Structural modifications of diarylpyrimidines (DAPYs) as HIV-1 NNRTIs: Synthesis, anti-HIV activities and SAR

Article abstract of DOI:10.1016/j.bmc.2017.03.009

30 new analogues of diarylpyrimidines were synthesized for further structural modifications, involving not only the linker but also the wing α of DAPYs. The anti-HIV-1 activities of all target molecules were evaluated, and most of them exhibited potent anti-HIV-1 (WT) activities and low cytotoxicities. Among which, compound 4g showed excellent activities against WT HIV-1 with an EC₅₀ value of 5.8?nM and SI of up to 26,034. Another compound 4ab bearing a novel pyridinyl Wing α also displayed attractive activities. The structure-activity relationship (SAR) study was also summarized.

Full text of DOI:10.1016/j.bmc.2017.03.009

Accepted Manuscript
Structural modifications of diarylpyrimidines (DAPYs) as HIV-1 NNRTIs: syn-
thesis, anti-HIV activities and SAR
Huan-Huan Lu, Ping Xue, Yuan-Yuan Zhu, Xiu-Lian Ju, Xiao-Jiao Zheng, Xun
Zhang, Ting Xiao, Christophe Pannecouque, Ting-Ting Li, Shuang-Xi Gu
PII:
S0968-0896(17)30218-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.009
BMC 13605
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2017
4 March 2017
5 March 2017
Please cite this article as: Lu, H-H., Xue, P., Zhu, Y-Y., Ju, X-L., Zheng, X-J., Zhang, X., Xiao, T., Pannecouque,
C., Li, T-T., Gu, S-X., Structural modifications of diarylpyrimidines (DAPYs) as HIV-1 NNRTIs: synthesis, anti-
HIV activities and SAR, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.03.009
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Structural modifications of diarylpyrimidines (DAPYs) as HIV-1 NNRTIs:
synthesis, anti-HIV activities and SAR
Huan-Huan Lua, Ping Xuea, Yuan-Yuan-Zhub,*, Xiu-Lian Jua, Xiao-Jiao Zhenga; Xun Zhanga, Ting
Xiaoa, Christophe Pannecouquec,*, Ting-Ting Li,d Shuang-Xi Gua,*
a
Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering &
Pharmacy, Wuhan Institute of Technology, Wuhan 430205, China
b School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, China
c
KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega
Institute for Medical Research, B-3000 Leuven, Belgium
d Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
*Corresponding Authors:
E-mail: yyzhu531@163.com; christophe.pannecouque@kuleuven.be; shuangxigu@163.com.
Abstract
30 new analogues of diarylpyrimidines were synthesized for further structural modifications, involving
not only the linker but also the wing α of DAPYs. The anti-HIV-1 activities of all target molecules were
evaluated, and most of them exhibited potent anti-HIV-1 (WT) activities and low cytotoxicities. Among
which, compound 4g showed excellent activities against WT HIV-1 with an EC50 value of 5.8 nM and
SI of up to 26,034. Another compound 4ab bearing a novel pyridinyl Wing α also displayed attractive
activities. The structure-activity relationship (SAR) study was also summarized.
Keywords: AIDS; anti-HIV; diarylpyrimidines; nonnucleoside reverse transcriptase inhibitors; SAR
1

1. Introduction
Acquired immunodeficiency syndrome (AIDS) is a global infectious disease caused by human
immunodeficiency virus (HIV) infection. According to the data from UNAIDS reported on July 11th,
2016, the situation is still unoptimistic: in 2015, 36.7 million people lived with HIV, 2.1 million people
were newly infected with HIV, and there were as many as 1.1 million of AIDS-related deaths.1 So far
there is no cure or effective vaccine against HIV/AIDS. Highly active antiretroviral therapy (HAART)
still occupies a dominant position in combating AIDS in spite of its existing limitations such as
complicated dosing, intolerable toxicities, caustic adherence.2-4 Nonnucleoside reverse transcriptase
inhibitors (NNRTIs), known as one of the indispensable components of HAART for specifically
inhibiting HIV-1 reverse transcriptase (RT), have received wide attention due to their potent antiviral
activity, high specificity and low cytotoxicity.5, 6
Among various structurally diverse classes of NNRTIs,7, 8 diarylpyrimidines (DAPYs) have been
recognized as one of the most successful families of NNRTIs developed so far owing to their excellent
potency against both HIV-1 wild-type (WT) and mutant strains.9 In DAPYs series, the most important
representatives etravirine (TMC125, 1, Fig. 1) and rilpivirine (TMC278, 2, Fig. 1) have been approved
by the U.S. Food and Drug Administration (FDA) in 2008 and 2011, respectively.10, 11 Since the right
wing of the DAPY structure was identified as the essential pharmacophore, the further modifications
were mainly focused on the structural diversity of the linker between the left phenyl ring and the
central pyrimidine ring. Until present, nearly all of the DAPY derivatives share a common
characteristic: the monoatomic linker (C, O, S, or N) between Wing II and the central pyrimidine
ring.12-16
In 2015, based on the concept of improving conformational flexibility and positional adaptability,
we designed a new family of DAPYs featuring a diatomic linker, named CH2NH-DAPYs (3, Fig. 1),17
Most of the designed molecules showed excellent activities against wild-type (WT) HIV-1. Herein, in a
continuing effort to develop new HIV-1 NNRTIs, we have made structural modifications of
CH2NH-DAPYs to design 30 compounds, involving not only the modifications of the linker but also of
the wing α of DAPYs.
Fig. 1 Structures of DAPYs
2. Results and discussion
2.1 Synthesis
The target compounds 4a-ac were prepared according to our published methods.17-19 The synthetic
route of target molecules is given in Scheme 1. In this route, the key intermediate
4-((4-chloropyrimidin-2-yl)amino)benzonitrile (8) was prepared in three successive steps from
thiouracil (5) including methylation, solvent-free nucleophilic substitution, and chlorination.
Subsequently, intermediate 8 reacted with the corresponding alcohols, amine and thiol to afford the
2

product 4a-x, 4aa-4ab and 4ac via reaction d, e, and f. Furthermore, we also tried to carry out the
amination reaction of 8 with aqueous ammonia in dimethylformamide (DMF) at 140 °C. However, the
unexpected by-product 4ad was generated, which was ascribed to the existence of dimethylamine
produced by the decomposition reaction of DMF under 140℃.20
Compd
4a
R-Ph
Compd
4h
R-Ph
Compd
4o
R-Ph
Compd
4v
R-Ph
3-CN-Ph
4-CN-Ph
Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-F-Ph
3-F-Ph
4-OCH3-Ph
2-CF3-Ph
4-F-Ph
4b
4i
4p
4w
2-CH3-Ph
3-CH3-Ph
4-CH3-Ph
2-OCH3-Ph
3-OCH3-Ph
3-CF3-Ph
4c
4j
4q
4x
3,5-diMeO-Ph
5-F-2-Br-Ph
2-F-4-Br-Ph
2-CN-Ph
1-Naph
2-Naph
4d
4k
4r
4y
4e
4l
4s
4z
4f
4m
4n
4t
4g
4u
Scheme 1. Synthesis of target molecules 4a-ad. Reagents and conditions: a) MeI, NaOH, H2O, rt, 14 h;
b) 4-cyanoaniline, 180–190℃, 10 h; c) POCl3, reflux, 0.5 h; d) R-PhCH2OH or naphthalenemethanol,
NaOH, DMSO, 80℃; e) pyridin-3-yl-methanol, or pyridin-2-yl-methanamine, NaOH, DMSO, 80℃; f)
4,6-dimethylpyrimidine-2-thiol, NaOH, DMSO, 80℃; g) aqueous ammonia, DMF, 140℃ (4ad was a
by-product).
2.2 Biological activity
The anti-HIV activity and cytotoxicity of compounds 4a-ad were assessed using the MT-4/MTT
method.21, 22 Five FDA-approved drugs, including nevirapine, delavirdine, efavirenz, azidothymidine,
and etravirine, were also evaluated as reference drugs. The compounds were assayed for their anti-HIV
activity against wild-type (WT) HIV-1 (strain IIIB), RT mutant E138K, double RT mutant (K103N +
Y181C), as well as HIV-2 (strain ROD). The results, expressed as CC50 (50% cytotoxic concentration),
EC50 (50% HIV-1 replication inhibitory concentration) and SI (selectivity index given by the
CC50/EC50 (IIIB) ratio) values were listed in Table 1.
As shown in Table 1, most of target molecules displayed good to excellent activities against
wild-type (WT) HIV-1. The four most potent compounds (4g, 4k, 4r, and 4ab) displayed rather potent
WT HIV-1 inhibitory activities with EC50 values lower than 9 nM, which were comparable to the
FDA-approved efavirenz, etravirine and azidothymidine, and lower than the other two reference drugs
nevirapine, and delavirdine. Moreover, 14 target molecules showed excellent selectivity index of higher
than 1000.
3

Table 1. Anti-HIV activities and cytotoxicity in MT-4 cells of 4a-ada
EC50 (μM)b
Compd
ROD (μM)c
CC50 (μM)d
SIe
IIIB
0.034
0.049
0.840
0.036
0.048
4.7
E138K
>146
>153
>156
>130
5.0
K103N+Y181C
>146
>153
>156
>130
>221
>16
>146
>153
>156
>130
>221
>16
146
153
156
130.00
221
16
4424
3122
186
3611
4604
3.4
4a
4b
4c
4d
4e
>16
4f
0.0058
0.014
0.92
0.30
1.88
>81
>151
>105
>81
>151
>105
>81
151
105
81
26034
7500
88
4g
4h
4i
0.16
>12
>12
>12
12
75
4j
0.0088
0.16
1.18
7.2
>30
>30
30
3409
495
356
4720
102
102
193
4944
4872
641
8071
23
4k
>81
>81
81
4l
0.027
0.025
1.0
2.1
>8.9
>8.9
>118
>102
>173
>118
>44
8.9
4m
1.1
>118
>102
>173
>118
>44
118
102
173
118
44.21
190
141
113
4.1
4n
>102
>173
>118
0.23
>190
35.81
>113
>4.1
>209
1.8
4o
1.7
4p
0.61
4q
0.0089
0.039
0.22
4r
>190
>141
>113
>4.1
>190
>141
>113
>4.1
>209
>13
4s
4t
0.014
0.18
4u
4v
4w
1.4
>209
>13
209
13
149
591
513
1247
8
0.022
0.015
0.17
4x
0.30
10
>7.7
>7.7
>212
>6.4
>8.5
>224
>34
7.7
4y
>212
>6.4
212
6.4
4z
0.77
>6.4
0.24
>224
>34
4aa
0.0076
27
>8.5
8.5
1118
8
4ab
>224
>34
224
34
4ac
>34
<1
4ad
Nevirapine
Efavirenz
Etravirine
Azidothymidine
Delavirdine
0.21
0.24
0.0052
0.017
0.0066
0.076
>15
—
>15
6.3
72
0.0073
0.0042
0.011
0.037
0.16
—
868
455
662
628
0.046
0.0089
>23
—
1.9
—
7.5
—
23
a Data represent the mean of at least three independent experiments.
b Compound concentration required to protect MT-4 cells against HIV-1-induced cytopathogenicity by 50%.
c Compound concentration required to protect MT-4 cells against HIV-2-induced cytopathogenicity by 50%.
d Compound concentration that decreases the uninfected MT-4 cell viability by 50%.
e Selectivity index: CC50/EC50 (IIIB) ratio .
4

The 30 target molecules are chemically and biologically diverse. Herein, their structure-activity of
relationship (SAR) against WT HIV-1 was summarized. Compound 4a-i were characterized by the
presence of a mono-halogen substituent such as Cl, Br, or F. Compared with the non-substituted
compound 4x, introduction of Cl or Br seemed to be unfavorable to the WT HIV-1 inhibitory activities,
being demonstrated by 4a-f with more or less reduced activities. Notably, the para-Cl, -Br and -F were
all hugely detrimental to the potency, and the activities of corresponding compounds 4c, 4f, and 4i were
decreased to micromolar level. Encouragingly, ortho-F (4g, EC50 = 5.8 nM, SI = 26034) and meta-F
(4h, EC50 = 14 nM, SI = 7500) enhanced the activities significantly, combined with their low
cytotoxicity, resulted in extremely high SI. It is not unexpected as it is well known that fluorine atom
24
usually plays a very important role in medicinal chemistry due to its versatility.23,
For all
mono-halogen-substituted compounds, the position priority abided by the rules as follows: ortho- >
meta- > -para, for example, 4a > 4b > 4c; 4d > 4e > 4f; 4g > 4h > 4i. Moreover, di-halogen substituents
(4s and 4t) seem to be unfavorable to anti-WT-HIV-1 activities as compared to their monofluorinated
analogues.
For compounds 4j-l bearing a methyl, the position priority is as follows, meta- > ortho- > para-.
Among which, meta-methyl derivative 4k displayed an excellent activity against WT HIV-1 (EC50 =
8.8 nM) and a high SI of 3409. The compounds 4m-o with a methoxyyl seemed to be inferior to the
corresponding compounds 4j-l with a methyl in the same position. Trifluoromethyl, another versatile
group in medicinal chemistry, 22, 23 was also introduced to ortho- and meta-position. However, it was
proven to be disadvantageous to activities by 4p (EC50 = 1.7 μM) bearing a ortho-CF3 and 4q (EC50 =
0.61 μM) bearing a meta-CF3. When the cyano group (-CN) was introduced at different positions, only
introduction of an ortho-CN engenders an activity superior to the unsubstituted phenyl derivative,
while meta- or para-CN both led to a loss of activities. Replacing the phenyl wing in 4x by a 1-naphtyl
neither changes the inhibitory activity nor the cytotoxicity markedly, whereas the 2-naphtyl is
approximately 8-fold less active than the phenyl derivative, but has the advantage of being 17 fold less
cytotoxic. The wing α of target molecules was also modified with heteroaryls such as pyridinyl and
pyrimidinyl. Unexpectedly, the compound 4ab (EC50 = 7.6 nM) bearing a 2-pyridinyl was over
100-fold more potent than 4aa (EC50 = 0.77 µM) with a 3-pyridinyl. As could be expected, the
byproduct 4ad, missing the wing I, was the weakest of all compounds.
To investigate the potency against drug-resistant virus, all compounds were evaluated for their
activity against the RT mutant (E138K), the primary mutation in resistance to rilpivirine and double RT
mutant (K103N + Y181C) HIV-1 strains which is cross-resistant to most of currently available
NNRTIs.25, 26 As shown in Table 1, four compounds (4g, 4r, 4y, 4ab) exhibited moderate activities with
EC50 values of less than 1 μM against E138K mutant. And nearly all compounds are devoid of
activities against double RT mutant (K103N + Y181C) HIV-1 strain.
In this work, all compounds were also evaluated for their activities against HIV-2 (ROD) in MT-4
cells. It is not unexpected that all compounds are lack of activity against HIV-2. As well-known most of
previously discovered NNRTIs9, 27-28 are only active against HIV-1 and lack of anti-HIV-2 activity,
though HIV-1 and HIV-2 reverse transcriptases are similar in structure and functionality.29-31
2.3 Molecular modeling analysis
To investigate the binding mode of our newly synthesized compounds, molecular modeling was
5

carried out using the software SYBYL Surflex-Dock program and crystal structure of WT HIV-1
RT/TMC125 (PDB code: 3MEC). Compounds 4r and 4ab were docked into the HIV-1 RT
nonnucleoside binding site (NNBS) (Fig. 2). As illustrated in Fig. 2, compounds 4r and 4ab displayed
similar binding mode as TMC125 and its analogue in NNBS，adopting the expected “horseshoe”
conformation. 1). The left ring of compound 4r and 4ab and the aromatic amino acid residues Tyr181,
Tyr188 and Trp229 generated π-π stacking interaction. 2) The NH group of compound 4r and 4ab
formed a key hydrogen bond with the mainchain carbonyl of Lys101 and Leu100. 3) The
para-substituted CN of right wing extended to surface of solvent and protein and interacted with amino
acid residue Pro236 and Lys103.
(d)
(c)
Fig. 2 (a) Docking studies of 4r into NNBS; (b) Binding mode of 4r (orange) and overlap with
TMC125 (yellow) in NNBS; (c) Docking studies of 4ab into NNBS; (d) Binding mode of 4ab (violet)
and overlap with TMC125 (yellow) in NNBS. (PDB code: 3MEC)
On the other hand, as illustrated in Fig. 2, the binding mode of 4ab (with no substitutes at the
left wing) is totally different from TMC125. The 2,6-dimethyl-4-cyano fragment of TMC125
could form extensive interactions with surrounding hydrophobic sub-pocket defined by Trp229
6

and Phe227 which may improve the binding affinity and thereby benefit for the anti-HIV-1 activity.
Methoxy groups substituted at left wing of 4r cannot form strong π-π stacking with amino acid
residues like 2,6-dimethyl-4-cyano, that might account for that 4ab and 4r series showed inferior
double mutants activity. It is clear that hydrogen bond formed between TMC125 and Glu138,
however, no similar hydrogen bond formed between 4r (or 4ab) and Glu138. That might account
for why 4r and 4ab were not active to double mutant strains.
3. Conclusion
In conclusion, as a continuing effort to find new NNRTIs, a series of structurally diverse
analogues of diarylpyrimidines were synthesized. The modifications involved not only the linker but
also the wing α of DAPYs. The anti-HIV-1 activities of all target molecules were evaluated, and some
of them exhibited excellent anti-HIV-1 (WT) activities and extremely low cytotoxicities. Among which,
compound 4g showed excellent activity against WT HIV-1 with an EC50 value of 5.8 nM, HIV-1
mutant (E138K) with an EC50 value of 0.30 μM and SI of up to 26108. And compound 4ab with a
novel pyridinyl Wing α displayed activites against WT HIV-1 with an EC50 value of 7.6 nM, HIV-1
mutant (E138K) with an EC50 value of 0.24 μM, and SI of 1113. The SAR was also summarized, which
constitutes a foundation for the further development of new DAPYs as NNRTIs.
4. Experimental
4.1 Chemistry
Melting points were measured on a SGW X-1 microscopic melting-point apparatus. Nuclear
magnetic resonance (NMR) spectra on two Varian spectrometers (400M or 600 MHz for 1H NMR; 100
MHz or 150 MHz for 13C NMR; 564MHz for 19F NMR) were recorded in DMSO-d6. Chemical shifts
were reported in δ (ppm) units relative to the internal standard tetramethylsilane. Mass spectra were
obtained on a Waters Quattro Micromass instrument using electrospray ionization (ESI) techniques.
Elemental analyses were performed on a Carlo Erba 1106 instrument. All chemicals and solvents were
of reagent grade and were purified and dried by standard methods before use. All the reactions were
monitored by thin layer chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV
lamp. Column chromatography separations were performed on silica gel (300–400 mesh).
4.1.1 Synthesis of the intermediate 2-(methylthio)pyrimidin-4(1H)-one(6)
Methyl iodide (85.16 g, 0.60 mol) was added dropwise to a solution of 2-thiouracil (64.07 g, 0.50
mol), sodium hydroxide (20.8 g, 0.52 mol) in H2O (400 mL) in 20 min at 10 °C. The mixture was
stirred at room temperature for 14 h, then cooled to 5 ℃. The precipitates were filtered, washed with
water, and dried to obtain 2-(methylthio)pyrimidin-4(1H)-one (6) as a white solid, 58.72 g, yield 82.6%,
mp 200–201 ℃(lit. 200–201 ℃).
4.1.2 Synthesis of the intermediate 4-((4-oxo-1,4-dihydropyrimidin-2-yl) amino)benzonitrile (7)
The mixture of 6 (45.50 g, 0.32 mol) and 4-cyanoaniline (113.41 g, 0.96 mol) was slowly heated
to 180–190 °C and maintained at this temperature for 10 h. After cooling, the hard mixture was crushed
by ultrasound treatment in CH3CN (300 mL). Then the solid was filtered off and washed with CH3CN
until no residual 4-cyanoaniline was detected by TLC to afford 4-((4-oxo-1,4-dihydropyrimidin-2-
yl)amino)benzonitrile (7) as a light yellow solid (48.96 g, yield 72.1%), which was used directly in
next step.
7

4.1.3 Synthesis of the intermediate 4-((4-chloropyrimidin-2-yl) amino)benzonitrile (8)
A mixture of 7 (42.44 g, 0.20 mol) and POCl3 (100 mL) was refluxed for 30 min, then cooled
slightly and poured slowly into crushed ice (450 g) under vigorous agitation. The resulting yellow
precipitate was filtered. The filter cake was added into chilly water (200 mL), neutralized with sodium
hydroxide to pH 7. The mixture was filtered, and the filter cake was dried to get yellow powder, which
was added into acetonitrile (90 mL) and stirred for 2 h. The mixture was filtered and dried to give light
yellow powder 4-((4-chloropyrimidin-2-yl)amino)benzonitrile (8) (32.48 g, 70.4%), which could be
used without further purification. Pure 8 as white solid could be afforded by column chromatography
using dichloromethane as eluent. Its NMR data can be found in the supplementary data.
4.1.4 Synthesis of target molecules 4a-ac
To a 100 mL two-necked flask was added substituted benzyl alcohol or 4,6-dimethylpyrimidine-
2-thiol (2.60 mmol), NaOH (6.40 g, 1.60 mmol) and dimethylsulfoxide (30 mL). The flask was
vacuumed and flushed three times with nitrogen. Then the solution of 8 (0.20 g, 0.87 mmol) in
dimethylsulfoxide (5～10 mL) was added, and the resulting mixture was heated to 80 °C and kept at
that temperature for approximately 12 h until the completion of the reaction (monitored by TLC).
Subsequently, the mixture was cooled to room temperature and diluted with ethyl acetate (100 mL).
The mixture was washed with brine to pH 7, and the organic phase was dried with anhydrous sodium
sulfate. The organic phase was concentrated to get crude product, which was purified by column
chromatography (ethyl acetate / petroleum ether 1:30 as eluent) to get 4a–ac in yields of 27.3–75.1%.
1
4a: white solid, yield 67.1%, mp 207-209 °C; H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H,
4-CNArNH-), 8.34 (d, 1H, PyH), 7.90-7.39 (m, 8H, ArH), 6.52 (d, 1H, PyH), 5.52 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.16, 159.40, 159.29, 145.17, 134.18, 133.35, 132.90, 130.40, 130.30,
129.84, 127.86, 119.95, 118.86, 102.93, 100.58, 65.30. MS(ESI) 337.1 (M+1) +.
1
4b: white solid, yield 40.1%, mp 203-205 °C; H NMR (600 MHz, DMSO-d6) δ 10.16 (s, 1H,
4-CNArNH-), 8.33 (d, 1H, PyH), 7.90-7.39 (m, 8H, ArH) , 6.50 (d, 1H, PyH), 5.44 (s, 2H, -CH2-); 13
C
NMR (150 MHz, DMSO-d6) δ 169.23, 159.28, 159.00, 145.05, 139.32, 133.53, 133.40, 130.84, 128.39,
128.04, 126.87, 119.95, 119.01, 103.01, 100.63, 67.03. MS(ESI) 337.1 (M+1)+.
1
4c: white solid, yield 66.7%, mp 209-211 °C; H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H,
4-CNArNH-), 8.32 (d, 1H, PyH), 7.93-7.45 (m, 8H, ArH), 6.48 (d, 1H, PyH), 5.43 (s, 2H, -CH2-).; 13
C
NMR (150 MHz, DMSO-d6) δ 169.21, 159.43, 159.19, 145.16, 135.83, 133.40, 133.09, 130.22, 128.91,
119.98, 118.88, 102.90, 100.60, 67.02. MS(ESI) 337.1 (M+1)+.
1
4d: white solid, yield 66.1%, mp 199-201 °C; H NMR (600 MHz, DMSO-d6) δ 10.16 (s, 1H,
4-CNArNH-), 8.35 (d, 1H, PyH), 7.91-7.30 (m, 8H, ArH), 6.53 (d, 1H, PyH), 5.49 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.13, 159.41, 159.31, 145.16, 135.77, 133.37, 133.05, 130.57, 130.22,
128.38, 122.99, 119.96, 118.84, 102.92, 100.57, 67.48. MS(ESI) 383.0 (M+2) +.
4e: white solid, yield 65.7%, mp 190-192 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.13 (s, 1H,
4-CNArNH-),8.32 (d, 1H, PyH), 7.92-7.35 (m, 8H, ArH), 6.50 (d, 1H, PyH), 5.44 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.16, 159.42, 159.24, 145.13, 139.61, 133.39, 131.28, 131.12, 130.92,
127.24, 122.11, 119.97, 118.93, 102.91, 100.60, 66.93.MS(ESI) 383.0 (M+2)+.
4f: white solid, yield 57%, mp 179-181 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H,
4-CNArNH-), 8.32 (d, 1H, PyH), 8.31-7.43 (m, 8H, ArH), 6.48 (d, 1H, PyH), 5.42 (s, 1H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.98, 169.20, 159.56, 159.42, 159.20, 158.79, 145.16, 136.26, 133.40,
131.83, 130.48, 121.62, 119.97, 118.87, 102.90, 100.59, 67.05, 54.09. MS(ESI) 383.0 (M+2)+ .
4g: white solid, yield 53.9%, mp 185-187 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.14 (s, 1H,
8

4-CNArNH-), 8.32 (d, 1H, PyH), 7.95-7.22 (m, 8H, ArH), 6.47 (d, 1H, PyH), 5.49 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.17, 161.70 (d, J =243.45 Hz), 159.43, 158.48, 145.24 (d, J =17.25
Hz), 132.88, 131.59 (d, J =8.85 Hz), 131.53, 130.60 (d, J =19.35 Hz), 124.47 (d, J =8.55 Hz), 123.58,
119.97, 119.20, 118.30, 102.98, 101.08, 100.06, 62.08. 19F NMR(564 MHz, DMSO-d6) δ -118.32.
MS(ESI) 321.1 (M+1)+.
4h: white solid, yield 69.5%, mp 224-226 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.13(s, 1H,
4-CNArNH-), 8.33（d, 1H, J = 5.4 Hz）, 7.93-7.19 (m, 8H, ArH), 6.50 (d, 1H, J = 5.4 Hz), 5.45 (s, 2H,
-CH2-); 13C NMR (150 MHz, DMSO-d6) δ 169.20, 163.38 (d, J =241.65Hz), 159.42, 159.22, 145.16,
139.69 (d, J =7.8 Hz), 133.39, 130.99 (d, J =8.4 Hz), 124.25 (d, J =2.4 Hz), 119.97, 118.90, 115.30 (d,
J =20.7 Hz), 115.05(d, J = 21.75 Hz), 102.89, 100.62, 67.02. 19F NMR(564 MHz, DMSO-d6)
δ
-113.13. MS(ESI) 321.1 (M+1)+.
4i: white solid, yield 57.1%, mp 179-180 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H,
4-CNArNH-), 8.31（d, 1H, J = 5.4 Hz）, 7.92-7.19 (m, 8H, ArH), 6.47 (d, 1H, J = 5.4 Hz), 5.42 (s, 2H,
-CH2-); 13C NMR (150 MHz, DMSO-d6) δ 169.27, 163.14 (d, J =242.7 Hz), 159.43,159.13, 145.20,
133.41,132.99 (d, J =2.85 Hz), 130.85 (d, J =8.4 Hz), 119.98, 118.88, 115.81 (d, J =21.0 Hz), 102.88,
100.62, 67.19. 19F NMR(564 MHz, DMSO-d6) δ -114.05. MS(ESI) 321.1 (M+1)+.
4j: white solid, yield 65.3%, mp 185-187 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H,
4-CNArNH-), 8.32 (d, 1H, PyH), 7.94-7.20 (m, 8H, ArH), 6.48(d, 1H, PyH), 5.43 (s, 2H, -CH2-),
2.33(s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 169.43, 159.46, 159.10, 145.24, 136.95, 134.66,
133.35, 130.58, 129.02, 128.68, 126.31, 119.98, 118.88, 102.87, 100.56, 66.45, 18.91. MS(ESI) 317.1
(M+1)+.
4k: white solid, yield 64.2%, mp 168-170 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.11 (s, 1H,
4-CNArNH-), 8.30 (d, 1H, PyH), 7.95-7.14 (m, 8H, ArH), 6.46 (d, 1H, PyH), 5.39 (s, 2H, -CH2-), 2.30
(s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 169.37, 159.46, 159.07, 145.22, 138.06, 136.62,
133.37, 129.10, 128.99, 128.80, 125.51, 119.99, 118.91, 102.85, 100.62, 67.93, 21.37. MS(ESI) 317.1
(M+1)+.
4l: white solid, yield 75.1%, mp 170-172 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.10 (s, 1H,
4-CNArNH-), 8.30 (d, 1H, PyH), 7.95- 7.19 (m, 8H, ArH), 6.45 (d, 1H, PyH), 5.39 (s, 2H, -CH2-), 2.29
(s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 169.38, 159.45, 159.05, 145.23, 137.81, 133.68,
133.39, 129.43, 128.55, 119.99, 118.88, 102.85, 100.64, 67.81, 21.20. MS(ESI) 317.1 (M+1)+.
4m: white solid, yield 69.2%, mp 175-177 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.10 (s, 1H,
4-CNArNH-), 8.31 (d, 1H, PyH), 7.95-6.96 (m, 8H, ArH), 6.47 (d, 1H, PyH), 5.42 (s, 2H, -CH2-), 3.82
(s, 3H, -OCH3); 13C NMR (150 MHz, DMSO-d6) δ 169.50, 159.46, 158.96, 157.53, 145.29, 133.34,
130.07, 129.67, 124.33, 120.70, 119.98, 118.85, 111.28, 102.84, 100.68, 63.53, 55.88. MS(ESI) 333.2
(M+1)+.
4n: white solid, yield 61.8%, mp 154-155 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.11 (s, 1H,
4-CNArNH-), 8.31 (d, 1H, PyH), 7.94-6.90 (m, 8H, ArH), 6.48 (d, 1H, PyH), 5.41 (s, 2H, -CH2-), 3.74
(s, 3H, -OCH3); 13C NMR (150 MHz, DMSO-d6) δ 169.33, 159.75, 159.44, 159.13, 145.20, 138.29,
133.38, 130.04, 120.47, 119.97, 118.89, 113.94, 113.89, 102.86, 100.63, 67.75, 55.48. MS(ESI) 333.2
(M+1) +.
4o: white solid, yield 70.1%, mp189-191 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.07 (s, 1H,
4-CNArNH-), 8.30 (d,1H, PyH), 7.97-6.94 (m, 8H, ArH), 6.40 (d, 1H, PyH), 5.36 (s, 2H, -CH2-), 3.75
(s, 3H, -OCH3); 13C NMR (150 MHz, DMSO-d6) δ 169.40, 159.64, 159.45, 159.00, 145.26, 133.41,
130.43, 128.55, 120.00, 118.88, 114.28, 102.84, 100.66, 67.75, 55.54. MS(ESI) 333.2 (M+1) +.
9

1
4p: white solid, yield 46.4%, mp168-170 °C; H NMR (600 MHz, DMSO-d6) δ 10.13 (s,
1H,4-CNArNH-), 8.34 (d, 1H), 7.92-7.57 (m, 8H, ArH), 6.48 (d, 1H, PyH), 5.59 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.03, 159.42, 159.27, 145.13, 134.58, 133.25, 130.75, 129.23, 127.34
(d, J =30.6 Hz), 126.59 (d, J =5.25 Hz), 126.55 (d, J =5.70 Hz), 125.62 (d, J =272.55 Hz), 119.91,
118.89, 102.98, 100.53, 64.44. 19F NMR(564 MHz, DMSO-d6) δ -58.75. MS(ESI) 371.2 (M+1)+ .
4q: white solid, yield48.1%, mp 168-170 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.13 (s, 1H,
4-CNArNH-), 8.33 (d,1H), 7.93-7.63 (m, 8H, ArH), 6.51 (d, 1H), 5.54 (s, 2H, -CH2-); 13C NMR (150
MHz, DMSO-d6) δ 169.17, 159.43, 159.26, 145.13, 138.30, 133.36, 132.38, 130.03, 129.75 (d, J =30.5
Hz), 125.45 (d, J =270.9 Hz), 125.17 (d, J =4.05 Hz), 124.86 (d, J =3.6 Hz),119.94, 118.94, 102.93,
100.56, 67.05. 19F NMR(564 MHz, DMSO-d6) δ -61.15. MS(ESI) 371.1 (M+1)+ .
4r: white solid, yield 71.6%, mp 155-157 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.11 (s, 1H,
4-CNArNH-), 8.31-6.62 (m, 10H, ArH+PyH), 5.36 (s, 2H, -CH2-), 3.73 (s, 6H, -OCH3);13C NMR (150
MHz, DMSO-d6) δ 169.31, 160.96, 159.43, 159.14, 145.20, 139.03, 133.37, 119.97, 118.91, 106.25,
102.86, 100.62, 100.06, 67.78, 55.62. MS(ESI) 363.3 (M+1)+ .
4s: white solid, yield 45.7%, mp 213-215 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.16 (s, 1H,
4-CNArNH-), 8.35-6.56 (m, 9H, ArH+PyH), 5.45 (s, 2H, -CH2-). 13C NMR (150 MHz, DMSO-d6) δ
168.93, 162.71 (d, J =243.75 Hz), 159.37, 145.13, 138.36 (d, J =7.95 Hz), 134.76 (d, J =7.95 Hz),
133.34, 119.93, 118.85, 117.46 (d, J =22.05 Hz), 117.08 (d, J =2.55 Hz), 116.91 (d, J =23.85 Hz),
102.98, 100.59, 66.94. 19F NMR(564 MHz, DMSO-d6) δ -114.12. MS(ESI) 401.0 (M+2)+
4t: white solid, yield 45.0%, mp 211-214 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.13 (s, 1H,
4-CNArNH-), 8.33 (d, 1H, PyH), 7.94-7.45 (m, 7H, ArH), 6.47 (d, 1H, PyH), 5.46 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.02, 161.53(d, J =250.35 Hz), 159.37, 159.26, 145.14, 133.37,
132.57 (d, J =4.50 Hz), 128.21 (d, J =3.45 Hz), 123.41 (d, J =14.85 Hz), 122.40 (d, J =9.3 Hz), 119.94,
119.41 (d, J =24.15 Hz), 118.91, 102.98, 100.56, 61.59. 19F NMR(564 MHz, DMSO-d6) δ -115.03.
MS(ESI) 401.0 (M+2)+ .
4u: white solid, yield 27.3%, mp 252-254 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.15 (s, 1H,
4-CNArNH-), 8.33 (d, 1H, PyH), 8.08-7.49 (m, 8H, ArH), 6.49 (d, 1H, PyH), 5.91 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.39, 159.48, 159.20, 145.19, 133.69, 133.30, 132.24, 129.27, 129.02,
127.08, 126.48, 125.85, 124.04, 118.88, 102.83, 100.64, 66.24. MS(ESI) 328.1 (M+1)+ .
4v: white solid, yield 34.5%, mp 220-222 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.13 (s, 1H,
4-CNArNH-), 8.33 (d, 1H, PyH), 8.00-7.51 (m, 8H, ArH), 6.51 (d, 1H, PyH), 5.61 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 168.98, 159.07, 158.77, 144.79, 133.97, 132.99, 132.75, 132.60, 128.14,
127.79, 126.65, 126.42, 125.84, 119.58, 118.51, 102.47, 100.26, 67.60.
4w: white solid, yield31.2%, mp 225-228 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H,
4-CNArNH-), 8.36 (d, 1H, PyH), 7.96-7.57 (m, 8H, ArH), 6.54 (d, 1H, PyH), 5.62 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 168.59, 158.99, 144.70, 139.56, 133.56, 133.25, 132.97, 129.25, 119.52,
118.49, 117.13, 110.94, 102.55, 100.11, 65.38.
1
4x: white solid, yield 70.4%, mp 178-180 °C; H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H,
4-CNArNH-), 8.32 (d, 1H, PyH), 7.94-7.35 (m, 9H, ArH), 6.49 (d, 1H, PyH), 5.44 (s, 2H, -CH2-); 13C
NMR (150 MHz, DMSO-d6) δ 169.28, 159.44, 159.20, 145.16, 136.99, 134.91, 133.41, 131.02, 128.88,
119.98, 118.87, 102.88, 100.64, 67.58. MS(ESI) 303.64 (M+1)+ .
4y: white solid, yield 37.7%, mp 168-170 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H,
4-CNArNH-), 8.33 (d, 1H, PyH), 8.09-7.49 (m, 11H, ArH), 6.50 (d, 1H, PyH), 5.92 (s, 2H, -CH2-).
MS(ESI) 353.2 (M+1)+ .
10

4z: white solid, yield 31.9%, mp 196-197 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H,
4-CNArNH-), 8.33 (d, 1H, PyH), 8.00-7.51 (m, 11H, ArH), 6.51 (d, 1H, PyH), 5.61 (s, 2H, -CH2-).
MS(ESI) 353.1(M+1)+ .
1
4aa: white solid, yield 50.8%, mp 207-209 °C; H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H,
4-CNArNH-), 8.72-7.42 (m, 9H, ArH+PyH), 6.48 (d, 1H, PyH), 5.48 (s, 2H, -CH2-). MS(ESI)
304.2(M+1)+ .
4ab: white solid, yield 43.2%, mp 193-195 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H,
4-CNArNH-), 8.57-7.24 (m, 10H, ArH+PyH), 6.19 (s, 1H, -CH2NH-), 4.65 (s, 2H, -CH2-). MS(ESI)
303.1 (M+1)+ .
4ac: white solid, yield 53.6%, mp 237-239 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H,
4-CNArNH-), 8.49 (d, 1H, PyH), 7.86-7.67 (d, 4H, ArH), 7.58 (d, 1H, PyH), 7.24 (s, 1H, PyH), 2.42 (s,
6H, -CH3). MS(ESI) 334.36 (M+1)+ .
4.1.5 Synthesis of byproduct 4ad
To a 50 mL necked flask was added ammonium hydroxide (6.5 mL), compound 8 (0.434mmol)
and dimethylformamide (8 mL). The mixture was heated 140 °C for approximate 6.5 h until the
completion of the reaction (monitored by TLC). Subsequently, the mixture was cooled to room
temperature and poured into water (100 mL), extracted with ethyl acetate. The organic phase was dried
with anhydrous sodium sulfate, and concentrated to get crude product, which was purified by column
chromatography (ethyl acetate / petroleum ether 1:30 as eluent) to get 4ad. White solid, yield 92.3%,
mp 200-203 °C; 1H NMR (400 MHz, CDCl3) δ 7.99 (d, 1H, PyH), 7.76 (d, 2H, ArH), 7.65 (s, 1H,
4-CNArNH-), 7.56 (d, 2H, ArH), 6.05 (d, 1H, PyH), 3.12 (s, 6H, -NCH3). MS(ESI) 240.1 (M+1)+ .
4.2 Anti-HIV activity assay
4.2.1 Anti-HIV activity assay in MT-4 cells
The anti-HIV activity and cytotoxicity of the target molecules were evaluated against wild-type
(WT) HIV-1 strain IIIB, RT mutant (E138K), double RT mutant (K103N + Y181C) HIV-1 (RES056)
and HIV-2 strain ROD in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method.21, 22 Briefly, stock solutions (10 x final concentration) of
test compounds were added in 25 µL volumes to two series of triplicate wells so as to allow
simultaneous evaluation of their effects on mock-and HIV-infected cells at the beginning of each
experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well
microtiter trays using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated control
HIV-and mock-infected cell samples were included for each sample. Virus stock (50 µL) at 100-300
CCID50 (50% cell culture infectious dose) or culture medium was added to either the virus-infected or
mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effect of test
compounds on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially
growing MT-4 cells were centrifuged for 5 min at 1,000 rpm (220 g) and the supernatant was discarded.
The MT-4 cells were resuspended at 6 x 105 cells/mL and 50-µL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was
examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (Acros Organics) by mitochondrial dehydrogenase activity in
metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The
absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at
11

two wavelengths (540 and 690 nm). All data were calculated using the median absorbance value of
three wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test
compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The
concentration achieving 50% protection against the cytopathic effect of the virus in infected cells was
defined as the 50% effective concentration (EC50).
4.2.2 Molecular docking
Molecular modeling was performed with the Tripos molecular modeling packages Sybyl-X 1.2.
All the molecules for docking were built using standard bond lengths and angles from Sybyl-X
1.2/base Builder and were then optimized using the Tripos force field for 2,000 generations two times
or more, until the minimized conformers of the ligand were the same. The flexible docking method,
called Surflex-Dock, docks the ligand automatically into the ligand binding site of the receptor by
using a protocol-based approach and an empirically-derived scoring function.32-34 The protocol is a
computational representation of a putative ligand that binds to the intended binding site and is a unique
and essential element of the docking algorithm. The scoring function in Surflex-Dock, which contains
hydrophobic, polar, repulsive, entropic, and solvation terms, was trained to estimate the dissociation
constant (Kd) expressed in -log (Kd)2. Prior to docking, the protein was prepared by removing water
molecules, the ligand (TMC120), and other unnecessary small molecules from the crystal structure of
the TMC125-HIV-1 RT complex (PDB code: 3MEC)35; simultaneously, polar hydrogen atoms were
added to the protein. Surflex-Dock default settings were used for other parameters, such as the number
of starting conformations per molecule (set to 0), the size to expand search grid (set to 8Å), the
maximum number of rotatable bonds per molecule (set to 100), and the maximum number of poses per
ligand (set to 20). During the docking procedure, all of the single bonds in residue side-chains inside
the defined RT binding pocket were regarded as rotatable or flexible, and the ligand was allowed to
rotate at all single bonds and move flexibly within the tentative binding pocket. The atomic charges
were recalculated using the Kollman all-atom approach for the protein and the Gasteiger–Hückel
approach for the ligand. The binding interaction energy was calculated to include van der Waals,
electrostatic, and torsional energy terms defined in the Tripos force field. The structure optimization
was performed for 20,000 generations using a genetic algorithm, and the 20-best-scoring
ligand–protein complexes were kept for further analyses. The －log (Kd)2 values of the 20-best-scoring
complexes, which represented the binding affinities of ligand with RT, encompassed a wide scope of
functional classes (10-2-10-9). Therefore, only the highest-scoring 3D structural model of the
ligand-bound RT was chosen to define the binding interaction36-39
.
Acknowledgements
The financial support from the National Natural Science Foundation of China (No. 21402148, No.
21602164), the Hubei Provincial Department of Education of China (No. XD2014154), Wuhan
Institute of Technology Scientific Research Fund (No. K201441, No. K201304), Wuhan Institute of
Technology Teaching Research Project (No. X2015014), and Wuhan Institute of Technology President
Fund (No. 2016079), is gratefully acknowledged. The authors wish to thank Kristien Erven, and Kris
Uyttersprot (Rega) for dedicated technical assistance.
Supplementary data
Supplementary data (1H NMR, 13C NMR, 19F NMR) associated with this article can be found in the
12

online version.
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Graphical abstract