Pharmacophore-fusing design of pyrimidine sulfonylacetanilides as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bioorg.2020.103595

Twenty-seven derivatives (40–66) were generated by pharmacophore fusing of sulfonylacetanilide-diarylpyrimidine (1) with rilpivirine or biphenyl-diarylpyrimidines. They displayed up to single-digit nanomolar activity against wild-type (WT) virus and vario

Full text of DOI:10.1016/j.bioorg.2020.103595

BioorganicChemistry96(2020)103595
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Bioorganic Chemistry
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Pharmacophore-fusing design of pyrimidine sulfonylacetanilides as potent
non-nucleoside inhibitors of HIV-1 reverse transcriptase
⁎
⁎
Yali Sanga,b, Christophe Pannecouqued, Erik De Clercqd, Chunlin Zhuanga,b, , Fener Chena,b,c,
a Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
b Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, People’s Republic of China
c Institute of Pharmaceutical Science and Technology, Zhejiang University of Technology, 18 Chao Wang Road, 310014 Hangzhou, People’s Republic of China
d Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty-seven derivatives (40–66) were generated by pharmacophore fusing of sulfonylacetanilide-diarylpyr-
imidine (1) with rilpivirine or biphenyl-diarylpyrimidines. They displayed up to single-digit nanomolar activity
against wild-type (WT) virus and various drug-resistant mutant strains in HIV-1-infected MT-4 cells, thereby
targeting the reverse transcriptase (RT) enzyme. Compound 51 displayed exceptionally potent activity against
WT virus (EC50 = 6 nM) and several mutant strains (L100I, EC50 = 8 nM, K103N, EC50 = 6 nM, Y181C,
EC50 = 26 nM, Y188L, EC50 = 122 nM, E138K, EC50 = 26 nM). The structure-activity relationships of the newly
obtained pyrimidine sulfonylacetanilides were also elucidated. Molecular docking analysis explained the activity
and provided a structural insight for follow-up research.
Biphenyl
Rilpivirine
Sulfonylacetanilide
DAPYs
HIV-1
Reverse transcriptase
1. Introduction
After the description of the first NNRTIs in the 1989–1990 [15,16],
six NNRTI-type drugs, nevirapine (NVP), delavirdine (DLV), efavirenz
Acquired immune deficiency syndrome (AIDS) has continued to be a
devastating globally health problem. With the development of new po-
tent antiviral drugs, the deaths (0.77 million) of AIDS have been globally
controlled in 2018 [1] compared to 1.9 million in 2003 [2]. Etiologically,
AIDS is primarily caused by human immunodeficiency virus (HIV) [3].
Reverse transcriptase (RT) plays a critical role in the HIV-1 infective life
cycle, and it has been recognized as an important therapeutic target of
HIV infection [4]. Efficient strategies were used to conduct the discovery
of potent anti-HIV-1 small molecules by inhibition of this enzyme, such
as conformation restriction strategy [5], molecular modelling [6,7] and
virtual screening approaches [8]. Among these small-molecule RT in-
hibitors, more than 50 kinds of non-nucleoside reverse transcriptase in-
hibitors (NNRTIs) were identified [9–10]. They specially bind to the RT
at an allosteric site, located about 10 Å away from the DNA polymerase
catalytic site [11]. During the process of ligand binding, conformational
changes occur in the orientation of some functional residues (e.g. Tyr181
and Tyr188), leading to the development of the NNRTI binding pocket,
namely NNIBP, accommodating the compounds [12]. These NNRTIs
employ a similar binding conformation in the NNIBP, described as a
“butterfly”, “horseshoe”, or “U” mode, providing valuable structural in-
formation for the design of new NNRTIs [13,14].
(EFV), etravirine (ETR), rilpivirine (RPV) and doravirine (DOR), have
been marketed [17]. The second generation NNRTIs, ETR and RPV,
bearing the diarylpyrimidine (DAPY) scaffold, form a flexible “U”
conformation with the RT enzyme, minimizing the loss of binding
stability [18]. Accordingly, they possess a higher genetic barrier than
the first generation NNRTIs (NVP, DLV, EFV), exhibiting excellent
pharmacological effects against mutant viral strains [19]. Un-
fortunately, they were confronted by high cytotoxicity (CC50 < 5 µM)
[20], and meanwhile, their effects are reduced by the emergence of new
drug-resistant mutants upon long-term application, such as K103N and
E138K [21]. Therefore, the development of new DAPY-type derivatives
with favorable physicochemical profiles and better activity, especially
toward mutant strains, is remains highly desirable.
2. Results and discussion
2.1. Design rationale
On the basis of the previous structure-activity relationships (SAR) of
DAPY analogues [19,20–22], a pharmacophore model could be deli-
neated for further optimizations (Fig. 1). In addition to the core
⁎ Corresponding authors at: Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University, Shanghai 200433,
People’s Republic of China.
E-mail addresses: zclnathan@163.com (C. Zhuang), rfchen@fudan.edu.cn (F. Chen).
https://doi.org/10.1016/j.bioorg.2020.103595
Received 1 December 2019; Received in revised form 14 January 2020; Accepted 18 January 2020
Availableonline22January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Y. Sang, et al.
BioorganicChemistry96(2020)103595
disclosed compound (3) favorably influenced the antiviral activity
against the drug-resistant strains [32]. The moiety can form π-π
stacking interactions with Y181, Y188, F227, and W229, making the
left ring region significantly adapt to the allosteric RT binding site.
Thus, this fragment might be favorable at the hydrophobic site of the
model. Collectively, the novel DAPY derivatives were designed by a
pharmacophore fusing strategy on the basis of the present model
(Fig. 2) in order to incorporate advantages to enhance antiviral potency
against WT and drug-resistant HIV-1 viruses.
2.2. Chemistry
The synthetic route of the desired compounds 40–56 is outlined in
Scheme 1. The key intermediates 9a-r were prepared from 4a-b by
protection with a triphenylmethyl group, condensation with amines
and deprotection. Compounds 11a-b were obtained via treating com-
mercially available 10 with the intermediates 4′-hydroxy-[1,1′-bi-
phenyl]-4-carbonitrile or (2E)-3-(4-hydroxy-3,5-dimethylphenyl)-2-
propenenitrile, which was prepared following our established proce-
dure [22,33]. Then, compounds 12a-b were easily prepared by oxida-
tion [34], and they were reacted with 9a-r via the substitution reaction,
leading to the formation of 13–39. Finally, 13–39 were reacted with 3-
chloroperoxybenzoic acid to obtain pyrimidine sulfonylacetanilide de-
rivatives 40–56. The desired compounds were determined by NMR and
MS characterization.
Fig. 1. A pharmacophore model of DAPYs.
diarylprimidine scaffold providing van der Waals and hydrophobic in-
teractions, two features are critical to the antiviral activity. First, a vinyl
or phenyl group is a suitable attachment on the left wing forming π-π
interactions with Y181, Y188 and W229. Second, a hydrogen-bond
(HB) acceptor or donor is favorable to produce hydrogen-bonding in-
teractions with K101 and K103 as well as to modulate the physico-
chemical properties of compounds.
Therefore, applying a pharmacophore fusing approach providing
two privileged scaffolds to these two sites may reveal new chemical
entities (NCE) and expand chemical space with more pharmacological
effects [23]. The sulfonylacetanilide, making the compounds less toxic
with favorable pharmacokinetic profiles, has been widely utilized, i.e.,
in matrix metalloproteinase inhibitors [24], gram-negative anti-
bacterial agents [25], noncompetitive CXCL8 inhibitors [26]. Ad-
ditionally, this motif has also been applied in potent HIV-1 inhibitors
[27,28]. The etravirine-VRX-480773 hybrid (1) with the sulfonylace-
tanilide group previously discovered by our group showed great anti-
HIV-1 activity [29]. This sulfonylacetanilide group was predicted to
form two hydrogen bonds with the main chain of residues K101 and
K103 [30–31], indicating its adaptation at the HB site of the model. The
4-cyanovinyl moiety of RPV (2) and a biphenyl group in our recent
2.3. Antiviral activity and binding prediction analysis
First, the acrylonitrile group of RPV was introduced to pyrimidine
sulfonylacetanilides (1). Eight new compounds (40–47) were synthe-
sized. They displayed promising anti-WT HIV-1 activity in the nano-
molar range (Table 1). Compounds 40–41 showed EC50 values of
5–7 nM, which were similar to those of 1 (EC50 = 2 nM), ETR
(EC50 = 6 nM) and RPV (EC50 = 1 nM). The results showed the ac-
rylonitrile moiety was a suitable attachment for improving anti-HIV-1
potency. Then, the anilide was extended by inserting one or two me-
thylenes to explore the chemical space in the P236 loop (Fig. 1).
Fig. 2. Design of new pyrimidine sulfonylacetanilides by a pharmacophore fusing strategy.
2

Y. Sang, et al.
BioorganicChemistry96(2020)103595
Scheme 1. Synthesis of compounds 40-66a. Reagents and conditions: (a) TFA, CHCl3, 30 °C, 3 h; (b) DIPEA, PyBOP, DCM, 25 °C, 15 h; (c) (Et)3SiH, TFA, DCM,
0–25 °C, 3 h; (d) 4′-hydroxy-[1,1′-biphenyl]-4-carbonitrile or (2E)-3-(4-hydroxy-3,5-dimethylphenyl)-2-propenenitrile, K2CO3, DMF, 80 °C, 4–6 h; (e) m-CPBA, DCM,
0–25 °C, 10 h; (f) 9a-r Na2CO3, acetone, H2O, 0–25 °C, 5 h; (g) m-CPBA, DCM, −45–25 °C, 12 h.
However, the structure-activity relationship (SAR) analysis highlighted
the importance of aniline with dramatically decreased activity of
compounds 42–47. Compounds containing one methylene (42–44) had
EC50 values of 0.944–1.854 µM. Those containing two methylene
(45–47) showed EC50 values of 0.163–0.794 µM. The cytotoxicity was
decreased to be lower than the reference compounds.
42, although the hydrogen bond between the sulphonyl oxygen and
K103 (O]S]O⋯HeN, distance: 2.5 Å) was observed, the extended
right wing missed these essential features especially the benzylamine
locating outside P236 loop (Fig. 3B).
Next, the biphenyl moiety was introduced to obtain biphenyl-pyr-
imidine sulfonylacetanilides (48–51). They exhibited great activity
with EC50 values of 6–34 nM against WT HIV-1 (Table 2). Compound 50
with a bromo group and 51 with a cyano group had similar single-digit
nanomolar activity and SI values of ~4000. Similar extension on the
P236 loop was performed with one more methylene (52–55), leading to
reduced activity (EC50 = 1.28- > 6.41 µM). Compound 55 with a cyano
group had the highest activity (EC50 = 1.28 µM) in these four new
DAPYs. Extending with two more methylenes (compounds 56–58) was
still unfavorable to the activity. If a methyl group was introduced at the
sulfone side into 49 and 51, the corresponding compounds 59–60 had
similar antiviral activity at low nanomolar range, and low cytotoxicity
was observed, indicating the tolerance of methyl moiety on sulfona-
mide fragment. However, the methyl group did not increase the activity
of compounds 61–63 or 64–66 compared with compounds 52–54.
Then, the compounds exhibiting anti-WT HIV-1 activity in the na-
nomolar range were selected to be further evaluated against single-
(L100I, K103N, Y181C, Y188L and E138K) and double-mutant viral
strains (F227L + V106A and RES056) in MT-4 cells, as well as for their
inhibitory ability against HIV-1 RT enzyme (Table 3). As expected,
these compounds exhibited increased inhibitory activities against
single-mutant virus strains with the exception of Y188L, while retaining
inhibitory activity against the HIV-1 RT enzyme. Compound 40 ex-
hibited great inhibitory activity against K103N with an EC50 of
0.006 μM, which was superior to the references NVP, EFV
(EC50 = 0.102, > 10.075 μM, respectively). Compound 41 was active
against L100I, K103N, Y181C, and E138K (EC50 = 0.009, 0.007, 0.021
and 0.015 μM, respectively), comparable to that of ETR (EC50 = 0.007,
0.003, 0.012 and 0.007 μM, respectively). Compound 51 showed low
nanomolar EC50 values toward all of the mutant strains. Notably, it had
an EC50 of 0.008 μM toward the L100I and 0.026 μM toward E138K,
which was more potent than the parent compound 1 (EC50 = 0.067 μM
and 0.310 μM, respectively). Meanwhile, 51 was the most potent in-
hibitor toward the Y188L mutant (EC50 = 0.122 μM) and dual mutant
Then, the binding modes of 40 (EC50 = 5 nM) and 42 with one
more methylene (EC50 = 1.854 µM) bound to wild-type RT were pre-
dicted. They exhibited several common features (Fig. 3). (1) These
compounds employed a similar “U” mode binding conformation in the
NNIBP. (2) Both of their left wing structures formed π-π stacking in-
teractions with the residues Y181, Y188, F227, and W229. The cyano
group additionally formed hydrogen interactions with Y188
(CN⋯HeO, distance = 3.2 Å for 40, 2.1 Å for 42) and residue K223
(CN⋯HeN, distance = 2.7 and 3.1 Å for 40, 3.4 Å for 42). Differently,
the performance of sulfonylacetamide fragment in the NNIBP offered an
explanation for their distinct activity. The sulfonylacetanilide carbonyl
group of 40 had a hydrogen-bonding interaction with K103 (C]
O⋯HeN, distance: 2.0 Å). And the anilineformed two hydrogen bonds
with the hydroxyl group of Y318 (NeH⋯OeH, distance: 3.4 Å) and
residue P236 (NeH⋯O]C, distance = 3.0 Å) (Fig. 3A). For compound
Table 1
Activity and cytotoxicity of compounds 40–47 against WT HIV-1 strain in MT-4
cells.
Compounds
n
X
HIV-1 (WT)
EC50 (µM)a
CC50 (µM)b
SIc
40
0
0
1
1
1
2
2
2
F
0.005
0.007
1.854
> 1.43
0.944
0.163
0.490
0.794
0.002
0.006
0.001
0.00
19.5
6.43
3900
613
3
41
Cl
F
0.00
0.08
4.29
0.97
0.35
0.54
1.25
42
5.29
43
Cl
Br
F
1.43
< 1
4
44
0.27
0.02
0.11
0.24
0.00
0.00
0.00
3.37
variants RES056 (EC50
= 0.642 μM) and F227L + V106A
45
55.53
24.46
39.23
> 6.01
> 4.61
> 3.98
47.19
340
50
(EC50 = 0.226 μM). This was comparable to EFV and better than NVP.
Docking analysis indicated that the best compound 51 formed
strong interactions with a number of key residues in the NNIBP (Fig. 4).
First, the biphenyl fragment fitted well into the hydrophobic site (Y181,
Y188, F227, and W229) and the hydrogen bonds between 51 and re-
sidues Y188 and K223 were retained, consistent with that of 40.
Second, the sulfonylacetanilide moiety protruded into the Pro236 loop.
A hydrogen-bonding interaction (NeH⋯O]C, distance = 2.2 Å) be-
tween the amino hydrogen of the sulfonylacetanilide fragment and the
46
Cl
Br
9.97
8.55
47
49
1
> 3005
> 768
3980
ETR
RPV [16]
a
EC50: The effective concentration required to protect MT-4 cells against
viral cytopathicity by 50%.
b
CC50: The cytotoxic concentration of the compound that reduced the
normal uninfected MT-4 cell viability by 50%.
c
SI: selectivity index, ratio CC50/EC50 (WT).
3

Y. Sang, et al.
BioorganicChemistry96(2020)103595
Fig. 3. Predicted binding modes of 40 (A), and 42 (B) with the WT HIV-1 RT crystal structure (PDB: 6C0N). Hydrogen bonds are indicated as yellow dashed lines. The
Figure was generated using PyMol (http://pymol.sourceforge.net/).
conformation, hydrogen bonds, hydrophobic and electrostatic interac-
Table 2
tions) were observed as in the wild-type RT. Especially, the N103 re-
Activity and cytotoxicity of compounds 48–66 against HIV-1 (WT) strain in MT-
sidue involved in additional hydrogen-bonding interactions with the
pyrimidine nitrogen (NeH⋯N, distance = 2.7 Å) and aniline group
(C]O⋯HeN, distance = 2.1 Å). In the Y181C and Y188L mutant, the
π-π interactions were reduced compared with the wild-type RT, which
together with the loss of the hydrogen bond between the cyano group
4 cells.
and Y188L contributed to the loss of their antiviral activity. For E138K
RT, an additional hydrogen bond between the pyrimidine core and
K101 was observed (distance = 3.2 Å) for maintaining good activity.
3. Conclusion
Compounds
n
R2
R3
X
HIV-1 (WT)
EC50 (µM)a
CC50 (µM)b
SIc
In this study, two series of novel cyanovinyl- and biphenyl-pyr-
imidine sulfonylacetanilides were designed and synthesized via a
pharmacophore fusing strategy. The most potent compound 51 dis-
played an EC50 of 0.006 µM against HIV-1 wt strain, and EC50 of 0.008,
0.006 µM, 0.026 µM, 0.122 µM, 0.026 µM against L100I, K103N,
Y181C, Y188L, and E138K mutants. Molecular modelling further ex-
plained their activity and provided an example for pharmacophore
fusing as a strategy in designing new HIV inhibitors.
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
1
0
0
0
0
1
1
1
1
2
2
2
0
0
1
1
1
1
1
1
H
–
F
0.023
0.034
0.007
0.01 9.15
3.95
3.18
1.79
1.63
398
H
–
Cl
Br
0.01 7.41
0.00 26.91
0.00 24.44
6.41
218
H
–
3844
4073
< 1
< 1
< 1
H
–
CN 0.006
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
F
> 6.41
0.96
H
Cl
Br
> 3.86
> 3.57
3.86
0.98
0.91
H
3.57
H
CN 1.28
0.69
1.86
51.23
15
13.48 40
A preliminary SAR was concluded (Fig. 5). (1) The cyanovinyl and
biphenyl groups on the left wing were beneficial to enhance the anti-
viral ability. (2) Compound 59 and 60 with potent inhibitory activity
against the HIV-1-infected cells showed the tolerance of methyl group
on the sulfonylacetanilide fragment. (3) Modifications of the sulfiny-
lacetamide group showed that extending the right wing was extremely
detrimental to the antiviral ability.
H
F
3.62
8.78
4
H
Cl
Br
Cl
> 8.27
> 8.44
0.025
8.26
8.44
1.94
0.94
< 1
H
< 1
654
Me
Me
Me
Me
Me
H
0.01 16.35
0.00 15.38
5.09
CN 0.009
7.45
2.40
5.17
1708
3
F
5.83
2.11
20.19
12.17
4.76
Cl
Br
F
> 12.18
> 4.77
> 4.56
> 3.82
> 3.21
0.002
< 1
0.89
< 1
4.55
0.75
1.12
0.92
< 1
H
Me Cl
Me Br
3.82
< 1
4. Experimental
H
3.21
< 1
0.00
0.00
0.00
> 6.01
> 4.61
> 3.98
> 3005
> 768
3980
ETR
RPV
0.006
4.1. General
0.001
All reagents and solvents were purchased from commercial sources
and used as received unless specified. Column chromatography was
performed on silica gel (300–400 mesh). Thin-layer chromatography
(TLC) was carried out on 0.25 mm silica gel plates and visualized with
UV light (λ = 254 nm) and/or by staining with ethanolic phospho-
molybdic acid (PMA) or iodine. Proton nuclear magnetic resonance (1H
NMR) and carbon nuclear magnetic resonance (13C NMR) spectra were
recorded in DMSO‑d6 on a Bruker AV-400 spectrometer with tetra-
methylsilane (TMS) as the internal standard. Chemical shifts (δ) are
given in ppm relative to TMS, coupling constants (J) in Hz. Melting
a
EC50: The effective concentration required to protect MT-4 cells against
viral cytopathicity by 50%.
b
CC50: The cytotoxic concentration of the compound that reduced the
normal uninfected MT-4 cell viability by 50%.
c
SI: selectivity index, ratio CC50/EC50 (WT).
carbonyl group of K103 was formed (Fig. 4A). These interactions to-
gether may contribute to the potent antiviral activity. As shown in
Fig. 4B-F, 51 was oriented into the pockets of the L100I, K103N, Y181C,
Y188L, and E138K mutants and several common features (“U” binding
4

Y. Sang, et al.
BioorganicChemistry96(2020)103595
Table 3
Inhibitory activity of the selected compounds toward a panel of clinically relevant HIV-1 mutant strains and HIV-1 RT enzyme.
Compounds
EC50 (µM)a
IC50 (µM)b
L100I
K103N
Y181C
Y188L
E138K
F227L + V106A
RES056
40
0.033
0.009
0.177
0.116
0.049
0.008
0.069
0.106
0.067
2.102
0.044
0.007
0.02
0.006
0.007
0.035
0.056
0.030
0.006
0.035
0.019
0.003
0.00
0.00
0.01
0.01
0.00
0.00
0.01
0.04
0.00
0.045
0.021
0.528
0.275
0.114
0.026
0.146
0.242
0.017
> 15.867
0.007
0.012
0.01
0.01
0.15
0.06
0.03
0.00
0.03
0.02
0.00
2.281
0.494
1.981
0.938
0.977
0.122
1.002
0.726
0.031c
> 15.867
0.292
0.020
1.07
0.10
1.00
0.17
0.14
0.04
0.09
0.01
0.023
0.015
0.112
0.197
0.091
0.026
0.114
0.041
0.310
0.210
0.006
0.007
0.01
0.01
0.01
0.04
0.01
0.01
0.00
0.01
0.07
0.03
0.00
0.01
2.198
0.76
0.47
0.16
0.440
0.228
3.536
4.209
4.999
0.226
3.294
1.433
0.425
> 15.867
0.273
0.045
0.17
0.04
1.00
0.80
0.68
0.02
0.04
0.32
0.31
0.197
0.186
0.162
0.078
1.666
0.142
1.886
2.606
0.065
0.404
0.013
0.011
0.03
0.01
0.12
0.01
1.47
0.01
0.33
0.74
0.00
0.09
0.01
0.01
41
0.00
0.06
0.04
0.01
0.00
0.02
0.00
0.00
0.75
0.01
0.00
48
4.102
4.333
4.301
0.642
2.490
1.303
0.127
> 15.867
0.279
0.020
2.33
0.53
1.60
0.09
0.42
0.13
0.01
49
50
51
59
60
1
NVP
EFV
ETR
> 10.075
0.102
0.003
0.05
0.00
0.00
0.01
0.04
0.01
0.10
0.01
0.03
0.02
a
b
c
EC50: The effective concentration required to protect MT-4 cells against viral cytopathicity by 50%.
IC50: inhibitory concentration of the test compound required to inhibit WT HIV-1 RT polymerase activity by 50%.
0.031: the average of two EC50 measurements.
Fig. 4. Predicted binding modes of 51 with the HIV-1 wt (A), L100I (B), K103N (C), Y188C (D), Y188L (E) and E138K (F) mutant RT crystal structure (PDB: 6C0N).
Mutated residues are depicted as green sticks. Hydrogen bonds are indicated as yellow dashed lines. The Figure was generated using PyMol (http://pymol.
sourceforge.net/).
5

Y. Sang, et al.
BioorganicChemistry96(2020)103595
4.6. General procedure for the preparation of compounds 12a-b
m-CPBA (150 mmol, 3.0 equiv.) was added to a solution of 11a-b
(50 mmol, 1.0 equiv.) in CH2Cl2 (150 mL) in small portions at 0 °C.
Stirring was continued for an additional 10 h at 25 °C until complete
consumption of starting material as judged by TLC. The reaction mix-
ture was washed by sat. Na2SO3, sat. Na2CO3, water and brine. The
organic layer was dried over anhydrous Na2SO4, filtered and condensed
under reduced pressure. The residue was then purified via column
chromatography on silica gel, and eluted with EtOAc/PE to obtain 12a-
b as white solid.
4.7. General procedure for the preparation of compounds 13–39
A solution of intermediate 9a-r (1 mmol, 1 equiv.) in acetone (3 mL)
was added dropwise to an ice-cold mixture of 12a-b (1 mmol, 1.0
equiv.) and Na2CO3 (2.5 mmol, 2.5 equiv.) in acetone/H2O (5/2 mL)
over 20 min. The reaction mixture was stirred overnight at 25 °C, after
which TLC analysis indicated the reaction was complete. The reaction
mixture was neutralized with concentrated hydrochloric acid, filtered
off, washed with cold water and dried to afford the desired product.
Fig. 5. A systematic summary of SAR.
points were measured on WRS-1B digital melting-point apparatus. EI-
MS were recorded on an Agilent 6890N/5975 spectrometer and ESI-MS
was recorded on a Waters Micromass Quattro Micro spectrometer.
High-resolution mass spectra were recorded on a Bruker ApeXIII 7.0
TESLA FTMS.
4.8. General procedure for the preparation of compounds 40–66
m-CPBA (1.25 mmol, 2.5 equiv.) was added to a solution of 13–39
(0.5 mmol, 1.0 equiv.) in CH2Cl2 (5 mL) in small portions at −45 °C.
When the temperature gradually increased, stirring was continued until
complete consumption of starting material as judged by TLC. The re-
action mixture was washed by sat. Na2SO3, sat. Na2CO3, water and
brine. The organic layer was dried over anhydrous Na2SO4, filtered and
condensed under reduced pressure. The crude product was purified by
column chromatography on silica gel (EtOAc/PE = 1:5) to provide
target compounds 40–66.
4.2. General procedure for the preparation of compounds 6a-b
Triphenylmethanol (38.4 mmol, 1.0 eq) and TFA (49.9 mmol,
1.3 eq) was added to 4a-b (38.4 mmol, 1.0 eq) in CHCl3 (40 mL). The
reaction mixture was stirred at 30 °C for 3 h. The volatiles were re-
moved in vacuum. Crystallization with hexane yielded 6a-b as white
solid.
4.3. General procedure for the preparation of compounds 8a-r
4.8.1. (E)-2-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)
sulfonyl)-N-(4-fluorophenyl)acetamide (40)
DIPEA (7.2 mmol, 2.5 eq), PyBOP (3.6 mmol, 1.25 eq) and 6a-b
(2.9 mmol, 1.0 eq) in CH2Cl2 (20 mL) was stirred at 25 °C for 2 h. then,
7a-l (3.2 mmol, 1.1 eq) was added. The mixture was stirred for another
13 h and quenched with H2O (100 mL). The aqueous layer was ex-
tracted with CH2Cl2 (30 mL X 3) and the combined organic layers were
washed with brine, and dried over anhydrous Na2SO4. Purification of
the crude reaction mixture was progressed by column chromatography
(5–30% EtOAc/PE).
Yield: 80%. mp: 140.1–142.7 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
10.63 (s, 1H), 9.06 (d, J = 5.6 Hz, 1H), 7.67 (d, J = 16.7 Hz, 1H),
7.61–7.55 (d, J = 12.4 Hz, 5H), 7.24 (t, J = 8.5 Hz, 2H), 6.50 (d,
J = 16.7 Hz, 1H), 4.65 (s, 2H), 2.13 (s, 6H). 13C NMR (101 MHz,
DMSO‑d6) δ: 169.07, 164.72, 161.29, 159.89, 158.87 (d,
J
F = 242.4 Hz) 150.97, 150.26, 135.06, 132.33, 131.35, 128.93, 121.4C9-
(d, J C-F = 7.9 Hz), 119.27, 115.97 (d, J C-F = 22.4 Hz), 111.15, 97.32,
57.57, 16.37. HRMS (ESI) calcd for C23H19FN4O4S: 466.11, found:
489.100 [M + Na]+
.
4.4. General procedure for the preparation of compounds 9a-r
4.8.2. (E)-N-(4-chlorophenyl)-2-((4-(4-(2-cyanovinyl)-2,6-
dimethylphenoxy)pyrimidin-2-yl)sulfonyl)acetamide (41)
TFA (19.0 mmol, 10.0 eq) and (Et)3SiH (3.8 mmol, 2.0 eq) were
added to 8a-r (1.9 mmol, 1.0 eq) in CH2Cl2 (15 mL) at 0 °C. The mixture
was stirred at 25 °C for 3 h. Volatiles were removed in vacuo. The crude
product was crystallized from cold hexane to obtain the desired com-
pounds as white solid.
Yield: 54%. mp: 152.5–154.8 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
10.59 (s, 1H), 8.98 (d, J = 5.7 Hz, 1H), 7.60 (d, J = 16.7 Hz, 1H),
7.53–7.47 (m, 5H), 7.39 (s, 1H), 7.37 (s, 1H), 6.43 (d, J = 16.7 Hz, 1H),
4.58 (s, 2H), 2.05 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.06,
164.68, 161.30, 160.14, 150.96, 150.26, 137.58, 132.33, 131.34,
129.29, 128.92, 128.17, 121.23, 119.26, 111.18, 97.32, 57.64, 16.37.
4.5. General procedure for the preparation of compounds 11a-b
HRMS (ESI) calcd for C23H19ClN4O4S: 482.08, found: 505.069
[M + Na]+
.
4′-hydroxy-[1,1′-biphenyl]-4-carbonitrile or (2E)-3-(4-hydroxy-3,5-
dimethylphenyl)-2-propenenitrile (50 mmol, 1.0 equiv.) was dissolved
in DMF (80 mL) in the presence of K2CO3 (150 mmol, 3.0 equiv.),
followed by addition of the 4-chloro-2-(methylthio)pyrimidine
(50 mmol, 1.0 equiv.). The reaction mixture was stirred at 80 °C for
4–6 h (monitored by TLC), and then the reaction was cooled to ambient
temperature and poured into 400 mL H2O yielding a precipitate. The
precipitate was collected by filtration and the residue was then purified
via column chromatography on silica gel, and eluted with EtOAc/PE to
obtain 11a-b as white solid.
4.8.3. (E)-2-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)
sulfonyl)-N-(4-fluorobenzyl)acetamide (42)
Yield: 77%. mp: 130.7–132.0 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.81 (t, J = 5.5 Hz, 1H), 7.61 (d, J = 16.7 Hz,
1H), 7.51 (s, 2H), 7.47 (d, J = 5.7 Hz, 1H), 7.31–7.21 (m, 2H), 7.14 (t,
J = 8.7 Hz, 2H), 6.44 (d, J = 16.7 Hz, 1H), 4.42 (s, 2H), 4.22 (d,
J = 5.7 Hz, 2H), 2.07 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.05,
164.80, 161.76 (d, J
= 244.4 Hz), 161.26, 161.16, 151.00, 150.24,
C-F
135.04 (d, J
= 3.0 Hz), 132.32, 131.98, 131.42, 129.69 (d, J
C-F
C-
6

Y. Sang, et al.
BioorganicChemistry96(2020)103595
= 8.1 Hz), 129.14, 128.91, 119.26, 115.52 (d, J
= 21.3 Hz),
4.8.9. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)pyrimidin-
F
C-F
111.02, 97.33, 56.63, 42.06, 16.37. LCMS (ESI) calcd for
2-yl)sulfonyl)-N-(4-fluorophenyl)acetamide (48)
C
24H21FN4O4S: 480.13, found: 503.609 [M + Na]+
.
Yield: 63%. mp: 158.3–160.8 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
10.47 (s, 1H), 8.98 (d, J = 5.7 Hz, 1H), 7.95–7.85 (m, 4H), 7.56 (s, 2H),
7.53–7.42 (m, 3H), 7.14 (t, J = 8.8 Hz, 2H), 4.60 (s, 2H), 2.12 (s, 6H).
13C NMR (101 MHz, DMSO‑d6) δ: 169.29, 164.83, 161.21, 158.87 (d, J
4.8.4. (E)-N-(4-chlorobenzyl)-2-((4-(4-(2-cyanovinyl)-2,6-
dimethylphenoxy)pyrimidin-2-yl)sulfonyl)acetamide (43)
Yield: 73%. mp: 148.7–151.5 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.85 (t, J = 5.8 Hz, 1H), 7.61 (d, J = 16.7 Hz,
1H), 7.51 (s, 2H), 7.47 (d, J = 5.7 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H),
7.25 (d, J = 8.4 Hz, 2H), 6.44 (d, J = 16.7 Hz, 1H), 4.42 (s, 2H), 4.23
(d, J = 5.8 Hz, 2H), 2.06 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ:
169.05, 164.77, 161.34, 161.17, 151.00, 150.24, 137.95, 132.32,
132.03, 131.42, 129.53, 128.91, 128.72, 119.26, 111.04, 97.35, 56.64,
42.08, 16.38. LCMS (ESI) calcd for C24H21ClN4O4S: 496.10, found:
C-F = 242.4 Hz), 159.90, 149.59, 144.22, 136.59, 135.04 (d, J
C-
C-
=
2.5 Hz), 133.20, 131.32, 128.15, 127.97, 121.48 (d, J
F
F
= 7.9 Hz), 119.33, 115.94 (d, J
= 22.3 Hz), 111.05, 110.51,
57.60, 16.51. HRMS (ESI) calcd foCr-FC27H21FN4O4S: 516.13, found:
539.116 [M + Na]+
.
4.8.10. N-(4-chlorophenyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (49)
Yield: 75%. mp: 170.0–172.6 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
10.55 (s, 1H), 9.02–8.94 (m, 1H), 7.95–7.79 (m, 4H), 7.56–7.42 (m,
5H), 7.35 (d, J = 7.5 Hz, 2H), 4.62 (s, 2H), 2.12 (s, 6H). 13C NMR
(101 MHz, DMSO‑d6) δ: 169.30, 164.82, 161.21, 160.15, 149.59,
144.21, 137.57, 136.59, 133.18, 131.30, 129.24, 128.20, 128.13,
127.95, 121.20, 119.33, 111.08, 110.51, 57.66, 16.52. HRMS (ESI)
497.555 [M + H]+
.
4.8.5. (E)-N-(4-bromobenzyl)-2-((4-(4-(2-cyanovinyl)-2,6-
dimethylphenoxy)pyrimidin-2-yl)sulfonyl)acetamide (44)
Yield: 60%. mp: 159.0–161.2 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.97 (d, J = 5.7 Hz, 1H), 8.88 (t, J = 5.7 Hz, 1H), 7.64 (d, J = 16.7 Hz,
1H), 7.53 (d, J = 6.7 Hz, 2H), 7.49 (d, J = 5.7 Hz, 1H), 7.21 (d,
J = 8.2 Hz, 1H), 6.47 (d, J = 16.7 Hz, 1H), 4.45 (s, 2H), 4.23 (d,
J = 5.8 Hz, 2H), 2.08 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.06,
164.79, 161.36, 161.20, 151.02, 150.27, 138.42, 132.35, 131.66,
131.44, 129.93, 128.94, 120.51, 119.29, 111.06, 97.40, 56.65, 42.16,
16.41. HRMS (ESI) calcd for C24H21BrN4O4S: 540.05, found: 563.036
calcd for C27H21ClN4O4S: 532.10, found: 555.086 [M + Na]+
.
4.8.11. N-(4-bromophenyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (50)
Yield: 66%. mp: 153.6–155.4 °C. 1H NMR (400 MHz, DMSO‑d6)
δ:10.54 (s, 1H), 8.98 (d, J = 5.7 Hz, 1H), 7.90 (q, J = 8.3 Hz, 4H), 7.56
(s, 2H), 7.52–7.41 (m, 5H), 4.60 (s, 2H), 2.12 (s, 6H). 13C NMR
(101 MHz, DMSO‑d6) δ: 169.28, 164.78, 161.23, 160.17, 149.59,
144.23, 137.98, 136.58, 133.22, 132.18, 131.31, 128.15, 127.97,
121.57, 119.34, 116.24, 111.11, 110.50, 57.66, 16.53. LCMS (ESI)
[M + Na]+
.
4.8.6. (E)-2-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)
sulfonyl)-N-(4-fluorophenethyl)acetamide (45)
calcd for C27H21BrN4O4S: 576.05, found: 599.901 [M + H]+
.
Yield: 75%. mp: 174.3–176.5 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.40 (d, J = 5.1 Hz, 1H), 7.60 (d,
J = 16.7 Hz, 1H), 7.51 (s, 2H), 7.47 (d, J = 5.7 Hz, 1H), 7.22 (dd,
J = 8.3, 5.8 Hz, 2H), 7.09 (t, J = 8.9 Hz, 2H), 6.43 (d, J = 16.7 Hz,
1H), 4.32 (s, 2H), 3.21 (dd, J = 12.8, 6.7 Hz, 2H), 2.65 (t, J = 7.0 Hz,
2H), 2.08 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.02, 164.82,
4.8.12. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
pyrimidin-2-yl)sulfonyl)-N-(4-cyanophenyl)acetamide (51)
Yield: 65%. mp: 138.5–141.2 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
10.82 (s, 1H), 8.99 (d, J = 5.7 Hz, 1H), 7.88 (dd, J = 18.4, 8.3 Hz, 4H),
7.75 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.52 (s, 2H), 7.48 (d,
J = 5.7 Hz, 1H), 4.67 (s, 2H), 2.11 (s, 6H). 13C NMR (101 MHz,
DMSO‑d6) δ:169.30, 164.76, 161.26, 160.87, 149.57, 144.18, 142.69,
136.57, 133.82, 133.18, 131.26, 128.12, 127.93, 119.75, 119.33,
119.28, 111.17, 110.51, 106.44, 57.70, 16.50. LCMS (ESI) calcd for
161.37 (d, J
= 242.4 Hz), 161.16, 151.01, 150.23, 135.65 (d, J
= 3.0 Hz)C,-F132.33, 131.42, 130.95 (d, J
= 7.9 Hz), 128.90,
C-
F
C-F
119.25, 115.45 (d, J
= 21.0 Hz), 110.97, 97.32, 56.54, 40.95,
C-F
34.26, 16.39. HRMS (ESI) calcd for C25H23FN4O4S: 494.14, found:
517.132 [M + Na]+
.
C
28H21N5O4S: 523.13, found: 524.672 [M + H]+
.
4.8.7. (E)-N-(4-chlorophenethyl)-2-((4-(4-(2-cyanovinyl)-2,6-
4.8.13. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
pyrimidin-2-yl)sulfonyl)-N-(4-fluorobenzyl)acetamide (52)
dimethylphenoxy)pyrimidin-2-yl)sulfonyl)acetamide (46)
Yield: 80%. mp: 177.1–179.6 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.38 (t, J = 5.5 Hz, 1H), 7.60 (d, J = 16.7 Hz,
1H), 7.51 (s, 2H), 7.47 (d, J = 5.7 Hz, 1H), 7.32 (d, J = 8.3 Hz, 2H),
7.21 (d, J = 8.3 Hz, 2H), 6.42 (d, J = 16.7 Hz, 1H), 4.32 (s, 2H), 3.22
(dd, J = 12.9, 6.8 Hz, 2H), 2.65 (t, J = 7.0 Hz, 2H), 2.08 (s, 6H). 13C
NMR (101 MHz, DMSO‑d6) δ: 169.01, 164.80, 161.18, 151.01, 150.22,
138.54, 132.33, 131.42, 131.33, 131.08, 128.90, 128.69, 119.24,
110.98, 97.32, 56.55, 40.70, 34.37, 16.39. LCMS (ESI) calcd for
Yield: 71%. mp: 156.7–159.0 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.6 Hz, 1H), 8.81 (d, J = 5.2 Hz, 1H), 7.92 (s, 4H), 7.60 (s,
2H), 7.45 (d, J = 5.5 Hz, 1H), 7.31–7.20 (m, 2H), 7.12 (t, J = 8.6 Hz,
2H), 4.45 (s, 2H), 4.23 (d, J = 5.2 Hz, 2H), 2.13 (s, 6H). 13C NMR
(101 MHz, DMSO‑d6) δ: 169.27, 164.90, 161.74 (d, J
= 265.1 Hz),
= 2.8 Hz),
C-F
161.30, 161.11, 149.63, 144.24, 136.59, 135.04 (d, J
133.26, 131.41, 129.70 (d, J
= 8.1 Hz), 128.16, 1C2-7F.99, 119.35,
C-F
115.50 (d, J
= 21.2 Hz), 110.93, 110.52, 56.62, 42.07, 16.52.
C-F
C
25H23ClN4O4S: 510.11, found: 511.555 [M + H]+
.
HRMS (ESI) calcd for
C28H23FN4O4S: 530.14, found: 553.131
[M + Na]+
.
4.8.8. (E)-N-(4-bromophenethyl)-2-((4-(4-(2-cyanovinyl)-2,6-
dimethylphenoxy)pyrimidin-2-yl)sulfonyl)acetamide (47)
4.8.14. N-(4-chlorobenzyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (53)
Yield: 81%. mp: 182.8–185.4 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.39 (t, J = 5.4 Hz, 1H), 7.60 (d, J = 16.7 Hz,
1H), 7.51 (s, 2H), 7.46 (t, J = 6.8 Hz, 3H), 7.15 (d, J = 8.3 Hz, 2H),
6.42 (d, J = 16.7 Hz, 1H), 4.32 (s, 2H), 3.22 (dd, J = 12.8, 6.7 Hz, 2H),
2.64 (t, J = 7.0 Hz, 2H), 2.08 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ:
169.01, 164.80, 161.18, 151.01, 150.22, 138.96, 132.33, 131.61,
131.49, 131.42, 128.90, 119.79, 119.25, 110.98, 97.33, 56.54, 40.64,
34.43, 16.40. HRMS (ESI) calcd for C25H23BrN4O4S: 554.06, found:
Yield: 79%. mp: 166.3–168.9 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.83 (t, J = 5.6 Hz, 1H), 7.93 (s, 4H), 7.61 (s,
2H), 7.46 (d, J = 5.7 Hz, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.24 (d,
J = 8.3 Hz, 2H), 4.45 (s, 2H), 4.23 (d, J = 5.7 Hz, 2H), 2.13 (s, 6H). 13
C
NMR (101 MHz, DMSO‑d6) δ: 169.25, 164.87, 161.36, 161.11, 149.62,
144.25, 137.95, 136.59, 133.28, 132.02, 131.40, 129.54, 128.70,
128.16, 128.00, 119.34, 110.95, 110.53, 56.64, 42.09, 16.53. LCMS
577.050 [M + Na]+
.
(ESI) calcd for C28H23ClN4O4S: 546.11, found: 569.697 [M + Na]+
.
7

Y. Sang, et al.
BioorganicChemistry96(2020)103595
4.8.15. N-(4-bromobenzyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (54)
121.42, 119.32, 111.21, 110.52, 63.31, 16.51, 12.74. HRMS (ESI) calcd
for C28H23ClN4O4S: 546.11, found: 569.101 [M + Na]+
.
Yield: 60%. mp: 169.4–171.0 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.84 (t, J = 5.6 Hz, 1H), 7.94 (d, J = 8.4 Hz,
4H), 7.60 (s, 2H), 7.47 (dd, J = 11.5, 7.0 Hz, 3H), 7.18 (d, J = 8.2 Hz,
2H), 4.45 (s, 2H), 4.21 (d, J = 5.7 Hz, 2H), 2.13 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6) δ: 169.25, 164.86, 161.37, 161.11, 149.62,
144.24, 138.38, 136.59, 133.28, 131.61, 131.41, 129.91, 128.15,
128.00, 120.49, 119.35, 110.96, 110.53, 56.63, 42.16, 16.53. LCMS
4.8.21. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
pyrimidin-2-yl)sulfonyl)-N-(4-cyanophenyl)propanamide (60)
Yield: 61%. mp: 105.8–107.2 °C. 1H NMR (400 MHz, DMSO‑d6) δ
10.44 (s, 1H), 8.97 (d, J = 5.7 Hz, 1H), 7.89 (q, J = 8.1 Hz, 4H), 7.62 –
7.49 (m, 4H), 7.45 (d, J = 5.6 Hz, 1H), 7.15 (t, J = 8.7 Hz, 2H), 4.68 (q,
J = 6.4 Hz, 1H), 2.12 (s, 6H), 1.48 (d, J = 6.8 Hz, 3H). 13C NMR
(101 MHz, DMSO‑d6) δ 169.36, 164.59, 163.22, 161.22, 160.08, 157.68,
149.65, 144.24, 136.69, 135.10, 133.23, 131.33, 128.17, 127.96, 121.75,
121.67, 119.32, 115.99, 115.77, 111.16, 110.52, 63.23, 16.49, 12.75.
(ESI) calcd for C28H23BrN4O4S: 590.06, found: 613.772 [M + Na]+
.
4.8.16. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
HRMS (ESI) calcd for C29H23N5O4S: 537.15, found: 536.14 [M - H]−
.
pyrimidin-2-yl)sulfonyl)-N-(4-cyanobenzyl)acetamide (55)
Yield: 76%. mp: 174.8–176.5 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.8 Hz, 2H), 7.91 (s, 4H), 7.77 (d, J = 8.0 Hz, 2H), 7.59 (s,
4.8.22. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
2H), 7.47–7.39 (m, 3H), 4.50 (s, 2H), 4.34 (d, J = 5.6 Hz, 2H), 2.12 (s,
pyrimidin-2-yl)sulfonyl)-N-(4-fluorobenzyl)propanamide (61)
13C
6H).
NMR (101 MHz, DMSO‑d6) δ:169.28, 164.84, 161.60, 161.11,
Yield: 60%. mp: 197.4–200.1 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.6 Hz, 1H), 8.83 (s, 1H), 7.90 (q, J = 8.1 Hz, 4H), 7.59 (s,
2H), 7.46 (d, J = 5.6 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.25 (d,
J = 8.1 Hz, 2H), 4.55 (q, J = 6.7 Hz, 1H), 4.31 (dd, J = 15.3, 5.9 Hz,
1H), 4.19 (dd, J = 15.3, 5.0 Hz, 1H), 2.14 (s, 6H), 1.41 (d, J = 6.9 Hz,
3H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.28, 164.73, 164.69, 161.75
149.60, 144.81, 144.21, 136.60, 133.26, 132.70, 131.39, 128.38, 128.14,
127.97, 119.35, 119.28, 110.98, 110.53, 110.22, 56.69, 42.49, 16.52.
LCMS (ESI) calcd for C29H23N5O4S: 537.15, found: 538.609 [M + H]+
.
4.8.17. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
pyrimidin-2-yl)sulfonyl)-N-(4-fluorophenethyl)acetamide (56)
(d, J
= 243.4 Hz), 161.08, 149.71, 144.27, 136.66, 135.15 (d, J
C-F
C-
Yield: 74%. mp: 155.3–157.2 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
9.05 (d, J = 5.7, 1H), 8.48 (s, 1H), 8.05–7.96 (m, 4H), 7.70 (s, 2H),
7.55 (d, J = 5.7, 1H), 7.35–7.26 (m, 2H), 7.16 (t, J = 8.7, 2H), 4.45 (s,
2H), 3.31 (dd, J = 12.9, 6.7, 2H), 2.74 (t, J = 7.0, 2H), 2.24 (s, 6H).
= 2.8 Hz), 133.25, 131.40, 129.61 (d, J
= 8.1 Hz), 128.15,
= 21.2 Hz),C1-F11.12, 110.52, 62.35,
F
127.97, 119.34, 115.44 (d, J
C-F
42.23, 16.52, 12.59. LCMS (ESI) calcd for C29H25FN4O4S: 544.16,
found: 567.742 [M + Na]+
.
13C NMR (101 MHz, DMSO‑d6) δ: 169.32, 165.00, 161.44 (d, J
C-
C-
F = 242.4 Hz), 161.28, 161.20, 149.73, 144.33, 136.67, 135.73 (d, J
4.8.23. N-(4-chlorobenzyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
= 3.0 Hz), 133.34, 131.49, 131.03 (d, J
= 7.9 Hz), 128.24,
F
= 21.0 Hz),C1-F10.98, 110.60, 56.65,
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)propanamide (62)
128.07, 119.43, 115.52 (d, J
C-F
Yield: 72%. mp: 160.6–162.5 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.94 (d, J = 5.7 Hz, 1H), 8.80 (t, J = 5.3 Hz, 1H), 7.91 (q, J = 8.2 Hz,
4H), 7.60 (s, 2H), 7.46 (d, J = 5.6 Hz, 1H), 7.31–7.21 (m, 2H), 7.11 (t,
J = 8.7 Hz, 2H), 4.54 (q, J = 6.7 Hz, 1H), 4.31 (dd, J = 15.1, 5.9 Hz,
1H), 4.18 (dd, J = 15.1, 5.0 Hz, 1H), 2.14 (s, 6H), 1.40 (d, J = 6.9 Hz,
3H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.28, 164.76, 164.71, 161.08,
149.71, 144.26, 138.05, 136.66, 133.25, 131.97, 131.39, 129.49,
128.65, 128.14, 127.96, 119.34, 111.14, 110.52, 62.35, 42.29, 16.53,
12.56. LCMS (ESI) calcd for C29H25ClN4O4S: 560.13, found: 583.570
41.04, 34.35, 16.63. LCMS (ESI) calcd for C29H25FN4O4S: 544.16,
found: 567.805 [M + Na]+
.
4.8.18. N-(4-chlorophenethyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (57)
Yield: 53%. mp: 162.9–164.7 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.39 (d, J = 5.4 Hz, 1H), 7.96–7.86 (m, 4H),
7.60 (s, 2H), 7.46 (d, J = 5.7 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.20 (d,
J = 8.4 Hz, 2H), 4.35 (s, 2H), 3.22 (dd, J = 12.7, 6.7 Hz, 2H), 2.65 (t,
J = 6.9 Hz, 2H), 2.14 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.22,
164.89, 161.22, 161.11, 149.64, 144.24, 138.54, 136.58, 133.25,
131.40, 131.31, 131.07, 128.66, 128.15, 127.98, 119.34, 110.91,
110.51, 56.55, 40.71, 34.37, 16.54. HRMS (ESI) calcd for
[M + Na]+
.
4.8.24. N-(4-bromobenzyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)propanamide (63)
Yield: 64%. mp: 132.1–133.9 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.82 (t, J = 5.6 Hz, 1H), 7.90 (q, J = 8.5 Hz,
4H), 7.59 (s, 2H), 7.52–7.41 (m, 3H), 7.19 (d, J = 8.3 Hz, 2H), 4.54 (q,
J = 6.9 Hz, 1H), 4.29 (dd, J = 15.4, 6.1 Hz, 1H), 4.17 (dd, J = 15.4,
5.3 Hz, 1H), 2.14 (s, 6H), 1.41 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ: 169.28, 164.76, 164.70, 161.08, 149.71, 144.26, 138.49,
136.66, 133.26, 131.57, 131.40, 129.87, 128.15, 127.97, 120.42,
119.34, 111.14, 110.53, 62.35, 42.34, 16.54, 12.56. HRMS (ESI) calcd
C
29H25ClN4O4S: 560.13, found: 583.117 [M + Na]+
.
4.8.19. N-(4-bromophenethyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (58)
Yield: 61%. mp: 165.3–167.9 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.95 (d, J = 5.7 Hz, 1H), 8.38 (t, J = 5.4 Hz, 1H), 7.91 (q, J = 8.4 Hz,
4H), 7.61 (s, 2H), 7.47 (d, J = 5.7 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H),
7.14 (d, J = 8.2 Hz, 2H), 4.34 (s, 2H), 3.22 (dd, J = 12.8, 6.6 Hz, 2H),
2.63 (t, J = 6.9 Hz, 2H), 2.15 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ:
169.21, 164.88, 161.21, 161.11, 149.65, 144.24, 138.96, 136.58,
133.26, 131.58, 131.49, 131.40, 128.15, 127.99, 119.77, 119.35,
110.92, 110.51, 56.56, 40.64, 34.42, 16.55. LCMS (ESI) calcd for
for C29H25BrN4O4S: 604.08, found: 627.067 [M + Na]+
.
4.8.25. 2-((4-((4′-cyano-3,5-dimethyl-[1,1′-biphenyl]-4-yl)oxy)
pyrimidin-2-yl)sulfonyl)-N-(1-(4-fluorophenyl)ethyl)acetamide (64)
Yield: 83%. mp: 101.8–103.5 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.93 (d, J = 5.7 Hz, 1H), 8.78 (d, J = 7.8 Hz, 1H), 7.98–7.88 (m, 4H),
7.60 (s, 2H), 7.43 (d, J = 5.7 Hz, 1H), 7.31 (dd, J = 8.5, 5.6 Hz, 2H),
7.13 (t, J = 8.9 Hz, 2H), 4.80 (t, J = 7.2 Hz, 1H), 4.42 (s, 2H), 2.11 (s,
6H), 1.32 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6) δ:
C
29H25BrN4O4S: 604.08, found: 627.832 [M + Na]+
.
4.8.20. N-(4-chlorophenyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)propanamide (59)
Yield: 55%. mp: 117.0–118.3 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
10.52 (s, 1H), 8.97 (d, J = 5.7 Hz, 1H), 7.88 (q, J = 8.4 Hz, 4H),
7.58–7.52 (m, 4H), 7.45 (d, J = 5.7 Hz, 1H), 7.36 (d, J = 8.8 Hz, 2H),
4.69 (d, J = 7.0 Hz, 1H), 2.12 (s, 6H), 1.48 (d, J = 6.9 Hz, 3H). 13C
NMR (101 MHz, DMSO‑d6) δ: 169.37, 164.52, 163.43, 161.22, 149.65,
144.23, 137.65, 136.68, 133.22, 131.32, 129.20, 128.16, 127.95,
169.23, 164.89, 161.56 (d, J
= 246.4 Hz), 161.08, 149.61, 144.25,
140.19 (d, J
= 3.0 Hz), C1-F36.58, 133.28, 131.40, 128.36 (d, J
= 8.1 Hz),C1-F28.16, 128.00, 119.34, 115.43 (d, J
= 21.2 HzC),-
F
110.86, 110.53, 56.59, 48.19, 22.70, 16.51. LCMS C(-FESI) calcd for
C
29H25FN4O4S: 544.16, found: 567.742 [M + Na]+
.
8

Y. Sang, et al.
BioorganicChemistry96(2020)103595
4.8.26. N-(1-(4-chlorophenyl)ethyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (65)
the dsDNA quantitation reagent PicoGreen. This reagent shows a pro-
nounced 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 [37]. This condition was met in the assay. A
poly(rA) template of approximately 350 bases long, and an oligo(dT)16
primer, were annealed in a molar ratio of 1:1.2 (60 min. at room
temperature). Fifty-two ng of the RNA/DNA was brought into each well
of a 96-well plate in a volume of 20 µL polymerization buffer (60
mMTris-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 reaction mixtures were incubated at 25 °C for 40 min
and then stopped by the addition of EDTA (15 mM fc). Heteroduplexes
were then detected by addition of PicoGreen. Signals were read using
an excitation wavelength of 490 nm and emission detection at 523 nm
using a spectrofluorometer (Safire 2, Tecan). To test the 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 contain 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.
Yield: 76%. mp: 140.6–142.9 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.93 (d, J = 5.7 Hz, 1H), 8.81 (d, J = 7.7 Hz, 1H), 7.98–7.88 (m, 4H),
7.60 (s, 2H), 7.43 (d, J = 5.7 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.30 (d,
J = 8.4 Hz, 2H), 4.88 – 4.71 (m, 1H), 4.42 (s, 2H), 2.11 (s, 6H), 1.32 (d,
J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.23, 164.87,
161.08, 160.41, 149.60, 144.25, 143.08, 136.59, 133.28, 131.85,
131.40, 128.68, 128.30, 128.14, 128.00, 119.34, 110.87, 110.54,
56.59, 48.30, 22.55, 16.51. LCMS (ESI) calcd for C29H25ClN4O4S:
560.13, found: 583.633 [M + Na]+
.
4.8.27. N-(1-(4-bromophenyl)ethyl)-2-((4-((4′-cyano-3,5-dimethyl-[1,1′-
biphenyl]-4-yl)oxy)pyrimidin-2-yl)sulfonyl)acetamide (66)
Yield: 74%. mp: 137.5–139.0 °C. 1H NMR (400 MHz, DMSO‑d6) δ:
8.93 (d, J = 5.7 Hz, 1H), 8.81 (d, J = 7.6 Hz, 1H), 7.98–7.89 (m, 4H),
7.60 (s, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 5.7 Hz, 1H), 7.24 (d,
J = 8.4 Hz, 2H), 4.84–4.70 (m, 1H), 4.42 (s, 2H), 2.10 (s, 6H), 1.31 (d,
J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6) δ: 169.22, 164.87,
161.08, 160.42, 149.60, 144.24, 143.53, 136.59, 133.29, 131.60,
131.40, 128.68, 128.14, 128.01, 120.32, 119.34, 110.88, 110.54,
56.58, 48.37, 22.51, 16.51. HRMS (ESI) calcd for C29H25BrN4O4S:
604.08, found: 629.064 [M + Na]+
4.9. Antiviral activity assays
.
4.10. Molecular modeling
4.9.1. In vitro anti-HIV assay
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 con-
tained 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 hydro-
phobic, polar, repulsive, entropic and solvation terms, was trained to
estimate the binding energy. Prior to docking, the protein was prepared
by removing water molecules, the ligand ETR, and other unnecessary
small molecules from the crystal structure of the HIV-1 RT complex.
Simultaneously, hydrogen atoms were added to the protein. Surflex-
Dock default settings were used for other parameters, such as the
number of starting conformations per molecule (set to 0), the size to
expand search grid (set to 8 Å), the maximum number of the rotatable
bonds per molecule (set to 100), and the maximum number of poses per
ligand (set to 20). During the docking procedure, all of the single bonds
in amino acid residue side-chains inside the defined RT binding pocket
were regarded as rotatable or flexible, and the ligand was allowed to
rotate at all single bonds and move flexibly within the tentative binding
pocket. The atomic charges were recalculated using the Kollman all-
atom approach for the protein and the Gasteiger-Hückel approach for
the ligand. The binding interaction energy was calculated, including
van der Waals, electrostatic, and torsional energy terms defined in the
Tripos force field. The structure optimization was performed for more
than 10,000 generations using a genetic algorithm, and the 20-best-
scoring ligand-protein complexes were kept for the further analyses.
The -log(Kd)2 values of the 20-best-scoring complexes, which re-
presented 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.
Evaluation of the antiviral activity of the compounds against wild-
type HIV-1, and mutante strains (L100I, K103N, Y181C, Y188L, E138K,
F227L + V106A, and RES056) in MT-4 cells was performed using the
MTT assay as previously described [35]. 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 ex-
periment. 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-in-
fected 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 micro-
titer 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 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) by mitochondrial dehydrogenase activity in metabo-
lically 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 wa-
velengths (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 re-
duced the absorbance (OD540) of the mock-infected control sample by
50%. The concentration achieving 50% protection against the cyto-
pathic effect of the virus in infected cells was defined as the 50% ef-
fective concentration (EC50).
4.9.2. HIV-1 RT inhibition assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and pur-
ified as previously described [36]. The RT assay was performed with the
EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, In-
vitrogen), as described by the Manufacturer. The assay was based on
9

Y. Sang, et al.
BioorganicChemistry96(2020)103595
Declaration of Competing Interest
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The authors declare that they have no known competing financial
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ence the work reported in this paper.
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Acknowledgments
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(2019) 7322–7339.
We gratefully acknowledge the generous support provided by the
National Natural Science Foundation of P. R. China (81872791,
21372050) and the Young Elite Scientists Sponsorship Program by the
China Association for Science and Technology (2017QNRC061). The
technical assistance of Mr. Kris Uyttersprot, Mrs. Kristien Erven, and
Mrs. Cindy Heens for the HIV experiments and HIV RT polymerase
assays is gratefully acknowledged.
[18] 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
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Appendix A. Supplementary material
[21] 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
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K103N and E138K mutants and pharmacokinetic profiles, ACS Infect. Dis. (2019),
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