured in RPMI 1640 supplemented
with 10% fetal calf serum and 1 mM L-glutamine. T84 cells were
maintained in a 1:1 mixture of Ham's F12 medium and DMEM with 2.5
mM L-glutamine and 10% fetal calf serum.
[0130] Human peripheral blood mononuclear cells (PBMC) were
isolated from heparinized blood samples of healthy volunteers using
a lymphocyte separation medium (Accuspin System-Histopaque-1077,
Sigma Diagnostics) and maintained in RPMI 1640 supplemented with
10% fetal calf serum, 1 mM L-glutamine, 1 mM Na pyruvate, 1% non
essential amino acids. Purified T-lymphocytes were prepared by
passage through a nylon wool column. Activated T cell blasts were
prepared by treating the resting cells with 1 .mu.g/ml
phytohemagglutinin (PHA-P, DIFCO, Detroit, Mich.).
[0131] Recordings from the Jurkat SK.sub.Ca channel were made in
K.sup.+ aspartate Ringer (Na.sup.+ was replaced by K.sup.+) with
the same internal pipette solution. Recordings form the RBL inward
rectifier (rKir2.1) were made in aspartate Ringer with a K.sup.+
aspartate based pipette solution containing 50 nM of free
Ca.sup.++. No ATP was added to the pipette solution, because within
15 min of recording we witnessed no significant channel rundown.
For both SK.sub.Ca and inward rectifier currents the reduction of
slope conductance at -110 mV from 200 ms voltage ramps from -120 mV
to 40 mV every 10 s was taken as a measure of channel block.
BK.sub.Ca currents were elicited by 200 ms voltage ramps from -80
to 80 mV every 30 s. In aspartate Ringer with 1 .mu.M of free
Ca.sup.++ in the pipette solution currents turned on at -20 mV and
the reduction of slope conductance at 35 mV was used to evaluate
channel block. For measurements of Kv currents cells were bathed in
normal Ringer solution (160 mM NaCl, 4.5 mM KCl, 2mM CaCl.sub.2, 1
mM MgCl.sub.2, 10 mM HEPES) and an internal pipette solution
containing 134 mM KF, 2 mM MgCl.sub.2, 10 mM HEPES, 10 mM EGTA,
adjusted to pH 7.2 with NaOH, with an osmolarity of 290-310 mosM,
was used. For recordings from Kv1.1, Kv1.3, Kv1.4, Kv1.5, Kv3.1 and
Kv4.2 the voltage was stepped to 40 mV from the holding potential
for 200 ms every 30 s. K.sub.d-values were determined by fitting
the Hill equation to the reduction of peak current. For Kv1.2,
because of its "use-dependent" activation, a different pulse
protocol was used: 300 ms every 10 sec, and the reduction of the
mean of the current between 80-100% of the pulse duration was
fitted to the Hill equation. For sodium channel recordings we
employed voltage steps to -15 mV every 10 sec. Series resistance
compensation (60-80%) was used if currents exceeded 1 nA.
Capacitative and leak currents were subtracted using the P/8
procedure. Whole-cell recordings of monovalent currents through
Jurkat CRAC channels with Na.sup.+ as the charge carrier were made
as previously described (Kerschbaum 1999, Science 283: 836). For
measurements of swelling-activated mini chloride currents (Ross
1994, Biophys. J. 66: 169) 3 days activated human T-lymphocytes
were bathed in normal Ringer solution (290 mosM) and a hypertonic
internal pipette solution containing 160 mM Cs glutamate, 2 mM
MgCl.sub.2, 10 mM HEPES, 0.1 mM CaCl.sub.2, 1.1 mM EGTA, 4 mM
Na.sub.2ATP and 100 mM sucrose, adjusted to pH 7.2 with CsOH, with
an osmolarity of 420 mosM, was used. Chloride currents were
elicited by the same voltage ramps as IK.sub.Ca currents and
blocking potency of the compounds on the slope conduction at -40 mV
was evaluated between 300 and 900 s after break-in. A simple
syringe-driven perfusion system was used to exchange the bath
solution in the recording chamber.
[0132] The results of this assay are provided in TABLE 9, below.
The effect of 100 nM of
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T-34 in Appendix A)
on cloned and native IKCa1 currents is shown in FIG. 1. As shown in
TABLE 9 and FIGS. 1a-1c,
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) is a highly
potent and selective blocker of both cloned and native IKCa1
currents. Contrary to Clotrimazole,
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) does not
inhibit the activity of CYP3A4, the major xenobiotic metabolizing
enzyme of human liver (see TABLE 9). TABLE-US-00011 TABLE 9 Channel
Clotrimazole [nM] T34 [nM] IK.sub.Ca hIKCa1 70 .+-. 10 (n = 9) 20
.+-. 3 (n = 15) lymphocyte IK 100 25 .+-. 5 (n = 9) T84 IK 90 .+-.
15 (n = 9) 22 .+-. 10 (n = 9) K mKv1.1 10 000 .+-. 850 (n = 9) 9500
.+-. 1000 (n = 9) rKv1.2 5000 .+-. 730 (n = 9) 4500 .+-. 520 (n =
9) mKv1.3 6000 .+-. 440 (n = 9) 5000 .+-. 350 (n = 9) hKv1.4 6000
.+-. 520 (n = 9) 7500 .+-. 410 (n = 9) hKv1.5 8000 .+-. 890 (n = 9)
7000 .+-. 620 (n = 9) mKv3.1 33 000 .+-. 4000 (n = 9) 30 000 .+-.
5000 (n = 9) rKv4.2 8000 .+-. 950 (n = 9) 6000 .+-. 870 (n = 9)
Jurkat-SK 22 000 .+-. 1200 (n = 6) 23 000 .+-. 2000 (n = 6) BK
(hSlo.alpha.) 24 000 .+-. 2000 (n = 9) 25 000 .+-. 1800 (n = 9)
rKir2.1 no effect at 10 .mu.M (n = no effect at 10 .mu.M (n = 3) Na
HSKM1 7000 .+-. 550 (n = 9) 8000 .+-. 600 (n = 9) Ca Jurkat-CRAC no
effect at 10 .mu.M (n = no effect at 10 .mu.M (n = 2) Cl Lymphocyte
swelling- not done 10 000 .+-. 3000 (n = 4) activated COS-7 no
effect at 10 .mu.M (n = no effect at 10 .mu.M (n = 5) EC.sub.50 for
inhibition 30 No inhibitory of CYP3A4 (99% inhibition at 100 effect
at 10 .mu.M (n = 2)
C) Inhibition of Cytochrome P450 3A4 Catalytic Activities by a
Single Concentration of 10 Test Substances
[0133] The test substances shown in Table 8 were evaluated for
their inhibitory effect on the catalytic activity of human
cytochrome P450 enzyme. Cytochrome P450 3A4 (CYP3A4) is involved in
many drug-drug interactions and in many other pathways in the body
including the catabolism of steroids and xenobiotics. Inhibition of
this enzyme was measured in 96 well plates with
7-benzyloxy-4-trifluoro-methylcoumarin (BFC) as the substrate and
cDNA-derived enzymes in microsomes prepared from
baculovirus-infected insect cells.
[0134] The inhibition study consisted of the determination of
inhibition by the test substance on CYP3A4 catalytic activity. A
single concentration of each model substrate (approximately the
apparent K.sub.m) and one test substance concentration (10 .mu.M),
were tested in duplicate. The compounds were prepared as 10 mM
stock solutions in acetonitrile. Metabolism of the model substrate
was assayed by the production of
7-hydroxy-4-trifluoromethylcoumarin metabolite. The metabolite was
detected by fluorescence.
[0135] Table 10 below provides a list of the name of enzymes
examined, the catalog number of the microsomes used as a source of
the enzyme, the concentration of the model substrate used, the
amount of enzyme used, the final buffer concentration, and the
positive control compound. TABLE-US-00012 TABLE 10 Enzyme CYP3A4
Catalog Number P202 Substrate BFC Substrate Concentration 50 .mu.M
Pmole Enzyme per Well 1-2 Potassium Phosphate Buffer Concentration
200 mM Positive Control (Concentration) ketoconazole (5 .mu.M)
[0136] Assays were conducted in 96 well microtiter plates. The BFC
substrate was initially prepared in acetonitrile. The final
concentration of the substrate was 50 .mu.M, which is below the
apparent K.sub.m. Six wells were used for one test. Wells 1 and 2
contained a 10 .mu.M concentration of the test substance (except
clotrimazole, which had 0.1 .mu.M), and wells 3 and 4 contained no
test substance and wells 5 and 6 were blanks for background
fluorescence (stop solution added before the enzyme). For the
positive controls, 12 wells in a row were used for one test. The
positive control inhibitor/enzyme combination was examined in
duplicate rows. Wells 1 to 8 contained serial 1:3 dilutions of the
inhibitors. The highest inhibitor concentration were as described
in the table above. Wells 9 and 10 contained no inhibitor and wells
11 and 12 were blanks for background fluorescence (stop solution
added before the enzyme).
[0137] After buffer, cofactors and inhibitor addition, the plates
were pre-warmed to 37.degree. C. Incubations were initiated by the
addition of pre-warmed enzyme and substrate. The final cofactor
concentrations were 1.3 mM NADP, 3.3 mM glucose-6-phosphate and 0.4
U/ml glucose-6-phosphate dehydrogenase. The final incubation volume
was 0.2 ml. Incubations were carried out for 30 minutes [CYP3A4
(BFC)] and stopped by the addition of 0.075 ml of 80%
acetonitrile-20% 0.5M Tris. Fluorescence per well was measured
using a BMG FLUOstar fluorescence plate reader. The BFC metabolite
7-hydroxy-4-trifluoromethyl-coumarin was measured using an
excitation wavelength of 410 nm and emission wavelength of 538
nm.
[0138] All results listed in Table 11 are consistent with a
properly functioning model with 0.1 .mu.M of clotrimazole producing
99% of inhibition. The ambivalent effects (e.g. inhibition or
activation) on CYP3A4 catalytic activity by some compounds is
commonly observed with CYP3A4. One explanation for this is that the
enzyme is capable of accommodating 2 or more compounds
simultaneously, one of which may activate, or inhibit metabolism of
the other (Thummel 1998, Ann. Rev. Pharmacol. Toxicol. 38: 389).
Although activation of CYP3A4 enzymatic activity is commonly
observed in vitro, to our knowledge, this has not been demonstrated
in vivo in humans. It appears that the compounds designated as T3,
T34, T58 and T75 on Appendix A have the potential for activation
metabolism of compounds that are substrates for CYP3A4.
TABLE-US-00013 TABLE 11 Percent of inhibition of catalytic activity
of CY3A4 Test compound concentration % inhibition T3 10 .mu.M -30.5
T34 10 .mu.M -77.5 T39 10 .mu.M 10 T40 10 .mu.M 9 T58 10 .mu.M -26
T66 10 .mu.M 86.5 T67 10 .mu.M 74 T74 10 .mu.M 4 T75 10 .mu.M -54
clotrimazole 0.1 .mu.M 99
D) In Vitro Toxicity
[0139] The in vitro toxicity was performed as follows. Jurkat E6-1,
MEL, C.sub.2F.sub.3, NGP, NLF cells and human T-lymphocytes were
seeded at 5.times.10.sup.5 cells/ml, and L929, COS-7, CHO and RBL
cells were seeded at 10.sup.5 cells/ml in twelve-well plates. Drug
(2 or 5 .mu.M) was added in a final DMSO concentration of 0.1%.
After 48 h of incubation at 37.degree. C. with 5% CO.sub.2, cells
were harvested by sucking them off the plates (suspension cells) or
by trypsinization (adherent cell lines). Cells were centrifuged,
resuspended in 0.5 ml PBS containing 1 .mu.g/ml propidium iodide
(PI), and red fluorescence measured on a FACScan flow cytometer
after 20 min, 10.sup.4 cells of every sample being analyzed. The
percentage of dead cells was determined by their PI uptake. Two
controls for every cell line (one in medium and one with 0.1% DMSO)
were also analyzed.
[0140] The results of this in vitro toxicity assay are shown in
TABLE 12. 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34 in
Appendix A), at a concentration 100-250 times the K.sub.d for IKCa1
channel block, did not reduce cell viability in any of the ten cell
lines examined. TABLE-US-00014 TABLE 12 Cells* Control control 0.1%
DMSO 2 .mu.M T34 5 .mu.M T34 T-lymphocytes 6.1 7.1 6.2 7.0 Jurkat
E6-1 3.2 2.3 2.5 3.2 MEL 5.1 4.8 6.2 5.8 L929 9.6 7.8 4.6 7.5 COS-7
4.5 4.0 3.6 4.5 CHO 4.6 5.6 5.6 4.7 RBL-2H3 5.5 7.6 7.9 8.8
C.sub.2F.sub.3 17.4 14.3 15.8 12.8 NLF 12.8 9.1 10.8 10.8 NGP 8.0
4.2 10.9 5.9 *MEL, murine erythroleukemia cells; L929, murine
fibrosarcoma cell; COS-7, SV40 transformed African green monkey
kidney; CHO, Chinese hamster ovary cells; RBL-2H3, rat basophilic
leukemia cells; Jurkat E6-1, human leukemic T-cell line;
C.sub.2F.sub.3 human myoblasts; NGP and NLF human neuroblastoma
cell lines.
E) Acute in Vivo Toxicity
[0141] An ISO acute systemic toxicity study was performed in CF-1
BR mice (17-19 g) according to the guidelines of the United States
Pharmacopeia XXII. Five mice were injected intravenously with a
single 0.9-1.0 ml (50 ml/kg) dose of 0.5 mg/kg
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) (29 .mu.M in
mammalian Ringer solution with 1% ethanol and 2.5% bovine serum
albumin). Mice were observed for adverse effects immediately after
dosing, at 4 h after injection and daily for 7 days. Five control
mice were injected with an equal volume of the vehicle. The mice
were weighed at the beginning of the study and at its
termination.
[0142] The results clearly showed that there was no mortality and
all animals appeared clinically normal during the 7-day study. The
body weight data of the test compound treated group (wt on day 1:
17.8 g; wt on day 7: 27.0 g) were similar to controls (day 1: 17.4
g; day 7: 23.4 g) during the study. These data, taken together with
the data from the cell viability assay, suggest that
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) was not toxic
at .about.100-1000 times the pharmacological dose thus exhibiting a
very good therapeutic index.
F) Hydrolytic Stability of
1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (T34) vs.
Clotrimazole
[0143] Applicants investigated the stability of
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34 in Appendix A)
against hydrolysis at pH 7.4 and at pH 5.0. The resulting
concentrations of compound T34 and its expected degradation product
(2-chlorophenyl)diphenyl methanol (T3) in analogy to clotrimazole
were determined by HPLC.
[0144] To access oral bioavailability the stability of
1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (T34 in Appendix A)
under acidic conditions was determined at pH 1 in artificial
stomach fluid (DAB9).
[0145] A Waters 590 liquid chromatograph with a Waters 746
integrator and a Waters 486 UV/Vis detector, and a 25 cm
Lichrospher RP18 column (5-.mu.M particle size) was used. The
mobile phase consisted of K.sub.2HPO.sub.4 25 mM, KH.sub.2PO.sub.4
30 mM, methanol at 262:13:725. The chromatographic separation was
performed at 20.degree. C. with a flow rate of 1.0 ml/min, the
absorbance of the elluent was monitored at 210 nm.
[0146] Hydrolysis profiles (10 .mu.M compound with 20%
acetonitrile) were carried out in Soerensen phosphate buffer (pH
7.4 and pH 5.0) in a total volume of 10 ml. 100 .mu.I samples were
collected 0, 1, 2, 3, 4, 5, 24, 48 and 72 hours after incubation,
with shaking, in a waterbath at 37.degree. C. and manually injected
into the HPLC over Rheodyne 7125 system. For profiles at pH 1.0
artificial stomach fluid (DAB 9), containing 2 g NaCl, 3.2 g pepsin
and 80 ml 1M HCL in 1.0 L of water was used. After 0, 1 and 2 hours
400 .mu.l of sample were collected and extracted three times with
500 .mu.l of ethyl ether by vortexing. The combined extracts were
than evaporated to dryness by passing nitrogen through the
solution. The residue was than dissolved in 400 .mu.l of mobile
phase of which 100 .mu.M were injected in the HPLC. The recovery
rate from this extraction procedure was 80%.
[0147] This chromatographic system gave retention times of 16.1 min
for T3, 20.6 min for clotrimazole and 23.4 min for T34. Whereas
clotrimazole slowly hydrolyzed at pH 5.0 (22% T3 after 48 h, 35% T3
after 72 h), T34 was completely stable against hydrolysis at pH 7.4
and pH 5.0 over 72 h (see FIG. 2).
[0148] As shown in FIG. 2, the results clearly show that compound
T34 was completely stable against hydrolysis at pH 10, pH 7.4 and
pH 5.0 over 72 h (pH 10.0 is not shown). Specifically, FIG. 2 shows
the hydrolysis of compound T34 and clotrimazole at pH 7.4 and pH
5.0 (for clotrimazole at pH 5.0 the corresponding amount of T3 can
be detected at the corresponding retention time (mean of 3
determinations). At pH 1.0 T34 rapidly breaks down to T3 with a
half-life of .about.45 min (FIG. 3).
[0149] These results demonstrate that T34 can be used in
T-lymphocyte proliferation and cytokine secretion assays. However,
for oral administration T34 should be used in an acid-resistant
coated formulation.
[0150] The compounds of this invention, or pharmaceutically
acceptable salts or hydrates thereof, can be delivered to a patient
using a wide variety of routes or modes of administration. Suitable
routes of administration include, but are not limited to,
inhalation, transdermal, oral, rectal, transmucosal, intestinal and
parenteral administration, including intramuscular, subcutaneous
and intravenous injections. The compounds described herein, or
pharmaceutically acceptable salts or hydrates thereof, may be
administered singly or in combination with other therapeutic
agents, e.g. analgesics, antibiotics and other immunosuppressive
drugs like cyclosporin A or Kv1.3 selective blockers. The active
compound (T34) may be administered per se or in the form of a
pharmaceutical composition wherein the active compound is in
admixture with one or more physiologically acceptable carriers,
excipients or diluents. Pharmaceutical compositions for use in
accordance with the present invention may be formulated in
conventional manner using one or more physiologically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Proper formulation is dependent on the route
of administration chosen. For parenteral administration (bolus
injection or continuous infusion), the agents of the invention may
be formulated in water soluble form in aqueous solutions,
preferably in physiologically compatible buffers such as Hank's
solution, Ringer's solution, or physiological saline buffer.
Additionally, suspensions of the compounds may be prepared as oily
injections with fatty oils, synthetic fatty acid esters, or
liposomes. The compounds may also be formulated as a depot
preparation. For oral administration, the compounds can be
formulated readily by combining the active compound with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the compounds of the invention to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions an the like, for oral ingestion for patients to be
treated. Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol:
cellulose preparations such as, for example maize starch, wheat
starch, rice strach, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropyl-methylcellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone.
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