U.S. patent application number 14/159566 was filed with the patent office on 2014-08-28 for antiviral compounds.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Joseph Anthony Bilotta, Zhi Chen, Qingjie Ding, Shawn David Erickson, Eric Mertz, Robert James Weikert.
Application Number | 20140242028 14/159566 |
Document ID | / |
Family ID | 50033474 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140242028 |
Kind Code |
A1 |
Bilotta; Joseph Anthony ; et
al. |
August 28, 2014 |
ANTIVIRAL COMPOUNDS
Abstract
The present invention discloses compounds of Formula I:
##STR00001## wherein the variables in Formula I are defined as
described herein. Also disclosed are pharmaceutical compositions
containing such compounds and methods for using the compounds of
Formula I in the prevention or treatment of HCV infection.
Inventors: |
Bilotta; Joseph Anthony;
(Nutley, NJ) ; Chen; Zhi; (Livingston, NJ)
; Ding; Qingjie; (Bridgewater, NJ) ; Erickson;
Shawn David; (Leonia, NJ) ; Mertz; Eric; (Fair
Lawn, NJ) ; Weikert; Robert James; (Basel,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Nutley |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Nutley
NJ
|
Family ID: |
50033474 |
Appl. No.: |
14/159566 |
Filed: |
January 21, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61755519 |
Jan 23, 2013 |
|
|
|
Current U.S.
Class: |
424/85.4 ;
514/340; 514/383; 546/272.4; 548/265.2 |
Current CPC
Class: |
A61P 1/16 20180101; A61K
31/4196 20130101; A61K 45/06 20130101; A61P 31/14 20180101; A61K
31/4439 20130101; C07D 249/14 20130101; C07D 401/12 20130101 |
Class at
Publication: |
424/85.4 ;
514/340; 514/383; 546/272.4; 548/265.2 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439; A61K 45/06 20060101 A61K045/06; C07D 249/14 20060101
C07D249/14; A61K 31/4196 20060101 A61K031/4196; C07D 401/12
20060101 C07D401/12 |
Claims
1. A compound of formula I ##STR00149## wherein: R.sup.1 is H or
lower alkyl; each R.sup.2 is independently halo or lower haloalkyl;
n is 0, 1, or 2; R.sup.3 is lower haloalkyl, cyano, lower alkoxy,
C(.dbd.O)OCH.sub.3, or S(.dbd.O).sub.2CH.sub.3; p is 0, or 1;
X.sup.1 is S, S(.dbd.O).sub.2, O, S(.dbd.O), NH, or OCH.sub.2; and
X.sup.2 is CH or N; or a pharmaceutically acceptable salt
thereof.
2. The compound of claim 1, wherein X.sup.2 is CH and R.sup.1 is
H.
3. The compound of claim 2, wherein X.sup.1 is S or 0.
4. The compound of claim 2, wherein X.sup.1 is S(.dbd.O).sub.2 or
S(.dbd.O).
5. The compound of any one of claim 1, wherein n is 1.
6. The compound of any one of claim 1, wherein n is 2.
7. The compound of claim 5, wherein R.sup.1 is Cl.
8. The compound of claim 6, wherein both R.sup.1 are Cl.
9. The compound of any one of claim 1, wherein p is 0.
10. The compound of any one of claim 1, wherein p is 1.
11. The compound of claim 10, wherein R.sup.3 is or
S(.dbd.O).sub.2CH.sub.3, cyano, or CF.sub.3.
12. A compound selected from the group consisting of:
N5-(3-Fluoro-4-phenylsulfanyl-5-trifluoromethyl-phenyl)-1H-[1,2,4]triazol-
e-3,5-diamine;
N3-(3-Chloro-4-phenylamine-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
N3-(3-Chloro-4-phenoxy-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
N3(3,5-Dichloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
N3(4-Benzenesulfinyl-3,5-dichloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
N3(4-Benzenesulfinyl-3,5-dichloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
N3-[3,5-Dichloro-4-(4-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4]t-
riazole-3,5-diamine;
N3-[3,5-Dichloro-4-(4-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1,2,4]-
triazole-3,5-diamine;
N3-[3,5-Dichloro-4-(4-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1,2,4]-
triazole-3,5-diamine;
N.sup.3-[3,5-Dichloro-4-(2-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,-
2,4]triazole-3,5-diamine;
N.sup.3-[3,5-Dichloro-4-(3-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1-
,2,4]triazole-3,5-diamine;
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-phenylsulfanyl]-b-
enzonitrile;
4-[4(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfinyl]-b-
enzonitrile;
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfonyl]--
benzonitrile;
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2-chloro-6-trifluoromethyl-phe-
nylsulfanyl]-benzoic acid methyl ester;
N.sup.3-[3,5-Dichloro-4-(4-methoxy-phenylsulfanyl)-phenyl]-1H-[1,2,4]tria-
zole-3,5-diamine;
N.sup.3-[3-Chloro-4-(4-methoxy-phenylsulfanyl)-5-trifluoromethyl-phenyl]--
1H-[1,2,4]triazole-3,5-diamine;
N.sup.3-[3,5-Dichloro-4(4-methoxy-benzenesulfonyl)-phenyl]-1H-[1,2,4]tria-
zole-3,5-diamine;
N.sup.3[3,5-Dichloro-4(4-methanesulfonyl-phenoxy)-phenyl]-1H-[1,2,4]triaz-
ole-3,5-diamine;
N.sup.3-[3-Chloro-4-(4-methanesulfonyl-phenoxy)-5-tiifluoromethyl-phenyl]-
-1H-[1,2,4]triazole-3,5-diamine;
N.sup.3(3-Benzyloxy-5-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
N.sup.3-(3-Chloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]triazole-3,5-diamine-
;
N.sup.3-(4-Benzenesulfonyl-3-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diami-
ne;
N.sup.3-(3-Chloro-4-phenylsulfanyl-phenyl)-N.sup.3-methyl-1H-[1,2,4]tr-
iazole-3,5-diamine;
N.sup.3-(3-Chloro-5-phenoxy-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
and
N.sup.3-[3,5-Dichloro-4-(pyridin-2-yloxy)-phenyl]-1H-[1,2,4]triazole-3,5--
diamine.
13. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 1.
14. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 2.
15. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 3.
16. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 4.
17. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 5.
18. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 6.
19. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 7.
20. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 8.
21. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 9.
22. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 10.
23. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 11.
24. A method for preventing a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 12.
25. The method of claim 1, further comprising administering to a
patient in need thereof a therapeutically effective amount of an
immune system suppressant.
26. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 1.
27. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 2.
28. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 3.
29. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 4.
30. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of any one of claim
5.
31. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 6.
32. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 6.
33. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 7.
34. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 8.
35. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 9.
36. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 10.
37. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 11.
38. A method for treating a Hepatitis C Virus (HCV) infection
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of claim 12.
39. The method of claim 1, further comprising administering a
combination of antiviral agents that inhibits replication of
HCV.
40. The method of claim 1, further comprising administering an
immune system modulator or an antiviral agent that inhibits
replication of HCV, or a combination thereof.
41. The method of claim 39, wherein the immune system modulator is
an interferon or a chemically derivatized interferon.
42. The method of claim 38, wherein the antiviral agent is selected
from the group consisting of a HCV protease inhibitor, a HCV
polymerase inhibitor, a HCV helicase inhibitor, a HCV NS5A
inhibitor, or any combination thereof.
43. A composition comprising a compound of claim 1 and a
pharmaceutically acceptable excipient.
Description
FIELD OF THE INVENTION
[0001] The present invention provides compounds of Formula I useful
as inhibitors of hepatitis C virus (HCV), as inhibitors of HCV
infection, and for the prevention and treatment of hepatitis C
infection.
[0002] Hepatitis C virus (HCV) infection is a major health problem
that affects 170 million people worldwide and 3-4 million people in
the United States (Armstrong, G. L., et al., Ann. Intern. Med.
2006, 144:705-714; Lauer, G. M., et al., N. Eng. J. Med. 2001,
345:41-52). HCV infection leads to chronic liver disease, such as
cirrhosis and hepatocellular carcinoma in a substantial number of
infected individuals. Chronic HCV infection associated liver
cirrhosis and hepatocellular carcinoma are also the leading cause
of liver transplantation in the United States. Current treatments
for HCV infection include immunotherapy with pegylated
interferon-.alpha. in combination with the nucleoside-analog
ribavirin. Pegylated interferon-.alpha. in combination with
ribavirin and one of the two recently approved HCV NS3 protease
inhibitors Incivek or Victrelis is the current standard of care for
the treatment of genotype 1HCV infected patients, the most
difficult to treat patient population. However, current HCV
treatments are compromised by suboptimal sustained virological
response rates and associated with severe side effects, as well as
resistance to the protease inhibitors. Therefore there is a clear
need for improved antiviral drugs with better efficacy, safety, and
resistance profiles.
[0003] The infection of human hepatocytes by HCV, also known as HCV
entry, is mediated by the functional interactions of
virally-encoded envelope glycoproteins E1 and E2 and host cell
co-receptors, followed by a receptor-mediated endocytosis
processes. This HCV entry step is a putative target for therapeutic
intervention. Several virally-encoded enzymes are also putative
targets for therapeutic intervention, including a metalloprotease
(NS2-3), a serine protease (NS3, amino acid residues 1-180), a
helicase (NS3, full length), an NS3 protease cofactor (NS4A), a
membrane protein (NS4B), a zinc metalloprotein (NS5A) and an
RNA-dependent RNA polymerase (NS5B).
[0004] Systems have been developed to study the biology of HCV
entry into host cells. Pseudotyping systems where the E1 and E2
glycoproteins are used to functionally replace the glycoproteins of
retroviruses have been developed (Bartosch, B., Dubuisson, J. and
Cosset, F.-L. J. Exp. Med. 2003, 197:633-642; Hsu, M. et al. Proc.
Natl. Acad. Sci. USA. 2003, 100:7271-7276). These systems yield HCV
pseudoparticles that bind to and enter host cells in a manner which
is believed to be analogous to the natural virus, thus making them
a convenient tool to study the viral entry steps as well as to
identify inhibitors blocking this process.
[0005] There is a clear and long-felt need to develop effective
therapeutics for treatment of HCV infection. Specifically, there is
a need to develop compounds that selectively inhibit HCV viral
entry and replication and that are useful for treating HCV-infected
patients and protecting liver transplant patients from HCV
re-infection. This application discloses novel compounds that are
effective in prevention of HCV infection. Additionally, the
disclosed compounds provide advantages for pharmaceutical uses, for
example, with respect to their mechanism of action, binding,
prevention of infection, inhibition efficacy, and target
selectivity.
SUMMARY OF THE INVENTION
[0006] The application provides compound of formula I
##STR00002##
wherein: R.sup.1 is H or lower alkyl; each R.sup.2 is independently
halo or lower haloalkyl; n is 0, 1, or 2; R.sup.3 is lower
haloalkyl, cyano, lower alkoxy, C(.dbd.O)OCH.sub.3, or
S(.dbd.O).sub.2CH.sub.3; p is 0, or 1;
X.sup.1 is S, S(.dbd.O).sub.2, O, S(.dbd.O), NH, or OCH.sub.2;
and
[0007] X.sup.2 is CH or N; [0008] or a pharmaceutically acceptable
salt thereof.
[0009] The application provides a method for preventing a Hepatitis
C Virus (HCV) infection comprising administering to a patient in
need thereof a therapeutically effective amount of a compound of
Formula I.
[0010] The application provides a method for treating a Hepatitis C
Virus (HCV) infection comprising administering to a patient in need
thereof a therapeutically effective amount of a compound of Formula
I.
[0011] The application provides a composition comprising a compound
of Formula I and a pharmaceutically acceptable excipient.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0012] The phrase "a" or "an" entity as used herein refers to one
or more of that entity; for example, a compound refers to one or
more compounds or at least one compound. As such, the terms "a" (or
"an"), "one or more", and "at least one" can be used
interchangeably herein.
[0013] As used in this specification, whether in a transitional
phrase or in the body of the claim, the terms "comprise(s)" and
"comprising" are to be interpreted as having an open-ended meaning.
That is, the terms are to be interpreted synonymously with the
phrases "having at least" or "including at least". When used in the
context of a process, the term "comprising" means that the process
includes at least the recited steps, but may include additional
steps. When used in the context of a compound or composition, the
term "comprising" means that the compound or composition includes
at least the recited features or components, but may also include
additional features or components.
[0014] As used herein, unless specifically indicated otherwise, the
word "or" is used in the "inclusive" sense of "and/or" and not the
"exclusive" sense of "either/or".
[0015] The term "independently" is used herein to indicate that a
variable is applied in any one instance without regard to the
presence or absence of a variable having that same or a different
definition within the same compound. Thus, in a compound in which
R'' appears twice and is defined as "independently carbon or
nitrogen", both R''s can be carbon, both R''s can be nitrogen, or
one R'' can be carbon and the other nitrogen.
[0016] When any variable occurs more than one time in any moiety or
formula depicting and describing compounds employed or claimed in
the present invention, its definition on each occurrence is
independent of its definition at every other occurrence. Also,
combinations of substituents and/or variables are permissible only
if such compounds result in stable compounds.
[0017] The symbols "*" at the end of a bond or ".sup.------" drawn
through a bond each refer to the point of attachment of a
functional group or other chemical moiety to the rest of the
molecule of which it is a part. Thus, for example:
##STR00003##
[0018] A bond drawn into ring system (as opposed to connected at a
distinct vertex) indicates that the bond may be attached to any of
the suitable ring atoms.
[0019] The term "optional" or "optionally" as used herein means
that a subsequently described event or circumstance may, but need
not, occur, and that the description includes instances where the
event or circumstance occurs and instances in which it does not.
For example, "optionally substituted" means that the optionally
substituted moiety may incorporate a hydrogen atom or a
substituent.
[0020] If a substituent is designated to be "absent", the
substituent is not present.
[0021] The term "about" is used herein to mean approximately, in
the region of, roughly, or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
20%.
[0022] Certain compounds may exhibit tautomerism. Tautomeric
compounds can exist as two or more interconvertable species.
Prototropic tautomers result from the migration of a covalently
bonded hydrogen atom between two atoms. Tautomers generally exist
in equilibrium and attempts to isolate an individual tautomers
usually produce a mixture whose chemical and physical properties
are consistent with a mixture of compounds. The position of the
equilibrium is dependent on chemical features within the molecule.
For example, in many aliphatic aldehydes and ketones, such as
acetaldehyde, the keto form predominates while; in phenols, the
enol form predominates. Common prototropic tautomers include
keto/enol (--C(.dbd.O)--.revreaction.--(--OH).dbd.CH--),
amide/imidic acid (--C(.dbd.O)--NH--.revreaction.--C(--OH).dbd.N--)
and amidine
(--C(.dbd.NR)--NH--.revreaction.--C(--NHR).dbd.N--)tautomers. The
latter two are particularly common in heteroaryl and heterocyclic
rings and the present invention encompasses all tautomeric forms of
the compounds.
[0023] Technical and scientific teens used herein have the meaning
commonly understood by one of skill in the art to which the present
invention pertains, unless otherwise defined. Reference is made
herein to various methodologies and materials known to those of
skill in the art. Standard reference works setting forth the
general principles of pharmacology include Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.thEd., McGraw Hill
Companies Inc., New York (2001). Any suitable materials and/or
methods known to those of skill can be utilized in carrying out the
present invention. However, preferred materials and methods are
described. Materials, reagents and the like to which reference are
made in the following description and examples are obtainable from
commercial sources, unless otherwise noted.
[0024] The definitions described herein may be appended to form
chemically-relevant combinations, such as "heteroalkylaryl,"
"haloalkylheteroaryl," "arylalkylheterocyclyl," "alkylcarbonyl,"
"alkoxyalkyl," and the like. When the term "alkyl" is used as a
suffix following another term, as in "phenylalkyl," or
"hydroxyalkyl," this is intended to refer to an alkyl group, as
defined above, being substituted with one to two substituents
selected from the other specifically-named group. Thus, for
example, "phenylalkyl" refers to an alkyl group having one to two
phenyl substituents, and thus includes benzyl, phenylethyl, and
biphenyl. An "alkylaminoalkyl" is an alkyl group having one to two
alkylamino substituents. "Hydroxyalkyl" includes 2-hydroxyethyl,
2-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl,
2,3-dihydroxybutyl, 2-(hydroxymethyl), 3-hydroxypropyl, and so
forth. Accordingly, as used herein, the term "hydroxyalkyl" is used
to define a subset of heteroalkyl groups defined below. The term
-(ar)alkyl refers to either an unsubstituted alkyl or an aralkyl
group. The term (hetero)aryl or (het)aryl refers to either an aryl
or a heteroaryl group.
[0025] The term "carbonyl" or "acyl" as used herein denotes a group
of formula --C(.dbd.O)R wherein R is hydrogen or lower alkyl as
defined herein.
[0026] The term "ester" as used herein denotes a group of formula
--C(.dbd.O)OR wherein R is lower alkyl as defined herein.
[0027] The term "alkyl" as used herein denotes an unbranched or
branched chain, saturated, monovalent hydrocarbon residue
containing 1 to 10 carbon atoms. The term "lower alkyl" denotes a
straight or branched chain hydrocarbon residue containing 1 to 6
carbon atoms. "C.sub.1-10alkyl" as used herein refers to an alkyl
composed of 1 to 10 carbons. Examples of alkyl groups include, but
are not limited to, lower alkyl groups include methyl, ethyl,
propyl, i-propyl, n-butyl, i-butyl, t-butyl or pentyl, isopentyl,
neopentyl, hexyl, heptyl, and octyl.
[0028] When the term "alkyl" is used as a suffix following another
term, as in "phenylalkyl," or "hydroxyalkyl," this is intended to
refer to an alkyl group, as defined above, being substituted with
one to two substituents selected from the other specifically-named
group. Thus, for example, "phenylalkyl" denotes the radical R'R''-,
wherein R' is a phenyl radical, and R'' is an alkylene radical as
defined herein with the understanding that the attachment point of
the phenylalkyl moiety will be on the alkylene radical. Examples of
arylalkyl radicals include, but are not limited to, benzyl,
phenylethyl, 3-phenylpropyl. The terms "arylalkyl" or "aralkyl" are
interpreted similarly except R' is an aryl radical. The terms
"(het)arylalkyl" or "(het)aralkyl" are interpreted similarly except
R' is optionally an aryl or a heteroaryl radical.
[0029] The terms "haloalkyl" or "halo lower alkyl" or "lower
haloalkyl" refers to a straight or branched chain hydrocarbon
residue containing 1 to 6 carbon atoms wherein one or more carbon
atoms are substituted with one or more halogen atoms.
[0030] The term "alkylene" or "alkylenyl" as used herein denotes a
divalent saturated linear hydrocarbon radical of 1 to 10 carbon
atoms (e.g., (CH.sub.2).sub.n) or a branched saturated divalent
hydrocarbon radical of 2 to 10 carbon atoms (e.g., --CHMe- or
--CH.sub.2CH(i-Pr)CH.sub.2--), unless otherwise indicated. Except
in the case of methylene, the open valences of an alkylene group
are not attached to the same atom. Examples of alkylene radicals
include, but are not limited to, methylene, ethylene, propylene,
2-methyl-propylene, 1,1-dimethyl-ethylene, butylene,
2-ethylbutylene.
[0031] The term "alkoxy" as used herein means an --O-alkyl group,
wherein alkyl is as defined above such as methoxy, ethoxy,
n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy,
pentyloxy, hexyloxy, including their isomers. "Lower alkoxy" as
used herein denotes an alkoxy group with a "lower alkyl" group as
previously defined. "C.sub.1-10alkoxy" as used herein refers to
an-O-alkyl wherein alkyl is C.sub.1-10.
[0032] The terms "haloalkoxy" or "halo lower alkoxy" or "lower
haloalkoxy" refers to a lower alkoxy group, wherein one or more
carbon atoms are substituted with one or more halogen atoms.
[0033] The term "hydroxyalkyl" as used herein denotes an alkyl
radical as herein defined wherein one to three hydrogen atoms on
different carbon atoms is/are replaced by hydroxyl groups.
[0034] The term "sulfinyl" as used herein denotes a --SO--
group.
[0035] The term "sulfonyl" as used herein denotes a --SO.sub.2--
group.
[0036] The terms "alkylsulfonyl" and "arylsulfonyl" as used herein
refers to a group of formula --S(.dbd.O).sub.2R wherein R is alkyl
or aryl respectively and alkyl and aryl are as defined herein. The
term "heteroalkylsulfonyl" as used herein refers herein denotes a
group of formula --S(.dbd.O).sub.2R wherein R is "heteroalkyl" as
defined herein.
[0037] The term "lower alkyl sulfonylamido" as used herein refers
to a group of formula --S(.dbd.O).sub.2NR.sub.2 wherein each R is
independently hydrogen or C.sub.1-3alkyl, and lower alkyl is as
defined herein.
[0038] The term "trifluoromethyl sulfonyl" as used herein refers to
a group of formula --S(.dbd.O).sub.2CF.sub.3.
[0039] The term "trifluoromethyl sulfinyl" as used herein refers to
a group of formula --S(.dbd.O)CF.sub.3.
[0040] The term "trifluoromethyl sulfanyl" as used herein refers to
a group of formula --SCF.sub.3.
[0041] The term "nitro" as used herein refers to a group of formula
--N.sup.+(.dbd.O)O.sup.-.
[0042] The term "carboxyl" as used herein refers to a group of
formula --C(.dbd.O)R.sub.2 wherein each R is independently hydrogen
or C.sub.1-3alkyl, and lower alkyl is as defined herein.
[0043] The term "cycloalkyl" denotes a monovalent saturated
monocyclic or bicyclic hydrocarbon group of 3 to 10 ring carbon
atoms. In particular embodiments cycloalkyl denotes a monovalent
saturated monocyclic hydrocarbon group of 3 to 8 ring carbon atoms.
Bicyclic means consisting of two saturated carbocycles having one
or more carbon atoms in common. Particular cycloalkyl groups are
monocyclic. Examples for monocyclic cycloalkyl are cyclopropyl,
cyclobutanyl, cyclopentyl, cyclohexyl or cycloheptyl. Examples for
bicyclic cycloalkyl are bicyclo[2.2.1]heptanyl, or
bicyclo[2.2.2]octanyl.
[0044] The term "amino" as used herein denotes a group of the
formula --NR'R'' wherein R' and R'' are independently hydrogen,
alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.
Alternatively, R' and R'', together with the nitrogen to which they
are attached, can form a heterocycloalkyl. The term "primary amino"
denotes a group wherein both R' and R'' are hydrogen. The term
"secondary amino" denotes a group wherein R' is hydrogen and R'' is
not. The term "tertiary amino" denotes a group wherein both R' and
R'' are not hydrogen. Particular secondary and tertiary amines are
methylamine, ethylamine, propylamine, isopropylamine, phenylamine,
benzylamine dimethylamine, diethylamine, dipropylamine and
diisopropylamine.
[0045] The term "amido" as used herein denotes a group of the
formula --C(.dbd.O)NR'R'' or --NR'C(.dbd.O)R'' wherein R' and R''
are independently hydrogen, alkyl, alkoxy, cycloalkyl,
heterocycloalkyl, aryl or heteroaryl.
[0046] The term "heteroaryl" denotes a monovalent aromatic
heterocyclic mono- or bicyclic ring system of 5 to 12 ring atoms,
comprising 1, 2, 3 or 4 heteroatoms selected from N, O and S, the
remaining ring atoms being carbon. Examples of heteroaryl moieties
include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl,
thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl,
pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl,
isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl,
benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl,
benzoisothiazolyl, benzooxadiazolyl, benzothiadiazolyl,
benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl,
or quinoxalinyl.
[0047] The term "heterocycloalkyl" denotes a monovalent saturated
or partly unsaturated mono- or bicyclic ring system of 3 to 9 ring
atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O
and S, the remaining ring atoms being carbon. In particular
embodiments, heterocycloalkyl is a monovalent saturated monocyclic
ring system of 4 to 7 ring atoms, comprising 1, 2, or 3 ring
heteroatoms selected from N, O and S, the remaining ring atoms
being carbon. Examples for monocyclic saturated heterocycloalkyl
are aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl,
tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl,
imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,
piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl,
morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl,
azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Examples for
bicyclic saturated heterocycloalkyl are 8-aza-bicyclo[3.2.1]octyl,
quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl,
9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, or
3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples for partly unsaturated
heterocycloalkyl are dihydrofuryl, imidazolinyl, dihydro-oxazolyl,
tetrahydro-pyridinyl, or dihydropyranyl.
Inhibitors of HCV Entry
[0048] The application provides a compound of formula I
##STR00004##
wherein: R.sup.1 is H or lower alkyl; each R.sup.2 is independently
halo or lower haloalkyl; n is 0, 1, or 2; R.sup.3 is lower
haloalkyl, cyano, lower alkoxy, C(.dbd.O)OCH.sub.3, or
S(.dbd.O).sub.2CH.sub.3; p is 0, or 1;
X.sup.1 is S, S(.dbd.O).sub.2, O, S(.dbd.O), NH, or OCH.sub.2;
and
X.sup.2 is CH or N;
[0049] or a pharmaceutically acceptable salt thereof.
[0050] The application provides a compound of formula I, wherein
X.sup.2 is CH and R.sup.1 is H.
[0051] The application provides a compound of formula I, wherein
X.sup.1 is S or O.
[0052] The application provides a compound of formula I, wherein
X.sup.1 is S(.dbd.O).sub.2 or S(.dbd.O).
[0053] The application provides a compound of formula I, wherein
X.sup.1 is S or O, X.sup.2 is CH and R.sup.1 is H.
[0054] The application provides a compound of formula I, wherein
X.sup.1 is S(.dbd.O).sub.2 or S(.dbd.O), X.sup.2 is CH and R.sup.1
is H.
[0055] The application provides any of the above compounds of
formula I, wherein n 1.
[0056] The application provides the above compound of formula I,
wherein R.sup.1 is Cl.
[0057] The application provides any of the above compounds of
formula I, wherein n is 2.
[0058] The application provides the above compound of formula I,
wherein both R.sup.1 are Cl.
[0059] The application provides any of the above compounds of
formula I, wherein p is 0.
[0060] The application provides any of the above compounds of
formula I, wherein p is 1.
[0061] The application provides any of the above compounds of
formula I, wherein R.sup.3 is or S(.dbd.O).sub.2CH.sub.3, cyano, or
CF.sub.3.
[0062] The application provides a compound selected from the group
consisting of: [0063]
N5-(3-Fluoro-4-phenylsulfanyl-5-trifluoromethyl-phenyl)-1H-[1,2,4]triazol-
e-3,5-diamine; [0064]
N3-(3-Chloro-4-phenylamino-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
[0065]
N3-(3-Chloro-4-phenoxy-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
[0066]
N3-(3,5-Dichloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
[0067]
N3-(4-Benzenesulfinyl-3,5-dichloro-phenyl)-1H-[1,2,4]triazole-3,5--
diamine; [0068] N3-(4-Benzenesulfinyl-3,5-di
chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine; [0069]
N3-[3,5-Dichloro-4-(4-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4]t-
riazole-3,5-diamine; [0070]
N3-[3,5-Dichloro-4-(4-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1,2,4]-
triazole-3,5-diamine; [0071]
N3-[3,5-Dichloro-4-(4-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1,2,4]-
triazole-3,5-diamine; [0072]
N.sup.3-[3,5-Dichloro-4-(2-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,-
2,4]triazole-3,5-diamine; [0073]
N.sup.3-[3,5-Dichloro-4-(3-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1-
,2,4]triazole-3,5-diamine; [0074]
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-phenylsulfanyl]-b-
enzonitrile; [0075]
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfinyl]--
benzonitrile; [0076]
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfonyl]--
benzonitrile; [0077]
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2-chloro-6-trifluoromethyl-phe-
nylsulfanyl]-benzoic acid methyl ester; [0078]
N.sup.3[3,5-Dichloro-4-(4-methoxy-phenylsulfanyl)-phenyl]-1H-[1,2,4]triaz-
ole-3,5-diamine; [0079]
N.sup.3-[3-Chloro-4-(4-methoxy-phenylsulfanyl)-5-trifluoromethyl-phenyl]--
1H-[1,2,4]triazole-3,5-diamine; [0080]
N.sup.3-[3,5-Dichloro-4-(4-methoxy-benzenesulfonyl)-phenyl]-1H-[1,2,4]tri-
azole-3,5-diamine; [0081] N.sup.3-[3,5-Di
chloro-4-(4-methanesulfonyl-phenoxy)-phenyl]-1H-[1,2,4]triazole-3,5-diami-
ne; [0082]
N.sup.3-[3-Chloro-4-(4-methanesulfonyl-phenoxy)-5-trifluorometh-
yl-phenyl]-1H-[1,2,4]triazole-3,5-diamine; [0083]
N.sup.3-(3-Benzyloxy-5-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
[0084]
N.sup.3-(3-Chloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]triazole-3,5--
diamine; [0085]
N.sup.3-(4-Benzenesulfonyl-3-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamin-
e; [0086]
N.sup.3-(3-Chloro-4-phenylsulfanyl-phenyl)-N.sup.3-methyl-1H-[1,-
2,4]triazole-3,5-diamine; [0087]
N.sup.3-(3-Chloro-5-phenoxy-phenyl)-1H-[1,2,4]triazole-3,5-diamine;
and [0088]
N.sup.3-[3,5-Dichloro-4-(pyridin-2-yloxy)-phenyl]-1H-[1,2,4]triazo-
le-3,5-diamine.
[0089] The application provides a method for preventing a Hepatitis
C Virus (HCV) infection comprising administering to a patient in
need thereof a therapeutically effective amount of a compound of
Formula I.
[0090] The application provides the above method, further
comprising administering to a patient in need thereof a
therapeutically effective amount of an immune system
suppressant.
[0091] The application provides a method for treating a Hepatitis C
Virus (HCV) infection comprising administering to a patient in need
thereof a therapeutically effective amount of a compound of Formula
I.
[0092] The application provides any of the above methods, further
comprising administering a combination of antiviral agents that
inhibits replication of HCV.
[0093] The application provides any of the above methods, further
comprising administering an immune system modulator or an antiviral
agent that inhibits replication of HCV, or a combination
thereof.
[0094] The application provides the above method, wherein the
immune system modulator is an interferon or a chemically
derivatized interferon.
[0095] The application provides any of the above methods, further
comprising administering an immune system modulator or an antiviral
agent that inhibits replication of HCV, or a combination thereof,
wherein the antiviral agent is selected from the group consisting
of a HCV protease inhibitor, a HCV polymerase inhibitor, a HCV
helicase inhibitor, a HCV NS5A inhibitor, or any combination
thereof.
[0096] The application provides a composition comprising a compound
of Formula I and a pharmaceutically acceptable excipient.
[0097] The application provides the use of the compound of Formula
I in the preparation of a medicament for the prevention of HCV.
[0098] The application provides the use of the compound of Formula
I in the preparation of a medicament for the treatment of HCV.
[0099] The application provides any compound, composition, method
or use as described herein.
Compounds
[0100] Examples of representative compounds encompassed by the
present invention and within the scope of the invention are
provided in the following Table. These examples and preparations
which follow are provided to enable those skilled in the art to
more clearly understand and to practice the present invention. They
should not be considered as limiting the scope of the invention,
but merely as being illustrative and representative thereof.
[0101] In general, the nomenclature used in this Application is
based on AUTONOMTM v.4.0, a Beilstein Institute computerized system
for the generation of IUPAC systematic nomenclature. If there is a
discrepancy between a depicted structure and a name given that
structure, the depicted structure is to be accorded more weight. In
addition, if the stereochemistry of a structure or a portion of a
structure is not indicated with, for example, bold or dashed lines,
the structure or portion of the structure is to be interpreted as
encompassing all stereoisomers of it.
[0102] Table I depicts examples of compounds according to generic
Formula I:
TABLE-US-00001 TABLE I # Nomenclature Structure 1
N5-(3-Fluoro-4-phenylsulfanyl-5- trifluoromethyl-phenyl)-1H-
[1,2,4]triazole-3,5-diamine ##STR00005## 2
N3-(3-Chloro-4-phenylamino-phenyl)- 1H-[1,2,4]triazole-3,5-diamine
##STR00006## 3 N3-(3-Chloro-4-phenoxy-phenyl)-1H-
[1,2,4]triazole-3,5-diamine ##STR00007## 4
N3-(3,5-Dichloro-4-phenylsulfanyl-
phenyl)-1H-[1,2,4]triazole-3,5-diamine ##STR00008## 5
N3-(4-Benzenesulfinyl-3,5-dichloro-
phenyl)-1H-[1,2,4]triazole-3,5-diamine ##STR00009## 6
N3-(4-Benzenesulfinyl-3,5-dichloro-
phenyl)-1H-[1,2,4]triazole-3,5-diamine ##STR00010## 7
N3-[3,5-Dichloro-4-(4- trifluoromethyl-phenylsulfanyl)-
phenyl]-1H-[1,2,4]triazole-3,5-diamine ##STR00011## 8
N3-[3,5-Dichloro-4-(4- trifluoromethyl-benzenesulfinyl)-
phenyl]-1H-[1,2,4]triazole-3,5-diamine ##STR00012## 9
N3-[3,5-Dichloro-4-(4- trifluoromethyl-benzenesulfinyl)-
phenyl]-1H-[1,2,4]triazole-3,5-diamine ##STR00013## 10
N.sup.3-[3,5-Dichloro-4-(2-trifluoromethyl-
phenylsulfanyl)-phenyl]-1H- [1,2,4]triazole-3,5-diamine
##STR00014## 11 N.sup.3-[3,5-Dichloro-4-(3-trifluoromethyl-
benzenesulfinyl)-phenyl]-1H- [1,2,4]triazole-3,5-diamine
##STR00015## 12 4-[4-(5-Amino-1H-[1,2,4]triazol-3-
ylamino)-2,6-dichloro-phenylsulfanyl]- benzonitrile ##STR00016## 13
4-[4-(5-Amino-1H-[1,2,4]triazol-3- ylamino)-2,6-dichloro-
benzenesulfinyl]-benzonitrile ##STR00017## 14
4-[4-(5-Amino-1H-[1,2,4]triazol-3- ylamino)-2,6-dichloro-
benzenesulfonyl]-benzonitrile ##STR00018## 15
4-[4-(5-Amino-1H-[1,2,4]triazol-3-
ylamino)-2-chloro-6-trifluoromethyl- phenylsulfanyl]-benzoic acid
methyl ester ##STR00019## 16 N.sup.3-[3,5-Dichloro-4-(4-methoxy-
phenylsulfanyl)-phenyl]-1H- [1,2,4]triazole-3,5-diamine
##STR00020## 17 N.sup.3-[3-Chloro-4-(4-methoxy-
phenylsulfanyl)-5-trifluoromethyl-
phenyl]-1H-[1,2,4]triazole-3,5-diamine ##STR00021## 18
N.sup.3-[3,5-Dichloro-4-(4-methoxy- benzenesulfonyl)-phenyl]-1H-
[1,2,4]triazole-3,5-diamine ##STR00022## 19
N.sup.3-[3,5-Dichloro-4-(4- methanesulfonyl-phenoxy)-phenyl]-
1H-[1,2,4]triazole-3,5-diamine ##STR00023## 20
N.sup.3-[3-Chloro-4-(4-methanesulfonyl-
phenoxy)-5-trifluoromethyl-phenyl]- 1H-[1,2,4]triazole-3,5-diamine
##STR00024## 21 N.sup.3-(3-Benzyloxy-5-chloro-phenyl)-
1H-[1,2,4]triazole-3,5-diamine ##STR00025## 22
N.sup.3-(3-Chloro-4-phenylsulfanyl-
phenyl)-1H-[1,2,4]triazole-3,5-diamine ##STR00026## 23
N.sup.3-(4-Benzenesulfonyl-3-chloro-
phenyl)-1H-[1,2,4]triazole-3,5-diamine ##STR00027## 24
N.sup.3-(3-Chloro-4-phenylsulfanyl-
phenyl)-N.sup.3-methyl-1H-[1,2,4]triazole- 3,5-diamine ##STR00028##
25 N.sup.3-(3-Chloro-S-phenoxy-phenyl)-1H-
[1,2,4]triazole-3,5-diamine ##STR00029## 26
N.sup.3-[3,5-Dichloro-4-(pyridin-2-yloxy)-
phenyl]-1H-[1,2,4]triazole-3,5-diamine ##STR00030##
Synthesis
General Schemes
[0103] The following schemes depict general methods for obtaining
compounds of Formula
##STR00031## ##STR00032##
##STR00033##
##STR00034##
Dosage and Administration:
[0104] The compounds of the present invention may be formulated in
a wide variety of oral administration dosage forms and carriers.
Oral administration can be in the form of tablets, coated tablets,
dragees, hard and soft gelatin capsules, solutions, emulsions,
syrups, or suspensions. Compounds of the present invention are
efficacious when administered by other routes of administration
including continuous (intravenous drip) topical parenteral,
intramuscular, intravenous, subcutaneous, transdermal (which may
include a penetration enhancement agent), buccal, nasal, inhalation
and suppository administration, among other routes of
administration. The preferred manner of administration is generally
oral using a convenient daily dosing regimen which can be adjusted
according to the degree of affliction and the patient's response to
the active ingredient.
[0105] A compound or compounds of the present invention, as well as
their pharmaceutically useable salts, together with one or more
conventional excipients, carriers, or diluents, may be placed into
the form of pharmaceutical compositions and unit dosages. The
pharmaceutical compositions and unit dosage forms may be comprised
of conventional ingredients in conventional proportions, with or
without additional active compounds or principles, and the unit
dosage forms may contain any suitable effective amount of the
active ingredient commensurate with the intended daily dosage range
to be employed. The pharmaceutical compositions may be employed as
solids, such as tablets or filled capsules, semisolids, powders,
sustained release formulations, or liquids such as solutions,
suspensions, emulsions, elixirs, or filled capsules for oral use;
or in the form of suppositories for rectal or vaginal
administration; or in the form of sterile injectable solutions for
parenteral use. A typical preparation will contain from about 5% to
about 95% active compound or compounds (w/w). The term
"preparation" or "dosage form" is intended to include both solid
and liquid formulations of the active compound and one skilled in
the art will appreciate that an active ingredient can exist in
different preparations depending on the target organ or tissue and
on the desired dose and pharmacokinetic parameters.
[0106] The term "excipient" as used herein refers to a compound
that is useful in preparing a pharmaceutical composition, generally
safe, non-toxic and neither biologically nor otherwise undesirable,
and includes excipients that are acceptable for veterinary use as
well as human pharmaceutical use. The compounds of this invention
can be administered alone but will generally be administered in
admixture with one or more suitable pharmaceutical excipients,
diluents or carriers selected with regard to the intended route of
administration and standard pharmaceutical practice.
[0107] "Pharmaceutically acceptable" means that which is useful in
preparing a pharmaceutical composition that is generally safe,
non-toxic, and neither biologically nor otherwise undesirable and
includes that which is acceptable for veterinary as well as human
pharmaceutical use.
[0108] A "pharmaceutically acceptable salt" form of an active
ingredient may also initially confer a desirable pharmacokinetic
property on the active ingredient which were absent in the non-salt
form, and may even positively affect the pharmacodynamics of the
active ingredient with respect to its therapeutic activity in the
body. The phrase "pharmaceutically acceptable salt" of a compound
means a salt that is pharmaceutically acceptable and that possesses
the desired pharmacological activity of the parent compound. Such
salts include: (1) acid addition salts, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like; or formed with organic acids
such as acetic acid, propionic acid, hexanoic acid,
cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic
acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric
acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary
butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic
acid, and the like; or (2) salts formed when an acidic proton
present in the parent compound either is replaced by a metal ion,
e.g., an alkali metal ion, an alkaline earth ion, or an aluminum
ion; or coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like.
[0109] Solid form preparations include powders, tablets, pills,
capsules, cachets, suppositories, and dispersible granules. A solid
carrier may be one or more substances which may also act as
diluents, flavoring agents, solubilizers, lubricants, suspending
agents, binders, preservatives, tablet disintegrating agents, or an
encapsulating material. In powders, the carrier generally is a
finely divided solid which is a mixture with the finely divided
active component. In tablets, the active component generally is
mixed with the carrier having the necessary binding capacity in
suitable proportions and compacted in the shape and size desired.
Suitable carriers include but are not limited to magnesium
carbonate, magnesium stearate, talc, sugar, lactose, pectin,
dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, a low melting wax, cocoa butter, and the
like. Solid form preparations may contain, in addition to the
active component, colorants, flavors, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners,
solubilizing agents, and the like.
[0110] Liquid formulations also are suitable for oral
administration include liquid formulation including emulsions,
syrups, elixirs, aqueous solutions, aqueous suspensions. These
include solid form preparations which are intended to be converted
to liquid form preparations shortly before use. Emulsions may be
prepared in solutions, for example, in aqueous propylene glycol
solutions or may contain emulsifying agents such as lecithin,
sorbitan monooleate, or acacia. Aqueous solutions can be prepared
by dissolving the active component in water and adding suitable
colorants, flavors, stabilizing, and thickening agents. Aqueous
suspensions can be prepared by dispersing the finely divided active
component in water with viscous material, such as natural or
synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other well-known suspending agents.
[0111] The compounds of the present invention may be formulated for
parenteral administration (e.g., by injection, for example bolus
injection or continuous infusion) and may be presented in unit dose
form in ampoules, pre-filled syringes, small volume infusion or in
multi-dose containers with an added preservative. The compositions
may take such forms as suspensions, solutions, or emulsions in oily
or aqueous vehicles, for example solutions in aqueous polyethylene
glycol. Examples of oily or nonaqueous carriers, diluents, solvents
or vehicles include propylene glycol, polyethylene glycol,
vegetable oils (e.g., olive oil), and injectable organic esters
(e.g., ethyl oleate), and may contain formulatory agents such as
preserving, wetting, emulsifying or suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form, obtained by aseptic isolation of sterile solid or by
lyophilization from solution for constitution before use with a
suitable vehicle, e.g., sterile, pyrogen-free water.
[0112] The compounds of the present invention may be formulated for
topical administration to the epidermis as ointments, creams or
lotions, or as a transdermal patch. Ointments and creams may, for
example, be formulated with an aqueous or oily base with the
addition of suitable thickening and/or gelling agents. Lotions may
be formulated with an aqueous or oily base and will in general also
containing one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents. Formulations suitable for topical administration
in the mouth include lozenges comprising active agents in a
flavored base, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base such as gelatin
and glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0113] The compounds of the present invention may be formulated for
administration as suppositories. A low melting wax, such as a
mixture of fatty acid glycerides or cocoa butter is first melted
and the active component is dispersed homogeneously, for example,
by stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool, and to solidify.
[0114] The compounds of the present invention may be formulated for
vaginal administration. Pessaries, tampons, creams, gels, pastes,
foams or sprays containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0115] The compounds of the present invention may be formulated for
nasal administration. The solutions or suspensions are applied
directly to the nasal cavity by conventional means, for example,
with a dropper, pipette or spray. The formulations may be provided
in a single or multidose form. In the latter case of a dropper or
pipette, this may be achieved by the patient administering an
appropriate, predetermined volume of the solution or suspension. In
the case of a spray, this may be achieved for example by means of a
metering atomizing spray pump.
[0116] The compounds of the present invention may be formulated for
aerosol administration, particularly to the respiratory tract and
including intranasal administration. The compound will generally
have a small particle size for example of the order of five (5)
microns or less. Such a particle size may be obtained by means
known in the art, for example by micronization. The active
ingredient is provided in a pressurized pack with a suitable
propellant such as a chlorofluorocarbon (CFC), for example,
dichlorodifluoromethane, trichlorofluoromethane, or
dichlorotetrafiuoroethane, or carbon dioxide or other suitable gas.
The aerosol may conveniently also contain a surfactant such as
lecithin. The dose of drug may be controlled by a metered valve.
Alternatively the active ingredients may be provided in a form of a
dry powder, for example a powder mix of the compound in a suitable
powder base such as lactose, starch, starch derivatives such as
hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The
powder carrier will form a gel in the nasal cavity. The powder
composition may be presented in unit dose form for example in
capsules or cartridges of e.g., gelatin or blister packs from which
the powder may be administered by means of an inhaler.
[0117] When desired, formulations can be prepared with enteric
coatings adapted for sustained or controlled release administration
of the active ingredient. For example, the compounds of the present
invention can be formulated in transdermal or subcutaneous drug
delivery devices. These delivery systems are advantageous when
sustained release of the compound is necessary and when patient
compliance with a treatment regimen is crucial. Compounds in
transdermal delivery systems are frequently attached to a
skin-adhesive solid support. The compound of interest can also be
combined with a penetration enhancer, e.g., Azone
(1-dodecylaza-cycloheptan-2-one). Sustained release delivery
systems are inserted subcutaneously into to the subdermal layer by
surgery or injection. The subdermal implants encapsulate the
compound in a lipid soluble membrane, e.g., silicone rubber, or a
biodegradable polymer, e.g., polylactic acid.
[0118] Suitable formulations along with pharmaceutical carriers,
diluents and excipients are described in Remington: The Science and
Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing
Company, 19th edition, Easton, Pa. A skilled formulation scientist
may modify the formulations within the teachings of the
specification to provide numerous formulations for a particular
route of administration without rendering the compositions of the
present invention unstable or compromising their therapeutic
activity.
[0119] The modification of the present compounds to render them
more soluble in water or other vehicle, for example, may be easily
accomplished by minor modifications (salt formulation,
esterification, etc.), which are well within the ordinary skill in
the art. It is also well within the ordinary skill of the art to
modify the route of administration and dosage regimen of a
particular compound in order to manage the pharmacokinetics of the
present compounds for maximum beneficial effect in patients.
[0120] The term "therapeutically effective amount" as used herein
means an amount required to reduce symptoms of the disease in an
individual. The dose will be adjusted to the individual
requirements in each particular case. That dosage can vary within
wide limits depending upon numerous factors such as the severity of
the disease to be treated, the age and general health condition of
the patient, other medicaments with which the patient is being
treated, the route and form of administration and the preferences
and experience of the medical practitioner involved. For oral
administration, a daily dosage of between about 0.01 and about 1000
mg/kg body weight per day should be appropriate in monotherapy
and/or in combination therapy. A preferred daily dosage is between
about 0.1 and about 500 mg/kg body weight, more preferred 0.1 and
about 100 mg/kg body weight and most preferred 1.0 and about 10
mg/kg body weight per day. Thus, for administration to a 70 kg
person, the dosage range would be about 7 mg to 0.7 g per day. The
daily dosage can be administered as a single dosage or in divided
dosages, typically between 1 and 5 dosages per day. Generally,
treatment is initiated with smaller dosages which are less than the
optimum dose of the compound. Thereafter, the dosage is increased
by small increments until the optimum effect for the individual
patient is reached. One of ordinary skill in treating diseases
described herein will be able, without undue experimentation and in
reliance on personal knowledge, experience and the disclosures of
this application, to ascertain a therapeutically effective amount
of the compounds of the present invention for a given disease and
patient.
[0121] The pharmaceutical preparations are preferably in unit
dosage forms. In such form, the preparation is subdivided into unit
doses containing appropriate quantities of the active component.
The unit dosage form can be a packaged preparation, the package
containing discrete quantities of preparation, such as packeted
tablets, capsules, and powders in vials or ampoules. Also, the unit
dosage form can be a capsule, tablet, cachet, or lozenge itself, or
it can be the appropriate number of any of these in packaged
form.
Indications and Method of Treatment
Indications
[0122] The application provides a method for preventing a Hepatitis
C Virus (HCV) infection comprising administering to a patient in
need thereof a therapeutically effective amount of a compound of
Formula I.
[0123] The application provides the above method, further
comprising administering to a patient in need thereof a
therapeutically effective amount of an immune system
suppressant.
[0124] The application provides a method for treating a Hepatitis C
Virus (HCV) infection comprising administering to a patient in need
thereof a therapeutically effective amount of a compound of Formula
I.
[0125] The application provides any of the above methods, further
comprising administering an immune system modulator or an antiviral
agent that inhibits replication of HCV, or a combination
thereof.
[0126] The application provides the above method, wherein the
immune system modulator is an interferon or a chemically
derivatized interferon.
[0127] The application provides any of the above methods, further
comprising administering an immune system modulator or an antiviral
agent that inhibits replication of HCV, or a combination thereof,
wherein the antiviral agent is selected from the group consisting
of a HCV protease inhibitor, a HCV polymerase inhibitor, a HCV
helicase inhibitor, a HCV NS5A inhibitor, or any combination
thereof.
Combination Therapy
[0128] The compounds of the invention and their isomeric forms and
pharmaceutically acceptable salts thereof are useful in treating
and preventing HCV infection alone or when used in combination with
other compounds targeting viral or cellular elements or functions
involved in the HCV lifecycle. Classes of compounds useful in the
invention include, without limitation, all classes of HCV
antivirals.
[0129] For combination therapies, mechanistic classes of agents
that can be useful when combined with the compounds of the
invention include, for example, nucleoside and non-nucleoside
inhibitors of the HCV polymerase, protease inhibitors, helicase
inhibitors, NS4B inhibitors, NS5A inhibitors and medicinal agents
that functionally inhibit the internal ribosomal entry site (IRES)
and other medicaments that inhibit HCV cell attachment or virus
entry, HCV RNA translation, HCV RNA transcription, replication or
HCV maturation, assembly or virus release. Specific compounds in
these classes and useful in the invention include, but are not
limited to, macrocyclic, heterocyclic and linear HCV protease
inhibitors such as telaprevir (VX-950), boceprevir (SCH-503034),
narlaprevir (SCH-9005 18), ITMN-191 (R-7227), TMC-435350 (a.k.a.
TMC-435), MK-7009, BI-201335, BI-2061 (ciluprevir), BMS-650032,
ACH-1625, ACH-1095 (HCV NS4A protease co-factor inhibitor), VX-500,
VX-8 13, PHX-1766, PHX2054, IDX-136, IDX-3 16, ABT-450 EP-0 13420
(and congeners) and VBY-376; the Nucleosidic HCV polymerase
(replicase) inhibitors useful in the invention include, but are not
limited to, R7128, PSI-785 1, IDX-184, IDX-102, R1479, UNX-08 189,
PSI-6130, PSI-938 and PSI-879 and various other nucleoside and
nucleotide analogs and HCV inhibitors including (but not limited
to) those derived as 2'-C-methyl modified nucleos(t)ides, 4'-aza
modified nucleos(t)ides, and T-deaza modified nucleos(t)ides.
Non-nucleosidic HCV polymerase (replicase) inhibitors useful in the
invention, include, but are not limited to, HCV-796, HCV-371,
VCH-759, VCH-916, VCH-222, ANA-598, MK-3281, ABT-333, ABT-072,
PF-00868554, BI-207127, GS-9190, A-837093, JKT-109, GL-59728 and
GL-60667.
[0130] In addition, compounds of the invention can be used in
combination with cyclophyllin and immunophyllin antagonists (e.g.,
without limitation, DEBIO compounds, NM-811 as well as cyclosporine
and its derivatives), kinase inhibitors, inhibitors of heat shock
proteins (e.g., HSP90 and HSP70), other immunomodulatory agents
that can include, without limitation, interferons (-alpha, -beta,
-omega, -gamma, -lambda or synthetic) such as Intron A, Roferon-A,
Canferon-A300, Advaferon, Infergen, Humoferon, Sumiferon MP,
Alfaferone, IFN-.beta., Feron and the like; polyethylene glycol
derivatized (pegylated) interferon compounds, such as PEG
interferon-.alpha.-2a (Pegasys), PEG interferon-.alpha.-2b
(PEGIntron), pegylated IFN-.alpha.-con1 and the like; long acting
formulations and derivatizations of interferon compounds such as
the albumin-fused interferon, Albuferon, Locteron, and the like;
interferons with various types of controlled delivery systems
(e.g., ITCA-638, omega-interferon delivered by the DUROS
subcutaneous delivery system); compounds that stimulate the
synthesis of interferon in cells, such as resiquimod and the like;
interleukins; compounds that enhance the development of type 1
helper T cell response, such as SCV-07 and the like; TOLL-like
receptor agonists such as CpG-10101 (actilon), isotorabine, ANA773
and the like; thymosin .alpha.-1; ANA-245 and ANA-246; histamine
dihydrochloride; propagermanium; tetrachlorodecaoxide; ampligen;
IMP-321; KRN-7000; antibodies, such as civacir, XTL-6865 and the
like and prophylactic and therapeutic vaccines such as InnoVac C,
HCV E1E2/MF59 and the like. In addition, any of the above-described
methods involving administering an NS5A inhibitor, a Type I
interferon receptor agonist (e.g., an IFN-.alpha.) and a Type II
interferon receptor agonist (e.g., an IFN-.gamma.) can be augmented
by administration of an effective amount of a TNF-.alpha.
antagonist. Exemplary, non-limiting TNF-.alpha. antagonists that
are suitable for use in such combination therapies include ENBREL,
REMICADE, and HUMIRA.
[0131] In addition, compounds of the invention can be used in
combination with antiprotozoans and other antivirals thought to be
effective in the treatment of HCV infection such as, without
limitation, the prodrug nitazoxanide. Nitazoxanide can be used as
an agent in combination with the compounds disclosed in this
invention as well as in combination with other agents useful in
treating HCV infection such as peginterferon .alpha.-2a and
ribavirin.
[0132] Compounds of the invention can also be used with alternative
forms of interferons and pegylated interferons, ribavirin or its
analogs (e.g., tarabavarin, levoviron), microRNA, small interfering
RNA compounds (e.g., SIRPLEX-140-N and the like), nucleotide or
nucleoside analogs, immunoglobulins, hepatoprotectants,
anti-inflammatory agents and other inhibitors of NS5A. Inhibitors
of other targets in the HCV lifecycle include NS3 helicase
inhibitors; NS4A co-factor inhibitors; antisense oligonucleotide
inhibitors, such as ISIS-14803, AVI-4065 and the like;
vector-encoded short hairpin RNA (shRNA); HCV specific ribozymes
such as heptazyme, RPI, 13919 and the like; entry inhibitors such
as HepeX-C, HuMax-HepC and the like; alpha glucosidase inhibitors
such as celgosivir, UT-231B and the like; KPE-02003002 and BIVN 401
and IMPDH inhibitors. Other illustrative HCV inhibitor compounds
include those disclosed in the following publications: U.S. Pat.
Nos. 5,807,876; 6,498,178; 6,344,465; and 6,054,472; PCT Patent
Application Publication Nos. WO97/40028; WO98/4038 1; WO00/56331,
WO02/04425; WO03/007945; WO03/010141; WO03/000254; WO01/32153;
WO00/06529; WO00/18231; WO00/10573; WO00/13708; WO01/85172;
WO03/037893; WO03/037894; WO03/037895; WO02/100851; WO02/100846;
WO99/01582; WO00/09543; WO02/18369; WO98/17679, WO00/056331;
WO98/22496; WO99/07734; WO05/073216, WO05/073195 and
WO08/021,927.
[0133] Additionally, combinations of, for example, ribavirin and
interferon, may be administered as multiple combination therapy
with at least one of the compounds of the invention. The present
invention is not limited to the aforementioned classes or compounds
and contemplates known and new compounds and combinations of
biologically active agents. It is intended that combination
therapies of the present invention include any chemically
compatible combination of a compound of this inventive group with
other compounds of the inventive group or other compounds outside
of the inventive group, as long as the combination does not
eliminate the anti-viral activity of the compound of this inventive
group or the anti-viral activity of the pharmaceutical composition
itself.
[0134] Combination therapy can be sequential, that is treatment
with one agent first and then a second agent (for example, where
each treatment comprises a different compound of the invention or
where one treatment comprises a compound of the invention and the
other comprises one or more biologically active agents) or it can
be treatment with both agents at the same time (concurrently).
Sequential therapy can include a reasonable time after the
completion of the first therapy before beginning the second
therapy. Treatment with both agents at the same time can be in the
same daily dose or in separate doses. Combination therapy need not
be limited to two agents and may include three or more agents. The
dosages for both concurrent and sequential combination therapy will
depend on absorption, distribution, metabolism and excretion rates
of the components of the combination therapy as well as other
factors known to one of skill in the art. Dosage values will also
vary with the severity of the condition to be alleviated. It is to
be further understood that for any particular subject, specific
dosage regimens and schedules may be adjusted over time according
to the individual's need and the judgment of the one skilled in the
art administering or supervising the administration of the
combination therapy.
[0135] The application provides a method for preventing a Hepatitis
C Virus (HCV) infection comprising administering to a patient in
need thereof a therapeutically effective amount of a compound of
Formula I.
[0136] The application provides the above method, further
comprising administering to a patient in need thereof a
therapeutically effective amount of an immune system
suppressant.
[0137] The application provides a method for treating a Hepatitis C
Virus (HCV) infection comprising administering to a patient in need
thereof a therapeutically effective amount of a compound of Formula
I.
[0138] The application provides any of the above methods, further
comprising administering an immune system modulator or an antiviral
agent that inhibits replication of HCV, or a combination
thereof.
[0139] The application provides the above method, wherein the
immune system modulator is an interferon or a chemically
derivatized interferon.
[0140] The application provides any of the above methods, further
comprising administering an immune system modulator or an antiviral
agent that inhibits replication of HCV, or a combination thereof,
wherein the antiviral agent is selected from the group consisting
of a HCV protease inhibitor, a HCV polymerase inhibitor, a HCV
helicase inhibitor, a HCV NS5A inhibitor, or any combination
thereof.
EXAMPLES
Abbreviations
[0141] Commonly used abbreviations include: acetyl (Ac),
azo-bis-isobutyrylnitrile (AIBN), atmospheres (Atm),
9-borabicyclo[3.3.1]nonane (9-BBN or BBN),
2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP),
tert-butoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or boc
anhydride (BOC.sub.2O), benzyl (Bn), butyl (Bu), Chemical Abstracts
Registration Number (CASRN), benzyloxycarbonyl (CBZ or Z), carbonyl
diimidazole (CDI), 1,4-diazabicyclo[2.2.2]octane (DABCO),
diethylaminosulfur trifluoride (DAST), dibenzylideneacetone (dba),
1,5-diazabicyclo[4.3.0]non-5-ene (DBN),
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
N,N'-dicyclohexylcarbodiimide (DCC), 1,2-dichloroethane (DCE),
dichloromethane (DCM), 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ), diethyl azodicarboxylate (DEAD),
di-iso-propylazodicarboxylate (DIAD), di-iso-butylaluminumhydride
(DIBAL or DIBAL-H), di-iso-propylethylamine (DIPEA), N,N-dimethyl
acetamide (DMA), 4-N,N-dimethylaminopyridine (DMAP),
N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
1,1'-bis-(diphenylphosphino)ethane (dppe),
1,1'-bis-(diphenylphosphino)ferrocene (dppf),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI),
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), ethyl (Et),
ethyl acetate (EtOAc), ethanol (EtOH),
2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (EEDQ), diethyl
ether (Et.sub.2O), ethyl isopropyl ether (EtOiPr),
O-(7-azabenzotriazole-1-yl)-N,N,N'N'-tetramethyluronium
hexafluorophosphate acetic acid (HATU), acetic acid (HOAc),
1-N-hydroxybenzotriazole (HOBt), high pressure liquid
chromatography (HPLC), iso-propanol (IPA), isopropylmagnesium
chloride (iPrMgCl), hexamethyl disilazane (HMDS), liquid
chromatography mass spectrometry (LCMS), lithium hexamethyl
disilazane (LiHMDS), meta-chloroperoxybenzoic acid (m-CPBA),
methanol (MeOH), melting point (mp), MeSO.sub.2-- (mesyl or Ms),
methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA),
mass spectrum (ms), methyl t-butyl ether (MTBE), methyl
tetrahydrofuran (MeTHF), N-bromosuccinimide (NBS), n-Butyllithium
(nBuLi), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS),
N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridinium
chlorochromate (PCC), Dichloro-((bis-diphenylphosphino)ferrocenyl)
palladium(II) (Pd(dppf)Cl.sub.2), palladium(II) acetate
(Pd(OAc).sub.2), tris(dibenzylideneacetone)dipalladium(0)
(Pd.sub.2(dba).sub.3), pyridinium dichromate (PDC), phenyl (Ph),
propyl (Pr), iso-propyl (i-Pr), pounds per square inch (psi),
pyridine (pyr),
1,2,3,4,5-Pentaphenyl-1'-(di-tert-butylphosphino)ferrocene
(Q-Phos), room temperature (ambient temperature, rt or RT),
sec-Butyllithium (sBuLi), tert-butyldimethylsilyl or t-BuMe.sub.2Si
(TBDMS), tetra-n-butylammonium fluoride (TBAF), triethylamine (TEA
or Et.sub.3N), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO),
triflate or CF.sub.3SO.sub.2-- (Tf), trifluoroacetic acid (TFA),
1,1'-bis-2,2,6,6-tetramethylheptane-2,6-dione (TMHD),
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate
(TBTU), thin layer chromatography (TLC), tetrahydrofuran (THF),
trimethylsilyl or Me.sub.3S i (TMS), p-toluenesulfonic acid
monohydrate (TsOH or pTsOH), 4-Me-C.sub.6H.sub.4SO.sub.2-- or tosyl
(Ts), and N-urethane-N-carboxyanhydride (UNCA). Conventional
nomenclature including the prefixes normal (n), iso (i-), secondary
(sec-), tertiary (tert-) and neo have their customary meaning when
used with an alkyl moiety. (J. Rigaudy and D. P. Klesney,
Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press,
Oxford.).
General Conditions
[0142] Compounds of the invention can be made by a variety of
methods depicted in the illustrative synthetic reactions described
below in the Examples section.
[0143] The starting materials and reagents used in preparing these
compounds generally are either available from commercial suppliers,
such as Aldrich Chemical Co., or are prepared by methods known to
those skilled in the art following procedures set forth in
references such as Fieser and Fieser's Reagents for Organic
Synthesis; Wiley & Sons: New York, 1991, Volumes 1-15; Rodd's
Chemistry of Carbon Compounds, Elsevier Science Publishers, 1989,
Volumes 1-5 and Supplementals; and Organic Reactions, Wiley &
Sons: New York, 1991, Volumes 1-40. It should be appreciated that
the synthetic reaction schemes shown in the Examples section are
merely illustrative of some methods by which the compounds of the
invention can be synthesized, and various modifications to these
synthetic reaction schemes can be made and will be suggested to one
skilled in the art having referred to the disclosure contained in
this application.
[0144] The starting materials and the intermediates of the
synthetic reaction schemes can be isolated and purified if desired
using conventional techniques, including but not limited to,
filtration, distillation, crystallization, chromatography, and the
like. Such materials can be characterized using conventional means,
including physical constants and spectral data.
[0145] Unless specified to the contrary, the reactions described
herein are typically conducted under an inert atmosphere at
atmospheric pressure at a reaction temperature range of from about
-78.degree. C. to about 150.degree. C., often from about 0.degree.
C. to about 125.degree. C., and more often and conveniently at
about room (or ambient) temperature, e.g., about 20.degree. C.
[0146] Various substituents on the compounds of the invention can
be present in the starting compounds, added to any one of the
intermediates or added after formation of the final products by
known methods of substitution or conversion reactions. If the
substituents themselves are reactive, then the substituents can
themselves be protected according to the techniques known in the
art. A variety of protecting groups are known in the art, and can
be employed. Examples of many of the possible groups can be found
in "Protective Groups in Organic Synthesis" by Green et al., John
Wiley and Sons, 1999. For example, nitro groups can be added by
nitration and the nitro group can be converted to other groups,
such as amino by reduction, and halogen by diazotization of the
amino group and replacement of the diazo group with halogen. Acyl
groups can be added by Friedel-Crafts acylation. The acyl groups
can then be transformed to the corresponding alkyl groups by
various methods, including the Wolff-Kishner reduction and
Clemmenson reduction. Amino groups can be alkylated to form mono-
and di-alkylamino groups; and mercapto and hydroxy groups can be
alkylated to form corresponding ethers. Primary alcohols can be
oxidized by oxidizing agents known in the art to form carboxylic
acids or aldehydes, and secondary alcohols can be oxidized to form
ketones. Thus, substitution or alteration reactions can be employed
to provide a variety of substituents throughout the molecule of the
starting material, intermediates, or the final product, including
isolated products.
PREPARATIVE EXAMPLES
Intermediate 1
Procedure 1
N.sup.5-(4-bromo-3-fluoro-5-trifluoromethylphenyl)-1H-[1,2,4]-triazole-3,5-
-diamine (Intermediate 3)
##STR00035##
[0147]
2-bromo-1-fluoro-5-isothiocyanato-3-trifluoromethylbenzene
##STR00036##
[0149] 4-bromo-3-fluoro-5-trifluoromethylaniline (4.22 g, 16.4
mmol, Eq: 1.00) and calcium carbonate (3.44 g, 1.17 ml, 34.3 mmol,
Eq: 2.1) were suspended in 50% aqueous dichloromethane (20 ml)
mixture. The thick suspension was stirred vigorously at 0.degree.
C. Thiophosgene (2.07 g, 1.38 ml, 18.0 mmol, Eq: 1.1) was added
slowly dropwise to the mixture. After the addition the mixture was
stirred at 0.degree. C. for 1.5 hr then stirred overnight at room
temperature. The solids were filtered and the filtrate was
extracted with dichloromethane. The combined organic phases were
washed with water, brine, dried over sodium sulfate and
concentrated in vacuo to afford 4.71 g (96%) of the desired
material as a light brown solid.
[0150] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 7.84 (s, 1H)
7.96 (dd, J=9.06, 2.27 Hz, 1H)
(4-Bromo-3-fluoro-5-trifluoromethyl-phenylamino)-(methyl-.lamda..sup.4sulf-
anylidene)-methyl-cyanamide
##STR00037##
[0152] 2-bromo-1-fluoro-5-isothiocyanato-3-trifluoromethylbenzene
(4.71 g, 15.7 mmol, Eq: 1.00) was dissolved in anhydrous methanol
(30 ml). Sodium hydrogencyanamide (1.00 g, 15.7 mmol, Eq: 1) was
added and the reaction was stirred for 1 hr at ambient temperature.
Methyl iodide (4.46 g, 1.96 ml, 31.4 mmol, Eq: 2) was added
dropwise and the reaction was stirred overnight at ambient
temperature. The light brown suspension was filtered to afford 1.91
g (34%) of the desired product as a pink solid.
[0153] MS +m/z: 357.7. (M+1)
[0154] .sup.1H NMR (300 MHz, DMSO-d.sub.5) .delta. ppm 2.78 (s, 3H)
7.87 (s, 1H) 7.97 (dd, J=1.00 Hz, 1H) 10.38 (br. s, 1H)
Preparation of
N.sup.5-(4-bromo-3-fluoro-5-trifluoromethylphenyl)-1H-[1,2,4]-triazole-3,-
5-diamine (Intermediate 1)
##STR00038##
[0156] Hydrazine (1.71 g, 53.4 mmol, Eq: 10) was added to a stirred
suspension of
(4-Bromo-3-fluoro-5-trifluoromethyl-phenylamino)-(methyl-X.sup.4sulfanyli-
dene)-methyl-cyanamide (1.9 g, 5.34 mmol, Eq: 1.00) in ethanol (30
ml). The mixture was heated to 70.degree. C. for 1 hr. The reaction
mixture was concentrated to a reduced volume (.about.5 ml) and
water (.about.10 ml) was added dropwise while stirring. The
suspension was stirred for 30 min. The precipitate was filtered and
washed with water (.about.50 ml), then dried under high vacuum at
70.degree. C. for two hours to filtered to afford 1.73 g (95%) of
the desired product as a light pink solid.
[0157] MS +m/z: 339.9. (M+1)
[0158] .sup.1H NMR (400 MHz, DMSO-d.sub.5) 8 ppm 6.03 (s, 2H) 7.81
(s, 1H) 7.86 (d, J=12.13 Hz, 1H) 9.52 (s, 1H) 11.40 (s, 1H)
Example 1
N.sup.5-(3-Fluoro-4-phenylsulfanyl-5-trifluoromethylphenyl)-1H-[1,2,4]-tri-
azole-3,5-diamine (Compound 1)
##STR00039##
[0160]
N.sup.5-(4-Bromo-3-fluoro-5-trifluoromethylphenyl)-1H-[1,2,4]-triaz-
ole-3,5-diamine (intermediate 1) (66 mg, 194 .mu.mol, Eq: 1.00),
1,1'-bis(diisopropylphosphino)ferrocene (12.2 mg, 29.1 Eq: 0.15)
and sodium tert-butoxide (24.2 mg, 252 .mu.mol, Eq: 1.3) were
suspended in dioxane (1.25 ml). Pd(OAc).sub.2 (6.54 mg, 29.1
.mu.mol Eq: 0.15) and benzenethiol (22.5 mg, 20.8 .mu.l 204 .mu.mol
Eq: 1.05) were added under an argon atmosphere. The reaction
mixture was heated to 130.degree. C. for 2 hours in the microwave.
1,1'-bis(diisopropylphosphino)ferrocene (24.4 mg, 58.2 .mu.mol, Eq:
0.30) and Pd(OAc).sub.2 (13 mg, 58.2 .mu.mol, Eq: 0.30) was added
and the reaction mixture was heated to 130.degree. C. for 1 hour.
The reaction mixture was diluted with water and dichloromethane.
The aqueous phase was extracted with dichloromethane and the
combined organic phases were washed with brine, dried over
Na.sub.2SO.sub.4and stripped in vacuo. The crude material was
purified by silica gel chromatography (0-10% methanol in
dichloromethane) to give a brown solid. The product was repurified
by preparative HPLC (20% ACN: 0.3% TFA in water to 100% ACN) to
afford 4 mg (4%) of the desired product as a white solid.
[0161] MS +m/z: 369.9. (M+1)
[0162] .sup.1H NMR (400 MHz, MeOD) .delta. ppm 7.09-7.30 (m, 5H)
7.70-7.79 (m, 2H)
N*3*-(3-Chloro-4-phenylamino-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(Compound 2)
##STR00040##
[0163] 2-chloro-4-nitro-N-phenylaniline
##STR00041##
[0165] Charged sodium hydride (688.6 mg, 17.2 mmol, Eq: 1.48) into
a 250-mL round-bottomed flask while purging with argon. Added
N,N'-dimethylformamide (60 mL) and cooled the mixture in an ice
bath. Added aniline (1.02 g, 1.00 mL, 11.0 mmol, Eq: 0.944) and
stirred in ice bath for .about.10 min; then charged
2-chloro-1-fluoro-4-nitrobenzene (2.04 g, 11.6 mmol, Eq: 1.00) into
the cold reaction mixture in one portion. Monitored the reaction by
HPLC: Stirred at 0.degree. C. for 1 h, stirred at room temperature
17 h, then heated the reaction at 65.degree. C. overnight. Cooled
reaction mixture in an ice bath. Using 1 N hydrochloric acid
solution the reaction was slowly quenched. Diluted the reaction
mixture with water and extracted with ethyl acetate (3.times.).
Combined organic layers and washed with water and saturated sodium
chloride. Dried over magnesium sulfate, filtered, and concentrated.
Obtained 2.94 g of crude material. Redissolved in dichloromethane
and concentrated onto silica. Purified using a 220 g silica gel
column on an Intelliflash 280; collected 28 mL fractions at 88
mL/min; equilibrated with hexanes; dry loaded; eluted 3 min with
hexanes; increased from 0-50% dichloromethane/hexanes over 45 min;
held at 50% dichloromethane/hexanes for 9 min. Obtained 700 mg
(20.3%) of impure 2-chloro-4-nitro-N-phenylaniline (84% purity) as
a yellow solid.
2-chloro-N-1-phenylbenzene-1,4-diamine
##STR00042##
[0167] To the 250-mL round-bottomed flask containing
2-chloro-4-nitro-N-phenylaniline (700 mg, 2.82 mmol, Eq: 1.00) was
added methanol (10 mL) and water (5 mL) followed by iron (786 mg,
14.1 mmol, Eq: 5.0) and ammonium chloride (1.51 g, 28.2 mmol, Eq:
10.0) all while purging with argon. Heat the mixture at reflux for
8 h. Filtered the reaction mixture through Celite, rinsing with
methanol. Concentrated the filtrate, then dissolved the residue in
ethyl acetate and added water. Added solid sodium bicarbonate until
neutral. Split layers and washed the organics with water and
saturated sodium chloride solution. Dried over sodium sulfate,
filtered, and concentrated. Obtained 630 mg (82% yield at 80%
purity) crude 2-chloro-N.sup.1-phenylbenzene-1,4-diamine as a brown
oil. Used as is.
2-chloro-4-isothiocyanato-N-phenylaniline
##STR00043##
[0169] Purged the 50-mL round-bottomed flask containing
2-chloro-N-1-phenylbenzene-1,4-diamine (630 mg, 2.88 mmol, Eq:
1.00) with argon. Added dichloromethane (30 mL) and cooled the
mixture to 0.degree. C. in an ice bath. Added
1,1'-thiocarbonyldiimidazole (770 mg, 4.32 mmol, Eq: 1.5) in one
portion then removed the ice bath an allowed the reaction mixture
to warm to room temperature. Stirred for 1 h. The reaction mixture
was diluted with dichloromethane and concentrated onto silica.
Purified using a 120 g silica gel column on an Intelliflash 280;
collected peaks only in 28 mL fractions at 53 mL/min; equilibrated
with hexanes; dry loaded; eluted for 3 min with hexanes; increased
from 0-25% dichloromethane/hexanes over 15 min; held at 25%
dichloromethane/hexanes for 2 min. Obtained 528.6 mg (59% yield at
84% purity) of 2-chloro-4-isothiocyanato-N-phenylaniline as a
yellow solid.
(Z)-methyl
N-3-chloro-4-(phenylamino)phenyl-N'-cyanocarbanthnidothioate
##STR00044##
[0171] Charged cyanamide (101 mg, 2.4 mmol, Eq: 1.51) into a 50-mL
round-bottomed flask while purging with argon. Added 0.5 M sodium
methoxide in methanol (5.0 mL, 2.5 mmol, Eq: 1.57) at room
temperature, then stirred for 15 min. Meanwhile, purged the 100-mL
round-bottomed flask containing
2-chloro-4-isothiocyanato-N-phenylaniline (520 mg, 1.6 mmol, Eq:
1.00) with argon and added methanol (10 mL) and began stirring.
Transferred the cyanamide mixture to the starting material mixture
via syringe at room temperature. The solids dissolved after a short
time of stirring. Stirred for a total of 1 h, then added
iodomethane (431 mg, 0.19 mL, 3.04 mmol, Eq: 1.9) and stirred
overnight at room temperature. Monitored the reaction by HPLC.
Diluted the reaction mixture with dichloromethane and methanol,
then concentrated onto silica gel. Purified using an 80 g silica
gel column on an Intelliflash 280; collected peaks only in 28 mL
fractions at 55 mL/min; equilibrated with hexanes; dry loaded;
eluted 2 min with hexanes; increased from 0-50% ethyl
acetate/hexanes over 22 min; held at 50% ethyl acetate/hexanes for
15 min. Obtained 302 mg (57% yield at 95% purity) of (Z)-methyl
N-3-chloro-4-(phenylamino)phenyl-N-cyanocarbamimidothioate as a
light brown solid.
N*3*-(3-Chloro-4-phenylamino-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(Compound 2)
##STR00045##
[0173] Purged the 2-neck, 100-mL round-bottomed flask, fitted with
a vigroux column, containing the starting material, (Z)-methyl
N-3-chloro-4-(phenylamino)phenyl-N-cyanocarbamimidothioate (302 mg,
953 .mu.mol, Eq: 1.00), with argon. Added ethanol (15 mL) and
hydrazine (306 mg, 0.30 mL, 9.56 mmol, Eq: 10.0). Heated at
65.degree. C. for .about.30 min, then removed an aliquot and took
an HPLC: no starting material remained. Cooled the reaction and
removed the solvent in vacuo. Obtained an oil that was subsequently
redissolved in dichloromethane/methanol (1:1) and concentrated onto
silica gel. Pre-purified using a 40 g silica gel column (0-10%
methanol/dichloromethane with 1% ammonium hydroxide). Purified by
prep-HPLC. Obtained 205 mg (71.5%) of
N*3*-(3-Chloro-4-phenylamino-phenyl)-1H-[1,2,4]triazole-3,5-diamine
as a light pink solid. MS calcd. for C.sub.14H.sub.13ClN.sub.6
[(M+H).sup.+] 301.1, obsd. 300.8
N*3*-(3-Chloro-4-phenoxy-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(Compound 3)
##STR00046##
[0174] 2-chloro-4-nitro-1-phenoxybenzene
##STR00047##
[0176] In a 250-mL round-bottomed flask,
2-chloro-1-fluoro-4-nitrobenzene (2.0 g, 11.4 mmol, Eq: 1.00),
phenol (1.07 g, 11.4 mmol, Eq: 1.00) and potassium carbonate (3.15
g, 22.8 mmol, Eq: 2.0) were combined with N,N-dimethylformamide
(22.8 mL). The reaction mixture was heated at 100.degree. C.
overnight. In the morning, the reaction mixture was poured over ice
water, then the mixture was extracted with ethyl acetate. The
organic phase was dried over sodium sulfate, filtered, then
concentrated to afford 3.07 g (108%) of
2-chloro-4-nitro-1-phenoxybenzene as a tan oil. .sup.1H NMR
indicates the presence of some excess N,N-dimethylformamide.
3-chloro-4-phenoxyaniline
##STR00048##
[0178] In a 250-mL round-bottomed flask,
2-chloro-4-nitro-1-phenoxybenzene (3.07 g, 12.3 mmol, Eq: 1.00),
iron (3.43 g, 61.5 mmol, Eq: 5.0) and ammonium chloride (6.58 g,
123 mmol, Eq: 10) were combined with methanol (32.8 ml) to give a
light brown suspension. Iron stuck to the magnetic stir bar. Water
(16.4 mL) was added, then the reaction mixture became a milky-white
suspension. The reaction mixture was refluxed for 6 h. After only
20 minutes, the reaction mixture was rust-colored. After 6 h at
reflux, reverse phase HPLC shows complete conversion of the
starting material to a more polar product. The reaction mixture was
filtered, then concentrated to remove most of the methanol. The
resulting crude suspension was extracted with ethyl acetate. The
organic phase was dried (magnesium sulfate), filtered, then
concentrated to give 2.5 g (92.5%) of 3-chloro-4-phenoxyaniline as
a slightly tan oil.
2-chloro-4-isothiocyanato-1-phenoxybenzene
##STR00049##
[0180] Purged the 1-L round-bottomed flask containing
3-chloro-4-phenoxyaniline (2.5 g, 11.4 mmol, Eq: 1.00) with argon.
Added dichloromethane (120 mL) and cooled the mixture to 0.degree.
C. in an ice bath. Added 1,1'-thiocarbonyldiimidazole (2.43 g, 13.7
mmol, Eq: 1.2) in one portion then removed the ice bath and allowed
the reaction mixture to warm to room temperature. After one hour
the reaction was complete according to HPLC. The reaction mixture
was diluted with dichloromethane and concentrated onto silica.
Purified using a 330 g silica gel column on an Intelliflash 280;
collected peaks only in 28 mL fractions at 86 mL/min; equilibrated
with hexanes; dry loaded; eluted for 5 min with hexanes; increased
from 0-25% dichloromethan/hexanes over 30 min. Obtained 1.69 g
(56.7%) of 2-chloro-4-isothiocyanato-1-phenoxybenzene as a clear,
colorless oil.
(Z)-methyl
N-3-chloro-4-phenoxyphenyl-N'-cyanocarbamimidothioate
##STR00050##
[0182] Charged cyanamide (350.6 mg, 8.34 mmol, Eq: 1.29) into a
50-mL round-bottomed flask while purging with argon. Added 0.5 M
sodium methoxide in methanol (15.5 mL, 7.75 mmol, Eq: 1.2) at room
temperature, then stirred for 15 min. Meanwhile, purged the 100-mL
round-bottomed flask containing
2-chloro-4-isothiocyanato-1-phenoxybenzene (1.69 g, 6.46 mmol, Eq:
1.00) with argon and added methanol (25 mL) and began stirring.
Transferred the cyanamide mixture to the starting material mixture
via syringe at room temperature. The mixture became homogeneous
after a short time of stirring. Stirred for a total of 1 h, then
added iodomethane (1.38 g, 0.606 mL, 9.69 mmol, Eq: 1.5) and
stirred overnight at room temperature. After 21 h, reaction was
complete according to HPLC. Diluted with dichloromethane and
methanol and concentrated onto silica gel. Purified using 120 g
silica gel column on an Intelliflash 280; collected peaks only in
28 mL fractions at 80 mL/min; equilibrated with hexanes; dry
loaded; eluted for 2.5 min with hexanes; increased from 0-50% ethyl
acetate/hexanes over 27.5 min; held at 50% for 10 min. Obtained
1.34 g (65% yield) of (Z)-methyl
N-3-chloro-4-phenoxyphenyl-N'-cyanocarbamimidothioate as a light
yellow solid.
N*3*-(3-Chloro-4-phenoxy-phenyl)-1H-[1,2,4]-triazole-3,5-diamine
(Compound 3)
##STR00051##
[0184] Purged the 3-neck, 250-mL round-bottomed flask, fitted with
a vigroux column, containing (Z)-methyl
N-3-chloro-4-phenoxyphenyl-N'-cyanocarbarnimidothioate (1.33 g,
4.19 mmol, Eq: 1.00), with argon. Added ethanol (50 mL) and
hydrazine (1.34 g, 1.31 mL, 41.9 mmol, Eq: 10.0). Heated at
65.degree. C. for .about.30 min, then removed an aliquot and took
an HPLC: No starting material remained. Cooled the reaction and
removed the solvent in vacuo. Obtained a foam that was subsequently
redissolved in a dichloromethane/methanol (1:1) mixture and
concentrated onto silica gel. Purified using an 80 g silica gel
column on an Intelliflash 280; collected peaks only in 28 mL
fractions at 53 mL/min; equilibrated with dichloromethane with 1%
ammonium hydroxide; dry loaded; eluted 2 min with dichloromethane
with 1% ammonium hydroxide; increased from 0-10%
methanol/dichloromethane with 1% ammonium hydroxide; held at 10%
for 2 min. Obtained 1.1165 g (88.4%) of
N*3*-(3-Chloro-4-phenoxy-phenyl)-1H-[1,2,4]triazole-3,5-diamine as
a white solid. MS calcd. for C.sub.14H.sub.12ClN.sub.5O
[(M+H).sup.+] 302.1, obsd. 302.3
N*3*-(3,5-Dichloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]-triazole-3,5-diamin-
e (Compound 4)
##STR00052##
[0185] (2,6-dichloro-4-nitrophenyl)(phenyl)sulfane
##STR00053##
[0187] In a 500-mL round-bottomed flask,
1,3-dichloro-2-fluoro-5-nitrobenzene (4.0 g, 19.0 mmol, Eq: 1.00),
potassium carbonate (6.6 g, 47.8 mmol, Eq: 2.51) and benzenethiol
(2.9 g, 2.7 ml, 26.3 mmol, Eq: 1.38) were combined with
N,N-dimethylformamide (40 ml) to give a white suspension. This
mixture was stirred at 100.degree. C. for 8 hours. After this time,
reverse-phase HPLC indicated complete conversion of the starting
material to a single new product. The reaction mixture was combined
with ice water, giving a yellow suspension. This suspension was
extracted with ethyl acetate. The organic phase was dried over
sodium sulfate, filtered, and concentrated to afford a brown oil.
.sup.1H NMR of the crude product was consistent with a new product,
but excess N,N-dimethylformamide and thiophenol also appeared to be
present. The crude product was loaded directly onto a 210 g
Analogix column. Flash chromatography (using 100% hexanes) provided
the purified product. Obtained 5.53 g (96.7%) of
(2,6-dichloro-4-nitrophenyl)(phenyl)sulfane as an oily yellow
solid.
3,5-dichloro-4-(phenylthio)aniline
##STR00054##
[0189] To the 250-mL round-bottomed flask containing
(2,6-dichloro-4-nitrophenyl)(phenyl)sulfane (2.09 g, 6.96 mmol, Eq:
1.00), charged iron (1.94 g, 34.8 mmol, Eq: 5.0), ammonium chloride
(3.72 g, 69.6 mmol, Eq: 10.0), methanol (42 mL), and water (21 mL).
Heated to reflux. After 1.5 h, removed an aliquot and took an HPLC:
the starting material had been consumed. Cooled reaction mixture
and filtered it through a bed of Celite, rinsing with a large
amount of methanol. Concentrated filtrate, then added ethyl acetate
and stirred for an hour. Filtered off the solids and concentrated
the filtrate. Obtained 1.846 g (82% yield at 84% purity) of
3,5-dichloro-4-(phenylthio)aniline as a light brown solid.
1,3-dichloro-5-isothiocyanate-2-phenylsulfanylbenzene
##STR00055##
[0191] To the 250-mL round-bottomed flask containing
3,5-dichloro-4-(phenylthio)aniline (1.846 g, 6.83 mmol, Eq: 1.00),
charged 1,1'-thiocarbonyldiimidazole (1.58 g, 8.88 mmol, Eq: 1.3)
and dichloromethane (40 mL). Stirred overnight at room temperature.
Diluted with dichloromethane and concentrated onto Celite. Purified
using an 80 g silica gel column on an Intelliflash 280; collected
peaks only in 28 mL fractions at 36 mL/min; equilibrated with
hexanes; dry loaded on Celite; eluted 3 min with hexanes; increased
from 0-10% dichloromethane/hexanes over 30 min. Obtained 1.64 g
(77%) of 1,3-dichloro-5-isothiocyanato-2-phenylsulfanylbenzene as
an orange solid.
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(phenylthio)phenyl)carbamimidothioat-
e
##STR00056##
[0193] Charged cyanamide (676 mg, 16.1 mmol, Eq: 3.08) into a 50-mL
round-bottomed flask while purging with argon. Added 0.5 M sodium
methoxide in methanol (16 mL, 8.00 mmol, Eq: 1.53) at room
temperature. Meanwhile, added methanol (30 mL) and toluene (10 nap
to the 250-mL round-bottomed flask containing
1,3-dichloro-5-isothiocyanato-2-phenylsulfanylbenzene (1.63 g, 5.22
mmol, Eq: 1.00). After 25 min of stirring the cyanamide mixture,
transferred the cyanamide mixture to the isothiocyanate mixture
using a syringe. Stirred at room temperature for 1 h, then removed
an aliquot: HPLC showed no starting material remaining. Added
iodomethane (1.14 g, 0.5 mL, 8.00 mmol, Eq: 1.53) to the reaction
mixture and stirred overnight at room temperature. LC/MS after 16 h
showed reaction was complete. Diluted the reaction mixture and
concentrated onto Celite. Purified using a 150 g silica gel column
on an Intelliflash 280; collected peaks only in 28 mL fractions at
76 mL/min; equilibrated with 10% ethyl acetate/hexanes; dry loaded;
eluted 3 min with 10% ethyl acetate/hexanes; increased from 10-50%
ethyl acetate/hexanes over 36 min; held at 50% for 10 min. Obtained
1.646 g (75% yield at 88% purity) of (Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(phenylthio)phenyl)carbamimidothioate as
a white solid.
N*3*-(3,5-Dichloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(Compound 4)
##STR00057##
[0195] Added ethanol (40 mL) and hydrazine (1.43 g, 1.4 mL, 44.6
mmol, Eq: 10.0) to the 100-mL round-bottomed flask containing
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(phenylthio)phenyl)carbamimidothioate
(1.64 g, 4.45 mmol, Eq: 1.00). Heated at reflux for 4 h; HPLC
showed no starting material remaining. Cooled to room temperature.
Concentrated onto Celite. Partially purified using a 120 g silica
gel column on an Intelliflash 280; collected peaks only in 28 mL
fractions at 76 mL/min; equilibrated using 5%
methanol/dichloromethane with 1% ammonium hydroxide; dry loaded;
eluted 4 min with 5% methanol/dichloromethane with 1% ammonium
hydroxide; increased from 5-10% methanol/dichloromethane with 1%
ammonium hydroxide over 24 min. Obtained 1.33 g (76.3% yield at 90%
purity) of impure product as a yellow foam. Removed 94 mg of
material and purified by prep-HPLC. Obtained 35.8 mg (38% recovery)
of
N*3*-(3,5-Dichloro-4-phenylsulfanyl-phenyl)-1H-[1,2,4]triazole-3,5-diamin-
e as an off-white solid. .sup.1H NMR (DMSO-d.sub.6) Shift: 11.41
(br. s., 1H), 9.53 (br. s., 1H), 7.81 (s, 2H), 6.91-7.38 (m, 5H),
6.07 (br. s., 2H). MS calcd. for C.sub.14H.sub.11Cl.sub.2N.sub.5S
[(M+H)+] 352.0, obsd. 351.8.
N*3*(4-Benzenesulfinyl-3,5-dichloro-phenyl)-1H-[1,2,4]-triazole-3,5-diamin-
e (Compound 5)
##STR00058##
[0197] Charged
N3-(3,5-dichloro-4-(phenylthio)phenyl)-1H-1,2,4-triazole-3,5-diamine
(compound 4) (500 mg, 1.42 mmol, Eq: 1.00) into a 100-mL
round-bottomed flask and added methanol (32 mL). To the mixture
added a solution of oxone (2.62 g, 4.26 mmol, Eq: 3.0) in water (16
mL). Stirred at room temperature for 20 h. HPLC showed a mixture of
sulfoxide and sulfone; all the starting material had been consumed.
Stirred for another 24 h. The ratio of sulfoxide to sulfone,
according to HPLC, was .about.1:1. Added a second 50 mg scale
reaction to the reaction mixture. Diluted the mixture with ethyl
acetate and added water. Split the layers, but a gummy orange solid
would not dissolve in either layer. Washed the organics with
saturated sodium chloride, dried over sodium sulfate, filtered, and
concentrated. Obtained an orange residue. Attempted to dissolve the
residue in ethyl acetate and acetone, but methanol was needed to
fully dissolve everything. Concentrated the mixture onto Celite.
Purified using a 23 g spherical silica gel column on an
Intelliflash 280; collected peaks only in 9 mL fractions at 32
mL/min (.about.1 min/CV); equilibrated with 2%
methanol/dichloromethane with 1% ammonium hydroxide; dry loaded;
eluted 2 min with 2% methanol/dichloromethane with 1% ammonium
hydroxide; increased from 2-8% methanol/dichloromethane with 1%
ammonium hydroxide over 16 min; held at 8% for 7 min. Obtained 31.4
mg (5.7%) of
N*3*-(4-Benzenesulfinyl-3,5-dichloro-phenyl)-1H-[1,2,4]triazole-3,5-diami-
ne as an orange solid. MS calcd. for C14H11C12N5OS [(M+H)+] 368.0,
obsd. 367.8.
N*3*44-Benzenesulfonyl-3,5-dichloro-phenyl)-1H-[1,2,4]-triazole-3,5-diamin-
e (Compound 6)
##STR00059##
[0199] Charged
N3-(3,5-dichloro-4-(phenylthio)phenyl)-1H-1,2,4-triazole-3,5-diamine
(compound 4) (300 mg, 852 .mu.mol, Eq: 1.00) into a 2-neck, 100-mL
round-bottomed flask. Added methanol (24 mL) and began stirring.
Meanwhile, dissolve the oxone (2.62 g, 4.26 mmol, Eq: 5.0) in water
(12 mL). Added the oxone solution to the starting material solution
via syringe. Immediately white solids crashed out. Stirred at
45.degree. C. overnight. Took HPLC after 21 h, no starting material
remained and the sulfone peak was the major peak. Cooled to room
temperature. Diluted the reaction mixture with a large amount of
ethyl acetate and washed with water. Split layers and washed with
saturated sodium chloride. Dried over sodium sulfate, filtered, and
concentrated. Redissolved in a small amount of methanol and diluted
with acetone, then concentrated onto Celite. Purified using a 50 g
spherical silica gel column on an Intellilflash 280; collected
peaks only in 9 mL fractions at 40 mL/min (.about.2 min/CV);
equilibrated with 1% methanol/dichloromethane with 1% ammonium
hydroxide; dry loaded; eluted 4 min with 1%
methanol/dichloromethane with 1% ammonium hydroxide; increased from
1-5% methanol/dichloromethane with 1% ammonium hydroxide over 38
min; held at 5% methanol/dichloromethane with 1% ammonium hydroxide
for 8 min, then increased to 6% methanol/dichloromethane with 1%
ammonium hydroxide over 10 min; held at 6% methanol/dichloromethane
with 1% ammonium hydroxide for 6 min. Obtained 66 mg (20%) of
N*3*-(4-Benzenesulfonyl-3,5-dichloro-phenyl)-1H-[1,2,4]triazole-3,5-diami-
ne as an orange solid. MS calcd. for C14H11C12N5O2S [(M+H)+] 384.0,
obsd. 384.0.
N3-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)-1H-1,2,4-triazol-
e-3,5-diamine (Compound 7)
##STR00060##
[0200]
(2,6-dichloro-4-nitrophenyl)(4-(trifluoromethyl)phenyl)sulfane
##STR00061##
[0202] In a 250-mL round-bottomed flask,
4-(trifluoromethyl)benzenethiol (1 g, 5.61 mmol, Eq: 1.00),
1,3-dichloro-2-fluoro-5-nitrobenzene (1.18 g, 5.61 mmol, Eq: 1.00)
and potassium carbonate (900 mg, 6.51 mmol, Eq: 1.16) were combined
with N,N-dimethylformamide (22 mL) to give a yellow suspension.
This mixture was heated at 100.degree. C. overnight. In the
morning, TLC indicated the presence of a new major product. TLC
also showed complete consumption of the thiophenol and only a trace
of the fluorobenzene remaining. The reaction mixture was poured
over ice. The product only oiled out, giving a yellowish
suspension. This suspension was extracted with ethyl acetate. The
organic phase was dried over magnesium sulfate, filtered, then
concentrated to a brown oil. This product was loaded directly on a
120 g SiliCycle column. Flash chromatography (5% ethyl
acetate-hexanes ramped to 10 ethyl acetate-hexanes) afforded 1.13 g
(47.7%) of
(2,6-dichloro-4-nitrophenyl)(4-(trifluoromethyl)phenyl)sulfane at
high purity as a yellow oil.
3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)aniline
##STR00062##
[0204] In a 250-mL round-bottomed flask,
(2,6-dichloro-4-nitrophenyl)(4-(trifluoromethyl)phenyl)sulfane
(1.13 g, 3.07 mmol, Eq: 1.00), iron (857 mg, 15.3 mmol, Eq: 5) and
ammonium chloride (1.64 g, 30.7 mmol, Eq: 10) were combined with
methanol (8 mL) to give a yellow suspension. Water (4.0 mL) was
added. The reaction mixture was refluxed at 85.degree. C. for 2
hours. After this time, TLC indicated complete conversion of the
starting material to a dominant new product spot. The reaction
mixture was cooled to room temperature, then it was partitioned
between saturated aqueous sodium bicarbonate and ethyl acetate. The
organic phase was dried over magnesium sulfate, filtered, then
concentrated. Obtained 1.04 g (quantitative) of
3,5-dichloro-4-(4-(trifluoromethyl)-phenylthio)aniline as yellow
crystals.
1,3-dichloro-5-isothiocyanato-2-(4-trifluoromethylphenylsulfanyl)benzene
##STR00063##
[0206] In a 1 L round-bottomed flask,
3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)aniline (1.04 g, 3.08
mmol, Eq: 1.00) and 1,1'-thiocarbonyldiimidazole (600 tug, 3.37
mmol, Eq: 1.09) were combined with methylene chloride (22 mL) to
give a light brown solution. The reaction mixture was stirred
overnight at room temperature. After this time, TLC was consistent
with new product formation (100% hexanes). The reaction mixture was
concentrated over silica gel. Flash chromatography (100% hexanes)
using an 80 gram SiliCycle column was used to purify the product.
Obtained 462 mg (39.5%) of
1,3-dichloro-5-isothiocyanato-2-(4-trifluoromethylphenylsulfanyl)benzene
as a yellow oil.
methyl
N'-cyano-N-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)-c-
arbamimidothioate
##STR00064##
[0208] Charged cyanamide (112 mg, 2.67 mmol, Eq: 2.2) into a 5-mL
round-bottomed flask while purging with argon. Added 0.5 M sodium
methoxide in methanol (2.8 mL, 1.4 mmol, Eq: 1.15) and stirred at
room temperature for 20 min. Meanwhile, purged the 100-mL
round-bottomed flask containing
1,3-dichloro-5-isothiocyanato-2-(4-trifluoromethylphenylsulfanyl)benzene
(462 mg, 1.22 mmol, Eq: 1.00) with argon, then added toluene (5 mL)
and methanol (10 mL). The cyanamide mixture transferred to the
isothiocyanate mixture via syringe. Stirred at room temperature for
75 min, then added iodomethane (345 mg, 152 .mu.L, 2.43 mmol, Eq:
2.0). Stirred at room temperature overnight. In the morning, no
solids had precipitated. TLC showed multiple spots. Concentrated
onto Celite and purified using a 40 g silica gel column on an
Intelliflash 280; collected peaks only in 9 mL fractions at 53
mL/min; equilibrated with hexanes; dry loaded; eluted 2 min with
hexanes; increased from 0-50% ethyl acetate/hexanes over 28 min;
held at 50% for 5 min. Obtained 509.5 mg (93%) of methyl
N'-cyano-N-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)carbamim-
idothioate as a white solid.
N3-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)-1H-1,2,4-triazol-
e-3,5-diamine (Compound 7)
##STR00065##
[0210] Added ethanol (25 mL) and hydrazine (204 mg, 200 .mu.L, 6.37
mmol, Eq: 5.46) to the 250-mL round-bottomed flask containing
methyl
N'-cyano-N-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)carbamim-
idothioate (509.5 mg, 1.17 mmol, Eq: 1.00). Equipped the flask with
a condenser and heated to reflux for 2 h. Cooled to room
temperature; HPLC showed no starting material remained. Removed the
solvent in vacuo. Obtained a white solid that turned reddish upon
exposure to air. The material was analyzed on HPLC: a 10% impurity
was present. Dissolved the crude product in methanol and
concentrated onto Celite. Purified using a 24 g silica gel column
on an Intelliflash 280; collected peaks only on 28 mL fractions at
32 mL/min; equilibrated with 2% methanol/dichloromethane with 1%
ammonium hydroxide; dry loaded on Celite; eluted 2 min with 2%
methanol/dichloromethane with 1% ammonium hydroxide; increased from
2-10% methanol/dichloromethane with 1% ammonium hydroxide over 13
min; held at 10% methanol/dichloromethane with 1% ammonium
hydroxide for 6.25 min. Obtained 353 mg (72%) of
N3-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)-1H-1,2,4-triazo-
le-3,5-diamine as a white solid. MS calcd. for C15H10C12F3N5S
[(M+H)+] 420.0, obsd. 419.8.
N3-(3,5-dichloro-4-(4-(trifluoromethyl)phenylsulfinyl)phenyl)-1H-1,2,4-tri-
azole-3,5-diamine (Compound 8)
##STR00066##
[0212] Charged
N3-(3,5-dichloro-4-(4-(trifluoromethyl)phenylthio)phenyl)-1H-1,2,4-triazo-
le-3,5-diamine (compound 7) (127 mg, 302 .mu.mol, Eq: 1.00) into a
25-mL round-bottomed flask and added methanol (5 mL) and oxone
(187.2 mg, 305 mmol, Eq: 1.01). Monitored the reaction by HPLC:
after 24 h, the reaction was mostly complete. Diluted the reaction
mixture with water and filtered off the solids. Air dried for
.about.1 h, then attempted to dissolve the solids in ethyl acetate,
but not all dissolved. Dissolved the remaining solids is
methanol/dichloromethane mixture. Combined the ethyl acetate and
methanol/dichloromethane mixtures together and concentrated onto
Celite. Purified using a 23 g spherical silica gel column on an
Intelliflash 280; collected peaks only in 9 mL fractions at 32
mL/min; equilibrated with 4% methanol/dichloromethane with 1%
ammonium hydroxide; dry loaded; eluted 2 min with 4%
methanol/dichloromethane with 1% ammonium hydroxide; increased from
4-10% methanol/dichloromethane with 1% ammonium hydroxide over 15
min; held at 10% methanol/dichloromethane with 1% ammonium
hydroxide for 10 min. Obtained 76.8 mg (58%) of
N3-(3,5-dichloro-4-(4-(trifluoromethyl)phenylsulfinyl)phenyl)-1H-1,2,4-tr-
iazole-3,5-diamine as a yellow solid. MS calcd. for C15H10C12F3N5OS
[(M+H)+] 436.0, obsd. 435.8.
N*3*-[3,5-Dichloro-4-(4-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4]-
triazole-3,5-diamine (Compound 9)
##STR00067##
[0213]
(2,6-dichloro-4-nitrophenyl)(3-(trifluoromethyl)phenyl)sulfane
##STR00068##
[0215] In a 250-mL round-bottomed flask,
1,3-dichloro-2-fluoro-5-nitrobenzene (1.0 g, 4.76 mmol, Eq: 1.00),
3-trifluoromethyl thiophenol (848 mg, 4.76 mmol, Eq: 1.00) and
potassium carbonate (658 mg, 4.76 mmol, Eq: 1.00) were combined
with N,N-dimethylformamide to give a light brown suspension. The
reaction mixture was heated at 100.degree. C. for 3 hr. After this
time, the reaction mixture was poured into ice to give a yellow
suspension. The suspension was extracted with ethyl acetate. The
organic phase was dried (sodium sulfate) filtered, then
concentrated to give a brown oil. Purified by loading the crude
directly onto a 120 g SiliCylcle column with a minimal amount of
methylene chloride. Eluted using 100% hexanes ramped to 10% ethyl
acetate hexanes. Obtained 1.62 g (92.4%) of impure product. The
material was purified a second time using an 80 g column on an
Intelliflash 280; collected peaks only in 28 mL fractions at 53
mL/min; equilibrated with hexanes; dry loaded; eluted 4 min with
hexanes; increased from 0-50% dichloromethane/hexanes over 20 min.
Obtained 730 mg (41.6%) of pure
(2,6-dichloro-4-nitrophenyl)(3-(trifluoromethyl)phenyl)sulfane as a
yellow oil.
3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)aniline
##STR00069##
[0217] Charged methanol (15 mL) to the 250-mL round-bottomed flask
containing
(2,6-dichloro-4-nitrophenyl)(3-(trifluoromethyl)phenyl)sulfane (730
mg, 1.98 mmol, Eq: 1.00). Added iron (565.6 mg, 10.1 mmol, Eq:
5.11) and ammonium chloride (1.06 g, 19.8 mmol, Eq: 10.0) in water
(5 mL). Heated at reflux for .about.1.5 h, then took an HPLC that
showed the reaction was complete. Cooled the reaction mixture to
room temperature, then filtered through a bed of Celite rinsing
with methanol. Concentrated the filtrate and added ethyl acetate.
Filtered off the solids and concentrated the filtrate. Obtained
578.8 mg (84%) of
3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)aniline as a yellow
solid.
1,3-dichloro-5-isothiocyanato-2-(3-trifluoromethylphenylsulfanyl)benzene
##STR00070##
[0219] Added 1,1'-thiocarbonyldiimidazole (397 mg, 2.22 mmol, Eq:
1.3) and dichloromethane (20 mL) to the round-bottomed flask
containing 3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)aniline
(578.8 mg, 1.71 mmol, Eq: 1.00) while purging with argon. Stirred
over weekend. HPLC showed two major peaks, but no starting
material. Diluted the reaction mixture with dichloromethane and
concentrated onto Celite. Purified using a 40 g silica gel column
on an Intelliflash 280; collected peaks only in 28 mL fractions at
53 mL/min; equilibrated with hexanes; dry loaded; eluted 2 min with
hexanes; increased from 0-25% dichloromethane/hexanes over 13 min;
held at 25% for 3 min. Obtained 267.5 mg (41%) of
1,3-dichloro-5-isothiocyanato-2-(3-trifluoromethylphenylsulfanyl)-benzene
as an orange solid.
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)pheny-
l)-carbamimidothioate
##STR00071##
[0221] Charged cyanamide (67.2 mg, 1.6 mmol, Eq: 2.34) into 10-mL
round-bottomed flask while purging with argon. Added 0.5 M sodium
methoxide in methanol (1.8 mL, 900 .mu.mol, Eq: 1.32) and stirred
at room temperature for 20 min. Meanwhile, dissolved
1,3-dichloro-5-isothiocyanato-2-(3-trifluoromethylphenylsulfanyl)benzene
(260 mg, 684 .mu.mol, Eq: 1.00) in toluene (2.5 mL), then added
methanol (5 mL) all while purging with argon. After the 20 min,
added the cyanamide mixture to the isothiocyanate mixture via
syringe. Stirred at room temperature for 1.5 h, then added
iodomethane (194 mg, 85.5 .mu.L, 1.37 mmol, Eq: 2.0) and stirred
overnight at room temperature. HPLC after 20 h, showed the reaction
was complete. Diluted the mixture with methanol and ethyl acetate.
Heated to a boil with a heat gun. Once all the solids were in
solution stopped heating and added Celite. Concentrated the
mixture. Purified using a 25 g SiliCycle HP silica gel column on an
Intelliflash 280; collected peaks only in 28 mL fractions at 32
mL/min; equilibrated with 25% ethyl acetate/hexanes; dry loaded;
eluted 2 min with 25% ethyl acetate/hexanes; increased from 25-60%
ethyl acetate/hexanes over 15 min. Obtained 194 mg (64%) of
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)phenyl)-carbami-
midothioate as a white solid.
N*3*-[3,5-Dichloro-4-(4-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4]-
-triazole-3,5-diamine (Compound 9)
##STR00072##
[0223] Added ethanol (20 mL) and hydrazine (71.2 mg, 69.8 .mu.L,
2.22 mmol, Eq: 5.00) to the 250-mL round-bottomed flask contained
the starting material, (Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)phenyl)-carbami-
midothioate (194 mg, 445 .mu.mol, Eq: 1.00). Heated at reflux for
1.25 h, then the reaction was complete according to HPLC. Cooled
the reaction mixture, then removed the solvent in vacuo. Placed in
a 50.degree. C. vacuum oven for 3 days. Obtained 175 mg (92%) of
N*3*-[3,5-Dichloro-4-(4-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4-
]triazole-3,5-diamine as an off-white solid. .sup.1H NMR
(DMSO-d.sub.6) Shift: 11.44 (br. s., 1H), 9.60 (br. s., 111), 7.84
(s, 2H), 7.42-7.61 (m, 2H), 7.12-7.40 (m, 2H), 6.08 (s, 2H). MS
calcd. for C.sub.15H.sub.10C12F3N5S [(M+H)+] 420.0, obsd.
419.8.
N*3*-[3,5-Dichloro-4-(2-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4]-
-triazole-3,5-diamine (Compound 10)
##STR00073##
[0224]
(2,6-dichloro-4-nitrophenyl)(2-(trifluoromethyl)phenyl)sulfane
##STR00074##
[0226] In a 250-mL round-bottomed flask,
1,3-dichloro-2-fluoro-5-nitrobenzene (1.22 g, 5.81 mmol, Eq: 1.00),
2-(trifluoromethyl)benzenethiol (1.01 g, 5.67 mmol, Eq: 0.976) and
potassium carbonate (0.98 g, 7.09 mmol, Eq: 1.22) were combined
with N,N-dimethylformamide (22 mL) to give a light brown
suspension. The reaction mixture was heated at 100.degree. C. for
3.5 hrs. After this time, the reaction mixture was partitioned
between ethyl acetate and water. The organic phase was dried over
sodium sulfate, filtered, and concentrated to a brown oil. This
crude product was dissolved in methylene chloride, then the mixture
was concentrated over silica gel. The silica gel supported crude
product was loaded onto a 120 gram silica gel column. Flash
chromatography (100% hexanes ramped to 5% ethyl acetate in hexanes)
provided 1.76 g (82.3%) of
(2,6-dichloro-4-nitrophenyl)(2-(trifluoromethyl)phenyl)sulfane as a
light yellow solid.
3,5-dichloro-4-(2-(trifluoromethyl)phenylthio)aniline
##STR00075##
[0228] Charged methanol (40 mL), iron (1.33 g, 23.8 mmol, Eq: 5.0),
ammonium chloride (2.55 g, 47.7 mmol, Eq: 10), and water (20 mL) to
the 250-mL round-bottomed flask containing
(2,6-dichloro-4-nitrophenyl)(2-(trifluoromethyl)phenyl)sulfane
(1.756 g, 4.77 mmol, Eq: 1.00). Stirred at reflux for 1.5 h: HPLC
showed the reaction was complete. Cooled the reaction mixture then
filtered through a bed of Celite, rinsing with methanol.
Concentrated and added ethyl acetate. Filtered off the solids,
rinsing with ethyl acetate, and concentrated the filtrate. Obtained
1.425 g (86%) of
3,5-dichloro-4-(2-(trifluoromethyl)phenylthio)aniline as a grey
solid.
1,3-dichloro-5-isothiocyanato-2-(2-trifluoromethylphenylsulfanyl)benzene
##STR00076##
[0230] Charged 1,1'-thiocarbonyldiimidazole (1.0 g, 5.61 mmol, Eq:
1.33) and dichloromethane (30 mL) to the 250-mL round-bottomed
flask containing
3,5-dichloro-4-(2-(trifluoromethyl)phenylthio)aniline (1.425 g,
4.21 mmol, Eq: 1.00), while purging with argon. Stirred at room
temperature overnight. In morning, the HPLC showed no starting
material. Diluted the mixture with dichloromethane and concentrated
onto Celite. Purified using a 40 g silica gel column on an
Intelliflash 280; collected peaks only in 28 mL fractions at 53
mL/min; equilibrated with hexanes; dry loaded; eluted 2 min with
hexanes, then increased from 0-25% dichloromethane/hexanes over 15
min; held at 25% for 3 min. Obtained 980 mg (60%) of
1,3-dichloro-5-isothiocyanato-2-(2-trifluoromethylphenylsulfanyl)benzene
as a yellow solid.
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(2-(trifluoromethyl)phenylthio)pheny- l)
carbamimidothioate
##STR00077##
[0232] Charged cyanamide (220.4 mg, 5.24 mmol, Eq: 2.04) into a
25-mL round-bottomed flask while purging with argon. Added 0.5 M
sodium methoxide in methanol (6.7 mL, 3.35 mmol, Eq: 1.31) and
began stirring. Meanwhile, dissolved the isothiocyanate (975 mg,
2.56 mmol, Eq: 1.00) in toluene (5 mL) and added methanol (10 mL)
all while purging with argon. After 10 min, the cyanamide mixture
was added to the starting material mixture via syringe. Stirred at
room temperature for 2 h, then added iodomethane (729 mg, 0.321 mL,
5.13 mmol, Eq: 2.00) and stirred for 4 days. Placed the reaction
mixture in the freezer for 2 h, then filtered off the solids. The
solids were air dried and the filtrate was concentrated onto
Celite. The solid was 660 mg of product. The filtrate was purified
using a 24 g silica gel column on an Intelliflash 280; collected
peaks only in 28 mL fractions at 32 mL/min; equilibrated with 25%
ethyl acetate/hexanes; dry loaded; eluted 2 min with 25% ethyl
acetate/hexanes; increased from 25-60% ethyl acetate/hexanes over
15 min; held at 60% for 5 min. Obtained 202 mg of product. Obtained
a total of 860 mg (76%) of (Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(2-(trifluoromethyl)phenylthio)phenyl)
carbamimidothioate as a white solid.
[0233]
N*3*-[3,5-Dichloro-4-(2-trifluoromethyl-phenylsulfanyl)-phenyl]-1H--
[1,2,4]-triazole-3,5-diamine (Compound 10)
##STR00078##
[0234] Added ethanol (30 mL) and hydrazine (306 mg, 300 .mu.L, 9.56
mmol, Eq: 4.91) to the 250-mL round-bottomed flask containing
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(2-(trifluoromethyl)phenylthio)phenyl)
carbamimidothioate (850 mg, 1.95 mmol, Eq: 1.00). Heated at reflux.
After 1.5 h the reaction was complete according to HPLC. Cooled
reaction and concentrated in vacuo. Placed in a 50.degree. C.
vacuum oven for 3 days. Obtained 860 mg of crude product. Dissolved
the solids in methanol and concentrated onto Celite. Purified using
a 40 g silica gel column on an Intelliflash 280; collected peaks
only in 28 mL fractions at 53 mL/min (1 min/CV); equilibrated with
5% methanol/dichloromethane with 1% ammonium hydroxide; dry loaded;
eluted 2 min with 5% methanol/dichloromethane with 1% ammonium
hydroxide; increased from 5-10% methanol/dichloromethane with 1%
ammonium hydroxide over 12 min. Obtained 647 mg (79%) of
N*3*-[3,5-Dichloro-4-(2-trifluoromethyl-phenylsulfanyl)-phenyl]-1H-[1,2,4-
]triazole-3,5-diamine as an off-white solid. MS calcd. for
C15H10C12F3N5S [(M+H)+] 420.0, obsd. 419.8.
N*3*-[3,5-Dichloro-4-(4-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1,2,4-
]-triazole-3,5-diamine (Compound 11)
##STR00079##
[0236] Added methanol (10 mL) and oxone (843 mg, 1.37 mmol, Eq:
4.0) to the 250-mL round-bottomed flask containing
N3-(3,5-dichloro-4-(3-(trifluoromethyl)phenylthio)phenyl)-1H-1,2,4-triazo-
le-3,5-diamine (compound 9) (144 mg, 343 .mu.mol, Eq: 1.00). Heated
the reaction at 60.degree. C. overnight. HPLC and LC/MS showed that
only sulfoxide was present. Cooled the mixture to room temperature.
Diluted with water and extracted with ethyl acetate. Split layers
and washed the organics with saturated sodium chloride. Dried
organics over sodium sulfate, filtered, and concentrated onto
Celite. Purified using a 24 g silica gel column on an Intelliflash
280; collected peaks only in 9 mL fractions at 32 mL/min (1
min/CV); equilibrated with 5% methanol/dichloromethane with 1%
ammonium hydroxide; dry loaded; eluted 2 min with 5%
methanol/dichloromethane with 1% ammonium hydroxide; increased from
5-10% methanol/dichloromethane with 1% ammonium hydroxide over 12
min; held at 10% methanol/dichloromethane with 1% ammonium
hydroxide for 2 min. Obtained 42 mg (26%) of
N*3*-[3,5-Dichloro-4-(4-trifluoromethyl-benzenesulfinyl)-phenyl]-1H-[1,2,-
4]triazole-3,5-diamine as a yellow solid. MS calcd. for
C15H10C12F3N5OS [(M+H)+] 436.0, obsd. 435.8.
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-phenylsulfanyl]-be-
nzonitrile (Compound 12)
##STR00080##
[0237] 4-(2,6-dichloro-4-nitrophenylthio)benzonitrile
##STR00081##
[0239] In a 250-mL round-bottomed flask,
1,3-dichloro-2-fluoro-5-nitrobenzene (1.5 g, 7.14 mmol, Eq: 1.00),
4-mercaptobenzonitrile (966 mg, 7.14 mmol, Eq: 1.00) and potassium
carbonate (1.09 g, 7.86 mmol, Eq: 1.1) were combined with
N,N-dimethylformamide (30 mL) to give a light brown suspension. The
reaction mixture was heated at 100.degree. C. for 3.5 hrs. After
this time the mixture was poured into water to give a yellow
suspension. The suspension was extracted with ethyl acetate, and
the organic phase was dried over sodium sulfate, filtered, and
concentrated to afford the crude product as a yellow oil. This
product was dissolved in methylene chloride and the resulting
solution was concentrated over silica gel. The silica-gel supported
crude product was loaded onto a 120 gram silica gel column. Flash
chromatography (100% hexanes ramped to 5% ethyl acetate in hexanes)
afforded 1.31 g (56.4%) of
4-(2,6-dichloro-4-nitrophenylthio)benzonitrile as a yellow
solid.
4-(4-amino-2,6-dichlorophenylthio)benzonitrile
##STR00082##
[0241] Added ammonium chloride (2.37 g, 44.3 mmol, Eq: 10.0), iron
(1.24 g, 22.1 mmol, Eq: 5.0), methanol (30 mL), and water (10 mL)
to the 250-mL round-bottomed flask containing
4-(2,6-dichloro-4-nitrophenylthio)benzonitrile (1.44 g, 4.43 mmol,
Eq: 1.00). Heated to reflux. After 1.25 h the reaction was complete
according to HPLC. Cooled the reaction mixture to room temperature,
then filtered the mixture through a bed of Celite, rinsing with
copious amounts of methanol. Concentrated the filtrate, then added
ethyl acetate and filtered off the solids. The filtrate was
concentrated and dried over the weekend in a 50.degree. C. vacuum
oven. Obtained 567 mg (40%) of
4-(4-amino-2,6-dichlorophenylthio)benzonitrile as a yellow
solid.
4-(2,6-Dichloro-4-isothiocyanatophenylsulfanyl)benzonitrile
##STR00083##
[0243] Added dichloromethane (10 mL) and
1,1'-thiocarbonyldiimidazole (440 mg, 2.47 mmol, Eq: 1.3) to the
250-mL round-bottomed flask containing
4-(4-amino-2,6-dichlorophenylthio)benzonitrile (560 mg, 1.9 mmol,
Eq: 1.00). Stirred at room temperature overnight. TLC in 1:3 ethyl
acetate/hexanes showed starting material remaining. Added more
1,1'-thiocarbonyldiimidazole (440 mg, 2.47 mmol, Eq: 1.3) and
stirred overnight. TLC again showed a large starting material spot,
but HPLC and LC/MS showed that it was not starting material.
Concentrated the reaction mixture onto Celite. Purified using a 24
g silica gel column on an Intelliflash 280; collected peaks only in
28 mL fractions at 32 mL/min; equilibrated with hexanes; dry
loaded; eluted 2 min with hexanes; increased from 0-15%
dichloromethane/hexanes over 10 min, but no peaks eluted. Increased
from 15-50% dichloromethane/hexanes over 20 min. Obtained 351 mg
(52%) of
4-(2,6-Dichloro-4-isothiocyanatophenylsulfanyl)benzonitrile as a
white solid.
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(4-cyanophenylthio)phenyl)-carbamimi-
dothioate
##STR00084##
[0245] While purging with argon, charged cyanamide (147 mg, 3.5
mmol, Eq: 3.42) into a 10 mL round-bottomed flask, then added 0.5 M
sodium methoxide in methanol (2.66 mL, 1.33 mmol, Eq: 1.3). Stirred
at room temperature for .about.15 min. Meanwhile, dissolved
4-(2,6-Dichloro-4-isothiocyanatophenylsulfanyl)benzonitrile (345
mg, 1.02 mmol, Eq: 1.00) in toluene (3 mL), then added methanol (7
mL). Added the cyanamide mixture to the isothiocyanate mixture via
syringe. Stirred at room temperature for 1 h, then removed an
aliquot for HPLC: very little starting material remained. Added
iodomethane (295 mg, 0.130 mL, 2.08 mmol, Eq: 2.03) at this time.
Stirred over weekend. A white precipitate formed. Placed the
reaction mixture in the freezer for 4 h. Filtered off the solids
rinsing with cold methanol. Air dried and obtained 204 mg of a
white solid, which was pure product according to .sup.1H NMR,
LC/MS, and HPLC. Also, concentrated the filtrate onto Celite and
purified using a 40 g silica gel column on an Intelliflash 280;
collected peaks only in 9 mL fractions at 53 mL/min; equilibrated
with 20% ethyl acetate/hexanes; dry loaded; eluted 2 min with 20%
ethyl acetate/hexanes; increased from 20-50% ethyl acetate/hexanes
over 15 min; held at 50% ethyl acetate/hexanes for 3 min. Obtained
40 mg of product. Obtained a total of 240 mg (59% yield) of
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(4-cyanophenylthio)phenyl)carbamimidothioate
as a white solid.
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-phenylsulfanyl]-be-
nzonitrile (Compound 12)
##STR00085##
[0247] Added ethanol (10 mL) and hydrazine (117 mg, 115 .mu.L, 3.66
mmol, Eq: 6.0) to the round-bottomed flask containing (Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(4-cyanophenylthio)phenyl)carbamimidothioate
(240 mg, 610 .mu.mol, Eq: 1.00). Heated at reflux for 1 h. Observed
that a larger amount of solids was present in the reaction mixture.
Removed an aliquot and tried to dissolve the solid in
acetonitrile/methanol mixture with heat, but not all would
dissolve. Cooled the mixture to room temperature. Placed the
reaction mixture in the freezer for 2 h. Filtered off the solid
rinsing with cold ethanol. Air dried overnight. Obtained 196 mg
(85%) of
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-phenylsulfanyl]-b-
enzonitrile as a white solid. MS calcd. for C15H10C12N6S [(M+H)+]
377.0, obsd. 376.8.
4-[4-(5-Amino-1H-[1,2,4]-triazol-3-ylamino)-2,6-dichloro-benzenesulfinyl]--
benzonitrile (Compound 13)
##STR00086##
[0249] Charged
4-(4-(5-amino-1H-1,2,4-triazol-3-ylamino)-2,6-dichlorophenylthio)benzonit-
rile (compound 12) (160 mg, 424 .mu.mol, Eq: 1.00; Joe Intermediate
G) into a 5-mL round-bottomed flask, then added methanol (10 mL)
and oxone (1.3 g, 2.12 mmol, Eq: 5.0). Heated at reflux over for 3
days. Cooled to room temperature and took HPLC, no starting
material remained. Filtered rinsing with methanol and concentrated
the filtrate onto Celite. Purified using a 25 g small particle
size, silica gel column on an Intelliflash 280; collected peaks
only in 9 mL fractions at 33 mL/min (.about.1 min/CV); equilibrated
with 5% methanol/dichloromethane with 1% ammonium hydroxide; dry
loaded; eluted 2 min with 5% methanol/dichloromethane with 1%
ammonium hydroxide; increased from 5-10% methanol/dichloromethane
with 1% ammonium hydroxide over 18 min; held at 10%
methanol/dichloromethane with 1% ammonium hydroxide for 4 min.
Obtained 5.5 mg (3%) of
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfinyl]--
benzonitrile as a brown solid. MS calcd. for C15H10C12N6OS [(M+H)+]
393.0, obsd. 392.8.
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfinyl]-b-
enzonitrile (Compound 14)
##STR00087##
[0251] Charged
4-(4-(5-amino-1H-1,2,4-triazol-3-ylamino)-2,6-dichlorophenylthio)benzonit-
rile (compound 12) (160 mg, 424 mmol, Eq: 1.00) into a 5-mL
round-bottomed flask, then added methanol (10 mL) and oxone (1.3 g,
2.12 mmol, Eq: 5.0). Heated at reflux over for 3 days. Cooled to
room temperature and took HPLC, no starting material remained.
Filtered rinsing with methanol and concentrated the filtrate onto
Celite. Purified using a 25 g small particle size, silica gel
column on an Intelliflash 280; collected peaks only in 9 mL
fractions at 33 mL/min (=1 min/CV); equilibrated with 5%
methanol/dichloromethane with 1% ammonium hydroxide; dry loaded;
eluted 2 min with 5% methanol/dichloromethane with 1% ammonium
hydroxide; increased from 5-10% methanol/dichloromethane with 1%
ammonium hydroxide over 18 min; held at 10%
methanol/dichloromethane with 1% ammonium hydroxide for 4 min.
Obtained 17.2 mg (9.6%) of
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2,6-dichloro-benzenesulfonyl]--
benzonitrile as a brown solid. MS calcd. for C15H10C12N6O2S
[(M+H)+] 409.0, obsd. 408.8.
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2-chloro-6-trifluoromethyl-phen-
ylsulfanyl]-benzoic acid methyl ester (Compound 15)
##STR00088##
[0252]
4-(2-chloro-4-nitro-6-(trifluoromethyl)phenylthio)benzoate
##STR00089##
[0254] In a 250-mL round-bottomed flask,
1-chloro-2-fluoro-5-nitro-3-(trifluoromethyl)benzene (1.5 g, 6.16
mmol, Eq: 1.00), methyl 4-mercaptobenzoate (1.1 g, 6.54 mmol, Eq:
1.06) and potassium carbonate (851 mg, 6.16 mmol, Eq: 1.00) were
combined with N,N-dimethylformamide (25 mL) to give a light brown
suspension. The reaction mixture was heated at 100.degree. C. for 7
hours. After this time, the reaction mixture was cooled room
temperature. The reaction mixture was partitioned between water and
ethyl acetate. The organic phase was dried (magnesium sulfate),
filtered, then concentrated. The crude product (brown oil) was
loaded directly onto a 120 gram ISCO column. Flash chromatography
afforded 5 product bands. The second eluting band was the desired
product by .sup.1H NMR. Obtained 1.0 g (41.4%) of
4-(2-chloro-4-nitro-6-(trifluoromethyl)phenylthio)benzoate as a
yellow solid.
methyl
4-(4-amino-2-chloro-6-(trifluoromethyl)phenylthio)benzoate
##STR00090##
[0256] In a 500-mL round-bottomed flask, methyl
4-(2-chloro-4-nitro-6-(trifluoromethyl)phenylthio)benzoate (1.0 g,
2.55 mmol, Eq: 1.00), iron (713 mg, 12.8 mmol, Eq: 5) and ammonium
chloride (1.37 g, 25.5 mmol, Eq: 10) were combined with methanol
(8.5 mL) to give a yellow suspension. Water was added. The reaction
mixture was heated at 100.degree. C. for 2 hr. After this time, the
reaction mixture was a brick-red suspension. The reaction mixture
was cooled to room temperature, then filtered. About half of the
methanol was evaporated off. The resulting solution was partitioned
between saturated aqueous sodium bicarbonate and ethyl acetate. The
organic phase was dried (magnesium sulfate) filtered, and
concentrated to afford 0.790 g (85.5%) of methyl
4-(4-amino-2-chloro-6-(trifluoromethyl)phenylthio)benzoate as a
yellow oily solid. This product was used without further
purification.
4-(2-chloro-4-isothiocyanato-6-trifluoromethylphenylsulfanyl)benzoic
acid methyl ester
##STR00091##
[0258] Added dichloromethane (30 mL) and
1,1'-thiocarbonyldiimidazole (0.57 g, 3.2 mmol, Eq: 1.45) to the
100-mL round-bottomed flask containing methyl
4-(4-amino-2-chloro-6-(trifluoromethyl)phenylthio)benzoate (800 mg,
2.21 mmol, Eq: 1.00). Stirred at room temperature for 1 h; took a
TLC in 1:3 ethyl acetate/hexanes: appeared only starting material
was present. Stirred for another 2 h, took a TLC and still starting
material Stirred overnight (18 h). Attempted to recover starting
material. Diluted with dichloromethane and concentrated onto
Celite. Purified using a 40 g silica gel column on an Intelliflash
280; collected peaks only in 28 mL fractions at 53 mL/min;
equilibrated with 10% ethyl acetate/hexanes; dry loaded; eluted 2
min with 10% ethyl acetate/hexanes; increased from 10-30%
dichloromethane/hexanes over 8 min; held at 30% for 2 min;
increased from 30-50% over 8 min. Obtained 379.7 mg (42%) of
4-(2-chloro-4-isothiocyanato-6-trifluoromethylphenylsulfanyl)benzoic
acid methyl ester as a yellow viscous oil, which partially
solidified overnight; no starting material was recovered only
product.
methyl
4-(2-chloro-4-((cyanoimino)(methylthio)methylamino)-6-(trifluoromet-
hyl)phenylthio)-benzoate
##STR00092##
[0260] Charged cyanamide (1.6 g, 38.1 mmol) into a 100-mL
round-bottomed flask; added 0.5 M sodium methoxide (37.5 mL, 17.75
mmol) and stirred at room temperature for 30 min. Meanwhile, added
toluene (3.8 mL) and methanol (4 mL) to the 100-mL round-bottomed
flask that contained
4-(2-chloro-4-isothiocyanato-6-trifluoromethylphenylsulfanyl)benzoic
acid methyl ester (380 mg, 941 .mu.mol, Eq: 1.00). Added the
cyanamide mixture (2.45 mL, 1.22 mmol, Eq: 1.3) to the starting
material via syringe. Stirred at room temperature and monitored by
TLC: 1:3 ethyl acetate/hexanes; after 2.5 h the starting material
had been consumed Added iodomethane (272 mg, 120 .mu.L, 1.92
.mu.mol, Eq: 2.04) and stirred at room temperature. After 1.5 h
took a TLC in 100% ethyl acetate: reaction was complete.
Transferred the reaction mixture to a 250-mL round-bottomed flask
and concentrated onto Celite. Purified using a 40 g silica gel
column on an Intelliflash 280; collected peaks only in 28 mL
fractions at 35 mL/min; equilibrated with 50% ethyl
acetate/hexanes; dry loaded; eluted 2 min with 50% ethyl
acetate/hexanes; increased from 50-100% ethyl acetate/hexanes over
20 min. Obtained 197 mg (45.5%) of methyl
4-(2-chloro-4-((cyanoimino)(methylthio)methylamino)-6-(trifluorome-
thyl)phenylthio)benzoate as a white solid.
4-[4-(5-Amino-1H-[1,2,4]-triazol-3-ylamino)-2-chloro-6-trifluoromethyl-phe-
nylsulfanyl]-benzoic acid methyl ester (Compound 15)
##STR00093##
[0262] Added ethanol (10 mL) and hydrazine (102 mg, 100 .mu.L, 3.19
mmol, Eq: 7.44) to the 100-mL round-bottomed flask containing
methyl
4-(2-chloro-4-((cyanoimino)(methylthio)methylamino)-6-(trifluoromethyl)ph-
enylthio)benzoate (197 mg, 428 .mu.mol, Eq: 1.00). Refluxed for 1
h; confirmed completion of the reaction by TLC in 100% ethyl
acetate and LC/MS. Concentrated the reaction mixture in vacuo.
Dried in a vacuum oven at 70.degree. C. under high vacuum. Obtained
184.5 mg (97%) of
4-[4-(5-Amino-1H-[1,2,4]triazol-3-ylamino)-2-chloro-6-trifluoromethyl-phe-
nylsulfanyl]-benzoic acid methyl ester as an off-white solid. MS
calcd. for C17H13ClF3NO2S [(M+H)+] 444.0, obsd. 408.8.
N*3*-[3,5-Dichloro-4-(4-methoxy-phenylsulfanyl)-phenyl]-1H-[1,2,4]triazole-
-3,5-diamine (Compound 16)
##STR00094##
[0263] (2,6-dichloro-4-nitrophenyl)(4-methoxyphenyl)sulfane
##STR00095##
[0265] In a 250-mL round-bottomed flask, 4-methoxybenzenethiol
(1.00 g, 633 .mu.l, 7.14 mmol, Eq: 1.00),
1,3-dichloro-2-fluoro-5-nitrobenzene (1.5 g, 7.14 mmol, Eq: 1.00)
and potassium carbonate (990 mg, 7.16 mmol, Eq: 1.00) were combined
with N,N-dimethylformamide (20 mL) to give a yellow suspension. The
reaction mixture was stirred overnight at room temperature. In the
morning TLC was consistent with the reaction having reached
completion. The reaction mixture was poured into water to give a
yellow cloudy suspension. This suspension was extracted with ethyl
acetate. The organic phase was dried over Sodium sulfate, filtered,
then concentrated to give a brown oil which was loaded directly
onto a 120 gram silica gel column. Flash chromatography 5-15% EtOAC
in hexanes was used to purify the product, however, this
purification was not very successful. The fractions containing the
product were concentrated. .sup.1H NMR showed that the product was
consistent with product with only a small impurity. Obtained 2.2 g
(93.3%) of (2,6-dichloro-4-nitrophenyl)(4-methoxyphenyl)sulfane as
a yellow oil.
3,5-dichloro-4-(4-methoxyphenylthio)aniline
##STR00096##
[0267] Charged iron (1.78 g, 31.8 mmol, Eq: 5.0), ammonium chloride
(3.4 g, 63.6 mmol, Eq: 10.0), and methanol (80 mL) into the 500-mL
round-bottomed flask containing
(2,6-dichloro-4-nitrophenyl)(4-methoxyphenyl)sulfane (2.1 g, 6.36
mmol, Eq: 1.00). Heated at reflux; after 2 h the reaction was
complete by TLC (1:3 ethyl acetate/hexanes) and LC/MS. Cooled the
mixture to room temperature, then filtered through a bed of celite
rinsing with a large amount of methanol. Concentrated the filtrate
in vacuo. Added ethyl acetate to the residue and filtered off the
solids. Concentrated the filtrate and obtained 1.87 g (98%) of
3,5-dichloro-4-(4-methoxyphenylthio)aniline as a dark brown oil
that solidified over time.
1,3-dichloro-5-isothiocyanato-2-(4-methoxyphenylsulfanyl)benzene
##STR00097##
[0269] Charged dichloromethane (50 mL) and
1,1'-thiocarbonyldiimidazole (1.44 g, 8.1 mmol, Eq: 1.3) to the
250-mL round-bottomed flask containing
3,5-dichloro-4-(4-methoxyphenylthio)aniline (1.87 g, 6.23 mmol, Eq:
1.00). Stirred at room temperature. Monitored the reaction by TLC
(1:3 ethyl acetate/hexanes). Stirred over the weekend. Diluted the
reaction with dichloromethane, added Celite, and concentrated.
Purified using a 40 g silica gel column on an Intelliflash 280;
collected peaks only in 28 mL fractions at 53 mL/min; equilibrated
with hexanes; dry loaded; eluted 2 min with hexanes; increased from
0-25% dichloromethane/hexanes over 10 min; held at 25% for 5.5 min;
stepped to 40% dichloromethane/hexanes and held for 5 min. Obtained
861 mg (40%) of
1,3-dichloro-5-isothiocyanato-2-(4-methoxyphenylsulfanyl)benzene as
an orange oil.
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(4-methoxyphenylthio)phenyl)carbamim-
idothioate
##STR00098##
[0271] Combined cyanamide (440 mg, 10.5 mmol) and 0.5 M sodium
methoxide (10.0 mL, 5.0 mmol). Then added some of the cyanamide
mixture (6.04 mL, 3.02 mmol, Eq: 1.2) to a mixture of
1,3-dichloro-5-isothiocyanato-2-(4-methoxyphenylsulfanyl)benzene
(861 mg, 2.52 mmol, Eq: 1.00) in methanol (15 mL) and toluene (3
mL). Stirred at room temperature. After 1 h TLC in 1:3 ethyl
acetate/hexanes showed starting material consumed. Added
iodomethane (714 mg, 315 mt, 5.03 mmol, Eq: 2.0) and stirred
overnight. During the night solids precipitated out. Placed the
reaction mixture in the freezer for 3 h. Filtered off the solids,
rinsing with cold methanol, and air dried for 2 h. Obtained 729 mg
(73%) of (Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(4-methoxyphenylthio)phenyl)carbamimidothioate
as a yellow solid.
N*3*-[3,5-Dichloro-4-(4-methoxy-phenylsulfanyl)-phenyl]-1H-[1,2,4-]triazol-
e-3,5-diamine (Compound 16)
##STR00099##
[0273] Added ethanol (15 mL) and hydrazine (292 mg, 0.286 mL, 9.11
mmol, Eq: 5.01) to the 50-mL round-bottomed flask containing
(Z)-methyl
N'-cyano-N-(3,5-dichloro-4-(4-methoxyphenylthio)phenyl)carbamimidothioate
(725 mg, 1.82 mmol, Eq: 1.00). Heated at reflux for 30 min (during
which time yellow solid dissolved, then white solids crashed out):
TLC in 10% methanol/dichloromethane with 1% ammonium hydroxide
showed that all the starting material had been consumed. Cooled to
room temperature. Placed the reaction mixture in the freezer for
.about.2 h. Filtered off the solids, rinsing with cold ethanol. Air
dried on the frit for 1 h. Obtained 596 mg (86%) of
N*3*-[3,5-Dichloro-4-(4-methoxy-phenylsulfanyl)-phenyl]-1H-[1,2,4]triazol-
e-3,5-diamine as a white fluffy solid. MS calcd. for C15H13C12N5OS
[(M+H)-1-] 382.0, obsd. 381.9.
N*3*-[3-Chloro-4-(4-methoxy-phenylsulfanyl)-5-trifluoromethyl-phenyl]-1H-[-
1,2,4]triazole-3,5-diamine (Compound 17)
##STR00100##
[0274]
2-chloro-4-nitro-6-(trifluoromethyl)phenyl)(4-methoxyphenyl)sulfane
##STR00101##
[0276] In a 250-mL round-bottomed flask, 4-methoxybenzenethiol
(1.00 g, 633 .mu.l, 7.14 mmol, Eq: 1.00),
1,3-dichloro-2-fluoro-5-nitrobenzene (1.5 g, 7.14 mmol, Eq: 1.00)
and potassium carbonate (990 mg, 7.16 mmol, Eq: 1.00) were combined
with N,N-dimethylformamide (20 mL) to give a yellow suspension. The
reaction mixture was stirred overnight at room temperature. In the
morning TLC was consistent with the reaction having reached
completion. The reaction mixture was poured into water to give a
yellow cloudy suspension. This suspension was extracted with ethyl
acetate. The organic phase was dried over sodium sulfate, filtered,
then concentrated to give a brown oil which was loaded directly
onto a 120 grain silica gel column. Flash chromatography 5-15%
ethyl acetate in hexanes was used to purify the product, however,
this purification was not very successful. The fractions containing
the product were concentrated. 111 NMR showed that the product was
consistent with product with only a small impurity. The product was
sufficiently pure to take onto the next step. Obtained 1.87 g
(83.5%) of
2-chloro-4-nitro-6-(trifluoromethyl)phenyl)(4-methoxyphenyl)sulfane.
3-chloro-4-(4-methoxyphenylthio)-5-(trifluoromethyl)aniline
##STR00102##
[0278] Charged iron (1.44 g, 25.7 mmol, Eq: 5.0), Ammonium chloride
(2.75 g, 51.4 mmol, Eq: 10.0), and methanol (80 mL) into the 500-mL
round-bottomed flask containing
(2-chloro-4-nitro-6-(trifluoromethyl)phenyl)(4-methoxyphenyl)sulfane
(1.87 g, 5.14 mmol, Eq: 1.00). Heated at reflux; after 2 h the
reaction was complete by TLC (1:3 ethyl acetate/hexanes) and LC/MS.
Cooled the mixture to room temperature, then filtered through a bed
of celite rinsing with a large amount of methanol. Concentrated the
filtrate in vacuo. Added ethyl acetate to the residue and filtered
off the solids. Concentrated the filtrate and obtained
3-chloro-4-(4-methoxyphenylthio)-5-(trifluoromethyl)aniline (1.32
g, 3.95 mmol, 76.9% yield) as a reddish-orange solid.
chloro-5-isothiocyanato-2-(4-methoxyphenylsulfanyl)-3-trifluoromethylbenze-
ne
##STR00103##
[0280] Charged dichloromethane (50 mL) and
1,1'-thiocarbonyldiimidazole (916 mg, 5.14 mmol, Eq: 1.3) to the
250-mL round-bottomed flask containing
3-chloro-4-(4-methoxyphenylthio)-5-(trifluoromethyl)aniline (1.32
g, 3.95 mmol, Eq: 1.00). Stirred at room temperature. Monitored the
reaction by TLC (1:3 ethyl acetate/hexanes) stirred over the
weekend at room temperature, and TLC confirmed the reaction was
complete. Diluted the reaction with dichloromethane, added Celite,
and concentrated. Purified using a 40 g silica gel column on an
Intelliflash 280; collected peaks only in 28 mL fractions at 53
mL/min; equilibrated with hexanes; dry loaded; eluted 2 min with
hexanes; increased from 0-40% dichloromethane/hexanes over 18 min.
Obtained 612 mg (41%) of
1-chloro-5-isothiocyanato-2-(4-methoxyphenylsulfanyl)-3-trifluoromethylbe-
nzene as an orange oil.
(Z)-methyl
N-3-chloro-4-(4-methoxyphenylthio)-5-(trifluoromethyl)phenyl-N'-
-cyanocarbamimidothioate
##STR00104##
[0282] Combined cyanamide (440 mg, 10.5 mmol) and 0.5 M sodium
methoxide (10.0 mL, 5.0 mmol). Added the cyanamide mixture (3.91
mL, 1.95 mmol, Eq: 1.2) to a mixture of
1-chloro-5-isothiocyanato-2-(4-methoxyphenylsulfanyl)-3-trifluoromethylbe-
nzene (612 mg, 1.63 mmol, Eq: 1.00) in methanol (10 mL) and toluene
(2 mL) Stirred at room temperature. After 1 h TLC in 1:3 ethyl
acetate/hexanes showed starting material consumed. Added
iodomethane (462 mg, 204 .mu.L, 3.26 mmol, Eq: 2.0) and stirred
overnight. During the night solids precipitated out. Placed the
reaction mixture in the freezer for 3 h. Filtered off the solids,
rinsing with cold methanol, and air dried for 2 h. Obtained 412 mg
(59%) of (Z)-methyl
N-3-chloro-4-(4-methoxyphenylthio)-5-(trifluoromethyl)phenyl-N'-cyanocarb-
amimidothioate as a light yellow solid.
N*3*-[3-Chloro-4-(4-methoxy-phenylsulfanyl)-5-trifluoromethyl-phenyl]-1H-[-
1,2,4]-triazole-3,5-diamine (Compound 17)
##STR00105##
[0284] Added ethanol (15 mL) and hydrazine (151 mg, 0.148 mL, 4.72
mmol, Eq: 5.00) to the 50-mL round-bottomed flask containing
(Z)-methyl
N-3-chloro-4-(4-methoxyphenylthio)-5-(trifluoromethyl)phenyl-N'-cyanocarb-
ainimidothioate (407 mg, 942 .mu.mol, Eq: 1.00). Stirred at room
temperature for 40 min, then heated at reflux for 20 min: TLC in
10% methanol/dichloromethane with 1% ammonium hydroxide showed that
all the starting material had been consumed. Cooled to room
temperature. Removed the solvent in vacuo. Obtained 367 mg (94%) of
N*3*-[3-Chloro-4-(4-methoxy-phenylsulfanyl)-5-trifluoromethyl-phenyl]-1H--
[1,2,4]triazole-3,5-diamine as a white solid. MS calcd. for
C.sub.16H.sub.13ClF.sub.3N.sub.50S [(M+H)+] 416.0, obsd. 415.9.
N*3*-[3,5-Dichloro-4-(4-methoxy-benzenesulfonyl)-phenyl]-1H-[1,2,4]-triazo-
le-3,5-diamine (Compound 18)
##STR00106##
[0286] Combined
N3-(3,5-dichloro-4-(4-methoxyphenylthio)phenyl)-1H-1,2,4-triazole-3,5-dia-
mine (compound 16) (150 mg, 392 .mu.mol, Eq: 1.00), oxone (1.21 g,
1.96 mmol, Eq: 5.00), and methanol (5 mL) together in a 25-mL
round-bottomed flask. Stirred overnight at room temperature open to
the air. TLC in 10% methanol/dichloromethane with 1% ammonium
hydroxide showed a new, more polar spot; LC/MS showed sulfoxide and
starting material only. Transferred the reaction mixture to a seal
tube and heated at 70.degree. C. for 4 h. LC/MS showed sulfoxide
and a very small sulfone peak. Heated overnight at 70.degree. C.
LC/MS showed slightly larger sulfone peak, but mostly sulfoxide
still. Heated at 90.degree. C. for 3 days: LC/MS showed
proportionately more sulfone. Added more oxone (605 mg, 98 mmol,
Eq: 2.5) and heated at 105.degree. C., still only a small amount of
sulfone. Decided to work-up: filtered off the solids, rinsing with
methanol. The filtrate was concentrated and purified by prep-HPLC.
Obtained 9.5 mg (6%) of
N*3*-[3,5-Dichloro-4-(4-methoxy-benzenesulfonyl)-phenyl]-1H-[1,2,4]tri-
azole-3,5-diamine as waxy, yellow solid. MS calcd. for
C15H13C12N5O3S [(M+MeCN+H)+] 455.0, obsd. 454.8.
N*3*-[3,5-Dichloro-4-(4-methanesulfonyl-phenoxy)-phenyl]-1H-[1,2,4]-triazo-
le-3,5-diamine (Compound 19)
##STR00107##
[0287]
1,3-Dichloro-2-(4-methanesulfonyl-phenoxy)-5-nitro-benzene
##STR00108##
[0289] To a stirred solution of
1,3-dichloro-2-fluoro-5-nitrobenzene (2.09 g, 10 mmol) in DMF (20
mL), was added K2CO3 (2.06 g, 15 mmol) and 4-(methylsulfonyl)
phenol (1.72 g, 10 mmol). The reaction mixture was stirred at
110.degree. C. for 3 hrs. The reaction was poured into water (100
mL), extracted with diethyl ether (3.times.25 mL) and dried with
sodium sulfate. Evaporation of solvent gave 3.48 g (96%) of desired
product as an off white solid.
1,3-Dichloro-2-(4-methanesulfonyl-phenoxy)-5-amino-benzene
##STR00109##
[0291] A suspension of
1,3-Dichloro-2-(4-(methylsulfonyl)phenoxy)-5-nitrobenzene (3.4 g,
9.39 mmol) and 10% Pd/C (0.5 g, 0.47 mmol) in EtOAc (100 mL) was
hydrogenated at 50 PSI for 3 hrs at rt. The reaction mixture was
filtered and the filtrate was concentrated to give 3.02 g (97%) of
desired product as a white solid. MS +m/z: 331.9 (M+H).sup.+
[0292] .sup.1H NMR (300 MHz, DMSO-d.sub.6) 8 ppm 3.19 (s, 3H) 5.74
(s, 2H) 6.56-7.22 (m, 4H) 7.89 (d, J=8.67 Hz, 2H)
(E)-Methyl
N'-cyano-N-(3,5-dichloro-4-(4-(methylsulfonyl)phenoxy)phenyl)ca-
rbamimidothioate
##STR00110##
[0294] To a solution of
1,3-dichloro-2-(4-methanesulfonyl-phenoxy)-5-amino-benzene (332 mg,
1 mmol) in pyridine (5 mL), was added dimethyl
cyanocarbonimidodithioate (146 mg, 1 mmol). The reaction mixture
was heated at reflux for 3 hours. The solvent was removed by
evaporation and the residue was chromatographed (methylene
chloride) to give 242 mg (56%) of desired product as a brown solid.
MS +m/z: 429.8 (M+H).sup.+
N.sup.*3*-[3,5-Dichloro-4-(4-methanesulfonyl-phenoxy)-phenyl]-1H-[1,2,4]-t-
riazole-3,5-diamine (Compound 19)
##STR00111##
[0296] To a solution of (E)-Methyl
N'-cyano-N-(3,5-dichloro-4-(4-methylsulfonyl)phenoxy)phenyl)
carbamimidothioate (240 mg, 0.558 mmol) in THF (4 mL) and methanol
(2 mL), was added hydrazine (35.7 mg, 0.035 uL, 1.12 mmol). The
reaction mixture was stirred overnight at room temperature. The
solvent was removed and the residue was chromatographed on a
combiflash machine (5% methanol/methylene chloride, then 5%
methanol/EtOAc) to give 178 mg (77%) of desired product as a white
solid. MS +m/z: 413.8 (M+H).sup.+
[0297] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 3.20 (s, 3H)
6.02 (br. s., 2 .mu.l) 7.07 (d, J=8.85 Hz, 2H) 7.67-8.00 (m, 4H)
9.29 (s, 1H) 11.32 (s, 1H)
N*3*-[3-Chloro-4-(4-methanesulfonyl-phenoxy)-5-trifluoromethyl-phenyl]-1H--
[1,2,4]-triazole-3,5-diamine (Compound 20)
##STR00112##
[0298]
Chloro-2-(4-methanesulfonyl-phenoxy)-5-nitro-3-trifluoromethyl-benz-
ene
##STR00113##
[0300] To a stirred solution of
1-chloro-2-fluoro-3-trifluoromethyl-5-nitrobenzene (1.42 g, 5.81
mmol) in DMF (20 mL), was added K2CO3 (2.06 g, 15 mmol) and
4-(methylsulfonyl) phenol (1.0 g, 5.81 mmol). The reaction mixture
was stirred at 110.degree. C. for 3 hrs. The reaction was poured
into water (100 mL) and the mixture was extracted with toluene
(3.times.25 mL) and dried with sodium sulfate. Evaporation of
solvent gave 1.82 g (79%) of desired product as an off-white
solid.
[0301] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 3.24 (s, 3H)
7.23 (d, J=8.85 Hz, 2H) 7.94 (d, J=8.85 Hz, 2H) 8.60 (d, J=2.45 Hz,
1H) 8.93 (d, J=2.45 Hz, 1H)
Chloro-2-(4-methanesulfonyl-phenoxy)-5-amino-3-trifluoromethyl-benzene
##STR00114##
[0303] A suspension of
1-Chloro-2-(4-(methylsulfonyl)phenoxy)-5-nitro-3-(trifluoromethyl)benzene
(1.8 g, 4.55 mmol) and Pd/C (10%, 120 mg, 0.115 mmol) in ethyl
acetate (50 mL) was hydrogenated at 50 PSI for 3 hrs. The reaction
mixture was filtered and the filtrate was concentrated to give 1.59
g (96%) of desired product as a white solid.
[0304] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 3.20 (s, 3H)
5.94 (s, 2H) 6.91-7.10 (m, 4H) 7.87 (d, J=8.85 Hz, 2H)
(E)-Methyl
N-3-chloro-4-(4-(methylsulfonyl)phenoxy)-5-(trifluoromethyl)phe-
nyl-N'-cyanocarbamimidothioate
##STR00115##
[0306] To a stirred solution of
3-chloro-4-(4-(methylsulfonyl)phenoxy)-5-(trifluoromethyl)aniline
(182 mg, 0.50 mmol) in THF (4 mL), was added 1M potassium
tert-butoxide in THF (0.50 mL, 0.50 mmol) drop wise. The reaction
mixture was stirred at rt for 15 min, after which dimethyl
cyanocarbonimidodithioate (73 mg, 0.50 mmol) was added, and the
resulting mixture was stirred at rt for 2 hrs. The solvent was
removed and the residue was chromatographed (100% methylene
chloride gradient to 5% methanol/methylene chloride) to give 60 mg
(26%) of desired product as an orange solid. MS +m/z: 463.63
(M+H).sup.+
N*3*-[3-Chloro-4-(4-methanesulfonyl-phenoxy)-5-trifluoromethyl-phenyl]-11'-
-[1,2,4]triazole-3,5-diamine (Compound 20)
##STR00116##
[0308] To a stirred solution of (Z)-methyl
N-3-chloro-4-(4-(methylsulfonyl)phenoxy)-5-(trifluoromethyl)phenyl-N'-cya-
nocarbamimidothioate (60 mg, 0.129 mmol) in methanol (3 mL) at rt,
was added hydrazine monohydrate (5 mg, 0.155 mmol). The reaction
mixture was stirred for 3 hrs, after which the solvent was reduced
to 1.5 mL and filtered through a membrane filter and then loaded on
a HPLC. Collection of the desired peak gave the 11 mg (19%) of
desired product as white solid. MS +m/z: 447.9 (M+H).sup.4.
[0309] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 3.20 (s,
12H) 6.05 (br. s., 2H) 7.07 (d, J=8.67 Hz, 2H) 7.88 (d, J=8.67 Hz,
2H) 7.95-7.98 (m, 1H) 8.10 (s, 1H) 9.47 (s, 1H) 11.38 (br. s.,
1H)
N*5*-(3-Benzyloxy-5-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(Compound 21)
##STR00117##
[0310] 1-(benzyloxy)-3-chloro-5-isothiocyanatobenzene
##STR00118##
[0312] 3-(benzyloxy)-5-chloroaniline (775 mg, 3.32 mmol) and
thiocarbonyldiimidazole (887 mg, 4.97 mmol) were stirred overnight
in a solution of CH.sub.2Cl.sub.2 (12 mL).
1-(Benzyloxy)-3-chloro-5-isothiocyanatobenzene (870 mg, 95%). was
purified directly from the reaction mixture by column
chromatography (8:1, hexane:EtOAc) as a brown oil.
(Z)-methyl 3-(benzyloxy)-5-chloro-N-cyanobenzimidate
##STR00119##
[0314] Sodium hydrogencyanamide (222 mg, 3.47 mmol) was added to a
solution of 1-(benzyloxy)-3-chloro-5-isothiocyanatobenzene (896 mg,
6.3 mmol) in CH.sub.3OH (20 mL). After stirring for 1.5 hours,
iodomethane (395 .mu.L, 6.31 mmol)) was added and the reaction
mixture was stirred for 60 hours. All volatiles were then removed
under reduced pressure and (Z)-methyl
3-(benzyloxy)-5-chloro-N-cyanobenzimidate (240 mg, 24%) was
isolated by column chromatography (4:1 to 1:1 hexane:EtOAc).
N*5*-(3-Benzyloxy-5-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(Compound 21)
##STR00120##
[0316] (Z)-methyl 3-(benzyloxy)-5-chloro-N-cyanobenzimidate (85 mg,
0.28 mmol) and hydrazine (200 .mu.L, 6.37 mmol) in ethanol (2 mL)
were heated to 85.degree. C. for 16 hours. After cooling, all
volatiles were removed under reduced pressure to yield a solid from
which
N*5*-(3-benzyloxy-5-chloro-phenyl)-1H-[1,2,4]triazole-3,5-diamine
(33 mg, 37%) was isolated as a white solid by column chromatography
(10% CH.sub.3OH in CH.sub.2Cl2). MH+=316.0
N3-(3-chloro-4-(phenylthio)phenyl)-1H-1,2,4-triazole-3,5-diamine
(Compound 22)
##STR00121##
[0317] 2-Chloro-4-nitro-1-phenylsulfanyl-benzene
##STR00122##
[0319] In a 250 mL round-bottom flask,
2-chloro-1-fluoro-4-nitrobenzene (4.0 g, 22.8 mmol, Eq: 1.00),
thiophenol (2.34 mL, 22.8 mmol, Eq: 1.00) and potassium carbonate
(6.3 g, 45.6 mmol, Eq: 2.0) were combined with DMF (45.6 mL) to
give a light brown suspension. The reaction mixture was heated at
100.degree. C. overnight. In the morning, the reaction mixture was
cooled to room temperature and partitioned between water and ethyl
acetate. The organic phase was dried (MgSO.sub.4), filtered, and
concentrated to afford a brown oil. The crude product was dissolved
in methylene chloride, and this solution was concentrated over
silica gel. The silica gel supported crude product was loaded onto
a 220 gram SiliCycle column. Flash chromatography (5-10% ethyl
acetate-hexanes) afforded 2-chloro-4-nitro-1-phenylsulfanyl-benzene
(7.5 g, 100%).
3-Chloro-4-(phenylthio)aniline
##STR00123##
[0321] In a 250 mL round-bottom flask,
2-chloro-4-nitro-1-phenylsulfanyl-benzene (3.31 g, 12.5 mmol, Eq:
1.00), iron (3.48 g, 55.85 mmol, Eq: 5.0) and ammonium chloride
(6.66 g, 53.49 mmol, Eq: 10) were combined with methanol (33 mL) to
give a light brown suspension. Water (16.6 mL) was added, and the
reaction mixture became a milky-white suspension. The reaction
mixture was refluxed for 6 hours. The reaction mixture was
filtered, and concentrated to remove most of the methanol. The
resulting crude suspension was extracted with ethyl acetate. The
organic phase was dried (MgSO.sub.4), filtered, and concentrated to
give 3-chloro-4-(phenylthio)aniline (2.47 g, 44%) as a slightly tan
oil.
(2-Chloro-4-isothiocyanatophenyl)(phenyl)sulfane
##STR00124##
[0323] In a 250 mL round-bottom flask, thiophosgene (884 .mu.L,
11.5 mmol, Eq: 1.1) and calcium carbonate (1.05 g, 10.5 mmol, Eq:
1.00) were combined with dichloromethane (37.4 mL) and water at
0.degree. C. to give a yellow suspension. The reaction mixture was
stirred at 0.degree. C. under argon for ten minutes. A mixture of
3-chloro-4-(phenylthio)aniline (2.47 g, 10.5 mmol, Eq: 1.00) in
methylene chloride (5 mL) was added dropwise to the cold suspension
via a syringe. The reaction mixture was stirred over four hours,
slowly warming to room temperature. The reaction mixture was
neutralized with 1.0 N aqueous HCl, and the resulting mixture was
extracted with methylene chloride. The organic phase was dried over
MgSO.sub.4, and concentrated onto silica gel. The silica gel
supported crude product was loaded onto a 200 g SiliCycle column.
Flash chromatography 0%-5% ethyl acetate-hexanes afforded
(2-chloro-4-isothiocyanatophenyl)(phenyl)sulfane (1.9 g, 65%) as a
clear oil.
(Z)-methyl
N-3-chloro-4-(phenylthio)phenyl-M-cyanocarbamimidothioate
##STR00125##
[0325] Cyanamide (150 mg, 3.56 mmol, Eq: 1.1) was added to a 50 mL
round-bottom flask while purging with argon. A 0.5 M solution of
sodium methoxide in methanol (1.22 mL, 3.95 mmol, Eq: 1.2) was
added at room temperature. The reaction mixture was then stirred
for 15 minutes. Separately,
(2-chloro-4-isothiocyanatophenyl)(phenyl)sulfane (0.9 g, 3.24 mmol,
Eq: 1.00) was combined with methanol (13 mL) with stirring. The
cyanamide mixture was transferred to the starting material mixture
via a syringe. The resulting mixture was stirred for a total of 1
hour, then iodomethane (0.304 mL, 4.86 mmol, Eq: 1.5) was added.
The reaction mixture was stirred at room temperature overnight. The
mixture was then diluted with methylene chloride and methanol. The
resulting solution was concentrated onto silica gel. The silica
gel-supported crude product was loaded onto a 200 g silica gel
column. Flash chromatography (100% hexanes) afforded (Z)-methyl
N-3-chloro-4-(phenylthio)phenyl-N'-cyanocarbamimidothioate (240 mg,
22%) as a white solid.
N3-(3-chloro-4-(phenylthio)phenyl)-1H-1,2,4-triazole-3,5-diamine
(Compound 22)
##STR00126##
[0327] In a 250 mL round-bottom flask, (Z)-methyl
N-3-chloro-4-(phenylthio)phenyl-N-cyanocarbamimidothioate (240 mg,
719 .mu.mol, Eq: 1.00) was combined with ethanol (10 mL) to give a
white suspension. Hydrazine (226 .mu.L, 7.19 mmol, Eq: 10) was
added. The reaction mixture was heated at reflux for 1 hour. The
reaction mixture was concentrated, giving an oily foam-like
substance. This product was dissolved in 15% methanol-chloroform,
and the solution filtered through a small filter plug. The liquid
filtrate was concentrated down to afford
N3-(3-chloro-4-(phenylthio)phenyl)-1H-1,2,4-triazole-3,5-diamine as
a brittle solid. MS cald. for C.sub.14H.sub.12ClN.sub.5S
[(M+H).sup.+]: 318, obsd. 318.0.
N3-(3-chloro-4-(phenylsulfonyl)phenyl)-1H-1,2,4-triazole-3,5-diamine
(Compound 23)
##STR00127##
[0328] (2-Chloro-4-nitrophenyl)(phenyl)sulfone
##STR00128##
[0330] In a 250 mL round-bottomed flask,
2-chloro-4-nitro-1-phenylsulfanyl-benzene (4.126 g, 15.5 mmol, Eq:
1.00) was combined with methylene chloride (100 mL) to give a
yellow solution. This mixture was cooled to 0.degree. C. in an ice
water bath. m-Chloroperoxybenzoic acid (10.7 g, 62.1 mmol, Eq: 4.0)
was added scoopwise. The reaction mixture was stirred overnight,
with slow warming to room temperature. In the morning, the reaction
mixture was washed with saturated aqueous sodium sulfite, then
aqueous sodium bicarbonate. The organic phase was dried over
MgSO.sub.4, filtered, and concentrated to afford a white solid. The
crude product was loaded onto silica gel. The silica gel supported
crude product was loaded onto a 200 g silacycle column. Flash
chromatography (40%-60% ethyl acetate-hexanes) provided
(2-chloro-4-nitrophenyl)(phenyl)sulfone (2.03 g, 44%) as a white
crystalline solid.
3-Chloro-4-(phenylsulfonyl)aniline
##STR00129##
[0332] In a 250 mL round-bottom flask,
(2-chloro-4-nitrophenyl)(phenyl)sulfone (2.03 g, 6.82 mmol, Eq:
1.00), iron (1.9 g, 34.1 mmol, Eq: 5.0) and ammonium chloride (3.65
g, 98.2 mmol, Eq: 10) were combined with methanol (22.7 mL) to give
a light brown suspension. Water (11.4 mL) was added, and the
reaction mixture became a milky-white suspension. The reaction
mixture was refluxed for 6 hours. After only 20 minutes, the
reaction mixture was rust-colored. After 6 hours heating at reflux,
the reaction mixture was cooled to room temperature. The mixture
was filtered, and concentrated to remove most of the methanol. The
resulting crude suspension was extracted with ethyl acetate. The
organic phase was dried (MgSO.sub.4), filtered, and concentrated to
give 3-chloro-4-(phenylsulfonyl)aniline (1.8 g, 99%) as a white
crystalline solid.
2-Chloro-4-isothiocyanato-1-)phenylsulfonyl)benzene
##STR00130##
[0334] In a 250 mL round-bottom flask, thiophosgene (567 .mu.L, 7.4
mmol, Eq: 1.1) and calcium carbonate (0.673 g, 6.72 mmol, Eq: 1.00)
were combined with dichloromethane (34 mL) and water (24 mL) at
0.degree. C. to give a yellow suspension. The reaction mixture was
stirred at 0.degree. C. under argon for ten minutes. A mixture of
3-chloro-4-(phenylsulfonyl)aniline (1.8 g, 6.72 mmol, Eq: 1.00) in
methylene chloride (5 mL) was added dropwise to the cold suspension
via a syringe. The reaction mixture was stirred over four hours,
being slowly allowed to warm to room temperature. The reaction
mixture was neutralized with 1.0 N aqueous HCl, and then extracted
with methylene chloride. The organic phase was dried over
MgSO.sub.4, and concentrated onto silica gel. The silica gel
supported crude product was loaded onto a 200 g silicylce column.
Flash chromatography (15%-40% ethyl acetate-hexane) afforded
2-chloro-4-isothiocyanato-1-)phenylsulfonyl)benzene (1.14 g, 55%)
as a white crystalline solid.
(Z)-Methyl
N-3-chloro-4-(phenylsulfonyl)phenyl-N'-cyanocarbamimidothioate
##STR00131##
[0336] A 50 mL round-bottom flask was charged with cyanamide (170
mg, 4.03 mmol, Eq: 1.1) and a 0.5 M solution of sodium methoxide in
methanol (8.1 mL, 4.05 mmol, Eq: 1.2), and this solution was
stirred for 15 minutes. Separately,
2-chloro-4-isothiocyanato-1-)phenylsulfonyl)benzene (1.136 g, 3.67
mmol, Eq: 1.00) and methanol (14.7 mL) were combined. The cyanamide
mixture was transferred to the starting material mixture via
syringe at room temperature. The mixture became homogeneous after a
short time of stirring. After stirring for one hour at room
temperature, iodomethane (0.794 g, 0.350 mL, 5.6 mmol, Eq: 1.5) was
added and the reaction mixture was stirred overnight at room
temperature. The reaction mixture was diluted with a 9:1 mixture
methylene chloride and methanol, and the solution was concentrated
onto silica gel. The silica gel supported crude product was loaded
onto a 120 gram silica gel column. Flash chromatography (25%-40%
ethyl acetate-hexanes) was used to isolate (Z)-methyl
N-3-chloro-4-(phenylsulfonyl)phenyl-N'-cyanocarbamimidothioate (84
mg, 6.3%) as a white solid.
N3-(3-chloro-4-(phenylsulfonyl)phenyl)-1H-1,2,4-triazole-3,5-diamine
(Compound 23)
##STR00132##
[0338] In a 200 mL round-bottomed flask, (Z)-methyl
N-3-chloro-4-(phenylsulfonyl)phenyl-N'-cyanocarbamimidothioate (84
mg, 230 .mu.mol, Eq: 1.00) and hydrazine (79 .mu.L, 2.51 mmol, Eq:
11.0) were combined with ethanol (3.5 mL) to give a colorless
solution. The reaction mixture was refluxed for 1 hour. The
reaction mixture was concentrated down on the rotary evaporator.
The crude product was a slightly yellow oil which was dissolved in
4:1 methylene chloride-methanol. This solution was carefully
filtered. The filtrate was dried down to give
N3-(3-chloro-4-(phenylsulfonyl)phenyl)-1H-1,2,4-triazole-3,5-diamine
as a brittle off-white powder. MS cald for
C.sub.14H.sub.12ClN.sub.5O.sub.2S [(M+H).sup.+]: 350, obsd.
349.9.
N3-(3-chloro-4-(phenylthio)phenyl)-N-3-methyl-1H-1,2,4-triazole-3,5-diamin-
e (Compound 24)
##STR00133##
[0339] (Z)-methyl
N-3-chloro-4-(phenylthio)phenyl-N'-cyano-N-methylcarbamimidothioate
##STR00134##
[0341] In a 50 mL round-bottomed flask, (Z)-methyl
N-3-chloro-4-(phenylthio)phenyl-N'-cyanocarbamimidothioate (53 mg,
159 .mu.mol, Eq: 1.00) was combined with DMF (1.59 mL) to give a
colorless solution. This mixture was cooled to 0.degree. C., then
sodium hydride (60% suspension in oil, 6.98 mg, 175 .mu.mol, Eq:
1.1) was added. The reaction mixture was stirred at 0.degree. C.
for 15 minutes, then methyl iodide (9.9 .mu.L, 0.159 .mu.mol) was
added. The reaction mixture was slowly warmed to room temperature
and then stirred at room temperature over 48 hours. The reaction
mixture was quenched with water, and extracted with ethyl acetate.
The organic phase was dried over MgSO.sub.4, filtered, and
concentrated in vacuo. The crude product was dissolved in methylene
chloride, and then concentrated over silica gel. The silica gel
supported crude product was loaded onto a 35 gram SiliCycle column.
Flash chromatography (0%-15% ethyl acetate in hexanes) afforded the
product (27 mg, 50%) as a clear oil.
N3-(3-chloro-4-(phenylthio)phenyl)-N-3-methyl-1H-1,2,4-triazole-3,5-diamin-
e (Compound 24)
##STR00135##
[0343] In a 50 mL round-bottomed flask, (Z)-methyl
N-3-chloro-4-(phenylthio)phenyl-N-cyano-N-methylcarbamimidothioate
(27.6 mg, 79.3 .mu.mol, Eq: 1.00) and hydrazine (50 .mu.L, 1.59
mmol, Eq: 20.1) were combined with ethanol (2.5 mL) to give a white
suspension. The reaction mixture was heated at reflux for 2 hours.
The reaction mixture was concentrated. Preparatory reverse-phase LC
purification provided
N3-(3-chloro-4-(phenylthio)phenyl)-N3-methyl-1H-1,2,4-triazole-3,5-diamin-
e (9 mg, 98%). MS cald. for C.sub.15H.sub.14ClN.sub.5S
[(M+H).sup.+]: 332, obsd. 331.9.
Example 2, Procedure E
N*3*-(3-Chloro-5-phenoxy-phenyl)-1H-[1,2,4]-triazole-3,5-diamine
(Compound 25)
##STR00136##
[0344] 1-bromo-3-chloro-5-phenoxybenzene
##STR00137##
[0346] In a 250 mL round-bottomed flask, 3-bromo-5-chlorophenol (2
g, 9.64 mmol, Eq: 1.00), phenylboronic acid (2.35 g, 19.3 mmol, Eq:
2.00) and triethylamine (9.76 g, 96.4 mmol, Eq: 10.00) were
combined with CH.sub.2Cl.sub.2 (100 ml) to give a colorless
solution. Powdered 4 .ANG. molecular sieves (200 mg) and copper(II)
acetate (2.63 g, 14.5 mmol, Eq: 1.50) were added. The reaction was
stirred at room temperature for 3 hours. Filtered out the solid,
filtrate was evaporated in vacuo. The compound was isolated by
column chromatography (Hexanes/EtOAc=70/30) to give the product 550
mg (20%).
5-Bromo-1-(4-methoxy-benzyl)-3-nitro-1H-[1,2,4]-triazole
##STR00138##
[0348] In a 250 mL round-bottomed flask,
5-bromo-3-nitro-1H-1,2,4-triazole (12 g, 62.2 mmol, Eq: 1.00),
1-(chloromethyl)-4-methoxybenzene (9.74 g, 62.2 mmol, Eq: 1) and
N-ethyl-N-isopropylpropan-2-amine (16.1 g, 124 mmol, Eq: 2) were
combined with acetonitrile (100 ml) to give a light yellow
solution. Potassium iodide (5.16 g, 31.1 mmol, Eq: 0.5) was added.
The reaction mixture was heated to reflux for 2 hours. The reaction
mixture was cooled and diluted with EtOAc (100 mL), washed with
H.sub.2O (50 mL) and brine (50 mL). The organic layer was dried
over anhydrous MgSO.sub.4, filtered and volatiles were removed
under reduced pressure to yield an oil from which the compound was
isolated by column chromatography (Hexanes/EtOAc=70/30) to product
7.8 g (40%).
Bis-(4-methoxy-benzyl)-[2-(4-methoxy-benzyl)-5-nitro-2,1-[1,2,4]triazol-3--
yl]-amine
##STR00139##
[0350] In a 10 mL sealed tube,
5-bromo-1-(4-methoxybenzyl)-3-nitro-1H-1,2,4-triazole (5.48 g, 17.5
mmol, Eq: 1.00) and bis(4-methoxybenzyl)amine (4.5 g, 17.5 mmol,
Eq: 1.00) were combined, the mixture was heated to 150.degree. C.
for overnight. Cool the reaction down, added CH.sub.2Cl.sub.2 (50
mL) washed with H.sub.2O (50 mL) and brine (50 mL), the organic
layer was dried over anhydrous MgSO.sub.4, filtered and volatiles
were removed under reduced pressure to afford the crude product 8.3
g (97% crude). MH+490.3
1,N*5*,N*5*-Tris-(4-methoxy-benzyl)-1H-[1,2,4]triazole-3,5-diamine
##STR00140##
[0352] In a 100 mL round bottle,
bis-(4-methoxy-benzyl)-[2-(4-methoxy-benzyl)-5-nitro-2H-[1,2,4]triazol-3--
yl]-amine (530 mg, 1.08 mmol, Eq: 1.00) and zinc (354 mg, 5.41
mmol, Eq: 5.00) were combined with the solution of saturated
NH.sub.4Cl aqueous solution/THF (1:1) (60.0 ml), the mixture was
stirred at room temperature for 1 hour. Filter out the solid,
extracted the mixture with CH.sub.2Cl.sub.2 (50 mL.times.2), the
organic layer was dried over anhydrous MgSO.sub.4, filtered and
volatiles were removed under reduced pressure. The compound was
isolated by column chromatography to give a light yellow solid 420
mg (84%). MH+460.3
N*3*-(3-Chloro-5-phenoxy-phenyl)-1,N*5*,N*5*-tris-(4-methoxy-benzyl)-1H-[1-
,2,4]triazole-3,5-diamine
##STR00141##
[0354] In a 25 mL sealed tube, sodium 2-methylpropan-2-olate (125
mg, 1.31 mmol, Eq: 1.20), bis(dibenzylideneacetone)palladium (62.6
mg, 109 mmol, Eq: 0.1) and
2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (46.2 mg,
109 mmol, Eq: 0.1) were combined with toluene (4.00 mL) to give a
dark brown suspension.
N5,N5,1-tris(4-methoxybenzyl)-1H-1,2,4-triazole-3,5-diamine (500
mg, 1.09 mmol, Eq: 1.00) and 1-bromo-3-chloro-5-phenoxybenzene (309
mg, 1.09 mmol, Eq: 1) were added. The reaction mixture was degassed
with argon for 15 min, and then heated to 110.degree. C. for 3
hours. The reaction mixture was cooled and diluted with EtOAc (50
mL), washed with H.sub.2O (25 mL) and brine (25 mL). The organic
layer was dried over anhydrous MgSO.sub.4, filtered and volatiles
were removed under reduced pressure to yield an oil from which the
compound was isolated by column chromatography
(Hexanes/EtOAc=70/30) to give an off-white solid 150 mg (21%).
MH+662.4
N*3*-(3-Chloro-5-phenoxy-phenyl)-1H-[1,2,4]-triazole-3,5-diamine
(Compound 25)
##STR00142##
[0356] In 10 mL round bottle,
N3-(3-chloro-5-phenoxyphenyl)-N5,N5,1-tris(4-methoxybenzyl)-1H-1,2,4-tria-
zole-3,5-diamine (150 mg, 227 .mu.mol, Eq: 1.00) was combined with
TFA (5.00 mL) to give a colorless solution. The resulting solution
was heated to 65.degree. C. overnight, the reaction mixture was
concentrated, and then diluted with EtOAc (30 mL). The solution was
washed with saturated NaHCO.sub.3, organic layer was dried over
anhydrous MgSO.sub.4, filtered and volatiles were removed under
reduced pressure. The compound was isolated by preparative TLC to
give an off-white solid 32 mg (47%). MH+302.0
Example 3, Procedure A
[0357]
N*3*-[3,5-Dichloro-4-(pyridin-2-yloxy)-phenyl]-1H-[1,2,4]triazole-3-
,5-diamine (Compound 26)
##STR00143##
2-(2,6-Dichloro-4-nitro-phenoxy)-pyridine
##STR00144##
[0359] In a 50 mL round-bottomed flask, potassium tert-butoxide
(3.53 g, 31.5 mmol, Eq: 1.50) and pyridin-2(1H)-one (2 g, 21.0
mmol, Eq: 1.00) were combined with DMF (25.0 ml) to give a light
brown suspension at 0.degree. C. under nitrogen.
1,3-Dichloro-2-fluoro-5-nitrobenzene (4.42 g, 21.0 mmol, Eq: 1.00)
was added. The reaction was stirred overnight at room temperature.
The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL),
washed with H.sub.2O (25 mL) and brine (25 mL) The organic layer
was dried over anhydrous MgSO.sub.4, filtered and volatiles were
removed under reduced pressure to yield an oil from which the
compound was isolated by column chromatography
(Hexanes/EtOAc=70/30) to give an off-white solid 2.4 g (40%).
MH+284.9
3,5-Dichloro-4-(pyridin-2-yloxy)-phenylamine
##STR00145##
[0361] In a 100 mL round bottle,
2-(2,6-dichloro-4-nitrophenoxy)pyridine (2.4 g, 8.42 mmol, Eq:
1.00) and zinc (2.75 g, 42.1 mmol, Eq: 5.00) were combined with a
solution of saturated NH.sub.4Cl aqueous solution/THF (1:1) (50
ml), the mixture was stirred at for overnight. Filter out the
solid, extracted with CH.sub.2Cl.sub.2 (50 mL.times.2), the organic
layer was dried over anhydrous Na.sub.2SO.sub.4; the solution was
concentrated under vacuum to afford the crude product 2.0 g (93%).
MH+254.9
2-(2,6-Dichloro-4-isothiocyanato-phenoxy)-pyridine
##STR00146##
[0363] In a 100 mL round-bottomed flask,
di(1H-imidazol-1-yl)methanethione (2.1 g, 11.8 mmol, Eq: 1.5) was
combined with CH.sub.2Cl.sub.12 (30 mL) to give a colorless
solution. Di(1H-imidazol-1-yl)methanethione (2.1 g, 11.8 mmol, Eq:
1.5) in CH.sub.2Cl.sub.2 (20 mL) was added dropwise at 0.degree. C.
The reaction was allowed to warm to room temperature, and allowed
to stir overnight. Concentrate the solution, the compound was
isolated by column chromatography (Hexanes/EtOAc=80/20) to give the
product 2.1 g (90%). MH+298.0
[3,5-Dichloro-4-(pyridin-2-yloxy)-phenylamino]-methylsulfanyl-methyl-cyana-
mide
##STR00147##
[0365] In a 100 mL round-bottomed flask,
2-(2,6-dichloro-4-isothiocyanatophenoxy)pyridine (1.2 g, 4.04 mmol,
Eq: 1.00) in MeOH (20 mL), sodium hydrogencyanamide (292 mg, 4.56
mmol, Eq: 1.13) was added. The suspension turned to clear after a
few minutes, the reaction was allowed to stir at room temperature
for 1 hour, iodomethane (1.15 g, 8.08 mmol, Eq: 2) was added, the
reaction mixture was allowed to stir at room temperature overnight.
Concentrate the solution, the compound was isolated by column
chromatography (CH.sub.2Cl.sub.2/MeOH=95/5) to give an off-white
solid 1.2 g (84%). MH+352.9
N*3*-[3,5-Dichloro-4-(pyridin-2-yloxy)-phenyl]-1H-[1,2,4]triazole-3,5-diam-
ine (Compound 26)
##STR00148##
[0367] In a 100 mL round-bottomed flask,
[3,5-dichloro-4-(pyridin-2-yloxy)-phenylamino]-methylsulfanyl-methyl-cyan-
amide (600 mg, 1.69 mmol, Eq: 1.00) in EtOH (30 mL), hydrazine (541
mg, 16.9 mmol, Eq: 10.00) was added. The reaction was heated to
65.degree. C. for 3 hours. The reaction mixture was concentrated,
added H.sub.2O (20 mL) to the residue, filtered out the solid and
washed the solid with H.sub.2O (30 mL) and CH.sub.2Cl.sub.2 (10
mL), air-dried the solid overnight to give an off-white solid 382
mg (67%). MH+336.9
Biological Examples
Determination of Compounds HCV GT1b and GT1a Entry Inhibitory
Activity Using the Pseudotyped HCV Particle (HCVpp) Reporter
Assay
Mammalian Expression Plasmids for the Generation of Pseudotyped
Virus Particles:
[0368] Plasmids expressing HCV E1 and E2 envelope proteins of GT1a
H77 strain (Proc Natl Acad Sci USA 1997 94:8738-43) or GT1b Con1
strain (Science 1999 285:110-3) were constructed by cloning the
nucleic acids encoding the last 60 amino acids of HCV core protein
and all of the HCV E1 and E2 proteins into pcDNA3.1(+) vector.
Plasmid pVSV-G expressing the glycoprotein G of the vesicular
stomatitis virus (VSV G) is from Clontech (cat #631530). The HIV
packaging construct expressing the firefly luciferase reporter gene
was modified based on the envelope defective
pNL.4.3.Luc-R.sup.-.E.sup.- vector (Virology 1995 206:935-44) by
further deleting part of the HIV envelope protein.
Generation of Pseudotyped Virus Particles in Transiently
Transfected HEK-293T Cells:
[0369] Pseudotyped HCV GT1a and GT1b particles (HCVpp) and the
pseudotyped VSV G particles (VSVpp) were generated from transiently
transfected HEK-293T cells (ATCC cat #CRL-573). For generating
HCVpp, the HEK-293T cells were transfected with equal amounts of
plasmids expressing the HCV envelope proteins and the HIV packaging
genome by using polyethylenimine (Polysciences cat #23966) as
transfection reagent. For generating VSVpp, the HEK-293T cells were
transfected with equal amounts of plasmids expressing VSV G and the
HIV packaging genome by using polyethylenimine. 24 hours after the
transfection, the cell culture medium containing the transfection
mixture was replaced with fresh Dulbecco's Modified Eagle Medium
(DMEM-Glutamax.TM.-I; Invitrogen cat #10569-010) supplemented with
10% Fetal Bovine Serum (Invitrogen cat #10082-147) and 2 mM
L-glutamine (Invitrogen cat #25030-081). The supernatant was
collected 48 hours after the transfection and filtered through a
sterile 0.45 .mu.m filter. Aliquots of the supernatant was frozen
and stored at -80.degree. C. until use.
[0370] Huh7-high CD81 cells with high CD81 expression level were
enriched by flow cytometry sorting using FITC-labeled CD81 antibody
JS-81 (BD Biosciences cat #561956) to allow more efficient HCV
entry. The Huh7-high CD81 cells were cultured in Dulbecco's
Modified Eagle Medium (DMEM-Glutamax.TM.-I; Invitrogen cat
#10569-010). The medium was supplemented with 10% Fetal Bovine
Serum (Invitrogen cat #10082-147) and 1% penicillin/streptomycin
(Invitrogen cat #15070-063). Cells were maintained at 37.degree. C.
in a humidified 5% CO.sub.2 atmosphere.
Determination of Compound HCVpp Entry Inhibitory Activity in
Huh7-High CD81 Cells
[0371] Huh7-high CD81 cells were plated at a cell density of 8000
cells per well in 96 well plates (Perkin Elmer, cat #6005660).
Cells were plated in 100 .mu.l of Dulbecco's Modified Eagle Medium
(DMEM-Glutamax.TM.-I, Invitrogen Cat #10569-010) supplemented with
10% Fetal Bovine Serum (Invitrogen Cat #10082-147) and 1%
penicillin/streptomycin (Invitrogen cat #15070-063). Cells were
allowed to equilibrate for 24 hours at 37.degree. C. and 5%
CO.sub.2 at which time compounds and pseudotyped viruses were
added. On the day of the assay, HCVpp aliquots were thawed in
37.degree. C. water bath and kept at 4.degree. C. until use.
Compounds (or medium as a control) were diluted in 3 fold dilution
series in DMEM-Glutamax.TM.-I with 2% DMSO and 2%
penicillin/streptomycin. The 100 plating medium in each culture
well was removed followed by the addition of 50 .mu.l compound
dilutions and 50 .mu.l thawed HCVpp. Firefly luciferase reporter
signal was read 72 hours after the addition of compounds and HCVpp
using the Steady-Glo luciferase Assay System (Promega, cat #E2520)
following the manufacturer's instruction. EC50 values were defined
as the compound concentration at which a 50% reduction in the
levels of firefly luciferase reporter was observed as compared to
control samples in the absence of compound and was determined by
non-linear fitting of compound dose-response data.
Determination of Compound Selectivity in Huh7-High CD81 Cells
[0372] Huh7 hCD81 cell assay plates and compound dilutions were set
up in the same format as in the HCVpp assay. 24 hours after cell
plating, thawed VSVpp was diluted by 800 fold in
DMEM-Glutamax.TM.-I supplemented with 10% fetal bovine serum. After
removal of the cell plating medium from the culture wells, 50 .mu.l
compound dilutions and 50 .mu.l diluted VSVpp were added to the
wells. Firefly luciferase reporter signal was read 72 hours after
the addition of compounds and VSVpp using the Steady-Glo luciferase
Assay System (Promega, cat #E2520). EC50 values were defined as the
compound concentration at which a 50% reduction in the levels of
firefly luciferase reporter was observed as compared to control
samples in the absence of compound and was determined by non-linear
fitting of compound dose-response data. The EC50 was approximated
if maximum percentage inhibition was less than 90% and more than
70%.
[0373] Representative assay data can be found in Table II
below:
TABLE-US-00002 TABLE II HCVpp GT-1a HCVpp GT-1b VSVpp Compound #
(EC.sub.50, .mu.M) (EC.sub.50, .mu.M) (EC.sub.50, .mu.M) 1 0.31
13.2 2 2.439 0.393 86.0 3 2.774 0.329 35.5 4 0.076 3.3 5 0.293 25.3
6 0.139 34.0 7 0.009 100.0 8 0.114 0.079 10.0 9 0.15 3.0 10 0.263
7.7 11 0.61 10.0 12 0.077 3.4 13 0.674 10.0 14 0.366 10.0 15 0.133
49.8 16 1.018 40.2 17 1.036 34.9 18 0.126 0.1 19 0.019 0.008 20.2
20 0.232 10.0 21 1.673 39.2 22 1.645 0.229 14.3 23 4.623 0.517 40.1
24 42.129 100.0 25 1.899 100.0 26 1.878 43.5
[0374] The foregoing invention has been described in some detail by
way of illustration and example, for purposes of clarity and
understanding. It will be obvious to one of skill in the art that
changes and modifications may be practiced within the scope of the
appended claims.
[0375] Therefore, it is to be understood that the above description
is intended to be illustrative and not restrictive. The scope of
the invention should, therefore, be determined not with reference
to the above description, but should instead be determined with
reference to the following appended claims, along with the full
scope of equivalents to which such claims are entitled.
[0376] All patents, patent applications and publications cited in
this application are hereby incorporated by reference in their
entirety for all purposes to the same extent as if each individual
patent, patent application or publication were so individually
denoted.
* * * * *