2-Amino-6-arylsulfonylbenzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1

Article abstract of DOI:10.1021/jm0004906

A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC₅₀ values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.

Full text of DOI:10.1021/jm0004906

1866
J . Med. Chem. 2001, 44, 1866-1882
2-Am in o-6-a r ylsu lfon ylben zon itr iles a s Non -n u cleosid e Rever se Tr a n scr ip ta se
In h ibitor s of HIV-1
J oseph H. Chan,* J ean S. Hong,‡ Robert N. Hunter III, G. Faye Orr, J ill R. Cowan, Douglas B. Sherman,§
Steven M. Sparks,| Barbara E. Reitter, C. Webster Andrews III, Richard J . Hazen, Marty St Clair,
Lawrence R. Boone, Rob G. Ferris, Katrina L. Creech, Grace B. Roberts, Steven A. Short, Kurt Weaver,
Ronda J . Ott, J ingshan Ren,3 Andrew Hopkins,3 David I. Stuart,3,∞ and David K. Stammers3,∞
Glaxo Wellcome, Inc., 5 Moore Drive, Research Triangle Park, North Carolina 27709, Structural Biology Division,
The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, U.K., and
Oxford Centre for Molecular Sciences, New Chemistry Building, University of Oxford, South Parks Road,
Oxford, OX1 3QT, U.K.
Received November 17, 2000
A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural
modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral
activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series
of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-
activity relationship (SAR) studies suggested that a meta substituent, particularly a meta
methyl substituent, invariably increased antiviral activities. However, optimal antiviral
activities were manifested by compounds where both meta groups in the arylsulfonyl moiety
were substituted and one of the substituents was a methyl group. Such a disubstitution led to
compounds 3v, 3w , 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range.
When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse
transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u -z
and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against
the V106A and P236L strains and submicromolar to low nanomolar activity against strains
E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and
Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v
(739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when
compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure
allowed for the rationalization of SAR data and potencies of the compounds against the mutants.
In tr od u ction
not utilize the biochemical machinery of the host cells,
NNRTIs usually manifest different toxicity profiles. Side
effects are usually milder than those resulting from
treatments with nucleosides.4 Generally, the most se-
vere and treatment-limiting NNRTI-related adverse
event seen in the clinic is rash.5
The first successful combination therapy for the
treatment of HIV-1 infections with the non-nucleoside
reverse transcriptase inhibitor (NNRTI) nevirapine1 as
a component in the treatment regimen has revived
interests in the search for novel and potent NNRTIs.
Unlike the nucleosides that act at the catalytic site of
HIV reverse transcriptase (RT) by terminating DNA
synthesis,2 NNRTIs bind in a region of the enzyme,
which is approximately 10 Å away from the catalytic
site. Their binding appears to result in a distortion of
the catalytic site because of changes in the position of
the key aspartic acid residues, which affects the ability
of the enzyme to carry out its catalytic functions.3
NNRTIs do not require anabolism for activation. Since
they are not analogues of natural compounds and do
Unfortunately, because of the rapid emergence of
resistant strains when used in monotherapy,6 NNRTIs
were initially considered to be of little therapeutic value.
Their potential for the treatment of HIV infections was
more broadly realized when nevirapine in combination
with ddI and AZT was found to lead to a sustained viral
load reduction.1 Thus, the approval by the FDA of
nevirapine for combination therapy was rapidly followed
by that of delavirdine7 and efavirenz.8
Combination therapy avoids or delays emergence of
resistant viral strains, particularly when such pressure
comes from potent drugs with different mechanisms of
inhibition. Such multiple-drug treatment approach has
indeed contributed to the declining morbidity and
mortality among HIV-infected patients.9,10 Yet, because
of a high pill burden, coupled with the high cost of
treatment and toxicity profile, much still remains to be
done to advance HIV chemotherapy. These limitations
could potentially be overcome by a treatment regimen
containing a potent NNRTI with a favorable side effect
profile.
* To whom correspondence should be addressed. Phone: 919-483-
2113. Fax: 919-483-6053. E-mail: jc25572@glaxowellcome.com.
3 The Wellcome Trust Centre for Human Genetics, University of
Oxford.
∞ Oxford Centre for Molecular Sciences, New Chemistry Building,
University of Oxford.
‡ Current address: Scynexis Chemistry & Automation, Inc., Re-
search Triangle Park, North Carolina 27709.
§ Current address: BD Technologies, 21 Davis Drive, Research
Triangle Park, North Carolina 27709.
| Current address: Department of Chemistry, University of Cali-
fornia, Irvine, California 92697.
Deceased September 1998.
10.1021/jm0004906 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/08/2001

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1867
Ta ble 1. Physical Constants for 6-Arylthio-2-nitrobenzonitriles
(8a -j)a
NNRTIs are generally noncompetitive potent inhibi-
tors of HIV-1 RT with drug substance of very low cost
and an acceptable toxicity profile. Indeed, an array of
structurally diverse compounds including MKC442
(emivirine),11,12 AG1549,13 PNU-142721,14 DPC961,15
and DPC96315 are currently in various stages of clinical
development.
Our own efforts in this area began with the random
screening of compounds from the Glaxo Wellcome
database and have resulted in the initial identification
of 6-arylthio-2-aminobenzonitriles (1) with micromolar
activity against HIV-1. Further modification of 1 yielded
nanomolar inhibitors in the 6-arylsulfinyl-2-aminoben-
zonitriles (2) and 6-arylsulfonyl-2-aminobenzonitriles
(3) classes of NNRTIs. While most analogues of 2
showed comparable antiviral activity to those of 1,
analogues in the 6-arylsulfonyl series 3 were generally
more potent than that in series 1 or 2.
Precedence exists in the literature on the antiviral
activity against HIV-1 of sulfones.16-21 For example, the
structure-activity relationship (SAR) of sulfones 416,17
and 518 had been previously pursued systematically.
Among these sulfones, NPPS (4a ) was possibly the first
to have received extensive evaluation as a potential
antiviral agent against HIV-1. One of the findings from
the SAR studies of 4 was that two ortho nitro groups
as in sulfone 4b, one on each phenyl ring, resulted in
an analogue with the most optimal antiviral activity.
However, the mononitrated 4a was shown to have better
in vivo bioavailability and was thus chosen as the
compound for further evaluation.16,17
%
empirical
formula
elemental
analysis
compd
R
yield mp (°C)
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
89
106-107
C
13H8N2O2S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
2-OCH3
3-OCH3
4-OCH3
3-CH3
3-F
3-CF3
3-NH2
3,5-Cl 2
70b 145-147 C14H10N2O3S
51c 144-147 C14H10N2O3S
93c 139-140 C14H10N2O3S
80c 129-130 C14H10N2O2S
86d 128-129 C13H7N2O2FS
76d 102-104 C14H7N2O2F3S C,H,N,S
88d 178-179 C13H9N3O2S
84d 157-158 C13H6N2O2Cl2S C,H,N,Cl,S
3,5-(CH3)2 80d 155-156 C15H12N2O2S
C,H,N,S
C,H,N,S
8j
a
Compounds were prepared according to method A described
in the Experimental Section. See also ref 23 for compounds 8a -
d . Solvent used for chromatography: EtOAc/hexane (2:3). c Sol-
b
d
vent used for chromatography: CH2Cl2. Solvent used for chro-
matography: EtOAc/hexane (1:1).
1o-q, 1t, and 1w -y, were obtained from the reaction
of the corresponding 2-arylthio-6-fluorobenzonitriles 10
in Table 3 with concentrated ammonium hydroxide or
ethanolic ammonia at 130-140 °C in a glass-lined Parr
bomb (Scheme 2).23,24 The details of this chemistry had
been previously described in connection with the syn-
thesis of a series of 2,4-diaminoquinazolines, and com-
pounds 1b-d , 1g-j, 1m , and 1r were reported as
chemical intermediates.23
Intermediates 8a -j were obtained from the reaction
at 0 °C of 2,6-dinitrobenzonitrile 6 with an appropriately
substituted arylthiol (7) and t-BuOK in DMSO or K2-
CO3 in DMF as the base (Scheme 1). Intermediates
10a -o, 10s-v, and 10z-bb were obtained by displac-
ing a fluoro group in 2,6-difluorobenzonitrile (9) by an
appropriately substituted arylthiol 7 under conditions
similar to those described above for the synthesis of the
nitro analogues 8. Compounds 10p -r were obtained
from the displacement of the bromo group in 10k -m ,
respectively, by copper cyanide in DMF.25 Details are
described in the Experimental Section.
The synthesis of compounds 10w -y is depicted in
Scheme 3 and started with the bromination of anilines
13a -c to their respective bromo analogues 14a -c.
Deamination through diazonium salts formation re-
sulted in intermediates 15a -c.26 Subsequently, a one-
pot reaction of these intermediates utilizing a bromolith-
ium exchange with sec-butyllithium and then reacting
the resultant aryllithium with sulfur resulted in their
respective thiolates that were then reacted in situ with
9 to give 10w -y. Table 8 summarizes the physical
constants of the intermediates 14a -c and 15a -c.
Oxidation of a select group of 6-arylthio-2-fluoroben-
zonitriles (10b-g, 10k -m , 10p -t, 10v, 10x, and 10z)
from Table 3 with 1 equiv of m-chloroperoxybenzoic acid
or OXONE27 led to 2-arylsulfinyl-6-fluorobenzonitriles
(11a -q ) (Scheme 2, Table 4). Oxidation of 10a -b b
using 2 equiv of the same oxidants gave 2-arylsulfonyl-
6-fluorobenzonitrile 12a -z and 12ee-ff (Scheme 2,
Table 6). Compound 12a a was obtained from the de-
methylation of 12y using boron tribromide in methylene
In this paper, we disclose our synthetic efforts and
structure-activity relationship (SAR) studies that led
to the identification of a group of analogues with the
6-arylsulfonyl-2-aminobenzonitrile (3) scaffold having
potent antiviral activity against HIV-1 and good thera-
peutic indices. Additionally, we have determined the
crystal structure of compound 3v (739W94) in complex
with HIV-1 RT, allowing some rationalization of both
structure-activity data as well as the effects of resis-
tance mutations on the binding potencies of the inhibi-
tor.
Ch em istr y
2-Amino-6-arylthiobenzonitriles 1a -d , 1f, 1n , 1r -s,
1u -v (Table 2) were obtained from the stannous
chloride reduction of the corresponding 2-arylthio-6-
nitrobenzonitriles 8a -j (Table 1),22,23 as depicted in
Scheme 1. The other analogues in Table 2, 1e, 1g-m ,

1868 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
Ta ble 2. Physical Constants for 2-Amino-6-arylthiobenzonitriles (1a -y)a
compd
R
% yield
mp (°C)
empirical formula
elemental analysis
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
94b
70c
51b
55d
42e
57f
21b
83b
58b
77b
62f
81f
88b
65f
50b
24b
44b
67f
49f
42f
76f
64f
64g
30b
79g
73-74
145-147
98-100
95-98
C13H10N2S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
3-F
2-CN
3-CN
4-CN
3-CF3
3-NH2
2,5-Cl2
3,5-Cl2
3,5-(CH3)2
3-Cl, 5-CH3
3-OCH3, 5-CH3
3-OCH3, 5-CF3
C14H12N2OS
C14H12N2OS
C14H12N2OS‚0.2H2O
C14H12N2S
C14H12N2S
C14H12N2S
C13H9N2ClS
C13H9N2ClS
C13H9N2ClS
C13H9N2BrS
C13H9N2BrS
C13H9N2BrS
C13H9N2FS
83-85
114-115
114-115
113-115
88-90
118-120
93-95
84-86
119-120
63-65
112-114
110-111
169-171
66-68
C14H9N3S
C14H9N3S
C14H9N3S‚0.2CH3OH
C14H9N2F3S
C13H11N3S‚0.1H2O
C13H8N2Cl2S
C13H8N2Cl2S
C15H14N2S
C14H11N2ClS
C15H14N2OS
C15H11N2OF3S
1s
1t
99-100
100-103
132-134
124-125
125-127
108-110
85-86
C,H,N,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,S
C,H,N,Cl,S
C,H,N,S
1u
1v
1w
1x
1y
C,H,N,S
a
Compounds 1a -d , 1f, 1n , 1r -s, and 1u ,v were prepared according to method B and compounds 1e, 1g-m , 1o-q, 1t, and 1w -y
b
according to method
G
described in the Experimental Section. Solvent used for chromatography: CH2Cl2. c Solvent used for
chromatography: EtOAc/hexane (2:3). Recrystallized from MeOH/H2O. e Recrystallized from EtOAc/hexane. f Solvent used for chroma-
d
g
tography: EtOAc/hexane (1:1). Solvent used for chromatography: CH2Cl2/hexane/EtOAc (4:5.5:0.5).
Sch em e 1a
resulting in 2-nitrophenyl phenylsulfide (17), as de-
picted in Scheme 5. Oxidation of the arylsulfide with
potassium permanganate gave NPPS. The synthesis of
the dinitro analogue 4b was accomplished using the
same procedure, starting with 2-nitrophenylthiolate.
Selective oxidation of the sulfide 18 (Scheme 5), how-
ever, was accomplished using OXONE27 to give sulfoxide
(19), which was further oxidized with hydrogen peroxide
to give 4b.
As depicted in Scheme 6, the n-butylamino and
cyclohexylamino analogues 20a and 20b were obtained
from the displacement of 12v by n-butylamine and
cyclohexylamine, respectively, using potassium carbon-
ate in DMF as the base. The 3-amino analogue 21 was
purchased from Sigma.
a
Conditions: (a) K2CO3, DMF; (b) SnCl2‚2H2O, HCl, diglyme.
Biologica l Resu lts, X-r a y Cr ysta llogr a p h y, a n d
Discu ssion
chloride, and 12bb-d d were obtained from the reaction
of the sodium salt of 12a a with alkyl bromide having
the appropriate chain lengths (Scheme 4). Amination
of 11a -q and 12a -ff using either concentrated am-
monium hydroxide or ethanolic ammonia in a Parr
bomb at 130-140 °C resulted in 2-amino-6-arylsulfi-
nylbenzonitriles 2a -q (Table 5) and 2-amino-6-arylsul-
fonylbenzonitriles 3a -z and 3bb-ff (Table 7), respec-
tively.Compound 3aa was obtained from thedemethylation
of 3y, a procedure similar to that used for the synthesis
of 12a a .
Compounds listed in Tables 2, 5, and 7 as well as
compounds 12u -v, 12y, 20a -b, and 21 were evaluated
in an assay to measure their antiviral activity in HIV-
1-infected MT4 cells. The assay assesses the reduction
of the cytopathic effect of HIV-1 on MT4 cells in the
presence of compounds, and the results are reported as
IC50 values, which are the concentrations of compounds
that would produce a 50% decrease in the cytopathic
effect. As a gauge of the therapeutic index of the
compounds assayed, compound-induced cytotoxicity of
MT4 cells was also measured in parallel with the
antiviral activity, and the results are reported as the
cell culture cytotoxicity IC50 (CCIC50) values. Addition-
The synthesis of the diarylsulfones 4a (NPPS) and
4b made use of a procedure slightly modified from that
reported.16 NPPS was synthesized by reacting 1-bromo-
2-nitrobenzene (16) with the anion of thiophenol (7a ),

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1869
Ta ble 3. Physical Constants for 2-Arylthio-6-fluorobenzonitriles (10a -bb)a
compd
R
% yield
mp (°C)
empirical formula
elemental analysis
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
35b
40c
13c
71e
47f
72c
70f
91g
51g
85g
57g
51h
84i
44e
47f
85g
58j
25g
72g
76b
34b
68k
35l
26k
46k
77m
104-106
d
C13H8NFS
C,H,N,S
d
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-Br
3-Br
C14H10NOFS
C14H10NOFS
C14H10NOFS
C14H10NFS
C14H10NFS
C14H10NFS
C13H7NClFS
C13H7NClFS
C13H7NClFS
C13H7NBrFS
C13H7NBrFS
C13H7NBrFS
C13H7NF2S‚0.2H2O
C13H7NF2S
132-134
83-85
58-60
96-98
93-95
85-87
80-81
98-100
88-90
104-107
80-82
83-85
95-97
118-120
110-112
92-94
d
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
d
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,S
C,H,N,Br,S
C,H,N,Cl,S
C,H,N,S
C,H,N,S
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
10w
10x
10y
10z
4-Br
2-F
3-F
2-CN
3-CN
4-CN
3-CF3
2,5-Cl2
3,5-Cl2
3,5-(CH3)2
3-Br, 5-CH3
3-Cl, 5-CH3
C14H7N2FS
C14H7N2FS‚0.2H2O
C14H7N2FS‚0.1H2O
C14H9N2F3S
C13H6NCl2FS
C13H6NCl2FS
C15H12NSF
C14H9NBrFS
C14H9NClFS
C15H12NOFS
C15H9NOF4S
d
74-77
98-99
87-90
d
94-95
78-80
3-OCH3, 5-CH3
3-OCH3, 5-CF3
10a a
10bb
79e
124-126
C17H10NSF
C17H10NSF
C,H,N,S
C,H,N,S
44e
a
Compounds 10a -o, 10s-v, and 10z-bb were prepared according to method C, 10p -r according to method D, and 10w -y according
to method J described in the Experimental Section. b Solvents used for chromatography: CH2Cl2/hexane (1:1). c Recrystallized from acetone/
water. Data not obtained. e Solvents used for chromatography: CH2Cl2/hexane (3:7). f Recrystallized from EtOAc/hexane. g Solvents used
d
h
i
for chromatography: CH2Cl2/hexane (7:3). Solvents used for chromatography: CH2Cl2/hexane (1:4). Solvents used for chromatography:
CH2Cl2/hexane (2:3). Solvents used for chromatography: CH2Cl2/hexane (1:1). Solvents used for chromatography: EtOAc/hexane (1:
4). Solvents used for chromatography: EtOAc/hexane (1:9). Solvents used for chromatography: EtOAc/hexane (1.5:8.5).
j
k
l
m
ally, these compounds were evaluated for their inhibi-
tory activity against HIV-1 RT. The results from the
cell-based and RT assays are summarized in Tables
9-12. As mentioned earlier, certain diarylsulfones have
previously been reported to have potent activity against
HIV-1. We have synthesized two such analogues, NPPS
(4a ) and its dinitro derivative 4b. These two analogues
were reported to be the most potent analogues in the
diarylsulfone series.18 Compounds 4a and 4b , the
precursors bisarylsulfide 18, and the bisarylsulfoxide 19
have been included in Tables 9-11 for comparison
purposes.
As shown in Table 9 for the sulfide series, the
moderate activity against HIV-1 of the parent compound
1a was in general enhanced by the presence of a
substituent in the C-3 position (compounds 1c, 1f, 1i,
1l, 1m , and 1o) of the arylthio ring. The 3-trifluoro-
methyl-substituted 1q, however, was almost equipotent
to 1a , and the 3-amino analogue 1r was 2-fold less active
against the virus than 1a . The reduced activity against
HIV-1 for 1r might suggest the detrimental effect of
having a polar substituent in the C-3 position of the
arylthio group.
The antiviral activity of the C-2 substituted analogues
appeared to vary considerably with the substituents.
The 2-methoxy analogue 1b and the 2-chloro analogue
1h were the most active in the series, and the 2-methyl
analogue 1e retained activity comparable to that of 1a .
On the other hand, the 2-bromo analogue 1k was much
less active against HIV-1 than that of 1a . Such a
disparity of activity could not simply be due to the
electronic properties of the substituents alone. The fact
that the 2-bromo analogue 1k was approximately 7-fold
less active than the 2-chloro 1h might suggest that the
size of the 2-substituent is important for maximizing
antiviral activity.
A select group of the more potent 6-arylsulfonyl-2-
aminobenzonitriles 3 were additionally evaluated against
a panel of mutant viruses genetically engineered to
contain RT mutations considered to be relevant to
NNRTIs. The results are included in Table 13. The
details of the procedures for all assays mentioned above
are described in the Experimental Section.
The initial SAR studies centered on substituent
variations on the arylthio moiety in 1. Subsequently,
the SAR studies were expanded to include sulfoxides 2,
sulfones 3, and modifications of the benzonitrile ring
system.

1870 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
Sch em e 2a
antiviral activity of these C-4 substituted analogues
compared to that of analogues having C-2 and C-3
substituents might suggest unfavorable interactions in
the region of HIV-1 RT into which the substituents
projected.
Disubstution on the arylthio group, each having a
meta methyl substituent, resulted in a marked increase
in antiviral activity. Thus, compounds 1t, 1v, and 1w
were about 14-, 4-, and 3-fold more active against HIV-1
than the corresponding monosubstituted 1f, 1i, and 1c.
On the other hand, if the additional meta substituent
were electron-withdrawing, such as the chloro and
trifluoromethyl groups in 1u and 1x, respectively, the
resultant compounds at best retained equipotency to the
corresponding monosubstituted analogues.
A number of the sulfides showed relatively low
cytotoxic IC50 values, resulting in marginal therapeutic
indices. These values were in direct contrast to those
found for the sulfoxide and sulfone series, which gener-
ally showed therapeutically acceptable indices (vide
infra). That the antiviral activity was due to the
inhibition of RT was well established by the correlation
of activity against HIV-1 RT of the sulfide analogues.
Compared to the bisnitrophenylsulfide 18, the 6-arylthio-
2-aminobenzonitriles were generally more potent against
HIV-1.
Utilizing the sulfides in Table 9 as the starting point
in an ensuing SAR study, we next synthesized a select
group of the sulfoxide analogues and an expanded group
of the sulfone analogues. The antiviral activities of these
compounds are summarized in Tables 10 and 11,
respectively.
Interestingly, in general, for a comparably substituted
compound, the antiviral activity of the sulfide and
sulfoxide analogues was quite similar. However, the
SAR for antiviral activity of the sulfoxides was not as
clear-cut as in the sulfide series. In the methoxy-
substituted analogues, the trend of activity was C-2
analogue 2a > C-3 analogue 2b > C-4 analogue 2c. This
a
Conditions: (a) t-BuOK, DMSO or K2CO3, DMF; (b) 1.2 equiv
of mCPBA, CH2Cl2, or 1.1 equiv of OXONE27 H2O; (c) concentrated
NH4OH or NH3, MeOH; (d) 2.2 equiv of mCPBA, CH2Cl2, or 2.2
equiv of OXONE, MeOH.
The least optimal position to place a substituent was
C-4 (compounds 1d , 1g, 1j, and 1p ). The much reduced
Sch em e 3a
a
Conditions: (a) Br2, AcOH, MeOH; (b) NaNO2, concentrated HCl, AcOH, H2O, H3PO2; (c) sec-BuLi, S8; (d) 2,6-difluorobenzonitrile,
THF, DMSO.

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1871
Ta ble 4. Physical Constants for 2-Arylsulfinyl-6-fluorobenzonitriles (11a -q)a
compd
R
% yield
mp (°C)
empirical formula
elemental analysis
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Br
3-Br
4-Br
2-CN
3-CN
4-CN
3-CF3
2,5-Cl2
3,5-(CH3)2
3-Cl, 5-CH3
3-OCH3, 5-CF3
40b
13b
50c
58d
72e
67e
83e
51e
67d
50f
89d
40d
60d
83g
68i
62j
77k
138-140
110-111
73-75
C14H10NO2FS
C14H10NO2FS
C14H10NO2FS
C14H10NOFS
C14H10NOFS
C14H10NOFS
C13H7NOBrFS
C13H7NOBrFS
C13H7NOBrFS
C14H7N2OFS‚0.3H2O
C14H7N2OFS‚0.2H2O
C14H7N2OFS
C14H7NOF4S
C13H6NO2ClFS
C15H12NOFS
C,H,N
C,H,N,S
C,H,N,S
C,H,N,S
h
C,H,N,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,Br,
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
h
87-88
96-98
200-202
105-107
104-107
121-124
152-160
153-155
160-162
85-87
h
98-99
131-133
127-128
C,H,N,S
C,H,N
C,H,N
C14H9NOClFS
C15H9NO2FS
a
b
All compounds were prepared according to method E described in the Experimental Section. Recrystallized from acetone/water.
c Solvent used for chromatography: CH2Cl2/MeOH (99:1). Solvent used for chromatography: CH2Cl2. e Solvent used for chromatography:
d
CH2Cl2/hexane/EtOAc (4:5:1). f Isolated analytically pure from the reaction medium. Yield is based on crude material isolated. hData
g
not obtained. i Solvents used for chromatography: EtOAc/hexane (1.:4). j Solvents used for chromatography: EtOAc/hexane (1:9). k Solvents
used for chromatography: CH2Cl2/hexane (1:9).
Ta ble 5. Physical Constants for 2-Amino-6-arylsulfinylbenzonitriles (2a -q)a
compd
R
% yield
mp (°C)
empirical formula
C14H12N2O2S
elemental analysis
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2-OCH3
3-OCH3
4-OCH3
2-CH3
40b
13b
67c
32d
72b
52c
55d
51e
42d
66d
78d
44c
18c
68f
59c
26g
77g
159-162
110-114
155-157
153-155
96-98
200-202
140-142
104-107
155-157
200-202
148-149
169-171
148-150
98-99
C,H,N
C14H12N2O2S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S,Cl
C,H,N,Cl,S
C,H,N,S
C14H12N2O2S‚0.1CH3OH
C14H12N2OS‚0.2EtOAc
C14H12N2OS
C14H12N2OS‚0.1CH3OH
C13H9N2OBrS
C13H9N2OBrS
C13H9N2OBrS
C14H9N3OS‚0.1H2O
C14H9N3OS‚0.8H2O
C14H9N3OS
3-CH3
4-CH3
2-Br
3-Br
4-Br
2-CN
3-CN
4-CN
3-CF3
C14H9N2OF3S‚0.3H2O
C15H14N2OS
C13H8N2OCl2S
C14H11N2OClS
3,5-(CH3)2
2,5-Cl2
3-Cl, 5-CH3
3-OCH3, 5-CF3
180-182
192-193
78-80
C15H11N2O2F3S
a
b
All compounds were prepared according to method G described in the Experimental Section. Recrystallized from acetone/water.
c Solvents used for chromatography: CH2Cl2/MeOH (99:1). Recrystallized from EtOH/H2O. e Solvents used for chromatography: CH2Cl2/
d
hexane (1:4). f Solvents used for chromatography: EtOAc/hexane (1:4). Solvents used for chromatography: EtOAc/hexane (1:9).
g
trend was similar for the cyano analogues (2j-l).
However, this trend was not observed for the methyl
(2d -f) or bromo (2g-i) analogues. As was observed
previously in the sulfide series, the addition of another
meta substituent also resulted in an increase in anti-
viral activity (compounds 2n , 2o, 2p , and 2q). Again,
the increase in antiviral activity was more pronounced
when a methyl group was the additional meta substitu-
ent (2n and 2p ). The sulfoxides in Table 10 were
synthesized as the racemates, and no attempt was made
to resolve them. Nonetheless, judging from the range
of antiviral activity, resolution of the racemates might
not result in a significant increase in antiviral activity
of the resultant optical isomers. The sulfoxides, in
contrast to the sulfides, showed relatively high cell
cytotoxic IC50 values, resulting in acceptable therapeutic
indices. Additionally, the correlation between antiviral
activity and RT activity was much more pronounced
than that in the sulfide series (Table 10). The bisnitro-
phenylsulfoxide 19 did not show any appreciable activity
against HIV-1 at concentrations up to 200 µM.
Previous work on the bisarylsulfones demonstrated
that for a similar substitution, the bisarylsulfones were
generally more potent than the corresponding bisaryl-

1872 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
Ta ble 6. Physical Constants for 6-Arylsulfonyl-2-fluorobenzonitriles (12a -ff)a
compd
R
% yield
mp (°C)
empirical formula
C13H8NO2FS
C14H10NO3FS
C14H10NO3FS
C14H10NO3FS
C14H10NO2FS
C14H10NO2FS
C14H10NO2FS
C13H7NO2ClFS
C13H7NO2ClFS
C13H7NO2ClFS
C13H7NO2BrFS
C13H7NO2BrFS
C13H7NO2BrFS
C13H7NO2F2S
elemental analysis
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
H
88b
4c
97-98
174-177
113-115
210-212
118-123
119-121
200-202
135-137
123-125
133-135
113-115
162-166
130-132
134-135
122-124
208-210
e
192-195
184-185
183-184
84-86
167-168
e
164-166
160-162
111-113
178-180
e
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
e
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-F
3-F
2-CN
3-CN
4-CN
3-CF3
2,5-Cl2
3,5-Cl2
23c
86b
50d
28d
83d
89f
44f
76f
37f
82d
73f
78b
64b
59c
63c
76c
94g
16h
75g
65i
78f
85f
83b
78d
50i
92j
49j
55j
C,H,N,S
C,H,N,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,S
C,H,N,Br,S
C,H,N,Cl,S
C,H,N,S
C,H,N,S
e
C13H7NO2F2S
C14H7N2O2FS‚0.2H2O
C14H7N2O2FS‚0.4H2O
C14H7N2O2FS‚0.4H2O
C14H7NO2F4S
C13H6NO2Cl2FS‚0.05CH2Cl2
C13H6NO2Cl2FS
C15H12NO2FS
C14H9NO2BrFS
C14H9NO2SClF
C15H12NO3FS
12s
12t
12u
12v
12w
12x
12y
12z
12a a
12bb
12cc
12d d
3,5-(CH3)2
3-Br, 5-CH3
3-Cl, 5-CH3
3-OCH3, 5-CH3
3-OCH3, 5-CF3
3-OH, 5-CH3
3-OCH2CH3, 5-CH3
3-O(CH2)2CH3, 5-CH3
3-O(CH2)3CH3, 5-CH3
C15H9NO3F4S
C14H10NO3FS
C16H14NO3FS
C17H16NO3FS
e
e
e
e
e
C18H18NO3FS
12ee
12ff
95c
155-158
110-112
C17H10NO2FS
C17H10NO2FS
e
e
90c
a
Compounds 12a -z and 12e-ff were prepared according to method F, 12a a according to method K, and 12bb-d d according to method
L described in the Experimental Section. Isolated analytically pure from the reaction medium. c Solvent used for chromatography:
b
CH2Cl2. Solvent used for chromatography: CH2Cl2/hexane/EtOAc (4:5:1). e Data not taken. f Solvent used for chromatography: CH2Cl2/
d
hexane (7:3). g Solvent used for chromatography: CH2Cl2/hexane (1:1). Solvent used for chromatography: CH2Cl2/hexane (3:7). Solvent
h
i
j
used for chromatography: EtOAc/hexane (1:1). Purified by silica preparative plate with CH2Cl2 as the eluent.
Sch em e 4a
a
Conditions: (a) BBr3, CH2Cl2; (b) NaH, RBr, DMF.
sulfide analogues.16 For example NPPS (4a ) was found
to be more active than nitrophenyl phenylsulfide 18
(antiviral IC50 values against HIV-1 of 2.2 and 93.5 µM,
respectively, in our assays). In addition, the NNRTI
pocket in the HIV-1 RT is very hydrophobic.3,28,29
Utilizing these two pieces of information in a continuing
effort to expand the SAR studies, we selected the
dimethyl analogue 1t and synthesized its sulfone coun-
terpart. The resultant compound 3v (739W94) was
approximately 43-fold more active against HIV-1 than
1t. Encouraged by such a potent level of antiviral
activity, we undertook an extensive SAR work around
the sulfone scaffold, and the antiviral activity of the
resultant compounds is listed in Table 11. In general,
the sulfones 3 showed much enhanced antiviral activity
versus both the sulfides 1 and the sulfoxides 2. The
pattern of activity generally mirrored that of the
sulfides. Potent antiviral activity required at least a

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1873
Ta ble 7. Physical Constants for 2-Amino-6-arylsulfonylbenzonitriles (3a -ee)a
compd
R
% yield
mp (°C)
empirical formula
elemental analysis
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
67b
35c
23c
61c
85b
80b
75b
80b
91b
68b
62b
19b
100d
85b
92b
73e
78e
77e
80c
58b
86b
56e
59c
76c
56h
92c
50f
60g
68g
56g
185-187
191-193
186-191
220-222
213-215
191-193
200-202
135-137
185-187
212-215
235-238
209-210
226-228
224-226
162.5-164
267-268
211-213
224-226
184-185
C13H10N2O2S‚0.2H2O
C14H12N2O3S
C14H12N2O3S
C14H12N2O3S
C14H12N2O2S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,Br,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-F
3-F
2-CN
3-CN
4-CN
3-CF3
2,5-Cl2
3,5-Cl2
3,5-(CH3)2
3-Br, 5-CH3
3-Cl, 5-CH3
3-OCH3, 5-CH3
3-OCH3, 5-CF3
3-OH, 5-CH3
3-OCH2CH3, 5-CH3
3-O(CH2)2CH3, 5-CH3
3-O(CH2)3CH3, 5-CH3
C14H12N2O2S
C14H12N2O2S‚0.2H2O
C13H9N2O2ClS
C13H9N2O2ClS
C13H9N2O2ClS
C13H9N2O2BrS
C13H9N2O2BrS
C13H9N2O2BrS
C13H9N2O2FS
C13H9N2O2FS
C14H9N3O2S‚0.1H2O
C14H9N3O2S‚0.2H2O
C14H9N3O2S
C14H9N2O2F3S
C13H8N2O2Cl2S
C13H8N2O2Cl2S
C15H14N2O2S
C14H11N2O2BrS
C14H11N2O2ClS
C15H14N2O3S
C15H11N2O3F3S
C14H12N2O3S
3s
3t
C,H,N,S
>200
C,H,N,Cl,S
C,H,N,Cl,S
C,H,N,S
C,H,N,Br,S
C,H,N,Cl,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
3u
3v
3w
3x
3y
3z
3a a
3bb
3cc
3d d
227-228
208-209
201-203
203-205
165-166
144-146
178-180
112-114
120-122
140-142
C16H16N2O3S‚0.2H2O
C17H18N2O3S
C18H20N2O3S‚0.4H2O
C,H,N,S
3ee
3ff
72c
228-232
C17H12N2O2S
C,H,N,S
C,H,N,S
75c
205-206
C17H12N2O2S
a
All compounds were prepared according to method G except for compound 3a a , which was synthesized according to method K. Details
are described in the Experimental Section. Solvent used for chromatography: CH2Cl2. c Solvent used for chromatography: CH2Cl2/
b
MeOH (49:1). Isolated analytically pure from the reaction medium. e Recrystallized from EtOH/H2O. f Solvents used for chromatography:
d
g
hexane/EtOAc (1:1). Purified on a silica gel preparative plate with CH2Cl2 as the eluent.
Ta ble 8. Physical Constants for Anilines (14a -c) and Arylbromide (15a -c)a
compd
R1
R2
R3
% yield
mp (°C)
empirical formula
elemental analysis
14a
14b
14c
Br
Cl
OCH3
4-CH3
4-CH3
6-CH3
6-Br
6-Br
4- Br
73b
74b
80d
71-73
c
55-56
C7H7NBr
C7H7NBrCl
C8H10NOBr
C,H,N,Br
C,H,N,Br,Cl
C,H,N,Br
15a
15b
15c
Br
Cl
CH3
CH3
CH3
OCH3
68e
70e
80f
37-40
oil
oil
C7H6Br2
C7H6 BrCl
C8H9OBr
C,H,Br
c
C,H,Br
a
Compounds 14a -c and 15a -c were prepared according to methods H and I, respectively. Details are described in the Experimental
Section. b Recrystallized from hexane. c Data not obtained. Solvents used for chromatography: CH2Cl2/hexane (19:1). e Isolated analytically
d
pure from the reaction medium. f Solvent used for chromatography: hexane.
lipophilic C-3 substituent in the phenylsulfonyl moiety
(3c, 3f, 3i, and 3l). Analogues with C-2 substituents (3e,
3h , 3k , and 3p ), although still showing reasonable
antiviral activity, were in general not as active against
HIV-1 as the C-3 substituted compounds. Two excep-
tions to this observation were the 2-methoxy analogue
3b and the 2-fluoro analogues 3n , both of which were
of equal potency to their 3-substituted counterparts. The
similarity in antiviral activity of the 2- and 3-methoxy
analogues 3b and 3c could possibly be explained by the

1874 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
Sch em e 5a
a
Conditions: (a) NaH, DMF; (b) OXONE,27 MeOH, H2O; (c) KMnO4, H2O, AcOH; (d) H2O2, trifluoroacetic acid.
Sch em e 6a
Ta ble 10. Activity against HIV-1 and RT of
2-Amino-6-arylsulfinylbenzonitriles (2a -q)a
anti-HIV-1
CCIC50
HIV-1 RT
compd
R
IC50 (µM)
(µM)
IC50 (µM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
19
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Br
3-Br
4-Br
2-CN
3-CN
4-CN
3-CF3
3,5-(CH3)2
2,5-Cl2
3-Cl, 5-CH3
3-OCH3, 5-CF3
4.8
16
>200
>200
>80
>200
>200
>200
>200
168
12
19
>20
>21
10
>21
>19
4.8
93
29.23
49
39.2
0.08
20.21
3.9
b
>200
>200
>200
>200
125
>21
a
9.9
Conditions: (a) RNH2, DMF.
14.2
>200
40
>15
>23
>22
0.5
Ta ble 9. Activity against HIV-1 and RT of
2-Amino-6-arylthiobenzonitriles (1a -x)a
0.34
9.85
0.32
2.07
c
200
anti-HIV-1
CCIC50 HIV-1 RT
>200
>200
108
6.2
compd
R
IC50 (µM)
(µM)
IC50 (µM)
0.52
0.90
b
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
14.6
4.3
6.0
b
16
6.1
115
4.1
7.4
>32
30.0
5.1
9.8
>6.4
1.73
43.8
12.8
31.5
>0.8
0.43
62% @5 µM
168
>200
>200
c
>40
79
>200
33
53
113
41
8.7
2.7
1.5
>17
c
0.96
5.7
7.2
16
b
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-Br
3-Br
3-F
2-CN
3-CN
4-CN
3-CF3
3-NH2
2,5-Cl2
3,5-(CH3)2
3,5-Cl2
3-Cl, 5-CH3
3-OCH3, 5-CH3
3-OCH3, 5-CF3
a
The protocols for obtaining the IC50 values are described in
the Experimental Section. Not tested. c No significant activity at
concentrations up to 200 µM.
b
properties of the substituents did not seem to have a
significant impact in the resultant antiviral activity.
Thus, the 3-methoxy analogue 3c had antiviral activity
similar to that of the electron-withdrawing 3-chloro
analogue 3i and the 3-bromo analogue 3l. However,
strong electron-withdrawing groups such as the cyano
group in 3q and the trifluoromethyl group in 3s led to
reduced antiviral activities.
As was the case with the sulfide series, C-4 substi-
tuted analogues were also generally the least active in
the sulfone series (3d , 3g, 3j, 3m , and 3r ). Similar to
the sulfide and sulfoxide series, the additional meta
methyl substituent enhanced antiviral activity in the
sulfone series. Compounds 3v (739W94) and 3w -y were
among the most potent in the current series, and they
were at least 20-fold more potent than that of the
corresponding monosubstituted 3c, 3f, 3i, and 3l against
HIV-1. Although not as pronounced as the addition of
a second meta methyl group, the addition of another
meta chloro group to a monosubstituted analogue 3i also
resulted in a more potent analogue 3u . The additive
effect of a C-3 substituent was also seen when a C-3
chloro group was added to the 2-chloro analogue 3h ,
resulting in a compound (3t) that was approximately
7-fold more active against HIV-1 than 3h .
12
>21
15
53
162
42
12
9.1
1.1
>19
7.1
>23
3.5
1.1
0.12
1.7
0.14
13
80
>200
34
168
4.3
>2
3
16
10
9
1s
1t
1u
1v
1w
1x
18
1.76
2
5.1
93.5
>200
>20
a
The protocols for obtaining the IC50 values are described in
b
the Experimental Section. Denotes no significant activity at
concentrations up to 200 µM. c Not tested.
flexibility of the methoxy groups, allowing for both
analogues to occupy the same space in the pocket of the
enzyme. The same level of antiviral activity shared by
the 2-fluoro and 3-fluoro analogues, 3n and 3o, respec-
tively, may be due to the small-sized fluoro group not
appreciably affecting the hydrophobic region of the
enzyme. This was further evidenced by the fact that
both 3n and 3o and the unsubstituted 3a showed
equipotency against HIV-1. Differences in the electronic
Demethylation of 3y resulted in a compound 3a a that,
although still active, was 9-fold less active than 3y,
again suggesting that the environment within the

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1875
Ta ble 11. Activity against HIV-1 of
However, a 2-naphthyl group (3ff) was detrimental to
antiviral activity.
2-Amino-6-arylsulfonylbenzonitriles (3a -ff)a
anti-HIV-1 CC IC50 HIV-1 RT
Similar to the sulfoxide series, the sulfone analogues
generally showed high IC50 values in the cell cytotoxic
assay, resulting in acceptable therapeutic indices. Anti-
viral potency in the sulfone series was well reflected in
the potent activity of the compounds against HIV-1 RT.
Compounds 3u -3z with submicromolar IC50 values
against the virus were also the most potent against
HIV-1 RT.
compd
R
IC50 (µM)
(µM)
IC50 (µM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
2
0.6
0.9
25
2.3
0.4
9.5
4.1
>5
146
>200
>200
>200
179
6.9
1.4
0.6
13
4.5
0.2
7.3
5.9
0.4
b
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-Cl
>200
>200
>200
>5
Finally, an array of the compounds in Table 11 was
more potent against HIV-1 than NPPS (4a) with 739W94
being >200-fold more potent than 4a against HIV-1.
3-Cl
0.59
3
5.0
3j
4-Cl
3k
3l
2-Br
3-Br
40
12
0.54
20
3.0
3.0
5.4
2.4
80.0
3.5
>200
>200
57
>200
>200
>200
>200
197
>200
>200
>200
133
0.2
>14
5.0
b
Our next SAR study focused on the aminocyanophenyl
ring system, and the antiviral results are summarized
in Table 12. Compounds 12u ,v and 12y are the chemical
precursors to the sulfones 3u ,v and 3y. The requirement
of the 3-amino group for antiviral activity was well
demonstrated by compounds 12u ,v and 12y. The only
analogue with an acceptable level of antiviral activity,
the 3,5-dimethyl-substituted 12v, showed antiviral po-
tency approximately 150-fold less than 3v. The need for
a 3-amino group was further amplified by the lack of
activity against HIV-1 of compounds 20a ,b. The n-
butylamino analogue 20a and the cyclohexylamino
analogue 20b were designed to probe spatial require-
ments, and as can be seen from Table 12, both com-
pounds showed a lack of antiviral activity. The need for
a 2-cyano group was demonstrated by compound 21,
which showed a lack of antiviral activity at concentra-
tions up to 50 µM. Thus, it was the cumulative effect of
the 2-cyano and 3-amino groups that imparted antiviral
activity.
3m
3n
3o
3p
3q
3r
4-Br
2-F
3-F
2-CN
3-CN
4-CN
3-CF3
2,5-Cl2
3,5-Cl2
3,5-(CH3)2
3-Br, 5-CH3
3-Cl, 5-CH3
3-OCH3, 5-CH3
3-OCH3, 5-CF3
3-OH, 5-CH3
3-OCH2CH3, 5-CH3
3-O(CH2)2CH3, 5-CH3
3-O(CH2)3CH3, 5-CH3
6.0
1.8
>18
5.3
0.3
0.03
0.007
0.003
0.005
0.01
0.04
b
3s
3t
0.3
3u
3v
3w
3x
3y
3z
3a a
3bb
3cc
3d d
0.07
0.01
0.02
0.03
0.05
0.09
0.43
0.06
0.06
0.6
>200
94
164
167
9
11
b
b
0.4
>2
3ee
3ff
3.32
0.05
>200
1.0
>200
0.03
4a
4b
8.6
2.2
b
b
16
14
Although there is a high degree of cross-resistance
among NNRTIs, the emergence of drug resistance is
generally treatment-specific. For example, in mono-
therapy with nevirapine, a rapid emergence of the
Y181C mutant was observed that rendered the drug
clinically ineffective. However, a therapy involving the
simultaneous use of nevirapine and AZT resulted in the
emergence of K103N, which was then followed by Y188C
or G190A.30 Likewise, the clinical failure of a therapy
involving efavirenz was attributed to the appearance
of the K103N.31 Therefore, although combination therapy
has been proven to be effective in prolonging the
emergence of NNRTI-resistant viruses, selection ulti-
mately occurs that leads to loss of clinical efficacy of the
NNRTI being used. It is well accepted that a clinically
useful next generation NNRTI should possess some
levels of potency against a group of mutant strains that
shows reduced susceptibility to currently approved
NNRTIs. Some of the key amino acid substitutions
around the NNRTI binding site that are selected in the
clinic are the K103N, Y181C, Y188C, V106A, V108I,
E138K, G190A, and P236L. In vitro and in vivo, a large
number of the currently available NNRTIs are ineffec-
tive against these mutants.30,32
a
The protocols for obtaining the IC50 values are described in
b
the Experimental Section. Not tested.
Ta ble 12. Anti-HIV-1 Activity of 2-Arylthio-6-fluoro and
6-Alkylaminobenzonitriles (10U-Y, 14b-d )a
Anti-HIV-1 CCIC50
compd
R
R1
IC50 (µM)
(µM)
12u
12v
12y
20a
3,5-Cl2
3,5-(CH3)2
3-OCH3, 5-CH3 2-CN, 3-F
2-CN, 3-F
2-CN, 3-F
42
1.5
51
188
>200
8
3,5-(CH3)2
3,5-(CH3)2
H
2-CN, 3-NH-
(CH2)3CH3
2-CN, 3-cyclo-
hexylamino
3-NH2
>12
>200
20b
>200
>50
>200
>50
21
a
The protocol for obtaining the IC50 values is described in the
Experimental Section.
enzyme that these meta substituents bind to was
hydrophobic. Increasing the lipophilicity by extending
the methylene group from one (3y) to two (3bb) and
three (3cc) did not appear to increase antiviral activity.
A four-methylene spacer unit (3d d ) caused a decrease
in antiviral activity. Introducing the bulky 1-naphthyl
group gave a compound 3ee that was still potent.
On the basis of the procedure of Kellam and Larder,34
we have created a panel of recombinant, isogenic viruses
containing the mutations mentioned above either singly
or in combination within the HIV reverse transcriptase
coding region. The genotypes corresponding to the
substituted amino acid residues for each of these
mutants are listed in Table 13, along with the antiviral

1876 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
Ta ble 13. Antiviral Activity of Representative 2-Amino-6-arylsulfonylbenzonitriles and Marketed NNRTIs against HIV-1 Wild-Type
and Mutant Viruses in the HeLa MAGI Assaya
IC50 values (µM) vs wild-type and mutant viruses
virus/compds
3u
3v
3w
3x
3y
3z
3ee
EFV
NEV
wild-type
K103N
V106A
Y181C
V106A/Y181C
E138K
G190A
V108I/Y181C
V108I
Y188C
K103N/L100I
P236L
0.037
8.3
0.0062
50
1.8
0.085
0.084
58
0.047
0.14
5.5
0.011
1.1
0.0024
14
0.49
0.017
0.030
13
0.017
0.21
3
0.009
0.97
0.0015
0.015
3.6
0.0028
14
0.21
0.015
0.040
13
0.017
0.4
20
0.01
0.014
1.8
0.0029
13
0.42
0.029
0.087
14
0.029
0.3
19
0.008
>50
16
7.8
0.063
0.014
2.3
0.0054
0.0009
0.024
0.089
7.1
10.5
0.013
36
2.1
0.08
0.66
36
0.12
0.4
0.0015
0.0018
0.0033
0.0008
0.0064
0.0033
0.0014
0.0012
1.6
∼10
10
29
>10
>10
0.077
0.015
0.031
18
0.011
1.7
16
0.011
>50
15
13
0.23
0.068
0.20
0.072
>10
>10
0.34
2.2
10
0.056
>50
21
10
66
0.03
0.18
1.7
5
0.0025
0.005
0.007
0.002
0.0005
0.042
0.074
K103N/Y181C
K103N/V108I
K103N/P225H
>50
>50
>50
33
19
>50
10
5.5
>50
4
3.8
4.8
3.3
3.5
2.8
0.13
a
The protocol for obtaining the IC50 values is described in the Experimental Section.
Ta ble 14. Statistics for Crystallographic Structure
Determinations
data of a select group of the most potent disubstituted
sulfones (3u -z, 3ee). Included in the table are marketed
drugs: the first generation nevirapine and efavirenz,
which is a recent addition to the armamentarium of
drugs against HIV.
Data Collection Details
data collection site
wavelength (Å)
KEK BL-6A2
1.000
unit cell (a,b,c in Å)
resolution range (Å)
observations
141.1, 110.9, 73.1
30.0-2.98
74 167
As seen in Table 13, sulfones 3u -z and 3ee showed
single-digit nanomolar activity against the V106A and
P236L strains and submicromolar to low nanomolar
activity against strains E138K, V108I, and Y188C.
Additionally, 3u -y manifested low nanomolar activity
against the G190A. These sulfones in general were more
potent than nevirapine against this panel of mutant
viruses. However, they were less potent than efavirenz
in this panel. Unfortunately, they showed a lack of
activity against two key resistant strains, the K103N
and Y181C, and double mutants based on those two
strains. As described below, we were able to use the
X-ray crystal structure of 3v (739W94) in complex with
HIV-1 RT to rationalize the activity (or lack of) of these
sulfones against the mutants described above.
The HIV-1 RT/739W94 complex has been refined to
an overall R factor of 0.211 for all data from 30 to 3.0 Å
resolution with the retention of excellent stereochem-
istry (Table 14). The binding mode of 739W94 to HIV-1
RT is revealed unambiguously from the omit map shown
in Figure 1a. Comparisons of the bound conformation
of 739W94 and the structurally related compound GCA-
186, an emivirine analogue,35 and the first generation
NNRTI, nevirapine,29 are shown in parts b and c of
Figure 1. The 3,5-dimethylphenyl of 739W94 overlapped
well with one of the pyridine rings of nevirapine at the
“top” of the NNRTI pocket but only very marginally with
the equivalent 3,5-dimethylphenyl ring of GCA-186.
There was also a greater degree of overlap of the
2-aminobenzonitrile ring of 739W94 and the second
pyridine ring of nevirapine than with the pyrimidine
ring of GCA-186. Thus, the 739W94 inhibitor was
significantly displaced from the binding position of GCA-
186 and showed a greater overlap with the more
structurally dissimilar nevirapine molecule. As a result
of this displacement, one of the sulfone oxygens of
739W94 was in a position to maintain the Tyr181 side
chain in the “up” position, a structural feature necessary
for tight binding for compounds of the HEPT/GCA-186
series.35,36 For this latter class of compounds Tyr181 is
unique reflections
completeness (%)
average I/σ(I)
23 124
96.1
7.49
14.6
rmerge (%)a
outer resolution shell
resolution range (Å)
unique reflections
completeness (%)
average I/ σ(I)
3.09-2.98
2219
93.7
0.86
Refinement Statistics
resolution range (Å)
30.0-3.0
unique reflections (working/test)
22606 (21508/1098)
b
R
(Rworking/Rfree
)
0.211 (0.221/0.265)
protein atoms
7706
inhibitor atoms
20
rms bond length deviation (Å)
rms bond angle deviation (°)
mean B factorc (Å2)
0.0096
1.61
87/91/57
a
b
Rmerge ) ∑|I - I |/∑ I . R ) ∑|Fo - Fc|/ ∑Fo. c Mean B factor
for main-chain, side chain, and inhibitor atoms, respectively.
positioned by an entirely different moiety, the 5-isopro-
pyl substituent of the pyrimidine ring. 739W94 formed
a hydrogen bond to the main-chain oxygen of Lys101
via its amino group and in this respect was more similar
to GCA-186 than nevirapine, which has no main-chain
hydrogen bonding. The contribution of this hydrogen
bond to the binding energy of the compound was
indicated by compound 12v, which has the amino group
replaced by fluorine, abolishing hydrogen bond and
reducing activity 150-fold. Despite the relative displace-
ment of 739W94 and GCA-186, the angle between the
two rings of each compound was similar, a feature noted
for many NNRTI compound series.29
The RT/739W94 model can be used to interpret some
of the structure-activity and drug-resistance data for
739W94 presented here (Tables 9-13) in terms of the
stereochemistry of inhibitor-protein interactions (Fig-
ure 1a). The weak binding of the sulfide (1) and
sulfoxide (2) analogues in contrast to the sulfone-

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1877
F igu r e 1. Position and orientation of 739W94 in the NNRTI pocket and comparison with nevirapine and GCA-186. (a) Stereoview
of simulated annealing that omits |Fo| - |Fc| maps contoured at 3σ, showing the electron density for 739W94 bound to HIV-1
reverse transcriptase. The 739W94 is shown with conventional atom colors, protein side chains as cyan ball-and-stick representation
and protein backbone as thin gray sticks. The H-bond is shown as a yellow dashed line. (b) Stereodiagram showing the relative
positions and orientations of RT-bound nevirapine and 739W94. The superimposition was carried out using the CR atoms of
amino acid residues around the NNRTI pocket to compensate for different crystal forms and domain rearrangements. Residues
used included 94-118, 156-215, and 225-243 from the palm domain, 317-319 from the connection domain of p66, and 137-139
from the fingers domain of p51. (c) Stereodiagram showing the relative positions and orientations of RT-bound GCA-186 and
739W94. The inhibitor and protein are shown in brown and dark-gray, respectively, for the RT/739W94 complex, cyan and light-
gray for RT/nevirapine in (b) and RT/GCA-186 in (c).
containing 739W94 can be explained by the absence of
a group to correctly position the side chain of Tyr181 in
the “up” position as discussed above. The detrimental
effect of most 4-position substituents on the phenyl ring
was due to a steric clash with adjacent residues,
particularly Pro95. Such a clash could lead to a dis-
placement of the inhibitor away from Tyr181 and thus
to weakened interactions that appeared to be a signifi-
cant contribution to the binding energy for this series.
It can be seen from Figure 1c that the positioning of
the phenyl ring of 739W94 meant that the 2-naphthyl-
containing compound (3ff) could be accommodated
within the site with only slight loss of potency because
the second ring would be positioned approximately
coincident with that of the GCA-186 phenyl ring.
The drug-resistant mutations that showed the great-
est effects on 739W94 binding were Y181C and K103N
(Table 13). Examination of the model of 739W94/RT
helped to explain these effects. 739W94 showed many
close contacts with Tyr181, and mutation to the smaller
nonaromatic cysteine will abolish most of these interac-
tions. We have previously reported that the emivirine
analogue GCA-186, which has 3,5-dimethyl substituents
on its phenyl ring, showed greater resilience to the
presence of the Y181C mutation than did emivirine
itself. Such substituents allowed GCA-186 to be in
contact with the highly conserved Trp229 at the top of
the NNRTI pocket.35 In the case of 739W94 its phenyl
ring was positioned lower in the pocket, and hence,
addition of 3,5-dimethyl groups to the ring did not give
rise to the same contacts with Trp229.
In contrast to its close contacts with Tyr181, 739W94
interacted with Tyr188 only via the â carbon; thus,
mutation at this position showed only a 10-fold weaker
binding compared to ∼100-fold reduction in binding for
the Tyr181 mutant. In the case of the K103N mutation,

1878 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
we observed van der Waals contacts between the nitrile
group of 739W94 and the aliphatic portion of the side
chain of Lys103. On mutation to the shorter asparagine
side chain, it seemed plausible that this favorable
interaction would be disrupted. An additional effect of
the K103N mutation might be derived from the poten-
tial of the asparagine side chain to hydrogen-bond to
the hydroxyl of Tyr188, leading to the stabilization of
unliganded reverse transcriptase.3 For two of the mu-
tant RTs studied (V106A and P236L) there was evidence
for hypersensitivity to 739W94 and analogues (Table
13). For the wild-type RT the side chain of Val106 had
a close contact between the 5′ position of the 2-amino-
benzonitrile ring and one sulfone oxygen (2.9 Å). The
reduction in bulk on mutation of V106A would relieve
this close contact and hence lead to tighter binding for
this mutant form. Pro236 had no van der Waals contact
with 739W94, while mutation of P236L would give a
longer and more flexible side chain that appeared to
point away from the inhibitor. It is possible that the
polypeptide chain might rearrange on mutation to
leucine, allowing some main-chain contacts to be made
with the inhibitor (this loop region has previously been
shown to be highly flexible).36,37
assay to gauge the antiviral activity of the more potent
sulfones in a panel of mutant viruses, 3u -z and 3ee
were generally active against the panel of mutants listed
in Table 13. However, these sulfones were inactive
against two key mutants, the K103N and Y181C, and
double mutants containing these strains. Their potency
against the panel of mutants in Table 13 was in general
better than that of the first-generation nevirapine but
was less than that of efavirenz. X-ray crystallographic
elucidation of the complex of 3v (739W94) in HIV-1 RT
suggested that the 6-arylsulfonyl-2-aminobenzonitriles
3 shared the same space in the RT binding pocket as
nevirapine. The lack of activity against the K103N and
Y181C and the activity of V106A and P236L mutants
to 739W94 could also be rationalized on the basis of the
crystal structure. Finally, bioavailability in rats ranged
from 4 to 18% for compounds 3u -y. Compound 3u had
the most acceptable pharmacokinetic profile with a
mean half-life of 5.2 h and a mean clearance of 18 mL
min-1 kg-1
.
Exp er im en ta l Section
Melting points were taken on a Thomas-Hoover or a Mel-
1
Temp apparatus and are uncorrected. H NMR spectra were
recorded on a Varian XL200 and Varian Unity Plus 400 or
200 MHz spectrometers. Elemental analyses were carried out
by Atlantic Microlabs, Inc., Atlanta, GA. 2,6-Dinitrobenzoni-
trile and 2,6-difluorobenzonitrile were purchased from Lan-
caster Synthesis, Inc., Windham, NH, and Aldrich, respec-
tively. All purchased starting materials were used without
further purifications. All solvents were reagent grades. Di-
methylformamide (DMF) and dimethylsulfoxide (DMSO) were
dried over 4 Å molecular sieves. Flash column chromatography
was performed according to ref 38. 3-Methoxy-5-methylthio-
phenol for the synthesis of 10y was prepared according to a
procedure described in ref 39.
Meth od A: 2-Ar ylth io-6-n itr oben zon itr ile (8). This
procedure has been reported in ref 23. Briefly, molar equiva-
lents of 2,6-dinitrobenzonitrile (6), arylthiol (7), and anhydrous
K2CO3 in DMF was cooled to 0 °C and stirred for 0.5 h. The
reaction mixture was made basic by the addition of pyridine
or 1 N NaOH, and this was followed by dilution with a copious
amount of water. The precipitate formed was collected by
filtration, washed with 1 N NaOH and water, and dried. 8 was
obtained either analytically pure or after purification by flash
column chromatography on silica gel using the solvent system
noted in Table 1.
Meth od B: 2-Am in o-6-a r ylth ioben zon itr ile (1). This
procedure followed that reported in ref 23. Briefly, intermedi-
ate 8 in diglyme was stirred with 3 molar equiv of SnCl2‚2H2O
in concentrated HCl (∼2 mL per mmol of 8) cooled in a water
bath. After being stirred for 0.5 h, the reaction mixture was
poured into a vigorously stirring mixture of 50% NaOH and
crushed ice (1:3 v/v, ∼5 mL per mmol of 8). Precipitate was
collected by filtration, washed with 1 N NaOH and water, and
dried. 1 was obtained after purification by methods noted in
Table 2.
Meth od C: 2-Ar ylth io-6-flu or oth ioben zon itr ile (10).
The procedure is similar to that reported in ref 23. Thus, to
an ice-bath-cooled suspension of potassium t-butoxide or
potassium carbonate in dry DMSO or DMF was added 1-1.1
molar equiv of arylthiol 7, followed by a molar equivalent of
2,6-difluorobenzonitrile (9). The resultant mixture was stirred
for 1 h and was then poured into H2O. This was followed by
stirring for about 30 min. Intermediate 10 was collected by
filtration and dried. This intermediate was either used without
purification or subjected to flash column chromatography on
silica gel using solvents summarized in Table 3.
The pharmacokinetic profiles of sulfones 3u -y were
next evaluated primarily to explore their potentials as
drug candidates. In a single 2.5 mg/kg dose by intra-
venous injection to male Wistar rats, analogue 3u had
the most acceptable pharmacokinetic profile (with a
mean half-life of 5.2 h and a mean clearance of 18 mL
min-1 kg-1). The other analogues, 3v-y were rapidly
cleared from the systemic circulation (mean clearance
values ranged from 40 to 70 mL min-1 kg-1; mean half-
life values ranged from 0.8 to 1.9 h). In a single 7.5 mg/
kg dose by oral administration to rats, analogues 3u -y
gave bioavailability values in the range 4-18% with 3v
having the lowest bioavailability and 3w and 3v the
highest bioavailability.
In summary, a series of 6-arylthio-2-aminobenzoni-
triles 1 and 6-arylsulfinyl-2-aminobenzonitriles 2 were
identified as having antiviral activity in a cell-based
assay against HIV-1. Modifications of these first groups
of compounds led to 6-arylsulfonyl-2-aminobenzonitriles
3, a series of compounds that showed potent antiviral
activity against HIV-1, some with IC50 values in the
nanomolar range. SAR studies on the arylsulfonyl ring
system of 3 suggested that the placement of substituent
for optimal antiviral activity was in the C-3 position.
Another hydrophobic meta substituent, particularly a
meta methyl group, added to an existing C-3 substituted
analogue invariably resulted in an enhancement of
antiviral activity. A good example of this was compound
3v (739W94), which was approximately 40-fold more
potent against HIV-1 than its monosubstituted analogue
3f was. The requirement for a C-3 amino group in the
other ring system of 3 was established by the lack of
antiviral activity of compounds 12u -y and 20a ,b.
Likewise, the need for a C-2 nitrile group for antiviral
activity was demonstrated by analogue 21, which showed
a lack of activity against HIV-1. A number of the
analogues within the 6-arylsulfonyl-2-aminobenzoni-
triles 3 were more potent than NPPS (4a ) and 4b, two
compounds in the bisarylsulfone series that received
extensive evaluation as agents against HIV-1. In an
Meth od D: 2-(2-Cya n op h en ylth io)-6-flu or oben zon i-
tr ile (10p ). A mixture of 10k (0.2 g, 0.6 mmol) and copper
cyanide (0.07 g, 0.8 mmol) was refluxed in DMF for 3 h. The

2-Amino-6-arylsulfonylbenzonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1879
reaction mixture was concentrated under high vacuum and
subjected to flash column chromatography on silica gel with
30% hexane in CH2Cl2 as the solvents. This gave 0.13 g (85%)
of 10p as a white solid, mp 118-120 °C. 1H NMR (Me2SO-d6):
δ 7.1 (d, 1H, aromatic), 7.5 (t, 1H, aromatic), 7.5-7.7 (m, 2H,
aromatic), 7.7-8 (m, 2H, aromatic), 8.0 (d, 1H, aromatic). Anal.
(C14H7N2FS) C, H, N, S.
washed with 1 N NaOH and H2O, dried (MgSO4), and
concentrated. Following flash column chromatography on silica
with 10% EtOAc in hexane, 1.03 g (36%) of 10w was obtained
1
as a white solid, mp 87-90 °C. H NMR (Me2SO-d6): δ 2.4 (s,
3H, CH3), 7.1 (d, 1H, aromatic), 7.4 (s, 1H, aromatic), 7.4-7.6
(m, 3H, aromatic), 7.7 (dd, 1H, aromatic). Anal. (C14H9NSBrF)
C, H, N, Br, S.
Meth od E: 2-Ar ylsu lfin yl-6-flu or oben zon itr ile (11). To
solution of 10 in CH2Cl2 was added 1.2 equiv of m-
Meth od K: 2-Am in o-6-(3-h yd r oxy-5-m eth ylp h en ylsu l-
fon yl)ben zon itr ile (3a a ). A stirred solution of 3y (0.1 g, 0.33
mmol) in 5 mL of CH2Cl2 was cooled to -78 °C under a
nitrogen atmosphere. BBr3 (0.27 g, 1.09 mmol) was added
dropwise via a syringe. The reaction mixture was gradually
brought to room temperature, and an additional 0.08 g of BBr3
was added. Stirring was continued for 1 h. The reaction
mixture was poured into ice-water, stirred for 10 minutes,
saturated with NaCl, and extracted with CH2Cl2. After drying
(MgSO4) and solvent removal, the crude product was purified
by flash column chromatography on silica gel with hexane/
EtOAc (1:1) as the solvents. This gave 0.05 g (50%) of 3a a as
a
chloroperbenzoic acid or OXONE.27 The resultant mixture was
stirred for 24 h, after which the mixture was diluted with
additional CH2Cl2 or EtOAc. This mixture was then washed
with concentrated sodium bisulfite, 1 N NaOH, water, and
brine. After drying over MgSO4 and solvent removal, the
resultant crude product 11 was purified by flash column
chromatography on silica using solvents summarized in Table
4.
Meth od F : 2-Ar ylsu lfon yl-6-flu or oben zon itr ile (12). To
a solution of 10 in CH2Cl2 was added 2-3 equiv of m-
chloroperbenzoic acid. The resultant mixture was stirred for
24 h, after which the mixture was diluted with additional CH2-
Cl2 or EtOAc. This mixture was then washed with concen-
trated sodium bisulfite, 1 N NaOH, water, and brine. After
drying over MgSO4 and solvent removal, the resultant crude
product 12 was purified by flash column chromatography on
silica using solvents summarized in Table 6.
Meth od G: 2-Am in o-6-ar ylth ioben zon itr ile (1), 2-Am in o-
6-a r ylsu lfin ylben zon itr ile (2), a n d 2-Am in o-6-a r ylsu lfo-
n ylben zon itr ile (3). Concentrated NH4OH or gaseous am-
monia was introduced into a solution of 10, 11, or 12 in EtOH,
EtOH/THF, or MeOH/EtOH. The mixture was sealed in a
glass-lined bomb and heated to 130-140 °C for 3-24 h. After
solvent removal, the resultant crude product was subjected to
flash column chromatography on silica as summarized in
Tables 2, 5, and 7.
Meth od H: 2,6-Dibr om o-4-m eth yla n ilin e (14a ). To an
ice-water-cooled solution of 2-bromo-4-methylaniline (13a ) (25
g, 134 mmol) in 60 mL of MeOH and 20 mL of AcOH was
added dropwise bromine (6.9 mL, 134 mmol) in 60 mL of
AcOH. The reaction mixture was allowed to stand for 2 h, after
which the solvents were removed in vacuo. The resultant
concentrate was suspended in 1 N NaOH (∼100 mL). This was
extracted with EtOAc, dried over MgSO4, and evaporated to
give a reddish crude compound. Recrystallization from hexane
gave 26 g (73%) of 14a as an off-white solid, mp 71-73 °C. 1H
NMR (Me2SO-d6): δ 2.2 (s, 6H, CH3), 5.1 (br s, 2H, NH2), 7.3
(s, 2H, aromatic). Anal. (C7H7NBr2) C, H, N, Br.
1
a white solid, mp 178-180 °C. H NMR (Me2SO-d6): δ 2.3 (s,
3H, CH3), 6.6 (br s, 2H, NH2), 6.9 (unresolved s, 1H, aromatic),
7-7.2 (m, 3H, aromatic), 7.3 (d, 1H, aromatic), 7.5 (t, 1H,
aromatic), 10.4 (s, 1H, OH). Anal. (C14H12N2O3S‚0.2H2O) C,
H, N, S.
Met h od L: 2-(3-E t h oxy-5-m et h ylp h en ylsu lfon yl)-2-
flu or oben zon itr ile (12bb). To a stirred suspension of sodium
hydride (50% oil dispersed, 0.03 g, 0.82 mmol) in 5 mL of DMF
was added 12a a (0.2 g, 0.7 mmol). The mixture was stirred
for 30 min, after which iodoethane (0.1 mL, 1.4 mmol) was
added. After being stirred for 1 h, the reaction mixture was
poured into water, which was in turn extracted with EtOAc.
This EtOAc solution was washed with water, dried (MgSO4),
and concentrated. Purification using a silica gel preparative
plate with CH2Cl2 gave 0.2 g (92%) of 12bb as a white solid.
1H NMR (Me2SO-d6): δ 1.4 (t, 3H, CH3), 2.3 (s, 3H, CH3), 4.0
(q, 2H, CH2), 7.2 (s, 1H, aromatic), 7.3 (s, 1H, aromatic), 7.4
(s, 1H, aromatic), 7.9 (t, 1H, aromatic), 8-8.1 (m, 1H, aro-
matic), 8.2 (d, 1H, aromatic).
Meth od M: 2-Cycloh exyla m in o-6-(3, 5-d im eth ylp h en -
ylsu lfon yl)ben zon itr ile (20b). A mixture of cyclohexylamine
(0.3 g, 3 mmol), 12v (0.5 g, 1.7 mmol), and K2CO3 (0.55 g, 4
mmol) in 10 mL of DMF was stirred at room temperature for
12 h. The reaction mixture was concentrated in vacuo, and
the resultant concentrate was subjected to flash column
chromatography on silica gel with hexane/EtOAc (1:1) as the
eluent. A total of 0.45 g (71%) of 20b was obtained as a white
1
solid, mp 152-153 °C. H NMR (Me2SO-d6): δ 1-2 (m, 10H,
cyclohexyl), 2.3 (s, 6H, 2CH3), 3.3-3.5 (m, 1H, cyclohexyl), 5.8
(d, 1H, NH), 7.2 (d, 1H, aromatic), 7.3-7.4 (m, 2H, aromatic),
7.5-7.7 (m, 3H, aromatic). Anal. (C21H24N2SO2) C, H, N, S.
Meth od I: 3,5-Dibr om otolu en e (15a ). To an ice-water-
cooled and stirred solution of 14a (10 g, 38 mmol) in a mixture
of 70 mL of acetic acid, 30 mL of H2O and 8 mL of concentrated
HCl was added dropwise a solution of NaNO2 (3.12 g, 45 mmol)
in 10 mL of H2O. The reaction mixture was stirred for 30 min
and was then added to an 80 mL solution of 50% H3PO2 cooled
at 0 °C. After being stirred for 8 h at 0 °C, the reaction mixture
was allowed to stand at room temperature for 72 h. Following
extraction with EtOAc (3 × 100 mL), drying (MgSO4), and
concentration of the solvent, 6.5 g (68%) of 15a was obtained
as off-white crystals, mp 37-40 °C. 1H NMR (Me2SO-d6): δ
2.4 (s, 3H, CH3), 7.5 (s, 2H, aromatic), 7.67 (s, 1H, aromatic).
Anal. (C7H6Br2) C, H, Br.
Meth od N: 2-(3, 5-Dim eth ylp h en ylsu lfon yl)-6-bu tyl-
a m in oben zon itr ile (20a ). Following the procedure described
for the synthesis of 20b above using n-butylamine, a 78% yield
of 20a was obtained as a white solid after purification by flash
column chromatography on silica gel with 40% EtOAc in
1
hexane as the eluent, mp 117-119 °C. H NMR (Me2SO-d6):
δ 0.9 (t, 3H, CH3), 1.1-1.4 (m, 2H, CH2), 1.4-1.6 (m, 2H, CH2),
3.2 (q, 2H, CH2), 6.4 (t, 1H, NH), 7.1 (d, 1H, aromatic), 7.3-
7.4 (m, 2H, aromatic), 7.5-7.7 (m, 3H, aromatic). Anal.
(C19H22N2SO2) C, H, N, S.
Meth od O: P h en yl Nitr op h en ylsu lfid e (17). A mixture
of 2-bromonitrobenzene (16) (4 g, 20 mmol), thiophenol (7a )
(2.17 g, 20 mmol), and NaH (0.8 g, 20 mmol) in 20 mL of DMF
was stirred for 12 h. The reaction mixture was concentrated
and dry-packed in silica gel. Flash column chromatography
on silica gel with 20% EtOAc in hexane gave 3.2 g (69%) of 17
as a yellow solid, mp 74-75 °C. 1H NMR (Me2SO-d6): δ 6.9
(d, 1H, aromatic), 7.4 (t, 1H, aromatic), 7.5-7.7 (m, 6H,
aromatic), 8.2 (d, 1H, aromatic). Anal. (C12H9NO2S) C, H, N,
S.
Met h od J : 2-(3-Br om o-5-m et h ylp h en ylt h io)-6-flu o-
r oben zon it r ile (10w ). A stirred solution of 15a (2.27 g, 9
mmol) in 20 mL of freshly distilled THF was cooled to -78 °C
under an atmosphere of nitrogen. To this solution was added
sec-BuLi (14 mL of a 1.3 M cyclohexane solution) via a syringe.
The reaction mixture was stirred for 10 min, after which sulfur
(0.32 g, 10 mmol) was added in one portion. The reaction
mixture was gradually brought to room temperature, and
stirring was continued for 12 h. The reaction mixture was
cooled in an ice-water bath. To this was added 2,6-difluo-
robenzonitrile (9) (1.27 g, 9 mmol) in 10 mL of dry DMSO.
After being stirred for 20 min, the mixture was poured into
water (100 mL) and this aqueous solution was extracted with
EtOAc (3 × 50 mL). The EtOAc extracts were combined and
Meth od P : P h en yln itr op h en ylsu lfon e (4a , NP P S). A
solution of KMnO4 (1.89 g, 12 mmol) in 23 mL of water was
added to a stirred solution of 17 in glacial AcOH. The reaction
mixture was stirred for 0.5 h and was then filtered through

1880 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Chan et al.
Celite with repeated washing with water. The filtrate was
basified with concentrated NH4OH. The precipitate formed
was collected by filtration. 4a was extracted from this pre-
cipitate with hot DMF. After treatment of the DMF solution
with charcoal and filtration through Celite, the solution was
concentrated to dryness. A total of 0.07 g (5%) of 4a was
obtained as a pale-yellow solid, mp 143-145 °C. 1H NMR (Me2-
SO-d6): δ 7.6-7.9 (m, 2H, aromatic), 7.9-8.1 (m, 6H, aro-
matic), 8.3-8.5 (d, 1H, aromatic). Anal. (C12H9NO4S) C, H, N,
S.
Meth od Q: Bisn itr op h en ylsu lfid e 18. This procedure is
similar to that described in method O using 2-nitrothiophenol.
Following flash column chromatography on silica gel with 30%
EtOAc in hexane as the eluent, a 60% yield of 18 was obtained
as a yellow solid, mp 121-122 °C. 1H NMR (Me2SO-d6): δ 7.4
(d, 2H, aromatic), 7.9-8.1 (m, 4H, aromatic), 8.32-8.3 (d, 2H,
aromatic). Anal. (C12H8N2O4S) C, H, N, S.
Meth od R: Bisn itr op h en ylsu lfoxid e 19. To a solution
of 18 (0.3 g, 1.1 mmol) in 50 mL of MeOH was added a solution
of OXONE27 (2.7 g, 4.3 mmol) in 50 mL of water. The resultant
mixture was stirred for 12 h. The reaction mixture was filtered,
and the precipitate was repeatedly washed with hot MeOH.
The filtrate was concentrated and subjected to column chro-
matography on silica gel with 4% MeOH in CH2Cl2 as the
eluent. This gave 0.2 g (77%) of 19 as a light-yellow solid, mp
185-187 °C. 1H NMR (Me2SO-d6): δ 7.8-7.9 (m, 2H, aro-
matic), 7.9-8.1 (m, 4H, aromatic), 8.2-8.3 (m, 2H, aromatic).
Anal. (C12H8N2O5S) C, H, N, S.
Meth od S: Bisn itr op h en ylsu lfon e 4b. To a solution of
19 (0.17 g, 0.6 mmol) in 2 mL of trifluoroacetic (0.6 mL, 6
mmol) acid was added dropwise 30% H2O2 (0.19 g, 5.7 mmol).
The resultant mixture was stirred for 12 h. Water was added
to the reaction mixture, and the precipitate formed was
collected by filtration. After the mixture was washed with a
copious amount of water and oven-dried at 60 °C, 0.14 g (82%)
of 4b was obtained as a white solid, mp 180-182 °C. 1H NMR
(Me2SO-d6): δ 8-8.1 (m, 4H, aromatic), 8.1-8.3 (m, 4H,
aromatic). Anal. (C12H8N2O6S) C, H, N, S.
HeLa MAGI Assa y. The assay described in this report is
a modified version of the HeLa MAGI system40 in which HIV-1
infection is detected by the activation of an HIV-LTR driven
â-galactosidase reporter in HeLa CD4-LTR-b-gal cells (ob-
tained from Dr. Michael Emerman through the AIDS Research
and Reference Reagent Program, Division of AIDS, NIAID,
NIH). Quantitation of â-galactosidase is achieved by measur-
ing the activation of a chemiluminescent substrate (Tropix) 3
days after infection. The concentration of each compound
required to inhibit 50% (IC50) of the HIV-1-induced â-galac-
tosidase signal, relative to untreated controls, was determined
for each isogenic recombinant virus.
Activity a ga in st HIV-1 in a n Acu te In fection Assa y
Usin g MT4 Cells. The human T-cell lymphotropic virus
type 1 transformed cell line MT4 was infected with HIV-1
(strain 3B) at 100 times the amount necessary to cause a
50% reduction in cell growth. The infected cells were incubated
for 5 days in the presence of various concentrations of each
of the compounds to be tested. HIV-1-mediated cytopathic
effect (CPE) is expressed as an inhibition of MT4 cell growth.
A compound that expresses activity against HIV-1 reverses
this CPE. The extent of the CPE reduction was monitored
by a propidium iodide stain for DNA (1), allowing for the
calculation of IC50 values (50% inhibitory concentration). The
antiviral effect of a compound is reported as an IC50 value,
which is the inhibitory concentration that would produce a
50% decrease in the HIV-induced CPE. This effect is mea-
sured by the amount of drug required to restore 50% of the
cell growth of HIV-1-infected MT4 cells, compared to unin-
fected MT4 cell controls. Each value represents the mean (
standard error (SE) from a single experiment performed in
triplicate.
reaction (sequence 1) was labeled with Eu-chelate (Wallac,
CR28-100) via a streptavidin-biotin linkage at the 5′ end of
the primer.
Sequence 1
DNA: 5′-Eu -SA-B-GTC ATA GCT GTT TCC TG-3′
RNA: 3′-CAG UAU CGA CAA AGG ACA CAC UUU A
Reactions (50 µL) contained 100 nM Cy5-dUTP (Amersham
Life Science, PA55022), 40 nM Eu-labeled primer template, 1
nM recombinant HIV reverse transcriptase, 50 mM Tris-HCl,
pH 8.0, 80 mM KCl, 10 mM MgCl2, 0.0032% NP40, 10 mM
DTT, test compound, and 2% DMSO. The reaction was
initiated by the addition of reverse transcriptase to each well.
Reaction progress was monitored by fluorescence energy
transfer from the Eu-chelate to the Cy5-dUMP incorporated
into the template primer. The Eu-chelate was excited at 340
nm, and time-resolved fluorescence emission of the incorpo-
rated Cy5-dUMP was monitored at 665 nm with a Victor41
1420 multilabel counter (Wallac) over 30 min.
Cr ysta lliza tion a n d X-r a y Da ta Collection . Crystals of
the complex of HIV-1 RT with compound 739W94 (3v) were
grown at 4 °C from citrate/phosphate buffer, pH 5.0, with
PEG3400 (6-10% w/v) as precipitant, using the protocols
described previously.42 Crystals were soaked in increasing
concentrations of PEG 3400, with citrate/phosphate buffer to
a maximum PEG 3400 concentration of 50% (w/v). This latter
procedure increases the order of the crystals and results in a
series of closely related cell forms.42,43 Crystals were resoaked
in 0.2 mM compound for 2 days prior to data collection. X-ray
diffraction data were collected using a Weissenberg camera
on beamline BL6-A244 at the Photon Factory, KEK, J apan,
utilizing procedures described previously29,45 Data were col-
lected at 16 °C with a 0.1 mm collimator, using 3.5° frames
with a coupling constant of 1.5°/mm on pairs of 200 mm ×
400 mm Fuji Bas-IIIB imaging plates positioned 429.7 mm
from the crystal. The exposure time was 112 s per frame. The
collimator, crystal enclosure, and camera cassette were flushed
with helium to reduce background scattering. Exposed image
plates were scanned off-line using Fuji BA 100 IP scanners. A
single large crystal of RT/739W94, exposed to X-rays at three
different positions, was used to collect a data set to 2.98 Å
resolution.
Data images were indexed and integrated using DENZO46
and merged with SCALEPACK.46 Statistics are given in Table
1.
Str u ctu r e Solu tion a n d Refin em en t. The structure was
solved by molecular replacement using the RT-1051U91
complex (refined at 2.2 Å resolution to an R factor of 0.214;
PDB 1RTH29). The molecular orientation and position were
optimized by rigid-body refinement (program CNS47). The
models were initially treated as single rigid bodies, then as
two subunits, and finally as separate rigid groups correspond-
ing to the nine domains of the heterodimer. The data used were
gradually extended from 12 to 6.0 to 3.0 Å. During the
refinement, anisotropic B factor scaling, solvent correction,
strong stereochemical restraints to all atoms, and positional
restraints to those lying greater than 25 Å from the CR atom
of residue 188 (approximately defining the NNRTI binding
pocket) were applied. Manual model rebuilding used the
program “O”48 on an SGI O2 workstation. Further rounds of
restrained refinement and manual rebuilding resulted in the
current models (Table 1). Illustrations were prepared using
BOBSCRIPT49 and RASTER3D.50
Ack n ow led gm en t . We thank Drs. Paul Feldman
and Karen Romines for valuable suggestions concerning
the manuscript. The valuable suggestions of Drs. Lee
Kuyper and J ames Kelley (previously of Burroughs
Wellcome Co.) at the initial stages of this investigation
are also much appreciated.
HIV Rever se Tr a n scr ip ta se Assa y. Assays were con-
ducted in black round-bottomed 96-well plates. Biotinylated
DNA primer and RNA template were custom-synthesized by
Oligos Etc. (Wilsonville, OR). The primer template used in the

2-Amino-6-arylsulfonylbenzonitriles
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