COMPOUNDS, COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING PNEUMOVIRUS INFECTION AND ASSOCIATED DISEASES

COMPOSES, COMPOSITIONS ET METHODES DE TRAITEMENT OU DE PREVENTION D'INFECTIONS PNEUMOVIRALES ET DE MALADIES ASSOCIEES

English Abstract

Compounds, compositions and methods are provided for the prophylaxis and
treatment of infections caused by viruses of the Pneumovirinae subfamily of
Paramyxoviridae and diseases associated with such infections.

French Abstract

L'invention concerne des composés, des compositions et des méthodes de prophylaxie et de traitement d'infections causées par les virus de la famille des Paramyxoviridae, et plus précisément de la sous-famille des Pneumovirinae, et de maladies associées à ces infections.

Claims

33
WHAT IS CLAIMED IS:
1. A compound having the formula:
<IMG>
wherein Het represents a substituted five to seven membered heterocyclic ring
containing at least one heteroatom selected from nitrogen, oxygen and sulfur,
said
heterocyclic ring substituents comprising at least one selected from the group
consisting
of alkyl, amino, monoalkylamino and dialkylamino, wherein alkyl comprises 1 to
6
carbon atoms;
R1 represents a radical selected from the group consisting of halogen;
perfluoroalkyl; alkoxyalkyl; amino; alkylamino; dialkylamino; amido;
alkylaminoalkyl;
an unsubstituted or substituted, saturated or unsaturated straight- or
branched-chain
alkyl radical, said alkyl chain substituent being at least one hydroxy group;
carboxy; an
unsubstituted or substituted phenyl radical (C6H5), said phenyl radical
substituent being
at least one selected from the group consisting of hydroxy, halogen,
perfluoroalkyl, thio,
nitro, carboxy, carboxyalkyl, carbalkoxy, carbalkoxyalkyl, carboxamide,
carboamidoalkyl, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkylthio,
alkylsulfinyl,
alkylsulfonyl, sulfonamide, amidino, cyano, amino, amido, alkylamino,
dialkylamino,
alkylaminoalkyl, and alkoxy monosubstituted with a substituent selected from
the group
consisting of carboxy, amino, alkylamino and dialkylamino; a cycloalkyl
radical
wherein all alkyl and alkoxy radicals comprise 1 to 6 carbon atoms; and a
heterocyclic
radical selected from the group consisting of pyridine, thiophene, oxazole,
oxadiazole,
thiadiazole, pyrazole, tetrazole, furan, pyrrole, isoxazole, imidazole,
triazole thiazole,
and all positional isomers of said heterocyclic radicals;

34
R2 represents a radical selected from the group consisting of hydrogen,
hydroxy,
thio, alkoxy, carboxy, carboxyalkyl, amino, alkylamino, dialkylamino,
carboxamide,
carboxamidoalkyl, sulfonamide and acetamido wherein alkyl and alkoxy comprise
1 to
6 carbon atoms;
X represents a divalent linking moiety selected from the group consisting of -
N=CH-, -CH=N-, -(CH2)n-NH-, -NH-(CH2)n-,
-(CH2)n-, -CH=CH- and -N=N-, n being an integer from 1 to 8; and
Z represents a substituent selected from the group consisting of formyl,
hydroxy
and -X-Het, wherein X and Het are as previously defined; the cis-and trans-
isomeric
forms of said compound and the pharmaceutically acceptable salts of said
compound.
2. A prodrug which undergoes conversion in vivo into a compound as claimed in
claim 1 and is selected from the group consisting of mono-, di- or tri-esters
of simple or
functionalized aliphatic carboxylic acids; esters of carbamic acids (R a-(O-CO-
NR b R c)n);
esters of amino acids (R a-(O-CO-CH(NH2)R b)n); esters of unsubstituted or
substituted
aromatic acids (R a-(O-CO-aryl)n), wherein the aryl ring may be substituted
with
hydroxy, carboxy, lower alkyl, alkylthio, alkylsulphinyl, alkylsulphonyl,
phosphoric
acid, amino, alkylamido and halogen groups; esters of derivatized phosphoric
acids;
(acyloxy)methyl or acyloxy(ethyl)ethers (R a-(O-CH2-O-CO-R b)n or R a-(O-
CH(CH3)-O-
CO-R b)n); (alkoxycarbonyloxy)methyl or (alkoxycarbonyloxy)ethyl ethers (R a-
(O-CH2-
O-CO-O-R b)n); and O-glycosides, wherein R a is a residue of a compound of the
invention, R b and R c are aliphatic radicals (C1-C10) and n=1-3.
3. The compound 5,5'-Bis[1-(((5-amino-1H-tetrazolyl)imino)methyl)]2,2',4"-
methylidynetrisphenol as claimed in claim 1.
4. The compound 5,5'-Bis[1-(((5-amino-1H-tetrazolyl)imino)methyl)]-4"-
methoxyphenyl-2,2'-benzylidenebisphenol as claimed in claim 1.
5. The compound 5,5'-Bis[1-(((5-amino-1H-1,2,4triazolyl)imino)methyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.

35
6. The compound 5,5'-Bis[4-(((3-amino-4H-1,2,4-triazolyl)imino)methyl)]-
2,2',4"-
methylidynetrisphenol as claimed in claim 1.
7. The compound 5,5'-Bis[2-(((5-amino-2H-tetrazolyl)imino)methyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.
8. The compound 5,5'-Bis[1-(((5-methyl-1H-tetrazolyl)imino)methyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.
9. The compound 5,5'-Bis[1-(((5-amino-1H-tetrazolyl)imino)methyl)]-2,2'-
benzylidenebisphenol as claimed in claim 1.
10. The compound 5,5'-Bis[1-(((5-amino-1H-tetrazolyl)imino)methyl)]2,2'-
methylidenebisphenol as claimed in claim 1.
11. The compound 5,5'-Bis[1-(2-(5-(1-methyl-1H-tetrazolyl))ethenyl)]-2,4',4"-
methylidynetrisphenol as claimed in claim 1.
12. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-(4-
propylphenyl)-2,2'-benzylidinebisphenol as claimed in claim 1.
13. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-(4-
propyloxyphenyl)-2,2'-benzylidenebisophenol as claimed in claim 1.
14. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-(4-
fluorophenyl)-2,2'-benzylidenebisphenol as claimed in claim 1.
15. The compound 5,5'-Bis[1-(2-(4-methylthiazolyl)ethenyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.
16. The compound 5,5'-Bis[1-(2-(5-(3-methylisoxazolyl))ethenyl)]-phenyl-2,2'-
benzylidenebisphenol as claimed in claim 1.

36
17. The compound 5,5'-Bis[2-(2-(5-methyl-2H-tetrazolyl)ethyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.
18. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)amino)methyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.
19. The compound 5-[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.
20. The compound 5-[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-2,4',4"-
methylidynetrisphenol as claimed in claim 1.
21. The compound 3-[5-[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-2,4'-
dihydroxydiphenylmethylene]-4-hydroxybenzaldehyde as claimed in claim 1.
22. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-[4-((2-
diethylamino)ethoxy)phenyl]-2,2'-benzylidenebisphenol as claimed in claim 1.
23. The compound 4-[5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-2,2'-
dihydroxydiphenylmethylene]phenoxyacetic acid as claimed in claim 1.
24. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-(4-
pyridinyl)-2,2'-benzylidenebisphenol as claimed in claim 1.
25. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-(4-
nitrophenyl)-2,2'-benzylidenebisphenol as claimed in claim 1.
26. The compound 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)methyl)]-(4-
aminophenyl)-2,2'-benzylidenebisphenol as claimed in claim 1.
27. The compound 5,5'-Bis[1-(2-(2-(1-methylimidazolyl)ethenyl)]-2,2',4"-
methylidynetrisphenol as claimed in claim 1.

37
28. The compound 5,5'-Bis[1-(((5-methyl-1H-tetrazolyl)imino)methyl)]phenyl-
2,2'-
benzylidenebisphenol as claimed in claim 1.
29. An intermediate for the preparation of a compound as claimed in claim 1,
said
intermediate having the formula:
<IMG>
wherein Q represents a reactive group selected from the group consisting of
5,5-
dimethyl-1,3-dioxan and formyl; R5 is a radical selected from the group
consisting of
hydrogen and hydroxy; and R6 is a radical selected from the group consisting
of
hydroxy, alkoxy (C1-C6), aryloxy (C1-C6) and aralkyloxy; and R7 is a radical
selected
from the group consisting of hydrogen,hydroxy, alkoxy, alkoxyalkyl, halogen,
perfluoroalkyl, thio, nitro, carboxy, carboxyalkyl, carbalkoxy,
carbalkoxyalkyl,
carboxamide, carboxamidoalkyl, alkyl, cycloalkyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, sulfonamide, amidino, cyano, amino, amido, alkylamino,
dialkylamino,
alkylaminoalkyl, and alkoxy monosubstituted with a substituent selected from
the group
consisting of carboxy, amino, alkylamino and dialkylamino, wherein all alkyl
and
alkoxy radicals comprise 1 to 6 carbon atoms.

38
30. A pharmaceutical composition for treating or preventing pneumovirus
infection,
said composition comprising a compound as claimed in claim 1 in an amount
effective
to attenuate infectivity of said virus, and a pharmaceutically acceptable
carrier medium.
31. A pharmaceutical composition as claimed in claim 30, further comprising at
least one supplemental active agent selected from the group consisting of
interferons,
ribavirin; immunomodulators, immunoglobulins, anti-inflammatory agents,
antibiotics,
anti-virals and anti-infectives.
32. A use of a therapeutically effective amount of a compound as claimed in
claim 1
or a prodrug of said compound for treatment of pneumovirus infection in a
patient in
need of said treatment.
33. The use as claimed in claim 32, wherein a precursor of said compound is in
the
form of a prodrug selected from the group consisting of mono-, di- or tri-
esters of
simple and functionalized aliphatic carboxylic acids; esters of carbamic acids
(R a (O-
CO-NR b R c)n); esters of amino acids (R a-(O-CO-CH(NH2)R b)n); esters of
unsubstituted
and substituted aromatic acids (R a (O-CO-aryl)n), wherein the aryl ring may
be
substituted with hydroxy, carboxy, lower alkyl, alkylthio, alkylsulphinyl,
alkylsulphonyl, phosphoric acid, amino, alkylamido and halogen groups; esters
of
derivatized phosphoric acids; (acyloxy)methyl or acyloxy(ethyl)ethers (R a-(O-
CH2-O-
CO-R b)n or R a-(O-CH(CH3)-O-CO-R b)n); (alkoxycarbonyloxy)methyl and
(alkoxycarbonyloxy)ethyl ethers (R a-(O-CH2-O-CO-O-R b)n); and O-glycosides,
wherein
R a is a residue of a compound of the invention, R b and R c are aliphatic
radicals (C1-C10)
and n=1-3.
34. A use of a prophylactically effective amount of a compound as claimed in
claim
1, or a prodrug of said compound for preventing pneumovirus infection in a
host
susceptible to said infection.
35. The use as claimed in claim 34, wherein a precursor of said compound is in
the
form of a prodrug.

39
36. A use of an effective amount of a compound as claimed in claim 1 for
treating
cells in culture that are susceptible to infection by, or infected or
contaminated with a
pneumovirus.
37. A use of an effective amount of a compound as claimed in claim 1 for
treating
biological materials that are susceptible to infection by, or infected or
contaminated
with a pneumovirus.
38. A method for preparing a compound as claimed in claim 1, said method
comprising causing a 3-halogen substituted-4-alkoxy-substituted benzaldehyde,
in
which the aldehyde moiety is protected with a protecting group, to undergo
reaction
with an alkylated alkali metal to effect a halogen-alkali metal exchange;
adding to the
reaction mixture an alkyl ester of an R-substituted benzoic acid under
conditions
yielding a dialkoxy-R-substituted triphenylcarbinol derivative including said
protecting
group; deprotecting and reducing said dialkoxy-R-substituted triphenylcarbinol
derivative to restore said aldehyde functional groups and convert the
triphenyl carbinol
moiety to a triphenylmethane moiety; dealkylating any alkoxy substituents to
hydroxy
substituents; and reacting said aldehyde functional groups with an amine-
substituted
heterocyclic reactant to produce the desired product, said R substituent being
selected
from the group consisting of hydrogen, alkoxy, alkoxyalkyl, hydroxy, halogen,
perfluoroalkyl, thio, nitro, carboxy, carboxyalkyl, carbalkoxy,
carbalkoxyalkyl,
carboxamide, carboxamidoalkyl, alkyl, cycloalkyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, sulfonamide, amidino, cyano, amino, amido, alkylamino,
dialkylamino,
alkylaminoalkyl, and alkoxy monosubstituted with a substituent selected from
the group
consisting of carboxy, amino, alkylamino and dialkylamino, wherein all alkyl
and
alkoxy radicals comprise 1 to 6 carbon atoms.
39. A method as claimed in claim 38, wherein said R-substituted benzoic acid
is
para-substituted.
40. A method for preparing a compound as claimed in claim 1, said method
comprising reacting a 4,4'-dihydroxy-3,3'-(4-R-substituted
phenyl)methylenebisbenzaldehyde, in which the hydroxy groups are etherified,
with the

40
anion of a methyl-substituted heterocyclic reactant to yield a heterocyclic-
hydroxyalkyl
derivative of etherified, R-substituted triphenylmethane as an intermediate
product; and
subjecting the intermediate product to dehydration and deetherification to
produce the
desired product, said R substituent being selected from the group consisting
of
hydrogen, alkoxy, alkoxyalkyl, hydroxy, halogen, perfluoroalkyl, thio, nitro,
carboxy,
carboxyalkyl, carbalkoxy, carbalkoxyalkyl, carboxamide, carboxamidoalkyl,
alkyl,
cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, sulfonamide, amidino,
cyano, amino,
amido, alkylamino, dialkylamino, alkylaminoalkyl, and alkoxy monosubstituted
with a
substituent selected from the group consisting of carboxy, amino, alkylamino
and
dialkylamino, wherein all alkyl and alkoxy radicals comprise 1 to 6 carbon
atoms.
41. A use of an effective amount of a compound as claimed in claim 1 in the
preparation of a medicament for treating cells in culture that are susceptible
to infection
by, or infected or contaminated with a pneumovirus.
42. A compound having the formula:
<IMG>
wherein X is a divalent linking moiety is -CH=CH- or N=CH-, the nitrogen of
said
divalent linking moiety being bound to R3; R is a radical selected from the
group
consisting of hydrogen, hydroxy, alkoxy, alkyl, halogen, nitro and alkoxy
monosubstituted with a substituent selected from the group consisting of
carboxyl,
amino, monoalkylamino, dialkylamino and acetamido; R2 is hydroxy; and R3 is an
unsubstituted heterocyclic radical selected from the group consisting of a 1-
pyrazolyl

41
radical, a 1-triazolyl radical, a 4-triazolyl radical, a 1-tetrazolyl radical,
a 2-tetrazolyl
radical, and a substituted heterocyclic radical selected from the group
consisting of
5-amino-1H-tetrazolyl,
3-amino-4H-2,4-triazolyl,
5-amino-1H-1,2,4-triazolyl,
5-amino-2H-tetrazolyl, and
5-methyl-1H-tetrazolyl; and
wherein all alkyl and alkoxy radicals comprise 1 to 6 carbon atoms; and
the isomeric forms of said compound and the pharmaceutically acceptable salts
of said
compound.
43. A compound as claimed in claim 42, wherein R3 represents a radical
selected
from the group consisting of a 1-tetrazolyl radical, a 5-amino-1H-tetrazolyl
radical and
a 5-methyl-1H-tetrazolyl radical.
44. A compound as claimed in claim 42, wherein X represents -N=CH-.
45. A compound as claimed in claim 42, wherein R represents hydroxy.

Description

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COMPOUNDS, COMPOSITIONS AND METHODS
FOR TREATING OR PREVENTING PNEUMOVIRUS
INFECTION AND ASSOCIATED DISEASES
Theodore J. Nitz
Daniel C. Pevear
FIELD OF THE INVENTION
The present invention relates to compounds, compositions and methods
for preventing and treating viral infections, and the diseases associated
therewith,
particularly those viral infections and associated diseases caused by viruses
of the
Pneumovirinae subfamily of the Paramyxoviridae.
BACKGROUND OF THE INVENTION
The Pneumovirinae subfamily of the Paramyxoviridae family consists of
pneumoviruses that cause significant disease in humans and a number of animal
species including cattle, goats, sheep, mice and in avian species.
Human respiratory syncytial virus (RSV), the prototypic member of the
pneumovirus group, is the major pediatric viral respiratory tract pathogen,
causing pneumonia and bronchiolitis in infants and young children. RSV disease
is seasonal, with outbreaks in the U.S. typically beginning in November and
continuing through April. During these yearly epidemics, approximately 250,000
infants contract RSV pneumonia, and up to 35% are hospitalized. Of those
hospitalized, mortality rates of up to 5% have been reported. Children with
underlying conditions such as prematurity, congenital heart disease,
bronchopulmonary dysplasia and various congenital or acquired
immunodeficiency syndromes are at greatest risk of serious RSV morbidity and
mortality. In adults, RSV usually causes upper respiratory tract
manifestations
but can also cause lower respiratory tract disease, especially in the elderly
and in
immunocompromised persons. Infection in elderly and immunocompromised
persons can be associated with high death rates. Natural infection with RSV
fails
to provide full protective immunity. Consequently, RSV causes repeated
symptomatic infections throughout life.

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The pneumoviruses of animals and avian species are similar to the human
virus antigenically, in polypeptide composition and in disease causation.
Attempts to develop vaccines for RSV are ongoing, but none have yet
been demonstrated to be safe and efficacious. Vaccine development has been
shadowed by adverse reactions exhibited by the initial formalin-inactivated
RSV
vaccine introduced in the late 1960s. Immunized children showed an increased
incidence of RSV lower respiratory tract disease and developed abnormally
severe illnesses, including death.
Chemotherapy with ribavirin [ 1-beta-D-ribofuranosyl-1 H-1,2,4-triazole-3-
carboxamide], an antiviral nucleoside which is the only pharmaceutical
approved
by the U.S. Food and Drug Administration (FDA) for treatment of RSV disease,
is considered only for certain RSV patients (e.g., those at high risk for
severe
complications or who are seriously ill with this infection). However, its
efficacy
and value are controversial. Recent studies have reported a failure to
demonstrate
either clinical or economic benefit to patients of ribavirin treatment.
Moreover,
ribavirin has certain toxic side-effects and, in order to minimize these, must
be
administred by inhalation as an aerosol in an enclosed environment.
A human intravenous immune globulin (IVIG) preparation is licensed for
prophylactic use in certain patients at high-risk for RSV disease.
Administration
of this drug requires intravenous infusion of a large volume over a 2 to 4
hour
period in children who have limited venous access due to prior intensive
therapy,
as well as compromised cardiopulmonary function. Moreover, intravenous
infusion necessitates monthly hospital visits during the RSV season, which in
turn places children at risk of nosocomial infections.
Thus, a need exists for new anti-viral agents and treatments for RSV
infection that overcome the shortcomings of existing pharmaceutical
preparations.

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SUMMARY OF THE INVENTION
In one aspect, the invention provides a compound of the formula:
Het\
X
z
0 0 ~I)
R2 \R2
R,
wherein Het represents an unsubstituted or substituted five to seven membered
heterocyclic ring containing one to three heteroatoms selected from nitrogen,
oxygen or sulfur, said heterocyclic ring substituents being at least one
selected
from those consisting of hydrogen, alkyl, amino, monoalkylamino or.
dialkylamino;
R, represents a radical selected from the group consisting of
hydrogen; halogen; perfluoroalkyl; alkoxyalkyl; amino; alkylamino;
dialkylamino; amido; alkylaminoalkyl; an unsubstituted or substituted,
saturated
or unsaturated, straight- or branched-chain alkyl radical, said alkyl chain
substituent being at least one hydroxy group; carboxy; an unsubstituted or
substituted phenyl radical (C6H5), said phenyl radical substituent being at
least
one selected from the group consisting of hydroxy, alkoxy, alkoxyalkyl,
halogen,
perfluoroalkyl, thio, nitro, carboxy, carboxyalkyl, carbalkoxy,
carbalkoxyalkyl,
carboxamide, carboxamidoalkyl, alkyl, cycloalkyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, sulfonamide, amidino, cyano, amino, amido, alkylamino,
dialkylamino, alkylaminoalkyl, or alkoxy monosubstituted with a substituent
selected from the group consisting of carboxy, amino, alkylamino or
dialkylamino; a cycloalkyl radical; or a heterocyclic radical selected from
the
group consisting of pyridine, thiophene, oxazole, oxadiazole, thiadiazole,

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4
pyrazole, tetrazole, furan, pyrrole, isoxazole, imidazole, triazole and
thiazole,
including all positional isomers of said heterocyclic radicals;
R2 represents a radical selected from the group consisting of
hydrogen, hydroxy, thio, alkoxy, carboxy, carboxyalkyl, amino, alkylamino,
dialkylamino, carboxamide, carboxamidoalkyl, sulfonamide acetamido;
X represents a valence bond or a divalent linking moiety selected
from the group consisting of -N=CH-, -CH=N-, -(CH2) -NH-, -NH-(CH2),; ,
-(CH2)n-, -CH=CH- or -N=N-, n being an integer from 1 to 8;
Z represents a substituent selected from the group consisting of
hydrogen, formyl, hydroxy or -X-Het, wherein X and Het are as previously
defined; the isomeric forms of said compound and the pharmaceutically
acceptable salts of said compound.
Particularly preferred are compounds having the formula:
R3 X"R
3
Rs
Y RZ QI1
R
wherein X is a divalent linking moiety selected from the group of -N=C- or
-CH=CH-; R is a radical selected from the group of hydrogen, hydroxy, alkoxy,
alkyl, halogen, nitro or alkoxy monosubstituted with a substituent selected
from
carboxy, amino, monoalkylamino, dialkylamino or acetamido; R2 is hydroxy; and
R3 is a heterocylic radical selected from the group consisting of 1-pyrazolyl
radicals, 1-triazolyl radicals (including the 1,2,3-; 1,2,4-; or 1,3,4-
isomers

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thereof), 4-triazolyl radicals, I -tetrazolyl radicals or 2-tetrazolyl
radicals
(including the isomers thereof) and the amino- and alkyl-derivatives of such
radicals, including, without limitation, 5-amino-lH-tetrazolyl, 3-amino-4H-
1,2,4
triazolyl, 5-amino-1H-1,2,4 triazolyl, 5-amino-2H-tetrazolyl and 5-methyl-lH-
5 tetrazolyl radicals.
In accordance with another aspect, the present invention provides a
class of novel intermediates that are useful in preparing the anti-viral
agents
described herein. These intermediates have the general formula:
a Q
11 O Q
(II)
Re Rs
0
R7
wherein Q represents a reactive group selected from those consisting of 5,5-
dimethyl-l,3-dioxan and formyl; R5 is a radical selected from those consisting
of
hydrogen and hydroxy; R6 is a radical selected from those consisting of
hydroxy,
alkoxy, aryloxy and aralkoxy and R7 is a radical selected from those
consisting of
hydrogen, hydroxy, alkoxy, alkoxyalkyl, halogen, perfluoroalkyl, thio, nitro,
carboxy, carboxyalkyl, carbalkoxy, carbalkoxyalkyl, carboxamide,
carboxamidoalkyl, alkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl,
sulfonamide, amidino, cyano, amino, amido, alkylamino, dialkylamino,
alkylaminoalkyl, or alkoxy monosubstituted with a substituent selected from
the
group consisting of carboxy, amino, alkylamino or dialkylamino.
The present invention also provides new synthetic methods for
preparation of the compounds described herein. One method comprises causing a
.3-halogen substituted-4-alkoxy-substituted benzaldehyde, in which the
aldehyde
moiety is protected with a protecting group, to undergo reaction with an
alkylated

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alkali metal to effect a halogen-alkali metal exchange; adding to the reaction
mixture an alkyl ester of an R-substituted benzoic acid under conditions
yielding
a dialkoxy-R-substituted triphenylcarbinol derivative including said
protecting
group; deprotecting and reducing the dialkoxy-R-substituted triphenylcarbinol
derivative to restore the aldehyde functional groups and convert the
triphenylcarbinol moiety to a triphenylmethane moiety; dealkylating any alkoxy
substituents to hydroxy substituents; and reacting the aldehyde functional
groups
with an amine-substituted heterocyclic reactant to produce the desired
product.
The R substituents on the benzoic acid ester are selected from the group
consisting of hydrogen, alkoxy, alkoxyalkyl, hydroxy, halogen, perfluoroalkyl,
thio, nitro, carboxy, carboxyalkyl, carbalkoxy, carbalkoxyalkyl, carboxamide,
carboxamidoalkyl, alkyl, cycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl,
sulfonamide, amidino, cyano, amino, amido, alkylamino, dialkylamino,
alkylaminoalkyl, or alkoxy monosubstituted with a substituent selected from
the
group consisting of carboxy, amino, alkylamino or dialkylamino.
Another method for preparing compounds of this invention comprises
reacting a 4,4'-dihydroxy-3,3'-(4-R-substituted
phenyl)methylenebisbenzaldehyde,
in which the hydroxy groups are etherified, with the anion of a methyl-
substituted
heterocyclic reactant to yield a heterocyclic hydroxyalkyl derivative of
etherified,
R-substituted triphenylmethane as an intermediate product; and subjecting the
intermediate product to dehydration and deetherification to produce the
desired
product.
According to still another aspect, the present invention provides
pharmaceutical compositions comprising one or more of the above-described
compounds in combination with a pharmaceutically acceptable carrier medium.
In accordance with a further aspect, the present invention provides a
method for preventing and treating pneumovirus infection and for preventing
and
treating diseases associated with pneumovirus infection in living hosts, by
administering to a living host susceptible to pneumovirus infection a
therapeutically effective amount of a compound of the above structures and/or
the

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7
isomers and pharmaceutically acceptable salts of said compounds, or
pharmaceutical compositions containing same.
DETAILED DESCRIPTION OF THE INVENTION
The compounds of the invention can be conveniently prepared from
known starting materials according to one of the synthetic scheme illustrated
below, wherein R and Het are as previously defined.
SCHEME A
Alk=alkyl; Z=halogen, e.g., Br or I; Rlk-alkyl; R and Het previously defined
0 0 COOM 0 0 0
( n-butylLi
+ IIW OH
Z
OAJk R HCOOH OAIk OAIk
CHO CHO R
HO
*OAik CHO C
~ I
BBr3
OAIk Y OH OH
R
R
Het-NH2
Het Het
N~
OH/ N
H OH
I
R

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Synthetic scheme A involves protection of the aldehyde moiety of a
bromobenzaldehyde followed by halogen-metal exchange and reaction of two
equivalents of the desired aryl lithium species with an ester group to provide
a tri-
aryl methanol. Reduction and regeneration of the aldehyde can be achieved with
formic acid. Liberation of the phenolic groups with boron tribromide (or
pyridine
hydrochloride) and condensation of the aldehyde groups with the appropriate
heterocyclic amine provides the compounds of the invention.
SCHEME B
Alk = alkyl; R and Het previously defined
CHO CHO
Het
+U-42C Het Het
OH HO
OAIk
qok
\ I \
CH3SOZCl OAIk OAIk
R
TEA I
Het Het
Het Het
BBr3 /
~, methylene chloride
OAIk OAIk 11
\ I
OH OH
R

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Synthetic scheme B involves the reaction of a bis aldehyde, prepared as
described in Scheme A, above, with the anion of a methyl heterocycle generated
from n-butyl lithium to give a heterocyclic hydroxyalkyl derivative of an
etherified, R-substituted triphenylmethane, as an intermediate product.
Dehydration of the intermediate with methane sulfonyl chloride provides the
unsaturated compound which is deetherified with boron tribromide to give the
desired compound.
The term "alkyl", as used herein, refers to aliphatic hydrocarbon radicals
of one to six carbon atoms in length. Similarly, the term "alkyl", or any
variation
thereof, used in combination form to name substituents, such as alkoxy (-0-
alkyl), alkylthio (-S-alkyl), alkylamino (-NH-alkyl), alkylsulfonyl (-S(O)2-
alkyl),
carboxyalkyl (-alkyl-COOH), or the like, also refers to aliphatic hydrocarbon
radicals of one to six carbon atoms in length, and preferably of one to four
carbon
atoms in length.
The designation "Het", as used herein, refers to an unsubstituted or
substituted 5-7 membered heterocyclic ring substituent on the compounds of the
invention, which substituent contains 1-3 heteroatoms selected from nitrogen,
oxygen or sulfur, in which the heterocyclic ring substituent is at least one
selected
from the group of hydrogen, alkyl, amino, alkylamino or dialkylamino.
Representative examples of such heterocyclic rings include, without
limitation,
those derived from pyrazole, triazole, tetrazole, oxadiazole, thiadiazole,
imidazole, oxazole, thiazole, isoxazole, pyridine, pyrimidine, triazine,
morpholine, piperidine, piperazine, 1,2,4-diazepine or the like.
The term "amido", as used herein, refers to a radical or substituent of the
formula -NR"C(=O)R', wherein R" and R"' represent hydrogen or alkyl.
The term "carboxamide", as used herein, refers to a radical or substituent
of the formula -C(=O)-NR"R', wherein R" and R"' are as previously defined.
The term "sulfonamide", as used herein, refers to a radical or substituent
of the formula -SO2NR"R' or -NR"SO2R', wherein R" and R"' are as
previously defined.

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The term "carbalkoxy", as used herein, refers to a radical or substituent
-C(=O)-OR", wherein R" is a previously defined.
Preparation of specific embodiments of anti-pneumovirus compounds
within the scope of the invention are exemplified below.
5 In vitro studies have been performed demonstrating the usefulness of
compounds described herein as antiviral agents against pneumoviruses.
Antiviral
activity was measured on the basis of activity against RSV in a cell culture
assay.
All possible isomers of the compounds described herein are within the
scope of the present invention. Representative examples of such isomers
include,
10 without limitation, cis and trans isomers.
The compounds described herein, their isomers and pharmaceutically
acceptable salts exhibit antiviral activity against pneumoviruses and are
within
the scope of the present invention.
The compounds of the invention can form useful salts with inorganic and
organic acids, including, for example, hydrochloric acid, hydrobromic acid,
methanesulfonic acid salts, or the like, as well as with inorganic bases, such
as
sodium or potassium salts.
The pharmaceutically acceptable salts of the compounds of the invention
are prepared following procedures which are familiar to those skilled in the
art.
The antiviral pharmaceutical compositions of the present invention
comprise one or more of the above-described compounds or precursors thereof,
as
the primary active ingredient in combination with a pharmaceutically
acceptable
carrier medium and, optionally one or more supplemental active agents.
The composition may be prepared in various forms for administration,
including tablets, caplets, pills or dragees, or can be filled in suitable
containers,
such as capsules, or, in the case of suspensions, filled into bottles. As used
herein, "pharmaceutically acceptable carrier medium" includes any and all
solvents, diluents, or other liquid vehicle, dispersion or suspension aids,
surface
active agents, isotonic agents, thickening or emulsifying agents,
preservatives,
solid binders, lubricants and the like, as suited to the particular dosage
form
desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin

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(Mack Publishing Co., Easton, PA, 1975) discloses various carriers used in
formulating pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional carrier medium is
incompatible with the antiviral compounds of the invention, such as by
producing
any undesirable biological effect or otherwise interacting in a deleterious
manner
with any other component(s) of the pharmaceutical composition, its use is
contemplated to be within the scope of this invention.
The compounds of the invention, any precursors thereof and their isomers
and pharmaceutically acceptable salts are also useful in treating and
preventing
pneumovirus infections and diseases when used in combination with
supplemental active agents, which may be optionally incorporated into the
pharmaceutical composition of the invention, or otherwise administered during
a
course of therapy. These include, without limitation, interferons, ribavirin,
and
immunomodulators, immunoglobulins, anti-flammatory agents, antibiotics, anti-
virals, anti-infectives, and the like, the combination of which with one or
more
compounds of the invention offers additive or synergistic therapeutic benefit.
In the pharmaceutical compositions of the invention, the active agent may
be present in any therapeutically effective amount, which is typically at
least
0.1 % and generally not more than 90% by weight, based on the total weight of
the composition, including carrier medium and/or supplemental active agent(s),
if
any. Preferably, the proportion of active agent varies between 1-50% by weight
of the composition.
Pharmaceutical organic or inorganic solid or liquid carrier media suitable
for enteral or parenteral administration can be used to make up the
composition.
Gelatine, lactose, starch, magnesium, stearate, talc, vegetable and animal
fats and
oils, gum, polyalkylene glycol, or other known carriers or excipients for
medicaments may all be suitable as carrier media.
Compounds of the invention are useful in treating and preventing
pneumovirus infections (and diseases) in humans, as well as in livestock, and
may be used to treat cattle, swine and sheep, or to treat avian species such
as

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turkeys, or for other animals susceptible to pneumovirus infection. Thus, the
term "patient" as used herein includes, without limitation, all of the
foregoing.
Compounds described herein are also useful in preventing or resolving
pneumoviral infections in cell cultures, tissue cultures and organ cultures,
as well
as other in vitro applications. For example, inclusion of compounds of the
invention as a supplement in cell or tissue culture growth media and cell or
tissue
culture components will prevent pneumoviral infections of cultures not
previously infected with pneumoviruses. Compounds described above may also
be used to eliminate pneumoviruses from cultures or other materials infected
or
contaminated with pneumoviruses, after a suitable treatment period, under any
number of treatment conditions as determined by the skilled artisan.
The compounds of the invention may be administered using any amount
and any route of administration effective for attenuating infectivity of the
pneumovirus. Thus, the expression "amount effective to attenuate infectivity
of
pneumovirus", as used herein, refers to a nontoxic but sufficient amount of
the
antiviral agent to provide the desired treatment of viral infection. The exact
amount required will vary from subject to subject, depending on the species,
age,
and general condition of the subject, the severity of the infection, the
particular
antiviral agent and its mode of administration, and the like.
The anti- pneumovirus compounds are preferably formulated in dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form
as used herein refers to a physically discrete unit of antiviral agent
appropriate for
the patient to be treated. Each dosage should contain the quantity of active
material calculated to produce the desired therapeutic effect either as such,
or in
association with the selected pharmaceutical carrier medium. Typically, the
antiviral compounds of the invention will be administered in dosage units
containing from about 0.1 g to about 50 mg of the antiviral agent, with a
range
of about 0.001 mg to about 25 mg being preferred.
The compounds of the invention, including their isomers and
pharmaceutically acceptable salts, may be administered as such, or in the form
of
a precursor from which the active agent can be derived, such as a prodrug. A

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prodrug is a derivative of a compound described herein, the pharmacologic
action
of which results from the conversion by chemical or metabolic processes in
vivo.
Prodrugs of the compounds of the invention may include, but are not limited to
mono-, di- or tri-esters of simple or functionalized aliphatic carboxylic
acids;
esters of carbamic acids (Ra-(O-CO-NRb&),J; esters of amino acids (Ra-(O-CO-
CH(NH2)Rb)õ ); esters of unsubstituted or substituted aromatic acids (Ra-(O-CO-
aryl)õ), wherein the aryl ring may be substituted with hydroxy, carboxy, lower
alkyl, alkylthio, alkylsulphinyl, alkylsulphonyl, phosphoric acid, amino,
alkylamido and halogen groups; esters of derivatized phosphoric acids;
(acyloxy)methyl or acyloxy(ethyl)ethers (Ra-(O-CH2-O-CO-Rb)n or Ra-(O-
CH(CH3)-O-CO-Rb)õ); (alkoxycarbonyloxy)methyl or (alkoxycarbonyloxy)ethyl
ethers (Ra-(O-CH2-O-CO-O-Rb)õ ); and O-glycosides, wherein Ra is a residue of
a
compound of the invention, Rb and & are aliphatic radicals (C1-C10) and n=1-3.
Such prodrugs may be prepared according to procedures well known in the field
of medicinal chemistry and pharmaceutical formulation science and are within
the
scope of the present invention.
The compounds of the invention may be administered orally,
parenterally, such as by intramuscular injection, intraperitoneal injection,
intravenous infusion or the like, or by inhalation, such as by aerosol, in the
form
of a solution or a dry powder, or the like, or by intubation, depending on the
nature and severity of the infection being treated. The compounds of the
invention may be administered orally, parenterally, or by inhalation or
intubation
at dosage levels of about 101 mg to about 1000 mg/kg, one or more times a day,
to obtain the desired therapeutic effect.
The compounds of the invention will typically be administered from I to
4 times a day so as to deliver the above-mentioned daily dosage. However, the
exact regimen for administration of the compounds and compositions described
herein will necessarily be dependent on the needs of the individual host being
treated, the type of treatment administered and the judgment of the attending
physician, veterinarian or medical specialist.

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In view of the inhibitory effect on pneumovirus replication in cell culture
produced by the compounds used in the method of the invention, it is
anticipated
that these compounds will be useful not only for therapeutic treatment of
pneumovirus infection, but for pneumovirus prophylaxis, as well. The dosages
will be essentially the same, whether for treatment or prophylaxis of
pneumovirus
infection.
The following examples are provided to describe the invention in further
detail. These examples, which set forth the preferred mode presently
contemplated for carrying out the invention, are intended to illustrate and
not to
limit the invention.
Examples 1-14 illustrate the chemical synthesis of representative
compounds of the invention.
EXAMPLE 1
Preparation of 5,5'-Bis[1-(((5-amino-lH-tetrazolyl)imino)methyl)]
2,2',4"-methylidynetrisphenol
a. 2-(3-Bromo-4-methoxyphenyl)-5.5-dimethvl-l.3-dioxane. A solution
of 3-bromo-4-methoxybenzaldehyde (74.65 g, 0.347 mol), neopentyl glycol
43.35 g, 0.416 mol), pyridinium p-toluenesulfonate (0.87 g, 0.035 mol), and
benzene (1.8 L) was refluxed with azeotropic removal of water for 6 hours. The
cooled reaction mixture was diluted with water. The aqueous phase was
extracted with ethyl acetate. The combined organic phases were washed with
brine, dried (Na2SO4), charcoaled, filtered through a short column of
FlorisilTM,
and concentrated in vacuo. There was obtained 102.8 g (98%) of ketal as a
peach
colored solid.
b. 5.5'-Bis(5.5 .5 dimethyl-1.3-dioxan-2-yl -2.2'.4"
trimethoxytriphenylmethanol. A solution of the dioxane derivative obtained in
step a., above (150.6 g, 0.500 mol) in anhydrous THE (2.0 L) was cooled to

CA 02319465 2009-02-23
-78 C. n-Butyllithium (50 mL of 10.0 M in hexanes) was added via syringe
pump at about 1.0 mL/min. After 15 minutes, a solution of methyl 4-
methoxybenzoate (33.24 g, 0.200 mot) in THE (350 mL) was added dropwise.
The mixture was stirred at -78-C for 15 minutes, at O C for two and one-half
5 hours, and quenched with 10% NH4C1(1 L). t-Butyl methyl ether (1 L) was
added and the layers separated. The aqueous phase was extracted with t-butyl
methyl ether (two times, 1 L). The combined organic phases were washed with
brine, dried (MgSO4), filtered through FlorisilTM and concentrated in vacuo.
The yellow oil obtained was dissolved in methanol (1L), seeded with a crystal
of
10 pure product, and chilled to 0 C. The resulting white solid was isolated,
washed
with cold methanol, and dried in vacuo to provide 102.8 g (88.8%) of the
desired
product.
c. 4 4'dimethoxy -3 3'-(4-methoxyphenyl)methylenebisbenzaldehyde.
15 The triarylcarbinol derivative (15.9 g, 0.0275 mot), produced as described
in step
b., above, was dissolved into formic acid (137 mL). The intense burgundy
colored solution was heated at 100 C for 13 hours, cooled to room temperature,
and concentrated in vacuo. The white solid obtained was suspended in water,
neutralized with saturated NaHCO3, filtered, washed with water and hexane
(removes neopentyl glycol bisformate) and dried in vacuo to provide 10.8 g
(-100 %) of nearly pure product as a faintly bluish powder.
d. 4 4'-dihydroxY 3 3'-(4-hydroxyphenyl)methylenebisbenzaldehyde.
Boron tribromide solution (80 mL, IM in methylene chloride) was added
dropwise to a solution of the trimethyl ether (5.23 g, 0.0 133 mot), resulting
from
the above-described deketalization, in dry methylene chloride. A mild exotherm
to -35 C was observed during the addition. After 18 hr at room temperature,
the
reaction mixture was poured onto crushed ice (500 g) and stirred for 1 hour at
room temperature. The resulting gray solid was extracted into ethyl acetate.
The
ethyl acetate solution was extracted three times with 10% Na2CO3. The
TM
combined aqueous extracts were treated with charcoal, filtered through Celite

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and carefully acidified with 6N HC1. The off-white precipitate was isolated,
washed with water, dried in vacuo, dissolved in THE (45 mL), diluted with t-
butyl methyl ether (45 mL), and filtered through FlorisilTM^ with THF/t-BME
1:1.
Concentration of the filtrate provided 3.85 g (85%) of pure dialdehyde which
contained a small amount of residual solvents.
e. Condensation with 1.5-Diaminotetrazole. A solution of the dialdehyde
obtained from the above-described demethylation reaction (3.00 g, 8.61 mmol),
dry N,N-dimethylformamide (120 mL), 1,5-diaminotetrazole (2.58 g, 25.8
mmol), and p-toluenesulfonic acid ( 0.33 g, 1.7 mmol) was stirred at 600C for
6
hours. The reaction mixture was cooled to room temperature and diluted with
water (400 mL). The resulting off-white precipitate was isolated, washed with
water, dissolved into tetrahydrofuran (150 mL), treated with charcoal,
filtered,
and concentrated in vacuo to provide 4.46 g of the title compound as a light
yellow powder.
EXAMPLE 2
Preparation of 5,5'-Bis[1-(((5-amino-1H-
tetrazolyl)imino)methyl)]-4"-methoxyphenyl-2,2'-benzylidenebisphenol
a. 3-Bromo-4-hydroxvbenzaldehyde. A mixture of 25.1 g (117
mmole) of 3-bromo-4-methoxybenzaldehyde and 54.47 g (471 mmole) of
pyridine hydrochloride was heated under nitrogen to 100 C for 2 hours. After
cooling to room temperature, the mixture was diluted with 1 liter of water and
500 ml of ethyl acetate. The organic layer was collected and the aqueous layer
was extracted with three 500 ml portions of ethyl acetate and the combined
organic layers were washed with water and dried. Removal of the solvent
provided 22 g of an orange solid.
b. 4-Phenylmethoxy-3-bromobenzaldehvde. To a solution of 22 g
(109 mmole) of 3-bromo-4-hydroxybenzaldehyde in 600 ml of acetone was added
at room temperature under nitrogen 24.3 g (161 mmole) of milled potassium .
carbonate and 17.0 ml (143 mmole) of benzyl bromide and the mixture heated to

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reflux with stirring for 2 hours. The reaction was quenched with water and the
volume was reduced to half in vacuo, and the mixture extracted three times
with
200 ml portions of ethyl acetate. The combined organic layers were
concentrated
to dryness. The residual solid was redissolved in 500 ml of acetone and passed
through a celite column. Water was added to the acetone solution. A yellow
solid
separated which was collected and dried to give 24.5 g of material.
c. 2-(3 -Bromo-4-P henvlmethoxyphenyl)-5.5-dimethyl-1.3 -dioxane.
A solution of the 24.5 g (84.2 mmole) of the benzaldehyde from the immediately
preceding step, 12.3 g (112 mmole) of neopentyl alcohol and 220 mg of p-
toluenesulfonic acid in 350 ml of benzene was heated to reflux for 5 hours. A
Dean Stark trap was used to collect the water which was generated during the
reaction. The reaction was quenched with 1 ml of triethylamine and stirred for
12
hours at room temperature. The mixture was poured into 300 ml of water and the
organic layer collected. The aqueous layer was extracted with three 100 ml
portions of ethyl acetate. The combined organic layers were dried and the
solvent
removed to give a yellow solid which was purified by recrystallization from
ethanol to give 19.2 g of an orange solid.
d. 5-5'-Bis(5.5-dimethyl-1.3-dio xan-2-yi)-4"-methoxy-2.2'-
diphenylmethoxytriphenylmethanol. To a solution of 1.9 g (5.04 mmole) of the
material obtained from the immediately preceding step in 15 ml of distilled
tetrahydrofuran, cooled to -100 C was added dropwise under nitrogen, 2.3 ml of
a 2.5 M solution of n-butyllithium in hexane. After the addition was
complete,a
solution of ethyl 4-methoxybenzoate, (2.5 mmole), was added and the solution
was stirred for 1.5 hours at -780C and stirred for 12 additional hours at OC
and
then quenched with water. After warming to room temperature, the volume was
reduced to half by concentration in vacuo and then the mixture was diluted
with
25 ml of water and 25 ml of ethyl acetate. The layers were separated and the
aqueous layer extracted with three 25 ml portions of ethyl acetate. The
combined
organic layers were dried and concentrated to dryness. The residual solid was
purified by column chromatography on silica by eluting with 80:20,
hexane/ethyl
acetate to give 60 mg of material.

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e. 4.4'-DihvdroU 3.3'(4-methoxyphenyl, methvlenebisbenzaldehvde.
The intermediate from the immediately preceding step, 50.4 mg (0.069 mmole)
was dissolved in 3 ml of formic acid and the solution heated for four hours at
100oC, cooled to room temperature, and then water, 3 ml, was added and a white
suspension appeared. The mixture was stirred overnight at room temperature.
The mixture was partioned between water and ethyl acetate and after drying and
removal of the ethyl acetate, the residual solid was purified by column
chromatography on silica eluting with 40:60 hexane/ethyl acetate to give a
white
solid.
f. Condensation with 1.5-Diaminotetrazole. A solution of the
dialdehyde obtained from the reaction described immediately above, 11.6 mg
(0.032 mmole), dry N,N-dimethylformanmide, 9.2 mg (0.0999 mmole) of 1,5-
diaminotetrazole and 0.25 ml of a 0.025 M solution ofp-toluenesulfonic acid
was
heated to 60 C for 17 hours. The solvent was removed in vacuo and the residue
was triturated with water to give a beige solid which was collected and dried
to
give 10 mg of the titled compound.
Furthermore, compounds of formula II, above, may be made with various
heterocyclic radicals (R3) by replacing the 1,5-diaminotetrazole with other
heterocyclic reactants, as described in Examples 3-6, below.
EXAMPLE 3
Preparation of 5,5'-Bis[l-(((5-amino-1H-1,2,4
triazolyl)imino)methyl))-2,2',4"-methylidyne trisphenol
The title compound was synthesized essentially according to the basic
procedure described in Example 1; however, 2,3-diamino-1,2,4 triazolyl was
used
instead of 1,5-diaminotetrazole.

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EXAMPLE 4
Preparation of 5,5'-Bis[4-(((3-amino-4H-1,2,4-
triazolyl)imino)methyl)]-2,2',4"-methylidyne trisphenol
The title compound was prepared essentially according to the basic
procedure described in Example 1, above; however, 3,4-diamino-1,2,4-triazole
was used instead of 1,5-diaminotetrazole.
EXAMPLE 5
Preparation of 5,5'-Bis[2-(((5-amino-2H-
tetrazolyl)imino)methyl)]-2,2',4"-methylidynetrisphenol
The title compound was prepared essentially according to the synthetic
procedure set out in Example 1; however, the 1,5-diaminotetrazole in Example 1
was replaced with 2,5-diaminotetrazole.
EXAMPLE 6
Preparation of 5,5'-Bis[1-(((5-methyl-lH-
tetrazolyl)imino)methyl)]-2,2',4"-methylidynetrisphenol
The title compound was synthesized essentially according to the basic
procedure described in Example 1; however, 1-amino-5-methyltetrazole was used
in place of 1,5-diaminotetrazole.
As described in the following example, compounds of formula I, above, in
which the R, radical is other than hydroxyphenyl may be prepared by
substitution
of a suitable ester for the methyl 4-methoxybenzoate in step b of the reaction
sequence of Example 1, above.
EXAMPLE 7
Preparation of 5,5'-Bis[l-(((5-amino-lH-tetrazolyl)imino)
methyl)]-2,2'-benzylidenebisphenol
The title compound was synthesized essentially according to the basic
procedure described above in Example 1; however, 4,4'-dihydroxy-3,3'-

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benzylidenebisbenzaldehyde was substituted for 4,4'-dihydroxy-3,3'-(4-
hydroxyphenyl)methylenebis-benzaldehyde. This intermediate was obtained by
using methylbenzoate in place of methyl 4-methoxybenzoate in step b. of
Example 1, above.
5 Other examples of substituted esters which may be used to prepare
additional compounds having the structure of formula I, above, include alkoxy,
halo, perfluoroalkyl, alkoxycarbonyl, alkylaminocarbonyl, alkylthio,
alkylsulfmyl, alkylsulfonyl, alkyl, alkoxyalkyl benzoates and esters of
pyridine,
thiophene, imidazole, furan, pyrole, oxazole, triazole, oxadiazole,
thiadiazole,
10 pyrazole, tetrazole, isoxazole, thiazole carboxylates.
EXAMPLE 8
Preparation of 5,5'-Bis[1-(((5-amino-1H-tetrazolyl)imino)
methyl)]2,2'-methylidenebisphenol
15 a. 4,4'-Dihydroxy-3.3'-methylenebisbenzaldehyde. To a solution of 5.0 g
(21.9 mmole) of 2,2'-methylenebis(4-methylphenol) in 100 ml of methanol was
added dropwise at -78 C under nitrogen with stirring, 19.89 g (87.6 mmole) of
2,3-dichloro-5,6-dicyanobenzoquinone in 100 ml of methanol. After 2 hours, the
solution was diluted with water and stirred for 30 minutes. The mixture was
20 extracted with two 100 ml portions of ethyl acetate. The combined organic
layers
were washed with saturated sodium chloride solution and dried over magnesium
sulfate. The mixture was filtered and the solution concentrated to dryness to
give
a brown solid. The material was dissolved in ethyl acetate and passed through
a
column containing FlorisilTM which was washed with ethyl acetate. The
fractions
were collected and the solvent removed to give 22 g of a yellow solid.
b. Condensation with 1 5-Diaminotetrazole. The title compound was
obtained according to step e. in Example 1, above.

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EXAMPLE 9
Preparation of 5,5'-Bis[1-(2-(5-(1-methyl-1H-tetrazolyl))
ethenyl)]-2,2',4"-methylidynetrisphenol
a. a a'-Bis[5-(1-metiyl-lH-tetrazolyll methyl]-4 4'-dimethoxy-3 3'
(4-methoxyphenyl) methvlenebisbenzenemethanol.
To a solution of 294 g (3.0 mmol) of 1,5-dimethyl-lH-tetrazole in
freshly distilled tetrahydrofuran chilled to -78 C was added dropwise over a 5
minute period 1.8 ml of a 1.7M solution of t-butyl lithium in pentane. The
solution was stirred for 50 minutes and to the yellow suspension was added 390
mg (1.0 mmoles) of 4, 4'-dimethoxy-3, 3'-(4 methoxyphenyl)
methylenebisbenzaldehyde in 15 ml of dry tetrahydrofuran over a 5 minute
period. To the reaction mixture was added 10 ml of a 10% ammonium chloride
solution. The mixture was warmed to room temperature and partitioned between
water (25 ml) and ethyl acetate (25 ml). The aqueous layer was collected and
extracted with 25 ml of ethyl acetate. The combined organic layers were washed
with a saturated sodium chloride solution and dried. Removal of the solvent
gave
346 mg of a pale yellow solid.
b. 5.5'-Bis[1-(2-(5-(1-methyl-lH-tetrazolyll ethenyl)1-2 2' 4"-
trimethoxytriphen lymethane.
A solution of 346 mg (.592 mmoles) of the material from step a,
above, 0.28 ml of triethylamine, 12 mg (0.1 mmol) of 4-dimethylaminopyridine
(DMAP) in 5 ml of dry methylene chloride was chilled in an ice bath. To the
solution was added 0.15 ml (2.0 mmole) of methanesulfonyl chloride and the
solution stirred for 2 hours in an ice bath and then allowed to slowly warm to
room temperature and left for 16 hours. To the solution was added 10 ml of
ethyl
acetate and the solution washed with two 10 ml portions of water, I N
hydrochloric acid and a saturated solution of sodium chloride, and then dried
over magnesium sulfate. Removal of the solvent gave 348 mg of a solid which
was dissolved in 5 ml of tetrahydrofuran. To this solution was added 90 mg of
1,8-diazobicyclo[5.4.0]-undec-7-ene. An oily material appeared and the mixture
stirred for 16 hours. The mixture was diluted with 15 ml of ethyl acetate and

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extracted with 15 ml of water. The organic layer was collected, washed with
water and dried. Removal of the solvent resulted in 348 g of the desired
product.
c. Demethvlation with boron tribromide
To a suspension of 110 mg (0.2 mmole) of the material prepared
in step b., above, dissolved in 1 ml of dry methylene chloride, cooled to 0 C
was
added 1.2 ml of 1.0 M boron tribromide in methylene chloride. The mixture was
stirred for 2 hours and the yellow solid which formed was collected, washed
with
water and suspended in 15 ml of water. To the suspension was added 5% sodium
hydroxide until a solution was obtained. The solution was treated with
charcoal
and the suspension filtered through Celite 503 and the solution acidified with
1 N
hydrochloric acid. The resulting white solid was collected by filtration,
washed
with water and dried to give 73 mg of product.
EXAMPLE 10
Preparation of 5,5'-Bis[((1-(5-methyl-1H-tetrazolyl)imino)
methyl)]-(4-propylphenyl)-2,2'-benzylidinebisphenol
a. 5 5'-Bis(5 5-dimet yl-1 3-dioxan-2-yl)-2 2'-dimethoxy-4"-
propyltriphenvlmethanol.
To a solution of 15.0 g (49.8 mmole) of 2-(3-bromo-4-
methoxyphenyl)-5,5-dimethyl-1,3-dioxane in 120 ml of dry THE at -78 C was
added dropwise 24 ml of 2.5 M n-butyllithium. After the addition was complete,
3.82 g (59.7 mmole) of ethyl 4-propyl benzoate in 30 ml of THE was added
dropwise and after the addition was complete, the mixture was allowed to warm
to
room temperature and stirred for 12 hours. One hundred ml of saturated
ammonium chloride was added followed by 100 ml of i-butyl methyl ether. The
organic layer was separated and washed with water, dried and the solvent
removed
to give 6.41 g of crude material. This was passed through silica and eluted
with
50% ethyl acetate-50% hexane, and the solvents removed to give 4.92 g of
product.
b. 4,4'-Dimethoxy-3.3'-(4-propylnhenvl)methvlene bisbenzaldehyde

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A solution of 4.3 g (7.28 mmole) of the material prepared in step
a., above, in 30 ml of formic acid was heated to reflux for 4 hours. After
cooling,
water (100 ml) was added and the mixture extracted with two 100 ml portions of
methyl t-butylether. The combined organic extracts were washed with water,
dried
and the solvent removed. The residual solid was passed through silica gel and
eluted with 50% ethyl acetate-50% hexane to give , after removal of the
solvent,
1.98 g of the desired solid.
c. 4.4'-Di ydroxy-3.3'-(4-propylphenyl methiylenebisbenzaldehyde
To a solution of 1.1 g (2.73 mmole) of the methyl ether from step b.,
above, in 15 ml of methylene chloride was added at room temperature 10.9 ml
(10.9 mmole) of boron tribromide over a 5 minute period and then stirred at
room
temperature for 12 hours. The reaction mixture was poured into ice water and
the
organic layer separated and dried. Removal of the solvent gave 750 mg of a
greenish-brown solid.
d. Condensation with 1-amino-5-methvltetrazole
This reaction was run in the same fashion as previously described.
EXAMPLE 11
Preparation of 5,5'-Bis[((1-(5-methyl-lH-tetrazolyl)imino)
methyl)]-(4'-propyloxyphenyl)-2,2'-benzylidenebisphenol-
a. 4.4'-Diallyloxy- '-(4-propyloxvnhenvll-
methvlenebisbenzaldehyde.
To a solution of 5.0 g (11.8 mmole) of 4,4'-Diallyloxy-3,3'(4-
hydroxyphenyl) methylenebisbenzaldehyde and 3.26 g (23.6 mmole) of potassium
carbonate in 40 ml of N-methylpyrrolidone was added 2.3 ml (23.6 mmole) of n-
propyliodide. The mixture was warmed to 90 C for 3 hours after which time an
additional 5 ml of n-propyliodide was added. The reaction was heated for an
additional 12 hours after which time it was diluted with 100 ml of water and
extracted 3 times with 50 ml. of t-butyl methyl ether. The combined organic
extracts were washed with water and dried to give 6.86 g of a crude product
which

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24
was passed through a silica gel column and eluted with 50% ethyl acetate and
50% hexane. After removal of the solvent, 4.43 g of yellow solid was obtained.
b. 4.4'-Dihvdroxy-3,3'-(4-propyloxyphenyl)-
methylenebisbenzaldehyde.
Ruthenium trichloride, 230 mg (.89 mmole) was added to a
refluxing solution of 4.12 g (8.95 mmole) of the diallyl protected ether,
prepared
in step a., above, in 120 ml of ethanol. After 90 minutes, an additional 100
mg of
ruthenium trichloride was added. After 6 hours, the solvent was removed and
the
residue dissolved in ethyl acetate and passed through silica gel and eluted
with
60% ethyl acetate-40% hexane. After removal of the solvent, 2.95 g of a brown
solid was obtained which was redissolved and again passed through a silica gel
column to give 1.73 g of product.
C. Condensation with 1-amino-5-methyltetrazole.
This reaction was run as previously described in example 6.
EXAMPLE 12
Preparation of 5,5'-Bis(((1-(5-methyl-lH-tetrazolyl)imino)
methyl)]-(4-fluorophenyl)-2,2'-benzylidenebisphenol -
a. 5.5-Bis 5.5'-dimethvl-1.3-dioxan-3-yl)-2.2'-dimethoxy-4"-
fluorotriphenvlnhenylmethanol.
The reaction was run as previously described using 2-(3-Bromo-4-
methoxyphenyl)-5,5'-dimethyl-1,3-dioxane and methyl 4-fluorobenzoate.
b. 4.4'-Dihydroxy-3.3'-(4-fluorophenyl)methvlenebisbenzaldehyde.
This compound was prepared as previously described from the compound
prepared in step a., above, and formic acid, followed by boron tribromide
demethylation
c. Condensation with 1-amino-5-meth ltazole.
This reaction was run as previously described.

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EXAMPLE 13
5,5'-Bis [ 1-(2-(4-methylthiazolyl)ethenyl))-2,2',4"-methylidynetrisphenol
a. 4.4'-Dibenzvloxy-3.3'-(4-benzvloxvphenyl)
methylenebisbenzaldehyde.
5 To a solution of 2.0 g (5.74 mmole) of 4,4'-dihydroxy-3,3'-(4-
hydroxyphenyl) methylenebisbenzaldehyde in 57 ml of DMF was added 7.95 g
(5.76 mmole) of potassium carbonate and 4.09 g (23.9 mmole) of benzylbromide.
The mixture was stirred for 12 hours at room temperature and then heated to
reflux for 2 hours. The reaction mixture was diluted with water (100 ml) and
then
10 extracted with ethyl acetate. The organic extracts were combined, dried and
the
solvent removed. The residue was purified by HPLC by eluting with 60-40 ethyl
acetate-hexane to give 3.25 g of product
b. a.a'-Bis[2-(4-methvlthiazolvl)methyll--4.4'-dibenzyloxv-3.3'-(4-
benzvloxyphenyl)methvlenebisbenzenemethanol.
15 A solution of 2.4 ml (21.3 mmole) of 2,4-dimethylthiazole in 48 ml
of dry THE was cooled to -78 C and to the solution was added dropwise 11.64 ml
of a 2.5 M solution of n-butyllithium in hexanes. After stirring for 1 hour,
6.0 g
(9.7 mmole) of the aldehyde prepared in step a., above, in 20 ml of THE was
added dropwise. The reaction mixture was stirred for an additional 2 hours and
20 then allowed to come to room temperature and stirred for an additional 12
hours.
The mixture was diluted with 60 ml of saturated ammonium chloride solution and
the THE was removed by concentration of the mixture in vacuuo. The residue was
extracted 3 times with ethyl acetate and the combined organic layers were
dried
and concentrated to dryness to give 8.72 g of crude material which was
purified
25 by HPLC, eluting with 70-30 hexane-ethyl acetate providing 2.49 g of
product.
c. 5.5'-Bis[1-L2_(24-methylthiazoly[))ethenyl)12.2'.4"-tribenzyloxv
triphenylmethane.
A solution of 500 mg (.592 mmole) of the alcohol from step b.,
above, in 16 ml of acetic anhydride was heated to reflux for 5 hours. After
cooling, the solution was diluted with water and extracted three times with
ethyl
acetate. The combined extracts were washed with water , dried and the solvent

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removed. The crude product was purified by HPLC, eluting with 70-30 hexane-
ethyl acetate to give 390 mg of product.
d.. 5.5'-Bis[ l -(2-(4-methvlthiazolyl)ethenvl)1-2.2'.4"-
methvlidinetrisp enol.
A solution of 570 mg (.705 mmole) of the material prepared in step
c., above, in 46 ml of formic acid was heated to reflux for 12 hours. The
cooled
solution was diluted with water and extracted three times with ethyl acetate.
The
combined organic extracts were washed with water, dried and the solvent
evaporated to dryness. The crude material was purified by recrystallization
from
methylene chloride.
EXAMPLE 14
Preparation of 5,5'-Bis[1-(2-(5-(3-methylisoxazolyl))ethenyl)]-
phenyl-2,2'-benzylidenebisphenol
a. a.a'-Bis[5-(3-methylisoxazolylmethvll-4.4'-dimethoxy-3.3'-
(phenyl)methylenebisbenzaldehde.
A solution of 2.9 ml (3.0 mmole) of 3,5-dimethylisoxazole in 150
ml of dry THE was cooled to -80 C. To this solution was added 12 ml of 2.5 M n-
butyllithium in hexanes. After the addition was complete, 3.6 g (1.0 mmole) of
4,4'-dimethoxy-3,3'-(phenyl)methylenebisbenzaldehyde was added over 1 hour
After the addition was complete, a saturated ammonium chloride solution was
added. The mixture was partitioned between water and methyl t-butylether. The
organic layer was collected, dried and the solvent removed to give 5.59 g of
product.
b. This reaction was performed in the same general manner as
described in Example 9, step b.
c. Demethylation.
A mixture of 260 mg (0.5 mmole) of the compound obtained in
step b., above, and 3.5 g of pyridine hydrochloride were heated to 220 C for 6
hours. The mixture was diluted with water and a solid separated. The solid was
dissolved in ethyl acetate and the solution extracted with water, treated with

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27
charcoal, filtered and the solvent removed to give , after drying, 128 mg of
the
desired product.
Other compounds of the invention having anti-pneumovirus
activity may be prepared following the various synthetic routes described
hereinabove. Additional examples include, without limitation, 5,5'-Bis[2-(2-(5-
methyl-2H-tetrazolyl)ethyl)]-2,2',4"-methylidynetrisphenol; 5,5'-bis[((1-(5-
methyl-1 H-tetrazolyl)amino)methyl)]-2,2',4"-methylidynetrisphenol;
5-[((1-(5-methyl-1 H-tetrazolyl)imino)methyl)]-2,2',4"-methylidynetrisphenol;
5-[((1-(5-methyl-1 H-tetrazolyl)imino)methyl)]-2,4',4"-methylidynetrisphenol;
3-[5-[((1-(5-methyl-1 H-tetrazolyl)imino)methyl)]-2,4'-
dihydroxydiphenylmethylene]-4-hydroxybenzaldehyde; 5,5'-bis[((1-(5-methyl-
1 H-tetrazolyl)imino)methyl)]-[4-((2-diethylamino)ethoxy)phenyl]-2,2'-
benzylidenebisphenol; 4-[5,5'-bis[((1-(5-methyl-IH-tetrazolyl)imino)methyl)]-
2,2'-dihydroxydiphenylmethylene]phenoxyacetic acid; 5,5'-bis[((1-(5-methyl-iH-
tetrazolyl) imino)methyl)]-(4-pyridinyl)-2,2'-benzylidenebisophenol; 5,5'-
bis[((1-
(5-methyl-1 H-tetrazolyl)imino)methyl)]-(4-nitrophenyl)-2,2'-
benzylidenebisphenol; 5,5'-bis[((1-(5-methyl-IH-tetrazolyl)imino)methyl)]-(4-
nitrophenyl)-2,2'-benzylidenenbisphenol; 5,5'-bis[l-(2-(2-(1-
methylimidazolyl))ethenyl)]-2,2',4"-methylidynetrisphenol; and 5,5'-Bis [ 1-
(((5-
methyl-1 H-tetrazolyl) imino) methyl)]phenyl-2,2'-benzylidenebisphenol.
Illustrative examples of the preparation of prodrugs in accordance with the
present invention are provided below.
EXAMPLE 15
Preparation of Prodrugs
a) A solution of 255 mg (.5 mmoles) of the compound
prepared as described in Example 1, above, in 2.5 ml of anhydrous pyridine and
.243 ml of acetic anhydride was left at room temperature overnight. The
solvent
was removed and to the residue was added 5 ml of water and the mixture was

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made slightly acidic by the addition of acetic acid. The solid was collected,
washed with water followed by hexane and then dried to give 240 mg of the
desired triacetate prodrug.
b) Following essentially the same procedure, 220 mg of the
triacetate derivative was obtained from 200 mg of the compound prepared as
described in Example 6, above.
Example 16 illustrates the effectiveness of the compounds used in the
method of the invention in inhibiting the viral replication of RSV in cell
culture.
EXAMPLE 16
Cell Culture Assay for Inhibition
of Pneumovirus Replication
The replication of many viruses may be quantitatively assessed in the
laboratory in various cell or tissue culture systems. Such in vitro culture
methodologies are available and useable by those skilled in the art for the
propagation and quantitative measurement of the replication of pneumoviruses.
The following procedure was used for the in vitro quantitative measure of RSV
replication.
Using the procedure described in this example, compounds of the present
invention were evaluated for their ability to inhibit the replication of the
virus in
cell culture. By adding compounds at various concentrations to the culture
medium, a dose response effect of the compound on virus replication was
determined. A useful quantitative measure of the inhibition of RSV replication
in
this assay is the concentration of the compound at which virus replication in
cell
culture is inhibited by 50% in comparison to that observed in the absence of
the
compound (50% Inhibitory Concentration, IC50). In the case of RSV, IC50 values
are defined as the concentration of compound that protected 50% of the cell
monolayer. from virus-induced cytopathic effect (syncytia formation).
Anti-pneumovirus compounds of the invention were screened for antiviral
activity against RSV (strain Long) on cultured HEp2 cells. Standard 96-well
culture plates were seeded with 4 x 104 HEp2 cells in 200 L of Minimal

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Essential Medium with Earles salts (EMEM) supplemented with 10% fetal bovine
serum (FBS). Twenty-four to 30 hours later, the cells were infected with a
dilution of RSV in Medium 199 (GIBCO/BRL) with 5% FBS that had been
titrated to yield > 85% destruction of the cell monolayer in 60 hours. After 1
hour
at 37oC, compounds were added to wells of the plate in a final DMSO
concentration of 0.5% as a series of 10 two-fold dilutions of the compound.
Virus control wells (VC, no test compound) and cell culture control wells (CC,
no
virus, no test compound) were also included on each plate. Plates were
incubated
in a humidified atmosphere at 370C and 5% carbon dioxide. After 60 hours, 100
L of a 5% solution of glutaraldehyde in water was added to each well, and the
wells were incubated at room temperature for 1 hour. The fixative was removed,
and the cells were stained with a 0.1% solution of crystal violet in water for
15-30
minutes. After rinsing and drying the plates, the optical density of the wells
was
measured at 570 nm (OD570).
To determine IC50 values for the test compounds,the mean value of the
OD570 readings of the virus control wells (VC) on a plate was substracted from
the
OD570 readings of all wells on that plate. The IC50 values were then
calculated
according to the following formula:
IC50= [(Y - B)/(A - B)] x (H - L) + L
where Y represents the mean ODS70 reading of the cell control wells (CC)
divided
by 2; B represents the mean ODS70 reading of wells of the compound dilution
nearest to and below Y; A represents the mean OD570 reading of wells of the
compound dilution nearest to and above Y; L represents the compound
concentration at B; and H represents the compound concentration at A.
A similar assay is useful for various strains of human RSV, including
subtype A and subtype B viruses, as well as other pneumoviruses.
The results of the cell culture assay for inhibition of the replication of
several pneumoviruses for representative compounds used in the method of the
invention are given in Table 1.
- - ---------------

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TABLE 1'
Example RSV-A RSV-B BRSV ORSV GRSV
5 1 0.001 0.008 0.003 0.002 0.001
2 0.001 0.008 0.001 n.d. n.d.
3 0.050 0.46 0.010 0.17 n.d.
4 0.110 0.15 0.270 n.d. n.d.
5 0.090 1.9 1.7 1.2 n.d.
10 6 0.001 0.002 0.001 0.001 0.001
7 0.001 n.d. n.d. n.d. n.d.
8 0.370 47.3 16.2 n.d. n.d.
Ribavirin 24.3 17.7 7.5 15.5 3.3
' All data represent IC50 values in M; abbreviations; RSV-A = human RSV
15 subtype A; human RSV-B = RSV subtype B; BRSV = bovine RSV; ORSV =
ovine RSV; GRSV=goat RSV; n.d. = not done.
The low concentrations of test compounds required to achieve 50%
inhibition of RSV replication in cell culture indicate that the compounds used
in
20 the method of the invention are effective at inhibiting the pneumovirus
replication
process. It is also demonstrated here that the compounds of the invention are
dramatically more potent than Ribavirin at inhibiting viral replication.
Example 17 demonstrates that the compounds of the invention are not
toxic or detrimental to the health of normal cells at concentrations well
above
25 those needed to inhibit pneumovirus replication.
EXAMPLE 17
Assay for Cytotoxicity of
Inhibitors of Pneumovirus Replication
30 To demonstrate that the compounds of the invention are not toxic or
detrimental to the health of normal cells, compounds of the invention were

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evaluated in an in vitro cytotoxicity assay. One useful assay for determining
the
cytotoxic effects of compounds on the growth of cells is a tetrazolium-based
colorimetric method (Mossman, T., J. Immun. Methods, 65 (1-2): 55-63 (1983)).
This assay measures cell viability, and therefore cytotoxicity, by
quantitatively
detecting the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) by viable cells. Cells are seeded in 96-well
plates in DMEM containing 5% FBS at a density of 4 x 103 cells per well. After
incubation for 4 hours at 37 C and 5% C02, 2-fold serial dilutions of compound
in 1% DMSO (or solvent alone) are added to quadruplicate wells and the plates
are incubated four an additional 68 hours at 37 C and 5% C02, which is
equivalent
to 3 to 4 cell doublings. The culture medium is removed, and the cells are
treated
with 1 mg/ml of MTT in phosphate-buffered saline, pH 7.2 for 4 hours at 37 C
and 5% CO2. After removal of the unreduced MTT, the reduced blue formazan
crystals produced by the viable cells are solubilized by the addition of 0.04N
HCI
in isopropanol. The optical density at 570 nm (ODS70) of each well is read
using a
suitable microplate reader. Cell viability is expressed as the percentage of
optical
density for compound-treated cells relative to the optical density of solvent
alone-
treated control wells. The highest compound concentration resulting in an
optical
density of > 75% of the control is represented as the cellular cytotoxicity
value
(CC75).
The results of the MTT cytotoxicity assay using compounds
prepared according to Examples 1 through 8 are given in Table 2.

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TABLE 2
Example CC73 (pM) ICse (pM)' SI
1 >12.5 0.001 >12,500
2 >150 0.001 >150,000
3 12.5 0.05 250
4- 18.8 0.112 171
5 >50.0 0.092 >556
6 3.1 0.001 3,100
7 >6.3 0.001 >6,250
- 8 9.4 0372 25
Ribavizira 9.4 24.3 <1
' Activity against human R$V subtype A-
2 protection from viral cywpat8ie effect of cell cultures achieved only 70-
90% at highest compound concentrations tested.
As shown in Table 2, the cellular cytotoxicity (C C) values for the
compounds of Examples 1 through 8 are considerably higher than the antiviral
(ICs0) values for these compounds. These results indicate that the compounds
of
the invention are highly selective and, at therapeutically effective doses,
they do
not detzimentally affect the health ofnonnal cells. A measure of this
selectivity
is provided by the high selective index value (Si), which is defined as CCõ
JIC..
The high SI values exhibited by compounds of the invention indicate very
desirable attributes of the compounds.
Although the Present invention has been described and
exemplified in terms of certain preferred embodiments, other embodiments will
be apparent to those skilled in the art