Design, synthesis, and biological evaluation of novel 4-aminopiperidinyl-linked 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione derivatives as HIV-1 NNRTIs

Article abstract of DOI:10.1111/cbdd.12468

Based on the hybridization of the privileged fragments in DABO and DAPY-typed HIV-1 NNRTIs, a novel series of 4-aminopiperidinyl-linked 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione derivatives were designed, synthesized, and evaluated for their in vitro

Full text of DOI:10.1111/cbdd.12468

Chem Biol Drug Des 2014
Research Article
Design, Synthesis, and Biological Evaluation of Novel
4
6
-Aminopiperidinyl-linked 3,5-Disubstituted-1,2,
-thiadiazine-1,1-dione Derivatives as HIV-1 NNRTIs
1
1
1
1
Tao Liu , Boshi Huang , Ye Tian , Xin Liang ,
clinical application of HIV-1 NNRTIs (1). Besides, the clinical
advancement of many promising NNRTIs was hindered by
poor aqueous solubility, low bioavailability and safety and
tolerability issues. In consequence, there is an urgent need
to develop novel NNRTIs with high potency, improved resis-
tance profiles, safety, excellent tolerability, and favorable
physicochemical properties.
1
3
1,
Hong Liu , Huiqing Liu , Peng Zhan *,
Erik De Clercq , Christophe Pannecouque and
Xinyong Liu *
2
2
1
,
1
Department of Medicinal Chemistry, Key Laboratory of
Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, Jinan, Shandong 250012, China
Rega Institute for Medical Research, K.U. Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Institute of Pharmacology, School of Medicine, Shandong
University, 44 West Culture Road, Jinan, Shandong
50012, China
Among the existing HIV-1 NNRTIs, 2-alkoxy-6-benzyl-3,4-
dihydro-4-oxopyrimidines (DABOs) and diarylprimidines
2
(
DAPYs) analogues represent two highly potent and prom-
ising classes (Figure S1). In the DABOs family, F -S-DA-
BOs (exemplified by MC1047) (2,3) and F -N,N-DABOs
exemplified by MC1220) (4) with high potency against
3
2
2
*
2
(
Corresponding authors: Peng Zhan,
zhanpeng1982@163.com; Xinyong Liu,
xinyongl@sdu.edu.cn
both wild-type (WT) and mutant strains have attracted
special attention over the past few years. On the other
hand, as two DAPY analogues TMC125 (Etravirine, ETR)
and TMC278 (Rilpivirine, RPV) have been approved by
FDA in 2008 and 2011, respectively (5), considerable
efforts have been devoted to the structural modification
and optimization of DAPYs.
Based on the hybridization of the privileged fragments
in DABO and DAPY-typed HIV-1 NNRTIs, a novel ser-
ies of 4-aminopiperidinyl-linked 3,5-disubstituted-1,2,6-
thiadiazine-1,1-dione derivatives were designed, syn-
thesized, and evaluated for their in vitro anti-HIV activi-
ties in MT-4 cells. Most of the target compounds
showed weak inhibitory activity against WT HIV-1. In
order to confirm the mode of action of the target com-
pounds, representative compounds Ba8 and Bb8 were
selected to perform the HIV-1 RT inhibitory assay. In
this assay, Ba8 and Bb8 displayed good activity with
IC50 values of 3.15 and 1.52 lM, respectively. Addition-
ally, preliminary structure–activity relationships (SARs)
analysis and molecular docking studies of newly syn-
thesized compounds are also discussed.
As a part of our continuing efforts directed towards the
development of potential DABO and DAPY derivatives (6),
herein we designed a novel series of 4-aminopiperidinyl-
linked 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione deriva-
tives as DABO–DAPY hybrid based on their structural
features, binding mode, and structure–activity relationships
(SARs) studies (Figure S2) (7–10).
Concretely, the rational design of 4-aminopiperidinyl-
linked 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione (Fig-
ure S2) was based on the following considerations:
Key words: 1,2,6-thiadiazine-1,1-dione, bioactivity, drug design,
HIV-1, NNRTIs, RT, synthesis
Received 24 July 2014, revised 31 August 2014 and
accepted for publication 15 October 2014
Nucleus (Domain I): According to the bioisosterism princi-
ple of rational drug design, 4-oxopyrimidine in DABOs was
replaced by the synthetically accessible 1,2,6-thiadiazine-
1
, 1-dione heterocyclic motif with an attempt to form addi-
As significant and indispensable components of highly
active antiretroviral therapy (HAART), HIV-1 non-nucleoside
reverse transcriptase (RT) inhibitors (NNRTIs) possess their
unique merits such as high potency, high selectivity, and rel-
atively low toxicity. Unfortunately, the rapid emergence of
drug resistance, caused by mutation of the amino acid resi-
dues in the binding pocket of HIV-1 RT severely impairs the
tional hydrogen bonds between the ligand and the sur-
rounding amino acids in RT. It should be noted that,
previous investigations in our laboratory using structure-
based isosterism drug design resulted in the discovery of
several novel series of thiadiazine-derived NNRTIs, which
proved to be effective in inhibiting HIV-1 replication at
micromolar concentration acting as RT inhibitors (6,11–
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12468
1

Liu et al.
3). Thus, this privileged structure should be further
1
To
10 mmol,
2.05 g, 10 mmol, 1 eqv.) in tetrahydrofuran (20 mL), N,
a
solution of 1-naphthaleneacetic acid (1.86 g,
exploited for the therapeutic benefits (14).
1
eqv.) or 2,4-dichlorophenyl acetic acid
(
Substituents at 3-position (Domain II): The privileged sub-
stituents of DABO family derivatives, such as 1-naphthylm-
ethyl or 2,4-dichlorobenzyl, were kept to remain the effect
of p–p interaction with hydrophobic amino acids Tyr181,
Tyr188, and Trp229.
N’-carbonyldiimidazole (CDI) (1.78 g, 11 mmol, 1.1 eqv.)
was added, and the solution was heated to 50 °C. Mel-
drum’s acid (1.58 g, 11 mmol, 1.1 eqv.) was added
carefully, and the reaction mixture was stirred for
another 5 h at 50 °C. The reaction mixture was concen-
trated under reduced pressure to evaporate the tetrahy-
drofuran and to obtain a red oil, then 100 mL water
and 100 mL dichloromethane (DCM) were added, and
the mixture was stirred vigorously. The aqueous phase
was adjusted to pH 2 with concentrated hydrochloric
acid. The organic phase was washed with 0.1 M hydro-
chloric acid (30 mL), water (30 mL), 0.1 M hydrochloric
acid (30 mL), water (30 mL) successively and was sub-
sequently dried (Na SO ), filtered, and concentrated
Substituents at 5-position (Domain III): Based on the con-
cept of molecular hybridization, Rotili and his partners have
identified novel diarylpyrimidine-dihydrobenzyloxopyrimidine
hybrids, which showed characteristic SAR profile and
nanomolar bioactivity at both the enzymatic and cellular level
(15). Inspired by this successful case of molecular hybridiza-
tion, the 4-aminopiperidinyl-linked substituted benzyl group
which belongs to a typical group of DAPYs was introduced
at this position to explore the effects of the linker and steric,
electronic, hydrophobic interaction. In addition, the benzyl
moiety was substituted by different substituents to explore
SARs in this domain.
2
4
under reduced pressure to obtain a crude product with-
out further purification.
2
,2-dimethyl-5-(2-(naphthalen-1-yl)acetyl)-1,3-dioxane-4,6-
dione (Bam1): 1.95 g crude yellow residue was obtained
with a yield of 62.5%.
Consequently, with the aim to achieve our goals described
above, novel 4-aminopiperidinyl-linked 3,5-disubstituted-
1
,2,6-thiadiazine-1,1-dione derivatives were designed, syn-
5
4
-(2-(2,4-dichlorophenyl)acetyl)-2,2-dimethyl-1,3-dioxane-
,6-dione (Bbm1): 2.16 g crude yellow–white residue was
thesized by a facile synthetic route and evaluated for their
anti-HIV activities in MT-4 cells using the MTT assay.
obtained with a yield of 69.2%.
Methods and Materials
5-(naphthalen-1-ylmethyl)-2H-1,2,6-thiadiazin-3(6H)-one
1,1-dioxide (Bam2) and 5-(2,4-dichlorobenzyl)-2H-1,2,
6-thiadiazin-3(6H)-one 1,1-dioxide (Bbm2) (17).
Chemistry
All melting points (mp) were determined on a micromelting
point apparatus and are uncorrected. Mass spectra were
taken on a LC Autosampler Device: Standard G1313A
instrument by electrospray ionization. H NMR spectra were
obtained on a Bruker Avance-400 NMR-spectrometer in the
1
0 mmol (1 eqv.) acylmeldrum’s acid and 1.15 g sulfamide
1
(12 mmol, 1.2 eqv.) were added in a mortar. After grinding
and mixing, the mixture was transferred into a reaction
flask, then heated to 100–110 °C in an oil bath, and fur-
ther stirred for 1.5 h. After cooling to room temperature,
the reaction mixture was dissolved in 30 mL ethyl acetate
indicated solvents. Chemical shifts (d) are reported in parts
13
per million relative to TMS as an internal standard. The
C
NMR spectra were recorded on a Bruker Avance-400
NMR-spectrometer and were reported accordingly. TLC
was performed on Silica Gel GF254 for TLC, and spots were
visualized by irradiation with UV light (254 nm). Flash column
chromatography was performed on a column packed with
silica gel 60 (200–300 mesh). Solvents were reagent grade
and, when necessary, were purified and dried by standard
methods. Concentration of the reaction solution involved
the use of rotary evaporator at reduced pressure.
(EA) and extracted using saturated sodium bicarbonate
solution for three times. The aqueous phases were com-
bined, then acidified with 2N diluted hydrochloric acid,
adjusted to pH 1, and then extracted with ethyl acetate
(
(
30 mL) for three times. Further, organic layer was dried
Na SO ), filtered, and concentrated to afford a crude
2
4
solid.
5
-(naphthalen-1-ylmethyl)-2H-1,2,6-thiadiazin-3(6H)-one
General synthetic procedure for compounds 3-(naphtha-
len-1-ylmethyl)-1, 1-dioxo-2H-1,2,6-thiadiazin-5-yl 4-meth-
ylbenzenesulfonate (Bam3) and 3-(2,4-dichlorobenzyl)-1,1-
dioxo-2H-1,2,6-thiadiazin-5-yl 4-methylbenzenesulfonate
1,1-dioxide (Bam2): 1.25 g crude yellow residue was
obtained with a yield of 43.5%. Mp 165–167 °C. H NMR
(DMSO-d ) d: 9.57 (s, 1H, NH), 7.40-8.06 (m, 7H, naph-
6
1
thalene), 4.37 (s, 1H, C=CH), 3.79 (s, 2H, CH
2
). EI-MS:
À
(
Bbm3).
m/z 287.2 [MÀH] . C14
12 2 3
H N O S (288.06).
2
4
2
,2-dimethyl-5-(2-(naphthalen-1-yl)
,6-dione (Bam1) and 5-(2-(2,4-dichlorophenyl) acetyl)-
,2-dimethyl-1,3-dioxane-4,6-dione (Bbm1) (16).
acetyl)-1,3-dioxane-
5-(2,4-dichlorobenzyl)-2H-1,2,6-thiadiazin-3(6H)-one 1,1-
dioxide (Bam2): 1.30 g crude yellow residue was obtained
1
with
a
yield of 43.5%. Mp 180–183 °C.
H NMR
2
Chem Biol Drug Des 2014

Disubstituted Thiadiazine Derivatives as HIV-1 NNRTIs
(
400 MHz, DMSO-d
NH), 7.69 (d, J = 2.0 Hz, 1H, Ph-H), 7.49 (dd, J = 8.3,
.0 Hz, 1H, Ph-H), 7.44 (d, J = 8.3 Hz, 1H, Ph-H), 5.01
s, 1H, C=CH), 3.80 (s, 2H, CH ). ESI-MS: m/z 305.3
6
) d 12.85 (s, 1H, NH), 12.12 (s, 1H,
concentrated to afford crude solid or oil Bm4 without
further purification (19,20).
2
(
[
A solution of Bm4 in 5 mL dichloromethane, 2 mL trifluo-
roacetic acid (TFA) was added and stirred for 12 h at room
temperature. The reaction mixture was adjusted to pH 8–9
2
À
-
MÀH] , 307.3 [M-H] . C10
8 2 2 3
H Cl N O S (305.96).
3-(naphthalen-1-ylmethyl)-1,1-dioxo-2H-1,2,6-thiadiazin
2 3
with saturated K CO aqueous solution, then 30 mL water
-
5-yl 4-methylbenzenesulfonate (Bam3) and 3-(2,4-dichlo-
was added, and the reaction mixture was extracted with
robenzyl)-1,1-dioxo-2H-1,2,6-thiadiazin-5-yl 4-methylben-
zenesulfonate (Bbm3) (18).
dichloromethane (3 9 40 mL). The organic phase was
2 4
washed with brine (3 9 40 mL), then dried (Na SO ), fil-
tered, concentrated to afford crude solid or oil Bm5 with-
19.32 mmol (1 eqv.) 5-(naphthalen-1-ylmethyl)-2H-1,2,6-
out further purification (21,22).
thiadiazin-3(6H)-one 1,1-dioxide (Bam2) or 5-(2,4-dichlo-
robenzyl)-2H-1,2,6-thiadiazin-3(6H)-one 1,1-dioxide (Bbm2)
in 50 mL acetonitrile (MeCN) was treated with 2.66 mL
General synthetic procedure for target compounds
(Ba1–Ba13, Bb1–Bb13)
triethylamine (TEA) (19.32 mmol,
1 eqv.) at ambient
temperature, and the resulting solution was stirred for 1 h.
p-toluenesulfonyl chloride (TsCl) 7.38 g (38.64 mmol, 2
eqv.) and 4-dimethylaminopyridine (DMAP) 0.12 g (cata-
lyst) were added into the solution and stirred for another
4-methylbenzenesulfonate thiadiazine derivatives Bam3 or
Bbm3 (1 mmol, 1 eqv.) and an excess of 1-substituted-
benzylpiperidin-4-amine (Bm5) were dissolved in acetoni-
trile, then TEA (1.5 mmol, 1.5 eqv.) was added, and the
mixture was refluxed for 48 h at 90 °C. The solution was
chromatographed on silica gel (1:2:0.01 EA/PE/
48 h at ambient temperature. The solution was chromato-
graphed on silica gel (2:1:0.01 DCM/PE/CH
eluent) to provide the title product.
3
COOH,
CH COOH, eluent) to provide the title target product.
3
3-(naphthalen-1-ylmethyl)-1,1-dioxo-2H-1,2,6-thiadiazin-5-
yl 4-methylbenzenesulfonate (Bam3): 3.96 g white solid
Anti-HIV activity evaluation
1
was obtained with a yield of 46.3%. Mp 160–162 °C;
NMR (400 MHz, DMSO-d ) d ppm: 9.96 (s, 1H, NH),
.98–7.93 (m, 2H, Naph-H), 7.90 (d, J = 8.2 Hz, 1H,
Naph-H), 7.78 (d, J = 8.4 Hz, 2H, Ph-H), 7.57–7.55
H
6
In vitro anti-HIV assay
7
The evaluation of target compounds for their activity
against HIV-1 strain (III ), mutant HIV-1 (RES056: RT
B
K103N/
(
(
m, 2H, Naph-H), 7.49 (d, J = 8.2 Hz, 1H, Naph-H), 7.43
d, J = 8.1 Hz, 2H, Ph-H), 7.40 (d, J = 7.3 Hz, 1H, Naph-
Y181C) and HIV-2 strain (ROD) in MT-4 cells was performed
using the MTT method as previously described (23). Stock
solutions (10 9 final concentration) of test compounds
were added in 25 lL volumes to two series of triplicate
wells to allow simultaneous evaluation of their effects on
mock- and HIV-infected cells at the beginning of each
experiment. Serial fivefold dilutions of test compounds
were made directly in flat-bottomed 96-well microtitre trays
by adding 100 lL medium to the 25 lL stock solution and
transferring 25 lL of this solution to another well that con-
tained 100 lL medium using a Biomek 3000 robot (Beck-
man Instruments, Fullerton, CA, USA). Untreated control
HIV- and mock-infected cell samples were included for
H), 5.26 (s, 1H, C=CH), 4.19 (s, 2H, CH
CH
2
), 2.41 (s, 3H,
À
3
); ESI-MS: m/z 441.4 [MÀH] . C21
18 2 5 2
H N O S (442.07).
3
-(2,4-dichlorobenzyl)-1,1-dioxo-2H-1,2,6-thiadiazin-5-yl 4-
methyl benzenesulfonate (Bbm3): 3.96 g white solid was
obtained with a yield of 44.4%. Mp 150–152 °C; H NMR
1
(400 MHz, DMSO-d ) d ppm: 10.21 (s, 1H, NH), 7.85 (d,
6
J = 8.3 Hz, 2H, Ph’-H), 7.64 (d, J = 2.1 Hz, 1H, Ph-H),
7
1
C=CH), 3.79 (s, 2H, CH
4
.49 (d, J = 8.2 Hz, 2H, Ph’-H), 7.44 (dd, J = 2.1, 8.3 Hz,
H, Ph-H), 7.36 (d, J = 8.3 Hz, 1H, Ph-H), 5.11 (s, 1H,
2
), 2.43 (s, 3H, CH
3
); ESI-MS: m/z
À
À
59.4 [MÀH] , 461.4 [MÀH] . C H Cl N O S (459.97).
each sample. HIV-1(III ) (24), mutant HIV-1 (RES056) or
17
14
2
2
5
2
B
HIV-2 (ROD) (25) stock (50 lL) at 100-300 CCID50 (50%
cell culture infectious dose-50%) or culture medium was
added in either the HIV-infected or mock-infected wells of
the microtitre plate. Mock-infected cells were used to eval-
uate the effect of test compound on uninfected cells to
assess the cytotoxicity of the test compound. Exponen-
tially growing MT-4 cells were centrifuged for 5 min at
220 g, and the supernatant was discarded. The MT-4 cells
General synthetic procedure for compound 1-substituted-
benzylpiperidin-4-amine (Bm5).
To a solution of 0.2 g 4-tert-butoxycarbonylaminopiperi-
dine (1 mmol, 1 eqv.) and substituted benzyl chloride or
bromide (1.2 mmol, 1.2 eqv.) in 10 mL dimethyl formam-
ide (DMF), 0.41 g K
sodium iodide (NaI) and tetrabutylammonium iodide
TBAI) were added, the mixture was then stirred for
2 3
CO (3 mmol, 3 eqv.), catalytic
5
were resuspended at 6 9 10 cells/mL, and 50 lL vol-
(
umes were transferred to the microtitre tray wells. Five
days after infection, the viability of mock- and HIV-
infected cells was examined spectrophotometrically by
1
1
2 h at 50 °C. After cooling to room temperature,
00 mL water was added and extracted with ethyl ace-
tate (3 9 40 mL). The organic phase was washed with
brine (3 9 40 mL), then dried (Na SO ), filtered, and
the MTT assay. The 50% cytotoxic concentration (CC50)
was defined as the concentration of the test compound
2
4
Chem Biol Drug Des 2014
3

Liu et al.
that reduced the viability of the mock-infected MT-4
cells by 50%. The concentration achieving 50% protec-
tion from the cytopathic effect of the virus in infected
cells was defined as the 50% effective concentration
nucleophilic manner by different substituted benzyls, then
the product was treated with TFA to give 1-benzyl-substi-
tuted 4-aminopiperidine Bm5. Finally, Bm5 participated in a
nucleophilic substitution at the C-5 position of the thiadi-
azine heterocycle (Bam3 or Bbm3), yielding 26 target com-
pounds. The synthesized compounds were confirmed by
(EC50).
1
physicochemical and spectral means, and the MS, H NMR
13
In vitro HIV-RT kit assay
and C NMR spectral data were found in agreement with
the assigned molecular structures.
Inhibition of HIV-1 RT was performed using homopolymer
template/primer (poly(A).oligo(dT)15), biotin-dUTP, and RT
with detection by ELISA for quantifying polymerase activity.
The incorporated quantities of the biotin-dUTP into the tem-
plate represented the activity of HIV-1 RT. IC50 values corre-
sponded to the concentration of target compound required
to inhibit biotin-dUTP incorporation by 50%. The procedure
for assaying RT inhibition was performed as described in the
kit (Roche, Mannheim, Germany) protocol (26).
Anti-HIV activity
All the synthesized compounds were evaluated for their
anti-HIV activity and cytotoxicity in MT-4 cell cultures using
the MTT assay. The used virus strains were WT HIV-1
(III ), mutant HIV-1 (RES056) and HIV-2 (ROD) (the
B
RES056 strain was prepared by site-directed mutagenesis,
provided by the University of Leuven in Belgium, Rega
Institute, Institute of Microbiology and Immunology). The
FDA approved drugs nevirapine (NVP), delavirdine (DLV),
efavirenz (EFV), etravirine (ETR), and zidovudine (AZT) were
used as controls. The experimental results of 26 com-
pounds were presented in Table S1.
Results and Discussions
Chemistry
The synthetic route of the 26 target compounds, 5-((1-
(
substitutedbenzyl)piperidin-4-yl)amino)-3-(naphthalen-1-ylme-
thyl)-2H-1,2,6-thiadiazine 1,1-dioxide (Ba1-Ba13) and
-(2,4-dichlorobenzyl)-5-((1-(substitutedbenzyl)piperidin-4-yl)
It was apparent that most of the compounds showed mod-
3
B
erate inhibitory activity against WT HIV-1 (III ) with an EC50
amino)-2H-1,2,6-thiadiazine 1,1-dioxide (Bb1-Bb13), is
depicted in Scheme 1.
value ranging from 22 to 35 lM and low selectivity index (SI)
values of 3–5. However, none of the target compounds was
actually active against mutant HIV-1 and HIV-2.
For the synthesis of these novel derivatives, we choose the
readily available Meldrum’s acid as starting material. 1-
Naphthaleneacetic acid or 2,4-dichlorophenylacetic acid
was first reacted with Meldrum’s acid followed by cyclization
with sulfamide to afford key intermediates 5-substituted-
Furthermore, the preliminary SARs analysis of these novel
thiadiazine derivatives could be summarized as follows:
ꢀ
On the whole, 2,4-dichlorobenzyl at C-3 position of
the thiadiazine heterocycle was more preferable than
-naphthylmethyl according to the number of active com-
pounds and EC50 values.
1
,2,6-thiadiazine-1,1,3-trione Bam2 or Bbm2, respectively.
Then Bam2 or Bbm2 was treated with TsCl to yield car-
bonyl activated thiadiazines Bam3 or Bbm3, respectively.
1
4
-Tert-butoxycarbonylaminopiperidine was substituted in a
Scheme 1: Reagents and Conditions: (1)
-Naphthaleneacetic acid (or 2,4-
Dichlorophenylacetic acid), CDI, THF, 0 °C,
1
2
1
0 min, then 50 °C, 5 h; (2) H
10 °C, 1 h; (3) TsCl, TEA, DMAP, MeCN,
CO , NaI, TBAI, DMF, 50 °C,
2 h; (5) TFA, DCM, rt, 10 h; (6) MeCN,
TEA, reflux, 48 h.
2 2 2
NSO NH ,
rt, 24 h; (4) K
1
2
3
4
Chem Biol Drug Des 2014

Disubstituted Thiadiazine Derivatives as HIV-1 NNRTIs
ꢀ
Both series of compounds had a similar activity profile.
nitrogen at the 6 position of 1,2,6-thiadiazine as well as the
NH group linked to the 5-position of the heterocycle could
form ternary hydrogen bonds with Lys101. In addition, the
hydrogen bond between NH at the 2 position of 1,2,6-thi-
adiazine and Glu138 could also be observed. Furthermore,
the aryl 1-naphthyl or 2,4,-dichlorophenyl which may form
p–p interactions with the hydrophobic amino acids locates
at the aromatic-rich binding pocket surrounded by the aro-
matic acids Tyr188, Phe227, and Trp229. The 4-aminopipe-
ridinyl-linked benzyl of the compounds extends to the
solvent/protein interface, which was surrounded by Pro235,
Pro236, and Val106. The nitryl of compounds Ba8 and Bb8
may form additional interactions with surrounding amino
acids so that they display the most potent activity against
HIV-1. Above all, the binding mode of representative com-
pounds Ba8 and Bb8 suggested that they could interact
with the key amino acids in a favorable conformation, which
is consistent with that of TMC125.
For instance, as illustrated by Ba1, Ba7, Bb1, and Bb7,
the 2,6-dichlorobenzyl or 2,4,6-trimethylbenzyl linked with
the 4-aminopiperidine was unfavorable for the activity. In
addition, Ba8 and Bb8 bearing 4-NO
were the most potent target compounds, with an EC50 of
2
2
substituted benzyl
5.26 and 22.94 lM, and SI of 4 and 4, respectively.
ꢀ
2-cyano and 4-cyano substituted benzyl were favorable
for anti-HIV activity while 3-cyano was unfavorable for the
Ba series compounds. In contrast, cyano substituted at
different positions of benzyl exhibited similar activity in the
Bb series.
ꢀ
It is also interesting to note that replacement of 4-bromo
on the benzyl present in Ba10 and Bb10 with 4-fluoro
yielded compounds Ba9 and Bb9, respectively, which
showed slightly decreased potency and cytotoxicity.
ꢀ
2-Methyl substituted benzyl was preferable, while 2,4,6-
trimethyl substituted benzyl resulted in total inactivity.
Conclusions
ꢀ
Regarding the cytotoxicity, 4-SO Me substituted benzyl
derivatives always displayed the lowest cytotoxicity.
Taken together, based on the bioisosterism and molec-
ular hybridization strategies in drug design, a novel
series of 4-aminopiperidinyl-linked 3,5-disubstituted-
2
1
,2,6-thiadiazine-1,1-dione derivatives were designed,
What not allow to ignore is that, the potencies of bioactive
molecules are also closely related to their physicochemical
properties including aqueous solubility and cell membrane
permeability, which will be fully taken into account in fur-
ther optimizations.
synthesized and evaluated as HIV-1 NNRTIs. Most
of the compounds displayed moderate inhibitory activ-
ity against HIV-1 with EC50 values ranging from 22
to 35 lM. Furthermore, the preliminary SAR of these
compounds based on the anti-HIV-1 activity has been
discussed. On the whole, the compounds pre-
sented in this paper do not exhibit the desired
characteristics as described above, that is, high
potency and improved resistance profile. Still, the
molecular modeling study further gave insight into
important interactions occurring between NNRTIs bind-
ing site and thiadiazines which provides useful informa-
tion for design of potent inhibitors with this heterocyclic
motif.
HIV-1-RT inhibitory activity evaluation
To further define the mechanism of action of our
newly designed compounds, Ba8 and Bb8, the best
compound from each series were evaluated for their
HIV-RT inhibitory activity using the HIV-RT kit assay. As
shown in Table S2, compound Ba8 and Bb8 exhib-
ited inhibition activity against HIV-1 RT with IC50 value
of 3.15 and 1.52 lM, respectively, comparable to
those of reference drugs NVP (IC50 = 2.38 lM) and
ETR (IC50 = 0.13 lM). It is reasonable that the newly
synthesized 1,2,6-thiadiazine NNRTIs probably target
HIV-1 RT.
Acknowledgments
The financial support from the National Natural Science
Foundation of China (No. 81273354, 81102320), Key
Project of NSFC for International Cooperation (No.
81420108027, 30910103908), Research Fund for the
Doctoral Program of Higher Education of China (No.
20110131130005), Natural Science Foundation of Shan-
dong Province (ZR2009CM016) and KU Leuven (GOA
10/014) are gratefully acknowledged.
Molecular modeling study
To predict the binding mode and validate the rationaliza-
tion of novel designed thiadiazine derivatives, representa-
tive compounds Ba8 and Bb8 were docked into the
NNRTIs binding pocket (NNIBP) of WT HIV-1 RT using
SYBYL-X 1.1, and the docking results were shown by
PyMOL.
The molecular modeling analysis demonstrated that com-
pounds Ba8 and Bb8 exhibit similar binding mode with
TMC125 and bind to the RT in a ‘U-shape’ (Figure S3).
According to the binding mode, the oxygen of 1-sulfuryl, the
Conflict of Interest
The authors declare no conflict of interest.
Chem Biol Drug Des 2014
5

Liu et al.
References
11. Lin Y., Liu X., Yan R., Li J., Pannecouque C., Witv-
rouw M., De Clercq E. (2008) Synthesis and anti-HIV
evaluation of novel 1,3-disubstituted thieno[3,2-c]
[1,2,6]thiadiazin-4(3H)-one 2,2-dioxides(TTDDs). Bioorg
Med Chem;16:157–163.
1
. Asahchop E., Wainberg M.A., Sloan R.D., Tremblay
C.L. (2012) Antiviral drug resistance and the need for
development of new HIV-1 reverse transcriptase
inhibitors.
000–5008.
Antimicrob
Agents
Chemother;56:
12. Liu X., Yan R., Wang Y., Zhan P., De Clercq E.,
Pannecouque C., Witvrouw M., Molina M.T., Vega S.
(2008) Synthesis and anti-HIV activity evaluation of
novel 2,4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihy-
dro-pyrazolo-[4,5-e][1,2]thiadiazines. Arch Pharm;341:
216–222.
13. Zhan P., Yan R., Liu X., Pannecouque C., Witvrouw
M., De Clercq E., Molina M.T., Vega S. (2011)
Synthesis and anti-HIV activity of novel 2,4-disubsti-
tuted-7-methyl-1,1,3-trioxo-2H,4H-pyrazolo[4,5-e][1,2,4]
thiadiazine derivatives. Drug Discov Ther;5:279–285.
14. Zhan P., Liu X., De Clercq E. (2008) Recent advances
in antiviral activity of benzo/heterothiadiazine dioxide
derivatives. Curr Med Chem;15:1529–1540.
15. Rotili D., Tarantino D., Artico M., Nawrozkij M.B.,
Gonzalez-Ortega E., Clotet B., Samuele A., Este J.A.,
Maga G., Mai A. (2011) Diarylpyrimidine-di-
hydrobenzyloxopyrimidine hybrids: new, wide-spec-
trum anti-HIV-1 agents active at (sub)-nanomolar level.
J Med Chem;54:3091–3096.
16. Sun P., Chen Y., Yu G. (2010) A process for preparing
R-beta-amino phenylbutyric acid derivatives. In
WO2010009630A1.
5
2
. Mugnaini C., Alongi M., Togninelli A., Gevariya H.,
Brizzi A., Manetti F., Bernardini C., Angeli L., Tafi A.,
Bellucci L. (2007) Dihydro-alkylthio-benzyl-oxopyrimi-
dines as inhibitors of reverse transcriptase: synthesis
and rationalization of the biological data on both wild-
type enzyme and relevant clinical mutants. J Med
Chem;50:6580–6595.
. Nawrozkij M.B., Rotili D., Tarantino D., Botta G., Erem-
iychuk A.S., Musmuca I., Ragno R., Samuele A., Zanoli
S., Armand-Ug oꢀ n M. (2008) 5-Alkyl-6-benzyl-2-(2-oxo-
3
2-phenylethylsulfanyl) pyrimidin-4 (3 H)-ones, a series
of anti-HIV-1 agents of the Dihydro-alkoxy-benzyl-oxo-
pyrimidine family with peculiar structure- activity rela-
tionship profile. J Med Chem;51:4641–4652.
. Mai A., Artico M., Rotili D., Tarantino D., Clotet-Codina
I., Armand-Ug oꢀ n M., Ragno R., Simeoni S., Sbardella
G., Nawrozkij M.B. (2007) Synthesis and biological
properties of novel 2-aminopyrimidin-4 (3 H)-ones
highly potent against HIV-1 mutant strains. J Med
Chem;50:5412–5424.
. Miller C., Crain J., Tran B., Patel N. (2011) Rilpivirine: a
new addition to the anti-HIV-1 armamentarium. Drugs
Today;47:5–15.
4
5
17. Iwataki I., Makisawa S., Saito K. (1986) Production of
heterocyclic compound. In JPS6242978.
6
. Tian Y., Rai D., Zhan P., Pannecouque C., Balzarini J.,
De Clercq E., Liu H., Liu X. (2013) Design, synthesis,
and biological evaluation of novel 3, 5-disubstituted-1,
18. Schindler W., Schwiebacher E. (2009) Transparent
polumer mixtures which contain alkoxysilane-termi-
nated polymers. In US20090156737A1.
2
, 6-thiadiazine-1, 1-dione derivatives as HIV-1 NNR-
19. Sato I., Morihira K., Inami H., Kubota H., Morokata T.,
Suzuki K., Hamada N., Iura Y., Nitta A., Imaoka T.
(2008) Synthesis and structure–activity relationships of
N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benza-
mide derivatives as novel CCR3 antagonists. Bioorg
Med Chem;16:144–156.
20. Nguyen T., Sakasegawa Y., Doh-ura K., Go M.-L.
(2011) Anti-prion activities and drug-like potential of
functionalized quinacrine analogs with basic phenyl
TIs. Chem Biol Drug Des;82:384–393.
7
. Yu M., Li Z., Liu S., Fan E., Pannecouque C., De
Clercq E., Liu X. (2011) Synthesis and biological evalu-
ation of 6-substituted 5-Alkyl-2-(phenylaminocarbon-
ylmethylthio) pyrimidin-4 (3H)-ones as potent HIV-1
NNRTIs. ChemMedChem;6:826–833.
. Zhang L., Zhan P., Chen X., Li Z., Xie Z., Zhao T., Liu
H., De Clercq E., Pannecouque C., Balzarini J. (2014)
Design, synthesis and preliminary SAR studies of novel
N-arylmethyl substituted piperidine-linked aniline deriva-
tives as potent HIV-1 NNRTIs. Bioorg Med
Chem;22:633–642.
8
residues at the 9-amino position. Eur
J
Med
Chem;46:2917–2929.
21. Beard R.L., Vuligonda V., Duong T.T., Vu T., Donello
J.E., Garst M.E., Rodrigues G.A. (2009) Bicyclic com-
pounds having activity at the CXCR-4 receptor. In
WO2009143058A1.
22. Dhanoa D.S., Becker O., Noiman S., Cheruku S.R.,
Marantz Y., Shachem S., Heifetz A., Inbal B., Kesavan
V., Bar-haim S., Reddy S.A., Melendez R.E., Sharad-
endu A., Chen D. (2004) New piperidinylamino-thieno
[2,3-d]pyrimidine compounds. In WO2004089312A2.
23. Pannecouque C., Daelemans D., De Clercq E. (2008)
Tetrazolium-based colorimetric assay for the detection
of HIV replication inhibitors: revisited 20 years later.
Nat Protoc;3:427–434.
9
. Chen X., Li Y., Ding S., Balzarini J., Pannecouque C.,
De Clercq E., Liu H., Liu X. (2013) Discovery of piperi-
dine-linked pyridine analogues as potent non-nucleo-
side
HIV-1
reverse
transcriptase
inhibitors.
ChemMedChem;8:1117–1126.
1
0. Chen X., Liu X., Meng Q., Wang D., Liu H., De Clercq
E., Pannecouque C., Balzarini J., Liu X. (2013) Novel
piperidinylamino-diarylpyrimidine derivatives with dual
structural conformations as potent HIV-1 non-nucleo-
side reverse transcriptase inhibitors. Bioorg Med Chem
Lett;23:6593–6597.
6
Chem Biol Drug Des 2014

Disubstituted Thiadiazine Derivatives as HIV-1 NNRTIs
2
2
2
4. Popovic M., Sarngadharan M.G., Read E., Gallo
R.C. (1984) Detection, isolation, and continuous pro-
duction of cytopathic retroviruses (HTLV-III) from
patients with AIDS and pre-AIDS. Science;224:497–
Figure S2. Structure-based design of target compounds.
Figure S3. (A) Molecular docking of Ba8 (cyan) into the
allosteric site of wt HIV-1 RT; (B) The structural overlap of
the docking pose of compound Ba8 (cyan) and TMC125
(yellow); (C) Molecular docking of Bb8 (orange) into the
allosteric site of wt HIV-1 RT; (D) The structural overlap of
the docking pose of compound Bb8 (orange) and
TMC125 (yellow). (PDB code: 3MEC. The docking was
performed using SYBYL-X 1.1, and the docking results were
shown by PYMOL. The hydrogen bonds are shown in red or
yellow dashed lines).
500.
5. Clavel F., Guetard D., Brun-Vezinet F., Chamaret S.,
Rey M.-A., Santos-Ferreira M., Laurent A.G., Dauguet
C., Katlama C., Rouzioux C. (1986) Isolation of a new
human retrovirus from West African patients with AIDS.
Science;233:343–346.
6. Roche Diagnostics GmbH, Roche Applied Science
(2010) Reverse Transcriptase Assay, Colorimetric.
Mannheim, Germany: Roche Diagnostics GmbH, Roche
Applied Science. http://lifescience.roche.com/shop/
products/reverse-transcriptase-assay-colorimetric.
Table S1. Anti-HIV activity, cytotoxicity and selectivity indi-
ces of target compounds.
Table S2. In vitro recombinant HIV-1 RT inhibitory assay
of representative compounds Ba8, Bb8 and reference
drugs NVP, ETR.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Representative analogues of the DABO and
DAPY derivatives.
Chem Biol Drug Des 2014
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