Scaffold hopping in discovery of HIV-1 non-nucleoside reverse transcriptase inhibitors: From CH(CN)-DABOs to CH(CN)-dapys

Article abstract of DOI:10.3390/molecules25071581

Scaffold hopping is a frequently-used strategy in the development of non-nucleoside reverse transcriptase inhibitors. Herein, CH(CN)-DAPYs were designed by hopping the cyano-methylene linker of our previous published CH(CN)-DABOs onto the etravirine (ETR). Eighteen CH(CN)-DAPYs were synthesized and evaluated for their anti-HIV activity. Most compounds exhibited promising activity against wild-type (WT) HIV-1. Compounds B4 (EC₅₀ = 6 nM) and B6 (EC₅₀ = 8 nM) showed single-digit nanomolar potency against WT HIV-1. Moreover, these two compounds had EC₅₀ values of 0.06 and 0.08 μM toward the K103N mutant, respectively, which were comparable to the reference efavirenz (EFV) (EC₅₀ = 0.08 μM). The preliminary structure-activity relationship (SAR) indicated that introducing substitutions on C2 of the 4-cyanophenyl group could improve antiviral activity. Molecular docking predicted that the cyano-methylene linker was positioned into the hydrophobic cavity formed by Y181/Y188 and V179 residues.

Full text of DOI:10.3390/molecules25071581

molecules
Article
Scaffold Hopping in Discovery of HIV-1
Non-Nucleoside Reverse Transcriptase Inhibitors:
From CH(CN)-DABOs to CH(CN)-DAPYs
Ting-Ting Li 1, Christophe Pannecouque 2, Erik De Clercq 2 , Chun-Lin Zhuang 3,4,
*
and
Fen-Er Chen 1,3,4,
*
1
Institute of Pharmaceutical Science and Technology, Zhejiang University of Technology, Hangzhou 310014,
China; toplis163@163.com
Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium;
christophe.pannecouque@kuleuven.be (C.P.); erik.declercq@kuleuven.be (E.D.C.)
Department of Chemistry, Fudan University, Shanghai 200433, China
2
3
4
Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, China
Correspondence: zclnathan@163.com (C.-L.Z.); rfchen@fudan.edu.cn (F.-E.C.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Paola Barraja, Alessandra Montalbano and Virginia Spanò
Received: 21 February 2020; Accepted: 24 March 2020; Published: 30 March 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Scaffold hopping is a frequently-used strategy in the development of non-nucleoside reverse
transcriptase inhibitors. Herein, CH(CN)-DAPYs were designed by hopping the cyano-methylene
linker of our previous published CH(CN)-DABOs onto the etravirine (ETR). Eighteen CH(CN)-DAPYs
were synthesized and evaluated for their anti-HIV activity. Most compounds exhibited promising
activity against wild-type (WT) HIV-1. Compounds B4 (EC50 = 6 nM) and B6 (EC50 = 8 nM) showed
single-digit nanomolar potency against WT HIV-1. Moreover, these two compounds had EC50 values
of 0.06 and 0.08
µM toward the K103N mutant, respectively, which were comparable to the reference
efavirenz (EFV) (EC50 = 0.08
µM). The preliminary structure–activity relationship (SAR) indicated
that introducing substitutions on C2 of the 4-cyanophenyl group could improve antiviral activity.
Molecular docking predicted that the cyano-methylene linker was positioned into the hydrophobic
cavity formed by Y181/Y188 and V179 residues.
Keywords: CH(CN)-DAPYs; HIV-1; NNRTI; scaffold hopping; strucutral optimization
1. Introduction
Human immunodeficiency virus-1 (HIV-1) non-nucleoside reverse transcriptase inhibitors
(NNRTIs) are key components of highly active antiretroviral therapy (HAART) for treating acquired
immune deficiency syndrome (AIDS) [
diverse classes of compounds have been reported as NNRTIs, which can be divided into the
following categories according to their structures: TIBO (tetrahydroimidazobenzodiazepinone), -APA
-anilinophenylacetamide), ITU (iminothiourea), DABO (dihydro-alkoxy-benzyl-oxopyrimidine),
DATA (diaryltriazine), DAPY (diarylpyrimidine), etc. [ ]. Diarylpyrimidine (DAPY) derivatives
are second-generation NNRTIs with remarkable anti-HIV-1 activity and favorable pharmacological
properties [ ]. Two representative DAPY analogues, etravirine (ETR) and rilpivirine (RPV), were
successfully approved by the U.S. Food and Drug Administration (FDA) and are widely used in the
clinic setting [ ]. Although RPV versus some NNRTIs exhibited high activity, there are some issues
such as high toxicity, poor water solubility, and low bioavailability for DAPY compounds used in
clinical practice [ 10]. Therefore, further optimization of DAPYs is still a the research focus in the
1]. Over the past decades, more than fifty structurally
α
(α
2,3
4
5–7
8–
medicinal chemistry community.
Molecules 2020, 25, 1581; doi:10.3390/molecules25071581
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1581
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The scaffold hopping strategy is widely applied in the development of NNRTIs. In the past 20 years,
our group published about multiple hybrids developed by the scaffold hopping strategy (Figure 1) [11],
such as cycloalkyl arylpyrimidines (CAPYs) [12], biphenyl-diarylpyrimidines (Biphenyl-DAPYs) [10
thiophene-biphenyl-DAPYs [ 14], diarylbenzopyrimidines (DABPs) [15], sulfinylacetamide-DABPs,
and etravirine-VRX-480773 hybrids [16 18]. As a result, these compounds exhibited remarkably
,13],
7,
–
increased antiviral activity compared with their parent lead compounds, indicating the efficacy of the
hopping strategy in developing the anti-HIV drug candidates.
Figure 1. Representative non-nucleoside reverse transcriptase inhibitors (NNRTIs) obtained by the
scaffold hopping strategy.
The dihydro-alkoxy-benzyl-oxopyrimidines (DABOs), first reported in 1992, were recognized to be
potent anti-HIV compounds. In our group, several potent analogues were obtained by structure-based
drug design (SBDD). In 2007, a cyano group was introduced into DABO as a linker to obtain a series
of novel CH(CN)-DABOs (Figure 2) with superior antiviral activity against wild-type (WT) HIV-1
(LAI strain IIIB) (EC50 = 2 nM). Molecular docking results showed that the 6-(α-cyanobenzyl) moiety
of CH(CN)-DABOs fitted into the aromatic-rich non-nucleoside binding pocket, surrounded by the
aromatic side chains of Y181, Y188, F227, and W229 [19]. In addition, the cyano group can also
form hydrophobic interactions with hydrophobic protein residues [20] together contributing to the
high potency.
Given that the oxygen linker of ETR is located at a small hydrophobic pocket formed by Y181/Y188
and V179, we continued with this study to design a new series of DAPY derivatives with the
cyano-methylene linker of CH(CN)-DABOs onto the ETR, employing the scaffold hopping strategy.
Furthermore, introducing a methyl into the pyrimidinyl ring that is located at the entrance channel
containing hydrophobic residues might establish nonpolar interactions with a reverse transcriptase
(RT) enzyme (Figure 2) and improve the activity [21,22].

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Figure 2. Molecular design of CH(CN)-DAPYs.
2. Results and Discussion
2.1. Chemistry
The synthetic route of the target compounds A1
–
A12 and B1
–
B6 are depicted in Scheme 1.
The key intermediates 4-(4-chloro-pyrimidin-2-ylamino)benzonitriles (10) were prepared from
compound ( ) according to our previously reported three-step protocol [23]. The substituted
7
4-cyanophenylacetonitriles 6a-l were synthesized via a one-pot procedure from 5a-l. Then, compounds
6a-l were reacted with key intermediates (10) to yield the target CH(CN)-DAPYs.
Scheme 1. Reagents and conditions: (
a
c
) methyl cyanoacetate, 60% NaH, DMSO, 90 ◦C; then H2O,
) 4-cyanoaniline, 180–190 ◦C, 8 h; (
) POCl3, reflux, 30 min;
reflux; ( ) MeI, NaOH, H2O, r.t., 24 h; (
b
d
(e) substituted-4-cyanophenylacetonitrile, 60% NaH, N2, anhydrous DMF, −15 ◦C to r.t., 48–72 h.

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2.2. Antiviral Activity of Compounds A1–A12 and Binding Conformation Analysis
First, we synthesized the CH(CN)-DAPY analogues (A1–A12) with methyl, methoxy,
trifluoromethyl, and halogen groups on the 4-cyanophenyl group. Their anti-WT HIV-1 activity
were evaluated and nevirapine (NVP), etravirine (ETR), efavirenz (EFV), and rilpivirine (RPV), four
drugs currently used in clinical treatment of HIV-1 infection, were selected as reference compounds.
As illustrated in Table 1, the target compounds A1
against WT HIV-1 (LAI strain IIIB) with EC50 values ranging from 0.059 to 11.74
exhibited low cytotoxicity with CC50 values ranging from 10.9 to 78.4 µM. Compound A1 with 4-CN
exhibited anti-HIV-1 activity with an EC50 value of 3.27 M. Compound A2 with 3-Me-4-CN exhibited
anti-HIV-1 activity with an EC50 value of 1.17 M. Compared with A2, the antiviral activity was
–
A12 exhibited significantly different antiviral activity
µM. Compound A2–A5
µ
µ
17-fold increased when the methyl substitution was moved to the 2-position (A3) of the 4-cyanophenyl
moiety. Compound A3 with 2-Me-4-CN displayed potency against WT HIV-1 with an EC50 value of
0.069
the activity decrease to an EC50 of 11.74
higher activity than the 3-substituted analogue (A4). Compound A5 with 2-OMe-4-CN displayed
potency against WT HIV-1 with an EC50 value of 0.059 M. Furthermore, compound A5 exhibited low
cytotoxicity with a CC50 of 25.8 M, which was about 5-fold higher than that of RPV (CC50 = 5.9 µM).
µM and low cytotoxicity with a CC50 of 10.9
µM. Replacing the methyl by methoxy (A4) made
µM. Similarly, the 2-methoxy analogue (A5) had a 200-fold
µ
µ
These results indicated that the 2-substituents of the 4-cyanophenyl moiety were more favorable to
the improvement in antiviral activity. Thus, we further introduced a trifluoromethyl (A6), fluoro
(
A7), chloro (A8), or bromo (A9) group at the 2-position of the 4-cyanophenyl group. Compound A6
with 2-CF3-4-CN and compound A7 with 2-F-4-CN were less potent with EC50 values of 0.49 and
0.24 M, respectively. Compound A8 with 2-Cl-4-CN and compound A9 with 2-Br-4-CN exhibited
potent anti-WT HIV-1 activity with EC50 values of 0.063 and 0.079 M, respectively. The activity of
compound A8 was comparable to that of compound A5 with the highest potency against WT HIV-1
among compounds A1 A9, which were 2~3-fold higher than that of NVP (EC50 = 0.20 M). Next, we
introduced di-substituents on the 4-cyanophenyl to obtain compounds A10 A12 with EC50 values
ranging from 0.082 to 0.30 M. Compared to the corresponding mono-substituted compounds on the
4-cyanophenyl ring (A8), 2,6-disubstituted compounds (A10) were less active. Compound A10 with
2,6-diCl-4-CN exhibited anti-HIV-1 activity with an EC50 value of 0.082 M. Compounds A11 A12 with
µ
µ
–
µ
–
µ
µ
–
di-substituents on the 4-cyanophenyl ring exhibited low activity. Compound A12 with 2-Me-4-CN-5-Br
exhibited low cytotoxicity with a CC50 value of 13.2 µM.
Molecular docking was carried out to predict the interaction of the best compound A5 with HIV-1
RT. As shown in Figure 3A,B, both S-A5 and R-A5 employed very similar “U”-like conformation
with RT. They formed two key hydrogen bonds between K101 and the pyrimidine N1 (N . . . HN
distance = 2.3 Å) and the p-cyanoaniline hydrogen (NH . . . O=C distance = 1.8 Å). The ring B of
A5 efficiently occupied the hydrophobic pockets. The small hydrophobic pockets in the regions of
Y181/Y188 and V179 were occupied by the cyano group and adjacent to the nonpolar group V179 (CN
. . . C distance = 3.3 Å), potentially enhancing the hydrophobic interactions (Figure 3C). Considering
their similar binding modes with RT, the racemic forms of the target compounds were selected for
biological evaluations.

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Table 1. Activity and cytotoxicity against HIV-1 (IIIB) strains in MT-4 cells of compounds A1–A12.
EC50 (µM) 2
HIV-1 IIIB
CC50 (µM) 3
SI 4
Compounds
R
1
A1
A2
A3
A4
A5
A6
A7
A8
A9
H
3-Me
2-Me
3-OMe
2-OMe
2-CF3
2-F
2-Cl
2-Br
3.27 ± 2.25
1.17 ± 0.34
32.7 ± 9.9
16.1 ± 3.6
10.9 ± 3.6
78.4 ± 10.4
25.8 ± 8.7
6.2 ± 1.4
10.3 ± 4.5
4.6 ± 2.7
3.4 ± 0.3
4.5 ± 1.3
6.1 ± 0.5
13.2 ± 1.6
>15.0
10
14
157
7
434
13
43
72
42
55
20
0.069 ± 0.008
11.74 ± 12.13
0.059 ± 0.011
0.49 ± 0.30
0.24 ± 0.16
0.063 ± 0.009
0.079 ± 0.02
0.082 ± 0.02
0.30 ± 0.03
A10
A11
A12
2,6-diCl
2-Me-3-Cl
2-Me-5-Br
0.24 ± 0.04
54
Nevirapine
Etravirine
Efavirenz
Rilpivirine
0.20 ± 0.10
>76
>1012
>3939
3747
0.005 ± 0.001
0.003 ± 0.001
0.0016 ± 0.0003
>4.6
>6.3
5.9 ± 0.3
1
2
IIIB means the wild type (WT) of HIV-1 virus strain. EC50: The effective concentration required to protect MT-4
3
cells against HIV-induced cytopathogenicity by 50%. CC50: The cytotoxic concentration of the compound that
reduced the normal uninfected MT-4 cell viability by 50%. SI: selectivity index, ratio CC50/EC50 (WT).
4
Figure 3. Predicted binding modes of compound A5 with reverse transcriptase (RT) (PDB: 2ZD1).
(
A
) RT with S-A5 (green); (
green or yellow. Residues involved in interactions are shown as gray sticks. Hydrogen bonds are
depicted as yellow dashed lines. ( ) The cyano linker of S-A5 (green) and R-A5 (yellow) docking with
the small hydrophobic pockets formed by Y181/Y188 and V179.
B) RT with R-A5 (yellow); The carbons of the compounds are depicted in
C

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2.3. Antiviral Activity of Compounds B1–B6 and Binding Conformation Analysis
Next, we introduced a methyl group to the C5-position in the central pyrimidine ring at the
entrance channel in order to improve the activity. The obtained new compounds B1 B6 were evaluated
for their anti-HIV activity (Table 2). All of them displayed low nanomolar EC50 values against the
WT HIV-1 strain and different cytotoxicity with CC50 values ranging from 6.6 to 108.6 M. Most
compounds displayed similar cytotoxicity with the reference EFV (CC50 = 6.3 M). Compared with the
non-methyl substituted analogues A1
A12, the methyl group at the C5-position (R1) of the pyrimidine
core significantly increased the anti-HIV-1 activity by 6~30-fold. The activity of compound B2 with
2-F-4-CN and compound B3 with 2-Cl-4-CN had EC50 values of 0.04 and 0.01 M, respectively.
Compound B5 with 2-Me-3-Cl showed an EC50 of 0.02 M. To our delight, B4 and B6 exhibited
single-digit nanomolar antiviral potency. The activity of B6 had an EC50 of 0.008 M, which was
comparable to the positive NNRTI drugs. Compound B4 with 2-Br-4-CN displayed the highest potency
against HIV-1 with an EC50 value of 0.006 M and a selectivity index (SI) value of 1086, which was
superior to the reference drug NVP (EC50 = 0.20 M, SI > 76) and similar to the references EFV and
–
µ
µ
–
µ
µ
µ
µ
µ
ETR (EC50 = 0.003 and 0.005 µM, SI > 3939 and >1012, respectively). These results suggested that a
methyl group at the C5-position (R1) of the pyrimidine core was favorable for antiviral potency.
Table 2. Activity and cytotoxicity against HIV-1 (IIIB) strains in MT-4 cells of compounds B1–B6.
EC50 (µM) 2
HIV-1 IIIB
CC50 (µM) 3
SI 4
Compounds
R
1
B1
B2
B3
B4
B5
H
2-F
2-Cl
0.30 ± 0.11
0.04 ± 0.02
108.6 ± 80.0
20.6 ± 3.6
9.6 ± 2.7
6.6 ± 1.3
8.6 ± 2.2
9.8 ± 3.5
>15.0
>4.6
>6.3
5.9 ± 0.3
356
586
920
1086
370
1215
>76
>1012
>3939
3747
0.01 ± 0.01
2-Br
0.006 ± 0.001
0.02 ± 0.008
0.008 ± 0.004
0.20 ± 0.10
2-Me-3-Cl
2-Me-5-Br
B6
Nevirapine
Etravirine
Efavirenz
Rilpivirine
0.005 ± 0.001
0.003 ± 0.001
0.0016 ± 0.0003
1
2
IIIB means the wild type (WT) of HIV-1 virus strain. EC50: The effective concentration required to protect MT-4
3
cells against HIV-induced cytopathogenicity by 50%. CC50: The cytotoxic concentration of the compound that
reduced the normal uninfected MT-4 cell viability by 50%. SI: selectivity index, ratio CC50/EC50 (WT).
4
Molecular docking predicted that the R-configuration of B4 had a similar binding conformation
with the S-configuration (Figure 4A,B). For the C-5 methyl group, nonpolar interactions between the
new methyl of compound B4 and hydrophobic side chains in the entrance channel cleft might be
established in addition to the typical interactions. The methyl group was predicted to be located
in the hydrophobic region of the protein (Figure 4C), conceivably contributing to the enhanced
inhibitory efficacy.

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Figure 4. Predicted binding modes of compound B4 with RT (PDB: 2ZD1). (
A) RT with S-B4 (green);
(
B
) RT with R-B4 (yellow); ( ) The space-filling model for the predicted binding mode of S-B4 (green)
C
and R-B4 (yellow). The carbons of the compounds are depicted in green or yellow. Residues involved
in interactions are shown as gray sticks. Hydrogen bonds are depicted as yellow dashed lines.
2.4. Antiviral Activity against Mutants of Compounds A1–A12 and B1–B6 and Enzymatic Assay of
Compounds B1–B6
Furthermore, we tested the antiviral activity against a panel of clinically observed drug-resistant
mutants (Table 3). Series A showed very low activity against the WT HIV-1. The compounds B1
with relatively higher anti-HIV-1 activity and SI values were more sensitive toward mutant strains.
Among them, compounds B3 B4 and B6 had better potency against the mutants than other analogues
in series A. Toward the K103N mutant, compound B6 had an EC50 of 0.06 M, which was more
potent than the reference standards NVP (EC50 =4.70 M) and EFV (EC50 = 0.08 M). The activities of
compounds B3 (EC50 = 0.08 M) and B4 (EC50 = 0.08 M) were comparable to those of EFV. Toward
the E138K mutant, compounds B3 (EC50 = 0.14 M) and B4 (EC50 = 0.11 M) were more potent
than NVP (EC50 = 0.20 M). They were inferior to the references EFV and ETR against the L100I
and Y181C mutant strains. Furthermore, compounds B3 B6 were superior to NVP (EC50 = 4.28 M)
toward the virus with double mutations F227L + V106A. Compound B4 showed an EC50 of 2.35 M.
Compound B6 exhibited the highest potency against the double mutant F227L + V106A among all
target compounds with EC50 values of 1.52 M. However, toward the virus with double mutations
K103N + Y181C, all target compounds exhibited low activity.
–B6
–
µ
µ
µ
µ
µ
µ
µ
µ
–
µ
µ
µ

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Table 3. Inhibitory activity of compounds A1–A12 and B1–B6 toward a panel of clinically relevant
HIV-1 mutant strains and inhibitory activity of compounds B1–B6 toward WT HIV-1 RT.
EC50(µM) 1
IC50 (µM) 2
Compounds
F227L +
V106A
K103N +
Y181C
L100I
K103N
Y181C
E138K
ND 3
ND
ND
ND
ND
ND
ND
ND
ND
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
B1
B2
B3
B4
B5
≥12.31
>16.07
>32.70
>16.07
11.26 ± 1.51
>16.07
>32.70
>16.07
>32.70
>16.07
>10.93
≥61.00
≥12.42
>6.23
>10.36
>4.59
>3.35
>32.70
>16.07
>10.93
≥65.72
>25.85
>6.23
>10.36
>4.59
>3.35
>4.49
>6.11
>13.23
>108.57
>20.63
>9.56
5.03 ± 0.55
1.96 ± 0.45
1.71 ± 0.13
3.04 ± 0.39
≥61.36
≥62.18
≥48.12
≥56.55
4.72 ± 0.79
>6.23
0.68 ± 0.16
>6.23
1.25 ± 0.58
2.29 ± 0.02
>10.36
1.63 ± 0.32
>6.23
>10.36
>10.36
>10.36
≥2.37
2.13 ± 0.22
1.87 ± 0.21
1.88 ± 0.42
2.20 ± 0.27
>6.11
2.20 ± 0.35
2.31 ± 0.02
2.19 ± 0.22
>6.11
>3.35
≥1.88
2.32 ± 0.48
2.32 ± 0.48
>13.23
1.78 ± 0.12
>6.11
>4.49
>6.11
ND
ND
ND
2.52 ± 0.42
3.95 ± 0.22
1.07 ± 0.37
0.08 ± 0.02
0.08 ± 0.03
0.39 ± 0.07
0.06 ± 0.01
4.70 ± 0.63
0.003 ± 0.001
0.08 ± 0.01
2.78 ± 0.36
7.05 ± 0.96
1.59 ± 0.18
0.26 ± 0.02
0.16 ± 0.17
1.02 ± 0.2
0.33 ± 0.10
6.07 ± 1.55
3.73 ± 0.59
4.92 ± 0.3
0.97 ± 0.14
0.14 ± 0.03
0.11 ± 0.04
0.60 ± 0.10
0.17 ± 0.06
0.20 ± 0.15
>13.23
≥58.97
>20.63
4.04 ± 0.70
2.35 ± 0.18
1.66 ± 0.27
1.52 ± 0.15
≥4.28
8.03 ± 4.43
3.04 ± 1.13
0.82 ± 0.15
0.51 ± 0.19
1.82 ± 0.4
0.86 ± 0.19
0.98 ± 0.39
1.46 ± 0.09
0.22 ± 0.05
0.05 ± 0.03
0.06 ± 0.03
0.07 ± 0.02
0.06 ± 0.02
0.45 ± 0.25
0.01 ± 0.009
ND
>6.62
>8.58
>9.79
B6
Nevirapine
Etravirine
Efavirenz
Rilpivirine
>15.02
0.04 ± 0.01
0.24 ± 0.06
0.008
±
0.002
0.017
0.006
±
±
±
0.003 0.008
0.001 0.005
0.001 0.003
±
±
±
0.004 0.014
0.002
0.001
±
0.005
0.28 ± 0.07
0.04 ± 0.008
0.002
±
0.001 0.001 ± 0.0002 0.003
0.04 ± 0.01
0.009
±
0.002
ND
1
EC520: The effective concentration required to protect MT-4 cells against HIV-induced cytopathogenicity by
50%. IC50: inhibitory concentration of the test compound required to inhibit biotin deoxyuridine triphosphate
3
(biotin-dUTP) incorporation into WT HIV-1 RT by 50%. ND: not determined.
Next, series B were selected to further test the binding affinity toward the RT enzyme. All the
novel DAPY derivatives displayed better inhibitory activity against WT HIV-1 RT than the reference
standards NVP (IC50 = 0.45
of 0.05 M, both compounds B4 and B6 had an IC50 of 0.06
(IC50 = 0.45 µM) by 7~9-fold.
µ
M) except compound B1. Moreover, compound B3 showed an IC50
µ
µM, the activity was higher than NVP
As shown in Figure 5A–C, B4 efficiently occupied the pockets of the single amino acid
mutations K103N and E138K and the double mutants F227L + V106A with an approximately “U”
conformation, which was one of the essential condition for the antiviral activity in the DAPY scaffold.
The hydrogen-bonding interactions between the NH linker and K101 was maintained. The N103
residue might be involved in additional hydrogen-bonding interactions with the linkage amide.
A water-mediated hydrogen bond between the cyano-methylene linkage and K138 was formed.
The F227L decreased the
compared to those of Etravirine and Rilpivirine. Docking of B4 with K103N + Y181C predicted that
several common features (“U” binding conformation, hydrogen bonds, and stacking interactions)
π-π stacking interaction with the p-CN-phenyl ring leading to lower activity
π-π
would be lost, which might contribute to the decrease in antiviral activity of compound B4 against the
HIV-1 double mutants K103N + Y181C.

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Figure 5.
compound B4 with E138K (PDB: 2ZD1). (
V106A. ( ) Predicted binding modes of compound B4 with K103N + Y181C. Mutated residues are
(
A
) Predicted binding modes of compound B4 with K103N. (
B) Predicted binding modes of
C) Predicted binding modes of compound B4 with F227L +
D
depicted as orange sticks. The carbons of the compounds are depicted in green. Residues involved in
interactions are shown as gray sticks. Hydrogen bonds are depicted as yellow dashed lines.
3. Materials and Methods
3.1. Synthesis
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, THF-d8, CDCl3, or Acetone-d6 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 a WRS-1B digital
melting point apparatus. Mass spectra and HRMS were obtained on a Waters Quattro Micromass
instrument and Brukersolari X-70 FT-MS instrument, respectively, using electrospray ionization (ESI)
techniques. The purities of the target compounds were
Waters 2695 system with UV detector and high-performance liquid chromatography (HPLC) (Agilent
1260) using a C18 column (Eslipse XDB, 4.6*150 mm, 5 m), eluting with a mixture of water (A) and
≥
95%, measured by LC-MS, performed on an
µ
methanol (B) (VA:VB = 50:50 to 10:90) over 30 min with a flow rate of 0.8 mL/min and detection at
254 nm.

Molecules 2020, 25, 1581
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3.1.1. General Procedure for the Preparation of Compounds 6a-l
Methyl cyanoacetate (20 mmol, 2 equiv.) was slowly added to a suspension of sodium hydride
(22.5 mmol, 60% dispersion in mineral oil, 2.25 equiv.) in DMSO (10 mL) at 0 ◦C. The mixture was
stirred for 1h at room temperature before 5a-l (10 mmol, 1 equiv.) was added as a solution in DMSO
◦
(10 mL) via cannula. The yellow solution was heated to 90 C for 2.5 h, H2O (40 mL) was added,
and the reaction was heated to reflux for 8 h. The mixture was cooled to r.t. and stirred for 10 h, the
resulting precipitate was filtered, washed with water, and dried to afford 6a-l.
3.1.2. General Procedure for the Preparation of Compounds A1–A12 and B1–B6
A solution of 10 (4 mmol, 1 equiv.), appropriate 4-cyanophenylacetonitrile (5.24 mmol, 1.3 equiv.),
and anhydrous DMF (10 mL) was stirred for 0.5 h at
dispersion in mineral oil, 2 equiv.) was added portion-wise at
at
15 ◦C for another 2 h, warmed to room temperature, and then reacted for 48–72 h (monitored by
−
15 ◦C under N2. Then, NaH (13.71 mmol; 60%
−
15 ◦C. Then, the mixture was stirred
−
TLC). The resulting mixture was poured into a saturated ammonium chloride solution. The precipitate
was collected by filtration and the residue was then purified via column chromatography on silica gel,
eluting with EtOAc/petroleum ether (1:3) to obtain compounds A1–A12 and B1–B6 as white or yellow
solids. Full characterization data for all final compounds can be found in the Supporting Information.
4-(4-(cyano-(4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A1) Yield = 87%. mp:
243.2–244.7 ◦C. 1H NMR (400 MHz, THF-d8)
δ
9.39 (s, NH), 8.40 (d, J = 5.0 Hz, 1H), 7.80 (d, J = 8.7 Hz,
2H), 7.68 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 5.0 Hz, 1H), 5.53
(s, 1H). 13C NMR (101 MHz, THF-d8)
163.6, 159.6, 159.3, 143.8, 139.0, 132.4 (2C), 132.2 (2C), 128.6 (2C),
δ
120.3, 118.2 (2C), 117.1, 116.8, 112.5, 110.4, 104.3, 43.5. HRMS (ESI): m/z calcd for C20H12N6 [M
335.1051, found 335.1053.
−
H]+
4-(4-(cyano-(3-methyl-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A2) Yield = 49%.
◦
1
mp: 223.7–224.9 C. H NMR (400 MHz, THF-d8)
J = 7.1 Hz, 2H), 7.60 (d, J = 7.9 Hz, 1H), 7.44–7.49 (m, 3H), 7.37 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 3.1 Hz,
1H), 5.46 (s, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, THF-d8)
163.7, 159.6, 159.2, 143.9, 142.4, 138.8, 132.7,
δ 9.38 (s, NH), 8.39 (d, J = 4.8 Hz, 1H), 7.82 (d,
δ
132.2 (2C), 129.4, 125.8, 118.9, 118.2 (2C), 116.8, 116.3, 112.9, 110.4, 104.3, 43.5, 19.0. HRMS (ESI): m/z
calcd for C21H14N6 [M + Na]+ 373.1172, found 373.1173. HPLC analysis: retention time = 11.71 min;
peak area, 97.24%.
4-(4-(cyano-(2-methyl-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A3) Yield = 47%.
mp: 213.5–214.6 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.43 (s, NH), 8.64 (d, J = 5.0 Hz, 1H), 7.99 (d,
J = 8.3 Hz, 2H), 7.80 (s, 3H), 7.68 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 4.5 Hz, 1H), 5.99 (s, 1H), 2.52 (s, 3H). 13
C
NMR (101 MHz, Acetone-d6) δ 163.5, 160.1, 159.8, 144.3, 138.6, 137.9, 134.5, 132.8 (2C), 130.5, 130.3, 118.9
(2C), 118.8, 117.9, 117.5, 112.7, 111.5, 104.4, 41.6, 18.6. HRMS (ESI): m/z calcd for C21H14N6 [M + Na]+
373.1172, found 373.1170.
4-(4-(cyano-(3-methoxy-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A4) Yield
= 63%. mp:156.2–157.0 ◦C.1H NMR (400 MHz, Acetone-d6)
δ
9.44 (s, 1H, NH), 8.60 (d, J = 5.0 Hz, 1H),
8.08 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.47 (s, 1H), 7.33 (d, J = 8.3 Hz,
1H), 7.12 (d, J = 5.0 Hz, 1H), 5.81 (s, 1H), 4.00 (s, 3H). 13C NMR (101 MHz, Acetone-d6)
163.9, 161.6,
δ
160.1, 159.8, 144.4, 141.3, 134.5, 132.9 (2C), 120.8, 118.9 (2C), 118.8, 117.5, 115.3, 112.1, 111.5, 104.4, 101.9,
56.1, 44.1. HRMS (ESI): m/z calcd for C21H14N6O [M + Na]+ 389.1121, found 389.1121.
4-(4-(cyano-(2-methoxy-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A5) Yield
= 54%. mp: 189.4–190.1 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.38 (s, NH), 8.60 (d, J = 5.0 Hz, 1H),
8.02 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.56 (m, 2H), 7.08 (d, J = 5.0
Hz, 1H), 5.93 (s, 1H), 4.00 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
163.8, 160.2, 159.6, 157.1, 144.9,
δ
133.3 (2C), 131.4, 127.9, 125.6, 119.6, 118.9 (2C), 118.8, 118.3, 115.9, 113.5, 111.8, 103.3, 57.1, 40.6. HRMS
(ESI): m/z calcd for C21H14N6O [M + Na]+ 389.1121, found 389.1123. HPLC analysis: retention time =
8.96 min; peak area, 96.35%.

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4-(4-(cyano-(2-trifluoromethyl-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A6)
Yield = 37%. mp: 236.7–237.5 ◦C. 1H NMR (400 MHz, THF-d8)
9.43 (s, NH), 8.43 (d, J = 4.8 Hz, 1H),
8.21 (s, 1H), 8.01 (m, 1H), 7.87 (m, 1H), 7.72 (d, J = 4.5 Hz, 2H), 7.47 (m, 2H), 6.85 (d, J = 4.8 Hz, 1H), 5.82
(s, 1H). 13C NMR (101 MHz, THF-d8)
163.2, 159.9, 159.8, 144.1, 137.2, 136.6, 132.7, 132.6 (2C), 130.7
δ
δ
(t, JC-F = 6 Hz), 129.3, 123.2 (t, JC-F = 273 Hz), 118.6 (2C), 118.5, 116.7, 116.4, 113.9, 111.3, 104.8, 40.4.
HRMS (ESI): m/z calcd for C21H11F3N6 [M + Na]+ 427.0889, found 427.0889. HPLC analysis: retention
time = 9.90 min; peak area, 97.54%.
4-(4-(cyano-(2-fluoro-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A7) Yield = 47%.
mp:165.7–166.5 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.43 (s, NH), 8.66 (d, J = 5.0 Hz, 1H), 7.96 (m,
J = 17.0, 8.0 Hz, 3H), 7.85 (m, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 5.0 Hz, 1H), 6.07 (s, 1H). 13
NMR (101 MHz, Chloroform-d) 162.7, 160.3, 159.8, 159.7 (d, JC-F = 251.7 Hz), 144.2, 132.8 (2C), 131.9
C
δ
(d, JC-F = 3.2 Hz), 129.4 (d, JC-F = 3.9 Hz), 127.3 (d, JC-F = 14.1 Hz), 120.0 (d, JC-F = 25 Hz), 118.9, 118.8
(2C), 116.8, 116.6, 114.5 (d, JC-F = 9.9 Hz), 111.3, 104.4, 38.4 (d, JC-F = 2.4 Hz). HRMS (ESI): m/z calcd for
C20H11FN6 [M − H]+ 353.0956, found 353.0941.
4-(4-(cyano-(2-chloro-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A8) Yield = 53%.
mp: 219.8–221.6 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.40 (s, NH), 8.64 (d, J = 4.8 Hz, 1H), 8.08 (s, 1H),
7.98 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 7.7 Hz, 2H), 7.16 (d, J = 4.8 Hz, 1H), 6.14 (s,
1H). 13C NMR (101 MHz, Acetone-d6)
162.6, 160.2, 159.8, 144.2, 137.2, 134.4, 133.6, 132.8 (2C), 132.0,
δ
131.7, 119.6, 118.9, 118.8 (2C), 116.7, 114.4, 111.9, 104.4, 41.9. HRMS (ESI): m/z calcd for C20H11ClN6
[M + Na]+ 393.0626, found 393.0628. HPLC analysis: retention time = 9.94 min; peak area, 98.79%.
4-(4-(cyano-(2-bromo-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A9) Yield = 59%.
mp: 223.5–224.7 ◦C. 1H NMR (400 MHz, THF-d8)
δ
9.43 (s, NH), 8.42 (m, 1H), 8.08 (s, 1H), 7.75–7.78
(m, 4H), 7.46 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 4.9 Hz, 1H), 5.90 (s, 1H). 13C NMR (101 MHz, THF-d8)
δ
162.4, 159.6, 159.3, 143.8, 138.5, 136.4, 132.2 (2C), 131.5, 131.1, 123.7, 118.1 (2C), 116.1, 115.8, 114.3, 111.0,
110.8, 104.3, 43.6. HRMS (ESI): m/z calcd for C20H11BrN6 [M + Na]+ 413.0156, found 423.0159. HPLC
analysis: retention time = 10.12 min; peak area, 95.35%.
4-(4-(cyano-(2,6-dichloro-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile (A10) Yield
= 73%. mp: 152.5–153.2 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ
8.55 (d, J = 5.0 Hz, 1H), 7.76 (s, 1H),
7.62 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.39 (s, 1H), 7.06 (d, J = 4.9 Hz, 1H), 6.18 (s, 1H). 13
C
NMR (101 MHz, Chloroform-d) δ 161.5, 159.7, 159.1, 142.7, 137.3 (2C), 134.9, 133.2 (2C), 132.1 (2C), 119.0,
118.5 (2C), 115.5, 115.3, 114.2, 111.0, 105.5, 40.1. HRMS (ESI): m/z calcd for C20H10Cl2N6 [M + Na]+
427.0236, found 427.0232. HPLC analysis: retention time = 10.29 min; peak area, 98.48%.
4-(4-(cyano-(2-methyl-3-chloro-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile
◦
(A11) Yield = 81%. mp:196.5–197.7 C.1H NMR (400 MHz, Acetone-d6)
δ
9.48 (s, NH), 8.65 (d,
J = 4.9 Hz, 1H), 7.97 (d, J = 8.4 Hz, 3H), 7.81 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.12 (d,
J = 4.8 Hz, 1H), 6.13 (s, 1H), 2.55 (s, 3H). 13C NMR (101 MHz, Acetone-d6)
163.2, 160.2, 159.8, 144.2,
δ
139.6, 137.1, 132.9, 132.2 (2C), 128.9, 118.9, 118.8 (2C), 118.7, 117.3, 115.6, 114.0, 111.6, 104.4, 42.4, 16.4.
HRMS (ESI): m/z calcd for C21H13ClN6 [M + Na]+ 407.0782, found 407.0784. HPLC analysis: retention
time = 11.24 min; peak area, 95.39%.
4-(4-(cyano-(2-methyl-5-bromo-4-cyanophenyl)methyl)-pyrimidin-2-ylamino)benzonitrile
◦
(A12) Yield = 65%. mp: 213.9–214.5 C.1H NMR (400 MHz, Acetone-d6)
δ
9.45 (s, NH), 8.65 (d,
J = 5.0 Hz, 1H), 8.03 (s, 1H), 7.97 (d, J = 8.5 Hz, 2H), 7.88 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.16 (d,
J = 5.0 Hz, 1H), 6.01 (s, 1H), 2.48 (s, 3H). 13C NMR (101 MHz, THF-d8)
162.7, 159.6, 159.4, 143.7, 139.0,
δ
137.2, 136.3, 133.2, 132.2 (2C), 121.8, 118.2 (2C), 118.1, 116.4, 115.7, 115.6, 110.5, 104.4, 41.0, 17.6. HRMS
(ESI): m/z calcd for C21H13BrN6 [M-H]+ 427.0312, found 427.0302. HPLC analysis: retention time =
11.30 min; peak area, 98.15%.
4-(4-(cyano-(4-cyanophenyl)methyl)-5-methylpyrimidin-2-ylamino)benzonitrile (B1) Yield =
71%. mp: 243.1–243.7 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.35 (s, NH), 8.46 (s, 1H), 8.06 (d, J = 8.5 Hz,
2H), 7.92 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.7 Hz, 2H), 6.04 (s, 1H), 2.31 (s, 3H).
13C NMR (101 MHz, Acetone-d6)
161.2, 160.7, 158.4, 144.7, 139.0, 133.1 (2C), 132.9 (2C), 129.5 (2C),
δ

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120.4, 119.0, 118.4 (2C), 118.3, 117.5, 112.6, 103.9, 41.5, 13.5. HRMS (ESI): m/z calcd for C21H14N6 [M +
Na]+ 373.1172, found 373.1165.
4-(4-(cyano-(2-fluoro-4-cyanophenyl)methyl)-5 methylpyrimidin-2-ylamino)benzonitrile (B2)
Yield = 47%. mp:211.9–212.9 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.32 (s, NH), 8.51 (s, 1H), 7.83–7.93
(m, 5H), 7.64 (d, J = 8.5 Hz, 2H), 6.20 (s, 1H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
161.3,
159.9(d, JC-F = 250.0 Hz), 159.9, 145.0, 133.3 (2C), 132.0 (d, JC-F = 4.0 Hz), 130.1 (d, JC-F = 3.0 Hz), 127.0 (d,
δ
J
C-F = 14.4 Hz), 120.7, 120.6, 120.5, 119.9, 118.5 (2C), 117,7 (d, JC-F = 3.0 Hz), 117.3, 113.9 (d, JC-F = 11 Hz),
103.0, 36.5 (d, JC-F = 1.0 Hz), 14.2. HRMS (ESI): m/z calcd for C21H13FN6 [M + Na]+ 391.1078, found
391.1070. HPLC analysis: retention time = 9.78 min; peak area, 95.05%.
4-(4-(cyano-(2-chloro-4-cyanophenyl)methyl)-5-methylpyrimidin-2-ylamino)benzonitrile (B3)
Yield = 62%. mp: 213.6–214.2 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
10.26 (s, NH), 8.53 (s, 1H), 8.26 (s,
1H), 8.07 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H),
6.37 (s, 1H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
161.3, 160.2, 158.2, 145.0, 137.3, 134.1, 134.0,
δ
133.2 (2C), 132.5, 131.8, 121.0, 119.9, 118.5 (2C), 117.5, 117.2, 113.8, 102.9, 40.8, 14.3. HRMS (ESI): m/z
calcd for C21H13ClN6 [M + Na]+ 407.0782, found 407.0778. HPLC analysis: retention time = 10.33 min;
peak area, 96.14%.
4-(4-(cyano-(2-bromo-4-cyanophenyl)methyl)-5-methylpyrimidin-2-ylamino)benzonitrile
(B4) Yield = 72%. mp: 238.9–240.1 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.27 (s, NH), 8.49 (s, 1H), 8.22
(d, J = 1.3 Hz, 1H), 8.05–8.08 (m, J = 8.1, 1.4 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.5 Hz, 2H),
7.57 (d, J = 8.7 Hz, 2H), 6.14 (s, 1H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
161.2, 160.4, 158.2,
δ
145.0, 139.1, 137.1, 133.2 (2C), 132.9, 131.7, 124.4, 121.1, 119.9, 118.5 (2C), 117.4, 117.3, 113.9, 102.9, 42.4,
14.4. HRMS (ESI): m/z calcd for C21H13BrN6 [M + Na]+ 451.0277, found 451.0276. HPLC analysis:
retention time = 10.75 min; peak area, 96.09%.
4-(4-(cyano-(2-methyl-3-chloro-4-cyanophenyl)methyl)-5-methylpyrimidin-2-ylamino)benzonitrile
(B5) Yield = 65%. mp: 205.4–206.5 ◦C. 1H NMR (400 MHz, Acetone-d6)
δ
9.32 (s, NH), 8.50 (s, 1H), 7.96
(d, J = 8.1 Hz, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 6.20 (s,
1H), 2.51 (s, 3H), 2.31 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
161.3, 160.4, 158.3, 145.1, 139.6, 137.1,
δ
136.9, 133.2 (2C), 132.9, 128.8, 120.8, 119.9, 118.5 (2C), 117.7, 116.5, 113.4, 102.9, 40.5, 16.9, 14.3. HRMS
(ESI): m/z calcd for C22H15ClN6 [M + Na]+ 421.0939, found 421.0937. HPLC analysis: retention time =
11.15 min; peak area, 95.15%.
4-(4-(cyano-(2-methyl-5-bromo-4-cyanophenyl)methyl)-5-methylpyrimidin-2-ylamino)benzonitrile
(B6) Yield = 76%. mp: 232.3–233.9 ◦C. 1H NMR (400 MHz, THF-d8)
δ
9.43 (s, NH), 8.42 (s, 1H), 7.79–7.85
(m, J = 11 Hz, 4H), 7.57 (d, J = 8.7 Hz, 2H), 5.94 (s, 1H), 2.35 (s, 3H), 2.25 (s, 3H). 13C NMR (101 MHz,
Acetone-d6) δ 160.9, 160.0, 158.4, 144.5, 139.3, 137.8, 136.7, 133.1, 132.8 (2C), 122.0, 120.8, 119.0, 118.3
(2C), 118.2, 116.5, 116.4, 115.6, 103.9, 39.1, 18.0, 13.5. HRMS (ESI): m/z calcd for C22H15BrN6 [M + Na]+
465.0434, found 465.0431.
3.2. In Vitro Anti-HIV Assay
Evaluation of the antiviral activity of the compounds against HIV in MT-4 cells was performed
using the MTT assay as previously described [24
of Professor Jan Balzarini from Rega Institute for Medical Research, KU Leuven. All used strains
were analyzed and reported earlier [26 28]. Prior to use in our assays, every freshly grown virus
,25]. All used NNRTI-resistant strains were a gift
–
stock was the subject of a genotypic analysis. In addition to the genotypic analysis, every stock
was subject to a phenotypic analysis using a panel of relevant reference compounds in MT-4 cells.
In the case of NNRTI-resistant strains the reference compounds used were NVP, EFV, and ETR. Stock
solutions (10 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 HIV- and mock-infected cell samples were included as controls. HIV stock (50 µL) at

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100–300 CCID50 (50% cell culture infectious doses) and culture medium (final 200
µL volume per well)
were added to either the infected or mock-infected wells of the microtiter tray. Mock-infected cells were
used to evaluate the effects 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 220 g and the
×
supernatant was discarded. The MT-4 cells were resuspended at 6 × 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 using 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, Geel, Belgium) 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, Männedorf, Switzerland) at
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).
3.3. Reverse Transcriptase Assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as described [29]. The RT
assay was performed with the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen),
as described by the Manufacturer. The assay is based on the dsDNA quantitation reagent PicoGreen.
This reagent shows a pronounced increase in fluorescence signal upon binding to dsDNA or RNA–DNA
heteroduplexes. Single-stranded nucleic acids generate only minor fluorescence signal enhancement
when a sufficiently high dye/base pair ratio is applied [30]. This condition is met in the assay. A poly(rA)
template of approximately 350 bases long and an oligo (dT)16 primer are annealed in a molar ratio of
1:1.2 (60 min at room temperature). Then, 52 ng of the RNA/DNA is brought into each well of a 96-well
plate in a volume of 20
µL polymerization buffer (60 mM Tris-HCl, 60 mM KCl, 8 mM MgCl2, 13 mM
DTT, 100 M dTTP, pH 8.1). Then, 5
µ
µL of RT enzyme solution, diluted to a suitable concentration in
enzyme dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT, pH 7.6), is added. The reactions are
incubated at 25 ◦C for 40 min and then stopped by the addition of EDTA (15 mM fc). Heteroduplexes
are then detected by addition of PicoGreen. Signals are read using an excitation wavelength of 490 nm
and emission detection at 523 nm using a spectrofluorometer (Safire 2, Tecan). To test the activity of
compounds against RT, 1 µL of compound in DMSO was added to each well before the addition of RT
enzyme solution. Control wells without compound contained the same amount of DMSO. Results
are expressed as relative fluorescence, i.e., the fluorescence signal of the reaction mix with compound
divided by the signal of the same reaction mix without compound.
3.4. 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 10,000 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. 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

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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 the 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 WBls, 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 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.
4. Conclusions
In summary, we have designed and synthesized a new series of substituted CH(CN)-DAPYs
by using a scaffold hopping strategy. All the target compounds displayed anti-HIV-1 activity with
micromolar to nanomolar EC50 values in HIV-1 infected MT-4 cells. Among the synthesized analogs,
compounds B4 (EC50 = 8 nM) and B6 (EC50 = 6 nM) were nearly as potent as EFV and ETR against
WT HIV-1. (1) The 2-position of 4-cyanophenyl was the most preferred position to improve antiviral
activity. (2) A methyl group at the C5-position (R1) of the pyrimidine core could increase the anti-HIV-1
activity (Figure 6). This information might be helpful to further DAPY optimizations.
Figure 6. A systematic summary of the structure–activity relationship (SAR).
Supplementary Materials: The following are available online, containing HRMS, 1H-NMR, and 13C-NMR
spectrum of final compounds.
Author Contributions: T.-T.L. designed and carried out the experiments and wrote the paper. C.P. and E.D.C.
carried out the bio-experiments. C.-L.Z. guided and modified the paper. F.-E.C. provided study design and
guidance. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Natural Science Foundation of P. R. China under Grant
No. 21372050, Shanghai Municipal Natural Science Foundation Grant No. 13ZR1402200, and the Young Elite
Scientists Sponsorship Program by the China Association for Science and Technology (2017QNRC061).
Acknowledgments: We gratefully acknowledge the technical assistance of Kristien Erven and Cindy Heens and
thank Kris Uyttersprot for the HIV experiments and HIV RT polymerase assays.
Conflicts of Interest: The authors declare no conflict of interest.

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