Conformational restriction design of thiophene-biphenyl-DAPY HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111603

Conformational restriction is a promising strategy in the development of DAPY-type non-nucleoside reverse transcriptase inhibitors (NNRTIs). Herein, eighteen thiophene-biphenyl-DAPY derivatives were designed and synthesized as potent HIV-1 NNRTIs in which halogen and methyl groups were introduced to explore the conformationally constrained effects. Molecular docking and dynamic simulation analysis indicated that substituents on different positions of the biphenyl ring induced different dihedral angles and binding conformations, further explaining their anti-viral activities. The 2′-fluoro and 3′-chloro substitutions could form electrostatic or halogen-bonding interactions with adjacent residues of the RT enzyme. The 2′-methyl group contributed to enlarge the dihedral angle of biphenyl ring and was positioned to a space-filling hydrophobic pocket. Notably, compounds 22 and 23 with two methyl groups exhibited potent biological activity against WT HIV-1-infected MT-4 cells (EC₅₀ = 14 and 17 nM, respectively) and RT enzyme (EC₅₀ = 27 and 42 nM, respectively). In particular, 23 exhibited much lower cytotoxicity (CC₅₀ = 264.19 μM) and higher selectivity index (SI = 18,564) than etravirine. Taken together, a rational conformational model for further design of DAPYs is proposed, providing a new guidance for the development of NNRTIs.

Full text of DOI:10.1016/j.ejmech.2019.111603

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Conformational restriction design of thiophene-biphenyl-DAPY HIV-1 non-nucleoside
reverse transcriptase inhibitors
Yali Sang, Sheng Han, Christophe Pannecouque, Erik De Clercq, Chunlin Zhuang,
Fener Chen
PII:
S0223-5234(19)30737-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111603
EJMECH 111603
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 June 2019
Revised Date: 2 August 2019
Accepted Date: 7 August 2019
Please cite this article as: Y. Sang, S. Han, C. Pannecouque, E. De Clercq, C. Zhuang, F. Chen,
Conformational restriction design of thiophene-biphenyl-DAPY HIV-1 non-nucleoside reverse
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graphic abstract

Conformational restriction design of thiophene-biphenyl-DAPY HIV-1
non-nucleoside reverse transcriptase inhibitors
Yali Sang,†, ‡ Sheng Han,†, ‡ Christophe Pannecouque,§ Erik De Clercq,§ Chunlin Zhuang,*,†, ‡ and Fener Chen*, †, ‡
†Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University,
Shanghai 200433, People’s Republic of China
‡Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, People’s
Republic of China
§Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
Corresponding Author
*(F.C.). E-mail: rfchen@fudan.edu.cn.; (C.Z.): zclnathan@163.com.
Notes
The authors declare no competing financial interest.

ABSTRACT
Conformational restriction is a promising strategy in the development of DAPY-type non-nucleoside reverse
transcriptase inhibitors (NNRTIs). Herein, eighteen thiophene-biphenyl-DAPY derivatives were designed and
synthesized as potent HIV-1 NNRTIs in which halogen and methyl groups were introduced to explore the
conformationally constrained effects. Molecular docking and dynamic simulation analysis indicated that substituents
on different positions of the biphenyl ring induced different dihedral angles and binding conformations, further
explaining their anti-viral activities. The 2’-fluoro and 3’-chloro substitutions could form electrostatic or
halogen-bonding interactions with adjacent residues of the RT enzyme. The 2’-methyl group contributed to enlarge the
dihedral angle of biphenyl ring and was positioned to a space-filling hydrophobic pocket. Notably, compounds 22 and
23 with two methyl groups exhibited potent biological activity against WT HIV-1-infected MT-4 cells (EC50 = 14 and
17 nM, respectively) and RT enzyme (EC50 = 27 and 42 nM, respectively). In particular, 23 exhibited much lower
cytotoxicity (CC50 = 264.19 µM) and higher selectivity index (SI = 18,564) than etravirine. Taken together, a rational
conformational model for further design of DAPYs is proposed, providing a new guidance for the development of
NNRTIs.
Keywords: conformational restriction, NNRTIs, biphenyl-DAPY, HIV-1, molecular modeling

Introduction
The human immunodeficiency virus (HIV) is the main infectious agent of the acquired immunodeficiency
syndrome (AIDS). The anti-HIV drugs targeting different steps of the viral life cycle are developed, including fusion
inhibitors (FIs), the coreceptor antagonist (maraviroc), integrase strand transfer inhibitors (INSTIs), protease inhibitors
(PIs), and reverse transcriptase inhibitors (RTIs) [1]. Diarylpyrimidines (DAPYs) (Fig 1), are a series of
non-nucleoside reverse transcriptase inhibitors (NNRTIs), displaying superior efficiency and pharmacological profile
toward clinically wild-type (WT) HIV-1 and drug-resistant mutant strains [2]. To date, two representative DAPYs,
etravirine (ETR) and rilpivirine (RPV) have been approved by FDA [3, 4]. They specifically interact with an allosteric
binding site of the RT, approximately 10 Å distant from the polymerase active site, triggering a conformational change
and leading to disturb RT catalytic function [5]. The torsional flexibility of DAPYs by the bridge-oxygen and -NH
rotation cause to positional adaptability in the hydrophobic pocket in a “butterfly” conformation, ascribing to their
powerful anti-HIV-1 activity [6-8]. However, point mutations are frequently occurred within the allosteric site to
block DAPY binding to RT because of the increased steric hindrances of the pocket or elimination of critical binding
interactions [9-11]. As a consequence, their long-term anti-HIV applications are still unsatisfactory due to the growing
number of new mutations in the HIV-1 RT [12]. Our group has made great efforts to enlarge the DAPY family by
developing more DAPY related compounds (Fig. 1), such as X-DAPYs [13-18], naphthyl-DAPYs [19, 20], and
biphenyl-DAPYs [21, 22]. They exhibited promising inhibitory activity toward WT and mutant HIV-1 with low
cytotoxicity and high selectivity.

Fig. 1 Families of DAPY and DAPY-related compounds
These derivatives were mostly designed with the purpose to strengthen the conformational restriction, which was
reported to improve HIV-1 RT inhibitory activity and selectivity [23, 24]. The classes of X-DAPYs with different
linkers were generated on the basis of stereochemical diversity-oriented conformational restriction, attributing to the
preferential “butterfly-like” conformation in NNRTI binding pocket of RT [25, 26]. The naphthyl and
biphenyl-DAPYs possessed conformations with planar conjugated systems and had significantly improved biological
activity [19-21, 27]. They were predicted to bind to HIV-1 RT by increasing π-π stacking interaction targeting the
highly conserved residue W229. Different from the naphthyl group, the planarity of biphenyl ring was, to some extent,
changed by the steric repulsion force between hydrogen atoms or different substitutions on the two phenyl rings [28,
29]. Thus, we assumed that the planarity change might have possibly contribute to the anti-viral activity. Recently, a
new series of thiophene-biphenyl-DAPYs was generated as potent HIV-1 NNRTIs [22]. However, the effects of
conformational restriction of biphenyl moiety on the anti-viral activity were still unknown. In this study, we
introduced halogens or methyl groups on the biphenyl rings to determine their influence on the dihedral angle, binding
conformation with HIV-1 RT and the anti-viral activity.

Design rationale
Based on the predicted mode of thiophene-biphenyl DAPY with RT (Fig. 2A and 2B), the thiophene[3,2 d]
pyrimidine group generated hydrophobic interactions with the residues V179/E138 and maintained hydrogen-bonding
interactions with the backbone of K101. The biphenyl system strengthened the π-π stacking interactions with
Y181/Y188, targeting the highly conserved residue W229. The two ortho-hydrogen atoms of the biphenyl ring made
an approximately planar dihedral angle (Fig. 2C), which might be increased up to ~90o with different substitutions on
the rings (Fig. 2D).
The fluorine and chlorine atoms may significantly influence the conformation and play an important role in lead
optimization [29, 30]. A highly polarized C−F bond has a strong tendency for C−F···H−Y interactions. In addition,
dipole and electrostatic interactions can be productively generated as the high electronegativity of fluorine atom (3.98
on the Pauling electronegativity scale) [31]. The chloro group has strong preferences for halogen-bond interactions
with the target protein residues [32]. The methyl group introduced on the ortho-position of an aryl ring can be
particularly beneficial to a conformational change [33]. This moiety has been commonly applied in the design of
DAPY derivatives, such as ETR and RPV [34, 35]. Moreover, ortho-methyl substitution in a biphenyl system changes
the interphenyl dihedral angle distribution, which is, for instance, applied in the discovery of potent p38α MAPK
inhibitors [33, 36, 37].
Taken together, introducing the above mentioned fluoro, chloro, or methyl groups on the biphenyl ring system
has potential conformational implications on improving the anti-viral activity of thiophene-biphenyl DAPY.

Fig. 2 Design rationale and optimization strategy of the thiophene-biphenyl DAPYs. (A), (B) Illustration of the
thiophene-biphenyl DAPYs in the NNRTI binding pocket. (C) Biphenyl with the planar conformation. (D) The
ortho-substitutions could cause the two phenyl rings to be oriented in a more orthogonal manner.
Results and discussion
Chemistry
The synthetic route of the desired compounds 6-23 is outlined in Scheme 1. The 4-methoxyphenylboronic acid 2
and different substituted 4-bromobenzonitriles in the aqueous phase at room temperature provided valuable
intermediates 3 by PdCl2 catalyzed Suzuki-Miyaura cross-coupling reaction [38]. The key intermediates,
4’-hydroxy-[1,1’-biphenyl]-4-carbonitrile 4a-r were prepared by classical demethylation [39, 40]. Finally, the target
derivatives 6-23 were obtained by a nucleophilic substitution reaction of the previous published intermediate 5 [22]
with 4a-r.
Scheme 1. Synthesis of 6-23.a

aReagents and conditions: (a) Triisopropyl borate, n-BuLi, THF, -78 oC-r.t., 14 h; (b) substituted 4-bromobenzonitrile,
PdCl2, K2CO3, PEG400, H2O, r.t., 16 h; (c) BBr3, DCM, N2, -45 oC-r.t., 12 h; (d) 5, K2CO3, DMF, 80 oC, 8 h.
Antiviral activity of compounds 6-11 and binding conformation analysis.
We firstly synthesized the thiophene-biphenyl-DAPY derivatives 6-11 with hydrogen as R4 and fluoro, chloro, or
methyl group as R5 substituents. Nevirapine (NVP), EFV, and ETR were used as references. As illustrated in Table 1,
the new DAPY derivatives displayed low nanomolar EC50 values against the WT HIV-1 strain; no apparent
cytotoxicity and promising SI values (>700~4184) were observed. Compound 6 with 2’-F had the best anti-WT HIV-1
activity (EC50 = 0.011 µM). When the fluoro group was moved to 3’-position (7), the anti-viral activity was ten-fold
decreased (EC50 = 0.114 µM). Replacing the fluorine by chloride (8) made the activity to a decreased EC50 of 0.376
µM. Differently, 3’-substitution was favorable for the chloride (9) and the activity was increased 5-fold to 0.075 µM as
compared to the 2’-substituted analogues. Finally, we changed the halogen to methyl group to obtain compounds 10
and 11. They maintained the activity at about 0.070 µM and the position effect was negligible.
Table 1. Activity and cytotoxicity against HIV-1 (IIIB) strains in MT-4 cells of compounds 6-11.
HIV-1 (IIIB)
Compounds
R4 R5
CC50 (µM)b
SI (IIIB)c
EC50 (µM)a
H
H
H
H
H
2'-F
3'-F
0.011 ± 0.008
0.114 ± 0.077
>53.98
>4184
>2365
>700
6
>269.92
>260.92
>260.92
>272.26
7
2'-Cl 0.376 ± 0.103
3'-Cl 0.075 ± 0.045
2'-Me 0.070 ± 0.021
8
>3434
>3860
9
10

H
3'-Me 0.074 ± 0.039
0.293 ± 0.107
>272.26
>15.03
>6.34
>3706
>51
11
Nevirapine
Efavirenz
Etravirine
0.007 ± 0.005
>863
>833
0.006 ± 0.002
>4.61
aEC50: The effective concentration required to protect the cell against HIV-induced cytopathogenic effect by 50% in
MT-4 cells.
bCC50: The cytotoxic concentration of the compound that reduced the normal uninfected MT-4 cell viability by 50%.
cSI: selectivity index, ratio CC50/EC50 (WT).
In order to explain the anti-viral activity, molecular docking was carried out to predict the interaction of the
thiophene-biphenyl-DAPY compounds with HIV-1 RT (Fig. 3A-D). Molecular dynamic (MD) simulation was
performed to support the dynamical nature of the essential interactions involved in molecular recognition and the
protein interactions with the ligand were monitored throughout the simulation (Fig. 3E-G). As shown in Fig. 3A and
3B, the 4-cyano-1,1’-biphenyl fragment interacted with the conserved viral residue W229 in the hydrophobic
subpocket. The thiophene[3,2-d]pyrimidine scaffold pointed to the backbones of V179 and E138 and formed a
hydrogen-bonding interaction with the backbone of residue K101. The para-cyanoaniline group inserted into the
pocket containing P236, L234, Y318 as well as attributing to a hydrogen-bonding interaction with K101 as a hydrogen
donor. Most importantly, the fluoro group at the 2’-position could form a productive electrostatic interaction with the
electropositive amide NH of W229 (distance: 2.8 Å) (Fig. 3C) and make the dihedral angle in ~±150 ̊ (Fig. 3F).
Although it was not a large angle for the biphenyl ring, the dihedral angle strengthened the π-π stacking interactions
with Y181/Y188. Differently, compound 7 with 3’-F bound to the RT by rotating the biphenyl ring and missing the
electrostatic interaction with W229 (Fig. 3D and Fig. 3G), which might be a predominant reason for its decreased
activity.

Fig. 3 (A), (B) Superimposition of 6 with the HIV-1 WT RT crystal structure (PDB: 2ZD1). (C), (D) The difference of
predicted binding mode of flouro-substituted biphenyls (orange, 6; purple, 7;) with 2ZD1. (E) Protein-Ligand
(2ZD1-6) root mean square deviation (RMSD) indicates the stability of the model. Left Y-axis shows the RMSD
evolution of a protein. Right Y-axis indicates how stable the ligand is with respect to the protein and its binding pocket.
(F) The conformation of the torsion throughout the course of the simulation (dial plot) and the probability density of
the torsion (bar plot). (G) Protein-ligand (2ZD1-6) contacts. The stacked bar charts are normalized over the course of
the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is
maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with
the ligand.

Compounds 8 and 9 with a chloride group showed a different trend of anti-viral activity compared with the
corresponding fluoro analogues (6 and 7). In addition to the common interactions as 6 (Fig. 4A and 4B), the docking
mode of 8 with RT predicted that the 3’-Cl made the biphenyl ring to have a favorable dihedral angle and form
potential halogen interactions with the carbonyl oxygen of L228 at a distance of 2.7 Å and backbone amide nitrogen
of L228, W229 (3.0 Å and 3.6 Å, respectively) (Fig. 4A and 4C). Although the 2’-Cl substitution was positive to the
dihedral angle of biphenyl ring, the halogen-bonding interaction was not observed in the docking model (Fig. 4D).
These chloro-oxygen/nitrogen interactions might result in different anti-viral activity and extremely high binding
selectivity.
Fig. 4 (A), (B) Superimposition of 9 with the HIV-1 WT RT crystal structure (PDB: 2ZD1). (C), (D) The difference of
predicted binding mode of chloride-substituted biphenyls (orange, 9; purple, 8;) with 2ZD1.

Fig. 5 (A), (B) Superimposition of 10 with the HIV-1 WT RT crystal structure (PDB: 2ZD1). (C), (D) The difference
of predicted binding mode of methyl-substituted biphenyls (orange, 10; purple, 11;) with 2ZD1. (E), (F) The
space-filling model for the predicted binding mode of 10 and 11 with 2ZD1.

Compounds 10 and 11 showed a similar anti-viral activity. However, the binding mode (Fig. 5A and 5B)
predicted that they had a different dihedral angle to build π-π interactions with Y181 and Y188 (Fig. 5C and 5D), as
ortho-methyl group had a stronger effect on the dihedral angle [33]. Further insights of the interactions revealed that
this conformation positioned the methyl group so that it caused to the proper space-filling in the hydrophobic pocket
(Fig. 5E and 5F), thus explaining their potent and similar anti-HIV-1 e cacy.
Antiviral activity of compounds 12-23 and binding conformation analysis.
Encouraged by the positive results of compounds 6-11, we further designed compounds 12-23 by introducing
fluoro, chloro, or a methyl group at R4 position and keeping the substitutions at R5. The new derivatives displayed low
nanomolar EC50 values against the WT HIV-1 strain (Table 2). First, we assessed R4 at 3-position of compounds 6 and
7, obtaining compounds 12-15. They showed the same trend in anti-viral activity as compounds 6 and 7 but the
cytotoxicity was unsatisfactory. More pronounced, compound 12 with 2’,3-difluoro had an EC50 of 0.139 µM.
Compound 13 with 3’,3-F showed 30-fold decrease in activity as compared with 12. Compound 14 with 3-Me-2’-F
showed an anti-HIV-1 activity of 0.029 µM, which was about 8-fold higher than that of compound 15 with 3-Me-3’-F
(EC50 = 0.231 µM). Introducing 3-Cl as R4 substituent in compounds 8 and 9 increased the activity of 16 and 17 by 6-
and 3-fold. Compound 17 was better than 16 as their non-substituted analogue at R4. Then, the fluoro group was
introduced in compounds 10-11. Compound 18 with 2’-Me-3-F and 19 with 3’-Me-3-F displayed similar activity as
compounds 10 and 11. Finally, we introduced the methyl group on the biphenyl ring to improve the anti-viral activity.
Compound 20 with 2’,3-dimethyl (EC50 = 0.095 µM) and compound 21 with 3’,3-dimethyl (EC50 = 0.146 µM) had
comparable activity against HIV-1. To our delight, compounds 22 and 23 with dimethyl group showed great potency
with EC50 values of 14 and 17 nM, which were comparable to EFV and ETR. In particular, compound 23 exhibited the
highest SI value of 18,564 and potent anti-viral activity with an EC50 value of 17 nM, which was superior to the
reference drug NVP (EC50 = 293 nM, SI >51) and slightly inferior to the references EFV and ETR (EC50 = 7 and 6 nM,
SI >863 and >833, respectively).

Table 2. Activity and cytotoxicity of compounds 12-23 against HIV-1 (IIIB) strains in MT-4 cells.
EC50 (µM)a
Compounds R4
R5
CC50 (µM)b
SI (IIIB)c
HIV-1 (IIIB)
0.139 ± 0.056
4.095 ± 0.91
0.029 ± 0.005
0.231 ± 0.143
0.060 ± 0.027
0.021 ± 0.010
3-F
3-F
2'-F
3'-F
42.23 ± 11.72 302
12
>259.83
>63
324
13
3-Me 2'-F
3-Me 3'-F
9.47 ± 0.85
14
46.02 ± 13.40 194
15
3-Cl
3-Cl
3-F
2'-Cl
3'-Cl
>243.65
>4013
≥963
16
≥20.21
17
2'-Me 0.065 ± 0.008
3'-Me 0.174 ± 0.094
245.87 ± 53.67 3806
35.25 ± 5.80 202
152.07 ± 6.91 1606
18
3-F
19
3-Me 2'-Me 0.095 ± 0.054
3-Me 3'-Me 0.146 ± 0.033
2-Me 2'-Me 0.014 ± 0.005
2-Me 3'-Me 0.017 ± 0.012
0.293 ± 0.107
20
6.15 ± 0.42
65.96 ± 4.5
42
21
4741
22
264.19 ± 17.23 18564
23
>15.03
>6.34
>4.61
>51
Nevirapine
Efavirenz
Etravirine
0.007 ± 0.005
>863
>833
0.006 ± 0.002
aEC50: The effective concentration required to protect the cell against HIV-induced cytopathogenic effect by 50% in
MT-4 cells.
bCC50: The cytotoxic concentration of the compound that reduced the normal uninfected MT-4 cell viability by 50%.
cSI: selectivity index, ratio CC50/EC50 (WT).
The molecular modelling study (Fig. 6A-D) and MD simulation (Fig. 6E-G) were performed to give a possible

explanation for the activity of compound 22. Docking result indicated that the molecule bound to the pocket with
common key features. Compared with compound 10, the additional ortho-methyl group theoretically increased the
dihedral angle of the biphenyl ring to an approximately perpendicular conformation, leading to powerful face to face
π-π interactions with Y181/Y188 (Fig. 6C) [37]. The dihedral angle of the biphenyl ring is enlarged to ~-80-90 ̊ by the
dimethyl group (Fig. 6F). Moreover, the 2’-methyl group representatively occupied the hydrophobic pocket in
F227/L234 region (Fig. 6D), together contributing to its potent and improved anti-HIV-1 e cacy as compared to that
of compounds 10-11.

binding mode of 22 with 2ZD1. (E) Protein-Ligand (2ZD1-22) root mean square deviation (RMSD). Left Y-axis
shows the RMSD evolution of a protein. Right Y-axis indicates how stable the ligand is with respect to the protein and
its binding pocket. (F) The conformation of the torsion throughout the course of the simulation (dial plot) and the
probability density of the torsion (bar plot). (G) Protein-ligand (2ZD1-22) contacts.
Antiviral activity against mutants and enzymatic assay of the selected compounds.
Furthermore, compounds 6, 14, 17, and 22-23 with good anti-HIV-1 activity, low cytotoxicity and high SI were
selected to investigate their inhibitory activity against a panel of clinically observed drug-resistant mutants and WT
HIV-1 RT enzyme (Table 3). Mutant strains were more sensitive to compounds 6 and 22, with higher anti-WT HIV-1
activity, than other analogues. Especially, compound 22 displayed low micromolar EC50 values toward all of the
mutant strains. Moreover, it had an EC50 of 0.02 µM toward the K103N mutant, which was more potent than the
reference standards NVP (EC50 = 9.55 µM) and EFV (EC50 = 0.102 µM). The five novel DAPY derivatives displayed
great inhibitory activity against WT HIV-1 RT with IC50 values ranging from 27 nM to 65 nM, which were 6~15-fold
higher than that of NVP (IC50 = 404 nM).
Table 3. Inhibitory activity of the selected compounds toward a panel of clinically relevant HIV-1 mutant strains and
HIV-1 RT enzyme.
EC50 (µM)a
Compounds
IC50 (µM)b
L100I
K103N
Y181C
E138K
0.61 ± 0.27
0.44 ± 0.45
0.94 ± 0.05
0.13 ± 0.08
1.97 ± 0.66
1.99 ± 0.75
0.09 ± 0.01
0.21 ± 0.13
20.21 ± 8.55
0.02 ± 0.01
0.13 ± 0.01
>9.55
0.63 ± 0.49
0.71 ± 0.01
0.66 ± 0.09
0.03 ± 0.01
0.13 ± 0.11
0.4 ± 0.07
0.04 ± 0.03
0.04 ± 0.01
0.065 ± 0.023
0.062 ± 0.015
0.031 ± 0.09
0.027 ± 0.018
0.042 ± 0.016
0.404 ± 0.066
6
14
17
22
195.72 ± 73.20 0.16 ± 0.12
>15.03 0.2 ± 0.026
23
Nevirapine

0.044 ± 0.07 0.102 ± 0.054
0.007 ± 0.003 0.003 ± 0.001
0.007 ± 0.001
0.012 ± 0.008
0.006 ± 0.001
0.006 ± 0.005
0.03 ± 0.022
Efavirenz
Etravirine
0.011 ± 0.009
aEC50: The effective concentration required to protect the cell against HIV-induced cytopathogenic effect by 50% in
MT-4 cells.
bIC50: inhibitory concentration of the test compound required to inhibit WT HIV-1 RT polymerase activity by 50%.
Conclusion
With a scope of determining the influence on the dihedral angle, conformation and anti-viral activity of the
biphenyl ring, we have designed and synthesized a new series of substituted thiophene-biphenyl-DAPYs. They had
good anti-WT-HIV-1 activity, maintained inhibitory ability against clinically observed mutant strains, and potent
anti-RT activities. The introduction of fluoro, chloro, or methyl group on the biphenyl ring exhibited different effects
on the anti-viral activity. The dimethyl-substituted agents (22, 23) exhibited highly potent inhibitory activity against
WT HIV-1 (EC50 = 14 and 17 nM, respectively). The predicted molecular models further supported our hypothesis
that two methyl substitutions could enlarge the dihedral angle and modulate the conformation of biphenyl group to fit
more tightly into the NNRTI binding pocket. On the basis of the structural insights, a rational conformational model
was generated and might be helpful to further DAPY optimizations.

Experimental Section
4.1. General
All reagents and solvents were purchased from commercial sources and used as received. Column
chromatography was performed on silica gel (300-400 mesh). TLC was carried out on 0.25 mm silica gel plates
visualized with UV light (λ = 254 nm) and/or by staining with ethanolic phosphomolybdic acid (PMA) or iodine.
Proton nuclear magnetic resonance (1H-NMR) and carbon nuclear magnetic resonance (13C-NMR) spectra were
recorded in DMSO-d6 or CF3COOD, CDCl3 on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as the
internal standard. Chemical shifts (δ) are given in ppm relative to TMS, coupling constants (J) in Hz. Melting points
were measured on WRS-1B digital melting-point apparatus. EI-MS were recorded on an Agilent 6890N/5975
spectrometer and ESI-MS were recorded on a Waters Micromass Quattro Micro spectrometer.
4.2. General procedure for the preparation of compounds 2a-e
To a solution of compound 1a-e (10.0 mmol, 1.0 eq) in anhydrous THF (20 mL) was slowly added n-BuLi (6 mL
o
of 2.5 M solution in hexane, 15.0 mmol, 1.5 eq) at -78 C. The mixture was stirred for 1 h and triisopropyl borate
o
(50.0 mmol, 5.0 eq) was added by syringe. The solution was stirred for another 1 h and allowed to warm to 25 C
o
overnight. The reaction was cooled to 0 C, followed by the sequential addition of water (5 mL) and 2N HCl (5 mL).
After 10 min, an additional 35 mL of 2N HCl was added and the mixture was further stirred for 30 min. The mixture
was extracted with ethyl acetate (20 mL * 3) and the combined organic layers were concentrated to a thick oil under
reduced pressure. Crystallization with cold hexane yielded a white solid.
4.3. General procedure for the preparation of compounds 3a-r
A reaction mixture of 2a-e (5.0 mmol, 1.0 equiv.), different substituted 4-bromobenzonitrile (6.0 mmol, 1.2 equiv.)
and potassium carbonate (12.5 mmol, 2.5 equiv.) in PEG400/H20 (10 mL/10 mL) was stirred at room temperature for
15 min, and PdCl2 (0.05 mmol, 0.01 equiv.) was subsequently added. Stirring was continued for an additional 15 h
until complete consumption of starting material as judged by TLC. Then the reaction mixture was poured into water
(80 mL) and extracted with ethyl acetate (30 mL*3). The organic layers were washed with brine, dried over anhydrous

Na2SO4, filtered and condensed under reduced pressure. The residue was then purified via column chromatography on
silica gel, eluting with EtOAc/petroleum ether to 3a-r as white solid.
4.3. General procedure for the preparation of compounds 4a-r
A solution of 3a-r (1.0 mmol, 1.0 equiv.) in dichloromethane (3 mL) was cooled to -45 oC under N2. BBr3 (6 mL
of 1 M solution in dichloromethane, 6.0 mmol, 6.0 equiv.) was added dropwise over 5 min. The solution was stirred
o
for another 30 min and allowed to warm to 25 C overnight. The mixture was then diluted with H2O (20 mL) and
extracted with ethyl acetate (10 mL*4). The organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered and condensed. The residue was then purified using column chromatography on silica gel, eluting with
EtOAc/petroleum ether to 4a-r as white solid.
4.4. General procedure for the preparation of the final compounds 6-23
Different substituted 4'-hydroxy-[1,1'-biphenyl]-4-carbonitrile (0.2 mmol, 1.0 equiv.) was dissolved in DMF (3
mL) in the presence of K2CO3 (0.6 mmol, 3.0 equiv.), followed by addition of the compound 5 (0.2 mmol, 1.0 equiv.).
The reaction mixture was stirred at 80 °C for 6-8 h (monitored by TLC), and then the reaction was cooled to ambient
temperature and poured into 15 mL H2O yielding a precipitate. The precipitate was collected by filtration and the
residue was then purified via column chromatography on silica gel, eluting with EtOAc/petroleum ether to obtain 6-23
as white solid.
4.4.1. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2’-fluoro-[1,1'-biphenyl]-4-carbonitrile (6)
Yield: 85%. mp: 272.5-273.6 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.44 (d, J = 7.1 Hz, 1H), 7.76 (d, J
= 8.2 Hz, 2H), 7.72-7.63 (m, 3H), 7.48 (d, J = 8.7 Hz, 2H), 7.41 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 6.91 (d,
J = 7.1 Hz, 1H). 13C NMR (101 MHz, CF3COOD, CDCl3) δ: 172.81, 159.21, 152.38, 151.78, 148.00, 139.75, 133.30,
131.69, 131.65, 130.92, 130.89, 129.09, 129.05, 121.78, 121.38, 120.69, 120.43, 118.43, 112.78, 109.96, 107.38,
101.57. LCMS (ESI) m/z C26H14FN5OS: calcd 463.09, found 464.31 [M + H]+.
4.4.2. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3’-fluoro-[1,1'-biphenyl]-4-carbonitrile (7)
Yield: 92%. mp: 258.8-260.3 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.43 (d, J = 5.4 Hz, 1H), 7.96 (d, J

= 8.2 Hz, 2H), 7.84 (d, J = 7.9 Hz, 2H), 7.74 (t, J = 8.5 Hz, 1H), 7.59-7.52 (m, 5H), 7.31 (t, J = 8.1 Hz, 2H). 13C NMR
(101 MHz, CF3COOD, CDCl3) δ: 172.84, 164.05, 152.40, 152.22, 147.97, 139.82, 137.48, 134.40, 133.20, 129.03,
123.43, 123.40, 122.22, 121.42, 118.44, 114.97, 114.76, 112.80, 109.98, 107.32, 101.59. LCMS (ESI) m/z
C26H14FN5OS: calcd 463.09, found 464.50 [M + H]+.
4.4.3. 2’-chloro-4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-[1,1'-biphenyl]-4-carbonitrile (8)
1
Yield: 74%. mp: > 300 °C. H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.45 (d, J = 7.1 Hz, 1H), 7.96 (s, 1H),
7.78 (d, J = 9.2 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8
13
Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 7.1 Hz, 1H). C NMR (101 MHz, CF3COOD, CDCl3) δ: 172.79,
152.44, 151.53, 147.98, 144.54, 139.79, 136.99, 134.01, 133.66, 133.39, 131.97, 131.12, 131.04, 121.36, 118.43,
112.78, 111.60, 109.96, 107.35, 101.63. LCMS (ESI) m/z C26H14ClN5OS: calcd 479.06, found 480.33 [M + H]+.
4.4.4. 3’-chloro-4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-[1,1'-biphenyl]-4-carbonitrile (9)
Yield: 81%. mp: 246.3-247.7 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.45 (d, J = 6.9 Hz, 1H), 7.91 (d, J
= 8.0 Hz, 1H), 7.85 (s, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.1 Hz, 1H), 7.43 (s, 4H), 7.38 (d, J = 8.0 Hz, 2H),
6.90 (d, J = 6.7 Hz, 1H). 13C NMR (101 MHz, CF3COOD, CDCl3) δ: 172.77, 152.39, 152.14, 148.08, 146.24, 139.82,
138.35, 137.43, 134.99, 133.23, 129.11, 128.53, 125.72, 122.25, 121.37, 118.43, 112.79, 111.03, 109.96, 107.38,
101.57. LCMS (ESI) m/z C26H14ClN5OS: calcd 479.06, found 502.34 [M + Na]+.
4.4.5. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2’-methyl-[1,1'-biphenyl]-4-carbonitrile (10)
Yield: 82%. mp: 278.4-279.5 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.45 (d, J = 7.0 Hz, 1H), 7.76 (s,
1H), 7.73 (d, J = 7.8 Hz, 1H), 7.57-7.51 (m, 6H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 6.9 Hz,
1H), 2.42 (s, 3H). 13C NMR (101 MHz, CF3COOD, CDCl3) δ: 173.00, 152.52, 151.03, 147.69, 146.03, 139.86, 139.58,
137.19, 134.42, 133.23, 130.53, 130.36, 130.05, 121.58, 121.21, 118.38, 112.75, 109.93, 101.64, 19.41. LCMS (ESI)
m/z C27H17N5OS: calcd 459.12, found 460.40 [M + H]+.
4.4.6. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3’-methyl-[1,1'-biphenyl]-4-carbonitrile (11)
Yield: 75%. mp: 256.5-257.9 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.07 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 7.95

(d, J = 7.6 Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 5.2 Hz,
1H), 7.39 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H), 2.30 (s, 3H). 13C NMR (101 MHz, CF3COOD, CDCl3) δ:
166.81, 151.58, 151.16, 150.35, 146.17, 142.62, 140.46, 139.51, 138.20, 133.14, 132.78, 131.04, 129.97, 123.29,
121.49, 119.06, 118.47, 117.45, 112.82, 111.73, 110.00, 109.69, 106.92, 19.88. LCMS (ESI) m/z C27H17N5OS: calcd
459.12, found 460.28 [M + H]+.
4.4.7.
4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2’,3-difluoro-[1,1'-biphenyl]-4-carbonitrile
(12)
Yield: 67%. mp: 295.3-297.0 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.41 (d, J = 5.4 Hz, 1H), 7.75-7.68
13
(m, 3H), 7.59 (d, J = 10.5 Hz, 1H), 7.58-7.51 (m, 5H), 7.45 (d, J = 8.7 Hz, 2H). C NMR (101 MHz, CF3COOD,
CDCl3) δ: 165.84, 159.13, 153.51, 151.76, 150.86, 143.18, 140.09, 139.02, 138.89, 134.99, 133.24, 131.55, 129.14,
125.86, 123.66, 121.83, 120.54, 118.46, 117.49, 112.82, 111.23, 109.99, 107.30. LCMS (ESI) m/z C26H13F2N5OS:
calcd 481.08, found 482.29 [M + H]+.
4.4.8. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,3'-difluoro-[1,1'-biphenyl]-4-carbonitrile (13)
1
Yield: 83%. mp: >300 °C. H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.42 (d, J = 5.4 Hz, 1H), 7.94-7.87 (m,
13
1H), 7.66-7.52 (m, 7H), 7.45 (s, 3H). C NMR (101 MHz, CF3COOD, CDCl3) δ: 165.80, 164.02, 154.01, 151.74,
150.90, 143.30, 140.16, 134.63, 133.12, 124.18, 124.02, 123.39, 121.84, 118.47, 117.51, 116.02, 115.83, 115.06,
114.86, 112.82, 111.27, 110.00, 107.26, 99.81, 99.65. LCMS (ESI) m/z C26H13F2N5OS: calcd 481.08, found 504.36 [M
+ Na]+.
4.4.9.
4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2’-fluoro-3-methyl-[1,1'-biphenyl]-4-carbonitrile (14)
Yield: 89%. mp: 268.5-271.0 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.38 (d, J = 5.4 Hz, 1H), 7.70 (d, J
= 5.5 Hz, 2H), 7.67-7.60 (m, 3H), 7.54 (d, J = 5.4 Hz, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.41-7.36 (m, 3H), 2.33 (s, 3H).
13C NMR (101 MHz, CF3COOD, CDCl3) δ: 166.75, 159.54, 151.91, 150.99, 143.02, 140.61, 133.79, 132.78, 132.07,
131.31, 129.37, 128.67, 122.33, 121.50, 120.99, 118.79, 117.94, 113.14, 111.72, 110.32, 107.26, 15.90. LCMS (ESI)

m/z C27H16FN5OS: calcd 477.11, found 500.33 [M + Na]+.
4.4.10.
4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3’-fluoro-3-methyl-[1,1'-biphenyl]-4-carbonitrile (15)
Yield: 81%. mp: 299.3-300.2 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.43 (d, J = 5.4 Hz, 1H), 7.91 (t, J =
7.2 Hz, 1H), 7.75-7.67 (m, 3H), 7.62 (d, J = 9.9 Hz, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.50-7.36 (m, 5H), 2.38 (s, 3H). 13C
NMR (101 MHz, CF3COOD, CDCl3) δ: 166.47, 159.08,165.38, 162.79, 151.60, 151.15, 150.55, 142.85, 140.38,
137.70, 133.16, 130.67, 126.42, 121.30, 118.49, 117.51, 114.94, 114.74, 112.84, 111.40, 110.02, 106.88, 98.74, 15.42.
LCMS (ESI) m/z C27H16FN5OS: calcd 477.11, found 478.44 [M + H]+.
4.4.11.
2’,3-dichloro-4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-[1,1'-biphenyl]-4-carbonitrile
(16)
1
Yield: 90%. mp: 298.4-300.9 °C. H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.52 (d, J = 7.0 Hz, 1H), 7.98 (s,
1H), 7.81 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.61-7.50 (m, 4H), 7.46-7.38 (m, 3H), 6.98 (d, J = 7.0 Hz, 1H). 13C NMR
(101 MHz, CF3COOD, CDCl3) δ: 172.01, 152.51, 148.51, 147.72, 143.29, 139.65, 138.24, 134.07, 133.61, 133.34,
131.84, 131.41, 131.14, 129.41, 126.86, 123.02, 121.66, 118.45, 112.80, 112.09, 109.98, 107.52, 101.30.
4.4.12.
3,3'-dichloro-4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-[1,1'-biphenyl]-4-carbonitrile
(17)
Yield: 75%. mp: 245.6-247.3 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.53 (d, J = 7.0 Hz, 1H), 7.93 (d, J
= 8.1 Hz, 1H), 7.86 (d, J = 1.8 Hz, 1H), 7.83 (s, 1H), 7.72-7.66 (m, 2H), 7.49-7.37 (m, 3H), 7.38 (d, J = 8.8 Hz, 2H),
6.97 (d, J = 7.0 Hz, 1H). 13C NMR (101 MHz, CF3COOD, CDCl3) δ: 171.96, 152.45, 148.71, 148.23, 144.91, 139.69,
138.79, 138.53, 135.13, 133.15, 129.52, 128.52, 127.88, 127.30, 125.71, 123.92, 121.62, 118.45, 112.80, 111.72,
109.98, 107.56, 101.22.
4.4.13.
4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2’-methyl-3-fluoro-[1,1'-biphenyl]-4-carbonitrile (18)
Yield: 86%. mp: 275.2-276.8 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.40 (d, J = 5.4 Hz, 1H), 7.76-7.69

(m, 2H), 7.57-7.46 (m, 6H), 7.42 (d, J = 7.9 Hz, 1H), 7.39-7.25 (m, 2H), 2.39 (s, 3H). 13C NMR (101 MHz,
CF3COOD, CDCl3) δ: 165.89, 153.26, 151.86, 150.79, 144.77, 143.18, 141.34, 140.15, 138.15, 138.02, 137.09,
133.17, 130.23, 125.71, 123.31, 122.04, 117.48, 112.88, 111.38, 110.05, 107.37, 19.61. LCMS (ESI) m/z
C27H16FN5OS: calcd 477.11, found 478.32 [M + H]+.
4.4.14.
4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3-fluoro-3’-methyl-[1,1'-biphenyl]
-4-carbonitrile (19)
Yield: 91%. mp: 252.5-254.2 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.12 (s, 1H, NH), 8.43 (d, J = 5.3 Hz, 1H),
13
7.99-7.88 (m, 3H), 7.82-7.69 (m, 5H), 7.56-7.44 (m, 3H), 2.60 (s, 3H). C NMR (101 MHz, DMSO-d6) δ:165.15,
163.48, 157.17, 155.93, 153.47, 145.24, 142.88, 142.82, 139.69, 138.55, 133.69, 133.13, 129.21, 125.67, 125.39,
124.53, 123.88, 119.91, 118.73, 118.38, 116.22, 116.03, 111.71, 108.92, 102.78, 20.53. LCMS (ESI) m/z
C27H16FN5OS: calcd 477.11, found 478.32 [M + H]+.
4.4.15. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2',3-dimethyl-[1,1'-biphenyl]-4-carbonitrile
(20)
1
Yield: 88%. mp: 262.4-263.7 °C. H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.40 (d, J = 5.4 Hz, 1H), 7.76 (s,
13
1H), 7.73 (d, J = 8.0 Hz, 1H), 7.57-7.46 (m, 6H), 7.44-7.36 (m, 3H), 2.44 (s, 3H), 2.35 (s, 3H). C NMR (101 MHz,
CF3COOD, CDCl3) δ: 166.62, 151.71, 150.39, 149.89, 146.26, 142.66, 140.43, 139.81, 137.22, 134.42, 133.22,
132.16, 130.60, 130.38, 130.02, 127.95, 121.52, 121.41, 118.45, 117.44, 112.81, 111.53, 109.99, 109.62, 106.90, 19.49,
15.26. LCMS (ESI) m/z C28H19N5OS: calcd 473.13, found 474.41 [M + H]+.
4.4.16. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,3'-dimethyl-[1,1'-biphenyl]-4-carbonitrile
(21)
Yield: 92%. mp: 259.4-261.1 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.41 (d, J = 5.4 Hz, 1H), 7.89 (d, J
= 8.0 Hz, 1H), 7.75-7.68 (m, 4H), 7.58 (d, J = 5.4 Hz, 1H), 7.45 (s, 4H), 7.40 (d, J = 8.3 Hz, 1H), 2.74 (s, 3H), 2.38 (s,
13
3H). C NMR (101 MHz, CF3COOD, CDCl3) δ: 166.61, 151.55, 150.67, 150.50, 145.13, 143.84, 142.58, 140.43,
139.18, 133.65, 133.17, 131.20, 130.70, 128.83, 126.48, 124.97, 122.19, 121.15, 118.39, 112.75, 109.93, 19.62, 15.32.

LCMS (ESI) m/z C28H19N5OS: calcd 473.13, found 474.28 [M + H]+.
4.4.17. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2,2'-dimethyl-[1,1'-biphenyl]-4-carbonitrile
(22)
1
Yield: 86%. mp: 274.3-275.8 °C. H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.43 (d, J = 7.1 Hz, 1H), 7.75 (s,
1H), 7.71 (d, J = 7.9 Hz, 1H), 7.56 (s, 4H), 7.30 (dd, J = 11.4, 8.1 Hz, 2H), 7.19 (s, 1H), 7.16 (d, J = 8.2 Hz, 1H), 6.88
13
(d, J = 7.1 Hz, 1H), 2.20 (s, 3H), 2.14 (s, 3H). C NMR (101 MHz, CF3COOD, CDCl3) δ: 172.87, 152.54, 150.88,
147.69, 146.01, 139.93, 139.07, 138.45, 137.82, 134.08, 133.26, 130.24, 130.18, 130.06, 122.51, 121.64, 118.53,
118.46, 115.63, 112.81, 109.99, 109.91, 107.37, 101.70, 19.25, 19.03. LCMS (ESI) m/z C28H19N5OS: calcd 473.13,
found 474.34 [M + H]+.
4.4.18. 4'-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-2,3’-dimethyl-[1,1'-biphenyl]-4-carbonitrile
(23)
Yield: 71%. mp: 264.9-266.2 °C. 1H NMR (400 MHz, CF3COOD, CDCl3) δ: 8.42 (d, J = 7.1 Hz, 1H), 7.83 (d, J
= 7.9 Hz, 1H), 7.51 (s, 4H), 7.45-7.39 (m, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 7.2 Hz, 2H), 6.88 (d, J = 7.1 Hz,
13
1H), 2.68 (s, 3H), 2.35 (s, 3H). C NMR (101 MHz, CF3COOD, CDCl3) δ: 172.99, 152.38, 151.08, 147.75, 146.08,
143.29, 139.99, 139.59, 138.24, 133.21, 133.09, 131.02, 130.99, 127.14, 122.91, 121.44, 118.67, 118.44, 112.79,
107.25, 101.56, 19.85, 19.80. LCMS (ESI) m/z C28H19N5OS: calcd 473.13, found 474.41 [M + H]+.
4.5. Antiviral activity assays
4.5.1. In vitro anti-HIV assay
The anti-HIV activity and cytotoxicity of the compounds were evaluated against WT HIV-1, and mutant strains
(L100I, K103N, E138K, and Y181C), in MT-4 cell cultures in MT-4 cell cultures using the method of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, virus stocks were titrated in MT-4 cells
and expressed as the 50% cell culture infective dose (CCID50). MT-4 cells were suspended in culture medium at
1×105 cells/mL and infected with HIV at a multiplicity of infection of 0.02. Immediately after viral infection,100 mL
of the cell suspension was placed in each well of a flat-bottomed micro-titer tray containing various concentrations of

the test compounds. The test compounds were dissolved in DMSO at 50 mM or higher. After 4 days of incubation at
37°C, the number of viable cells was determined using the MTT method. Compounds were tested in parallel for
cytotoxic effects in uninfected MT-4 cells.
4.5.2. HIV-1 RT inhibition assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as previously described. The RT assay
was performed with the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen), as described by
the manufacturer. The assay was based on the dsDNA quantitation reagent Pico-Green. This reagent showed a
pronounced increase in fluorescence signal upon binding to dsDNA or RNA-DNA hetero-duplexes. Single-stranded
nucleic acids generated only minor fluorescence signal enhancement when a sufficiently high dye: base pair ratio was
applied. This condition was met in the assay.
A poly(rA) template of approximately 350 bases long and anoligo(dT)16 primer were annealed in a molar ratio of
1:1.2 (60 min, at room temperature). 52 ng of the RNA/DNA was brought into each well of a 96-well plate in a
volume of 20 mL polymerization buffer (60 mM Trise HCl, 60 mM KCl, 8 mM MgCl2, 13 mM DTT, 100 mM dTTP,
pH = 8.1). 5.0 mL of RT enzyme solution, diluted to a suitable concentration in an enzyme dilution buffer (50 mM
Trise-HCl, 20% glycerol, 2mM DTT, pH = 7.6), was added. The reaction mixture was incubated at 25 for 40 min
and then stopped by the addition of EDTA (15 mM). Hetero duplexes were then detected by addition of Pico-Green.
Signals were read using an excitation wavelength of 490 nm and emission detection at 523 nm using a
spectrofluorometer (Safire 2, Tecan). To test the biological activity of compounds against RT, 1.0 mL of compound in
DMSO was added to each well before the addition of the RT solution. Control wells without compound contained the
same amount of DMSO. Results were expressed as relative fluorescence, i.e. the fluorescence signal of the reaction
mixed with compound divided by the signal of the same reaction mixed without compound.
4.9. Molecular modeling
Molecular modelling research work was performed with the Tripos molecular modelling software package

(Sybyl-X 2.0). All the molecules for docking analysis were built using the standard bond lengths and angles from
Sybyl-X 2.0/base Builder before being optimized using the Tripos force field for 10000 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. 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. The scoring function in Surflex-Dock, which contains hydrophobic,
polar, repulsive, entropic and solvation terms, was trained to estimate the binding energy. Prior to docking, the protein
was prepared by removing water molecules, the ligand ETR, and other unnecessary small molecules from the crystal
structure of the HIV-1 RT complex. Simultaneously, 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 8 Å), the maximum number of the 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 amino acid
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, including van der Waals, electrostatic, and torsional energy terms
defined in the Tripos force field. The structure optimization was performed for more than 10,000 generations using a
genetic algorithm, and the 20-best-scoring ligand-protein complexes were kept for the further analyses. The -log(Kd)2
values of the 20-best-scoring complexes, which represented the binding affinities of the ligand with RT, encompassed
a wide scope of the functional classes (10-2-10-9). Only the highest scoring 3D structural model of the ligand-bound
RT was chosen to define the binding interaction.
MD simulations were carried out for RT in complex with the structurally solved inhibitors. Each system was

solvated in a cubic box with explicit TIP3P water and counter ions consisting of a 10Å solvent buffer region from the
edge of the complex. The long range electrostatic interactions were evaluated by the Particle-Mesh Ewald method
under the periodic boundary condition. 10 ns simulation was carried out for each docked model using Desmond [42]
with OPLS-AA 2005 force field under the isobaric isothermal (NPT) condition at 300K and 1 atm. The stability of the
simulation was assessed by monitoring the RMSD with respect to the minimized starting structure.
Abbreviations
HIV, human immunodeficiency virus
FIs, fusion inhibitors
INSTIs, integrase strand transfer inhibitors
PIs, protease inhibitors
RTIs, reverse transcriptase inhibitors
DAPY, diarylpyrimidine
NNRTI, non-nucleoside reverse transcriptase inhibitor
WT, wild-type
ETR, etravirine
RPV, rilpivirine
EFV, efavirenz
RT, reverse transcriptase
NVP, nevirapine
MD, molecular dynamics
Acknowledgments
We gratefully acknowledge the generous support provided by the National Natural Science Foundation of P. R. China

under Grant No. 21372050, Shanghai Municipal Natural Science Foundation under Grant No. 13ZR1402200 and the
Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (2017QNRC061).
The technical assistance of Kristien Erven, Cindy Heens and of Kris Uyttersprot for the HIV experiments and HIV RT
polymerase assays is gratefully acknowledged.
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