U.S. patent application number 10/349741 was filed with the patent office on 2003-10-09 for substituted 11-phenyl-dibenzazepine compounds useful for the treatment or prevention of diseases characterized by abnormal cell proliferation.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. Invention is credited to Bellott, Emile M. JR., Brugnara, Carlo, Clifford, John J., Fluckiger, Rudolf, Gao, Ying-Duo, Haidar, Reem M., Halperin, Jose, Kelleher, Eugene W., Lombardy, Richard John, Moussa, Adel M., Sachdeva, Yesh P., Sun, Minghua, Taft, Heather N., Zeldin, Michael H..
Application Number | 20030191111 10/349741 |
Document ID | / |
Family ID | 25523175 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030191111 |
Kind Code |
A1 |
Brugnara, Carlo ; et
al. |
October 9, 2003 |
Substituted 11-phenyl-dibenzazepine compounds useful for the
treatment or prevention of diseases characterized by abnormal cell
proliferation
Abstract
The present invention provides substituted
11-phenyl-dibenzazepine compounds which are specific, potent and
safe inhibitors of mammalian cell proliferation. The compounds can
be used to inhibit mammalian cell proliferation in situ as a
therapeutic approach towards the treatment or prevention of
diseases characterized by abnormal cell proliferation, such as
cancer.
Inventors: |
Brugnara, Carlo; (Newton
Highlands, MA) ; Halperin, Jose; (Brookline, MA)
; Fluckiger, Rudolf; (Brookline, MA) ; Bellott,
Emile M. JR.; (Beverly, MA) ; Lombardy, Richard
John; (Littleton, MA) ; Clifford, John J.;
(Arlington, MA) ; Gao, Ying-Duo; (Edison, NJ)
; Haidar, Reem M.; (Woburn, MA) ; Kelleher, Eugene
W.; (Medford, MA) ; Moussa, Adel M.;
(Burlington, MA) ; Sachdeva, Yesh P.; (Concord,
MA) ; Sun, Minghua; (Libertyville, IL) ; Taft,
Heather N.; (Littleton, MA) ; Zeldin, Michael H.;
(Cambridge, MA) |
Correspondence
Address: |
GRAY CARY WARE & FREIDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
|
Family ID: |
25523175 |
Appl. No.: |
10/349741 |
Filed: |
January 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10349741 |
Jan 21, 2003 |
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09554848 |
Sep 22, 2000 |
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6534497 |
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09554848 |
Sep 22, 2000 |
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PCT/US98/24787 |
Nov 20, 1998 |
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PCT/US98/24787 |
Nov 20, 1998 |
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08975592 |
Nov 20, 1997 |
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Current U.S.
Class: |
514/217 ;
540/587 |
Current CPC
Class: |
C07D 223/20 20130101;
A61P 35/00 20180101; A61P 17/08 20180101; A61P 17/16 20180101; A61P
43/00 20180101; A61P 17/02 20180101; A61P 9/10 20180101; A61P 17/04
20180101 |
Class at
Publication: |
514/217 ;
540/587 |
International
Class: |
A61K 031/55; C07D
223/18 |
Claims
What is claimed is:
1. A process for preparing a compound having the formula: 12or a
pharmaceutically acceptable salt or hydrate thereof, wherein:
R.sub.1 is --R', (C.sub.6-C.sub.20) aryl or substituted
(C.sub.6-C.sub.20) aryl; R.sub.2 is --R', --OR', --SR', halogen or
trihalomethyl; R.sub.3 is --R', --OR', --SR', halogen or
trihalomethyl or, when taken together with R.sub.4, is
(C.sub.6-C.sub.20) aryleno; R.sub.4 is --R', --OR', --SR', halogen
or trihalomethyl or, when taken together with R.sub.3, is
(C.sub.6-C.sub.20) aryleno; each of R.sub.5, R.sub.6, R.sub.7,
R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13 and
R.sub.14 is independently selected from the group consisting of
--R', halogen and trihalomethyl; R.sub.15 is --R", --C(O)R",
--C(S)R", --C(O)OR", --C(S)OR", --C(O)SR", --C(S)SR",
--C(O)N(R").sub.2, --C(S)N(R").sub.2, --C(O)C(O)R" --C(S)C(O)R",
--C(O)C(S)R", --C(S)C(S)R", --C(O)C(O)OR", --C(S)C(O)OR",
--C(O)C(S)OR", --C(O)C(O)SR", --C(S)C(S)OR", --C(S)C(O)SR",
--C(O)C(S)SR", --C(S)C(S)SR", --C(O)C(O)N(R").sub.2,
--C(S)C(O)N(R").sub.2, --C(O)C(S)N(R").sub.2 or
--C(S)C(S)N(R").sub.2; each R' is independently selected from the
group consisting of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.2-C.sub.6)
alkenyl and (C.sub.2-C.sub.6) alkynyl; each R" is independently
selected from the group consisting of --H, (C.sub.1-C.sub.6) alkyl,
(C.sub.2-C.sub.6) alkenyl, (C.sub.2-C.sub.6) alkynyl,
(C.sub.6-C.sub.20) aryl, (C.sub.6-C.sub.20) substituted aryl,
(C.sub.6-C.sub.26) alkaryl and substituted (C.sub.6-C.sub.26)
alkaryl; and the aryl and alkaryl substituents are each
independently selected from the group consisting of --CN, --OR',
--SR', --NO.sub.2, --NR'R', halogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.2-C.sub.6) alkenyl, (C.sub.2-C.sub.6) alkynyl and
trihalomethyl, with the provisos that when R.sub.1 and R.sub.15 are
each --H, at least one of R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13
or R.sub.14 is other than --H, R.sub.8 is other than --H or --Cl
and at least three of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
other than --OCH.sub.3; when R.sub.1-R.sub.7 and R.sub.9-R.sub.14
are --H, and R.sub.8 is --H or --Cl, then R.sub.15 is other than
--H or --CH.sub.3; when R.sub.1-R.sub.7 and R.sub.9-R.sub.14 are
--H and R.sub.8 is --F or --I or --Br, then R.sub.15 is other than
--H; when R.sub.1 and R.sub.6-R.sub.15 are --H, then three of
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are not --OCH.sub.3 at the
same time; when R.sub.1-R.sub.5, R.sub.8 and R.sub.10-R.sub.15 are
--H, then at least one of R.sub.6, R.sub.7 or R.sub.9 is other than
--Cl; when R.sub.1 and R.sub.5-R.sub.14 are --H, and R.sub.15 is
--CH.sub.3, then R.sub.2, R.sub.3 and R.sub.4 are not --OCH.sub.3
at the same time; said method comprising: a) cyclizing a diphenyl
methanol compound according to Formula II: 13 under acidic
conditions; and optionally b) if R.sub.15 is hydrogen, said method
further comprising a step for derivatizing the nitrogen of the
azepine ring.
2. The method of claim 1 wherein said acidic conditions comprise
one or more compounds selected from the group of sulfuric acid,
acetic acid, methanesulfonic acid and phosphorus pentoxide.
3. The method of claim 1 wherein the derivatizing step comprises
reacting a compound of the formula: 14wherein R.sub.1-R.sub.14 are
defined as above, with an acylating agent having the formula
R"C(O)X, wherein X is Br, Cl or I and R" is defined as above.
4. The method of claim 1 wherein the derivatizing step comprises
reacting a compound of the formula: 15wherein R.sub.1-R.sub.4 are
defined as above, with an alkylating agent having the formula R"X,
wherein X is Br, Cl or I and R" is defined as above.
5. The method of claim 1 wherein the derivatizing step comprises
reacting a compound of the formula: 16wherein R.sub.1-R.sub.4 are
defined as above, with a chloroformate reagent of the formula:
17wherein X is Br or Cl and R" is defined as above.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to aromatic organic compounds
which are specific, potent and safe inhibitors of the
Ca.sup.2+-activated potassium channel (Gardos channel) of
erythrocytes and/or of mammalian cell proliferation. More
particularly, the invention relates to substituted 11-phenyl
dibenzazepine compounds capable of inhibiting the Gardos channel of
sickle erythrocytes and/or mitogen-induced mammalian cell
proliferation. The compounds can be used to reduce sickle
erythrocyte dehydration and/or delay the occurrence of erythrocyte
sickling or deformation in situ as a therapeutic approach towards
the treatment or prevention of sickle cell disease. The compounds
can also be used to inhibit mammalian cell proliferation in situ as
a therapeutic approach towards the treatment or prevention of
diseases characterized by abnormal cell proliferations.
2. BACKGROUND OF THE INVENTION
[0002] Sickle cell disease has been recognized within West Africa
for several centuries. Sickle cell anemia and the existence of
sickle hemoglobin (Hb S) was the first genetic disease to be
understood at the molecular level. It is recognized today as the
morphological and clinical result of a glycine to valine
substitution at the No. 6 position of the beta globin chain
(Ingram, 1956, Nature 178:792-794). The origin of the amino acid
change and of the disease state is the consequence of-a single
nucleotide substitution (Marotta et al., 1977, J. Biol. Chem.
252:5040-5053).
[0003] The major source of morbidity and mortality of patients
suffering from sickle cell disease is vascular occlusion caused by
sickled erythrocytes, which causes repeated episodes of pain in
both acute and chronic form and also causes ongoing organ damage
with the passage of time. It has long been recognized and accepted
that the deformation and distortion of sickle cell erythrocytes
upon complete deoxygenation is caused by polymerization and
intracellular gelation of sickle hemoglobin, hemoglobin S (Hb S).
The phenomenon is well reviewed and discussed by Eaton and
Hofrichter, 1987, Blood 70:1245. The intracellular gelation and
polymerization of Hb S can occur at any time during erythrocyte's
journey through the vasculature. Thus, erythrocytes in patients
with sickle cell disease containing no polymerized hemoglobin S may
pass through the microcirculation and return to the lungs without
sickling, may sickle in the veins or may sickle in the
capillaries.
[0004] The probability of each of these events occurring is
determined by the delay time for intracellular gelation relative to
the appropriate capillary transit time (Eaton et al., 1976, Blood
47:621). In turn, the delay time is dependent upon the oxygenation
state of the hemoglobin, with deoxygenation shortening the delay
time. Thus, if it is thermodynamically impossible for intracellular
gelation to take place, or if the delay time at venous oxygen
pressures is longer than about 15 seconds, cell sickling will not
occur. Alternatively, if the delay time is between about 1 and 15
seconds, the red cell will likely sickle in the veins. However, if
the delay time is less than about 1 second, red cells will sickle
within the capillaries.
[0005] For red cells that sickle within the capillaries, a number
of possible consequent events exist, ranging from no effect on
transit time, to transient occlusion of the capillary, to a more
permanent blockage that may ultimately result in ischemia or
infarction of the surrounding cells and in destruction of the red
cell.
[0006] It has long been recognized that the cytoplasm of the normal
erythrocyte comprises approximately 70% water. Water crosses a
normal erythrocyte membrane in milliseconds; however, the loss of
cell water causes an exponential increase in cytoplasmic viscosity
as the mean cell hemoglobin concentration (MCHC) rises above about
32 g/dl. Since cytoplasmic viscosity is a major determinate of
erythrocyte deformability and sickling, the dehydration of the
erythrocyte has substantial Theological and pathological
consequences. Thus, the physiological mechanisms that maintain the
water content of normal erythrocytes and the pathological
conditions that cause loss of water from erythrocytes in the blood
circulation are critically important. Not surprisingly, regulation
of erythrocyte dehydration has been recognized as an important
therapeutic approach towards the treatment of sickle cell disease.
Since cell water will follow any osmotic change in the
intracellular concentration of ions, the maintenance of the red
cell's potassium concentration is of particular importance (Stuart
and Ellory, 1988, Brit J. Haematol. 69:1-4).
[0007] Many attempts and approaches to therapeutically treating
dehydrated sickle cells (and thus decreasing polymerization of
hemoglobin S by lowering the osmolality of plasma) have been tried
with limited success, including the following approaches:
intravenous infusion of distilled water (Gye et al., 1973, Am. J.
Med. Sci. 266:267-277); administration of the antidiuretic hormone
vasopressin together with a high fluid intake and salt restriction
(Rosa et al., 1980, M. Eng. J. Med. 303:1138-1143; Charache and
Walker, 1981, Blood 58:892-896); the use of monensin to increase
the cation content of the sickle cell (Clark et al., 1982, J. Clin.
Invest. 70:1074-1080; Fahim and Pressman, 1981, Life Sciences
29:1959-1966); intravenous administration of cetiedil citrate
(Benjamin et al., 1986, Blood 67:1442-1447; Berkowitz and orringer,
1984, Am. J. Hematol. 17:217-223; Stuart et al., 1987, J. Clin.
Pathol. 40:1182-1186); and the use of oxpentifylline (Stuart et
al., 1987, J. Clin. Pathol. 40:1182-1186).
[0008] Another approach towards therapeutically treating dehydrated
sickle cells involves the administration of imidazole,
nitroimidazole and triazole antimycotic agents such as Clotrimazole
(U.S. Pat. No. 5,273,992 to Brugnara et al.). Clotrimazole, an
imidazole-containing antimycotic agent, has been shown to be a
specific, potent inhibitor of the Gardos channel of normal and
sickle erythrocytes, and to prevent Ca.sup.2+-dependent dehydration
of sickle cells both in vitro and in vivo (Brugnara et al., 1993,
J. Clin. Invest. 92:520-526; De Franceschi et al., 1994, J. Clin.
Invest. 93:1670-1676). When combined with a compound which
stabilizes the oxyconformation of Hb S, Clotrimazole induces an
additive reduction in the clogging rate of a micropore filter and
may attenuate the formation of irreversibly sickled cells (Stuart
et al., 1994, J. Haematol. 86:820-823). Other compounds that
contain a heteroaryl imidazole-like moiety believed to be useful in
reducing sickle erythrocyte dehydration via Gardos channel
inhibition include miconazole, econazole, butoconazole, oxiconazole
and sulconazole. Each of these compounds is a known antimycotic.
Other imidazole-containing compounds have been found to be
incapable of inhibiting the Gardos channel and preventing loss of
potassium.
[0009] As can be seen from the above discussion, reducing sickle
erythrocyte dehydration via blockade of the Gardos channel is a
powerful therapeutic approach towards the treatment and/or
prevention of sickle cell disease. Compounds capable of inhibiting
the Gardos channel as a means of reducing sickle cell dehydration
are highly desirable, and are therefore an object of the present
invention.
[0010] Cell proliferation is a normal part of mammalian existence,
necessary for life itself. However, cell proliferation is not
always desirable, and has recently been shown to be the root of
many life-threatening diseases such as cancer, certain skin
disorders, inflammatory diseases, fibrotic conditions and
arteriosclerotic conditions.
[0011] Cell proliferation is critically dependent on the regulated
movement of ions across various cellular compartments, and is
associated with the synthesis of DNA. Binding of specific
polypeptide growth factors to specific receptors in growth-arrested
cells triggers an array of early ionic signals that are critical in
the cascade of mitogenic events eventually leading to DNA synthesis
(Rozengurt, 1986, Science 234:161-164). These include: (1) a rapid
increase in cystolic Ca.sup.2+, mostly due to rapid release of
Ca.sup.2+ from intracellular stores; (2) capacitative Ca.sup.2+
influx in response to opening of ligand-bound and
hyperpolarization-sensitive Ca.sup.2+ channels in the plasma
membrane that contribute further to increased intracellular
Ca.sup.2+ concentration (Tsien and Tsien, 1990, Annu. Rev. Cell
Biol. 6:715-760; Peppelenbosch et al., 1991, J. Biol. Chem.
266:19938-19944); and (3) activation of Ca.sup.2+-dependent K.sup.+
channels in the plasma membrane with increased K.sup.+ conductance
and membrane hyperpolarization (Magni et al., 1991, J. Biol. Chem.
261:9321-9327). These mitogen-induced early ionic changes,
considered critical events in the signal transduction pathways, are
powerful therapeutic targets for inhibition of cell proliferation
in normal and malignant cells.
[0012] One therapeutic approach towards the treatment of diseases
characterized by unwanted or abnormal cell proliferation via
alteration of the ionic fluxes associated with early mitogenic
signals involves the administration of Clotrimazole. As discussed
above, Clotrimazole has been shown to inhibit the
Ca.sup.2+-activated potassium channel of erythrocytes. In addition,
Clotrimazole inhibits voltage- and ligand-stimulated Ca.sup.2+
influx mechanisms in nucleated cells (Villalobos et al., 1992,
FASEB J. 6:2742-2747; Montero et al., 1991, Biochem. J. 277:73-79)
and inhibits cell proliferation both in vitro and in vivo
(Benzaquen et al., 1995, Nature Medicine 1:534-540). Recently,
Clotrimazole and other imidazole-containing antimycotic agents
capable of inhibiting Ca.sup.2+-activated potassium channels have
been shown to be useful in the treatment of arteriosclerosis (U.S.
Pat. No. 5,358,959 to Halperin et al.), as well as other disorders
characterized by unwanted or abnormal cell proliferation.
[0013] As can be seen from the above discussion, inhibiting
mammalian cell proliferation via alteration of ionic fluxes
associated with early mitogenic signals is a powerful therapeutic
approach towards the treatment and/or prevention of diseases
characterized by unwanted or abnormal cell proliferation. Compounds
capable of inhibiting mammalian cell proliferation are highly
desirable, and are therefore also an object of the present
invention.
3. SUMMARY OF THE INVENTION
[0014] These and other objects are provided by the present
invention, which in one aspect provides a novel class of organic
compounds which are potent, selective and safe inhibitors of the
Ca.sup.2+-activated potassium channel (Gardos channel) of
erythrocytes, particularly sickle erythrocytes, and/or of mammalian
cell proliferation. The compounds are generally substituted
11-phenyl-dibenzazepine compounds. In one illustrative embodiment,
the compounds capable of inhibiting the Gardos channel and/or
mammalian cell proliferation according to the invention are
compounds having the structural formula: 1
[0015] or pharmaceutically acceptable salts of hydrates thereof,
wherein:
[0016] R.sub.1 is --R', (C.sub.6-C.sub.20) aryl or substituted
(C.sub.6-C.sub.20) aryl;
[0017] R.sub.2 is --R', --OR', --SR', halogen or trihalomethyl;
[0018] R.sub.3 is --R', --OR', --SR', halogen or trihalomethyl or,
when taken together with R.sub.4, is (C.sub.6-C.sub.20)
aryleno;
[0019] R.sub.4 is --R', --OR', --SR', halogen or trihalomethyl or,
when taken together with R.sub.3, is (C.sub.6-C.sub.20)
aryleno;
[0020] each of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9,
R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14 is
independently selected from the group consisting of --R', halogen
and trihalomethyl;
[0021] R.sub.15 is --R", --C(O)R", --C(S)R", --C(O)OR", --C(S)OR",
--C(O)SR", --C(S)SR", --C(O)N(R").sub.2, --C(S)N(R").sub.2,
--C(O)C(O)R", --C(S)C(O)R", --C(O)C(S)R", --C(S)C(S)R",
--C(O)C(O)OR", --C(S)C(O)OR", --C(O)C(S)OR", --C(O)C(O)SR",
--C(S)C(S)OR", --C(S)C(O)SR", --C(O)C(S)SR", --C(S)C(S)SR",
--C(O)C(O)N(R").sub.2, --C(S)C(O)N(R").sub.2, --C(O)C(S)N(R").sub.2
or --C(S)C(S)N(R").sub.2;
[0022] each R' is independently selected from the group consisting
of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl and
(C.sub.1-C.sub.6) alkynyl;
[0023] each R" is independently selected from the group consisting
of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl,
(C.sub.1-C.sub.6) alkynyl, (C.sub.6-C.sub.20) aryl, substituted
(C.sub.6-C.sub.20) aryl, (C.sub.6-C.sub.26) alkaryl and substituted
(C.sub.6-C.sub.26) alkaryl; and
[0024] the aryl and alkaryl substituents are each independently
selected from the group consisting of --CN, --OR', --SR',
--NO.sub.2, --NR'R', halogen, (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl and
trihalomethyl.
[0025] In another aspect, the present invention provides
pharmaceutical compositions comprising one or more compounds
according to the invention in admixture with a pharmaceutically
acceptable carrier, excipient or diluent. Such a preparation can be
administered in the methods of the invention.
[0026] In still another aspect, the invention provides a method for
reducing sickle erythrocyte dehydration and/or delaying the
occurrence of erythrocyte sickling or deformation in situ. The
method involves contacting a sickle erythrocyte in situ with an
amount of at least one compound according to the invention, or a
pharmaceutical composition thereof, effective to reduce sickle
erythrocyte dehydration and/or delay the occurrence of erythrocyte
sickling or deformation. In a preferred embodiment, the sickle cell
dehydration is reduced and erythrocyte deformation is delayed in a
sickle erythrocyte that is within the microcirculation vasculature
of a subject, thereby preventing or reducing the vaso-occlusion and
consequent adverse effects that are commonly caused by sickled
cells.
[0027] In still another aspect, the invention provides a method for
the treatment and/or prevention of sickle cell disease in a
subject, such as a human. The method involves administering a
prophylactically or therapeutically effective amount of at least
one compound according to the invention, or a pharmaceutical
composition thereof, to a patient suffering from sickle cell
disease. The patient may be suffering from either acute sickle
crisis or chronic sickle cell episodes.
[0028] In yet another aspect, the invention provides a method for
inhibiting mammalian cell proliferation in situ. The method
involves contacting a mammalian cell in situ with an amount of at
least one compound according to the invention, or a pharmaceutical
composition thereof, effective to inhibit cell proliferation. The
compound or composition may act either cytostatically,
cytotoxically or a by a combination of both mechanisms to inhibit
proliferation. Mammalian cells that can be treated in this manner
include vascular smooth muscle cells, fibroblasts, endothelial
cells, various types of pre-cancer cells and various types of
cancer cells.
[0029] In still another aspect, the invention provides a method for
treating and/or preventing unwanted or abnormal cell proliferation
in a subject, such as a human. In the method, at least one compound
according to the invention, or a pharmaceutical composition
thereof, is administered to a subject in need of such treatment in
an amount effective to inhibit the unwanted or abnormal mammalian
cell proliferation. The compound and/or composition may be applied
locally to the proliferating cells, or may be administered to the
subject systemically. Preferably, the compound and/or composition
is administered to a subject that has a disorder characterized by
unwanted or abnormal cell proliferation. Such disorders include,
but are not limited to, cancer, epithelial precancerous lesions,
non-cancerous angiogenic conditions or arteriosclerosis.
[0030] In a final aspect, the invention provides a method for the
treatment and/or prevention of diseases that are characterized by
unwanted and/or abnormal mammalian cell proliferation. The method
involves administering a prophylactically or therapeutically
effective amount of at least one compound according to the
invention, or a pharmaceutical composition thereof, to a subject in
need of such treatment. Diseases that are characterized by abnormal
mammalian cell proliferation which can be treated or prevented by
way of the methods of the invention include, but are not limited
to, cancer, blood vessel proliferative disorders, fibrotic
disorders and arteriosclerotic conditions.
[0031] 3.1 Definitions
[0032] As used herein, the following terms shall have the following
meanings:
[0033] "Alkyl:" refers to a saturated branched, straight chain or
cyclic hydrocarbon radical. Typical alkyl groups include, but are
not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
t-butyl, pentyl, isopentyl, hexyl, and the like. In preferred
embodiments, the alkyl groups are (C.sub.1-C.sub.6) alkyl, with
(C.sub.1-C.sub.3) being particularly preferred.
[0034] "Substituted Alkyl:" refers to an alkyl radical wherein one
or more hydrogen atoms are each independently replaced with other
substituents. Typical substituents include, but are not limited to,
--OR, --SR, --NRR, --CN, --NO.sub.2, -halogen and -trihalomethyl,
where each R is independently --H, alkyl, alkenyl, alkynyl, aryl or
alkaryl as defined herein.
[0035] "Alkenyl:" refers to an unsaturated branched, straight chain
or cyclic hydrocarbon radical having at least one carbon-carbon
double bond. The radical may be in either the cis or trans
conformation about the double bond(s). Typical alkenyl groups
include, but are not limited to, ethenyl, propenyl, isopropenyl,
butenyl, isobutenyl, tert-butenyl, pentenyl, hexenyl and the like.
In preferred embodiments, the alkenyl group is (C.sub.1-C.sub.6)
alkenyl, with (C.sub.1-C.sub.3) being particularly preferred.
[0036] "Substituted Alkenyl:" refers to an alkenyl radical wherein
one or more hydrogen atoms are each independently replaced with
other substituents. Typical substituents include, but are not
limited to, --OR, --SR, --NRR, --CN, --NO.sub.2, -halogen and
-trihalomethyl, where each R is independently --H, alkyl, alkenyl,
alkynyl, aryl or alkaryl as defined herein.
[0037] "Alkynyl:" refers to an unsaturated branched, straight chain
or cyclic hydrocarbon radical having at least one carbon-carbon
triple bond. Typical alkynyl groups include, but are not limited
to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and
the like. In preferred embodiments, the alkynyl group is
(C.sub.1-C.sub.6) alkynyl, with (C.sub.1-C.sub.3) being
particularly preferred.
[0038] "Substituted Alkynyl:" refers to an alkynyl radical wherein
one or more hydrogen atoms are each independently replaced with
other substituents. Typical substituents include, but are not
limited to, --OR, --SR, --NRR, --CN, --NO.sub.2, -halogen and
-trihalomethyl, where each R is independently --H, alkyl, alkenyl,
alkynyl, aryl or alkaryl as defined herein.
[0039] "Aryl:" refers to an unsaturated cyclic hydrocarbon radical
having a conjugated .eta. electron system. Typical aryl groups
include, but are not limited to, penta-2,4-diene, phenyl, naphthyl,
anthracyl, azulenyl, indacenyl, and the like. In preferred
embodiments, the aryl group is (C.sub.5-C.sub.20) aryl, with
(C.sub.5-C.sub.10) being particularly preferred.
[0040] "Substituted Aryl:" refers to an aryl radical wherein one or
more hydrogen atoms are each independently replaced with other
substituents. Typical substituents include, but are not limited to,
--OR, --SR, --NRR, --CN, --NO.sub.2, -halogen and -trihalomethyl
where each R is independently --H, alkyl, alkenyl, alkynyl, aryl or
alkaryl as defined herein.
[0041] "Aryleno:" refers to an aryl radical that is capable of
fusing to another aryl group. Typical aryleno groups include, but
are not limited to, benzeno, naphthaleno, anthracaleno and the
like. In preferred embodiments, the aryleno group is
(C.sub.6-C.sub.20) aryleno.
[0042] "Substituted Aryleno:" refers to an aryleno group wherein
one or more hydrogen atoms are each independently replaced with
other substituents. Typical substituents include, but are not
limited to, --OR, --SR, --NRR, --CN, --NO.sub.2, -halogen and
-trihalomethyl, where each R is independently --H, alkyl, alkenyl,
alkynyl, aryl or alkaryl as defined herein.
[0043] "Alkaryl:" refers to a straight-chain alkyl, alkenyl or
alkynyl group wherein one of the hydrogen atoms bonded to a
terminal carbon is replaced with an aryl moiety. Typical alkaryl
groups include, but are not limited to, benzyl, benzylidene,
benzylidyne, benzenobenzyl, naphthalenobenzyl and the like. In
preferred embodiments, the alkaryl group is (C.sub.6-C.sub.26)
alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl
group is (C.sub.1-C.sub.6) and the aryl moiety is
(C.sub.5-C.sub.20). In particularly preferred embodiments the
alkaryl group is (C.sub.6-C.sub.13), i.e., the alkyl, alkenyl or
alkynyl moiety of the alkaryl group is (C.sub.1-C.sub.3) and the
aryl moiety is (C.sub.5-C.sub.10).
[0044] "Substituted Alkaryl:" refers to an alkaryl radical wherein
one or more hydrogen atoms on the aryl moiety of the alkaryl group
are each independently replaced with other substituents. Typical
substituents include, but are not limited to, --OR, --SR, --NRR,
--CN, --NO.sub.2, -halogen and -trihalomethyl, where each R is
independently --H, alkyl, alkenyl, alkynyl, aryl or alkaryl as
defined herein.
[0045] "In Situ:" refers to and includes the terms"in vivo," "ex
vivo," and"in vitro" as these terms are commonly recognized and
understood by persons ordinarily skilled in the art. Moreover, the
phrase"in situ" is employed herein in its broadest connotative and
denotative contexts to identify an entity, cell or tissue as found
or in place, without regard to its source or origin, its condition
or status or its duration or longevity at that location or
position.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 provides a reaction scheme for synthesizing
11-aryl-5,6-dihydro-11H-dibenz[b,e]azepine compounds according to
the invention; and
[0047] FIG. 2 provides a reaction scheme for synthesizing
11-aryl-11-substituted-5,6-dihydro-dibenz[b,e]azepine compounds
according to the invention.
5. DETAILED DESCRIPTION OF THE INVENTION
[0048] As discussed in the Background section, blockade of sickle
dehydration via inhibition of the Gardos channel is a powerful
therapeutic approach towards the treatment and/or prevention of
sickle cell disease. Studies have shown that antimycotic agents
such as Clotrimazole block Ca.sup.2+-activated K.sup.+ transport
and reduce cell dehydration in sickle erythrocytes in vitro
(Brugnara et al., 1993, J. Clin. Invest. 92:520-526), and also
inhibit the Gardos channel of erythrocytes, increase red cell
K.sup.+ content, decrease the mean cell hemoglobin concentration
(MCHC) and decrease red cell density in vivo in a transgenic mouse
model for sickle cell disease (SAD mouse, Trudel et al., 1991, EMBO
J. 11:3157-3165; De Franceschi et al., 1994, J. Clin. Invest.
93:1670-1676). Moreover, therapy with oral Clotrimazole induces
inhibition of the Gardos channel and reduces erythrocyte
dehydration in human patients with sickle cell disease (Brugnara et
al., 1996, J. Clin. Invest. 97:1227-1234). Other antimycotic agents
which inhibit the Gardos channel in vitro include miconazole,
econazole, butoconazole, oxiconazole and sulconazole (U.S. Pat. No.
5,273,992 to Brugnara et al.). All of these compounds contain an
imidazole-like ring, i.e., a heteroaryl ring containing two or more
nitrogens.
[0049] Also as discussed in the Background section, the modulation
of early ionic mitogenic signals and inhibition of cell
proliferation are powerful therapeutic approaches towards the
treatment and/or prevention of disorders characterized by abnormal
cell proliferation. It has been shown that Clotrimazole, in
addition to inhibiting the Gardos channel of erythrocytes, also
modulates ionic mitogenic signals and inhibits cell proliferation
both in vitro and in vivo.
[0050] For example, Clotrimazole inhibits the rate of cell
proliferation of normal and cancer cell lines in a reversible and
dose-dependent manner in vitro (Benzaquen et al., 1995 Nature
Medicine 1:534-540). Clotrimazole also depletes the intracellular
Ca.sup.2+ stores and prevents the rise in cystolic Ca.sup.2+ that
normally follows mitogenic stimulation. Moreover, in mice with
severe combined immunodeficiency disease (SCID) and inoculated with
MM-RU human melanoma cells, daily administration of Clotrimazole
resulted in a significant reduction in the number of lung
metastases observed (Benzaquen et al., supra).
[0051] It has now been discovered that substituted 11-phenyl
dibenzazepine compounds also inhibit the Gardos channel of
erythrocytes and/or mammalian cell proliferation. Thus, in one
aspect, the present invention provides a new class of organic
compounds that are capable of inhibiting the Ca.sup.2+-activated
potassium channel (Gardos channel) of erythrocytes, particularly
sickle erythrocytes and/or of inhibiting mammalian cell
proliferation, particularly mitogen-induced cell proliferation.
[0052] The discovery that 11-phenyl dibenzazepine compounds inhibit
the Gardos channel and/or mammalian cell proliferation was quite
surprising. Significantly, the compounds of the invention do not
contain an imidazole or imidazole-like moiety. The imidazole or
imidazole-like moiety is well-recognized as the essential
functionality underlying the antimycotic and other biological
activities of Clotrimazole and the other above-mentioned
anti-mycotic agents. Thus, the 11-phenyl dibenzazepine compounds of
the invention provide an entirely new class of compounds that are
capable of effecting inhibition of the Gardos channel and/or
mammalian cell proliferation and that are therefore Useful for the
treatment of sickle cell disease and/or diseases related to
abnormal or unwanted cell proliferation.
[0053] In another aspect, the invention provides a method of
reducing sickle cell dehydration and/or delaying the occurrence of
erythrocyte sickling in situ as a therapeutic approach towards the
treatment of sickle cell disease. In its broadest sense, the method
involves only a single step--the administration of at least one
pharmacologically active compound of the invention, or a
composition thereof, to a sickle erythrocyte in situ in an amount
effective to reduce dehydration and/or delay the occurrence of cell
sickling or deformation.
[0054] While not intending to be bound by any particular theory, it
is believed that administration of the active compounds described
herein in appropriate amounts to sickle erythrocytes in situ causes
nearly complete inhibition of the Gardos channel of sickle cells,
thereby reducing the dehydration of sickle cells and/or delaying
the occurrence of cell sickling or deformation. In a preferred
embodiment, the dehydration of a sickle cell is reduced and/or the
occurrence of sickling is delayed in a sickle cell that is within
the microcirculation vasculature of the subject, thereby reducing
or eliminating the vaso-occlusion that is commonly caused by
sickled cells.
[0055] Based in part on the surmised importance of the Gardos
channel as a therapeutic target in the treatment of sickle cell
disease, the invention is also directed to methods of treating or
preventing sickle cell disease. In the method, an effective amount
of one or more compounds according to the invention, or a
pharmaceutical composition thereof, is administered to a patient
suffering from sickle cell disease. The methods may be used to
treat sickle cell disease prophylactically to decrease
intracellular Hb S concentration and/or polymerization, and thus
diminish the time and duration of red cell sickling and
vaso-occlusion in the blood circulation. The methods may also be
used therapeutically in patients with acute sickle cell crisis, and
in patients suffering chronic sickle cell episodes to control both
the frequency and duration of the crises.
[0056] The compounds of the invention are also potent, specific
inhibitors of mammalian cell proliferation. Thus, in another
aspect, the invention provides methods of inhibiting mammalian cell
proliferation as a therapeutic approach towards the treatment or
prevention of diseases characterized by unwanted or abnormal cell
proliferation. In its broadest sense, the method involves only a
single step--the administration of an effective amount of at least
one pharmacologically active compound according to the invention to
a mammalian cell in situ. The compound may act cytostatically,
cytotoxically, or by a combination of both mechanisms to inhibit
cell proliferation. Mammalian cells treatable in this manner
include vascular smooth muscle cells, fibroblasts, endothelial
cells, various pre-cancer cells and various cancer cells. In a
preferred embodiment, cell proliferation is inhibited in a subject
suffering from a disorder that is characterized by unwanted or
abnormal cell proliferation. Such diseases are described more fully
below.
[0057] Based in part on the surmised role of mammalian cell
proliferation in certain diseases, the invention is also directed
to methods of treating or preventing diseases characterized by
abnormal cell proliferation. In the method, an effective amount of
at least one compound according to the invention, or a
pharmaceutical composition thereof, is administered to a patient
suffering from a disorder that is characterized by abnormal cell
proliferation. While not intending to be bound. by any particular
theory, it is believed that administration of an appropriate amount
of a compound according to the invention to a subject inhibits cell
proliferation by altering the ionic fluxes associated with early
mitogenic signals. Such alteration of ionic fluxes is thought to be
due to the ability of the compounds of the invention to inhibit
potassium channels of cells, particularly Ca.sup.2+-activated
potassium channels. The method can be used prophylactically to
prevent unwanted or abnormal cell proliferation, or may be used
therapeutically to reduce or arrest proliferation of abnormally
proliferating cells. The compound, or a pharmaceutical formulation
thereof, can be applied locally to proliferating cells to arrest or
inhibit proliferation at a desired time, or may be administered to
a subject systemically to arrest or inhibit cell proliferation.
[0058] Diseases which are characterized by abnormal cell
proliferation that can be treated or prevented by means of the
present invention include blood vessel proliferative disorders,
fibrotic disorders, arteriosclerotic disorders and various
cancers.
[0059] Blood vessel proliferation disorders refer to angiogenic and
vasculogenic disorders generally resulting in abnormal
proliferation of blood vessels. The formation and spreading of
blood vessels, or vasculogenesis and angiogenesis, respectively,
play important roles in a variety of physiological processes such
as embryonic development, corpus luteum formation, wound healing
and organ regeneration. They also play a pivotal role in cancer
development. Other examples of blood vessel proliferative disorders
include arthritis, where new capillary blood vessels invade the
joint and destroy cartilage and ocular diseases such as diabetic
retinopathy, where new capillaries in the retina invade the
vitreous, bleed and cause blindness and neovascular glaucoma.
[0060] Another example of abnormal neovascularization is that
associated with solid tumors. It is now established that
unrestricted growth of tumors is dependent upon angiogenesis and
that induction of angiogenesis by liberation of angiogenic factors
can be an important step in carcinogenesis. For example, basic
fibroblast growth factor (bFGF) is liberated by several cancer
cells and plays a crucial role in cancer angiogenesis. The
demonstration that certain animal tumors regress when angiogenesis
is inhibited has provided the most compelling evidence for the role
of angiogenesis in tumor growth. Other cancers that are associated
with neovascularization include hemangioendotheliomas, hemangiomas
and Kaposi's sarcoma.
[0061] Proliferation of endothelial and vascular smooth muscle
cells is the main feature of neovascularization. The invention is
useful in inhibiting such proliferation, and therefore in
inhibiting or arresting altogether the progression of the
angiogenic condition which depends in whole or in part upon such
neovascularization. The invention is particularly useful when the
condition has an additional element of endothelial or vascular
smooth muscle cell proliferation that is not necessarily associated
with neovascularization. For example, psoriasis may additionally
involve endothelial cell proliferation that is independent of the
endothelial cell proliferation associated with neovascularization.
Likewise, a solid tumor which requires neovascularization for
continued growth may also be a tumor of endothelial or vascular
smooth muscle cells. In this case, growth of the tumor cells
themselves, as well as the neovascularization, is inhibited by the
compounds described herein.
[0062] The invention is also useful for the treatment of fibrotic
disorders such as fibrosis and other medical complications of
fibrosis which result in whole or in part from the proliferation of
fibroblasts. Medical conditions involving fibrosis (other than
atherosclerosis, discussed below) include undesirable tissue
adhesion resulting from surgery or injury.
[0063] Other cell proliferative disorders which can be treated by
means of the invention include arteriosclerotic conditions.
Arteriosclerosis is a term used to describe a thickening and
hardening of the arterial wall. An arteriosclerotic condition as
used herein means classical atherosclerosis, accelerated
atherosclerosis, atherosclerotic lesions and any other
arteriosclerotic conditions characterized by undesirable
endothelial and/or vascular smooth muscle cell proliferation,
including vascular complications of diabetes.
[0064] Proliferation of vascular smooth muscle cells is a main
pathological feature in classical atherosclerosis. It is believed
that liberation of growth factors from endothelial cells stimulates
the proliferation of subintimal smooth muscle which, in turn,
reduces the caliber and finally obstructs the artery. The invention
is useful in inhibiting such proliferation, and therefore in
delaying the onset of, inhibiting the progression of, or even
halting the progression of such proliferation and the associated
atherosclerotic condition.
[0065] Proliferation of vascular smooth muscle cells produces
accelerated atherosclerosis, which is the main reason for failure
of heart transplants that are not rejected. This proliferation is
also believed to be mediated by growth factors, and can ultimately
result in obstruction of the coronary arteries. The invention is
useful in inhibiting such obstruction and reducing the risk of, or
even preventing, such failures.
[0066] Vascular injury can also result in endothelial and vascular
smooth muscle cell proliferation. The injury can be caused by any
number of traumatic events or interventions, including vascular
surgery and balloon angioplasty. Restenosis is the main
complication of successful balloon angioplasty of the coronary
arteries. It is believed to be caused by the release of growth
factors as a result of mechanical injury to the endothelial cells
lining the coronary arteries. Thus, by inhibiting unwanted
endothelial and smooth muscle cell proliferation, the compounds
described herein can be used to delay, or even avoid, the onset of
restenosis.
[0067] Other atherosclerotic conditions which can be treated or
prevented by means of the present invention include diseases of the
arterial walls that involve proliferation of endothelial and/or
vascular smooth muscle cells, such as complications of diabetes,
diabetic glomerulosclerosis and diabetic retinopathy.
[0068] The compounds described herein are also potent
antineoplastic agents and are therefore useful in treating or
preventing various types of neoplastic diseases. Neoplastic
diseases which can be treated by means of the present invention
include, but are not limited to, biliary tract cancer; brain
cancer, including glioblastomas and medulloblastomas; breast
cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial
cancer; esophageal cancer; gastric cancer; hematological neoplasms,
including acute and chronic lymphocytic and myelogenous leukemia,
multiple myeloma, AIDS associated leukemias and adult T-cell
leukemia lymphoma; intraepithelial neoplasms, including Bowen's
disease and Paget's disease; liver cancer; lung cancer; lymphomas,
including Hodgkin's disease and lymphocytic lymphomas;
neuroblastomas; oral cancer, including squamous cell carcinoma;
ovarian cancer, including those arising from epithelial cells,
stromal cells, germ cells and mesenchymal cells; pancreas cancer;
prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma,
rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin
cancer, including melanoma, Kaposi's sarcoma, basocellular cancer
and squamous cell cancer; testicular cancer, including germinal
tumors (seminoma, non-seminoma (teratomas, choriocarcinomas)),
stromal tumors and germ cell tumors; thyroid cancer, including
thyroid adenocarcinoma and medullar carcinoma; and renal cancer
including adenocarcinoma and Wilms tumor.
[0069] The compounds of the invention are useful with hormone
dependent and also with nonhormone dependent cancers. They also are
useful with prostate and breast cancers. They further are useful
with multidrug resistant strains of cancer.
[0070] In addition to the particular disorders enumerated above,
the invention is also useful in treating or preventing
dermatological diseases including keloids, hypertrophic scars,
seborrheic dermatosis, papilloma virus infection (e.g., producing
verruca vulgaris, verruca plantaris, verruca plan, condylomata,
etc.), eczema and epithelial precancerous lesions such as actinic
keratosis; other inflammatory diseases including proliferative
glomerulonephritis; lupus erythematosus; scleroderma; temporal
arthritis; thromboangiitis obliterans; mucocutaneous lymph node
syndrome; and other pathologies mediated by growth factors
including uterine leiomyomas.
[0071] The compounds and methods of the invention provide myriad
advantages over agents and methods commonly used to treat sickle
cell disease and/or cell proliferative disorders. The compounds and
methods of the invention also provide myriad advantages over the
treatment of sickle cell disease and/or cell proliferative
disorders with Clotrimazole or other antimycotic agents. For
example, many of the compounds of the invention are more potent
than Clotrimazole in in vitro tests, and therefore may provide
consequential therapeutic advantages in clinical settings.
[0072] Most significantly, the compounds of the invention have
reduced toxicity as compared with Clotrimazole and other
antimycotic agents. For Clotrimazole, it is well-known that the
imidazole moiety is responsible for inhibiting a wide range of
cytochrome P-450 isozyme catalyzed reactions, which constitutes
their main toxicological effects (Pappas and Franklin, 1993,
Toxicology 80:27-35; Matsuura et al., 1991, Biochemical
Pharmacology 41:1949-1956). Analogues and metabolites of
Clotrimazole do not induce cytochrome P-450 (Matsuura et al., 1991,
Biochemical Pharmacology 41:1949-1956), and therefore do not share
Clotrimazole's toxicity.
[0073] 5.1 The Compounds
[0074] The compounds which are capable of inhibiting the Gardos
channel and/or mammalian cell proliferation according to the
invention are generally substituted 11-phenyl dibenzazepine
compounds. In one illustrative embodiment, the compounds capable of
inhibiting the Gardos channel and/or mammalian cell proliferation
according to the invention are compounds having the structural
formula: 2
[0075] wherein:
[0076] R.sub.1 is --R', (C.sub.6-C.sub.20) aryl or substituted
(C.sub.6-C.sub.20) aryl;
[0077] R.sub.2 is --R', --OR', --SR', halogen or trihalomethyl;
[0078] R.sub.3 is --R', --OR', --SR', halogen or trihalomethyl or,
when taken together with R.sub.4, is (C.sub.6-C.sub.20)
aryleno;
[0079] R.sub.4 is --R', --OR', --SR', halogen or trihalomethyl or,
when taken together with R.sub.3, is (C.sub.6-C.sub.20)
aryleno;
[0080] each of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9,
R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14 is
independently selected from the group consisting of --R', halogen
and trihalomethyl;
[0081] R.sub.15 is --R", --C(O)R", --C(S)R", --C(O)OR", --C(S)OR",
--C(O)SR", --C(S)SR", --C(O)N(R").sub.2, --C(S)N(R").sub.2,
--C(O)C(O)R", --C(S)C(O)R", --C(O)C(S)R", --C(S)C(S)R",
--C(O)C(O)OR", --C(S)C(O)OR", --C(O)C(S)OR", --C(O)C(O)SR",
--C(S)C(S)OR", --C(S)C(O)SR", --C(O)C(S)SR", --C(S)C(S)SR",
--C(O)C(O)N(R").sub.2, --C(S)C(O)N(R").sub.2, --C(O)C(S)N(R").sub.2
or --C(S)C(S)N(R").sub.2,
[0082] each R' is independently selected from the group consisting
of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl and
(C.sub.1-C.sub.6) alkynyl;
[0083] each R" is independently selected from the group consisting
of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl,
(C.sub.1-C.sub.6) alkynyl, (C.sub.6-C.sub.20) aryl,
(C.sub.6-C.sub.20) substituted aryl, (C.sub.6-C.sub.26) alkaryl and
substituted (C.sub.6-C.sub.26) alkaryl; and
[0084] the aryl and alkaryl substituents are each independently
selected from the group consisting of --CN, --OR', --SR',
--NO.sub.2, --NR'R', halogen, (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl and
trihalomethyl.
[0085] In a preferred embodiment of the invention, the compounds
are those of structure (I) wherein the chalcogens are each
oxygen.
[0086] In another preferred embodiment, the compounds are those of
structure (I) wherein the halogens are each independently --F,
--Cl, --Br or --I.
[0087] In another preferred embodiment, the alkyl, alkenyl and
alkynyl groups are each independently (C.sub.1,-C.sub.3) and/or the
aryl groups are phenyl and/or the aryleno groups are benzeno.
[0088] In another preferred embodiment, R.sub.5, R.sub.6, R.sub.7,
R .sub.9, R.sub.10,R.sub.11 and R.sub.13 are each independently
--R'.
[0089] In another preferred embodiment, the substituted aryl and
alkaryl are mono-substituted.
[0090] In another preferred embodiment, R.sub.15 is --R", --C(O)R",
--C(O)OR", --C(O)N(R").sub.2, --C(O)C(O)R", --C(O)C(O)OR" or
--C(O)C(O)N(R").sub.2.
[0091] In another preferred embodiment of the invention, the
compounds are those of structural formula (I) wherein:
[0092] R.sub.1 is --R' or (C.sub.6-C.sub.20) aryl;
[0093] R.sub.2 is --R' or --OR';
[0094] R.sub.3 is --R' or --OR' or, when taken together with
R.sub.4, is (C.sub.6-C.sub.20) aryleno;
[0095] R.sub.4 is --R' or --OR' or, when taken together with
R.sub.3, is (C.sub.6-C.sub.20) aryleno;
[0096] each of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9,
R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14 is
independently selected from the group consisting of --R' and
halogen;
[0097] R.sub.15 is --R", --C(O)R", --C(O)OR", --C(O)N(R").sub.2,
--C(O)C(O)R", --C(O)C(O)OR" or --C(O)C(O)N(R").sub.2;
[0098] each R' is independently selected from the group consisting
of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl and
(C.sub.1-C.sub.6) alkynyl;
[0099] each R" is independently selected from the group consisting
of --H, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl,
(C.sub.1-C.sub.6) alkynyl, (C.sub.6-C.sub.20) aryl, substituted
(C.sub.6-C.sub.20) aryl, (C.sub.6-C.sub.26) alkaryl and substituted
(C.sub.6-C.sub.26) alkaryl; and
[0100] the aryl and alkaryl substituents are each independently
selected from the group consisting of --CN, --OR', --NO.sub.2,
--NR'R', halogen, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6)
alkenyl and (C.sub.1-C.sub.6) alkynyl.
[0101] In another preferred embodiment, the compounds are those of
formula (I) wherein:
[0102] R.sub.1 is --R' or (C.sub.6-C.sub.10) aryl;
[0103] R.sub.2 is --R' or --OR';
[0104] R.sub.3 is --R' or --OR' or, when taken together with
R.sub.14 is (C.sub.6-C.sub.10) aryleno;
[0105] R.sub.4 is --R' or --OR' or, when taken together with
R.sub.3, is (C.sub.6-C.sub.10) aryleno;
[0106] each of R.sub.5, R.sub.6 and R.sub.7 is --H;
[0107] R.sub.8 is --R', --F, --Cl, --Br or --I;
[0108] each of R.sub.9, R.sub.10 and R.sub.11 is --H;
[0109] R.sub.12 is --R', --F, --Cl, --Br or --I;
[0110] R.sub.13 is --H;
[0111] R.sub.14 is --R', --F, --Cl, --Br or --I;
[0112] R.sub.15 is --R", --C(O)R", --C(O)OR", --C(O)N(R").sub.2,
--C(O)C(O)R", --C(O)C(O)OR" or --C(O)C(O)N(R").sub.2;
[0113] each R' is independently selected from the group consisting
of --H, (C.sub.1-C.sub.3) alkyl, (C.sub.1-C.sub.3) alkenyl and
(C.sub.1-C.sub.3) alkynyl;
[0114] each R" is independently selected from the group consisting
of --H, (C.sub.1-C.sub.3) alkyl, (C.sub.1-C.sub.3) alkenyl,
(C.sub.1-C.sub.3) alkynyl, (C.sub.6-C.sub.10) aryl, substituted
(C.sub.6-C.sub.10) aryl, (C.sub.6-C.sub.13) alkaryl or substituted
(C.sub.6-C.sub.13) alkaryl; and
[0115] the aryl and alkaryl substituents are each independently
selected from the group consisting of --OR',--NO.sub.2, --NR'R',
--F, --Cl, --Br, --I, (C.sub.1-C.sub.3) alkyl, (C.sub.1-C.sub.3)
alkenyl and (C.sub.1-C.sub.3) alkynyl.
[0116] In still another preferred embodiment, the compounds are
those of structural formula (I) wherein:
[0117] R.sub.1 is --R' or phenyl;
[0118] R.sub.2 is --R' or --OR';
[0119] R.sub.3 is --R' or --OR' or, when taken together with
R.sub.4, is benzeno;
[0120] R.sub.4 is --R' or --OR' or, when taken together with
R.sub.3, is benzeno;
[0121] each of R.sub.5, R.sub.6 and R.sub.7 is --H;
[0122] R.sub.9 is --R', --Cl or --Br;
[0123] each of R.sub.9, R.sub.10 and R.sub.11 is --H;
[0124] R.sub.12 is --R', --F or --Cl;
[0125] R.sub.13 is --H;
[0126] R.sub.14 is --R' or --Cl;
[0127] R.sub.15 is --R", --C(O)R", --C(O)OR", --C(O)NHR",
--C(O)C(0)R" or --C(O)C(O)OR";
[0128] each R' is independently selected from the group consisting
of --H, (C.sub.1-C.sub.3) alkyl, (C.sub.1-C.sub.3) alkenyl and
(C.sub.1-C.sub.3) alkynyl;
[0129] each R" is independently selected from the group consisting
of --H, (C.sub.1-C.sub.3) alkyl, (C.sub.1-C.sub.3) alkenyl,
(C.sub.1-C.sub.3) alkynyl, (C.sub.6-C.sub.10) aryl,
mono-substituted (C.sub.6-C.sub.10) aryl, (C.sub.6-C.sub.13)
alkaryl or mono-substituted (C.sub.6-C.sub.13) alkaryl; and
[0130] the aryl and alkaryl substituents are each independently
selected from the group consisting of --OR', --NO.sub.2, --NR'R',
--Cl, (C.sub.1-C.sub.3) alkyl, (C.sub.1-C.sub.3) alkenyl and
(C.sub.l-C.sub.3)alkynyl.
[0131] In still another preferred embodiment, the compounds of the
invention are as follows: 34567891011
[0132] In yet another preferred embodiment, the compounds are those
of structural formula (I), with the proviso that when R.sub.1 and
R.sub.15 are each --R', at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.9, R.sub.10,R.sub.11,
R.sub.12, R.sub.13 or R.sub.14 is other than --R', R.sub.8 is other
than --R' or halogen and at least three of R.sub.2, R.sub.3,
R.sub.4 and R.sub.5 are other than --OR'.
[0133] In yet another preferred embodiment, the compounds are those
of structural formula (I), with the proviso that when R.sub.1 and
R.sub.15 are each --H, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.9, R.sub.10, R.sub.11,
R.sub.12, R.sub.13 or R.sub.14 is other than --H, R.sub.8 is other
than --H or --Cl and at least three of R.sub.2, R.sub.3, R.sub.4,
and R.sub.5 are other than --OCH.sub.3.
[0134] In a final preferred embodiment, the compounds of the
invention are not 11-phenyl-5,6-dihydro-11H-dibenz[b,e]azepine,
11-phenyl-9-halo-5,6-di- hydro-11H-dibenz[b,e]azepine,
11-phenyl-9-chloro-5,6-dihydro-11H-dibenz[b,- e]azepine,
11-phenyl-5,6-dihydro-1,2,3-trialkoxy-11H-dibenz[b,e]azepine,
11-phenyl-5,6-dihydro-1,2,3-trimethoxy-11H-dibenz[b,e]azepine,
11-phenyl-5,6-dihydro-2,3,4-trialkoxy-11H-dibenz[b,e]azepine and/or
11-phenyl-5,6-dihydro-2,3,4-trimethoxy-11H-dibenz[b,e]azepine.
[0135] The chemical formulae referred to herein may exhibit the
phenomena of tautomerism, conformational isomerism, stereo
isomerism or geometric isomerism. As the formulae drawings within
this specification can represent only one of the possible
tautomeric, conformational isomeric, enantiomeric or geometric
isomeric forms, it should be understood that the invention
encompasses any tautomeric, conformational isomeric, enantiomeric
or geometric isomeric forms which exhibit biological or
pharmacological activity as described herein.
[0136] The compounds of the invention may be in the form of free
acids, free bases or pharmaceutically effective salts thereof. Such
salts can be readily prepared by treating a compound with an
appropriate acid. Such acids include, by way of example and not
limitation, inorganic acids such as hydrohalic acids (hydrochloric,
hydrobromic, etc.), sulfuric acid, nitric acid, phosphoric acid,
etc.; and organic acids such as acetic acid, propanoic acid,
2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid,
propandioic acid, butandioic acid, etc. Conversely, the salt can be
converted into the free base form by treatment with alkali.
[0137] In addition to the above-described compounds and their
pharmaceutically acceptable salts, the invention may employ, where
applicable, solvated as well as unsolvated forms of the compounds
(e.g. hydrated forms).
[0138] The compounds described herein may be prepared by any
processes known to be applicable to the preparation of chemical
compounds. Suitable processes are well known in the art. Preferred
processes are illustrated by the representative examples.
Additional methods are described in copending application Ser. No.
______, entitled "SYNTHESIS OF
11-ARYL-5,6-DIHYDRO-11H[b,e]AZEPINES, filed concurrently herewith
(Attorney Docket No. PEL97-06), which is incorporated herein by
reference in its entirety. Necessary starting materials may be
obtained commercially or by standard procedures of organic
chemistry. Moreover, many of the compounds are commercially
available.
[0139] An individual compound's relevant activity and potency as an
agent to affect sickle cell dehydration or deformation and/or
mammalian cell proliferation may be determined using standard
techniques. Preferentially, a compound is subject to a series of
screens to determine its pharmacological activity.
[0140] In most cases, the active compounds of the invention exhibit
two pharmacological activities: inhibition of the Gardos channel of
erythrocytes and inhibition of mammalian cell proliferation.
However, in some cases, the compounds of the invention may exhibit
only one of these pharmacological activities. Any compound
encompassed by formula (I) which exhibits at least one of these
pharmacological activities is considered to be within the scope of
the present invention.
[0141] In general, the active compounds of the invention are those
which induce at least about 25% inhibition of the Gardos channel of
erythrocytes (measured at about 10 .mu.M) and/or about 25%
inhibition of mammalian cell proliferation (measured at about 10
.mu.M), as measured using in vitro assays that are commonly known
in the art (see, e.g., Brugnara et al., 1993, J. Biol. Chem.
268(12):8760-8768; Benzaquen et al., 1995, Nature Medicine
1:534-540). Alternatively, or in addition, the active compounds of
the invention generally will have an IC.sub.50 (concentration of
compound that yields 50% inhibition) for inhibition of the Gardos
channel of less than about 10 .mu.M and/or an IC.sub.50 for
inhibition of cell proliferation of less than about 10 .mu.M, as
measured using in vitro assays that are commonly known in the art
(see, e.g., Brugnara et al., 1993, J. Biol. Chem.
268(12):8760-8768; Benzaquen et al., 1995, Nature Medicine
1:534-540).
[0142] Representative active compounds according to the invention
include Compounds 1 through 35, as illustrated above.
[0143] In certain embodiments of the invention, compounds which
exhibit only one pharmacological activity, or a higher degree of
one activity, may be preferred. Thus, when the compound is to be
used in methods to treat or prevent sickle cell disease, or in
methods to reduce sickle cell dehydration and/or delay the
occurrence of erythrocyte sickling or deformation in situ, it is
preferred that the compound exhibit at least about 75% Gardos
channel inhibition (measured at about 10 .mu.M) and/or have an
IC.sub.50 for Gardos channel inhibition of less than about 1 .mu.M,
with at least about 90% inhibition and/or an IC.sub.50 of less than
about 0.1 .mu.M being particularly preferred. Even more preferred
are compounds which meet both the % inhibition and IC.sub.50
criteria.
[0144] Exemplary preferred compounds for use in methods related to
Gardos channel inhibition and sickle cell disease include Compounds
1, 2, 3, 4, 6, 9, 18, 29 and 35, with Compounds 2, 3, 4, 6, 9, 29
and 35 being particularly preferred.
[0145] When the compound is to be used in methods to treat or
prevent disorders characterized by abnormal cell proliferation or
in methods to inhibit cell proliferation in situ, it is preferable
that the compound exhibit at least about 75% inhibition of
mitogen-induced cell proliferation (measured at about 10 .mu.M)
and/or have an IC.sub.50 of cell proliferation of less than about
3.5 .mu.M, with at least about 90% inhibition and/or an IC.sub.50
of less than about 1 .mu.M being particularly preferred. Even more
preferred are compounds which meet both the % inhibition and
IC.sub.50 criteria.
[0146] Exemplary preferred compounds for use in methods of
inhibiting mammalian cell proliferation or for the treatment or
prevention of diseases characterized by abnormal cell proliferation
include Compounds 2, 3, 4, 7, 8, 9, 13, 14, 16, 17, 18, 19, 20, 21,
22, 26, 27, 28, 29, 30, 31, 32, 33 and 34, with Compounds 14, 26,
28, 29, 30 and 31 being particularly preferred.
[0147] 5.2 Formulation and Routes of Administration
[0148] The compounds described herein, or pharmaceutically
acceptable addition 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.
[0149] The compounds described herein, or pharmaceutically
acceptable salts and/or hydrates thereof, may be administered
singly, in combination with other compounds of the invention,
and/or in cocktails combined with other therapeutic agents. of
course, the choice of therapeutic agents that can be
co-administered with the compounds of the invention will depend, in
part, on the condition being treated.
[0150] For example, when administered to patients suffering from
sickle cell disease, the compounds of the invention can be
administered in cocktails containing agents used to treat the pain,
infection and other symptoms and side effects commonly associated
with sickle cell disease. Such agents include, e.g., analgesics,
antibiotics, etc. The compounds can also be administered in
cocktails containing other agents that are commonly used to treat
sickle cell disease, including butyrate and butyrate derivatives
(Perrine et al., 1993, N. Enql. J. Med. 328(2):81-86); hydroxyurea
(Charache et al., 1995, N. Enql. J. Med. 323(20):1317-1322);
erythropoietin (Goldberg et al, 1990, N. Enql. J. Med. 323(6):
366-372); and dietary salts such as magnesium (De Franceschi et
al., 1996, Blood 88(648a):2580).
[0151] When administered to a patient undergoing cancer treatment,
the compounds may be administered in cocktails containing other
anti-cancer agents and/or supplementary potentiating agents. The
compounds may also be administered in cocktails containing agents
that treat the side-effects of radiation therapy, such as
anti-emetics, radiation protectants, etc.
[0152] Anti-cancer drugs that can be co-administered with the
compounds of the invention include, e.g., Aminoglutethimide;
Asparaginase; Bleomycin; Busulfan; Carboplatin; Carmustine (BCNU);
Chlorambucil; Cisplatin (cis-DDP); Cyclophosphamide; Cytarabine
HCl; Dacarbazine; Dactinomycin; Daunorubicin HCl; Doxorubicin HCl;
Estramustine phosphate sodium; Etoposide (VP-16); Floxuridine;
Fluorouracil (5-FU); Flutamide; Hydroxyurea (hydroxycarbamide);
Ifosfamide; Interferon Alfa-2a, Alfa 2b, Lueprolide acetate
(LHRH-releasing factor analogue); Lomustine (CCNU); Mechlorethamine
HCI (nitrogen mustard); Melphalan; Mercaptopurine; Mesna;
Methotrexate (MTX); Mitomycin; Mitotane (o.p'-DDD); Mitoxantrone
HCl; Octreotide; Plicamycin; Procarbazine HCl; Streptozocin;
Tamoxifen citrate; Thioguanine; Thiotepa; Vinblastine sulfate;
Vincristine sulfate; Amsacrine (m-AMSA); Azacitidine;
Hexamethylmelamine (HMM); Interleukin 2; Mitoguazone (methyl-GAG;
methyl glyoxal bis-guanylhydrazone; MGBG); Pentostatin; Semustine
(methyl-CCNU); Teniposide (VM-26); paclitaxel and other taxanes;
and Vindesine sulfate.
[0153] Supplementary potentiating agents that can be
co-administered with the compounds of the invention include, e.g.,
Tricyclic anti-depressant drugs (e.g., imipramine, desipramine,
amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline,
protriptyline, amoxapine and maprotiline); non-tricyclic and
anti-depressant drugs (e.g., sertraline, trazodone and citalopram);
Ca.sup.+ antagonists (e.g., verapamil, nifedipine, nitrendipine and
caroverine); Amphotericin (e.g., Tween 80 and perhexiline maleate);
Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g.,
quinidine); antihypertensive drugs (e.g., reserpine); Thiol
depleters (e.g., buthionine and sulfoximine); and calcium
leucovorin.
[0154] The active compound(s) may be administered per se or in the
form of a pharmaceutical composition wherein the active compound(s)
is in admixture with one or more pharmaceutically 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 upon the
route of administration chosen.
[0155] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0156] For oral administration, the compounds can be formulated
readily by combining the active compound(s) 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 by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. 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,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0157] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0158] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0159] For buccal administration,the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0160] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0161] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0162] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0163] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0164] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0165] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation or
transcutaneous delivery (for example subcutaneously or
intramuscularly), intramuscular injection or a transdermal patch.
Thus, for example, the compounds may be formulated with suitable
polymeric or hydrophobic materials (for example as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0166] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0167] 5.3 Effective Dosages
[0168] Pharmaceutical compositions suitable for use with the
present invention include compositions wherein the active
ingredient is contained in a therapeutically effective amount,
i.e., in an amount effective to achieve its intended purpose. Of
course, the actual amount effective for a particular application
will depend, inter alia, on the condition being treated. For
example, when administered in methods to reduce sickle cell
dehydration and/or delay the occurrence of erythrocyte sickling or
distortion in situ, such compositions will contain an amount of
active ingredient effective to achieve this result. When
administered in methods to inhibit cell proliferation, such
compositions will contain an amount of active ingredient effective
to achieve this result. When administered to patients suffering
from sickle cell disease or disorders characterized by abnormal
cell proliferation, such compositions will contain an amount of
active ingredient effective to, inter alia, prevent the development
of or alleviate the existing symptoms of, or prolong the survival
of, the patient being treated. For use in the treatment of cancer,
a therapeutically effective amount further includes that amount of
compound which arrests or regresses the growth of a tumor.
Determination of an effective amount is well within the
capabilities of those skilled in the art, especially in light of
the detailed disclosure herein.
[0169] For any compound described herein the therapeutically
effective amount can be initially determined from cell culture
arrays. Target plasma concentrations will be those concentrations
of active compound(s) that are capable of inducing at least about
25% inhibition of the Gardos channel and/or at least about 25%
inhibition of cell proliferation in cell culture assays, depending,
of course, on the particular desired application. Target plasma
concentrations of active compound(s) that are capable of inducing
at least about 50%, 75%, or even 90% or higher inhibition of the
Gardos channel and/or cell proliferation in cell culture assays are
preferred. The percentage of inhibition of the Gardos channel
and/or cell proliferation in the patient can be monitored to assess
the appropriateness of the plasma drug concentration achieved, and
the dosage can be adjusted upwards or downwards to achieve the
desired percentage of inhibition.
[0170] Therapeutically effective amounts for use in humans can also
be determined from animal models. For example, a dose for humans
can be formulated to achieve a circulating concentration that has
been found to be effective in animals. A particularly useful animal
model for sickle cell disease is the SAD mouse model (Trudel et
al., 1991, EMBO J. 11:3157-3165). Useful animal models for diseases
characterized by abnormal cell proliferation are well-known in the
art. In particular, the following references provide suitable
animal models for cancer xenografts (Corbett et al., 1996, J. Exp.
Ther. Oncol. 1:95-108; Dykes et al., 1992, Contrib. Oncol. Basel.
Karqer 42:1-22), restenosis (Carter et al., 1994, J. Am. Coll.
Cardiol. 24(5):1398-1405), atherosclerosis (Zhu et al., 1994,
Cardiology 85(6):370-377) and neovascularization (Epstein et al.,
1987, cornea 6(4):250-257). The dosage in humans can be adjusted by
monitoring Gardos channel inhibition and/or inhibition of cell
proliferation and adjusting the dosage upwards or downwards, as
described above.
[0171] A therapeutically effective dose can also be determined from
human data for compounds which are known to exhibit similar
pharmacological activities, such as Clotrimazole and other
antimycotic agents (see, e.g., Brugnara et al., 1995, JPET
273:266-272; Benzaquen et al., 1995, Nature Medicine 1:534-540;
Brugnara et al., 1996, J. Clin. Invest. 97(5):1227-1234). The
applied dose can be adjusted based on the relative bioavailability
and potency of the administered compound as compared with
Clotrimazole.
[0172] Adjusting the dose to achieve maximal efficacy in humans
based on the methods described above and other methods as are
well-known in the art is well within the capabilities of the
ordinarily skilled artisan.
[0173] Of course, in the case of local administration, the systemic
circulating concentration of administered compound will not be of
particular importance. In such instances, the compound is
administered so as to achieve a concentration at the local area
effective to achieve the intended result.
[0174] For use in the prophylaxis and/or treatment of sickle cell
disease, including both chronic sickle cell episodes and acute
sickle cell crisis, a circulating concentration of administered
compound of about 0.001 .mu.M to 20 .mu.M is considered to be
effective, with about 0.1 .mu.M to 5 .mu.M being preferred.
[0175] Patient doses for oral administration of the compounds
described herein, which is the preferred mode of administration for
prophylaxis and for treatment of chronic sickle cell episodes,
typically range from about 80 mg/day to 16,000 mg/day, more
typically from about 800 mg/day to 8000 mg/day, and most typically
from about 800 mg/day to 4000 mg/day. Stated in terms of patient
body weight, typical dosages range from about 1 to 200 mg/kg/day,
more typically from about 10 to 100 mg/kg/day, and most typically
from about 10 to 50 mg/kg/day. Stated in terms of patient body
surface areas, typical dosages range from about 40 to 8000
mg/m.sup.2/day, more typically from about 400 to 4000
mg/m.sup.2/day, and most typically from about 400 to 2000
mg/m.sup.2/day.
[0176] For use in the treatment of disorders characterized by
abnormal cell proliferation, including cancer, arteriosclerosis and
angiogenic conditions such as restenosis, a circulating
concentration of administered compound of about 0.001 .mu.M to 20
.mu.M is considered to be effective, with about 0.1 .mu.M to 5
.mu.M being preferred.
[0177] Patient doses for oral administration of the compounds
described herein for the treatment or prevention of cell
proliferative disorders typically range from about 80 mg/day to
16,000 mg/day, more typically from about 800 mg/day to 8000 mg/day,
and most typically from about 800 mg/day to 4000 mg/day. Stated in
terms of patient body weight, typical dosages range from about 1 to
200 mg/kg/day, more typically from about 10 to 100 mg/kg/day, and
most typically from about 10 to 50 mg/kg/day. Stated in terms of
patient body surface areas, typical dosages range from about 40 to
8000 mg/m.sup.2/day, more typically from about 400 to 4000
mg/m.sup.2/day, and most typically from about 400 to 2000
mg/m.sup.2/day.
[0178] For other modes of administration, dosage amount and
interval can be adjusted individually to provide plasma levels of
the administered compound effective for the particular clinical
indication being treated. For example, if acute sickle crises
are-the most dominant clinical manifestation, a compound according
to the invention can be administered in relatively high
concentrations multiple times per day. Alternatively, if the
patient exhibits only periodic sickle cell crises on an infrequent
or periodic or irregular basis, it may be more desirable to
administer a compound of the invention at minimal effective
concentrations and to use a less frequent regimen of
administration. This will provide a therapeutic regimen that is
commensurate with the severity of the sickle cell disease
state.
[0179] For use in the treatment of tumorigenic cancers, the
compounds can be administered before, during or after surgical
removal of the tumor. For example, the compounds can be
administered to the tumor via injection into the tumor mass prior
to surgery in a single or several doses. The tumor, or as much as
possible of the tumor, may then be removed surgically. Further
dosages of the drug at the tumor site can be applied post removal.
Alternatively, surgical removal of as much as possible of the tumor
can precede administration of the compounds at the tumor site.
[0180] Combined with the teachings provided herein, by choosing
among the various active compounds and weighing factors such as
potency, relative bioavailability, patient body weight, severity of
adverse side-effects and preferred mode of administration, an
effective prophylactic or therapeutic treatment regimen can be
planned which does not cause substantial toxicity and yet is
entirely effective to treat the clinical symptoms demonstrated by
the particular patient. Of course, many factors are important in
determining a therapeutic regimen suitable for a particular
indication or patient. Severe indications such as cancer may
warrant administration of higher dosages as compared with less
severe indications such as sickle cell disease.
[0181] 5.4 Toxicity
[0182] The ratio between toxicity and therapeutic effect for a
particular compound is its therapeutic index and can be expressed
as the ratio between LD.sub.50 (the amount of compound lethal in
50% of the population) and ED.sub.50 (the amount of compound
effective in 50% of the population). Compounds which exhibit high
therapeutic indices are preferred. Therapeutic index data obtained
from cell culture assays and/or animal studies can be used in
formulating a range of dosages for use in humans. The dosage of
such compounds preferably lies within a range of plasma
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
(See e.g. Fingl et al., 1975, In: The Pharmacological Basis of
Therapeutics, 8Ch. 1 p1).
[0183] The invention having been described, the following examples
are intended to illustrate, not limit, the invention.
6. EXAMPLE
[0184] Compound Syntheses
[0185] This Example demonstrates general methods for synthesizing
the compounds of the invention, as well as preferred methods for
synthesizing certain exemplary compounds of the invention. In all
the reaction schemes described herein, suitable starting materials
are either commercially available or readily obtainable using
standard techniques of organic synthesis. Where necessary, suitable
groups and schemes for protecting the various functionalities are
well-known in the art, and can be found, for example, in Kocienski,
Protecting Groups, Georg Thieme Verlag, New York, 1994 and Greene
& Wuts, Protective Groups in Organic Chemistry, John Wiley
& Sons, New York, 1991.
[0186] In FIGS. 1 and 2, the various substituents are as defined
for structure (I).
[0187] 6.1 Synthesis of 11-Aryl-5,6-dihydro-11H-dibenz[b,e]
azepines
[0188] This example provides a general method for synthesizing
substituted 11-aryl-5,6-dihydro-11H-dibenz[b,e]azepine compounds
according to the invention. A general reaction scheme is provided
in FIG. 1. In FIG. 1, R.sub.2-R.sub.15 are as previously defined
for structural formula (I).
[0189] Referring to FIG. 1, a mixture of an appropriately
substituted 2-aminobenzophenone 100 (1 equivalent), an
appropriately substituted benzyl chloride 102 (1 equivalent),
potassium carbonate (2 equivalents) and sodium iodide (1
equivalent) in acetonitrile is refluxed for 12 hours. The reaction
mixture is cooled to room temperature and water added. The mixture
is extracted with ethyl acetate. The combined ethyl acetate
extracts are washed with water then dried over sodium sulfate.
Evaporation of the solvent followed by column chromatography gives
the substituted N-alkyl-2-aminobenzophenone derivative 104 in about
55-80% yield.
[0190] The substituted N-alkyl-2-aminobenzophenone derivative 104
(1 equivalent) is dissolved in a 3:1 mixture of
tetrahydrofuran:methanol. Sodium borohydride (10 equivalents) is
slowly added and the reaction mixture is stirred at room
temperature for 12 hours. The reaction is quenched by adding 2 N
aqueous hydrochloric acid solution. The reaction mixture is
neutralized by adding 4 N aqueous sodium hydroxide solution and
extracted with ethyl acetate. The combined ethyl acetate extracts
are dried over sodium sulfate. Evaporation of the solvent followed
by column chromatography gives the substituted
N-alkyl-2-amino-benzyl alcohol derivative 106 in about 40-60%
yield.
[0191] A mixture of the substituted N-alkyl-2-amino-benzylalcohol
derivative 106 (1 equivalent), phosphorous pentoxide (5
equivalents) and methanesulfonic acid (5 equivalents) in
dichloromethane is stirred at room temperature for 12 hours. The
mixture is neutralized by adding aqueous sodium carbonate and then
extracted with dichloromethane. The organic solution is dried over
sodium sulfate. Evaporation of the solvent followed by column
chromatography gives the substituted
11-aryl-5,6-dihydro-11H-dibenz[b,e]azepine derivative 108 in about
45-70% yield.
[0192] The substituted 11-aryl-5,6-dihydro-11H-dibenz[b,e]azepine
derivative 108 (1 equivalent) is combined with potassium carbonate
(3.5 equivalents) and alkyl or acyl halide (3 equivalents) in
acetonitrile and stirred at room temperature for two days. Water is
added and the mixture is stirred for 15 min. at room temperature
and extracted with ethyl acetate. Evaporation of the solvent gives
the crude product as an oil. Tituration of the product from ethanol
followed by washing with hexane gives the pure N-substituted
11-aryl-5,6-dihydro-11H-dibenz[b,e]azepine product 110 as a white
solid in 30-80% yield.
[0193] Alternatively, the substituted
11-aryl-5,6-dihydro-11H-dibenz[b,e]a- zepines can be synthesized
from appropriate starting materials according to the methods
described in Sasakura and Sugasawa, 1981, Heterocycles
15:421-425.
[0194] 6.2 Synthesis of
11-Aryl-11-substituted-5,6-dihydro-dibenz[b,e]azep- ines
[0195] This example provides a general method for synthesizing
11-aryl-11-substituted-5,6-dihydro-dibenz[b,e]azepine compounds
according to the invention. A general reaction scheme is provided
in FIG. 2. In FIG. 2, R.sub.1-R.sub.15 are as previously defined
for structural formula (I).
[0196] Referring to FIG. 2, the substituted
N-alkyl-2-aminobenzophenone derivative 104 is prepared as described
in Section 6.1, supra. To a solution (0.25 M) of an approriate
grignard reagent in diethyl ether at -40.degree. C. is added a
solution (0.1 M) of the substituted N-alkyl-2-aminobenzophenone
derivative 104 in diethyl ether. The mixture is stirred at
-40.degree. C. for 30 min., warmed to room temperature and quenched
with water. Extraction with ethyl acetate and evaporation of the
solvent gives the crude alcohol product 112 as an oil. The alcohol
product 112 is purified by column chromatography to give the pure
alcohol product 112 as a white solid in 85-90% yield.
[0197] Compound 112 (1 equivalent) is dissolved in dichloromethane
to 0.5-1.0 M. Phosphorous pentoxide (4 equivalents) and methane
sulfonic acid (4 equivalents) are added and the mixture is stirred
at room temperature for 2 hours. The reaction is quenched with
saturated aqueous sodium bicarbonate and extracted with ethyl
acetate. Evaporation of the solvent followed by column
chromatography gives the
11-aryl-11-substituted-5,6-dihydro-dibenz[b,e]azepine 114 in about
90% yield.
[0198] The 11-aryl-11-substituted-5,6-dihydro-dibenz[b,e]azepine
114 can be converted to the corresponding
N-substituted-11-aryl-11-substituted-5,-
6-dihydro-dibenz[b,e]azepine 116 as described in Section 6.1,
supra.
[0199] 6.3 Synthesis of N-Methoxycarbonyl
-11-(2'-chlorophenyl)-5,6-dihydr- o-11H-dibenz[b,e]azepine
(Compound 9)
[0200] A preferred method of synthesis of N-methoxycarbonyl
-11-(2'-chlorophenyl)-5,6-dihydro-11H-dibenz[b,e]azepine (Compound
9) is as follows: A mixture of 0.3 g (0.00098 mole) of
11-(2'-chlorophenyl)-5,6- -dihydro-11H-dibenz[b,e]azepine, 1.08 g
(0.0078 mole) of potassium carbonate and 1.54 g (0.016 mole) of
methyl chloroformate in 10 mL of acetonitrile, was refluxed for 12
hours. The mixture was then allowed to cool to room temperature and
stirred with 15 mL of water for 10 minutes. The reaction mixture
was extracted with ethyl acetate (2.times.15 mL). The organic layer
was dried over magnesium sulfate. Evaporation gave the crude
product as a brown solid. Trituration of the crude product with
ethanol and washing the obtained solid with hexane gave 0.172 g
(48% yield) of a white solid having a melting point of
159-161.degree. C.
[0201] The product. gave the following analytical data: NMR
(CDCl.sub.3): .delta.3.10 ppm (3H, s, OCH.sub.3); .delta. 4.45 ppm
(1H, d, J=10 Hz, CH.sub.2N); .delta.5.48 ppm (1H, s, CH);
.delta.5.82 ppm (1H, d, J=10 Hz, CH.sub.2N); .delta.6.94 ppm (1H,
m, aryl); .delta.7.10 ppm (4H, m, aryl); .delta.7.28 ppm (6H, m,
aryl); .delta.7.69 ppm (1H, m, aryl).
[0202] 6.4 Synthesis of
N-Phenoxycarbonyl-11-phenyl-5,6-dihydro-11H-dibenz- [b,e]azepine
(Compound 14)
[0203] A preferred method of synthesis of
N-phenoxycarbonyl-11-phenyl-5,6-- dihydro-11H-dibenz[b,e]azepine
(Compound 14) is as follows: A mixture of 0.25 g (0.00092 mole) of
11-phenyl-5,6-dihydro-11H-dibenz[b,e]azepine, 0.318 g (0.0023 mole)
of potassium carbonate and 0.318 g (0.002 mole) of phenyl
chloroformate in 10 mL of acetonitrile, was stirred at room
temperature for 2 days. The mixture was stirred with 15 mL of water
for 15 minutes and then extracted with ethyl acetate (2.times.35
mL). The organic layer was dried over magnesium sulfate.
Evaporation gave the crude product as an oily material. Trituration
of the obtained oil with ethanol then washing it with hexane gave
0.285 g (80% yield) of a white solid having a melting point of
155-165.degree. C.
[0204] The product gave the following analytical data: NMR
(CDCl.sub.3): .delta.4.46 ppm (1H, d, J=7 Hz, CH.sub.2N);
.delta.5.28 ppm (1H, s, CH); .delta.5.69 ppm (1H, d, J=7 Hz,
CH.sub.2N); .delta.6.52 ppm (2H, m, aryl); .delta.6.98 ppm (2H, m,
aryl); .delta.7.14-7.42 ppm (13H, m, aryl); .delta.7.58 (1H, m,
aryl).
[0205] 6.5 Synthesis of
N-Phenoxycarbonyl-11-(2'-chlorophenyl)-5,6-dihydro-
-11H-dibenz[b,e]azepine (Compound 26)
[0206] A preferred method of synthesis of N-phenoxycarbonyl
-11-(2'-chlorophenyl)-5,6-dihydro-11H-dibenz[b,e]azepine (Compound
26) is as follows: A mixture of 0.2 g (0.00065 mole) of
11-(2'-chlorophenyl)-5,6- -dihydro-11H-dibenz[b,e]azepine, 0.18 g
(0.0013 mole) of potassium carbonate and 0.204 g (0.0013 mole) of
phenyl chloroformate in 10 mL of acetonitrile, was stirred at room
temperature for 2 days. The mixture was stirred with 15 mL of water
for 10 minutes and then extracted with ethyl acetate (2.times.35
mL). The organic layer was dried over magnesium sulfate, filtered
and the solvent was evaporated. Trituration of the obtained residue
with ethanol then washing it with hexane gave 0.082 g (30% yield)
of a white solid having a melting point of 95-99.degree. C.
[0207] The product gave the following analytical data: NMR
(CDCl.sub.3): .delta.4.50 ppm (1H, d, J=7 Hz, CH.sub.2N);
.delta.5.58 ppm (1H, s, CH); .delta.5.80 ppm (1H, d, J=7 Hz,
CH.sub.2N); .delta.6.52 ppm (2H, m, aryl); .delta.7.06-7.38 ppm
(14H, m, aryl); .delta.7.79 ppm (1H, m, aryl).
[0208] 6.6 Synthesis of
N-(4'-Nitrobenzoyl)-11-(2'-chlorophenyl)-5,6-dihyd-
ro-11H-dibenz[b,e]azepine (Compound 28)
[0209] A preferred method of synthesis of
N-(4'-nitrobenzoyl)-11-(2'-chlor-
ophenyl)-5,6-dihydro-11H-dibenz[b,e]azepine (Compound 28) is as
follows: A mixture of 0.2 g (0.00065 mole) of
11-(2'-chlorophenyl)-5,6-dihydro-11H-d- ibenz[b,e]azepine, 0.179 g
(0.0013 mole) of potassium carbonate and 0.133 g (0.00072 mole) of
4-nitrobenzoyl chloride in 10 mL of acetonitrile, was stirred at
room temperature for 12 hours. The mixture was stirred with 15 mL
of water for 10 minutes. The reaction mixture was extracted with
ethyl acetate (2.times.15 mL). The organic layer was dried over
magnesium sulfate. Evaporation gave the crude product as a sticky
solid. Trituration of the crude product with ethanol and washing
the obtained solid with hexane gave 0.148 g (50% yield) of a white
solid having a melting point of 178-181.degree. C.
[0210] The product gave the following analytical data: NMR
(CDCl.sub.3): .delta.4.42 ppm (1H, d, J=7 Hz, CH.sub.2N);
.delta.5.68 ppm (1H, s, CH); .delta.6.36 ppm (1H, d, J=7 Hz,
CH.sub.2N); .delta.6.52 ppm (3H, m, aryl); .delta.7.06 ppm (3H, m,
aryl); .delta.7.12 ppm (1H, m, aryl); .delta.7.26 ppm (6H, m,
aryl); .delta.7.79 ppm (3H, m, aryl).
[0211] 6.7 Other Compounds
[0212] Other compounds of the invention can be synthesized by
routine modification of the above-described syntheses, or by other
methods that are well known in the art. Appropriate starting
materials are commercially available or can be synthesized using
routine methods.
7. EXAMPLE
[0213] In Vitro Activity
[0214] This Example demonstrates the ability of several exemplary
compounds of formula (I) to inhibit the Gardos channel of
erythrocytes (Gardos channel assay) and/or mitogen-induced cell
proliferation (mitogenic assay) in vitro. The assays are generally
applicable for demonstrating the in vitro activity of other
compounds of formula (I).
[0215] 7.1 Experimental Protocol
[0216] The percent inhibition of the Gardos channel (10 .mu.M
compound) and the IC.sub.50 were determined as described in
Brugnara et al., 1993, J. Biol. Chem. 268(12):8760-8768. The
percent inhibition of mitogen-induced cell proliferation (10 .mu.M
compound) and the IC.sub.50 were determined as described in
Benzaquen et al. (1995, Nature Medicine 1:534-540) with NIH 3T3
mouse fibroblast cells (ATCC No. CRL 1658). Other cell lines, e.g.,
cancer cells, endothelial cells and fibroblasts, as well as many
others, may be used in the cell proliferation assay. Selection of a
particular cell line will depend in part on the desired
application, and is well within the capabilities of an ordinarily
skilled artisan.
[0217] 7.2 Results
[0218] The results of the experiment are provided in TABLE 1,
below. Clotrimazole is reported for purposes of comparison.
1TABLE 1 IN VITRO DATA FOR EXEMPLARY COMPOUNDS Mitogenic Assay
Gardos Channel Assay IC.sub.50 Inhibition IC.sub.50 Inhibition
Compound (.mu.M) (%) (.mu.M) (%) Clotrimazole 0.626 93.0 0.046 99.3
(1) 56.0 0.775 75.2 (2) 5.20 99.0 1.30 99.0 (3) 2.40 99.0 0.886
97.4 (4) 1.5 89.0 0.384 98.1 (5) 91.0 >10.0 14.4 (6) 87.0 0.236
97.5 (7) 1.60 99.0 >10.0 35.8 (8) 2.20 84.0 0 (9) 2.10 99.0
0.0850-0.093 97.3 (10) 53.0 2.533-1.940 63.0 (11) 32.0 >10.0 9.5
(12) 13.0 >10.0 54.8 (13) 1.7 97.0 0 (14) 0.04 98.0 >10.0
14.8 (15) 40.0 >10.0 9.50 (16) 1.7 99.0 >10.0 0.45 (17) 1.6
99.0 >10.0 20.6 (18) 2.6 99.0 0.502-0.692 81.5 (19) 1.6 99.0
>10.0 52.0 (20) 1.7 95.0 >10.0 13.6 (21) 2.7 93.0 >10.0
2.1 (22) 3.6 99.0 >10.0 14.9 (23) 55.0 >10.0 18.2 (24) 89.0
>10.0 32-55 (25) 75.0 >10.0 8.5 (26) 0.04-0.90 99.0 >10.0
0.8 (27) 2.20 99.0 >10.0 3.0 (28) 0.04-0.50 99.0 0 (29) 0.800
99.0 0.414-0.433 95.1 (30) 0.600 99.0 >10 14.6 (31) 0.400 99.0
>10 12.3 (32) 1.100 99.0 0 (33) 2.400 99.0 >10 67.5 (34) 4.00
99.0 >10 12.0 (35) 0 0.071-0.099 98.3
8. EXAMPLE
[0219] Activity in Cancer Cell Lines
[0220] This Example demonstrates the antiproliferative effect of
several exemplary compounds of formula (I) against a variety of
cancer cell lines. The assays are generally applicable for
demonstrating the antiproliferative activity of other compounds of
formula (I).
[0221] 8.1 Growth of Cells
[0222] The antiproliferative assays described herein were performed
using standard aseptic procedures and universal precautions for the
use of tissues. Cells were propagated using RPMI 1640 media (Gibco)
containing 2% or 5% fetal calf serum (FCS) (Biowhittaker) at
37.degree. C., 5% CO.sub.2 and 95% humidity. The cells were
passaged using Trypsin (Gibco). Prior to addition of test compound,
the cells were harvested, the cell number counted and seeded at
10,000 cells/well in 100 .mu.l 5% fetal calf serum (FCS) containing
RPMI medium in 96-well plates and incubated overnight at 37.degree.
C., 5% CO.sub.2 and 95% humidity.
[0223] On the day of the treatment, stock solutions of the test
compounds (10 mM compound/DMSO) were added in 100 .mu.l FCS
containing medium to a final concentration of 10-0.125 .mu.M and
the cells were incubated for 2, 3 or 5 days at 37.degree. C., 5%
CO.sub.2 and 95% humidity.
[0224] Following incubation, the cellular protein was determined
with the Sulforhodamine B (SRB) assay (Skehan et al., 1990, J.
Natl. Cancer Inst. 82:1107-1112). Growth inhibition, reported as
the concentration of test compound which inhibited 50% of cell
proliferation (IC.sub.50) was determined by curve fitting.
[0225] Values for VP-16, a standard anti-cancer agent, are provided
for comparison.
[0226] Except for MMRU cells, all cancer cell lines tested were
obtained from the American Type Culture Collection (ATCC,
Rockville, Md.). The ATCC assession numbers were as follows: HeLa
(CCL-2); CaSki (CRL-1550); MDA-MB-231 (HTB-26); MCF-7 (HTB-22);
A549 (CCL-185); HTB-174 (HTB-174); HEPG2 (HB-8065); DU-145
(HTB-81); SK-MEL-28 (HTB-72); HT-29 (HTB-38); HCT-15 (CCL-225);
ACHN (CRL-1611); U-118MG (HTB-15); SK-OV-3 (HTB-77).
[0227] MMRU cells (Stender et al., 1993, J. Dermatology 20:611-617)
were a gift of one of the authors.
[0228] 8.2 Results
[0229] The results of the cell culture assays are presented in
TABLES 2 and 3, below.
2TABLE 2 RESULTS OF SRB ASSAY 5% FCS/5 DAY INCUBATION Cancer Cell
Test Compound IC.sub.50 (.mu.M) Type Line VP-16 13 16 17 20 21 22
23 27 28 30 31 33 34 35 Cervical HeLa <1.25 0.6 0.2 0.3 0.5 0.2
CaSki 1.8 0.9 0.9 0.5 0.7 0.5 Breast MDA-MB-23 <1.25 >1 >1
0.5 0.9 0.4 MCF7 <1.25 2.7 4.2 4.5 >1 3.5 1.8 3.1 0.5 0.6 1.3
0.7 2.1 2 2.4 Lung A549 <1.25 3.1 >5 >5 >1 4.2 1.9 3.4
1.1 0.8 1.5 0.6 3.1 >5 >5 HTB174 <1.25 1.4 2.6 3.6 0.7 1.9
1.4 1.8 0.3 0.1 0.5 0.2 1.4 0.9 4 Hepatocel HEPG2 <1.25 1.8 3.4
2.6 0.9 1.9 1.4 2.3 0.4 0.3 0.9 0.4 1.4 1.5 4.5 Prostate DU-145
<1.25 >1 >1 0.9 >1 0.9 Melanoma SK-MEL-28 <1.25 0.9
0.7 0.4 0.6 0.3 MMRU <1.25 1.5 2.7 2.5 0.5 1.5 0.9 2.2 0.2 0.2
0.5 0.2 1.3 1.2 3.1 Colon HT29 <1.25 1.5 2.6 1.7 0.7 1.8 0.7 2.1
0.4 0.3 0.6 0.2 1.2 1.6 4.8 HCT-15 1.3 0.9 0.4 0.4 0.5 0.3 Renal
ACHN <1.25 >1 >1 0.7 >1 0.8 CNS U118MG 2.2 0.9 0.4 0.5
0.8 0.4 Ovary SK-OV-3 >1 >1 >1 >1 0.6 Normal HUVEC
<1.25 >1 >1 0.7 >1 0.8 human GM 1.4 >1 >1 0.9
>1 >1 3T3 >1 >1 >1 >1 >1 mouse L929 <1.25
0.9 >1 0.6 >1 0.9
[0230]
3TABLE 3 RESULTS OF SRB ASSAY Conditions Test Compound IC.sub.50
(.mu.M) Value in Cell Lines Compound % FCS/days A549 HT29 MMRU MCF7
HEPG2 U118MG VP-16 2%/3 days 2.3 20 <2.5 <2.5 4 2%/3 days
11.4 9.7 4.6 1.7 5 5%/2 days 1.4 1 >10 6 5%/2 days >10 >10
10 7 5%/2 days >10 >10 >10 14 5%/2 days >10 10 >10
26 5%/3 days 7 7.2 4.9 5.9 5.4 5.7 27 5%/3 days <1.25 <1.25
<1.25 <1.25 <1.25 1.9 28 5%/3 days 1.8 <1.25 <1.25
2.1 <1.25 1.3 29 5%/2 days >10 >10 >10 30 5%/3 days 1.7
<1.25 <1.25 1.6 <1.25 <1.25 31 5%/3 days <1.25
<1.25 <1.25 <1.25 <1.25 <1.25 32 5%/3 days 6.9 6.3
4.9 5.4 6.2 6.8 33 5%/3 days 6.9 6.3 4.9 5.4 6.2 6.8 34 5%/2 days
>10 6.8 >10 35 5%/2 days >10 10 >10
[0231] As can be seen in Tables 2 and 3, compounds which exhibited
significant activity in the mitogenic assay described in Section 7,
supra, exhibit significant antiproliferative activity against a
variety of cancer cell lines and cancer types (IC.sub.50 of less
than about 10 .mu.M).
[0232] Many of the compounds exhibit comparable or even greater
antiproliferative activity against a variety of cancer cell types
than VP-16, a known anti-cancer agent.
9. EXAMPLE
[0233] Formulations
[0234] The following examples provide exemplary, not limiting,
formulations for administering the compounds of the invention to
mammalian, especially human, patients. Any of the compounds
described herein, or pharmaceutical salts or hydrates thereof, may
be formulated as provided in the following examples.
[0235] 9.1 Tablet Formulation
[0236] Tablets each containing 60 mg of active ingredient are made
up as follows:
4 Active Compound 60 mg Starch 45 mg Microcrystalline 45 mg
Cellulose Sodium carboxymethyl 4.5 mg starch Talc 1 mg
Polyvinylpyrrolidone 4 mg (10% in water) Magnesium Stearate 0.5 mg
150 mg
[0237] The active ingredient, starch and cellulose are passed
through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution
of polyvinylpyrrolidone is mixed with the resultant powders which
are then passed through a No. 14 mesh U.S. sieve. The granules are
dried at 50.degree.-60.degree. C. and passed through a No. 18 mesh
U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and
talc, previously passed through a No. 60 mesh U.S. sieve, are then
added to the granules, which, after mixing are compressed by a
tablet machine to yield tablets each weighing 150 mg.
[0238] Tablets can be prepared from the ingredients listed by wet
granulation followed by compression.
[0239] 9.2 Gelatin Capsules
[0240] Hard gelatin capsules are prepared using the following
ingredients:
5 Active Compound 250 mg/capsule Starch dried 200 mg/capsule
Magnesium Stearate 10 mg/capsule
[0241] The above ingredients are mixed and filled into hard gelatin
capsules in 460 mg quantities.
[0242] 9.3 Aerosol Solution
[0243] An aerosol solution is prepared containing the following
components:
6 Active Compound 0.25% (w/w) Ethanol 29.75% (w/w) Propellant 22
77.00% (w/w) (Chlorodifluoromethane)
[0244] The active compound is mixed with ethanol and the mixture
added to a portion of the propellant 22, cooled to -30.degree. C.
and transferred to a filling device. The required amount is then
fed to a stainless steel container and diluted with the remainder
of the propellant. The valve units are then fitted to the
container.
[0245] 9.4 Suppositories
[0246] Suppositories each containing 225 mg of active ingredient
are made as follows:
7 Active Compound 225 mg Saturated fatty acid 2,000 mg
glycerides
[0247] The active ingredient is passed through a No. 60 mesh U.S.
sieve and suspended in the saturated fatty acid glycerides
previously melted using the minimum heat necessary. The mixture is
then poured into a suppository mold of nominal 2 g capacity and
allowed to cool.
[0248] 9.5 Suspensions
[0249] Suspensions each containing 50 mg of medicament per 5 mL
dose are made as follows:
8 Active Compound 50 mg Sodium 50 mg carboxymethylcellulose Syrup
1.25 mL Benzoic acid solution 0.10 mL Flavor q.v. Color q.v.
Purified water to 5 mL
[0250] The active ingredient is passed through a No. 45 mesh U.S.
sieve and mixed with the sodium carboxymethyl cellulose and syrup
to form a smooth paste. The benzoic acid solution, flavor and some
color are diluted with some of the water and added, with stirring.
Sufficient water is then added to produce the required volume.
[0251] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the above-described modes for
carrying out the invention which are obvious to those skilled in
the pharmaceutical arts or related fields are intended to be within
the scope of the following claims.
[0252] All cited references are hereby incorporated in their
entireties by reference herein.
* * * * *