Discovery of novel piperidine-substituted indolylarylsulfones as potent HIV NNRTIs via structure-guided scaffold morphing and fragment rearrangement

Article abstract of DOI:10.1016/j.ejmech.2016.10.009

To further explore the chemical space around the entrance channel of HIV-1 reverse transcriptase (RT), a series of novel indolylarylsulfones (IASs) bearing N-substituted piperidine at indole-2-carboxamide were identified as potent HIV NNRTIs by structure-

Full text of DOI:10.1016/j.ejmech.2016.10.009

Accepted Manuscript
Discovery of novel piperidine-substituted indolylarylsulfones as potent HIV NNRTIs
via structure-guided scaffold morphing and fragment rearrangement
Xiao Li, Ping Gao, Boshi Huang, Zhongxia Zhou, Zhao Yu, Zheng Yuan, Huiqing Liu,
Christophe Pannecouque, Dirk Daelemans, Erik De Clercq, Peng Zhan, Xinyong Liu
PII:
S0223-5234(16)30871-6
10.1016/j.ejmech.2016.10.009
EJMECH 8974
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 July 2016
Revised Date: 3 October 2016
Accepted Date: 4 October 2016
Please cite this article as: X. Li, P. Gao, B. Huang, Z. Zhou, Z. Yu, Z. Yuan, H. Liu, C. Pannecouque, D.
Daelemans, E. De Clercq, P. Zhan, X. Liu, Discovery of novel piperidine-substituted indolylarylsulfones
as potent HIV NNRTIs via structure-guided scaffold morphing and fragment rearrangement, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.009.
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Graphical Abstract:
A series of novel indolylarylsulfones (IASs) bearing N-substituted piperidine at
indole-2-carboxamide were identified as potent HIV NNRTIs by structure-guided
scaffold morphing and fragment rearrangement.

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Discovery of Novel Piperidine-substituted Indolylarylsulfones as Potent HIV
NNRTIs via Structure-guided Scaffold Morphing and Fragment Rearrangement
Xiao Li1, Ping Gao1, Boshi Huang1, Zhongxia Zhou1, Zhao Yu1, Zheng Yuan3, Huiqing Liu3,
Christophe Pannecouque2, Dirk Daelemans2, Erik De Clercq2, Peng Zhan1,*, Xinyong Liu1,*
1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
250012 Jinan, Shandong, P.R. China
2Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
3Department of Pharmacology, Shandong University School of Medicine, Shandong University, 44
West Culture Road, 250012 Jinan, Shandong, P.R. China
Abstract: To further explore the chemical space around the entrance channel of
HIV-1 reverse transcriptase (RT), a series of novel indolylarylsulfones (IASs) bearing
N-substituted piperidine at indole-2-carboxamide were identified as potent HIV
NNRTIs by structure-guided scaffold morphing and fragment rearrangement. All the
IASs exhibited moderate to excellent potency against wild-type HIV-1 with EC50
values ranging from 0.62 ꢀM to 0.006 ꢀM. 8 (EC50 = 6 nM) and 18 (EC50 = 9 nM)
were identified as the most potent compounds, which were more active than NVP and
DLV, and reached the same order of EFV and ETV. Furthermore, most compounds
maintained high activity agaist various single HIV-1 mutants (L100I, K103N, E138K,
Y181C) as well as one double mutant (F227L/V106A) with EC50 values in
low-micromolar to double-digit nanomolar concentration ranges. Especially, 8
displayed outstanding potency against L100I (EC50 = 17 nM with a 2.8-fold resistance
ratio) and 18 was relatively more potent to E138K mutant (EC50 = 43 nM with a
4.7-fold resistance ratio). Preliminary SARs and molecular modeling studies were
also discussed in detail, which may provide valuable insights for further optimization.
*Corresponding authors. Tel: +86 531 88380270; fax: +86 531 88382731; E-mail
addresses:zhanpeng1982@sdu.edu.cn & xinyongl@sdu.edu.cn.

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Keywords: HIV, NNRTIs, Indolylarylsulfones, Antiviral activity, SAR, Molecular
modeling, Chemical space, Drug design.
1. Introduction
Highly active antiretroviral therapy (HAART) in general has significantly
changed the progression and outcome of the infection with HIV-1 [1-5].
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have received a lot of
attentions, and have been shown to be a standard component of HAART, because of
their favorable potency and low cytotoxicity. So far, five NNRTIs have been approved
for AIDS treatment, and about 50 classes of structurally diverse NNRTIs are
being widely investigated [6-12]. Over the past few years, considerable efforts have
been devoted to the structural modification of diarylpyrimidines (DAPYs), a family of
NNRTIs with remarkable anti-HIV-1 activity, leading to the approval of Etravirine
(ETV) and Rilpivirine (RPV) (Figure 1) as new generation of anti-HIV drugs
(approved by US FDA in 2008, 2011, respectively) with significantly improved drug
resistance profiles, compared with the first generation NNRTIs [13, 14]. However,
hyper sensitivity reactions or other adverse effects have been reported with
second-generation NNRTIs [15]. Also, although drug resistance is delayed, it
continues to emerge in patients receiving second-generation NNRTIs regimens [16,
17]. Therefore, there is a substantial need for the identification of new inhibitors,
especially in additional NNRTIs families, that display a broad spectrum of activity
against clinically relevant HIV-1 mutant strains and lower adverse effects [18, 19].
Figure 1
Figure 1. The structures of representative DAPYs (Etravirine, Rilpivirine and 5h) and IASs
(L-737,126 and 7e), and the newly designed N-substituted piperidine-IASs via the combination of
structure-guided scaffold morphing and fragment rearrangement approaches.
Figure 2

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Figure 2. Schematic diagram of the binding modes of IASs and DAPYs with NNIBP: (a) HIV RT
in complex with inhibitor 7e (pink) (PDB code: 2RF2); (b) HIV RT in complex with inhibitor 5h
(dark blue) (PDB code: 3M8Q). These figures were generated using PyMOL (www.pymol.org).
Residues important for binding are shown as sticks. Hydrogen bonds are indicated by yellow
dashes.
Indolylarylsulfones (IASs), a potent class of NNRTIs developed from the Merck
prototype compound L-737,126 (Figure 1), have endowed with inhibitory activities
against wild-type (WT) and drug-resistant HIV-1 in nanomolar range [20-26]. Based
on the crystallography and molecular modeling study (exemplified by 7e, Figure 2), it
suggested that the side chain at 2-position of the indole nucleus located into the
entrance channel (the protein/solvent interface between the p66 and p51 RT subunits)
of NNRTI binding pocket (NNIBP), mainly formed by Leu100, Glu138 and Val179.
Additionaly, the groups at this position could build potential hydrogen bonds with
around amino acids such as Glu138 [27]. In recent years, to further improve
anti-HIV-1 activity and optimize physicochemical properties and pharmacokinetics of
IASs, most work on the modification of IASs was focused on introducing different
substituents (natural and unnatural amino acids, cyclic and nitrogen containing
substituents) into the 2-carboxamide nitrogen by taking advantage of the solvent
exposed region, affording derivatives endowed with activities against WT HIV-1 and
NNRTI-resistant mutants superior to that of the parent L-737,126 [21-29]. These
promising results motivated us to put more efforts in in-depth exploration of untapped
chemical space around the entrance channel through chemical modifications at
2-position of indole (Figure 1).
The “Pro236 hairpin loop” is another protein/solvent interface in NNIBP that
provide broad space that are potentially available to accommodate novel NNRTIs [8].
A systematic exploration of the chemical space around the “Pro236 hairpin loop” led
to discovery of novel piperidine-linked aminopyrimidine derivatives with broad
activity against WT as well as drug-resistant mutant viruses [30,31]. X-ray
crystallographic analysis of early leads showed a water molecule linking the

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piperidine nitrogen with the backbone NH of Lys103 (exemplified by 5h, Figure 2).
Guided by crystallography and studies in silico, to identify new DAPYs by
establishing novel binding interactions with this solvent-accessible region and to
further expand the chemical scope of the core ring system in DAPYs families, we
made considerable efforts on chemically modification using the privileged
N-substituted piperidine group, which has given a number of highly active NNRTIs
with excellent anti-HIV-1 activity and significantly improved water solubility, which
is an urgent issue of existing DAPY drugs [32-44].
In continuation of our research project, inspired by the analyses of this two
categories of NNRTIs and based on the concept of “mixed sites inhibitor” [45,46], we
planned the structure-based design, synthesis of new IASs characterized by
structurally diverse N-substituted piperidine groups at the 2-carboxamide to explore
the detail of the conformational and space-filling requirements for optimum
occupation of the entrance channel (Figure 1). Meanwhile, the preferred combination
of the 5-halogen-indole core, 3-arylsulfonyl and 2-carboxamide group of original
IASs were preserved. Therefore, the present study will highlight the synthesis,
biological evaluation of novel IAS derivatives, and the preliminary structure-activity
relationships (SARs) and molecular modeling results will also be discussed in detail.
2. Results and Discussion
2.1 Chemistry
To achieve the target compounds 7-30, the straightforward synthetic route
adopted is depicted in Scheme 1. Briefly, the commercial available ethyl
5-chloro/bromo-1H-indole-2-carboxylate (1a or 1b) was converted to intermediate 2a
and 2b by reacting with commercial 3,5-dimethylbenzenethiol in the presence of
1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(tetrafluoroborate)
(SelettflourTM
)
[47]. Then treatment of intermediate 2a or 2b with
m-chloroperoxybenzoic acid and lithium hydroxide orderly gave intermediate 4a or
4b, which underwent acylation reaction with1-Boc-4-(aminomethyl)-piperidine or
1-Boc-4-amino-piperidine to generate intermediates 5a, 5b and 5c [21-23]. The Boc
group of 5a, 5b and 5c was removed in the presence of trifluoroacetic acid (TFA) at

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room temperature to afford intermediates 6a, 6b and 6c in good yield [48]. Finally, the
target compounds 7-30 was obtained by alkylating at the N atom of piperidine
under two different reaction conditions [49]. All the synthesized compounds
1
(displayed in Table 1) were confirmed by their spectral data (ESI-MS, H NMR and
13C NMR). Spectral data are in accordance with the assumed structures.
Scheme 1.
Scheme 1. Reagents and conditions: (i) 3,5-dimethylbenzenethiol, SelettflourTM, MeCN; (ii)
M-chloroperoxybenzoic acid, CH2Cl2, 0°C; (iii) LiOH, H2O, THF, 50°C; (iv)
1-Boc-4-(aminomethyl)-piperidine or 1-Boc-4-aminopiperidine, HATU, EDC.HCl, Et3N, DMF; (v)
CF3COOH, CH2Cl2; (vi) K2CO3, DMF, substituted benzyl; (vii) substituted-aldehyde AcOH,
NaBH3CN, THF/MeCN.
2.2 Anti-HIV activity evaluation
The newly prepared IAS derivatives were evaluated for their activity against WT
HIV-1 strain (IIIB) and HIV-2 strain (ROD) in MT-4 cell cultures using MTT method,
respectively. What’s more, these compounds were also assayed against frequently
encountered single (L100I, K103N, Y181C, Y188L, E138K) or (F227L/V106A,
K103N/Y181C (RES056)) double mutant HIV-1 strains for the sake of defining the
resistance profiles. The FDA-approved drugs nevirapine (NVP), delavirdine (DLV),
efavirenz (EFV) and Etravirine (ETV) were chosen as reference drugs. The biological
results are represented as EC50 values (anti-HIV activity), CC50 values (cytotoxicity)
and SI values (selectivity index, given by the CC50/EC50 ratio). All the data were
illustrated in Table 1.
The biological testing results clearly indicated that all the novel IASs exhibited
moderate to excellent potency against WT HIV-1 with EC50 values in the range of
0.006-0.62 ꢀM and SI values ranging from 93 to 9391. Totally, 18 compounds were
much better than those of NVP and reach the same or a higher level of DLV. Thereinto,
compounds 8 (EC50 = 6 nM, SI = 1005) and 18 (EC50 = 9 nM, SI = 1467) were the
most active derivatives, which were about 20 and 13 times more active than NVP, 4
and 3 times more active than DLV, respectively, and reached the same order of EFV

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and ETV. None of the newly synthesized compounds were active at subtoxic
concentration against HIV-2 (ROD), thus the detailed date were not listed.
Against these mutant strains, except for Y188L mutant, most compounds
maintained high activity against various single mutants with EC50 values in
low-micromolar to two-digit nanomolar concentration ranges. Notably, 8 displayed
outstanding potency against L100I (EC50 = 17 nM with a 2.8-fold resistance ratio,
namely, EC50(mutant strain)/EC50(WT strain) ratio) and K103N (EC50 = 84 nM with a 14-fold
resistance ratio) mutant strains, which were more active than the reference drugs NVP,
DLV and EFV. More interestingly, compared with 8, compound 18 bearing a
1,2,4-triazol-3(4H)-one tail displayed different drug resistance profile, which is
relatively more potent against E138K mutant (EC50 = 43 nM with a 4.7-fold resistance
ratio) and L100I mutant HIV-1 strain (EC50 = 49 nM with a 5.4-fold resistance ratio),
but weak potency against the K103N mutant strain (EC50 = 0.18 ꢀM with a 20-fold
resistance ratio). Regarding the Y188L mutant, the most sensitive single mutant for
IASs, 18 was also found to be the most active one (EC50 = 0.84 ꢀM). Besides, fifteen
compounds can inhibit F227L/V106A double mutant replication at low-micromolar
concentration while only compound 20 demonstrated remarkable inhibitory activty to
,
K103N/Y181C double mutant (RES056) with EC50 value of 4.4 ꢀM.
Based on the analysis of above anti-HIV results, preliminary SARs of these IASs
could be delineated as follows (Figure 3):
Firstly, we focused on modifications at the terminal groups (R) substituted in the
N atom of the piperidine ring. The SAR suggests that the nature of the R substituents
have dramatically effects on antiviral activity against WT and mutiple HIV-1 mutant
strains. Generally, polar groups substituted benzenes, alkanes or a set of commonly
used small heterocycles, with ability to form hydrogen bond, seem to be more
favorable at this region, exemplified by
8
(hydroxymethyl) and 18
(1,2,4-triazol-3(4H)-one) as the most two potent compounds. In contrast, some other
modifications at this region may lead to a deleterious effect on anti-HIV potency and
drug resistance profiles, such as halogene at benzenes 10 (4-Cl, EC50(IIIB) = 0.13 ꢀM),
11 (3-Cl, EC50(IIIB) = 0.30 ꢀM), 23 (4-CF3, EC50(IIIB) = 0.17 ꢀM), 28 (3-F, EC50(IIIB) =

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0.18 ꢀM
= 0.62 ꢀM
halo substituents, exemplified by 11 (3-Cl, CC50(IIIB) = 103.0 ꢀM), 10 (4-Cl, CC50(IIIB)
= 67.3 ꢀM) and 28 3-F, CC50(IIIB) = 61.3 ꢀM).
), cyano-derivatived 29 (EC50(IIIB) = 0.14 ꢀM), and nitro analogue 25 (EC50(IIIB)
)
. Moreover, lower cytotoxicities were usually observed in compounds with
(
More specifically, for the phenyl-substituted series, amide or sulfamide appeared
to be the most favorable groups, closely followed by substituted phenyls in the
sequence 3-COMe > 4-Cl > 3-Cl (in 7-14 sub-series); 4-CF3 > 4-NO2 (in 15-25
sub-series); 3-F = 3-CN (in 26-30 sub-series). Moreover, the replacement of the
substituted phenyls by a set of aromatic heterocycles resulted in comparable level of
anti-HIV (WT and mutants) activities, indicating structurally diverse heterocycles, as
hydrogen bond donors or receptors, can also better accommodate the chemical
environment in this region of RT and have a favorable interactions with surrounding
residues. This SAR conclusion is consistent with our design hypothesis.
Interestingly, no substantial difference in antiviral activity against WT and
mutant strains can be observed between pyridin-3-yl and pyridin-4-yl counterparts
(12/13, 16/17). By contrast, it is noteworthy that thiophene-2-yl (21) was always
preferred over thiophene-3-yl (22) against WT and all the tested mutant strains,
especially for L100I, Y181C and F227L/V106A.
Then we focused on substituent effects at 5-position (X substitutions) of indole
ring. The results demonstrated that the inhibitory activities (EC50 values) against WT
HIV-1 seemed only marginally affected by the substitutions (Br or Cl) at the position
5 of the indole nucleus, through the pairwise comparison of the EC50 values, namely,
9 (X = Br, EC50(IIIB) = 14 nM) and 19 (X = Cl, EC50(IIIB) = 13 nM); 12 (X = Br,
EC50(IIIB) = 19 nM) and 16 (X = Cl, EC50(IIIB) = 12 nM); 13 (X = Br, EC50(IIIB) = 17 nM)
and 17 (X = Cl, EC50(IIIB) = 16 nM); 14 (X = Br, EC50(IIIB) = 12 nM) and 20 (X = Cl,
EC50(IIIB) = 11 nM). Similarly, in general, the influence on the activity against mutant
strains was also negligible.
Lastly, for purpose of exploring the influence of flexibility on anti-HIV activity,
piperidine (n = 0) and methylenylpiperidine (n = 1) groups were primarily introduced
to
indole-2-carboxamide.
Piperidine appears
to be
preferred
over

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methylenylpiperidine counterpart against WT as well as mutiple mutant HIV-1 strains
[e.g. 20 (n = 0, EC50(IIIB) = 11 nM) and 27 (n =1, EC50(IIIB) = 24 nM); 8 (n = 0, EC50 (IIIB)
= 6 nM) and 26 (n = 1, EC50(IIIB) = 32 nM)].
Figure 3.
Figure 3. A illustrative figure of the SARs.
Table1. Activity against HIV-1 strains, cytotoxicity and selectivity indexin MT-4 cells.
Table 1
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against
HIV-1-induced cytotoxicity, as determined by the MTT method.
bCC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
determined by the MTT method.
cSI: selectivity index, the ratio of CC50/EC50.
Table 1 (Continued). Activity against HIV-1 strains, cytotoxicity and selectivity index in MT-4
cells.
Table 1. (Continued)
2.3 HIV-1 RT inhibition assay
To further confirm that the title compounds target HIV-1 RT, the representative
compounds 18 and 30 were selected to be evaluated in recombinant HIV-1 RT
inhibitory assays, which use hybrid hybrid poly (A)·oligo (dT)15 (9A260 nm/mL),
lyophilizate as template primer (RT kit, Roche). As shown in Table 2, 18 and 30
exhibited moderate inhibition of enzymatic activity with an IC50 value of 7.0 ꢀM and
10.1 ꢀM respectively, which is slightly lower than the reference drugs NVP (IC50
=
2.8 ꢀM) and ETV (IC50 = 1.1 ꢀM). The results further indicated that the newly
synthesized IAS derivatives, represented by 18 and 30, do indeed act as typical HIV-1
NNRTIs.
It is noteworthy that the high and low order of the enzyme-inhibitory potencies in
this study was inconsistent with that of their antiviral activities to some extent (18, 30

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& NVP). These differences usually ascribed to template-specific variation in the
relationship between polymerase processivity and HIV-RT-RNA binding affinity,
which have been observed in other NNRTI families [50]. Besides, compared with
NVP, these piperidine-substituted IASs have multiple hydrogen bond donor and
acceptor sites, there is a high probability of formation of cocrystals or salts. Therefore,
the differences between the two assay systems can be attributed at least in part to
differences in the physiochemical properties (especially the intrinsic solubility).
Table 2. Inhibitory activity of compounds18, 30, NVP and ETV against HIV-1 RT.
Table 2.
aIC50: inhibitory concentration of test compound required to inhibit biotin deoxyuridine
triphosphate (biotin-dUTP) incorporation into HIV-1 (WT) RT by 50%.
2.4. Molecular modeling analysis
With the aim to shed light on the preferred binding mode in the NNIBP and
rationalize SARs results of novel IASs, molecular modeling of the most potent
compounds 8 and 18 were performed by using software Surflex-Dock moduleo
Sybyl-X 1.1 [51] and the docking results were shown by PyMOL 1.5.
The theoretical binding mode of compounds 8 and 18 to the NNIBP is shown in
Figure 4. The similar binding mode as previously reported IASs can be observed and
notable features can be summarized as follows: (i) The 3,5-dimethylphenyl moiety
fitted into an aromatic-rich binding subpocket formed by side chains of Tyr181,
Tyr188 and Trp229, exhibiting crucial hydrophobic interactions. (ii) Double hydrogen
bonds were formed between the indole NH and the carbonyl oxygen of
Lys101, remarkablely improving the stability of RT-inhibitor complex. (iii) The
chlorine or bromine atom at the position 5 of indole was very closed to Val106 and
probably established halogen bond interactions.
From analysis of the results, the piperidine moiety could well occupy the
entrance channel of NNIBP and the substituent on it (the triazolone moiety of
compound 18) located toward the protein/solvent interface (Figure 4c). Specifically,
extra H-bond interaction was built between the nitrogen atom of the triazolone and

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Glu138, which is important in the binding of this compound (Figure 4b).
Overall, the molecular modeling analysis confirmed our the initial design
assumption that introducing a N-substituted piperidine at indole-2-carboxamide of
IASs could not only make terminal substituent of new compounds to extend more
closer to the protein/solvent interface of HIV-1 RT, but also built potential interactions
with surrounding residues (especially Glu138). These preliminary conclusions will be
taken into account for the next round rational structural optimization.
Figure 4.
Figure 4. Predicted binding modes of novel IASs in the allosteric site of HIV-1 WT RT (PDB
code: 2rf2): (a) Ribbon model of the 8 binding; (b) Ribbon model of the 18 binding (c) Surface
presentation of 18 located toward a solvent exposure region. Hydrogen bonds are indicated with
dashed lines in red, and hydrogen atoms are omitted for clarity.
3. Conclusion
In summary, to explore the uncharted chemical space around the entrance
channel in RT, a series of novel IASs bearing N-substituted piperidine at
indole-2-carboxamide were identified as potent HIV NNRTIs by the structure-based
scaffold morphing and fragment rearrangement approaches. All the novel IASs
exhibited moderate to excellent potency against WT HIV-1 in MT-4 cells. Especially,
compounds 8 and 18 were identified as the most promising leads with EC50 value of 6
nM and 9 nM, respectively, which were more active than NVP and DLV, and reached
the same order of EFV and ETV. Furthermore, most compounds maintained high
activity against various single mutants as well as F227L/V106A double mutant with
EC50 values in low-micromolar to double-digit nanomolar concentration range.
Preliminary SARs based on anti-HIV-1 activities were also discussed in detail and
molecular modeling studies were carried out to investigate the interactions between
IASs and HIV-1 RT. Taking full advantage of these valuable information, further
efforts to improve drug resistance profile of these IAS derivatives is underway in our
laboratory and will be reported in due course.

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4. Experimental Section
4.1. Synthetic procedures and analytical data
Mass spectrometry was performed on an API 4000 triple-quadrupole mass
spectrometer (Applied Biosystems/MDS Sciex, Concord, ON, Canada). 1H-NMR and
13C-NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker BioSpin,
Switzerland), using solvents as indicated (DMSO-d6). Chemical shifts were reported
in δ values (ppm) with tetramethylsilane as the internal reference, and J values were
reported in hertz (Hz). Melting points (mp) were determined on a micromelting point
apparatus (Tian Jin Analytical Instrument Factory, Nankai, Tianjin, China). Flash
column chromatography was performed on columns packed with silica gel 60
(200–300 mesh) (Qingdao waves silica gel desiccant co., Ltd, Qingdao, China). Thin
layer chromatography was performed on pre-coated HUANGHAI® HSGF254,
0.15-0.2 mm TLC-plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai,
Shandong, China).
4.1.1 General procedure for the synthesis of 5-chloro-3-(3,5-dimethylphenyl-
sulfonyl)-1H-indole-2-carboxylic acid (6a)
Ethyl 5-bromo-3-(3,5-dimethylphenylthio)-1H-indole-2-carboxylate(2a): To a
solution of ethyl 5-bromo-1H-indole-2-carboxylate (1a, 0.10 g, 0.373 mmol) in
MeCN (10 mL) at ambient temperature was successively added
3,5-dimethylbenzenethiol
(51.6
mg,
0.373
mmol,
50
ꢀL)
and
1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (0.10
g, 0.373 mmol) and the resulting mixture was stirred for 6 h (monitored by TLC). The
solvent was removed under reduced pressure and water (30 mL) was added and
extracted with CH2Cl2 (3×10 mL). Combined organic phase was washed with brine,
dried over anhydrous MgSO4, filtered and concentrated. Purification on silica gel
(ethyl acetate: petroleum (EA:PE) = 1:8) gave 2a as a white solid. Yield: 50%.
Ethyl 5-bromo-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxylate (3a):
3-Chloroperoxybenzoic acid (0.128 g, 0.742 mmol) was added to an ice-cooled
solution of crude intermediate 2a (0.10 g, 0.247 mmol) in anhydrous CH2Cl2. The

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reaction mixture was stirred at room temperature for 3 h, then poured on crushed ice,
and extracted with CH2Cl2 (3×10 mL). The organic phase was shaken with saturated
solution of sodium hydrogen carbonate and then brine, dried over anhydrous MgSO4,
filtered and concentrated under reduced pressure. The obtained crude product was
recrystallized using EA to give the pure compound 3a as a white solid. Yield: 68.8%,
mp: 205-207 °C. ESI-MS: m/z 436.3 (M+1). C19H18BrNO4S [435.01].
5-Bromo-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxylic acid (4a):
Crude intermediate 3a (0.10 g, 0.229 mmol) was dissolved in the THF-H2O mixture
(6 mL/6 mL) at room temperature followed by addition of lithium hydroxide (16.5 mg,
0.687 mmol). The reaction mixture was stirred at 50 °C for 24 hours (monitored by
TLC), then the THF was removed under reduced pressure. The residue was diluted
with water and modulated to pH 5-6 by adding diluted hydrochloric acid. The
precipitated compound 4a was filtered by suction filtration and air-dried to a white
powder. Yield: 66.5%, mp: 215-217 °C. ESI-MS: m/z 425.3 (M+18). C17H14BrNO4S
[406.98].
tert-Butyl-4-(5-bromo-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxamido
)piperidine-1-carboxylate (5a): The reaction mixture of intermediate 4a (1.0 g, 2.45
mmol), HATU (1.39 g, 3.67 mmol), 4-amino-1-Boc-piperidine (0.735 g, 3.67 mmol)
and N,N-diisopropylethyl-amine (0.949 g, 7.35 mmol, 1.3 mL) in 10 mL DMF was
stirred at ambient temperature for 24 h. After removal of the solvent under reduced
pressure, the residues was redissolved in THF (100 mL) and washed with saturated
sodium hydroxide aqueous solution. Then organic phase was dried over anhydrous
MgSO4, filtered and concentrated. Purification on silica gel (EA:PE = 1:4) gave 5a as
a white solid. Yield: 47.5%.
5-Chloro-3-(3,5-dimethylphenyl-sulfonyl)-1H-indole-2-carboxylic acid (6a):
Trifluoroacetic acid (1 mL) was added dropwise under stirring to a solution of
intermediate 5a (0.5 g, 0.848 mmol) in CH2Cl2 (2 mL) at room temperature and
stirred for 4 h. To the reaction mixture was added water and neutralized with 2 N
NaOH to pH 8, and the organic layer was separated and dried over anhydrous MgSO4,
filtered and concentrated under reduced pressure to give the product 6a as white solid.

ACCEPTED MANUSCRIPT
Yield: 75.4%, mp: 230-231 °C. ESI-MS: m/z 490.4 (M+1). C22H24BrN3O3S [489.07].
4.1.2 General procedure for the synthesis of 5-chloro-3-(3,5-dimethylphenyl-
sulfonyl)-N-(piperidin-4-yl)-1H-indole-2-carboxamide (6b)
Using starting material ethyl 5-chloro-1H-indole-2-carboxylate and following the
procedure as in the preparation of 6a to give 6b.
Ethyl 5-chloro-3-(3,5-dimethylphenylthio)-1H-indole-2-carboxylate (2b): Yield:
58.4%, mp: 135-136°C. ESI-MS: m/z 360.4 (M+1), 377.5 (M+18), 382.4 (M+23).
C19H18ClNO2S [359.07].
Ethyl 5-chloro-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxylate (3b):
Yield: 58.8%, mp: 210-211 °C. ESI-MS: m/z 392.0706 (M+1). C19H18ClNO4S
[391.06].
5-Chloro-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxylic acid (4b):
Yield: 65.8%, mp: 270 °C, decomposition. ESI-MS: m/z 386.5 (M+1). C17H14ClNO4S
[363.03].
tert-Butyl-4-(5-chloro-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxamido
)piperidine-1-carboxylate (5b): Yield: 52.2%, mp: 240-242 °C. ESI-MS: m/z 545.14
(M+1), 563.4 (M+18). C27H32ClN3O5S [545.18].
5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(piperidin-4-yl)-1H-indole-2-carbox
amide (6b): Yield: 70.1%, mp: 235-236 °C. ESI-MS: m/z 446.3 (M+1).
C22H24ClN3O3S [445.12].
4.1.3 General procedure for the synthesis of5-chloro-3-(3,5-dimethylphenyl-
sulfonyl)-N-(piperidin-4-ylmethyl)-1H-indole-2-carboxamide (6c)
Using intermediate 4b and 1-Boc-4-aminomethylpiperidineand following the
procedure as in the preparation of 6b to give 6c.
tert-Butyl-4-((5-chloro-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxamid
o)methyl)piperidine-1-carboxylate (5c): Yield: 48.2%, mp: 246-248 °C.
5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(piperidin-4-ylmethyl)-1H-indole-2-
carboxamide (6c): Yield: 76.3%, mp: 239-241 °C. ESI-MS: m/z 460.3(M+1).
C23H26ClN3O3S [459.14].

ACCEPTED MANUSCRIPT
4.1.4 General procedure for the synthesis of target compounds (7-20, 26-30)
Intermedite 6a, 6b or 6c (0.65mmol, 1 equiv), K2CO3 (0.10 g, 0.76 mmol, 1.2
equiv), and different appropriate RCH2X (X = Cl, Br) (1.1 equiv) were dissolved in
DMF (10 mL). The reaction mixture was stirred at ambient temperature for 4 hours
and then evaporated under reduced pressure. Water (30 mL) was added and extracted
with EA (3×10 mL). Combined organic phase was washed with brine, dried over
anhydrous MgSO4, filtered and concentrated. The residue was purified on silica gel
using EA-PE system and pure fractions were collected, concentrated and
recrystallized, giving the desired compounds (7-20, 26-30).
4.1.5.N-(1-(3-Methoxybenzyl)piperidin-4-yl)-5-bromo-3-(3,5-dimethylphenyl-sulfonyl
)-1H-indole-2-carboxamide (7)
1
White solid, yield: 43.2%. mp: 175-177 °C. H NMR (400MHz, DMSO-d6,
ppm)δ: 13.02 (s, 1H, indole-NH), 8.95 (s, 1H, CONH), 8.09 (s, 1H, indole-H), 7.62 (s,
2H, Ph-H), 7.50 ( q, 2H, J = 8.00 Hz, indole-H), 7.26 ( t, 2H, J = 8.00 Hz, Ph’-H),
6.88 ( s, 2H, Ph’-H), 6.83 ( d, 1H, J = 8.00 Hz, Ph-H), 3.86 (s, 1H, piperidine-H), 3.73
(s, 3H, OCH3), 3.46 (s, 2H, benzyl-CH2), 2.79 (s, 2H, piperidine-H), 2.31 (s, 6H,
2×CH3), 2.14 (s, 2H, piperidine-H), 1.93 (s, 2H, piperidine-H), 1.59 (s, 2H,
piperidine-H). 13C NMR (100MHz, DMSO-d6, ppm) δ: 159.73 (C=O), 158.99, 143.05,
140.74, 139.43 (2×C), 137.73, 135.14, 133.58, 129.67, 127.60, 126.34, 124.10 (2×C),
122.36, 121.34, 115.70 (2×C), 114.58, 112.74, 111.53, 62.49 (CH2), 55.40 (2×CH2),
52.07(O-CH3), 47.28, 31.65 (2×CH2), 21.24 (2×CH3). ESI-MS: m/z 610.3 (M+1).
C30H32BrN3O4S [609.13].
4.1.6. 5-Bromo-3-(3,5-dimethylphenylsulfonyl)-N-(1-(2-hydroxyethyl)piperidin-4-yl)
-1H-indole-2-carboxamide (8)
White solid, yield: 40.1%. mp: 220-222 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.20-12.59 (b, 1H, indole-NH), 8.95 (d, 1H, J = 8.00 Hz, CONH), 8.08 (s, 1H,
Indole-H), 7.63 (s, 2H, Ph-H), 7.50 (q, 2H, J = 8.00 Hz, Indole-H), 7.26 (s, 1H, Ph-H),
4.44(s, 1H, OH), 3.86 (s, 1H, piperidine-H), 3.34 (s, 2H, CH2), 2.91 (s, 2H,
piperidine-H), 2.45 (s, 2H, CH2), 2.31 (s, 6H, 2×CH3), 2.22 (s, 2H, piperidine-H),

ACCEPTED MANUSCRIPT
1.91 (s, 2H, piperidine-H), 1.60 (s, 2H, piperidine-H). 13C NMR (100MHz, DMSO-d6,
ppm) δ: 159.08 (C=O), 143.10, 139.41(2×C), 137.89, 135.11, 133.66, 127.53, 126.34,
124.11 (2×C), 122.33, 115.73, 115.65, 111.49, 60.59 (CH2), 58.98 (CH2), 52.54
(2×CH2), 47.13, 31.47 (2×CH2), 21.25 (2×CH3). HRMS: m/z 536.1040 (M+3).
C24H28BrN3O4S [533.1].
4.1.7. N-(1-(4-Sulfonamido-benzyl)piperidin-4-yl)-5-bromo-3-(3,5-dimethylphenyl-
sulfonyl)-1H-indole-2-carboxamide (9)
White solid, yield: 42.1%. mp: 170-171 °C. 1H NMR (40 0MHz, DMSO-d6, ppm)
δ: 13.01 (s, 1H, indole-NH), 8.97 (s, 1H, CONH), 8.09 (s, 1H, indole-H), 7.80 ( d, 2H,
J = 8.00 Hz, Ph’-H), 7.62 (s, 2H, Ph-H), 7.51-7.44 (m, 4H, indole-H, Ph’-H), 7.32 (s,
2H, SONH2), 7.27 (s, 1H, Ph-H), 3.87 (s, 1H, piperidine-H), 3.56 (s, 2H, benzyl-CH2),
2.81 (d, 2H, J = 12.00 Hz, piperidine-H), 2.31 (s, 6H, 2×CH3), 2.20 (t, 2H, J = 12.00
Hz, piperidine-H), 1.93 (d, 2H, J = 8.00 Hz, piperidine-H), 1.63 (q, 2H, J = 12.00 Hz,
piperidine-H). 13C NMR (100MHz, DMSO-d6, ppm) δ: 158.99 (C=O), 143.39, 143.20,
143.03, 139.44 (2×C), 137.70, 135.16, 133.54, 129.41 (2×C), 127.63, 126.30, 126.11
(2×C), 124.10 (2×C), 122.36, 115.71, 115.69, 111.55, 61.88 (CH2), 52.10 (2×CH2),
47.23 (CH), 31.65 (2×CH2), 21.25 (2×CH3). ESI-MS: m/z 659.4 (M+1).
C29H31BrN4O5S2 [658.09].
4.1.8.N-(1-(4-Chlorobenzyl)piperidin-4-yl)-5-bromo-3-(3,5-dimethylphenylsulfonyl)-1
H-indole-2-carboxamide (10)
White solid, yield: 46.2%. mp: 229-230 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.07-12.94 (br, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 8.09 (s, 1H,
indole-H), 7.62 (s, 2H, Ph-H), 7.50 (q, 2H, J = 8.00 Hz, indole-H), 7.30 (d, 2H, J =
8.00 Hz, Ph’-H), 7.35 (d, 2H, J = 8.00 Hz, Ph’-H), 7.26 (s, 1H, Ph-H), 3.86 (s, 1H,
piperidine-H), 3.48 (s, 2H, benzyl-CH2), 2.80 (d, 2H, J = 12.00 Hz, piperidine-H),
2.31 (s, 6H, 2×CH3), 2.17 (t, 2H, J = 12.00 Hz, piperidine-H), 1.93 (d, 2H, J = 12.00
Hz, piperidine-H), 1.61 (q, 2H, J = 12.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 158.99 (C=O), 143.02, 139.43 (2×C), 138.12, 137.71, 135.15,
133.54, 131.84, 130.93 (2×C), 128.62 (2×C), 127.62, 126.29, 124.10 (2×C), 122.35,
115.71, 115.68, 111.55, 61.60 (CH2), 51.99 (2×CH2), 47.24 (CH), 31.64 (2×CH2),

ACCEPTED MANUSCRIPT
21.24 (2×CH3). HRMS: m/z 615.1481 (M+1). C29H29BrClN3O3S [613.08].
4.1.9.N-(1-(3-Cyanobenzyl)piperidin-4-yl)-5-bromo-3-(3,5-dimethylphenylsulfonyl)-
1H-indole-2-carboxamide (11)
White solid, yield: 42.3%. mp: 223-236 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.03 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 8.09 (s, 1H, indole-H),
7.76 (d, 2H, J = 12.00 Hz, Ph’-H), 7.69 ( d, 1H, J = 8.00 Hz, Ph’-H), 7.63 (s, 2H,
Ph-H), 7.58 (t, 1H, J = 8.00 Hz, Ph’-H), 7.50 (q, 2H, J = 8.00 Hz, indole-H), 7.26 (s,
1H, Ph-H), 3.87 (s, 1H, piperidine-H), 3.56 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J =
12.00 Hz, piperidine-H), 2.31 (s, 6H, 2×CH3), 2.21 (t, 2H, J = 12.00 Hz, piperidine-H),
1.94 (d, 2H, J = 12.00 Hz, piperidine-H), 1.63 (q, 2H, J = 12.00 Hz, piperidine-H).
13C NMR (100MHz, DMSO-d6, ppm) δ: 158.98 (C=O), 143.01, 139.43 (2×C), 137.67,
135.16, 134.05, 133.58, 133.52, 132.48, 131.28, 129.95, 127.64, 126.29, 124.11 (2×C),
122.36, 119.38, 115.72, 115.67, 111.69, 111.57, 61.36 (CH2), 51.95 (2×CH2), 47.18
(CH), 31.61 (2×CH2), 21.24 (2×CH3). ESI-MS: m/z 605.4 (M+1). C30H29BrN4O3S
[604.11].
4.1.10.5-Bromo-3-(3,5-dimethylphenylsulfonyl)-N-(1-(pyridin-3-ylmethyl)piperidin-4-
yl)-1H-indole-2-carboxamide (12)
1
White solid, yield: 47.8%. mp: 220-221 °C. H NMR (400MHz, DMSO-d6,
ppm)δ: 13.02 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 8.51 (s, 1H,
pyridine-H), 8.48 (d, 1H, J = 4.00 Hz, pyridine-H), 8.09 (s, 1H, indole-H), 8.48 (d, 1H,
J = 8.00 Hz, pyridine-H), 7.63 (s, 2H, Ph-H), 7.50 (q, 2H, J = 8.00 Hz, indole-H),
7.38 (dd, 1H, J1 = 8.00 Hz, J2 = 4.00 Hz, pyridine-H), 7.26 (s, 1H, Ph-H), 3.86 (s, 1H,
piperidine-H), 3.52 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz, piperidine-H),
2.31 (s, 6H, 2×CH3), 2.19 (t, 2H, J = 12.00 Hz, piperidine-H), 1.93 (d, 2H, J = 12.00
Hz, piperidine-H), 1.61 (q, 2H, J = 12.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 159.01 (C=O), 150.43, 148.74, 143.02, 139.42 (2×C), 137.73,
136.90, 135.14, 134.38, 133.54, 127.61, 126.28, 124.11 (2×C), 123.90, 122.35, 115.70,
115.68, 111.55, 59.67 (CH2), 51.97 (2×CH2), 47.22 (CH), 31.62 (2×CH2), 21.24
(2×CH3). ESI-MS: m/z 581.1 (M+1). C28H29BrN4O3S [580.11].
4.1.11.5-Bromo-3-(3,5-dimethylphenylsulfonyl)-N-(1-(pyridin-4-ylmethyl)piperidin-4-

ACCEPTED MANUSCRIPT
yl)-1H-indole-2-carboxamide (13)
White solid, yield: 46.6%. mp: 243-244 °C.1H NMR (400MHz, DMSO-d6,
ppm)δ: 13.03 (s, 1H, Indole-NH), 8.98 (d, 1H, J = 8.00 Hz, CONH), 8.53(d, 2H, J =
4.00 Hz, pyridine-H), 8.09 (s, 1H, Indole-H), 7.63 (s, 2H, Ph-H), 7.50 (q, 2H, J = 8.00
Hz, Indole-H), 7.35 (d, 2H, J = 8.00 Hz, pyridine-H), 7.27 (s, 1H, Ph-H), 3.88 (s, 1H,
piperidine-H), 3.53 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz, piperidine-H),
2.31 (s, 6H, 2×CH3), 2.21 (t, 2H, J = 8.00 Hz, piperidine-H), 1.94 (d, 2H, J = 8.00 Hz,
piperidine-H), 1.61 (q, 2H, J = 8.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 159.01 (C=O), 150.02 (2×C), 148.27, 143.01, 139.43 (2×C),
137.70, 135.16, 133.52, 127.64, 126.27, 124.11 (4×C), 122.35, 115.72, 115.67, 111.57,
61.15 (CH2), 52.13 (2×CH2), 47.14 (CH), 31.60 (2×CH2), 21.14 (2×CH3). ESI-MS:
m/z 581.3 (M+1). C28H29BrN4O3S [580.11].
4.1.12.N-(1-(4-Carbamoylbenzyl)piperidin-4-yl)-5-bromo-3-(3,5-dimethylphenyl-
sulfonyl)-1H-indole-2-carboxamide (14)
White solid, yield: 48.1%. mp: 175-176 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.02 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 8.09 (s, 1H, indole-H),
7.94 (s, 1H, NH2), 7.86 (d, 2H, J = 8.00 Hz, Ph’-H), 7.63 (s, 2H, Ph-H), 7.50 (q, 2H, J
= 8.00 Hz, indole-H), 7.40 (d, 2H, J = 8.00 Hz, Ph’-H), 7.33 (s, 1H, NH2), 7.27 (s, 1H,
Ph-H), 3.86 (s, 1H, piperidine-H), 3.54 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz,
piperidine-H), 2.31 (s, 6H, 2×CH3), 2.18 (t, 2H, J = 8.00 Hz, piperidine-H), 1.93 (d,
2H, J = 8.00 Hz, piperidine-H), 1.62 (q, 2H, J = 8.00 Hz, piperidine-H). 13C NMR
(100MHz, DMSO-d6, ppm) δ: 168.23 (C=O), 158.99 (C=O), 143.02, 142.50, 139.43
(2×C), 137.71, 135.15, 133.53, 133.46, 128.86 (2×C), 127.93 (2×C), 127.62, 126.29,
124.11 (2×C), 122.36, 115.71, 115.68, 111.55, 60.23 (CH2), 52.09 (2×CH2), 47.25
(CH), 31.65 (2×CH2), 21.24 (2×CH3). HRMS: m/z 625.1344 (M+3). C30H31BrN4O4S
[622.12].
4.1.13.N-(1-(4-(Methylsulfonyl)benzyl)piperidin-4-yl)-5-chloro-3-(3,5-dimethylphenyl
sulfonyl)-1H-indole-2-carboxamide (15)
1
White solid, yield: 55.1%. mp: 179-180 °C. H NMR (400MHz, DMSO-d6,
ppm)δ: 13.01 (s, 1H, indole-NH), 8.98 (d, 1H, J = 8.00 Hz, CONH), 7.94 (s, 1H,

ACCEPTED MANUSCRIPT
indole-H), 7.91(d, 2H, J = 8.00 Hz, Ph’-H), 7.63 (s, 2H, Ph-H), 7.61(d, 2H, J = 8.00
Hz, Ph’-H), 7.55 (d, 1H, J = 8.00 Hz, indole-H), 7.56 (d, 1H, J = 8.00 Hz, indole-H),
7.26 (s, 1H, Ph-H), 3.87 (s, 1H, piperidine-H), 3.61 (s, 2H, benzyl-CH2), 3.21 (s, 2H,
SO2CH3), 2.81 (d, 2H, J = 8.00 Hz, piperidine-H), 2.31 (s, 6H, 2×CH3), 2.22 (t, 2H, J
= 12.00 Hz, piperidine-H), 1.94 (d, 2H, J = 8.00 Hz, piperidine-H), 1.61 (q, 2H, J =
12.00 Hz, piperidine-H). 13C NMR (100MHz, DMSO-d6, ppm) δ: 159.03(C=O),
145.49, 143.04, 139.86, 139.44 (2×C), 137.87, 135.15, 133.29, 129.76 (2×C), 127.68,
127.43 (2×C), 125.71, 125.10, 124.12 (2×C), 119.34, 115.33, 111.72, 61.80 (CH2),
52.11 (2×CH2), 47.19 (CH), 44.08 (CH3), 31.65 (2×CH2), 21.25 (2×CH3). ESI-MS:
m/z 614.3.4 (M+1). C30H32ClN3O5S2 [613.15].
4.1.14.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(1-(pyridin-3-ylmethyl)piperidin-4-
yl)-1H-indole-2-carboxamide (16)
1
White solid, yield: 50.0%. mp: 224-225 °C. H NMR (400MHz, DMSO-d6,
ppm)δ: 13.01 (s, 1H, Indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 8.51 (s, 1H,
pyridine-H), 8.48 (d, 1H, J = 4.00 Hz, pyridine-H), 7.95 (s, 1H, indole-H), 7.73 (d, 1H,
J = 8.00 Hz, pyridine-H), 7.63 (s, 2H, Ph-H), 7.55 ( d, 1H, J = 8.00 Hz, pyridine-H),
7.38 (q, 2H, J = 8.00 Hz, indole-H), 7.26 (s, 1H, Ph-H), 3.86 (s, 1H, piperidine-H),
3.53 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 8.00 Hz, piperidine-H), 2.31 (s, 6H,
2×CH3), 2.19 (t, 2H, J = 8.00 Hz, piperidine-H), 1.93 (d, 2H, J = 8.00 Hz,
piperidine-H), 1.62 (q, 2H, J = 12.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 159.02 (C=O), 150.43, 148.74, 143.04, 139.42 (2×C), 137.87,
136.90, 135.14, 134.39, 133.29, 127.68, 125.71, 125.09, 124.12 (2×C), 123.89, 119.35,
115.33, 111.73, 59.67 (CH2), 51.97 (2×CH2), 47.22 (CH), 31.62 (2×CH2), 21.24
(2×CH3). ESI-MS: m/z 537.4 (M+1) . C28H29ClN4O3S [536.16].
4.1.15.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(1-(pyridin-4-ylmethyl)piperidin-4-
yl)-1H-indole-2-carboxamide (17)
1
White solid, yield: 50.5%. mp: 240-241 °C. H NMR (400MHz, DMSO-d6,
ppm)δ: 13.02 (s, 1H, indole-NH), 8.98 (d, 1H, J = 8.00 Hz, CONH), 8.52 (d, 2H, J =
4.00 Hz, pyridine-H) 7.95 (s, 1H, indole-H), 7.63 (s, 2H, Ph-H), 7.55 (d, 1H, J = 8.00
Hz, indole-H), 7.35 (d, 3H, J = 8.00 Hz, indole-H, pyridine-H ), 7.26 (s, 1H, Ph-H),

ACCEPTED MANUSCRIPT
3.87 (s, 1H, piperidine-H), 3.53 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz,
piperidine-H), 2.31 (s, 6H, 2×CH3), 2.22 (t, 2H, J = 12.00 Hz, piperidine-H), 1.93 (d,
2H, J = 12.00 Hz, piperidine-H), 1.62 (q, 2H, J = 12.00 Hz, piperidine-H).13C NMR
(100MHz, DMSO-d6, ppm) δ: 159.03 (C=O), 150.02 (2×C), 148.26, 143.03, 139.43
(2×C), 137.85, 135.15, 133.28, 127.69, 125.71, 125.11, 124.12 (2×C), 124.09 (2×C),
119.55, 115.33, 111.74, 61.16 (CH2), 52.13 (2×CH2), 47.15 (CH), 31.62 (2×CH2),
21.24 (2×CH3). ESI-MS: m/z 537.6 (M+1) . C28H29ClN4O3S [536.16].
4.1.16.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(1-((4-methyl-5-oxo-4,5-dihydro-1
H-1,2,4-triazol-3-yl)methyl)piperidin-4-yl)-1H-indole-2-carboxamide (18)
White solid, yield: 53.1%. mp: 274-275 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.02 (s, 1H, indole-NH), 11.52 (s, 1H, triazole-NH), 8.95 (d, 1H, J = 8.00 Hz,
CONH), 7.94 (s, 1H, indole-H), 7.63 (s, 2H, Ph-H), 7.55 (d, 1H, J = 8.00 Hz,
indole-H), 7.36 (d, 1H, J = 8.00 Hz, indole-H), 7.26 (s, 1H, Ph-H), 3.85 (s, 1H,
piperidine-H), 3.41 (s, 2H, benzyl-CH2), 3.17 (s, 3H, CH3), 2.81 (d, 2H, J = 12.00 Hz,
piperidine-H), 2.31 (s, 6H, 2×CH3), 2.24 (t, 2H, J = 12.00 Hz, piperidine-H), 1.93 (d,
13
2H, J = 12.00 Hz, piperidine-H), 1.62 (q, 2H, J = 12.00 Hz, piperidine-H). C NMR
(100MHz, DMSO-d6, ppm) δ: 159.04 (C=O), 155.89 (C=O), 145.35, 143.03, 139.43
(2×C), 137.84, 135.15, 133.28, 127.68, 125.70, 125.09, 124.12 (2×C), 119.35, 115.32,
111.73, 53.41 (CH2), 51.79 (CH2), 49.07 (CH2), 47.05 (CH), 31.52 (2×CH2), 27.28
(CH3), 21.23 (2×CH3). HRMS: m/z 557.1745 (M+1). C26H29ClN6O4S [556.17].
4.1.17. N-(1-(4-Sulfonamido-benzyl)piperidin-4-yl)-5-chloro-3-(3,5-dimethylphenyl-
sulfonyl)-1H-indole-2-carboxamide (19)
White solid, yield: 52.1%. mp: 215-217 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.01 (s, 1H, Indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 7.94 (s, 1H, indole-H),
7.81(d, 2H, J = 8.00 Hz, Ph’-H), 7.63 (s, 2H, Ph-H), 7.55-7.50 (m, 3H, indole-H,
SO2NH2), 7.36-7.32 (m, 3H, Ph’-H, SO2NH2), 7.26(s, 1H, Ph-H), 3.87 (s, 1H,
piperidine-H), 3.57 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz, piperidine-H),
2.31 (s, 6H, 2×CH3), 2.20 (t, 2H, J = 12.00 Hz, piperidine-H), 1.94 (d, 2H, J = 12.00
Hz, piperidine-H), 1.63 (q, 2H, J = 12.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 159.01 (C=O), 143.20, 143.03, 139.44 (2×C), 137.84, 135.15,

ACCEPTED MANUSCRIPT
133.29, 129.41 (2×C), 127.69, 126.11 (2×C), 125.72, 125.10, 124.11 (2×C), 119.35,
115.34, 111.72, 61.87 (CH2), 52.10 (2×CH2), 47.22 (CH), 31.65 (2×CH2), 21.24
(2×CH3). ESI-MS: m/z 615.3 (M+1). C29H31ClN4O5S2 [614.14].
4.1.18.N-(1-(4-Carbamoylbenzyl)piperidin-4-yl)-5-chloro-3-(3,5-dimethylphenyl-
sulfonyl)-1H-indole-2-carboxamide (20)
White solid, yield: 56.6%. mp: 153-155 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.01 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 7.94 (s, 2H, indole-H),
7.86 (d, 2H, J = 8.00 Hz, Ph’-H), 7.63 (s, 2H, Ph-H), 7.55-7.53 (d, 1H, indole-H),
7.40-7.32 (m, 4H, Ph’-H, CONH2), 7.26 (s, 1H, Ph-H), 3.87 (s, 1H, piperidine-H),
3.54 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz, piperidine-H), 2.31 (s, 6H,
2×CH3), 2.18 (t, 2H, J = 12.00 Hz, piperidine-H), 1.94 (d, 2H, J = 12.00 Hz,
piperidine-H), 1.63 (q, 2H, J = 12.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 168.25 (C=O), 159.01 (C=O), 143.03, 139.43 (2×C), 137.87,
135.15, 133.46, 133.28, 128.87, 127.93 (4×C), 127.68, 125.70, 125.10, 124.12 (2×C),
119.35, 115.33, 111.72, 60.22 (CH2), 52.09 (2×CH2), 47.25 (CH), 31.64 (2×CH2),
21.24 (2×CH3). HRMS: m/z 579.1874 (M+1). C30H31ClN4O4S [578.18].
4.1.19. General procedure for the synthesis of target compounds (21-25)
A solution of intermediate 6b (0.10 g, 0.178 mmol, 1 equiv) in THF/MeOH (1/2,
30 mL) was treated with glacial acetic acid under stirring to adjust the pH value of 4-5.
Then the aromatic aldehydes (RCHO) (0.535 mmol, 3 equiv) was added and the
mixture was stirred for 40 minutes followed by addition of sodium cyanoborohydride
(0.056 g, 0.89 mmol, 5 equiv). After additional 10 h, the solvent was evaporated under
reduced pressure and the residues were dissovled with a small amount CH2Cl2 and
chromatographed on silica gel using EA-PE system. Pure fractions were collected,
concentrated and recrystallized, giving the desired compounds 21-25.
4.1.20.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(1-(thiophen-3-ylmethyl)-
piperidin-4-yl)-1H-indole-2-carboxamide (21)
1
White solid, yield: 70.2%. mp: 230-231 °C. H NMR (400MHz, DMSO-d6,
ppm)δ: 12.92 (s, 1H, indole-NH), 8.99 (d, 1H, J = 8.00 Hz, CONH), 7.94 (s, 1H,

ACCEPTED MANUSCRIPT
indole-H), 7.63-7.54 (m, 4H, thiophene-H,Ph-H), 7.44(d, 1H, J = 8.00 Hz, indole-H),
7.24(d, 1H, J = 8.00 Hz, indole-H), 7.15-7.13 (m, 2H, thiophene-H,Ph-H), 4.21 (s, 2H,
thiophene-CH2), 3.99 (s, 1H, piperidine-H), 3.34 (s, 2H, piperidine-H), 3.00 (s, 2H,
piperidine-H), 2.20 (s, 6H, 2×CH3), 2.04 (s, 2H, piperidine-H), 1.64 (s, 2H,
piperidine-H). ESI-MS: m/z 542.4 (M+1). C27H28ClN3O3S2 [541.13].
4.1.21.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(1-(thiophen-3-ylmethyl)
piperidin-4-yl)-1H-indole-2-carboxamide (22)
White solid, yield: 69.6%. mp: 225-256 °C. ESI-MS: m/z 542.4 (M+1).
C27H28ClN3O3S2 [541.13].
4.1.22.N-(1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)-5-chloro-3-(3,5-dimethylphen-
ylsulfonyl)-1H-indole-2-carboxamide (23)
White solid, yield: 69.8%. mp: 209-210 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.00 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 7.95 (s, 1H, indole-H),
7.70(d, 2H, J = 8.00 Hz, Ph’-H), 7.63(s, 2H, Ph-H), 7.56-7.53(m, 3H, J = 8.00 Hz,
indole-H, Ph’-H), 7.35(d, 1H, J = 8.00 Hz, indole-H), 7.26(s, 1H, Ph-H), 3.88 (s, 1H,
piperidine-H), 3.59 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 12.00 Hz, piperidine-H),
2.31 (s, 6H, 2×CH3), 2.21 (t, 2H, J = 12.00 Hz, piperidine-H), 1.94 (d, 2H, J = 12.00
Hz, piperidine-H), 1.64 (q, 2H, J = 12.00 Hz, piperidine-H). 13C NMR (100MHz,
DMSO-d6, ppm) δ: 159.02 (C=O), 144.21, 143.05, 139.43 (2×C), 137.87, 135.14,
133.31, 129.71 (2×C), 128.18, 127.87, 127.68, 126.61, 125.73, 125.47, 125.08, 124.11
(2×C), 119.35, 115.34, 111.72, 61.83 (CH2), 52.08 (2×CH2), 47.21 (CH), 31.66
(2×CH2), 21.23 (2×CH3). ESI-MS: m/z 604.4 (M+1). C30H29ClF3N3O3S [603.16].
4.1.23.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-(1-(furan-3-ylmethyl)piperidin-4-
yl)-1H-indole-2-carboxamide (24)
White solid, yield: 68.8%. mp: 223-224 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 12.91 (s, 1H, indole-NH), 8.98 (d, 1H, J = 8.00 Hz, CONH), 7.79 (s, 1H, indole-H),
7.72 (s, 1H, furan-H), 7.53 (s, 2H, Ph-H), 7.44 (d, 1H, J = 8.00 Hz, indole-H), 7.24 (d,
1H, J = 8.00 Hz, indole-H), 7.14 (s, 1H, Ph-H), 6.57 (s, 1H, furan-H), 6.46 (s, 1H,
furan-H), 4.24 (s, 2H, thiophene-CH2), 3.98 (s, 1H, piperidine-H), 3.31 (s, 2H,
piperidine-H), 2.98 (s, 2H, piperidine-H), 2.20 (s, 6H, 2×CH3), 2.00 (s, 2H,

ACCEPTED MANUSCRIPT
piperidine-H), 1.65 (s, 2H, piperidine-H). HRMS: m/z 526.1573 (M+1).
C27H28ClN3O4S [525.15].
4.1.24.N-(1-(4-Nitrobenzyl)piperidin-4-yl)-5-chloro-3-(3,5-dimethylphenylsulfonyl)-
1H-indole-2-carboxamide (25)
1
White solid, yield: 65.6%. mp: 258-259 °C, decomposition. H NMR (400MHz,
DMSO-d6, ppm) δ: 13.01 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 8.21
(d, 2H, J = 8.00 Hz, Ph’-H), 7.94 (s, 1H, indole-H), 7.63-7.60 (m, 4H, Ph-H, Ph’-H),
7.55 (d, 1H, J = 8.00 Hz, indole-H), 7.35 (d, 1H, J = 8.00 Hz, indole-H), 7.26 (s, 1H,
Ph-H), 3.88 (s, 1H, piperidine-H), 3.63 (s, 2H, benzyl-CH2), 2.81 (d, 2H, J = 8.00 Hz,
piperidine-H), 2.31 (s, 6H, 2×CH3), 2.23 (t, 2H, J = 8.00 Hz, piperidine-H), 1.95 (d,
2H, J = 12.00 Hz, piperidine-H), 1.64 (q, 2H, J = 8.00 Hz, piperidine-H). 13C NMR
(100MHz, DMSO-d6, ppm) δ: 159.01(C=O), 147.59, 147.01, 143.03, 139.43 (2×C),
137.84, 135.15, 133.29, 130.02 (2×C), 127.68, 125.71, 125.10, 124.11 (2×C), 123.85
(2×C), 119.35, 115.53, 111.72, 61.55 (CH2), 52.08 (2×CH2), 47.14 (CH), 31.65
(2×CH2), 21.24(2×CH3). ESI-MS: m/z 581.4 (M+1). C29H29ClN4O5S [580.15].
4.1.25.5-Chloro-3-(3,5-dimethylphenylsulfonyl)-N-((1-(2-hydroxyethyl)piperidin-4-yl)
methyl)-1H-indole-2-carboxamide (26)
White solid, yield: 50.0%. mp: 255-256 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 13.02 (s, 1H, indole-NH), 9.08 (d, 1H, J = 4.00 Hz, CONH), 7.88 (s, 1H, indole-H),
7.65 (s, 2H, Ph-H), 7.55 (s, 1H, J = 8.00 Hz, indole-H), 7.35 (s, 1H, J = 8.00 Hz,
indole-H), 7.27 (s, 1H, Ph-H), 5.28 ( s, 1H, OH), 3.37 (s, 2H, CH2), 3.50 (s, 2H, CH2),
3.40 (s, 2H, CH2), 3.11 (s, 2H, CH2), 2.31 (s, 6H, 2×CH3), 2.29 (s, 2H, CH2), 1.98 (s,
2H, CH2), 1.86 (s, 1H, CH), 1.53 (s, 2H, CH2). HRMS: m/z 504.1772 (M+1).
C25H30ClN3O4S [503.16].
4.1.26.N-((1-(4-Carbamoylbenzyl)piperidin-4-yl)methyl)-5-chloro-3-(3,5-dimethyl-
phenylsulfonyl)-1H-indole-2-carboxamide (27)
White solid, yield: 52.3%. mp: 259-260 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ: 12.93 (s, 1H, indole-NH), 8.98 (d, 1H, J = 8.00 Hz, CONH), 7.92-7.90 (m, 2H,
indole-H, NH2), 7.83 (d, 2H, J = 8.00 Hz, Ph’-H), 7.61 (s, 2H, Ph-H), 7.54 (d, 1H, J =
8.00 Hz, indole-H), 7.37-7.52 (m, 5H, indole-H, NH2, Ph-H, Ph’-H ), 3.50 (s, 2H,

ACCEPTED MANUSCRIPT
benzyl-CH2), 3.32-3.24 (m, 2H, CONH-CH2), 2.83 (d, 2H, J = 12.00 Hz,
piperidine-H), 2.30 (s, 6H, 2×CH3), 1.98 (t, 2H, J = 8.00 Hz, piperidine-H), 1.76 (d,
2H, J = 8.00 Hz, piperidine-H), 1.57 (s, 1H, piperidine-H), 1.62 (q, 2H, J = 8.00 Hz,
piperidine-H). 13C-NMR (100MHz, DMSO-d6, ppm) δ: 168.26 (C=O), 159.86 (C=O),
143.07, 142.49, 139.40 (2×C), 137.95, 135.12, 133.38, 133.34, 128.89 (2×C), 127.86
(2×C), 127.64, 125.77, 125.05, 124.09 (2×C), 119.33, 115.36, 111.53, 62.38 (CH2),
53.40 (2×CH2), 45.40 (CH), 36.07 (CH2), 30.09 (2×CH2), 21.24 (2×CH3). ESI-MS:
m/z 593.5 (M+1). C31H33ClN4O4S [592.19].
4.1.27.N-((1-(3-Fluorobenzyl)piperidin-4-yl)methyl)-5-chloro-3-(3,5-dimethylphenyl
-sulfonyl)-1H-indole-2-carboxamide (28)
White solid, yield: 46.5%. mp: 193-194 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ:13.00 (s, 1H, indole-NH), 8.97 (d, 1H, J = 8.00 Hz, CONH), 7.92 (d, 1H, J = 12.00
Hz, indole-H), 7.61 (s, 2H, Ph-H), 7.54 (d, 1H, J = 8.00 Hz, indole-H), 7.37-7.31 (m,
2H, indole-H, Ph’-H), 7.26 (s, 1H, Ph-H), 7.14-7.04 (m, 3H, Ph’-H), 3.47 (s, 2H,
benzyl-CH2), 3.27 (t, 2H, J = 8.00 Hz, CONH-CH2), 2.82 (d, 2H, J = 12.00 Hz,
piperidine-H), 2.30 (s, 6H, 2×CH3), 1.97 (t, 2H, J = 12.00 Hz, piperidine-H), 1.94 (d,
2H, J = 2.00 Hz, piperidine-H), 1.57 (s, 1H, piperidine-H), 1.30 (q, 2H, J = 12.00 Hz,
piperidine-H). 13C-NMR (100MHz, DMSO-d6, ppm) δ: 161.26 (C-F), 159.86 (C=O),
143.10, 139.38 (2×C), 137.98, 135.09, 133.40, 130.50, 130.42, 127.61, 125.81,
125.10, 124.10 (2×C), 119.33, 115.63, 115.39, 114.14, 113.93, 111.53, 62.05 (CH2),
53.40 (2×CH2), 45.37 (CH), 36.06 (CH2), 30.08 (2×CH2), 21.23 (2×CH3). ESI-MS:
m/z 568.5 (M+1). C30H31ClFN3O3S [567.18].
4.1.28.N-((1-(3-Cyanobenzyl)piperidin-4-yl)methyl)-5-chloro-3-(3,5-dimethylphenyl
-sulfonyl)-1H-indole-2-carboxamide (29)
White solid, yield: 52.6%. mp: 213-214 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ:13.00 (s, 1H, indole-NH), 8.96 (d, 1H, J = 8.00 Hz, CONH), 7.92 (s, 1H, indole-H),
7.80 (d, 1H, J = 4.00 Hz, Ph’-H), 7.68 (t, 1H, J = 8.00 Hz, Ph’-H), 7.61 (s, 2H, Ph-H),
7.57-7.30 (m, 4H, indole-H, Ph’-H), 7.24 (s, 1H, Ph-H), 3.63 (s, 2H, benzyl-CH2),
3.26 (t, 2H, J = 8.00 Hz, CONH-CH2), 2.82 (d, 2H, J = 12.00 Hz, piperidine-H), 2.30
(s, 6H, 2×CH3), 2.06 (t, 2H, J = 12.00 Hz, piperidine-H), 1.76 (d, 2H, J = 12.00 Hz,

ACCEPTED MANUSCRIPT
piperidine-H), 1.59 (s, 1H, piperidine-H), 1.29 (q, 2H, J = 12.00 Hz, piperidine-H).
13C-NMR (100MHz, DMSO-d6, ppm) δ: 159.86 (C=O), 143.10, 142.95, 139.35 (2×C),
138.11, 138.06, 135.06, 133.50, 133.41, 130.51, 128.35, 127.56, 125.86, 124.95,
124.12 (2×C), 119.32, 118.19, 115.43, 112.51, 111.51, 60.56 (CH2), 53.36 (2×CH2),
45.33 (CH), 35.97 (CH2), 30.06 (2×CH2), 21.24 (2×CH3). ESI-MS: m/z 575.5(M+1).
C31H31ClN4O3S [574.18].
4.1.29.Ethyl3-(4-((5-chloro-3-(3,5-dimethylphenylsulfonyl)-1H-indole-2-carboxamido
)methyl)piperidin-1-yl)propanoate (30)
White solid, yield: 45.6%. mp: 194-195 °C. 1H NMR (400MHz, DMSO-d6, ppm)
δ:13.01 (s, 1H, indole-NH), 8.96 (s, 1H,CONH), 7.92 (s, 1H,indole-H), 7.61 (s, 2H,
Ph-H), 7.80 (d, 1H, J = 8.00 Hz, indole-H), 7.34 (d, 1H, J = 8.00 Hz, indole-H), 7.26
(s, 1H, Ph-H), 4.07 (q, 2H, J = 4.00 Hz, O-CH2), 3.25 (t, 2H, J = 4.00 Hz, CO-CH2),
2.82 (d, 2H, J = 12.00 Hz, piperidine-H), 2.57 (d, 2H, J = 8.00 Hz, CH2), 2.46 (d, 2H,
J = 8.00 Hz, CH2), 2.31 (s, 6H, 2×CH3), 1.98 (t, 2H, J = 12.00 Hz, piperidine-H), 1.75
(d, 2H, J = 12.00 Hz, piperidine-H), 1.56 (s, 1H, piperidine-H), 1.25-1.15 (m, 5H,
piperidine-H, CH3). 13C-NMR (100MHz, DMSO-d6, ppm) δ: 172.46 (C=O), 159.85
(C=O), 143.06, 139.41 (2×C), 137.94, 135.12, 133.34, 127.63, 125.75, 125.05, 124.10
(2×C), 119.32, 115.35, 111.51, 60.19 (CH2), 53.92 (2×CH2), 53.20, 45.38 (CH), 36.02
(CH2), 32.37, 30.03 (2×CH2), 21.24 (2×CH3), 14.59. ESI-MS: m/z 560.4(M+1).
C28H34ClN3O5S [559.19].
4.2. In vitro anti-HIV assay
Evaluation of the antiviral activity and cytotoxicity of the compounds in MT-4
cells was performed using the MTT assay as previously described [33,52]. Stock
solutions (10×final concentration) of test compounds were added in 25 µL volumes to
two series of triplicate wells so as to allow simultaneous evaluation of their effects on
mock- and HIV-infected cells at the beginning of each experiment. Serial five-fold
dilutions of test compounds were made directly in flat-bottomed 96wells microtiter
trays using a Biomek 3000 robot (Beckman instruments, Fuller-ton, CA). Untreated
control HIV- and mock-infected cell samples were included for each sample.

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HIV-1(IIIB), single and double RT mutant strains (L100I, K103N, Y181C,
Y188L, E138K, F227L/V106A, RES056) or HIV-2 (ROD) [53] stock (50µL) at
100-300 CCID50 (cell culture infectious dose) or culture medium was added to either
the infected or mock-infected wells of the microtiter tray. Mock-infected cells were
used to evaluate the effect of test compounds on uninfected cells in order to assess the
cytotoxicity of the test compound. Exponentially growing MT-4 cells were
cen-trifuged for 5 minutes at 1000 rpm and the supernatant was discarded. The MT-4
cells were resuspended at 6×105 cells/mL, and 50-µL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of mock- and
HIV-infected cells was examined spectrophotometrically by the MTT assay. The 50%
cytotoxic concentration (CC50) was defined as the concentration of the test compound
that reduced the absorbance (OD540) of the mock-in-fected control sample by 50%.
The concentration achieving 50% protection from the cytopathic effect of the virus in
infected cells was defined as the 50% effective concentration (EC50).
4.3. HIV-1 RT inhibition assay
A reverse transcriptase (RT) assay kit produced by Roche was selected for the RT
inhibition assay. All the reagents for performing the RT reaction came with the kit and
the ELSIA procedures for RT inhibition assay was carried out following the
description in the kit protocol [54,55]. Briefly, the reaction mixture containing
template/primer complex, viral nucleotides (dNTPs) and RT in the incubation buffer
with or without inhibitors was incubated for 1hours at 37 °C. After that, the reaction
mixture was transferred to a streptavidine-coated microtiter plate and incubated for
another 1hours at 37 °C to make sure retranscriptional cDNA chain that consisted
biotin-labeled dNTPs bound to streptavidine. Then removed un-bound dNTPs using
washing buffer and added anti-DIG-POD working solution. After incubation for
1hours at 37 °C, the DIG-labeled dNTPs incorporated in cDNA were bound to the
anti-DIG-POD antibody. The unbound anti-DIG-PODs were removed and the
peroxidesubstrate (ABST) solution was added to the MTPs. A colored reaction
proceed during cleavage of the substrate catalyzed by POD. The absorbance of the

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sample was determined at OD 405 nm using a microtiter plate ELISA reader. The
percentage inhibitory activity of RT inhibitors was calculated byformula as given
below:
%Inhibition = [OD value with RT but without inhibitors - OD value with RT and
inhibitors]/[OD value with RT and inhibitors - OD value without RT and inhibitors]
The IC50 values corresponded to the concentrations of the in-hibitors required to
inhibit biotin-dUTP incorporation by 50%.
4.4. Molecular simulation experiment
The molecules 8 and 18 for docking was optimized for 2000-generations until
the maximum derivative of energy became 0.005 kcal/(mol*A), using the Tripos force
field. Charges were computed and added according to Gasteiger-Huckel parameters.
The published three dimensional crystal structures of RT complexes (PDB code: 2rf2)
were retrieved from the Protein Data Bank and were used for the docking experiment
by means of surflex-docking module of Sybyl-X 1.1. The protein was prepared by
using the Biopolymer application accompanying Sybyl: The bound ligand was
extracted from the complexes, water molecules were removed, hydrogen atoms were
added, side chain amides and side chains bumps were fixed, and charges and atom
types were assigned according to AMBER 99. After the protomol was generated, the
optimized 8 and 18 were surflex-docked into the binding pocket of NNRTIs, with the
relevant parameters set as defaults. Top-scoring pose was shown by the software of
PyMOL version 1.5 (www.pymol.org.). The secondary structure of RT is shown in
cartoons, and only the key residues for interactions with the inhibitor were shown in
sticks and labeled. The potential hydrogen bonds were presented by dashed lines.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We thank K. Erven, K. Uyttersprot and C. Heens for technical assistance with the
HIV assays. The financial support from the National Natural Science Foundation of
China (NSFC Nos. 81273354, 81573347), Key Project of NSFC for International

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Cooperation (No. 81420108027), The Natural Science Foundation of Shandong
Province (No. ZR2009CM016), Young Scholars Program of Shandong University
(YSPSDU, No. 2016WLJH32, to P.Z.), Major Project of Science and Technology of
Shandong Province (No. 2015ZDJS04001) and KU Leuven (GOA 10/014).
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