Lead optimization to improve the antiviral potency of 2-aminobenzamide derivatives targeting HIV-1 Vif-A3G axis

Article abstract of DOI:10.1016/j.ejmech.2021.113680

The viral infectivity factor (Vif)–apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) axis has been recognized as a valid target for developing novel small-molecule therapies for acquired immune deficiency syndrome (AIDS) or for enhancing innate immunity against viruses. Our previous work reported the novel Vif antagonist 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide (2) with strong antiviral activity. In this work, through optimizations of ring C of 2, we discovered the more potent compound 6m with an EC₅₀ of 0.07 μM in non-permissive H9 cells, reflecting an approximately 5-fold enhancement of antiviral activity compared to that of 2. Western blotting indicated that 6m more strongly suppressed the defensive protein Vif than 2 at the same concentration. Furthermore, 6m suppressed the replication of various clinical drug-resistant HIV strains (FI, NRTI, NNRTI, IN and PI) with relatively high efficacy. These results suggested that compound 6m is a more potent candidate for treating AIDS.

Full text of DOI:10.1016/j.ejmech.2021.113680

European Journal of Medicinal Chemistry 224 (2021) 113680
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Lead optimization to improve the antiviral potency of 2-
aminobenzamide derivatives targeting HIV-1 Vif-A3G axis
,
,
,
Xinxin Zhong a 1, Ronghua Luo b 1, Guoyi Yan c 1, Kai Ran a, Huifang Shan a, Jie Yang a,
Yuanyuan Liu a, Su Yu a, Chunlan Pu a, Yongtang Zheng b **, Rui Li a
,
, *
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China
b Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of
Zoology,Chinese Academy of Sciences, Kunming, Yunnan, 650223, PR China
c School of Pharmacy, Henan University, Kaifeng, Henan, 475001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The viral infectivity factor (Vif)eapolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G
(APOBEC3G) axis has been recognized as a valid target for developing novel small-molecule therapies for
acquired immune deficiency syndrome (AIDS) or for enhancing innate immunity against viruses. Our
previous work reported the novel Vif antagonist 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sul-
fonyl)benzamide (2) with strong antiviral activity. In this work, through optimizations of ring C of 2, we
Received 28 December 2020
Received in revised form
27 April 2021
Accepted 27 June 2021
Available online 1 July 2021
discovered the more potent compound 6m with an EC50 of 0.07 mM in non-permissive H9 cells, reflecting
an approximately 5-fold enhancement of antiviral activity compared to that of 2. Western blotting
indicated that 6m more strongly suppressed the defensive protein Vif than 2 at the same concentration.
Furthermore, 6m suppressed the replication of various clinical drug-resistant HIV strains (FI, NRTI,
NNRTI, IN and PI) with relatively high efficacy. These results suggested that compound 6m is a more
potent candidate for treating AIDS.
Keywords:
Vif mediated A3G degradation
Vif antagonists
Antiviral activity
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
hA3G converts cytosine to uracil in newly synthesized viral DNA,
thus causing a G to A hyper-mutation [8e10] and inactivation of the
Acquired immune deficiency syndrome (AIDS) caused by human
immunodeficiency virus-1 (HIV-1) infection has been a serious
public health problem for more than 30 years [1,2]. According to
statistics reported by UNAIDS on July 16th, 2019, approximately
74.9 million people have been infected by HIV globally, and 32.0
million people have died from AIDS-related illnesses over the past
30 years [3]. Despite the remarkable benefit of highly active anti-
retroviral therapy, toxicity and frequent drug resistance caused by
rapid viral replication have limited its clinical applications [4e6].
Therefore, novel therapeutics targeting other viral proteins in HIV-1
are urgently needed.
new viral genome [11,12]. To neutralize the function of A3G, HIV-1
produces the protein viral infectivity factor (Vif) to overcome the
effective antiviral function of hA3G. Vif binds to hA3G and recruits
an E3 ubiquitin ligase complex consisting of Elongin B and C, Cullin
5, Rbx2 and CBF-b, followed by the polyubiquitination and subse-
quent proteasomal degradation of hA3G [13e19]. Because of the
absolute dependence on Vif for viral replication in the host and
non-existing cellular homologues, the development of small-
molecule inhibitors that inhibit Vif-mediated degradation of A3G
is an extremely attractive approach for anti-HIV drug discovery.
Early studies had demonstrated that targeting Vif-A3G axis is an
effective approach for implementing anti-HIV-1 activity, and the
reported lead compounds included RN-18 [20], IMB-26 [21], 1,2,3-
triazole derivatives [22], Zif-15 [23], and CV-3 [24]. In our previous
work, a potent Vif antagonist 12c (compound 2 in this work) was
derived from the first Vif antagonist RN-18 (compound 1 in this
work) [25]. The 150-fold greater antiviral activity of compound 2
was attributed to the introduction of an amino group on the central
ring of RN-18, which might form an H-bond interaction with
Human apolipoprotein
B mRNA-editing enzyme, catalytic
polypeptide-like 3G (Human APOBEC3G, hA3G), a DNA-editing
cytidine deaminase expressed in “non-permissive” host cells, acts
as an innate weapon against retroviruses [7]. During viral infection,
* Corresponding author.
** Corresponding author.
E-mail addresses: Zhengyt@mail.kiz.ac.cn (Y. Zheng), lirui@scu.edu.cn (R. Li).
These authors contribute equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113680
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
Gln136 of Vif. Further structure-activity relationship (SAR) studies
of compound 2 indicated that the replacement of the nitro group
with other groups (e.g., COOH, F, CF3) can only reduce cytotoxicity
without further enhancing antiviral activity [26]. However, the
ring B and Gln136 is essential for its antiviral activity [25], and
replacement of the nitro group of ring A with other electro-
withdrawing groups would decrease the antiviral activity [26].
Furthermore, after observing the binding mode of compound 2
with Vif, we found that the ortho-methoxyl substituted ring C did
not sufficiently match the hydrophobic pocket formed by Phe115,
Cys133, Tyr148, Ile155, and Pro157 [25]. Based on these findings
(Fig. 1), we introduced different substitutions to ring C to generate a
series of novel derivatives and investigated their activities.
Firstly, the methoxyl group of ring C of compound 2 was moved
from ortho position to meta and para position to yield compound 6a
and 6b, respectively. However, they all showed decreased antiviral
activity in nonpermissive H9 cells (reduced HIV-1 p24 antigen
production by 50%, Table 1). Then the methoxyl group of ring C of
6a and 6b was substituted with hydroxyl group to obtain 6c and 6d,
respectively. 6c still showed reduced activity, while the para-hy-
droxyl substituted 6d displayed moderate antiviral potency with an
moderate activity of compound 2 (EC50 ¼ 0.34
mM in H9 cells in this
work) is still inadequate for further development as a clinical
candidate. Analysis of the binding mode of compound 2 with Vif
revealed that the hydrophobic pocket formed by Phe115, Cys 133,
Tyr 148, Ile155, and Pro157 was sufficient to accommodate ring C of
compound 2 [25]; in other words, ring C of compound 2 has not
adequately occupied the pocket. Therefore, further optimization of
ring C could lead to the development of better Vif inhibitors.
Furthermore, the introduction of heteroatoms into ring B was
attempted to reduce the potential toxicity caused by the aniline
group [27] (Fig. 1).
2. Results and discussion
EC50 of 4.59 mM. Next, to expand the hydrophobic volume of ring C,
some disubstituted groups including methoxyl, methyl, halogen,
hydroxyl and their cross were introduced on ring C to produce 2,4-
dimethoxyl substituted 6e, 2,5-dimethoxyl substituted 6f, 3,4-
dimethoxyl substituted 6g, 3,5-dimethoxyl substituted 6h, 3-
2.1. Synthetic chemistry
The general synthetic steps of 5f, 5m, 5n, 5o, 6ae6p, and 7ae7c
are outlined in Scheme 1. Briefly, commercially available 2-amino-
6-bromobenzoic acid (3) was reacted with 4-nitrobenzenethiol via
a thiolation reaction using Cu/Cu2O as the catalyst to give the in-
termediate 4, which was subsequently condensed with different
substituted anilines to provide 5ae5p. Then, oxidation of 5ae5p by
hydrogen peroxide generated the sulfones 6ae6p. In addition,
nucleophilic substitution of the phenol 5m with different alkyl
halides under basic conditions yielded compounds 7ae7c.
hydroxy-4-methoxyl
substituted
6i,
2-methyl-3-bromo
substituted 6j, 3,5-dimethyl substituted 6k and 2-fluoro-4-chloro
substituted 6l, respectively. Among these disubstituted de-
rivatives, only 6e and 6f bearing 2-methoxyl substitution on ring C
displayed good antiviral effect with EC50 values of 3.58 and 3.97 mM,
respectively, and other compounds exhibited sharply reduced
efficacy.
Therefore, the 2-methoxyl group on ring C was retained in our
following optimization. When an additional hydroxyl group was
introduced to the C-5 position of ring C, compound 6m exhibited
remarkable improvement in antiviral potency with an EC50 of
13ae13d were prepared in four steps starting from 4,6-
dichloropyrimidine-5-carbaldehyde (8), as presented in Scheme
2. Firstly, amination of 8 in the presence of ammonia afforded 9.
Oxidation of the aldehyde 9 with NaClO2 followed by condensation
with different substituted anilines led to 11ae11d. The final com-
pounds 13ae13d were obtained through a SNAr reaction between
11ae11d and 4-nitrothiophenol under basic conditions.
0.07
mM, meanwhile, it also possesses low toxicity with CC50 at
258.69
mM and therapeutic index up to 3695.57. Inspired by this
result, a carboxyl group (6n) and fluorine (6o) were introduced at
the same position. However, 6n showed reduced activity and the
activity of 6o decreased about 28 fold when compared with 6m.
Besides, moving the hydroxyl group from C-5 to C-4 position (6p)
also resulted in an apparent loss in potency. At the same time, we
retrospectively tested the antiviral potency of non-oxidized
2.2. SAR study
The SARs of compound 2 in our previous investigation revealed
that the formation of a hydrogen bond between the amino group on
Fig. 1. Design strategies for compound 2 focusing on ring C with different substitutions and ring B featuring heteroatoms.
2

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
Scheme 1. Reagents and conditions: (i) 4-Nitrothiophenol, K2CO3, Cu/Cu2O, 2-ethoxyethanol, 95 BC, overnight; (ii) substituted Ar-NH2, EDCI, THF, rt, 5 h; (iii) 30% H2O2, AcOH, 55
BC, 6 h; (iv) alkyl halide, K2CO3, DMF, 60 BC, 5 h.
Scheme 2. Reagents and conditions: (i) NH3 (7 M solution in MeOH), toluene, 60 BC, 4 h; (ii) aminosulfonic acid, NaClO2, THF, 0 BCert; (iii) substituted Ar-NH2, EDCI, THF, rt, 5 h;
(iv) 4-Nitrothiophenol, Et3N, n-butanol, 120 BC.
compounds 5f, 5m, 5n and 5o which bearing both 2-methoxyl and
C-5 substitution on ring C, and most of them (5f, 5m and 5o)
manifested slightly lower antiviral activity compared with the
oxidized counterpart (6f, 6m and 6o) with EC50 values of
hydrophilic groups were linked to compound 6m via the 5-
hydroxyl bridge. Nevertheless, the resulting compounds 7a, 7b
and 7c exhibited a dramatic loss in antiviral activity, which might
be ascribed to the bulky volume of hydrophilic substituent.
To reduce the potential toxicity of aniline, a heteroatom nitrogen
was incorporated into ring B. Although the newly synthesized
0.42e4.05
mM, which is coincident with our previous QSAR
research [25]. In addition, to increase aqueous solubility, some
3

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
Table 1
(TI ¼ 0.11e3.38, Table 2). Hence, these analogues were not con-
ducted for further investigation.
In vitro antiviral activity of 2, 5f, 5m, 5n, 5o, 6ae6p, and 7ae7c in H9 cells.
Based on its highest antiviral activity and acceptable therapeutic
index, compound 6m was chosen for further evaluation.
2.3. Binding mode analysis
To elucidate the interaction mode of 6m with Vif, we conducted
molecular docking analysis using a reported crystal structure of
HIV-1 Vif (PDB ID: 4N9F) [28]. The binding site selection referenced
our previous research [25]. As illustrated in Fig. 2, compound 6m
adopted a similar binding conformation with 2 and maintained the
key H-bonding interaction of amino group with Vif. The original o-
methoxyl group formed hydrogen bond with Tyr 148. Apart from
that, the extra hydroxyl group on ring C of compound 6m forms
two new hydrogen bonds with Cys133 and Lys156, which increased
the binding affinity and can interpret the phenomenon that 6m
bearing more potent antiviral activity than compound 2.
Compd
X
R1
R2
EC50
0.34
>100
>100
>100
4.59
(
m
M) CC50
(
m
M) TIa
>1376.47
2
SO2 2-OCH3
SO2 3-OCH3
SO2 4-OCH3
SO2 3-OH
H
H
H
H
H
>468
N.Ab
N.A.
6a
6b
6c
6d
6e
6f
6g
6h
6i
N.A.
N.A.
N.A.
21.79
>27.93
>50.44
>6.45
N.A.
N.A.
SO2 4-OH
32.53
>100
>200
>100
N.A.
N.A.
N.A.
>100
>100
258.69
N.A.
89.63
54.71
>200
>100
>100
>100
N.A.
SO2 2-OCH3 4-OCH3
SO2 2-OCH3 5-OCH3
SO2 3-OCH3 4-OCH3
SO2 3-OCH3 5-OCH3
SO2 3-OH
SO2 2-CH3
SO2 3-CH3
SO2 2-F
3.58
3.97
2.4. 6m protects A3G from Vif-mediated degradation
15.51
>100
>100
>100
26.37
10.3
0.07
>100
1.93
5.77
4.05
0.47
0.42
4-OCH3
3-Br
5-CH3
4-Cl
N.A.
N.A.
Because Vif was reported to mediate the degradation of A3G,
western blotting was performed to measure the effect of 6m on Vif-
mediated A3G degradation (Fig. 3). The results indicated that 6m
induced the decrease of Vif protein in a dose-dependent manner,
6j
6k
6l
>3.79
>9.71
3695.57
N.A.
46.44
9.48
>49.38
>212.77
>238.10
>47.17
N.A.
6m
6n
6o
6p
5f
5m
5n
5o
7a
SO2 2-OCH3 5-OH
SO2 2-OCH3 5-COOH
SO2 2-OCH3 5-F
and the reduction of Vif level in the presence of 50 mM 6m was
more obvious than that in the presence of the same concentration
of compound 2. Meanwhile, the degradation of A3G mediated by
Vif was reversed by the addition of 6m in a concentration-
dependent manner. Unexpectedly, the recovery of A3G in the
SO2 2-OCH3 4-OH
S
S
S
S
S
2-OCH3 5-OCH3
2-OCH3 5-OH
2-OCH3 5-COOH
2-OCH3 5-F
2-OCH3
2.12
>100
presence of 50 mM 6m was merely equivalent to that in the pres-
ence of compound 2, which not matched with the distinct reduc-
tion of Vif expression. The possible reason for this observation was
that Vif can also bind to other proteins and mediated their degra-
dation, such as the reported substrate protein phosphatase 2A
(PP2A) [29]. All these results suggested that 6m more potently
inhibited Vif-mediated A3G degradation than the lead compound 2.
7b
7c
S
S
2-OCH3
2-OCH3
>100
>100
N.A.
N.A.
N.A.
N.A.
Lamivudine
0.21
>200
>975.61
a
TI: therapeutic index, TI ¼ CC50/EC50
.
b
NA: a CC50 values could not be obtained at the tested concentration range.
2.5. Inhibitory effects of 6m on drug-resistant HIV-1 strains
A common problem in current anti-HIV therapy is the devel-
opment of drug resistant. Subsequently, we determined the anti-
viral activity of 6m in TZM-bl cells infected with different clinical
drug-resistant HIV strains including pNL4-3gp41(36G)V38A,N42T (FI-
resistant), HIV-14755-5 (NRTI-resistant), HIV-1A17 (NNRTI-resistant),
pYU2G140S/Q148H (IN-resistant) and HIV-1L10R/M46I/L63P/V82T/I84V (PI-
resistant). As presented in Table 3, 6m inhibited the replication of
different drug-resistant HIV-1 strains effectively with EC50 values of
Table 2
In vitro antiviral activity of 13ae13d in H9 cells.
3.02e8.40
replication
(EC50 ¼ 46.43
m
M, although the compound only slightly inhibited the
of the NNRTI-resistant strain HIV-1A17
M).
m
3. Conclusion
Compd
R2
EC50
9.73
40.61
41.13
20.04
0.21
(
m
M)
CC50
(m
M)
TIa
Through further structural optimization of compound 2, we
developed the novel HIV-1 Vif antagonist 6m bearing additional 5-
hydroxyl group on ring C. Compared to 2, 6m displayed 5-fold
13a
13b
13c
13d
H
-OH
-F
32.91
35.83
26.57
2.22
3.38
0.88
0.65
0.11
-Cl
greater antiviral activity with an EC50 of 0.07
mM in non-
Lamivudine
>200
>975.61
permissive H9 cells. Molecular docking studies revealed that the
extra 5-hydroxyl group might form two new hydrogen bonds with
Cys133 and Lys156, which reinforces the ligand and Vif interaction.
Meanwhile, 6m more strongly reduced Vif expression than 2 at the
same concentration in Western blot analysis. Further biological
activity assays illustrated the promising inhibitory effects of 6m
against various clinical drug-resistant HIV strains. Thus, its
a
TI: therapeutic index, TI ¼ CC50/EC50
.
amino-pyrimidine compounds 13ae13d maintained moderate
antiviral activities, remarkable cytotoxicity (CC50 ¼ 2.22e35.83 M)
was also observed, leading to low therapeutic indexes
m
4

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
Fig. 2. Binding mode analysis of 6m with Vif (PDB ID: 4N9F). (a) The possible binding pose of 6m with Vif. (b) 2D diagram of 6m bound to Vif.
improved antiviral activity makes compound 6m a more effective
candidate for treating HIV.
at room temperature for 5 h. The reaction was quenched with water
and extracted with ethyl acetate twice. The combined extracts were
washed with brine, dried over anhydrous Na2SO4 and concentrated.
The residue was purified by flash column chromatography (ethyl
acetate/hexanes ¼ 1:2) to afford the desired compound 5a-5p.
4. Experimental
4.1. Chemistry
4.1.2.1. 2-amino-N-(2,5-dimethoxyphenyl)-6-((4-nitrophenyl)thio)
benzamide (5f). 1H NMR (400 MHz, DMSO‑d6)
d 9.21 (s, 1H), 8.09 (d,
All reagents and drugs are commercially available analytical
reagents, which can be used directly without treatment. Purity of
the intermediates and compounds was monitored using thin layer
chromatography (TLC) on precoated silica gel 60 F254 plates
(0.25 mm, Qingdao Haiyang Inc.) and components were visualized
under ultraviolet light (254 nm). The 1H NMR and 13C NMR spectra
were measured on a Bruker Avance spectrometer in DMSO‑d6 or
CDCl3, and analyzed by MestReNova Software. High resolution
mass spectrometry (HRMS) was performed on an Agilent LC/MSD
TOF system G3250AA. Silicycle silica gel 300e400 (particle size
J ¼ 8.9 Hz, 2H), 7.51 (d, J ¼ 2.9 Hz,1H), 7.31 (d, J ¼ 8.9 Hz, 2H), 7.23 (t,
J ¼ 7.9 Hz, 1H), 6.91 (dd, J ¼ 8.5, 3.5 Hz, 2H), 6.77 (d, J ¼ 7.5 Hz, 1H),
6.66 (dd, J ¼ 8.9, 3.0 Hz, 1H), 5.41 (s, 2H), 3.67 (s, 3H), 3.63 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 165.65, 153.37, 148.86, 147.50,
145.09, 131.26, 128.05, 127.27, 123.91, 123.39, 117.54, 112.54, 109.67,
56.28. HRMS (ESI) calcd for C21H19N3O5S [MþH]þ 426.1119, found:
426.1120; Purity > 96%.
4.1.2.2. 2-amino-N-(5-hydroxy-2-methoxyphenyl)-6-((4-
nitrophenyl)thio)benzamide (5m). 1H NMR (400 MHz, DMSO‑d6)
40e63 mm) mesh was used for all column chromatography exper-
iments. All final compounds were purified to >95% purity as
determined by HPLC analyses were carried out on an Agilent 1260
Infinity II LC system (Waldbronn, Germany) with a fixed wave-
length UV detector at 254 nm. An InertSustain®C18 Column
d
9.41 (s, 1H), 9.19 (s, 1H), 8.07 (d, J ¼ 13.0 Hz, 2H), 7.30 (d, J ¼ 6.7,
2H), 7.26 (d, J ¼ 8.5 Hz,1H), 7.20 (t, J ¼ 7.9 Hz,1H), 6.88 (d, J ¼ 7.7 Hz,
1H), 6.75 (d, J ¼ 7.0 Hz, 1H), 6.43 (d, J ¼ 2.4 Hz, 1H), 6.30 (dd, J ¼ 8.5,
2.4 Hz, 1H), 5.36 (s, 2H), 3.65 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
(Waldbronn, Germany) (250 mm ꢀ 4.6 mm, 5
flow rate of 1.0 mL/min for the sample. The injection volume was
L. Mobile phase was consisted of methanol (C) and water with
mM) was used with a
d
165.47, 151.35, 148.93, 147.41, 145.25, 143.75, 131.22, 127.59,
124.44, 123.44, 117.53, 112.86, 111.33, 110.69, 56.70. HRMS (ESI)
calcd for
Purity > 97%.
20H17N3O5S [MþH]þ 412.0963, found: 412.0960;
5
m
C
0.1% of formic acid (D) with gradient elution timing programs:
0e20 min (50% C and 50% D).
4.1.2.3. 2-amino-N-(5-hydroxy-2-methoxyphenyl)-6-((4-
nitrophenyl)thio)benzamide (5n). 1H NMR (400 MHz, DMSO‑d6)
4.1.1. Synthesis of 2-amino-6-((4-nitrophenyl)thio)benzoic acid (4)
A mixture of commercially available 2-amino-6-bromobenzoic
acid (3, 2370 mg, 11 mmol), 4-nitrobenzenethiol (2050 mg,
13.2 mmol), Cu (130 mg, 2 mmol), Cu2O (140 mg, 1 mmol) and
K2CO3 (3036 mg, 22 mmol) in 2-ethoxyethanol (50 mL) was stirred
at 95 BC for 12 h. Upon completion, the reaction mixture was
cooled to room temperature, and the resulting precipitate was
filtered off, while the filter cake was washed with ethyl acetate.
Then the filtrate was acidified with 5 N HCl aqueous solution,
extracted with ethyl acetate twice. The combined extracts were
washed with bine, dried over anhydrous Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography us-
ing ethyl acetate/hexanes as eluent to give compound 4.
d
9.35 (s,1H), 8.24 (s,1H), 8.07 (d, J ¼ 13.8 Hz, 2H), 7.69 (d, J ¼ 8.2 Hz,
1H), 7.32 (d, J ¼ 8.9 Hz, 2H), 7.22 (t, J ¼ 7.9 Hz,1H), 6.96 (d, J ¼ 8.5 Hz,
1H), 6.90 (d, J ¼ 8.1 Hz, 1H), 6.78 (d, J ¼ 8.8 Hz, 1H), 5.40 (s, 2H), 3.73
(s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 165.72, 154.11, 148.89,
147.40, 145.26, 131.23, 127.66, 127.46, 127.30, 126.64, 124.98, 124.43,
123.36, 117.46, 111.06, 56.36. HRMS (ESI) calcd for C21H17N3O6S
[MþNa]þ 462.0730, found: 462.0729; Purity > 99%.
4.1.2.4. 2-amino-N-(5-fluoro-2-methoxyphenyl)-6-((4-nitrophenyl)
thio)benzamide (5o). 1H NMR (400 MHz, DMSO‑d6)
d 9.42 (s, 1H),
8.08 (d, J ¼ 8.1 Hz, 2H), 7.83 (dd, J ¼ 10.6, 3.0 Hz, 1H), 7.31 (d,
J ¼ 8.9 Hz, 2H), 7.23 (t, J ¼ 7.9 Hz, 1H), 6.98 (dd, J ¼ 16.9, 8.5 Hz, 1H),
6.94e6.84 (d, J ¼ 8.5 Hz, 2H), 6.77 (d, J ¼ 7.5 Hz, 1H), 5.43 (s, 2H),
4.1.2. General procedure A for the preparation of 5a-5p
3.67 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 165.94, 157.27, 154.94,
A mixture of 4 (50 mg, 0.13 mmol), different substituted aniline
(0.13 mmol) and EDCI (30 mg, 0.16 mol) in THF (10 mL) was stirred
148.82, 147.49, 146.77, 145.27, 131.34, 127.42, 126.93, 124.43, 123.32,
117.55, 112.50, 109.63, 56.67. HRMS (ESI) calcd for C20H16FN3O4S
5

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
Table 3
Anti-HIV-1 activities of 6m in drug-resistant strainsa.
Cell
Virus
Subtype
EC50(m
M)b
TZM-bl
pNL4-3gp41(36G)V38A,N42T
HIV-14755-5
HIV-1A17
pYU2G140S/Q148H
HIV-1L10R/M46I/L63P/V82T/I84V
FI-resistant
3.02 1.07
8.40 0.74
46.43 2.70
3.74 0.32
4.39 2.66
NRTI-resistant
NNRTI-resistant
IN-resistant
PI-resistant
a
All data represent means SD of three separate experiments.
EC50, 50% effective concentration.
b
[MþNa]þ 436.0738, found: 436.0737; Purity > 98%.
4.1.3. General procedure B for the preparation of compounds 6a-6p
To a solution of compounds 5a-5p (0.15 mmol) in acetic acid
(10 mL) was added 30% H2O2 (0.75 mmol) slowly. The reaction
mixture was heated in an oil bath at 55 BC and monitored using
TLC. Next, the reaction was cooled to room temperature and
quenched with MnO2 (0.5 mmol). The insoluble solid was filtered
off. The filtrate was dried over anhydrous Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography us-
ing ethyl acetate/hexanes as eluent to afford compound 6a-6p.
4.1.3.1. 2-amino-N-(3-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)
benzamide (6a). 1H NMR (400 MHz, CDCl3)
d 9.43 (s, 1H), 8.36 (d,
J ¼ 8.8 Hz, 2H), 8.13e8.08 (d, J ¼ 8.8 Hz, 2H), 7.36 (d, J ¼ 9.1, Hz, 2H),
7.29 (dd, J ¼ 9.0, 2.0 Hz, 2H), 7.08 (d, J ¼ 2.5 Hz, 1H), 6.83 (d,
J ¼ 7.8 Hz, 1H), 6.76 (t, J ¼ 7.5 Hz, 1H), 5.47 (s, 2H), 3.76 (s, 3H). 13
C
NMR (101 MHz, CDCl3)
d 164.42, 159.89, 150.46, 147.37, 140.79,
136.98, 130.45, 129.68, 124.89, 121.54, 117.13, 112.83, 109.37, 106.53,
55.51. HRMS (ESI) calcd for C20H17N3O6S [MþNa]þ 450.0730, found:
450.0732; Purity > 97%.
4.1.3.2. 2-amino-N-(4-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)
benzamide (6b). 1H NMR (400 MHz, DMSO‑d6)
d 10.26 (s, 1H), 8.37
(d, J ¼ 8.3 Hz, 2H), 8.19e8.07 (d, J ¼ 8.8 Hz, 2H), 7.57 (m, 2H),
7.41e7.25 (m, 2H), 7.08 (d, J ¼ 8.0 Hz, 1H), 6.91 (d, J ¼ 15.7 Hz, 2H),
5.45 (s, 2H), 3.77e3.74 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
163.88,156.08,150.45,147.39,147.16,137.01,132.77,130.36,129.61,
126.77, 124.87, 121.85, 121.59, 117.15, 115.27, 114.16, 55.72. HRMS
(ESI) calcd for C20H17N3O6S [MþNa]þ 450.0730, found: 450.0731;
Purity > 98%.
4.1.3.3. 2-amino-N-(3-hydroxyphenyl)-6-((4-nitrophenyl)sulfonyl)
benzamide (6c). 1H NMR (400 MHz, DMSO‑d6)
d 10.30 (s, 1H), 9.39
(s, 1H), 8.39e8.33 (d, J ¼ 9.1 Hz, 2H), 8.16e8.10 (d, J ¼ 9.1 Hz, 2H),
7.35 (t, J ¼ 7.9 Hz, 1H), 7.32e7.25 (m, 2H), 7.13e7.06 (m, 2H),
7.04e7.00 (d, J ¼ 8.1 Hz, 1H), 6.54e6.49 (dd, J ¼ 8.5, 1.8 Hz, 1H), 5.45
(s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 164.25, 157.96, 150.46,
147.62, 147.12, 140.62, 136.94, 130.39, 129.59, 124.86, 121.63, 117.18,
111.24, 107.70. HRMS (ESI) calcd for
436.0574, found: 436.0579; Purity > 96%.
C
19H15N3O6S [MþNa]þ
4.1.3.4. 2-amino-N-(4-hydroxyphenyl)-6-((4-nitrophenyl)sulfonyl)
benzamide (6d). 1H NMR (400 MHz, DMSO‑d6)
8.79 (s, 1H),
d
8.32e8.24 (d, J ¼ 9.1 Hz, 2H), 7.99e7.90 (d, J ¼ 9.0 Hz, 2H), 7.67 (d,
J ¼ 14.0 Hz, 1H), 7.61 (t, J ¼ 7.9 Hz, 1H), 7.54 (dd, J ¼ 7.7, 1.0 Hz, 1H),
7.22 (dd, J ¼ 10.0, 3.1 Hz, 1H), 6.96e6.87 (m, 2H), 6.15 (dt, J ¼ 6.9,
3.4 Hz, 1H), 6.07 (dd, J ¼ 10.0, 2.1 Hz, 1H), 5.75 (s, 1H). 13C NMR
(101 MHz, DMSO‑d6)
d 184.58, 162.48, 155.17, 148.83, 147.71, 147.57,
147.10, 134.85, 128.50, 127.78, 126.66, 124.57, 118.18, 113.75, 109.48,
64.47. HRMS (ESI) calcd for C19H15N3O5S [MþK]þ436.0545, found:
436.0543; Purity > 97%.
6

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
4.1.3.5. 2-amino-N-(2,4-dimethoxyphenyl)-6-((4-nitrophenyl)sulfo-
8.39e8.33 (d, J ¼ 8.8 Hz, 2H), 8.16e8.09 (d, J ¼ 8.7 Hz, 2H), 7.35 (t,
J ¼ 7.9 Hz, 1H), 7.31e7.22 (m, 3H), 7.08 (d, J ¼ 6.0 Hz, 1H), 6.74 (s,
1H), 5.43 (s, 2H), 2.27 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
nyl)benzamide (6e). 1H NMR (400 MHz, DMSO‑d6)
d 9.64 (s, 1H),
8.39e8.32 (d, J ¼ 8.9 Hz, 2H), 8.20e8.11 (d, J ¼ 8.9 Hz, 2H), 7.48 (d,
J ¼ 8.6 Hz, 1H), 7.35 (t, J ¼ 7.9 Hz, 1H), 7.28 (dd, J ¼ 7.8, 1.1 Hz, 1H),
6.69e6.62 (m, 2H), 6.61e6.58 (d, J ¼ 2.8 Hz, 1H), 5.57 (s, 2H),
d
164.26, 150.43, 147.56, 147.17, 139.48, 137.88, 136.93, 129.98,
129.59,125.60,124.90, 121.63,117.86,117.12, 21.62. HRMS (ESI) calcd
3.80e3.77 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
d
164.79, 158.86,
for C21H19N3O5S [MþH]þ 426.1119, found: 426.1121; Purity > 98%.
154.24, 150.42, 147.76, 147.12, 137.07, 129.67, 127.13, 126.50, 124.80,
121.66, 119.66, 117.22,112.52, 105.10, 99.51, 56.24, 55.90. HRMS (ESI)
4.1.3.12. 2-amino-N-(4-chloro-2-fluorophenyl)-6-((4-nitrophenyl)
calcd for
Purity > 98%.
C
21H19N3O7S [MþNa]þ 480.0836, found: 480.0840;
sulfonyl)benzamide (6l). 1H NMR (400 MHz, DMSO‑d6)
d 10.53 (s,
1H), 8.39 (d, J ¼ 8.5 Hz, 2H), 8.16 (d, J ¼ 8.8 Hz, 2H), 8.04 (t,
J ¼ 8.6 Hz, 1H), 7.50 (dd, J ¼ 8.9, 4.4 Hz, 1H), 7.36 (t, J ¼ 8.0 Hz, 2H),
7.27 (d, J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 8.1 Hz, 1H), 5.51 (s, 2H). 13C NMR
4.1.3.6. 2-amino-N-(2,5-dimethoxyphenyl)-6-((4-nitrophenyl)sulfo-
nyl)benzamide (6f). 1H NMR (400 MHz, DMSO‑d6)
9.59 (s, 1H),
d
(101 MHz, DMSO‑d6) d 165.26, 155.82, 153.33, 150.50, 147.68, 147.11,
8.36 (d, J ¼ 8.9 Hz, 2H), 8.15 (d, J ¼ 8.9 Hz, 2H), 7.49 (d, J ¼ 3.1 Hz,
1H), 7.36 (t, J ¼ 7.9 Hz, 1H), 7.29 (d, J ¼ 7.1 Hz, 1H), 7.06 (d, J ¼ 8.0 Hz,
1H), 7.00 (d, J ¼ 9.0 Hz,1H), 6.76 (dd, J ¼ 9.3, 4.7 Hz,1H), 5.55 (s, 2H),
3.75 (s, 3H), 3.72 (s, J ¼ 6.2 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
137.07, 130.62, 129.56, 126.82, 126.38, 124.93, 121.60, 120.81, 117.23,
114.19. HRMS (ESI) calcd for C19H13ClFN3O5S [MþNa]þ 472.0140,
found: 472.0141; Purity > 97%.
d
164.71, 153.52, 150.44, 147.67, 147.18, 146.04, 137.00, 130.48,
4.1.3.13. 2-amino-N-(5-hydroxy-2-methoxyphenyl)-6-((4-
129.57, 127.96, 124.86, 121.43, 117.22, 112.72, 111.35, 110.11, 56.34.
HRMS (ESI) calcd for C21H19N3O7S [MþNa]þ 480.0836, found:
480.0840; Purity > 97%.
nitrophenyl)sulfonyl)benzamide (6m). 1H NMR (400 MHz, DMSO‑d6)
d
9.51 (s, 1H), 9.15e8.98 (s, 1H), 8.36 (d, J ¼ 8.6 Hz, 2H), 8.11 (d,
J ¼ 8.3 Hz, 2H), 7.35 (dd, J ¼ 9.3, 5.5 Hz, 2H), 7.31e7.20 (d, J ¼ 7.5 Hz,
1H), 7.06 (d, J ¼ 7.9 Hz, 1H), 6.88 (d, J ¼ 8.8 Hz, 1H), 6.58 (dd, J ¼ 8.6,
2.5 Hz, 1H), 5.55 (s, 2H), 3.77e3.55 (s, 3H). 13C NMR (101 MHz,
4.1.3.7. 2-amino-N-(3,4-dimethoxyphenyl)-6-((4-nitrophenyl)sulfo-
nyl)benzamide (6g). 1H NMR (400 MHz, DMSO‑d6)
d
10.21 (s, 1H),
DMSO‑d6) d 164.54, 151.48, 150.45, 147.70, 147.18, 145.03, 136.98,
8.44e8.29 (d, J ¼ 9.1 Hz, 2H), 8.10 (d, J ¼ 9.1 Hz, 2H), 7.41e7.32 (t,
J ¼ 7.8 Hz, 2H), 7.31 (d, J ¼ 7.3 Hz, 1H), 7.22 (dd, J ¼ 13.9, 7.0 Hz, 1H),
7.09 (t, J ¼ 8.6 Hz, 1H), 6.97e6.93 (m, 1H), 5.45 (s, 2H), 3.75 (s, 6H).
130.45, 129.57, 127.60, 124.83, 121.47, 117.23, 113.07, 112.27, 56.73.
HRMS (ESI) calcd for C20H17N3O7S [MþNa]þ 466.0679, found:
466.0678; Purity > 98%.
13C NMR (101 MHz, DMSO‑d6)
d 163.87, 150.44, 148.95, 147.58,
147.19, 145.75, 136.97, 133.27, 130.35, 129.62, 124.87, 121.62, 117.12,
112.60, 105.89, 56.36, 55.94. HRMS (ESI) calcd for C21H19N3O7S
[MþNa]þ 480.0836, found: 480.0840; Purity > 99%.
4.1.3.14. 3-(2-amino-6-((4-nitrophenyl)sulfonyl)benzamido)-4-
methoxybenzoic acid (6n). 1H NMR (400 MHz, DMSO‑d6)
d 9.81 (s,
1H), 8.42 (d, J ¼ 2.1 Hz, 1H), 8.36 (d, J ¼ 8.9 Hz, 2H), 8.16 (d,
J ¼ 8.9 Hz, 2H), 7.83 (dd, J ¼ 8.5, 2.0 Hz, 1H), 7.36 (t, J ¼ 8.0 Hz, 1H),
7.27 (d, J ¼ 7.7 Hz,1H), 7.18 (d, J ¼ 8.7 Hz,1H), 7.10e7.05 (d,J ¼ 7.9 Hz,
1H), 5.56 (s, 2H), 3.86 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
4.1.3.8. 2-amino-N-(3,5-dimethoxyphenyl)-6-((4-nitrophenyl)sulfo-
nyl)benzamide (6h). 1H NMR (400 MHz, DMSO‑d6)
d 9.41 (s, 1H),
8.40e8.34 (d,J ¼ 8.8 Hz, 2H), 8.13e8.10 (d, J ¼ 8.8 Hz, 2H), 7.36 (t,
J ¼ 7.9 Hz, 1H), 7.30 (dd, J ¼ 7.7, 0.9 Hz, 1H), 7.09 (d, J ¼ 3.7 Hz, 1H),
6.95 (d, J ¼ 7.2 Hz, 2H), 6.28 (d, J ¼ 2.2 Hz, 1H), 5.46 (s, 2H), 3.74 (d,
d 172.48, 168.94, 167.25, 156.58, 151.97, 138.31, 128.66, 127.84,
126.37, 124.78, 123.82, 118.90, 114.70, 113.87, 112.47, 56.41. HRMS
(ESI) calcd for C21H17N3O8S [MþNa]þ 494.0628, found: 494.0634;
Purity > 99%.
J ¼ 1.4 Hz, 6H).13C NMR (101 MHz, DMSO‑d6)
d 165.11,150.51,147.50,
147.14, 137.90, 137.00, 132.84, 131.90, 129.84, 129.71, 127.55, 126.41,
125.03, 124.99, 121.74, 121.38, 117.40, 49.07. HRMS (ESI) calcd for
4.1.3.15. 2-amino-N-(5-fluoro-2-methoxyphenyl)-6-((4-nitrophenyl)
C
21H19N3O7S [MþNa]þ 480.0836, found: 480.0840; Purity > 97%.
sulfonyl)benzamide (6o). 1H NMR (400 MHz, DMSO‑d6)
d 9.76 (s,
1H), 8.40e8.33 (d, J ¼ 8.9 Hz, 2H), 8.18e8.12 (d, J ¼ 8.9 Hz, 2H), 7.86
(dd, J ¼ 10.4, 3.1 Hz, 1H), 7.36 (t, J ¼ 8.0 Hz, 1H), 7.31e7.24 (dd,
J ¼ 7.9, 1.1 Hz, 1H), 7.09e7.03 (m, 2H), 7.01 (d, J ¼ 3.2 Hz, 1H), 5.53 (s,
4.1.3.9. 2-amino-N-(3-hydroxy-4-methoxyphenyl)-6-((4-
nitrophenyl)sulfonyl)benzamide (6i). 1H NMR (400 MHz, DMSO‑d6)
d
10.15 (s, 1H), 9.03 (s, 1H), 8.38e8.32 (d, J ¼ 8.8 Hz, 2H), 8.16e8.08
2H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 165.01, 150.46,
(d, J ¼ 8.9 Hz, 2H), 7.35 (t, J ¼ 7.9 Hz,1H), 7.29 (dd, J ¼ 7.8,1.5 Hz, 2H),
147.59, 147.19, 136.99, 130.55, 130.17, 129.56, 124.90, 121.60, 121.02,
117.20, 112.75, 110.77, 56.77. HRMS (ESI) calcd for C20H16FN3O6S
[MþH]þ 446.0636, found: 446.0634; Purity > 98%.
6.95e6.87 (m, 2H), 5.40 (s, 2H), 3.75 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6)
d 163.74, 150.45, 147.64, 147.12, 146.81, 144.64, 136.95,
133.25, 129.60, 126.73, 124.84, 121.68, 112.96, 111.39, 109.13, 56.52.
HRMS (ESI) calcd for C20H17N3O7S [MþNa]þ 466.0679, found:
466.0678; Purity > 99%.
4.1.3.16. 2-amino-N-(4-hydroxy-2-methoxyphenyl)-6-((4-
nitrophenyl)sulfonyl)benzamide (6p). 1H NMR (400 MHz, DMSO‑d6)
d
9.53 (s, 1H), 8.40e8.32 (d, J ¼ 8.8 Hz, 2H), 8.21e8.14 (d, J ¼ 8.9 Hz,
4.1.3.10. 2-amino-N-(3-bromo-2-methylphenyl)-6-((4-nitrophenyl)
2H), 7.84 (d, J ¼ 6.1 Hz, 1H), 7.32 (d, J ¼ 3.2 Hz, 1H), 7.30e7.27 (m,
1H), 7.05 (d, J ¼ 8.0 Hz, 1H), 6.51 (d, J ¼ 2.4 Hz, 1H), 6.42 (dd, J ¼ 8.5,
2.4 Hz, 1H), 5.58 (s, 2H), 3.72 (s, 3H). HRMS (ESI) calcd for
sulfonyl)benzamide (6j). 1H NMR (400 MHz, DMSO‑d6)
d 10.22 (s,
1H), 8.39 (d, J ¼ 8.9 Hz, 2H), 8.19 (d, J ¼ 8.9 Hz, 2H), 7.74 (d,
J ¼ 7.9 Hz, 1H), 7.53e7.45 (d, J ¼ 7.8 Hz, 1H), 7.36 (t, J ¼ 7.6 Hz, 1H),
7.29 (d, J ¼ 7.2 Hz,1H), 7.21 (t, J ¼ 8.0 Hz,1H), 7.08 (d, J ¼ 11.1 Hz,1H),
5.64e5.39 (s, 2H), 2.40 (s, 3H).13C NMR (101 MHz, DMSO‑d6)
C
20H16FN3O6S [MþH]þ 446.0679, found: 446.0678; Purity > 97%.
4.1.4. General procedure C for the preparation of compounds 7a-7c
To a stirring mixture of compound 6m (0.2 mmol) and anhy-
drous potassium carbonate (0.2 mmol) in DMF (10 mL) was added
different kinds of alkyl halide (0.24 mmol). The reaction was heated
at 60 BC for 8 h and monitored using TLC. After cooling, the mixture
was extracted with ethyl acetate twice. The combined extracts were
washed with bine, dried over anhydrous Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography
d
165.11, 150.51, 147.78, 147.22, 137.90, 137.00, 132.84, 130.51, 129.84,
129.58, 127.55, 126.41, 125.03, 124.95, 121.74, 121.38, 117.40, 113.30,
18.55. HRMS (ESI) calcd for C20H16BrN3O5S [MþNa]þ 511.9886,
found: 511.9890; Purity > 98%.
4.1.3.11. 2-amino-N-(3,5-dimethylphenyl)-6-((4-nitrophenyl)sulfo-
nyl)benzamide (6k). 1H NMR (400 MHz, DMSO‑d6)
d 10.24 (s, 1H),
7

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
using ethyl acetate/hexanes as eluent to give compound 7a-7c.
resulting residue was purified by silica gel column chromatography
using ethyl acetate/hexanes as eluent to give compound 13a-13d.
4.1.5.3.1. 4-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)thio)
pyrimidine-5-carboxamide (13a). 1H NMR (400 MHz, DMSO‑d6)
4.1.4.1. 2-amino-N-(5-(2-hydroxyethoxy)-2-methoxyphenyl)-6-((4-
nitrophenyl)thio)benzamide (7a). 1H NMR (400 MHz, DMSO‑d6)
d
8.14 (d, J ¼ 9.0 Hz, 2H), 7.37 (d, J ¼ 8.9 Hz, 2H), 6.97 (m, 2H),
d
9.88 (s, 1H), 8.23 (s, 1H), 8.20 (d, J ¼ 2.5 Hz, 2H), 7.99 (m, 1H), 7.74
6.85e6.58 (m, 2H), 6.54 (d, J ¼ 7.6 Hz, 1H), 6.37 (d, J ¼ 8.0 Hz, 1H),
5.16 (s, 2H), 3.94e3.62 (s, 3H), 3.57 (t, J ¼ 15.5 Hz, 2H), 2.98 (d,
J ¼ 16.0 Hz, 2H). HRMS (ESI) calcd for C22H21N3O6S [MþNa]þ
478.1043, found: 478.1044; Purity > 99%.
(d, J ¼ 8.3 Hz, 2H), 7.15 (dd, J ¼ 11.3, 4.2 Hz, 1H), 7.07 (d, J ¼ 7.5 Hz,
2H), 6.96 (t, J ¼ 7.5 Hz, 1H), 3.81 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6)
d 160.46, 157.79, 147.31, 140.52, 134.27, 127.14, 125.88,
124.17, 123.83, 120.70, 113.62, 112.00, 56.30. HRMS (ESI) calcd for
C
18H15N5O4S [MþH]þ 398.0917, found: 398.0919; Purity > 96%.
4.1.4.2. 2-amino-N-(5-(3-hydroxypropoxy)-2-methoxyphenyl)-6-
4.1.5.3.2. 4-amino-N-(5-hydroxy-2-methoxyphenyl)-6-((4-
((4-nitrophenyl)thio)benzamide (7b). 1H NMR (400 MHz, DMSO‑d6)
nitrophenyl)thio)pyrimidine-5-carboxamide
(400 MHz, DMSO‑d6)
(13b). 1H
NMR
d
9.27 (s,1H), 8.03 (d, J ¼ 9.1 Hz, 2H), 7.37 (d, J ¼ 9.3 Hz, 2H), 6.91 (m,
d
9.75 (s, 1H), 9.02 (s, 1H), 8.19 (d, J ¼ 12.0 Hz,
4H), 6.51 (d, J ¼ 7.6 Hz, 1H), 6.38 (d, J ¼ 8.1 Hz, 1H), 5.26 (s, 2H),
4.83e4.17 (m, 2H), 4.10e3.85 (m,1H), 3.67 (s, 3H), 3.51 (t, J ¼ 5.8 Hz,
2H), 2.94 (s, 2H), 1.79e1.63 (t, J ¼ 6.5 Hz, 2H). HRMS (ESI) calcd for
3H), 7.74 (d, J ¼ 8.7 Hz, 2H), 7.62 (s, 1H), 7.07 (s, 2H), 6.87 (d,
J ¼ 8.8 Hz, 1H), 6.52 (dd, J ¼ 8.8, 2.9 Hz, 1H), 3.73 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6)
d 160.99, 160.44, 157.77, 151.41, 147.30, 140.56,
C
23H23N3O6S [MþH]þ 492.1200, found: 492.1205; Purity > 97%.
134.23, 127.94, 127.27, 125.04, 124.16, 113.71, 113.21, 111.47, 110.66,
57.05. HRMS (ESI) calcd for C18H15N5O5S [MþH]þ 414.0868, found:
414.0873; Purity > 97%.
4.1.4.3. 2-amino-N-(2-methoxy-5-(2-morpholinoethoxy)phenyl)-6-
((4-nitrophenyl)thio)benzamide (7c). 1H NMR (400 MHz, DMSO‑d6)
4.1.5.3.3. 4-amino-N-(5-fluoro-2-methoxyphenyl)-6-((4-
d
9.25 (s, 1H), 8.03 (d, J ¼ 9.1 Hz,2H), 7.36 (d, J ¼ 9.3 Hz, 2H), 6.95 (d,
nitrophenyl)thio)pyrimidine-5-carboxamide
(400 MHz, DMSO‑d6)
8.03 (d, J ¼ 10.0 Hz, 1H), 7.72 (d, J ¼ 8.1 Hz, 2H), 7.60e7.46 (m, 1H),
7.06 (dd, J ¼ 9.0, 5.2 Hz, 1H), 6.95 (dd, J ¼ 8.6, 3.0 Hz, 1H), 3.80 (s,
3H). HRMS (ESI) calcd for C18H14FN5O4S [MþH]þ 416.0825, found:
416.0834; Purity > 98%.
(13c). 1H
NMR
J ¼ 16.8 Hz, 2H), 6.76 (s, 2H), 6.54 (d, J ¼ 7.7 Hz, 1H), 6.40 (d,
J ¼ 8.1 Hz, 1H), 5.26 (s, 2H), 4.19 (m, 1H), 3.86e3.59 (s, 3H),
3.59e3.53 (t, J ¼ 4.6 Hz 4H), 3.17 (d, J ¼ 3.6 Hz, 1H), 3.11e2.86 (t,
J ¼ 7.3 Hz, 2H), 2.43e2.33 (t, J ¼ 4.1 Hz, 4H). HRMS (ESI) calcd for
d
8.44 (d, J ¼ 4.5 Hz, 1H), 8.28e8.16 (m, 3H),
C
26H28N4O6S [MþH]þ 525.1804, found: 525.1806; Purity > 98%.
4.1.5.3.4. 4-amino-N-(5-chloro-2-methoxyphenyl)-6-((4-
4.1.5. General procedure D for the preparation of compounds 13a-
13d
nitrophenyl)thio)pyrimidine-5-carboxamide
(400 MHz, DMSO‑d6)
(13d). 1H
NMR
d
10.07 (s, 1H), 8.20 (d, J ¼ 7.0 Hz, 3H), 7.73 (d,
4.1.5.1. Synthesis of 4-amino-6-chloropyrimidine-5-carbaldehyde
(9). A mixture of 4,6-dichloro-5-pyrimidine formaldehyde (8,
1050 mg, 6 mmol) and ammonia solution (7 M in methanol,
12 mmol) in toluene was heated 60 BC for 4 h. The reaction mixture
was concentrated under vacuum to give the crude product 9
(800 mg), which was used in next step without further purification.
J ¼ 8.7 Hz, 2H), 7.19 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.14 (s, 1H), 7.08 (d,
J ¼ 8.8 Hz, 1H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 163.75,
160.76, 160.39, 157.86, 149.50, 147.29, 140.54, 134.13, 128.63, 124.88,
124.19, 122.55, 113.74, 113.28, 56.69. HRMS (ESI) calcd for
C
18H14ClN5O4S [MþH]þ 432.0529, found: 432.0534; Purity > 97%.
4.2. In vitro antiviral assays
4.1.5.2. Synthesis of 4-amino-6-chloropyrimidine-5-carboxylic acid
(10). To a solution of 9 (800 mg, 5.0 mmol) in THF (15 mL) at 0 B
C
The antiviral activities of the compounds were determined by
the HIV-1 p24 assay. H9 cells were infected with HIVe1IIIB at a
multiplicity of infection (MOI) of 0.03 for 2 h, then washed three
times to remove free virus and resuspended with RPMI-1640. Serial
diluted compounds and 4 ꢀ 104 cells/well infected cells were added
into 96-well cell culture plates and incubated at 37 BC with 5% CO2
for 7 days. The p24 level of the culture supernatant was measured
by in-house ELISA assay [30]. The 50% effective concentrations
(EC50) were calculated.
was added aminosulfonic acid (593 mg, 6 mmol) and NaClO2
(900 mg, 10.2 mmol) sequentially and slowly. After being stirred at
0 BC for 0.5 h, the reaction was warmed to room temperature and
monitored using TLC. Upon completion, the reaction mixture was
quenched with water, then extracted with dichloromethane
(3 ꢀ 25 mL). The combined extracts were washed with brine, dried
over anhydrous Na2SO4 and concentrated. The resulting residue
was purified by silica gel column chromatography using ethyl ac-
etate/hexanes as eluent to afford compound 10 (600 mg,
yield ¼ 67%).
4.3. Cytotoxicity assays
4.1.5.3. General procedure D for the preparation of compounds 13a-
13d. A mixture of 4-amino-6-chloropyrimidine-5-carboxylic acid
10 (52 mg, 0.3 mmol), different substituted Ar-NH2 (0.3 mmol) and
EDCI (71 mg, 0.36 mmol) in THF (10 mL) was stirred at room
temperature for 5 h. Upon completion, the reaction mixture was
quenched with water, and then extracted with ethyl acetate twice.
The combined extracts were washed with brine, dried over anhy-
drous Na2SO4 and concentrated. The residue was purified by silica
gel column chromatography using ethyl acetate/hexanes as eluent
to give compound 11a-11d.
The cytotoxicity of the compounds on H9 were determined by
MTT colorimetric assay described previously [31]. The prepared
compounds with different concentrations were added into 96 well
culture plate and maintained in RPMI-1640 complete medium
(containing 10% FBS). 2 ꢀ 105 cell/well H9 were added and co-
incubated at 37 BC, 5% CO2 for seven days. The cell viability was
determined by using MTT, and the 50% cytotoxicity concentration
(CC50) was calculated.
4.4. Molecular docking
A mixture of 11a-11d (0.2 mmol), 4-nitrobenzenethiol (31 mg,
0.2 mmol) and Et3N (40 mg, 0.4 mmol) in n-butanol (10 mL) was
heated in oil bath at 120 BC for 4 h and monitored using TLC. After
cooling, the reaction mixture was poured in to water and extracted
with ethyl acetate (3 ꢀ 10 mL). The combined extracts were washed
with brine, dried over anhydrous Na2SO4 and concentrated. The
The crystal structure of HIV-1 Vif was gained from Protein Data
Bank (PDB ID 4N9F), and the binding site (X ¼ 72.881, Y ¼ ꢁ69.580,
Z ¼ ꢁ181.951) prepared for docking was chosen based on our
previous work [25]. Docking studies were performed with the
AutoDock (4.2) suite of programs. Binding sites were defined from
8

X. Zhong, R. Luo, G. Yan et al.
European Journal of Medicinal Chemistry 224 (2021) 113680
receptor cavities by using “define and edit binding site” method of
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Declaration of competing interest
The authors declare no conflicts of interest.
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Science and Technology Department of Sichuan Province of China
(grants 2021YJ0220).
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