Design, synthesis, and evaluation of diarylpyridines and diarylanilines as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm100738d

On the basis of the structures and activities of our previously identified non-nucleoside reverse transcriptase inhibitors (NNRTIs), we designed and synthesized two sets of derivatives, diarylpyridines (A) and diarylanilines (B), and tested their anti-HIV

Full text of DOI:10.1021/jm100738d

J. Med. Chem. 2010, 53, 8287–8297 8287
DOI: 10.1021/jm100738d
Design, Synthesis, and Evaluation of Diarylpyridines and Diarylanilines as Potent Non-nucleoside
HIV-1 Reverse Transcriptase Inhibitors
Xingtao Tian,†,# Bingjie Qin,† Zhiyuan Wu,† Xiaofeng Wang,† Hong Lu,‡ Susan L. Morris-Natschke, Chin Ho Chen,§
Shibo Jiang,‡ Kuo-Hsiung Lee, ,^ and Lan Xie*,†
†Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China, ‡Lindsley F. Kimball Research Institute,
New York Blood Center, New York, New York 10065, United States, §Duke University Medical Center, Box 2926, Surgical Oncology Research
Facility, Durham, North Carolina 27710, United States, Natural Products Research Laboratories, Eshelman School of Pharmacy,
University of North Carolina, Chapel Hill, North Carolina 27599-7568, United States, and ^Chinese Medicine Research and Development Center,
China Medical University and Hospital, Taichung, Taiwan. # Current address: Institute of Chemical Defense, P.O. Box 1048,
Beijing 102205, China
Received June 18, 2010
On the basis of the structures and activities of our previously identified non-nucleoside reverse
transcriptase inhibitors (NNRTIs), we designed and synthesized two sets of derivatives, diarylpyridines
(A) and diarylanilines (B), and tested their anti-HIV-1 activity against infection by HIV-1 NL4-3 and
IIIB in TZM-bl and MT-2 cells, respectively. The results showed that most compounds exhibited potent
anti-HIV-1 activity with low nanomolar EC50 values, and some of them, such as 13m, 14c, and 14e,
displayed high potency with subnanomolar EC50 values, which were more potent than etravirine
(TMC125, 1) in the same assays. Notably, these compounds were also highly effective against infection
by multi-RTI-resistant strains, suggesting a high potential to further develop these compounds as a
novel class of NNRTIs with improved antiviral efficacy and resistance profile.
Introduction
HIV-1 reverse transcriptase (RT) is one of the most
important viral enzymes and plays a unique role in the HIV-1
life cycle. It has two known drug-target sites, the substrate
catalytic site and an allosteric site that is distinct from, but
located closely to, the substrate site.1,2 Non-nucleoside reverse
transcriptase inhibitors (NNRTIs) interact with the allosteric
site in a noncompetitive manner to distort the enzyme’s active
conformation and thus disrupt the function of the enzyme.
The first-generation NNRTI drugs (nevirapine, delavirdine,
and efavirenz) exhibit very potent anti-HIV-1 activity and
low toxicity. However, rapid drug-resistance emergence, due
to single point mutations (especially K103 mutant)3 in the
NNRTI binding site, compromises their clinical usefulness.
Etravirine (TMC125, 1),4 a diarylpyrimidine (DAPY, Figure 1),
was recently approved as a next-generation NNRTI for
AIDS therapy. It exhibits high potency against wild-type and
a number of mutatedviralstrains withnanomolarEC50 values
and has a higher genetic barrier5 to delay the emergence of
drug-resistance. The success of 1 greatly encouraged further
research to explore additional novel NNRTIs.6-8 The most
advanced NNRTI in development is another DAPY deriva-
tiverilpivirine(TMC278, 2)9 in phase III, which showed better
potency and pharmacological profiles than 1, such as once-
daily administration.10
Acquired immunodeficiency syndrome (AIDSa), caused by
human immunodeficiency virus (HIV), threatens human
health and life, spreading rapidly worldwide and resulting in
more than 60 million people infected by HIV and about
25 million patients dying of AIDS (www.unaids.org). Because
there is no effective vaccine to prevent HIV infection, devel-
opment of anti-HIV therapeutics is critical to improve the
quality and save the lives of HIV infected individuals. To date,
26 anti-HIV drugs have been approved for the clinical treat-
ment of HIV infection and AIDS (www.fda.gov/oashi/aids/
virals.html), including reverse transcriptase inhibitors (RTIs),
protease inhibitors(PIs), integrase inhibitors, fusion inhibitor,
and entry inhibitor (CCR5 coreceptor antagonist). Highly
active antiretroviral therapies (HAART), which use a com-
bination of three to four drugs, can significantly reduce the
morbidity and mortality of AIDS. However, as a result of
emerging drug-resistant HIV mutants, increasing numbers of
HIV-infected patients cannot use or fail to respond to HAART.
Therefore, the development of new anti-HIV drugs is urgently
required.
*To whom correspondence should be addressed. Phone/Fax: 86-10-
66931690. E-mail: lanxieshi@yahoo.com.
a Abbreviations: AIDS, acquired immunodeficiency syndrome; CC50,
concentration for 50% cytotoxicity; DAPYs, diarylpyrimidines; DAPDs,
diarylpyridines; DAANs, diarylanilines; EC50, effective concentration
for 50% inhibition; HAART, highly active antiretroviral therapies;
HIV, human immunodeficiency virus, NNRTI, non-nucleoside reverse
transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibi-
tor; PDB, protein database; RF, resistant fold; RT, reverse transcrip-
tase; RTI, reverse transcriptase inhibitor; SAR, structure-activity
relationship; SI, selective index (ratio of CC50/EC50).
By using an isosteric replacement strategy on the central
pyrimidine ring of DAPYs, our prior studies discovered two
series of active compounds, diarylpyridine (A, DAPD),11 with
one pyrimidine nitrogen replaced by carbon, and diarylaniline
(B, DAAN),12 with both pyrimidine nitrogens replaced by
carbon. Exemplary di-para-cyanophenylpyridine compound 3
and di-para-cyanophenylaniline compound 4 (Figure 1)
r
2010 American Chemical Society
Published on Web 11/04/2010
pubs.acs.org/jmc

8288 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Tian et al.
Figure 1. Drugs (1 and 2), leads (3 and 4), and new target compounds.
Scheme 1a
exhibited high potency against HIV-1 wild-type and RT-
resistant viral strains. Previous SAR studies also indicated
that the para-cyanoaniline moiety (A-ring) is necessary and
theaminogroup onthe centralring, either pyridineorbenzene
(B-ring), ortho to the A-ring, is quite crucial for enhancing
anti-HIV activity in both the A and B series. On the other
hand, the para substituent on the phenoxy ring (C-ring) was
modifiable and could greatly affect the anti-HIV potency. We
hypothesized that the amino group on the central ring might
form additionalH-bondswiththe key amino acidK101on the
NNRTI binding site and thus compensate for the loss of the
H-bond between K101 and the nitrogen on the pyrimidine
ring in DAPYs.13 Our previous molecular modeling results
provided rational support for our hypothesis.12 In our con-
tinuing studies, wehave made further structural modifications
on the C-ring moiety of A and B series of derivatives. The aims
are to investigate how the C-ring moiety will affect anti-HIV
activity and develop novel classes of NNRTIs with improved
antiviral efficacy. Thus, we report herein the design, synthesis,
and anti-HIV activity of two sets of new target A and B
compounds (Figure 1).
The crystal structures of complexes HIV-1 RT/2 (Protein
Database (PDB) code: 2zd1) and K103N/Y181C HIV-1 RT/2
(PDB: 3bgr)14 reveal that the linear cyanovinyl in 2 is embed-
ded deep into a cylindrical hydrophobic tunnel, which con-
nects the NNRTI-binding pocket to the nucleic acid-binding
cleft and is favored to interact with the more conservable
amino acids W229 and Y183. Thus, the para-cyanovinyl
group on the C-ring of 2 is thought to be an important moiety
for enhancing activity. On the basis of the information about
the binding site and our previous SAR results, we designed
new target compounds in the A and B series with diverse para
substituents (R2) on the phenoxy ring (C-ring). The R2 groups
included cyanovinyl and other linear substituents, which
could likely insert into the cylindrical hydrophobic tunnel of
the binding site and reach the conserved amino acid W229.
The R1 and R3 substituents on the central pyridine or benzene
ring were limited to NH2, NO2, and H.
a (a) Heating 140 ꢀC without solvent, 4 h; (b) t-BuOK/DMF, rt, 24 h;
(c) K2CO3/DMF, 130-140 ꢀC, 6-8 h; (d) K2CO3/DMF, 190 ꢀC, MW,
15 min.
Six of them (11a, 11d, 11f, 13a, 13d, and 13f) have been
previously described,11 but without the full experimental
details presented herein. The starting materials 4-aminobenzo-
nitrile, 2,6-dichloro-3-nitro pyridine (5), 2,4-dichloronitro-
benzene (6), and 2,4-dichloro-1,5-dinitrobenzene (7) are
inexpensive and commercially available. Coupling of 4-amino-
benzonitrile and 5 was performed by heating at 140 ꢀC for
4 h without solvent under nitrogen to provide intermediate
4-(6-chloro-3- nitropyridin-2-ylamino)benzonitrile (8) in 73%
yield. The same reaction of dichloronitrobenzene 6 or 7 with
4-aminobenzonitrile took place in DMF in the presence of
triethylamine or potassium tert-butoxide (t-BuOK) at room
temperature to produce corresponding intermediates N-(4-
cyanophenyl)-5-chloro-2-nitroaniline (9) or N-(4-cyanophenyl)-
5-chloro-2,4-dinitroaniline (10) in 64% and 89% yields,
respectively. Subsequently, intermediate 8 was reacted with a
trisubstituted phenol in DMF in the presence of potassium
carbonate by traditional heating at 130-140 ꢀC for 6-8 h to
afford corresponding 2,6-diaryl-3-nitrophyridines 11a-11c
and 11g with yields ranging from 56 to 82%. Alternatively,
N1,5-diarylnitroanilines 12a and 12b were prepared from 9 or
10 by coupling with a substituted phenol under microwave
irradiation in DMF in the presence of potassium carbonate.12
As shown in Scheme 2, a Sonogashira coupling reaction between
11a and 2-methyl-3-butyn-2-ol or ethynylcyclopropane took
place in the presence of triethylamine and palladium-copper
catalyst to give corresponding 11d and 11e, respectively, with
different elongated para-ethynyl substituents on the C-ring.
Next, the para substituent on the C-ring of 11d was removed
Chemistry
Totally, 34 target compounds (A and B series, Figure 1)
were synthesized via the short routes detailed in Schemes 1-4.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8289
Scheme 2a
a (a) Pd(PPh3)Cl2, CuI, DMF/Et3N, N2 protection; (b) NaOH, toluene, reflux, 16 h; (c) Na2S2O4, NH3 H2O, THF/H2O (v/v 1:1), rt; (d) NiCl2 6H2O/
NaBH4, THF/MeOH (v/v 1:1).
3
3
Scheme 3a
a (a) CH3NO2, NaOH/THF, rt; (b) CH2(COOH)2, piperidine/Py, 2 h, reflux; (c) (EtO)2P(O)CH2CN, t-BuOK/THF, 0 ꢀC to rt, 48 h; (d) Na2S2O4,
NH3 H2O, THF/H2O (1:1), rt, 3 h; (e) 20% HCl/ether in acetone; (f) acetone, NaOH, rt; (g) NaBH4, SbCl3; (h) HCOOH/Pd-C, Et3N/CH3CN, reflux,
3
1-3 h; (i) H2, Pd-C/THF, 6 h.
Scheme 4a
a (a) MW, 100-150 ꢀC, 10-20 min; (b) Na2S2O4, NH3 H2O, THF or 1,4-dioxane/H2O (1:1), rt, 3 h; (c) HCOOH/Pd-C, Et3N/CH3CN, reflux, 1-3 h.
3
to convert into 11f by refluxing in dry toluene in the presence
of powdered sodium hydroxide in a 69% yield. The nitro-
containing compounds 11a-11f were reduced by using
sodium hydrosulfite dehydrate15 or nickel(II) chloride hydrate
and sodium borohydride to afford corresponding amino-
compounds 13a-13f, with yields ranging from 43 to 82%.
Moreover, the synthesis of other compounds with different
elongated R2 substituents is indicated in Scheme 3. The con-
densation of 11g with nitromethane, malonic acid, or acetone
under basic condition afforded 11i, 11j, or 11k, respectively,
with a para-linear side chain on the phenoxy ring (Scheme 3).
The cyanovinyl compounds 11m, 12c, and 12e were prepared
by the condensation of diethyl cyanomethyl phosphonate
with aldehyde compounds 11g, 12a, or 12b, respectively,

8290 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Tian et al.
Table 1. Data of Target Compounds 11-14 against HIV-1 Wild-Type Strain Replication
NL4-3 in TZM-bl cell line
IIIB in MT-2 cell line
R1
R2
R3
EC50b (μM)
CC50c (μM)
SI d(μM)
EC50
CC50 (μM)
SI
11a*a
13a*a
11b
13b
11c
13c
11d*a
13d*a
11e
13e
11f*a
13f*a
11i
H
I
NO2 0.0607 ( 0.024
NH2 0.0076 ( 0.0023
NO2 0.0502 ( 0.0068
NH2 0.00729 ( 0.0020
NO2 NA
>41.15
16.72
>675
2200
0.079 ( 0.002
0.054 ( 0.004
0.045 ( 0.016
0.033 ( 0.01
1.34 ( 0.70
22.93
7.24
283
134
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
Br
Br
>45.66
15.54
>909
2132
65.11
9.61
1447
291
CH2CHdCH2
CH2CHdCH2
CtCC(OH)Me2
CtCC(OH)Me2
CtCCH(CH2)2
CtCCH(CH2)2
CtCH
39..68
14.02
23.28
3.54
27.37
209
NH2 0.00689 ( 0.0014
NO2 41.35
8.46
45.25
8.73
1228
0.067 ( 0.003
1.09 8.91 ( 0.00
36
2.61
2.25
NH2 0.241 ( 0.0147
NO2 >47.17
1.58 ( 0.097
NA
NH2 0.0912 ( 0.037
NO2 0.111 ( 0.086
NH2 0.0067 ( 0.0010
NO2 0.308 ( 0.098
7.45
>52.08
9.94
82
>469
1484
>145
695
0.41 ( 0.05
0.247 ( 0.040
0. 042 ( 0.004
1.71 ( 0.134
0.068 ( 0.015
5.71
14
>405
221
>100
CtCH
CH(OH)CH2NO2
9.28
27.06
20.57
34.79
15.69
>47.90
18.35
>100
5.63
>44.54
7.02
16
303
13i
11j
CH(OH)CH2NO2 HCl NH2 0.0101 ( 0.0026
3
CHdCHCOOH
CHdCHCOOH
CHdCHCOMe
CH2CH2COMe
CHdCHCN
NO2 14.92
NH2 0.60 ( 0.043
27.84
18.0
1.87 8.12 ( 0.42
4
25
13j
30
>1915
4744
0.63 ( 0.22
11k
13k
11m
13m
12c
14c
12d
14d
12e
14e
14f
NO2 0.0244 ( 0.020
NH2 0.00195 ( 0.00053
NO2 0.0080 ( 0.0020
NH2 0.00071 ( 0.00058
NO2 0.0104 ( 0.00063
NH2 0.00055 ( 0.00008
NO2 0.146 ( 0.040
NH2 0.00633 ( 0.0018
NO2 0.0971 ( 0.0178
NH2 0.00038 ( 0.00007
NH2 0.0043 ( 0.0005
NH2 0.00068 ( 0.00003
0.0014 ( 0.0004
>46.73
9.25
0.608 ( 0.267
0.055 ( 0.000
0.049 ( 0.01
<0.014 ( 0.004
0.037 ( 0.021
<0.014 ( 0.002
0.35 ( 0.17
0.047 ( 0.023
0.37 ( 0.04
<0.014 ( 0.002
<0.014 ( 0.002
0.015 ( 0.001
0.041 ( 0.002
>79
1835
>2041
>402
>2702
>631
>286
481
>48.66
9.75
>6082
13732
4690
CHdCHCN
CHdCHCN
CHdCHCN
>48.78
25.0
>100
8.83
45455
167
929
NO2 CHdCHCOMe
NO2 CHdCHCOMe
NO2 CHdCHCN
NO2 CHdCHCN
NH2 CHdCHCN
24.36
5.88
>100
22.63
21.69
88.17
18.94
>100
50.73
>43.96
>47.06
14.43
8.98
>453
>123842
3356
59.35
>6298
>1353
>6667
1237
3
H
CtN
13206
6400
1
8.96
a Data were reported in ref 11. b p24 ELISA was used to determine 50% effective concentration (EC50) against HIV-1 strains. c XTT assay was used to
determine the CC50 value that causes cytotoxicity to 50% cells. d SI (selective index) = CC50/EC50, NA = not active at the highest concentration tested.
in the presence of potassium terbutoxide16 in 53-91% yields.
Subsequently, diaryl-nitropyridines 11i-11m and diaryl-
mononitroaniline 12c were reduced by sodium hydrosulfite
dehydrate to provide corresponding diaryl-pyridinamines 13i,
13j, and 13 m, diarylaniline 14c, and an unexpected reduction
product 13k, in which both the nitro group and the para-
conjugated double bond on the phenoxy ring were reduced,
albeit in low yield. To improve the yield, 13k was then
prepared from 11k by using catalytic hydrogenation in THF
in 79% yield. Alternatively, selective reduction of diaryl-
dinitroanilines 12d and 12e was achieved by using formic
acid in the presence of Pd-C (10%) and triethylamine to give
corresponding diaryl-4-nitrobenzene-1,2-diamines 14d and
14e, respectively, in which the reduced amino group was
identified as ortho to the NH-linked aniline.12 Additionally,
12e was reduced completely by using sodium borohydride in
the presence of antimony(III) chloride (SbCl3) to provide
diamino compound 14f. Scheme 4 shows the syntheses of
compounds 15-20 with a 6-cyanonaphthoxy moiety as the
C-ring. The coupling of 8 and 10 with 6-cyano-2-naphthol or
1-bromo-6-cyano-2-naphthol by using microwave irradiation
followed by the nitro reduction or selective reduction afforded
corresponding diarylpyridines 15-16 and diarylanilines
17-20, respectively, in which the para-cyanovinyl group was
incorporated into a fused ring system.
Results and Discussion
The synthesized diarylpyridines 11, 13, 15, and 16 (A series)
and diarylanilines 12c-12e, 14, and 17-20 (B series) were first
tested against infection by wild-type HIV-1 strains NL4-3 and
IIIB of TZM-bl and MT-2 cells, respectively, with 1 tested in
parallel. HIV-1 was generally quite sensitive to the com-
pounds (low EC50 values), and both assays provided similar
patterns of antiviral activity. As shown in Table 1, most
compounds, except 11d, 11e, and 11j (EC50 >3 μM), were
highly potent in both assays. Notably, most amino-com-
pounds (R3 = NH2), including 13a-13f, 13i-13m, and
14c-14f, were much more potent (at least 10-fold) than the
corresponding nitropyridine and nitrobenzene compounds
(11 and 12, R3 = NO2). The most potent compounds were
13m, 14c, and 14e, with EC50 values e0.7 nM against HIV-1
NL4-3 infection of TZM-bl cells and <14 nM against HIV-1
IIIB replication in MT-2 cells. These three compounds exhib-
ited great potency than 1 (EC50 = 1.4 and 41 nM against
NL4-2 and IIIB, respectively) and comparable potency to 3.
These results further support our previous hypothesis that
the amino group on the central ring at the ortho position to
the 4-cyanoaniline A-ring is crucial for enhancing anti-HIV
activity.
Like the DAPY analogue 2, newly synthesized DAPD
compounds 13m, 14c, and 14e all contain a cyanovinyl group

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8291
Table 2. Data against HIV Drug-Resistant Viral Strain Replicationa
HIV-1 RTMDRb in TZM-bl cell line
A17 c in MT-2 cell line
EC50(μΜ) RF
>10
R1
R2
R3
EC50 (μΜ)
RF d
11a
13a
11b
13b
11f
13f
11m
13m
13c
13e
13k
12c
14c
14e
14f
3
H
I
NO2
NH2
NO2
NH2
NO2
NH2
NO2
NH2
NH2
NH2
NH2
NO2
NH2
NH2
NH2
NH2
0.0943 ( 0.009
0.00513 ( 0.00115
0.0776 ( 0.0137
0.0068 ( 0.0030
0.215 ( 0.033
0.0090 ( 0.002
0.0148 ( 0.0024
0.00059 ( 0.00009
0.0154 ( 0.0016
0.121 ( 0.0177
0.0032 ( 0.0008
0.0170 ( 0.0011
0.00179 ( 0.0013
0.00087 ( 0.0001
0.0020 ( 0.0003
0.00096 ( 0.0003
0.0010 ( 0.0004
1.55
0.67
1.55
0.93
1.94
1.34
1.85
0.83
2.23
1.33
1.64
1.63
3.25
2.29
0.47
1.41
0.71
>127
36
H
I
1.93 ( 0.06
>10
H
Br
Br
>222
106
H
3.49 ( 0.46
>10
H
CtCH
CtCH
>40
34
H
1.43 ( 0.26
>10
H
CHdCHCN
CHdCHCN
CH2CHdCH2
CtCCH(CH2)2
CH2CH2COMe
CHdCHCN
CHdCHCN
CHdCHCN
CHdCHCN
CtN
>204
14
H
0.20 ( 0.01
>10
>10
H
>149
>24
13
H
H
0.73 ( 0.058
>10
H
>270
43
H
0.60 ( 0.09
0.095 ( 0.001
3.05 ( 0.03
0.39 ( 0.065
0.33 ( 0.02
NO2
NH2
H
6.79
128
26
1
6.00
a Each compound was tested at least in triplicate. b HIV-1 RTMDR (obtained from AIDS Research and Reference Reagent Program, Division
of AIDS, NIAID, NIH), which contains mutations in RT amino acid residues L74 V, M41L, V106A, and T215Y, is resistant to AZT, ddI, nevirapine,
and other non-nucleoside RT inhibitors. c The multi-NRTI-resistant strain A17 from NIH with mutations at amino acids K103N and Y181C in the viral
RT domain is highly resistant to NRTIs.19 d Resistant fold.
as the R2 substituent at the para position on the C-ring. This
common feature suggests that the para-cyanovinyl moiety
may be a favorable structural moiety for anti-HIV activity.
This postulate was further confirmed by the high potency of
cyanovinyl compound 11m relative to corresponding com-
pounds withthe same R1 and R3 groups but many different R2
substituents (11a-11f, 11i, 11j, and 11k) and even our prior
active lead 3. However, incorporation of the cyanovinyl group
into a fused ring system, such as the 6-cyanonaphthol moiety
in 15-20, resulted in obviously reduced anti-HIV activity
(EC50 > 0.1 μM in both assays), much less than most com-
pounds in Table 1. These results suggest that flexibility of the
R2 group might alsobenecessary. Furtheranalysisofdifferent
R2 groups indicated that hydrophobic and more linear sub-
stituents, such as bromo (Br, 13b), allyl (CH2CHdCH2, 13c),
ethynyl(CtCH, 13f), andconjugated R,β-unsaturatedketone
(CHdCHCOMe, 14d), were favorable for anti-HIV activity
regardless of substituent length. Compound 13k with a more
flexible para substituent (-CH2CH2COCH3) on the C-ring
exhibited potent anti-HIV-1 activity comparable to that of 1
but was slightly less active than 13m, 14c, and 14e. In contrast,
R2 substituents with increased bulk (11d, 11e, 13d, 13e) or
polarity (11g, 11i, 11j, 13i, 13j) led to reduced antiviral
potency. Thus, the hydrophobic tunnel on the western wing
of the NNRTI binding site might be very narrow and only
allow a straight linear, more flexible, hydrophobic substituent
to enter and adjust to an appropriate conformation.
compound pairs: 11a vs 13a, 11b vs 13b, 11f vs 13f, 11m vs
13m, and 12c vs 14c. Comparison of 14e with 14c and 14f
indicated that an additional nitro-group (R1) on the central
ring was obviously preferable to NH2 or H at the same
position against multi-RTI-resistant HIV-1 strains. Addition-
ally, 14e was also the least cytotoxic of the three compounds
(see Table 1). The most potent compounds 13m and 14e
against wild-type HIV still exhibited high potency against
multidrug-resistant viral strains HIV-1RTMDR1 and A17 with
low EC50 values (0.59 and 0.87 nM; 0.20 and 0.095 μM,
respectively) and were more potent than our prior lead 3
(EC50 0.96 nM and 0.39 μM, respectively) and 1 (EC50 1.0 nM
and 0.33 μM, respectively). Notably, 14e showed a low
resistant fold (RF) of 6.79 against viral strain A17 with double
mutant K103N/Y181C. This value is similar to that of 1
(RF = 6) in the same assay and in the literature (RF = 5),17
thus suggesting that 14e would be a promising new lead for
further development.
To understand the binding mode of the newly active
compounds, molecular modeling was conducted by using
the software Discovery Studio 2.5 and crystal structures of
the wild-type RT/1 (PDB: 3mec) complex18 and the K103N/
Y181C RT/2 (PDB: 3bgr) complex. As shown in Figure 2,
the most active compound (14e) and 1 were superposed into
both wild-type and mutant NNRTI binding sites. The
compounds showed similar binding orientations, horseshoe
conformational shapes, and hydrogen bonds between the
secondary amine linker and the main-chain carbonyl of
Furthermore, 15 active compounds from the A and B series
were tested against two HIV-1 strains resistant to multiple
NRTI/NNRTIs, including RTMDR and A17. The data in
Table 2 indicate that, in regard to the R3 substituent, the
amino-compounds (13 and 14) were again more effective than
the nitro-compounds (11 and 12) against the multi-RTI-
resistant HIV-1 strains, as shown by comparing the following
˚
K101 (1.88 and 1.85 A, respectively). However, an additional
hydrogen bond was observed between the amino group on
˚
the central ring of 14e and the K101 carbonyl (2.31 A) in both
sites, which is consistent with our previous results.12 When
compared to 1 bound with the wild-type RT structure,
the cyanovinyl group of 14e is protruded deeply into a

8292 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Tian et al.
Figure 2. Binding mode of 14e (green) with (A) HIV-1 wild-type RT (PDB: 3mec) and (B)K103N/Y181C mutant RT (PDB: 3bgr),
respectively. Drug 1 (yellow) was superposed into the same pocket for comparison. The hydrogen bonds between the inhibitors and K101
are illustrated with dashed lines (pink). The side chains of key amino acids on the binding site surface are indicated with solid lines (gray).
cylindrical open hydrophobic tunnel, which is formed by
the side chains of amino acid residues Y188, F227, W229,
and L234 and connected to the nucleic acid-binding cleft.
Accordingly, the cyanovinyl group might disrupt the viral
transcription from RNA to DNA more effectively. As ob-
served in Figure 2B and reported about 2,14 the interactions
of the cyanovinyl with the hydrophobic tunnel were con-
served despite rearrangements in mutated RT. The cyano-
vinyl also is likely involved in supplementary interaction
highly potent against wild-type and multidrug resistant viral
strains. The promising data results and readily accessible
synthetic methods greatly encourage us to perform further
studies for developing and identifying new drug candidates
with desirable drug-like properties.
Experimental Section
Chemistry. Melting points were measured with an RY-1
melting apparatus without correction. The proton nuclear
magnetic resonance (1H NMR) spectra were measured on a
JNM-ECA-400 (400 MHz) spectrometer using tetramethyl-
silane (TMS) as the internal standard. The solvent used was
DMSO-d6 unless otherwise indicated. Mass spectra (MS) were
measured on an ABI Perkin-Elmer Sciex API-150 mass spectrom-
eter with electrospray ionization, and the relative intensity of
each ion peak is presented as percent (%). The purities of target
compounds were g95%, measured by HPLC analyses, which
were performed by Agilent 1100 HPLC system with a UV
detector, using an Agilent TC-C18 column (250 mm ꢀ 4.6 mm,
5 μm) eluting with a mixture of solvents A and B (condition 1:
acetonitrile/water with 0.1% formic acid 80:20, flow rate
1.2 mL/min; condition 2: MeOH/water with 0.1% formic acid
80:20, flow rate 1.0 mL/min; UV 254 nm). The microwave
reactions were performed on a microwave apparatus from
Biotage, Inc. Thin-layer chromatography (TLC) and prepara-
tive TLC were performed on silica gel GF254 plates. Silica gel
(200-300 mesh) from Qingdao Haiyang Chemical Company
was used for column chromatography. Medium-pressure col-
umn chromatography was performed using a CombiFlash
Companion purification system. All chemicals were obtained
from Beijing Chemical Works or Sigma-Aldrich, Inc.
˚
with the shifted Y183 (distance 3.53 A) to compensate the
affinity loss of Y181C mutation. Therefore, the extensive
interactions of the cyanovinyl group and additional H-bond
may explain why 14e, as well as 13m and 14c, is more potent
than 1. On the other hand, modeling results indicated that
the nitro group was superposed very well with the bromine
atom of 1, although no interaction of these groups (NO2 or
Br) with the binding site was observed, implying that this
position on the central ring would be modifiable for explor-
ing more SAR and new compounds with high potency
against both wild-type and mutant viral strains.
Conclusion
Thirty-five target compounds of A and B series (11-20)
were newly synthesized and evaluated for anti-HIV activity.
Most of them showed potent anti-HIV activity (EC50 <1 μM),
but amino compounds, 2,6-diarylpyridin-3-amine (13) and
2,4-diarylbenzen-1,2-diamine (14) derivatives, were much more
potent against wild-type and multidrug-resistant viral strains
than corresponding nitro compounds. The most promising
compounds 13m and 14e exhibited high potency against wild-
type and multidrug-resistant viral strains with EC50 values at
subnanomolar level and were more potent than 1 in the same
assays. The modifications of the C-ring and substituents on
the central B-ring provided more structure-activity relation-
ship (SAR) correlations as follows: (1) both 2-pyridinamine
and aniline could serve as the central moieties in our com-
pounds, (2) an appropriately positioned amino (R3) group on
the central B-ring is crucial for achieving high potency, (3)
para-cyanovinyl (R2) on the phenoxy C-ring is a beneficial
substituent, (4) a favorable para-R2 substituent on the C-ring
should be hydrophobic, flexible, and more linear to insert into
the narrow tunnel on the NNRTI binding site, and (5) an
additional nitro group (R1) on the central ring ortho to the
C-ring could benefit inhibitory activity and reduce cytotoxity.
Therefore, 2,6-diarylpyridin-3-amines and 2,4-diarylbenzen-
1,2-diamines have been discovered as novel NNRTI agents
6-Chloro-2-(40-cyanoaniline)-3-nitropyridine (8). A mixture of
2,6-dichloro-3-nitropyridine (5, 1.93 g, 10 mmol) and 4-amino-
benzonitrile (1.42 g, 12 mmol) was heated at 140 ꢀC without
solvent under N2 protection for 4 h. After cooling to room tem-
perature, DMF (ca. 15 mL) was added into the flask to dissolve
the mixture. The solution was then poured into ice-water and
the pH adjusted to 2-3 with aq HCl (5%). The resulting solid
was collected and recrystallized from 95% EtOH to give pure 8
(2.01 g, 73% yield), yellow solid, mp 175-178 ꢀC. 1H NMR δ
ppm 10.47 (1H, br s, NH), 8.53 (1H, d, J = 8.4 Hz, ArH-4), 7.86
(2H, d, J=8.8Hz, ArH-20, 60), 7.70(2H, d, J=8.8Hz, ArH-30, 50),
6.96 (1H, d, J = 8.4 Hz, ArH-5). MS m/z (%) 275 (M þ 1, 100),
276 (M þ 2, 28).
5-Chloro-N1-(40-cyanophenyl)-2-nitroaniline (9). To a mixture
of 2,4-dichloronitrobenzene (6, 1.92 g, 10 mmol) and 4-amino-
benzonitrile (1.30 g, 11 mmol) in DMF (20 mL) was added
t-BuOK (2.24 g, 20 mmol) slowly in an ice bath and then stirred
at room temperature for 24 h and monitored by TLC. The
mixture was poured into ice-water and pH was adjusted to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8293
2-3 with 5% aq HCl. The red solid was collected, washed to
neutral, and dried to afford 1.75 g of 9 in 64% yield, mp 221-
224 ꢀC. 1H NMRδppm9.56(1H, br, NH), 8.20(1H, d, J=9.0Hz,
ArH-3), 7.72 (2H, d, J = 9.0 Hz, ArH-20, 60), 7.38 (1H, d, J =
2.0 Hz, ArH-6), 7.36 (2H, d, J = 9.0 Hz, ArH-30, 50), 6.91 (1H, d,
J=8.4Hz, ArH-4). MSm/z(%) 272(M- 1, 100), 274 (M þ 1, 28).
5-Chloro-N1-(40-cyanophenyl)-2,4-dinitroaniline (10). To a
solution of 1,3-dichloro-4,6-dinitrobenzene (7, 237 mg, 1 mmol)
and 4-cyanoaniline (130 mg, 1.1 mmol) in DMF (3 mL) was
slowly added potassium tert-butoxide (2 equiv) at ice-water
bath temperature with stirring and then at room temperature for
about 1 h, monitored by TLC until the reaction was completed.
The mixture was poured into ice-water, pH was adjusted to
6 with 5% aq HCl, and solid was collected and washed with
water, The crude product was purified by recrystallization from
EtOH to afford 284 mg of 10 in an 89% yield, yellow solid, mp
174-176 ꢀC. 1H NMR (CDCl3) δ ppm 7.41 (1H, s, ArH-6), 7.57
(2H, d, J=8.8Hz, ArH-20, 60), 7.91(2H, d, J=8.8Hz, ArH-30, 50),
8.90 (1H, s, ArH-3), 10.07 (1H, s, NH). MS m/z (%) 319
(M þ 1, 100), 321 (M þ 3, 23).
(420 mg, 5 mmol) slowly under N2 protection at room tempera-
ture with stirring for 7 h. The mixture was poured into water and
extracted with CH2Cl2 three times. After removal of solvent
under reduced pressure, residue was purified by flash silica chro-
matography (gradient eluent: EtOAc/petroleum ether 0-40%)
to provide 362 mg of pure 11d, 78% yield, yellow solid, mp
114-116 ꢀC. 1H NMR δ ppm 10.37 (1H, br, NH), 8.67 (1H, d,
J = 8.8 Hz, ArH-4), 7.40 and 7.34 (each 2H, d, J = 8.8 Hz,
ArH), 7.31 (2H, s, ArH), 6.83 (1 H, d, J = 8.8 Hz, ArH-5), 5.47
(1H, s, OH), 2.00 (6H, s, ArCH3 ꢀ 2), 1.53 (6H, s, CH3 ꢀ 2).
MS m/z (%) 465 (M þ Na, 25), 425 (M - OH, 100). HPLC
purity: 98.25%.
N-(40-Cyanophenyl)-6-[400-(2-cyclopropylethynyl)-200,600-dimethyl]-
phenoxy-3-nitro-pyridin-2-amine (11e). The procedure was the
same as for synthesis of 11d, starting with 11a (486 mg, 1 mmol)
and ethynylcyclopropane (330 mg, 5 mmol), to provide 290 mg
of 11e in a 68% yield, yellow solid, mp 188-192 ꢀC. 1H NMR
(CDCl3) δ ppm 10.65 (1H, br, NH), 8.61 (1H, d, J = 9.2 Hz,
ArH-4), 7.36 (2H, d, J = 8.8 Hz, ArH), 7.22 (2H, s, ArH), 7.19
(2H, d, J = 8.8 Hz, ArH), 6.64 (1H, d, J = 9.2 Hz, ArH-5), 2.05
(6H, s, CH3 ꢀ 2), 1.52 (1H, m, CH), 0.89 (4H, m, CH2 ꢀ 2).
MS m/z (%) 425 (M þ 1, 100). HPLC purity: 96.95%.
General Procedure for the Preparation of N2-(40-Cyanophenyl)-
3-nitro-6-(400-substituted-200,600-dimethyl)phenoxypyridin-2-amines
(11a-11c, 11g). A mixture of 8 (1 equiv) and a para-substituted
2,6-dimethylphenol (1.2 equiv) in DMF in the presence of
anhydrous K2CO3 (excess, 3.5 equiv) was heated at 130-140 ꢀC
for 6-8 h. The mixture was poured into ice-water and pH was
adjusted to 2-3 with 5% aq HCl. Crude solid product was
collected and washed to neutral, followed by silica gel column
purification (eluant: CH2Cl2) to give corresponding pure target
compounds.
N2-(40-Cyanophenyl)-6-(200,600-dimethyl-400-ethynyl)phenoxy-
3-nitropyridin-2-amine (11f). A solution of 11d (422 mg, 1 mmol)
in toluene (20 mL) in the presence of powdered NaOH (16 mg,
0.4 mmol) was refluxed for 24 h under nitrogen protection. After
cooling to room temperature, 2 drops of HOAc was dropped
into the flask. The organic phase was washed with water and
solvent was evaporated. The residue was purified on a flash
silica column (gradient eluent: EtOAc/petroleum ether 0-40%)
to provide 265 mg of 11f in 69% yield, yellow solid, mp 186-
188 ꢀC. 1H NMR (CDCl3) δ ppm 10.66 (1H, br, NH), 8.63
(1H, d, J = 8.8 Hz, ArH-4), 7.35 (2H, d, J = 8.8 Hz, ArH), 7.34
(2H, s, ArH), 7.19 (2H, d, J = 8.8 Hz, ArH), 6.66 (1 H, d, J =
8.8 Hz, ArH-5), 3.17 (1H, s, tCH), 2.09 (6H, s, CH3 ꢀ 2). MS m/z
(%) 385 (M þ 1, 100). HPLC purity: 96.63%.
N2-(40-Cyanophenyl)-6-(200,600-dimethyl-400-iodo)phenoxy-
3-nitropyridin-2-amine (11a). Yield 82%, starting with 275 mg
(1.0 mmol) of 8 and 297 mg (1.2 mmol) of 2,6-dimethyl-
4-iodophenol to afford 399 mg of 11a, yellow solid, mp
162-165 ꢀC. 1H NMR δ ppm 10.65 (1H, br, NH), 8.63 (1H, d,
J = 8.8 Hz, ArH-4), 7.54 (2H, s, ArH-300, 500), 7.38 (2H, d, J =
8.8 Hz, ArH), 7.21 (2H, d, J = 8.8 Hz, ArH), 6.65 (1 H, d, J =
8.8 Hz, ArH-5), 2.06 (6H, s, CH3 ꢀ 2). MS m/z (%) 487 (M þ 1,
100). HPLC purity: 98.63%.
N-(40-Cyanophenyl)-6-[200,600-dimethyl-400-(2-nitroethan-l-o1)]-
phenoxy-3-nitropyridin-2-amine (11i). To a solution of 11g (388mg,
1 mmol) and CH3NO2 (2 mL, excess) in THF (20 mL) was added
dropwise 33% aq NaOH (1 mL) at ice-water bath and stirred for
additional 1 h and then at room temperature for 12 h. The mixture
was poured into ice-water, pH was adjusted with 5% aq HCl to
neutral, and the mixture extracted with CH2Cl2. After removal of
organic solvent in vacuo, residue was purified on a silica gel column
(eluent: CH2Cl2/MeOH = 60/1) to afford 306 mg of 11i in a 68%
yield, yellow solid, mp 230-234 ꢀC. 1H NMRδ ppm 10.43 (1H, br,
NH), 8.67 (1H, d, J = 8.8 Hz, ArH-4), 7.49 and 7.40 (each 2H, d,
J = 8.4 Hz, ArH on A-ring), 7.33 (2H, s, ArH on C-ring), 6.81
(2H, d, J = 8.8 Hz, ArH-5), 6.32 (1H, d, J= 5.6 Hz, OH), 5.33 and
4.94 (each 1H, m, CH2NO2), 4.66 (1H, m, CHOH), 2.06 (6H, s,
CH3 ꢀ 2). MS m/z (%) 450 (M þ 1, 100), 472 (M þ Na, 95). HPLC
purity: 97.39%.
6-(400-Bromo-200,600-dimethyl)phenoxy-N2-(40-cyanophenyl)-
3-nitropyridin-2-amine (11b). Yield 64%, starting with 275 mg
(1.0 mmol) of 8 and 241 mg (1.2 mmol) of 2,6-dimethyl-
4-bromophenol to afford 280 mg of 11b, yellow solid, mp
193-194 ꢀC. 1H NMR δ ppm 10.65 (1H, br, NH), 8.63 (1H, d,
J = 9.0 Hz, ArH-4), 7.37 (4H, m, ArH), 7.21 (2H, d, J =
8.8 Hz, ArH), 6.65 (1H, d, J = 9.0 Hz, ArH-5), 2.08 (6H, s,
CH3 ꢀ 2). MS m/z (%) 439 (M þ 1, 100), 441 (M þ 3, 90). HPLC
purity: 97.24%.
6-(400-Allyl-200,600-dimethyl)phenoxy-N-(40-cyanophenyl)-
3-nitropyridin-2-amine (11c). Yield 56%, starting with 275 mg
(1.0 mmol) of 8 and 194 mg (1.2 mmol) of 4-allyl-2,6- dimethyl-
phenol to afford 225 mg of 11c, yellow solid, mp 193-195 ꢀC. 1H
NMR δ ppm 10.67 (1H, br, NH), 8.61 (1H, d, J = 8.8 Hz, ArH-4),
7.27 (4H, m, ArH), 7.01 (2H, s, ArH), 6.63 (1H, d, J = 8.8 Hz,
ArH-5), 6.05 (1H, m, CHd), 5.24 (2H, m, CH2d), 3.44 (2H, d,
J = 3.2 Hz, CH2), 2.08 (6H, s, CH3 ꢀ 2). MS m/z (%) 401 (M þ 1,
100). HPLC purity: 95.22%.
N-(40-Cyanophenyl)-6-(200,600-dimethyl-400-formyl)phenoxy-
3-nitropyridin-2-amine (11g). Yield 66%, starting with 275 mg
(1.0 mmol) of 8 and 180 mg (1.2 mmol) of 4-hydroxy-3,5-
dimethylbenzaldehyde to afford 257 mg of 11g, yellow solid, mp
226-228 ꢀC. 1H NMR δ ppm 10.68 (1H, br, NH), 10.08 (1H, s,
CHO), 8.66 (1H, d, J = 8.8 Hz, ArH-4), 7.75 (2H, s, ArH), 7.20
(4H, s, ArH), 6.69 (1H, d, J = 8.8 Hz, ArH-5), 2.21 (6H, s,
CH3 ꢀ 2). MS m/z (%) 389 (M þ 1, 100). HPLC purity: 97.90%.
N-(40-Cyanophenyl)-6-[200,600-dimethyl-400-(3-methylbut-3-ol-1-yn)-
phenoxy]-3-nitropyridin-2-amine (11d). To a mixture of 11a
(486 mg, 1 mmol), Pd(PPh3)2Cl2 (69 mg, 0.1 mmol), and cuprous
iodide (19 mg, 0.1 mmol) in anhydrous DMF (5 mL) in the pre-
sence of triethylamine (0.6 mL) was added 2-methylbut-3-yn-2-ol
(E)-6-(400-(2-Carboxyvinyl)-200,600-dimethyl)phenoxy-N-(40-
cyanophenyl)-3-nitropyridin-2-amine (11j). A mixture of 11g
(388 mg, 1 mmol) in pyridine (5 mL) and malonic acid (416 mg,
4 mmol) was refluxed in the presence of piperidine (0.15 mL) for
2 h. Then the mixture was poured into ice-water and pH was
adjusted to 2-3 with 5% aq HCl. After standing overnight, the
solid was collected and washed with cold water to afford 377 mg
of 11j in 88% yield, yellow solid, mp 258-260 ꢀC. 1H NMR δ
ppm 12.43 (1H, br, COOH), 10.38 (1H, br, NH), 8.68 (1H, d,
J = 8.8 Hz, ArH-4), 7.65 (1H, d, J = 16.0 Hz, dCHCOOH),
7.62 (2H, s, ArH on C-ring), 7.37 and 7.32 (each 2H, d, J = 8.8
Hz, ArH on A-ring), 6.70 (1H, d, J = 8.8 Hz, ArH-5), 6.60 (1H, d,
J = 16.0 Hz, ArCHd), 2.07 (6H, s, CH3 ꢀ 2). MS m/z (%) 431
(M þ 1, 30), 453 (M þ Na, 70), 413 (M - OH, 100). HPLC
purity: 98.73%.
(E)-N2-(40-Cyanophenyl)-6-(400-(r,β-unsaturated-but-3-)one-
200,600-dimethyl)phenoxy-3-nitropyridin-2-amine (11k). To a solu-
tion of 11g (388 mg, 1 mmol) in acetone (20 mL) and MeOH

8294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Tian et al.
(5 mL) was added 10% aq NaOH (2 mL) slowly at ice-water
bath temperature. Then the mixture was stirred at room tem-
perature for 1 h, poured into ice-water, and pH adjusted to 4-5
with 5% aq HCl. The solid was filtered, washed with water, and
purified on a silica gel column (gradient eluent: EtOAc/petro-
leum ether 0-50%) to obtain 155 mg of 11k in 36% yield, yellow
solid, mp 194-197 ꢀC. 1H NMR δ ppm (CDCl3) 10.66 (1H, br,
NH), 8.64 (1H, d, J = 9.2 Hz, ArH-4), 7.56 (1H, d, J = 16.0 Hz,
ArCHd), 7.39 (2H, s, ArH on C-ring), 7.22 (4H, s, ArH on
A-ring) 6.78 (2H, 1H, d, J = 16.0 Hz, dCHCO), 6.66 (1H, d,
J = 9.2 Hz, ArH-5), 2.46 (3H, s, COCH3), 2.14 (6H, s, CH3 ꢀ 2).
MS m/z (%) 429 (M þ 1, 100), 387 (M - 43, 50). HPLC purity:
97.99%.
(E)-N-(40-Cyanophenyl)-6-(400-cyanovinyl-200,600-dimethyl)-
phenoxy-3-nitropyridin-2-amine (11m). To a solution of diethyl
cyanomethyl phosphonate (266 mg, 1.5 mmol) in THF (15 mL)
was added t-BuOK (168 mg, 1.5 mmol) at ice-water bath
temperature with stirring for 30 min. Compound 11g (388 mg,
1 mmol) in THF (15 mL) was added dropwise into the above
mixture at room temperature and then stirring continued for
2 days. The reaction was then quenched with water and extracted
with EtOAc. After removal of solvent under reduced pressure,
the residue was purified by silica gel column chromatography
(eluent: CH2Cl2/MeOH = 30/1) to afford 333 mg of 11m in an
81% yield, yellow solid, mp 231-233 ꢀC. 1H NMR δ ppm 10.37
(1H, br, NH), 8.68 (1H, d, J = 8.8 Hz, ArH-4), 7.72 (1H, d, J =
16.8 Hz, ArCHdC), 7.56 (2H, s, ArH on C-ring), 7.34 (4H, s,
ArH on A-ring), 6.84 (1H, d, J = 8.8 Hz, ArH-5), 6.53 (1H, d,
J = 16.8 Hz, dCHCN), 2.06 (6H, s, CH3 ꢀ 2). MS m/z (%) 412
(M þ 1, 100), 434 (M þ Na, 30). HPLC purity: 98.08%.
(E)-N2-(40-Cyanophenyl)-4-(400-cyanovinyl-200,600-dimethyl)-
phenoxy-1-nitroaniline (12c). The same method as the prepara-
tion of 11m, starting with 387 mg (1 mmol) of 12a and 266 mg
(1.5 mmol) of (EtO)2P(O)CH2CN to afford 372 mg of 12c, yield
91%, yellow solid, mp 178-181 ꢀC. 1H NMR δ ppm 9.55 (1H,
br, NH), 8.18 (1H, d, J = 9.2 Hz, ArH-6), 7.73 (1H, d, J =
8.8 Hz, ArH-30,50), 7.60 (1H, d, J = 16.8 Hz, ArCHd), 7.51 (2H,
s, ArH-300, 500), 7.32 (2H, d, J = 8.8 Hz, ArH-20, 60), 6.62 (1H, d,
J = 2.8 Hz, ArH-3), 6.51 (1H, dd, J = 9.2 and 2.8 Hz, ArH-4),
6.44 (1H, d, J = 16.8 Hz, dCHCN), 2.08 (6H, s, CH3 ꢀ 2).
MS m/z (%) 411 (M þ 1, 100), 433 (M þ Na, 85). HPLC purity:
95.40%
The mixture was then poured into ice-water and extracted with
EtOAc. After removal of solvent in vacuo, the residue was
purified on a silica gel column (eluent: CH2Cl2/MeOH = 30/1)
to obtain corresponding diarylpyridinamines. Method B: To
a solution of a diaryl-nitropyridine or diaryl-nitrobenzene
(1 equiv) in THF and MeOH (30 mL, v/v 1:1) was added
NiCl2 6H2O (0.3 equiv) and NaBH4 (4 equiv) successively at
3
ice-water bath temperature. Then the mixture was stirred in the
ice-water bath for another 30 min until the starting material
disappeared, as monitored by TLC. The mixture was poured
into water, pH adjusted to 4-5, warmed to 40-50 ꢀC for 10 min,
and then extracted with EtOAc. After removal of solvent in
vacuo, crude product was purified on a silica gel column (eluent:
CH2Cl2/MeOH = 30/1) to provide corresponding products.
N2-(40-Cyanophenyl)-6-(200,600-dimethyl-400-iodo)phenoxypyridin-
2,3-diamine (13a). Method A, yield 49%, starting with 486 mg
(1.0 mmol) of 11a to afford 225 mg of 13a, brown solid, mp
186-188 ꢀC. 1H NMR (CDCl3) δ ppm 7.50 (2H, s, NH2), 7.33
(2H, d, J = 8.8 Hz, ArH), 7.22 (2H, d, J = 8.8 Hz, ArH), 6.98
(1H, d, J = 8.4 Hz, ArH-4), 6.44 (1H, d, J = 8.4 Hz, ArH-5),
2.09 (6H, s, CH3 ꢀ 2). MS m/z (%) 457 (M þ 1, 100). HPLC
purity: 95.72%.
6-(400-Bromo-200,600-dimethyl)phenoxy-N2-(40-cyanophenyl)-
pyridin-2,3-diamine (13b). Method B, yield 82%, starting with
439 mg (1.0 mmol) of 11b to afford 336 mg of 13b, brown solid,
mp 190-193 ꢀC. 1H NMR δ ppm 8.35 (1H, br s, NH), 7.42 (2H,
s, ArH), 7.32 (4H, s, ArH), 7.12 (1H, d, J = 8.4 Hz, ArH-4), 6.44
(1H, d, J = 8.4 Hz, ArH-5), 4.84 (2H, br, NH2), 2.03 (6H, s,
CH3 ꢀ 2). MS m/z (%) 409 (M þ 1, 100), 411 (M þ 3, 90). HPLC
purity: 97.36%.
6-(400-Allyl-200,600-dimethyl)phenoxy-N2-(40-cyanophenyl)pyridin-
2,3-diamine (13c). Method A, yield 43%, starting with 400 mg
(1.0 mmol) of 11c to afford 160 mg of 13c, brown solid,
mp 150-152 ꢀC. 1H NMR δ ppm 8.30 (1H, br, NH), 7.36 and
7.29 (each 2H, d, J = 8.8 Hz, ArH), 7.11 (1H, d, J = 8.0 Hz,
ArH-4), 6.98 (2H, s, ArH), 6.39 (1H, d, J = 8.0 Hz, ArH-5), 6.03
(1H, m, CHd), 5.14 (2H, m, CH2d), 4.76 (2H, br, NH2), 3.36
(2H, d, J = 3.6 Hz, CH2), 2.01 (6H, s, CH3 ꢀ 2). MS m/z 371
(M þ 1, 100). HPLC purity: 97.40%.
N2-(40-Cyanophenyl)-6-[200,600-dimethyl-400-(3-methylbut-3-ol-
1-yn)phenoxy]pyridin-2,3-diamine (13d). Method A, yield 48%,
starting with 442 mg (1.0 mmol) of 11d to afford 198 mg of 13d,
1
(E)-N1-(40-Cyanophenyl)-5-[200,600-dimethyl-400-(3-oxobut-1-enyl)]-
phenoxy-2,4-dinitro-aniline (12d). The same method as the pre-
paration of 11k, starting with 432 mg of 12b to afford 152 mg of
12d, yield 32%, brown solid, mp 213-215 ꢀC. 1H NMR δ ppm
(CDCl3) 9.94 (1H, br, NH), 9.18 (1H, s, ArH-3), 7.53 (2H, d, J =
8.4 Hz, ArH-30,50), 7.43 (1H, d, J = 16.4 Hz, ArCHd), 7.31 (2H, s,
ArH-300, 500), 7.11 (2H, d, J = 8.4 Hz, ArH-20,60), 6.68 (1H, d,
J = 16.4 Hz, dCHCN), 6.25 (1H, s, ArH-6), 2.42 (3H, s,
COCH3), 2.16 (6H, s, CH3 ꢀ 2). MS m/z (%) 473 (M þ 1,
100), 495 (M þ Na, 80). HPLC purity: 99.47%.
brown solid, mp 155-157 ꢀC. H NMR δ ppm 8.33 (1H, br,
NH), 8.01 (1H, br, OH), 7.41 (1H, d, J = 8.8 Hz, ArH-4), 7.29
(4H, m, ArH), 7.23 (2H, s, ArH), 6.55 (1 H, d, J = 8.8 Hz, ArH-5),
4.83 (2H, br, NH2), 2.08(6H, s, Ar-CH3 ꢀ 2), 1.51(6H, s, CH3 ꢀ 2).
MS m/z (%) 413 (M þ 1, 100). HPLC purity: 97.16%.
N2-(40-Cyanophenyl)-6-[400-(2-cyclopropylethynyl)-200,600-
dimethylphenoxy]pyridin-2,3-diamine (13e). Method A, yield
49%, starting with 424 mg (1.0 mmol) of 11e to afford 193 mg
1
of 13e, brown solid, mp 82-85 ꢀC. H NMR (CDCl3) δ ppm
7.33 (2H, d, J = 8.4 Hz, ArH-30, 50), 7.22 (2H, s, ArH), 7.18 (3H,
m, ArH-20, 60 and ArH-4), 6.36 (1H, d, J = 8.0 Hz, ArH-5), 2.06
(6H, s, CH3 ꢀ 2), 1.51 (1H, m, CH), 0.89 (4H, m, CH2 ꢀ 2).
MS m/z (%) 395 (M þ 1, 100). HPLC purity: 96.99%.
(E)-N1-(40-Cyanophenyl)-5-(400-cyanovinyl-200,600-dimethyl)-
phenoxy-2,4-dinitroaniline (12e). The same method as the pre-
paration of 11m, starting with 432 mg (1 mmol) of 12b and 266
mg (1.5 mmol) of (EtO)2P(O)CH2CN to afford 240 mg of 12e,
yield 53%, yellow solid, mp 242-244 ꢀC. 1H NMR (CDCl3) δ
ppm 9.97 (1H, br, NH), 9.19 (1H, d, J = 9.2 Hz, ArH-3), 7.55
(1H, d, J = 8.8 Hz, ArH-30,50), 7.32 (1H, d, J = 16.4 Hz,
ArCHd), 7.21 (2H, s, ArH-300, 500), 7.12 (2H, d, J = 8.8 Hz,
ArH-20, 60), 6.26 (1H, d, s, ArH-6), 5.86 (1H, d, J = 16.4 Hz,
dCHCN), 2.18 (6H, s, CH3 ꢀ 2). MS m/z (%) 456 (M þ 1, 100).
HPLC purity: 97.15%.
N2-(40-Cyanophenyl)-6-(200,600-dimethyl-400-ethynyl)phenoxy-
pyridin-2,3-diamine (13f). Method A, yield 61%, starting with
384 mg (1.0 mmol) of 11f to afford 217 mg of 13f, brown solid,
mp 79-82 ꢀC. 1H NMR δ ppm 8.34 (1H, br, NH), 7.37 (2H, s,
ArH on C-ring), 7.03 (4H, m, ArH on A-ring), 6.56 (1H, d, J =
8.4 Hz, ArH-4), 6.44 (1H, d, J = 8.4 Hz, ArH-5), 4.84 (2H, br,
NH2), 3.36 (1H, s, CHt), 2.02 (6H, s, CH3 ꢀ 2). MS m/z (%) 355
(M þ 1, 100). HPLC purity: 98.03%.
General Reduction Methods for Nitro Group(s) on the Central
Ring (Pyridine or Benzene) to Prepare 13a-13f, 13i, 13j, 13m,
and 14c. Method A: To a solution of a diaryl-nitropyridine
(1 mmol) in THF and water (40 mL, v/v 1:1) was added ammonia
(0.5 mL) and Na2S2O4 (90%, 10 mmol) successively. The
mixture was stirred at room temperature for 2 h monitored
by TLC (CH2Cl2/MeOH: 15/1) until reaction was completed.
N2-(40-Cyanophenyl)-6-[200,600-dimethyl-400-(2-nitroethan-l-o1)]-
phenoxypyridin-2,3-diamine Hydrochloride (13i). Method A,
yield 42%, starting with 90 mg (0.2 mmol) of 11i to provide
amino compound, which was treated with 20% HCl-ether
solution in acetone to afford 38 mg of 13i, white solid, dec
>230 ꢀC. 1H NMR δ ppm 9.28 (1H, br, NH), 7.58 (1H, d, J =
8.4 Hz, ArH-4), 7.43 and 7.38 (each 2H, d, J = 9.2 Hz, ArH on

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8295
A-ring), 7.27 (2H, s, ArH on C-ring), 6.59 (1H, d, J = 8.4 Hz,
ArH-5), 5.29 (1H, dd, J = 3.6 and 9.8 Hz, CH2NO2), 4.90 (1H,
dd, J = 3.6 and 12.4 Hz, CH2NO2), 4.62 (1H, dd, J = 9.8 and
12.4 Hz, CH), 3.57 (2H, br, NH2), 2.05 (6H, s, CH3 ꢀ 2). MS m/z
(%) 420 (M þ 1, 100). HPLC purity: 99.44%.
ArH-300, 500), 6.76 (2H, d, J = 8.8 Hz, ArH-20,60), 6.31 (1H, s, ArH-
6), 5.82 (1H, d, J = 16.8 Hz, dCHCN), 3.55 (2H, br, NH), 2.18
(6H, s, CH3 ꢀ 2). MS m/z (%) 426 (M þ 1, 100), 448 (M þ Na, 20).
HPLC purity: 97.10%
(E)-N1-(40-Cyanophenyl)-5-(400-cyanovinyl-200,600-dimethyl)-
phenoxybenzene-1,2,4-triamine (14f). To the solution of 12e
(100 mg, 0.22 mmol) in EtOH (15 mL) in the presence of SbCl3
(201 mg, 0.33 mmol) was added NaBH4 (168 mg, 4.4 mmol) in
portions at ice-bath temperature, and then stirring continued at
room temperature for 40 min. The mixture was then poured into
ice-water, pH adjusted to 4 with 5% aq HCl, and stirring
continued for an additional 1 h. After the pH was adjusted to
9 with Na2CO3, the mixture was extracted with EtOAc three
times. After removal of solvent under reduced pressure, the
residue was purified on a silica gel column (eluent: CHCl3/
CH3OH = 40:1) to afford 56 mg of 14f, yield 64%, red solid, mp
160-162 ꢀC. 1H NMR (CDCl3) δ ppm 7.37 (2H, d, J = 8.4 Hz,
ArH-30,50), 7.28 (1H, d, J = 16.4 Hz, ArCHd), 7.16 (2H, s,
ArH-300, 500), 6.47 (2H, d, J = 8.4 Hz, ArH-20,60), 6.30 (1H, s,
ArH-3), 5.78 (1H, d, J = 16.4 Hz, dCHCN), 5.32 (1H, s, ArH-6),
4.01 and 3.51 (each 2H, br, NH2), 2.18 (6H, s, CH3 ꢀ 2).
MS m/z (%) 396 (M þ 1, 100). HPLC purity: 100.0%.
(E)-6-[400-(2-Acrylic)-200,600-dimethyl)phenoxy]-N2-(40-cyano-
phenyl)pyridin-2,3-diamine (13j). Method A, yield 80%, starting
with 86 mg (0.2 mmol) of 11j to afford 64 mg of 13j, deep-brown
solid, mp 245-7 ꢀC. 1H NMR δ ppm 8.41 (1H, s, NH), 7.38 (2H,
d, J = 9.2 Hz, ArH), 7.37 (2H, s, ArH-300, 500), 7.28 (1H, d, J =
15.6 Hz, ArCHd), 7.24 (2H, d, J = 9.2 Hz, ArH-20, 60), 7.11
(1H, d, J = 8.4 Hz, ArH-4), 6.43 (1H, d, J = 15.6 Hz, dCHCN),
6.39 (1H, d, J = 8.4 Hz, ArH-5), 4.83 (2H, br, NH2), 2.05 (6H, s,
CH3 ꢀ 2). MS m/z (%) 401 (M þ 1, 100), 383 (M - OH, 93).
HPLC purity: 99.48%.
(E)-N2-(40-Cyanophenyl)-6-(400-cyanovinyl-200,600-dimethyl)-
phenoxypyridin-2,3-diamine (13m). Method A, with a 50% yield,
starting with 411 mg (1 mmol) of 11m to afford 192 mg of 13m,
brown solid, mp 162-164 ꢀC. 1H NMR δ ppm 8.35 (1H, br, NH),
7.68 (1H, d, J = 16.8 Hz, ArCHdC), 7.50 (2H, s, ArH on
C-ring), 7.37 and 7.25 (each 2H, d, J = 8.8 Hz, ArH on A-ring),
7.13 (1H, d, J = 8.0 Hz, ArH-4), 6.45 (1H, d, J = 8.0 Hz, ArH-5),
6.45 (1H, d, J = 16.8 Hz, dCHCN), 4.84 (2H, br s, NH2), 2.06
(6H, s, CH3 ꢀ 2). MS m/z (%) 382 (M þ 1, 100). HPLC purity:
98.44%.
6-(6-Cyano-2-naphthoxy)-N2-(4-cyanophenylamino)-3-nitro-
pyridine (15). A mixture of 8 (54.9 mg, 0.2 mmol), 6-hydroxy-
2-naphthonitrile (50.8 mg, 0.3 mmol), and Cs2CO3 (227.5 mg,
0.7 mmol) in DMF (1 mL) was heated at 100 ꢀC for 10 min under
microwave irradiation. Then the mixture was poured into water
and pH was adjusted to 2-3 with aq HCl (1N). The solid was
collected and purified on a silica gel column (gradient eluent:
petroleum ether/CH2Cl2 0-60%) to afford 51 mg of 15, yield
63%, yellow solid, mp 264-6 ꢀC. 1H NMR δ ppm 10.27 (1H, s,
NH), 8.67 (1H, s, ArH-500), 8.61 (1H, d, J = 8.8 Hz, ArH-800),
8.17 (1H, d, J = 8.8 Hz, ArH-4), 8.04 (1H, d, J = 8.8 Hz, ArH-700),
7.87 (1H, d, J = 2.4 Hz, ArH-100), 7.81 (1H, d, J = 8.8 Hz,
ArH-400), 7.55 (1H, dd, J = 2.4 and 8.8 Hz, ArH-300), 7.26 (2H, d,
J = 8.8 Hz, ArH-30, 50), 6.93 (2H, d, J = 8.8 Hz, ArH-20, 60), 6.78
(1H, d, J = 8.8 Hz, ArH-5). MS m/z (%) 408 (M þ 1, 100).
HPLC purity: 99.60%.
(E)-N2-(40-Cyanophenyl)-4-(400-cyanovinyl-200,600-dimethyl)-
phenoxybenzene-1,2-diamine (14c). Method A, starting with 12c
(410 mg, 1 mmol) to afford 226 mg of 14c, yield 59%, brown
1
solid, mp 215-218 ꢀC. H NMR (CDCl3) δ ppm 7.46 (2H, d,
J = 8.8 Hz, ArH-30,50), 7.32 (1H, d, J = 16.8 Hz, ArCHd), 7.18
(2H, s, ArH-300, 500), 6.74 (1H, d, J = 9.2 Hz, ArH-6), 6.64 (2H,
d, J = 8.8 Hz, ArH-20,60), 6.58 (1H, d, J = 2.8 Hz, ArH-3), 6.51
(1H, dd, J = 9.2 and 2.8 Hz, ArH-5), 5.80 (1H, d, J = 16.8 Hz,
dCHCN), 3.52 (2H, br, NH2), 2.15 (6H, s, CH3 ꢀ 2). MS m/z
(%) 381 (M þ 1, 100). HPLC purity: 97.94%.
6-[(400-Butan-2-one)-200,600-dimethyl]phenoxy-N2-(40-cyanophenyl)-
pyridin-2,3-diamine (13k). A mixture of 11k (50 mg, 0.11 mmol)
in THF (20 mL) and excess Pd-C (10%) was hydrogenated at
65 psi for 6 h. After removal of Pd-C and solvent successively,
the residue was purified by flash silica gel column chromatog-
raphy (eluent: CH2Cl2/MeOH=100:1) to provide 37 mg of pure
13k, yield 79%, gray solid, mp 84-86 ꢀC. 1H NMR δ ppm 8.32
(1H, br, NH), 7.38 and 7.32 (each 2H, d, J = 9.2 Hz, ArH), 7.10
(1H, d, J = 8.0 Hz, ArH-4), 6.99 (2H, s, ArH), 6.37 (1H, d,
J = 8.0 Hz, ArH-5), 4.80 (2H, br, NH2), 4.79 (4H, m, CH2CH2),
2.13 (3H, s, COCH3), 2.00 (6H, s, CH3 ꢀ 2). MS m/z (%) 401
(M þ 1, 100). HPLC purity: 97.35%.
6-(6-Cyano-2-naphthoxy)-N2-(4-cyanophenylamino)pyridin-
3-amine (16). To a solution of 15 (51 mg, 0.125 mmol) in 1,4-
dioxane (10 mL) was added ammonia (0.5 mL) in water (10 mL)
with stirring for 30 min at rt. Then Na2S2O4 (386 mg, excess) was
added and stirring continued for an additional 2 h, monitored by
TLC until reaction was completed. The mixture was poured into
ice-water and extracted with diethyl ether. After removal of
solvent in vacuo, the residue was purified by PTLC to obtain
27 mg of 16 in 57% yield, gray solid, mp 204-6 ꢀC. 1H NMR
δ ppm 8.66 (1H, s, ArH-500), 8.50 (1H, s, NH), 8.18 (1H, d, J =
8.8 Hz, ArH-800), 8.09 (1H, d, J = 8.8 Hz, ArH-700), 7.82 (1H, dd,
J = 1.6 and 8.8 Hz, ArH-300), 7.66 (1H, d, J = 2.0 Hz, ArH-100),
7.51-7.54 (3H, m, ArH-30, 50, 400), 7.30 (2H, d, J = 8.8 Hz, ArH-
20, 60), 7.24 (1H, d, J = 8.4 Hz, ArH-4), 6.64 (1H, d, J = 8.4 Hz,
ArH-5), 5.11 (2H, s, NH2). MS m/z (%) 378 (M þ 1, 100). HPLC
purity: 97.53%
(E)-N1-(40-Cyanophenyl)-5-[200,600-dimethyl-400-(3-oxobut-1-enyl)]-
phenoxy-4-nitrobenzene-1,2-diamine (14d). To a solution of 12d
(165 mg, 0.35 mmol) in acetonitrile (3 mL) and triethylamine
(3 mL) in the presence of Pd-C (10%, 30 mg) was added formic
acid (85%, 2 mL) in acetonitrile (2 mL) keeping the temperature
under -15 ꢀC and then stirred for another 1 h. Next it was
heated to reflux for 1 h. After filtration of Pd-C, the mixture
was poured into ice-water and pH adjusted to neutral, and red
solid was collected. The crude product was purified on a silica
gel column (eluent: CHCl3/MeOH = 40:1) to yield 81 mg of 14d,
yield 53%, red solid, mp 210-212 ꢀC. 1H NMR (CDCl3) δ ppm
9.94 (1H, br, NH), 9.18 (1H, s, ArH-3), 7.31 (2H, s, ArH-300, 500),
7.53 (2H, d, J = 8.8 Hz, ArH-30,50), 7.44 (1H, d, J = 16.4 Hz,
ArCHd), 7.27 (2H, d, J = 8.8 Hz, ArH-20, 60), 6.68 (1H, d, J =
16.4 Hz, dCHCO), 6.25 (1H, s, ArH-6), 2.39 (3H, s, COCH3),
2.16 (6H, s, CH3 ꢀ 2). MS m/z (%) 443 (M þ 1, 100). HPLC
purity: 98.21%.
5-(6-Cyano-2-naphthoxy)-N1-(40-cyanophenyl)-2,4-dinitro-
aniline (17). Starting 160 mg (0.5 mmol) of 10 and 102 mg (0.75
mmol) of 6-hydroxy-2-naphthonitrile in DMF under micro-
wave irradiation at 150 ꢀC for 20 min to obtain 147 mg of 17 as
yellow solid with a 65% yield, mp 230-232 ꢀC. 1H NMR
(CDCl3) δ ppm 10.00 (1H, s, NH), 9.22 (1H, s, ArH-3), 8.27
(1H, s, ArH-500), 7.98, 7.82, 7.68 (each 1H, d, J = 8.8 Hz, ArH on
C-ring), 7.55 (2 H, d, J = 8.8 Hz, ArH on A-ring), 7.39 (1H, s,
ArH-10), 7.37 (1H, d, J = 8.8 Hz, ArH-30), 7.25 (2H, d, J = 8.8
Hz, ArH on A-ring), 6.80 (1H, s, ArH-6). MS m/z (%) 450 (M - 1,
100). HPLC purity: 97.28%.
5-(1-Bromo-6-cyano-2-naphthoxy)-N-(40-cyanophenyl)-2,4-
dinitroaniline (18). The same method as the preparation of 17,
starting with 10 (319 mg, 1.0 mmol) and 5-bromo-6-hydroxy-
2-naphthonitrile (248 mg, 1.0 mmol) to afford 390 mg of 18 as
yellow solid in a 74% yield, mp 254-256 ꢀC. 1H NMR (CDCl3)
(E)-N1-(40-Cyanophenyl)-5-(400-cyanovinyl-200,600-dimethyl)-
phenoxybenzene-4-nitro-1,2-diamine (14e). Using the same method
as preparation of 14d, starting with 150 mg (0.35 mmol) of 12e to
afford 120 mg of 14e as red solid, yield 81%, mp 252-254 ꢀC.
1H NMR (CDCl3) δ ppm 7.63 (1H, s, ArH-3), 7.47 (2H, d, J =
8.8 Hz, ArH-30,50), 7.32 (1H, d, J = 16.8 Hz, ArCHd), 7.20 (2H, s,

8296 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Tian et al.
δ ppm 9.95 (1H, s, NH), 9.24 (1H, s, ArH-3), 8.40 (1H, d, J =
8.8 Hz, ArH on C-ring), 8.29 (1H, s, ArH-500), 7.95 and 7.86
(each 1H, d, J = 8.8 Hz, ArH on C-ring), 7.44 (2 H, d, J =
8.4 Hz, ArH-30,50), 7.34 (1H, d, J = 8.8 Hz, ArH on C-ring), 7.17
(2H, d, J = 8.4 Hz, ArH-20,60), 6.45 (1H, s, ArH-6). MS m/z (%)
528 (M - 1, 100), 530 (Mþ1, 98). HPLC purity: 96.45%.
4-(6-Cyano-2-naphthoxy)-N2-(40-cyanophenyl)-5-nitrobenzene-
1,2-diamine (19). The method was the same as preparation of
14d, starting with 17 (188 mg, 0.35 mmol) to obtain 88 mg of 19
as red solid with a 60% yield, mp 196-198 ꢀC. 1H NMR
(CD3COCD3) δ ppm 10.43 (1H, s, NH), 8.39 (1H, s, ArH-3),
8.06 and 7.94 (each 1H, d, J = 8.8 Hz, ArH on C-ring), 7.72 (1H, s,
ArH-500), 7.64 (1H, dd, J = 2.0 and 8.8 Hz, ArH on C-ring),
7.54 (2 H, d, J = 8.8 Hz, ArH on A-ring), 7.45 (1H, dd, J = 2.0
and 8.8 Hz, ArH on C-ring),7.35 (1H, d, J = 2.0 Hz, ArH on
C-ring),7.19 (1H, s, ArH-6), 7.15 (2H, d, J = 8.8 Hz, ArH on
A-ring), 5.18 (2H, s, NH2). MS m/z (%) 422 (M þ 1, 100). HPLC
purity: 98.87%.
structures of wild-type RT/1 complex (PDB: 3mec) and K103N/
Y181C mutant RT/2 complex (PDB: 3bgr). Following the
“Guide-Docking ligands” in the software tool, the proteins
and ligands were prepared, the dockings were performed by
the CDOCK module, and results were analyzed in the DS 2.5
Base with relevant modules. The conformation energies of
ligands, 1 and 14e, were minimized with the modified para-
meters algorithm (smart minimizer), max steps (2000), and rms
gradient (0.05). The radius of the binding site sphere was defined
˚
as 10 A. Comparing to crystal structure, the root mean squared
deviation (rmsd) of the modeling was 0.29, thus ensuring the
docking method was reliable.
Acknowledgment. This investigation was supported by
grants 30930106, 20472114, and 7052057 from the Natural
Science Foundation of China (NSFC) and Beijing municipal
government, respectively, awarded to L. Xie, and U.S. NIH
grants awarded to S. Jiang (AI46221), C. H. Chen (AI65310),
and K. H. Lee (AI33066).
4-(1-Bromo-6-cyano-2-naphthoxy)-N1-(40-cyanophenyl)-5-nitro-
benzene-1,2-diamine (20). The method was the same as prepara-
tion of 14d, starting with 186 mg (0.35 mmol) of 18 to provide
120 mg of 20 as red solid, yield 60%, mp 190-192 ꢀC. 1H NMR
(CDCl3) δ ppm 8.38 (1H, d, J = 8.8 Hz, ArH on C-ring), 8.19
(1H, s, ArH on C-ring), 7.79 and 7.75 (each 1H, d, J = 8.8 Hz,
ArH on C-ring), 7.67 (1H, s, ArH-3), 7.52 (2 H, d, J = 8.8 Hz,
ArH on A-ring), 7.09 (1H, d, J = 8.8 Hz, ArH on C-ring), 6.97
(2H, d, J = 8.8 Hz, ArH on A-ring), 6.95 (1H, s, ArH-6). MS m/z
(%) 500 (M þ 1, 100), 502 (M þ 3, 99). HPLC purity: 100.0%.
Assay for Measuring the Inhibitory Activity of Compounds on
HIV-1 IIIB Replication in MT-2 Cells. Inhibitory activity of
compounds against infection by HIV-1 IIIB and its variant A17,
which is resistant to multiple RTIs, was determined as pre-
viously described.20 Briefly, MT-2 cells (104 well) were infected
with an HIV-1 strain (100 TCID50) in 200 μL of RPMI 1640
medium containing 10% FBS in the presence or absence of a test
compound at graded concentrations overnight. Then the culture
supernatants were removed and fresh media containing no
test compounds were added. On the fourth day postinfection,
100 μL of culture supernatants were collected from each well,
mixed with equal volumes of 5% Triton X-100, and assayed for
p24 antigen, which was quantitated by ELISA, and the percen-
tage of inhibition of p24 production was calculated as previously
described.20 The effective concentrations for 50% inhibition
(EC50) were calculated using a computer program CalcuSyn.21
HIV-1 Infection Assay Using TZM-bl as Reporter Cells.
Inhibition of HIV-1 infection was measured as reduction in
luciferase gene expression after a single round of virus infection
of TZM-bl cells as described previously.22 Briefly, 200 TCID50
of virus (NL4-3) was used to infect TZM-bl cells in the presence
of various concentrations of compounds. Two days after infec-
tion, the culture medium was removed from each well and
100 μL of Bright Glo reagent (Promega, Luis Obispo, CA) was
added to the cells for measurement of luminescence using a
Victor 2 luminometer. The 50% inhibitory concentration (IC50)
was defined as the concentration that caused a 50% reduction of
luciferase activity (Relative Light Units) compared to virus
control wells.
Supporting Information Available: HPLC conditions and
summary of HPLC purity data for final target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Esnouf, R.; Ren, J.; Ross, C.; Jones, Y.; Stammers, D.; Stuart, D.
Mechanism of inhibition of reverse transcriptase by non-nucleoside
inhibitors. Nature Struct. Biol. 1995, 2, 303–308.
(2) Pauwels, R. New non-nucleoside reverse transcriptase inhibitors
(NNRTIs) in development for the treatment of HIV infections.
Curr. Opin. Pharmacol. 2004, 4, 437–446.
(3) Sweeney, Z. K.; Klumpp, K. Improving non-nucleoside reverse
trancriptase inhibitors for first-line treatment of HIV infection: the
development pipeline and recent clinical data. Curr. Opin. Drug
Discovery Dev. 2008, 11 (4), 458–470.
(4) Minudo, J. J.; Haubrich, R. Entravirine: a second-generation
NNRTI for treatment- experienced adults with resistant HIV-1
infection. Futur. HIV Ther. 2008, 2 (6), 525–537.
(5) Sarafianos, S. G.; Marchand, B.; Das, K.; Himmel, D. M.; Parniak,
M. A.; Hughes, S. H.; Arnold, E. Structure and function of HIV-1
reverse transcriptase: molecular mechanisms of polymerization
and inhibition. J. Mol. Biol. 2009, 385, 693–713.
(6) Rossotti, R.; Rusconi, S. Efficacy and resistance of recently devel-
oped non-nucleoside reverse transcriptase inhibitors for HIV-1.
HIV Ther. 2009, 3 (1), 63–77.
(7) Lai, M. T.; Munshi, V.; Touch, S.; Tynebor, R. M.; Tucker, T. J.;
McKenna, P. M.; Williams, T. M.; DiStefano, D. J.; Hazuda, D. J.;
Miller, M. D. Antiviral activity of MK-4965, a novel non-nucleoside
reverse transcriptase inhibitor. Antimicrob. Agents Chemother. 2009, 53
(6), 2424–2431.
(8) Tucker, T. J.; Sisko, J. T.; Tynebor, R. M.; Williams, T. M.; Felock,
P. J.; Flynn, J. A.; Lai, M. T.; Liang, Y. X.; Gaughey, G. M.; Liu,
M. Q.; Miller, M.; Moyer, G.; Munshi, V.; Perlow-Poehnelt, R.;
Prasad, S.; Reid, J. C.; Sanchez, R.; Torrent, M.; Vacca, J. P.; Wan,
B. L.; Yan, Y. W. Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]-
pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile
(MK-4965): a potent, orally bioavailable HIV-1 non-nucleoside re-
verse transcriptase inhibitor with improved potency against key mutant
viruses. J. Med. Chem. 2008, 51, 6503–6511.
(9) Ripamonti, D.; Maggiolo, F. Rilpivirine, a non-nucleoside reverse
transcriptase inhibitor for the treatment of HIV infection. Curr.
Opin. Invest. Drugs 2008, 9 (8), 899–912.
Assessment of in Vitro Cytotoxicity in MT-2 Cells. The in vitro
cytotoxicity of compounds on MT-2 cells was measured using a
colorimetric XTT assay.23 Briefly, 100 μL of the test compound
at graded concentrations were added to equal volumes of cells
(5 ꢀ 105/mL) in wells of a 96-well plate. After incubation at
37 ꢀC for 4 days, 50 μL of XTT solution (1 mg/mL) containing
0.02 μM of phenazine methosulphate (PMS) was added. After
4 h, the absorbance at 450 nm was measured with an ELISA
reader. The CC50 (concentration for 50% cytotoxicity) values
were calculated using the CalcuSyn computer program as
described above.
(10) Pecora Fulco, P.; McNicholl, I. R. Ereavirine and rilpivirine: non-
nucleoside reverse transcriptase inhibitors with activity against
human immunodeficiency virus type 1 strains resistant to previous
non-nucleoside agents. Pharmacotherapy 2009, 29 (3), 281–294.
(11) Tian, X. T.; Qin, B. J.; Lu, H.; Lai, W.; Jiang, S.; Lee, K. H.; Chen,
C. H.; Xie, L. Discovery of diarylpyridine derivatives as novel non-
nucleoside HIV-1 reverse transcriptase inhibitors. Bioorg. Med.
Chem. Lett. 2009, 19, 3482–5485.
(12) Qin, B. J.; Jiang, X. K.; Lu, H.; Tian, X. T.; Barbault, F.; Huang,
L.; Qian, K.; Chen, C. H.; Huang, R.; Jiang, S.; Lee, K. H.; Xie,
L. Diarylaniline derivatives as a distinct class of HIV-1 non-
nucleoside reverse transcriptase inhibitors. J. Med. Chem. 2010,
53, 4906–4916.
Molecular Modeling. The docking studies were conducted by
using software Discovery Studio 2.5 (DS 2.5) and two crystal

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8297
(13) De Corte, B. L. From 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk]-
(1,4)benzodiazepine-2(1H)-one (TIBO) to etravirine (TMC125):
fifteen years of research on non-nucleoside inhibitors of HIV-1
reverse transcriptase. J. Med. Chem. 2005, 48, 1689–1696.
(14) Das, K.; Bauman, J. D.; Clark, A. D. J.; Frenkel, Y. V.; Lewi, P. J.;
Shatkin, A. J.; Hughes, S. H.; Arnold, E. High-resolution struc-
tures of HIV-1 reverse transcriptase/TMC-278 complexes: strate-
gic flexibility explains potency against resistance mutations. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105 (5), 1466–1471.
(18) Lansdon, E. B.; Broendza, K. M.; Hung, M.; Wang, R.; Mukund,
S.; Jin, D.; Birkus, G.; Kutty, N.; Liu, X. CrystalstructuresofHIV-1
reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278):
implications for drug design. J. Med. Chem. 2010, 53, 4295–4299.
(19) Obiol-Pardo, C.; Rubio-Martinez, J. Comparative evaluation
of MMPBSA and XSCORE to compute binding free energy in
XIA-peptide complexes. J. Chem. Inf. Model. 2007, 47, 134–142.
(20) Jiang, S.; Lu, H.; Liu, S.; Zhao, Q.; He, Y.; Debnath, A. K.
N-Substituted pyrrole derivatives as novel human immunodeficiency
virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle
formation and block virus fusion. Antimicrob. Agents Chemother. 2004,
48, 4349–4359.
(15) Redemann, C. T.; Redemann, C. E. 5-Amino-2,3-dihydro-1,4-
phthalazinedione. Org. Synth. 1949, 29, 8–9.
(16) Mordant, C.; Schmitt, B.; Pasquier, E.; Demestre, C.; Queguiner,
L.; Masungi, C.; Peeters, A.; Smeulders, L.; Bettens, E.; Hertogs,
K.; Heeres, J.; Lewi, P.; Guillemont, J. Synthesis of novel diaryl-
pyrimidine analogues of TMC278 and their antiviral activity
against HIV-1 wild-type and mutant strains. E. J. Med. Chem.
2007, 42, 567–569.
(17) Andries, K.; Azijn, H.; Thielemans, T.; Ludovici, D.; Kukla, M.;
Heeres, J.; Janssen, P.; De Corte, B.; Vingerhoets, J.; Pauwels, R.;
de Bethune, M. P. TMC125, a novel next-generation non-nucleoside
reverse transcriptase inhibitor active against non-nucleoside reverse
transcriptase inhibitor-resistant human immunodeficiency virus type 1.
Antimicrob. Agents Chemother. 2004, 48, 4680–4686.
(21) Chou, T. C.; Talalay, P. Quantitative analysis of dose-effect
relationships: the combined effects of multiple drugs or enzyme
inhibitors. Adv. Enzyme Regul. 1984, 22, 27–55.
(22) Dang, Z.; Lai, W.; Qian, K.; Lee, K. H.; Chen, C. H.; Huang, L.
Betulinic Acid Derivatives as Human Immunodeficiency Virus
Type 2 (HIV-2) Inhibitors. J. Med. Chem. 2009, 52, 7887–7891.
(23) Zhao, Q.; Ma, L.; Jiang, S.; Lu, H.; Liu, S.; He, Y.; Strick,
N.; Neamati, N.; Debnath, A. K. Identification of N-phenyl-
N0-(2,2,6,6-tetramethylpiperidin-4-yl)-oxalamides as a new class
of HIV-1 entry inhibitors that prevent gp120 binding to CD4.
Virology 2005, 339, 213–225.