Privileged scaffold inspired design of novel oxime-biphenyl-DAPYs in treatment of HIV-1

Article abstract of DOI:10.1016/j.bioorg.2020.103825

Oxime is a key pharmacophore in drug development. The biphenyl diarylpyrimidines (DAPYs) have been developed by our group as novel non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, fourteen oxime-biphenyl-DAPYs were designed and synthesized through a privileged scaffold inspired design strategy. They exhibited promising activity toward wild type HIV-1 and single mutant strains. Compound 7d was found to be the most potent one against both wild type (EC₅₀ = 12.1 nM) and E138K mutant strains (EC₅₀ = 0.0270 μM). It also had a much lower cytotoxicity (CC₅₀ > 292 μM) and higher selective index (SI > 24105) than those of the FDA-approved drugs efavirenz and etravirine. Molecular docking and dynamics simulation predicted and disclosed the binding mode of compound 7d with the RT, providing the explanation on the antiviral activity. These results were helpful for subsequent structural optimizations in anti-HIV-1 drug discovery.

Full text of DOI:10.1016/j.bioorg.2020.103825

Journal Pre-proofs
Privileged scaffold inspired design of novel oxime-biphenyl-DAPYs in treat-
ment of HIV-1
Yang Yang, Christophe Pannecouque, Erik De Clercq, Chunlin Zhuang, Fen-
Er Chen
PII:
S0045-2068(20)30140-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103825
YBIOO 103825
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Bioorganic Chemistry
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11 March 2020
5 April 2020
Please cite this article as: Y. Yang, C. Pannecouque, E. De Clercq, C. Zhuang, F-E. Chen, Privileged scaffold
inspired design of novel oxime-biphenyl-DAPYs in treatment of HIV-1, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.103825
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Privileged scaffold inspired design of novel oxime-biphenyl-
DAPYs in treatment of HIV-1
Yang Yang, a Christophe Pannecouque,e Erik De Clercq,e Chunlin Zhuang *b,c and Fen-Er Chen *a,b,c,d
a.
Sichuan Research Center for Drug Precision Industrial Technology, West China School of
Pharmacy, Sichuan University, Chengdu 610041, People's Republic of China
b.
Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry,
Fudan University, Shanghai 200433, People's Republic of China
c.
Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai
200433, People's Republic of China
d.
Institute of Pharmaceutical Science and Technology, Zhejiang University of Technology
Hangzhou 310014, People's Republic of China
e.
Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
* Corresponding authors. E-mail addresses: rfchen@fudan.edu.cn (F. Chen), zclnathan@163.com
(C. Zhuang)

Abstract
Oxime is a key pharmacophore in drug development. The biphenyl diarylpyrimidines (DAPYs) have been
developed by our group as novel non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study,
fourteen oxime-biphenyl-DAPYs were designed and synthesized through a privileged scaffold inspired
design strategy. They exhibited promising activity toward wild type HIV-1 and single mutant strains.
Compound 7d was found to be the most potent one against both wild type (EC50 = 12.1 nM) and E138K
mutant strains (EC50 = 0.0270 µM). It also had a much lower cytotoxicity (CC50 > 292 µM) and higher
selective index (SI > 24105) than those of the FDA-approved drugs efavirenz and etravirine. Molecular
docking and dynamics simulation predicted and disclosed the binding mode of compound 7d with the RT,
providing the explanation on the antiviral activity. These results were helpful for subsequent structural
optimizations in anti-HIV-1 drug discovery.
Keywords: HIV-1; Oxime; Reverse transcriptase; NNRTIs; DAPY; Privileged scaffold; Molecular
modelling.

Introduction
Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus type 1 (HIV-
1), is still a serious public health problem throughout the world [1]. Since first description of the highly
active antiretroviral therapy (HAART) by Dr. Dayi He, AIDS has been effectively controlled. HAART, a
regimen combining three or more kinds of antiretroviral drugs, could increase antiviral potency and
reduce the development of resistance [2]. The non-nucleoside reverse transcriptase inhibitor (NNRTI) is a
critical component of the HAART [3]. NNRTIs are attracting more and more focuses in the medicinal and
organic chemistry communities due to its high specificity and low toxicity [4]. However, two critical
issues of NNRTIs are requiring to be resolved: 1) The low solubility and poor bioavailability [3] are the
main drawbacks limiting the prescription in clinic. 2) The rapid development of extensive cross-resistant
strains of HIV-1 reduces the clinical efficacy of the NNRTIs.
In the last two decades, diarylpyrimidines (DAPYs) have been recognized as a series of lead
compounds for structural optimization with the disclosure of etravirine (ETR) and rilpivirine (RPV)
bearing a DAPY scaffold. Among them, biphenyl-DAPYs, developed by our group, were a novel series
of analogues with highly potent antiviral activity against wild-type (WT) HIV-1 and a panel of clinically
observed mutant strains [5-8]. Besides, the biphenyl moiety was predicted to form strong π-π stacking
interactions with W229 conserved hydrophobic loop and might contribute to stabilize the conformations
in the reverse transcriptase (RT) allosteric pocket [5, 7]. However, these compounds were highly
hydrophobic, making the aqueous solubility still unsatisfactory [9].
Oxime, a key pharmacophore in a number of marketed drugs (Fig. 1), such as cholinesterase
inhibitors, pyraloxime methiodine (2-PAM) [10], and fifth-generation cephalosporin antibiotic,
ceftobiprole [11]. In addition, oxime bearing chemicals were reported to perform antiviral activities with
respect to influenza virus A [12], and HIV-1 [13]. In the view of chemical structure, the oxime
characterizes with both electron withdrawing group (C=N) and donating group (-O-), easily binding with

various protein residues and, to some extent, improving physiochemical properties [14]. These
interactions might be attributed to improve the biological activity as well [15].
NH2
S
N
N
H
O
N
N
S
N
OH
HO
N
OH
NH
O
N
N
O
I
HO
O
N
O
OH
Oxime of oleanonic acid
Pyraloxime Methiodide
Ceftobiprole
Fig. 1 The structures of oxime-containing marketed drugs and lead compounds
Considering the strengths of the biphenyl and oxime pharmacophores, we decided to combine them
into one molecule through a fusing strategy (Fig. 2). On the basis of the binding mode of the biphenyl-
DAPY 1, a water-mediated hydrogen-bonding interaction was observed at the E138 region, which should
be replaced by an oxime moiety to form a similar binding interaction. The newly obtained oxime-
biphenyl-DAPYs (7a-n) were synthesized and their structure-activity relationships (SARs) were
discussed.
CN
R1
CN
CN
Privileged scaffold
inspired design
R2
HO
N
NH
HN
N
NH
N
N
N
1
H-bond with
E138(K)
OH
Fig. 2 The optimization strategy of biphenyl-DABPs

Results and discussion
Chemistry
The synthetic route of the target compounds is depicted in Scheme 1. Compound 4a-n were synthesized
following our established procedure (Scheme 1) [5-7]. Then, the key intermediates 6a-n were synthesized
from 5 and 4a-n using N-heterocyclic carbene catalyzing and NaH in DMSO at room temperature under
N2 atmosphere [16, 17]. Oximation of 6a-n with hydroxylamine sulfate in the presence of NaOH in
1
EtOH/H2O provided final target compounds 7a-n [18, 19]. The final structures were characterized by H
NMR and 13C NMR spectroscopy and HRMS.
R1
R1
CN
NH
a
b
R1
O
H
F
Cl
N
N
Br
B
N
HO
OH
F
O
F
N
CN
O
N
3
5
6a-n
4a-n
2a-n
R1
R1=H
R1=2-F
R1=3-F
R1=4-F
R1=2-CH3
R1=3-CH3
R1=4-CH3
R1=2-OCH3
R1=3-OCH3
R1=4-OCH3
R1=2-Cl
R1=4-Cl
R1=2-CF3
R1=4-CF3
7a
7b
7c
7d
7e
7f
7h
7i
CN
7j
7k
7l
c
F
HO
N
NH
N
N
7m
7n
7g
7a-n
Scheme 1. Synthesis of target molecules 7a-n.
Reagents and conditions: (a) PdCl2, K2CO3, PEG400-H2O, r.t., 0.5 h, N2 protection; (b) 4-((4-
chloropyrimidin-2-yl)amino)benzonitrile, NaH, 1,3-dimethyl imidazole iodide, DMSO, r.t., N2 protection;
(c) NaOH, (NH2OH)2·H2SO4, ethyl alcohol, 65°C.

Biological activities of compounds 7a-n
Fourteen oxime derivatives were evaluated by the MTT method using HIV-1 infected MT-4 cells, along
with nevirapine (NEV), etravirine (ETR), and efavirenz (EFV) as the reference drugs (Table 1). The
inhibitory activities of the oxime derivatives against the WT HIV-1 was promising ranging from 0.0121
µM to 0.369 µM with the selectivity index (SI) ranging from 71 to 24105. Most compounds, except 7f, 7i
and 7n, were more potent than NVP. Compound 7a with non-substitution on the biphenyl ring had an
EC50 of 16.9 nM, and a CC50 of 29.4 µM. Fluoro-substituted analogues (7b-d) exhibited better activities
than 7a except 7c with 3-F. Compound 7d with R1 substituted by 4-F exhibited the most potent HIV-1
inhibition (EC50=12.1 nM), lowest cytotoxicity (CC50 > 292 µM) and best SI over 24105. Compounds 7e-
g with methyl group and 7h-j with methoxy group exhibited similar activities to 7a with exception of the
meta-substitution. Next, we further synthesized chloro and trifluoromethyl group on ortho- and para-
positions of the phenyl ring to obtain compounds 7k-n. The activity decreased compared with 7a. SAR
analysis indicated that ortho- and para-substituted R1 showed much better activities than meta-substituted
analogues. The substitutions with moderate electron-withdrawing effects (e.g. F, Cl) were positive with
regard to the antiviral activity. The analogues substituted with electron-donating effects (e.g. CH3, OCH3)
or high electron-withdrawing effects (e.g. CF3) were less active.
Table 1. Activities and cytotoxicities of compounds 7a-n against HIV-1 (IIIB) strains in MT-4 cells
SI (ⅢB)c
compounds
R1
EC50 (µM)a
CC50 (µM)b
7a
7b
7c
7d
7e
7f
H
0.0169 ± 0.009
0.0149 ± 0.003
0.0448 ± 0.02
0.0121 ± 0.002
0.0511 ± 0.02
0.194 ± 0.08
29.4 ± 5
32.0 ± 0.5
30.9 ± 0.5
>292
1740
2152
690
2-F
3-F
4-F
>24105
511
2-CH3
3-CH3
26.1 ± 6
32.2 ± 0.1
166

7g
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-Cl
0.0483 ± 0.01
0.0634 ± 0.02
0.194 ± 0.07
21.8 ± 8
26.3 ± 6
23.6 ± 9
22.5 ± 11
30.8 ± 2
29.9 ± 4
28.3 ± 0.8
26.3 ± 3
>15.0
452
7h
415
7i
121
7j
0.0340 ± 0.01
0.0387 ± 0.01
0.0237 ± 0.01
0.0565 ± 0.01
0.369 ± 0.1
663
7k
797
7l
4-Cl
1259
500
7m
7n
2-CF3
4-CF3
71
NVP
EFV
ETR
RPV
0.199 ± 0.09
>76
0.00320 ± 0.001
0.00460 ± 0.001
0.00140 ± 0.0001
>6.30
>3939
>1012
3747
>4.60
5.40 ± 0.3
aEC50: The effective concentration required to protect MT-4 cells against virus-induced cytopathicity by
50%.
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).
As proposed that these oxime-DAPYs might exhibit interaction with E138, we first evaluated the
inhibitory activity toward the E138K mutant strain (Table 2). Consistent with the activity toward WT
HIV-1, they exhibited EC50 values of 27.0 nM to 1.08 µM and the ortho- and para-substituted analogues
showed better activities than meta-substituted analogues. Compound 7d had the best activity toward the
E138K mutant. However, they showed submicromolar inhibitory activities toward other single mutant
strains including L100I, K103N, Y181C and double mutants (F227L + V106A, K103N + Y181C).

Table 2. Inhibitory activity toward a panel of clinically relevant HIV-1 mutant strains and HIV-1 RT
enzyme.
EC50 (µM)
comp
ound
s
RES056(=
K103N
F227L +
V106A
E138K
L100I
K103N
Y181C
Y188L
+Y181C)
0.0530 ±
0.02
0.390 ±
0.06
7a
7b
7c
7d
7e
7f
1.08 ± 0.2
1.95 ± 0.4
3.89 ± 0.6
4.78 ± 1
9.63 ± 2
7.14 ± 3
4.43 ± 1
0.0470 ±
0.02
0.483 ±
0.02
0.673 ±
0.03
0.217 ±
0.05
3.40 ± 0.2 3.77 ± 0.1
3.67 ±
0.144 ±
0.9
2.46 ± 0.3
3.03 ± 0.3
0.855 ± 0.2 3.87 ± 0.3
0.09
0.0270 ±
0.01
0.400 ±
0.06
0.449 ±
0.08
0.182 ±
3.43 ± 0.2 4.59 ± 0.6 22.7 ± 14
0.04
0.259 ±
0.1
1.58 ± 0.5
3.77 ± 0.1
2.92 ± 1
3.30 ± 0.5
3.94 ± 0.08
2.81 ± 0.8
3.91 ± 0.7
5.45 ± 3
0.316 ± 0.1 4.11 ± 0.6 3.41 ± 0.2 4.09 ± 0.3
0.497 ±
0.08
3.04 ± 0.3
1.19 ± 0.3
4.73 ± 1
5.93 ± 1
4.76 ± 1
7.04 ± 1
7.70 ± 2
6.42 ± 1
0.160 ±
0.03
7g
7h
7i
0.360 ±
0.09
2.94 ± 0.4
5.07 ± 2
0.766 ± 0.2 7.71 ± 0.7 7.52 ± 0.8 7.13 ± 0.9
0.690 ±
0.06
2.71 ± 0.7
7.91 ± 1
8.20 ± 0.8
>7.01

0.166 ±
0.06
7j
7k
7l
1.44 ± 0.5
1.23 ± 0.2
0.746 ± 0.1
1.74 ± 0.3
2.24 ± 0.3
0.371 ± 0.1
0.314 ± 0.1
0.576 ± 0.3
5.16 ± 2
4.88 ± 1
3.25 ± 1
3.57 ± 0.4
5.23 ± 2
6.17 ± 1
3.92 ± 0.8
4.30 ± 1
4.52 ± 1
5.04 ± 1
4.24 ± 1
0.158 ±
0.02
0.0700 ±
0.02
0.699 ±
0.06
0.205 ±
0.02
0.300 ±
0.09
3.37 ±
0.08
7m
0.888 ± 0.3
3.43 ± 0.4
0.976 ± 0.4
1.14 ± 0.3
2.97 ± 0.2
4.69 ± 0.6
7n
1.08 ± 0.4
2.48 ± 0.5
4.73 ± 2
3.52 ± 0.4 5.11 ± 0.5 7.12 ± 0.6
NVP
0.199 ±
0.1
≧4.62
≧4.28
>15.0
EFV 0.00510 ±
0.002
0.0348 ±
0.006
0.0824 ±
0.01
0.00570 ±
0.001
0.241 ±
0.1
0.276 ±
0.07
0.241 ±
0.06
ETR 0.00760 ±
0.004
0.00830 ±
0.002
0.00300 ±
0.001
0.0170 ±
0.003
0.0168 ±
0.006
0.0138 ±
0.005
0.0391 ±
0.009
RPV 0.00300 ±
0.001
0.00150 ±
0.001
0.00110 ±
0.0001
0.00300 ±
0.001
0.0273 ±
0.007
0.0397 ±
0.01
0.00770 ±
0.002
Enzymatic assay of the selected compounds and Molecular docking and dynamics analysis
Compounds 7b, 7d and 7l were chosen to test inhibitory activity against WT HIV-1 RT enzyme (Table
3). They exhibited activities from 40.6 to 42.1 nM that were better than reference drugs NVP. As a result,
the oxime-biphenyl-DAPYs effected anti-HIV activity by targeting HIV RT enzyme.
Table 3. The activity of selected compounds against WT HIV-1 RT enzyme.

Compounds
7b
7d
7l
NVP
± 0.450 ± 0.00800
0.2 0.003
EFV
dIC50 (WT RT enzyme, 0.0421
± 0.0421
0.002
± 0.0406
0.005
±
µM)
0.002
dIC50: inhibitory concentration of test compound required to inhibit WT HIV-1 RT polymerase activity by
50%.
A
B
D
C
Fig. 3 Protein (PDB: 2ZD1)-Ligand (A, RT-7d; B, E138K-RT-7d) root mean square deviation (RMSD)
indicates the stability of the model. Left Y-axis shows the RMSD evolution of a protein. (C) The
predicted binding mode of 7d with WT HIV-1 RT (PDB: 2ZD1). (D) The predicted binding mode of 7d
with RT (E138K). Mutated residues are depicted as pink sticks. Hydrogen bonds are depicted as yellow
dashed lines.

Compound 7d was chosen for molecular modelling study and prediction of the molecular insight after
being stabilized by molecular dynamics (Fig. 3, RMSD ≈ 2 Å in 3 ns). The compound was located in the
NNRTI binding pocket (NNIBP) as a typical “U” conformation [20], and formed several strong
interactions with crucial amino acids of RT. The K101 residue interacted with the compound by two
hydrogen bonds with the pyrimidine and the linker NH. The biphenyl fragment increased the π–π
interactions with amino acid residues W229 and Y188. The newly introduced oxime, as proposed, could
form a hydrogen bond with E138 (Fig. 3C) instead of the water-mediated hydrogen bond in the lead
compound 1 (Fig. 2). In the E138K mutated enzyme, which was generated and minimized using
BioLuminate, the compound 7d showed very similar conformation and the hydrogen bonds with adjacent
residues were maintained, especially with the K138 (Fig. 3D). The above result provided an explanation
for the potency of the oxime-biphenyl-DAPYs.
As shown in Fig. 4, compound 7d docking with NNIBP showed the common features in the other
single mutant strains and double mutants, including U-shaped conformation, the hydrogen bond (K101,
Fig. 4A and E138 except K103N + Y181C mutant) and hydrophobic interactions. It was worth noting that
the linkage amide could form a hydrogen bond with N103 making a 10-fold higher EC50 than that of NVP
(Fig. 4B). In the Y181C and Y188L mutants, both of the π-π interactions were reduced compared with the
WT RT (Fig. 4C and Fig. 4D). In the F227L + V106A mutant, the interactions with Y181 and Y188
residues were also decreased as the mutant residues made the benzyl ring moved down or outside the
hydrophobic pocket (Fig. 4E). In the K103N + Y181C mutant, the crucial hydrogen bond with E138 was
lost (Fig. 4F).

A
B
D
C
E
F

Fig. 4 Predicted binding mode of compound 7d with HIV-1 mutants (PDB: 2ZD1). (A) L100I; (B)
K103N; (C) Y181C; (D) Y188L; (E) F227L + V106A; (F) K103N + Y181C.
Conclusions
A series of oximes have been designed and synthesized by pharmacophore fusion strategy. They were
demonstrated to possess nanomolar-level antiviral activity and low cytotoxicity. SAR studies led to the
discovery of compound 7d possessing 4-F, which exhibited the best antiviral activity. This compound
could be fitted in the binding pocket of WT and mutants in suitable conformations and interactions. The
hydrogen-bonding interactions formed by the oxime with E138 replaced the common water-mediated
hydrogen bond that maintained the better activity toward WT and E138K mutant viral strains. However,
the compounds exhibited much less activity toward other clinically observed single mutants and double
mutants. Further optimizations based on the oxime-biphenyl-DAPYs as lead compounds are ongoing.
Experimental Procedures
Chemistry
General. All reagents and solvents were purchased from commercial sources and used without further
purification. All reactions and column chromatography (silica gel of 300-400 mesh) were monitored by
silica gel plates in UV light (λ = 254 nm). Melting points were measured on a WRS-1 digital melting
1
point apparatus and are uncorrected. H NMR spectra were recorded in DMSO-d6 or Acetone-d6 using a
Bruker AV 400 MHz spectrometer, chemical shifts (δ) using ppm as units relative to the internal standard
tetramethylsilane (TMS). HRMS were obtained on Brukersolari X-70 FT-MS instrument. The purity of
the compounds 7a-n were detected by high-performance liquid chromatography (HPLC) (Agilent 1260)
using a C18 column (Eslipse XDB, 4.6*150 mm, 5 μm) with gradient methanol/water as the mobile phase
at a flow rate of 0.8 mL/min: (a) 0-12 min, 50%-95% MeOH; (b) 12-20 min, 95% MeOH; (c) 20-25 min,
95%-50% MeOH; (d) 25-30 min, 50% MeOH.

General procedure for the synthesis of compounds 4a-n.
Compounds 2a-n (1.0 mmol, 1.0 equiv.), different substituted 4-bromo-2-fluorobenzaldehyde (1.0 mmol,
1.0 equiv.), PdCl2 (0.005 mmol, 0.005 equiv.) and potassium carbonate (3.5 mmol, 3.5 equiv.) were
mixed in PEG400/H2O (25 mL/25 mL) and stirred at room temperature for 30 min. The reaction was
monitored by TLC. When the reactant disappearing, the reaction mixture was poured into water (80 mL)
and extracted with ethyl acetate. The organic layer was 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 4a-n as white solid.
General procedure for the synthesis of compounds 6a-n.
To a solution of 5 (1 mmol, 1.0 equiv) in anhydrous DMSO (10 mL) was added appropriate 4a-n (1.2
mmol, 1.2 equiv) and 1,3-dimethyl imidazole iodide (0.5 mmol, 0.5 equiv). After stirred at room
temperature for about 30 min, 60% NaH (2.5 mmol, 2.5 equiv) was added in portion under a nitrogen
atmosphere and reacted for 4-6 h. The resulting mixture was poured into 200 mL saturated ammonium
chloride solution. The precipitate was collected and purified by column chromatography, eluting with
EtOAc/petroleum ether/ dichloromethane obtaining 6a-n.
4-((4-(3-fluoro-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6a).
1
Yield: 216 mg, 55%, yellow solid, mp 180-182.3°C. H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H),
8.89 (d, J = 4.9 Hz, 1H), 7.84 (t, J = 8.4 Hz, 5H), 7.78 - 7.72 (m, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (t, J
13
= 7.3 Hz, 2H), 7.47 (t, J = 7.2 Hz, 1H), 7.42 (d, J = 4.9 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ
191.9, 162.32 (d, JC-F = 94.6 Hz), 161.3, 160.3, 159.5, 147.4 (d, JC-F = 8.5 Hz), 144.8, 138.1, 133.3 (2C),
132.4, 129.6 (2C), 129.5, 127.6 (2C), 124.0 (d, JC-F = 13.2 Hz), 123.3, 119.8, 119.0 (2C), 114.8 (d, JC-F
=
22.4 Hz), 111.0, 103.4. HRMS (ESI) calcd for [M-H]- C24H14FN4O-: 393.1157, found: 393.1145 .

4-((4-(2',3-difluoro-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6b).
1
Yield: 255mg, 62%, yellow solid, mp 233.1-235.2°C. H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H),
8.92 (d, J = 4.9 Hz, 1H), 7.93 - 7.82 (m, 3H), 7.74 - 7.58 (m, 5H), 7.58 - 7.50 (m, 1H), 7.50 - 7.32 (m,
13
3H). C NMR (101 MHz, DMSO-d6) δ 192.0, 162.4 (d, JC-F = 125.3 Hz), 161.4, 159.6, 159.4 (d, JC-F
=
120.4 Hz), 144.8, 142.0, 133.3 (2C), 132.0, 131.5, 131.3, 125.7, 125.7, 124.7 (d, JC-F = 13.7 Hz), 119.8,
119.0 (2C), 117.1, 117.0, 116.9, 116.8, 111.0, 103.4. HRMS (ESI) calcd for [M-H]- C24H13F2N4O-:
411.1063, found: 411.1056.
4-((4-(3,3'-difluoro-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6c).
1
Yield: 230 mg, 56%, yellow solid, mp 218.2-219.2°C. H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H),
8.89 (d, J = 4.9 Hz, 1H), 7.88 - 7.81 (m, 3H), 7.82 - 7.66 (m, 4H), 7.61 - 7.53 (m, 3H), 7.42 (d, J = 4.9
Hz, 1H), 7.33 - 7.27 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 191.9, 163.6 (d, JC-F = 171.5 Hz), 161.6,
161.3, 161.1 (d, JC-F = 83.0 Hz), 159.6, 145.71 (d, JC-F = 7.7 Hz), 144.8, 140.5, 133.3, 132.4 (2C), 131.6
(d, JC-F = 8.4 Hz), 124.6 (d, JC-F = 13.4 Hz), 123.7, 123.5, 119.8, 119.0 (2C), 116.3 (d, JC-F = 20.8 Hz),
115.0 (d, JC-F = 22.9 Hz), 114.5 (d, JC-F = 22.9 Hz), 111.0, 103.4. HRMS (ESI) calcd for [M+H]+
C24H15F2N4O+: 413.1208, found: 413.1208.
4-((4-(3,4'-difluoro-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6d).
1
Yield: 268 mg, 65%, yellow solid, mp 194.1-195.6°C. H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H),
8.91 (d, J = 4.8 Hz, 1H), 7.89 (ddd, J = 20.4, 8.1, 5.3 Hz, 5H), 7.77 (t, J = 11.6 Hz, 2H), 7.62 (d, J = 8.5
13
Hz, 2H), 7.44 (d, J = 4.8 Hz, 1H), 7.38 (t, J = 8.7 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 191.9,
163.6 (d, JC-F = 168.9 Hz), 161.8, 161.3, 161.1 (d, JC-F = 174.5 Hz), 159.5, 146.2 (d, JC-F = 8.9 Hz), 144.8,
134.6, 133.4 (2C), 132.4, 129.9, 129.8, 123.9 (d, JC-F = 13.3 Hz), 123.2, 119.8, 119.0 (2C), 116.7, 116.4,

114.7 (d, JC-F = 22.6 Hz), 111.0, 103.4. HRMS (ESI) calcd for [M-H]- C24H13F2N4O-: 411.1063, found:
411.1051.
4-((4-(3-fluoro-2'-methyl-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6e).
1
Yield: 232 mg, 57%, yellow solid, mp 195.6-197.0°C. H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H),
8.92 (d, J = 4.9 Hz, 1H), 7.85 (dd, J = 10.6, 8.1 Hz, 3H), 7.63 (d, J = 8.8 Hz, 2H), 7.50 - 7.44 (m, 2H),
13
7.44 - 7.28 (m, 5H), 2.32 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 192.1, 161.8 (d, JC-F = 32.9 Hz),
161.4, 159.6, 159.4, 148.7 (d, JC-F = 8.6 Hz), 144.8, 139.6, 135.3, 133.3 (2C), 131.7, 131.1, 129.8, 128.9,
126.7, 126.0, 123.8 (d, JC-F = 13.3 Hz), 119.8, 119.0 (2C), 117.3 (d, JC-F = 21.6 Hz), 111.1, 103.4, 20.5.
HRMS (ESI) calcd for [M+H]+ C25H18FN4O+: 409.1459, found: 409.1458.
4-((4-(3-fluoro-3'-methyl-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6f).
1
Yield: 175 mg, 43%, yellow solid, mp 195.1-197.4°C. H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H),
8.91 (d, J = 4.9 Hz, 1H), 7.89 - 7.82 (m, 3H), 7.77 - 7.71 (m, 2H), 7.68 - 7.58 (m, 4H), 7.43 (dd, J = 7.9,
6.4 Hz, 2H), 7.30 (d, J = 7.5 Hz, 1H), 2.42 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 191.9, 162.3 (d, JC-
F = 95.3 Hz), 161.3, 160.3, 159.6, 147.5 (d, JC-F = 8.6 Hz), 144.8, 139.0, 138.1, 133.3 (2C), 132.3, 130.2,
129.6, 128.2, 124.7, 123.9 (d, JC-F = 13.5 Hz), 123.3, 119.8, 119.0 (2C), 114.7 (d, JC-F = 22.5 Hz), 111.0,
103.4, 21.5. HRMS (ESI) calcd for [M+H]+ C25H18FN4O+: 409.1459, found: 409.1473.
4-((4-(3-fluoro-4’-methyl-[1,1’-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6g).
1
Yield: 261 mg, 64%, yellow solid, mp 215.8-216.2°C. H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H),
8.91 (d, J = 4.9 Hz, 1H), 7.91 - 7.78 (m, 3H), 7.78 - 7.69 (m, 4H), 7.65 - 7.58 (m, 2H), 7.43 (d, J = 4.9
Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 2.38 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 191.8, 162.4 (d, JC-F
=
92.3 Hz), 161.2, 160.4, 159.5, 147.4 (d, JC-F = 8.6 Hz), 144.9, 139.2, 135.2, 133.3 (2C), 132.4, 130.3

(2C), 127.4 (2C), 123.6 (d, JC-F = 13.1 Hz), 122.9, 119.8, 119.0 (2C), 114.4 (d, JC-F = 22.6 Hz), 111.0,
103.4, 21.2. HRMS (ESI) calcd for [M+H]+ C25H18FN4O+: 409.1459, found: 409.1473.
4-((4-(3-fluoro-2’-methoxy-[1,1’-biphenyl]-4-carbonyl)pyrimidi-n-2-yl)amino)benzonitrile (6h).
1
Yield: 170 mg, 40%, yellow solid, mp 217.6-219.0°C. H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H),
8.92 (d, J = 4.9 Hz, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.81 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.58 -
7.51 (m, 2H), 7.45 (dd, J = 6.5, 4.0 Hz, 3H), 7.22 (d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 3.86 (s,
13
3H). C NMR (101 MHz, DMSO-d6) δ 192.2, 161.8 (d, JC-F = 15.5 Hz), 161.4, 159.6, 159.3, 156.6,
145.5, 144.9, 133.3 (2C), 131.3, 130.9, 130.8, 130.1, 126.0, 123.8 (d, JC-F = 13.4 Hz), 121.5, 119.9, 119.0
+
(2C), 117.3 (d, JC-F = 22.3 Hz), 112.5, 110.9, 103.4, 56.1. HRMS (ESI) calcd for [M+H]+ C25H18FN4O2 :
425.1408, found: 425.1409.
4-((4-(3-fluoro-3’-methoxy-[1,1’-biphenyl]-4-carbonyl)pyrimidi-n-2-yl)amino)benzonitrile (6i).
1
Yield: 148 mg, 35%, yellow solid, mp 187.3-188.7°C. H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H),
8.91 (d, J = 4.9 Hz, 1H), 7.90 - 7.82 (m, 3H), 7.82 - 7.74 (m, 2H), 7.61 (d, J = 8.7 Hz, 2H), 7.46 (dd, J =
10.0, 6.2 Hz, 2H), 7.43 - 7.34 (m, 2H), 7.06 (ddd, J = 8.2, 2.6, 1.2 Hz, 1H), 3.87 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 191.9, 162.2 (d, JC-F = 96.7 Hz), 161.3, 160.4, 160.2, 159.6, 147.2 (d, JC-F = 8.7 Hz),
144.8, 139.6, 133.3 (2C), 132.3, 130.8, 124.1 (d, JC-F = 13.5 Hz), 123.4, 119.9, 119.8, 119.0 (2C), 115.3,
+
114.9 (d, JC-F = 22.6 Hz), 112.9, 111.0, 103.4, 55.8. HRMS (ESI) calcd for [M+H]+ C25H18FN4O2 :
425.1408, found: 425.1425.
4-((4-(3-fluoro-4’-methoxy-[1,1’-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6j).
Yield: 178 mg, 42%, yellow solid, mp 216.5-218.3°C. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H),
8.90 (d, J = 4.9 Hz, 1H), 7.93 - 7.78 (m, 5H), 7.76 - 7.68 (m, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.42 (d, J =
4.9 Hz, 1H), 7.15 - 7.05 (m, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 191.7, 162.6 (d, JC-F
=

85.7 Hz), 161.2, 160.6, 160.5, 159.5, 147.2 (d, JC-F = 8.9 Hz), 144.9, 133.4 (2C), 132.4, 130.2, 128.9
(2C), 123.0 (d, JC-F = 12.9 Hz), 122.5, 119.9, 119.0 (2C), 115.1 (2C), 113.9 (d, JC-F = 22.4 Hz), 111.0,
+
103.4, 55.8. HRMS (ESI) calcd for [M+H]+ C25H18FN4O2 : 425.1408, found: 425.1404.
4-((4-(2'-chloro-3-fluoro-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6k).
Yield: 137 mg, 32%, yellow solid, mp 265-266.7°C. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.93
(d, J = 4.9 Hz, 1H), 7.87 (dd, J = 12.8, 8.2 Hz, 3H), 7.69 - 7.65 (m, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.59-
13
7.50 (m, 5H), 7.48 (d, J = 4.9 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 192.1, 161.5 (d, JC-F = 35.8
Hz), 161.5, 159.6, 159.2, 145.6 (d, JC-F = 8.8 Hz), 144.8, 138.1, 133.4 (2C), 131.9, 131.7, 131.6, 130.8,
130.6, 128.3, 126.3, 124.7 (d, JC-F = 13.4 Hz), 119.8, 119.0 (2C), 117.7 (d, JC-F = 22.5 Hz), 111.0, 103.4.
HRMS (ESI) calcd for [M+H]+ C24H15ClFN4O+: 429.0913, found: 429.0923.
4-((4-(4'-chloro-3-fluoro-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile (6l).
1
Yield: 141 mg, 33%, yellow solid, mp 221.3-224.1°C. H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H),
8.89 (d, J = 4.9 Hz, 1H), 7.86 (td, J = 7.7, 7.1, 3.7 Hz, 5H), 7.80 - 7.72 (m, 2H), 7.59 (dd, J = 8.7, 3.7 Hz,
4H), 7.42 (d, J = 4.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 191.9, 162.2 (d, JC-F = 100.7 Hz), 161.3,
160.2, 159.6, 145.9 (d, JC-F = 8.7 Hz), 144.8, 136.9, 134.5, 133.3 (2C), 132.5, 129.6 (2C), 129.4 (2C),
124.3 (d, JC-F = 13.3 Hz), 123.3, 119.8, 119.0 (2C), 114.8 (d, JC-F = 22.8 Hz), 111.0, 103.4. HRMS (ESI)
calcd for [M+H]+ C24H15ClFN4O+: 429.0913, found: 429.0924.
4-((4-(3-fluoro-2'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile
(6m).
1
Yield: 171 mg, 37%, yellow solid, mp 175.8-177.4°C. H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H),
8.93 (d, J = 4.9 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.89 - 7.82 (m, 3H), 7.80 (d, J = 7.6 Hz, 1H), 7.72 (t, J
= 7.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.49 - 7.44 (m, 2H), 7.39 (d, J = 8.1 Hz,

1H). 13C NMR (101 MHz, DMSO-d6) δ191.9, 161.5, 161.3 (d, JC-F = 17.7 Hz), 159.6, 158.9, 146.3 (d, JC-
F = 8.7 Hz), 144.8, 138.7, 133.3 (2C), 133.0, 132.2, 131.5, 129.5, 127.1 (d, JC-F = 29.6 Hz), 126.8 (q, JC-F
= 5.3 Hz), 125.9, 124.7 (d, JC-F = 13.2 Hz), 123.2, 119.8, 119.0 (2C), 117.4 (d, JC-F = 22.4 Hz), 111.1,
103.4. HRMS (ESI) calcd for [M+H]+ C25H15F4N4O+: 463.1177, found: 463.1169.
4-((4-(3-fluoro-4'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbonyl)pyrimidin-2-yl)amino)benzonitrile
(6n).
1
Yield: 152 mg, 33%, yellow solid, mp 210.7-211.4°C. H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H),
8.93 (d, J = 4.9 Hz, 1H), 8.08 (d, J = 8.0 Hz, 2H), 7.93 - 7.83 (m, 7H), 7.62 (d, J = 8.4 Hz, 2H), 7.46 (d, J
13
= 4.8 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ192.0, 162.1 (d, JC-F = 110.8 Hz), 161.4, 160.1, 159.6,
145.5 (d, JC-F = 8.3 Hz), 144.8, 142.1, 133.3 (2C), 132.5, 129.6 (d, JC-F = 31.6 Hz), 128.5 (2C), 126.5 (q,
JC-F = 3.8 Hz), 126.0, 125.0 (d, JC-F = 13.5 Hz), 123.8, 123.3, 119.8, 119.0 (2C), 115.4 (d, JC-F = 23.2 Hz),
111.0, 103.4. HRMS (ESI) calcd for [M+H]+ C25H15F4N4O+: 463.1177, found: 463.1174.
General procedure for the synthesis of compounds 7a-n.
Compounds 6a-n (0.5 mmol, 1.0 equiv) was solved in EtOH (25 mL) and followed by dropwise adding
an aqueous solution (H2O, 5 mL) containing NaOH (5 mmol, 10.0 equiv) and hydroxylamine sulphate
(1.5 mmol, 3.0 equiv). Then, the mixture was refluxed for 1 h until complete consumption of starting
material monitored by TLC. The resulting solution was poured into 100 mL ice water and neutralized
with 1 N HCl. The filtrated precipitation was dried and purified by column chromatography to give
compounds 7a-n.
((4-((3-fluoro-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrim-idin-2-yl)amino)benzonitrile (7a).
1
Yield: 112 mg, 52%, white solid, mp 192.4-193.6°C. H NMR (400 MHz, DMSO-d6) δ 12.58 (s, 1H),
10.21 (s, 1H), 8.63 (d, J = 5.1 Hz, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.74 - 7.60 (m, 5H), 7.56 (t, J = 7.6 Hz,

2H), 7.51-7.43 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 162.1, 159.6, 159.9
(d, JC-F = 243.8 Hz), 159.3, 150.8, 145.1, 143.5 (d, JC-F = 8.1 Hz), 139.0, 132.9 (2C), 131.9 (d, JC-F = 5
Hz), 129.6 (2C), 128.8, 127.3 (2C), 122.9, 119.8, 119.6, 118.7 (2C), 114.0 (d, JC-F = 22.4 Hz), 109.1,
102.7. HRMS (ESI) calcd for [M+Na]+ C24H16FN5NaO+: 432.1231, found: 432.1219. HPLC analysis: tR =
12.9 min; peak area, 99.7%.
((4-((2',3-difluoro-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)benzonitrile
(7b).
1
Yield: 103 mg, 48%, white solid, mp 214.4-216.2°C. H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H),
10.20 (s, 1H), 8.61 (d, J = 5.2 Hz, 1H), 7.61 (d, J = 8.5 Hz, 3H), 7.57 - 7.50 (m, 3H), 7.48 (t, J = 5.8 Hz,
13
3H), 7.42-7.34 (m, 5H). C NMR (101 MHz, DMSO-d6) δ 162.0, 159.62, 159.6 (d, JC-F = 245.2 Hz),
159.3,159.3 (d, JC-F = 244.2 Hz), 150.7, 145.1, 138.2 (d, JC-F = 8.2 Hz), 132.9 (2C), 131.7 (d, JC-F = 4.7
Hz), 131.3, 131.3, 130.9 (d, JC-F = 8.6 Hz), 125.7 (d, JC-F = 3.7 Hz), 125.2 (d, JC-F = 17.8 Hz), 119.8,
118.7 (2C), 116.9, 116.7, 109.1, 102.7. HRMS (ESI) calcd for [M+H]+ C24H16F2N5O+: 428.1317, found:
428.1325. HPLC analysis: tR = 13.1 min; peak area, 99.2%.
((4-((3,3'-difluoro-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)benzonitrile
(7c).
Yield: 111 mg, 52%, white solid, mp >250°C. 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 10.20 (s,
1H), 8.63 (d, J = 5.2 Hz, 1H), 7.76 (dd, J = 16.2, 8.6 Hz, 3H), 7.70 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.8
Hz, 2H), 7.52 - 7.45 (m, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.34 - 7.27 (m, 1H). 13C NMR (101 MHz, DMSO)
δ 162.77 (d, JC-F = 341.9 Hz), 162.1, 159.7 (d, JC-F = 122.4 Hz), 159.6, 159.3, 150.7, 145.1, 142.0 (d, JC-F
= 8.1 Hz), 141.3 (d, JC-F = 7.7 Hz), 132.9 (2C), 132.0 (d, JC-F = 4.7 Hz), 131.6 (d, JC-F = 8.7 Hz), 123.38,
122.99, 120.35 (d, JC-F = 18 Hz), 119.76, 118.7 (2C), 115.6 (d, JC-F = 21.2 Hz), 114.2 (d, JC-F = 22.9 Hz),

114.1 (d, JC-F = 22.2 Hz), 109.2, 102.7. HRMS (ESI) calcd for [M+H]+ C24H16F2N5O+: 428.1317, found:
428.1307. HPLC analysis: tR = 13.2 min; peak area, 98.9%.
4-((4-((2',3-difluoro-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)benzonitrile
(7d).
1
Yield: 124 mg, 58%, white solid, mp: 204.4-206.4°C. H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H),
10.18 (s, 1H), 8.63 (d, J = 5.2 Hz, 1H), 7.93 - 7.84 (m, 2H), 7.74 - 7.60 (m, 4H), 7.46 (dd, J = 9.2, 6.3 Hz,
2H), 7.37 (dt, J = 8.6, 4.4 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 162.83 (d, JC-F = 298.2 Hz), 162.1,
160.4 (d, JC-F = 244.1 Hz), 159.3, 158.6, 150.8, 145.1, 142.4 (d, JC-F = 7.8 Hz), 135.4, 132.9 (2C), 131.98,
129.5, 129.4, 122.8, 119.8, 119.6, 118.7 (2C), 116.6, 116.3, 113.9 (d, JC-F = 22.8 Hz), 109.2, 102.7.
HRMS (ESI) calcd for [M+H]+ C24H16F2N5O+: 428.1317, found: 428.1310. HPLC analysis: tR = 12.8 min;
peak area, 98.4%.
((4-((3-fluoro-2'-methyl-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7e).
Yield: 91 mg, 43%, white solid, mp: 208.6-210.2°C. 1H NMR (400 MHz, DMSO- d6) δ 12.56 (s, 1H),
10.21 (s, 1H), 8.63 (d, J = 5.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.50 - 7.32 (m, 10H), 2.33 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 162.2, 159.6, 159.3, 159.1 (d, JC-F = 244.6 Hz) 150.8, 145.1, 144.4 (d, JC-F
= 47.8 Hz) 140.1, 135.3, 133.0 (2C), 131.2, 131.1, 130.0, 128.4, 126.6, 125.4, 119.8, 119.2 (d , JC-F
=
17.3 Hz), 118.7 (2C), 116.4 (d, JC-F = 21.9 Hz), 109.3, 102.7, 20.6. HRMS (ESI) calcd for [M+H]+
C25H19FN5O+: 424.1568, found: 424.1575. HPLC analysis: tR = 13.9 min; peak area, 98.8%.
((4-((3-fluoro-3'-methyl-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7f).

1
Yield: 83 mg, 39%, white solid, mp: 196.2-197.4°C. H NMR (400 MHz, DMSO-d6) δ 12.58 (s, 1H),
10.21 (s, 1H), 8.63 (d, J = 5.2 Hz, 1H), 7.64 (ddd, J = 16.9, 13.1, 5.6 Hz, 7H), 7.50 - 7.47 (m, 1H), 7.43 (t,
13
J = 8.0 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 7.6 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ
162.1, 159.8 (d, JC-F = 243.9 Hz), 159.6, 159.3, 150.9, 145.1, 143.7 (d, JC-F = 8 Hz), 139.0, 138.9, 132.9
(2C), 131.9, 129.5, 129.4, 128.0, 124.4, 122.9, 119.8, 119.6 (d, JC-F = 17.9 Hz), 118.6 (2C), 114.0 (d, JC-F
= 22.3 Hz), 109.1, 102.7, 21.6. HRMS (ESI) calcd for [M+H]+ C25H19FN5O+: 424.1568, found: 424.1565.
HPLC analysis: tR = 14.4 min; peak area, 99.0%.
((4-((3-fluoro-4'-methyl-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7g).
1
Yield: 95 mg, 45%, white solid, mp: 218.1-220.3°C. H NMR (400 MHz, DMSO-d6) δ 12.52 (s, 1H),
10.16 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.66 - 7.58 (m, 4H), 7.47 - 7.39 (m, 2H),
13
7.34 (dd, J = 10.7, 8.2 Hz, 4H), 2.37 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 162.2, 159.6 (d, JC-F
=
244.1 Hz), 159.2, 158.7, 150.8, 145.2, 143.4 (d, JC-F = 7.8 Hz), 138.3, 136.1, 132.9 (2C), 131.9, 130.2
(2C), 127.1 (2C), 122.5, 119.8, 119.4, 118.7 (2C), 113.6 (d, JC-F = 22.5 Hz), 109.2, 102.7, 21.2. HRMS
(ESI) calcd for [M+H]+ C25H19FN5O+: 424.1568, found: 424.1566. HPLC analysis: tR = 14.1 min; peak
area, 99.1%.
((4-((3-fluoro-2'-methoxy-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7h).
1
Yield: 103 mg, 47%, white solid, mp: 183-185.7°C. H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H),
10.24 (s, 1H), 8.63 (d, J = 5.1 Hz, 1H), 7.63 (d, J = 8.5 Hz, 2H), 7.52 - 7.47 (m, 3H), 7.46 - 7.37 (m, 5H),
7.23 (d, J = 8.3 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 3.88 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.2
(d, JC-F = 195.3 Hz), 159.6, 159.3, 157.88, 156.5, 151.0, 145.2, 141.3 (d, JC-F = 8.2 Hz), 133.0 (2C),
131.0, 130.2, 130.1, 128.4, 125.5, 121.4, 119.9, 119.2 (d, JC-F = 17.7 Hz), 118.6 (2C), 116.5 (d, JC-F
=

+
22.4 Hz), 112.3, 109.0, 102.6, 56.0. HRMS (ESI) calcd for [M+H]+ C25H19FN5O2 : 440.1517, found:
440.1518. HPLC analysis: tR = 13.2 min; peak area, 97.5%.
((4-((3-fluoro-3'-methoxy-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7i).
1
Yield: 86 mg, 39%, white solid, mp: 201.5-202.7°C. H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H),
10.17 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.73 - 7.63 (m, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.49-7.39 (m, 3H),
13
7.39-7.29 (m, 4H), 7.01 (dd, J = 8.3, 2.4 Hz, 1H).3.86(s, 3H). C NMR (101 MHz, DMSO-d6) δ 162.1,
160.4, 159.8 (d, JC-F = 246.8 Hz), 159.6, 159.3, 150.8, 145.2, 143.4 (d, JC-F = 7.8 Hz), 140.5, 132.9 (2C),
131.9, 130.7, 123.0, 119.8, 119.7, 119.6, 118.7 (2C), 114.7, 114.1 (d, JC-F = 22.6 Hz), 112.5, 109.1, 102.7,
+
55.7. HRMS (ESI) calcd for [M+H]+ C25H19FN5O2 : 440.1517, found: 440.1519. HPLC analysis: tR =
13.3 min; peak area, 98.6%.
((4-((3-fluoro-4'-methoxy-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7j).
1
Yield: 88 mg, 40%, white solid, mp: 207.9-209.0°C. H NMR (400 MHz, DMSO-d6) δ 12.52 (s, 1H),
10.17 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.66 - 7.57 (m, 4H), 7.47 - 7.33 (m, 4H),
7.10 - 7.04 (m, 2H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.2, 160.0, 160.0 (d, JC-F = 234.9
Hz), 159.6, 159.2, 150.9, 145.2, 143.2 (d, JC-F = 8.1 Hz), 132.9 (2C), 131.9, 131.2, 128.5 (2C), 122.2,
119.8, 118.6 (2C), 115.0 (2C), 115.0, 113.3 (d, JC-F = 22.1 Hz), 109.2, 102.7, 55.7. HRMS (ESI) calcd for
+
[M+H]+ C25H19FN5O2 : 440.1517, found: 440.1536. HPLC analysis: tR = 13.0 min; peak area, 97.6%.
((4-((2'-chloro-3-fluoro-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7k).

Yield: 78 mg, 35%, white solid, mp: 222.5-223.8°C. 1H NMR (400 MHz, Acetone-d6) δ 11.80 (s, 1H),
9.27 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.78 (d, J = 8.9 Hz, 2H), 7.65 (d, J = 7.2 Hz, 1H), 7.61-7.54 (m,
3H), 7.54 - 7.49 (m, 3H), 7.49 - 7.44 (m, 3H). 13C NMR (101 MHz, Acetone-d6) δ 161.23 (d, JC-F = 177.1
Hz), 159.62, 158.67, 157.89, 151.1, 144.6, 142.0 (d, JC-F = 8.3 Hz), 138.7, 132.7 (2C), 131.8, 131.6, 131.0
(d, J = 4.6 Hz), 130.3, 129.7, 127.6, 125.1 (d, JC-F = 3.1 Hz), 118.9, 118.4 (2C), 118.3, 116.4 (d, JC-F
=
22.6 Hz), 109.0, 103.5. HRMS (ESI) calcd for [M+H]+ C24H16ClFN5O+: 444.1022, found: 444.1015.
HPLC analysis: tR = 13.0 min; peak area, 97.6%.
((4-((4'-chloro-3-fluoro-[1,1'-biphenyl]-4-yl)(hydroxyimino)methyl)pyrimidin-2-yl)amino)
benzonitrile (7l).
1
Yield: 93 mg, 42%, white solid, mp >250°C. H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H), 10.17 (s,
1H), 8.60 (d, J = 5.2 Hz, 1H), 7.89 - 7.82 (m, 2H), 7.74 - 7.64 (m, 2H), 7.64 - 7.54 (m, 4H), 7.49-7.42 (m,
2H), 7.36 (d, J = 8.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 162.1, 159.9 (d, JC-F = 244.2 Hz), 159.6,
159.3, 150.7, 145.1, 142.1 (d, JC-F = 8.1 Hz), 137.7, 133.7, 132.9 (2C), 132.1, 129.6 (2C), 129.1 (2C),
122.8, 120.1 (d, JC-F = 17.8 Hz), 119.8, 118.7 (2C), 114.0 (d, JC-F = 22.8 Hz), 109.2, 102.7. HRMS (ESI)
calcd for [M+H]+ C24H16ClFN5O+: 444.1022, found: 444.1029. HPLC analysis: tR = 14.0 min; peak area,
97.5%.
((4-((3-fluoro-2'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)(hydroxyl-imino)methyl)pyrimidin-2-yl)
amino)benzonitrile (7m).
1
Yield: 83 mg, 35%, white solid, mp: 217.5-218.3°C. H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H),
10.23 (s, 1H), 8.64 (d, J = 5.2 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.73 - 7.65 (m,
13
3H), 7.47 (dd, J = 8.9, 4.8 Hz, 5H), 7.40 (d, J = 10.3 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H). C NMR (101
MHz, DMSO-d6) δ162.0, 159.7, 159.4, 158.8 (d, JC-F = 245.1 Hz), 150.5, 145.1, 142.4 (d, JC-F = 8.2 Hz),
139.4, 133.1 (2C), 133.0, 132.6, 131.1, 129.2, 127.3 (d, JC-F = 29.3 Hz), 126.8 (q, JC-F = 5.5 Hz), 126.0,

125.2, 123.2, 120.0, 119.8, 118.6 (2C), 116.4 (d, JC-F = 22.8 Hz), 109.3, 102.7. HRMS (ESI) calcd for
[M+H]+ C25H16F4N5O+: 478.1285, found: 478.1271. HPLC analysis: tR = 13.9 min; peak area, 99.2%.
((4-((3-fluoro-4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)(hydroxyl-imino)methyl)pyrimidin-2-yl)
amino)benzonitrile (7n).
1
Yield: 98 mg, 41%, white solid, mp: 210.7-211.4°C. H NMR (400 MHz, DMSO-d6) δ 12.62 (s,
1H), 10.20 (s, 1H), 8.63 (d, J = 5.2 Hz, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.1 Hz, 2H), 7.79 (ddd,
13
J = 18.5, 9.4, 1.7 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.56 - 7.46 (m, 2H), 7.38 (d, J = 8.5 Hz, 2H). C
NMR (101 MHz, DMSO-d6) δ162.0, 159.9 (d, JC-F = 244.6 Hz), 159.6, 159.3, 150.6, 145.1, 142.9, 141.8
(d, JC-F = 8.0 Hz), 133.0 (2C), 132.2, 130.1, 129.1 (d, JC-F = 31.6 Hz), 128.2, 126.4 (q, JC-F = 3.8 Hz),
126.1, 124.8, 123.3, 120.8 (d, JC-F = 17.6 Hz), 119.8, 118.7 (2C), 114.5 (d, JC-F = 22.8 Hz), 109.2, 102.72.
HRMS (ESI) calcd for [M+H]+ C25H16F4N5O+: 478.1285, found: 478.1268. HPLC analysis: tR = 13.3
min; peak area, 99.7%.
Biological assays
In vitro anti-HIV assay
Evaluation of the antiviral activity of the compounds against WT HIV-1, and mutant strains (L100I,
K103N, E138K, Y188L and Y181C) in MT-4 cells was performed using the MTT assay [21]. 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 HIV- and mock-infected cell samples were included as controls. HIV stock (50 µL) at 100-300
CCID50 (50% cell culture infectious doses) or culture medium was added to either the infected or mock-
infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effects of test

compound on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially
growing MT-4 cells were centrifuged for 5 minutes at 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 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) 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 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).
Reverse transcriptase assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as previously described [22]. The
RT assay is 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 [23]. 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). Fifty-two 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 µM dTTP, pH 8.1). 5 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 minutes and then stopped by the addition of EDTA (15 mM).
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 is added to each well before the
addition of RT enzyme solution. Control wells without compound contain the same amount of DMSO.
Results are expressed as relative fluorescence. The fluorescence signal of the reaction mix with
compound divided by the signal of the same reaction mix without compound.
Molecular modelling
The modelling study was carried out by Schrödinger Maestro 11.4 [24]. The first step was the protein,
HIV-1 RT (PDB entry: 2ZD1) preparation, which followed a standard protocol. Before the preparation,
the protein was prepared by removing water molecules, the ligand, and other unnecessary molecules from
the crystal structure of the HIV-1 RT complex. After that the docking study was performed using Glide.
The parameters choose the default without any constraints. Final scoring was then obtained on the
energy-minimized poses. MD simulations were carried out for RT in complex with the compound scoring
lowest one. Each system was solvated in a cubic box with explicit TIP3P water and counter ions
consisting of a 10 Å 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. 3
ns simulation was carried out for each docked model using Desmond with OPLS-AA 2005 force field.
The stability of the simulation was assessed by monitoring the RMSD with respect to the minimized
starting
structure.
The
molecular
docking
result
was
generated
using
PyMol
(http://pymol.sourceforge.net/) [6-8, 25, 26].
Acknowledgements

We gratefully acknowledge the support provided by 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.
References
[1] WHO website. HIV/AIDS fact sheet, 15 November,2019. https://www.who.int/en/news-room/fact-
sheets/detail/hiv-aids.
[2] H. Liu, Y. Ma, Y. Su, M.K. Smith, Y. Liu, Y. Jin, H. Gu, J. Wu, L. Zhu, N. Wang, Emerging trends of
HIV drug resistance in Chinese HIV-infected patients receiving first-line highly active antiretroviral
therapy: a systematic review and meta-analysis, Clin. Infect. Dis. 59 (2014) 1495-502.
[3] D. Li, P. Zhan, E. De Clercq, X. Liu, Strategies for the Design of HIV-1 Non-Nucleoside Reverse
Transcriptase Inhibitors: Lessons from the Development of Seven Representative Paradigms, J. Med.
Chem. 55 (2012) 3595-3613.
[4] Y. Mehellou, E. De Clercq, Twenty-Six Years of Anti-HIV Drug Discovery: Where Do We Stand and
Where Do We Go?, J. Med. Chem. 53 (2010) 521-538.
[5] K. Jin, H. Yin, E. De Clercq, C. Pannecouque, G. Meng, F. Chen, Discovery of biphenyl-substituted
diarylpyrimidines as non-nucleoside reverse transcriptase inhibitors with high potency against wild-type
and mutant HIV-1, Eur. J. Med. Chem. 145 (2018) 726-734.
[6] 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 transcriptase inhibitors, Eur. J. Med.
Chem. 182 (2019) 111603.
[7] Y. Sang, S. Han, C. Pannecouque, E. De Clercq, C. Zhuang, F. Chen, Ligand-Based Design of
Nondimethylphenyl-Diarylpyrimidines with Improved Metabolic Stability, Safety, and Oral
Pharmacokinetic Profiles, J. Med. Chem. 62 (2019) 11430-11436.
[8] K. Jin, M. Liu, C. Zhuang, E. De Clercq, C. Pannecouque, G. Meng, F. Chen, Improving the
positional adaptability: structure-based design of biphenyl-substituted diaryltriazines as novel non-
nucleoside HIV-1 reverse transcriptase inhibitors, Acta Pharm. Sin. B 10 (2020) 344-357.
[9] C. Zhuang, C. Pannecouque, E. De Clercq, F. Chen, Development of non-nucleoside reverse
transcriptase inhibitors (NNRTIs): Our past twenty years, Acta Pharm. Sin. B (2019), doi:
10.1016/j.apsb.2019.11.010.
[10] M. Katalinić, G. Šinko, N. Maček Hrvat, T. Zorbaz, A. Bosak, Z. Kovarik, Oxime-assisted
reactivation of tabun-inhibited acetylcholinesterase analysed by active site mutations, Toxicology 406-
407 (2018) 104-113.
[11] D.L. Hughes, Patent Review of Manufacturing Routes to Fifth-Generation Cephalosporin Drugs.
Part 2, Ceftaroline Fosamil and Ceftobiprole Medocaril, Org. Process Res. Dev. 21 (2017) 800-815.
[12] O.B. Flekhter, E.I. Boreko, L.R. Nigmatullina, N.I. Pavlova, N.I. Medvedeva, S.N. Nikolaeva, E.V.
Tret'yakova, O.V. Savinova, L.A. Baltina, L.T. Karachurina, F.Z. Galin, F.S. Zarudii, G.A. Tolstikov,
Synthesis and Pharmacological Activity of Acylated Betulonic Acid Oxides and 28-Oxo-Allobetulone,
Pharm. Chem. J. 38 (2004) 148-152.
[13] C. M. Ma, N. Nakamura, M. Hattori, T. Kawahata, T. Otake, Inhibitory effects of triterpene-
azidothymidine conjugates on proliferation of human immunodeficiency virus type 1 and its protease,
Chem. Pharm. Bull. 50 (2002) 877-880.
[14] L.-L. Wang, N. Battini, R. R. Y. Bheemanaboina, M. F. Ansari, J.-P. Chen, Y.-P. Xie, G.-X. Cai, S.-

L. Zhang, C.-H. Zhou, A new exploration towards aminothiazolquinolone oximes as potentially multi-
targeting antibacterial agents: Design, synthesis and evaluation acting on microbes, DNA, HSA and
topoisomerase IV, Eur. J. Med. Chem. 179 (2019) 166-181.
[15] B. Bednarczyk-Cwynar, L. Zaprutko, Recent advances in synthesis and biological activity of
triterpenic acylated oximes, Phytochem. Rev. 14 (2015) 203-231.
[16] V. P. Mehta, A. Sharma, S. G. Modha, S. Sharma, T. Meganathan, V. S. Parmar, E. Van der Eycken,
N-heterocyclic carbene catalyzed aroylation of 3,5-dichloro-2(1H)-pyrazinones, J. Org. Chem. 76 (2011)
2920-5.
[17] Y. Suzuki, N. Iwata, K. Dobashi, R. Takashima, S. Arulmozhiraja, E. Ishitsubo, N. Matsuo, H.
Tokiwa, Alkanoylation of quinazoline by nucleophilic aromatic substitution: Combined experimental and
computational study, Tetrahedron 74 (2018) 392-400.
[18] B. Chakravarti, T. Akhtar, B. Rai, M. Yadav, J. Akhtar Siddiqui, S.K. Dhar Dwivedi, R. Thakur,
A.K. Singh, A.K. Singh, H. Kumar, K. Khan, S. Pal, S.K. Rath, J. Lal, R. Konwar, A.K. Trivedi, D.
Datta, D.P. Mishra, M.M. Godbole, S. Sanyal, N. Chattopadhyay, A. Kumar, Thioaryl
naphthylmethanone oxime ether analogs as novel anticancer agents, J. Med. Chem. 57 (2014) 8010-8025.
[19] M.L. Liu, X.Y. Pan, T. Yang, W.M. Zhang, T.Q. Wang, H.Y. Wang, H.X. Lin, C.G. Yang, Y.M.
Cui, The synthesis and antistaphylococcal activity of dehydroabietic acid derivatives: Modifications at C-
12, Bioorg. Med. Chem. Lett. 26 (2016) 5492-5496.
[20] Y. Sang, S. Han, C. Pannecouque, E. De Clercq, C. Zhuang, F. Chen, Ligand-Based Design of
Nondimethylphenyl-Diarylpyrimidines with Improved Metabolic Stability, Safety, and Oral
Pharmacokinetic Profiles, J. Med. Chem. 62 (2019) 11430-11436.
[21] E.P. Gillis, K.J. Eastman, M.D. Hill, D.J. Donnelly, N.A. Meanwell, Applications of Fluorine in
Medicinal Chemistry, J. Med. Chem. 58 (2015) 8315-8359.
[22] C. Pannecouque, D. Daelemans, E. De Clercq, Tetrazolium-based colorimetric assay for the
detection of HIV replication inhibitors: revisited 20 years later, Nat. Protoc. 3 (2008) 427-434.
[23] J. Auwerx, T.W. North, B.D. Preston, G.J. Klarmann, E. De Clercq, J. Balzarini, Chimeric human
immunodeficiency virus type 1 and feline immunodeficiency virus reverse transcriptases: Role of the
subunits in resistance/sensitivity to non-nucleoside reverse transcriptase inhibitors, Mol. Pharmacol. 61
(2002) 400-406.
[24] C. Zhuang, S. Narayanapillai, W. Zhang, Y.Y. Sham, C. Xing, Rapid Identification of Keap1–Nrf2
Small-Molecule Inhibitors through Structure-Based Virtual Screening and Hit-Based Substructure Search,
J. Med. Chem. 57 (2014) 1121-1126.
[25] S. Han, Y. Sang, Y. Wu, Y. Tao, C. Pannecouque, E. De Clercq, C. Zhuang, F.E. Chen, Molecular
Hybridization-Inspired Optimization of Diarylbenzopyrimidines as HIV-1 Nonnucleoside Reverse
Transcriptase Inhibitors with Improved Activity against K103N and E138K Mutants and Pharmacokinetic
Profiles, ACS Infect. Dis. (2019), doi:10.1021/acsinfecdis.9b00229.
[26] S. Han, Y. Lei, C. Pannecouque, E. De Clercq, C. Zhuang, F. Chen, Fragment-based discovery of
sulfur-containing diarylbenzopyrimidines as novel nonnucleoside reverse transcriptase inhibitors, Chin.
Chem. Lett. 31 (2020) 764-768.