U.S. patent application number 16/930581 was filed with the patent office on 2020-11-05 for microbicidal pyrimidine or triazine containing compounds.
The applicant listed for this patent is Janssen Sciences Ireland UC. Invention is credited to Marie-Pierre T.M.M.G. De Bethune, Paul Stoffels, Jens Marcel Van Roey.
Application Number | 20200345733 16/930581 |
Document ID | / |
Family ID | 1000004959809 |
Filed Date | 2020-11-05 |
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United States Patent
Application |
20200345733 |
Kind Code |
A1 |
Van Roey; Jens Marcel ; et
al. |
November 5, 2020 |
Microbicidal Pyrimidine or Triazine Containing Compounds
Abstract
The present invention concerns the microbicidal activity of
certain pyrimidine or triazine containing non-nucleoside reverse
transcriptase inhibitors. The compounds of the present invention
inhibit the systemic infection of a human being with HIV, in
particular, the present compounds prevent sexual HIV transmission
in humans.
Inventors: |
Van Roey; Jens Marcel;
(Roeselare, BE) ; De Bethune; Marie-Pierre T.M.M.G.;
(Everberg, BE) ; Stoffels; Paul; (Hoogstraten,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Sciences Ireland UC |
Little Island |
|
IE |
|
|
Family ID: |
1000004959809 |
Appl. No.: |
16/930581 |
Filed: |
July 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15389662 |
Dec 23, 2016 |
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16930581 |
|
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13043174 |
Mar 8, 2011 |
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15389662 |
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10514361 |
May 31, 2005 |
7935710 |
|
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PCT/EP03/50158 |
May 13, 2003 |
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13043174 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/535 20130101;
A61K 47/10 20130101; A61K 9/0014 20130101; A61K 9/06 20130101; A61K
45/06 20130101; A61K 9/0034 20130101; A61K 47/02 20130101; A61K
31/505 20130101; A61K 47/14 20130101; A61K 9/0036 20130101; A61K
31/506 20130101; A61K 47/38 20130101; A61K 47/12 20130101 |
International
Class: |
A61K 31/505 20060101
A61K031/505; A61K 31/535 20060101 A61K031/535; A61K 31/506 20060101
A61K031/506; A61K 9/00 20060101 A61K009/00; A61K 9/06 20060101
A61K009/06; A61K 45/06 20060101 A61K045/06; A61K 47/02 20060101
A61K047/02; A61K 47/10 20060101 A61K047/10; A61K 47/12 20060101
A61K047/12; A61K 47/14 20060101 A61K047/14; A61K 47/38 20060101
A61K047/38 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2002 |
EP |
02076897.4 |
Claims
1-27. (canceled)
28. A method for treating chancroids in a patient in need thereof,
comprising administering to the patient a compound that is:
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-2-pyrimidinyl]amino]benzonitri-
le;
4-[[4-[(2,4-dichlorophenyl)methyl]-6-[(4-hydroxybutyl)amino]-2-pyrimid-
inyl]amino]-benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(3-hydroxypropyl)amino]-2-pyrimidin-
yl]amino]-benzonitrile;
N-[2-[(4-cyanophenyl)amino]-6-[(2,6-dichlorophenyl)methyl]-4-pyrimidinyl]-
acetamide;
N-[2-[(4-cyanophenyl)amino]-6-[(2,6-dichlorophenyl)methyl]-4-py-
rimidinyl]butanamide;
4-[[2-amino-6-(2,6-dichlorophenoxy)-4-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(2-hydroxy-2-phenylethyl)amino]-2-p-
yrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[3-(2-oxo-1-pyrrolidinyl)propyl]ami-
no]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[2-(2-hydroxyethoxy)ethyl]amino]-2--
pyrimidinyl]amino]benzonitrile monohydrochloride;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(2,3-dihydroxypropyl)amino]-2-pyrim-
idinyl]-amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-(hydroxyamino)-2-pyrimidinyl]amino]b-
enzonitrile;
4-[[4-[(2-cyanoethyl)amino]-6-[(2,6-dichlorophenyl)methyl]-2-pyrimidinyl]-
amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[2-(1-pyrrolidinyl)ethyl]amino]-2-p-
yrimidinyl]-amino]benzonitrile;
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-5-methyl-2-pyrimidinyl]amino]b-
enzonitrile;
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
4-[[2-[(2,4,6-trimethylphenyl)amino]-4-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-(2,4,6-trimethylphenoxy)-2-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)thio]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-2-pyrimidinyl]amino]be-
nzonitrile;
4-[[4-[[2,6-dichloro-4-(trifluoromethyl)phenyl]amino]-2-pyrimidinyl]amino-
]benzonitrile;
4-[[4-[(2,4-dichloro-6-methylphenyl)amino]-2-pyrimidinyl]amino]benzonitri-
le;
4-[[2-[(cyanophenyl)amino]-4-pyrimidinyl]amino]-3,5-dimethylbenzonitri-
le;
4-[[4-[(2,4-dibromo-6-fluorophenyl)amino]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[methyl(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzo-
nitrile;
4-[[4-[(2,4,6-trichlorophenyl)amino]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[(2,4,6-trimethylphenyl)thio]-2-pyrimidinyl]amino]benzonitrile-
;
4-[[4-[(2,4,6-trimethylphenyl)amino-2-pyrimidinyl]amino]benzonitrile;
4-[[4-amino-6-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[2-amino-6-[(2,4,6-trimethylphenyl)amino]-4-pyrimidinyl]amino]ben-
zonitrile;
4-[[4-(2-bromo-4-chloro-6-methylphenoxy)-2-pyrimidinyl]amino]be-
nzonitrile;
4-[[4[(4-chloro-2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitril-
e;
3,5-dichloro-4-[[2-[(4-cyanophenyl)amino]-4-pyrimidinyl]amino]benzonitr-
ile;
4-[[4-[[2,6-dichloro-4-(trifluoromethoxy)phenyl]amino]-2-pyrimidinyl]-
amino]-benzonitrile;
4-[[4-[(2,4-dibromo-3,6-dichlorophenyl)amino]-2-pyrimidinyl]amino]benzoni-
trile;
4-[[4-[[2,6-dibromo-4-propylphenyl]amino]-2-pyrimidinyl]amino]benzo-
nitrile;
4-[[4-[(4-(1,1-dimethylethyl)-2,6-dimethylphenyl)amino]-2-pyrimid-
inyl]amino]-benzonitrile;
4-[[2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethylbenzonitrile;
4-[[4-[(4-chloro-2,6-dimethylphenyl)amino]-5-methyl-2-pyrimidinyl]amino]
benzonitrile;
4-[[2-[(4-cyanophenyl)amino]-5-methyl-4-pyrimidinyl]amino-3,5-dimethyl
benzonitrile;
4-[[4-[[4-(1,1-dimethylethyl)-2,6-dimethylphenyl]amino]-5-methyl-2-pyrimi-
dinyl]-amino]benzonitrile;
4-[[4-[(4-bromo-2,6-dimethylphenyl)amino]-5-methyl-2-pyrimidinyl]amino]
benzonitrile;
4-[[5-methyl-4-[(2,4,6-trimethylphenyl)thio]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[(2,6-dibromo-4-propylphenyl)amino]-5-methyl-2-pyrimidinyl]ami-
no]benzonitrile;
4-[[4-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-5-methyl-2-pyrimidinyl-
]amino]-benzonitrile;
4-[[2-[(4-cyanophenyl)amino]-5-methyl-4-pyrimidinyl]amino]-3,5-dimethyl
benzonitrile;
4-[[4-[(phenylmethyl)amino]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-amino-5-chloro-6-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino-
]-benzonitrile;
4-[[5-chloro-4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzoni-
trile;
4-[[5-bromo-4-(4-cyano-2,6-dimethylphenoxy)-2-pyrimidinyl]amino]ben-
zonitrile;
4-[[4-amino-5-chloro-6-[(4-cyano-2,6-dimethylphenyl)amino]-2-py-
rimidinyl]amino]-benzonitrile;
4-[[5-bromo-6-[(4-cyano-2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]ben-
zonitrile;
4-[[4-amino-5-chloro-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimi-
dinyl]amino]-benzonitrile; or
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile; or an N-oxide, a pharmaceutically acceptable
addition salt, a stereochemically isomeric form thereof, or a
quaternary amine thereof.
29. The method of claim 28, wherein the compound is:
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile, or
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
or an N-oxide thereof, a pharmaceutically acceptable addition salt
thereof, or a quaternary amine thereof.
30. The method of claim 29, wherein the compound is
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile,
or an N-oxide thereof, a pharmaceutically acceptable addition salt
thereof, or a quaternary amine thereof.
31. The method of claim 30, wherein the compound is
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile.
32. The method of claim 29, wherein the compound is
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile, or an N-oxide thereof, a pharmaceutically
acceptable addition salt thereof, or a quaternary amine
thereof.
33. The method of claim 31, wherein the compound is
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile.
34. The method of claim 28, wherein the compound is administered to
the vagina.
35. A method for treating chancroids in a patient in need thereof,
comprising administering to the patient a composition comprising a
compound that is:
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-2-pyrimidinyl]amino]benzonitri-
le;
4-[[4-[(2,4-dichlorophenyl)methyl]-6-[(4-hydroxybutyl)amino]-2-pyrimid-
inyl]amino]-benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(3-hydroxypropyl)amino]-2-pyrimidin-
yl]amino]-benzonitrile;
N-[2-[(4-cyanophenyl)amino]-6-[(2,6-dichlorophenyl)methyl]-4-pyrimidinyl]-
acetamide;
N-[2-[(4-cyanophenyl)amino]-6-[(2,6-dichlorophenyl)methyl]-4-py-
rimidinyl]butanamide;
4-[[2-amino-6-(2,6-dichlorophenoxy)-4-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(2-hydroxy-2-phenylethyl)amino]-2-p-
yrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[3-(2-oxo-1-pyrrolidinyl)propyl]ami-
no]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[2-(2-hydroxyethoxy)ethyl]amino]-2--
pyrimidinyl]amino]benzonitrile monohydrochloride;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(2,3-dihydroxypropyl)amino]-2-pyrim-
idinyl]-amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-(hydroxyamino)-2-pyrimidinyl]amino]b-
enzonitrile;
4-[[4-[(2-cyanoethyl)amino]-6-[(2,6-dichlorophenyl)methyl]-2-pyrimidinyl]-
amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[2-(1-pyrrolidinyl)ethyl]amino]-2-p-
yrimidinyl]-amino]benzonitrile;
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-5-methyl-2-pyrimidinyl]amino]b-
enzonitrile;
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
4-[[2-[(2,4,6-trimethylphenyl)amino]-4-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-(2,4,6-trimethylphenoxy)-2-pyrimidinyl]amino]benzonitrile;
4-[[4-[(2,6-dichlorophenyl)thio]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-2-pyrimidinyl]amino]be-
nzonitrile;
4-[[4-[[2,6-dichloro-4-(trifluoromethyl)phenyl]amino]-2-pyrimidinyl]amino-
]benzonitrile;
4-[[4-[(2,4-dichloro-6-methylphenyl)amino]-2-pyrimidinyl]amino]benzonitri-
le;
4-[[2-[(cyanophenyl)amino]-4-pyrimidinyl]amino]-3,5-dimethylbenzonitri-
le;
4-[[4-[(2,4-dibromo-6-fluorophenyl)amino]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[methyl(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzo-
nitrile;
4-[[4-[(2,4,6-trichlorophenyl)amino]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[(2,4,6-trimethylphenyl)thio]-2-pyrimidinyl]amino]benzonitrile-
;
4-[[4-[(2,4,6-trimethylphenyl)amino-2-pyrimidinyl]amino]benzonitrile;
4-[[4-amino-6-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[2-amino-6-[(2,4,6-trimethylphenyl)amino]-4-pyrimidinyl]amino]ben-
zonitrile;
4-[[4-(2-bromo-4-chloro-6-methylphenoxy)-2-pyrimidinyl]amino]be-
nzonitrile;
4-[[4-[(4-chloro-2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitri-
le;
3,5-dichloro-4-[[2-[(4-cyanophenyl)amino]-4-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[[2,6-dichloro-4-(trifluoromethoxy)phenyl]amino]-2-pyrimidinyl-
]amino]-benzonitrile;
4-[[4-[(2,4-dibromo-3,6-dichlorophenyl)amino]-2-pyrimidinyl]amino]benzoni-
trile;
4-[[4-[(2,6-dibromo-4-propylphenyl]amino]-2-pyrimidinyl]amino]benzo-
nitrile;
4-[[4-[(4-(1,1-dimethylethyl)-2,6-dimethylphenyl)amino]-2-pyrimid-
inyl]amino]-benzonitrile;
4-[[2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethylbenzonitrile;
4-[[4-[(4-chloro-2,6-dimethylphenyl)amino]-5-methyl-2-pyrimidinyl]amino]
benzonitrile;
4-[[2-[(4-cyanophenyl)amino]-5-methyl-4-pyrimidinyl]amino-3,5-dimethyl
benzonitrile;
4-[[4-[[4-(1,1-dimethylethyl)-2,6-dimethylphenyl]amino]-5-methyl-2-pyrimi-
dinyl]-amino]benzonitrile;
4-[[4-[(4-bromo-2,6-dimethylphenyl)amino]-5-methyl-2-pyrimidinyl]amino]
benzonitrile;
4-[[5-methyl-4-[(2,4,6-trimethylphenyl)thio]-2-pyrimidinyl]amino]benzonit-
rile;
4-[[4-[(2,6-dibromo-4-propylphenyl)amino]-5-methyl-2-pyrimidinyl]ami-
no]benzonitrile;
4-[[4-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-5-methyl-2-pyrimidinyl-
]amino]-benzonitrile;
4-[[2-[(4-cyanophenyl)amino]-5-methyl-4-pyrimidinyl]amino]-3,5-dimethyl
benzonitrile;
4[[4-[(phenylmethyl)amino]-2-pyrimidinyl]amino]benzonitrile;
4-[[4-amino-5-chloro-6-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino-
]-benzonitrile;
4-[[5-chloro-4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzoni-
trile;
4-[[5-bromo-4-(4-cyano-2,6-dimethylphenoxy)-2-pyrimidinyl]amino]ben-
zonitrile;
4-[[4-amino-5-chloro-6-[(4-cyano-2,6-dimethylphenyl)amino]-2-py-
rimidinyl]amino]-benzonitrile;
4-[[5-bromo-6-[(4-cyano-2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]ben-
zonitrile;
4-[[4-amino-5-chloro-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimi-
dinyl]amino]-benzonitrile; or
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
] benzonitrile; or an N-oxide, a pharmaceutically acceptable
addition salt, a stereochemically isomeric form thereof, or a
quaternary amine thereof.
36. The method of claim 35, wherein the compound is:
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile, or
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
or an N-oxide thereof, a pharmaceutically acceptable addition salt
thereof, or a quaternary amine thereof.
37. The method of claim 36, wherein the compound is
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
or an N-oxide thereof, a pharmaceutically acceptable addition salt
thereof, or a quaternary amine thereof.
38. The method of claim 37, wherein the compound is
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile.
39. The method of claim 36, wherein the compound is
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile, or an N-oxide thereof, a pharmaceutically
acceptable addition salt thereof, or a quaternary amine
thereof.
40. The method of claim 39, wherein the compound is
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]-benzonitrile.
41. The method of claim 35, wherein the composition is administered
to the vagina.
42. The method of claim 35, wherein the composition is administered
in the form of an intravaginal ring.
43. The method of claim 35, wherein the composition is administered
in a topical form.
44. The method of claim 35, wherein the composition is administered
in the form of a bioadhesive.
45. The method of claim 35, wherein the composition is administered
is in the form of an immediate release drug delivery system.
46. The method of claim 35, wherein the composition is administered
in the form of a sustained release drug delivery system.
47. The method of claim 35, wherein the composition is a gel and
further comprises: a gel-forming compound, a buffer, a
pharmaceutically acceptable diluent, optionally a humectant, and
optionally a preservative.
48. The method of claim 35, wherein the composition comprises
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]-benzonitrile,
hydroxyethyl cellulose, glycerol, methyl paraben, propyl paraben,
lactic acid, sodium hydroxide, and water.
49. The method of claim 35, wherein the composition comprises one
or more additional antiretroviral compounds.
50. The method of claim 35, wherein the composition comprises one
or more components selected from an antibody, a detergent or
surfactant, a coating for the pathogen, a coating for the site of
transmission, an antibiotic peptide or a pH regulator.
51. The method of claim 35, wherein the composition comprises a
spermicidal compound.
Description
[0001] The present invention concerns the microbicidal activity of
certain pyrimi dine or triazine containing non-nucleoside reverse
transcriptase inhibitors (NNRTIs), in particular, the present
invention concerns the use of pyrimidine or triazine derivatives in
the manufacture of a medicament for the prevention of HIV (Human
Immunodeficiency Virus) transmission or infection in humans, in
particular sexual transmission. It also concerns pharmaceutical
compositions adapted to be applied at the site where sexual
intercourse or related intimate contact takes place.
[0002] Worldwide, the heterosexual route is the prevalent mode of
transmission of AIDS. Therefore, demands have been raised for
measures that block sexual spreading of the HIV infection. As there
is no effective treatment or vaccine against AIDS, preventive
measures are the only tools that can presently reduce transmission
of Human Immunodeficiency virus (HIV). The consistent and correct
use of condoms represents an effective barrier to prevent HIV
transmission. However, the reduction of acquiring infection can
only be significantly reduced if condoms are used for almost all
sexual intercourses; a result that can not be achieved despite
intensive prevention programs to increase condom use.
[0003] Development of microbicides for topical use may represent an
efficacious alternative to condoms. A microbicide is any agent that
kills or deactivates disease-causing microbes. According to the
International Association of Physicians in AIDS CARE (IAPAC), the
definition of microbicides also includes interventions that can
block or prevent infection, as well as amplification of the body's
natural defenses to prevent infection through sexual acts.
[0004] Ideally, microbicides should have little or no side effects
at an effective microbicidal concentration. One aspect in this
respect is that the drug used as microbicide should have little or
no immunosuppressive activity at an effective microbicidal
concentration. In addition, the ideal microbicide should
sufficiently withstand varying temperatures and acceptably function
within varied pH ranges (ranges of alkaline and acidic levels in
the vagina). Further, it should not eliminate the natural
beneficial lactobacilli that reside in the vagina and regulate
vaginal health.
[0005] Studies have demonstrated that HIV transmission through
direct, biological mechanisms are facilitated in a person already
infected with a sexually transmitted disease (STD) (Fleming et al.
Sexually Transmitted Infections (1999 February), 75(1), 3-17).
[0006] Sores, lesions lesions and inflammations caused by STDs
compromise certain physical barriers to disease, For these reasons,
taking measures to prevent STD transmission is a valuable strategy
in the fight against HIV infection. Several microbicides in human
clinical trials contain detergent-type ingredients, which may cause
lesions at vaginal and cervical epithelia. Spermicidal products
containing biodetergents can inactivate HIV in vitro. However, it
has been shown that such biodetergents may exacerbate genital
ulcers and facilitate HIV transmission when tested in vivo.
[0007] Besides surfactants, which directly act on the virus
particle, drugs that block the early steps of HIV multiplication
such as antiretroviral drugs are undergoing preclinical assessment.
Various antiretrovirals including non-nucleoside reverse
transcriptase inhibitors (NNRTIs) have been tested in vitro with
varying results. To date, no evidence has been published on the in
vivo effectiveness of NNRTIs as microbicidal agents.
[0008] It has now been found that the pyrimidine and triazine
compounds of the present invention exhibit microbicidal activity in
that these compounds have the ability to prevent the infection by
HIV.
[0009] In addition, the pyrimidine and triazine compounds of the
present invention also have shown microbicidal activity against STD
pathogens such as Haeinophilus dicreyi, while maintaining their
compatibility with lactobacilli and normal vagina flora. The
derived healing effect of the present compounds on the chancroids
caused by Haemophilus ducreyi, significantly contributes to the
prevention of systemic HIV infection.
[0010] EP 1002795, WO 99/50250, WO 99/50256 and WO 00/27828
disclose compounds inhibiting the replication of the HIV virus in
the human T-4 cells via an interaction with the HIV reverse
transcriptase enzyme.
DESCRIPTION OF THE INVENTION
[0011] The present invention concerns the use of compounds having
the formula (I), (II) and (III) wherein a compound of formula (I)
corresponds to
##STR00001##
[0012] a N-oxide, a pharmaceutically acceptable addition salt or a
stereochernically isomeric form thereof, wherein
[0013] Y is CR.sup.5 or N,
[0014] A is CH, CR.sup.4 or N;
[0015] n is 0, 1, 2, 3 or 4;
[0016] Q is --NR.sup.1R.sup.2 or when Y is CR.sup.5 then Q nay also
be hydrogen; [0017] R.sup.1 and R.sup.2 are each independently
selected from hydrogen, hydroxy, C.sub.1-12alkyl,
C.sub.1-12alkyloxy, C.sub.1-12alkylcarbonyl,
C.sub.1-12alkyloxycarbonyl, aryl, amino, mono- or
di(C.sub.1-12alkyl)amino, mono- or di(C.sub.1-12alkyl)aminocarbonyl
wherein each of the aforementioned C.sub.1-12alkyl groups may
optionally and each individually be substituted with one or two
substituents each independently selected from hydroxy,
C.sub.1-6alkyloxy, hydroxyC.sub.1-6alkyloxy, carboxyl,
C.sub.1-6alkyloxycarbonyl, cyano, amino, imino, aminocarbonyl,
aminocarbonylamino, mono- or di(C.sub.1-6alkyl)amino, aryl and Het;
or [0018] R.sup.1 and R.sup.2 taken together may form pyrrolidinyl,
piperidinyl, morpholinyl, azido or mono- or
di(C.sub.1-12alkyl)aminoC.sub.1-4alkylidene;
[0019] R.sup.3 is hydrogen, aryl, C.sub.1-6alkylcarbonyl,
C.sub.1-6alkyloxycarbonyl, C.sub.1-6alkyl substituted with
C.sub.1-6alkyloxycarbonyl; and [0020] each R.sup.4 independently is
hydroxy, halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, cyano,
aminocarbonyl, nitro, amino, trihalomethyl, trihalomethyloxy, or
when Y is CR.sup.5 then R.sup.4 may also represent C.sub.1-6alkyl
substituted with cyano or aminocarbonyl; [0021] R.sup.5 is hydrogen
or C.sub.1-4alkyl; [0022] L is --X.sup.1--R.sup.6 or
-X.sup.2-Alk-R.sup.7 wherein [0023] R.sup.6 and R.sup.7 each
independently are phenyl or phenyl substituted with one, two,
three, four or five substituents each independently selected from
halo, hydroxy, C.sub.1-6alkyl, C.sub.1-6alkyloxy,
C.sub.1-6alkylcarbonyl , C.sub.1-6alkyloxycarbonyl, formyl, cyano,
nitro, amino, and trifluoromethyl; or when Y is CR.sup.5 then
R.sup.6 and R.sup.7 may also be selected from phenyl substituted
with one, two, three, four or five substituents each independently
selected from aminocarbonyl, trihalomethyloxy and trihalomethyl; or
when Y is N then R.sup.6 and R.sup.7 may also be selected from
indanyl or indolyl, each of said indanyl or indolyl may be
substituted with one, two, three, four or five substituents each
independently selected from halo, hydroxy, C.sub.1-6alkyl,
C.sub.1-6alkyloxy, C.sub.1-6alkylcarbonyl,
C.sub.1-6alkyloxycarbonyl, formyl, cyano, nitro, amino, and
trifluoromethyl; when R.sup.6 is optionally substituted indanyl or
indolyl, it is preferably attached to the remainder of the molecule
via the fused phenyl ring. For instance, R.sup.6 is suitably 4-,
5-, 6- or 7-indolyl; [0024] X.sup.1 and X.sup.2 are each
independently --NR.sup.3--, --NH--NH--, --N.dbd.N--, --O--, --S--,
--S(.dbd.O)-- or --S(.dbd.O).sub.2--;
[0025] Alk is C.sub.1-4alkanediyl; or [0026] when Y is CR.sup.5
then L may also be selected from C.sub.1-10alkyl,
C.sub.3-10alkenyl, C.sub.3-10alkynyl, C.sub.3-7cycloalkyl, or
C.sub.1-10alkyl substituted with one or two substituents
independently selected from C.sub.3-7cycloalkyl, indanyl, indolyl
and phenyl, wherein said phenyl, indanyl and indolyl may he
substituted with one, two, three, four or where possible five
substituents each independently selected from halo, hydroxy,
C.sub.1-6alkyl, C.sub.1-6alkyloxy, cyano, aminocarbonyl,
C.sub.1-6alkyloxycarbonyl, formyl, nitro, amino, trihalomethyl,
trihalomethyloxy and C.sub.1-6alkylearbonyl; [0027] aryl is phenyl
or phenyl substituted with one, two, three, four or five
substituents each independently selected from halo, C.sub.1-6alkyl,
C.sub.1-6alkyloxy, cyano, nitro and triflouromethyl; [0028] Het is
an aliphatic or aromatic heterocyclic radical; said aliphatic
heterocyclic radical is selected from pyrrolidinyl, piperidinyl,
homopiperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl and
tetrahydrothienyl wherein each of said aliphatic heterocyclic
radical may optionally be substituted with an oxo group; and said
aromatic heterocyclic radical is selected from pyrrolyl, furanyl,
thienyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl wherein
each of said aromatic heterocyclic radical may optionally be
substituted with hydroxy; [0029] and, wherein a compound of formula
(II) corresponds to
##STR00002##
[0030] a N-oxide, a pharmaceutically acceptable addition salt,
quaternary amine and the stereochemically isomeric forms thereof,
wherein
[0031] -b.sup.1=b.sup.2-C(R.sup.2a)=b.sup.3-b.sup.4= represents a
bivalent radical of formula
--CH.dbd.CH--C(H.sup.2a).dbd.CH--CH.dbd. (b-1);
--N.dbd.CH--C(R.sup.2a).dbd.CH--CH.dbd. (b-2);
--CH.dbd.N--C(R.sup.2a).dbd.CH--CH.dbd. (b-3);
--N.dbd.CH--C(R.sup.2a).dbd.N--CH.dbd. (b-4);
--N.dbd.CH--C(R.sup.2a).dbd.CH--N.dbd. (b-5);
--CH.dbd.N--C(R.sup.2a).dbd.N--CH.dbd. (b-6);
--N.dbd.N--C(R.sup.2a).dbd.CH--CH.dbd. (b-7);
[0032] q is 0, 1, 2; or where possible q is 3 or 4; [0033] R.sup.1
is hydrogen, aryl, formyl, C.sub.1-6alkylcarbonyl, C.sub.1-6alkyl,
C.sub.1-6alkyloxycarbonyl, C.sub.1-6alkyl substituted with formyl,
C.sub.1-6alkylcarbonyl, C.sub.1-6alkyloxycarbonyl; [0034] R.sup.2a
is cyano, aminocarbonyl, mono- or di(methyl)aminocarbonyl,
C.sub.1-6alkyl substituted with cyano, aminocarbonyl or mono- or
di(methyl)aminocarbonyl, C.sub.2-6 alkenyl substituted with cyano,
or C.sub.2-6alkynyl substituted with cyano; [0035] each R.sup.2
independently is hydroxy, halo, C.sub.1-6alkyl optionally
substituted with cyano or --C(.dbd.O)R.sup.6, C.sub.3-7cycloalkyl,
C.sub.2-6alkenyl optionally substituted with one or more halogen
atoms or cyano, C.sub.2-6alkynyl optionally substituted with one or
more halogen atoms or cyano, C.sub.1-6alkyloxy,
C.sub.1-6alkyloxycarbonyl, carboxyl, cyano, nitro, amino, mono- or
di(C.sub.1-6alkyl)amino, polyhalomethyl, polyhalomethyloxy,
polyhalomethylthio, --S(.dbd.O).sub.pR.sup.6,
--NH--S(.dbd.O).sub.pR.sup.6, --C(.dbd.O)R.sup.6, --NHC(.dbd.O)H,
--C(.dbd.O)NHNH.sub.2, --NHC(.dbd.O)R.sup.6, --C(.dbd.NH)R.sup.6 or
a radical of formula
[0035] ##STR00003## [0036] wherein each A independently is N, CH or
CR.sup.6); [0037] B is NH, O, S or NR.sup.6, [0038] p is 1 or 2;
and [0039] R.sup.6 is methyl, amino, mono- or dimethylamino or
polyhalomethyl; [0040] L is C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl, C.sub.3-7cycloalkyl, whereby each of said
aliphatic group may be substituted with one or two substituents
independently selected from [0041] C.sub.3-7cycloalkyl, [0042]
indolyl or isoindolyl, each optionally substituted with one, two,
three or four substituents each independently selected from halo,
C.sub.1-6allkyl, hydroxy, C.sub.1-6alkyloxy, cyano, aminocarbonyl,
nitro, amino, polyhalomethyl, polyhalomethyloxy and
C.sub.1-6alkylcarbonyl, [0043] phenyl, pyridinyl, pyrimidinyl,
pyrazinyl or pyridazinyl, wherein each of said aromatic rings may
optionally be substituted with one, two, three, four or five
substituents each independently selected from the substituents
defined in R.sup.2; or [0044] L is --X--R.sup.3 wherein [0045]
R.sup.3 is phenyl, pyridinyl, pyrimidinyl, pyrazinyl or
pyridazinyl, wherein each of said aromatic rings may optionally be
substituted with one, two, three, four or five substituents each
independently selected from the substituents defined in R.sup.2;
and [0046] X is --NR.sup.1--, --NH--NH--, --N.dbd.N--, --O--,
--C(.dbd.O)--, --CHOH--, --S--, --S(.dbd.O)-- or
--S(.dbd.O).sub.2--; [0047] Q represents hydrogen, C.sub.1-6alkyl,
halo, polyhaloC.sub.1-6alkyl or --NR.sup.4R.sup.5; and [0048]
R.sup.4 and R.sup.5 are each independently selected from hydrogen,
hydroxy, C.sub.1-6alkyl, C.sub.1-12alkyloxy,
C.sub.1-12alkylcarbonyl, C.sub.1-12alkyloxycarbonyl, aryl, amino,
mono- or di(C.sub.1-12alkyl)amino, mono- or
di(C.sub.1-12alkyl)aminocarbonyl, wherein each of the
aforementioned C.sub.1-12alkyl groups may optionally and each
individually be substituted with with one or two substituents each
independently selected from hydroxy, C.sub.1-6alkyloxy,
hydroxyC.sub.1-6alkyloxy, carboxyl, C.sub.1-6alkyloxycarbonyl,
cyano, amino, imino, mono- or di(C.sub.1-6alkyl)amino,
polyhalomethyl, polyhalomethyloxy, polyhalomethylthio,
--S(.dbd.O).sub.pR.sup.6, --NH--S(.dbd.O).sub.pR.sup.6,
--C(.dbd.O)R.sup.6, --NHC(.dbd.O)H, --C(.dbd.O)NHNH.sub.2,
--NHC(.dbd.O)R.sup.6, --C(.dbd.NH)R.sup.6, aryl and Het; or [0049]
R4 and R.sup.5 taken together may form pyrrolidinyl, piperidinyl,
morpholinyl, azido or mono- or
di(C.sub.1-12alkyl)aminoC.sub.1-4alkylidene; [0050] Y represents
hydroxy, halo, C.sub.3-7cycloalkyl, C.sub.2-6alkenyl optionally
substituted with one or more halogen atoms, C.sub.2-6alkynyl
optionally substituted with one or more halogen atoms,
C.sub.1-6alkyl optionally substituted with cyano or
--C(.dbd.O)R.sup.6, C.sub.1-6alkyloxy, C.sub.1-6alkyloxycarbonyl,
carboxyl, cyano, nitro, amino, mono- or di(C.sub.1-6alkyl)amino,
polyhalomethyl, polyhalomethyloxy, polyhalomethylthio,
--S(.dbd.O).sub.pR.sup.6, --NH--S(.dbd.O).sub.pR.sup.6,
--C(.dbd.O)R.sup.6, --NHC(.dbd.O)H, --C(.dbd.O)NHNH.sub.2,
--NHC(.dbd.O)R.sup.6, --C(.dbd.NH)R.sup.6 or aryl; [0051] aryl is
phenyl or phenyl substituted with one, two, three, four or five
substituents each independently selected from halo, C.sub.1-6alkyl,
C.sub.3-7cycloalkyl, C.sub.1-6alkyloxy, cyano, nitro,
polyhaloC.sub.1-6alkyl and polyhaloC.sub.1-6alkyloxy;
[0052] Het is an aliphatic or aromatic heterocyclic radical; said
aliphatic heterocyclic radical is selected from pyrrolidinyl,
piperidinyl, homopiperidinyl, piperazinyl, morpholinyl,
tetrahydrofuranyl and tetrahydrothienyl wherein each of said
aliphatic heterocyclic radical may optionally be substituted with
an oxo group; and said aromatic heterocyclic radical is selected
from pyrrolyl, furanyl, thienyl, pyridinyl, pyrimidinyl, pyrazinyl
and pyridazinyl wherein each of said aromatic heterocyclic radical
may optionally be substituted with hydroxy; Het is meant to include
all the possible isomeric forms of the heterocycles mentioned in
the definition of Het, for instance, pyrrolyl also includes
2H-pyrrolyl; the Het radical may be attached to the remainder of
the molecule of formula (II) through any ring carbon or heteroatom
as appropriate, thus, for example, when the heterocycle is
pyridinyl, it may be 2-pyridinyl, 3-pyridinyl or 4-pyridinyl;
[0053] and, wherein a compound of formula (III) corresponds to
##STR00004##
[0054] a N-oxide, a pharmaceutically acceptable addition salt,
quaternary amine and the stereochemically isomeric forms thereof,
wherein
[0055] -a.sup.1=a.sup.2-a.sup.3=a.sup.4- represents a bivalent
radical of formula
--CH.dbd.CH--CH.dbd.CH-- (a-1);
--N.dbd.CH--CH.dbd.CH-- (a-2);
--N.dbd.CH--N.dbd.CH-- (a-3);
--n.dbd.CH--CH.dbd.N-- (a-4);
--N.dbd.N--CH.dbd.CH-- (a-5); [0056] n is 0, 1, 2, 3 or 4; and in
case -a.sup.1=a.sup.2-a.sup.3=a.sup.4- is (a-1), then n may also be
5; [0057] R.sup.1 is hydrogen, aryl, formyl,
C.sub.1-6alkylcarbonyl, C.sub.1-6alkyl, C.sub.1-6alkyloxycarbonyl,
C.sub.1-6alkyl substituted with formyl, C.sub.1-6allkylcarbonyl,
C.sub.1-6alkyloxycarbonyl, and [0058] each R.sup.2 independently is
hydroxy, halo, C.sub.1-6alkyl optionally substituted with cyano or
--C(.dbd.O)R.sup.4, C.sub.3-7cycloalkyl, C.sub.2-6alkenyl
optionally substituted with one or more halogen atoms or cyano,
C.sub.2-6alkynyl optionally substituted with one or more halogen
atoms or cyano, C.sub.1-6alkyloxy, C.sub.1-6alkyloxycarbonyl,
carboxyl, cyano, nitro, amino, mono- or di(C.sub.1-6alkyl)amino,
polyhalomethyl, polyhalomethyloxy, polyhalomethylthio,
--S(.dbd.O).sub.pR.sup.4, --NH--S(.dbd.O).sub.pR.sup.4,
--C(.dbd.O)R.sup.4, --NHC(.dbd.O)H, --C(.dbd.O)NHNH.sub.2,
--NHC(.dbd.O)R.sup.4, --C(.dbd.NH)R.sup.4 or a radical of
formula
[0058] ##STR00005## [0059] wherein each A independently is N, CH or
CR.sup.4; [0060] B is NH, O, S or NR.sup.4; [0061] p is 1 or 2; and
[0062] R.sup.4 is methyl, amino, mono- or dimethylamino or
polyhalomethyl; [0063] L is C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl, C.sub.3-7cycloalkyl, whereby each of said
aliphatic group may be substituted with one or two substituents
independently selected from [0064] C.sub.3-7cycloalkyl, [0065]
indolyl or isoindolyl, each optionally substituted with one, two,
three or four substituents each independently selected from halo,
C.sub.1-6alkyl, hydroxy, C.sub.1-6alkyloxy, cyano, aminocarbonyl,
nitro, amino, polyhalomethyl, polyhalomethyloxy and
C.sub.1-6allkylcarbonyl, [0066] phenyl, pyridinyl, pyrimidinyl,
pyrazinyl or pyridazinyl, wherein each of said aromatic rings may
optionally be substituted with one, two, three, four or five
substituents each independently selected from the substituents
defined in R.sup.2; or [0067] L is --X--R.sup.3 wherein [0068]
R.sup.3 is phenyl, pyridinyl, pyrimidinyl, pyrazinyl or
pyridazinyl, wherein each of said aromatic rings may optionally be
substituted with one, two, three, four or five substituents each
independently selected from the substituents defined in R.sup.2;
and [0069] X is --NR.sup.1--, --NH--NH--, --N.dbd.N--, --O--,
--C(.dbd.O)--, --CHOH--, --S--, --S(.dbd.O)-- or
--S(.dbd.O).sub.2--; [0070] aryl is is phenyl or phenyl substituted
with one, two, three, four or five substituents each independently
selected from halo, C.sub.1-6alkyl, C.sub.3-7cycloalkyl,
C.sub.1-6alkyloxy, cyano, nitro, polyhaloC.sub.1-6alkyl and
polyhaloC.sub.1-6alkyloxy; [0071] with the proviso that compounds
of formula (III) wherein [0072] L is C.sub.1-3alkyl; R.sup.1 is
selected from hydrogen, ethyl and methyl;
-a.sup.1=a.sup.2-a.sup.3=a.sup.4- represents a bivalent radical of
formula (a-1); n is 0 or 1 and R.sup.2 is selected from fluoro,
chloro, methyl, trifluoromethyl, ethyloxy and nitro; or [0073] is
--X--R.sup.3, X is --NH--; R.sup.1 is hydrogen;
-a.sup.1=a.sup.2-a.sup.3=a.sup.4- represents a bivalent radical of
formula (a-1); n is 0 or 1 and R.sup.2 is selected from chloro,
methyl, methyloxy, cyano, amino and nitro and R.sup.3 is phenyl,
optionally substituted with one substituent selected from chloro,
methyl, methyloxy, cyano, amino and nitro; [0074] and the compounds
[0075] N,N'-dipyridinyl-(1,3,5)-triazine-2,4-diamine; [0076]
(4-chloro-phenyl)-(4(1-(4-isobutyl-phenyl)-ethyl)-(1,3,5)
triazin-2-yl)-amine are not included;
[0077] in the manufacture of a medicament useful for preventing the
transmission of or infection with HIV, particularly via sexual
intercourse or related intimate contact between partners. In
particular, the use of a compound of formula (I), (II) or (III) in
the manufacture of a topical medicament useful for preventing the
transmission of or infection with HIV.
[0078] Thus, the present invention also concerns a method to
prevent the transmission of or infection with HIV, particularly via
sexual intercourse or related intimate contact between partners,
which method comprises administering, in particular topically
administering, to a human an effective amount, in particular a
microbicidal effective amount, of a microbici dal compound of
formula (I), formula (II) or formula (III).
[0079] Suitably, the present invention concerns the use of a
compound of formula (I), (II) or (III) in the manufacture of a
microbicidal medicament useful for preventing the transmission of
HIV wherein Y in the compound of formula (II) represents hydroxy,
halo, C.sub.3-7cycloalkyl, C.sub.2-6alkenyl optionally substituted
with one or more halogen atoms, C.sub.2-6alkynyl optionally
substituted with one or more halogen atoms, C.sub.1-6alkyl
substituted with cyano or --C(.dbd.O)R.sup.6, C.sub.1-6alkyloxy,
C.sub.1-6alkyloxycarbonyl, carboxyl, cyano, nitro, amino, mono- or
di(C.sub.1-6alkyl)amino, polyhalomethyl, polyhalomethyloxy,
polyhalomethylthio, --S(.dbd.O).sub.pR.sup.6,
--NH--S(.dbd.O).sub.pR.sup.6, --C(.dbd.O)R.sup.6, --NHC(.dbd.O)H,
--C(.dbd.O)N H.sub.2, --NHC(.dbd.O)R.sup.6, --C(.dbd.NH)R.sup.6 or
aryl.
[0080] The term sexual intercourse or related intimate contact
between partners comprises vaginal sex, anal sex, oral sex and
contact of body sites with HIV infected fluids of the sexual
partner, in particular semen. Particularly, the term sexual
intercourse or related intimate contact between partners
constitutes vaginal, anal or oral sex, more particularly vaginal
sex.
[0081] The contact sites believed to be most responsible for the
transmission of HIV via sexual intercourse or related intimate
contact between partners are the genitals, rectum, mouth, hands,
lower abdomen, upper thighs.
[0082] The term "partners" as mentioned hereinbefore or hereinafter
defines two or more warm-blooded animals, in particular humans, who
are sexually active with each other, ie. who have sexual
intercourse with each other or who have intimate contact with each
other related to sexual activities.
[0083] In an embodiment, the present invention concerns the use of
compounds having the formula (IV), wherein a compound of formula
(IV) corresponds to
##STR00006##
[0084] a N-oxide, a pharmaceutically acceptable addition salt,
quaternary amine and the stereochemically isomeric forms thereof,
wherein
[0085] n is 0, 1, 2, 3 or 4; [0086] R.sup.1 is hydrogen, aryl,
formyl, C.sub.1-6alkylcarbonyl, C.sub.1-6alkyl,
C.sub.1-6alkyloxycarbonyl, C.sub.1-6alkyl substituted with formyl,
C.sub.1-6alkylcarbonyl, C.sub.1-6alkyloxycarbonyl; and [0087]
R.sup.2a is cyano; aminocarbonyl; mono- or dimethylaminocarbonyl;
C.sub.1-6alkyl optionally substituted with cyano, aminocarbonyl, or
mono- or dimethylaminocarbonyl; C.sub.2-6alkenyl substituted with
cyano; and C.sub.2-6alkynyl substituted with cyano; [0088] each
R.sup.2 independently is hydroxy, halo, C.sub.1-6alkyl optionally
substituted with cyano or --C(.dbd.O)R.sup.4, C.sub.3-7cycloalkyl,
C.sub.2-6alkenyl optionally substituted with one or more halogen
atoms or cyano, C.sub.2-6alkynyl optionally substituted with one or
more halogen atoms or cyano, C.sub.1-6alkyloxy,
C.sub.1-6alkyloxycarbonyl, carboxyl, cyano, nitro, amino, mono- or
di(C.sub.1-6alkyl)amino, polyhalomethyl, polyhalomethyloxy,
polyhalomethylthio, --S(.dbd.O).sub.pR.sup.4,
--NH--S(.dbd.O).sub.pR.sup.4, --C(.dbd.O)R.sup.4, --NHC(.dbd.O)H,
--C(.dbd.O)NHNH.sub.2, --NHC(.dbd.O)R.sup.4, --C(.dbd.NH)R.sup.4 or
a radical of formula
[0088] ##STR00007## [0089] wherein each A independently is N, CH or
CR.sup.4; [0090] B is NH, O, S or NR.sup.4; [0091] p is 1 or 2; and
[0092] R.sup.4 is methyl, amino, mono- or dimethylamino or
polyhalomethyl; [0093] L is C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl, C.sub.3-7cycloalkyl, each of said aliphatic
group substituted with phenyl, which may optionally be substituted
with one, two, three, four or five substituents each independently
selected from the substituents defined in R.sup.2; or [0094] L is
--X--R.sup.3 wherein [0095] R.sup.3 is phenyl, optionally
substituted with one, two, three, four or five substituents each
independently selected from the substituents defined in R.sup.2;
and [0096] X is --NR.sup.1--, --NH--NH--, --N.dbd.N--, --O--,
--C(.dbd.O)--, --CHOH--, --S--, --S(.dbd.O)-- or
--S(.dbd.O).sub.2--; [0097] aryl is phenyl or phenyl substituted
with one, two, three, four or five substituents each independently
selected from halo, C.sub.1-6alkyl, C.sub.3-7cycloalkyl,
C.sub.1-6alkyloxy, cyano, nitro, polyhaloC.sub.1-6alkyl and
polyhaloC.sub.1-6alkyloxy;
[0098] with the proviso that the compound
2,4-di-p-cyanoanilino-1,3,5-triazine is not included;
[0099] in the manufacture of a microbicidal medicament useful for
preventing the transmission of or infection with HIV.
[0100] Thus, the present invention also concerns a method to
prevent the transmission of or infection with HIV, which method
comprises administering, in particular topically administering, to
a human an effective amount, in particular a microbicidal effective
amount, of a microbicidal compound of formula (IV).
[0101] As used in the foregoing definitions and hereinafter halo
defines fluoro, chloro, bromo and iodo; polyhalomethyl as a group
or part of a group is defined as mono- or polyhalosubstituted
methyl, in particular methyl with one or more fluoro atoms, for
example, difluoromethyl or trifluoromethyl; polyhaloC.sub.1-6alkyl
as a group or part of a group is defined as mono- or
polyhalosubstituted C.sub.1-6alkyl, for example, the groups defined
in halomethyl, 1,1-difluoro-ethyl and the like; in case more than
one halogen atoms are attached to an alkyl group within the
definition of polyhalomethyl or polyhaloC.sub.1-6alkyl, they may be
the same or different; C.sub.1-4alkyl as a group or part of a group
encompasses the straight and branched chained saturated hydrocarbon
radicals having from 1 to 4 carbon atoms such as, for example,
methyl, ethyl, propyl, butyl and the like; C.sub.1-6alkyl as a
group or part of a group encompasses the straight and branched
chained saturated hydrocarbon radicals as defined in C.sub.1-4alkyl
as well as the higher homologues thereof containing 5 or 6 carbon
atoms such as, for example pentyl or hexyl; C.sub.1-10alkyl as a
group or part of a group encompasses the straight and branched
chained saturated hydrocarbon radicals as defined in C.sub.1-6alkyl
as well as the higher homologues thereof containing 7 to 10 carbon
atoms such as, for example, heptyl, octyl, nonyl or decyl;
C.sub.1-12alkyl as a group or part of a group encompasses the
straight and branched chained saturated hydrocarbon radicals as
defined in C.sub.1-10alkyl as well as the higher homologues thereof
containing 11 or 12 carbon atoms such as, for example, undecyl,
dodecyl and the like; C.sub.1-4alkylidene as a group or part of a
group defines bivalent straight and branched chained hydrocarbons
having from 1 to 4 carbon atoms such as, for example, methylene,
ethylidene, propylidene, butylidene and the like;
C.sub.1-4alkanediyl as a group or part of a group encompasses those
radicals defined under C.sub.1-4alkylidene as well as other
bivalent straight and branched chained hydrocarbons having from 1
to 4 carbon atoms such as, for example, 1,2-ethanediyl,
1,3-propanediyl, 1,4-butanediyl and the like; C.sub.3-7cycloalkyl
as a group or part of a group is generic to cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl;
C.sub.3-10alkenyl as a group or part of a group defines straight
and branch chained hydrocarbon radicals containing one double bond
and having from 3 to 10 carbon atoms such as, for example,
2-propenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl,
3-hexenyl, 3-heptenyl, 2-octenyl, 2-nonenyl, 2-decenyl and the
like, whereby the carbon atom attached to the pyrimidine ring is
preferably an aliphatic carbon atom; C.sub.3-10alkynyl as a group
or part of a group defines straight and branch chained hydrocarbon
radicals containing one triple bond and having from 3 to10 carbon
atoms such as, for example, 2-propynyl, 2-butynyl, 2-pentynyl,
3-pentynyl, 3-methyl-2-butynyl, 3-hexynyl, 3-heptynyl, 2-octenyl,
2-nonynyl, 2-decenyl and the like, whereby the carbon atom attached
to the pyrimidine ring is preferably an aliphatic carbon atom;
C.sub.2-6alkenyl defines straight and branched chain hydrocarbon
radicals having from 2 to 6 carbon atoms containing a double bond
such as ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like;
C.sub.2-10alkenyl defines straight and branched chain hydrocarbon
radicals having from 2 to 10 carbon atoms containing a double bond
such as the groups defined for C.sub.2-6alkenyl and heptenyl,
octenyl, nonenyl, decenyl and the like; C.sub.2-6alkynyl defines
straight and branched chain hydrocarbon radicals having from 2 to 6
carbon atoms containing a triple bond such as ethynyl, propynyl,
butynyl, pentynyl, hexynyl and the like; C.sub.2-10alkynyl defines
straight and branched chain hydrocarbon radicals having from 2 to
10 carbon atoms containing a triple bond such as the groups defined
for C.sub.2-6alkynyl and heptynyl, octynyl, nonynyl, decynyl and
the like; C.sub.1-3alkyl as a group or part of a group encompasses
the straight and branched chain saturated hydrocarbon radicals
having from 1 to 3 carbon atoms such as, methyl, ethyl and propyl;
C.sub.4-10alkyl encompasses the straight and branched chain
saturated hydrocarbon radicals as defined above, having from 4 to
10 carbon atoms. The term C.sub.1-6a defines straight or branched
chain saturated hydrocarbon radicals such as methoxy, ethoxy,
propyloxy, butyloxy, pentyloxy, hexyloxy, 1-methylethyloxy,
2-methylpropyloxy, 2-methylbutyloxy and the like;
C.sub.3-6cycloalkyloxy is generic to cyclopropyloxy, cyclobutyloxy,
cyclopentyloxy and cyclohexyloxy.
[0102] As used herein before, the term (.dbd.O) forms a carbonyl
moiety when attached to a carbon atom, a sulfoxide group when
attached once to a sulfur atom, and a sulfonyl group when attached
twice to a sulfur atom.
[0103] When any variable (e.g. aryl etc.) occurs more than one time
in any constituent, each definition is independent.
[0104] Lines drawn into ring systems from substituents indicate
that the bond may be attached to any of the suitable ring atoms.
For instance for compounds of formula (I), R.sup.4 can be attached
to any available carbon atom of the phenyl or pyridyl ring.
[0105] For use in the presently described medicaments and methods,
salts of the compounds of the present invention are those wherein
the counted on is pharmaceutically acceptable. However, salts of
acids and bases which are non-pharmaceutically acceptable may also
find use, for example, in the preparation or purification of a
pharmaceutically acceptable compound. All salts, whether
pharmaceutically acceptable or not are included within the ambit of
the present invention.
[0106] The pharmaceutically acceptable addition salts as mentioned
hereinabove are meant to comprise the microbicidal active non-toxic
addition salt forms which the compounds of the present invention
are able to form. The latter can conveniently be obtained by
treating the base form with such appropriate acids as inorganic
acids, for example, hydrohalic acids, e.g. hydrochloric or
hydrobromic acid and the like; sulfuric acid; nitric acid;
phosphoric acid and the like; or organic acids, for example,
acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic,
succinic, maleic, fumaric, malic, tartaric, citric,
methanesulfonic, ethanesulfonic, benzenesulfonic,
p-toluene-sulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic
and the like acids. Conversely the salt form can be converted by
treatment with alkali into the free base form.
[0107] The pharmaceutically acceptable addition salts as mentioned
hereinabove are also meant to comprise the microbicidal active
non-toxic base forms, in particular, metal or amine addition salt
forms which the compounds of the present invention are able to
form. Said salts can conveniently be obtained by treating the
compounds of the present invention containing acidic hydrogen atoms
with appropriate organic and inorganic bases such as, for example,
the ammonium salts, the alkali and earth alkaline metal salts, e.g.
the lithium, sodium, potassium, magnesium, calcium salts and the
like, salts with organic bases, e.g. primary, secondary and
tertiary aliphatic and aromatic amines such as methylamine,
ethylamine, propylamine, isopropylamine, the four butylamine
isomers, dimethylamine, diethylamine, diethanolamine,
dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine,
piperidine, morpholine, trimethylamine, triethylamine,
tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline,
the benzathine, N-methyl-D-glucamine,
2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrabamine salts, and
salts with amino acids such as, for example, arginine, lysine and
the like. Conversely said salt forms can be converted by treatment
with acid into the free acid form.
[0108] The term addition salts comprises as well the hydrates and
the solvent addition forms which the compounds of the present
invention are able to form. Examples of such forms are e.g.
hydrates, alcoholates and the like.
[0109] The term "quaternary amine" as used hereinbefore defines the
quaternary ammonium salts which the compounds are able to form by
reaction between a basic nitrogen of a compound and an appropriate
quaternizing, agent, such as, for example, an optionally
substituted alkylhalide, arylhalide or arylalkylhalide, e.g.
methyliodide or benzyliodide. Other reactants with good leaving
groups may also be used, such as alkyl trifluoromethanesulfonates,
alkyl methanesulfonates, and alkyl p-toluenesulfonates. A
quaternary amine has a positively charged nitrogen.
Pharmaceutically acceptable counterions include chloro, bromo,
iodo, trifluoroacetate and acetate. The counterion of choice can be
introduced using ion exchange resins.
[0110] The N-oxide forms of the present compounds are meant to
comprise the compounds wherein one or several tertiary nitrogen
atoms are oxidized to the so-called N-oxide.
[0111] The term stereochemically isomeric forms of the compounds of
the present invention, their N-oxides, addition salts, quaternary
amines, as used hereinbefore, defines all possible compounds made
up of the same atoms bonded by the same sequence of bonds but
having different three-dimensional structures which are not
interchangeable, which the compounds of the present invention may
possess. Unless otherwise mentioned or indicated, the chemical
designation of a compound encompasses the mixture of all possible
stereochemically isomeric forms that said compound may possess.
Said mixture may contain all diastereomers and/or enantiomers of
the basic molecular structure of said compound. All
stereochemically isomeric forms of the compounds both in pure form
or in admixture with each other are intended to be embraced within
the scope of the present invention.
[0112] In particular, stereogenic centers may have the R- or
S-configuration; substituents on bivalent cyclic (partially)
saturated radicals may have either the cis- or trans-configuration.
Compounds encompassing double bonds can have an E (entgegen) or Z
(zusammen)-stereochemistry at said double bond. The terms cis,
trans, R, S, E and Z are well known to a person skilled in the
art.
[0113] Some of the present compounds may also exist in their
tautomeric forms. Such forms although not explicitly indicated in
the above formula are intended to be included within the scope of
the present invention.
[0114] Whenever used hereinafter, the term "compounds", the term
"compounds of the present invention" is meant to include any
subgroup thereof, also the N-oxide forms, the pharmaceutically
acceptable addition salts, the quaternary amines and all
stereochemically isomeric forms. Of special interest are those
compounds which are stereochemically pure.
[0115] Whenever substituents can be selected each independently
from a list of numerous definitions, such as for example for
R.sup.6 and R.sup.7, all possible combinations are intended which
are chemically possible and which lead to chemically stable
molecules.
[0116] Suitable compounds of formula (I) are those wherein Y is
CR.sup.5 or N; A is CH, CR.sup.4 or N; n is 0, 1, 2, 3 or 4; Q is
--NR.sup.1R.sup.2; R.sup.1 and R.sup.2 are each independently
selected from hydrogen, hydroxy, C.sub.1-12alkyl,
C.sub.1-12allkyloxy, C.sub.1-12alkylcarbonyl,
C.sub.1-12allkyloxycarbonyl, aryl, amino, mono- or
di(C.sub.1-12alkyl)amino, mono- or di(C.sub.1-12alkyl)aminocarbonyl
wherein each of the aforementioned C.sub.1-12alkyl groups may
optionally and each individually be substituted with one or two
substituents each independently selected from hydroxy,
C.sub.1-6alkyloxy, hydroxyC.sub.1-6alkyloxy, carboxyl,
C.sub.1-6alkyloxycarbonyl, cyano, amino, amino, aminocarbonyl,
aminocarbonylamino, mono- or di(C.sub.1-6alkyl)amino, aryl and Het;
or R.sup.1 and R.sup.2 taken together may form pyrrolidinyl,
piperidinyl, morpholinyl, azido or mono- or
di(C-.sub.1-12alkyl)aminoC.sub.1-4alkylidene; R.sup.3 is hydrogen,
aryl, C.sub.1-6alkylcarbonyl, C.sub.1-6alkyl,
C.sub.1-6alkyloxycarbonyl, C.sub.1-6alkyl substituted with
C.sub.1-6alkyloxycarbonyl; each R.sup.4 independently is hydroxy,
halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, cyano, aminocarbonyl,
nitro, amino, trihalomethyl, trihalomethyloxy; R.sup.5 is hydrogen
or C.sub.1-4alkyl; L is --X.sup.1--R.sup.6 or -X.sup.2-Alk-R.sup.7
wherein R.sup.6 and R.sup.7 each independently are phenyl or phenyl
substituted with one, two, three, four or five substituents each
independently selected from halo, hydroxy, C.sub.1-6alkyl,
C.sub.1-6alkyloxy, C.sub.1-6alkylcarbonyl,
C.sub.1-6alkyloxycarbonyl, formyl, cyano, nitro, amino, and
trifluoromethyl, X.sup.1 and X.sup.2 are each independently
--NR.sup.3--, --NH--NH--, --N.dbd.N--, --O--, --S--, --S(.dbd.O)--
or --S(.dbd.O).sub.2--, and Alk is C.sub.1-4alkanediyl; aryl is
phenyl or phenyl substituted with one, two, three, four or five
substituents each independently selected from halo, C.sub.1-6alkyl,
C.sub.1-6alkyloxy, cyano, nitro and triflouromethyl; Het is an
aliphatic or aromatic heterocyclic radical; said aliphatic
heterocyclic radical is selected from pyrrolidinyl, piperidinyl,
hornopiperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl and
tetrahydrothienyl wherein each of said aliphatic heterocyclic
radical may optionally be substituted with an oxo group; and said
aromatic heterocyclic radical is selected from pyrrolyl, furanyl,
thienyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl wherein
each of said aromatic heterocyclic radical may optionally be
substituted with hydroxy.
[0117] Most preferred compounds of formula (I) are
[0118]
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-2-pyrimidinyl]amino]benz-
onitrile;
[0119]
6-[(2,6-dichlorophenyl)methyl]-N2-(4-fluoropheny)-2,4-pyrimidinedia-
mine;
[0120]
4-[[4-[(2,4-dichlorophenyl)methyl]-6-[(4-hydroxybutyl)amino]-2-pyri-
midinyl]amino]-benzonitrile;
[0121]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(3-hydroxypropyl)amino]-2-pyr-
imidinyl]-amino]benzonitrile;
[0122]
N-[2-[(4-cyanophenyl)amino]-6-[(2,6-dichlorophenyl)methyl]-4-pyrimi-
dinyl]-acetamide,
[0123]
N-[2-[(4-cyanophenyl)amino]-6-[(2,6-dichlorophenyl)methyl]-4-pyrimi-
dinyl]-butanamide;
[0124]
4-[[2-amino-6-(2,6-dichlorophenoxy)-4-pyrimidinyl]amino]benzonitril-
e;
[0125]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(2-hydroxy-2-phenylethyl)amin-
o]-2-pyrimidinyl]amino]benzonitrile;
[0126]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[3-(2-oxo-1-pyrrolidinyl)prop-
yl]amino]-2-pyrimidinyl]amino]benzonitrile;
[0127]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[2-(2-hydroxyethoxy)ethyl]ami-
no]-2-pyrimidinyl]amino]berizontrile monohydrochloride;
[0128]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[(2,3-dihydroxypropyl)amino]-2-
-pyrimidinyl]-amino]benzonitrile;
[0129]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-(hydroxyamino)-2-pyrimidinyl]a-
mino]-benzonitrile;
[0130]
4-[[4-[(2-cyanoethyl)amino]-6-[(2,6-dichlorophenyl)methyl]-2-pyrimi-
dinyl]amino]-benzonitrile;
[0131]
4-[[4-[(2,6-dichlorophenyl)methyl]-6-[[2-(1-pyrroldinyl)ethyl]amino-
]-2-pyrimidinyl]-amino]benzonitrile;
[0132]
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-5-methyl-2-pyrimidinyl]a-
mino]-benzonitrile;
[0133]
N2-(4-bromophenyl)-6-[(2,6-dichlorophenyl)methyl]-5-methyl-2,4-pyri-
midinediamine;
[0134]
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitri-
le;
[0135]
4-[[2-[(2,4,6-trimethylphenyl)amino]-4-pyrimidinyl]amino]benzonitri-
le;
[0136]
4-[[4-[(2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile;
[0137]
4-[[4-(2,4,6-trimethylphenoxy)-2-pyrimidinyl]amino]benzonitrile;
[0138]
4-[[4-[(2,6-dichlorophenyl)thio]-2-pyrimidinyl]amino]benzonitrile;
[0139]
4-[[4-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-2-pyrimidinyl]am-
ino]benzonitrile;
[0140]
4-[[4-[[2,6-dichloro-4-(trifluoromethyl)phenyl]amino]-2-pyrimidinyl-
]amino]benzonitrile;
[0141]
4-[[4-[(2,4-dichloro-6-methylphenyl)amino]-2-pyrimidinyl]amino]benz-
onitrile;
[0142]
4-[[2-[(cyanophenyl)amino]-4-pyrimidinyl]amino]-3,5-dimethylbenzoni-
trile;
[0143]
4-[[4-[(2,4-dibromo-6-fluorophenyl)amino]-2-pyrimidinyl]amino]benzo-
nitrile;
[0144]
4-[[4-amino-6-[(2,6-dichlorophenyl)methyl]-5-methyl-2-pyrimidinyl]a-
mino]-benzeneacetonitrile;
[0145]
4-[[4-[methyl(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benz-
onitrile;
[0146]
4-[[4-[(2,4,6-trichlorophenyl)amino]-2-pyrimidinyl]amino]benzonitri-
le;
[0147]
4-[[4-[(2,4,6-trimethylphenyl)thio]-2-pyrimidinyl]amino]benzonitril-
e;
[0148]
4-[[4-[(2,4,6-trimethylphenyl)amino-2-pyrimidinyl]amino]benzonitril-
e;
[0149]
4-[[4-amino-6-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]be-
nzonitrile;
[0150]
4-[[2-amino-6-[(2,4,6-trimethylphenyl)amino]-4-pyrimidinyl]amino]be-
nzonitrile;
[0151]
4-[[4-(2-bromo-4-chloro-6-methylphenoxy)-2-pyrimidinyl]amino]benzon-
itrile;
[0152]
4-[[4-[(4-chloro-2,6-dimethylphenyl)amino]-2-pyrimidinyl]amino]benz-
onitrile;
[0153]
3,5-dichloro-4-[[2-[(4-cyanophenyl)amino]-4-pyrimidinyl]amino]benzo-
nitrile;
[0154]
4-[[4-[[2,6-dichloro-4-(trifluoromethoxy)phenyl]amino]-2-pyrimidiny-
l]amino]-benzonitrile;
[0155]
4-[[4-[(2,4-dibromo-3,6-dichlorophenyl)amino]-2-pyrimidinyl]amino]b-
enzonitrile;
[0156]
4-[[4-[(2,6-dibromo-4-propylphenyl]amino]-2-pyrimidinyl]amino]benzo-
nitrile;
[0157]
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzamide;
[0158]
4-[[4-[(4-(1,1-dimethylethyl)-2,6-dimethylphenyl)amino]-2-pyrimidin-
yl]amino]benzonitrile;
[0159]
4-[[2-[(4-cyanophenyl)amino]4-pyrimidinyl]oxy]-3,5-dimethylbenzonit-
rile;
[0160]
4-[[4-[(4-chloro-2,6-dimethylphenyl)amino]-5-methyl-2-pyrimidinyl]a-
mino]-benzonitrile;
[0161]
4-[[2-[(4-cyanophenyl)amino]-5-methyl-4-pyrimidinyl]amino-3,5-dimet-
hylbenzonitrile;
[0162]
4-[[4-[[4-(1,1-dimethylethyl)-2,6-dimethylphenyl]amino]-5-methyl-2--
pyrimidinyl]-amino]benzonitrile;
[0163]
4-[[4-[(4-bromo-2,6-dimethylphenyl)amino]-5-methyl-2-pyrimidinyl]am-
ino]-benzonitrile;
[0164]
4-[[5-methyl-4-[(2,4,6-trimethylphenyl)thio]-2-pyrimidinyl]amino]be-
nzonitrile;
[0165]
4-[[4-[(2,6-dibromo-4-propylphenyl)amino]-5-methyl-2-pyrimidinyl]am-
ino]-benzonitrile;
[0166]
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzamide,
N3-oxide;
[0167]
N2-(4-chlorophenyl)-N4-(2,4,6-trimethylphenyl)-2,4-pyrimidinediamin-
e;
[0168]
4-[[4-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-5-methyl-2-pyrim-
idinyl]amino]-benzonitrile;
[0169]
4-[[2-[(4-cyanophenyl)amino]-5-methyl-4-pyrimidinyl]amino]-3,5-dime-
thyl benzonitrile;
[0170]
4-[[4-[(phenylmethyl)amino]-2-pyrimidinyl]amino]benzonitrile;
[0171]
4-[[4-amino-6-(2,6-dimethylphenoxy)-1,3,5-triazin-2-yl]amino]benzon-
itrile;
[0172]
4-[[4-amino-6-[(2-chloro-6-methylphenyl)amino]-1,3,5-triazin-2-yl]a-
mino]benzonitrile;
[0173]
4-[[4-amino-6-[(2,4,6-trimethylphenyl)amino]-1,3,5-triazin-2-yl]ami-
no]benzonitrile;
[0174]
4-[[4-(hydroxyamino)-6-[(2,4,6-trimethylphenyl)amino]-1,3,5-triazin-
-2-yl]amino]-benzonitrile;
[0175]
4-[[4-amino-6-[(2-ethyl-6-methylphenyl)amino]-1,3,5-triazin-2-yl]am-
ino]benzonitrile;
[0176]
4-[[4-amino-6-[(2,6-dichlorophenyl)thio]-1,3,5-triazin-2-yl]amino]b-
enzonitrile;
[0177]
4-[[4-(hydroxyamino)-6-[(2,4,6-trichlorophenyl)amino]-1,3,5-triazin-
-2-yl]amino]-benzonitrile;
[0178]
4-[[4-amino-6-(2,4,6-trimethylphenoxy)-1,3,5-triazin-2-yl]amino]ben-
zonitrile;
[0179]
4-[[4-(hydroxyamino)-6-(2,4,6-trimethylphenoxy)-1,3,5-triazin-2-yl]-
amino]-benzonitrile;
[0180] 4-[[4-amino-6-[(2,4-dichloro-6-methylphenyl)amino]-1
3,5-triazin-2-yl]amino]-benzonitrile;
[0181]
4-[[4-[(2,4-dichloro-6-methylphenyl)amino]-6-(hydroxyamino)-1,3,5-t-
riazin-2-yl ]-amino]benzonitrile;
[0182]
4-[[4-(hydroxyamino)-6-(2,4,6-trichlorophenoxy)-1,3,5-triazin-2-yl]-
amino]benzonitrile trifluoroacetate (1:1);
[0183]
4-[[4(4-acetyl-2,6-dimethylphenoxy)-6-amino-1,3,5-triazin-2-yl]amin-
o]benzonitrile;
[0184]
4-[[4-amino-6-(2,4,6-tribromophenoxy)-1,3,5-triazin-2-yl]amino]benz-
onitrile;
[0185]
4-[[4-amino-6-(4-nitro-2,6-dimethylphenoxy)-1,3,5-tiazin-2-yl]amino-
]benzonitrile;
[0186]
4-[[4-amino-6-(2,6-dibromo-4-methylphenoxy)-1,3,5-triazin-2-yl]amin-
o]benzonitrile;
[0187]
4-[[4-amino-6-(4-formyl-2,6-dimethylphenoxy)-1,3,5-triazin-2-yl]ami-
no]benzonitrile;
[0188]
4-[[4-amino-6-[(2,4-dichlorophenyl)thio]-1,3,5-triazin-2-yl]amino]b-
enzonitrile;
[0189] 4-[[4-[(5-acetyl
-2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-6-amino-1,3,5-triazin-2-yl]-amin-
o]benzonitrile;
[0190]
4-[[4-amino-6-[(4-bromo-2-chloro-6-methylphenyl)amino]-1,3,5-triazi-
n-2-yl]amino]-benzonitrile;
[0191]
4-[[4-amino-6-[(2-chloro-4,6-dimethylphenyl)amino]-1,3,5-triazin-2--
yl]amino]-benzonitrile;
[0192]
4-[[4-amino-6-[[2,4-dichloro-6-(trifluoromethyl)phenyl]amino]-1,3,5-
-triazin-2-yl]-amino]benzonitrile;
[0193]
4-[[4-amino-6-[methyl(2,4,6-trimethylphenyl)amino]-1,3,5-triazin-2--
yl]-amino]-benzonitrile;
[0194]
4-[[4-amino-6-[(2,6-dibromo-4-methylphenyl)amino]-1,3,5-triazin-2-y-
l]-amino]-benzonitrile;
[0195]
4-[[4-amino-6-[[2,6-dibromo-4-(1-methylethyl)phenyl]amino]-1,3,5-tr-
iazin-2-yl]-amino]benzonitrile,
[0196] the N-oxides, the pharmaceutically acceptable addition salts
and the stereochemically isomeric forms thereof.
[0197] Suitable compounds of forint/la (II) are those wherein one
or more of the following restrictions apply: [0198]
-b.sup.1=b.sup.2-C(R.sup.2a)=b.sup.3-b .sup.4= is a radical of
formula (b-1); [0199] q is 0; [0200] R.sup.2a is cyano or
--C(.dbd.O)NH.sub.2, preferably R.sup.2a is cyano; [0201] Y is
cyano, --C(.dbd.O)NH.sub.2 or a halogen, preferably a halogen;
[0202] Q is hydrogen or --NR.sup.4R.sup.5 wherein R.sup.4 and
R.sup.5 are preferably hydrogen; [0203] L is --X--R.sup.3 wherein X
is preferably --NR.sup.1--, --O-- or --S--, most preferably X is
--NH--, and R.sup.3 is substituted phenyl with C.sub.1-6alkyl,
halogen and cyano as preferred substituents.
[0204] Another interesting group of compounds of formula (II) are
those compounds wherein L is --X--R.sup.3 wherein R.sup.3 is
2,4,6-trisubstituted phenyl, each substituent independently
selected from chloro, bromo, fluoro, cyano or C.sub.1-4alkyl.
[0205] Also interesting are those compounds of formula (II) wherein
Y is chloro or bromo and Q is hydrogen or amino.
[0206] Particular compounds are those compounds of formula (II)
wherein the moiety in the 2 position of the pyrimidine ring is a
4-cyano-anilino group.
[0207] Preferred compounds are those compounds of formula (II)
wherein the moiety in the 2 position of the pyrimidine ring is a
4-cyano-anilino group, L is --X--R.sup.3 wherein R.sup.3 is a
2,4,6-trisubstituted phenyl, Y is a halogen and Q is hydrogen or
NH.sub.2,
[0208] Most preferred compounds of formula (II) are:
[0209]
4-[[4-amino-5-chloro-6-[(2,4,6-trimethylpheny)amino]-2-pyrimidinyl]-
amino]-benzonitrile;
[0210]
4-[[5-chloro-4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]b-
enzonitrile;
[0211]
4-[[5-bromo-4-(4-cyano-2,6-dimethylphenoxy)-2-pyrimidinyl]amino]ben-
zonitrile;
[0212]
4-[[4-amino-5-chloro-6-[(4-cyano-2,6-dimethylphenyl)amino]-2-pyrimi-
dinyl]amino]-benzonitrile;
[0213]
4-[[5-bromo-6-[(4-cyano-2,6-dimethylphenyl)amino]-2-pyrimidinyl]ami-
no]benzonitrile;
[0214]
4-[[4-amino-5-chloro-6-(4-cyano-26-dimethylphenyloxy)-2-pyrimidinyl-
]amino]-benzonitrile; and
[0215]
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl-
]amino]-benzonitrile; the N-oxides, the pharmaceutically acceptable
addition salts, quaternary amities and the stereochemically
isomeric forms thereof.
[0216] An interesting group of compounds are those compounds of
formula (III) wherein one or more of the following conditions are
met: [0217] n is 1; [0218] -a.sup.1=a.sup.2-a.sup.3=a.sup.4-
represents a bivalent radical of formula (a-1); [0219] R.sup.1 is
hydrogen or C.sub.1-6alkyl; [0220] R.sup.2 is cyano; aminocarbonyl;
mono- or di(methyl)aminocarbonyl; C.sub.1-6alkyl substituted with
cyano, aminocarbonyl or mono- or di(methyl)aminocarbonyl; and more
in particular, R.sup.2 is on the 4 position relative to the
--NR.sup.1-- moiety; [0221] L is --X--R.sup.3 wherein X is
preferably --NR.sup.1--, --O-- or --S--, most preferably X is
--NH--, and R.sup.3 is substituted phenyl with C.sub.1-6alkyl,
halogen and cyano as preferred substituents.
[0222] Preferred compounds are those compounds of formula (III)
wherein L is --X--R.sup.3 wherein R.sup.3 is a disubstituted phenyl
group or a trisubstituted phenyl group, each substituent
independently selected from chloro, bromo, fluoro, cyano or
C.sub.1-4alkyl.
[0223] Most preferred compound of formula (III) is
4-[[4-[(2,4,6-trimethylphenyl)amino]-1,3,5-triazin-2-yl]amino]benzonitril-
e.
[0224] Particular compounds compounds of the present invention
include
4-[[4-amino-5-bromo-6-(4-cyano-2,6-dimethylphenyloxy)-2-pyrimidinyl]amino-
]benzonitrile (compound A) and
4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile
(compound B), their N-oxides, pharmaceutically acceptable salts and
stereoisomers thereof.
[0225] The compounds of the present invention can be prepared
according to art-known procedures. In particular, they are prepared
according to the procedures described in EP 1002795, WO 99/50250,
WO 99/50256 and WO 00/27828.
[0226] The compounds of the present invention have microbici dal
activity and have the ability to prevent the transmission of HIV.
In particular, they can prevent sexual or vaginal transmission of
HIV by preventing either the production of infectious viral
particles or infection of uninfected cells. If infected cells in
sperm can reach the mucosa, the compounds of the present invention
can prevent HIV infection of host cells, such as macrophages,
lymphocytes, Langerhans and M cells. Thus, the present compounds
prevent systemic HIV infection of a human being, exhibiting a
prophylactic action against HIV. Evidence for this microbicidal
activity is given in the experimental part and is based on in vivo
activity of compound B in a human SCID (Severe Combined Immune
Deficiency) animal model (Di Fabio et al., AIDS 2001, 15,
2231-2238) and on in vitro activity of Compound B in a model based
on immature monocyte derived dendritic cells.
[0227] In addition, it has been found that the compounds of this
invention have a killing effect on the Haemophilus ducreyi
bacteria. As such, the compounds of this invention may be used in
the prevention and treatment of chancroids, the venereal disease
caused by this bacteria. These additional effects will even improve
the effectiveness of the present compounds in preventing infection
with HIV.
[0228] The compounds of the invention may be formulated into
pharmaceutical compositions that can be used to apply microbicides
to effectively prevent transmission of pathogens through mucosae
and/or skin, more particularly to prevent the sexual or vaginal
transmission of HIV. Thus, the compositions are in forms adapted to
be applied to the site where sexual intercourse or related intimate
contact takes place, such as the genitals, vagina, vulva, cervix,
rectum, mouth, hands, lower abdomen, upper thighs, especially the
vagina, vulva, cervix, and ano-rectal mucosae.
[0229] The compounds of the present invention may be formulated
into pharmaceutical compositions designed for immediate release or
sustained or slow release.
[0230] As appropriate topical compositions there may be cited for
example gels, jellies, creams, pastes, emulsions, dispersions,
ointments, films, sponges, foams, aerosols, powders, intravaginal
rings or other intravaginal drug delivery systems, cervical caps,
implants, patches, suppositories or pessaries for rectal, or
vaginal application, vaginal or rectal or buccal tablets,
mouthwashes.
[0231] To prepare the pharmaceutical compositions of this
invention, an effective amount of the particular compound,
optionally in addition salt form, as the active ingredient may be
combined in intimate admixture with a pharmaceutically acceptable
carrier, which carrier may take a wide variety of forms depending
on the form of administration. For example, in preparing the
compositions for topical oral administration, any of the usual
pharmaceutical media may be employed such as, for example, water,
glycols, oils, alcohols and the like, which are suitable for oral
liquid preparations such as mouthwashes in the form of suspensions,
emulsions and solutions. Solid carriers such as starches, sugars,
kaolin, diluents, lubricants, binders, disintegrating agents and
the like will be adequate in the case of tablets. Also included are
solid form preparations which are intended to be converted, shortly
before use, to liquid form preparations. In the compositions
suitable for topical cutaneous administration, the carrier
optionally comprises a suitable wetting agent, optionally combined
with suitable additives of any nature in minor proportions, which
additives do not introduce a significant deleterious effect on the
skin. Said additives may facilitate the administration to the skin
and/or may be helpful for preparing the desired compositions. These
compositions may be administered in various ways, e.g., as a cream
or gel.
[0232] The active ingredient may be present in the pharmaceutical
formulations as a free agent or alternatively, encapsulated into
drug carriers like liposomes, nanoparticles or cyclodextrins, which
encapsulation results in an increased concentration of the
compounds within the microbe target site. The active ingredient may
also be present as nanoparticles.
[0233] Liposomes may be present in the formulation which include,
amongst others, distearoylphosphatidylcholine (DSPC),
distearoylphosphatidyglycerol (DSPG),
distearoylphosphatidylethanolamine-polyethylene-glycol (DSPE-PEG),
dipalmitoylphosphatidylcholine (DPPC), dicetylphosphate (DP),
cholesterol (CHOL), dipalmitoylphosphatidylglycerol (DPPG), and
combinations thereof, such as distearoylphosphatidylcholine (DSPC):
distearoylphosphatidylglycerol (DSPC); within which the active
ingredient is entrapped.
[0234] Appropriate cyclodextrins are .alpha.-, .beta.-,
.gamma.-cyclodextrins or ethers and mixed ethers thereof wherein
one or more of the hydroxy groups of the anhydroglucose units of
the cyclodextrin are substituted with C.sub.1-6alkyl, particularly
methyl, ethyl or isopropyl, e.g. randomly methylated .beta.-CD;
hydroxyC.sub.1-6alkyl, particularly hydroxyethyl, hydroxypropyl or
hydroxybutyl; carboxy C.sub.1-6alkyl, particularly carboxymethyl or
carboxyethyl; C.sub.1-6alkylcarbonyl, particularly acetyl.
Especially noteworthy as complexants and/or solubilizers are
.beta.-CD, randomly methylated .beta.-CD, 2,6-dimethyl-.beta.-CD,
2-hydroxyethyl-.beta.-CD, 2-hydroxyethyl-.beta.-CD,
2-hydroxypropyl-.beta.-CD and (2-carboxymethoxy)propyl-.beta.-CD,
and in particular 2-hydroxypropyl-62 -CD (2-HP-.beta.-CD).
Cyclodextrins are additionally useful in enhancing the solubility
of the compounds.
[0235] The term mixed ether denotes cyclodextrin derivatives
wherein at least two cyclodextrin hydroxy groups are etherified
with different groups such as, for example, hydroxy-propyl and
hydroxyethyl.
[0236] Particularly, the present compounds may be formulated as a
gel formulation comprising: [0237] a topically effective amount of
a compound of the present invention; [0238] a gel-forming compound;
[0239] a buffer; [0240] a pharmaceutically acceptable diluent,
preferably water; [0241] optionally a humectant; and [0242]
optionally a preservative.
[0243] Typical gel formulations can be prepared using natural or
synthetic polymers as gelifying agents, and hydrophobic or
hydrophilic liquids. Examples of gel-forming compounds commonly
employed in gel formulations include polysaccharides which include
cellulose derivatives, glycosaminoglycans, gums, starch (a-amylose
or amylopectin), and chitosan; carboxyvinylic derivates, vinyl
polymers such as polyethylenes, polyehtyelene glycols, e.g.
polyethylene glycol 4500, Plastibase.RTM. (Plasticized Hydrocarbon
Gel), polyacrylic acid, (Carbopols.RTM. family, e.g. Carbopol.RTM.
940), polymethacrylic acid, polyvinyl pyrrolidone and polyvinyl
alcohol; polyacrylamide or polymethacrylamide polymers including
clays such as bentonite, Veegum.RTM. (R.T Vanderbilt) and
Laponite.RTM. (Laporte Industries);
polyoxyethylene-polyoxypropylene or polyethylene oxides copolymers
such as poloxamers, e.g. poloxamer 407, poloxamines; proteins,
colloidal silica, soaps, silicones such as dimethylpolysiloxanes or
dimeticone, hydrocarbonated bases (mixtures of parafine and
yaselines).
[0244] Useful cellulose derivates include methyl cellulose,
hydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxypropylmethyl cellulose, carboxymethyl cellulose. Useful
glycosaminoglycans include hyaluronic acid, chondroitin,
chondroitin-4-sulfate, heparan sulfate and heparin. Useful gums
include natural and artificial gums, tragacanth, carrageenan,
pectin, agar, alginic acid, dextrans. The glycosaminoglycans may be
used to enhance wound healing in combination with any other
gel-forming polymer such as, for example, collagen, gelatin,
fibronectin. A preferred gelifying agent is hydroxyethyl cellulose,
which has additionally bioadhesive properties.
[0245] Concentrations of the gel-forming compounds may be varied
upon conditions such as the liquid/gel transition temperature, the
physical properties sought for the gel and the pH used in the
making of the formulations.
[0246] Gel forming compounds employed in the present invention are
typically water-soluble polymers capable of forming a viscous
aqueous solution, or non-water soluble, water-swellable polymers
(e. g., collagen) that can also form a viscous solution and that
gel upon contact with skin. Gelling agents suitable for use in the
present invention should be stable over a wide pH range, especially
over the normal acidic pH values found in the vagina.
[0247] Buffering agents are used in the gel formulation of this
invention to maintain the pH of the vagina within its healthy
acidic range (i.e., a pH of less than about 5 and more preferably
in the range of about 3.2 to about 4.5) even in the presence of
normal amounts of ejaculate. A normal acidic range in the vaginal
milieu and environment assists in diminishing the activity of
certain STD-causing microbes, including the HIV virus. Maintaining
the normal vaginal milieu also assists in maintaining the body's
natural defenses against certain STD-causing microorganisms.
Examples of buffering agents include, without being limited to,
lactic acid, phosphoric acid, sodium citrate, sodium hydroxide,
sodium phosphate, sodium phosphate dibasic anhydrous, tartaric
acid, triethanolamine, citric acid, potassium acid tartrate,
benzoic acid, alginic acid, sorbic acid, fumaric acid, ascorbic
acid, stearic acid, oleic acid, edetic acid,
ethylenediaminetetracetic acid, acetic acid, malic acid, and the
like, preferably sodium hydroxide and lactic acid, the latter being
additionally a preservative and having certain antimicrobial
activity.
[0248] The acids acids may be added as free acids, hydrates, or
pharmaceutically acceptable salts. Free acids can be converted to
the corresponding salts in situ (i.e., within the vagina). It is
generally preferred that several buffering agents are included in
the gel of this invention to provide increased buffering capacity.
Even more preferably, buffering agents comprise a combination of
acid and hydrogen-accepting substance that occur naturally in the
human female body that, when applied to the surface of the vagina,
maintains the pH level thereon at approximately the pH level of a
healthy vagina. Said acid(s) may be selected from the froup
consisting of acetic acid, lactic acid, phosphoric acid and
sulfuric acid, and combinations thereof. One of the characteristics
common to each of said member in said group, is that each acid
occurs naturally in the female body. Another common characteristic
is each readily contributes to the formation of a buffering system,
by temporarily donating hydrogen ion and accepting a cation to form
a salt.
[0249] Said hydrogen-accepting substance may be selected from the
group consisting of potassium hydroxide, sodium hydroxide, calcium
hydroxide, potassium carbonate, sodium carbonate and calcium
carbonate, and combinations thereof. One of the characteristics
common to each of said member in said group, is that each
substances is found naturally in the female body. Another common
characteristic is each readily contributes to the formation of a
buffering system, by temporarily accepting hydrogen ion and
donating a cation to form a salt. Such salts may be selected from
the group consisting of acetate, lactate, phosphate and sulfate, in
combination with said cation from said hydrogen-accepting
substance.
[0250] The gels of this invention may also include, and preferably
do include, humectants. Suitable humectants include, for example,
glycerol, polyethylene glycols, propylene glycols, sorbitol,
triacetin, and the like. Glycerol, which is the preferred
humectant, is a buffer activating component due to its capability
of water absortion, or other fluid, from the vaginal environment
into the gel. It is believed that such fluid intake prevents the
formation of a dry film on the gel when placed within the vagina,
providing additional solvent to enhance the application of the gel
formulation, or to otherwise enhance its functioning.
[0251] The gels of this invention may also include, and preferably
do include, a preservative, which amongst other properties, extends
the shelf life of the gel formulations. Suitable preservatives
include, for example, benzoic acid, sodium benzoate, methyl
paraben, ethyl paraben, butyl paraben, propyl paraben,
benzylalkonium chloride, phenylmercuric nitrate, chlorhexidine,
benzyl alcohol, phenethyl alcohol, propylene glycol, and the like.
The preferred preservatives are methyl paraben, and propyl paraben,
which both also contribute to the antimicrobial capacity of the
gel.
[0252] The gels of this invention are prepared using conventional
gel preparation techniques. It is desirable, however, to ensure
that the buffering agents are solubilized in the final product and
that the entrapment of air in the gel is avoided or at least kept
to a minimum. To reduce the entrapment of air in the gel, it is
generally preferred that the less hydrophilic agents are added in
small increments. Alternatively, the gels of this invention can
also be prepared in readily dispersable solid forms (e.g., powders,
tablets, and the like) which can be converted to the desired gel
consistency by action of aqueous based fluids external to or within
the vagina when desired. As those skilled in the art will realize,
the methods for preparing the gels of this invention can be
modified for batch, semi continuous, or continuous operation so
long as the resulting gels have the desired and beneficial
properties described herein.
[0253] The gel formulations can be combined with other active
ingredients such as microbicides, antimicrobials, chemotherapeutic
agents, antiinflammatory agents, spermicides or other appropriate
drugs. Furthermore, microbicides or spermicides or both can be
combined with liposomes (or other drug carriers) to prevent any
disease of mucosae and/or skin. In addition, gel or liposome or
other drug carriers formulations can also be used as carriers of
vaccines against infections caused by pathogens or any disease. If
desired, flavorants, scents, fragrances, and colorants can be
incorporated into the gel so long as they do not interfere with the
protection afforded by the gel. Indeed, incorporation of such
flavorants, scents, fragrances, and colorants into the compositions
of this invention may provide further protection by increasing the
probability that the gel will be used during sexual activity.
[0254] In one embodiment, the gel formulation is composed of
compound B, hydroxyethyl cellulose (HEC), glycerol, methyl paraben,
propyl paraben, lactic acid, sodium hydroxide (for reaching a pH
around 4.5), and water.
[0255] In another embodiment, the gel formulation comprises
compound B, HEC with a concentration from about 0.5 to about 5%
(w/w), glycerol with a concentration from about 1 to about 15%
(w/w), methyl paraben with a concentration from about 0.02 to about
0.5% (w/w), propyl paraben with a concentration from about 0.005 to
about 0.2% (w/w), lactic acid with a concentration from about 0.005
to about 0.5% (w/w), sodium hydroxide in sufficient quantity to
achieve a pH of 4.5, and water.
[0256] In another embodiment, the gel formulation comprises
compound B, HEC with a concentration from about 1 to about 3%
(w/w), glycerol with a concentration from about 3 to about 7%
(w/w), methyl paraben with a concentration from about 0.1 to about
0.3% (w/w), propyl paraben with a concentration from about 0.01 to
about 0.03% (w/w), lactic acid with a concentration from about 0.03
to about 0.07% (w/w), sodium hydroxide in sufficient quantity to
achieve a pH of 4.5, and water.
[0257] In another embodiment, any of the above gel formulations
comprise compound A as a microbicide.
[0258] The present topical formulations such as the gel
formulations described herein are to be used for coating different
types of mucosae such as vulvar, vaginal, cervical, ano-rectal,
mouth, or skin to prevent the penetration of pathogens such as
viruses, bacteria, fungi, parasites, ectoparasites and
mycoplasmas.
[0259] The present topical formulations such as the gel
formulations described herein could, for example, be applied into
the vagina by hand, suppositories, or conventional tampon or
syringe techniques. The method of administering or delivering the
gel into the vagina is not critical so long as an effective amount
of the gel is delivered into the vagina. The present topical
formulations such as the gel formulations described herein may also
be used for protection during anal intercourse and can be applied
using similar techniques.
[0260] For vaginal heterosexual intercourse, the present topical
formulations such as the gel formulations described herein may be
applied into the vagina prior to intercourse. For anal intercourse
(heterosexual or homosexual), the present topical formulations such
as the gel formulations described herein may be inserted into the
rectum prior to intercourse. For either vaginal or anal
intercourse, the present topical formulations such as the gel
formulations described herein may also act as a lubricant. For
added protection it is generally preferred that the present topical
formulations such as the gel formulations described herein be
applied before intercourse or other sexual activity and that, if
appropriate, a condom be used. For even further protection, the
present topical formulations such as the gel formulations described
herein can be applied as soon as possible after completion of the
sexual activity. Although application only after the sexual
activity is less recommended, it would still be desirable
afterwards if the application was not performed prior to the sexual
activity for any reason (e.g., in cases of rape).
[0261] The present topical formulations such as the gel
formulations described herein are highly suited for the protection
of women (as well as their partners) with or without requiring the
partner's knowledge of the application of these gels. In addition,
reliance on the partner's claim of being STD-free, concretely
HIV-free, would not be necessary, neither the agreement to use
condoms or other barrier devices for protection.
[0262] The gel formulations of the present invention are
additionally advantageous because they do not significantly affect
or inhibit the growth characteristics of the normal vaginal flora
or otherwise significantly irritate the vaginal tissue when used at
inhibitory, noncytotoxic, or clinical concentrations. Significant
inhibition or modifications of the vaginal flora or other
irritations can lead to increased risks of infections (both STD and
non-STD types) frequently mediated by ulcerations in the vagina,
unusual discharges, general discomforts, and the like.
[0263] Intravaginal rings (IVR) are as well suitable drug delivery
systems for the vaginal administration of the compounds of the
present invention. IVRs comprise the compound(s) dispersed
throughout a biocompatible elastomeric system that forms the
delivery device, which preferentially takes the form of a ring.
These elastomers preferably include hydrophobic material, such as
silicones (organo polysiloxanes including dimethylpolysiloxanes),
polyethylene-co-poly (vinyl acetate), styrene-butadiene-styrene
block copolymers, polyphosphazenes, poly(isoprene), poly
(isobutylene), polybutadienes, polyurethanes, nitrile rubbers,
neoprene rubbers or mixtures thereof. Said IVRs can be formulated
as sustained-released microbicides, resulting in an extended and
stable contact time between the compound and target pathogens and
cells. IVR formulations have already been described in the
literature, WO02076426 all of which is herein incorporated by
reference.
[0264] In order to increase the residence time of the topical
pharmaceutical composition at the site of administration, it may be
advantageous to include a bioadhesive in the different drug
delivery systems, in particular a bioadhesive polymer. A
bioadhesive may be defined as a material that adheres to a living
biological surface such as for example a mucus membrane or skin
tissue. The term bioadhesive is well-known to the person skilled in
the art. Thus, the present invention also relates to a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and as active ingredient a microbicidal effective amount of
a compound of the invention characterized in that the
pharmaceutical composition is bioadhesive to the site of
application. Preferably, the site of application is the vagina,
vulva, cervix, rectum, mouth or skin, most preferred is the vagina
and the vulva.
[0265] Examples of bioadhesives which may be used in the
pharmaceutical compositions of the present invention comprise
polyacrylic acid derivatives, such as for example carbopol or
polycarbophil, e.g. carbopol 934P, carbopol 940, polycarbophil AA1;
cellulose ether derivatives such as for example hydroxypropyl
methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose,
sodium carboxymethyl cellulose, chitosan; natural polymers such as
for example alginates, tragacanth, inulin; pregelatinized starch;
polysaccharide gums such as xanthan gum, and the like.
[0266] Alternatively, formulations of the present invention may be
in the form of implants, patches, pads, injections or other
preparations for achieving a percutaneous and subcutaneous delivery
of the compounds to the cervical, vaginal and rectal tissues.
[0267] As already indicated within the gel specifications, the
present compounds may be used in all the suitable formulations,
alone or in combination with other active ingredients, such as
antivirals, antibiotics, immunomodulators or vaccines. They may
also be used alone or in combination with other prophylactic agents
for the prevention of viral infections. The present compounds may
be used in vaccines and methods for protecting individuals against
viral infections over an extended period of time. The compounds may
be employed in such vaccines either alone or together with other
compounds of this invention or together with other anti-viral
agents in a manner consistent with the conventional utilization of
reverse transcriptase inhibitors in vaccines. Thus, the present
compounds may be combined with pharmaceutically acceptable
adjuvants conventionally employed in vaccines and administered in
prophylactically effective amounts to protect individuals over an
extended period of time against HIV infection.
[0268] Antiviral compounds which may be used in combination with
the compounds of the invention may be known antiretroviral
compounds such as suramine, pentamidine, thymopentin,
castanospermine, dextran (dextran sulfate), foscarnet-sodium
(trisodium phosphono formate); nucleoside reverse transcriptase
inhibitors, e.g. zidovudine (3'-azido-3'-deoxythymidine, AZT),
didanosine (2',3'-dideoxyinosine; ddI), zalcitabine
(dideoxycytidine, ddC) or lamivudine
(2'-3'-dideoxy-3'-thiacytidine, 3TC), stavudine
(2',3')-didehydro-3'-deoxythymidine, d4T), abacavir and the like;
non-nucleoside reverse transcriptase inhibitors such as nevirapine
(11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido-[3,2-b:2',3-e][1,4]
diazepin-6-one), efavirenz, delavirdine, and the like; phosphonate
reverse transcriptase inhibitors, e.g. tenofovir and the like;
compounds of the TIBO
(tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepine-2(1H)-one and
thione)-type
(S)-8-chloro-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo-[4-
,5,1-jk][1,4]benzo-diazepine-2(1H)-thione, compounds of the
.alpha.-APA (.alpha.-anilino phenyl acetamide) type e.g.
.alpha.-[(2-nitrophenyl)amino]-2,6-dichlorobenzene-acetamide and
the like; inhibitors of trans-activating proteins, such as
TAT-inhibitors, e.g. RO-5-3335, or REV inhibitors, and the like;
protease inhibitors e,g indinavir, ritonavir, saquinavir, lopinavir
(ABT-378), nelfinavir, amprenavir, TMC-126, BMS-232632, VX-175 and
the like; fusion inhibitors, e.g. T-20, T-1249 and the like; CXCR4
receptor antagonists, e.g. AMD-3100 and the like; inhibitors of the
viral integrase; ribonucleotide reductase inhibitors, e.g.
hydroxyurea and the like.
[0269] Combinations may as well exert a synergistic effect in
inhibiting HIV replication when components of the combination act
on different or same sites of HIV replication, preferably on
different sites. The use of such combinations may reduce the dosage
of a given conventional antiretroviral agent which would be
required for a desired prophylactic effect as compared to when that
agent is administered as a single active ingredient. These
combinations reduce potential of resistance to single agent, while
minimizing any associated toxicity. These combinations may also
increase the efficacy of the conventional agent without increasing
the associated toxicity.
[0270] Thus, the compounds of the present invention may also be
administered in combination with art-known microbicides,
consequently potentiating the prophylactic effect. They can block
the infection by creating a barrier between the pathogen, in this
case the Human Immunodeficiency Virus, and the site at which
transmission will take place, e.g. vulva, vagina; they can kill or
immobilize the pathogen; they can prevent a virus from replicating
once it has infected the cells lining the site of transmission,
e.g. the cells that line the vaginal wall. Examples of microbicides
are: [0271] Antibiotic peptides: small protein molecules that form
part of the body's first line of defense against infection. These
peptides line every surface of the body--eyes, skin, lungs, tongue
and intestinal tract--and kill bacteria within minutes of contact.
Thus, if applied at the site of potential infection of HIV,
peptides may kill pathogens before they cause infection. [0272]
Antibodies: isolated antibodies that counteract HIV are available
in the literature, They may be appropriately combined with the
compounds of the present invention to prevent HIV infection. [0273]
pH regulators, especially for the vagina. A natural vaginal
environment is too acidic for HIV to survive, but semen decreases
its acidity, allowing HIV to survive. pH regulators regulate the
natural acidity of the vagina making it inhospitable for the HIV,
Said regulators encompass the use of Lactobacillus bacteria that
produce hydrogen peroxide and thereby help to keep the vaginal
environment healthy and acidic, The acidic polymer BufferGel
(ReProtect, LLC) is another example of a pH regulator which has in
addition spermicidal activity. [0274] Detergents and surfactants:
these compounds are able to disrupt the outer shell of viruses and
therefore are useful as microbicide and they can be combined with
the compounds to prevent HIV infection. Examples of such detergents
and surfactants are nonoxynol-9 and octoxynol-9, but all detergents
and surfactants that are commonly used in shampoos, toothpastes and
cleaning solutions, contact lens solutions may be equally suitable.
[0275] Coatings for the pathogen, such as Pro-2000 Gel which
contains a synthetic polymer that binds to HIV, disrupting the
binding of the virus to target cells. [0276] Coatings for the site
of transmission, such as for example gels. These products may
prevent HIV from entering the cells by covering the site of
transmission, e.g. the vaginal and vulvar epithelium. Examples,
including the gel preparations described above, encompass sulphated
and sulphonated polymers such as PC-515 (carrageenan), dextrin 2
sulphate, secretory leukocyte protease inhibitor (SLPI), which
binds to the target cells so that they are not accessible to the
virus, cyanovirin-N which also binds to the cell, prohibiting cell
fusion with HIV.
[0277] In the compositions of the present invention, one or more or
all of the above-listed microbicides may be combined with a
compound of the invention. Thus, the present invention also relates
to a pharmaceutical composition comprising a compound of the
present invention and further comprising one or more components
wherein the components are selected from antibiotic peptides,
antibodies, pH regulators, detergents or surfactants, coatings for
the pathogen, coatings for the site of administration.
[0278] One particular example of the combination of microbicides is
the combination of compounds of the invention with cellulose
acetate phthalate (CAP) and/or hydroxypropyl methylcellulose
phthalate (HPMCP). CAP and its derivates are excipients which
exhibit an additional microbicide effect, CAP formulations have
already been described in the literature, EP1030547, U.S. Pat. No.
6,165,493, by Neurath et al., all of which are herein incorporated
by reference.
[0279] The present invention relates also to a pharmaceutical
composition as outlined hereinabove further comprising a
spermicidal compound. Said compositions are able to prevent at the
same time conception and HIV infection. Suitable spermicides are
for example nonoxynol-9, octoxynol-9, menfegol, benzalkoniuni
chloride, N-docasanol.
[0280] Those of skill in the prophylaxis of HIV-infection could
determine the microbicidal effective amount from the test results
presented here and may range from about 1 ng to about 10 mg, in
particular from about 10 ng to about 1 mg, more in particular from
about 100 ng to about 100 .mu.g and preferably from about 500 ng to
about 50 .mu.g of active ingredient per application or unit dose,
in particular an application or unit dose of an immediate release
formulation.
[0281] It may be appropriate to apply the required dose as unit
dosage forms. The volume of a unit dose, in particular the unit
dose of an immediate release formulation, whether or not in a unit
dosage form, may range in the case of a topical formulation from
about 10 .mu.l up to about 25 ml of topical formulation and in
particular from about 1 ml up to about 10 ml of topical
formulation. In the case of a gel or a cream for instance, a
convenient unit dose could range between about 1 ml and about 5
ml.
[0282] For instance in the case of topical formulations, in
particular topical formulations for immediate release, as mentioned
herein, e.g. a gel, a cream and the like, the active ingredient may
be present in a concentration ranging from about 1 nM up to about
10 mM, in particular from about 10 nM up to about 1 mM, more in
particular from about 100 nM up to about 100 .mu.M and preferably
from about 1 .mu.M to about 100 .mu.M.
[0283] It is evident that said effective amount may be lowered or
increased depending on the particular compound being used, on the
response of the treated subject and/or depending on the evaluation
of the physician prescribing the compounds of the instant
invention.
EXAMPLES
[0284] The following examples are intended to illustrate the
present invention.
Example 1: In Vitro Evaluation of the Non-Nucleoside Reverse
Transcriptase Inhibitors Compound B as HIV Microbicide
[0285] Cell-Free and Cell-Associated HIV Strains
[0286] For experiments with CEM T cells, we used the lymphotropic
SI/X4 HIV strain HTLV-III.sub.B, originally obtained from R. C.
Gallo and M. Popovic (NIH, Bethesda, Md.). For experiments with
monocyte-derived dendritic cells (MO-DC), the monotropic NSI/R5 HIV
strain Ba-L, kindly provided by the NIH AIDS Research and Reference
Reagent Program (Rockville, Md) was used. Ba-L stocks were grown
and tittered on PHA/IL-2-stimulated peripheral blood mononuclear
cells (PBMC) in complete medium, containing RPMI-1640
(Bio-Whittaker, Verviers, Belgium) and 10% bovine calf serum
(Hyclone, Utah, US) (Peden K. Virological and Molecular Genetic
Techniques for Studies of Established HIV Isolates. 1995, 21-45).
The supernatant of these cultures were directly used as cell-free
virus to infect MO-DC. To prepare cell-associated HIV Ba-L,
non-stimulated PBMC (2.times.10.sup.6 cells/ml) were incubated
overnight with 10.sup.-2 MOI (=multiplicity of infection) of HIV
Ba-L in complete medium. Afterwards, cells were extensively washed,
frozen in liquid nitrogen and thawed on the day of infection.
[0287] HIV Antigen Detection After Primary Culture and Calculation
of EC50 Value
[0288] HIV antigen was detected using an in-house developed ELISA
assay, of which the characteristics have been described (Beirnaert
E, Willems B, Peeters M, Bouckaert A, Heyndrickx L, Zhong P et al.
Design and Evaluation of an in-House HIV-1 (Group M and O), SIVmnd
and SIVcpz Antigen Capture Assay. J Virol Methods 1998, 73: 65-70).
The lower detection limit is about 200 pg/ml and the upper limit is
about 25,000 pg/ml, as determined using a standard curve of Ba-L
stock dilutions with known p24 content. The 50% Effective
Concentration (EC50) was calculated by plotting HIV Ag
concentration against drug concentration, followed by regression
analysis on the linear part of the curve,
[0289] Measuring 50% Effective and 50% Ctotoxic Concentrations in
CEM T Cells
[0290] As a reference system, CEM T cells (obtained from the
American Type culture Collection in Rockville, Md.) were used under
previously standardized conditions (Balzarini J, et al. AIDS Res
Hum Retroviruses 10 Apr. 2000, 16: 517-528). Briefly, cells were
suspended at 250,000 cells/ml in RPMI-1640, supplemented with 10%
fetal calf serum, 2 mM L-glutamine and 0.075% NaHCO.sub.3 and
infected with HTLV-III.sub.B at .about.20 TCID.sub.50. 100 .mu.l of
a 5-fold dilution series of the drugs were immediately added to 100
.mu.l of the infected cells in 200-.mu.l well plates. After 4 to 5
days of incubation at 37.degree. C., the cultures were examined for
syncytium formation. The EC.sub.50 is the concentration required to
inhibit syncytium formation by 50%. Cytotoxicity was evaluated and
given as CC50 values, which is the concentration at which the
viability of the CEM cells is decreased by 50%.
[0291] Generation of Monocvte-Derived Interstitial-Type Dendritic
Cells (MO-DC) and T Cells
[0292] Monocytes and lymphocytes were separated from buffy coat
PBMC by counter-flow elutriation, as previously described (Van
Herrewege et al. AIDS Res Hum Retroviruses 10 Oct. 2002, 18:
1091-1102). Monocytes were further differentiated to MO-DC by
culture at 37.degree. C. and 5% CO2 during 7 days in complete
medium, supplemented with 20 ng/ml GM-CSF and IL-4 (Lmmunosource,
Zoersel, Belgium) (Sallusto et al. J Exp Med 1 Apr. 1994, 179:
1109-1118. Romani et al. J Exp Med 1 Jul. 1994, 180: 83-93.
Geissmann F, et al. Exp Med 16 Mar. 1998, 187: 961-966). The
lymphocyte fraction was frozen in liquid nitrogen and thawed on the
day of infection. CD4(+) T cells were purified by positive
selection, using a CD4(+) isolation kit (Dynal, Oslo, Norway), as
described (Vanham et al. AIDS 20 Oct. 2000, 14: 2299-231 1. Vanham
et al. AIDS 18 Aug. 2000, 14: 1874-1876).
[0293] Pre-treatment of HIV with Drugs, with or without Continued
Treatment of MO-DC/CD4(+) T Cell Co-Cultures After Infection
[0294] Cell-free HIV Ba-L was pre-incubated with a serial dilution
of drug, ranging from 10,000 to 0.1 nM (final concentration), for 1
hour at 37.degree. C. MO-DC were infected with drug-treated HIV at
a multiplicity of infection (MOI) of 10.sup.-3. After 2 hours (at
37.degree. C.), MO-DC were washed (6.times.) and suspended at
4.times.10.sup.5 cells/ml. 50 .mu.l of MO-DC were dispensed in a
96-well cup, together with 50 .mu.l of autologous CD4(+) T cells
(2.times.10.sup.6 cells/ml) and 100 .mu.l of complete medium or 100
.mu.l of drug (at the same concentration as for the
pre-incubation). Half of the culture medium was refreshed twice
weekly with complete medium, with or without drug. After 2 weeks of
primary culture, supernatants were analysed by ELISA and cells were
used for secondary cultures to check viral rescue. For experiments
with cell-associated HIV, a similar set-up was used except that
pre-incubated, cell-associated virus was washed before addition of
MO-DC/CD4(+) T cells and remained present during the MO-DC/CD4(+) T
cell co-culture.
[0295] 24 Hours Drug Treatment of MO-DC/CD4(+) T Cell Co-Cultures
During HIV Infection
[0296] To evaluate the effect of a 24-hours treatment, MO-DC and
autologous CD4(+) T cells were suspended in complete medium at
4.times.10.sup.4, resp. 2.times.10.sup.5 cells/ml. Fifty .mu.l of
MO-DC and 50 .mu.l of CD4(.+-.) T cells were dispensed in a 96-well
cup, together with 50 .mu.l of cell-associated or cell-free virus
(10.sup.-3 MOI) and 50 .mu.l of complete medium or 50 .mu.l of a
serial dilution of drug. After 24-hours (37.degree. C., 5%
CO.sub.2), cells were washed (3.times.) and incubated for 2 weeks.
Half of the culture medium was refreshed twice weekly with complete
medium (without drug). After 2 weeks of primary culture,
supernatants were analysed by ELISA and cells were used for
secondary cultures to check viral rescue.
[0297] Detection of Viral Rescue: Secondary Culture and PCR
Analysis
[0298] PBMC were isolated from donor buffy coats and cultured for 2
days in complete medium supplemented with 5 ng/ml IL-2
(Immunosource, Zoersel, Belgium) and 0.5 .mu.g/ml PHA (Murex,
Dartford, England). After 2 weeks of primary culture, MO-DC/CD4(+)
T cell co-cultures were washed (3.times.) and secondary cultures
were set-up by adding 1.times.10.sup.5 PHA/IL-2 activated PBMC per
cup. Half of the culture medium was replaced every 3-4 days with
IL-2 containing medium (without drug) and supernatants as well as
cells were harvested after 2 additional weeks. Supernatants were
tested for HIV-Ag in ELISA. Cells were processed for HIV DNA
measurement, using a PCR-based HIV proviral DNA quantitation kit,
developed from Amplicor HIV-1 Monitor.TM. Test, version 1.5 (Roche
Molecular Systems, Branchburg, USA), the modifications of which
have been described (Christopherson et al. J Clin Microbiol 2000,
38: 630-634). A lower threshold of 10 HIV copies per 10.sup.6 cells
was confirmed by using 8E5/LAV cells, containing 1 copy of proviral
DNA per cell (kindly provided by the Centralized Facility for AIDS
Reagents, Potters Bar, UK)
[0299] Evaluation of the Immune Suppressive Activity and Cellular
Toxicity of Compound B in MO-DC/CD4(+) T Cell Co-Cultures
[0300] The immune suppressive activity of compound B was measured
in mixed leukocyte cultures (MLC), with MO-DC as stimulators and
allogenic CD4(+) T cells as responders. Cultures of
3.times.10.sup.3 MO-DC and 100.times.10.sup.3 T cells were set-up
in 6-fold in a 96-well plate, in the presence or absence of a
dilution series of compound B. In a first part of experiments,
compound B was removed after 24 hours by washing and cells were
cultured for an additional 4 days. In a second part of experiments,
compound B remained present during the 5-day culture period. In
both set-ups, 1 .mu.Ci of [methyl-.sup.3H] thymidine (TRA. 120 from
Amersham Pharmacia, Buckinghamshire, U.K.) was added to each well
at the fifth day of culture. Plates were harvested 7 hours later
and [methyl-.sup.3H] thymidine incorporation was measured in a
scintillation counter (Top Count.TM., Canberra-Packard, Zellik,
Belgium) and expressed as counts per minute (CPM). The
[0301] Immune Suppressive Concentration (ISC.sub.50) is defined as
the drug concentration inhibiting 50% of the lymphocyte
proliferation. Cellular toxicity was evaluated microscopically by
cosine staining of co-cultures of MO-DC and allogeneic CD4(+) T
cells , cultured for 5 days in the presence of a dilution series of
drug. Part of the harvested cells was also used for flowcytometric
analysis of lymphocyte blast formation and apopoptosis, based on
forward and side scatter.
[0302] Reference Data on Antiviral Activity and Cellular Toxicity
of Compound B
[0303] The CEM T cell line was used as a reference to determine the
antiviral activity of compound B. As shown in Table 1, compound B
was active in the nanomolar range and showed a low toxicity. Next
to antiviral activity in CEM T cells, inhibition of HIV-1 reverse
transriptase activity was measured in a cell-free assay, in which
the 50% inhibitory concentration (IC50) of said compound is
indicated (FIG. 1). The CEM system, using a lab T cell-line and the
SI/X4labstrain HTLV-IIIb, was not directly relevant to sexual
transmission, where primary T cells, dendritic cells and NSI/R5
viruses are involved. Therefore, we focussed on prevention of
NSI/R5 HIV infection in MO-DC/CD4(+) T cell co-cultures.
TABLE-US-00001 TABLE 1 Antiviral activity, cytotoxicity and HIV-1
RT inhibitory capacity of compound B EC50 CC50 IC50 Drug Treatment
HIV (nM) .sup.a (nM) .sup.b (nM) .sup.c compound B Continuous HTLV-
1 1.367 24 IIIb .sup.a EC50: 50% Effective Concentration,
concentration required to inhibit syncytium formation of HTLV-IIIb
infected CEM T cells by 50% .sup.b CC50: 50% Cytotoxic
Concentration, concentration at which the viability of CEM T cells
is decreased by 50% .sup.c IC50: 50% Inhibitory Concentration,
concentration that inhibits HIV-1 reverse transcriptase activity by
50%.
[0304] Drug Treatment of MO-DC/CD4(+) T Cell Co-Cultures Prevented
HIV Integration
[0305] In preliminary experiments the HIV Ba-L virus was pretreated
for 1 hour with up to 10,000 nM of compound B. The drug remained
present during the 2 hours incubation of the virus with the MO-DC,
but it was thoroughly washed away before addition of autologous
CD4(+) T cells.
[0306] In order to study the maximal effect of the drug,
pre-treatment of the virus and treatment of the cells during
infection was combined with further treatment during the entire
primary culture period of 2 weeks. An example of the inhibitory
effects of compound B on infection with cell-associated virus is
shown in Table 2. Compound B blocked infection in the primary
cultures already at 10 nM, but addition of PHA/IL-2 blasts revealed
that 100 nM was needed to completely block infection and prevent
proviral integration. When cell-free virus was used for infection,
continuous treatment with 10 nM of compound B sufficed to
completely block HIV infection, also during secondary culture
(Table 3).
[0307] Next, it was investigated whether drug treatment during the
first 24 hours of the primary culture could suffice to prevent
viral infection and integration, as measured by ELISA and PCR
respectively. As compared to continuous treatment, compound B
showed to block infection at 1 log higher concentrations as used
for the continuous treatment (Table 3).
TABLE-US-00002 TABLE 2 Inhibition of infection of MO-DC/CD4(+) T
Cell co-cultures with cell-associated HIV Ba-L HIV Antigen (number
of positive wells).sup.c HIV proviral DNA .sup.d Drug Conc (nM)
1.degree. Cult..sup.a 2.degree. Cult..sup.b (2.degree. Cult.)
compound B 10,000 0 0 Neg 1,000 0 0 Neg 100 0 0 Neg 10 0 3 4.74 No
drug 0 6 6 4.85 .sup.aCell-associated HIV Ba-L was pre-incubated
with drug, washed, and added to co-cultures of MO-DC and autologous
CD4+ T cells. Cells were cultured for 2 weeks, in the continuous
presence of drug (Primary (1.degree.) Culture) .sup.bAfter primary
culture, cells were washed and PHA/IL-2 activated PBMCs were added
and maintained in IL-2 containing medium during a secondary
(2.degree.) culture of 2 weeks (no drug present). .sup.cCulture
supernatant was tested for HIV antigen by ELISA. The number of
antigen-positive microcultures (out of 6) is represented. .sup.d
After secondary culture, cells were analysed in PCR for the
presence of proviral DNA, results are expressed as Log(number of
DNA copies/10.sup.6 cells)
TABLE-US-00003 TABLE 3 Conditions for Prevention of HIV infection
in MO-DC/CD4(+) T Cell co-cultures Drug Treatment HIV Conc. (nM)
.sup.a compound B 24 Hours Cell-free 100 Cell-associated 1,000
Continuous Cell-free 10 Cell-associated 100 .sup.a MO-DC/CD4(+) T
cell co-cultures were incubated with cell-free or cell-associated
HIV and concurrently drug treated during 24 hours or continuously
during 1.degree. culture. After 1.degree. culture, cells were
washed and used for 2.degree. cultures (no drug present). Drug
concentrations that prevent replicative HIV infection, as measured
by ELISA of culture supernatants and PCR of cells after 2.degree.
culture, are shown. .sup.b The concentration of 10,000 nM was not
used in this part of the experiment
[0308] Compound B had a Low Cellular Toxicity in CEM T Cells and a
Favourable Therapeutic Index in MO-DC/CD4(+) T Cell Co-Cultures
[0309] Cellular toxicity (CC50 value) in reference GEM T cells was
at 1,367 nM for compound B (Table 1).
[0310] The immune suppressive activity of compound B was evaluated
in mixed leukocyte cultures with N40-DC as stimulators and
allogenic CD4(+) T cells as responders. If drug was present during
the whole culture period, the 50% Immune Suppressive Concentration
(ISC50) was about 1,500 nM. If the drug was only present during the
first 24 hours, the ISC50 increased to almost 25,000 nM (Table 4).
Thus, the immune suppressive activity of compound B was clearly
less suppressive in the 24 hours treatment as compared to
continuous treatment. In order to fully evaluate the relation of
anti-viral and immune suppressive activity, the 50% antiviral
activity (or EC50) values were calculated on primary drug-treated
cultures of HIV-infected autologous MO-DC/CD4(+) T cell co-cultures
and the therapeutic indices (TI) were determined. The data of Table
4 shows that compound B has a favourable TI as measured in this
model of primary target cells of sexual transmission.
TABLE-US-00004 TABLE 4 Overview of the Antiviral and Immune
Suppressive Activity of compound B in co-cultures of MO-DC/CD4(+) T
cells EC50 ISC50 Drug Treatment HIV (nM) .sup.a (nM) .sup.b TI
.sup.c Comp. B 24 hours Cell-free 42 24,886 592 Cell-associated 63
395 Continuous Cell-free <0.1 1,515 >15,150 Cell-associated
<1 .sup.d >1,515 .sup.a EC50: 50% Effective Concentration:
drug concentration inhibiting 50% of HIV Ba-L replication .sup.b
ISC50: 50% Immune Suppressive Concentration: drug concentration
inhibiting 50% of T-lymphocyte proliferation. .sup.c TI:
Therapeutic Index: ISC50/EC50
[0311] Additional experiments were done to evaluate if inhibition
of DNA synthesis corresponded to increased mortality of T cells or
only to decreased blast formation. Fifty percent inhibition of
blast formation was observed at 3,916 nM. The 50% death rate of T
cells was calculated at 54,222 nM.
[0312] In conclusion, prevention of HIV infection was possible
against both cell-free and cell-associated NSI/R5 virus and a 24
hours treatment was sufficient. Compound B showed a high
therapeutic index, based on its relative weak immune suppressive
and potent anti-viral activity. These results confirmed the use of
compound B as microbicide.
Example 2: Human SCID Animal Model
[0313] In order to mimic the in vivo transmission that occurs in
humans, a hu-SCID animal model of vaginal transmission of HIV for
the evaluation of vaginal microbicides was used (Di Fabio et al.,
AIDS 2001, 15, 2231-2234 Gels made up of carbopol 940 or
hydroxyethyl cellulose (HEC), two water soluble polymers, were
prepared containing Compound B in different concentrations (0.225
mM; 0.0225 mM or 0.00225 mM). Animals received a single
intravaginal application of 25 .mu.l of gel containing Compound B,
15-20 minutes prior to a non invasive vaginal challenge with
2.times.10.sup.6 human peripheral blood lymphocytes PBL, (hu-PBL)
previously infected in vitro with non-syncitium (NSI) strains of
HIV-1 (SF162 and 1/BX08). Cell to cell transmission was assessed by
p24 production and by quantitative PCR. As a result of this study
with Compound B, systemic infection was successfully inhibited.
Example 3: In Vitro Model Based on T-Cell Derived Jurkat-Tat Cells
(PM-1)
[0314] Direct antiviral activity was demonstrated in a model using
Jurkat-tat cells. HIV-1.sup.RF (10.sup.3TCID.sub.50) immobilised
into 96-well coated plates were treated with test compound B for 1
hour at 37.degree. C., compound was subsequently removed by washing
with 4 volumes of PBS before co-culture with indicator cells for 8
days. Protection of infection was provided at the 100 nM
concentration. Additionally, protection of infection was
demonstrated at 10 nM in a parallel setup were virus was pretreated
prior to addition of cells and without removal of the compound.
Example 4: Cervical Explant Model
[0315] Compound B, at a concentration of 10 nM, was able to block
infection of the tissue and at a concentration of 100 nM the
compound could prevent transfer of infectious virus from migratory
dendritic cells to co-cultured T-cells.
[0316] Compound demonstrated good efficacy against primary HIV
strains (X4, CCR5 and X41R5) in relevant cell lines for a
vaginal/rectal microbicide indication: In cervical epithelial cells
(MEI180) exposed to the compound during either 1 hr, 24 hrs or 5
days after which drug was removed by washing, viability of the
cells was assessed with an MTT assay. Data showed no toxicity at
the 50 .mu.M concentration and some reduction of viability at 100
nM.
Example 5: Biochemical Characterization of the Interaction Between
Compound B and HIV-1 Reverse Transcriptase
[0317] In order to investigate the nature of the interaction
between compound B and HIV-1 reverse transcriptase (HIV-1 RT), the
inhibition of the RNA-dependent DNA polymerization reaction was
investigated under steady state conditions. In a first experiment,
the reaction velocity was determined in the presence of different
concentrations of compound B and different concentrations of dGTP
while the concentration of the p(rC)p(dG) complex was constant. The
result showed that the binding of compound B to HIV-1 RT is
non-competitive against dGTP with a Km value of 2.51 .mu.M and a Ki
of 0.033 .mu.M.
[0318] In a second experimental the reaction velocity was
determined in the presence of different concentrations of compound
B and different concentrations of p(rC)p(dG) while the
concentration of the substrate was constant. The result showed that
the binding of compound B to HIV-1 RT is also non-competitive
against p(rC)p(dG) with a Km value of 10.3 .mu.M and a Ki of 0.028
.mu.M. Taken together this means that the binding of compound B on
HIV-1 RT is independent from the binding of nucleotide and
independent from the binding of primer/template.
Example 6: Compatibility with Lactobacilli and Normal Vaginal
Flora
[0319] Compatibility with lactobacilli and normal vaginal flora is
an important requirement for a vaginal microbicide. Activity on
pathogens of sexual transmitted diseases is an additional
advantage. In in vitro tests for antibacterial activity of compound
B, the following susceptibilities were found: [0320] 33 different
Lactobacillus species isolated from recto-vaginal cultures of
pregnant women were studied. Results showed a minimum inhibitory
concentration (MIC.sub.50) of >32 mg/L [0321] Hemophilus ducreyi
was inhibited by the compound with a MIC.sub.50 of 1 mg/L and a
MIC.sub.50 of 2 mg/L [0322] Some Neisseria gonorrhoea strains were
inhibited at clinical relevant concentrations.
Example 7: Rabbit Vaginal Irritation Test
[0323] Several formulations of the compound were used to exclude
any irritation in a Rabbit Vaginal irritation test. 24 hours after
the application of 1 ml of gellcream at different concentrations
(0.1M, 0.9 mM, 0.45 mM and 0.225 mM), vaginal epithelium was
carefully examined macroscopically and microscopically. Microscopic
slides of parafinized samples of different parts of the
cervico-vagina were stained and analysed histologically.
Macroscopic and microscopic scores obtained indicated that the
formulations tested were well tolerated.
Example 8: White Rabbit Vaginal Irritation and Toxicity Study
[0324] Gel formulations of the compound B at different
concentrations were used to study the exclusion of any irritation
in a Rabbit Vaingal Irritation test. 10 days after the application
of the gel formulations onto 6 different rabbit groups, vaginal and
cervical epithelium were carefully examined by a pathologist
macroscopically and histologically. The following scores were
determined.
TABLE-US-00005 TABLE 5 Vaginal examination Epithelial loss and
atrophy accompanied by minimal or slight epithelial inflammatory
cell infiltration was only seen in all female rabbits treated with
Nonoxynol-9, 4%. Group/Sex 1F 2F 3F 4F 5F 6F Treatment Sham Placebo
gel gel gel Nonoxynol Control (HEC-gel) 22.5 .mu.M 225 .mu.M 10 mM
9, 4% Epithelial loss Marked 0 0 0 0 0 2 and atrophy Severe 0 0 0 0
0 3 Total 0 0 0 0 0 5b Epithelial Minimal 0 0 0 0 0 3 inflammatory
Slight 0 0 0 0 0 2 infiltrate Total 0 0 0 0 0 5a ap < 0.05
(Fisher's exact two-tailed probability test) bp < 0.01 (Fisher's
exact two-tailed probability test)
TABLE-US-00006 TABLE 6 Cervical examination Loss and atrophy of the
epithelia and luminal inflammatory cells and cellular debris were
only seen in females treated with Nonoxynol-9, 4%. Group/Sex 1F 2F
3F 4F 5F 6F Treatment Sham Placebo gel gel gel Nonoxynol Control
(HEC-gel) 22.5 .mu.M 225 .mu.M 10 mM 9, 4% Epithelial loss Slight 0
0 0 0 0 1 and atrophy Marked 0 0 0 0 0 3 Total 0 0 0 0 0 4a Luminal
Present 0 0 0 0 0 4 Inflammatory Total 0 0 0 0 0 4a cells ap <
0.05 (Fisher's exact two-tailed probability test)
Example 9: Microbicide Gels
[0325] This example illustrates various gels with different dosages
of active principle that can be prepared and used as microbicides
in the prevention of HIV infection for topical administration.
TABLE-US-00007 TABLE 7 22.5 .mu.M gel compound B 0.74 mg HEC 2 g
Glycerol 5 g Methyl paraben 180 mg Propyl paraben 20 mg Lactic acid
50 mg Sodium hydroxide q.s. for pH 4.5 Water q.s. 100 g
TABLE-US-00008 TABLE 8 225 .mu.M gel compound B 7.40 mg HEC 2 g
Glycerol 5 g Methyl paraben 180 mg Propyl paraben 20 mg Lactic acid
50 mg Sodium hydroxide q.s. for pH 4.5 Water q.s. 100 g
TABLE-US-00009 TABLE 9 1 mM gel Compound B 32.94 mg HEC 2 g
Glycerol 5 g Methyl paraben 180 mg Propyl paraben 20 mg Lactic acid
50 mg Sodium hydroxide q.s. for pH 4.5 Water q.s. 100 g
TABLE-US-00010 TABLE 10 10 mM gel compound B 329.40 mg HEC 2 g
Glycerol 5 g Methyl paraben 180 mg Propyl paraben 20 mg Lactic acid
50 mg Sodium hydroxide q.s. for pH 4.5 Water q.s. 100 g
[0326] Various modifications and alterations to the present
invention may be appreciated based on a review of this disclosure,
These changes and additions are intended to be within the scope and
spirit of this invention as defined by the following claims.
* * * * *