Synthesis, biological evaluation and molecular docking study of N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent HIV-1 Vif antagonists

Article abstract of DOI:10.1016/j.ejmech.2017.01.010

Viral infectivity factor (Vif) is protective against APOBEC3G (A3G)-mediated viral cDNA hypermutations, and development of molecules that inhibit Vif mediated A3G degradation is a novel strategy for blocking HIV-1 replication. Through optimizations of the central ring of N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (RN-18), we found a potent compound 12c with EC₅₀value of 1.54?μM, enhancing the antiviral activity more than 150-fold compared with RN-18 in nonpermissive H9 cells. 12c protected A3G from degradation by inhibiting Vif function. Besides, 12c suppressed different HIV-1 clinical strains (HIV-1_KM018, HIV-1_TC-1and HIV-1_WAN) and drug-resistant strains (NRTI, NNRTI, PI, and FI) with relatively high activities. Amidation of 12c with glycine gave a prodrug 13a, improving the water solubility about 2600-fold compared with 12c. Moreover, 13a inhibited the virus replication efficiently with an EC₅₀value of 0.228?μM. These results suggested that the prodrug 13a is a promising candidate agent for the treatment of AIDS.

Full text of DOI:10.1016/j.ejmech.2017.01.010

Accepted Manuscript
Synthesis, biological evaluation and molecular docking study of N-(2-
methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent HIV-1 Vif
antagonists
Meng Zhou, Rong-Hua Luo, Xue-Yan Hou, Rui-Rui Wang, Guo-Yi Yan, Huan Chen,
Rong-Hong Zhang, Jian-You Shi, Yong-Tang Zheng, Rui Li, Yu-Quan Wei
PII:
S0223-5234(17)30018-1
10.1016/j.ejmech.2017.01.010
EJMECH 9161
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 December 2016
Revised Date: 6 January 2017
Accepted Date: 8 January 2017
Please cite this article as: M. Zhou, R.-H. Luo, X.-Y. Hou, R.-R. Wang, G.-Y. Yan, H. Chen, R.-H. Zhang,
J.-Y. Shi, Y.-T. Zheng, R. Li, Y.-Q. Wei, Synthesis, biological evaluation and molecular docking study of
N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent HIV-1 Vif antagonists,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and molecular
docking study of N-(2-methoxyphenyl)-6-((4-
nitrophenyl)sulfonyl)benzamide derivatives as
potent HIV‑1 Vif antagonists
a,c,d,1
b,1
a,1
b
a
b
Meng Zhou
, Rong-Hua Luo , Xue-Yan Hou , Rui-Rui Wang , Guo-Yi Yan , Huan Chen ,
e
a
b,*
a,*
a
Rong-Hong Zhang , Jian-You Shi , Yong-Tang Zheng , Rui Li Yu-Quan Wei
a
State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West
China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. 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, P. R. China.
c
Engineering Research Center for the Development and Application of Ethnic Medicine and
TCM, Ministry of Education, Guizhou Medical University, Guiyang 550004, Guizhou, China.
d
National Engineering Research Center of Miao‘s Medicines, Guizhou Medical University,
Guiyang 550004, Guizhou, China.
e
Tissue Engineering and Stem Cell Research Center, Guizhou Medical University, Guiyang
5
50004, Guizhou, P. R. China.
1
These authors contributed equally to this work.
*
Corresponding authors. E-mail addresses: lirui@scu.edu.cn (L. Rui), zhengyt@mail.kiz.ac.cn
Y-T, Zheng).
(
1

ACCEPTED MANUSCRIPT
Abstract. Viral infectivity factor (Vif) is protective against APOBEC3G (A3G)-mediated viral
cDNA hypermutations, and development of molecules that inhibit Vif mediated A3G
degradation is a novel strategy for blocking HIV-1 replication. Through optimizations of the
central ring of N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (RN-18), we found a
potent compound 12c with EC value of 1.54 ꢀM, enhancing the antiviral activity more than
5
0
1
50-fold compared with RN-18 in nonpermissive H9 cells. 12c protected A3G from degradation
by inhibiting Vif function. Besides, 12c suppressed different HIV-1 clinical strains (HIV-1KM018
,
HIV-1TC-1 and HIV-1WAN) and drug-resistant strains (NRTI, NNRTI, PI, and FI) with relatively
high activities. Amidation of 12c with glycine gave a prodrug 13a, improving the water
solubility about 2,600-fold compared with 12c. Moreover, 13a inhibited the virus replication
efficiently with an EC value of 0.228 ꢀM. These results suggested that the prodrug 13a is a
50
promising candidate agent for the treatment of AIDS.
Keywords. Vif antagonists, Vif mediated A3G degradation, nonpermissive, antiviral activity.
1
. Introduction
A major cellular host defense factor, APOBEC3G (A3G), which is an APOBEC3 family
member, is reported to protect host cells from retroviral damage and severely weaken infectivity
of human immunodeficiency virus type I (HIV-1) in nonpermissive cells (e.g., H9, CEM) [1, 2].
Antiviral effects of A3G include deaminase-dependent and -independent mechanisms. In the
absence of viral infectivity factor (Vif), approximately seven copies of A3G are incorporated into
budding virions by binding to the viral genomic (+) RNA or Gag [3, 4]. To initiate reverse
transcription, A3G is released from RNA-induced multimer by RNase H and induces
deamination of C-to-U on the first minus-strand viral cDNA [5, 6], which subsequently
2

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inactivates viral proteins and produces noninfectious virions [7-10]. Also, A3G can inhibit
numerous HIV-1 replication steps via a deaminase-independent mechanism. A3G inhibits tRNA
Lys
3
primer annealing, strand transfer, and nascent HIV-1 cDNA elongation [11, 12]. However, Vif
recruits an E3 ubiquitin ligase complex composed of elongin B (ELOB) and C (ELOC), cullin 5
CUL5), Rbx2 and CBF-β and mediates A3G degradation [13-19]. Thus, inhibition of Vif may
(
be an effective approach for preventing A3G from degradation and implementing antiviral
activity [20].
RN-18 (N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide, 1, Fig. 1), a novel Vif
antagonists discovered by Rana’s group [21], offered an innovative approach to treating HIV.
The derivatives of 1 reduce viral infectivity by enhancing A3G-dependent Vif degradation,
increasing A3G incorporation into virions, and enhancing cytidine deamination of the viral
genome [21-23]. Recent researches showed that replacement of amide functionality of 1 with a
1
,2,3-trizole improved the potency [24], indicating the potential of 1 optimizations. Although
structure-activity relationship of 1 on two branched chains (ring A and C, Fig. 1) has been well
studied [23], the role of the central ring is remain unclear. Therefore, this study describes the
design, synthesis and biological evaluation of a range of novel derivatives on this moiety of 1.
2
. Results and discussion
2
2
.1. Chemistry
.1.1. Optimizing strategies on the central ring of 1 (Strategy I, II, and III)
The central ring B of 1 was optimized with two strategies: replacement of the benzene ring
with a heteroaromatic ring (Strategy I, Fig. 1) and introduction of substituents to ring B (Strategy
II, Fig. 1). Firstly, replacement of ring B with a heterocyclic thiophene (2 and 3) decreased EC50
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5
-fold compared to the reported Vif inhibitor (1) in nonpermissive H9 cells (reduced HIV-1 p24
antigen production by 50%, Table 1). It should be noted that the different EC50 values of 1
1
, 21
reported by Rana
and this study might derive from the different testing systems. When
replaced with a pyridine (4) [23], EC value decreased to 147 ꢀM. Then, addition of halogens or
5
0
electron withdrawing groups to C5, such as a chlorine atom (5), fluorine (6), or a trifluoromethyl
7), nitro group (8), were investigated. Antiviral activities of 6, 7, and 8 were similar to 1, but the
(
activity of 5 was improved to 18.3 ꢀM. Interestingly, introduction of a chlorine atom to C5 of the
pyrimidine ring of 4 got a similar result, and the activity of 9 was 3-fold greater than 4,
indicating the existence of a potential halogen bond (X-bond) [25]. The heteroatom serving as
the X-bond acceptor was speculated to be a carbonyl oxygen of the peptide backbone, the most
common X-bond acceptors in protein-inhibitor complexes [26]. The decreased activity of 8
confirmed that a hydrogen bond (H-bond) acceptor such as carbonyl should be used instead of an
H-bond donor (hydroxyl, amide or thiol group); otherwise, the nitro group of 8 would form an H-
bond with the target, conferring a greater activity. The activity of thiophene (2 and 3) was 3-fold
greater than that of pyridine (4), which could be explained by an intermolecular C-S σ*
interaction with the carbonyl oxygen atom [27].
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Fig. 1. Optimization strategies of 1 (I and II), 11 (III), 12 (IV), and 12c (V).
Table 1. Antiviral activities of compound 1 derivatives in H9 cells.
a
Compound CC50
EC50
263
62.2
51.2
147
18.3
249
223
235
54.7
235
6.63
240
233
457
1.87
94.6
10.5
1.54
278
226
TI
b
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
N.A.
472
518
525
483
502
N.A.
N.A.
480
N.A.
506
302
182
171
387
202
335
462
200
439
N.A.
7.59
10.1
3.57
26.4
2.02
N.A.
N.A.
8.78
N.A.
76.3
1.26
0.781
0.374
207
0
1
1a
1b
1c
2
2a
2b
2c
2d
2e
2.14
31.9
300
0.719
1.94
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1
1
1
1
1
1
1
1
1
1
1
1
1
1
2f
38.2
211
265
45.4
214
43.6
216
41.8
221
8.24
242
187
176
30.6
172
0.248
1.46
0.841
0.986
6.08
0.713
0.849
N.A.
3.71
N.A.
N.A.
N.A.
11.9
N.A.
790
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
3a
3b
154
35.5
N.A.
30.6
N.A.
N.A.
N.A.
364
N.A.
196
216
148
a
b
TI: therapeutic index, TI = CC /EC . NA: a CC values could not be obtained at the tested
5
0
50
50
concentration range.
To verify our hypothesis, an amino group with two H-bond donors, was introduced to C5 (11)
of the central ring, and we noted an increased activity (EC 6.63 ꢀM), which might be ascribed
5
0
to the H-bond interaction between the amino group and the nearby carbonyl group of the target.
To understand the role of the amino group with respect to antiviral activity, the amino group of
1
1 was mono (11a)/dimethylated (11b), acetylated (11c) or translocated to the C6 position,
respectively (Strategy III, Fig. 1). 11a, 11b and 11c showed reduced activities, perhaps due to
partial or total loss of the H-bond and increased steric hindrance (Table 1). However, when the
amino group was transferred from C5 to C6 (12), activity obviously increased to 1.87 ꢀM.
Subsequently, the sulfide bridge of 12 was replaced by an oxygen (12a), a sulfoxide (12b) or a
sulphone (12c) (Strategy IV, Fig. 1). Results revealed that atom types and the sulfur oxidation
states could influence the activity. When sulfur (12) was replaced by oxygen (12a), activity
decreased ~50-fold; when sulfur was oxidized to sulfoxide (12b), activity decreased to 10.5 ꢀM;
while activity increased to 1.54 ꢀM when sulfoxide was oxidized to sulphone (12c).
2
.1.2. Lowest conformation analysis of 12, 12a, 12b and 12c
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To analyse the structure-activity relationship (SAR) of 12 and its analogues (12a, 12b, and
2c), the conformation of each compound was then studied. It was widely accepted that drugs
1
bound to an enzyme in a particular conformation which was probably the lowest energy
conformation or one close in energy to it [28-30]. Thus, the lowest energy conformation was
usually used in quantitative structure activity relationship (QSAR) and docking researches [31-
3
3]. The global minimum conformations of structure 12, 12a, 12b, and 12c were studied through
a conformational search method by using Discovery Studio software (3.1) [34] with a systematic
search method, which were then fully optimized using DFT at the B3LYP/6-31G(d) level, as
implemented in the Gaussian 03 program package [35]. All of them displayed no imaginary
frequencies. Results showed that the lowest conformation of four compounds (12, 12a, 12b and
1
2c) all tended to form two intramolecular H-bonds: the amino group with the amide carbonyl
group and the heteroatom of A-B ring bridge with the hydrogen of amide bond, conferring them
structural rigidity (Fig. 2). Through superposition of their common aniline group, the lowest
conformation of 12a was unique (Supplementary Fig. S1), which was interpreted from the
smaller radius of oxygen compared to sulfur. Regarding the torsion of ring B to C, with the
torsion angle of the four compounds decreasing, the activities declined (Table 1 and 2),
indicating that the direction in which ring B extended contributed to the activity. Compounds
with better activities (12 and 12c) had similar angles between ring B and A, and hence the angle
of 12c might be ideal in the active conformation (104.97 degrees). Deviation of 12a to the ideal
angle was larger than that of 12b by 10.40 degrees, which explained the obviously improved
antiviral effect of 12b. Thus, for 12 and 12c, the active conformations might be related to their
lowest conformations.
7

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Fig. 2. The lowest conformations and intramolecular H-bonds of 12, 12a, 12b and 12c,
respectively.
Table 2. The lowest conformational parameters of 12, 12a, 12b, and 12c, respectively.
a
b
Compound B-C torsion Deviation
A-B angle
104.97
104.67
100.37
119.97
Deviation
0
0.30
4.60
15.00
1
1
1
1
2c
2
2b
2a
53.82
46.23
44.48
27.85
0
7.59
9.34
25.97
a
b
Deviation means the difference torsion between 12c and 12, 12b, 12a, respectively;
Deviation means the difference angle between 12c and 12, 12b, 12a, respectively.
2
.1.3. Optimizing strategies of 12 (Strategy IV, V, and VI)
Next, the amino group of 12c was monomethylated (12d), and dimethylated (12e), and the
activity declined for each (Strategy IV, Fig. 1), indicating that the two hydrogens of the amino
group were important to antiviral activity as in the case of derivatives 11a, b. One hydrogen was
used to confer rigidity via an intramolecular H-bond with the carbonyl oxygen, and the other
might interact with Vif. Thus, to study additional interaction sites in the vicinity, one hydrogen
atom of the amino group was substituted. First, aminoethyl (12f, 12g), xethyl (12h, 12i) or
acetamide (12j) groups were used to explore the possible neighboring H-bond. Whereas the
antiviral activity of each modified compound was reduced (Table 1). Next, bromoethyl (12k),
8

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piperazine (12l), morpholine (12m), benzenesulfonamide (12n, 12o) and p-
methylbenzenesulfonamide (12p, 12q) were used to explore a possible X-bond and hydrophobic
interaction, and again activity decreased for all compounds, indicating a narrow cavity in which
the amino group was located. Interestingly, when the two hydrogens of the amino group were
replaced by piperazine, 12l retained activity (8.24 ꢀM), perhaps due to rigidity of the piperazine
group that translocated the structure outward to ensure H-bond interaction between polar
hydrogen on the piperazine group with the carbonyl oxygen. In contrast, with an amino group
replaced by morpholine 12m exhibited a reduced potency, confirming the presense of a carbonyl
group in the amino group interaction region.
2
.1.4. Binding mode analysis
Since the complicated activity of Vif, the crystal structure of Vif complex was not resolved
until recently [36], and the binding site of 1 was still unclear. Thus, to better understand the
SARs, a binding mode of the most potent inhibitor 12c with Vif (PDB ID 4N9F) was studied by
using Autodock (4.2) [37]. We noted five major binding pockets on the surface of Vif
(Supplementary Fig. S2, Table S1), and pocket 1 was the largest one but with no biological role.
Pocket 2 was smaller than the former and it had a definite physiological function, interacting
with ELOC and CUL5 [36, 38, 39]. Although pockets 3, 4 and 5 also bound A3G and CBF-β
[
17, 36, 40, 41], they were too small to accomadate 12c. Therefore, we focused on pocket 2 and
after 100 repeated dockings on pocket 2 with 12c, three major binding modes were found
Supplementary Fig. S3 and S4). Basing on the SAR of 12 derivatives, we speculated that mode
, with the lowest binding energy (−7.31 Kcal/mol), the highest occurrance rate (44%) and the
(
1
most (five) intermolecular H-bonds, was the possible binding mode of 12c (Table S2). Moreover,
9

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its active conformation adopted the lowest conformation, bearing two intramolecular H-bonds
(Fig. 3a). The ring C was buried in the hydrophobic cavity formed by Phe115, Tyr148, Ile155,
and Pro157 and the oxygen of the o-methoxyl group formed an H-bond with Cys133. The amino
group of central ring B formed H-bonds with Tyr148 and Gln136, and Tyr148 formed another H-
bond with the carbonyl group of 12c. With the intramolecular H-bond between the amino and the
amide carbonyl group, a pseudo-four-membered ring structure formed, reinforcing the ligand
interaction. Another carbonyl group was noted on His139 near the C-5 position of ring B (3.43
Å), which was not observed in the other two binding modes, indicating an origin of the X-bond
with Cl and the C−S σ* interaction with thiophene. The nitro group of ring A was near the main
chain polar hydrogen of Ala153 and tended to form an H-bond. Although the sulphone linker
was a little far from Lys156 (distance between sulphone and amino group on Lys156 = 3.91 Å),
due to the flexibility of the side chain of Lys, the H-bond could be easily formed via an induced-
fit process.
Fig. 3. Binding pose analysis of 1, 12a and 12c. (a) The possible binding pose of 12c with Vif.
(b) Binding mode difference of 12a (gray carbon) and 12c (green carbon) with Vif. (c) The
binding mode comparison of 1 (gray carbon) and 12c (green carbon).
10

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Through docking of 12a to pocket 2, a similar active conformation with 12c was observed,
with B and C rings well superimposed (Fig. 3b). Due to the larger angle between ring A and B of
1
2a, the whole ring A of 12a moved downward, rendering the nitro group totally lose the H-bond
interaction with Ala153. Besides, shorter C-O bond and smaller radius of oxygen increased the
distance of ether oxygen to Lys156 (distance between ether oxygen and amino group on Lys156
=
5.02 Å), thus decreasing the possibility of H-bond formation.
As for 1, this molecule adopted a similar active conformation with 12c, with ring C buried into
the hydrophobic cavity mentioned above and an H-bond was formed between the oxygen of o-
methoxyl group and Cys133 (Fig. 3c). Differently, without the amino group, the H-bond between
1
and Tyr148 or Gln136 was not observed. Besides, without the restriction of amino
intramolecular H-bond, torsion occurred on the amide bond, making carbonyl oxygen atom far
away from Tyr148 and thereby losing another H-bond with Tyr148. Apart from that, A and B
rings moved outward, and the distance between the nitro and Ala153 increased, which impaired
the H-bond. When the sulfide bridge was replaced by ether oxygen, due to the smaller radius of
oxygen and shorter C-O bond, the whole ring A moved inward and the distance of the nitro and
Ala153 was decreased, which might partially restore the H-bond interaction. That is in
agreement with the slightly improved activity of oxygen ether analogue of 1 in the reported
researches (Supplementary Fig. S5) [22, 23].
2
.1.5. Synthetic routes of representative compounds
Processes for the preparation of compounds in this study were similar, and six typical
compounds (2, 11a, 12, 12b, 12c, and 13a) were described in Scheme 1. Synthetic procedure of
other analogues was described in Experimental section. Intermediate 2I was obtained through
thiolation between 3-bromothiophene-2-carboxylic acid and 4-nitrobenzenethiol using base and
11

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copper nanopowder, which was then converted to the corresponding acyl chloride and was
further reacted with 2-methoxyaniline to afford 2 (Scheme 1a). For molecule 12, in the presence
of condensing agents, the intermediate 12I was obtained by the amidation reaction between 2-
amino-6-bromobenzoic acid and o-anisidine. Subsequently, 12I was mixed with 4-
nitrothiophenol in the presence of copper nanopowder and base to generate 12. Afterwards, 12
was oxidized by hydrogen peroxide dissolving in methanol or acetic acid to give sulfoxide 12b
or sulfone 12c, respectively (Scheme 1b). As for optimizations of the amino group of ring B,
substituents were introduced under base condition. For example, 11a was obtained in the
presence of potassium carbonate and iodomethane (Scheme 1c). While for the synthesis of
glycine prodrug 13a, 12c was reacted with boc-glycine under the condition of condensation
agents to afford the intermediate, which was then generated 13a through deprotection (Scheme
1
d).
12

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a
Scheme 1. Synthetic of representative compounds. Reagents and conditions: (a) 4-
Nitrothiophenol, copper nanopowder, K CO , DMF, 55 °C, 8 h. (b) SOCl , cat. DMF, 80 °C, 2 h.
2
3
2
(
c) 2-Methoxyaniline, Et N, benzene, 80 °C, 2 h. (d) 2-Methoxyaniline, EDCI, THF, RT, 2 h. (e)
3
4
-Nitrothiophenol, K CO , copper nanopowder, DMF, 65 °C, 8 h. (f) 35% H O , CH OH, 66 °C,
2 3 2 2 3
4
0min. (g) 35% H O , acetic acid (HAc), 55 °C, 3 h. (h) Iodomethane, K CO , DMF, 50 °C, 10
2
2
2
3
h. (i) Gly-Boc, DCC, HOBt, DCM, RT. (j) TFA/DCM (1:3), RT, 2 h.
13

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2
2
.2. Biological activity
.2.1. 12c protects APOBEC3G from Vif-mediated degradation.
Since Vif was reported to mediate the degradation of A3G [42], a fluorescent primary
screening assay was used to measure 12c effects on Vif-mediated A3G degradation. Results
showed that the EYFP fluorescence of 12c-treated cells were greater than those in DMSO-treated
cells (Fig. 4a). Also, compared with 1, 12c protected A3G better at the same concentration, and
1
2c protection of A3G against Vif-mediated degradation was dose-dependent (Fig. 4a and 4b).
To verify that 12c inhibited Vif-A3G interaction, TREX-hvif-15 cells were transfected with
pcDNA3.1-APOBEC3G-HA and total cellular proteins were extracted 48 hours post-transfection
for Vif and HA tag analysis. Results showed that A3G decreased in the presence of Vif and this
was reversed with the addition of 12c, and A3G increased with increasing concentrations of 12c
(Fig. 4c). In nonpermissive PBMCs infected with clinical strain HIV-1TC-1, Vif-mediated
degradation of A3G was inhibited by 12c and Vif expression was decreased with increasing
concentration of 12c, also, p24 expression was blocked (Fig. 4d). All these results proved that
1
2c had the ability of inhibiting Vif-mediated A3G degradation.
14

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Fig. 4. Effects of 12c on the expression of A3G, Vif, and p24. TREX-hvif-15 cells were
transfected with EYFP-N1-hA3G, and Vif was induced by 1 ꢀg/mL Dox. The cells were treated
with or without 12c (a) or 1 (b), respectively. The EYFP fluorescence was determined by flow
cytometry and no Vif expression was set to 100%. (c) pcDNA3.1-APOBEC3G-HA was
transfected into TREX-hvif-15 and Vif expression was induced by 0.1 ꢀg/mL Dox. (d) PBMCs
were infected with HIV-1TC-1 in the presence or absence of 12c. The total cellular protein was
extracted, and the expression of A3G, Vif, HA tag, p24 and β-actin (internal control) were
determined by western blot.
15

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2
.2.2. 12c inhibits HIV-1 replication in different cell lines with relatively low cytotoxicity
The antiviral activity of 12c was measured in H9 cells and PHA-stimulated PBMCs. H9 cells
were infected with NL4-3 and PBMCs were infected with clinical strains HIV-1KM018, HIV-1TC-1
,
HIV-1WAN, and HIV-1SF162, the former three of which are prevalent in China [43, 44]. Result
showed that 12c effectively inhibited replication of different wild-type HIV-1 strains in different
cell lines (Table 3). EC values of 12c against lab-adaptive strains ranged from 0.522-17.8 ꢀM.
50
Moreover, 12c had low cytotoxicity against H9 cells and PBMCs, with CC values higher than
50
4
62 ꢀM.
a
Table 3. Anti-HIV-1 activities of 12c in cell cultures
b
c
Cells
H9
PBMC
Virus
NL4-3
HIV-1KM018
HIV-1SF162
HIV-1TC-1
HIV-1WAN
Subtype
CC50 (µM)
> 462
> 468
EC50 (µM)
B
B/C
1.40 ± 0.13
17.8 ± 6.30
17.8 ± 4.20
0.522 ± 0.11
d
B
CRF01-AE
CRF01-AE
2.95 ± 1.14
a
b
All data represent means ± standard deviation for three separate experiments. CC , 50%
5
0
c
d
cytotoxic concentration. EC , 50% effective concentration. B/C, B/C recombinant.
50
2
.3 Solubility optimizations of 12c
The aqueous and organic solvent solubilities, partition coefficient (log p), and plasma protein
binding of 12c were measured. Data showed that aqueous solubility of 12c was poor but organic
solubility was high (Supplementary Table S3, S4, and S5). 12c was readily bound to plasma
protein (Fig. 5a), which might lead to potentially low activity after absorbed in vivo. In order to
improve the aqueous solubility of 12c, water-soluble glycine and phenylalanine prodrugs of 12c
were then synthesised (13a and 13b, respectively, Strategy VII, Fig. 1) [45-50]. The water
solubility was increased significantly, and the solubility of 13a and 13b were improved to 1730
16

ACCEPTED MANUSCRIPT
and 120 ꢀg/mL, respectively (Supplementary Table S3). Moreover, 13a and 13b showed
obviously enhanced activity in H9 cells (0.248 and 1.46 ꢀM, respectively, Table 1), probably
because of the increased aqueous solubility. Protein analyses showed that 13a reduced Vif level
and improved A3G expression in a dose-dependent manner like 12c (Fig. 5b). Considering
potent antiviral activity, low-cost and synthetic efficiency, the glycine prodrug 13a was chosen
as a candidate prodrug of 12c.
The forced degradation studies of 13a were then studied using different pH buffer solutions
and human liver microsomes (HLM). 13a was stable at pH 4 and 7 (Fig. 5c), and about 50% of
1
3a remained 180 h after exposure to pH 4 buffer. However, degradation of 13a accelerated with
increasing pH, and > 90% of 13a was hydrolyzed at pH 9 in 15 h. Then, HLM metabolism
studies were used to examine 13a enzymatic degradation. Data showed that 13a was degraded
within 15 min (10 min, 76.3%; 15 min, 99.9%) to the parent 12c (Fig. 5d, Supplementary Fig. S6
and S7). After 20 min, the prodrug was undetectable.
17

ACCEPTED MANUSCRIPT
Fig. 5. Degradation researches and effects on the expression of A3G, Vif, and p24 of 13a. (a)
Plasma protein binding rate of 12c at different concentrations (5, 20 and 32 ꢀg/mL). (b) 293T
cells were co-transfected with pcDNA3.1-APOBEC3G-HA and pNL4-3 in the presence or
absence of 13a. The total cellular protein was extracted, and the expression of A3G, Vif, HA tag,
p24 and β-actin (internal control) were determined by western blot. (c) Degradation research of
1
3a at pH 4, pH 7, and pH 9. (d) Degradation research of 13a in human liver microsome (HLM)
in 0.1 M phosphate buffer (pH 7.4) containing 36 ꢀL NADPH generating system at 37 °C for 0,
5
, 10, 15, 20, 40 min, respectively.
3
. Conclusions
In summary, through the optimizations on the central B ring of 1, a new HIV-1 Vif antagonist
2c was found. The SAR of 12c derivatives revealed that two hydrogens of the amino group
1
were crucial to improve the antiviral activity, with one hydrogen forming an H-bond with Vif,
and the other hydrogen conferring rigidity via an intramolecular H-bond with the carbonyl
oxygen. In nonpermissive H9 cells, 12c showed improved activity about 150-fold compared with
1
. 12c protected the expression of A3G more effectively than 1 at the same concentration.
Meanwhile, 12c significantly inhibited Vif-mediated degradation of A3G, and Vif expression
was decreased with the concentration increasing, as well as p24 expression. Further biological
activity assays showed a promising inhibitory effect of 12c against various wild-type strains with
low cytotoxicities. To improve the water solubility, amino acids (glycine and phenylalanine) was
conjugated with 12c and the glycine prodrug 13a enhanced the aqueous solubility more than
2
,600 times. In addition, the antiviral activity of 13a was increased obviously compared with 12c
0.228 ꢀM). The efficient antiviral effect and good solubility make 13a a potential drug for
treating AIDS.
(
18

ACCEPTED MANUSCRIPT
4
. Experimental
4
.1. General information
1
Proton magnetic resonance ( H NMR) spectra was recorded on a Bruker (Avance III 400 MHz)
spectrometer. Chemical shifts were reported in parts per million (ppm, δ scale) relative to
tetramethylsilane (δ 0.00). Data are represented as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiple resonances, br = broad, app = apparent),
integration, and coupling constant (J) in Hertz. Splitting patterns that could not be easily
interpreted are designated as multiplet (m) or broad (br). Carbon nuclear magnetic resonance
1
3
(
C NMR) spectra was recorded on a Bruker (Avance III 400 MHz) (100 MHz) spectrometer.
High resolution Mass Spectroscopy (HRMS) was performed on an ESI-QTOF mass
spectrometer (Micromass, UK) equipped with an interchangeable ESI Z-spray source. All final
compounds were purified to > 95% purity as determined by HPLC (Dionex Ultimate 3000, USA)
analysis using the following methods. Purity analysis of final compounds was performed through
a C Acclaim 120 (particle size = 5 ꢀm, pore size = 12 nm, dimensions = 250 × 4.6 mm) column.
1
8
The injection volume was 10 ꢀL and the flow rate was 1.0 mL/min. Each analysis lasted for 20
minutes. The percentage of each solvent and retention times (RT,HPLC) and purity data (%) are
displayed in the analytical data of the respective compounds in Experimental Section.
4
.2. General procedure for the synthesis of 2-13.
4
.2.1. 3-((4-nitrophenyl)thio)thiophene-2-carboxylic acid (2I)
3
-bromothiophene-2-carboxylic acid (2070 mg, 10 mmol), 4-nitrobenzenethiol (1860 mg, 12
mmol), copper nanopowder (320 mg, 5 mmol) and anhydrous potassium carbonate (2760 mg, 20
19

ACCEPTED MANUSCRIPT
mmol) was dissolved in DMF (50 mL), and the mixture was stirred at 55 °C for 8 hours the
reaction was monitored using TLC. The reaction mixture was cooled to room temperature,
filtered and washed with DMF (2 × 5 mL). The filtrate was acidified using 5N HCl leading to the
precipitation of crude product. The crude residue was diluted with ethyl acetate (150 mL) and
washed with water (2 × 50 mL). The organic layer was dried over anhydrous Na SO , filtered,
2
4
and concentrated. The oily residue was applied to silica gel column chromatography (1:4 v/v
1
ethyl acetate/petroleum ether) to afford a yellow solid (2166 mg, 77%). H NMR (400 MHz,
DMSO-d ) δ = 6.79 (d, J = 7.2Hz, 1H), 7.65 (d, J = 10Hz, 2H), 7.90 (d, J = 7.2Hz, 1H), 8.24 (d,
6
J = 8.8Hz, 2H), 13.42 (s, 1H).
4
.2.2. N-(2-methoxyphenyl)-3-((4-nitrophenyl)thio)thiophene-2-carboxamide (2)
A solution of 3-((4-nitrophenyl)thio)thiophene-2-carbonyl chloride (prepared by stirring of 2I
(
281 mg, 1 mmol) in SOCl (15 mL) at 78 °C for 2 h followed by concentration of the mixture
2
under reduced pressure) in anhydrous THF (25 mL) was added slowly to a stirred mixture of 2-
methoxyaniline (98.4 mg, 0.8 mmol) and Et N (0.416 mL, 3 mmol) dissolved in 10 mL THF at
3
0
°C. The reaction mixture was stirred for 2 hours at room temperature and concentrated under
reduced pressure. The oily residue was diluted with ethyl acetate (50 mL) and washed with water
2 × 20 mL), dried over anhydrous Na SO , filtered, and concentrated under reduced pressure.
(
2
4
The residue was applied to silica gel column chromatography (1:6 v/v ethyl acetate/petroleum
1
ether) to afford a white solid (300.6 mg, 77.8%). m. p. 138.4–138.9 °C; H NMR (400 MHz,
CDCl ) δ = 3.75 (s, 3H), 6.86 (d, J = 8.0Hz, 1H), 6.98 (t, J = 8.0Hz, 1H), 7.07 (m, 1H), 7.16 (d,
3
J = 5.2Hz, 1H), 7.26 (d, J = 8.8Hz, 2H), 7.67 (d, J = 4.8Hz, 1H), 8.14 (d, J = 8.4Hz, 2H), 8.45
+
(
dd, J = 8.0,1.6Hz, 1H), 10.11 (s, 1H); HRMS (ESI) calcd for C H N O S [M+H] : 387.0473,
1
8
15
2
4 2
20

ACCEPTED MANUSCRIPT
found: 387.0476; RT,HPLC = 11.077 min, Purity > 99%, MeOH:H O = 75:25, 30 °C. The same
2
procedure was also followed for the syntheses of 3, 4, 5, 6, 7, 8, 9 and 10.
4
.2.3. N-(2-methoxyphenyl)-4-((4-nitrophenyl)thio)thiophene-3-carboxamide (3)
1
Yellow solid; 63.7% yield; m. p. 137.0–137.9 °C; H NMR (400 MHz, CDCl ) δ = 3.80 (s, 3H),
3
6
.85 (d, J = 8.0Hz, 1H), 6.95 (t, J = 8.0Hz, 1H), 7.05 (m, 1H), 7.18 (d, J = 6.8Hz, 2H), 7.75 (d, J
=
3.6Hz, 1H), 8.08 (d, J = 6.8Hz, 2H), 8.41 (dd, J = 8.0,1.2Hz, 1H), 8.44 (d, J = 3.6Hz, 1H),
+
9
.68 (s, 1H); HRMS (ESI) calcd for C H N O S [M+H] : 387.0473, found: 387.0479; R
1
8
15
2
4
2
T,HPLC
=
15.677 min, Purity > 99%, MeOH:H O = 75:25, 30 °C.
2
4
.2.4. N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)nicotinamide (4)
1
White solid; 67% yield; m. p. 132.9–133.2 °C; H NMR (400 MHz, CDCl ) δ = 3.91 (s, 3H),
3
6
.94 (d, J = 8.0Hz, 1H), 7.04 (t, J = 7.6Hz, 1H), 7.14 (m, 1H), 7.27 (d, J = 12.0Hz, 2H), 7.66 (d,
J = 8.4Hz, 1H), 7.98 (d, J = 7.6Hz, 1H), 8.20 (d, J = 8.8Hz, 2H), 8.49 (brs, 3H); HRMS (ESI)
+
calcd for C H N O S [M+H] : 382.0862, found: 382.0864; R
= 6.587 min, Purity > 99%,
1
9
16
3
4
T,HPLC
MeOH:H O = 75:25, 30 °C.
2
4
.2.5. 5-chloro-N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (5)
1
Yellow solid; 72% yield; m. p. 117.4–118.4 °C; H NMR (400 MHz, CDCl ) δ = 3.80 (s, 3H),
3
6
2
.87 (d, J = 8.0Hz, 1H), 6.67 (t, J = 8.0Hz, 1H), 7.08 (m, 1H), 7.29 (d, J = 8.4Hz, 2H), 7.49 (m,
H), 7.77 (s, 1H), 8.07 (d, J = 8.8Hz, 2H), 8.36 (s, 1H), 8.38 (s, 1H); HRMS (ESI) calcd for
+
C H ClN O S [M+H] : 415.0519, found: 415.0520; R
= 14.180 min, Purity > 99%,
20
16
2
4
2
T,HPLC
MeOH:H O = 75:25, 30 °C.
2
21

ACCEPTED MANUSCRIPT
4
.2.6. 5-fluoro-N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (6)
1
Yellow solid; 69.3% yield; m. p. 110.0–111.2 °C; H NMR (400 MHz, CDCl ) δ = 3.77 (s,
3
3
8
H), 6.86 (d, J = 8.0Hz, 1H), 6.97 (t, J = 7.6Hz, 1H), 7.08 (m, 1H), 7.25 (m, 3H), 7.55 (dd, J =
.8,2.8Hz, 1H), 7.62 (m, 1H), 8.06 (d, J = 8.8Hz, 2H), 8.37 (d, J = 7.2Hz, 1H), 8.45 (s, 1H);
+
HRMS (ESI) calcd for C H FN O S [M+H] : 399.0815, found: 399.0818; R
= 10.170
20
16
2
4
T,HPLC
min, Purity > 99%, MeOH:H O = 75:25, 30 °C.
2
4
.2.7. N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)-5-(trifluoromethyl)benzamide (7)
1
Yellow solid; 77.9% yield; m. p. 99.9–101.7 °C; H NMR (400 MHz, CDCl ) δ = 3.85 (s, 3H),
3
6
7
.90 (d, J = 8.1Hz, 1H), 6.99 (t, J = 7.6Hz, 1H), 7.11 (td, J = 8.0, 1.5, 1H), 7.44 (d, J = 8.8, 2H),
.52 (d, J = 8.3Hz, 1H), 7.67 (dd, J = 8.3, 1.5, 1H), 7.97 (s, 1H), 8.14 (t, J = 5.7, 2H), 8.39 (d, J
+
=
=
6.2, 2H); HRMS (ESI) calcd for C H F N O S [M+H] : 449.0783, found: 449.0782; R
2
1
16
3
2
4
T,HPLC
14.587 min, Purity > 98%, MeOH:H O = 75:25, 30 °C.
2
4
.2.8. N-(2-methoxyphenyl)-5-nitro-2-((4-nitrophenyl)thio)benzamide (8)
1
Yellow solid; 52% yield; H NMR (400 MHz, DMSO-d ): δ = 3.938 (S, 3H), 6.76 (d, J=8.4
6
Hz, 1H), 7.06 (t, J=8 Hz, 1H), 7.18 (t, J=7.6 Hz, 1H), 7.70 (m, 2H), 8.15 (d, J=8.8 Hz, 2H), 8.23
+
(
m, 3H), 8.39 (m, 2H); HRMS (ESI) calcd for C H N NaO S [M+Na] : 448.0579, found:
20
15
3
6
4
48.0584; RT,HPLC = 12.427 min, Purity > 99%, MeOH:H O = 75:25, 30 °C.
2
4
.2.9. N-(2-chloro-5-((4-nitrophenyl)thio)pyrimidin-4-yl)-2-methoxybenzamide (9)
1
White solid; 59.2% yield; m. p. 84.9–87.2 °C; H NMR (400 MHz, CDCl ) δ = 4.00 (s, 3H),
3
7
.05 (m, 3H), 7.56 (t, J = 7.6Hz, 1H), 7.71 (t, J = 7.6Hz, 2H), 7.86 (d, J = 8.8Hz, 1H), 8.28 (d, J
22

ACCEPTED MANUSCRIPT
+
=
8.24, 2H); HRMS (ESI) calcd for C H ClN O S [M+H] : 417.0424, found: 417.0426; R
1
8
14
4
4
T,HPLC
=
12.073 min, Purity > 98%, MeOH:H O = 75:25, 30 °C.
2
4
.2.10. N-(2-aminophenyl)-2-nitro-6-((4-nitrophenyl)thio)benzamide (10)
1
White solid; 68.5% yield; m. p. 200.7–201.6 °C; H NMR (400 MHz, CDCl ) δ = 3.57 (brs,
3
2
2
1
H), 6.83 (d, J = 8.0Hz, 2H), 7.14 (t, J = 7.2Hz, 1H), 7.22 (d, J = 8.0Hz, 2H), 7.40 (d, J = 8.0Hz,
H), 7.68 (t, J = 7.6Hz, 1H), 7.85 (d, J = 8.0Hz, 1H), 7.09 (d, J = 8.0Hz, 2H), 8.31 (d, J = 8.0Hz,
+
H); HRMS (ESI) calcd for C H N O S [M+H] : 411.0763, found: 411.0761; R
= 4.287
1
9
15
4
5
T,HPLC
min, Purity > 99%, MeOH:H O = 75:25, 30 °C.
2
4
.2.11. 5-amino-N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (11)
1
Yellow solid; 84% yield; m. p. 141.0–142.3 °C; H NMR (400 MHz, CDCl ) δ = 3.71 (s, 3H),
3
4
.20 (brs, 2H), 6.82 (m, 2H), 6.95 (t, J = 7.6Hz, 1H), 7.04 (t, J = 8.0Hz, 1H), 7.14 (m, 3H), 7.42
(d, J = 8.0Hz, 1H), 8.03 (d, J = 9.2Hz, 2H), 8.40 (d, J = 8.0Hz, 1H), 8.45 (brs, 1H); HRMS (ESI)
+
calcd for C H N O S [M+H] : 396.1018, found: 396.1021; R
= 7.207 min, Purity > 99%,
2
0
18
3
4
T,HPLC
MeOH:H O = 75:25, 30 °C.
2
4
.2.12. N-(2-methoxyphenyl)-5-(methylamino)-2-((4-nitrophenyl)thio)benzamide (11a)
1
1 (395 mg, 1 mmol) was dissolved in DMF (40 mL) and treated with anhydrous potassium
carbonate (276 mg, 2 mmol) at room temperature. To this mixture was added iodomethane (56
L, 0.9 mmol) via a micro syringe. After being stirred for 10 hours at 50 °C, the reaction mixture
ꢀ
was poured into ice-cold water (200 mL) to get crude product. The crude solid was filtered and
washed several times with water and purified by silica gel column chromatography (1:6 v/v ethyl
acetate/petroleum ether) to provide a yellow solid (176.3 mg, 43% yield). m. p. 150.4–151.6 °C;
23

ACCEPTED MANUSCRIPT
1
H NMR (400 MHz, CDCl ) δ = 2.93 (s, 3H), 3.69 (s, 3H), 4.24 (brs, 1H), 7.72 (d, J = 8.4Hz,
3
1
7
H), 6.82 (d, J = 8.0Hz, 1H), 6.94 (t, J = 6.4Hz, 1H), 7.03 (m, 2H), 7.13 (d, J = 8.8Hz, 2H),
.43 (d, J = 8.4Hz, 1H), 8.02 (d, J = 8.8Hz, 2H), 8.41 (d, J = 8.0Hz, 1H), 8.45 (brs, 1H); HRMS
+
(
ESI) calcd for C H N O S [M+H] : 410.1175, found: 410.1179; R
= 11.117 min, Purity >
T,HPLC
2
1
20
3
4
9
9%, MeOH:H O = 75:25, 30 °C. The same procedure was also followed for the syntheses of
2
1
2d, 12f, 12h, 12j and 12k.
4
.2.13. 5-(dimethylamino)-N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (11b)
1
1 (395 mg, 1 mmol) was dissolved in DMF (40 mL) and treated with anhydrous potassium
carbonate (552 mg, 4 mmol) at room temperature. To this mixture was added iodomethane (156
L, 2.5 mmol) via a micro syringe. After being stirred for 10 hours at 60 °C, the reaction mixture
ꢀ
was poured into ice-cold water (200 mL) to get crude product. The crude solid was filtered and
washed several times with water and purified by silica gel column chromatography (1:8 v/v ethyl
1
acetate/petroleum ether) to provide a yellow solid (330.8 mg, 78.1% yield). H NMR (400 MHz,
CDCl ) δ = 3.09 (s, 6H), 3.69 (s, 3H), 6.83 (m, 2H), 6.95 (t, J = 6.8Hz, 1H), 7.04 (t, J = 7.6Hz,
3
1
8
=
H), 7.13 (m, 3H), 7.47 (d, J = 8.4Hz, 1H), 8.03 (d, J = 9.2Hz, 2H), 8.43 (dd, J = 8.0,1.2Hz, 1H),
+
.48 (brs, 1H); HRMS (ESI) calcd for C H N O S [M+H] : 424.1331, found:424.1336; R
2
2
22
3
4
T,HPLC
6.953 min, Purity > 98%, MeOH:H O = 75:25, 30 °C. The same procedure was also followed
2
for the syntheses of 12e.
4
.2.14. 5-acetamido-N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (11c)
1
1 (395 mg, 1 mmol) was dissolved in DCM (30 mL) at room temperature. To this mixture
was added acetic anhydride (122 mg, 1.2 mmol), followed by the addition of DMAP (61 mg, 0.5
mmol). After being stirred for 5 hours at room temperature, the reaction mixture was
24

ACCEPTED MANUSCRIPT
concentrated under reduced pressure. The crude residue was diluted ethyl acetate (60 mL),
washed with water (2 × 15 mL), dried over anhydrous Na SO , filtered, and concentrated under
2
4
reduced pressure. The residue was purified by silica gel column chromatography (1:6 v/v ethyl
acetate/petroleum ether) to provide a yellow solid (420 mg, 96% yield). m. p. 191.4–193.2 °C;
1
H NMR (400 MHz, CDCl ) δ = 2.18 (s, 3H), 3.73 (s, 3H), 6.85 (d, J = 8.4Hz, 1H), 6.95 (t, J =
3
7
.6Hz, 1H), 7.07 (t, J = 8.0Hz, 1H), 7.19 (d, J = 8.8Hz, 2H), 7.56 (d, J = 8.8Hz, 1H), 7.83 (d, J
=
1.6Hz, 1H), 7.94 (d, J = 8.0Hz, 1H), 7.98 (brs, 1H), 8.04 (d, J = 9.2Hz, 2H), 8.35 (d, J =
+
7
4
.6Hz, 1H), 8.51 (brs, 1H); HRMS (ESI) calcd for C H N O S [M+H] : 438.1124, found:
22
20
3
5
38.1127; RT,HPLC = 7.240 min, Purity > 99%, MeOH:H O = 75:25, 30 °C.
2
4
.2.15. Synthesis of 2-amino-6-bromo-N-(2-methoxyphenyl)benzamide (12I)
2
-methoxyaniline (369 mg, 3 mmol) and EDCI (230 mg, 1.2 mmol) were mixed in
tetrahydrofuran (30 mL) with a magnetic stirrer for 5 min at room temperature (RT), and to this
mixture was slowly added a solution of 2-amino-6-bromobenzoic acid (215 mg, 1 mmol) in
tetrahydrofuran (20 mL). After stirred for 2 hours at RT, the reaction mixture was concentrated
under reduced pressure and then was diluted with water (30 mL). The aqueous layer was
extracted with ethyl acetate (3 × 15 mL) and the combined organic layers were dried over
anhydrous sodium sulfate, filtered, and concentrated. The oily residue was applied to silica gel
column chromatography (1:5 v/v ethyl acetate/petroleum ether) to afford 12I as a white
1
amorphous solid (243 mg, 76% yield). m. p. 124.1–125.3 °C; H NMR (400 MHz, DMSO-d ) δ
6
=
=
3.80 (s, 3H), 5.33 (s, 2H), 6.72 (d, J = 8Hz, 1H), 6.78 (d, J = 8Hz, 1H), 6.99 (m, 2H), 7.07 (d, J
1
3
8.4Hz, 1H), 7.18 (t, J = 7.6Hz, 1H), 7.85 (t, J = 7.2Hz, 1H), 9.43 (s, 1H); C NMR (100 MHz,
DMSO-d ) δ = 55.8, 111.5, 114.3, 119.2, 119.6, 120.3, 123.5, 123.9, 125.8, 126.6, 130.7, 147.2,
6
25

ACCEPTED MANUSCRIPT
+
1
51.2, 165.1; HRMS (ESI) calcd for C H BrN O [M+H] : 321.0239, found: 321.0240; R
1
4
14
2
2
T,HPLC
=
6.187 min, Purity > 98%. MeOH:H O = 70:30, 30 °C.
2
4
.2.16. Synthesis of 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)thio)benzamide (12)
1
2I (320 mg, 1 mmol), 4-nitrothiophenol (310 mg, 2 mmol), copper nanopowder (32 mg, 0.5
mmol) and anhydrous potassium carbonate (414 mg, 3 mmol) were mixed in DMF (40 mL) at
RT. Then the reaction flask was heated in an oil bath at 65 °C. After 8 hours, the heating bath
was removed and the reaction flask was allowed to cool to RT. The reaction mixture was filtered
and the liquid layer was diluted with water (40 mL), extracted with ethyl acetate (3 × 20 mL),
washed with water (6 × 15 mL), dried over anhydrous Na SO , filtered, and concentrated under
2
4
reduced pressure. The residue was purified by silica gel column chromatography (1:3 v/v ethyl
acetate/petroleum ether) to provide the product 12 as a yellow amorphous solid (296.3 mg, 75%
1
yield); m. p. 135.0–136.0 °C; H NMR (400 MHz, CDCl ) δ = 3.69 (s, 3H), 4.54 (br, 2H), 6.84
3
(d, J = 8Hz, 2H), 6.95 (m, 2H), 7.06 (t, J = 8Hz, 1H), 7.22 (m, 3H), 8.03 (d, J = 7.6Hz, 2H), 8.23
1
3
(
s, 1H), 8.32 (d, J = 7.6Hz, 1H); C NMR (100 MHz, DMSO-d ) δ = 55.6, 111.3, 117.0, 120.2,
6
1
22.9, 123.3, 123.9, 125.4, 126.6, 126.9, 130.7, 144.7, 146.9, 148.5, 150.7, 165.1; HRMS (ESI)
+
calcd for C H N O S [M+H] : 396.1018, found: 396.1020; R
= 12.583 min, Purity > 99%,
2
0
18
3
4
T,HPLC
MeOH:H O = 60:40, 30 °C. The same procedure was also followed for the syntheses of 11 and
2
1
2a.
4
.2.17. 2-amino-N-(2-methoxyphenyl)-6-(4-nitrophenoxy)benzamide (12a)
1
Yellow solid; 38% yield; m. p. 136.3–137.9 °C; H NMR (400 MHz, CDCl ) δ = 3.77 (s, 3H),
3
5
.88 (brs, 2H), 6.28 (d, J = 8Hz, 1H), 6.60 (d, J = 8Hz, 1H), 6.90 (m, 2H), 7.67 (t, J = 8Hz, 1H),
.00 (d, J = 8Hz, 2H), 7.18 (t, J = 8Hz, 1H), 8.21 (d, J = 8.8Hz, 2H), 8.42 (d, J = 8Hz, 1H), 9.49
7
26

ACCEPTED MANUSCRIPT
+
(
s, 1H); HRMS (ESI) calcd for C H N NaO [M+Na] : 402.1066, found: 402.1052; R
=
T,HPLC
2
0
17
3
5
9
.533 min, Purity > 98%, MeOH:H O = 75:25, 30 °C.
2
4
.2.18. 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfinyl)benzamide (12b)
1
2 (395 mg, 1 mmol) was dissolved in CH OH (30 mL) with a magnetic stirrer at RT, and 35%
3
H O (0.258 mL, 3 mmol) was added slowly to this solution. This reaction mixture was heated in
2
2
an oil bath at 66 °C and monitored using TLC. The reaction was quenched with MnO at RT.
2
The mixture was filtered and the residue was washed with ethyl acetate (3 × 5 mL). The liquid
layer was concentrated under reduced pressure and the oily residue was purified by silica gel
column chromatography (1:4 v/v ethyl acetate/petroleum ether) to provide a yellow solid; 44.3%
1
yield; m. p. 192.7–194.2 °C; H NMR (400 MHz, CDCl ) δ = 3.90 (s, 3H), 4.46 (brs, 2H), 6.88
3
(d, J = 8.0Hz, 1H), 6.96 (d, J = 8.0Hz, 1H), 7.04 (t, J = 7.6Hz, 1H), 7.17 (t, J = 8.0Hz, 1H), 7.41
(t, J = 8.0Hz, 1H), 7.47 (d, J = 7.2Hz, 1H), 7.91 (d, J = 8.8Hz, 2H), 8.21 (d, J = 8.8Hz, 2H),
+
8
.28 (d, J = 7.2Hz, 1H), 8.75 (brs, 1H); HRMS (ESI) calcd for C H N NaO S [M+Na] :
2
0
17
3
5
4
34.0787, found: 434.0784; RT,HPLC = 4.913 min, Purity > 96%, MeOH:H O = 75:25, 30 °C.
2
4
.2.19. 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide (12c)
1
2 (395 mg, 1 mmol) was dissolved in acetic acid (30 mL) with a magnetic stirrer at RT, and
H O (0.233 mL, 3 mmol) was added to this solution. This reaction mixture was heated in an oil
2
2
bath at 55 °C and the reaction was monitored using TLC. The reaction was quenched with MnO2
305 mg, 3.5 mmol) at RT. The mixture was filtered and the residue was washed with ethyl
(
acetate (3 × 10 mL). The liquid layer was concentrated under reduced pressure and the oily
residue was purified by silica gel column chromatography (1:2 v/v ethyl acetate/petroleum ether)
to provide the product 12c as a yellow amorphous solid (162.4 mg, 38% yield). m. p. 217.0–
27

ACCEPTED MANUSCRIPT
1
2
7
=
18.8 °C; H NMR (400 MHz, DMSO-d ) δ = 3.79 (s, 3H), 5.57 (s, 2H), 7.01 (t, J = 7.6Hz, 1H),
6
.08 (m, 2H), 7.22 (t, J = 7.6Hz, 1H), 7.29 (d, J = 7.6Hz, 1H), 7.36 (t, J = 7.6Hz, 1H), 7.76 (d, J
1
3
7.6Hz, 1H), 8.16 (d, J = 8.8Hz, 2H), 8.36 (d, J = 8.8Hz, 2H), 9.71 (s, 1H); C NMR (100 MHz,
DMSO-d ) δ = 55.7, 111.5, 116.8, 120.4, 121.0, 124.4, 124.9, 126.2, 126.5, 129.2, 130.0, 136.6,
6
+
1
46.7, 147.2, 149.9, 151.9, 164.2; HRMS (ESI) calcd for C H N O S [M+H] : 428.0916,
2
0
18
3
6
found: 428.0920; RT,HPLC = 9.797 min, Purity > 97%, MeOH:H O = 60:40, 30 °C. The same
2
procedure was also followed for the syntheses of 12g, 12i, 12o, and 12p.
4
.2.20. N-(2-methoxyphenyl)-2-(methylamino)-6-((4-nitrophenyl)sulfonyl)benzamide (12d)
1
White solid; 85.2% yield; m. p. 262.9–265.7 °C; H NMR (400 MHz, CDCl ) δ = 3.29 (s, 3H),
3
4
.03 (s, 3H), 4.73 (brs, 1H), 6.66 (dd, J = 8.0,0.8Hz, 1H ), 7.04 (td, J = 8.0,1.2Hz, 1H), 7.15 (dd,
J = 8.0,1.2Hz, 1H), 7.24 (m, 3H), 7.41 (m, 2H), 7.57 (m, 2H), 7.95 (d, J = 12.0Hz, 2H); HRMS
+
(
ESI) calcd for C H N NaO S [M+Na] : 464.0892, found: 464.0894; R
= 3.910 min,
T,HPLC
2
1
19
3
6
Purity > 98%, MeOH:H O = 80:20, 30 °C.
2
4
.2.21. 2-(dimethylamino)-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide (12e)
1
Yellow solid; 77.2% yield; m. p. 118.5–119.2 °C; H NMR (400MHz, CDCl ) δ = 2.76 (d, J =
3
4
.8Hz, 3H), 3.28 (s, 3H), 4.08 (s, 3H), 6.61 (d, J = 8.0Hz, 1H), 7.09 (t, J = 7.6Hz, 1H), 7.13 (d, J
=
8.0Hz, 1H), 7.21 (d, J = 7.6Hz, 2H), 7.36 (m, 2H), 7.43 (t, J = 6.4Hz, 1H), 7.60 (d, J = 9.2Hz,
+
2
4
H), 7.91 (d, J = 8.0Hz, 2H); HRMS (ESI) calcd for C H N NaO S [M+Na] : 478.1049, found:
2
2
21
3
6
78.1047; RT,HPLC = 4.487 min, Purity > 98%, MeOH:H O = 80:20, 30 °C.
2
4
.2.22. 2-((2-aminoethyl)amino)-N-(2-methoxyphenyl)-6-((4-nitrophenyl)thio)benzamide (12f)
28