Synthesis, structure-activity relationships, and docking studies of N-phenylarylformamide derivatives (PAFAs) as non-nucleoside HIV reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.03.032

A series of N-phenylarylformamide derivatives (PAFAs) with anti-wild-type HIV-1 activity (EC₅₀ values) ranging from 0.3 nM to 5.1 nM and therapeutic index (TI) ranging from 10 616 to 271 000 were identified as novel non-nucleoside reverse trans

Full text of DOI:10.1016/j.ejmech.2012.03.032

European Journal of Medicinal Chemistry 58 (2012) 504e512
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structureeactivity relationships, and docking studies
of N-phenylarylformamide derivatives (PAFAs) as non-nucleoside
HIV reverse transcriptase inhibitors
,
Xiao-Dong Ma a, Qiu-Qin He a, Xuan Zhang b c, Shi-Qiong Yang a, Liu-Meng Yang b, Shuang-Xi Gu a,
,
d
,
e,
**
Yong-Tang Zheng b, Fen-Er Chen a , Hui-Fang Dai
*
a Department of Chemistry, Fudan University, Shanghai 200433, 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 650223, PR China
c Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
d Institute of Biomedical Science, Fudan University, Shanghai 200031, PR China
e School of Pharmacy, Fudan University, Shanghai 200031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-phenylarylformamide derivatives (PAFAs) with anti-wild-type HIV-1 activity (EC50 values)
ranging from 0.3 nM to 5.1 nM and therapeutic index (TI) ranging from 10 616 to 271 000 were identified
as novel non-nucleoside reverse transcriptase inhibitors. Among them, compound 13g (EC50 ¼ 0.30 nM,
TI ¼ 184 578), 13l (EC50 ¼ 0.37 nM, TI ¼ 212 819), 13m (EC50 ¼ 0.32 nM, TI ¼ 260 617) and 13r
(EC50 ¼ 0.27 nM, TI ¼ 271 000) displayed the highest activity against this type virus nearly as potent as
lead compound GW678248. Moreover, all of them were also active to inhibit the double mutant strain
Received 2 November 2011
Received in revised form
15 March 2012
Accepted 15 March 2012
Available online 31 March 2012
A17 (K103N þ Y181C) with EC50 values of 0.29
particular, compound 13m, which showed broad-spectrum anti-HIV activity, was also effective to inhibit
the HIV-2 ROD replication within 4.37 M concentration.
Ó 2012 Elsevier Masson SAS. All rights reserved.
mM, 0.14 mM, 0.10 mM and 0.27 mM, respectively. In
Keywords:
Anti-HIV activity
NNRTIs
Benzophenone derivatives
N-Phenylarylformamide
Structureeactivity relationship
m
1. Introduction
Furthermore, cross resistance between the approved NNRTIs is
quite common, and patients are generally forced to abandon the
approved NNRTIs altogether once they have developed resistance
to one of its members [11,12]. Therefore, an urgent need has arisen
for more potent NNRTIs that possess both a broad spectrum of
antiviral activity against key mutant strains and a high genetic
barrier to the selection of new mutant strains.
Benzophenone derivatives (BPs, Fig. 2), originated in a high-
throughput screening in 1995 [13], are typical NNRTIs and very
efficacious against both wild-type and clinically relevant NNRTI-
resistant mutant HIV-1 strains [13e19]. For searching more active
BPs as NNRTIs, considerable efforts on the modifications of BPs
have been made and led to identify several highly potent inhibitors,
such as GW564511 (7, Fig. 2) [13] and GW678248 (8, Fig. 2) [15,16].
The initial SARs of BPs showed that the keto group between A- and
B-rings was important for maintaining their high anti-HIV activity
[19,20]. Therefore, few modifications on the keto template were
performed in the following structure optimizations. A flexible
amido linker between A- and B-rings might not only improve the
Since the identification of the human immunodeficiency virus
(HIV) as the causative agent of AIDS [1e4], the search for safe and
effective agents for HIV treatments has become a major focus for
drug discovery groups worldwide. Although the highly active
antiretroviral therapy (HAART) combination regimens such as
nevirapine (NVP, 1, Fig. 1) [5], delavirdine (DLV, 2, Fig. 1) [6], efa-
virenz (EFV, 3, Fig. 1) [7], etravirine (ETV, 4, Fig. 1) [8] and rilpivirine
(RPV, 5, Fig. 1) [9,10], which have been approved by US FDA as novel
HIV-1 NNRTIs, are proving to be effective for AIDS therapy. Unfor-
tunately, the genetic barrier of current NNRTIs is relatively low, and
single mutations begin to reduce the susceptibility of virus to drug.
* Corresponding authors. Department of Chemistry, Fudan University, Shanghai
200433, PR China. Tel./fax: þ86 21 65643811.
** Corresponding author. Tel./fax: þ86 21 65643811.
E-mail address: rfchen@fudan.edu.cn (F.-E. Chen).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.032

X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
505
H
OH
O
N
O
OH
Cl
O
O
O
O2N
NO2
O
O2N
O
O2N
c
a
b
SO2NH2
d
Cl
Cl
Cl
9
10
11
H
H
N
N
O
O
H
f
e
O
O
N
O
H2N
SO2NH2
SO2NH2
R
Cl
13a-y
Cl
12
Scheme 1. Synthesis of N-phenylarylformamide analogues 13aey. Reagents and conditions: (a) BrCH2COOEt, K2CO3, n-Bu4NBr, acetone,
2
h, 50 ꢀC, 91%; (b) LiOH$H2O,
THFeH2OeEtOH, r.t, 2 h, 89%; (c) SOCl2, 80 ꢀC, 1 h; (d) 4-amino-3-methylbenzenesulfonamide, NaHCO3, acetone, 80 ꢀC, 2 h, 85%; (e) Fe, NH4Cl, acetone-H2O, 80 ꢀC, 12 h, 82%; (f)
RCOCl, NaHCO3, acetone, r.t, 30 min, 70e89%.
adaptation of the inhibitor to RT, but also promote the H-bond
bindings with key mutant amino acids Tyr188 or Tyr181. Herein,
the important aromatic residues Tyr181, Tyr188 and Trp229 was
carried out [20]. It was found that relatively small changes in
structure did prove to have a significant impact on the anti-wild-
type potency. In general, placement of a small group at the meta
position (13c) was more favourable than at the ortho (analogue
13b) or at the para (analogue 13d) position, but for the nitro group,
the ortho-substituted analogue 13g was 2-fold higher than the
meta-substituted analogue 13h. In the case of meta-substituted
analogues 13bej, the order of the potency against wild-type virus
was NO2 > Me > Cl > F > CF3, but the trifluoro-substituted
analogue 13i almost lost 1000-fold activity in particular.
a
series of N-phenylarylformamide derivatives (PAFAs) were
synthesized and evaluated for their anti-HIV activity. Moreover,
their preliminary structureeactivity relationships (SARs) and the
possible binding modes with RT were also explored in this
manuscript.
2. Results and discussion
2.1. Chemistry
Additionally, several analogues with di-meta substitutions at
different positions (analogue 13keu) were also synthesized and
As depicted in Scheme 1, alkylation of the staring material 4-chloro-
2-nitrophenol with ethyl bromoacetate [21], and subsequent hydro-
lysis gave the acid derivative 10 [22]. Treatment acid chloride of 10 with
4-amino-3-methylbenzenesulfonamide in basic condition provided
the compound 11 [8]. After reducing the nitro-compound 11 in the
presence of FeeNH4Cl, amino compound 12 was obtained in 82% yield.
Finally, coupling 12 with the appropriately substituted of benzoyl
chloride was accomplished by mixing with NaHCO3 in the solvent of
acetone at room temperature to provide the corresponding target
compounds 13aey in 70e89% yield.
H3C
N
CH3
NH
CH3
CH3
N
H
N
O
N
F3C
HN
S
O
Cl
O
O
N
N
N
H
O
N
N
H
O
2. Delavirdine
1. Nevirapine
3. Efavirenz
2.2. Biological activity
CN
CN
O
CN
NH
All title molecules were evaluated for the activity against wild-
type HIV-1 strain IIIB, the double mutant HIV-1 strain A17
(K103N þ Y181C) and HIV-2 strain ROD in C8166 cells [23,24]. For
comparation, lead compound GW678248 and the FDA-approved
drug, zidovudine (AZT) were also tested as reference compounds,
and the activity data is interpreted in CC50 (cytotoxicity), EC50 (anti-
HIV activity) and TI (therapeutic index, given by the CC50/EC50
ratio).
CN
NH
H3C
CH3
N
H3C
CH3
N
HN
N
Br
N
NH2
5. Rilpivirine
4. Etravirine
As illustrated in Tables 1and 2, more than half of the newly
synthesized compounds displayed strong potency against the wild-
type HIV-1 strain IIIB with EC50 values ranging from 0.30 nM to
5.10 nM, and therapeutic index values from 10 616 to 271 000. In
particular, analogues 13g (EC50 ¼ 0.30 nM, TI ¼ 184 578), 13l
(EC50 ¼ 0.37 nM, TI ¼ 212 819), 13m (EC50 ¼ 0.32 nM, TI ¼ 260 617)
and 13r (EC50 ¼ 0.27 nM, TI ¼ 271 000) were identified as the
maximum inhibitors nearly as potent as GW678248 against this
type of virus, indicating that the flexibility of amide linkage might
improve the adaptation of inhibitor to RT.
Fig. 1. Structures of currently marketed NNRTIs.
H
N
H
N
O
O
O
O
C
R1
O
O
O
SO2NH2
A
B
O
R2
N
Cl
6
7, R1 = F, R2 = CF3 ( GW 564511)
8, R1 = Cl, R2 = CN ( GW 678248)
To explore the potential SARs, great efforts on the modifications
of the A-ring, which placed in the top hydrophobic pocket lined by
Fig. 2. Structures of potent benzophenones.

506
X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
Table 1
Biological activity of compounds 13aey against wild-type HIV-1 and HIV-2 ROD in C8166 cells.a
M)c
TId
b
Compd
R
EC50
CC50
(
m
HIV-1 IIIB (nM)
HIV-2 (mM)
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
13r
Ph
2-MeePh
3-MeePh
4-MeePh
3-ClePh
3-FePh
2-NO2ePh
3-NO2ePh
3-CF3ePh
4-MeOePh
2,4-DiMeePh
2,5-DiMeePh
3,4-DiMeePh
3,5-DiMeePh
2,4-DiClePh
2,5-DiClePh
3,4-DiClePh
3,5-DiClePh
3,5-Cl,BrePh
3,5-DiBrePh
3,5-Cl,CNePh
PhCH]CH
2,6-DiClePhCH2
1-Naphthyl
2-Naphthyl
3.67
17.93
1.46
5.10
3.19
3.53
0.30
0.66
1505.73 ꢁ 1002.08
204.38 ꢁ 8.42
0.56
0.37
0.32
1.16
2.97
2.34
51.40
72.10
55.51
29.60
57.33
52.47
185.87
11.85
36.10
81.31
58.76
41.36
27.68
4.37
25.87
14.42
21.57
55.52
18.01
61.49
56.49
23.16
77.45
160.17
97.68
31.59
2.1
207.37 ꢁ 16.98
67.27 ꢁ 9.40
52.90 ꢁ 6.48
54.17 ꢁ 7.72
59.66 ꢁ 12.85
242.98 ꢁ 25.37
54.78 ꢁ 8.87
58.84 ꢁ 10.86
123.63 ꢁ 9.92
69.53 ꢁ 7.83
47.60 ꢁ 3.93
79.70 ꢁ 2.48
84.11 ꢁ 8.48
36.45 ꢁ 4.04
61.14 ꢁ 12.40
42.12 ꢁ 4.55
157.50 ꢁ 21.95
72.39 ꢁ 1.11
113.92 ꢁ 2.55
89.66 ꢁ 13.36
131.08 ꢁ 10.96
163.39 ꢁ 6.42
>359.16
56 548
3751
36 155
10 616
18 722
68 811
184 578
89 425
82
340
85 339
212 819
260 617
31 327
20 615
18 004
3064
271 000
37
1954
136 287
2
>2
>3497
373
0.27
13s
13t
3088.87 ꢁ 1820.53
45.89
13u
13v
13w
13x
13y
GW678248
AZT
0.96
95395.72 ꢁ 3663.03
248504.98 ꢁ 97472.42
109.16 ꢁ 51.82
100.77 ꢁ 21.05
0.103 ꢁ 0.052
12.80 ꢁ 3.12
>381.69
37.60 ꢁ 10.82
>386.8
>37 773 585
39.0
5401.4 ꢁ 345.5
422 067
a
Data represent the mean of at least two separate experiments.
b
c
Compound concentration required to protect C8166 cells against viral cytopathogenicity by 50%.
Compound concentration that decreases the uninfected C8166 cell viability by 50%.
Therapeutic index: CC50/EC50 ratio (wildetype).
d
evaluated for anti-HIV activity to further explore the SARs of A-ring.
In fact, most of the di-substituted analogues showed strong anti-
HIV-1 activity with EC50 values less than 3.00 nM except for 3,4-
dichlo analogue (13q, EC50 ¼ 51.40 nM), 3,5-Cl,Br analogue (13s,
EC50 3088.87 nM) and 3,5-dibromo analogue (13t,
EC50 ¼ 45.89 nM), and notably, 3,5-dichloro analogue 13r was
¼
Table 2
Biological activity of compounds 13aey against mutant HIV-1 virus A17 (K103N þ Y181C) in C8166 cells.a
Compd
R
EC50
(
m
M)b
CC50
(
m
M)c
TId
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
13r
Ph
2-MeePh
3-MeePh
4-MeePh
3-ClePh
3-FePh
2-NO2ePh
3-NO2ePh
3-CF3ePh
4-MeOePh
2,4-DiMeePh
2,5-DiMeePh
3,4-DiMeePh
3,5-DiMeePh
2,4-DiClePh
2,5-DiClePh
3,4-DiClePh
3,5-DiClePh
3,5-Cl,BrePh
3,5-DiBrePh
3,5-Cl,CNePh
PhCH]CH
2,6-DiClePhCH2
1-Naphthyl
2-Naphthyl
1.59 ꢁ 0.13
2.29 ꢁ 0.40
0.46 ꢁ 0.02
0.93 ꢁ 0.08
1.49 ꢁ 0.70
1.02 ꢁ 0.31
0.29 ꢁ 0.21
0.46 ꢁ 0.32
52.94
207.37 ꢁ 16.98
67.27 ꢁ 9.40
52.90 ꢁ 6.48
54.17 ꢁ 7.72
59.66 ꢁ 12.85
242.98 ꢁ 25.37
54.78 ꢁ 8.87
58.84 ꢁ 10.86
123.63 ꢁ 9.92
69.53 ꢁ 7.83
47.60 ꢁ 3.93
79.70 ꢁ 2.48
84.11 ꢁ 8.48
36.45 ꢁ 4.04
61.14 ꢁ 12.40
42.12 ꢁ 4.55
157.50 ꢁ 21.95
72.39 ꢁ 1.11
113.92 ꢁ 2.55
89.66 ꢁ 13.36
131.08 ꢁ 10.96
163.39 ꢁ 6.42
>359.16
130.17
29.37
114.73
58.16
39.93
239.29
188.25
128.07
2.34
18.07
3.85
0.09 ꢁ 0.03
0.14 ꢁ 0.01
0.10 ꢁ 0.05
0.33 ꢁ 0.04
0.33 ꢁ 0.09
0.42 ꢁ 0.13
1.14 ꢁ 0.37
0.27 ꢁ 0.02
27.75
1.39 ꢁ 0.38
0.29 ꢁ 0.03
153.62
240.55
21.54
543.07
559.58
836.04
109.55
186.99
100.29
137.67
268.23
4.11
64.66
453.99
1.06
>1.49
>17.72
4.22
>1 369 863
215 627
13s
13t
13u
13v
13w
13x
13y
GW678248
AZT
>381.69
8.91
37.60 ꢁ 10.82
0.00028 ꢁ 0.000038
>386.8
0.0228
4913.8
a
Data represent the mean of at least two separate experiments.
b
c
Compound concentration required to protect C8166 cells against viral cytopathogenicity by 50%.
Compound concentration that decreases the uninfected C8166 cell viability by 50%.
Therapeutic index: CC50/EC50 ratio.
d

X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
507
Fig. 3. A: Superimposition of the docked conformations of GW678248 (purple) and 13u (red). B: Superimposition of the docked conformations of GW678248 (purple) and 13r
(cyan). C: Superimposition of the docked conformations of GW678248 (purple) and 13g (green). D: Superimposition of the docked conformations of GW678248 (purple) and 13s
(red). E: Superimposition of the docked conformations of GW678248 (purple) and 13i (green). F: Superimposition of the docked conformations of GW678248 (purple) and 13j
(green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

508
X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
Table 3
sulfonamide group with Val106; c) the pep interactions formed by
Selected compounds assayed against HIV-1 RT (wild-
type).
the A-ring with Tyr188, and B-ring with Tyr181. Apparently, all of
the docked conformations of 13g, 13r and 13u were superimposed
well onto that of GW678248, indicating that their interactions with
RT were comparable to GW678248. However, some of the impor-
tant H-bonds with the key mutant amino acid residues of RT were
disappeared (13g lost the H-bond with Val106, 13r lost the H-bond
with Lys103, and 13u lost the H-bond with Lys104), which might
lead to the falling activity against mutant HIV virus A17
(K103N þ Y181C). While for the analogue 13i, 13j and 13s, which
showed less potent against HIV, were superimposed badly onto
GW678248 for their docked conformations. Moreover, all of them
lost the H-bond with Lys103, which was a key mutant amino acid
for producing drug resistance. In summary, all these analyses were
in accordance with the test result.
Compd
IC50 (nM)a
13m
13u
GW678248
1.14
2.05
2.69
a
Compound concentration required to inhibit HIV-
1 RTwt activity by 50%.
identified as the most active inhibitor with an EC50 value of
0.27 nM. As seen from the result, it was found that introducing
another group on the A-ring of the mono-substituted analogues
strengthen the interaction with RT.
In addition, four peculiar structure molecules 13vey were
prepared with the aim to enhance the pep interactions with amino
acid resides Tyr188 and Tyr181 [25], Unfortunately, the test result
indicated that compound 13v and 13w completely lost the ability to
3. Conclusion
inhibit HIV-1 within more than 25
mM concentration. While
In summary, we report a novel series of N-phenylarylformamide
derivatives (PAFAs) endowed with high anti-HIV activity as potent
NNRTIs. Among these strong inhibitors, more than half showed
relative low concentration activity against wild-type HIV-1 virus
within a range of 0.3 nMe5.1 nM. In particular, compound 13g
interestingly, both 1-naphthyl analogues 13x and 2-naphthyl
analogues 13y displayed moderate inhibitory activity with EC50
values of 109.16 nM and 100.77 nM, respectively.
Furthermore, all of the compounds were also evaluated for
activityagainst the double mutant HIV-1 strain A17 (K103N þ Y181C)
in parallel with the HIV-2 strain ROD. In these assays, most of the
derivatives exhibited moderate potency against the drug-resistant
(EC50
¼
0.30 nM, TI
¼
184 578), 13l (EC50
¼
0.37 nM,
TI ¼ 212819), 13m (EC50 ¼ 0.32 nM, TI ¼ 260617) and 13r
(EC50 ¼ 0.27 nM, TI ¼ 271000) displayed the most activity against
this type virus nearly as potent as GW678248. Furthermore, all of
them were also effective to inhibit the double mutant HIV-1
viral strain within less than
1 mM concentration. Notably,
analogues 13g, 13l, 13m and 13r, the most active inhibitors against
wild-type HIV-1 virus, also showed the strongest potency to inhibit
the mutant type virus A17 (K103N þ Y181C) with EC50 values of
strain A17 (K103N þ Y181C) with EC50 values of 0.29
mM, 0.14 mM,
0.10 M and 0.27 M, respectively. In addition, both the preliminary
m
m
0.29 mM, 0.14 mM, 0.10 mM and 0.27 mM, respectively. Nevertheless,
SARs and the binding modes with RT were also discussed in this
manuscript. Overall, the designed PAFAs exhibited promising
activity against the anti-wild-type HIV-1 virus, although were not
effective to inhibit the A17 (K103N þ Y181C) virus.
almost all of them lost the ability to inhibit the HIV-2 strain ROD
(EC50 > 4.37 M), indicating that N-
m
M) except for 13m (EC50 ¼ 4.37
m
phenylarylformamide derivatives might be HIV-1 non-nucleoside
reverse transcriptase inhibitors only.
To directly prove the target of N-phenylarylformamide deriva-
tives was HIV-1 RT, compound 13m and 13u, the most active
inhibitors tested in C8166 cell cultures, were also chosen to eval-
uate the potency against HIV-1 RTwt at molecular level in parallel
with the reference compound GW678248. A poly (rA)/oligo (dT)15
homopolymer template with the HIV antigen detection ELISA was
used for quantifying expression of HIV-1 RTwt in culture medium
[26]. As shown in Table 3, apparently, both 13m and 13u exhibited
strong capacity to inhibit the HIV-1 RTwt with IC50 values of 1.14 nM
and 2.05 nM, respectively.
4. Experimental section
4.1. Chemistry
All chemicals used were purchased from commercial sources,
were of analytical grade and were used without further purifica-
tion. Melting points were measured on a SGW X-1 microscopic
melting-point apparatus and were uncorrected. Mass spectra were
obtained on a Waters Quattro Micromass instrument using elec-
trospray ionization (ESI) techniques. 1H NMR and 13C NMR spectra
on a Brucker AV 400 MHz spectrometer were recorded in [D]DMSO.
2.3. Molecular simulation
Chemical shifts are reported in d (ppm) units relative to the internal
standard tetramethylsilane (TMS). Elemental analyses were per-
formed on a Carlo Erba 1106 instrument and the results of
elemental analyses for C, H and N were within ꢁ0.4% of the theo-
retical values. TLC analyses were run on silica gel 60 F254 plates
(Merck) using a variety of solvent systems and a fluorescent indi-
cator for visualization. Spots were visualized under 254 nm UV
illumination. Flash chromatography separations were obtained on
Silica Gel (300e400 mesh) using EtOAc/hexane as eluents.
To investigate the binding modes of these derivatives with HIV-
1 RT (wild-type), several representative analogues 13g, 13i, 13j, 13r,
13s and 13u (13g, 13r and 13u were the most active inhibitors,
while 13i, 13j and 13s showed the least potency) were docked into
the non-nucleoside binding site (NNBS) of HIV-1 RTwt (PDB code:
3DLG) [20] according to the basis of the protocols, using Base-
builder program and Surflex-Dock program interfaced with
SYBYL-1.2 [27,28]. The default SYBYL-X 1.2 parameters were used.
For comparation, the lead compound GW678248 was also docked
to the NNBS and superimposed onto the reference compound
according to the same the protocols [29e31].
4.2. General procedure for the synthesis of N-[4-(aminosulfonyl)-2-
methylphenyl]-2-(4-chloro-2-nitrophenoxy)acetamide (11)
As seen in Fig. 3, a panel of tightening interactions were formed
by GW678248 with RT, such as: a) the van der Waals interactions
between the cyano group at meta-position of the A-ring and the
amino acid residue Trp229; b) the H-bond interactions formed by
the NH group that linking the B- and C-rings with Lys103, the NH of
the sulfonamide group with Lys104 and the oxygen atom of the
Thionyl chloride (21.8 mL, 0.30 mol) was added dropwise to 10
(23.19 g, 0.10 mol), the mixture was stirred at 80 ꢀC for 2 h, and then
concentrated in vacuo. The crude product was used immediately
without further purification or characterization.
A solution of the crude product (0.10 mol) in acetone (50 mL)
was added dropwise to the mixture of 4-amino-3-methylbenzenes

X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
509
ulfonamide (18.62 g, 0.10 mol), acetone (100 mL), sodium
bicarbonate (25.20 g, 0.30 mol) and water (1 mL) over 20 min, and
the resulting mixture was stirred at room temperature for 2 h. The
reaction mixture was then poured into water (150 mL) and
extracted with CH2Cl2 (2 ꢂ 100 mL). The aqueous layer was then
filtered to give crude product as a beige solid. The crude material
was suspended in 1 N HCl and then filtered and washed with
CH2Cl2, MeOH, and hexane to give 11 as an off-white solid (33.92 g,
85%). mp 253.7e254.8 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.31 (s, 3H,
CH3), 5.04 (s, 2H, CH2), 7.41 (d, J ¼ 8.8 Hz, 1H, ArH), 7.64 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.69 (s, 1H, ArH), 7.81 (dd, J ¼ 8.4 Hz, J ¼ 8.8 Hz,
140.9, 147.8, 166.6, 168.0; MS (ESIꢃ) m/z: 486 [MeH]ꢃ. Anal. calcd
for C23H22ClN3O5S: C 56.61, H 4.54, N 8.61, Cl 7.27, S 6.57, found: C
56.60, H 4.52, N 8.60, Cl 7.29, S 6.56.
4.4.3. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
methylbenzoyl)aminophenoxy]acetamide (13c)
Yield 87%; mp 240.2e241.8 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.13
(s, 3H, CH3), 2.36 (s, 3H, CH3), 4.93 (s, 2H, CH2), 7.31 (m, 2H,
SO2NH2), 7.19e7.98 (m,10H, ArH), 9.83 (s,1H, NH), 10.02 (s, 1H, NH);
13C NMR ([D]DMSO)
d
¼ 17.6, 20.9, 68.4, 115.1, 123.3, 123.7, 124.8,
125.0, 125.1, 125.2, 127.7, 128.1, 128.5, 128.9, 132.4, 132.6, 134.0,
138.0, 138.3, 140.9, 148.3, 165.6, 167.0; MS (ESIꢃ) m/z: 486 [MeH]ꢃ.
Anal. calcd for C23H22ClN3O5S: C 56.61, H 4.54, N 8.61, Cl 7.27, S 6.57,
found: C 56.58, H 4.52, N 8.60, Cl 7.29, S 6.58.
2H, ArH), 8.09 (s, 1H, ArH); 13C NMR ([D]DMSO)
d
¼ 17.7, 68.0, 117.3,
123.5, 123.8, 124.7, 124.8, 124.9, 127.7, 131.0, 134.1, 138.4, 140.3,
149.6, 165.7, MS (ESIþ) m/z: 400 [M þ H]þ. Calcd for C15H14ClN
3O6S 399.
4.4.4. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(4-
methylbenzoyl)aminophenoxy]acetamide (13d)
4.3. General procedure for the synthesis of N-[4-(aminosulfonyl)-2-
methylphenyl]-2-(4-chloro-2-aminophenoxy)acetamide (12)
Yield 85%; mp 239.9e241.0 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.15
(s, 3H, CH3), 2.39 (s, 3H, CH3), 4.93 (s, 2H, CH2), 7.28 (m, 2H,
SO2NH2), 7.19e7.99 (m, 10H, ArH), 9.81 (s, 1H, NH), 9.94 (s, 1H, NH);
A mixture of compound 11 (39.98 g, 0.10 mol) and ammonium
chloride (13.37 g, 0.25 mol) in acetoneewater (330 mL, acetone:
H2O ¼ 2: 1) solution was heated to 80 ꢀC, then iron powder (22.30 g,
0.40 mol) was added slowly, and the resulting mixture was stirred
at this temperature for 12 h. After completion, filtrated, the blank
reside was washed with DMSO (3 ꢂ 50 mL), and the combined
filtration was poured into water (1000 mL), filtered and washed
with H2O (3 ꢂ 200 mL) and CH2Cl2 (100 mL), then dried to give
crude product as a white solid 12 (30.26 g, 82%). mp 251.1e252.9 ꢀC;
1H NMR ([D]DMSO) dppm ¼ 2.32 (s, 3H, CH3), 5.04 (s, 2H, CH2), 7.30
(s, 2H, SO2NH2), 7.41e8.09 (m, 6H, ArH), 9.53 (1H, NH); 13C NMR
13C NMR ([D]DMSO)
d
¼ 17.5, 21.0, 68.3, 114.9, 123.2, 123.7, 124.9,
125.1, 125.2, 127.6 (2C), 127.7, 129.0, 129.1 (2C), 131.2, 132.3, 138.3,
140.8, 142.2, 148.2, 165.2, 167.0; MS (ESIꢃ) m/z: 486 [MeH]ꢃ. Anal.
calcd for C23H22ClN3O5S: C 56.61, H 4.54, N 8.61, Cl 7.27, S 6.57,
found: C 56.63, H 4.52, N 8.59, Cl 7.28, S 6.58.
4.4.5. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
chlorobenzoyl)aminophenoxy]acetamide (13e)
Yield 79%; mp 244.3e245.4 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.13
(s, 3H, CH3), 4.92 (s, 2H, CH2), 7.29 (m, 2H, SO2NH2), 7.20e8.03
(m,10H, ArH), 9.76 (s, 1H, NH), 10.18 (s, 1H, NH); 13C NMR ([D]
([D]DMSO)
d
¼ 17.7, 68.0, 117.3, 123.5, 123.9, 124.7, 124.9, 127.8,
131.0, 134.2, 138.5, 139.6, 140.3, 149.6, 165.7. MS (ESIþ) m/z: 370
[M þ H]þ. Calcd for C15H16ClN3O4S 369.
DMSO)
d
¼ 17.5, 68.4, 115.2, 123.7, 123.8, 125.0, 125.2, 125.4, 126.5,
127.5,127.7,128.6,130.6,131.8,132.3,133.4,136.1,138.3,140.8,148.6,
164.2, 167.0; MS (ESIꢃ) m/z: 507 [MeH]ꢃ. Anal. calcd for
4.4. General procedure for the synthesis of N-phenylarylformamide
C22H19Cl2N3O5S: C 51.98, H 3.77, N 8.27, Cl 13.95, S 6.31, found: C
derivatives (13aey)
51.99, H 3.75, N 8.25, Cl 13.97, S 6.32.
A mixture of 12 (3.70 g, 0.01 mol), acetone (50 mL) and sodium
bicarbonate (3.36 g, 0.04 mol) was cooled to 0 ꢀC in an ice bath, then
acyl chloride (0.02 mol) was added dropwise over 10 min, and the
resulting mixture was allowed to warm to room temperature and
stirred for another 30 min. The reaction mixture was then poured
into water (150 mL), filtered and washed with CH2Cl2, MeOH, and
hexane to give crude product as a beige solid, which was purified by
crystallization from acetonitrile to prepare the target compounds
13aey as a white solid.
4.4.6. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
fluorobenzoyl)aminophenoxy]acetamide (13f)
Yield 79%; mp 242.2e243.1 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.12
(s, 3H, CH3), 4.97 (s, 2H, CH2), 7.62 (m, 2H, SO2NH2), 7.19e7.93 (m,
10H, ArH); MS (ESIꢃ) m/z: 490 [MeH]ꢃ. Anal. calcd for
C22H19ClFN3O5S: C 53.72, H 3.89, N, Cl 7.21, F 3.86, S 6.52, found: C
53.72, H 3.89, N, Cl 7.20, F 3.88, S 6.51.
4.4.7. N-[4-(aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2-
nitrobenzoyl)aminophenoxy]acetamide (13g)
4.4.1. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-
(benzoyl)aminophenoxy]acetamide (13a)
Yield 77%; mp 245.7e246.9 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.10 (s,
3H, CH3), 4.86 (s, 2H, CH2), 7.28 (m, 2H, SO2NH2), 7.19e8.19 (m, 10H,
Yield 83%; mp 237.8e238.5 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.14
(s, 3H, CH3), 4.93 (s, 2H, CH2), 7.29 (m, 2H, SO2NH2), 7.19e7.99 (m,
11H, ArH), 9.81(s, 1H, NH), 10.03 (s, 1H, NH); 13C NMR ([D]DMSO)
ArH), 9.60 (s, 1H, NH), 10.36 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.5,
68.0, 114.6, 122.5, 123.7, 124.3, 124.8, 124.9, 125.0, 127.6, 128.2, 129.3,
131.1, 132.2, 132.3, 134.2, 138.2, 140.8, 146.2, 147.4, 164.8, 166.4; MS
(ESIꢃ) m/z: 516 [MeH]ꢃ. Anal. calcd for C22H19ClN4O7S: C 50.92, H 3.69,
N 10.80, Cl 6.83, S 6.18, found: C 50.90, H 3.71, N 10.78, Cl 6.85, S 6.17.
d
¼ 17.5, 68.3,114.9,123.4,123.7,125.0,125.1,125.2,127.6 (2C),127.8,
128.6 (2C), 128.9, 132.0, 132.4, 134.0,138.3,140.8,148.3, 166.5, 167.0;
MS (ESIꢃ) m/z: 472 [MeH]ꢃ. Anal. calcd for C22H20ClN3O5S: C 55.75,
H 4.25, N 8.87, Cl 7.48, S 6.77, found: C 55.74, H 4.26, N 8.85, Cl 7.51, S
6.75.
4.4.8. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
nitrobenzoyl)aminophenoxy]acetamide (13h)
Yield 74%; mp 236.5e237.7 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.11
(s, 3H, CH3), 4.92 (s, 2H, CH2), 7.62 (m, 2H, SO2NH2), 7.22e8.79 (m,
10H, ArH), 9.74 (brs, 1H, NH), 10.43 (brs, 1H, NH); 13C NMR ([D]
4.4.2. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2-
methylbenzoyl)aminophenoxy]acetamide (13b)
Yield 85%; mp 239.6e240.5 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.11
(s, 3H, CH3), 2.44 (s, 3H, CH3), 4.88 (s, 2H, CH2),7.28 (m, 2H, SO2NH2),
7.20e8.05 (m, 10H, ArH), 9.76 (s, 1H, NH), 9.89 (s, 1H, NH); 13C NMR
DMSO)
d
¼ 17.4, 68.3, 115.2, 122.5, 123.7, 124.3, 124.8, 125.1, 125.8,
126.5, 127.6, 128.3, 130.3, 132.0, 134.1, 135.6, 138.2, 140.7, 147.8,
148.9, 163.6, 166.9; MS (ESIꢃ) m/z: 516 [MeH]ꢃ. Anal. calcd for
([D]DMSO)
d
¼ 17.5,19.4, 68.0,114.4,123.0,123.7,124.8,125.0,125.2,
C
22H19ClN4O7S: C 50.92, H 3.69, N 10.80, Cl 6.83, S 6.18, found: C
125.8, 127.3, 127.6, 128.6, 130.0, 130.7, 132.5, 135.6, 136.3, 138.2,
50.91, H 3.70, N 10.82, Cl 6.81, S 6.16.

510
X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
4.4.9. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
trifluoromethylbenzoyl)aminophenoxy]acetamide (13i)
4.4.15. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2,4-
dichlorobenzoyl)aminophenoxy]acetamide (13o)
Yield 72%; mp 257.2e258.1 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.11 (s,
Yield 73%; mp 253.1e254.3 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.14 (s,
3H, CH3), 4.92 (s, 2H, CH2), 7.66 (m, 2H, SO2NH2), 7.22e8.32 (m, 10H,
3H, CH3), 4.86 (s, 2H, CH2), 7.65 (s, 2H, SO2NH2), 7.19e8.09 (m, 9H, ArH),
ArH), 9.73 (s, 1H, NH), 10.34 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.4,
9.68 (s, 1H, NH), 10.22 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.6, 68.0,
68.4, 115.3, 122.5e129.5 (q, 1C, 122.5, 125.3, 129.2, 129.5), 123.7, 124.1,
124.3 (q, 1C), 124.8, 125.2, 125.3, 125.6, 127.9, 128.5 (2C), 129.9, 131.8,
132.1,135.1,138.3,140.8,148.8,164.3,167.0; MS(ESIꢃ)m/z:539[MeH]ꢃ.
Anal. calcd for C23H19ClF3N3O5S: C 50.97, H 3.53, N 7.75, Cl 6.54, F 10.52,
S 5.92, found: C 50.98, H 3.52, N 7.77, Cl 6.51, F 10.50, S 5.90.
114.6, 122.7, 123.7, 124.9, 125.0, 125.1, 127.4, 127.7, 128.0, 129.3, 130.6,
131.4, 132.4, 135.1, 135.2, 136.2, 140.9, 147.5, 164.4, 166.4; MS (ESIꢃ) m/z:
540 [MeH]ꢃ.Anal.calcdforC22H18Cl3N3O5S: C48.68, H 3.34, Cl 19.59, N
7.74, S 5.91, found: C 48.70, H 3.35, Cl 19.60, N 7.75, S 5.90.
4.4.16. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2,5-
dichlorobenzoyl)aminophenoxy]acetamide (13p)
4.4.10. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(4-
methoxybenzoyl)aminophenoxy]acetamide (13j)
Yield 71%; mp 253.3e254.1 ꢀC; dppm:1H NMR ([D]DMSO) dppm ¼ 2.15
Yield 82%; mp 241.1e242.2 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.15
(s, 3H, CH3), 3.83 (s, 3H, CH3O), 4.93 (s, 2H, CH2), 7.30 (s, 2H,
SO2NH2), 7.05e7.98 (m, 10H, ArH), 9.84 (s, 1H, NH), 9.90 (s, 1H, NH);
(s, 3H, CH3), 4.85 (s, 2H, CH2), 7.29 (m, 2H, SO2NH2), 7.20e8.08 (m, 9H,
ArH), 9.67 (s, 1H, NH), 10.28 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.6,
68.1, 114.7, 122.9, 123.7, 125.0, 125.1 (2C), 127.7, 127.9, 128.8, 128.9, 131.1,
131.5, 131.9, 132.4, 137.8, 138.2, 140.9, 147.6, 163.9, 166.4; MS (ESIꢃ) m/z:
540 [MeH]ꢃ. Anal. calcd for C22H18Cl3N3O5S: C 48.68, H 3.34, Cl 19.59, N
7.74, S 5.91, found: C 48.69, H 3.35, Cl 19.58, N 7.72, S 5.90.
13C NMR ([D]DMSO)
d
¼ 17.6, 55.5, 68.3, 113.8 (2C), 114.9, 123.4,
123.7, 124.8, 125.1, 125.2, 126.1, 127.7, 129.1, 129.6 (2C), 132.4, 138.1,
140.8, 148.3, 162.3, 164.9, 167.0; MS (ESIꢃ) m/z: 502 [MeH]ꢃ. Anal.
calcd for C23H22ClN3O6S: C 54.82, H 4.40, N 8.34, Cl 7.03, S 6.36,
found: C 54.80, H 4.41, N 8.32, Cl 7.05, S 6.33.
4.4.17. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3,4-
dichlorobenzoyl)aminophenoxy]acetamide (13q)
4.4.11. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2,4-
dimethylbenzoyl)aminophenoxy]acetamide (13k)
Yield 74%; mp 249.8e251.7 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.13
(s, 3H, CH3), 4.91 (s, 2H, CH2), 7.28 (m, 2H, SO2NH2), 7.21e8.23 (m,
9H, ArH), 9.74 (s, 1H, NH), 10.24 (s, 1H, NH); 13C NMR ([D]DMSO)
Yield 84%; mp: 248.5e249.6 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.12
(s, 3H, CH3), 2.32 (s, 3H, CH3), 2.41 (s, 3H, CH3), 4.88 (s, 2H, CH2), 7.28
(s, 2H, SO2NH2), 7.09e8.05 (m, 9H, ArH), 9.78 (s, 2H, 2NH); 13C NMR
d
¼ 17.5, 68.4, 123.7, 123.9, 124.9, 125.1, 127.6, 127.7, 128.0, 128.4,
129.6 (2C, 129.59, 129.65), 130.8, 131.4, 132.2, 134.5, 134.8, 138.2,
140.8, 148.7, 163.4, 166.9; MS (ESIꢃ) m/z: 541 [MeH]ꢃ. Anal. calcd
for C22H18Cl3N3O5S: C 48.68, H 3.34, Cl 19.59, N 7.74, S 5.91, found: C
48.71, H 3.30, Cl 19.57, N 7.75, S 5.89.
([D]DMSO)
d
¼ 17.5, 19.5, 20.8, 68.1, 114.4, 122.8, 123.7, 124.6, 125.0,
126.1, 127.6, 127.7, 128.7, 131.3, 131.4, 132.5, 133.3, 135.8, 138.2, 139.8,
140.9, 147.7, 166.7, 167.9; MS (ESIꢃ) m/z: 501 [MeH]ꢃ. Anal. calcd for
C
24H24ClN3O5S: C 57.42, H 4.82, N 8.37, Cl 7.06, S 6.39, found: C
57.41, H 4.81, N 8.36, Cl 7.05, S 6.40.
4.4.18. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3,5-
dichlorobenzoyl)aminophenoxy]acetamide (13r)
4.4.12. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2,5-
dimethylbenzoyl)aminophenoxy]acetamide (13l)
Yield 82%; mp 254.7e255.8 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.15 (s,
3H, CH3), 4.90 (s, 2H, CH2), 7.28 (m, 2H, SO2NH2), 7.21e8.14 (m, 9H,
Yield 85%; mp 244.7e245.8 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.10
(s, 3H, CH3), 2.29 (s, 3H, CH3), 2.38 (s, 3H, CH3), 4.88 (s, 2H, CH2), 7.30
(s, 2H, SO2NH2), 7.17e8.03 (m, 9H, ArH), 9.79 (s, 1H, NH), 9.90 (s, 1H,
ArH), 9.73(s, 1H, NH), 10.30 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.6,
68.6,115.5,122.7 (2C),123.7,124.0,124.9,125.2,125.7,127.7,128.3,129.7
(2C), 132.2, 136.6, 137.7, 138.3, 140.8, 143.8, 162.8, 167.0; MS (ESIꢃ) m/z:
541 [MeH]ꢃ. Anal. calcd for C22H18Cl3N3O5S: C 48.68, H 3.34, Cl 19.59,
N 7.74, S 5.91, found: C 48.69, H 3.36, Cl 19.60, N 7.75, S 5.90.
NH); 13C NMR ([D]DMSO)
d
¼ 17.5, 19.0, 20.4, 68.1, 114.5, 123.0,
123.7, 124.8, 125.0, 125.2, 127.7, 127.8, 128.6, 130.6, 130.7, 132.5,
132.6, 134.8, 136.2, 138.3, 140.9, 147.8, 166.7, 168.1; MS (ESIꢃ) m/z:
501 [MeH]ꢃ. Anal. calcd for C24H24ClN3O5S: C 57.42, H 4.82, N 8.37,
Cl 7.06, S 6.39, found: C 57.40, H 4.81, N 8.38, Cl 7.07, S 6.40.
4.4.19. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
chloro-5-bromobenzoyl)aminophenoxy]acetamide (13s)
Yield 72%; mp 265.4e266.3 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.14 (s,
4.4.13. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3,4-
dimethylbenzoyl)aminophenoxy]acetamide (13m)
3H, CH3), 4.90 (s, 2H, CH2), 7.67 (m, 2H, SO2NH2), 7.20e8.10 (m, 9H,
ArH); 13C NMR ([D]DMSO)
d
¼ 17.6, 68.6, 115.5, 122.5, 123.7, 124.0,
Yield 87%; mp 248.8e249.7 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.10
(s, 3H, CH3), 2.29 (s, 6H, 2CH3), 4.87 (s, 2H, CH2), 7.28 (s, 2H,
SO2NH2), 7.19e8.06 (m, 9H, ArH), 9.76 (s, 1H, NH), 9.88 (s, 1H, NH);
125.0,125.2,125.5,126.9,127.7,128.9,129.3,132.2,133.8,134.5,137.8,
138.3, 140.8, 148.8, 162.9, 167.0; MS (ESIꢃ) m/z: 586 [MeH]ꢃ. Anal.
calcd for C22H18Cl2BrN3O5S: C 44.99, H, 3.09 Br 13.61, Cl 12.07, N 7.16,
S 5.46, found: C 45.00, H, 3.10, Br 13.60, Cl 12.05, N 7.18, S 5.48.
13C NMR ([D]DMSO)
d
¼ 16.0, 17.5, 19.8, 68.0, 114.4, 123.0, 123.7,
124.7 (2C), 124.9, 125.3, 125.2, 127.6, 128.5, 131.0, 132.5, 133.6, 137.3
(2C), 138.2, 140.9, 147.7, 166.6, 168.7; MS (ESIꢃ) m/z: 500 [MeH]ꢃ.
Anal. calcd for C24H24ClN3O5S: C 57.42, H 4.82, N 8.37, Cl 7.06, S
6.39, found: C 57.41, H 4.80, N 8.38, Cl 7.10, S 6.36.
4.4.20. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3,5-
bromobenzoyl)aminophenoxy]acetamide (13t)
Yield 75%; mp 297.6e298.5 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.16 (s,
3H, CH3), 4.92 (s, 2H, CH2), 7.29 (m, 2H, SO2NH2), 7.21e8.13 (m, 9H, ArH),
4.4.14. N-[4-(aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3,5-
dimethylbenzoyl)aminophenoxy]acetamide (13n)
9.74 (s, 1H, NH), 10.29 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.6, 68.7,
115.6, 122.8 (2C), 123.8, 124.0, 124.9, 125.2, 125.7, 127.7, 128.4, 129.7 (2C),
132.2, 136.6, 137.7, 138.3, 140.8, 148.7, 162.9, 167.3; MS (ESIꢃ) m/z: 631
[MeH]ꢃ. Anal. calcd for C22H18ClBr2N3O5S: C 41.83, H 2.87, Br 25.30, Cl
5.61, N 6.65, S 5.08, found: C 41.81, H 2.86, Br 25.32, Cl 5.60, N 6.63, S 5.07.
Yield 89%; mp: 252.4e253.7 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.15
(s, 3H, CH3), 2.32 (s, 6H, 2CH3), 4.93 (s, 2H, CH2), 7.31 (s, 2H,
SO2NH2), 7.19e7.99 (m, 9H, ArH), 9.84 (s, H, NH), 9.98 (s, H, NH); 13
C
NMR ([D]DMSO)
d
¼ 17.6, 20.8 (2C), 68.6, 115.2, 123.2, 123.7, 124.9,
125.0, 125.4 (3C), 127.8, 129.1, 132.4, 133.4, 134.1, 137.9 (2C), 138.4,
140.9, 148.3, 165.7, 167.1; MS (ESIꢃ) m/z: 500 [MeH]ꢃ. Anal. calcd
for C24H24ClN3O5S: C 57.42, H 4.82, N 8.37, Cl 7.06, S 6.39, found: C
57.41, H 4.83, N 8.38, Cl 7.07, S 6.40.
4.4.21. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-
chloro-5-cyanobenzoyl)aminophenoxy]acetamide (13u)
Yield 65%; mp 255.7e256.8 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.14
(s, 3H, CH3), 4.91 (s, 2H, CH2), 7.28 (m, 2H, SO2NH2), 7.22e8.37 (m,

X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
511
9H, ArH), 9.73 (s, 1H, NH), 10.37 (s, 1H, NH); 13C NMR ([D]DMSO)
for drug control. After 3 days of culture, the number of syncytia
(multinucleated giant cells) was scored under an inverted micro-
scope; 50% effective concentration to blocking syncytia formation
(EC50) was calculated [24].
d
¼ 17.5, 68.5, 113.5, 115.4, 117.0, 123.7, 123.9, 124.8, 125.2, 125.7,
127.7,128.2,130.3,132.1,132.4,134.4,134.8,137.1,138.2,140.8,148.6,
162.7, 166.9; MS (ESIꢃ) m/z: 532 [MeH]ꢃ. Anal. calcd for
C
23H18Cl2N4O5S: C 51.79, H 3.40, Cl 13.29, N 10.50, S 6.01, found: C
51.80, H 3.41, Cl 13.30, N 10.52, S 6.00.
4.6. RT inhibition assay
4.4.22. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-{[4-chloro-2-
[(E)-cinnamoyl]aminophenoxy}acetamide (13v)
According to the method reported by Liu [26], assays against
HIV-1 RTwt were performed using a poly(rA)/oligo (dT)15 homo-
polymer template with HIV antigen detection ELISA for quantifying
expression of HIV-1 RT in culture medium. Oligo (dT) was immo-
bilized via its 50-terminal phosphate to Covalink-NH microtiter
plates. The biotin-dUTP was incorporated by reverse transcriptase.
The reaction mixture contained 50 mM TriseHCl (pH 8.3), 3 mM
MgCl2, 75 mM KCl, 5 mM DTT (d,l-dithiothreitol), 0.13 mg/mL BSA,
10 mg/mL poly (A), 0.75 mM biotin-11-dUTP, and 1.5 mM dTTP.
After incubation at 37 ꢀC for 1 h, the plate was washed three times
with a wash buffer containing 50 mM TriseHCl (pH 7.5), 0.15 M
NaCl, 0.05 mM MgCl2, and 0.02% Tween-20. After 100 mL of 1% BSA
was added to each well and incubated for 30 min at room
temperature, the plate was washed with the same buffer. Subse-
quently 50 mL of SA-ALP (alkaline phosphatase streptavidin)
solution (100 ng/mL) was added per well and then incubated for 1 h
at 37 ꢀC. The plate was washed as above and then 50 mL of PNPP (p-
nitrophenyl phosphate, disodium) (1 mg/mL, pH 9.5) was added,
after 30 min at 37 ꢀC the reaction was stopped by addition of 0.5 M
NaOH. The products were detected and quantified using a colori-
metric streptavidin alkaline phosphatase reporter system.
Yield 68%; mp 263.2e264.3 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 1.99
(s, 3H, CH3), 4.89 (s, 2H, CH2), 7.21 (d, 1H, J ¼ 8.8 Hz, CH), 7.54 (d, 1H,
J ¼ 8.8 Hz, CH), 7.60 (m, 2H, SO2NH2), 7.29e8.30 (m, 11H, ArH); 13
C
NMR ([D]DMSO)
d
¼ 17.4, 68.1, 114.6, 123.4, 123.6, 125.1 (2C), 125.7,
126.4, 127.1, 127.6(2C), 128.3(2C), 129.7, 130.5, 132.4, 133.2, 134.2,
138.3, 140.8, 148.0, 166.6, 167.6; MS (ESIꢃ) m/z: 499 [MeH]ꢃ. Anal.
calcd for C24H22CN3O5S: C 57.66, H 4.44, Cl 7.09, N 8.40, S 6.41,
found: C 57.68, H 4.45, Cl 7.10, N 8.41, S 6.40.
4.4.23. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2,6-
dichlorophenylacetyl)aminophenoxy]acetamide (13w)
Yield 77%; mp 297.5e298.3 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.28
(s, 3H, CH3), 4.18 (s, 2H, CH2), 4.90 (s, 2H, CH2), 7.30 (s, 2H, SO2NH2),
7.16e8.01 (m, 9H, ArH), 9.73 (s, 1H, NH), 9.96 (s, 1H, NH); 13C NMR
([D]DMSO)
d
¼ 17.7, 38.5, 68.0, 114.4,121.5, 123.7, 123.9,124.8,125.0,
127.7, 128.1(2C), 128.7, 129.5, 131.9, 131.1, 135.6 (2C), 138.4, 140.7,
146.8, 166.6, 167.2; MS (ESIꢃ) m/z: 555 [MeH]ꢃ. Anal. calcd for
C
23H20Cl3N3O5S: C 49.61, H 3.62, Cl 19.10, N 7.55, S 5.76, found: C
49.60, H 3.60, Cl 19.13, N 7.54, S 5.75.
4.4.24. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(1-
naphthoyl)aminophenoxy]acetamide (13x)
4.7. Molecular simulation
Yield 74%; mp 262.4e263.1 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 1.99
(s, 3H, CH3), 4.90 (s, 2H, CH2), 7.61 (s, 2H, SO2NH2), 7.22e8.31 (m,
Molecular modelling was carried out with the Tripos molecular
modelling packages Sybyl-1.2. All the molecules for docking were
built by using the standard bond lengths and angles from the Sybyl-
X 1.2/base Builder, and then were optimized for 2000-generations
two until the maximum derivative of energy became
0.1kcal$molꢃ1 Åꢃ1, using the Tripos force field. The flexible docking
method, called Surflex-Dock program docks ligand automatically
into a receptor’s ligand binding site using a protomol based
approach and an empirically derived scoring function [27,28]. The
protomol 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. Surflex-Dock’s scoring function,
which contains hydrophobic, polar, repulsive, entropic, and salva-
tion terms, was trained to estimate the dissociation constant (Kd)
expressed in ꢃlog (Kd)2 unit. Prior to docking, the protein was
prepared by removing water molecules, ligand and other useless
small molecules from the complex of HIV-1 RT with GW564511
(PDB code: 3DLG) [20], in parallel with adding polar hydrogen
atoms to protein. Other parameters were set as defaults for Surflex-
Dock, such as the starting conformations per molecule of 0, the
angstroms to expand search grid of 8, the max number of rotatable
bonds per molecule of 100, and the maximum number of poses per
ligand of 20. During the docking procedure, all of the single bonds
of residue side chains inside the defined RT binding pocket were
regarded as rotatable or flexible bonds, and the ligand was allowed
to rotate on all single bonds and move flexibly within the tentative
binding pocket. The atomic charges were recalculated by using the
Kollman all-atom approach for the protein and the
GasteigereHückel approach for the ligand. The binding interaction
energy was calculated to include van der Waals, electrostatic, and
torsional energy terms defined in the Tripos force field. The struc-
ture optimization was performed for 20000-generations using
a genetic.
13H, ArH); 13C NMR ([D]DMSO)
d
¼ 17.4, 68.2, 114.6, 123.3, 123.6,
125.0 (3C), 125.1 (2C), 125.7, 126.4, 127.1, 127.6, 128.3, 128.7, 129.7,
130.5, 132.4, 133.1, 134.1, 138.4, 140.8, 148.0, 166.6, 167.6; MS (ESIꢃ)
m/z: 522 [MeH]ꢃ. Anal. calcd for C26H22ClN3O5S: C 59.60, H 4.23, Cl
6.77, N 8.02, S 6.12, found: C 59.62, H 4.25, Cl 6.75, N 8.00, S 6.14.
4.4.25. N-[4-(Aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(2-
naphthoyl)aminophenoxy]acetamide (13y)
Yield 71%; mp 281.1e282.3 ꢀC; 1H NMR ([D]DMSO) dppm ¼ 2.13
(s, 3H, CH3), 4.96 (s, 2H, CH2), 7.67 (s, 2H, SO2NH2), 7.22e8.63 (m,
13H, ArH), 9.86 (s, 1H, NH), 10.24 (s, 1H, NH); 13C NMR ([D]DMSO)
d
¼ 17.6, 68.4, 115.1, 123.3, 123.7, 124.2, 125.1 (2C), 125.2, 127.0, 127.7
(2C), 128.1, 128.2 (2C), 128.9, 129.0, 131.4, 132.1, 132.4, 134.4, 138.3,
140.9, 148.4, 165.6, 167.0; MS (ESIꢃ) m/z: 522 [MeH]ꢃ. Anal. calcd
for C26H22ClN3O5S: C 59.60, H 4.23, Cl 6.77, N 8.02, S 6.12, found: C
59.61, H 4.25, Cl 6.79, N 8.02, S 6.09.
4.5. Anti-HIV assays
4.5.1. Cytotoxicity assay
The cytotoxicity of compounds on C8166 cells were assessed by
MTT colorimetric assay as described previously [23]. The absor-
bance at 570 nm/630 nm (A570/630) was read in an ELISA reader
(Elx800, Bio-Tek Instrument Inc., USA). The minimum cytotoxic
concentration that caused the reduction of viable cells by 50%
(CC50) was determined from dose response curve.
4.5.2. Syncytium reduction assay
In the presence of 100 mL various concentrations of compounds,
C8166 cells (4 ꢂ 105/mL) were infected with viruses (HIV-1IIIB, HIV-
1
A17, and HIV-2ROD) at a multiplicity of infection (M.O.I) of 0.06. The
final volume per well was 200 mL. AZT and GW678248 were used

512
X.-D. Ma et al. / European Journal of Medicinal Chemistry 58 (2012) 504e512
[11] R.M. Grant, F.M. Hecht, M. Warmerdam, L. Liu, T. Liegler, C.J. Petropoulos,
Acknowledgements
N.S. Hellmann, M. Chesney, M.P. Busch, J.O. Kahn, JAMA 288 (2002) 181e188.
[12] Z. Zhang, R. Hamatake, Z. Hong, Antivir. Chem. Chemother. 15 (2004)
121e134.
[13] J.H. Chan, G.A. Freeman, J.H. Tidwell, K.R. Romines, L.T. Schaller, J.R. Cowan,
S.S. Gonzales, G.S. Lowell, C. Andrews III, D.J. Reynolds, J. Med. Chem. 47
(2004) 1175e1182.
[14] R.J. Hazen, R.J. Harvey, M.H. St Clair, R.G. Ferris, G.A. Freeman, J.H. Tidwell,
L.T. Schaller, J.R. Cowan, S.A. Short, K.R. Romines, Antimicrob. Agents Che-
mother. 49 (2005) 4465e4473.
[15] R.G. Ferris, R.J. Hazen, G.B. Roberts, M.H. St Clair, J.H. Chan, K.R. Romines,
G.A. Freeman, J.H. Tidwell, L.T. Schaller, J.R. Cowan, Antimicrob. Agents Che-
mother. 49 (2005) 4046e4051.
This work was supported in part by grants from the National
Natural Science Foundation of China (Nos. 20872018 and
30672536), the National Natural Science Foundation of Shanghai
(No. 10ZR1401900), the Knowledge Innovation Program of CAS
(KSCX2-YW-R-185), 973 program (2009CB522306) and Eleventh
Five-Year Key Scientific and Technological Program of China
(2009ZX09501-029).
[16] K.R. Romines, G.A. Freeman, L.T. Schaller, J.R. Cowan, S.S. Gonzales,
J.H. Tidwell, C.W. Andrews III, D.K. Stammers, R.J. Hazen, R.G. Ferris, J. Med.
Chem. 49 (2006) 727e739.
[17] T.J. Tucker, S. Saggar, J.T. Sisko, R.M. Tynebor, T.M. Williams, P.J. Felock,
J.A. Flynn, M.T. Lai, Y. Liang, G. McGaughey, Bioorg. Med. Chem. Lett. 18 (2008)
2959e2966.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.03.032.
[18] A.A. San Juan, Bioorg. Med. Chem. Lett. 18 (2008) 1181e1194.
[19] J. Ren, P.P. Chamberlain, A. Stamp, S.A. Short, K.L. Weaver, K.R. Romines,
R. Hazen, A. Freeman, R.G. Ferris, C.W. Andrews, J. Med. Chem. 51 (2008)
5000e5008.
[20] P.G. Wyatt, R.C. Bethell, N. Cammack, D. Charon, N. Dodic, B. Dumaitre,
D.N. Evans, D.V.S. Green, P.L. Hopewell, J. Med. Chem. 38 (1995)
1657e1665.
[21] F.A. Macías, J.M. De Siqueira, N. Chinchilla, D. Marín, R.M. Varela,
J.M.G. Molinillo, J. Agr. Food Chem. 54 (2006) 9843e9851.
[22] J.P. Brown, E.B. McCall, J. Chem. Soc. 4 (1955) 3681e3687.
[23] Y.T. Zheng, K.L. Ben, S.W. Jin, Acta Pharmacol. Sin. 21 (2000) 179e182.
[24] R.R. Wang, L.M. Yang, Y.H. Wang, W. Pang, S.C. Tam, P. Tien, Y.T. Zheng, Bio-
chem. Biophys. Res. Commun. 382 (2009) 540e544.
References
[1] a) F. Barré-Sinoussi, J.C. Chermann, F. Rey, M.T. Nugeyre, S. Chamaret, J. Gruest,
C. Dauguet, C. Axler-Blin, F. Vézinet-Brun, C. Rouzioux, Science 220 (1983)
868e871;
b) R.C. Gallo, S.Z. . Salahuddin, M. Popovic, G. Shearer, M. Kaplan, B. Haynes,
T. Palker, R. Redfield, J. Oleske, B. Safai, Science 224 (1984) 500e503.
[2] F.J. Palella, K.M. Delaney, A.C. Moorman, M.O. Loveless, J. Fuhrer, G.A. Satten,
D.J. Aschman, S.D. Holmberg, New Engl. J. Med. 338 (1998) 853e860.
[3] E. De Clercq, Curr. Med. Chem. 8 (2001) 1543e1572.
[4] M.-P. de Béthune, Antivir. Res. 58 (2010) 75e90.
[5] R.A. Koup, V.J. Merluzzi, K.D. Hargrave, J. Adams, K. Grozinger, R.J. Eclmer,
J.L. Sullivan, J. Infect. Dis. 163 (1991) 966e970.
[25] a) X.Q. Feng, Y.H. Liang, Z.S. Zeng, F.E. Chen, J. Balzarini, C. Pannecouque, E. De
Clercq, ChemMedChem 4 (2009) 219e224;
[6] W.W. Freimuth, Adv. Exp. Med. Biol. 394 (1996) 279e289.
[7] a) S.D. Young, S.F. Britcher, L.O. Tran, L.S. Payne, W.C. Lumma, T.A. Lyle,
J.R. Huff, P.S. Anderson, D.B. Olsen, S.S. Carroll, Antimicrob. Agents Chemother.
39 (1995) 2602e2605;
b) J.J. . Minuto, R. Haubrich, Future HIV Ther. 2 (2008) 525e537;
c) L.B. Johnson, L.D. Saravolatz, Clin. Infect. Dis. 48 (2009) 1123e1128.
[8] D. Ripamonti, F. Maggiolo, Curr. Opin. Invest. Drugs 9 (2008) 899e912;
b) P. Pecora Fulco, I.R. McNicholl, Pharmacotherapy 29 (2009) 281e294.
[9] a) K. Andries, H. Azijn, T. Thielemans, D. Ludovici, M. Kukla, J. Heeres,
P. Janssen, B. De Corte, J. Vingerhoets, R. Pauwels, M.-P. de Béthune, Anti-
microb. Agents Chemother. 48 (2004) 4680e4686;
b) Y.P. He, F.E. Chen, G.F.
. Sun, Y.P. . Wang, E. De Clercq, J. Balzarini,
C. Pannecouque, Bioorg. Med. Chem. Lett. 14 (2004) 3173e3176;
c) L. Ji, F.E. Chen, E. De Clercq, J. Balzarini, C. Pannecouque, J. Med. Chem. 50
(2007) 1778e1786;
d) Y.H. Liang, X.Q. . Feng, Z.S. Zeng, F.E. Chen, J. Balzarini, C. Pannecouque, E. De
Clercq, ChemMedChem 4 (2009) 1537e1545;
e) Y.P. . Wang, F.E. . Chen, E. De Clercq, J. Balzarini, C. Pannecouque, Bioorg.
Med. Chem. 16 (2008) 3887e3894.
[26] M.Y. Yu, Z.Y. Li, S. Liu, E.K. Fan, C. Pannecouque, E. De Clercq, X.Y. Liu,
ChemMedChem 6 (2011) 826e833.
[27] a) J. Park, N.-D. Sung, Bull. Korean Chem. Soc. 31 (2011) 1241e1248;
b) Q. Liu, B. Masek, K. Smith, J. Smith, J. Med. Chem. 50 (2007) 5392e5402.
[28] a) A.N. Jain, J. Comput. Aided Mol. Des. 10 (1996) 427e440;
b) A.N. Jain, J. Comput. Aided Mol. Des. 21 (2007) 281e306;
c) A.N. Jain, J. Med. Chem. 46 (2003) 499e511.
b) P.A. Janssen, P.J. Lewi, E. Arnold, F. Daeyaert, M. de Jonge, J. Heeres,
L. Koymans, M. Vinkers, J. Guillemont, E. Pasquier, M. Kukla, D. Ludovici,
K. Andries, M.-P. de Béthune, R. Pauwels, K. Das, A.D. Clark Jr., Y.V. Frenkel,
S.H. Hughes, B. Medaer, F. De Knaep, H. Bohets, F. De Clerck, A. Lampo,
P. Williams, P. Stoffels, J. Med. Chem. 48 (2005) 1901e1909;
[29] a) J.Z. Chen, J. Wang, X.Q. Xie, J. Chem. Inform. Model. 47 (2007) 1626e1637;
b) S. Raduner, A. Majewska, J.Z. Chen, X.Q. Xie, J. Hamon, B. Faller,
K.H. Altmann, J. Gertsch, J. Biol. Chem. 281 (2006) 14192e14206.
[30] J. Ruppert, W. Welch, A.N. Jain, Protein Sci. 6 (1997) 524e533.
[31] W. Welch, J. Ruppert, A.N. Jain, Chem. Biol. 3 (1996) 449e462.
c) H. Azijn, I. Tirry, J. Vingerhoets, M.-P. de Béthune, G. Kraus, K. Boven,
D. Jochmans, E. Van Craenenbroeck, G. Picchio, L.T. Rimsky, Antimicrob.
Agents Chemother. 54 (2010) 718e727.
[10] S. Moreno, J. López Aldeguer, J.R. Arribas, P. Domingo, J.A. Iribarren, E. Ribera,
A. Rivero, F. Pulido, J. Antimicrob. Agents Chemother. 65 (2010) 827e835.