Note: Descriptions are shown in the official language in which they were submitted.
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USE OF SUBSTITUTED DIPHENYL INDANONE, INDANE AND INDOLE COMPOUNDS FOR THE
TREATMENT OR
PREVENTION OF SICKL&CELL DISEASE, INFLAMMATORY DISEASES CHARACTERIZED BY
ABNORMAL CELL
PROLIFERATION, DIARRHE AND SCOURS
Field of the Invention
The present invention relates to aromatic organic compounds which are
specific,
potent and safe inhibitors of the Caz+-activated potassium channel (Gardos
channel) of
1 o erythrocytes, of mammalian cell proliferation, and/or of secretagogue-
stimulated
transepithelial electrogenic chloride secretion in intestinal cells. 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
15 therapeutic approach towards the treatment or prevention of diseases
characterized by
abnormal cell proliferation. Furthermore, the compounds can be used to inhibit
chloride
secretion as a therapeutic approach towards the treatment of diarrhea and
scours.
Background of the Invention
2o 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
25 state is the consequence of a single nucleotide substitution (Marotta et
al., 1977, ~. Biol.
Chem. 252:5040-5053).
The major source of morbidity and mortality of patients suffering from sickle
cell
disease is vascular occlusion caused by the sickled cells, which causes
repeated episodes of
pain in both acute and chronic form and also causes ongoing organ damage with
the passage
30 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, l~ ood 70:1245. The intracellular
gelatin and
polymerization of Hb S can occur at any time during erythrocyte's journey
through the
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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.
The probability of each of these events is determined by the delay time for
intracellular gelation relative to the appropriate capillary transit time
(Eaton ~., 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.
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.
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
2o 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 a 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, l~rit J. Haematol. ~~:1-4).
3o 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
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distilled water (Gye et al., 1973, Am. J. Med. Sci. xøø: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. 0:1138-1143; Charache and Walker, 1981, Blood
5$:892-896);
the use of monensin to increase the canon content of the sickle cell (Clark et
al., 1982, J.J. Clin.
Invest. 70:1074-1080; Fahim and Pressman, 1981, ~~e Sciences x:1959-1966);
intravenous
administration of cetiedil citrate (Benjamin et al., 1986, Blood ~1:1442-1447;
Berkowitz and
Ornnger, 1984, Am. J. Hematol. x:217-223; Stuart et al., 1987, J. Clin.
Pathol.
4Q,:1182-1186); and the use of oxpentifylline (Stuart et al., 1987, J. Clin.
Pathol.
4_Q:1182-1186).
1o Another approach towards therapeutically treating dehydrated sickle cells
involves the
administration of imidazole, nitroimidazole and triazole antimycotic agents
such as
Clotrimazole (U.S. Patent 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 prevents Ca2~-dependent
dehydration of sickle
cells both in vitro and in vivo (Brugnara et al., 1993, J. Clin. Invest. x:520-
526; De
Franceschi et al., 1994, J. Clin. Invest. x: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. $x:820-823). Other compounds that
contain a
2o 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.
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.
3o 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
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diseases, fibrotic conditions and arteriosclerotic conditions.
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 X4:161-164). These include
( 1 ) a rapid
increase in cystolic Ca2+, mostly due to rapid release of Ca2+ from
intracellular stores; (2)
capacitative Caz+ influx in response to opening of ligand-bound and
hyperpolarization-
sensitive Caz+ channels in the plasma membrane that contribute further to
increased
to intracellular Ca2+ concentration (Tsien and Tsien, 1990, Annu. Rev. Cell
Biol. 6_:715-760;
Peppelenbosch et al., 1991, J. Biol. Chem. 26:19938-19944); and (3) activation
of Caz+-
dependent K+ channels in the plasma membrane with increased K+ conductance and
membrane hyperpolarization {Magni et al., 1991, ,1. Biol. Chem. x: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.
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. Clotrimazole has been
shown to inhibit
2o the Ca2+-activated potassium channel of erythrocytes. In addition,
Clotrimazole inhibits
voltage- and ligand-stimulated Ca2+ influx mechanisms in nucleated cells
(Villalobos et al.,
1992, FASEB J.J. x:2742-2747; Montero et al., 1991, Biochem. J. ?71:73-79) and
inhibits cell
proliferation both in vitro and in vivo (Benzaquen et al., 1995, Nature
Medicine x:534-540).
Recently, Clotrimazole and other imidazole-containing antimycotic agents
capable of
inhibiting Ca2+-activated potassium channels have been shown to be useful in
the treatment of
arteriosclerosis (U.S. Patent No. 5,358,959 to Halperin et al.), as well as
other disorders
characterized by unwanted or abnormal cell proliferation.
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
3o 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.
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Acute and chronic diarrheas represent a major medical problem in many areas of
the
world. Diarrhea is both a significant factor in malnutrition and the leading
cause of death
(5,000,000 deaths/year) in children less than five years old. Secretory
diarrheas are also a
dangerous condition in patients of acquired imrnunodeficiency syndrome (AIDS}
and chronic
inflammatory bowel disease (IBD). 16 million travelers to developing countries
from
industrialized nations every year develop diarrhea, with the severity and
number of cases of
diarrhea varying depending on the country and area of travel. The major
medical
consequences of diarrheal diseases include dehydration, acidosis, death and
impaired growth.
Diarrhea in barn animals and pets such as cows, pigs and horses, sheep, goats,
cats and
l0 dogs, also known as scours, is a major cause of death in these animals.
Diarrhea can result
from any major transition, such as weaning or physical movement. One form of
diarrhea is
characterized by diarrhea in response to a bacterial or viral infection and
generally occurs
within the first few hours of the animal's life.
Although the major consequences of diarrheal diseases are very similar, there
are
numerous causes of diarrhea. Secretory and exudative diarrhea are primarily
caused by
bacterial or viral infections. The most common diarrheal causing bacteria is
enterotoxogenic
E-coli (ETEC) having the K99 pilus antigen. Common viral causes of diarrhea
include
rotavirus and coronavirus. Other infectious agents include cryptosporidium,
giardia lamblia,
and salmonella, among others.
2o The treatment for diarrhea depends on the patient and the infection source.
Diarrhea
which is found in travelers to industrialized nations (travelers diarrhea)
frequently is caused
by bacterial pathogens which are acquired through ingestion of fecally
contaminated food
and/or water. Approximately 50-75% of these cases are attributed to ETEC.
Although
traveler's diarrhea is painful, it is generally not life-threatening and often
the symptoms last
only 3-5 days. The symptoms include urgent diarrhea, abdominal cramps, nausea
and fever.
The most effective course of treatment for traveler's diarrhea is the
administration of
antibiotics in conjunction with oral rehydration. It has been shown that
prophylactic
administration of antibiotics drastically reduces the number of travelers
experiencing
symptoms of diarrhea. However, routine administration of antibiotics is not
suggested as it
3o may cause resistant strains of a bacteria to develop. Other treatment
methods include
administration of bismuth subsalicylate, often taken in the form of Pepto-
Bismal,
diphenoxylate and loperamide.
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Diarrhea in AIDS patients is a very serious condition which causes wasting and
may
be an important factor in the decline of these patients. AIDS patients often
develop diarrhea
due to enteric infections which their immune system is not capable of fighting
off, but AIDS
patients may also develop diarrhea by AIDS enteropathy. AIDS enteropathy is a
disorder
characterized by diarrhea without the involvement of secondary infections. It
is caused by the
human immunodeficiency virus (HIV) infection of the small bowel mucosal cells
and colonic
mucosal cells. The most common infective agent causing diarrhea due to enteric
infection in
AIDS patients in cryptosporidium. The methods for treating diarrhea in AIDS
patients
include administration of antibiotics and administration of immunoglobulins or
an
1 o immunoglobulin enriched fraction of bovine colostrum. Colostrum, which is
the first milk
produced by mammals after birthing is enriched with antibodies.
Acute diarrhea or scours, is a main cause of death in many newborn barn
animals such
as calves and pigs. Scours is often caused by ETEC with a K99 pilus antigen.
Infection with
the ETEC causes hypersecretion of fluid and electrolytes. Hypersecretion in
turn causes
~ 5 dehydration and pH imbalance which may result in death of the newborn calf
or pig.
Newborn barn animals are also susceptible to viral infectious agents causing
scours.
Infections with rotavirus and coronavirus are common in newborn calves and
pigs. Rotavirus
infection often occurs within 12 hours of birth. Symptoms of rotaviral
infection include
excretion of watery feces, dehydration and weakness. Coronavirus which causes
a more
2o severe illness in the newborn animals, has a higher mortality rate than
rotaviral infection.
Often, however, a young animal may be infected with more than one virus or
with a
combination of viral and bacterial microorganisms at one time. This
dramatically increases
the severity of the disease.
Generally the best protection for a newborn barn animal from viral or
bacterial
25 infection is the consumption of colostrum. If the mother animal has been
exposed to these
infectious agents then the colostrum will contain antibodies, which are often
sufficient to
protect the newborn from contracting the diseases. Sometimes, however, this is
not sufficient
and the animals need further protection. A common method of treatment includes
administration of a concentrated colostrum solution or an immunoglobulin
fraction isolated
3o from a colostrum solution. This oral treatment may be combined with
rehydration salts.
Although these methods have improved the morbidity and mortality rate of
newborn animals
having scours, there still exists a need for more effective treatments.
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_7_
Certain imidazoles such as clotrimazole are agents which have been used both
topically and systemically as antifungals. More recently, studies have
identified other uses for
such imidazoles. U.S. patent no. 5,273,992 revealed that these imidazoles
regulate Ca++
activated K+ channels in erythrocytes, and are thus useful in treating sickle
cell anemia, which
involves the inhibition of potassium transport. These imidazoles have also
been found to be
effective in inhibiting endothelial and/or vascular smooth muscle cell
proliferation. The
results of this finding are described in U.S, patent no. 5,358,959 and U.S.
serial no.
08/018,840, which discloses using clotrimazole for treating atherosclerotic
and angiogenic
conditions, respectively. Nonimidazole metabolites and analogs of the
foregoing compounds
Io also have been described as useful in treating the foregoing conditions
(see U.S. serials nos.
08/307,874 and 08/307,887).
Su~v of the Invention
These and other objects are provided by the present invention, which in one
aspect
provides a class of organic compounds which are potent, selective and safe
inhibitors of the
Caz+-activated potassium channel (Gardos channel) of erythrocytes, of
mammalian cell
proliferation andlor of secretagogue-stimulated transepithelial electrogenic
chloride secretion
in intestinal cells. 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
2o 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
proliferation. Furthermore,
the compounds can also be used to inhibit chloride secretion in intestinal
cells as a therapeutic
approach towards the treatment of diarrhea and scours. The compounds are
generally
substituted 3,3-diphenyl indanone, indane or (3-I~ indole compounds, as well
as analogues
thereof.
In one illustrative embodiment, the compounds capable of inhibiting the Gardos
channel, mammalian cell proliferation and/or secretagogue-stimulated
transepithelial
electrogenic chloride secretion in intestinal cells according to the
invention, are compounds
3o having the structural formula:
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_g_
~R8)n
~R5)n
(1) 1R7)m ~ ~ I ~Y R4
or pharmaceutically acceptable salts or hydrates thereof, wherein:
mis0, 1,2,3or4;
1o each n is independently 0, l, 2, 3, 4 or 5;
XisCorN;
Y is absent, (C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl;
R, is absent, -OR, -SR, =O, =S, =N-OR, -O-C(O)R, -S-C(O)R, -O-C(S)R, -S-C(S)R,
or when taken together with RZ is a 3-8 membered heterocycloalkyl or a
substituted 3-8
membered heterocycloalkyi;
RZ is absent or -H;
R3 is absent or -H;
R4 is -H, -OR', -SR', -NR'z, -CN, -NO2, (C3-C8) cycloalkyl, 3-8 membered
heterocycloalkyl, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', -
C(O)NR'2 or
-C(S)NR'2;
each R5, R6 and R., is independently selected from the group consisting of -
halogen,
-R', -OR', -SR', -NR'2, -ONR'2, -SNR'2, -NO2, -CN, -C(O)R', -C(S)R', -C(O)OR',
-C(O)SR',
-C(S)OR', -CS(S)R', -C(O)NR'2, -C(S)NR'2, -C(O)NR'(OR'), -C(S)NR'(OR'); -
C(O)NR'(SR'),
-C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R']~, -CH[C(S)R']2, -CH[C(O)OR']z, -
CH[C(S)OR']Z,
-CH[C(O)SR']2 and -CH[C(S)SR']~;
each R is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (CS-CZa) aryl, substituted (CS-C2o) aryl,
(C6-Czb) alkaryl and
substituted (C6-Cz6) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group
3o consisting of -CN, -NO2, -NR'z, -OR', -C(O)NR'z, -C(S)NR'2, -C(O)OR', -
C(S)OR', -C(O)SR',
-C(S)SR' and trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group
R~ R2
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consisting of halogen, -C(O)R', -C(S)R', -C(O)OR', -C(S}OR', -C(O)SR', -
C{S)SR',
-C(O)NR'z, -C(S}NR'Z and trihalamethyl;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl, (C,-
C6) alkenyl and (C,-C6) alkynyl; and
--- designates a single or double bond.
In a preferred embodiment of the invention, the chalcogens in the compounds of
formula (I) are each oxygen.
In one embodiment, the substituents of the aromatic compounds of structural
formula
(I) are as follows: m is 0, 1, 2, 3 or 4; each n is independently 0, l, 2, 3,
4 or 5; X is C or N; Y
is absent, (C,-C6) alkyl, (C,-Cb) alkenyl or (C,-C6) alkynyl; R, is absent, -
OR, =O, =N-OR,
-O-C(O)R, or when taken together with RZ is a 3-8 membered oxirane or a
substituted 3-8
membered oxirane; RZ is absent or -H; R3 is absent or -H; Rq is -H, -OR', -
NR'2, -CN, -NOz,
(C3-C8) cycloalkyl, 3-8 membered oxiranyl, 5-8 membered dioxycycloalkyl, -
C(O)R',
-C(O)OR' or -C(O)NR'2; each R5, R.~ and R, is independently selected from the
group
15 consisting of -halogen, -R', -OR', -NR'2, -ONR'Z, -NO2, -CN, -C(O)R', -
C(O)OR', -C(O)NR'z,
-C(O)NR'(OR'), -CH(CN)2, -CH[C(O)R']2 and -CH[C(O)OR']Z; each R is
independently
selected from the group consisting of -H, (C,-C6) alkyl, {C,-C6) alkenyl, (C,-
C6) alkynyl, (CS-
CZO) aryl, substituted (CS-CZO) aryl, (C6-C26) alkaryl and substituted (C6-
Cz6) alkaryl; the
oxirane substituents are each independently selected from the group consisting
of -CN, -NOz,
20 -NR'2, -OR', -C(O)NR'2, -C(O)OR' and trihalamethyl; the aryl and alkaryl
substituents are
each independently selected from the group consisting of halogen, -C(O)R', -
C(O)OR',
-C(O)NR'2 and trihalomethyl; each R' is independently selected from the group
consisting of
-H, (C,-C6) alkyl, (C,-C6) alkenyl and (C,-C6) alkynyl; and/or --- designates
a single or double
bond.
25 In another preferred embodiment, the compounds are those of structural
formula (I)
wherein: m is 0 or l; each n is independently 0 or 1; X is C or N; Y is
absent, (C,-C3) alkyl,
(C,-C3) alkenyl or (C,-C3) alkynyl; R, is absent -H, -OR, =O, -NR2, =N-OR, -O-
C(O)R, or
when taken together with RZ is 3-5 membered oxirane or 3-5 membered
substituted oxirane;
RZ is absent or -H; R3 is absent or -H; R4 is -H, -OR, -NR2, -CN, -C(O)OR, -
C(O}NRZ or 5-6
3o membered dioxoycycloalkyl; each R5, Rs and R, is independently selected
from the group
consisting of -R', -F, -Cl or -Br; each R is independently selected from the
group consisting of
-H, (C,-C3) alkyl, (C,-C3) alkenyl, (C,-C3) alkynyi, (CS-C,o) aryl,
substituted (CS-C,o) aryl, (C6-
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C,3) alkaryl, substituted C6-C,3) alkaryl; the oxirane substituent is -CN, -
NO2, -NR'Z, -OR' and
trihalomethyl; the aryl and alkaryl substituents are each independently
selected from the group
consisting of -F, -Cl, -Br, -CN, -NO2, -NR'z, -C(O)R', -C(O)OR' and
trihalomethyl; R' is -H,
(C,-C3) alkyl, (C,-C3) alkenyl or (C,-C3) alkynyl; and/or --- is a single or
double bond.
In still another preferred embodiment, the compounds are those of structural
formula
(I) wherein: m is 0, 1, 2, 3 or 4; each n is independently 0, 1, 2, 3, 4 or S;
X is C or N; Y is
absent, (C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl; R, is absent, -OR, -
SR, =O, =S,
=N-OR, -O-C(O)R, -S-C(O)R, -O-C(S)R, -S-C(S)R, or when taken together with RZ
is a 3-8
membered heterocycloalkyl or a substituted 3-8 membered heterocycloalkyl; RZ
is absent or
-H; R3 is absent or -H; R4 is -H, -OR', -SR', -NR'z, -CN, -NO2, (C3-C8)
cycloalkyl, 3-8
membered heterocycloalkyl, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C{O)SR', -
C(S)SR',
-C(O)NR'z or -C(S)NR'z; each R5, R6 and R, is independently selected from the
group
consisting of -halogen, -R', -OR', -SR', -NR'z, -ONR'2, -SNR'2, -NO~, -CN, -
C(O)R', -C(S)R',
-C(O)OR', -C(O)SR', -C(S)OR', -CS(S)R', -C(O)NR'z, -C(S)NR'2, -C(O)NR'(OR'),
-C(S)NR'(OR'); -C(O)NR'(SR'), -C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R')Z, -
CH[C(S)R')z,
-CH[C(O)OR']2, -CH[C(S)OR']2, -CH[C(O)SR']2 and -CH[C(S)SR']2; each R is
independently selected from the group consisting of -H, {C,-C6) alkyl, (C,-C6)
alkenyl, (C,-C6)
alkynyl, (C5-CZO) aryl, substituted {C5-CZO) aryl, (C6-C26) alkaryl and
substituted {C6-C26)
alkaryl; the heterocycloalkyl substituents are each independently selected
from the group
consisting of -CN, -NO2, -NR'2, -OR', -C(O)NR'z, -C(S)NR'2, -C(O)OR', -
C(S)OR', -C(O)SR',
-C(S)SR' and trihalomethyl; the aryl and alkaryl substituents are each
independently selected
from the group consisting of halogen, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -
C(O)SR',
-C(S)SR', -C(O)NR'Z, -C{S)NR'2 and trihalomethyl; each R' is independently
selected from the
group consisting of -H, (C,-C6) alkyl, (C,-C6) alkenyl and (C,-C6) alkynyl; ---
designates a
single or double bond; and wherein when X is C and R, is =O, =S or -OR', at
least one of R5,
Rs or R~ is other than -R', preferably other than -H, or Y is present or R~ is
other than -H; and
when X is N, --- is a double bond and R,, R2, R3 and Y are absent, R4 is other
than -NR'z,
preferably other than -NH2.
In still another preferred embodiment, the compounds are those of structural
formula
(I) wherein: m is 0, 1, 2, 3 or 4; each n is independently 0, 1, 2, 3, 4 or 5;
X is C; Y is absent,
(C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl; R, is absent, -OR, -SR, =O,
=S, =N-OR,
-O-C(O)R, -S-C(O)R, -O-C(S)R, -S-C(S)R, or when taken together with R~ is a 3-
8
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membered heterocycloalkyl or a substituted 3-8 membered heterocycloalkyl; RZ
is absent or
-H; R3 is absent or -H; R4 is -H, -OR', -SR', -NR'Z, -CN, -NO2, (C3-Cg)
cycloalkyl, 3-8
membered heterocycloalkyl, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -
C(S)SR',
-C(O)NR'2 or -C(S)NR'2; each R5, Itfi and R, is independently selected from
the group
consisting of -halogen, -R', -OR', -SR', -NR'z, -ONR'Z, -SNR'2, -NO2, -CN, -
C(O)R', -C(S)R',
-C(O)OR', -C(O)SR', -C(S)OR', -CS(S)R', -C{O)NR'2, -C(S)NR'z, -C(O)NR'(OR'),
-C(S)NR'(OR'); -C(O)NR'(SR'), -C(S)NR'(SR'), -CH(CN)~, -CH[C(O)R']2, -
CH[C(S)R']2,
-CH[C(O)OR'J2, -CH[C(S)OR']2, -CH[C(O)SR']2 and -CH[C(S)SR']2; each R is
independently selected from the group consisting of -H, (C,-C6) alkyl, {C,-C6)
alkenyl, (C,-C6)
1o alkynyl, (CS-Czo) aryl, substituted (CS-CZO) aryl, (C6-C26) alkaryl and
substituted (C6-C26)
alkaryl; the heterocycloalkyl substituents are each independently selected
from the group
consisting of -CN, -N02, -NR'Z, -OR', -C(O)NR'2, -C(S)NR'z, -C(O)OR', -
C(S)OR', -C(O)SR',
-C(S)SR' and trihalomethyl; the aryl and alkaryl substituents are each
independently selected
from the group consisting of halogen, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -
C{O)SR',
-C(S)SR', -C(O)NR'2, -C(S)NR'z and trihalomethyl; each R' is independently
selected from the
group consisting of -H, (C,-C6) alkyl, (C,-C6) alkenyl and (C,-C6) alkynyl; ---
designates a
single or double bond; and wherein when R, is =O or -OH, at least one of R5,
R6 or R~ is other
than -R', preferably other than -H, or Y is present or R4 is other than -H.
In still another aspect, the invention provides a method for reducing sickle
erythrocyte
2o 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.
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
3o 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
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chronic sickle cell episodes.
In one aspect of the invention a method is provided for inhibiting unwanted
cellular
proliferation associated with an inflammatory disease. The method includes the
step of
contacting a cell the proliferation of which contributes to inflammation in
situ with an amount
s of a compound having the above described formula (I) effective to inhibit
proliferation of the
cell. In one embodiment the method of administration is selected from the
group consisting of
oral, parenteral, intravenous, subcutaneous, transdermal and transmucosal for
a living human.
In one embodiment the mammalian cell is a fibrotic cell or a lymphocyte.
According to another aspect of the invention a method is provided for treating
or
t o preventing an inflammatory disease. The method includes the step of
administering to a
subject in need of such treatment a therapeutically effective amount of a
compound of the
above-described formula (I). In one embodiment the inflammatory disease is
diarrhea.
Preferably the diarrhea is caused by inflammatory bowel disease. In another
embodiment the
inflammatory disease is an autoimmune disease. In other embodiments the
inflammatory
is disease is selected from the group consisting of proliferative
glomerulonephritis; lupus
erythematosus; scleroderma; temporal arteritis; thromboangiitis obliterans;
mucocutaneous
lymph node syndrome; asthma; host versus graft; inflammatory bowel disease;
multiple
sclerosis; rheumatoid arthritis; thyroiditis; Grave's disease; antigen-induced
airway
hyperactivity; pulmonary eosinophilia; Guillain-Bane syndrome; allergic
rhinitis; myasthenia
2o gravis; human T-lymphotrophic virus type 1-associated myelopathy; herpes
simplex
encephalitis; inflammatory myopathies; atherosclerosis; and Goodpasture's
syndrome. In
certain embodiments the administration is parenteral or per oral.
In yet another aspect, the invention provides a method for inhibiting
mammalian cell
proliferation in situ. Preferably, the mammalian cell proliferation is not
associated with a
25 proliferative disease selected from the group consisting of cancer, actinic
keratosis, and
Kaposi's sarcoma. 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.
3o Mammalian cells in this manner include vascular smooth muscle cells,
fibroblasts and
endothelial cells.
In still another aspect, the invention provides a method for treating and/or
preventing
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unwanted or abnormal cell proliferation in a subject, such as a human.
Preferably, the
unwanted or abnormal cell proliferation is not associated with a proliferative
disease selected
from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
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, and
preferably the
1o unwanted or abnormal cell proliferation is not associated with a
proliferative disease selected
from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
Such disorders
include, but are not limited to, non-cancerous angiogenic conditions or
arteriosclerosis.
In yet another aspect, the invention provides a method for the treatment
and/or
prevention of diseases that are characterized by unwanted and/or abnormal
mammalian cell
I5 proliferation. Preferably, the unwanted or abnormal cell proliferation is
not associated with a
proliferative disease selected from the group consisting of cancer, actinic
keratosis, and
Kaposi's sarcoma. 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
20 abnormal mammalian cell proliferation which can be treated or prevented by
way of the
methods of the invention include, but are not limited to, blood vessel
proliferative disorders,
fibrotic disorders and arteriosclerotic conditions.
According to another aspect of the invention, a method for treating diarrhea
of diverse
etiology is provided. The method involves administering to a subject who is in
need of such
25 treatment, an aromatic compound of the invention in an amount effective to
inhibit the
diarrhea. Preferably the compound is administered orally in conjunction with
oral rehydration
fluids. The aromatic compounds useful in the invention are generally
substituted 3,3-diphenyl
indanone, indane or (3-I~ indole compounds, or analogues thereof.
In one embodiment of the invention the foregoing aromatic compounds may be
3o administered in combination with other non-formula (I) anti-diarrheal
agents. In another
embodiment the aromatic compounds may be administered in combination with
other non-
formula {I) anti-scours agents.
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According to one embodiment of the invention the subject in need of such
treatment is
a subject who has symptoms of diarrhea or scours. In another embodiment of the
invention,
the subject in need of such treatment is a subject at risk of developing
diarrhea or scours.
In general diarrhea is a secretory disorder, which is caused by at least one
of several
mechanisms. In one embodiment the diarrhea is an exudative form of diarrhea;
In one
embodiment the diarrhea is a nonexudative form of diarrhea; In another
embodiment the
diarrhea is a decreased absorption form of diarrhea; In another embodiment the
diarrhea is a
non-decreased absorption form of diarrhea; In yet another embodiment the
diarrhea is a
secretory form of diarrhea. In yet another embodiment the diarrhea is a
nonsecretory form of
1 o diarrhea. In still another embodiment the diarrhea is a noninflammatory
foam of diarrhea.
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.
According to another aspect of the invention, pharmaceutical preparations are
provided, comprising one or more of the aromatic compounds of the invention in
admixture
with a pharmaceutically acceptable carrier, excipient or diluent, wherein the
aromatic
compounds) of the invention is (are) in an amount effective for treating: (i)
unwanted or
abnormal cell proliferation, preferably not a proliferative disease selected
from the group
2o consisting of cancer, actinic keratosis, and Kaposi's sarcoma; (ii) an
inflammatory disease;
(iii) sickle cell disease; and (iv) diarrhea or scours. In one embodiment, the
aromatic
compounds useful according to the invention have the general formula (I)
provided above. In
certain other embodiments, the pharmaceutical preparations include the
aromatic compounds
of the invention together with a non-formula (I) agent selected from the group
consisting of an
anti-proliferative agent; (ii) an anti-inflammatory agent; (iii) anti-sickle
cell agent; and (iv} an
anti-dian hea or anti-scours agent.
According to another aspect, the use of aromatic compounds of the invention in
the
manufacture of medicaments is provided. The medicaments are useful for
treating: (i)
unwanted or abnormal cell proliferation, preferably not a proliferative
disease that includes
cancer, actinic keratosis, and Kaposi's sarcoma; (ii) an inflammatory disease;
(iii) sickle cell
disease; and (iv) diarrhea or scours.
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According to another aspect of the invention, pharmaceutical preparations are
provided. These pharmaceutical preparations include the aromatic compounds of
the
invention together with an anti-diarrheal agent. In one embodiment, the
aromatic compounds
useful according to the invention have the general formula (I) provided above.
In other
embodiments the aromatic compounds useful according to the invention are the
preferred
compounds described above. Preferably the pharmaceutical composition of the
invention may
be administered orally.
The invention also provides the aromatic compounds of the invention in the
manufacture of a medicament for the treatment of diarrhea or scours. In one
embodiment, the
to aromatic compounds useful according to the invention have the general
formula (I) provided
above. In other embodiments the aromatic compounds useful according to the
invention are
the preferred compounds described above.
According to another aspect of the invention, veterinary preparations are
provided.
These veterinary preparations include the aromatic compounds useful according
to the
invention together with an anti-scours preparation. In one embodiment, the
aromatic
compounds useful according to the invention have the general formula (I)
provided above. In
other embodiments the aromatic compounds useful according to the invention are
the
preferred compounds described above.
2o Each of the limitations of the invention can encompass various embodiments
of the
invention. It is, therefore, anticipated that each of the limitations of the
invention involving
any one element or combinations of elements can be included in each aspect of
the invention.
Brief Description of the Drawings
FIG. 1 is a general reaction scheme for synthesizing certain compounds
according to
the invention;
FIG. 2 is a general reaction scheme for synthesizing certain compounds
according to
the invention;
3o FIG. 3 is a bar graph depicting the effect of clotrimazole in the
inhibition of cAMP and
Ca++ dependent CY secretion in T84 cells; and
FIG. 4 is a graph showing the effect of clotrimazole on the inhibition of base
line and
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Ca++ - stimulated 86 Rb efflux from T84 monolayers.
Detailed Description of the Invention
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. In vitro studies have shown that
Clotrimazole, an imidazole-
containing antimycotic agent, blocks Caz+-activated K+ transport and cell
dehydration in sickle
erythrocytes (Brugnara et al., 1993, J. Clin. Invest. 92:520-526). Studies in
a transgenic
mouse model for sickle cell disease (SAD mouse, Trudel et al., 1991, EMBO J.J.
~:3157-
3165) show that oral administration of Clotrimazole leads to inhibition of the
red cell Gardos
channel, increased red cell K+ content, a decreased mean cell hemoglobin
concentration
(MCHC) and decreased cell density (De Franceschi et al., 1994, J. Clin.
Invest. ~:1670-
1676). Moreover, therapy with oral Clotrimazole induces inhibition of the
Gardos channel
and reduces erythrocyte dehydration in patients with sickle cell disease
(Brugnara et al., 1996,
J. Clin. Invest. Q2:1227-1234). Other antimycotic agents which inhibit the
Gardos channel in
vitro include miconazole, econazole, butoconazole, oxiconazole and sulconazole
(U.S. Patent
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.
Also as discussed in the Background section, the modulation of early ionic
mitogenic
2o 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.
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 etet
al., 1995 Nature
)~,dicine j :534-540). Clotrimazole also depletes the intracellular Caz+
stores and prevents the
rise in cystolic Ca2+ 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
3o number of lung metastases observed (Benzaquen et al., supra).
It has now been discovered that substituted 3,3-diphenyl indanone, indane and
(3-l~
indole compounds, as well as analogues of these classes of compounds, also
inhibit the
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Gardos channel of erythrocytes, mammalian cell proliferation and/or Cl-
secretion from
intestinal cells. Thus, in one aspect, the present invention provides a new
class of organic
compounds that are capable of inhibiting the Gardos channel of erythrocytes,
mammalian cell
proliferation, particularly mitogen-induced cell proliferation, and/or Cl-
secretion from
intestinal cells.
The activities of these compounds are 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
to anti-mycotic agents. Thus, the substituted 3,3-diphenyl indanone, indane or
(3-I~ indole
compounds and analogues of the invention provide an entirely new class of
compounds
capable of effecting inhibition the Cap+-activated potassium channel (Gardos
channel) of
erythrocytes, particularly sickle erythrocytes, mammalian cell proliferation,
particularly
mitogen-induced cell proliferation, and/or Cl- secretion from intestinal
cells.
In another aspect, the invention provides a method of reducing sickle cell
dehydration
andlor delaying the occurrence of erythrocyte sickling in situ as a
therapeutic approach
towards the treatment of sickle cell disease. In its broad.P~~ sense, the
method ir. solves only a
single step -- the administration of at ;;,asi one pharmacologically active
compound of the
invention, ~r a composition thereof, to a sickle erythrocyte in situ in an
amount effective to
reduce dehydration andlor delay the occurrence of cell sickling or
deformation.
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 sickiing 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.
Based in part on the surmised importance of the Gardos channel as a
therapeutic target
3o 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
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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.
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
to 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.
Based in part on the surmised role of mammalian cell proliferation in certain
diseases,
2o 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 Ca2+-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
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a subject systemically to arrest or inhibit cell proliferation.
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.
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 arteritis,
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.
Another example of abnormal neovascularization is that associated with solid
tumors.
~ 5 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
2o evidence for the role of angiogenesis in tumor growth. Other cancers that
are associated with
neovascularization include hemangioendotheliomas, hemangiomas and Kaposi's
sarcoma.
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
25 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
3o 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.
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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.
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
1o muscle cell proliferation, including vascular complications of diabetes.
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
~ 5 therefore in delaying the onset of, inhibiting the progression of, or even
halting the
progression of such proliferation and the associated atherosclerotic
condition.
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
20 obstruction of the coronary arteries. The invention is useful in inhibiting
such obstruction and
reducing the risk of, or even preventing, such failures.
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
25 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.
30 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
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glomerulosclerosis and diabetic retinopathy.
The compounds described herein are also useful in treating or preventing
various types
of cancers. Cancers 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
1o 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 c~.~.~Pr including
adenocarcinoma and
Wilms ~'~.-nar.
The compounds of the invention are useful with hormone dependent and also with
2o nonhormone dependent cancers. They also are useful with prostate and
nonprostate cancers
and with breast and nonbreast cancers. They further are useful with multidrug
resistant strains
of cancer.
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.), and eczema and epithelial
precancerous lesions
such as actinic keratosis. It also is useful with pathologies mediated by
growth factors such as
uterine leiomyomas.
In addition to the particular disorders enumerated above, the invention is
particularly
3o useful in treating or preventing inflammatory diseases associated with
cellular proliferation.
An "inflammatory disease associated with cellular proliferation" as used
herein is a disease in
which lymphoproliferation contributes to tissue or organ damage leading to
disease. For
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instance, excessive T cell proliferation at the site of a tissue or organ will
cause damage to the
tissue or organ. Inflammatory disease are well known in the art and have been
described
extensively in medical textbooks (See, e.g., Harnson's Principles of
Experimental Medicine,
13th Edition, McGraw-Hill, Inc., N.Y.).
Inflammatory diseases associated with cellular proliferation include but are
not limited
to proliferative glomerulonephritis; lupus erythematosus; scleroderma;
temporal arteritis;
thromboangiitis obliterans; mucocutaneous lymph node syndrome; asthma; host
versus graft;
inflammatory bowel disease; multiple sclerosis; rheumatoid arthritis;
thyroiditis; Grave's
disease; antigen-induced airway hyperactivity; pulmonary eosinophilia;
Guillain-Barre
syndrome; allergic rhinitis; myasthenia gravis; human T-lymphotrophic virus
type 1-
associated myelopathy; herpes simplex encephalitis; inflammatory myopathies;
atherosclerosis; and Goodpasture's syndrome. Some examples of inflammatory
diseases
associated with cellular proliferation as well as animal models for testing
and developing the
compounds are set forth in Table 1 below.
15 Table 1
Disease ProliferatingReference Animal ModelReference
Cells
Asthma T cells Hogg 1997 Airway inflammationHenderson
APMIS et al.
100:105(10):735-45and 1997 J Clin
Invest
hyperresponsiveness100( 12):3083-3092.
in Ovalbumin-
sensitized
mice or
guinea pigs.
GlomerulonephritisMesangial Nitta et al. NZB/NZW crossedClynes et
1998 al. 1998
(glomerular) Eur J Pharmacolmice developScience 279(5353):
cells
344:107-110 glomerular 1052-54.
disease
and lupus-like
syndrome.
Host versus T cells Schorlemmer Renal allogra8Lazarivuts
Graft et al. et al. 1996
B cells 1997 Int J rejection Nature 380(6576)
Tissue in mice.
React 19:157-61. 717-720.
Sedgwick et
al. 1998
J Immunol
160:5320-30.
20 Inflammatory Epithelial Bajaj-ElliottTrinitrobenzeneBoughton-Smith
Bowel cells et al. et
Disease 1997 Am J. sulphonic al. 1988 Br
Pathol. acid J
151:1469-76. induced bowelPharmacol
94:65-72.
inflammation
in rats.
Systemic LupusGlomerular Kodera et NZB1NZW crossedPeng et al.
cells al. 1997 1996
ErythematosisLymphocytes Am J Nephol mice developMol Biol Rep
17:466- 23(3-
. 70. glomerular 4):247-S 1.
disease
Akashi et and lupus-like
al. 1998
Immunology syndrome.
93:238-
48
CA 02311129 2000-OS-19
WO 99/26624
- 23 -
PCT/US98I24968
Multiple SclerosisT cells Constantinesecu et Experimental aller
ic D
g
rescher et al. 1998
al. 1998 Immunol encephalomyelitis.
J Clin Invest
Res 17(1-2):217-27. 101(8):1765-74.
Rheumatoid T cells Ceponis et al. 1998 Rat adjuvant arth
Arthritis iti
r
Synovial cellss Anderson et al. 1996
Br J Rheumatol
assay J Clin Invest
37(2):170-8
97( I 1 ):2672-9.
Thyroiditis T cells and Rose et al. 1997 HLA transgenic mice
Taneja et
l
1998
Epithelial a
cells .
Crit Rev Immunol immunized with J Clin
Investig
17:511-7. thyroglobulin. 101 (5):921-6
.
Schumm-Draeger et
al. 1996 Verh Dtsch
Ges Pathol 80:297-
301.
Grave's DiseaseThyroid cellsDiPaola et al. 1997 Thiouracil-fed r
t
a
s. Viglietto et al. 1997
J Clin Endocrinol
Oncogene 15:2687-
Metab 82:670-3
. 98.
Disease Proliferating Reference Model
Cells
Antigen-induced T cells Wolyniec et al
airway 1998
hyperactivity .
Am J Respir Cell
Mol
Biol 18:777-85
Pulmonary eosinophiliaT cells Wolyniec et al.
1998
Am J Respir Cell
Mol
Biol 18:777-85
Guillain-Barre T cells Hartung et al. Ex
S 1991 eriment
l
yndrome Ann Neurol. 30:48-53p
(inflammatory a
autoimmune neuritis
(immunization
demyelinating with PNS
disease)
myelin and Freunds
complete adjuvant)
Giant cell arteritisT cells Brack et al
(a 1997
i5 form of systemic .
Mol Med 3:530-43
vascuiitis) Inflammation
of large arteries
Allergic RhinitisT cells Baraniuk et al.
1997
J Allergy Clin
Immunol
99:5763-72
Myasthenia gravisT cells Hartung et al.
1991
Ann Neurol 30:48-53
Human T-lymphotropicT cells Nakamura et al.
1996
virus type I Intern Mede 35:195-99
- associated
myelopathy
Herpes simplex T cells Hartung et al.
1991
encephalitis
Ann Neurol 30:48-53
Inflammatory T cells Hartung et al.
1991
myopathies (ie. Ann Neurol 30:48-53
.
Polymyositis, Lindberg et al
1995
dermatomyocitis) .
Scand J Immunol
41:421-26
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ArtherosclerosisT cells Rosenfeld et
al. 1996
Diabetes Res
Clin Pract
30 suppl.: I-11
Goodpasture's Macrophages Lan et al. 1995
syndrome
Am J Pathol 147:1214-
20
The compounds and methods of the invention provide myriad advantages over
agents
and methods commonly used to treat cell proliferative disorders. 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.
Most significantly, the compounds of the invention have reduced toxicity as
compared
with these other agents. For Clotrimazole, it is well-known that the imidazole
moiety is
I O responsible for inhibiting a wide range of cytochrome P-450 isozyme
catalyzed reactions,
which constitutes their main toxicological effects (Pappas and Franklin, 1993,
Toxicoloev_
>~:27-35; Matsuura et al., 1991, Biochemical PharmacotoEV 41:1949-1956).
Analogues and
metabolites of Clotrimazole do not induce cytochrome P-450 (Matsuura et al.,
1991,
Biochemical PharmacolQgv x:1949-1956), and therefore do not share
Clotrimazole's toxicity.
The invention in another aspect also involves methods and products for
reducing the
symptoms of diarrhea or preventing diarrhea in a subject at risk for
developing diarrhea, using
the compounds of the invention. The aromatic compounds useful according to the
invention
may be provided in a pharmaceutical preparation or a veterinary preparation.
The aromatic
compounds of the invention are also useful in a method for treating diarrhea
and scours as
well as a method for preventing diarrhea and scours.
Diarrhea, as used herein, indicates a medical syndrome which is characterized
by the
symptoms of diarrhea or scours. In general, diarrhea is a disorder resulting
in a secretory
imbalance. For purposes of this patent application diarrhea is divided into
three categories
based on the underlying mechanism: exudative, decreased absorption, and
secretory and the
term diarrhea as used herein encompasses each of these categories. Exudative
diarrheas result
from inflammatory processes leading to impaired colonic absorption, and
outpouring of cells
and colloid caused by such disorders as ulcerative colitis, shigellosis, and
amebiasis.
Disorders of decreased absorption include osmotic, anatomic derangement, and
motility
disorders. Osmotic diarrhea can occur as a result of digestive abnormalities
such as lactose
intolerance. Anatomic derangement results in a decreased absorption surface
caused by such
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procedures as subtotal colectomy and gastrocolic fistula. Motility disorders
result from
decreased contact time resulting from such diseases as hyperthyroidism and
irritable bowel
syndrome. Secretory diarrhea is characterized by the hypersecretion of fluid
and electrolytes
from the cells of the intestinal wall. In classical form, the hypersecretion
is due to changes
which are independent of the permeability, absorptive capacity and exogenously
generated
osmotic gradients within the intestine. As discussed above, however, all foams
of diarrhea
may actually manifest a secretory component.
The methods and products of the invention are particularly useful in treating
diarrhea
which is secretory. However, the methods and products of the invention may
also be used in
combination with other treatment methods which are known in the art to treat
diarrhea caused
by decreased absorption or inflammation. The compounds of the invention are
involved in
regulating Cf secretion and can function alone or when used in combination
with other
treatment methods to decrease net fluid secretion even when this is due
primarily to
abnormalities in absorption or inflammation.
1 s The methods and products of the invention are useful in preventing
diarrhea and
scours in subjects at risk of developing these disorders. Subjects at risk of
developing
diarrhea and scours are those subjects which have a high likelihood of
exposure to the
bacterial and viral microorganisms which cause these symptoms. For example,
approximately
1/3 of travelers to developing countries will develop diarrhea; infection with
rotavirus is one
20 of the leading causes of death in infants in developing countries; subjects
with HIV have a
greater than 50% chance of developing diarrhea, and many newborn calves and
pigs develop
scours. Subjects with inflammatory bowel disease develop recurrent diarrhea.
The methods and products of the invention are also useful in treating subjects
who
already exhibit the symptoms of diarrhea and scours. Once a subject has been
exposed to a
25 microorganism causing the symptoms, the subject may be treated with the
methods and
products of the present invention in order to reduce the symptoms. The
symptoms of diarrhea
include bowel irregularity, fecal fluid rich in sodium or potassium, fluid
feces, dehydration,
fever, Loss of body weight, headache, anorexia, vomiting, malaise and myalgia.
The
symptoms of scours include a loss of body weight or failure to grow,
dehydration, malodorous
3o feces, fluid feces, feces containing pieces of partially digested milk or
semisolid material, and
feces of a yellow-white or gray color.
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The o poLndc
PCTNS98/24968
The compounds which are potent, selective and safe inhibitors of Caz+-
activated
potassium channel (Gardos channel) of erythrocytes, particularly sickle
erythrocytes,
mammalian cell proliferation, particularly mitogen-induced cell proliferation,
and/or
secretagogue-stimulated transepithelial electrogenic chloride secretion in
intestinal cells
according to the invention, are generally substituted 3,3-diphenyl indanone,
indane and (3-I~
indole compounds, as well as analogues of these classes of compounds wherein
the atoms at
ring positions l and 2 are connected via a double bond.
In one illustrative embodiment, the compounds capable of inhibiting the Gardos
to channel, mammalian cell proliferation and/or chloride secretion in
intestinal cells according to
the invention are compounds having the structural formula:
(Rs)o
(I) ~R7)m ~ I Y-R4
Rs
t Rz
or pharmaceutically acceptable salts or hydrates thereof, wherein:
2o m is 0, I, 2, 3 or 4;
each n is independently 0, l, 2, 3, 4 or 5;
XisCorN;
Y is absent, (C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl;
R, is absent, -OR, -SR, =O, =S, =N-OR, -O-C(O)R, -S-C(O)R, -O-C(S)R, -S-C(S)R,
or when taken together with Rz is a 3-8 membered heterocycloalkyl or a
substituted 3-8
membered heterocycloalkyl;
RZ is absent or -H;
R3 is absent or -H;
R4 is -H, -OR', -SR', -NR'2, -CN, -NO2, (C3-C8) cycloalkyl, 3-8 membered
3o heterocycloalkyl, -C(O)R', -C(S)R', -C(O}OR', -C(S)OR', -C(O)SR', -C(S)SR',
-C(O)NR'z or
-C(S)NR'2;
each R5, R.~ and R, is independently selected from the group consisting of -
halogen,
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-R', -OR', -SR', -NR's, -ONR'2, -SNR'2, -NOz, -CN, -C(O)R', -C(S)R', -C(O)OR',
-C(O)SR',
-C(S)OR', -CS(S)R', -C(O)NR'2, -C(S)NR'2, -C(O)NR'(OR'), -C(S)NR'(OR'); -
C(O)NR'(SR'),
-C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R']~, -CH[C(S)R']2, -CH[C(O)OR']2, -
CH[C(S)OR']2,
-CH[C(O)SR']z and -CH[C(S)SR']2;
each R is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (CS-CZO) aryl, substituted (CS-C2o) aryl,
(C6-CZ6) alkaryl and
substituted (C6-CZ6) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group
consisting of -CN, -NO2, -NR'2, -OR', -C(O)NR'2, -C(S)NR'2, -C(O)OR', -
C(S)OR', -C(O)SR',
-C(S)SR' and trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group
consisting of halogen, -C(O)R', -C(S)R', -C(O)OR', -C{S)OR', -C(O)SR', -
C(S)SR',
-C(O)NR'z, -C(S)NR'2 and trihalomethyl;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl, (C~-
15 C6) alkenyl and (C,-C6) alkynyl; and
--- designates a single or double bond.
In the compounds of structural formula (I), the bond between the atoms at ring
positions l and 2 (designated ---) can be either a single or double bond. It
will be recognized
by those of skill in the art that when the bond is a double bond, certain of
the substituents
2o must be absent. It will also be recognized that the identity of X also
influences the presence
or absence of certain substituents. Thus, it is to be understood that when X
is N and --- is a
double bond, R,, Rz and R3 are absent; when X is C and --- is a double bond,
RZ and R3 are
absent. When X is N and --- is a single bond, one of R, and RZ is present and
the other is
absent and R3 is present; when X is C and --- is a single bond, R,, R2 and R3
are each present.
25 In a preferred embodiment of the invention, the chalcogens in the compounds
of
formula (I) are each oxygen.
In another preferred embodiment of the invention, the compounds are those of
structural formula (I) wherein:
mis0,1,2,3or4;
3o each n is independently 0, 1, 2, 3, 4 or 5;
X is C or N;
Y is absent, (C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl;
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R, is absent, -OR, =O, =N-OR, -O-C(O)R, or when taken together with RZ is a 3-
8
membered oxirane or a substituted 3-8 membered oxirane;
RZ is absent or -H;
R3 is absent or -H;
R4 is -H, -OR', -NR'2, -CN, -NO2, (C3-Cg) cycloalkyl, 3-8 membered oxiranyl, 5-
8
membered dioxycycloalkyl, -C(O)R', -C(O)OR' or -C(O)NR'z;
each R5, Rs and R, is independently selected from the group consisting of -
halogen,
-R', -OR', -NR'2, -ONR'2, -NO2, -CN, -C(O)R', -C(O)OR', -C(O)NR'z, -
C(O)NR'(OR'),
-CH(CN)2, -CH[C(O)R'JZ and -CH[C(O)OR']Z;
to each R is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (CS-CZO) aryl, substituted (CS-CZo) aryl,
(C6-C26) alkaryl and
substituted (C6-C26) alkaryl;
the oxirane substituents are each independently selected from the group
consisting of
-CN, -NOZ, -NR'2, -OR', -C(O)NR'2, -C(O)OR' and trihalomethyl;
15 the aryl and alkaryl substituents-are each independently selected from the
group
consisting of halogen, -C(O)R', -C(O)OR', -C(O)NR'z and trihalomethyl;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl, (C,-
C6) alkenyl and (C,-C6) alkynyl; and/or
--- designates a single or double bond.
2o In another preferred embodiment, the compounds are those of structural
formula (I)
wherein:
mis0orl;
each n is independently 0 or 1;
XisCorN;
25 Y is absent, (C,-C3) alkyl, (C,-C3) alkenyl or (C,-C3) alkynyl;
Rl is absent -H, -OR, =O, -NRz, =N-OR, -O-C(O)R, or when taken together with
RZ is
3-5 membered oxirane or 3-5 membered substituted oxirane;
Rz is absent or -H;
R3 is absent or -H;
3o R4 is -H, -OR, -NR2, -CN, -C(O)OR, -C(O)NRz or 5-6 membered
dioxoycycloalkyl;
each R5, R6 and R~ is independently selected from the group consisting of -R',
-F, -Cl
or -Bx;
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each R is independently selected from the group consisting of -H, (C,-C3)
alkyl,
(C,-C3) alkenyl, (C,-C3) alkynyl, (CS-C,o) aryl, substituted (CS-C,o) aryl,
(C6-C,3) alkaryl,
substituted C6-C,3) alkaryl;
the oxirane substituent is -CN, -NOz, -NR'Z, -OR' and trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group
consisting of -F, -Cl, -Br, -CN, -NO2, -NR'2, -C(O)R', -C(O)OR' and
trihalomethyl;
R' is -H, (C,-C3) alkyl, (C,-C3) alkenyl or (C,-C3) alkynyl; and/or
--- is a single or double bond.
In still another preferred embodiment, the compounds are those of structural
formula
(I) wherein:
m is 0, 1, 2, 3 or ~;
each n is independently 0, 1, 2, 3, 4 or 5;
X is C or N;
Y is absent, (C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl;
R, is absent, -OR, -SR, =O, =S, =N-OR, -O-C(O)R, -S-C(O)R, -O-C(S)R, -S-C(S)R,
or when taken together with Rz is a 3-8 membered heterocycloalkyl or a
substituted 3-8
membered heterocycloalkyl;
RZ is absent or -H;
R3 is absent or -H;
2o R4 is -H, -OR', -SR', -NR'Z, -CN, -NO2, (C3-Cg) cycloalkyl, 3-8 membered
heterocycloalkyl, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', -
C(O)NR'z or
-C(S)NR'2;
each R5, R6 and R., is independently selected from the group consisting of -
halogen,
-R', -OR', -SR', -NR'z, -ONR'2, -SNR'2, -NOZ, -CN, -C(O)R', -C(S)R', -C(O)OR',
-C(O)SR',
-C(S)OR', -CS(S)R', -C(O)NR'2, -C(S)NR'2, -C(O)NR'(OR'), -C(S)NR'(OR'); -
C(O)NR'(SR'),
-C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R')2, -CH[C(S)R')z, -CH[C(O)OR']2, -
CH[C(S)OR']2,
-CH[C(O)SR']Z and -CH[C(S)SR']2;
each R is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (CS-CZo) aryl, substituted (CS-CZO) aryl,
(C6-C26) alkaryl and
3o substituted (C6-Cz6) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group
consisting of -CN, -N02, -NR'2, -OR', -C(O)NR'2, -C(S)NR'2, -C(O)OR', -
C(S)OR', -C(O)SR',
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-C(S)SR' and trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group
consisting of halogen, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -
C(S)SR',
-C(O)NR'2, -C(S)NR'2 and trihalomethyl;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl, (C~-
C6) alkenyl and (C,-C6) alkynyl;
--- designates a single or double bond; and
wherein when X is C and R, is =O, =S or -OR', at least one of RS, R6 or R~ is
other
than -R', preferably other than -H, or Y is present or R4 is other than -H;
and when X is N, ---
1o is a double bond and R,, R2, R3 and Y are absent, R4 is other than -NR'Z,
preferably other than
-NH2.
In still another preferred embodiment, the compounds are those of structural
formula
(I) wherein:
m is 0, 1, 2, 3 or 4;
15 each n is independently 0, 1, 2, 3, 4 or 5;
XisC;
Y is absent, (C,-C6) alkyl, (C,-C6) alkenyl or (C,-C6) alkynyl;
R, is absent, -OR, -SR, =O, =S, =N-OR, -O-C(O)R, -S-C(O)R, -O-C(S)R, -S-C(S)R,
or when taken together with RZ is a 3-8 membered heterocycloalkyl or a
substituted 3-8
2o membered heterocycloalkyl;
Rz is absent or -H;
R3 is absent or -H;
R4 is -H, -OR', -SR', -NR'2, -CN, -NO2, (C3-Cg) cycloalkyl, 3-8 membered
heterocycloalkyl, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', -
C(O}NR'2 or
25 -C(S)NR'2;
each R5, R6 and R~ is independently selected from the group consisting of -
halogen,
-R', -OR', -SR', -NR'2, -ONR'2, -SNR'Z, -NO2, -CN, -C(O)R', -C(S)R', -C(O)OR',
-C(O)SR',
-C(S)OR', -CS(S)R', -C(O)NR'2, -C(S)NR'2, -C(O)NR'(OR'), -C(S)NR'(OR'); -
C(O)NR'(SR'),
-C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R']2, -CH[C(S)R']z, -CH[C(O)OR']2, -
CH[C(S)OR']2,
30 -CH[C(O)SR']2 and -CH[C(S)SR'J2;
each R is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (CS-CZO) aryl, substituted (CS-CZO) aryl,
(C6-Czb) alkaryl and
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substituted (C6-C26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group
consisting of -CN, -NO2, -NR'2, -OR', -C(O)NR'z, -C(S)NR'2, -C(O)OR', -
C(S}OR', -C(O)SR',
-C(S)SR' and trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group
consisting of halogen, -C(O)R', -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -
C(S)SR',
-C(O)NR'2, -C(S)NR'2 and trihalomethyl;
each R' is independently selected from the group consisting of -H, {C,-C6)
alkyl, (C,-
C6) alkenyl and (C,-C6) alkynyl;
--- designates a single or double bond; and
wherein when R, is =O or -OH, at least one of 85,126 or R, is other than -R',
preferably
other than -H, or Y is present or R4 is other than -H.
As used herein, the term "alkyl" refers to a saturated branched, straight
chain or cyclic
hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl,
isopropyl,
cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, pentyl, isopentyl,
cyclopentyl, hexyl,
cyclohexyl and the like.
As used herein, the term "heterocycloalkyl" refers to a saturated cyclic
hydrocarbon
radical wherein one or more of the carbon atoms is replaced with another atom
such as Si, Ge,
N, O, S or P. Typical heterocycloalkyl groups include, but are not limited to,
morpholino,
2o thiolino, piperidyl, pyrrolidinyl, piperazyl, pyrazolidyl, imidazolidinyl,
and the like.
As used herein, the term "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 ethenyl, propenyl, isopropenyl, cyclopropenyl, butenyI, isobutenyl,
cyclobutenyl, tert-
butenyl, pentenyl, hexenyl and the like.
As used herein, the term "alkynyl" refers to an unsaturated branched, straight
chain or
cyclic hydrocarbon radical having at least ane carbon-carbon triple bond.
Typical alkynyl
groups include ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and
the Iike.
As used herein, the term "alkoxy:" refers to an -OR radical, where R is alkyl,
alkenyl
or alkynyl, as defined above.
As used herein, the term "aryl" refers to an unsaturated cyclic hydrocarbon
radical
having a conjugated ~ electron system. Typical aryl groups include, but are
not limited to,
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penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, indacenyl, and the
like.
As used herein, the term "heteroaryl" refers to an aryl group wherein one or
more of
the ring carbon atoms is replaced with another atom such as N, O or S. Typical
heteroaryl
groups include, but are not limited to,furanyl, imidazole, pyridinyl,
thiophenyl, indolyl,
imidazolyl, quinolyl, thienyl, indolyl, pyrrolyl, pyranyl, pyridyl, pyrimidyl,
pyrazyl,
pyridazyl, and the like.
As used herein, the term "heteroarylium" refers to a heteroaryl group wherein
one or
more hydrogens has been added to any position of the neutral parent ring.
Typical
heteroarylium groups include, but are not limited to, pyridinium, pyrazinium,
pyrimidinium,
to pyridazinium, 1,3,5-triazinium, and the like.
As used herein, the term "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.
In still another preferred embodiment, the compounds of structural formula (I)
are
selected from the group of compounds set forth below:
Hz
OH
(1) (2)
(3)
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\ /
CHZCH=CH2
O-CHZCH=CH2
N-OH CN
(4) (5) (6)
\ / / ' I
/
O N
O-'~ CI
CH3 O
(7) (8) (9)
ri _
I
/ w
NHZ
C CI O O
N-OH
(10) (11) (12)
/ ~ _CH3
I NHZ
N
OH O
(13) (14) (15)
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\ / ~
1
O
O I
/ \
/ \
(is) (1~) (
(19) (20)
The compounds will be referred to herein by way of compound numbers as
presented
above.
In still another preferred embodiment, the compounds of structural formula (I)
are
selected from the group consisting of Compounds 4, 5, 6, 7, 8, 9, 10, 11,12,
13, 14, 15, 16,
17, 18, 19 and 20.
The chemical formulae referred to herein may exhibit the phenomena of
tautomerism
l0 or conformational isomerism. As the formulae drawings within this
specification can
represent only one of the possible tautomeric or conformational isomeric
forms, it should be
understood that the invention encompasses any tautomeric or conformational
isomeric forms
which exhibit biological or pharmacological activity as described herein.
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
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treatment with alkali.
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).
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.
Necessary starting
materials may be obtained commercially or by standard procedures of organic
chemistry.
Synthetic protocols for these compounds are described in US application serial
no.
08/975,595, filed November 20, 1997, entitled "Methods for Treatment or
Prevention of
Sickle Cell Disease with Substituted biphenyl Indane and Indole Compounds and
Analogs
Thereof', the disclosure of which is incorporated herein by reference.
An individual compound's relevant activity and potency as an agent to affect
sickle
cell dehydration or deformation, mammalian cell proliferation and/or
secretagogue-stimulated
transepithelial electrogenic chloride secretion in intestinal cells may be
determined using
standard techniques. Preferentially, a compound is subject to a series of
screens to determine
its pharmacological activity.
In most cases, the active compounds of the invention exhibit three
pharmacological
activities: inhibition of the Gardos channel of erythrocytes, inhibition of
secretagogue-
2o stimulated transepithelial electrogenic chloride secretion in intestinal
cells 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.
In general, the active compounds of some embodiments of the invention are
those
which induce at least about 25% inhibition of the Gardos channel of
erythrocytes (measured at
about 10 ~M), at least about 25% inhibition of secretagogue-stimulated
transepithelial
electrogenic chloride secretion in intestinal cells (measured at about 10 ~,M)
andlor about 25%
inhibition of mammalian cell proliferation (measured at about 10 g.M), as
measured using in
3o vitro assays that are commonly known in the art (see, e.g., Brugnara et
al., 1993, J.J. Biol.
Chem. x$(12):8760-8768; Benzaquen et al., 1995, Nature Medicine x:534-540).
Alternatively, or in addition, the active compounds of the invention generally
will have an
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ICso {concentration of compound that yields 50% inhibition) for inhibition of
the Gardos
channel of erythrocytes of less than about 10 pM, an ICSO for secretagogue-
stimulated
transepithelial electrogenic chloride secretion in intestinal cells of less
than about 10 ~.M,
and/or an ICS° for inhibition of cell proliferation of less than about
10 pM, as measured using
in vitro assays that are commonly known in the art (see, e.g., Brugnara et
al., 1993, J.~,'~Ql_.
Chem. xø$(12):8760-8768; Benzaquen et al., 1995, N~,zre Medicine 1_:534-540)
and the
Examples section below. Other assays for assessing the activity andlor potency
of an agent
with respect to the uses of the invention are described below with respect to
an effective
amount of the compounds.
to Representative active compounds according to the invention are Compounds 1
through
20, as illustrated above.
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 wM).
Exemplary preferred compounds for use in methods related to Gardos channel
inhibition and sickle cell disease include Compounds 1, 2, 3, 4, 7, 9, 12, 13
and 14.
2o When the compound is to be used in methods to treat or prevent diarrhea
and/or
scours, it is preferred that the compound exhibit at least about 75%
inhibition of Cl~ secretion
from intestinal cells (measured at about 10 ~M) and/or have an ICSp of
inhibition of CY
secretion from intestinal cells of less than about 1 pM, with at least about
90% inhibition
andlor an ICS° of less than about 0.1 ~M being particularly preferred.
2s 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 pM) and/or have an ICS° of
cell proliferation of less
than about 3.5 pM, with at least about 90% inhibition and/or an ICSO of less
than about 1 pM
3o being particularly preferred. Even more preferred compounds meet both the %
inhibition and
ICS° criteria.
Exemplary preferred compounds for use in methods inhibiting mammalian cell
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proliferation or for the treatment or prevention of diseases characterized by
abnormal cell
proliferation include compound numbers 1, 2, 3, 4, 6, 7, 8, 10, 11, 15, 16,
17, 19 and 20.
formulation and Route~of Administration
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.
to The compounds described herein, or pharmaceutically acceptable salts andlor
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.
15 A subject as used herein, means humans, primates, horses, cows, sheep,
pigs, goats,
cats and dogs.
For example, when administered to subjects 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
2o containing agents that treat the side-effects of radiation therapy, such as
anti-emetics, radiation
protectants, etc.
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
25 HCI; Dacarbazine; Dactinomycin; Daunorubicin HCI; Doxorubicin HCI;
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 HCl
(nitrogen
mustard); Melphalan; Mercaptopurine; Mesna; Methotrexate (MTX); Mitomycin;
Mitotane
30 (o.p'-DDD); Mitoxantrone HCI; Octreotide; Plicamycin; Procarbazine HCI;
Streptozocin;
Tamoxifen citrate; Thioguanine; Thiotepa; Vinblastine sulfate; Vincristine
sulfate; Amsacrine
(m-AMSA); Azacitidine; Hexamethylmelamine {HMM); Interleukin 2; Mitoguazone
(methyl-
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GAG; methyl glyoxal bis-guanylhydrazone; MGBG); Pentostatin; Semustine (methyl-
CCNLJ); Teniposide (VM-26); paclitaxel and other taxanes; and Vindesine
sulfate.
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++ 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);
1o Thiol depleters (e.g., buthionine and sulfoximine); and calcium leucovorin.
The active compounds) 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
~ 5 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.
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,
20 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.
For oral administration, the compounds can be formulated readily by combining
the
active compounds) with pharmaceutically acceptable carriers well known in the
art. Such
25 carriers enable the compounds of the invention to be formulated as tablets,
pills, dragees,
capsules, liquids, gels, syrups, slurnes, suspensions and the like, for oral
ingestion by a
subject 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
3o 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
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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.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar
solutions rnay 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.
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, andlor 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.
For buccal administration,the compositions may take the form of tablets or
lozenges
formulated in conventional manner.
2o 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.
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
3o 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
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dispersing agents.
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.
Alternatively, the active ingredient may be in powder form for constitution
with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
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.
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.
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.
One product of the invention is a veterinary preparation of an aromatic
compound of
the invention, used alone or combined with an anti-scours agent. An anti-
scours agent is a
composition which is known to be useful in preventing or inhibiting the
symptoms of scours.
3o Known compositions include, for example, colostral extracts, such as those
described in U.S.
patent no. 4,377,569 and Canadian patent no. 1,175,352 and widely commercially
available
(e.g. Soluble Colostrum Powder, by VedCo, Inc., St. Joseph MO; Colostrum Bolus
II, by RX
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Veterinary Products, Kansas City MO, etc.); an immunological preparation of
colostrum
isolated from milk-producing mammals which may have been immunized against
certain
diarrheal causing microorganisms, such as those described in U.S. patent no.
4,834,974,
Australian patent no. 39340/89, Australian patent no. 52547/90, and German
patent no.
1,560,344; microorganism specific immunological preparations, including
microorganism
specific hybridoma-derived monoclonal antibodies such as those described in
Sherman et al.,
Infection and Immunity, V. 42 (2), P. 653-658 (1983) and a bovine
immunoglobulin fraction
prepared from bovine plasma or clear bovine serum such as the fraction
described in U.S.
patent no. 3,984,539; oral rehydration fluids and/or replacement electrolyte
compositions
1o which are widely commercially available in the form of dry compositions or
liquid solutions
prepared for oral or intravenous administration (e.g. Electrolyte H, by Agri-
Pet Inc., Aubrey
TX; Electrolyte Powder 8x, by Phoenix Pharmaceutical Inc, St. Joseph MO;
Electrolyte
Solution Rx, by Lextron Inc., Greeley CO, ProLabs LTD, St. Joseph MO, and
VetTek Inc.,
Blue Springs MO; Calf Rehydrate, by Durvet Inc., Blue Springs MO, etc.) and
antibiotic
compositions which are commercially available (e.g. BIOSOL~ Liquid, by The
UpJohn
Company Animal Health Division, Kalamazoo MI; AMOXI-BOL~, by SmithKline-
Beecham
Animal Health, Exton PA; 5-WAY CALF SCOUR BOLUSTM, by Agri Laboratories LTD,
St.
Joseph MO; 1-A-DAY CALF SCOUR BOLUS, by A.H.A.; GARACIN~ PIG PUMP, by
Schering-Plough Animal Health Corporation, Kenilworth NJ, etc.).
2o In one embodiment, the veterinary preparation is a dry preparation of the
aromatic
compound of the invention and an antiscours agent. The dry preparation may be
administered
directly or may be hydrated and/or diluted in a liquid solution prior to
administration. In
another embodiment, the veterinary preparation is a liquid solution of the
compound of the
invention and an anti-scours agent.
Another product of the invention is a pharmaceutical preparation of an
aromatic
compound of the invention and an anti-diarrheal agent. An anti-diarrheal agent
includes, for
example, an immunoglobulin preparation from bovine colostrum; lomotil; an
intravenous or
oral rehydration fluid; a dry rehydration composition salt; an electrolyte
replacement
composition (in dry or liquid form); an oral or intravenous sugar-electrolyte
solution or dry
3o composition; an antibiotic such as tetracycline, trirmethoprim or
sulfamethoxazole; a
quinolone drug such as norfloxacin or ciprofloxacin, bismuth subsalicylate,
diphenoxylate;
and loperamide.
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In one embodiment the pharmaceutical preparation is a dry preparation of the
aromatic
compound of the invention and an anti-diarrheal agent. The dry preparation may
be
administered directly or may be hydrated and/or diluted in a liquid solution
prior to
administration. In another embodiment the pharmaceutical preparation is a
liquid solution of
the aromatic compound of the invention and an anti-diarrheal agent.
The time of administration of the aromatic compounds useful according to the
invention varies depending upon the purpose of the administration. When the
compounds of
the invention are administered in order to prevent the development of diarrhea
in subjects
traveling to areas with high risk of exposure to infectious agent or subjects
otherwise exposed
1 o to diarrhea causing agents, the compounds should be administered at about
the time that the
subject is exposed to the risk or the high risk area. When the compounds are
administered to
subjects in order to prevent the development of scours, the veterinary
preparation should be
administered within the first 12 hours after birth, and preferably within the
first 4 hours after
birth. When the compounds of the invention are used to treat subjects having
symptoms of
diarrhea or scours, the compounds may be administered at any point while the
subject is
experiencing symptoms, and preferably as soon as the symptoms develop. Other
considerations will be apparent when the compounds are used to treat
inflammatory diseases,
proliferative diseases, etc.
When administered, the formulations of the invention are applied in
pharmaceutically
2o acceptable amounts and in pharmaceutically acceptable compositions. Such
preparations may
routinely contain salts, buffering agents, preservatives, compatible carriers,
and optionally
other therapeutic ingredients. When used in medicine the salts should be
pharmaceutically
acceptable, but non-pharmaceutically acceptable salts may conveniently be used
to prepare
pharmaceutically acceptable salts thereof and are not excluded from the scope
of the
invention. Such pharmacologically and pharmaceutically acceptable salts
include, but are not
limited to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric,
nitric, phosphoric, malefic, acetic, salicylic, p-toluene sulfonic; tartaric,
citric, methane
sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene
sulfonic. Also,
pharmaceutically acceptable salts can be prepared as alkaline metal or
alkaline earth salts,
3o such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric
acid and a
salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and
a salt
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(0.8-2% W/V).
Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V);
chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V} and thimerosal (0.004-
0.02%
W/V).
The active compounds of the present invention may be pharmaceutical
compositions
having a therapeutically effective amount of an aromatic compound of the
general formula
provided above in combination with a non-formula (I) active agent, optionally
included in a
pharmaceutically-acceptable carrier. The active compounds of the present
invention also may
be veterinary compositions having a therapeutically effective amount of an
aromatic
1o compound of the general formula provided above in combination with a non-
formula (I) active
agent, optionally included in a pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" as used herein means one or more
compatible solid or
liquid filler, dilutants or encapsulating substances which are suitable for
administration to a
human or other animal. The term "carrier" denotes an organic or inorganic
ingredient, natural
15 or synthetic, with which the active ingredient is combined to facilitate
the application. The
components of the pharmaceutical compositions also are capable of being
commingled with
the compound of the present invention, and with each other, in a manner such
that there is no
interaction which would substantially impair the desired pharmaceutical
efficacy.
A common administaration vehicle (e.g., pill, tablet, bolus, powder or
solution for
2o dilution, pig pump, implant, injectable solution, etc.) would contain both
the compounds
useful in this invention and a non-formula (I) active agent. Thus, the present
invention
provides pharmaceutical or veterinary compositions, for medical or veterinary
use, which
comprise the active compounds of the invention together with one or more
pharmaceutically
acceptable carriers thereof and other therapeutic ingredients.
25 A variety of administration routes are available. The particular mode
selected will
depend of course, upon the particular drug selected, the severity of the
condition being treated
and the dosage required for therapeutic efficacy. The methods of this
invention, generally
speaking, may be practiced using any mode of administration that is medically
acceptable,
meaning any mode that produces effective levels of the active compounds
without causing
3o clinically unacceptable adverse effects. Such modes of administration
include oral, rectal,
topical, nasal, transdermal or parenteral routes. The term "parenteral"
includes subcutaneous,
intravenous, intramuscular, or infusion. Intravenous and intramuscular routes
are not
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particularly suited for long term therapy and prophylaxis. They could,
however, be preferred
in emergency situations. Oral administration will be preferred for
prophylactic treatment
because of the convenience to the subject as well as the dosing schedule.
The compositions may conveniently be presented in unit dosage form and may be
prepared by any of the methods well known in the art of pharmacy. All methods
include the
step of bringing the active compounds into association with a carrier which
constitutes one or
more accessory ingredients. In general, the compositions are prepared by
uniformly and
intimately bringing the active compounds into association with a liquid
carrier, a finely
divided solid Garner, or both, and then, if necessary, shaping the product.
Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of the active compound, which is preferably isotonic with
the blood of
the recipient. This aqueous preparation may be formulated according to known
methods
using those suitable dispersing or wetting agents and suspending agents. The
sterile injectable
preparation may also be a sterile injectable solution or suspension in a non-
toxic
parenterally-acceptable diluent or solvent, for example as a solution in l, 3-
butane diol.
Among the acceptable vehicles and solvents that may be employed are water,
Ringer's
solution, and isotonic sodium chloride solution. In addition, sterile, fixed
oils are
conventionally employed as a solvent or suspending medium. For this purpose
any bland
fixed oil may be employed including synthetic mono or di-glycerides. In
addition, fatty acids
such as oleic acid find use in the preparation of injectables. Carrier
formulations suitable for
oral, subcutaneous, intravenous, intramuscular, etc. can be found in
Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
Other delivery systems can include time-release, delayed release or sustained
release
delivery systems. Such systems can avoid repeated administrations of the
active compounds
of the invention, increasing convenience to the subject and the physician.
Many types of
release delivery systems are available and known to those of ordinary skill in
the art. They
include polymer based systems such as polylactic and polyglycolic acid,
polyanhydrides and
polycaprolactone; nonpolymer systems that are lipids including sterols such as
cholesterol,
cholesterol esters and fatty acids or neutral fats such as mono-, di- and
triglycerides; hydrogel
3o release systems; silastic systems; peptide based systems; wax coatings,
compressed tablets
using conventional binders and excipients, partially fused implants and the
like. Specific
examples include, but are not limited to: (a) erosional systems in which an
agent of the
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invention is contained in a form within a matrix such as those described in
U.S. Patent Nos.
4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an
active
component permeates at a controlled rate from a polymer such as described in
U.S. Patent
Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware
delivery
systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for
treatment
of diarrhea in immunodeficient subjects, who need continuous administration of
the
compositions of the invention. "Long-term" release, as used herein, means that
the implant is
constructed and arranged to deliver therapeutic levels of the active
ingredient for at least 30
1 o days, and preferably 60 days. Long-term sustained release implants are
well known to those
of ordinary skill in the art and include some of the release systems described
above.
Effective Dosages
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 subjects suffering from
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 subject 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.
3o For any compound described herein the therapeutically effective amount can
be
initially determined from cell culture assays. Target plasma concentrations
will be those
concentrations of active compounds) that are capable of inducing at least
about 25%
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inhibition of the Gardos channel, at least about 25% inhibition of Cl-
secretion in intestinal
cells 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 compounds) that are capable of inducing at least about 50%, 75%, or
even 90% or
higher inhibition of the Gardos channel, of Cf secretion in intestinal cells,
and/or of cell
proliferation in cell culture assays are preferred. The percentage inhibition
of the Gardos
channel, of CY secretion in intestinal cells, andlor cell proliferation in the
subject 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.
1 o 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. Useful animal
models for
diseases characterized by abnormal cell proliferation are well-known in the
art. A particularly
useful animal model for sickle cell disease is the SAD mouse model (Trudel et
al., 1991,
t s ~MB(,~. L: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. x:95-108;
Dykes et al., 1992, Contrib. Qncol. Basel. Kar~er ~:1-22), restenosis (Carter
et al., 1994, ~
Am. Coll. Cardiol. x(5):1398-1405), atherosclerosis (Zhu et al., 1994,
Cardioloav 85(6):370-
20 377) and neovascularization (Epstein et al., 1987, Cornea x(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.
Additional in vivo assays are well known in the art. For instance, the
following assays
are useful for assessing effective amounts of compounds for treating
inflammatory diseases
25 associated with cellular proliferation: Airway inflammation and
hyperresponsiveness in
Ovalbumin-sensitized mice or guinea pigs; NZB/NZW crossed mice develop
glomerular
disease and lupus-like syndrome; Renal allograft rejection in mice;
Trinitrobenzene sulphonic
acid induced bowel inflammation in rats; NZB/NZW crossed mice develop
glomerular disease
and lupus-like syndrome; Experimental allergic encephalomyelitis; Rat adjuvant
arthritis
3o assay; HLA transgenic mice immunized with thyroglobulin; and Thiouracil-fed
rats.
A therapeutically effective dose can also be determined from human data for
compounds which are known to exhibit similar pharmacological activities, such
as
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Clotrimazole and other antirnycotic agents (see, e.g., Brugnara et al., 1995,
JPET ~:266-
272; Benzaquen et al., 1995, Nature Medicine 1:534-540; Brugnara et al., 1996,
lin.
Invest. QZ(5):1227-1234). The applied dose can be adjusted based on the
relative
bioavailability and potency of the administered compound as compared with
Clotrimazole.
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.
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.
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 ~M to 20 wM is considered to be
effective, with about
0.1 ~.M to 5 pM being preferred.
Subject 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.
2o Stated in terms of subject body weight, typical dosages range from about 1
to 200 mg/kg/day,
more typically from about 10 to 100 mg/kglday, and most typically from about
10 to 50
mg/kg/day. Stated in terms of subject body surface areas, typical dosages
range from about
40 to 8000 mg/m2/day, more typically from about 400 to 4000 mglm2lday, and
most typically
from about 400 to 2000 mg/m2/day.
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 uM to 20 ~M is
considered to be
effective, with about 0.1 ~M to 5 ~.M being preferred.
Subject doses for oral administration of the compounds described herein for
the
3o 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 subject bpdy weight,
typical
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dosages range from about 1 to 200 mg/kglday, more typically from about 10 to
100
mg/kg/day, and most typically from about 10 to 50 mglkg/day. Stated in terms
of subject
body surface areas, typical dosages range from about 40 to 8000 mg/m2/day,
more typically
from about 400 to 4000 mg/mZ/day, and most typically from about 400 to 2000
mg/m2/day.
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 subject 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.
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
2o administration of the compounds at the tumor site.
Combined with the teachings provided herein, by choosing among the various
active
compounds and weighing factors such as potency, relative bioava.ilability,
subject 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 subject. Of course, many factors are important in determining a
therapeutic
regimen suitable for a particular indication or subject. Severe indications
such as cancer may
warrant administration of higher dosages as compared with less severe
indications such as
sickle cell disease.
3o The formulations of the invention are also administered in effective
amounts when
treating diarrhea or scours. An effective amount is one sufficient to inhibit
or prevent diarrhea
or scours and is thus sufficient to inhibit the CY secretion of intestinal
epithelial cells. An
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amount which is sufficient to inhibit the CI- secretion of intestinal
epithelial cells thereby
effectively decreases the secretory response, thereby resulting in a decrease
in diarrhea or
scours andlor the symptoms thereof. Effective amounts will depend, of course,
on the
particular condition being treated; the severity of the condition; individual
subject parameters
including age, physical condition, size and weight; concurrent treatment;
frequency of
treatment; and the mode of administration. These factors are well known to
those of ordinary
skill in the art and can be addressed with no more than routine
experimentation.
An effective amount for an individual compound may be assesed using any method
known in the art which reliably determines the amount of CY secretion from
intestinal cells.
A compound may be subject to a series of standard assays or screens to
determine its
pharmacological activity and effective amounts.
In general, the active compounds of the invention are those which induce at
least about
25% inhibition of the Cl- secretion, as measured using in vitro assays that
are commonly
known in the art (see, e.g., Example 4). Alternatively, or in addition, the
active compounds of
the invention generally will have an ICso (concentration of compound that
yields 50%
inhibition) for inhibition of the CY secretion of less than about 10 pM as
measured using in
vitro assays.
It is preferred generally that a maximum dose be used, that is, the highest
safe dose
according to sound medical judgment, particularly if acute diarrhea or scours
are the dominant
2o clinical manifestation. Dosage may be adjusted appropriately to achieve
desired drug plasma
levels. Generally, daily oral doses of active compounds will be from about
0.01
milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral
doses in the
range of 50 to 500 milligrams/kg, in one or several administrations per day,
will yield the
desired results. In the event that the response in a subject is insufficient
at such doses, even
higher doses (or effective higher doses by a different, mare localized
delivery route) may be
employed to the extent that subject tolerance permits. Multiple doses per day
are
contemplated to achieve appropriate systemic levels of compounds.
Toxicity
The ratio between toxicity and therapeutic effect for a particular compound is
its
3o therapeutic index and can be expressed as the ratio between LDS°
(the amount of compound
lethal in 50% of the population) and EDSO (the amount of compound effective in
SO% of the
population). Compounds which exhibit high therapeutic indices are preferred.
Therapeutic
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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 EDso 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 subject's condition. (See
e.g. Fingl et al.,
1975, In: The Pharmacological Basis of Thera ep utics, Ch. 1 pl).
The invention having been described, the following examples are intended to
illustrate,
not limit, the invention. Some of the following examples depict tests that are
employed to
determine the effects on C 1- secretion. Clotrimazole, which is outside of the
scope of the
present claims is used to exemplify how the compounds of the present invention
are tested for
certain conditions. The compounds of the present invention are structurally
distinct from the
structure of clotrimazole. Nevertheless, the compounds of the present
invention act on
chloride secretion in the same manner as clotrimazole and, therefore, are
useful in the methods
and products of the present invention.
~xamules
Example 1. Com on and S3rntheses
This Example demonstrates general methods for synthesizing the compounds of
the
invention, as well as preferred methods of synthesizing certain exemplary
compounds of the
2o invention. In all of 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
funtionalities 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 Grou sp in Organic
Wig, John Wiley & Sons, New York, 1991.
In FIGS. 1 and 2, the various substituents are defined as for structure (I),
supra.
1. ~ynthe~i ~ of Suhstituted 3 3-Dinhenyl Indanones
Referring to FIG. 1, substituted 3,3-diphenyl indanone compounds are
synthesized as
follows: substituted triphenylpropionic acid ~QO (0.25-0.50 M in sulfuric
acid) is stirred at
3o room temperature for 1 hour and then poured into an equal volume of cold
water. The
aqueous mixture is extracted with an equal volume of ethyl acetate and the
organics dried over
sodium sulfate. Evaporation gives the desired substituted 3,3-diphenyl
indanone compound
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~l in about 60-75% yield.
2. synthesis of Substituted 1-H d~X-3 3-Dinhenyl Indane Compounds
Referring to FIG. 1, substituted 1-hydroxy-3,3-diphenyl indane compounds are
synthesized as follows: a solution of substituted 3,3-diphenylindanone ~Q~,
(0.25 M in
tetrahydrofuran) is added dropwise to 0.25 volume of a 1.0 M solution of
lithium aluminum
hydride in tetrahydrofuran at 0-5 °C. The mixture is warmed to reflux
and refluxed for 2.5 h,
cooled to 0-5 °C and an equal volume of 1 M HCl added slowly. The
mixture is then
extracted three times with an equal volume of ethyl acetate. The combined
organic extracts
1 o are washed with a saturated aqueous solution of sodium bicarbonate and
dried over sodium
sulfate. Evaporation gives the desired substituted 1-hydroxy-3,3-diphenyl
indane compound
~ in about 45-90 % yield.
3. Synthesis of Substituted 1-N-Oxime-3 3-Di enyl Inr~anes
Referring to FIG. 1, substituted 1-N-oxime-3,3-diphenyl indane compounds are
synthesized as follows: substituted 3,3-diphenylindanone 112 (1 equivalent) is
combined with
5 equivalents of hydroxylarnine hydrochloride and 10 equivalents of sodium
acetate and
dissolved in methanol. The solution is stirred at room temperature for 16 h
and then an equal
volume of water is added. The mixture is extracted three times with an equal
volume of ethyl
2o acetate and the combined organic extracts are dried over sodium sulfate.
Evaporation gives
the desired substituted 1-N-oxime-3,3-diphenyl indane compound 1~ (as a
mixture of cis and
traps isomers) in about 90-98 % yield.
4. ~ nthesis of Substituted 2-A~~Cy~~-3.3-Diphen l~ndanones
Refernng to FIG. 1, substituted 2-alkyl-3,3-diphenyl indanone compounds are
synthesized as follows: substituted 3,3-diphenyl indanone ~ (1 equivalent) is
dissolved in
tetrahydrofuran (0.4 - 1.0 M) and 1.2 equivalents of potassium hydride is
added. The mixture
is stirred at room temperature until the gas evolution subsides and then the
bromoalkane (1.2
equivalents) is added. The mixture is stirred at room temperature and
monitored by TLC.
3o The reaction is quenched with water and the mixture extracted with ethyl
acetate. The desired
substituted 2-alkyl-3,3-diphenyl indanone compound ~Q$ is isolated by silica
gel
chromatography in about 50-75% yield.
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5. ~vnthesis of Substituted 1-Alkox ~~-3,3-Di henyl Indanes
Referring to FIG. 1, substituted I-alkoxy-3,3-diphenyl indane compounds are
synthesized as follows: substituted 1-hydroxy-3,3-diphenylindanone 1(~4 (i
equivalent) is
combined with 2 equivalents of sodium hydride in N,N-dimethylformamide and
stirred at
room temperature until the gas evolution subsides. The haloalkane (2
equivalents) is added
and stirred at room temperature for 16-20 hours. An equal volume of water is
added and the
mixture extracted four times with twice the volume of ethyl acetate. The
combined organic
extracts are dried over sodium sulfate and the solvent removed in vacuo. The
desired
substituted 1-alkoxy-3,3-diphenyl indane compound 1~"Q is isolated by vacuum
distillation.
to
1.6 ~, nt~ hesis of Substituted 3.3-Diphenyl-3H Indoles
Referring to FIG. 2, substituted 3,3-diphenyl-3H indole compounds are
synthesized as
follows: substituted phenyl hydrazine ~ is combined with an equimolar amount
of
substituted 1,1-diphenyl-2-ketone ~ in phosphoric acid. This mixture is
stirred at 100-120
°C until the reaction is complete as determined by TLC. The reaction is
cooled to 60-70 °C
and diluted with twice the volume of water while stirring. After cooling to
room temperature,
the mixture is filtered, washed with water, and the crude solid substituted
3,3-diphenyl indole
compound ~ is purified by column chromatography or crystallization.
1.7 synthesis of S~~stituted 3,,3-D' hoe yl-3H Indolines
Referring to FIG. 2, substituted 3,3-diphenyl-3H indoline compounds are
synthesized
as follows: substituted 3,3-diphenyl indole compound ,~ is reduced with sodium
borohydride or sodium cyanoborohydride in a suitable solvent to yield the
substituted 3,3-
diphenyl-3H indoline compound ~ø.
1.8 ~" nthy esis o_f ~ybstituted N-Substituted-3.3-Dinhenyl Indolines
Referring to FIG. 2, substituted N-substituted-3,3-diphenyl indoline compounds
are
synthesized as follows: substituted 3,3-diphenyl indoline jib (1 equivalent)
is combined with
an alkyl halide (1 equivalent) and potassium carbonate (3-4 equivalents) in
acetonitrile. The
3o mixture is stirred at reflux until the reaction is complete as determined
by TLC. Water and
ethyl acetate are added and the mixture is extracted with ethyl acetate.
Evaporation of the
combined ethyl acetate extracts gives the crude substituted N-substituted-3,3-
diphenyl
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indoline compound ~$, which is purified by column chromatography.
1.9 Synthesis of 3.3-Di~henylindanone (ColIl on and 2)
3,3-Diphenylindanone (Compound 2) was synthesized as follows:
Triphenylpropionic
acid (12 g, 0.04 mol) was stirred in 50 ml concentrated sulfuric acid for 1
hour. The reaction
mixture was cooled in an ice bath and diluted with 50 ml water. This mixture
was extracted
three times with ethyl acetate. The ethyl acetate extracts were combined,
dried over sodium
sulfate and the solvent removed in vacuo to yield 9.0 g (78% yield) of 3,3-
Diphenylindanone
(Compound 2) as a white solid having a melting point of 119-123 °C.
1.10 synthesis of 1-H,~y-3.3-Di henylin ~~l,Compoa_nd_3_l
1-Hydroxy-3,3-Diphenylindane (Compound 3) was synthesized as follows: A
solution
of 2 g (0.007 mol) 3,3-diphenylindanone (Compound 2) in 20 ml of
tetrahydrofuran was
added dropwise to a solution of 0.34 g (0.009 mol) LiAlH4 in 10 ml
tetrahydrofuran at 0-5 °C.
The mixture was warmed to reflux and refluxed for 3 hr., cooled to 0-5
°C and 30 ml of 1 M
HCl added slowly. The mixture was then extracted three times with 60 ml ethyl
acetate. The
ethyl acetate extracts were combined, washed with a saturated aqueous solution
of sodium
bicarbonate and dried over sodium sulfate. Evaporation of the solvent gave 0.9
g (45% yield)
of 1-Hydroxy-3,3-Diphenylindane (Compound 3) as white crystals with a melting
point of
133-135°C.
1.11 Synthesis of 1-N-Oxime-3.3-Dinhenvlindane (Compound 41
1-N-Oxime-3,3-Diphenylindane (Compound 4) was synthesized as follows: 3,3-
Diphenylindanone (Compound 2) (2.0 g, 0.007 mol) was combined with 2.4 g
(0.035 mol) of
hydroxylamine hydrochloride and 5.8 g (0.07 mol) of sodium acetate and
dissolved in 30 ml
of methanol. The solution was stirred at room temperture for 16 hr and then
100 ml of water
was added. The mixture was extracted with 100 ml ethyl acetate and the organic
layer dried
over sodium sulfate. Evaporation of the solvent gave 1.9 g (90% yield) of 1-N-
Oxime-3,3-
Diphenylindane (Compound 4) as a white solid having a melting point of 138-141
°C.
1.12 ~ynthesis of Sp~,ro[~~3-di enyl-2.3-dihydro(,1H)indene-1_3'-2'-
~yanooxirane~
(Com on and 51 and 2-Cvanomethyl-3"3-dinhenylindanone (Compound 97
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Spiro[3,3-diphenyl-2,3-dihydro(1F-I)indene-1,3'-2'-cyanooxirane] {Compound 5)
and
2-cyanomethyl-3,3-diphenylindanone (Compound 9) were synthesized as follows:
3,3-
diphenylindanone (Compound 2), 5.0 g (0.0176 mole) and 2.62 g (0.0229 mole) of
potassium
hydride were stirred at room temperature in 40 mL of tetrahydrofuran. After
the gas evolution
subsided (approx. 45 min), 1.5 mL (0.0215 mole) of bromoacetonitrile was
added. The dark
red mixture was stirred for 1 hour and then SO mL of water was added. The
mixture was
extracted three times with 75 mL of ethyl acetate. The combined organic
extracts were
concentrated in vacuo, loaded onto a silica gel column and eluted with I O%
ethyl acetate in
hexane. Three fractions were collected. After evaporation of the solvent, the
first fraction
yielded unreacted starting material (3.5 g). The second fraction yielded 0.49
g (9% yield) of
spiro[3,3-diphenyl-2,3-dihydro(1H)indene-1,3'-2'-cyanooxirane] (Compound 5) as
a white
solid. The third fraction yielded 1.05 g (18% yield) of 2-cyanomethyl-3,3-
diphenylindanone
(Compound 9) as a yellow oil.
1.13 Svnthesis~2-l2'-Pro~~l-1-(2'-pro e~yl-3.3-diphenvlindane lCo~uound 61
2-(2'-Propenyl)-1-(2'-propenoxy)-3,3-diphenylindane (Compound 6) was
synthesized
as follows: 3,3-diphenylindanone (Compound 2) 2.0 g (0.007 mole) and 0.28 g
(0.0084 mole)
sodium hydride were stirred at room temperature in 40 mL of dimethylformamide
for 1 hour.
The reaction mixture was then added drop-wise to 0.64 mL (0.0078 mole) of
allyl bromide at
-50 °C. The mixture was then warmed to reflux and refluxed for 1 hour.
After cooling to
room temperature, 50 mL of water was added. The mixture was extracted with
ethyl acetate,
dried over sodium sulfate and concentrated in vacuo. 2-(2'-propenyl)-1-(2'-
propenoxy)-3,3-
diphenylindane (Compound 6) was isolated in 30% yield as the first fraction
from a silica gel
column using 10% dichloromethane in hexane as eluate.
1.14 ~"~thesis of 1-Acetoxy-3,3-dinhenylindane (Compound 71
1-Acetoxy-3,3-diphenylindane (Compound 7) was synthesized as follows: 1-
~Iydroxy-3,3-diphenylindane (Compound 3) (0.06 g, 0.0021 mol) was combined
with 0.3 mL
(0.0022 mol) triethylamine in 10 mL of dichloromethane. The mixture was warmed
to reflux
3o with stirring to dissolve all of the starting material. The heat was
removed and 0.16 mL
(0.0022 mol) of acetyl chloride was added to the warm solution. The mixture
was returned to
reflux and stirred at reflux for 1 h. After cooling to room temperature, the
reaction was
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quenched by adding 5 mL of water. The reaction mixture was extracted with
dichloromethane
and the organic layer dried over sodium sulfate. Evaporation of the solvent
gave 0.008 g
(11% yield) of 1-acetoxy-3,3-diphenylindanone (Compound 7) as an off white
solid with a
melting point of 90°C.
1.15 S~~nthesis of 6-Chloro-3.3-di(4-chlorophenyllindanone (Compound 81
6-Chloro-3,3-di(4-chlorophenyl)indanone (Compound 8) was synthesized as
follows:
3,3,3-Tris(4-chlorophenyl) propionic acid (1.5 g, 0.004 mol) was stirred in 10
mL of
concentrated sulfuric acid at room temperature for 1.5 h. The reaction mixture
was then
to poured into 10 mL of ice water and the mixture extracted with
dichloromethane. The solvent
was evaporated and 0.8 g (54% yield) of 6-Chloro-3,3-di(4-
chlorophenyl)indanone
(Compound 8) was collected as an off white solid having a melting point of
134°C.
1.16 Synthesis of 6-Chloro-2-cyanomethyl-3.3-di(4'-chloronhenyl)indanone
(Compound 10)
15 6-Chloro-2-cyanomethyl-3,3-di(4'-chlorophenyl)indanone (Compound 10) was
synthesized as follows: 6-Chloro-3,3-di(4'-chlorophenyl)indanone (Compound 8)
(1.0 g,
0.0026 mol) was dissolved in S mL of tetrahydrofuran and 0.124 g (0.0031 mol)
of sodium
hydride was added. The reaction mixture was stirred at room temperature for
1.5 h before
0.22 mL (0.0215 mol) of bromoacetonitrile was added. After stirring overnight
the reaction
2o was quenched with water and extracted with ethyl acetate. The extracts were
combined and
the solvent removed in vacuo. The residue was purified on a silica gel column
using 5% ethyl
acetate in hexane as the eluent. The first fraction from the column was
recovered starting
material (1.05 g). The second fraction contained undesired side reaction
product. The third
fraction contained the desired product. After evaporation of the solvent,
0.179 g (16% yield)
25 6-Chloro-2-cyanomethyl-3,3-di(4'-chlorophenyl)indanone (Compound 10) as a
pale yellow
solid was obtained.
1.17 ~ynthes,'_s ~f f-Chloro-3.3-dif4'-chlorophen~l-2-N-oxime-3.3-
diphenvlindane
~Com~ound 11),
30 6-Chloro-3,3-di(4'-chlorophenyl)-2-N-oxime-3,3-diphenylindane (Compound 11)
was
synthesized as follows: 6-Chloro-3,3-di(4'-chlorophenyl)indanone (compound 8)
(0.80 g,
0.0021 mol) was combined with 0.72 g (0.0103 moI) of hydroxylamine
hydrochloride and
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1.69 g (0.0206 mol) of sodium acetate and dissolved in 25 mL of methanol. The
solution was
stirred at room temperature for 16 h and then water was added. The mixture was
extracted
with ethyl acetate and the organic layer was dried over magnesium sulfate.
Evaporation of the
solvent gave 0.85 g (100% yield) of 6-Chloro-3,3-di(4'-chlorophenyl)-2-N-oxime-
3,3-
diphenylindane (Compound 11) as a white solid having a melting point of 85
°C.
1.18 synthesis of 2-Acetamide-3.3-di ~nhe_n,-~rlindanone (Compound 12)
2-Acetamide-3,3-diphenylindanone {Compound 12) was synthesized as follows: 2-
Cyanomethyl-3,3-diphenylindanone (0.685 g, 0.0021 mol) was combined with 10 mL
of
1 o concentrated sulfuric acid and 10 mL of glacial acetic acid. The solution
was stirred at room
temperature for 3 h and then water was added. The mixture was cooled in an ice
bath and
neutralized to pH 7 with concentrated ammonium hydroxide and then extracted
with ethyl
acetate. The organic layer was dried over magnesium sulfate. Evaporation of
the solvent
gave 0.77 g of a light orange solid. This solid was crystallized from a
mixture of ethyl acetate
and hexane. 2-Acetamide-3,3-diphenylindanone (Compound 12) was obtained as off
white
crystals, 0.527g (73% yield), having a melting point of 169 - 171 °C.
1.19 Synthesis of 2-Cyanometh~.3-diphenvlindanol lComnound 131
2-Cyanomethyl-3,3-diphenylindanol (Compound 13) was synthesized as follows: 2-
2o Cyanomethyl-3,3-diphenylindanone (Compound 2) (0.311 g, 0.001 mol) was
dissolved in 5
mL of ethanol at room temperature. Sodium borohydride (0.437 g, 0.011 mol) was
added and
the mixture was stirred at room temperature for 15 min. The mixture was
diluted with ethyl
acetate and the pH was adjusted to 2 with 2N hydrochloric acid. The layers
were separated
and the aqueous layer extracted twice with ethyl acetate. The combined
extracts were
evaporated in vacuo and the crude product was purified on a silica gel column
using 20%
ethyl acetate in hexane. The first fraction was unreacted starting material.
The second
fraction, when the solvent was evaporated, gave 0.16 g (51% yield) of 2-
Cyanomethyl-3,3-
diphenylindanol (Compound 13) as a white solid having a melting point of 79 -
85 °C.
1.20 Synthesis of 2-Acetamide-3.3-dinhenylindanol (Comnound 141
2-Acetamide-3,3-dipheriylindanol (Compound 14) was synthesized as follows: 2-
Acetamide-3,3-diphenylindanone (Compound 12) (0.100 g, 0.0003 mol) was
dissolved in 2
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mL of ethanol and 0.5 mL of methanol at room temperature. Sodium borohydride
(0.136 g,
0.0004 mol) was added and the mixture was stirred at room temperature for 3
hours. The
mixture was quenched with 2N hydrochloric acid to pH 1. The mixture was
extracted with
ethyl acetate and the combined extracts dried over magnesium sulfate.
Evaporation of the
solvent gave an off white solid which was crystallized from a mixture of ethyl
acetate/hexane.
2-Acetamide-3,3-diphenylindanol (Compound 14) was collected by filtration as a
white solid
(0.026 g, 25% yield) having a melting point of 218 - 220 °C.
1 21 Syythesis of 3 3-Diphenylindanone-2-methyl acetalg_(,Comnound 15~
3,3-Diphenylindanone-2-methyl acetate (Compound 15) was synthesized as
follows:
3,3-Diphenylindanone (Compound 2) (3.84 g, 0.0135 mol) was dissolved in 30 mL
of
tetrahydrofuran at room temperature. Potassium hydride (1.85 g, 0.0162 mol)
was added and
the mixture was stirred at room temperature for 1 hour. Methyl chloroformate
(1.25 mL,
0.0162 mol) was added and the mixture was stirred at room temperature for 1
hour. The
I5 mixture was quenched with water and extracted with ethyl acetate. The
combined extracts
were dried over magnesium sulfate. Evaporation of the solvent gave an dark
brown solid
which was purified on a silica gel column using 5% ethyl acetate in hexane as
eluent. The
product was collected in the second fraction off the column. Evaporation of
the solvent gave
a slightly wet, pink solid which was stirred in hexane. 3,3-Diphenylindanone-2-
methyl
2o acetate (Compound 15) was collected by filtration as an off white solid
(2.06 g, 45% yield)
having a melting point of 140 - 142°C.
1.22 S~vnt_h_esis of 3.3-Diphenvl-1-indan l~ ht lmet yl ether (Compound 16)
3,3-biphenyl-1-indanyl 2-naphthylmethyl ether (Compound 16) was synthesized as
25 follows: 1-Hydroxy-3,3-diphenylindane (Compound 3) (0.25 g, 0.87 mmol) was
dissolved in
10 mL of dimethylformamide and cooled to 0°C with stirring. Sodium
amide (0.042 g, 1.04
mmol) was added and the reaction stirred for 0.5 h at 0 °C before 0.23
g ( 1.04 mmol) of 2-
bromomethylnaphthalene was added. The reaction mixture was allowed to warm to
room
temperature and stirred for 15h. An equal volume of water was added to the
mixture and this
3o was extracted twice with 50 mL of ethyl acetate. After drying over
magnesium sulfate the
solvent was evaporated and the resultant solid was purified on a silica gel
column using 2%
ethyl acetate in hexane as the eluent. The second fraction collected was the
desired product.
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Evaporation of the solvent gave 0.300 g (81% yield) of 3,3-biphenyl-1-indanyl
2-
naphthylmethyl ether {Compound 16) as an off white, sticky solid.
1.23 Synthesis of 3.3-biphenyl-1-indan3r~4-methvltoluatel ether (Compound 17~
3,3-biphenyl-1-indanyl a-{4-methyltoluate) ether (Compound 17) was synthesized
as
follows: 1-Hydroxy-3,3-diphenylindane (Compound 3) (0.505 g, 1.8 mmol} was
combined
with 0.069 g (2.9 mmol) of sodium amide in 10 mL of dimethylformamide and
stirred at room
temperature for 1.5 h before 0.667 g (2.9 mmol) of methyl 4-
(bromomethyl)benzoate was
added. The reaction mixture was stirred for 18h. The reaction mixture was
poured into 50
f o mL of water and extracted four times with 25 mL of ethyl acetate. The
combined extracts
were washed with brine, dried over sodium sulfate and the solvent evaporated
to yield a
yellow oil. The oil was purified by vacuum distillation to give 0.370 g (47%
yield) of 3,3-
Diphenyl-1-indanyl a-(4-methyltouate) ether (Compound 17) as a yellow solid
having a
melting point of 50-52 °C.
1.24 Synthesis of 3,3-biphenyl-1-indan, lea-(2-chlorotoluy~l ether (,Compound
18)
3,3-biphenyl-1-indanyl a-(2-chlorotoluyl) ether (Compound 18) was synthesized
as
follows: 1-Hydroxy-3,3-diphenylindane (Compound 3) (0.503 g, 1.8 mmol) was
combined
with 0.075 g (3.1 mmol) of sodium amide in 10 mL of dimethylformamide and
stirred at room
2o temperature for 1.5 h before 0.40 mL (3.2 mmol) of 2-chlorobenzyl chloride
was added. The
reaction mixture was stirred for 21 h. The reaction mixture was poured into 50
mL of water
and extracted four times with 25 mL of ethyl acetate. The combined extracts
were washed
with brine, dried over sodium sulfate and the solvent evaporated to yield a
yellow oil. The oil
was purified by vacuum distillation to give 0.520 g (70% yield) of 3,3-
biphenyl-1-indanyl a-
(2-chlorotoluyl) ether (Compound 18) as a solid having a melting point of 27 -
29°C.
1.25 S~vnthesis of 3-f"3'.3'-dinhenYl-2'-indar~vl-1'-one ronanol f~"o~~pound 1
3-(3',3'-diphenyl-2'-indanyl-1'-one}propanol (Compound 19) was synthesized as
follows: 3,3-Diphenylindanone (Compound 2) (2 g, 0.007 mol) was dissolved in
10 mL of
3o tetrahydrofuran, cooled in an ice bath, and 0.97 g {0.0085 mol) of
potassium hydride was
added. The reaction mixture was stirred at room temperature for 0.5 h before
0.72 mL
(0.0077 mol) of 3-bromo-1-propanol was added. After stirnng overnight the
reaction was
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quenched with water and extracted with ethyl acetate. The combined extracts
were dried over
magnesium sulfate and the solvent removed in vacuo. The residue was purified
on a silica gel
column using 15% ethyl acetate in hexane as the eluent. The first fraction
from the column
was recovered starting material ( 1.05 g). The second fraction contained the
product. After
evaporation of the solvent, 0.84 g (35% yield) of 3-(3',3'-diphenyl-2'-indanyl-
1'-one)propanol
(Compound 19) as a beige solid having a melting point of 98°C was
obtained.
1.26 is f 2- -2'- 1 'ox 1 -1- -3 3-di h lin Com
2-(Ethyl-2'-{1,3-dioxolane))-1-hydroxy-3,3-diphenylindene (Compound 20) was
to synthesized as follows: 3,3-Diphenylindanone (Compound 2) (4.0 g, 0.0141
mol) was
dissolved in 30 rnL of tetrahydrofuran at room temperature. Potassium hydride
(2.4 g, 0.0175
mol) was added and the mixture was stirred at room temperature for 0.5 h. 2-(2-
Bromoethyl)-
1,3-dioxolane (2.0 mL, 0.0170 mol) was added and the mixture was continued
stirring
overnight at room temperature. The mixture was quenched with water and
extracted with
ethyl acetate. The combined extracts were purified on a silica gel column
using 8% ethyl
acetate in hexane followed by 10% ethyl acetate in hexane as eluent. The
product was
collected in the second fraction off the column. Evaporation of the solvent
gave 2-(Ethyl-2'-
(1,3-dioxolane))-1-hydroxy-3,3-diphenylindene (Compound 20) as an off white
solid (0.47 g,
9% yield) having a melting point of 124-126°C.
1.27 Other Compounds
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.
Compound 1
is available from Maybridge Chemical Company (distributor: Ryan Scientific,
South
Carolina).
~xamnle 2. In Yitro Activity
This Example demonstrates the ability of several exemplary compounds of
structural
formula (I) to inhibit the Gardos channel of erythrocytes (Gardos Channel
Assay) and/or
3o mitogen-induced cell proliferation (Mitogenic Assay) in vitro. The assays
are generally
applicable for demonstrating the in vitro activity of other compounds of
structural formula (I).
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ethos The percent inhibition of the Gardos channel ( 10 ~M compound) and the
ICso were determined as described in Brugnara et al., 1993, J. Biol. Chem. ~$(
12):8760-
8768. The percent inhibition of mitogen-induced cell proliferation (10 ~M
compound) and
the ICso were determined or described in Benzaquen et al. (1995, ~l~ture
Medicine ~: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.
$~,~~.lts. The results of the experiment are provided in Table 2, below.
Clotrimazole is
1 o reported for purposes of comparison. Most of the compounds tested
exhibited significant
activity in both assays. All of the compounds tested exhibited significant
activity in at least
one of the assays.
Table 2
Pharmacological Activities of Various Compounds
(% Inhibition measured at 10 ~.M)
Mitogenic Gardos Channel
Assay Assay
Compound ICS Inhibition ICSO Inhibition
2o Number (per (%) (~M) {%)
Clotrimazole 0.626 93.0 0.046 99.3
(1) 0.700 97.0 0.419 98.0
(2) 1.300 99.0 1.006 100.0
(3) 1.100 90.0 0.819 100.0
(4) 2.600 99.0 1.350 100.0
(5) -- 29.0 -- 67.3
(6) 3.400 90.0 -- 3 5.0
(7) 3.400 98.0 1.152 88.0
(8) 2.000 97.0 0.176 30.0
(9) -- 45.0 0.505 100.0
(10) 3.300 98.0 -- 49.5
(11) 3.400 99.0 -- 50.0
(12) -- 31.0 0.189 99.5
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Mitogenic Gardos
Assay Channel
Assay
Compound ICSO Inhibition ICS Inhibition
2o Number (~M) (%) (pM) (%)
(13) -- 12.0 1.590 99.5
(14) -- 3.0 2.961 90.5
(15) 7.500 80.0 2.901 54.8
(16) -- 75.0 -- 0
(17) -- 76.0 -- 0
(Ig) __ 73.0 -- 0
(19) 1.500 99.0 5.952 43.7
(20) -- 81.0 -- 0
t o Example 3. Activity In Cancer Cell Lines
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).
Methods. growth ~ Sells 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% N 5% fetal
calf serum
(Biowhittaker) at 37°C, 5% COz 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 x,15% fetal calf serum (FCS) containing
RPMI medium
2o in 96-well plates and incubated overnight at 37°C, 5% COZ and 95%
humidity.
On the day of the treatment, stock solutions of the test compounds ( 10 mM
compound/DMSO) were added in 100 ul FCS containing medium to a final
concentration of
10-0.125 ~,M and the cells were incubated for 2, 3 or 5 days at 37°C,
5% COZ and 95%
humidity.
Following incubation, the cellular protein was determined with the
ulforhodamine B
(SRB) assay (Skehan P 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
(ICS°) was determined by curve fitting.
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Values for VP-16, a standard anti-cancer agent, are provided for comparison.
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).
MMRU cells (Stender et al., 1993, J. Dermatoloev 20:611-617) were a gift of
one of
the authors.
1 o Re . The results of the cell culture assays are presented in Tables 3 and
4, below.
Table 3
SRB ASSAY RESULTS
(5% FCS, 5 Day Incubation)
Test Comp ound ICsoM)
(p
2o Cancer Type Cell Line VP-16 8 11
Cervical HeLa <1.25 >10 5.1
CaSki 1.8 6.8 7
Breast MDA-MB-23 < 1.25 > 10 > 10
MCF7 <1.25 5.5 4.4
Lung A549 < I .25 8.9 8.8
HTB 174 < 1.25 > 10 5.9
Hepatocel HEPG2 <1.25 6.4 5.8
Prostate DU-145 <1.25 >10 >10
Melanoma SK-MEL-28 <1.25 >10 5.5
3o MMRU <1.25 >10 6.2
Colon HT29 <1.25 8.3 6.8
HCT-15 1.3 >10 6.6
Renal ACHN <1.25 > 10 > 10
CNS U118MG 2.2 >10 >10
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Ovary SK-OV-3 > 10
Normal HUVEC <1.25 >10 6.4
human GM 1.4 > 10 > 10
3T3 >10 >10
mouse L929 < 1.25 > 10 8.6
Table 4
SRB RESULTS
CompoundConditionsTest
Compound
ICS
(wM)
is
Various
Cell
Lines
%FCS/day
s A549 HT29 MMRU MCF7 HEPG2 U118MG
VP-16 2%/3 2.3 20 <2.5 <2.5
days
3 5%I2 >10 >10 5.8
days
4 2%J3 8.5 <2.5 8.2 <2.5
days
8 5%/3 >10 >10 3.3 >10 7.8 >10
days
Example 4. Formulations
The following examples provide exemplary, not limiting, formulations for
administering the compounds of the invention to mammalian, especially human,
subjects.
2o Any of the compounds described herein, or pharmaceutical salts or hydrates
thereof, may be
formulated as provided in the following examples.
4.1 Tablet Formulation
Tablets each containing 60 mg of active ingredient are made up as follows:
Active Compound 150 mg
Starch 150 mg
Microcrystalline Cellulose 150 mg
Sodium carboxymethyl starch 4.5 mg
Talc 1 mg
3o Polyvinylpyrrolidone (10% in 4 mg
water)
Magnesium Stearate 0.5 me
160 mg
The active ingredient, starch and cellulose are passed through a No. 45 mesh
U.S.
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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
SO°-60°C and passed through aNo. 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 1 SO mg.
Tablets can be prepared from the ingredients listed by wet granulation
followed by
compression.
4.2 Gelatin Capsules
to Hard gelatin capsules are prepared using the following ingredients:
Active Compound 250 mg/capsule
Starch dried 200 mglcapsule
Magnesium Stearate 10 mg/capsule
The above ingredients are mixed and filled into hard gelatin capsules in 460
mg
quantities.
4.3 ~~r~sol Solution
An aerosol solution is prepared containing the following components:
2o Active Compound 0.25% (w/w)
Ethanol 29.75% (w/w)
Propellant 22 77.00% (w/w)
(Chlorodifluoromethane)
The active compound is mixed with ethanol and the mixture added to a portion
of the
propellant 22, cooled to -30°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.
4.4 Suppositories
Suppositories each containing 225 mg of active ingredient are made as follows:
Active Compound 225 mg
Saturated fatty acid glycerides 2,000 mg
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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.
4.5 Sys~ensions
Suspensions each containing SO mg of medicament per 5 mL dose are made as
follows:
Active Compound 50 mg
Sodium carboxymethylcellulose 50 mg
Syrup 1.25 mL
1o Benzoic acid solution 0.10 mL
Flavor q.v.
Color q.v.
Purified water to 5 mL
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.
~x ~e S: Clotrimazole inhibits water and electrolyte secretion in intestinal
epithelial cells.
The biochemical basis of secretory diarrhea involves intestinal Cf secretion
in
intestinal crypt cells. Under normal conditions, CY ions are maintained within
intestinal crypt
cells at levels above their electrochemical potential by primarily and
secondarily active
transport mechanisms such as the Na/K ATPase pumps and Na/K/2Cl
cotransporters. Cf is
transported into the lumen from the intestinal crypt cells through apical CI-
channels.
Intracellular levels of K+, cAMP, cGMP, and Ca++ are all involved in
regulating the secretory
response.
T84 cells were used to determine whether clotrimazole regulates Cf secretion
in
intestinal crypt cells. T84 cells form confluent monolayers of columnar
epithelia that exhibit
3o high transepithelial resistances, polarized apical and basilateral
membranes, and cAMP and
Ca++ regulated Cf secretory pathways analogous to those found in native
intestine.
Methods. Growth of T84 cells. T84 cells obtained from ATCC were cultured and
passaged in equal parts of dulbecco's modified eagle's medium (DMEM), lg/1D-
glucose) and
Hams F-12 nutrient mixture, supplemented with 5% newborn calf serum, 15 mM
HEPES, 14
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mM Na HC03, 40mg/I penicillin, 8mg/1 ampicillin, 0.90 mg/1 streptomycin. Cells
were
seeded at confluent density onto 0.33 cm2 or Scmz Transwell inserts (Costar,
Cambridge, MA)
coated with dilute rat collagen solution as previously described (Lencer et
al., J. Clin. Invest.,
92: 2941-2951 (1993); Lencer et al., J. dell Biol. 117: 1197-1209 (1992).
Transepithelial
resistances attain stable levels {> 1000 Ohms.cm2) after 7 days. The
development of high
transepithelial resistances correlated with the formation of confluent
monolayers with well-
developed tight junctions as assessed by morphological analysis, and with the
ability of
monolayers to secrete Cf {Madara et al., Gastr~ 92: 1133-1145 (1987).
~lectroph s~gy (mesurement of electrogenic CY secretion). Confluent monolayers
to were transferred to Hanks Buffered Salt Solution {HBSS) containing 0.185
gll CaCl2, 0.098
g/1 MgS04, 0.4 g/1 KCI, 0.06 g/1 KHzP04, 8 NaCI, 0.048 g/1 NaZHP04, 1 g/1
glucose, and
l OnM HEPES, pH 7.4. Serosal and mucosal reservoirs were interfaced with
Calomel and Ag-
Ag Cl electrodes via 5% agar bridges made with Ringer's buffer.
Transepithelial resistance
was measured using a dual voltage clamp device to apply 25 or SO~A current
pulses. Short
circuit current (ISC) was calculated using Ohms law as previously described
{Lencer et al., J_.
~l~n. Invest. 92: 2941-2951 (1993); Lencer et al. J. Cell B~QI 117: 1197-1209
(1992).
Results. Clotrimazole reversibly inhibits CY secretion elicited by Ca~+- or
cAMP-
dependant agonists in T84 cells. Previous studies have shown that Cf secretion
in T84 cells
is controlled by K+ efflux pathways which are biophysically and
pharmacologically distinct
from one another. One pathway participates in the secretory response to cAMP-
dependent
agonists and displays sensitivity to Ba++ salts (McRoberts, et al., J. Biol.
Chem. 260: 14163-
14172 (1985); Reenstra, Am J. Physiol. 264: C161-168 (1993}). The other
mediates the
response to Ca++-dependent agonists, and is Ba++-insensitive. Several pathway
specific
agonists of K+ channels are useful for determining whether a particular
compound is
functioning through a cAMP or Ca++ specific pathway. For instance, vasoactive
intestinal
peptide (VIP} and cholera toxin are cAMP mediated agonists of the K+ channel,
whereas,
carbachol is a Ca++-dependent agonist of the Ca++ regulated K+ channels. The
pathway by
which a particular inhibitor of Cf secretion in T84 cells is functioning may
be identified by
measuring the ability of the inhibitor to modify transepithelial resistances
in T84 cells which
3o have been treated with VIP or carbachol to stimulate Cl- secretion.
T84 cells were grown as described above and CY secretion was stimulated by the
addition to the media of either carbachol (100mM) or VIP (SnM). The cells were
then treated
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with BaCI (3mM), charybdotoxin (100nM), or clotrimazole (33mM). The short
circuit current
(ISC) was determined for the various inhibitor treatments as a percentage of
the control in the
absence of inhibitor (Fig. 3). BaCI strongly inhibited the secretory response
to the cAMP
mediated agonist VIP, but had no apparent affect on the secretory response
elicited by the
Ca++-dependent agonist carbachol. In contrast, the scorpion venom
Charybdotoxin strongly
inhibited the secretory response elicited by carbachol, but had minimal
affects on Cl- secretion
elicited by VIP. However, clotrimazole inhibited the CY secretory responses to
both agonists.
Inhibition of Cl- secretion by clotrimazole was fully reversible {962%, n = 4)
after 60 min
recovery in the presence of 0.01 mg/ml bovine serum albumin.
1o To examine possible effects of clotrimazole on the synergy between cAMP and
Ca++-
mediated agonists, monolayers, initially stimulated with VIP were allowed to
reach steady-
state levels of secretion and then additionally exposed to carbachol (100 pM).
Clotrirnazole
was slightly more effective in inhibiting the secretory response to carbachol
than to cAMP
with IC50 values of 3 and 8 pM, respectively. When the effects of clotrimazole
on cAMP-
and Ca++-dependent secretory pathways were examined on the same monolayers.,
inhibition of
the synergistic response to VIP plus carbachol was found to parallel the
inhibition of secretion
promoted by Ca++ agonists alone. In low doses (=10-' or less), clotrimazole
potentiated
slightly (by S-10%) the CI-secretory responses to either agonist. clotrimazole
inhibited
effectively the secretory response to cholera toxin (20 nM, a cAMP-dependent
agonist) and E
2o Coli heat-stabile toxin (100 nm, a cGMP-agonist) (ICSO values of 10 pM and
15 ~M,
respectively).
The effect of clotrimazole on K+ conductances was also examined by isotopic
flux
studies using 86RB. T84 cells were grown in the presence of a cAMP agonist,
VIP, or a Ca++
mediated agonist {Thapsigargin). Clotrimazole was added and 86RB efflux was
measured.
Clotrimazole significantly inhibited baseline and Ca++ stimulated FRB efflux
in the presence
of both cAMP and Ca++ mediated agonists compared to those cells which were not
treated
with clotrimazole.
Other aromatic compunds of the invention were found to inhibit chloride
secretion.
Although clotrimazole was the most potent inhibitor tested of cAMP and Ca++
elicited Cl
secretion, ketoconazole, econazole, miconazole, and 2-chlorophenyl-bis-phenyl
methanol also
were effective at inhibiting chloride secretion.
Taken together, these studies indicate that clotrimazole inhibits CY secretion
elicited
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by cAMP or Ca++ mediated K+ channels in T84 cells .
Exam In a 6: Clotrimazole acts at distal steps in the cAMP and Ca++-dependent
signal
transduction aoa thw~Ys_.
To determine the site of clotrimazole action, the effects of clotrimazoIe
pretreatment
were examined on monolayers stimulated with agonists that initiate Cl-
secretion at sequential
steps in the cAMP signalling cascade. T84 monolayers were preincubated in HBSS
in the
presence or absence of clotrimazole (33 ~M) and then stimulated with either 5
~.M VIP
(which activates adenylate cyclase through heterotrimeric GTPase-linked cell
surface
receptors), 10 wM forskolin (which activates adenylate cyclase directly), or 3
mM 8Br-cAMP
(a direct stimulator of protein kinase A). Clotrimazole inhibited the
secretory response to
each of these agonists. These data provide evidence that clotrimazole acts at
a step distal to
the activation of Protein Kinase A.
Ca++-dependent intracellular signaling in T84 and other non-exciteable cells
involves
recruitment of inositol trisphosphate (IP3)-dependent intracellular Ca++
stores (Halm and
Frizzell, Textbook of Secretory Diarrhea, Raven Press, 47-58 ( 1990); Mandel
et al., J. Biol.
Chem. 267: 704-712 (1986); Halm et al., Am. .I. Physiod. (Cell Physiol. 23)
254:C505-C511
(1988)), and subsequent activation of plasma membrane Ca++ influx pathways
(Barrett, Am.
J. Physiol. (Cell Physiol. 34): C859-C868 (1993)}. Downstream events may be
mediated by
[Ca++~i, IP3, diacylglycerol, or as yet unidentified diffusable factors
(Putney and Bird, Cell
75:199-201 (1993)). To examine the site of clotrimazole action alone, this
signalling,
cascade, T84 monolayers pretreated in the presence or absence of clotrima:zole
(33 pM) were
stimulated with the Ca++-dependent agonists carbachol (100 ~.M which elicits
both Ca++ and
IP3 signals), thapsigargin (5 ~M, which elevates cytoplasrnic Ca++ via
inhibition of ER Ca++-
ATPase) (Vandorpe et al., Biophys. J. 66:46-58 (1994)), or the Ca++ ionophore
ionomycin
(10 ~,M). Clotrimazole inhibited strongly the Cl-secretory response to each to
these reagents.
These data suggest that clotrimazole acts at steps in the secretory response
distal to the release
of intracellular Ca++ stores.
3o Exan~nle 7: Clotrimazole does not affect ayical membrane anion conductance
or basolateral
NaK2C1 cotrans otters.
Methods. 'ZSI Efflux Studies Confluent monolayers on 5 cmz Transwell inserts
were
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used 10-14 days after plating. 'ZSI was measured as an indicator of apical Cl-
, channel and
basolateral K+ channel activity as previously described (Venglarik, et al, Am.
J. Physiol. (Cell
Physiol. 28):C358-C364 (1990). Monolayers were preincubated at 37° C
with 4 pCi/mI'ZSI
in HBSS for 90 minutes, with 33 pM clotrimazole absent or present during the
final 30
minutes of this 90 min preincubation period. Clotrimazole pretreatment did not
alter'ZSI
loading of the cells. After washing twice in fresh HBSS, 0.5 ml samples were
obtained every
two min from the apical reservoir and replaced with fresh HBSS. After four
baseline samples
were obtained, the cells were treated (at t = 8 minutes) with vasoactive
intestinal peptide (VIP,
5 pnM) or thapsigargin (5 p.M) to stimulate Cl- secretion, and an additional
15 timed samples
were obtained. Finally, the cell monolayer was rinsed, cut with its support
from the
polystyrene ring, and the residual cell-associated radioactivity was
determined. Monolayers
were maintained at 37 ° C in room air throughout the study. 'z5I was
counted by gamma
counting and normalized to percent total uptake as previously described
(Venglarik, et al, Am.
J. Physiol. (Cell Physiol. 28):C358-C364 (1990).
~Rb Uptake tudies Confluent monolayers on 5 cm2 Transwell inserts were
incubated
for 30 minutes in HBSS at 37° C. A group of control and CLT treated (33
pM, for 30 min)
monolayers were treated with bumetanide (10 pM for 12 min). All monolayers
were then
treated with VIP (5nM and shifted to HBSS containing 1 uCi/ml $6Rb for 3
minutes at 37° C.
g6Rb uptake was terminated by washing the inserts in an ice-cold solution
containing 100mM
2o MgCI2, and l OmM TRIS-CL, pH 7.4. Monolayers were cut from their inserts,
placed into
scintillation vials, and counted using standard methods.
Results. Studies were conducted to determine whether the inhibition of
electrogenic
CI- secretion might occur by blockade of apical membrane Cl-channels, or
blockade of
basolaterally situated NaK2C1 cotransporters. To determine if clotrimazole
affected ion
conductance through apical membrane Cl-channels, we examined the time course
of'ZSI efflux
from T84 monolayers pretreated in the presence or absence of clotrimazole
(Venglarik, et al,
Am. J. Physiol. (Cell Physiol. 28):C358-C364 (1990). Clotrimazole had little
or no effect on
the time course of''-SI efflux from monolayers treated with VIP. Rate
constants for 125I
efflux from monolayers treated or not treated with clotrimazole were
indistinguishable
(0.0637 vsØ0645 % uptake/minute, n=2 in duplicate). Clotrimazole had similar
lack of effect
on'zsI efflux stimulated by thapsigargin.
We next tested the effect of clotrimazole on basolateral NaK2CI
cotransporters, as
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assessed by bumetanide-sensitive 86Rb uptake (Matthews et al., J. l3iol. Chem.
269:15703-
15709 (1994)). CIotrimazole treatment reduced the total amount of 86Rb uptake
by 53.615.8%
(meantSEM. n=6), but had no effect on the fractional component that was
bumetanide-
sensitive (8813.2 vs 75.2112.7% total uptake, meanfSEM). Taken together, these
data
strongly suggest that clotrimazole does not affect CI- secretion in T84 cells
via inhibition of
either apical membrane Cl- channels or basolateral membrane NaK2Cl
cotransporters.
Exam~e 8: Clotrimazole inhibits Chloride secretioa~v inhibitinE K+ efflux
through
basolateral K+ channels i,~'j'84 cells.
1o
8.1 Clotrimazole inhibits chloride secretion ~v blockade of K+ transport
through both
Ba++-sensitive and charybdotoxin-sensitive channels
ethods. 86Rb Efflux Studies Confluent monolayers on 5 cm2 Transwell inserts
were
used 10-14 days after plating. B6Rb flux was measured as an indicator of
apical Cl-, channel
and basolateral K+ channel activity as previously described (Venglarik, et al,
Am. J. Physiol.
(Cell Physiol. 28):C358-C364 (1990). Monolayers were preincubated at
37° C with 4 pCi/ml
B6Rb in HBSS for 90 minutes, with 33 ~M clotrimazole absent or present during
the final 30
minutes of this 90 min preincubation period. clotrimazole pretreatment did not
alter 86Rb
loading of the cells. One ml samples were obtained and replaced from the
basolateral
2o reservoir. After four baseline samples were obtained, the cells were
treated (at t = 8 minutes)
with vasoactive intestinal peptide (VIP, 5 ~nM) or thapsigargin (5 pM) to
stimulate Cl-
secretion, and an additional 15 timed samples were obtained. Finally, the cell
monolayer was
rinsed, cut with its support from the polystyrene ring, and the residual cell-
associated
radioactivity was determined. Monolayers were maintained at 37 ° C in
room air throughout
the study. 86Rb was counted by scintillation counting and normalized to
percent total uptake
as previously described (Venglarik, et al, Am. J. Physiol. (Cell Physiol.
28):C358-C364
( 1990).
$~S,~t . K+ channel activity was estimated by measurement of g6Rb efflux.
Clotrimazole was found to significantly inhibit the rate of 86Rb efflux after
treatment with the
3o cAMP agonist VIP (5 pM). The rate constant for VIP-stimulated 86Rb efflux
was reduced by
87% in monolayers treated with clotrimazole (0.0062 vs. 0.0465 %
uptake/minute, n=2 in
triplicate). clotrimazole inhibited to a similar degree 86Rb efflux from
monolayers stimulated
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with thapsigargin (panel B, rate constants 0.011 vs. 0.048% uptake/minute,
n=2), suggesting
that clotrimazole can inhibit Cl- secretion by blockade of K+ transport
through both Ba++-
sensitive and charybdotoxin-sensitive channels.
8 2 Clotrimazole inhibits chloride secretion through distinct cAMP and Ca++
sensitive
basolateral K+ channels
Methods. Selective mebrane Permeabilization and Measurement of Potassium
Conductanc~Qf the Basolateral Membrane. The basolateral potassium conductance
was
measured using the technique developed by Dawson and co-workers. A potassium
gradient
to (mucosal to serosal) was first established across the monolayer using
asymmetric mucosal and
serosal buffers containing K+ as the sole permeant ion. The addition of
amphotericin B (20
~M) to the mucosal reservoir forms conductive pores in the apical membrane,
and thus
removes all resistance to transepithelial potassium movement across this
membrane. Thus,
under the conditions of the experiment, in which the monolayer is short
circuited (i.e.,
voltage-clamped at zero potential) and the transepithehal potassium gradient
is constant, the
amphotericin-dependent Isc becomes a measure of the rate of the
transepithelial potassium
flux across basolateral membranes. Changes in short circuit current (Isc),
then represent
changes in basolateral K+ conductances (gK). Isc and K+ conductances were
measured using
calomel electrodes, 3M KCI-agar bridges, and a voltage clamp (University of
Iowa, Iowa
2o City). To generate a voltage-current channel relationships, currents were
elicited by 1 sec test
potentials from -80 to +80 in 10 mV increments in the asymmetrical high K+
gluconate
solution.
Calculation of basolateral membrane K+ germeabilitv. Membrane permeabilities
were
calculated according to the formula:
PK= (cm/s)-'K (mM/cm2~s)/~[ K+] (mMlcm3)
where 0 [K+] is equal to the difference in K+ concentration (135 mM) between
the asymmetric
apical and basolateral bathing solutions. Maximal Isc values were converted
into K+ fluxes by
dividing by the Faraday constant F (96,500 coulombs/mol) as previously
described {Huflejt et
al., J. Clin. Invest. 93: 1900-1910 (1994)).
3o RP~lts. Basolateral K+ transport was examined in T84 monolayers
permeabilized
apically by pretreatment with amphotericin B. Apical and basolateral buffers
contained K+ as
the sole permeant ion. All studies were performed with a I35 mM basolaterally
directed K+
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gradient. This method has been utilized previously to examine both CI- and K+
transport in
T84 cells and fIT29-C1.16E cells. Briefly, ion conductances in the luminal or
basolateral
membranes of confluent T84 cell monolayers can be assessed separately by
selectively
permeabilizing the apical or basolateral membrane using the ionophore
amphotericin B. This
artificially removes all electrical resistance to ion transport across the
plasma membrane
containing pores formed by amphotericin B. As a result, the intact
contralateral plasma
membrane becomes rate limiting for transepithelial ion transport. Agonist-
dependent changes
in ion conductances can be assessed directly either as transepithelial short
circuit current (Isc)
in the presence of established ion gradients, or as transepithelial
conductance (G) in the
to presence of established transepithelial potentials.
K+ transport was measured at baseline and after the ordered additions of cAMP-
and
Ca++ -agonists. The initial permeabilization with amphotericin B was
associated with 49 +
19% increase in conductance. Pores formed by amphotericin B display
selectivity for
monovalent cations. Ca++ remained relatively impermeant as evidenced by the
small steady
state increase in Isc and GK caused by apical permeabilization with
amphotericin B. Given
this low baseline Isc and GK, both cAMP- and Ca~'-sensitive K+ permeabilities
(PK) were
readily apparent after agonist stimulation. Treatment with the cAMP-agonist
forskolin (10
~.M) caused a brisk increase in K+ transport through apparently low-
conductance pathway(s),
as evidenced by symmetrical increases in Isc and G. Carbachol also increased
K+ currents.
2o The magnitude of the carbachol-induced IscK, however, was similar whether
carbachol was
added alone or after forskolin (111.7 ~ 7.4 vs. 180.7 t 15.7pA/cm2
respectively. Thus, there
was no clear evidence of synergy between cAMP and Ca++ mediated K+ pathways,
as would
be expected in an apically permeabilized cell system. Analagous to our
previous findings in
intact T84 monolayers, the forskolin-induced changes in Isc were sustained
while the effect of
carbachol was short-lived. Both IscK and GK returned to baseline values within
5 min after
addition of carbachol.
Formal current/voltage (I/V) relations were defined before and after agonist
stimulation to confirm that both cAMP- and Ca++-dependent currents were
elicited at
physiologic membrane potentials. Thapsigargin was used in place of carbacol as
a Ca++-
3o agonist in these studies because the K+ transients elicited by thapsigargin
achieve steady state
conductances of much longer duration, as in intact monolayers. It was found
that under
conditions of basolaterally directed K+ gradients, both forskolin and
thapsigargin activate
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macroscopic outwardly rectified (mucosal to serosal) currents at positive
transepithelial
voltages. Experimental IIV relations obtained after forskolin and thapsigargin
stimulation
displayed reversal potentials (- 40 mV) that approximated the calculated
Nernst-potential (-85
mV calculated as RT/zQo log [Na]o"t/[Na];"). These results are consistent with
the activation
of distinct cAMP- and Ca++-sensitive basolateral membrane K+ conductances in
conjunction
with one or more nonspecific transepithelial ion shunts, possibly occurnng
through
intercellular tight junctions or basolateral membrane "leaks."
To confirm that the observed changes in Isc and G represented K+ transport
through
K+ selective pathways, the effect of forskolin and carbachol on T84 monolayer
conductances
1 o were examined using buffers containing Na+ as the sole permeant cation.
These studies were
performed using an analogous 135 mM basolaterally directed cation (Na+)
gradient. Increases
in Isc and G were not detectable in the absence of K+. Thus, the increases in
cation
conductances induced by agonist stimulation are specific to K+ transport.
Two pharmacologically distinct K+ efflux pathways have been previously
identified in
intact T84 cells. One pathway participates in the secretary response to cAMP-
dependent
agonists and displays sensitivity to Ba++ salts. The other K+ efflux pathway
mediates the
response to Ca++-dependent agonists, and is Ba++-insensitive. These findings
were confirmed
in the permeabolized cell model. The cAMP-sensitive IK (elicited by treatment
with forskolin,
10 ~,M) was inhibited by greater than 70% by the addition of BaCl2 (3 mM) to
basolateral
2o reservoirs. Ba++ , however, had no detectable effect on K+ transport
induced by the
subsequent addition of carbachol (100 ~,M) to the same monolayers. In
contrast, when
permeabilized monolayers were treated first with carbachol, the induced Ca~'~*
IK was inhibited
by 50% by pretreatment with the scorpion venom charybdotoxin (100 nM).
Charybdotoxin,
however, had no detectable effect on K+ transport induced by the subsequent
addition of
forskoiin. Thus in permeabilized cells, the differential sensitivity of K+
transport to inhibition
by the K+ channel blockers BaCl2 and charybdotoxin paralleled exactly the
effect of these
channel selective inhibitors on K+ transport in intact cells (measured
indirectly as a Cl-
current).
Taken together, these studies define the permeabilized T84 cell model, and
provide
3o strong evidence that under the defined conditions both Isc and G represent
K+ transport
through distinct cAMP- and Ca++-sensitive basolateral K+ channels.
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i 2- r le
metabolite. inhibit K+transport through both cAMP- and Ca++-dependent K+
channels
We next tested the hypothesis that clotrimazole may inhibit directly
basolateral
membrane K+ channels in human intestinal T84 cells, as it does in the red
cell. clotrimazole
significantly inhibited the time course of K+ transport after treatment with
the cAMP agonist
forskolin {I0 ~,M) and the Ca++ agonist carbachol (100 ~M). Formal IV
relations taken at
steady state after cAMP or Ca++ stimulation confirm that clotrimazole affected
both cAMP-
and Ca++- sensitive channels. Nearly identical results were obtained with 2-
chlorophenyl-bis-
phenyl methanol. clotrimazole and its metabolite 2-chlorophenyl-bis-phenyl
methanol inhibit
I o directly both CAMP- and Ca++-sensitive intestinal K+ channels indicating
that the ring
structure in the absence of the imidazole ring sufficient (and perhaps
necessary) for this
bioactivity.
8.4. Clotrimazole targets the basolateral rather than the apical surface of
T84 cells
Methods. Measurement of Cf Conductance of the ~nical_ Plasma Membrane. To
examine apical Clconductances, Cl- was used as the sole permeant ion using
identical apical
and basolateral buffer solutions. Monolayers were pemeabilized basolaterally
by the addition
of 100 ~M Amphotericin B to the serosal reservoir. Generation of voltage-
current curves of
channel currents were elicited by 1 sec test potentials from -80 to + 80 mV in
10 mV
2o increments in symmetrical high Choline Cl- buffers.
Results. Studies were performed to determine whether the primary target of
clotrimazole was located on the basoolateral or apical cell surfaces. Most
rapid inhibition was
achieved by incubation with clotrimazole on both sides of the monolayer.
However,
basolateral application alone was almost as effective as incubation on both
sides.
Additionally, the apparent potency of inhibition of clotrimazole at a fixed
time point was
found to be greater when applied basolaterally than apically. This
preferential action of
clotrimazole at the basolateral surface of the cell is consistent with the
hypothesis that its
principal targets are basolateral K+ channels.
To confirm these findings, we examined Cl- transport in T84 cell monolayers
permeabilized basolaterally with pores formed by amphotericin B. These studies
were
performed with Cl- as the only permeant anion, and with symmetrical apical and
basolateral
Cl- concentrations (142 mM). In monolayers not treated with clotrimazole, the
addition of
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forskolin (10 ~M) to basolateral reservoirs increased CI- conductances
significantly over
baseline, presumably via activation of the cystic fibrosis transmembrane
regulator (CFTR) CI-
channel. In contrast to the clear inhibitory effects of clotrimazole on
basolateral K+
conductances, however, clotrimazole had no detectable effect on either
forskoIin- or
thapsigargin-stimulated Cl-conductances. I/V relations for CI- transport were
nearly identical
in monolayers treated or not treated with clotrimazole. These data provide
further evidence
that clotrimazole inhibits Cl- secretion in intact T84 cell rnonolayers by
affecting specifically
basolateral K+ channels. Apical membrane Cl-channels are not inhibited.
Example 9. Clotrimazole inhibits Cl- secretion in vivo.
~.1. Chamber studies using rabbit colonic mucosa.
Metho s. 4 male, New Zealand rabbits (2.5 kg) were anesthetized by an
intravenous
injection of pentobarbital {0.5 ml/kg). A 15 cm length of distal colon was
removed and
opened longitudinally. External muscle layers were removed by blunt dissection
and colonic
mucosal preparations were mounted in an Ussing chamber (DCTSYS; Precision
Instrument
Design, CA; 10.3 cm2 surface area) and incubated with buffer solution
containing (in mM):
NaCI 122.0, CaCl2, 2.0; MgS04,1.3; KCI, 5.0, glucose, 20; NaHC03, 25.0 (pH
when gassed
with 95% 02/5 CO2; temperature was maintained at 37°C) with and without
clotrimazole
(30pM). The volume of fluid on each side of the mucosa was 7 ml.
2o Potential difference and Isc were monitored continuously and registered
every 10
minutes. Luminal and serosal buffer solutions were interfaced via Ag-AgCI
electrodes
(Voltage/Current Clamp, Model VCC600, Physiologic Instruments, Inc., San
Diego, CA,
USA) and Ringer/agar bridge to voltage clamp device {model DVC-1000;
Voltage/Current
Clamp, World Precision Instruments, Inc.). Resistance (R) was calculated using
Ohm's law
and the Isc and is given in Cl x cm2. After stable baseline resistance and Isc
values had been
obtained, mucosal preparations were incubated in the presence or absence of
serosal
clotrimazole (30 uM) for 30 min, and then stimulated by the addition of
forskolin (10 ~.M) or
carbachol {10 ~M) to the serosal reservoir.
Results. To test the ability of clotrimazole to block K+ channels and thus Cl-
secretion
3o in native intestinal tissue, we mounted isolated preparations of rabbit
colonic mucosa in
Ussing chambers containing modified Ringer's solution with or without
clotrimazole (30 ~M).
After Isc had stabilized, successive additions of forskolin (10 pM) and then
cubachol (100
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~,M) were applied to serosal reservoirs, and Isc and G were monitored
continuously.
clotrimazole inhibited strongly the time course of forskolin induced Isc.
Carbachol had no
further effect on Isc in this system.
9.2. Murine model of secre~totorv diarrhea.
a ods. Treated and control, untreated, mice were gavage fed either
clotrimazole
(150 mg/kglday divided in two equal doses, dissolved in peanut oil at a
concentration of 20
mg/ml) ox vehicle control over a 7 day loading period. Mice were then
challenged by gavage
with either 25 pg purified cholera toxin (Calbiochem, San Diego, CA) in PBS,
vehicle control
1o alone (PBS without cholera toxin), or cholera toxin in PBS containing 30 ~M
clotrimazole.
Animals were sacrificed after 5 hours in an uncrowded COZ hood. The carcass
was weighed,
the abdomen was opened, and ligatures were tied at the proximal duodenum and
distal rectum.
The intestinal block was dissected free of supporting structures and removed
as a single unit
and weighed. Small and large intestinal segments were normalized to body
weight (intestinal
weight/carcass weight) for each animal.
Results. To examine whether clotrimazole may inhibit intestinal secretion in
vivo, we
utilized a murine model of secretary diarrhea. Balb/C mice were gavage fed 150
rnglkg/day
clotrimazole, divided into two equal doses, or vehicle control every 12 h for
7 days and
subsequently challenged orally with purified cholera toxin (25 fig). Five
hours after treatment
2o with cholera toxin, the mice were sacrificed and intestinal fluid secretion
assessed
gravimetrically. Pretreatment with clotrimazole reduced by 86% intestinal
fluid secretion
induced by cholera toxin. Clotrimazole had no effect on intestinal secretion
in the absence of
cholera toxin. Thus, clotrimazole effectively treated secretory diarrhea in
vivo, presumably
by inhibiting basolateral K+ channels of crypt epithelial cells.
The foregoing written specification is considered to be sufficient to enable
one skilled
in the art to practice the invention. The present invention is not to be
limited in scope by
examples provided, since the examples are intended as a single illustration of
one aspect of
the invention and other functionally equivalent embodiments are within the
scope of the
invention. The advantages and objects of the invention are not necessarily
encompassed by
3o each embodiment of the invention.
Each of the foregoing patents, patent applications and references is herein
incorporated
by reference in its entirety.
