Diarylaniline derivatives as a distinct class of HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm1002952

By using structure-based drug design and isosteric replacement, diarylaniline and 1,5-diarylbenzene-1,2-diamine derivatives were synthesized and evaluated against wild type HIV-1 and drug-resistant viral strains, resulting in the discovery of diarylaniline derivatives as a distinct class of next-generation HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) agents. The most promising compound 37 showed significant EC₅₀ values of 0.003-0.032 mn; against HIV-1 wild-type strains and of 0.005-0.604 μM against several drug-resistant strains. Current results also revealed important structure-activity relationship (SAR) conclusions for diarylanilines and strongly support our hypothesis that an NH₂ group on the central benzene ring ortho to the aniline moiety is crucial for interaction with K101 of the NNRTI binding site in HIV-1 RT, likely by forming H-bonds with K101. Furthermore, molecular modeling studies with molecular mechanism/general Born surface area (MM/GBSA) technology demonstrated the rationality of our hypothesis.

Full text of DOI:10.1021/jm1002952

4906 J. Med. Chem. 2010, 53, 4906–4916
DOI: 10.1021/jm1002952
Diarylaniline Derivatives as a Distinct Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors
Bingjie Qin,† Xingkai Jiang,† Hong Lu,§ Xingtao Tian,† Florent Barbault, Li Huang,^ Keduo Qian,‡ Chin-Ho Chen,^
Rong Huang,‡ Shibo Jiang,§ Kuo-Hsiung Lee,‡ and Lan Xie*,†
†Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China, ‡Natural Products Research Laboratories,
University of North Carolina, Chapel Hill, North Carolina 27599, §Lindsley F. Kimball Research Institute, New York Blood Center, New York,
New York 10065, ITODYS, Universiteꢀ Paris Diderot;CNRS UMR 7086, 15 rue Jean de Baıf, 75205 Paris, France, and ^Duke University
Medical Center, Box 2926, Surgical Oncology Research Facility, Durham, North Carolina 27710
¨
Received March 5, 2010
By using structure-based drug design and isosteric replacement, diarylaniline and 1,5-diarylbenzene-1,2-
diamine derivatives were synthesized and evaluated against wild type HIV-1 and drug-resistant viral strains,
resulting in the discovery of diarylaniline derivatives as a distinct class of next-generation HIV-1 non-
nucleoside reverse transcriptase inhibitor (NNRTI) agents. The most promising compound 37 showed
significant EC50 values of 0.003-0.032 μM against HIV-1 wild-type strains and of 0.005-0.604 μM against
several drug-resistant strains. Current results also revealed important structure-activity relationship (SAR)
conclusions for diarylanilines and strongly support our hypothesis that an NH2 group on the central benzene
ring ortho to the aniline moiety is crucial for interaction with K101 of the NNRTI binding site in HIV-1 RT,
likely by forming H-bonds with K101. Furthermore, molecular modeling studies with molecular mechanism/
general Born surface area (MM/GBSA) technology demonstrated the rationality of our hypothesis.
Introduction
reverse transcriptase inhibitors (NNRTIs) that interact with
the allosteric binding site, a highly hydrophobic cavity, in a non-
competitive manner to cause distortion of the three-dimensional
structure of the enzyme and thus inhibit RT catalytic function.
NNRTIs currently approved for AIDS therapy include nevir-
apine (1), delavirdine (2), efavirenz (3), and etravirine (TM-
C125, 4) (Figure 1).3 In general, NNRTIs exhibit high inhibitory
potency and low toxicity, but drug resistance to NNRTIs has
emerged rapidly as a result of mutations in amino acid residues
that are in or surround the NNRTI binding site. Compound 4 is
the most recently approved NNRTI and is active against many
drug-resistant HIV-1 strains. The related riplivirine (TMC278,
5)4 is now undergoing phase III clinical trials as a promising new
drug candidate. Compounds 4, 5, and TMC120 (6),5 a prior
clinical candidate, belong to the diarylpyrimidine (DAPY)
family (Figure 1) and all are very potent against wild-type and
many drug-resistant HIV-1 strains with nanomolar EC50 values.
They have excellent pharmacological profiles, which has en-
couraged more research to explore next-generation NNRTI
agents.6-8 In this study, we used isosteric replacements to syn-
thesize new NNRTIs and consequently discovered a series of
diarylaniline compounds with high potency against both wild-
type and RT-resistant viral strains.
According to UNAIDS statistics, more than 60 million people
worldwide have been infected by the human immunodeficiency
virus (HIVa) and about 25 million patients have died of AIDS. In
the absence of an effective vaccine, there is a need to develop
effective anti-HIV therapeutics to prolong the lives of HIV-
infected individuals. Thus far, more than 20 anti-HIV drugs
have been approved by the U.S. FDA (www.fda.gov/oashi/aids/
virals.html) including reverse transcriptase inhibitors (RTIs),
protease inhibitors (PIs), fusion inhibitors, integrase inhibitors,
and entry inhibitors (CCR5 coreceptor antagonist). Highly ac-
tive antiretroviral therapy (HAART), which uses a combination
of three to four drugs, can significantly reduce the morbidity and
mortality of HIV-1 infected patients. However, as a result of
emerging drug-resistant HIV mutants, increasing numbers of
HIV-infected patients fail to respond to HAART. Thus, the
development of new anti-HIV drugs is urgently required.
To address this need, we have synthesized compounds
targeting HIV-1 reverse transcriptase (RT), one of the most
important enzymes in the HIV-1 life cycle. It has two known
drug-target sites, the substrate binding site and an allosteric
site, which is distinct from, but closely located to, the substrate
binding site.1,2 Specifically, we focused on non-nucleoside
Design
*To whom correspondence should be addressed. Phone/Fax: 86-10-
66931690. E-mail: lanxieshi@yahoo.com.
Prior studies4,9 on DAPY derivatives have resulted in infor-
mative SAR conclusions, including (1) a “U” or horseshoe
binding conformation in contrast to the typical butterfly like
binding shape of 1-3, (2) a proper positioning of two phenyl
rings in the eastern and western wings of the NNRT binding
pockets, (3) a para-substituted aniline moiety in the eastern
wing, and (4) two hydrogen bonds between K101 of HIV-1
RT to the NH linker and the nitrogen atom on the pyrimidine
a Abbreviations: CC50, concentration for 50% cytotoxicity; DAPY,
diarylpyrimidine; EC50, effective concentration for 50% inhibition;
HAART, highly active antiretroviral therapy; HIV, human immunodefi-
ciency virus, MM/GBSA, molecular mechanism/general Born surface area;
NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside
reverse transcriptase inhibitor; PDB, protein database; PI, protease inhi-
bitor; RF, resistant fold; rmsd, root-mean-square deviation; RT, reverse
transcriptase; RTI, reverse transcriptase inhibitor; SAR, structure-activity
relationship; SI, selective index (ratio of CC50/EC50).
r
pubs.acs.org/jmc
Published on Web 06/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4907
ring.10 Crystal structures of complexes K103N HIV-1 RT/4
(protein database (PDB) code: 1sv5), HIV-1 RT/5 (PDB:
2zd1), K103N/Y181C HIV-1 RT/5 (PDB: 3bgr), and HIV-1
RT/6(PDB:1s6q)11,12 furtherindicatethat twoflexible linkers
between rings allow the inhibitors to adopt bound conforma-
tions with either wild-type or mutant HIV-1 RT, resulting in
high potency against both wild-type and a wide range of drug-
resistant mutant HIV-1 RT. Therefore, the molecular flex-
ibility of DAPY compounds is considered to be very crucial
for next-generation NNRTI drugs.
On the basis of these SAR data, our strategy was to synthe-
size new compounds by using isosteric replacement in the cen-
tral B-ring of DAPY compounds. Unlike the reported DAPY
derivatives,13-15 the new analogues designed in this study
(Figure 2) have a central benzene ring rather than the pyri-
midine inDAPY compounds. The new compoundsalso retain
two phenyl rings in the eastern and western wings but have
different para-substituents from the DAPY compounds. Be-
cause the presence of a central benzene ring does not change
molecular topology and flexibility, these new compounds are
expected to have similar binding orientations and conforma-
tions to those of DAPYs. However, they will lose the H-bond
between K101 and the nitrogen on the pyrimidine ring of
DAPY family compounds.10 To compensate for this loss, we
introduced a nitro or amino group (R4) on the central benzene
ring atthe ortho-position to the aniline ring to serve as H-bond
acceptor or donor, respectively, and to interact with K101
on the binding site. Furthermore, we investigated how an
additional nitro or amino group (R2) on the opposite side of
the central phenyl ring would affect anti-HIV activity.
Chemistry
All target compounds were synthesized via the short routes
detailed in Schemes 1 and 2. The starting material, 1,3-dichloro-
4,6-dinitrobenzene or 2,4-dichloro-1-nitrobenzene, is inexpensive
and commercially available. The dichloronitrobenzene was reac-
ted with a para-substituted aniline in a 1:1.1 molar ratio in DMF
at room temperature for less than 40 min in the presence of
triethylamine to provide corresponding N-aryl-5-chloro- 2,4-dini-
troanilines (7-11) or in the presence of potassium tert-butoxide
(t-BuOK) to provide corresponding N-aryl-5-chloro-2-nitroani-
lines (12). The nucleophilic substitution took place between the
aromatic amine and the chloride at the ortho-position to the nitro
group in 71-94% yields. Compounds 7-12 were then coupled
with a substituted phenol by using microwave irradiation in
DMF (or DMSO) in the presence of potassium carbonate with
stirring at 190 ꢀC for 15 min to afford diaryl-dinitroanilines
(13-23) and diaryl-mononitroanilines (24-28).16,17 Some com-
pounds could also be prepared by the traditional method of heat-
ing at 130 ꢀC for 5 h with yields ranging from 74 to 94%. As
shown in Scheme 2, dinitro (19-22) and mononitro (24, 26-27)
compounds were reduced completely by using hydrazine hydrate
Figure 1. HIV-1 NNRTI agents (1-6).
in isopropyl alcohol in the presence of FeCl3 6H2O and activated
3
carbon at reflux for 20-30 min to prepare corresponding dia-
mino (32-35) or monoamino (29-31) compounds, respectively,
in 83-95% yields . Alternatively, 19, 21, and 22 were reduced
Figure 2. Design of Target Compounds.
Scheme 1. Synthesis of Target Compounds 13-28a
a “c or d” indicates two different reaction conditions, the former is under microwave irradiation and the later is a traditional heating method. (a) Et3N/
DMF, rt 40 min; (b) t-BuOK/DMF, rt 1 h; (c) K2CO3/DMF or DMSO, 190 ꢀC, MW, 10-15 min; (d) K2CO3/DMF, 130 ꢀC, 5 h.

4908 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Qin et al.
Scheme 2a
a (a) FeCl3 6H2O/C, N2H4 H2O, (CH3)2CHOH, reflux, 20-30 min; (b) Et3N/HCOOH, Pd/C, CH3CN, reflux, 1 h; (c) triethyl orthoformate, HCl
(1N in diethyl ether), rt 3 h; (d) CH3COCH3, HCl (18% in diethyl ether).
3
3
selectively by formic acid in the presence of Pd-C (10%) and
triethylamine in acetonitrile under reflux for 1 h to give corres-
ponding 1,5-diaryl-4-nitrobenzene-1,2-diamines 36-38, respec-
tively.18 Next, 37 was reacted with triethyl orthoformate under
acidic conditions to provide the benzoimidazole product 39.
This reaction validated that the nitro group ortho to the NH-
linked aniline was reduced selectively in the prior step. Further-
more, active compound 36 was converted to hydrochloride salt
40 (shown in Scheme 2) in acetone to investigate the effect of
improved molecular water solubility on anti-HIV activity.
Table 1. Antiviral and Cytotoxicity Data of 13-38 and 40a
HIV-1 IIIB in H9 cells
b
c
EC50
(μM)
CC50
(μM)
compd
R1
R2
R3
R4
SId
Results and Discussion
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
40
AZT
OMe NO2
H
NO2
NO2
NO2
NO2
NO2
3.840 >61.12
2.990 >59.10
>16
>20
14
OMe NO2 Me
OMe NO2 Br
All target compounds were first tested against wild-type HIV-
1 (IIIB strain) replication in the H9 cell line, and the results are
summarized in Table 1. The most potent compound 37 had an
EC50 value of 0.003 μM and an extremely high selective index
(SI) of 20887 in this initial assay. Compound 36 and its hydro-
chloride salt 40 were also very potent, with EC50 values of 0.016
and 0.012 μM, respectively; SI values of both compounds
were >2800. Other active compounds 27, 29-31, 34-35, and
38 had EC50 values ranging between 0.030 and 0.073 μM and SI
values between 520-1698. In addition, compounds 19, 20, 22,
24, 26, and 32 showed potency at the submicromolar level and
had a SI range of >113-313. These promising results demon-
strated that the isosteric replacement of carbon for nitrogen in
the central B-ring of DAPY compounds was successful and
resulted in the discovery of a series of 1,5-diarylbenzene-1,2-dia-
mine analogues as potent NNRTI agents.
Some SAR results for this compound series can be concluded
from the data in Table 1. (1) For the para-substituent on the
A-ring (R1 moiety), the cyano (CN) group appears to be nece-
ssary at this position for increased anti-HIV activity. Among
compounds 13-19, only 19 (R1=CN) exhibited high anti-HIV-
1 activity with an EC50 value of 0.172 μM and SI of >301, while
13-18 (R1 = MeO, Me, Cl, or NO2) were much less active
(EC50 >2.99 μM) or inactive. Except for compounds 21, 23, 28,
and 33, all the other compounds containing the active p-cya-
noanilino A-ring (19-20, 22, 24-27, 29-32, 34-38, and 40)
exhibited potent inhibitory activity against wild-type HIV-1
replication. (2) The presence of NH2 (R4 moiety) on the central
B-ring ortho to anilino A-ring position in 29-38 and 40 generally
enhanced the anti-HIV-1 activity of the analogues (EC50:0.003-
0.161 μM), except for 33. The NH2-substituted compounds (32,
34, 35, 29, 30, 31) were as or more potent than the corresponding
NO2-substituted compounds (19, 21, 22, 24, 26, 27), respectively.
This result is consistent with that in our previous report on
diarylpyridine compounds19 and therefore supports our hypoth-
esis that an amino group at the R4 position of the central
3.630
4.310
51.23
52.97
Me
Cl
NO2 Br
NO2 Br
NO2 Br
NO2 Br
12
NA
NO2
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
NO2 >49.7
>49.70
>1
>301
>113
>14
>214
38
NO2
NO2
NO2
NO2
0.172 >51.76
0.545 >61.88
4.190 >58.28
0.280 >59.81
NO2
H
NO2 CN
NO2 Me
NO2 CHO NO2
1.530
0.317 >57.08
3.147 69.64
0.208 >65.10
57.87
H
H
H
H
H
H
H
H
Br
H
NO2
NO2
NO2
NO2
>180
22
>313
CN
Me
0.067 >67.02 >1000
CHO NO2
2.190
0.047
0.070
0.073
0.161
3.226
0.030
0.070
0.016
0.003
0.062e
0.012
14.20
44.61
49.77
51.02
38.38
53.20
50.95
50.00
45.47
62.66
32.22e
36.42
6
949
Br
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
CN
Me
711
699
NH2 Br
NH2
238
16
H
NH2 CN
NH2 Me
NO2 Br
NO2 CN
NO2 Me
1698
714
2,842
20887
520
hydrochloride salt of 36
3,035
36019
0.052 1873
a Assays using H9 cells were performed at Panacos Pharmaceuticals, Inc.,
Gaithersburg, Maryland. Results are averages of three independent assays.
Standard deviations were not provided by Panacos. b Concentration of a
compound that causes 50% inhibition of HIV-1 IIIB replication. c Concen-
tration of a compound that causes cytotoxicity to 50% cells. d SI (selective
index)=CC50/EC50. e Data from Duke University. NA: not active.
B-ring is crucial for interaction with K101 on the NNRTI
binding site and can form additional H-bonds to the protein.
(3) On the central B-ring ortho to the phenoxy C-ring position
(R2 substituent), NO2 moiety (36-38) is more favorable
compared with H (29-31) or NH2 (32, 34-35) for achieving

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4909
Table 2. Data against HIV-1IIIB and Multi-RTI-Resistant Viral Strainsa
EC50 (μM) in MT-2 cell line
NNRTI-resistant
mutantb
multi-RTI-resistant
mutantb
wild type
A17 (Y181C/
K103N)
compd
IIIB
Y181C
8605MR
6005MR
34
36
37
1
0.065
0.008
0.005
0.053
1.734
1.298
0.504
1.244
0.254
0.231
0.138
0.020
0.005
0.033
0.007
0.005
>3.700
>3.700
>3.700
>3.700
a Data provided by Panacos Pharmaceuticals Inc.. Results are average of
three independent assays. Standard deviations were not provided by Pana-
cos. Compound 1was used as control due to the fact that 4was not approved
for market at the testing time. b NNRTI-resistant mutant and multimutated
viral strains were from Panacos Inc. 8605MR: multimutated-RT with M41L,
D67N, L210W, T215Y, M184 V, K103N. 6005MR: multimutated-RT with
M41L, L74 V, M184 V, L210W, T215Y, ins SS, A98G.
Figure 3. RT inhibition activity of diarylanaline analogues 34 and
37. Data provided by Dr. Chin-Ho Chen, Duke University Medical
Center. Results are average of three independent assays. The RT
used in the assays is from HIV-1DH012 lysate.
with mutations in the viral RT domain that is highly resistant
to NRTIs. These results provide further support that the
DAPY pyrimidine ring can be replaced by a benzene ring to
generate new potent compounds as a distinct class of next-
generation NNRTIs. Meanwhile, although compound 34
(R2=NH2) was not as potent as 4 and 37, it had a higher resis-
tantfoldchange(RF>73, ratioof EC50 against mutant strain/
EC50 against wild-type strain) against A17 compared with
other compounds, suggesting that the R2 substituent on the
central B-ring may play an important role in the anti-HIV-1
activity against drug-resistant virus.
To confirm RT is the biological target of the newly synthe-
sized diarylaniline analogues, compounds 34 and 37 were
evaluated against viral enzyme RT (from HIV-1DH012 lysate)
and compared with 4. From Figure 3, we can see all three
compounds potently inhibited RT activity. Inhibition of
DH012 RT activity by 34 and 37 suggests that HIV-1 RT is
a target of the compounds. However, the anti-RT potency of
these two compounds was much weaker than 4 even though
compound 37 was more potent than 4 against HIV-1 replica-
tion (Table 3). The discrepancy in potency between anti-HIV-
1 replication and anti-RT activity could be due to the fact that
different viral strains were used in the assays. The T cell-
adapted virus HIV-1 IIIB was used in the virus replication
assay (Table 3), whereas the primary isolate DH012 was used
for the RT assay. It is also possible that 34 and 37 inhibited
HIV-1 RT in a different manner than 4. In addition to
inhibition of the RNA-dependent DNA polymerase activity
of HIV-1 RT, compound 37 might also inhibit DNA-depen-
dent DNA polymerase activity that could not be optimally
detected by the RT assay used in this study.
Table 3. Data against HIV-1IIIB and A17 in the MT-2 Cell Linea
EC50 (μM) in MT-2 cell line
compd
IIIB (wild-type)
A17 (Y181C/K103N)b
RF
34
36
37
4
0.211 ( 0.053
0.052 ( 0.005
0.032 ( 0.005
0.058 ( 0.001
15.469 ( 9.035
1.055 ( 0.088
0.604 ( 0.179
0.575 ( 0.093
73.3
20.3
18.9
9.9
a Data provided by Dr. Shibo Jiang, New York Blood Center. Results
are average of three independent assays. b The multi-NRTI-resistant
strain A17 from NIH with mutations at amino acids 103 (KfN) and 181
(YfC) in the viral RT domain is highly resistant to NRTIs21
high anti-HIV-1 potency. (4) Br, CN, and CH3 para-substi-
tuent (R3 moiety) on the C-ring showed better anti-HIV
activity, but CHO and H (23, 28, and 33) did not. In addition,
compound 40, the hydrochloride salt of 36, displayed more
potent inhibitory activity than 36, suggesting a possibility to
improve water solubility in active compounds.
The most active compounds 34, 36, and 37 were further
evaluated in comparison with the anti-HIV-1 NNRTI drugs 1
and 4 against wild-type HIV-1 and several RTI-resistant viral
strains in the MT-2 cell line. These data are summarized in
Tables 2 and 3. As shown in Table 2, we found that both 37
and 36 showed better EC50 values (0.005 and 0.008 μM,
respectively) against HIV-1IIIB wild-type virus than 1. While
drug 1 lost its antiviral replication activity completely against
multi-RTI-resistant mutants, compounds 34, 36, and 37
retained theirhigh potencytowardtheseviruses. Impressively,
compound 37 still showed the most potent EC50 value of 0.005
μM against multi-RTI-resistant mutants 8605MR and
6005MR. Compound 36 also was highly active against these
mutants, with similar or slightly lower potency than 37. Com-
pound 37 kept some of its antiviral activity against NNRTI-
resistant mutant Y181C and A17 (Y181C/K103N) with EC50
values of 0.504 and 0.231 μM, respectively, while 1 completely
lost its potency. Compound 36 also showed moderate inhibi-
tion activity toward NNRTI-resistant mutants, although it is
less potent than 37.
Molecular Modeling Studies
Computational studies of three compounds 21, 34, and 37
were carried out to understand how the substituents on the
central B-ring of these diarylaniline analogues might interact
with HIV-1 RT. Because of the high structural similarity with
DAPY, the ligand-protein complexes of the three com-
pounds were created with the help of the experimental crystal
structure of HIV-1 in interaction with DAPY compound
(PDB: 1s6q).11
After energetic optimizations, the ligand positions in the
NNRTI binding pocket were carefully checked. To under-
stand the so-called “induced fit effect,” all complexes were
compared to HIV-RT alone, which was computed by the
same protocol. The root mean square deviation (rmsd)
permits the determination of geometrical variations bet-
ween the free and complex protein; all values are compiled
The antiviral activity of 34, 36, and 37 were further tested
against wild-type virus and A17 resistant mutant in parallel
with drug 4 after it was approved by US FDA and became
available on the market (Table 3). The viral inhibition activity
of 37 was slightly better than that of 4 against HIV-1IIIB (wild-
type) in this screening, which double-confirmed our results in
Table 2. Compounds 37 and 4 showed similar activity (EC50:
0.604 and 0.575 μM, respectively) against A17 (from NIH)

4910 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Qin et al.
Table 4. RMSD Values of the HIV-1 RT Protein Interacting with 21,
34, and 37, when Compared to the Protein Alonea
˚
rmsd (A)
ligand
global
backbone
side-chain
21
34
37
0.666
0.654
0.635
0.501
0.493
0.480
0.738
0.725
0.703
a Values are divided into global (all protein atoms), backbone
(peptidic bonds), and side-chain (only side-chain atoms).
Table 5. MM/GBSA Energetic Values of the Three Ligand/HIV-1 RT
Complexesa
kcal/mol
energy
ΔEelec
ΔEvdw
ΔEint
ligand 21
ligand 34
ligand 37
31.4
-53.8
14.4
-23.8
-61.3
7.2
-56.5
-55.5
-8.3
ΔEgas
-8.0
-6.2
1.5
-77.9
-6.9
35.3
-120.3
-6.4
54.6
ΔGsolvent_np
ΔGsolvent_p
ΔGsolvent
ΔG
-4.7
-12.7
28.4
48.2
-49.6
-72.1
a Eelec, Evdw, and Eint signify: electrostatic, van der Waals, and
internal (bonds þ angles þ dihedrals) energies in gas phase (in vacuo),
respectively. Egas is the sum of the first three energy terms. Gsolvent_p,
Gsolvent_np, and Gsolvent are the polar and nonpolar contributions of
the solvent free energy, and the sum of these two terms (solvent free
energy), respectively; G is the free energy of binding (Egas þ Gsolvent).
in Table 4. From this table, it appears that, globally, the
ligand-protein structure is kept and that only small adapta-
tions are made by the protein. Comparison of the backbone
and side-chain RMSDs shows that the side-chain residues
have moved much more in the optimization process than the
peptidic chain, indicating that the secondary enzyme structure
was conserved and only small adaptations of residues oc-
curred. In comparison, the less biologically active compound
21 induced more “deformation” (i.e., a higher rmsd) of the
protein than either 34 or 37, denoting that the protein needs a
more significant deformation for adaptation to 21, which is
energetically unfavorable for binding.
The molecular mechanics/general Born surface area (MM/
GBSA) technique is one of the best computational methods to
evaluate free energy of binding, generally from molecular dyna-
mics simulations, for ligands in biological macromolecules.20,21
Recently, it has been shown on a large number of enzymatic
systems (including HIV-1 RT) that the MM/GBSA method is
able to rescore docking results, even with only one minimized
structure.22 Therefore, we employed this technique on our three
compounds, and the results are compiled in Table 5. To take
into account the protein adaptation to the ligands, the calcula-
tions were made by considering the three species individually
(protein, ligand, and complex). The computed free energies of
binding paralleled the experimental antiviral activity data, i.e.,
the ranking of EC50 values. Furthermore, the energetic decom-
positions indicated which contributions are the most important
for the binding affinity and which are not. Comparison of values
in Table 5 indicated that the differences of binding free energies
come mainly from the gas phase energy and, more precisely,
from the electrostatic contribution of the ligand to the protein.
However, the nonpolar solvent effect is almost identical for the
three compounds. This result may reflect the fact that, in the
X-ray structure, the NNRTI binding pocket is deeply inserted
into the HIV-1 RT enzyme, and no water molecules are close to
the DAPY ligand. Indeed, nonpolar solvent free energy of
Figure 4. Compounds 21 (top), 34 (middle), and 37 (bottom) in
interaction with HIV-1 RT.
binding comes from the entropic desolvation of water around
the ligand interacting site.
On the basis of the above computational studies, the docking
of three ligands with HIV-1 RT is illustrated more clearly in
Figure 4. Compounds 21, 34, and 37 differ structurally only in
the identity of the R2 and R4 substituents on the central aro-
matic ring (21: NO2, NO2; 34: NH2, NH2; 37: NO2, NH2).
Therefore, our analyses focused on these moieties. As shown in
Figure 4, a hydrogen bond between the NH linker and the
peptidic carbonyl oxygen of K101, like that for DAPY com-
pounds, is observed with all three compounds. However, the
neighboring amino group (R4) present in 34 and 37 provides two
more hydrogen bonds with K101: one is between the peptidic
carbonyl oxygen of the protein residue and one NH vector of the
ligand NH2 group and the second one involves the NH atoms of
K101 and targets the nitrogen atom of the ligand NH2 group.
The most active compound 37 also has a nitro group as the R2
substituent on the central ring. This group provides a small
electrostatic interaction with the positively charged K172 (ligand
˚
nitrogen-protein nitrogen distance of 6.68 A; see Figure 3,
bottom). When the nitro group in 37 was replaced by an amino
group in 34, the corresponding ligand-protein electrostatic
interaction decreased (see Figure 3, middle). Indeed, the distance
between the nitrogen atom of the amino moiety of 34 and the
˚
K101 nitrogen increased from 6.68 to 7.04 A, indicating that
repulsion has occurred. Moreover, this electrostatic effect was
also observed computationally (Table 5) with electrostatic
energy values of -56.5 kcal/mol and -23.8 kcal/mol for 37

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4911
and 34, respectively. In comparison with 34 and 37, the least
activecompound21has two nitro groups on the central B-ring
(see Figure 3, top). Thus, the rightmost (R2) nitro group of 21
has the same electrostatic interaction with K172 found in 37,
and the ligand nitrogen-protein nitrogen distance shortens to
eluting with a mixture of solvents A and B (condition 1: aceto-
nitrile/water 80:20, flow rate 0.2 mL/min; condition 2: MeOH/
water 80:20, flow rate 0.2 mL/min; UV 254 nm). The microwave
reactions were performed on a microwave reactor from Biotage,
Inc. Thin-layer chromatography (TLC) was performed on silica
gel GF254 plates. Silica gel GF254 (200-300 mesh) from Qingdao
Haiyang Chemical Company was used for TLC, preparative
TLC, and column chromatography. Medium-pressure column
chromatography was performed using a CombiFlash Compa-
nion purification system. All chemicals were obtained from
Beijing Chemical Works or Sigma-Aldrich, Inc.
˚
6.56 A. However, the presence of the leftmost (R4) nitro group
in 21 completely disrupts the hydrogen bond network with
K101. The NH atoms of K101 face the nitrogen atom of the
nitro moiety identically as for the amino group, but the
nitrogen of a nitro group is electropositive while that of an
amino group is electronegative. Thus, for 21, electrostatic
repulsion occurs between the R4 nitro group and the NH of
K101, which is also observed in the computational electro-
static energy value of 31.4 kcal/mol (Table 5). Because a
hydrogen bond is mainly electrostatic, the two supplementary
H-bonds found with 34 and 37 could explain the experimental
EC50 differences, which correlated with the computed free
energies of binding. Therefore, these molecular modeling
results also strongly support our hypothesis and demonstrate
why the amino-substituted compounds 29-38 are more po-
tent than nitro-substituted compounds 19-28.
General Procedure for the Preparation of N1-Aryl-5-chloro-2-
nitroanilines (7-12). Method A: A para-sustituted aniline (1.1
equiv) was added slowly into a solution of 2,4-dichloronitro-
benzene (1 equiv) in DMF (3 mL) and triethylamine (1.5 mL,
excess). The yellow mixture was stirred at rt for 40 min. The
mixture was then poured into water (ca. 30 mL) and stirred for
an additional 30 min. The solid product was collected and
purified by recrystallization from EtOH. Method B: To a
solution of 2,4-dichloronitrobenzene (1 equiv) and a substituted
aniline (1.1 equiv) in DMF (3 mL), was slowly added potassium
tert-butoxide (2 equiv) at ice-water bath temperature, then
stirred at rt for about 1 h until the reaction was completed as
monitored by TLC. The mixture was poured into ice-water,
and pH was adjusted to 6 with 5% aq HCl. The solid was
collected, washed with water three times, and purified by
recrystallization from EtOH.
Conclusion
Onthebasis of ourisostericreplacementstrategy, diarylani-
line derivatives were discovered as a distinct class of HIV-1
NNRTI agents. Current results supported our hypothesis that
the NH2 group on the central B-ring ortho to the anilino ring
position is crucial for interaction with K101 on the NNRTI
binding site by forming additional H-bonds. In the series of
new compounds, active diarylaniline derivatives are highly
potent against both wild-type and drug resistant HIV-1
strains. Among them, the most promising compound is 37
with low EC50 values against HIV-1 wild-type (0.003-0.005
μM) and several multidrug-resistant strains (0.005-0.604
μM). Generally speaking, 36 and 37 showed slightly better
EC50 values (0.052 and 0.032 μM, respectively) than 4 (EC50
0.058 μM), a newly marketed HIV-1 NNRTI drug. In addi-
tion, they can be synthesized more conveniently than the
DAPY derivatives 4 and 5.23,24 The present biological and
modeling studies further revealed some important SARs for
this series of diarylanilines: (1) the new scaffold can maintain a
similar binding orientation and conformation to those of
DAPY derivatives, (2) a para-cyanoaniline moiety is neces-
sary for anti-HIV activity, (3) an amino group on the central
B-ring ortho totheanilinemoietyposition iscrucial for achiev-
ing high potency, likely by forming H-bonds with K101, (4) an
additional nitro group (R2) on the central aromatic ring is
helpful for enhancing affinity of the inhibitor by interaction
with K172, and (5) the para-substituent (R3) on the phenoxy
ring is changeable but can greatly affect the anti-HIV activity.
5-Chloro-N-(4-methoxyphenyl)-2, 4-dinitroaniline (7). Method
A, yield 82%, starting with 237 mg (1 mmol) of 1,3-dichloro-4,6-
dinitrobenzene and 136 mg (1.1 mmol) of 4-methoxyaniline
to afford 226 mg of 7, yellow solid, mp 150-151 ꢀC. H NMR
1
(CDCl3) δ ppm 3.88 (3H, s, OCH3), 7.03 (2H, d, J = 8.8 Hz, ArH-
30, 50), 7.05 (1H, s, ArH-6), 7.21 (2H, d, J = 8.8 Hz, ArH-20, 60),
9.09 (1H, s, ArH-3), 9.74 (1H, s, NH). MS m/z (%) 324
(M þ 1, 100), 326 (M þ 3, 30).
5-Chloro-N-(4-methylphenyl)-2,4-dinitroaniline (8). Method
A, yield 94%, starting with 237 mg (1 mmol) of 1,3-dichloro-
4,6-dinitrobenzene and 118 mg (1.1 mmol) of 4-methylaniline to
1
afford 324 mg of 8, yellow solid, mp 148-150 ꢀC. H NMR
(CDCl3) δ ppm 2.43 (3H, s, CH3), 7.12 (1H, s, ArH-6), 7.17 (2H,
d, J = 8.0 Hz, ArH-30, 50), 7.32 (2H, d, J = 8.0 Hz, ArH-20, 60),
9.09 (1H, s, ArH-3), 9.80 (1H, br NH). MS m/z (%): 308 (M þ 1,
100), 310 (M þ 3, 31).
5-Chloro-N-(4-chlorophenyl)-2,4-dinitroaniline (9). Method
A, yield 80%, starting with 237 mg (1 mmol) of 1,3-dichloro-
4,6-dinitrobenzene and 166 mg (1.3 mmol) of 4-chloroaniline at
80 ꢀC for 20 min to afford 263 mg of 9, yellow solid, mp 136-
138 ꢀC. 1H NMR (CDCl3) δ ppm 7.11 (1H, s, ArH-6), 7.25 (2H,
d, J = 8.8 Hz, ArH-30, 50), 7.50 (2H, d, J = 8.8 Hz, ArH-20, 60),
9.08 (1H, s, ArH-3), 9.77 (1H, s, NH). MS (negative charge) m/z
(%): 326 (M - 1, 100), 328 (M þ 1, 80).
5-Chloro-N-(4-nitrophenyl)-2,4-dinitroaniline (10). Method B,
yield 83%, starting with 237 mg (1 mmol) of 1,3-dichloro-4,6-
dinitrobenzene and 166 mg (1.2 mmol) of 4-nitroaniline to
1
afford 280 mg of 10, yellow solid, mp 140-141 ꢀC. H NMR
δ ppm 7.55 (1H, s, ArH-6), 7.60 (2H, d, J = 8.8 Hz, ArH-20, 60),
8.29 (2H, d, J = 8.8 Hz, ArH-30, 50), 8.90 (1H, s, ArH-3), 10.19
(1H, s, NH). MS m/z (%): 308 (M þ 1, 100), 310 (M þ 3, 30).
5-Chloro-N-(4-cyanophenyl)-2,4-dinitroaniline (11). Method
B, yield 89%, starting with 237 mg (1 mmol) of 1,3-dichloro-
4,6-dinitrobenzene and 130 mg (1.1 mmol) of 4-cyanoaniline to
Experimental Section
Chemistry. Melting points were measured with an RY-1
melting apparatus without correction. The proton nuclear mag-
netic resonance (1H NMR) spectra were measured on a JNM-
ECA-400 (400 MHz) spectrometer using tetramethylsilane
(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 spectrometer with
electrospray ionization, and the relative intensity of each ion
peak is presented as percent (%). The purities of target com-
pounds were g95%, measured by HPLC analyses, which were
performed by Agilent 1100 HPLC system with a UV detector,
using a Grace Alltima HP C18 column (100 mm ꢀ 2.1 mm, 3 μm)
1
afford 284 mg of 11, yellow solid, mp 174-176 ꢀC. H NMR
(CDCl3) δ ppm 7.41 (1H, s, ArH-6), 7.57 (2H, d, J = 8.8 Hz, ArH-
20, 60), 7.91 (2H, d, J = 8.8 Hz, 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).
5-Chloro-N-(4-cyanophenyl)-2-nitroaniline (12). Method B,
yield 71%, starting with 576 mg (3 mmol) of 2,4-dichloro-1-
nitrobenzene and 425 mg (3.6 mmol) of 4-cyanoaniline to afford
1
583 mg of 12, red-yellow solid, mp 123-124 ꢀC. H NMR δ
ppm 7.17 (1H, d, J = 9.0 Hz, ArH-4), 7.40 (2H, d, J = 8.8 Hz,

4912 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Qin et al.
ArH-20, 60), 7.45 (1H, s, ArH-6), 7.78 (2H, d, J = 8.8 Hz, ArH-
30, 50), 8.14 (1H, d, J = 9.0 Hz, ArH-3), 9.48 (1H, s, NH). MS m/
z (%): 272 (M - 1, 100), 321 (M þ 1, 22).
10.13 (1H, s, NH). MS m/z (%): 503 (M þ 1, 100), 505 (M þ 3,
98). HPLC purity: 97.7%.
5-(400-Bromo-200,600-dimethylphenoxy)-N-(40-cyanophenyl)-
2,4-dinitroaniline (19). Method D, yield 85%, starting with
3.18 g (10 mmol) of 11 and 2.4 g (12 mmol) of 2,6-dimethyl-4-
bromophenol in the presence of K2CO3 (excess, 3.0 g, 21.7
General Preparation of Diphenylether (13-28). Method C
(microwave): A mixture of N-aryl-5-chloro-2-nitroaniline
(1 equiv) and 2,6-dimethylphenol (1.2 equiv) in DMF (3 mL)
in the presence of anhydrous potassium carbonate (2 equiv) was
irradiated under microwave with stirring at 190 ꢀC for 15 min.
The mixture was poured into ice-water, pH adjusted to neutral
with 5% HCl aq, and the mixture extracted with EtOAc three
times. After removal of organic solvent in vacuo, crude product
was purified by PTLC or a silica column (eluent: petroleum
ether/EtOAc). Method D (traditional): A mixture of N-aryl-5-
chloro-2-nitroaniline (11 or 12, 1 equiv) and a para-substituted
2,6-dimethylphenol (1.2 equiv) in DMF in the presence of
anhydrous potassium carbonate (excess) was heated at 130 ꢀC
for 5 h. Workup was the same as above, and crude product was
purified by a silica gel column.
1
mmol) to afford 4.3 g of 19, yellow solid, mp 276-278 ꢀC. H
NMR δ ppm 2.05 (6H, s, 2 ꢀ CH3), 5.93 (1H, s, ArH-6), 7.28
(2H, d, J = 8.8 Hz, ArH-20, 60), 7.42 (2H, s, ArH-300, 500), 7.73
(2H, d, J = 8.8 Hz, ArH-30, 50), 8.96 (1H, s, ArH-3), 10.07 (1H, s,
NH). MS (negative charge) m/z (%): 481 (M - 1, 100), 483 (M þ
1, 99). HPLC purity: 98.5%.
N-(40-Cyanophenyl)-5-(200,600-dimethylphenoxy)-2,4-dinitroa-
niline (20). Method C, yield 81%, starting with 319 mg (1.0
mmol) of 11 and 146.4 mg (1.2 mmol) of 2,6-dimethylphenol to
afford 327 mg of 20, orange solid, mp 229-230 ꢀC. 1H NMR δ
ppm 2.06 (6H, s, 2 ꢀ CH3), 6.01 (1H, s, ArH-6), 7.09 (2H, d, J =
8.8 Hz, ArH-20, 60), 7.11 (1H, t, J = 6.8 Hz, ArH-400), 7.28 (2H,
d, J = 8.8 Hz, ArH-30, 50), 7.69 (2H, d, J = 6.8 Hz, ArH-300, 500),
8.96 (1H, s, ArH-3), 10.07 (1H, s, NH). MS m/z (%): 405 (M þ 1,
100); HPLC purity: 99.9%.
5-(200,600-Dimethylphenoxy)-N-(40-methoxyphenyl)-2,4-dinitro-
aniline (13). Method C, yield 75%, starting with 129 mg (0.4
mmol) of 7 and 59 mg (0.48 mmol) of 2,6-dimethylphenol to
5-(400-Cyano-200,600-dimethylphenoxy)-N-(40-cyanophenyl)-
2,4-dinitroaniline (21). Method D, yield 94%, starting with 1.27
g (4.0 mmol) of 11 and 705 mg (4.8 mmol) of 2,6-dimethyl-4-
cyanophenol in DMF (4 mL) in the presence of anhydrous
potassium carbonate (966 mg, 7 mmol) to afford 1.61 g of 21,
1
afford 123 mg of 13, yellow solid, mp 169-170 ꢀC. H NMR
(CDCl3) δ ppm 2.06 (6H, s, 2 ꢀ CH3), 3.78 (3H, s, OCH3), 5.91
(1H, s, ArH-6), 6.75 (2H, d, J = 8.8 Hz, ArH-20, 60), 6.89 (2H, d,
J = 8.8 Hz, ArH-30, 50), 7.11 (1H, t, J = 6.8 Hz, ArH-400), 7.69
(2H, d, J = 6.8 Hz, ArH-300, 500), 8.96 (1H, s, ArH-3), 9.70 (1H, s,
NH). MS m/z (%): 410 (M þ 1, 100). HPLC purity: 99.4%.
N1-(40-Methoxyphenyl)-5-(200,400,600-trimethylphenoxy)-2,4-dini-
troaniline (14). Method C, yield 33%, starting with 162 mg
(0.5 mmol) of 7 and 82 mg (0.6 mmol) of 2,4,6-trimethylphenol
1
orange solid, mp >300 ꢀC. H NMR δ ppm 2.08 (6H, s, 2 ꢀ
CH3), 5.90 (1H, s, ArH-6), 7.29 (2H, d, J = 8.8 Hz, ArH-20, 60),
7.73 (2H, s, ArH-300, 500), 7.74 (2H, d, J = 8.8 Hz, ArH-30, 50),
8.97 (1H, s, ArH-3), 10.07 (1H, s, NH). MS m/z (%): 430 (M þ 1,
100). HPLC purity: 98.1%.
1
to afford 70 mg of 14, red solid, mp 155-158 ꢀC. H NMR
N-(40-Cyanophenyl)-2,4-dinitro-5-(200,400,600-trimethylphenoxy)-
aniline (22). Method C, yield 70%, starting with 637 mg
(2 mmol) of 11 and 286 mg (2.1 mmol) of 2,4,6-trimethylphenol
to afford 580 mg of 22, yellow solid, mp 223-225 ꢀC. 1H NMR δ
ppm 2.00 (6H, s, 2 ꢀ CH3), 2.22 (3H, s, CH3), 5.92 (1H, s, ArH-6),
6.95 (2H, s, ArH-300, 500), 7.27 (2H, d, J = 8.8 Hz, ArH-20, 60), 7.72
(2H, d, J = 8.8 Hz, ArH-30, 50), 8.95 (1H, s, ArH-3), 10.04 (1H, s,
NH). MS m/z (%): 419 (M þ 1, 100). HPLC purity: 98.3%.
N-(40-Cyanophenyl)-5-(200,600-dimethyl-400-formylphenoxy)-
2,4-dinitroaniline (23). Method C, yield 74%, starting with
160 mg (0.5 mmol) of 11 and 113 mg (0.6 mmol) 4-hydroxy-
3,5-dimethylbenzaldehyde to afford 144 mg of 23, yellow solid,
mp 260-263 ꢀC. 1H NMR (CDCl3) δ ppm 2.23 (6H, s, 2 ꢀ CH3),
6.24 (1H, s, ArH-6), 7.10 (2H, d, J = 8.8 Hz, ArH-20, 60), 7.54
(2H, d, J = 8.8 Hz, ArH-30, 50), 7.67 (2H, s, ArH-300, 500), 9.20
(1H, s, ArH-3), 9.97 (2H, s, NH and CHO). MS m/z (%) 433
(M þ 1, 100). HPLC purity: 98.7%.
(CDCl3) δ ppm 1.98 (6H, s, 2 ꢀ CH3), 2.16 (3H, s, CH3), 3.73
(3H, s, OCH3), 5.82 (1H, s, ArH-6), 6.69 (2H, s, ArH), 6.72 (2H,
d, J = 8.8 Hz, ArH-20, 60), 6.84 (2H, d, J = 8.8 Hz, ArH-30, 50),
9.11 (1H, s, ArH-3), 9.60 (1H, s, NH). MS m/z (%): 424 (M þ 1,
100). HPLC purity: 98.3%.
5-(400-Bromo-200,600-dimethylphenoxy)-N-(40-methoxyphenyl)-
2,4-dinitroaniline (15). Method C, yield 53%, starting with
647 mg (2 mmol) of 7 and 422 mg (2.1 mmol) of 4-bromo-2,6-
dimethylphenol to afford 517 mg of 15, yellow solid, mp
221-223 ꢀC. 1H NMR (CDCl3) δ ppm 2.07 (6H, s, 2 ꢀ CH3),
3.78 (3H, s, OCH3), 5.91 (1H, s, ArH-6), 6.75 (2H, d, J = 9.0 Hz,
ArH-20, 60), 6.89 (2H, d, J = 9.0 Hz, ArH-30, 50), 7.34 (2H, s,
ArH-300, 500), 9.17 (1H, s, ArH-3), 9.68 (1H, s, NH). MS m/z (%):
488 (M þ 1, 100), 490 (M þ 3, 98). HPLC purity: 95.8%.
5-(400-Bromo-200,600-dimethylphenoxy)-N-(40-methylphenyl)-
2,4-dinitroaniline (16). Method C, yield 52%, starting with
154 mg (0.5 mmol) of 8 and 121 mg (0.6 mmol) of 4-bromo-2,
6-dimethylphenol to afford 123 mg of 16, yellow solid, mp
200-202 ꢀC. 1H NMR (CDCl3) δ ppm 2.08 (6H, s, 2 ꢀ CH3),
2.36 (3H, s, CH3), 5.89 (1H, s, ArH-6), 6.86 (2H, d, J = 8.8 Hz,
ArH-30, 50), 7.10 (2H, d, J = 8.8 Hz, ArH-20, 60), 7.16 (2H, s,
ArH-300, 500), 9.17 (1H, s, ArH-3), 9.71 (1H, s, NH). MS m/z (%):
472 (M þ 1, 100), 474 (M þ 3, 92). HPLC purity: 97.5%.
5-(400-Bromo-200,600-dimethylphenoxy)-N-(40-chlorophenyl)-
2,4-dinitroaniline (17). Method C, yield 64%, starting with 328
mg (1.0 mmol) of 9 and 201 mg (1.0 mmol) of 4-bromo-2,6-
dimethylphenol to afford 315 mg of 17, yellow solid, mp
206-208 ꢀC. 1H NMR δ ppm 2.01 (6H, s, 2 ꢀ CH3), 5.70 (1H,
s, ArH-6), 7.15 (2H, d, J = 8.8 Hz, ArH-30, 50), 7.35 (2H, d, J =
8.8 Hz, ArH-20, 60), 7.37 (2H, s, ArH-300, 500), 8.96 (1H, s, ArH-
3), 10.01 (1H, s, NH). MS m/z (%): 492 (M þ 1, 70), 494 (M þ 3,
100). HPLC purity: 96.3%.
5-(400-Bromo-200,600-dimethylphenoxy)-N-(40-cyanophenyl)-2-
nitroaniline (24). Method C, yield 93%, starting with 12 (100 mg,
0.36 mmol) and potassium 4-bromo-2,6-dimethylphenoxide
(105 mg, 0.44 mmol) in DMF (2 mL) were irradiated under
microwave with stirring at 192 ꢀC for 10 min to get 147 mg of 24,
1
light-yellow solid, mp 143 ꢀC. H NMR (CDCl3) δ ppm 2.03
(6H, s, 2 ꢀ CH3), 6.19 (1H, dd, J = 9.2 and 2.4 Hz, ArH-6), 6.74
(1H, d, J = 2.4 Hz, ArH-4), 7.21 (2H, d, J = 8.8 Hz, ArH-20, 60),
7.30 (2H, s, ArH-300, 500), 7.57 (2H, d, J = 8.8 Hz, ArH-30, 50),
8.13 (1H, d, J = 9.2 Hz, ArH-3), 9.69 (1H, s, NH). MS m/z (%):
438 (M þ 1, 100), 440 (M þ 3, 98). HPLC purity: 95.7%.
N-(40-Cyanophenyl)-5-(200,600-dimethylphenoxy)-2-nitroani-
line (25). Method C, yield 31%, starting with 12 (50 mg, 0.18
mmol) and 2,6-dimethylphenol (31 mg, 0.25 mmol) in the
presence of potassium carbonate (58 mg, 0.42 mmol) in DMSO
(2 mL) to afford 20 mg of 25, yellow solid, mp 158-60 ꢀC. 1H
NMR (CDCl3) δ ppm 2.12 (6H, s, 2 ꢀ CH3), 6.35 (1H, q,
J1 = 9.2 Hz, J2 = 2.4 Hz, ArH-4), 6.73 (1H, d, J = 2.4 Hz, ArH-
6), 7.10 (3H, m, ArH-300, 400, 500), 7.24 (2H, d, J = 8.4 Hz, ArH-
20, 60), 7.59 (2H, d, J = 8.4 Hz, ArH-30, 50), 8.21 (1H, d, J = 9.2
Hz, ArH-3), 9.76 (1H, s, NH). MS m/z (%): 360 (Mþ, 100).
HPLC purity: 96.6%.
5-(400-Bromo-200,600-dimethylphenoxy)-N-(40-nitrophenyl)-2,4-
dinitroaniline (18). Method C, yield 78%, starting with 154 mg
(0.44 mmol) of 10 and 107 mg (0.53 mmol) of 4-bromo-2,
6-dimethylphenol to afford 173 mg of 18, yellow solid, mp
190-191 ꢀC. 1H NMR δ ppm 2.05 (6H, s, 2 ꢀ CH3), 6.06 (1H,
s, ArH-6), 7.31 (2H, d, J = 8.8 Hz, ArH-20, 60), 7.41 (2H, s, ArH-
300, 500), 8.13 (2H, d, J = 8.8 Hz, ArH-30, 50), 8.96 (1H, s, ArH-3),

Article
5-(400-Cyano-200,600-dimethylphenoxy)-N-(40-cyanophenyl)-2-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4913
(2H, d, J=8.8 Hz, ArH-20, 60), 7.30 (2H, s, ArH-300, 500), 7.38
(2H, d, J=8.8 Hz, ArH-30, 50), 7.71 (1H, s, NH). MS m/z (%):
423 (M þ 1, 100), 425 (M þ 3, 98). HPLC purity: 96.1%.
N1-(40-Cyanophenyl)-5-(200,600-dimethylphenoxy)benzene-1,2,
4-triamine (33). Yield 30%, starting with 152 mg (0.31 mmol) of
20 to afford 39 mg of 33, brown solid, mp 156-158 ꢀC. 1H NMR
δ ppm 2.08 (6H, s, 2 ꢀ CH3), 4.22 (2H, s, NH2-4), 4.84 (2H,s,
NH2-2), 5.70 (1H, s, ArH-3), 6.23 (1H, s, ArH-6), 6.43 (2H, d,
J = 8.8 Hz, ArH-20, 60), 6.99 (2H, d, J = 6.8 Hz, ArH-300, 500),
7.07 (1H, t, J = 6.8 Hz, ArH-400), 7.36 (2H, d, J = 8.8 Hz, ArH-
30, 50), 7.67 (1H, s, NH). MS m/z (%): 345 (Mþ, 100). HPLC
purity: 99.6%.
nitroaniline (26). Method D, yield 79%, starting with 821 mg
(3 mmol) of 12 and 530 mg (3.6 mmol) of 2,6-dimethyl-4-cya-
nophenol in the presence of K2CO3 (excess, 1.45 g, 10.5 mmol)
1
to afford 0.92 g of 26, yellow solid, mp186-188 ꢀC. H NMR
(CDCl3) δ ppm 2.16 (6H, s, 2 ꢀ CH3), 6.15 (1H, dd, J = 9.2 and
2.4 Hz, ArH-4), 6.87 (1H, d, J=2.4 Hz, ArH-6), 7.31 (2H, d, J=
8.8 Hz, ArH-20, 60), 7.45 (2H, s, ArH-300, 500), 7.66 (2H, d, J=8.8
Hz, ArH-30, 50), 8.21 (1H, d, J=9.2 Hz, ArH-3), 9.75 (1H, s,
NH). MS m/z (%): 385 (M þ 1, 100). HPLC purity: 99.0%.
N-(40-Cyanophenyl)-5-(200,400,600-trimethylphenoxy)-2-nitroa-
niline (27). Method D, yield 74%, starting with 274 mg (1 mmol)
of 12 and 241 mg (1.2 mmol) of 2,4,6-trimethylphenol in the
presence of excess K2CO3 to afford 225 mg of 27, yellow solid,
mp 168-170 ꢀC. 1H NMR (CDCl3) δ ppm 2.00 (6H, s, 2 ꢀ CH3),
2.23 (3H, s, CH3), 6.25 (1H, d, J = 9.2 Hz, ArH-4), 6.69 (1H, s,
ArH-6), 6.84 (2H, s, ArH-300, 500), 7.18 (2H, d, J = 8.8 Hz, ArH-
20, 60), 7.53 (2H, d, J=8.8 Hz, ArH-30, 50), 8.12 (1H, d, J=9.2 Hz,
ArH-3), 9.67 (1H, s, NH). MS m/z (%) 474 (M þ 1, 100). HPLC
purity: 95.9%.
5-(400-Cyano-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)ben-
zene-1,2,4-triamine (34). Yield 86%, starting with 300 mg (0.7
mmol) of 21 to afford 223 mg of 34, brown solid, mp 144 ꢀC. 1H
NMR δ ppm 2.08 (6H, s, 2 ꢀ CH3), 4.33 (2H, s, NH2), 4.97 (2H,
s, NH2), 5.71 (1H, s, ArH-3), 6.23 (1H, s, ArH-6), 6.43 (2H, d,
J = 8.8 Hz, ArH-20, 60), 7.38 (2H, d, J = 8.8 Hz, ArH-30, 50), 7.61
(2H, s, ArH-300, 500), 7.71 (1H, s, NH). MS m/z (%) 370 (M þ 1,
100). HPLC purity: 100.0%.
N1-(40-Cyanophenyl)-5-(200,400,600-trimethylphenoxy)benzene-
1,2,4-triamine (35). Yield 85%, starting with 150 mg (0.36 mmol)
of 22 to afford 110 mg of 35, light-brown solid, mp 152-154 ꢀC.
1H NMR (CDCl3) δ ppm 2.08 (6H, s, 2 ꢀ CH3), 2.26 (3H, s,
CH3), 3.50(2H, s, NH2), 4.0(2H, s, NH2), 5.33 (1H, s, ArH-6), 5.97
(1H, s, ArH-3), 6.29 (1H, s, NH), 6.47 (2H, d, J = 8.8 Hz, ArH-20,
60), 6.86 (2H, s, ArH-300, 500), 7.36 (2H, d, J=8.8 Hz, ArH-30, 50).
MS m/z (%): 359 (M þ 1, 100). HPLC purity: 100.0%.
N-(40-Cyanophenyl)-5-(200,600-dimethyl-400-formylphenoxy)-2-
nitroaniline (28). Method C, yield 70%, starting with 137 mg (0.5
mmol) of 12 and 90 mg (0.6 mmol) of 4-hydroxy-3,5- dimethyl-
benzaldehyde to afford 135 mg of 28, yellow solid, mp 152-
1
155 ꢀC. H NMR (CDCl3) δ ppm 2.21 (6H, s, 2 ꢀ CH3), 6.23
(1H, dd, J = 8.8 and 2.4 Hz, ArH-4), 6.84 (1H, d, J = 2.4 Hz,
ArH-6), 7.30 (2H, d, J = 8.8 Hz, ArH-20, 60), 7.64 (2H, d, J = 8.8
Hz, ArH-30, 50), 7.67 (2H, s, ArH), 8.22 (1H, d, J = 8.8 Hz, ArH-
3), 9.76 (1H, br s, NH), 9.97 (1H, s, CHO). MS m/z (%): 338
(M þ 1, 100). HPLC purity: 98.6%.
5-(400-Bromo-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)-4-
nitrobenzene-1,2-diamine (36). Pd-C (10%, 30 mg) was added to
a solution of 19 (338 mg, 0.7 mmol) in acetonitrile (6 mL) and
triethylamine (6 mL). The mixture was cooled to -15 ꢀC, and
then formic acid (95%, 1 mL, excess) in acetonitrile (4 mL) was
slowly added at the low temperature. The mixture was then
heated to reflux for 1 h, following which the Pd-C and solvent
were removed. The residue was purified by PTLC (petroleum
ether/EtOAc 4:1) to obtain 269 mg of 36, yield 85%, deep-red
solid, mp 218-220 ꢀC. 1H NMR δ ppm 2.07 (6H, s, 2 ꢀ CH3),
5.13 (2H, s, NH2), 6.16 (1H, s, ArH-6), 6.82 (2H, d, J = 8.4 Hz,
ArH-20, 60), 7.40 (2H, s, ArH-300, 500), 7.55 (2H, d, J = 8.4 Hz,
ArH-30, 50), 7.95 (1H, s, ArH-3), 8.45 (1H, s, NH). MS m/z (%)
453 (M þ 1, 95), 455 (M þ 3, 100). HPLC purity: 97.0%.
5-(400-Cyano-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)-4-
nitrobenzene-1,2-diamine (37). The preparation was the same as
that of 36. Yield 86%, starting with 150 mg (0.35 mmol) of 21 to
afford 237 mg of 37, red solid, mp 224-226 ꢀC. 1H NMR δ ppm
2.07 (6H, s, 2 ꢀ CH3), 5.17 (2H, s, NH2), 6.15 (1H, s, ArH-6),
6.81 (2H, d, J = 8.8 Hz, ArH-20, 60), 7.55 (2H, d, J = 8.8 Hz,
ArH-30, 50), 7.71 (2H, s, ArH), 8.06 (1H, s, ArH-3), 8.45 (1H, s,
NH). MS m/z (%): 400 (M þ 1, 100). HPLC purity: 96.8%.
N1-(40-Cyanophenyl)-5-(200,400,600-trimethylphenoxy)-4-nitro-
benzene-1,2-diamine (38). The preparation was the same as that
of 36. Yield 79%, starting with 300 mg (0.72 mmol) of 22 to
afford 220 mg of 38, red solid, mp 110-112 ꢀC. 1H NMR
(CDCl3) δ ppm 2.07 (6H, s, 2 ꢀ CH3), 2.28 (3H, s, CH3), 6.36
(1H, s, ArH-6), 6.76 (2H, d, J = 8.8 Hz, ArH-20, 60), 6.89 (2H, s,
ArH-300, 500), 7.45 (2H, d, J = 8.8 Hz, ArH-30, 50), 7.62 (1H, s,
ArH-3). MS m/z (%): 389 (M þ 1, 100); HPLC purity: 98.3%.
6-(4-Cyano-2,6-dimethylphenoxy)-1-(4-cyanophenyl)-5-nitro-
1H-benzoimidazole (39). Triethyl orthoformate (1 mL, excess)
and HCl in diethyl ether (1N, 1 mL) were added successively to a
solution of 37 (50 mg, 0.125 mmol) in DMF (3 mL) under N2.
The mixture was stirred at rt for 3 h, poured into water with pH
adjusted to 6-7, and left to stand overnight. The resulting
yellow solid was collected and washed with water. The crude
product was purified by a silica gel column (eluent: CH2Cl2/
EtOAc 10:1) to afford 42 mg of 39, yield 88%, light-yellow solid,
mp 250-252 ꢀC. 1H NMR (CDCl3) δ ppm 2.21 (6H, s, 2 ꢀ CH3),
6.43 (1H, s, ArH-7), 7.43 (2H, d, J=8.8 Hz, ArH-30, 50), 7.46
(2H, s, ArH-300, 500), 7.86 (2H, d, J=8.8 Hz, ArH-20, 60), 8.16
General Preparation of 1,5-Diarylbenzene-1,2-diamines or
1,2,4-Triamines (29-38). To a solution of a diaryl-nitrobenzene
(24, 26-27, or 19-22, 1 equiv) in 15 mL of isopropyl alcohol was
added FeCl3 6H2O (1 equiv), activated carbon (2 equiv), and
3
N2H4 H2O (10 equiv), successively. The mixture was refluxed
3
for 20-30 min. After removal of carbon and solvent, the residue
was purified by a silica gel column (eluent: CHCl3/MeOH 40:1)
to obtain corresponding diarylbenzene-1,2-diamine or -1,2,4-
triamines, respectively.
5-(400-Bromo-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)ben-
zene-1,2-diamine (29). Yield 83%, starting with 150 mg (0.34
mmol) of 24 to afford 115 mg of 29, brown solid, mp 154-155
ꢀC. 1H NMR δ ppm 2.07 (6H, s, 2 ꢀ CH3), 4.55 (2H, s, ArNH2),
6.34 (1H, s, ArH-6), 6.47 (1H, d, J = 8.8 Hz, ArH-4), 6.65 (2H,
d, J = 8.8 Hz, ArH-20, 60), 6.73 (1H, d, J = 8.8 Hz, ArH-3), 7.34
(2H, s, ArH-300, 500), 7.49 (2H, d, J = 8.8 Hz, ArH-30, 50), 8.08
(1H, s, NH). MS m/z (%) 408 (M þ 1, 100), 410 (M þ 3, 98).
HPLC purity: 96.8%.
5-(400-Cyano-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)ben-
zene-1,2-diamine (30). Yield 83%, starting with 130 mg (0.34
mmol) of 26 to afford 100 mg of 30, brown solid, mp 154-
156 ꢀC. 1H NMR (CDCl3) δ ppm 2.18 (6H, s, 2ꢀCH3), 3.0-4.0
(2H, NH2), 5.67 (1H, s, NH), 6.47 (1H, q, J = 8.8 and 2.8 Hz,
ArH-4), 6.58 (1H, d, J = 2.8 Hz, ArH-6), 6.70 (2H, d, J = 8.8
Hz, ArH-20, 60), 6.74 (1H, d, J = 8.8 Hz, ArH-3), 7.40 (2H, s,
ArH-300, 500), 7.47 (2H, d, J = 8.8 Hz, ArH-30, 50). MS m/z (%):
355 (M þ 1, 100). HPLC purity: 99.2%.
N1-(40-Cyanophenyl)-5-(200,400,600-trimethylphenoxy)benzene-
1,2-diamine (31). Yield 84%, starting with 150 mg (0.4 mmol) of
27 to afford 116 mg of 31, mp152-154 ꢀC. 1H NMR δ ppm 2.02
(6H, s, 2 ꢀ CH3), 2.22 (3H, s, CH3), 4.48 (2H, s, NH2), 6.29 (1H,
d, J = 2.8 Hz, ArH-6), 6.48 (1H, dd, J = 8.4 and 2.8 Hz, ArH-4),
6.64 (1H, d, J = 8.4 Hz, ArH-3), 6.72 (2H, d, J = 8.8 Hz, ArH-
20, 60), 7.48 (2H, d, J = 8.8 Hz, ArH-30, 50), 8.07 (1H, s, NH). MS
m/z (%): 344 (M þ 1, 100). HPLC purity: 99.8%.
5-(400-Bromo-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)ben-
zene-1,2,4-triamine (32). Yield 95%, starting with 150 mg (0.31
mmol) of 19 to afford 125 mg of 32, brown solid, mp 181-
183 ꢀC. 1H NMR δ ppm 2.08 (6H, s, 2ꢀCH3), 4.28 (2H, s, NH2),
4.91 (2H, s, NH2), 5.70 (1H, s, ArH-3), 6.22 (1H, s, ArH-6), 6.43

4914 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Qin et al.
(1H, s, ArH-2), 8.54 (1H, s, ArH-4). MS m/z (%): 410 (Mþ1,
100). HPLC purity: 96.0%.
of inhibition of p24 production was calculated as previously
described.25 The effective concentrations for 50% inhibition
(EC50) were calculated using a computer program, designated
CalcuSyn,26 kindly provided by Dr. T. C. Chou (Sloan-Kettering
Cancer Center, New York, NY).
(400-Cyano-200,600-dimethylphenoxy)-N1-(40-cyanophenyl)-4-
nitrobenzene-1,2-diamine Hydrochloride (40). To a solution of 36
(100 mg) in acetone (10 mL) was slowly added HCl diethyl ether
solution (18%, 3 mL), and a light-yellow solid appeared. The
solid was filtered out and recrystallized from anhydrous MeOH
to afford 52 mg of 40, yield 48%, yellow solid, mp 170-171 ꢀC.
1H NMR δ ppm 2.07 (6H, s, 2 ꢀ CH3), 6.17 (1H, s, ArH-6), 6.83
(2H, d, J = 8.8 Hz, ArH-20, 60), 7.40 (2H, s, ArH-300, 500), 7. 54
(1H, s, ArH-3), 7.56 (2H, d, J = 8.8 Hz, ArH-30, 50), 8.50 (1H, s,
NH). MS m/z (%) 453 (M þ 1, 100), 455 (M þ 3, 100). HPLC
purity: 97.6%.
HIV-1 Infectivity Assay with Mutant Viral Strains in MT-2
Cell Line. A diluted drug resistant HIV-1 stock, mutated viral
strain (8605MR or 6005MR from Panacos, Inc.), at a multi-
plicity of infection (MOI) of 0.001 TCID50/cell was used to
infect MT-2 cells. First, 20 μL of the virus and 20 μL of
compounds at various concentrations in RPMI 1640 containing
10% fetal bovine serum were added to 20 μL of MT4 cells at 6 ꢀ
105 cells/mL in a 96-well microtiter plate. The cell/virus/com-
pound mixture was then incubated at 37 ꢀC in a humidified CO2
incubator. Fresh medium (180 μL) containing an appropriate
concentration of the compound was added to each well of the
cultures on day 2. On day 4 postinfection, supernatant samples
were harvested and assayed for P24 using an ELISA kit from
ZeptoMetrix Corporation, Buffalo, NY.
Assessment of In Vitro Cytotoxicity in MT-2 Cells. The in
vitro cytotoxicity of compounds on MT-2 cells was measured by
XTT assay.27 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.
HIV Growth Inhibition Assay in H9 Lymphocytes from Pana-
cos, Inc. The evaluation of HIV-1 inhibition was carried out as
follows according to established protocols. The human T-cell
line, H9, was maintained in continuous culture with complete
medium (RPMI 1640 with 10% fetal calf serum supplemented
with L-glutamine at 5% CO2 and 37 ꢀC). Test samples were first
dissolved in dimethyl sulfoxide at a concentration of 10 mg/mL
to generate master stocks with dilutions made into tissue culture
media to generate working stocks. The following drug concen-
trations were routinely used for screening: 100, 20, 4, and 0.8 μg/
mL. For agents found to be active, additional dilutions were
prepared for subsequent testing so that an accurate EC50 value
could be determined. Test samples were prepared and to each
sample well was added 90 μL of media containing H9 cells at 3 ꢀ
105 cells/mL and 45 μL of virus inoculum (HIV-1 IIIB isolate)
containing 125 TCID50. Control wells containing virus and cells
only (no drug) and cells only (no virus or drug) were also
prepared. A second set of samples were prepared identical to
the first and were added to cells under identical conditions
without virus (mock infection) for toxicity determinations
(IC50 defined below). In addition, AZT was also assayed during
each experiment as a positive drug control. On days 1 and 4
postinfection (PI), spent media was removed from each well and
replaced with fresh media. On day 6 PI, the assay was terminated
and culture supernatants were harvested for analysis of virus
replication by p24 antigen capture. Compound toxicity was
determined by XTT using the mock-infected samples.
Assay for Measuring the Inhibitory Activity of Compounds on
HIV-1 IIIB Replication in MT-2 Cells. The inhibitory activity of
compounds on HIV-1 IIIB replication was determined as pre-
viously described. In brief, 1 ꢀ 104 MT-2 cells 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 cul-
ture 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. Briefly, wells of
polystyrene plates (Immulon 1B, Dynex Technology, Chantilly,
VA) were coated with HIV immunoglobulin (HIVIG) in
0.085 M carbonate-bicarbonate buffer (pH 9.6) at 4 ꢀC over-
night, followed by washes with washing buffer (0.01 M PBS
containing 0.05% Tween-20) and blocking with PBS containing
1% dry fat-free milk (Bio-Rad, Inc., Hercules, CA). Virus
lysates were added to the wells and incubated at 37 ꢀC for 1 h.
After extensive washes, anti-p24 mAb (183-12H-5C), biotin-
labeled antimouse IgG1 (Santa Cruz Biotechnology, Santa
Cruz, CA), streptavidin-labeled horseradish peroxidase (Zymed,
S. San Francisco, CA), and the substrate 3,30,5,50-tetramethyl-
benzidine (Sigma Chemical Co., St. Louis, MO) were added
sequentially. Reactions were terminated by addition of 1N
H2SO4. Absorbance at 450 nm was recorded in an ELISA reader
(Ultra 386, TECAN, Research Triangle Park, NC). Recombi-
nant protein p24 purchased from US Biological (Swampscott,
MA) was included for establishing standard dose response
curves. Each sample was tested in triplicate. The percentage
Assay for RT Enzymatic Inhibition. The RT activity of HIV-
1DH012 (a primary isolate viral strain) was determined in the
presence of various concentrations of the tested compounds
using a Roche colorimetric HIV-1 RT assay kit following the
protocol provided by the manufacturer.
Computational Study. Three-dimensional constructions of
compounds 21, 34, and 37 were achieved by using the Sybyl
version 7.2 software with the Tripos force-field and Gasteiger-
Huckel atomic partial charges.28 To get the correct orientation
€
of the three compounds inside the HIV-1 RT enzyme, the
constructions were made by modifying the experimental coor-
dinates of the DAPY compound in the HIV-1 RT enzyme (PDB:
1s6q). Then the compounds were optimized with 20 simplex
iterations followed by 1000 steps of Powell algorithm.
Energy minimizations were obtained with the Amber 9
software.29 Ligand parameters were created according to the
antechamber procedure and the partial charges were evaluated
following the AM1 bond charge correction (AM1-BCC)
method.30-32 The general Amber force field33 (gaff) was set
for the ligand, whereas the ff03 force field34 was used for the
protein. Minimizations were done with 1000 steps of steepest
descent followed by 4000 steps of conjugate gradient. Solvent
effects were taken into account through general Born model
implicit solvent model.35-38 The molecular mechanics/general
Born surface area (MM/GBSA) method39-41 permitted the
evaluation of the ligand-protein interaction free energies, and
calculation details may be found elsewhere.42 Finally, VMD
software43 was employed to visualize and analyze the 3D
structures resulting from AMBER calculations.
Acknowledgment. We thank Nicole Kilgore at Panacos
Pharmaceuticals, Inc., for providing data listed in Tables 1
and 2. HIV-1 mutated viral strains 8605MR (multimutated-
RT: M41L, D67N, L210W, T215Y, M184 V, K103N) and
6005MR (multimutated-RT: M41L, L74 V, M184 V, L210W,
T215Y, ins SS, A98G) were from Panacos, Inc., and HIV-1
strains IIIB, A17 were provided by the NIH AIDS Reagent
and Reference Program, by contributions from Drs. R. Gallo,
E. Emini, and Trimeris, Inc. This investigation was supported

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4915
by grants 20472114, 30930106, and 7052057 from the National
Natural Science Foundation of China and Beijing govern-
ment, respectively, awarded to L. Xie, as well as U.S. NIH
grants awarded to K. H. Lee (AI33066), S. Jiang (AI46221),
and C. H. Chen (AI65310).
non-nucleoside reverse transcriptase inhibitors that are highly
potent and effective against wild-type and drug-resistant HIV-1
variants. J. Med. Chem. 2004, 47, 2550–2560.
(12) Das, K.; Bauman, J. D.; Clark, A. D.; Frenkel, Y. V.; Lewi, P. J.;
Shatkin, A. J.; Hughes, S. H.; Arnold, E. High-resolution struc-
tures of HIV-1 reverse transcriptase/TMC278 complexes: strategic
flexibility explains potency against resistance mutations. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 1466–1471.
(13) Heeres, J.; de Jonge, M.; Koymans, L. M. H.; Daeyaert, F. F. D.;
Vinkers, M.; Van Aken, K. J. A.; Arnold, E.; Das, K.; Kilonda, A.;
Hoornaert, G. J.; Compernolle, F.; Cegla, M.; Azzam, R. A.;
Supporting Information Available: HPLC conditions and
summary of HPLC purity data for final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
ꢀ
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