Note: Descriptions are shown in the official language in which they were submitted.
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COMPOUNDS AND METHODS FOR USE THEREOF
IN THE TREATMENT OF CANCER OR VIRAL INFECTIONS
TECHNICAL FIELD
The present invention relates to benzimidazole derivatives and their use for
the
treatment of cancer or a viral infection in warm blooded animals, particularly
in humans and
other mammals. The methods may use such a compound in combination with a
potentiator or
a chemotherapeutic agent.
BACKGROUND OF THE INVENTION
Cancers are a leading cause of death in animals and humans. The exact cause of
cancer is not known, but links between certain activities such as smoking or
exposure to
carcinogens and the incidence of certain types of cancers and tumors has been
shown by a
number of researchers.
Many types of chemotherapeutic agents have been shown to be effective against
cancers and tumor cells, but not all types of cancers and tumors respond to
these agents.
Unfortunately, many of these agents also destroy normal cells. The exact
mechanism for the
action of these chemotherapeutic agents are not always known.
Despite advances in the field of cancer treatment the leading therapies to
date are
surgery, radiation and chemotherapy. Chemotherapeutic approaches are said to
fight cancers
that are metastasized or ones that are particularly aggressive. Such cytocidal
or cytostatic
agents work best on cancers with large growth factors, i.e., ones whose cells
are rapidly
dividing. To date, horniones, in particular estrogen, progesterone and
testosterone, and some
antibiotics produced by a variety of microbes, alkylating agents, and anti-
metabolites form the
bulk of therapies available to oncologists. Ideally cytotoxic agents that have
specificity for
cancer and tumor cells while not affecting normal cells would be extremely
desirable.
Unfortunately, none have been found and instead agents that target especially
rapidly dividing
cells (both tumor and normal) have been used.
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2
Clearly, the development of materials that would target cancer cells due to
some
unique specificity for them would be a breakthrough. Alternatively, materials
that were
cytotoxic to cancer cells while exerting mild effects on normal cells would be
desirable.
Human Immunodeficiency Virus (HIVE, the etiological agent for AIDS (acquired
immune deficiency syndrome), is a member of the lentiviruses, a subfamily of
retroviruses.
HN integrates its genetic information into the genome of the host. Most
importantly, HIV
infects and invades cells of the immune system; it breaks down the body's
immune system
and renders the patient susceptible to opportunistic infections and neoplasms.
HIV-1 is
cytopathic for T4 lymphocytes, cells of the immune system that express the
cell surface
differentiation antigen CD4. In addition to CD4+ T cells, the host range of
HIV includes cells
of the mononuclear phagocytic lineage, including blood monocytes, tissue
macrophages,
Langerhans cells of the skin and dendritic reticulum cells within lymph nodes.
Precursor cells in the bone marrow are released into the blood in an immature
circulating form known as monocytes. Monocytes use the blood strictly as a
transport
medium. Once they arrive where they're going to be used, they leave the blood
and complete
differentiation into macrophages. Cells of the monocyte/macrophage lineage are
a major
target population for infection with HIV in the body and are thought to
provide reservoirs of
virus for disseminating infection throughout the body. HIV is also
neurotropic, capable of
infecting monocytes and macrophages in the central nervous system causing
severe
neurologic damage. They can interact and fuse with CD4-bearing T cells,
causing T cell
depletion and thus contributing to the pathogenesis of AIDS.
Progression from HN infection to AIDS is primarily determined by the effects
of
HIV on the cells that it infects, including CD4+ T lymphocytes and
macrophages. In tum,
cell activation, differentiation and proliferation regulate HIV infection and
replication in those
cells. HIV and other lentiviruses can proliferate in nonproliferating,
terminally differentiated
macrophages and growth-arrested T lymphocytes. This ability of lentiviruses,
including HN,
to replicate in nonproliferating cells, particularly in macrophages, is
believed to be unique
among retroviruses.
Due to the above-mentioned problems in the art, the present inventors have
sought
improvements and provide such improvements herein.
Carbendazim, or 2-methoxycarbonylaminobenzimidazole, has been studied as a
cancer treatment. See US 5,767,138 issued June 16, 1998 to J. B. Camden.
Carbendazim
metabolizes in the body through the hydroxylation of the benzene ring,
primarily in the 5-
position. -The metabolite is not as active in the treatment of cancer as the 2-
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methoxycarbonylaminobenzimidazole. Moreover, this compound is not very
soluble. A
derivative that maintains the activity of the benzimidazole but is more
soluble is highly
preferred. It has been found that substituted benzimidazole carbamates, and in
particular,
those claimed herein are more soluble, yet maintain cytotoxic behavior.
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SUMMARY OF TI3E INVENTION
The compounds of the present invention are represented by the following
formula A:
Xn
Y \/ I ~-IVHR~
N
I
R
A
wherein,
R is hydrogen,-COORz or-CONHRz;
Rl is hydrogen, -COORS or -CONHR3;
each of RZ and R3 is independently alkyl, haloalkyl, allcenyl, haloalkenyl,
cycloallcyl,
cycloalkalkyl, heterocycloalkyl, heterocycloalkalkyl, substituted or
unsubstituted benzyl,
hydroxyalkyl, alkoxyalkyl, poly(alkoxy)alkyl, hydroxyalkoxyalkyl,
hydroxypoly(alkoxy)alkyl, haloalkoxyalkyl, halopoly(alkoxy)alkyl, or
aminoalkyl;
each of X and Y is independently hydrogen, alkyl, alkenyl, cycloalkyl,
haloalkyl,
haloalkenyl, bromo, chloro, fluoro, vitro, or amino; and
nisl,2or3.
The benzyl group may optionally be substituted with one or more vitro,
carboxy,
hydroxy, alkyl, alkoxy, or halide substituents.
Pharmaceutically acceptable salts of the benzimidazole derivatives of formula
A are
also included in the present invention. Further included in the invention are
the prodrugs of
the compounds of formula A.
In one presently preferred embodiment of the invention, the benzimidazole
derivatives of the invention are of the formula A-1 or A-2:
N Xn\i N
NH2 Y ( I >-NHR~
N \ N
R I
A-1 A2
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and more preferably the compounds are of formula A-1 or A-2 where each of X,
and Y is
hydrogen.
In another presently preferred embodiment of the invention, the benzimidazole
derivatives are of the formula A-3:
Xn ~i N.
[, I ~-N o~
Y /~ N Rs
H
O
A-3
and more preferably, the compounds are of formula A-3 where X is hydrogen and
Y is
hydrogen or halogen; the halogen is preferably chloro.
In another presently preferred embodiment of the invention, the benzimidazole
derivatives are of the formula A-4;
X~ ~ N H H
w I ~ NCR
Y H s
O
A-4
and more preferably, the compounds are of formula A-4 where X is hydrogen and
Y is
hydrogen or halogen; the halogen is preferably chloro.
Methods are provided by the present invention for treatment of warm blooded
animals, and in particular, humans and other mammals, that are affected by
cancer or viral
infection, the methods comprising administering a therapeutically effective
amount of a
benzimidazole derivative of formula A, or a salt or a prodrug thereof, to the
animal.
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DETAILED DESCRIPTION OF THE INVENTION
A. Definitions:
The term "alkyl" refers to a fully saturated monovalent hydrocarbon radical of
1 to 12
carbon atoms. It may be straight-chain or branched. Preferred are those alkyl
groups
containing 1 to 10 carbon atoms, with 2 to 8 carbon atoms particularly
preferred.
The term "alkenyl" refers to an unsaturated monovalent hydrocarbon radical of
2 to
12 carbon atoms containing only carbon and hydrogen and having one or more
double bonds.
It may be straight-chain or branched. Preferred are those alkenyl groups
containing 2 to 10
carbon atoms, with 2 to 8 carbon atoms particularly preferred.
The term "alkoxy" means the group -0R' wherein R' is alkyl. Preferred are
alkoxy
groups having 1 to 10 carbon atoms, more preferably 2 to 8 carbon atoms.
The term "alkoxyalkyl" refers to an alkoxy group covalently attached to an
alkyl
group. The alkoxy group contains from 1 to 12, preferably from 1 to 6 carbon
atoms. The
alkoxy group may be substituted with one or more hydroxyl groups (an.
"hydroxyalkoxyalkyl") or with one or more halogen atoms (a "haloalkoxyalkyl");
preferably
the hydroxyl or halogen is on the terminal end of the alkoxyalkyl substituent.
The term "poly(allcoxy)alkyl" denotes 2 to 200, preferably 2 to 20, alkoxy
groups
covalently linked in either a linear or a branched configuration and attached
to an alkyl group.
Linear poly(alkoxy)alkyl moieties have a structure such as
-(CHZ)",-O--(CHZ)m O-(CHZ)m-O-(CHZ)m ...--O-CmH2m+i, where "m" is an integer,
the same or different along the length of the chain. Branched moieties have
two or more (-O-
(CH~)m ) groups bound to a common third (-O-(CHi)m ) group, where "t" has a
value that is
independently selected from 0, I and 2 for each (CH~)m group. Linear
configurations are
preferred. The number of repeating (-O-(CH,)m ) groups within a substituent
may be up to
200, preferably from 2 to 20, more preferably from 2 to 7, and most preferably
is 2-5. The
individual alkoxy groups may be the same or different, and individual alkoxy
groups
preferably contain from 1 to 6 carbon atoms each, and most preferably from 1
to 3 carbon
atoms each. A presently preferred poly(alkoxy)allcyl is -(CHZ)y (OCHzCHz)X-
0CH3 or
-(CHZ)Y (OCHZCHZ)X-0CHZCH3, where y = 1-4 and x = 1-100, preferably 1-10, and
more preferably, 2-5. The individual alkoxy groups may be substituted with one
or more
hydroxyl groups (an "hydroxypoly(alkoxy)alkyl") or with one or more halogen
atoms (a
"halopoly(alkoxy)alkyl"); preferably the hydroxyl or halogen is on the
terminal end of the
poly(alkoxy)alkyl substituent.
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"Heterocyclo" designates a heterocyclic group; that is, a closed-ring
structure, usually
of either 5 or 6 members, in which one or more of the atoms in the ring is an
element other
than carbon, such as for example sulfur, nitrogen, or oxygen. A heterocyclic
group may be,
but is not limited to, pyridine, pyrrole, furan, thiophene, morpholine, and
purine, optionally
substituted with one or more nitro, carboxy, sulfonic acid, hydroxy, alkyl,
alkoxy, or halide
substituents.
The term "amino" refers to primary amines (-NHz), secondary amines (-NHR'),
and
tertiary amines (-NR'R"), where R' and R" are the same or different
substituent groups, such
as alkyl, alkenyl, halogen, hydroxy, and the like.
"Independently" signifies that two or more of the groups immediately preceding
the
term are either identical or different; i.e., selection of one from the list
following the term
does not affect selection of the other(s).
"Substituted" encompasses both single and multiple substitutions, the latter
including
multiple substitutions by the same substituent as well as mixtures of
different substituents.
The term "optional" or "optionally" means that the subsequently described
event or
circumstance may or may not occur, and that the description includes instances
in which said
event or circumstance occurs and instances in which it does not. For example,
"optionally
substituted phenyl" means that the phenyl may or may not be substituted and
that the
description includes both unsubstituted phenyl and phenyl wherein there is
substitution. As
another example, "optionally" followed by "converting the free base to the
acid addition salt"
means that such conversion may or may not be carried out in order for the
process described
to fall within the invention, and the invention includes those processes
wherein the free base
is converted to the acid addition salt and those processes in which it is not.
As used herein, "a therapeutically effective amount" means the concentration
or
quantity or level of the compound of the present invention that can attain a
particular medical
end, such as control or destruction of cancer cells, virally-infected cells,
or viruses without
producing unacceptable toxic symptoms. The term "safe and effective amount"
refers to the
quantity of a component which is sufficient to yield a desired therapeutic
response without
undue adverse side effects (such as toxicity, irritation, or allergic
response) commensurate
with a reasonable benefit/risk ratio when used in the manner of this
invention. The specific
"safe and effective amount" will vary with such factors as the particular
condition being
treated, the physical condition of the patient, the type of mammal being
treated, the duration
of the treatment, the nature of concurrent therapy (if any), and the specific
formulations
employed and the structure of the compounds or its salts.
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As used herein, a "pharmaceutical addition salt" or "pharmaceutically
acceptable salt"
is a salt of the benzimidazole derivative compound with an organic or
inorganic acid. These
preferred acid addition salts are chlorides, bromides, sulfates, nitrates.,
phosphates, sulfonates,
formates, tartrates, maleates, malates, citrates, benzoates, salicylates,
ascorbates, and others
known to those of ordinary skill in the art.
As used herein, the term "prodrug" refers to a form of a benzimidazole
derivative
compound that has minimal therapeutic activity until it is converted to its
desired biologically
active form. A prodrug is a compound liaving one or more functional groups or
carriers
covalently bound thereto, which functional groups or carriers are removed from
the
compound by metabolic processes within the body to form the respective
bioactive
compound.
As used herein, the term "metabolite" refers to the break-down or end product
of a
benzimidazole derivative compound or its salt produced by metabolism or
biotransformation
in the animal or human body; e.g., biotransformation to a more polar molecule
such as by
oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and
Gilman, "The
Pharmacological Basis of Therapeutics" 8th Ed., Pergamon Press, Gilman et al.
(eds.), 1990
for a discussion of biotransformation). As used herein, the metabolite of a
benzimidazole
derivative compound or its salt may be the biologically active form of the
compound in the
body. An assay for activity of a metabolite of a benzimidazole derivative of
the present
invention is known to one of skill in the art in light of the present
disclosure, for example,
testing for efficacy against a virus ifz vitro or in vivo.
As used herein, a "subject in need thereof' is a warm blooded animal having
cancer
or having a viral infection.
As used herein, "cancer" refers to all types of cancers, or neoplasms or
benign or
malignant tumors. In one embodiment, those cancers that attack normal healthy
blood cells or
bone marrow are contemplated by the present invention. Preferred cancers for
treatment
using methods provided herein include carcinoma, sarcoma, lymphoma, or
leukemia. By
"carcinoma" is meant a benign or malignant epithelial tumor and includes, but
is not limited
to, breast carcinoma, prostate carcinoma, non-small cell lung carcinoma, colon
carcinoma,
CNS carcinoma, melanoma carcinoma, ovarian carcinoma, or renal carcinoma. A
preferred
host is a human host.
A "viral infection" as used herein means an infection due to a DNA virus or an
RNA
virus (retrovirus). Examples of a double-stranded DNA virus are the Herpes
virus and the
influenza virus. Human immunodeficiency virus (HIV) is a prototype for
retroviruses, i.e.,
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viruses that use reverse transcription to replicate. However, certain DNA
viruses use, in part,
reverse transcription mechanisms to replicate such as, for example, the
Hepatitus B virus.
"Viruses" include retroviruses such as HIV or HTLV, influenza, rhinoviruses,
herpes,
hepatitis, or the like.
As used herein, a benzimidazole derivative of formulas A and A-1 through A-4,
or a
pharmaceutical salt thereof or a prodrug thereof, are "compounds of the
present invention."
Such compounds are further set forth under B infra.
As used herein, "chemotherapeutic agents" includes DNA-interactive agents,
antimetabolites, tubulin-interactive agents, hormonal agents and others, such
as asparaginase
or hydroxyurea, and are as further set forth under D infra.
As used herein, "potentiators" are materials that affect the immune system or
enhance
the effectiveness of a compound of the present invention and are further set
forth under E
herein.
Following long-standing patent law convention, the terms "a" and "an" mean
"one or
more" when used in this application, including the claims.
B. Benzimidazole Derivatives
The benzimidazole derivatives of the present invention are those of formula A,
as set
forth above. In one embodiment of the invention, presently preferred compounds
are those of
formulas A-1 and A-2. In another embodiment, presently preferred compounds are
those of
formulas A-3 and A-4.
Pharmaceutically acceptable salts of the benzimidazole compounds are
considered
within the scope of compounds of the present invention. They are salts with an
organic or
inorganic acid. Preferred acid addition salts are chlorides, bromides,
sulfates, nitrates,
phosphates, sulfonates, formates, tartrates, maleates, malates, citrates,
benzoates, salicylates,
ascorbates, or the like. Such salts may be synthesized from the compound of
the present
invention, or derivative thereof, that contains a basic or acidic moiety, by
conventional
chemical methods. Generally, such salts may be prepared by reacting a free
acid or base form
of the compound, or derivative thereof, with a stoichiometric amount of the
appropriate base
or acid in water or in an organic solvent, or in a mixture of the two.
Generally, nonaqueous
media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are
preferred. Further
suitable salts may be found in Remington: The Scierace and Practice
ofPharrnacy, 19th ed.,
Mack Publishing Company, Easton, PA., 1995, p. 1457.
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Pharmaceutically acceptable salts of the compounds of the present invention
include
conventional non-toxic salts or the quaternary ammonium salts of the compounds
or
derivatives formed, for example, from non-toxic inorganic or organic acids.
For example,
such conventional non-toxic salts include those derived from inorganic acids
such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, or the
like; and salts
prepared from organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic,
tartaric, citric, ascorbic, malefic, hydroxymaleic, phenylacetic, glutamic,
benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,
ethane disulfonic,
oxalic, isethionic, or the like. Preferred acid addition salts are chlorides,
bromides, sulfates,
nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates,
citrates, benzoates,
salicylates, ascorbates, or the like. A presently preferred salt is the
hydrochloride salt.
Further included within the scope of the compound, or salts thereof, useful
for the
present invention are prodrugs thereof. As used herein, a "prodrug" is a drug
covalently
bonded to a Garner wherein release of the drug occurs iiz vivo when the
prodrug is
administered to a mammalian subject. Prodrugs of the compounds of the present
invention
are prepared by modifying functional groups present in the compounds in such a
way that the
modifications are cleaved, either in routine manipulation or in vivo, to yield
the desired
compound. Prodrugs include compounds wherein hydroxy, amine, or sulfhydryl
groups are
bonded to any group that, when administered to a mammalian subject, is cleaved
to form a
free hydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugs
include, but
are not limited to, acetate, formate, or benzoate derivatives of alcohol or
amine functional
groups in the compounds of the present invention; phosphate esters,
dimethylglycine esters,
aminoalkylbenzyl esters, aminoalkyl esters or carboxyalkyl esters of alcohol
or phenol
functional groups in the compounds of the present invention; or the like.
Synthetic Reaction Parameters
The terms "solvent", "inert organic solvent" and "inert solvent" mean a
solvent that is
passive or non-reactive under the conditions of the reaction being described
in conjunction
therewith (including, for example, benzene, toluene, acetonitrile,
tetrahydrofuran ("THF"),
dimethylformamide ("DMF"), chloroform, methylene chloride (or
dichloromethane), diethyl
ether, methanol, pyridine, and the like). Unless specified to the contrary,
the solvents used in
the reactions of the present invention are inert organic solvents.
The reaction temperature can vary widely depending on the reactivity of the
reactants.
However, the temperature should not be so high as to decompose the reactants
or so low as to
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cause inhibition of the condensation or freezing of the solvent. Unless
specified to the
contrary, the reactions described herein take place at atmospheric pressure
over a temperature
range from the temperature of dry ice to about 100°C, more preferably
from about 10°C to
about 50°C, and most preferably at "room" or "ambient" temperature
("RT"), e.g. about 20°C.
Unless otherwise specified, the reaction times and conditions are intended to
be
approximate.
The time required for the reactions herein will depend to a large extent on
the
temperature being used and the relative reactivities of the starting
materials. Therefore, the
reaction time can vary greatly, for example from about five minutes to about
two days.
Various known techniques such as different types of chromatography, especially
thin layer
chromatography ("TLC"), gas chromatography ("GC"), or spectroscopy can be used
to follow
the progress of the reaction by the disappearance of the starting compound(s).
Isolation and purification of the compounds and intermediates described herein
can
be effected, if desired, by any suitable separation or purification procedure
such as, for
example, filtration, extraction, crystallization, column chromatography, thin-
layer
chromatography, thick-layer chromatography, centrifugal chromatography, or
preparatory
HPLC, or a combination of these procedures. Specific illustrations of suitable
separation and
isolation procedures can be found by reference to the examples hereinbelow.
However, other
equivalent separation or isolation procedures can, of course, also be used.
Synthesis of Compounds of Formula A
The compounds of the invention are prepared by modification to the
benzimidazole
nucleus:
6/ N
~NH2
s\ N
H3
In one synthetic process, commercially available 2-aminobenzimidazole (101)
can be
reacted with a variety of chloroformates or isocyanates in pyridine to form,
respectively,
carbamates (105) or ureas (103) at the N3 position. In this manner, N3-
substituted
benzimidazole derivatives of formula A-1 (105) or A-2 (103) with a free 2-
amino group are
synthesized (Reaction Scheme 1).
Reaction Scheme 1:
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12
N
~~NH2
N
H
R'NCO R'OCOCI
Isocyanates 1 ~~~ Chloroformates
N~-NH2 ~ ~ N~-NHz
N ~ N
~R ~ ,R~
O N O O
H
103 105
By heating the N3-acyl analogs (103, 105) in pyridine, the acyl groups can be
induced to
migrate to the 2-amino position to give benzimidazole derivatives of formula A-
3 or A-4 (205
and 203, respectively) (Reaction Scheme 2).
N
I ~~--NH2 ~ I N~--NHZ
N ~ N
~R ~ ,R.
O N O O
H
103 105
Pyridine,
Heat
W I N~ N \ ~ I N~ O\
H ~ R ~ H ~ R.
O O
203 205
Reaction Scheme 2:
Where it is desired to synthesize a compound of formula A having a chlorine
atom at
the 5(6) position, 4-chloro-1,2-phenylenediamine 301 is condensed with
cyanogen bromide
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(Zou, R. et al., J. Med. Claem. 40:811-818 (1997)) to give the corresponding
chloro 2-
aminobenzimidazole 303. The benzimidazole 303 can then be heated with an
isocyanate in
pyridine to form the chloro 2-aminourea analogs 305 (Reaction Scheme 3).
Reaction Scheme 3:
NH2 CNBr ~ N R3NC0 / , N' H H
I NH CH~ ~ ~ N~NHZ PYridine, ~ ~N~'~ N~
Cl 2 CI H heat CI H ~ R3
O
30'1 303 305
In another method for preparing the compounds of formula A-3 or A-4, the
commercially-available fungicide methyl 2-aminobenzimidazole carbamate
(carbendazim,
401) is treated with an appropriate alcohol or amine with or without added
toluene as the
solvent in the presence of aluminum isopropoxide catalyst to give new
carbamates (403) and
areas (405), respectively (Reaction Scheme 4).
Reaction Scheme 4:
N
\ H ~ \ Rs
O
403
N R30H or R3NH2
\ I \~ O~ AI(OiPr)
H ~ heat
O
401 \ ~ N~ N
R3
O
405
Presently Preferred Embodiments
In one embodiment of the present invention, presently preferred benzimidazole
derivatives are those where R, and X are hydrogen, and Y is hydrogen or chloro
and is in the
5(6)-position. Further preferred are those compounds where R~, X and Y are
hydrogen and R
is selected from those groups listed in Table 1: Solubility is based on a
standard measure
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used in medicinal chemistry, the octanol-water partition coefficient, Loge,
Lower Log)
values indicate higher aqueous solubility. There are a variety of methods to
estimate this
value for a proposed structure using computer calculations. The below Loge
solubility values
were determined using the atom typing method of Ghose, Pritchett et al. (J.
Comput. Chem
9(1): 80-90, 1988). Carbendazim has a Loge of 1.302.
Table 1
C d. No. R Lo
1-1 -COOCH2CH=CHz 2.408
I-2 -COOCHZCC13 3.202
1-3 -CONHCH2CHZCl 1.721
1-4 -CONHCHZPh 2.794
I-5 -COOCHZPh 3.444
1-6 -CONHCHZCH2CH3 1.829
1-7 -COOCHZCHZCH3 2.479
I-8 -COOCH2CHzCH2N CH3 z --
1-9 -CONHCH2Ph -2-OH
1-10 -COOCHZCHz-mo holino --
1-11 -COOCH CH3 c clo ro 1 --
In another embodiment of the invention, presently preferred benzimidazole
derivatives are those where R is hydrogen, X is hydrogen, and Y is hydrogen or
chloro and is
in the 5(6)-position. Further preferred are those compounds where R and X are
hydrogen, Y
is hydrogen or chloro in the 5(6) position, and R~ is selected from those
groups listed in Table
2:
Table 2
C d. No. R1 Lo P
2-1 -CONHCH2CHzN CH3 z 0.631
2-2 -COOCHzCH=CHz 2.042
2-3 -COO CHzCH20 zCHZCH3 1.315
2-4 -CONHCHZCHZOCHzCHZOH 0.044
.2-5 -COOCHZCHZOCHZCHZCI 1.841
2-6 --COOCH2CHZC1 ~ 2.005
2-7 -COOCHZCHZOCH2CH20H 0.694
2-8 -COOCHZCHZCH3 2.113
2-9 -COOCHZPh 3.078
2-10 -CONHCHZCH2Cl 1.355
2-10* -CONHCH2CHzC1 1.873
2-11 -CONHCH2Ph 2.428
2-I2 -CONHCH2CH2CH3 1.463
2-12* -CONHCH2CHZCH3 1.981
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C d. No. R~ Lo P
2-13 -CONHCH3 --
2-13* -CONHCH3 1.170
2-14 -CONHCHZPh -2-OH) --
2-15 -COOCHzCH2-mo holino --
2-16 -COOCH CH3 c clo ro 1 --
* Y = Cl, at the 5(6) position
C. Screening Assays
Screening assays for determining those cancers susceptible to treatment using
compounds of the present invention include incubating cell line models
representing specific
cancers as set forth, for example, by the National Cancer Institute, in the
presence and
absence of such compounds. Viability of cells may be determined by the MTT
assay
(Promega Corp., Madison, WI 53711), or the SRB (sulforhodamine B) assay
(Skehan, et al.,
JNCI, 82:13,1107,1990). Susceptibility to said compounds exists when viability
in the
presence of a compound of the present invention is less than viability in the
absence of such
compound.
Exemplary cell line models representing specific cancers include, but are not
limited
to, the following:
Non-small cell lung cancer: NCIH23, NCIH324, NCIH522, A549/ATCC,
A549(ASC), CALU1, EKVX, NCIH125M, NCIH226, NCIH520, SKMES1, NCIH322M,
NCIH358M, NCIH460, NCIH292, HOP62, HOP18, HOP19, HOP92, LXFL 529, SW1573,
LXFS 650L, ML1019, ML1076, ML1045, or UABLG22;
Small cell lung cancer: NCIH69, NCIH146, NCIH82, NCIH524, DMS 114, DMS
273, HOP27, SHP77, or RHOS;
Colon cancer: HT29, HCC2998, HCT116, LOVO, SW1116, SW620, COLD 205,
DLD1, WIDR, COLO 320DM, HCT15, CXF 280, KM12, KM20L2, COLO 741, CXF 264L,
COLD 746, UABC02, MLI059, CAC02, HT29/PAR, HT29/MDRl, or NB4;
Breast cancer: MCF7, MCF7/ADRRES, ZR751, ZR7S30, MDAMB231/ATCC, HS
578T, UISOBCA1, MCF7/ATCC, SKBR3, MDAMB435, MDAN, BT549, T47D,
MDAMB231, MAXF 401, BT474, or MDAMB468;
Ovarian cancer: OVCAR3, OVCAR4, OVCARS, OVCARB, A2780, IGROVl,
SKOV3, OVXF 899, A1336, or ES2;
Leukemia: P388, P3888/ADR, CCRFCEM, CCRFSB, K562, MOLT4, L1210,
HL60(TB), RPMI8226, SR, or K562/ADR;
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Fibroblast: Mt90, or CCD19LU;
Renal cancer: U031, SN12C, SN12S1, SN12K1, SN12L1, SN12A1, A498, A704,
CAKI1, RXF 393, RXF631, 7860, SW156, TK164, 769P, SS78, ACHN, TK10, RXF 486L,
UOK57, or UOK57LN;
Melanonza: LOX IMVI, MALME3M, RPMI7951, SKMEL2, SKMELS, SKMEL28,
SKMEL31, UCSD 242L, UCSD 354L, M14, M19MEL, UACC62, UACC257, MEXF 514L,
or UABMEL3;
Prostate cancer: PC3, PC3M, DU145, LNCAP, 1013L, UMSCP1, WIS, JE, RER,
MRM, DHM, AG, RB, RVP, FC, WAE, DB/SMC, JCA1, ND1, WMF, TSUPRI, JECA,
GDP, T10, WBW, RVP1, or WLL;
CNS cancer: . SNB7, SNB I9, SNB44, SNB56, SNB75, SNB78, U251, TE671,
SF268, SF295, SF539, XF 498, SW 1088, SW 1783, U87 MG, SF767, SF763, A172, or
SMSKCNY;.
Bonelmuscle: A204/ATCC, OHS, TE85, A673, CHA59, MHM 25, RH18, RH30, or
RD; and
Lymphoma: AS283, HT, KD488, PA682, SUDHL7, RL, DB, SUDHL1, SUDHL4,
SUDHL 10, NUDUL 1, or HUT 102.
D. Chemotherapeutic Agents
Chemotherapeutic agents are generally grouped as DNA-interactive agents,
antimetabolites, tubulin-interactive agents, hormonal agents, other agents
such as
asparaginase or hydroxyurea, and agents as set forth in Table 3. Each of the
groups of
chemotherapeutic agents can be further divided by type of activity or
compound.
Chemotherapeutic agents used in combination with a compound of the present
invention, or
salts thereof of the present invention may be selected from any of these
groups but are not
limited thereto. For a detailed discussion of the cliemotherapeutic agents and
their method of
administration, see Don, et al, Cancer Chenzotlzerapy Handbook, 2d edition,
pages 15-34,
Appleton & Lange (Connecticut, 1994) herein incorporated by reference.
DNA-interactive agents include alkylating agents, e.g. cisplatin,
cyclophosphamide,
altretamine; DNA strand-breakage agents, such as bleomycin; intercalating
topoisomerase II
inhibitors, e.g., dactuiomycin and doxorubicin); nonintercalating
topoisomerase II inhibitors
such as, etoposide and teniposide; and the DNA minor groove binder plicamydin,
for
example.
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17
The alkylating agents form covalent chemical adducts with cellular DNA, RNA,
or
protein molecules, or with smaller amino acids, glutathione, or similar
chemicals. Generally,
alkylating agents react with a nucleophilic atom in a cellular constituent,
such as an amino,
carboxyl, phosphate, or sulfhydryl group in nucleic acids, proteins, amino
acids, or in
glutathione. The mechanism and the role of these alkylating agents in cancer
therapy is not
well understood.
Typical alkyladng agents include, but are not limited to, nitrogen mustards,
such as
chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil
mustard;
aziridine such as thiotepa; methanesulphonate esters such as busulfan; nitroso
ureas, such as
carmustine, lomustine, streptozocin; platinum complexes, such as cisplatin,
carboplatin;
bioreductive alkylator, such as mitomycin, and procarbazine, dacarbazine and
altretamine.
DNA strand breaking agents include bleomycin, for example.
DNA topoisomerase II inhibitors include the following intercalators, such as
amsacrine, dactinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin,
and
mitoxantrone; nonintercalators, such as etoposide and teniposide, for example.
A DNA minor groove binder is plicamycin, for example.
Antimetabolites interfere with the production of nucleic acids by one of two
major
mechanisms. Certain drugs inhibit production of deoxyribonucleoside
triphosphates that are
the immediate precursors for DNA synthesis, thus inhibiting DNA replication.
Certain of the
compounds are analogues of purines or pyrimidines and are incorporated in
anabolic
nucleotide pathways. These analogues are then substituted into DNA or RNA
instead of their
normal counterparts.
Antimetabolites useful herein include, but are not limited to, folate
antagonists such
as methotrexate and trimetrexate; pyrimidine antagonists, such as
fluorouracil,
fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine
antagonists
include mercaptopurine, 6-thioguanine, fludarabine, pentostatin; and
ribonucleotide reductase
inhibitors include hydroxyurea.
Tubulin interactive agents act by binding to specific sites on tubulin, a
protein that
polymerizes to form cellular microtubules. Microtubules are critical cell
structure units.
When the interactive agents bind the protein, the cell can not form
microtubules. Tubulin
interactive agents include vincristine and vinblastine, both alkaloids and
paclitaxel (Taxol),
for example.
Hormonal agents are also useful in the treatment of cancers and tumors. They
are
used in hormonally susceptible tumors and are usually derived from natural
sources.
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Hormonal agents include, but are not limited to, estrogens, conjugated
estrogens and ethinyl
estradiol and diethylstilbesterol, chlortrianisen and idenestrol; progestins
such as
hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and
androgens such as
testosterone, testosterone propionate; fluoxymesterone, and
methyltestosterone.
Adrenal corticosteroids are derived from natural adrenal cortisol or
hydrocortisone.
They are used because of their anti-inflammatory benefits as well as the
ability of some to
inhibit mitotic divisions and to halt DNA synthesis. These compounds include,
but are not
limited to, prednisone, dexamethasone, methylprednisolone, and prednisolone.
Leutinizing hormone releasing hormone agents or gonadotropin-releasing hormone
antagonists are used primarily the treatment of prostate cancer. These include
leuprolide
acetate and goserelin acetate. They prevent the biosynthesis of steroids in
the testes.
Antihormonal antigens include, for example, antiestrogenic agents such as
tamoxifen,
antiandrogen agents such as flutamide; and antiadrenal agents such as mitotane
and
aminoglutethimide.
Further agents include the following: hydroxyurea appears to act primarily
through
inhibition of the enzyme ribonucleotide reductase, and asparaginase is an
enzyme which
converts asparagine to nonfunctional aspartic acid and thus blocks protein
synthesis in the
tumor.
Taxol (paclitaxel) is a preferred chemotherapeutic agent.
A listing of currently available chemotherapeutic agents according to class,
and
including diseases for which the agents are indicated, is provided as Table 3.
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19
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CA 02422916 2003-03-13
WO 02/26716 PCT/US01/29261
23
E. Potentiators
A "potentiator," as used herein, is a material that improves or increases the
efficacy of
the benzimidazole derivatives or a salt or a prodrug thereof, or that acts on
the immune
system as an immunomodulator. Potentiators can be used in combination with a
compound of
the present invention. A potentiator may be an antiviral agent. One such
potentiator is
triprolidine or its cis-isomer. Triprolidine is described in U.S. 5,114,951
(I992, the patent is
incorporated by reference herein). A further potentiator is procodazole, (also
named 1H-
benzimidazole-2-propanoic acid, or 13-(2-benzimidazole) propionic acid or 2-(2-
carboxyethyl)benzimidazole or propazol). Procodazole is a non-specific
immunoprotective
agent active against viral and bacterial infections and may be used in
combination with the
compounds set forth herein. Pxocodazole is effective with a compound of the
present
invention, alone in treating cancers, tumors, leukemia or viral infections, or
combined with a
chemotherapeutic agent.
Further potentiators include, but are not limited to, propionic acid, salts
thereof, ox
I5 esters thereof; antioxidant vitamins such as vitamins A, C, E, or beta-
carotene; abacavir; AL-
721 (lipid mixture); amprenavir; Amphotericin $ methyl ester; Ampligen
(mismatched
RNA); anti-AIDS antibody; anti-human interferon-a antibody; anti-AIDS
antibody, ascorbic
acid and derivatives thereof; AS-I01 (heavy metal based immunostimulant);
azidothymidine;
(3-interferon; Bropirimine; butylated hydroxytoluene; Ciamexon, Cimetidine; CL-
246,738,
colony stimulating factors, including GM-CSF; Creme Pharmatex (benzalkonium
chloride);
CS-82 (5-unsubstituted derivative of Zidovudine); Cyclosporin; D-penicillamine
(3-mercapto-
D-valine); delavirdine; dextran sulphate; dinitrochlorobenzene; efavirenz;
erythropoietin;
Foscarnet (trisodium phosphonoformate); fusidic acid; ganciclovir; glucan;
glycyrrhizin,
HPA-23 (ammonium-21-tungsto-9-antimonate); human immunevirus antiviral;
hyperimmune
gamma-globulin,11VE2EG-1, IMREG-2; indinavir; interferon-a; interferon-gamma;
interleukin-I or interleukin-2; isoprinosine; Krestin; LC-9018; lamivudine;
Ientilart; LF-1695;
methionine-enkephalin; Minophagen C; muramyl tripeptide; naltrexone;
nelfinavir;
Neutropin; nevirapine; Nonoxinol; Ornidyl (eflornithine); non-nucleoside
inhibitors of
reverse transcriptase; nucleoside analogues (ddA, ddC, ddI, ddT, ddG, AZT, and
the like);
pentamidine isethionate; Phenytoin; polymannoacetate; Peptide T (octapeptide
sequence);
protease inhibitors; R.ibavirin; R.ifabutin (ansamycin); ritonavir; RNA
irnmunomodulator;
rsT4 (recombinant soluble T4); saquinavir; shosaikoto and ginseng; SK-818
(germanium-
derived antiviral); sodium diethylthiocarbarmate; stavudine; stearic acid
derivative; suramin
and analogues thereof; thymic humoral factor; TP-5; Thymosin fraction 5 and
Thymosin 1;
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Thymostimulin; TNF (tumor necrosis factor), vitamin B preparations;
Trimetrexate; UA001;
alpha-interferon; or acyclovir, for example.
- A compound, or a salt or a prodrug thereof, of the present invention may be
combined
with a potentiator and with a chemotherapeutic agent in the methods of the
present invention.
S F. Dosage
Any suitable dosage may be administered in the methods of the present
invention.
The compound or salt or prodrug thereof chosen for a particular application,
the carrier and
the amount will vary widely depending on the species of the warm blooded
animal or human,
the type of cancer, or the particular viral infection being treated, and
depending upon the
effective inhibitory concentrations observed in trial studies. The dosage
administered will, of
course, vary depending upon known factors, such as the pharmacodynamic
characteristics of
the particular compound, salt, or combination and its mode and route of
administration; the
age, health, or weight of the subj ect; the nature and extent of symptoms; the
metabolic
characteristics of the drug and patient, the kind of concurrent treatment; the
frequency of
1 S treatment; or the effect desired.
Generally a dosage of as little as about 1-2 milligram (mg) per kilogram (kg)
of body
weight is suitable, but preferably as little as 10 mg/kg and up to about
10,000 mg/kg may be
used. Preferably, a dosage from 15 rng/kg to about 5000 mg/kg is used. Most
preferably, the
dose is between 150 mglkgto about 1000 mg/kg. Doses useful in the treatment of
cancer or
viral infections are 250 mg/kg, 500 mg/lcg, 800 mg/kg, 1000 mg/kg, 1500 mglkg,
2500
mg/kg, 3500 mg/kg, 4000 mg/kg, 5000 mglkg, or 6000 mg/kg. Any range of doses
can be
used. Generally, a compound, salt thereof, prodrug thereof, or combination of
the present
invention can be administered on a daily basis one or more times a day, or one
to four times a
week, either in a single dose or separate doses during the day. Twice-weekly
dosing over a
period of at least several weeks is preferred, and often dosing will be
continued over extended
periods of time and possibly for the lifetime of the patient. However, the
dosage and the
dosage regimen will vary depending on the ability of the patient to sustain
the desired and
effective plasma levels of the compounds of the present invention, or salt or
prodrug thereof,
in the blood.
The compound, salt thereof, prodrug thereof, or combination may be micronized
or
powdered so that it is more easily dispersed and solubilized by the body.
Processes for
grinding or pulverizing drugs are well known in the art. For example, a hammer
mill or
similar milling device can be used. The preferred particle size is less than
about 100p. and
preferably less than 50p..
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Intravenously, the most preferred doses may range from about 1 to about 10
mg/kg/minute during a constant rate infusion.
The dosage for humans is generally less than that used in mice and is
typically about
1/12 of the dose that is effective in mice. Thus, if 500 mg/kg was effective
in mice, a dose of
5 42 mg/kg would be used in humans. For a 60 kg man, this dose would be 2520
mg.
The compounds and salts and prodrugs thereof of the present invention are
generally
safe. The LDso is high, about 1500 mg/kg given orally in mice and there are no
special
handling requirements. The compounds can be given orally, and since they are
not very
soluble, they are preferably given in tablet form or as a suspension.
Alternatively, when
10 micronized to sufficiently small size, they may be and preferably are given
as an injection.
The compounds and salts and prodrugs thereof of the present invention may be
administered in a unit dosage form which may be prepared by any methods (mown
to one of
skill in the art in light of the present disclosure. Unit dosages may include
from 1 milligram
to 1000 milligrams of active ingredient. Preferably the dosage unit will
contain from about
15 10 mg to about 500 mg active ingredient. The active ingredient is generally
present in an
amount of about 0.5% to about 95% by weight based on the total weight of the
dosage unit.
For intravenous use, preferred dosages may range from about 1 to about 10
mg/kg/minute during a constant rate infusion.
A dosage unit may comprise a single compound, or mixtures thereof, with other
20 compounds or other cancer- or viral-inhibiting compounds. The dosage unit
may comprise
diluents, extenders, carriers, liposomes, or the like. The unit may be in
solid or gel form such
as pills, tablets, capsules and the like or in liquid form suitable for oral,
rectal, topical,
intravenous injection or parenteral administration or injection into or around
the treatment
site. The range and ratio of benzimidazole derivative, or salt or prodrug
thereof, to
25 chemotherapeutic agent or to potentiator will depend on the type of cancer
or viral infection
being treated and the particular chemotherapeutic agent or potentiator.
G. Formulations
Formulations of the present invention include the compound of the present
invention,
a salt thereof or a prodrug thereof and, optionally, a chemotherapeutic agent
and, optionally,
a potentiator, generally mixed with a pharmaceutically acceptable carrier. A
"pharmaceutical
carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle
for delivering a
compound of the present invention to the animal or human. The carrier may be
liquid or solid
and is selected with the planned manner of administration in mind. A
"pharmaceutically
acceptable" component is one that is suitable for use with humans and/or
animals without
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26
undue adverse side effects (such as toxicity, irritation, and allergic
response) commensurate
with a reasonable benefitlrisk ratio.
Oral formulations suitable for use in the practice of the present invention
include
capsules, gels, cachets, tablets, effervescent or non-effervescent powders or
tablets, powders
or granules; as a solution or suspension in aqueous or non-aqueous liquid; or
as an oil-in-
water liquid emulsion or a water-in-oil emulsion. The compounds of the present
invention
may also be presented as a bolus, electuary, or paste.
Generally, formulations are prepared by uniformly mixing the active ingredient
with
liquid carnets or finely divided solid carriers or both, and then if necessary
shaping the
product. A pharmaceutical carrier is selected on the basis of the chosen route
of
administration and standard pharmaceutical practice. Each carrier must be
"acceptable" in the
sense of being compatible with the other ingredients of the formulation and
not injurious to
the subject. This carrier can be a solid or liquid and the type is generally
chosen based on the
type of administration being used. Examples of suitable solid carriers include
lactose,
sucrose, gelatin, agar and bulk powders. Examples of suitable liquid carriers
include water,
pharmaceutically acceptable fats and oils, alcohols or other organic solvents,
including esters,
emulsions, syrups or elixirs, suspensions, solutions andlor suspensions, and
solution and or
suspensions reconstituted from non-effervescent granules and effervescent
preparations
reconstituted from effervescent granules. Such liquid carriers may contain,
for example,
suitable solvents, preservatives, emulsifying agents, suspending agents,
diluents, sweeteners,
thickeners, and melting agents. Preferred carriers are edible oils, for
example, corn or canola
oils. Polyethylene glycols, e.g. PEG, are also preferred carriers.
The formulations for oral administration may comprise a non-toxic,
pharmaceutically
acceptable, inert Garner such as lactose, starch, sucrose, glucose, methyl
cellulose, magnesium
stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol,
cyclodextrin, cyclodextrin
derivatives, or the like.
Capsule or tablets can be easily formulated and can be made easy to swallow or
chew.
Tablets may contain suitable carriers, binders, lubricants, diluents,
disintegrating agents,
coloring agents, flavoring agents, flow-inducing agents, or melting agents. A
tablet may be
made by compression or molding, optionally with one or more additional
ingredients.
Compressed tables may be prepared by compressing the active ingredient in a
free flowing
form (e.g., powder, granules) optionally mixed with a binder (e.g., gelatin,
hydroxypropylmethylcellulose), lubricant, inert diluent, preservative,
disintegrant (e.g.,
sodium starch glycolate, cross-linked carboxymethyl cellulose) surface-active
or dispersing
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agent. Suitable binders include starch, gelatin, natural sugars such as
glucose or beta-lactose,
corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or
sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes, or the like. Lubricants
used in these
dosage forms include sodium oleate, sodium stearate, magnesium stearate,
sodium benzoate,
sodium acetate, sodium chloride, or the like. Disintegrators include, for
example, starch,
methyl cellulose, agar, bentonite, xanthan gum, or the like. Molded tablets
may be made by
molding in a suitable machine a mixture of the powdered active ingredient
moistened with an
inert liquid diluent.
The tablets may optionally be coated or scored and may be formulated so as to
provide slow- or controlled-release of the active ingredient. Tablets may also
optionally be
provided with an enteric coating to provide release in parts of the gut other
than the stomach.
Exemplary pharmaceutically acceptable carriers and excipients that may be used
to
formulate oral dosage forms of the present invention are described in US. Pat.
No. 3,903,297
to Robert, issued Sept. 2, 1975, incorporated by reference herein. Techniques
and
compositions for making dosage forms useful in the present invention are
described in the
following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker &
Rhodes,
Editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981);
and Ansel,
Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976).
. Formulations suitable for topical administration in the mouth wherein the
active
ingredient is dissolved or suspended in a suitable carrier include lozenges
which may
comprise the active ingredient in a flavored carrier, usually sucrose and
acacia or tragacanth;
gelatin, glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in
a suitable liquid carrier.
Topical applications for administration according to the method of the present
invention include ointments, cream, suspensions, lotions, powder, solutions,
pastes, gels,
spray, aerosol or oil. Alternately, a formulation may comprise a transdermal
patch or dressing
such as a bandage impregnated with an active ingredient and optionally one or
more carriers
or diluents. To be administered in the form of a transdermal delivery system,
the dosage
administration will, of course, be continuous rather than intermittent
throughout the dosage
3 0 regimen.
The topical formulations may desirably include a compound that enhances
absorption
or penetration of the active ingredient through the skin or other affected
areas. Examples of
such dermal penetration enhancers include dimethylsulfoxide and related
analogues.
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The oil phase of an emulsion used to treat subjects in the present invention
may be
constituted from ingredients known to one of skill in the art in light of the
present disclosure.
An emulsion may comprise one or more emulsifiers. For example, an oily phase
may
comprise at least one emulsifier with a fat or an oil, with both a fat and an
oil, or a hydrophilic
emulsifier may be included together with a lipophilic emulsifier that acts as
a stabilizer.
Together, the emulsifier(s), with or without stabilizer(s), make up an
emulsifying wax, and
the wax together with the oil and/or fat make up the emulsifying ointment base
that forms the
oily dispersed phase of the cream formulations.
Emulsifiers and emulsion stabilizers suitable for use in the formulation
include
Tween 60, Span 80, cetosteryl alcohol, myristyl alcohol, glyceryl monostearate
and sodium
lauryl sulphate, paraffin, straight or branched chain, mono-or dibasic alkyl
esters, mineral oil.
The choice of suitable oils or fats for the formulation is based on achieving
the desired
cosmetic properties, the properties required and compatibility with the active
ingredient.
Compounds of the present invention may also be administered vaginally, for
example, as pessaries, tampons, creams, gels, pastes, foams or spray
formulations containing
appropriate carriers in addition to the active ingredient. Such carriers are
known in the art in
light of the present disclosure.
Formulations for rectal administration may be presented as a suppository with
a
suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for nasal administration may be administered in a liquid
form,
for example, nasal spray, nasal drops, or by aerosol administration by
nebulizer, including
aqueous or oily solutions of the active ingredient. Formulations for nasal
administration,
wherein the Garner is a solid, include a coarse powder having a particle size,
for example, of
less than about 100 microns, preferably less than about SO microns, which is
administered in
the manner in which snuff is taken, i.e., by rapid inhalation through the
nasal passage from a
container of the powder held close up to the nose.
Formulations suitable for parenteral administration include aqueous and non-
aqueous
formulations isotonic with the blood of the intended recipient; and aqueous
and non-aqueous
sterile suspensions which may include suspending systems designed to target
the compound
to blood components or one or more organs. The formulations may be presented
in unit-dose
or mufti-dose sealed containers, for example, ampoules or vials.
Extemporaneous injections
solutions and suspensions may be prepared from sterile powders, granules and
tablets of the
kind previously described. Parenteral and intravenous forms may also include
minerals and
other materials to make them compatible with the type of injection or delivery
system chosen.
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In general, water, a suitable oil, saline, aqueous dextrose (glucose), or
related sugar
solutions and glycols such as propylene glycol or polyethylene glycols are
suitable Garners for
parenteral solutions. Solutions for parenteral administration preferably
contain a water
soluble salt of the active ingredient, suitable stabilizing agents and, if
necessary, buffer
substances. Antioxidizing agents, such as sodium bisulfite, sodium sulfite, or
ascorbic acid,
either alone or combined, are suitable stabilizing agents. Also used are
citric acid salts
thereof, or sodium EDTA. In addition, parenteral solutions may contain
preservatives, such
as benzalkonium chloride, methyl- or propyl-paraben, or chlorobutanol.
Suitable
pharmaceutical carriers are described in Remington, cited supra.
The present invention additionally contemplates administering compounds of the
herein described invention for use in the form of veterinary formulations,
which may be
prepared, for example, by methods that are conventional in the art in light of
the present
disclosure.
Useful pharmaceutical dosage formulations for administration of the compounds
of
the present invention are illustrated as follows:
Capsules: A large number of unit capsules are prepared by filling standard two-
piece
hard gelatin capsules each with 100 milligrams of powdered active ingredient,
150 milligrams
of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.
Soft Gelatizz Capsules: A mixture of active ingredient in a digestible oil
such as
soybean oil,' cottonseed oil or olive oil is prepared and injected by means of
a positive
displacement pump into gelatin to form soft gelatin capsules containing 100
milligrams of
the active ingredient. The capsules are washed and dried.
Tablets: A large number of tablets are prepared by conventional procedures so
that
the dosage unit was 100 milligrams of active ingredient, 0.2 milligrams of
colloidal silicon
dioxide, 5 milligrams of magnesium stearate, 275 milligrams of
microcrystalline cellulose; 11
milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings can
be applied to
increase palatability or delay absorption.
Injectable: A parenteral composition suitable for administration by injection
is
prepared by stirring 1.5% by weight of active ingredient in 10% by volume
propylene glycol
and water. The solution is made isotonic with sodium chloride and sterilized.
Suspezzsion: An aqueous suspension is prepared for oral administration so that
each 5
ml contains 100 mg of finely divided active ingredient, 200 mg of sodium
carboxymethyl
cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and
0.025 ml of .
vanillin.
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Compounds of the present invention may be administered in the form of liposome
delivery systems, such as small unilamellar vesicles, large unilamellar
vesicles, and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as
cholesterol, stearylamine, or phosphatidylcholines.
5 Compounds of the present invention may be coupled with soluble polymers as
targetable drug carriers. Such polymers can include polyvinylpyrrolidone,
pyran copolymer,
polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or
poiyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore,
the
compounds of the present invention can be coupled to a class of biodegradable
polymers
10 useful in achieving controlled release of a drug, for example, polylactic
acid, polyglycolic
acid, copolymers of polylactic and polyglycolic acid, polyepsilon
caprolactone, polyhydroxy
butyric acid, polyorthoesters, polyacetals, polydihydropyrans,
polycyanoacylates, and
crosslinked or amphipathic block copolymers of hydrogels.
15 H. Method of Treatment
The method of treahnent can be any suitable method which is effective in the
treatment of the particular cancer or viral infection that is being treated.
Treatment includes
administering a therapeutically effective amount of the compounds of the
present invention in
a form described hereinabove, to a subject in need of treatment.
20 Compounds of the present invention can be administered by any means that
produces
contact of the active agent with the agent's site of action in the body, for
example, suitable
means including, but not limited to,~oral, rectal, nasal, topical (including
transdermal, aerosol,
buccal or sublingual), vaginal, parenteral (including subcutaneous,
intramuscular, intravenous
or intradermal), intravesical, or injection into or around the cancer or site
of viral infection.
25 They can be administered by any conventional means available for use in
conjunction with
pharmaceuticals, either as individual therapeutic agents or in a combination
of therapeutics.
Preferably, compounds of the present invention are administered as a
pharmaceutical
formulation comprising at least one compound of the present invention, as
defined above,
together with one or more pharmaceutically acceptable carriers. It can be co-
administered in
30 the form of a tablet or capsule, as an agglomerated powder, or in a liquid
form, or as a
liposome.
The preferred route will vary with the condition and age of the recipient,
virus or
cancer being treated nature of the disorder, or severity of disorder. It is
believed that oral
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administration, or parenteral treatment is the preferred method of
administering the
compounds to subjects in need thereof.
In each of the above-described methods, the administering may be in vivo, or
may be
ex vivo. Ira vivo treatment is useful for treating diseases in a mammal,
preferably the mammal
is a human; and ex vivo treatment is useful for purging body fluids, such as
blood, plasma,
bone marrow, and the like, for return to the body. The nation's blood supply
is currently
tested for antibodies to HIV. However, the test is still imperfect and samples
that yield
negative tests can still contain HN virus. Treating blood and blood products
with the
compounds of the present invention can add an extra margin of safety to kill
any retrovirus
that may have gone undetected. Body tissue may be internal or external to an
animal body,
or, for example, may be the surface skin of the animal.
I. Combination Therapy
Compounds of the present invention may additionally be combined with
chemotherapeutic agents and/or potentiators to provide a combination therapy.
Combination
therapy is intended to include any chemically compatible combination of a
compound of the
present invention with otlier compounds of the present invention or other
compounds outside
of the present invention, as long as the combination does not eliminate the
activity of the
compound of the present invention. For example, one or more compounds may be
combined
with a potentiator or with a chemotherapeutic agent. In the case of retroviral
infection, a
combination therapy with nucleoside analogues such as AZT, nonnucleoside
reverse
transcriptase inhibitors, TC-3, or protease inhibitors is contemplated by the
present invention.
In the case of hepatitis, acyclovir, famciclovir or valacyclovir, Ribavirin,
interferon, or
combinations of Ribavirin and interferon or beta globulin is contemplated as a
combination
therapy. For herpes, a recombinant alpha uiterferon can be used as a
combination therapy.
The active agent can be coadministered, for example, in the form of a tablet
or capsule,
liposome, as an agglomerated powder, or in a liquid form. The amount of
chemotherapeutic
agent or potentiator used can be lower than that of the benzimidazole
derivative. It will be
present in a dosage unit in an amount that provides an operative combination
with the
benzimidazole derivative. The dosage of the chemotherapeutie agent or the
potentiator can
range from about O.S mg/kg body weight to about 400 mg/kg body weight.
Combination therapy can be sequential, that is the treatment with one agent
first and
then the second agent, or it can be treatment with both agents at the same
time. The
sequential therapy can be within a reasonable time after the completion of the
first therapy
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before beginning the second therapy. The treatment with both agents at the
same time can be
in the same daily dose or in separate doses. For example, treatment with one
agent on day 1
and the other on day 2. The exact regimen will depend on the disorder being
treated, the
severity of the disorder, and the response to the treatment.
In addition to the use of chemotherapeutic agents and potentiators, a
benzimidazole
derivate or a salt or a prodrug thereof can be combined with a fungicide or an
herbicide.
Preferred herbicides and fungicides include carbendazim, fluoconazole,
benomyl, glyphosate,
and propicodazole.
J. Pharmaceutical Kits
The present invention also includes pharmaceutical kits useful, for example,
for the
treatment of cancer or viral infection. The kits comprise one or more
containers containing a
pharmaceutical composition comprising a therapeutically effective amount of a
compound of
the present invention. Such kits can further include, if desired, one or more
of various
1 S conventional pharmaceutical kit components, such as, for example,
containers with one or
more pharmaceutically acceptable Garners, additional containers, etc., as will
be readily
apparent to those skilled in the art. Instruction, such as printed
instructions for example,
either as inserts or as labels, the instruction indicating quantities of the
components to be
administered, guidelines for administration, and/or guidelines for mixing the
components, can
also be included in the kit.
K. Studies
The following studies were performed to test the effectiveness of the
benzirnidazole
derivatives of the present invention against certain cancers and.viral
infections.
Colon and Melanoma Tumor Cells Test:
The following cell culture tests were performed to test the toxicity of
benzimidazole
derivative compounds of the present invention on colon and melanoma tumor
cells. The
viability of the cells were tested by looking at MTT (3-[4,5-dimethylthiazol-2-
yl] -2,5-
diphenyltetrazolium bromide) reduction. MTT assay is a well known measure of
cell
viability.
The colon tumor cells (HT29) and the melanoma cells (B 16 murine melanoma)
were
seeded (1000-2000 cells) into each well of a 96-well microtiter plate and
allowed to grow.
Twenty-four hours later, increasing concentrations of each benzimidazole
derivative were
~
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added to the plates and the cells were allowed to incubate for 3-6 days in the
presence of the
drugs. MTT reagent was then added to the wells and allowed to incubate for 4
hours at 37°C,
after which the formazan metabolite was solubilized with acidic isopropanol
and the
absorbance was read. Experimental controls included blank wells to which no
cells were
added (zero point of formazan absorbance) and control wells to which no drug
was added
(highest level of formazan absorbance). Each drug concentration was tested in
duplicate and
the resulting average absorbance was plotted against drug concentration using
the EZ-ED50
computer program on a personal computer interfaced to the microtiter plate
reader. The
program fit the resulting curves to a four-parameter equation:
,= Amax - Arvin
Arvin
~~ ~' X~ IC50)n
where Am~ and Am;" are, respectively, the absorbances in the absence and
presence of highest
drug concentration, X is the drug concentration and Y is the absorbance at
that concentration,
n is the slope of the curve, and ICSO is the concentration of drug that gives
SO% growth
inhibition. The ICso value is then derived from this equation by EZ-ED50. The
ICso values
for growth inhibition of both B 16 and HT29 cancer cell lines are reported in
Table 4 below (Y
= H unless otherwise indicated).
TABLE 4
MTT Assay - Growth Inhibitory Activity
ICso M
C d. No. Murine Melanoma Human Colon Carcinoma
1-1 14.8 23.6
1-2 29.5 38.4
1-3 8.9 27.1
1-4 73.5 66.0__
1-5. 32.5 52.0
1-6 33.9 31.9
1-7 115.6 80.6
2-1 181.9 252.8
2-2 1.7 2.0
2-3 14.2 11.1
2-4 200.6 212.6
2-5 25.6 31.3
2-6 292.6 374.7
2-7 159.5 119.2
2-8 1.8 1.9
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34
ICSO M
C d. No. Murine Melanoma Human Colon Carcinoma
2-9 >100 >100
2-10 771.5 83.8
2-10* 112.8 91.5
2-11 13.7 45.5
2-12 3.1 4.1
2-12* 0.98 1.9
2-13 * 0.63 0.29
* Y = Cl, at the 5(6) position
Microtubule Inhibition Assay:
The benzimidazole derivatives of this invention were evaluated for their
ability to
inhibit formation of microtubules, following the procedure of Luduena, R.F.
and M. C. Roach
(Pharmacol. Tlzer. 49: 133-152 (199i)). Tubulin was purified from bovine brain
by a cycle
of assembly and disassembly, followed by phosphocellulose chromatography and
gel
filtration (Fellous, A. et al., Eur. J. Biochem. 78(1): 167-174 (1977)). The
assay was
performed by preparing a sample of tubulin protein under conditions which
promote
polymerization into microtubules (Prasad, V. et al., J. Protein Chem. 11(5):
509-515 (1992)),
then adding the benzimidazole derivatives, at 2pM, and following the time
course of
polymerization. Microtubule polymerization was followed by performing the
reaction in a
cuvette and monitoring UV absorbance at 350 nm, allowing direct visualization
of the time
course of the polymerization. After 30 minutes, polymerization was complete
and the percent
inhibition of polymerization was calculated as the final absorbance of the
reaction taken as a
percentage of the final absorbance of a control polymerization reaction run in
parallel without
drug added. These data are reported in Table 5 below.
TABLE 5
Tubulin Polymerization Inhibition
C d. No. % Inhibition
1-1 25
1-2 28
1-3 20
1-4 63
1-5 38
1-6 31
1-7 15
2-1 17
2-2 3
2-3 19
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C d. No. % Inhibition
2-4 10
2-5 31
2-6 0
2-7 21
2-8 15
2-9 18
2-10 36
2-10* 23
2-11 48
2-12 0
2-12* 22
2-13* 50
* Y = Cl, at the 5(6) position
DNA Binding Assay:
The DNA binding assay was performed in a manner very similar to that of the
MTT
5 cell growth inhibition assay above. The assay was based upon the ability of
the test
compounds to displace methyl green (Sigma) from DNA and the resultant
hydration of the
free methyl green molecule to a colorless derivative (Burres, N.S. et al., J.
Nat. Prod. 55(11):
1582-1587 (1992); Kim and Norden, FEBSLett. 315(1): 61-64 (1993)). As for the
MTT
assay, each drug concentration was assayed in duplicate and the data were
averaged before
10 analysis. Experimental controls in this assay included wells containing
free methyl green
solution with no DNA, for fully decolorized background absorbance, and methyl
green-DNA
complex (Sigma) without drug, for the highest possible methyl green
absorbance. Methyl
green-DNA in buffer was added to each well to obtain Abss of approximately
0.7. Serial
dilutions of drug in buffer were added and the plates were mixed, then allowed
to stand at
15 room temperature for 24 hours to allow for displacement and hydration of
the free methyl
green. The absorbance at 655 nm was measured as a function of added drug
concentration in
an automated microplate reader, and the data were reduced by the computer
program EZ-
ED50 to derive ICso values (concentration at which 50% of the methyl green is
displaced from
DNA).
20 None of the benzimidazole derivatives listed in Table 4 or 5 showed any DNA
interaction by this assay.
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EXAMPLES
Example 1: Synthesis of Cpd. No. 1-4
0.200 Gram (1.5 mmol) of 2-aminobenzimidazole was dissolved in 4 mL dry
pyridine
and one equivalent (1.5 mmol, 200 mg) of benzyl isocyanate was added under
argon. The
mixture was stirred for 16 hours under argon at 23°C and another
equivalent of benzyl
isocyanate was then added. After four more hours stirring under argon at
23°C, the mixture ~,
was poured into 20 mL water to precipitate the product. The product was
collected by
filtration, washed with water and dried under reduced pressure for 16 hours to
provide 0.234 g
(0.879 mmol, 58.6% yield) of white powder.
Example 2: Synthesis of Cpd. No. l-5
0.200 Gram (1.5 mmol) of 2-aminobenzimidazole was dissolved in 4 mL dry
pyridine
and one equivalent (1.5 mmol, 256 mg) of benzyl chloroformate was added under
argon. The
mixture was stirred for 16 hours under argon at 23°C and another
equivalent of benzyl
chloroformate was then added. After four more hours stirring under argon at
23°C, the
mixture was poured into 20 mL water to precipitate the product. The product
was collected
by filtration, washed with water and dried under reduced pressure for 16 hours
to provide
0.168 g (0.629 mol, 41.9% yield) of white powder.
Example 3: Synthesis of Cpd. No. 2-11
0.112 Gram (0.421 mmol) of Cpd. No. 1-4, from Example 1, was dissolved in 1 mL
dry pyridine and heated to 80°C for 5 hours with stirring. Six mL water
was added to the
mixture with stirring and the solid precipitate was collected by filtration,
washed with water,
and dried under reduced pressure for 16 liours. 0.074 g (0.277 mmol, 65.8%
yield) of off
white powdery product was obtained.
Example 4: Synthesis of Cpd. No. 2-9
0.040 Gram (0.150 mmol) of Cpd. No. 1-5, from Example 2, was dissolved in 4 mL
toluene and heated to 110°C with stirring for 24 hours. The mixture was
cooled in an ice-
water bath and the precipitated solid collected by filtration to provide 0.024
g (0.090 mmol,
60% yield) of product as a white powder.
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Example 5: Synthesis of Cpd. No. 2-7
0.025 Gram (0.131 mmol) of methyl 2-aminobenzimidazole carbamate was mixed
with 2 mL of toluene and 0.696 g (6.55 mmol) of diethylene glycol. Three mg of
aluminum
isopropoxide was added and the mixture was heated to 110°C for 15
hours. The toluene was
evaporated under a stream of argon with continued heating, then the heat was
removed and
the residue was treated with 4 mL of boiling water. The resulting mixture was
cooled to
23°C, then stored 18 hours at 4°C. The precipitate that formed
was collected by filtration and
dried under reduced pressure to provide 0.029 g (0.109 mmol, 83.2% yield) of
product as off
white flakes.
Example 6: Synthesis of Cpd. No. 2-5
0.025 Gram (0.131 mmol) of methyl 2-aminobenzimidazole carbamate was mixed
with 2 mL of toluene and 0.817 g (6.55 mmol) of 2-(2-chloroethoxy)ethanol.
Three mg of
aluminum isopropoxide was added and the mixture was heated to 110°C for
15 hours. The
toluene was evaporated under a stream of argon with continued heating, then
the heat was
removed and the residue was treated with 4 ml of boiling water. The resulting
mixture was
cooled to 23°C, then stored 18 hours at 4°C. The precipitate
that formed was collected by
filtration and dried under reduced pressure to provide 0.026 g (0.09 mmol,
68.7% yield) of
product as off white powder.
Example 7: Synthesis of Cpd. No. 2-4
0.025 g (0.131 mmol) of methyl 2-aminobenzimidazole carbamate was mixed with 2
ml of toluene and 0.689 g (6.55 mmol) of 2-(2-aminoethoxy)ethanol. Three mg of
aluminum
isopropoxide was added and the mixture was heated to 110°C for 15
hours. The toluene was
evaporated under a stream of argon with continued heating, then the heat was
removed and
the residue was treated with 4 ml of boiling water. The resulting mixture was
cooled to 23°C,
then stored 18 hours at 4°C The precipitate that formed was collected
by filtration and dried
under reduced pressure to provide 0.009 g (0.034 mmol, 25.9% yield) of product
as colorless
platelike crystals.
