Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-VIRAL COMPOUNDS
The present invention is in the field of human
medicine, particularly in the treatment of viral infections.
More particularly, the present invention relates to the
treatment of rhinovirai, enteroviral and flaviviral
nventlons.
The incidence of viral upper respiratory disease, the
common cold, is immense. It has been estimated that nearly
a billion cases annually appear in the United States alone.
Rhinovirus, a member of the picornaviridae family, is the
major cause of the common cold in humans. Because more than
110 strains of rhinoviruses have been identified, the
development of a practical rhinovirus vaccine is not
feasible, and chemotherapy appears to be the more desirable
approach. Another member of the picornavirus family is the
enterovirus, which includes approximately eighty human
pathogens. Many of these enteroviruses cause cold-like
symptomsi others can cause more serious diseases such as
polio, conjunctivitis, aseptic meningitis and myocarditis.
Illness related to rhinovirus infection is evidenced by
nasal discharge and obstruction. Furthermore, it has been
implicated in otitis media, predisposes the development of
bronchitis, exacerbates sinusitis, and has been implicated
in the precipitation of asthmatic altoclis. Although it is
considered by many to be a mere nuisance, its frequent
occurrence in otherwise healthy individuals and the
resulting economic importance in terms of employee
absenteeism and physician visits have made it the subject of
extensive investigation.
The ability of chemical compounds to suppress the
growth of viruses in vitro may be readily demonstrated using
a virus plaque suppression test or a cytopathic effect test
(CPE). Cf Siminoff, Applied Microbiology, 9(1), 66 (1961).
Although a number of chemical compounds that inhibit
picornaviruses such as rhinoviruses have been identified,
many are unacceptable due to 1) limited spectrum of
activity, 2) undesirable side effects or 3) inability to
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prevent infection or illness in animals or humans. See
Textbook of ~uman Viroloav, edited by Robert B. Belshe,
chapter 16, "Rhinoviruses," Roland A. Levandowski, 391-405
(1985). Thus, despite the recognized therapeutic potential
associated with a rhinovirus inhibitor and the research
efforts expended thus far, a viable therapeutic agent has
not yet emerged. For example, antiviral benzimidazole
compounds have been disclosed in U.S. Pat. Ser. Nos.
4,008,243, 4,018,790, 4,118,573, 4,118,742, 4,174,454 and
4,492,708.
In general, the compounds disclosed in the above
patents do not have a desirable pharmacological profile for
use in treating rhinoviral infections. Specifically, these
compounds do not possess satisfactory oral bioavailability
or a high enough inhibitory ac~ivity to compensate for their
relatively low oral bioavailability to permit their
widespread use. In addition, it is widely accepted in the
art that compounds used to treat rhinoviral infections
should be very safe from a toxicological standpoint.
Accordingly, it is a primary object of this invention
to provide novel benzimidazole compounds which inhibit the
growth of picornaviruses, such as rhinoviruses,
enteroviruses such as polioviruses, coxsackieviruses of the
A and B groups, or echo virus and which have a desirable
pharmacological profile
The present invention provides compounds of formula I
(R)~ R
R~ I
O=C
IR4R5
wherein:
a is 0, 1, 2 or 3;
. .
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each R is independently hydrogen, halo, cyano, amino,
halo(C1-C6)alkyl, di(C1-C4)alkylamino, azido, C1-C6 alkyl,
carbamoyl, carbamoyloxy, carbamoylamino, C1-C6 alkoxy, C1-C4
alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl,
pyrrolidino, piperidino or morpholino;
R0 is hydrogen, halo, C1-C4 alkyl or C1-C4 alkoxy;
Rl is halo, cyano, hydroxy, methyl, ethyl, methoxy,
ethoxy, methylthio, methylsulfinyl or methylsulfonyl;
R2 is hydrogen, amino or -NHC(O)(C1-C6 alkyl);
R3 is dimethylamino, C1-C1o alkyl, C3-C7 cycloalkyl,
substituted C3-C7 cycloalkyl, halo(C1-C6)alkyl, phenyl,
substituted phenyl, furyl, thienyl, thiazolyl,
thiazolidinyl, pyrrolidino, piperidino, morpholino or a
group of the formula:
S ~ N
~
R4 and R5 are independently hydrogen or C1-C4 alkyl;
or a pharmaceutically acceptable salt thereof.
The present invention also provides pharmaceutical
formulations comprising a compound of the present invention,
or a pharmaceutically acceptable salt thereof, in
combination with a pharmaceutically acceptable carrier,
diluent or excipient therefor.
The present invention also provides a method for
inhibiting a picornavirus comprising administering to a host
in need thereof, an effective amount of a compound of
formula I, or a pharmaceutically acceptable salt thereof,
wherein a, R, R0, Rl, R2, R3, R4 and R5 are as defined above.
The present invention also provides a method for
inhibiting a flavivirus comprising administering to a host
in need thereof, an effective amount of a compound of
formula I, or a pharmaceutically acceptable salt thereof,
wherein a, R, R0, Rl, R2, R3, R4 and R5 are as defined above.
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All temperatures stated herein are in degrees Celsius
(~C). All units of measurement employed herein are in
weight units except for liquids which are in volume units.
As used herein, the term "C1-C1o alkyl" represents a
straight or branched alkyl chain having from one to ten
carbon atoms. Typical C1-C1o alkyl groups include methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
t-butyl, pentyl, neo-pentyl, hexyl, 2-methylhexyl, heptyl
and the like. The term "C1-C1o alkyl" includes within its
definition the terms "C1-C6 alkyl" and "C1-C4 alkyl.~
"Halo" represents chloro, fluoro, bromo or iodo.
"Halo(C1-C6)alkyl" represents a stralght or branched
alkyl chain having from one to six carbon atoms with 1, 2 or
3 halogen atoms attached to it. Typical halo(C1-C6)-alkyl
groups include chloromethyl, 2-bromoethyl, 1-
chloroisopropyl, 3-fluoropropyl, 3-bromobutyl, 3-
chloroisobutyl, iodo-t-butyl, trichloromethyl,
trifluoromethyl, 2,2-chloro-iodoethyl, 2,3-dibromopropyl and
the like.
"C1-C4 alkylthio" represents a straight or branched
alkyl chain having from one to four carbon atoms attached to
a sulfur atom. Typical C1-C4 alkylthio groups include
methylthio, ethylthio, propylthio, isopropylthio, butylthio
and the like.
~C1-C6 alkoxy" represents a straight or branched alkyl
chain having from one to six carbon atoms attached to an
oxygen atom. Typical Cl-c6 alkoxy groups include methoxy,
ethoxy, propoxy, isopropoxy, butoxy and the like. The term
"C1-C6 alkyl" includes within its definition the term
"C1-C4 alkyl."
"Di(C1-C4)alkylamino" represents two straight or
branched alkyl chains having from one to four carbon atoms
attached to a common amino group. Typical di(C1-C4)alkyl-
amino groups include dimethylamino, ethylmethylamino,
methylpropylamino, ethylisopropylamino, butylmethylamino,
sec-butylethylamino and the like.
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IlCl-C4 alkylsulfinyl~' represents a straight or branched
alkyl chain having from one to four carbon atoms attached to
a sulfinyl moiety. Typical Cl-C4 alkylsulfinyl groups
include methylsulfinyl, ethylsulfinyl, propyl-sulfinyl,
isopropylsulfinyl, butylsulfinyl and the like.
IlCl-C4 alkylsulfonyl" represents a straight or branched
alkyl chain having from one to four carbon atoms attached to
a sulfonyl moiety. Typical Cl-C4 alkylsulfonyl groups
include methylsulfonyl, ethylsulfonyl, propyl-sulfonyl,
isopropylsulfonyl, butylsulfonyl and the like.
"Substituted phenyl" represents a phenyl ring
substituted with 1-3 substituents selected from the
following: halo, cyano, Cl-C4 alkyl, Cl-C4 alkoxy, amino or
halo(Cl-C4)alkyl.
"Substituted C3-C7 cycloalkyl" represents a cycloalkyl
ring substituted with 1-3 substituents selected from the
following: halo, cyano, Cl-C4 alkyl, Cl-C4 alkoxy, amino or
halo(Cl-C~)alkyl.
The claimed compounds can occur in either the cis or
trans conformation. For the purposes of the present
application, cis refers to those compounds where the
carboxamide moiety is cis to the benzimidazo~e ring and
trans refers to those compounds where the carboxamide moiety
is trans to the benzimidazole ring. Both isomers are
included in the scope of the claimed compounds.
As mentioned above, the invention includes the
pharmaceutically acceptable salts of the compounds defined
by formula I. A compound of this invention can possess a
sufficiently acidic, a sufficiently basic, or both
functional groups, and accordingly react with any of a
number of inorganic bases, and inorganic and organic acids,
to form a pharmaceutically acceptable salt.
The term "pharmaceutically acceptable salt~ as used
herein, refers to salts of the compounds of the above
formula which are substantially non-toxic to living
organisms. Typical pharmaceutically acceptable salts
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include those salts prepared by reaction of the compounds of
the present invention with a mineral or organic acid or an
inorganic base. Such salts are known as acid addition and
base addition salts.
Acids commonly employed to form acid addition salts are
inorganic acids such as hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, phosphoric acid, and the
like, and organic acids such as p-toluenesulfonic,
methanesulfonic acid, ethansulfonic acid, oxalic acid, p-
bromophenylsulfonic acid, carbonic acid, succinic acid,
citric acid, benzoic acid, acetic acid, and the like.
Examples of such pharmaceutically acceptable salts are
the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide,
acetate, propionate, decanoate, caprylate, acrylate,
formate, isobutyrate, caproate, heptanoate, propiolate,
oxalate, malonate, succinate, suberate, sebacate, fumarate,
maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate,
chlorobenzoate, methylbenzoate, dinitrobenzoate,
hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,
xylenesulfonate, phenylacetate, phenylpropionate,
phenylbutyrate, citrate, lactate, ~ -hydroxybutyrate,
glycollate, tartrate, methanesulfonate, ethanesulfonate,
propanesulfonate, naphthalene-l-sulfonate, napththalene-2-
sulfonate, mandelate and the like. Preferred
pharmaceutically acceptable acid addition salts are those
formed with mineral acids such as hydrochloric acid and
sulfuric acid, and those formed with organic acids such as
maleic acid and methanesulfonic acid.
Base addition salts include those derived from
inorganic bases, such as ammonium or alkali or alkaline
earth metal hydroxides, carbonates, bicarbonates, and the
like. Such bases useful in preparing the salts of this
invention thus include sodium hydroxide, potassium
hydroxide, ammonium hydroxide, potassium carbonate, sodium
carbonate, sodium bicarbonate, potassium bicarbonate,
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calcium hydroxide, calcium carbonate, and the like. The
potassium and sodium salt forms are particularly preferred.
It should be recognized that the particular counterion
forming a part of any salt of this invention is not of a
critical nature, so long as the salt as a whole is
pharmacologically acceptable and as long as the counterion
does not contribute undesired qualities to the salt as a
whole.
Preferred compounds of this invention are those
compounds
(R)a R~
~ R~
Rl ) R3
O=C
IR4R5
where:
a is 0, 1 or 2;
each R is independently hydrogen, halo, Cl-C4 alkyl,~5 Cl-C4 alkoxy or di(Cl-C4)alkylamino;
R0 is hydrogen;
~2 is amino;
R3 is dimethylamino, Cl-C6 alkyl, halo(Cl-C6)alkyl,
phenyl, substituted phenyl, C3-C7 cycloalkyl, substituted
C3-C7 cycloalkyl, thienyl, thiazolidinyl, pyrrolidino,
piperidino or morpholino;
R4 is hydrogen, methyl or ethyl;
R5 is hydrogen, methyl or ethyl;
or a pharmaceutically acceptable salt thereof.~5
Of these preferred compounds, more preferred are those
compounds of formula I where:
a is 0 or 1;
each R is independently hydrogen, fluoro, methyl,~0 ethyl, methoxy, ethoxy, dimethylamino;
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R3 is C1-C4 alkyl, phenyl, substituted phenyl, C3-C7
cycloalkyl or substituted C3-C7 cycloalkyli
or a pharmaceutically acceptable salt thereof.
Of these compounds, the most preferred compounds are:
-N~ NH2
o=lC ~HCl
NH~
NH~
o=lC HCl
NH2
'~-NH
o=C ~ HCl or
NH2
~ I '~-NH~,
~I ~
o=C ~ Hcl
NH-CH3
or a pharmaceutically acceptable salt thereof.
The compounds of formula I may be prepared by reacting
a suitably substituted acetamide with a base to provide the
corresponding anion which is then reacted with a suitably
substituted ketone of formula IA to provide a carbinol
intermediate. The reactions are typically carried out in an
organic solvent for one to twelve hours at a temperature of
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from about -90~C to room temperature using an excess of the
base and acetamide reactant relative to the ketone reactant.
The acetamide is preferably protected with a suitable
- protecting group prior to use in the reaction. Typical
bases include sodium hydride, lithium diisopropylamide (LDA)
and n-butyllithium. A preferred base is n-butyllithium.
Solvent choice is not critical so long as the solvent
employed is inert to the ongoing reaction and the reactants
are sufficiently solubilized to effect the desired reaction.
A solvent that is suitable for use in this reaction is
tetrahydrofuran although the acetamide reactant can also be
used as a solvent. The carbinol intermediate is generally
prepared in from about one to eighteen hours when the
reaction is initiated at -78~C and allowed to slowly warm to
room temperature. The reaction may be monitored by HPLC and
quenched by the addition of an acid when it is substantially
complete. Typical acids include hydrochloric acid,
hydrobromic acid, formic acid and the like. A preferred
acid is concentrated hydrochloric acid. The resultant
carbinol intermediate is preferably dehydrated without prior
isolation or purification.
In particular, the carbinol intermediate is reacted
with an acid for thirty minutes to twelve hours at a
temperature of from about room temperature to the reflux
temperature of the mixture to provide the desired compound
of formula I. Typical acids include hydrochloric acid,
hydrobromic acid, formic acid, acetic acid and combinations
of acids. A preferred acid combination is formic acid
containing concentrated hydrochloric acid. The desired
compound is generally prepared in from about thirty minutes
to seven hours when the reaction is carried out at just
below the reflux temperature of the mixture. The reaction
is preferably monitored by HPLC, for example, to ensure that
the reaction goes to completion.
The compounds of formula I are preferably isolated and
the resulting cis/trans isomers separated using procedures
known in the art. For example, the cis and trans forms of
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the isolated compounds may be separated using column
chromatography, for example reverse phase HPLC. The
compounds may be eluted from the column using an appropriate
ratio of acetonitrile and water or methanol and water. The
cis form of the compound may be converted to a cis/trans
mixture by exposure to h~ irradiation and recycled through
the above-mentioned purification process.
The ketone intermediates of formula IA may be prepared
according to procedures detailed in the art. For example,
the ketone intermediates may be prepared according to the
following Reaction Scheme I.
Reaction Scheme I
(R) a R~
1. base oxidizing
Rl l X' NH agent
X R~
(R) a R~)
2. Reduction
Rl ~ R3
(R)a R~
3. Cyclization
Rl ~ R3
(R)~ R~
R2 IA
Rl ~ R3
where:
X is cyano or -COOR', where R' is C1-C4 alkyl;
X' is halo;
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a, R, R0, R1, R2 and R3 are defined above.
Reaction Scheme I, above, is accomplished by carrying
out reactions 1-4. Once a reaction is complete, the
intermediate compound may be isolated, if desired, by
procedures known in the art. For example, the compound may
be crystallized and then collected by filtration, or the
reaction solvent may be removed by extraction, evaporation
or decantation. The intermediate compound may be further
purified, if desired, by common techniques such as
crystallization or chromatography over solid supports such
as silica gel or alumina, before carrying out the next step
of the reaction scheme.
Reaction I.1 is accomplished by first exposing an
appropriately substituted halo-nitroaniline and an
appropriately substituted phenylacetonitrile or benzoate to
a base in an organic solvent for one to twenty four hours at
a temperature of from about -10~C to about 40~C to provide a
ketone precursor. The reaction is typically carried out
using e~uimolar proportions of the reactants in the presence
of two equivalents of the base. Typical bases include
sodium hydride, potassium t-butoxide, lithium
diisopropylamide (LDA). A preferred base is potassium t-
butoxide. Examples of solvents suitable for use in this
reaction include dimethylformamide, dimethylacetamide and
the like. Solvent choice is not critical so long as the
solvent employed is inert to the ongoing reaction and the
reactants are sufficiently solubilized to effect the desired
reaction. The ketone precursor is generally prepared in
from about one to fifteen hours when the reaction is
initiated at 0~C and allowed to progress at room
temperature. The ketone precursor is preferably oxidized in
the same reaction mixture without prior isolation or
purification.
In particular, the ketone precursor is reacted with an
oxidizing agent for 30 minutes to 15 hours at a temperature
of from about 0~C to about 30~C to provide the corresponding
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ketone compound. Typical oxidizing agents include hydrogen
peroxide, oxygen and air. The oxygen and air are typically
bubbled through the reaction mixture. A preferred oxidizing
agent is hydrogen peroxide, preferably in a 30% solution.
The ketone is generally prepared in from about thirty to
five hours when the reaction is carried out between 0~C and
room temperature. The reaction is preferably monitored by
TLC, for example, to ensure that the reaction goes to
completion.
In reaction I.2, the nitro substituent on the ketone is
reduced according to procedures known in the art to provide
the corresponding diaminobenzophenone compound. For
example, the nitro substituent may be reduced by catalytic
hydrogenation, for example by combining the ketone isolated
from reaction I.1 with hydrogen gas in ethanol or
tetrahydrofuran and a catalyst. A preferred catalyst is
palladium-on-carbon or Raney nickel. Solvent choice is not
critical so long as the solvent employed is inert to the
ongoing reaction and the nitro reactan~ is sufficiently
solubilized to effect the desired reaction. The hydrogen
gas is typically used at a pressure of up to 60 psi,
preferably at or about 30 psi. The reaction is generally
substantially complete after about 1 to 24 hours when
conducted at a temperature in the range of from about 0~C to
about 40~C. The reaction is preferably conducted at a
temperature in the range of from about 20~C to about 30~C
for about 2 to 5 hours.
In reaction I.3, the compound isolated from reaction
I.3 is cyclized via a nitrile intermediate by reacting the
benzophenone compound with cyanogen ~romide in ar. alcoholic
solvent such as isopropanol. Typically, the reaction is
carried out at a temperature of from about 0~C to about
30~C. When the benzophenone is completely dissolved, the
resultant solution is combined with cyanogen bromide. The
cyanogen bromide is typically added in the form of a
solution (3-7M for example in acetonitrile). The reaction
is generally complete after one to eighteen hours when the
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reaction mixture is stirred at room temperature. However,
in certain instances nitrile intermediate will precipitate
out of the reaction mixture. In order to form the desired
ketone, this precipitate is isolated and then refluxed in an
alcoholic solvent such as isopropanol for one to four hours
to provide the desired ketone compound of formula I.
The compounds of the formula:
R~
X' NO R3
where:
X', Ro and R3 are as defined above;
are prepared by displacing the chloro or fluoro substituent
on a compound of the formula
R(J
~ N~2
X~ Y
where Y is chloro or fluoro, with the proviso that Y cannot
be chloro when X~ is fluoro, with a primary amine of the
formula NH2R3, where R3 is as defined above, in an organic
solvent. The reaction is optionally carried out in the
presence of an acid scavenger such as potassium carbonate or
a large excess of the primary amine. Typical solvents
include tetrahydrofuran, dimethylformamide,
dimethylacetamide and the like. The reaction is generally
complete in one to twenty hours when carried out at a
temperature of from about 20~C to about 80~C. The resultant
alkylated halo nitroaniline is then reacted as described in
Reaction Scheme I, above.
The compounds of formula I where R2 is -NHC(O)(C1-C6
alkyl) may be prepared by acylating the ketone intermediate
or the corresponding compound of formula I, where R2 is
amino, according to procedures known in the art. For
example, the amine compound may be acylated with a suitable
acyl halide, isocyanate or chloroformate, preferably in the
presence of an acid scavenger such as a tertiary amine,
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preferably triethylamine. A preferred acylating agent is
acetic anhydride. The reaction is typically carried out at
a temperature of from about -20~C to about 25~C. Typical
solvents for this reaction include ethers and chlorinated
hydrocarbons, preferably diethylether, chloroform or
methylene chloride. The amine reactant is generally
employed in equimolar proportions relative to the acylating
reactant, and preferably in the presence of equimolar
quantities of an acid scavenger such as a tertiary amine. A
preferred acid scavenger for this reaction is N-
methylmorpholine (NMM).
The compounds employed as initial starting materials in
the synthesis of the compounds of this invention are known
in the art, and, to the extent not commercially available
are readily synthesized by standard procedures commonly
employed in the art.
It will be understood by those in the art that in
performing the processes described above it may be desirable
to introduce chemical protecting groups into the reactants
in order to prevent secondary reactions from taking place.
Any amine, alcohol, alkylamine or carboxy groups which may
be present on the reactants may be protected using any
standard amino-, alcohol- or carboxy- protecting group which
does not adversely affect the remainder of the moleculels
ability to react in the manner desired. The various
protective groups may then be removed simultaneously or
successively using methods known in the art.
The pharmaceutically acceptable salts of the invention
are typically formed by reacting a compound of formula I
with an equimolar or excess amount of acid or base. The
reactants are generally combined in a mutual solvent such as
diethyl ether, tetrahydrofuran, methanol, ethanol,
isopropanol, benzene and the like, for acid addition salts,
or water, an alcohol or a chlorinated solvent such as
methylene chloride for base addition salts. The salts
normally precipitate out of solution within about one hour
,
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- 15 -
to about ten days and can be isolated by filtration or other
conventional methods.
The following Preparations and Examples further
illustrate specific aspects of the present invention. It is
to be understood, however, that these examples are included
for illustrative purposes only and are not intended to limit
the scope of the invention in any respect and should not be
so construed.
In the following Preparations and Examples, the terms
melting point, nuclear magnetic resonance spectra, electron
impact mass spectra, field desorption mass spectra, fast
atom bombardment mass spectra, infrared spectra, ultraviolet
spectra, elemental analysis, high performance liquid
chromatography, and thin layer chromatography are
abbreviated "m.p.", "NMR", "EIMS", "MS(FD)", "MS(FAB)",
"IR", " W", "Analysis", "HPLC", and "TLC", respectively.
The MS(FD~ data is presented as the mass number unless
otherwise indicated. In addition, the absorption maxima
listed for the IR spectra are only those of interest and not
all of the maxima observed.
In conjunction with the NMR spectra, the following
abbreviations are used: "s" is singlet, "d" is doublet,
"dd" is doublet of doublets, "t" is triplet, "q" is quartet,
~m" is multiplet, "dm" is a doublet of multiplets and
"br.s", "br.d", "br.t", and "br.m" are broad singlet,
doublet, triplet, and multiplet respectively. "J" indicates
the coupling constant in Hertz (Hz). Unless otherwise
noted, NMR data refers to the free base of the subject
compound.
The MMR spectra were obtained on a Bruker Corp. 250 MHz
instrument or on a General Electric QE-300 300 MHz
instrument. The chemical shifts are expressed in delta,
values (parts per million downfield from tetramethyl-
silane). The MS(FD) spectra were taken on a Varion-MAT 731
Spectrometer using carbon dendrite emitters. EIMS spectra
were obtained on a CEC 21-110 instrument from Consolidated
Electrodynamics Corporation. IR spectra were obtained on a
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Perkin-Elmer 281 instrument. W spectra were obtained on a
Cary 118 instrument. TLC was carried out on E. Merck silica
gel plates. Melting points are uncorrected.
Exam~le 1
A. 2-Iso~ro~ylamino-4-fluoro-nitrobenzene
To a cold (0~C) mixture of 43.35 ml (400 mmol) of 2,4-
difluoronitrobenzene and 55 g (approx. 400 mmol) of
potassium carbonate in 400 ml of tetrahydrofuran, was added
approximately 34.4 ml of isopropylamine (400 mmol). The
reaction mixture was warmed to room temperature and reacted
for 60 hours and then filtered. The potassium carbonate was
washed with ethyl acetate and the organics were then
concentrated in vacuo resulting in the crystallization of
the desired compound which was then isolated by filtration
and washed with a small volume of hexane.
Yield: 66.37 g, yellow crystals (84%).
B. 3-Iso~ro~vlamino-4-nitro-2,3-difluorobenzo~henone
To a cold (0~C) mixture of 7.65 g (50 mmol) of 2,3-
difluorophenylacetonitrile and 9.9 g (50 mmol) of the
compound of Example lA in 80 ml of dimethylformamide, was
added 11.22 g (100 mmol) of potassium t-butoxide. The
reaction mixture was warmed to room temperature and reacted
for approximately l hour. When the reaction was
substantially complete, as determined by TLC, the mixture
was cooled to 0~C, followed by the addition of 15 ml of a
30% solution of hydrogen peroxide. The mixture was warmed
to room temperature, stirred overnight and then poured into
1 liter of lN hydrochloric acid resulting in the formation
of 16 g of an orange solid which was used without further
purification.
C. 3-Iso~ro~vlamino-4-amino-2~3-difluorobenzo~henone
The compound of Example lB was hydrogenated in 250 ml
of tetrahydrofuran using 2.1 g of Raney nickel catalyst
under 60 psi of hydrogen (gas) for six hours. The reaction
. , .
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mixture was filtered and the filtrate was concentrated in
vacuo to provide 14 g of a solid which was used without
further purification.
D. F ~ ~ ~ NH~
F o HBr
To a cold (0~C) mixture of 14 g of Example lC in 125 ml
of isoproyl alcohol, was added one equivalent of cyanogen
bromide (9.6 ml of a 5M solution in acetonitrile). The
resultant mixture was warmed to room temperature and stirred
for 2 days and then concentrated in vacuo to provide a
residue. ThiS residue was redissolved in ethyl acetate and
then sonicated resulting in the formation of 13.0 g of
crystals.
Analysis for C17H16N3OBrF2:
Calcd: C, 51.53; H, 4.07; N, 10.61; Br, 20.17,
Found: C, 51.64; H, 4.17; N, 10.51; Br, 20.41.
MS(FD): 315 (M+).
H NMR (300 MHz; d6-DMSO): ~ 1.56 (d, 6H); 4.85 (septet,
lH); 7.41 (m, 2H); 7.33 (d, lH)i 7.67 (d, lH); 7.74 (m,
lH)i 8.01 (s, lH) and 8.87 ~s, 2H).
IR(CHCl3): ~ 3088, 2984, 1663, 1626, 1481, 1304 and
1276 cm-l.
W/VIS (95% EtOH): ~max = 318 nm (E=11480); 223 nm
(E=24524).
F
E. ~ ~ '~ NH~
O ~
The desired compound was obtained by adding lN sodium
hydroxide to Example lD in ethyl acetate. The resulting
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- 18 -
layers were separated and the organic phase was concentrated
in vacuo.
Yield: 9.34 g (62%).
Analysis for C17H1sN3OF2:
Calcd: C, 64.76; H, 4.80; N, 13.33;
Found: C, 64.97; H, 4.78; N, 13.40.
MS(FD): 315 (M+).
H NMR (300 MHz; d6-DMSO): ~ 1.38 (d, 6H); 3.67 (septet,
lH); 7.01 ~s, 2H); 7.18 (d, lH); 7.35 (m, 3H); 7.66 (s,
lH) and 7.77 (s, lH).
IR(CHCl3): ~ 3380, 2910, 1652, 1608, 1522, 1307, 1276 and
1264 cm~1.
W/VIS (95% EtOH): ~maX = 341 nm (E=21011); 220.5 nm
(E=26966).
F
~ ~ N
J' )\
o=f . Hcl
NH2
To a cold (-78~C) solution of 18.8 ml (76 mmol) of
bis(trimethylsilyl)acetamide in 200 ml of tetrahydrofuran,
was slowly added 30.4 ml of 2.5M n-butyllithium in hexane
(76 mmol), followed by the addition of 3.0 g (9.5 mmol) of
of Example lE. The reaction mixture was stirred for 8 hours
at -78~C and then allowed to warm to room temperature. When
the reaction was substantially complete, as indicated by
HPLC, the reaction was quenched by the ad~ition of 6.4 ml
(76 mmol) of concentrated hydrochloric a l and then
concentrated in vacuo to provide an oil wnich was then
redissolved in formic acid containing 1% concentrated
hydrochloric acid. The mixture was allowed to react for 4
hours at 95~C. When the reaction was substantially
complete, as indicated by HPLC, the mixture was concentrated
in vacuo to provide an oil. This oil was separated using
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- 19 --
reverse phase HPLC (eluent of acetonitrile in water) to
provide the cis and trans isomers of the subtitled compound.
cis
not characterized
trans
Analysis for ClgH21N4O2F2Cl:
Calcd: C, 55.55; H, 5.15; N, 13.64;
Found: C, 54.21; H, 4.93; N, 12.98.
MS(FD): 356 (M+).
lH NMR (300 MHz; d6-DMSO): ~ 1.48 (d, 6H); 4.73 (septet,
lH); 6.71 (s, lH); 7.93 (m, 3H); 7.18 (m, 2H); 7.25 (d,
lH); 7.35 (m, lH); 7.42 (s, lH); 7.52 (s, lH); 7.79 (s,
2H).
IR (KBr): ~ 3152, 2982, 1662, 1596, 1483, 1474 and
1269 cm~l.
W /VIS (95~ EtOH): ~maX = 310 nm (E=9665); 223 nm
(E=24308).
ExamPle 2
NH-
o=
NH-CH~~0
The compound was prepared substantially as described in
Example lF using N-methyl-N-trimethylsilylacetamide.
cis
not characterized
trans
Analysis for C20H23N4o2F2cl:
Calcd: C, 56.54; H, 5.46; N, 13.19;
Found: C, 56.32; H, 5.10; N, 13.06.
MS(FD): 370.3 (M+).
lH NMR (300 MHz; d6-DMSO): ~ 2.52 (d, 6H); 2.59 (d, 3H);
4.81 (septet, lH); 6.78 (s, lH); 6.94 (m, 2H); 7.2 (m,
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lH); 7.4 (m, 2H); 7.6 (s, lH); 8.19 (m, lH); 8.75 (m,
lH); 8.79 (s, lH).
IR (KBr): V 3068, 1669, 1627, 1589, 1482 and 1474 cm~l.
W/VIS (95% EtOH): ~max = 305 nm (E=13688); 223 nm
(E=30904).
Exam~le 3
NH~
A
o-lC ~HCl
NH-CH~CH3
The compound was prepared substantially ln accordance
with Example lE, with the exception that n-butyllithium
(15.85 mmol) was slowly added to a solution that was
prepared as follows. A cold (-78~C) solution of
bis(trimethylsilyl)amide (1 equivalent) and N-ethylacetamide
(1 equivalent) in tetrahydrofuran was stirred for 1 hour
followed by the addition of chlorotrimethylsilane (1
equivalent). The resultant solution was stirred for 15
minutes and then allowed to warm slowly to room temperature.
NOTE: The solution was cooled to -78~C again before the
addition of the n-butyllithium.
cis
not characterized
trans
Analysis for C21H22N4OF2-HCl-H2O:
Calcd: C, 57.47; H, 5.74; N, 12.76;
Found: C, 57.25; H, 5.65; N, 12.74.
MS(FD): 384.2 (M+).
H MMR (300 MHz; d6-DMSO): ~ 0.96 (t, 3H); 1.49 (d, 6H);
3.0 (p, 2H); 4.80 (septet, lH); 6.74 (s, lH); 6.88 (t,
lH); 6.94 (d, lH); 7.16 (q, lH); 7.30-7.44 (m, 2H);
7.55 (s, lH); 8.17 (t, lH); 8.75 (s, 2H); and 12.8 (s,
lH).
IR (CHCl3): ~ 2986, 1664, 1602, 1514 and 1482 cm~l.
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W097/46237 PCT~S97/08848
W/VIS (95% EtOH): ~maX = 304.00 nm (E=13407.11); 224.00
nm (E=31891.73).
The following compounds were prepared substantially as
described in Example lA-F.
ExamPle 4
NH2
o=C ~HCl
NH~
cis
MS(FD): 338 (M+).
Analysis for ClgHlgN4OF HCl:
Calcd: C, 60.88; H, 5.38; N, 14.95;
Found: C, 60.62; H, 5.66; N, 14.78.
trans
MS(FD): 338 (M+).
Analysis for ClgHlgN4OF HCl 1.2H20:
Calcd: C, 57.56; H, 5.70; N, 14.13;
Found: C, 57.26; H, 5.28; N, 13.75.
ExamPle 5
F ~ N
NH2
o=lC~ ~
NH2 OCH3
ClS
Analysis for C23Hl8N4o2F2:
Calcd: C, 65.71; H, 4.32; N, 13.33;
25Found: C, 65.44i H, 4.30; N, 13.05.
MS(FD): 420 (M+).
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H NMR (250 MHz; d6-DMSO): ~ 3.83 (s, 3H); 6.08 (s, lH);
6.29 (s, 2H); 6.78 (m, 2H); 7.13 (m, 6H); 7.33 (m, 4H).
IR (CHC13): V 3390, 3013, 1664, 1514 and 1254 cm~l.
W /VIS (95% EtOH): ~maX = 217 nm (E=40891).
trans
AnalysiS for C23Hl8N4o2F2:
Calcd: C, 65.71; H, 4.32; N, 13.33;
Found: C, 63.22; H, 4.63; N, 12.63.
MS(FD): 420 (M+).
lH NMR (250 MHz; d6-DMSO): ~ 3.86 (s, 3H); 6.40 (s, 2H);
6.49 (s, lH); 6.71 (d, lH); 6.84 (m, 3H); 7.13 (m, 4H);
7.32 (m, 3H) and 7.36 (d, lH).
IR (KBr): ~ 3416, 3314, 3201, 1664, 1582, 1543, 1513 and
1271 cm~l.
W/VIS (95% EtOH): ~maX = 330 nm (E=16300); 226 nm
(E=33818).
Exam~le 6
NH2
o=lC ~HCl
NH~
cis
not characterized
trans
MS(FD): 382 (M~).
Analysis for C21H20N4oF2-Hcl-l.2H2o:
Calcd: C, 57.26; H, 5.35; N, 12.72;
Found: C, 57.21; H, 5.08; N, 12.47.
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Exam~le 7
NH2
b
o= IC ~ HCl
NH2
cis
MS (FD): 364 (M+) .
Analysis for C21H21N4OF- 1.2HCl:
Calcd: C, 61.79; H, 5.48; N, 13.73;
Found: C, 61.68; H, 5.60; N, 13.56.
trans
MS (FD): 364 (M+) .
Analysis for C2 lH2 lN40F~ HC l:
Calcd: C, 62.92; H, 5.53; N, 13.98;
Found: C, 62.78; H, 5.55; N, 13.68.
The following compounds were prepared
substantially as described above in Example 2.
Exam~le 8
N~
b
o=c
~HCl
NH-CH3
cis
MS (FD): 378 (M+) .
Analysis for C22H23N40F-HCl-0.5H20:
Calcd: C, 62.33; H, 5.94; N, 13.22;
Found: C, 62.33; H, 5.74; N, 12.98.
trans
MS (FD): 378 (M+) .
Analysis for C22H23N4OF ~ HCl ~ 0.2H2O:
Calcd: C, 63.14; H, 5.88; N, 13.39;
Found: C, 63.00; H, 5.92; N, 13.33.
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Exam~le 9
NH
o=lC ~HCl
NH-CH3
ClS
MS(FD): 350 (M+).
Analysis for C20HlgN4oF-l.5Hcl-l.5H2o:
Calcd: C, 55.59; H, 5.48; N, 12.97;
Found: C, 55.92; H, 5.24; N, 12.80.
trans
MS(FD): 350 (M+).
Analysis for C2oHlgN4OF 1.6HCl:
Calcd: C, 58.77; H, 5.08; N, 13.71;
Found: C, 58.89; H, 5.42; N, 12.55.
Exam~le 10
NH~
I ~HCl
NH-CH3
cis
not characterized
trans
MS(FD): 366 (M+).
Analysis for C21H23N4OF-1.4HCl:
Calcd: C, 60.42; H, 5.89; N, 13.42;
Found: C, 60.28; H, 6.15; N, 13.24.
The following compounds are made substantially as
detailed in Example lA-F.
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Exam~le 11
- F ~ ~ ~ NH~
o=C
I ~HCl
NH2
Exam~le 12
CH30 ~ ~ ~ NH~
o=C
I HCl
NH~
The present compounds appear to inhibit replication of
plus-strand viral RNA by interfering with the structure
and/or function of the viral replication complex (a
membrane-bound complex of viral and cellular proteins).
Mutant rhinovirus and enterovirus have been isolated which
demonstrate very low levels of drug tolerance. These
mutants contain a single amino acid substitution in the
protein that is expressed by the viral gene known as "3A".
Therefore, the compounds of the present invention inhibit
the rhinovirus and enterovirus by inhibiting a 3A function.
The 3A gene encodes a hydrophobic protein which serves as
the scaffolding protein that attaches the proteins of the
replication complex to intracellular membranes.
The replicative strategy of flaviviruses such as
hepatitis C virus (HCV) and bovine diarrheal virus (BVDV) is
similar to that of the rhinovirus and enterovirus, discussed
above. In particular, both families of virus contain
single-stranded, messenger-sense RNA that replicates in a
cytoplasmic complex via a minus-strand RNA intermediate. In
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addition, both families of virus translate their genome into
a polyprotein that is subsequently cleaved. Furthermore,
the replication complexes of both viruses are tightly
associated with intracellular membranes. Finally, both
families of virus have analogous genomic structures
including the presence of a 5' and 3' non-translated region
which are re~uired by the viruses for replication. There
are two HCV proteins that have been implicated with this
intracellular association: NS2 and NS4. It is postulated
that either NS2 or NS4 is analogous to the picornavirus 3A
protein.
Accordingly, another embodiment of the present
invention is a method of treating or preventing a flavivirus
infection comprising administering to a host in need thereof
an effective amount of a compound of formula I or a
pharmaceutically acceptable salt thereof. It is preferred
to inhibit hepatitis C.
As noted above, the compounds of the present invention
are useful as antiviral agents. They have shown inhibitory
activity against various enterovirus and rhinovirus. An
embodiment of the present invention is a method of treating
or preventing a picornavirus infection comprising
administering to a host in need thereof an effective amount
of a compound of formula I or a pharmaceutically acceptable
salt thereof.
The term "effective amount" as used herein, means an
amount of a compound of formula T which is capable of
inhibiting viral replication. The picornavirus inhibition
contemplated by the present method includes either
therapeutic or prophylactic treatment, as appropriate. The
specific dose of compound administered according to this
invention to obtain therapeutic or prophylactic effects
will, of course, be determined by the particular
circumstances surrounding the case, including, for example,
the compound administered, the route of administration, the
condition being treated and the individual being treated. A
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typical daily dose will contain a dosage level of from about
0.01 mg/kg to about 50 mg/kg of body weight of an active
compound of this invention. Preferred daily doses generally
will be from about 0.05 mg/kg to about 20 mg/kg and ideally
from about 0.1 mg/kg to about 10 mg/kg.
The compounds can be administered by a variety of
routes including oral, rectal, transdermal, subcutaneous,
intravenous, intramuscu~ar and intranasal. The compounds of
the present invention are preferably formulated prior to
administration. Therefore, another embodiment of the
present invention is a pharmaceutical formulation comprising
an effective amount of a compound of formula I or a
pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier, diluent or excipient
therefor.
The active ingredient in such formulations comprises
from 0.1% to 99.9~ by weight of the formulation. By
"pharmaceutically acceptable" it is meant that the carrier,
diluent or excipient is compatible with the other
ingredients of the formulation and not deleterious to the
recipient thereof.
The present pharmaceutical formulations are prepared by
known procedures using well-known and readily available
ingredients. In making the compositions of the present
invention, the active ingredient will usually be admixed
with a carrier, or diluted by a carrier, or enclosed within
a carrier which may be in the form of a capsule, sachet,
paper or other container. When the carrier serves as a
diluent, it may be a solid, semi-solid or liquid material
which acts as a vehicle, excipient or medium for the active
ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets,
elixirs, suspensions, emulsions, solutions, syrups,
aerosols, (as a solid or in a liquid medium), ointments
containing, for example, up to 10~ by weight of the active
compound, soft and hard gelatin capsules, suppositories,
,
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sterile injectable solutions, sterile packaged powders and
the like.
The following formulation examples are illustrative
only and are not intended to limit the scope of the
invention in any way. The term "active ingredient" means a
compound according to formula I or a pharmaceutically
accepta~le salt thereof.
Formulation 1
10Hard gelatin capsules are prepared using the following
ingredients:
Quantity
(mq/ca~sule)
Active ingredient 250
Starch, dried 200
Magnesium stearate 10
Total 460 mg
Formulation 2
20A tablet is prepared using the ingredients below:
Quanti ty
(m~/ca~sule)
Active ingreaient 250
Cellulose, microcrystalline400
Silicon dioxide, fumed 10
Stearic acid 5
Total 665 mg
The components are blended and compressed to form
tablets each weighing 665 mg.
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Formulation 3
An aerosol solution is prepared containing the
following components:
Weiqht
Active ingredient 0.25
Methanol 25.75
Propellant 22
(Chlorodi~luoromethane) 70.00
Total 100.00
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.
Formulation 4
Tablets, each containing 60 mg of active ingredient,
are made as follows:
Quantity
(mq/tablet)
Active ingredient 60
Starch 45
Microcrystalline cellulose 35
Polyvinylpyrrolidone
(as 10% solution in water) 4
Sodium carboxymethyl starch 4.5
Magnesium stearate 0.5
Talc
Total 150
The active ingredient, starch and cellulose are passed
through a No. 45 mesh U.S. sieve and mixed thoroughly. The
aqueous solution containing polyvinylpyrrolidone is mixed
with the resultant powder, and the mixture then is passed
through a No. 14 mesh U.S. sieve. The granules so produced
are dried at 50~C and passed through a No. 18 mesh U.S.
sieve. The sodium carboxymethyl starch, magnesium stearate
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and talc, previously passed through a No. 60 mesh U.S.
sieve, are then added to the granules which, after mixing,
are compressed on a tablet machine to yield tablets each
weighing 150 mg.
Formulation 5
Capsules, each containing 80 mg of active ingredient,
are made as follows:
Quantity
(mq/ca~sule)
Active ingredient 80 mg
Starch 59 mg
Microcrystalline cellulose 59 mg
Magnesium stearate 2 mg
Total 200 mg
The active ingredient, cellulose, starch and magnesium
stearate are blended, passed through a No. 45 mesh U.S.
sieve, and filled into hard gelatin capsules in 200 mg
quantities.
Formulation 6
Suppositories, each containing 225 mg of active
ingredient, are made as follows:
Active ingredient 225 mg
Saturated fatty acid glycerides 2,000 mg
Total 2,225 mg
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.
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Formulation 7
Suspensions, each containing 50 mg of active ingredient
per 5 ml dose, are made as follows:
Active ingredient 50 mg
Sodium carboxymethyl cellulose 50 mg
Syrup 1.25 ml
Benzoic acid solution 0.10 ml
Flavor q.v.
Color q.v.
Purified water to total 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 color are diluted with a portion of the water and
added, with stirring. Sufficient water is then added to
produce the required volume.
Formulation 8
An intravenous formulation may be prepared as follows:
Active ingredient 100 mg
Isotonic saline 1,000 ml
The solution of the above ingredients generally is
administered intravenously to a subject at a rate of 1 ml
per minute.
The following experiment was carried out to demonstrate
the ability of the compounds of formula I to inhibit certain
vlrus .
Test Method for Anti-Picornaviral Assav
African green monkey kidney cells (BSC-l) or Hela cells
(5-3) were grown in 25 CC Falcon flasks at 37~C in medium
199 with 5 percent inactivated fetal bovine serum (FBS),
penicillin (1~0 units 1 ml) and streptomycin ( 150 micrograms
per milliliter (~g/ml)). When confluent monolayers were
formed, the supernatant growth medium was removed and 0. 3 ml
of an appropriate dilution of virus (echo, Mengo, Coxsackie,
polio or rhinovirus) were added to each flask. After
, . .
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absorption for one hour at room temperature, the virus
infected cell sheet was overlaid with a medium comprising
one part of 1 percent Ionagar No. 2 and one part double
strength Medium lg9 with FBS, penicillin and streptomycin
which contains drug at concentrations of 100, 50, 25, 12, 6,
3 and 0 ~g/ml. The flask containing no drug served as the
control for the test. The stock solutions of vinyl
acetylene benzimidazole compounds were diluted with
dimethylsulfoxide to a concentration of 104 ~g/ml. The
flasks were then incubated for 72 hours at 37~C for polio,
Coxsackie, echo and Mengo virus and 120 hours at 32~C for
rhinovirus. Virus pla~ues were seen in those areas were the
virus infected and reproduced in the cells. A solution of
10 percent formalin and 2 percent sodium acetate was added
to each flask to inactivate the virus and fix the cell sheet
to the surface of the flask. The virus plaques,
irrespective of size, were counted after staining the
surrounding cell areas with crystal violet. The plaque
count was compared to the control count at each drug
concentration. The activity of the test compound was
expressed as percentage plaque reduction, or percent
inhibition. Alternatively, the drug concentration which
inhibits plaque formation by 50 percent can be used as a
measure of activity. The 50 percent inhibition is indicated
by the symbol IC50.
In vitro CPE/XTT anti-BV~V AssaY
MDBK cells were dispersed in the 96-wells microtiter
plate at 10,000 cells per well with Minimum Essential Medium
containing Earl's balanced salt solution (EBSS), 2~ horse
serum, penicillin (100 units/ml) and streptomycin (100
ug/ml). Plates were grown at 37~C CO. incubator overnight.
The MDBK cells were then infected with ~0.02 moi
(multiplicity of infection) of bovine viral diarrhea virus
(BVDV, ATCC VR-534). After allowing the virus to adsorb to
the cells for 1-2 hours, medium containing serial dilutions
of drug or medium alone was added to the wells. After
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further incubating for 3-4 days (when extensive cpe was
apparent in medium alone wells), the antiviral effect of
testing drugs were assessed by performing a XTT assay as
described below.
XTT [2,3-bis(methoxy-4-nitro-5-sulfophenyl)-2H-
tetraazolium-5-carboxanilide, inner salt, sodium salt] at
lmg/ml for warm medium without FBS were freshly prepared and
used immediately. For each 5 ml of the XTT solution, 25 ul
of 5mM of PMS (phenazine methosulfate) in phosphate buffer
saline was added. Then 50 jul of the freshly prepared
XTT/PMS mixture was added to each of the microtiter wells.
Incubate at 37~C (CO~) for 3-4 hours or until color change
is prominent. Read absorptance at 450 nm/ref. 650 nm in a
spectrophotometer. The concentration of drug required to
cause 50% cytotoxic effect as compared to the no drug no
virus control (TC50) and which to inhibit the development of
virus cytopathic effect (cpe) by 50% (IC50) was then
determined from the liner portion of each dose response
curve.
