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

Article abstract of DOI:10.1111/cbdd.12160

On the basis of structural features, binding mode, and structure-activity relationship studies of two pyrimidine-derived non-nucleoside reverse-transcriptase inhibitors, DABOs, and diaryl pyrimidines, a novel class of 1,2,6-thiadiazine-1,1-dione derivatives were rationally designed using the strategies of bioisosterism and molecular hybridization, synthesized, and evaluated for their anti-HIV activity in MT4 cell cultures. Three compounds were found to have moderate activity against HIV-1 replication with EC₅₀ values ranging from 23 to 32 μm. To further confirm the binding target, compound IIg was selected to conduct an HIV-1 reverse-transcriptase inhibitory assay. In addition, preliminary structure-activity relationship analysis among the newly synthesized compounds was discussed, and the binding mode of the active compound IIg was rationalized by molecular docking and physicochemical studies.

Full text of DOI:10.1111/cbdd.12160

Received Date : 07-Feb-2013
Revised Date : 29-Mar-2013
Accepted Date : 23-Apr-2013
Article type
: Research Article
Design, synthesis and biological evaluation of novel
3,5-disubstituted-1,2,6-thiadiazine-1,1-dione derivatives as HIV-1
NNRTIs
Ye Tian1, Diwakar Rai1, Peng Zhan1, Christophe Pannecouque2, Jan Balzarini2, Erik De
Clercq2, Huiqing Liu3, Xinyong Liu1
1 Department of medicinal Chemistry, Key Laboratory of Chemical Biology (Educational Ministry
of China), School of Pharmaceutical Sciences, Shandong University, No.44 Wenhuaxi Road, Jinan
250012, China
2Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
3Institute of Pharmacology, School of Medicine, Shandong University, 44, West Culture Road,
250012 Jinan, Shandong, PR China
*Phone: +86-531-88380270. Fax: +86-531-88382731. E-mail: xinyongl@sdu.edu.cn.
Abstract
On the basis of structural features, binding mode and SAR studies of two
pyrimidine-derived
NNRTIs,
DABOs
and
DAPYs,
a
novel
class
of
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an 'Accepted Article', doi: 10.1111/cbdd.12160
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1,2,6-thiadiazine-1,1-dione derivatives were rationally designed using the strategies of
bioisosterism and molecular hybridization, synthesized and evaluated for their anti-HIV
activity in MT4 cell cultures. Three compounds were found to have moderate activity against
HIV-1 replication with EC50 values ranging from 23 to 32 µM. In order to further confirm the
binding target, compound IIg was selected to conduct an HIV-1 RT inhibitory assay. In
addition, preliminary SAR analysis among the newly synthesized compounds was discussed
and the binding mode of the active compound IIg was rationalized by molecular docking and
physicochemical studies.
Key words HIV-1, RT, NNRTIs, 1,2,6-thiadiazine-1,1-dione, synthesis, activity assay
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been widely used
in the clinic for the treatment of AIDS patients, owing to their potent activity, high selectivity
and relatively low toxicity. However, NNRTI therapy suffers from the emergence of
drug-resistant viral strains due to rapidly emerging mutations of the amino acid residues in
1
the binding pocket of RT . Therefore, further development of novel NNRTIs with high
potency and improved resistance resilience profiles is essential for the successful application
of NNRTIs in drug combination regimens of anti-HIV therapy .
DABO (2-Alkoxy-6-benzyl-3,4-dihydro-4-oxopyrimidines) and DAPY (Diaryl
pyrimidines) analogues represent two important and promising classes of HIV-1 NNRTIs
(Fig. 1). They have very potent activities against not only wild-type virus but also against
single or even double mutant strains 2-6. Recently, two compounds belonging to the DAPY
series, viz. TMC125 (etravirine) and TMC278 (rilpivirine), have been approved by the FDA
for the treatment of AIDS patients 7.
Figure 1. Representative analogues of the DABO and DAPY series
As part of our research interest on these two pyrimidine-bearing NNRTIs 8-11, based on
their structural features, binding mode and SAR studies 8, 10, 12, we made core refinements and
several modifications on the substituents of the DABO series trying to obtain improved
anti-HIV activity (Fig. 2):
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(i) Nucleus: According to the bioisosterism of drug design, we replaced the
4-oxopyrimidine by a 1,2,6-thiadiazine-1,1-dione ring with an attempt to form additional
hydrogen bonds between the ligand and amino acids of RT.
(ii) Substituents at the 3-position: Inspired by the concept of molecular hybridization, we
adopted the groups which are similar to the right ‘wing’ of DAPYs (substituted anilines) to
keep the key structural requirements that were responsible for highly potent enzyme
inhibition. We also used those typical groups of DABOs (various substituted benzylamines,
phenylethylamines and aliphatic amines) in order to explore the effects of the linker and
steric, electrical, hydrophobic factors.
(iii) Substituents at the 5-position: Benzyl and 1-naphtylmethyl were used as
substituents at the C-5 position of thiadiazine to investigate the effect of π-π interaction with
Tyr181, Tyr188 and, especially, the hydrophobic interaction with the highly conserved
Trp229.
To achieve our goal, two novel series of 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione
derivatives were designed, synthesized and evaluated for their anti-HIV activities in
MT4-cells using the MTT assay.
Figure 2. Structure-based biosterism design of 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione
derivatives
Materials and Methods
Chemistry
All melting points (mp) were determined on a micromelting point apparatus and are
uncorrected. Infrared spectra (IR) were recorded with a Nexus 470FT-IR Spectro-meter. Mass
spectra were taken on a LC Autosampler Device: Standard G1313A instrument by
electrospray ionization. High-resolution mass spectra were conducted by LTQ Orbitrap Mass
1
Spectrometer. H-NMR spectra were obtained on a Brucker Avance-600 NMR-spectrometer
in the indicated solvents. Chemical shifts are reported in δ units and TMS as an internal
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reference. The 13C NMR spectra were recorded on a Brucker Avance-600 NMR-spectrometer
and are 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.
General
synthetic
procedure
for
compounds
&
6-H-5-benzyl-3-chloro-1,2,6-thiadiazine-1,1-dione
(T1e)
6-H-5-(naphthalen-1-ylmethyl)-3-chloro-1,2,6-thiadiazine-1,1-dione (T2e)
13
2,2-Dimethyl-5-(2-phenylacetyl)-1,3-dioxane-4,6-dione (T1c).
To a solution of
Meldrum’s acid (T1b, 5.45 g, 37.81 mmol, 1 eqv.) and pyridine (6.2 mL) in dichloromethane
(70 mL) at 0 °C, phenyl acetyl chloride (T1a, 5.0 mL, 37.81 mmol, 1 eqv.) was added
dropwise. The solution was stirred for another 30 min at 0 °C, and then warmed slowly and
allowed to reach room temperature within overnight stirring. The reaction mixture was
washed with 10% aqueous HCl (2×50 mL) and H2O (50 mL). The organic phase was dried
(MgSO4), filtered and concentrated under reduced pressure. 7.54 g crude white residue was
obtained with a yield of 76%.
2,6-2H-5-benzyl-1,2,6-thiadiazine-1,1,3-trione (T1d) 14: 1.31 g T1c (5 mmol, 1.25 eqv.)
and 0.38 g sulfamide (4 mmol, 1 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 the oil bath and
stirred for 1.5 hours. After cooling to room temperature, the reaction mixture was dissolved in
30 mL ethyl acetate, and extracted by sodium bicarbonate solution at a pH of 8~9 for three
times. The aqueous phases were combined, and then acidified with 2N diluted hydrochloric
acid and adjusted to pH 3~4. Then again extracted with ethyl acetate (30mL) for three times.
Further, organic layer was dried (MgSO4), filtered and concentrated to afford 0.86 g crude
1
yellow solid with yield of 90%. H-NMR (DMSO-d6) δppm: 3.66 (s, 2H, CH2), 5.32 (s, 1H,
C=CH), 7.27-7.36 (m, 5H, benzene), 11.99 (br, 1H, NH), 12.81 (br, 1H, NH).
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6-H-5-benzyl-3-chloro-1,2,6-thiadiazine-1,1-dione (T1e) 15: 5.1g T2d (21.4 mmol，1
eqv.) and 0.83 mL triethylamine (40.7 mmol, 1.9 eqv.) were dissolved in 25 mL acetonitrile.
To the solution, 10.7 mL of phosphorus oxychloride (115.6 mmol, 5.4 eqv.) was added
dropwise in an ice bath condition. Then heated to 80 °C in an oil bath and stirred for 8~10
hours under nitrogen atmosphere. On cooling to room temperature, 5 g of crushed ice was
added to the reaction mixture . Subsequently the mixture was extracted with ethyl acetate (30
mL) for three times. The organic layer was dried (MgSO4), filtered and concentrated. A red
brown oil (3.3 g) was obtained after silica gel column chromatography using PE/EA as eluent
1
with a yield of 60%. H-NMR (DMSO-d6) δppm: 3.71 (s, 2H, CH2), 5.91 (s, 1H, C=CH),
7.28-7.36 (m, 5H, benzene), 9.61 (br, 1H, NH); EI-MS: 257.1 [M+H].
2,2-dimethyl-5-(2-(naphthalen-1-yl)acetyl)-1,3-dioxane-4,6-dione (T2c): To a well
stirred mixture of 1.86 g commercially available phenylacetyl chloride (T2a) (10 mmol, 1
eqv.) and 10 ml of tetrahydrofuran at room temperature was slowly added 1.78 g of
carbonyldimidazole (11 mmol, 1.1 eqv.). Then the temperature was raised up to 50 °C. The
reaction mixture was stirred for 3 hours after the addition of 1.59 g of Meldrum’s acid (11
mmol, 1.1 eqv.). Subsequently, water (10 mL) and CH2Cl2 (14 mL) were added to the
reaction mixture, then the water phase was acidified with 2N diluted hydrochloric acid to pH
2~3. Consequently, organic layer was washed with 0.1N aqueous HCl and H2O for three
times, dried (MgSO4), filtered and concentrated under reduced pressure to afford 2.63 g of
crude yellow solid with a yield of 84%.
2,6-2H-5-(naphthalen-1-ylmethyl)-1,2,6-thiadiazine-1,1,3-trione (T2d): The procedure
was similar to the synthesis of T1d except for using of T2c as the reactant (yield: 93%).
1H-NMR (DMSO-d6) δppm: 3.79 (s, 2H, CH2), 4.37 (s, 1H, C=CH), 7.40-8.06 (m, 7H,
naphthalene), 9.57 (s, 1H, NH); EI-MS: 287.2 [M-H].
6-H-5-(naphthalen-1-ylmethyl)-3-chloro-1,2,6-thiadiazine-1,1-dione
(T2e):
The
procedure was similar to the synthesis of T1e except for using of T2d as the reactant (yield:
23%). 1H-NMR (DMSO-d6) δppm: 4.17 (s, 2H, CH2), 5.57 (s, 1H, C=CH), 7.43-8.03 (m, 7H,
naphthalene), 11.60 (br, 1H, NH); EI-MS: 305.3 [M-H].
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General synthetic procedure for target compounds (Ia-g, i-n, p, q, t-v &
IIa-w)
Chlorothiadiazine derivatives (T1e or T2e, 1 eqv.) and appropriate aniline, benzylamine
or phenylethyl amine (1 eqv.) were dissolved in an adequate amount of ethanol and refluxed
for 12~48 hours under continued stirring. After completion of the reaction as revealed by
TLC, the pure product was obtained by recrystallization from ethanol or by silica gel column
chromatography using PE/EA as eluant.
General synthetic procedure for target compounds (Ih, o, r, s, w and x)
T1e (1 eqv.) and appropriate aniline or benzylamine (1 eqv.) were dissolved in a
minimal amount of ethanol and then heated to 100-120 °C under stirring. After 5 min on
stirring, the solvent was evaporated and the pure products were obtained from the
residues by recrystallization with appropriate solvent or by silica gel column chromatography.
General synthetic procedure for target compounds (IIx and z)
T2e (1eqv) and appropriate aliphatic amine (1eqv) were dissolved in an adequate
volume of ethanol and refluxed overnight under stirring. After concentration, the residue was
dissolved in dichloromethane, washed with water for three times, dried (MgSO4) and
concentrated again. Title compounds were obtained by silica gel column chromatography.
Synthetic procedure for target compound (IIy)
T2e (1 eqv.), dimethylamine hydrochlorate (2 eqv.) and anhydrous K2CO3 (2.4 eqv.)
were dissolved in adequate DMF and refluxed overnight under stirring. After addition of
water, extracted with ethyl acetate. The combined organic phases were washed with water for
three times, dried (MgSO4) and concentrated. Title compounds were obtained by silica gel
column chromatography.
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Anti-HIV activity Evaluation
In vitro Anti-HIV assay
The evaluation of target compounds for their activity against HIV-1 strain IIIB and
HIV-2 strain ROD in MT-4 cells was performed using the MTT method as previously
described 16. Stock solutions (10 × final concentration) of test compounds were added in
25-μl 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 five-fold
dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays by
adding 100 µl medium to the 25 µl stock solution and transferring 25 µl of this solution to
another well that contained 100 µl medium using a Biomek 3000 robot (Beckman
instruments, Fullerton, CA). Untreated control HIV- and mock-infected cell samples were
included for each sample.
17
18
HIV-1(IIIB) or HIV-2 (ROD) stock (50 μL) at 100-300 CCID50 (50% cell culture
infectious dose-50%) or culture medium was added to either the infected or mock-infected
wells of the microtiter plate. Mock-infected cells were used to evaluate the effect of test
compound on uninfected cells in order to assess the cytotoxicity of the test compound.
Exponentially growing MT-4 cells were centrifuged for 5 min 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 viability of the mock-infected MT-4 cells 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).
In vitro HIV-RT kit assay
Inhibition of HIV-1 RT was performed by using homopolymer template/primer linked to
a microplate, biotin-dUTP and RT with detection by ELISA for quantifying expression. The
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incorporated quantities of the biotin-dUTP into the template represented the activity of HIV-1
RT. IC50 values corresponded 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) protocol19.
Result and Discussion
Chemistry
The synthetic route of the target compounds, 6-H-5-benzyl-3-(substituted
amino)-1,2,6-thiadiazine-1,1-dione (Ia-x) and 6-H-5-(naphthalen-1-ylmethyl)-3-(substituted
amino)-1,2,6-thiadiazine-1,1-dione (IIa-z) is depicted in Scheme 1.
For the preparation of the two designed series, we chose phenylacetyl chloride (T1a)
and 1-naphthlacetic acid (T2a) as the starting material, respectively. T1a or T2a were first
reacted with Meldrum's acid followed by cyclization with sulfonamide, yielded key
13-15
intermediates 5-substituted-1,2,6-thiadiazine-1,1,3-trione respectively for both series
.
Then, T1d and T2d were subjected to chlorination using POCl3, and further, it participated in
a nuclephilic substitution reaction at its C-3 position with different aromatic or aliphatic
amines, and afforded 50 title compounds Ia-Ix, IIa-IIz . The synthesized compounds were
characterized by physicochemical and spectral means and the IR, MS, 1H-NMR spectral data
were found in agreement with the assigned molecular structures. HRMS and 13C-NMR data
are reported for compounds Ia and IIa as representatives to confirm their skeleton.
Sheme 1. Reagents and conditions: (i)Py, CH2Cl2; (ii)CDI, THF; (iii)Sulfuric diamide;
(iv)POCl3, CH3CN; (v)NH2R or NHR1R2, EtOH or K2CO3/DMF.
Anti-HIV-1 (IIIB) activity
All the synthesized compounds were evaluated for their antiviral activity based on the
potency of inhibition of the replication of wild-type HIV-1 (IIIB), HIV-2 (ROD) strains in
MT4 cell cultures using the MTT assay.
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The FDA approved drugs nevirapine (NVP) and delavirdine (DLV) were used as
controls. The experimental results of 50 compounds are presented in Table 1. On the whole,
these compounds did not demonstrate excellent anti-HIV activity as we expected in
comparison to the controls. Then, 3 of them, IIe-IIg, showed moderate potency against
wild-type HIV-1 (IIIB) and satisfactory selectivity index (SI) values of 5-6 be similar to other
NNRTIs, none of these compounds were active against HIV-2.
Further, the structure-activity relationship (SAR) was derived from these results.
Compound IIe with a F atom at the 4-position of the phenyl ring showed activity (EC50 = 32
µM, SI = 5), and the replacement of F by Cl (IIf, EC50 = 26 µM) or Br (IIg, EC50 = 23 µM )
kept the moderate potency. The anti-HIV activities for these three compounds might be
correlated with the electron withdrawing effects and hydrophobic property of the halogen
atoms. Interestingly, the introduction of the CN group at the 4-position of the phenyl ring led
to a significant loss of activity (IIh, EC50: 250 µM), indicating that the CN group is crucial
for the DAPY series but not for this novel class. Besides, the compounds with a benzylamino,
phenylethyl amino or aliphatic amino at 3 position did not show the desired potency against
HIV replication.
As shown in Table 1, naphtylmethyl substituted thiadiazines IIe-IIg have a better
antiviral activity in comparison to their benzyl counterparts Ie-Ig. Thus, a preliminary finding
was obtained that the substitution of the 1-naphtylmethyl ring at the C-5 position of
1,2,6-thiadiazine-1,1-dione have a better interaction with the binding pocket by forming
tighter π-π stacking with Tyr181 and Tyr188 than the benzyl group.
Regarding the cytotoxicity, the CC50 of these compounds are, to a certain extent,
proportional to the EC50. For example, the I series has higher CC50 than II series; compounds
with an halogen atom on the right ‘wing’ are relatively more toxic (Br > Cl > F) while the
ones with CN or CONH2 have almost the highest CC50 in their series.
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Table 1. Inhibitory action of compounds Ia-Ix and IIa-IIz against HIV-1 and HIV-2
replication in MT-4 cells.
a
Compd
R1
EC50(µM)b
HIV-1 IIIB
CC50(µM)c
SId
HIV-2
ROD
>292
HIV-1
IIIB
HIV-2
ROD
<1
Phenyl
>292
>182
>268
>189
>255
>179
>163
>369
>286
>318
>318
>256
307 61
>177
>175
>179
>198
>191
>310
292 43
182 8.8
268 68
189 8.2
255
<1
Ia
Ib
Ic
Id
Ie
If
2-Chlorophenyl
>182
>268
>189
>255
>179
>163
>369
>286
>318
>318
>256
>250
>177
>175
>179
>198
>191
>310
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
><1
><1
<1
<1
<1
<1
<1
<1
<1
><1
2-Methoxyphenyl
3-Chlorophenyl
4-Fluorophenyl
<1
4-Chlorophenyl
179 13
163 2.5
369
<1
<1
4-Bromophenyl
Ig
Ih
Ii
4- Cyanophenyl
<1
4-Carbamoylphenyl
4-Sulfamoylphenyl
4-Methoxyphenyl
4-(Dimethylamino)phenyl
3,4-Difluorophenyl
3,4-Dimethylphenyl
3-Chloro-4-methylpheny
4-Chloro-3-fluorophenyl
2,3,4-Trifluorophenyl
2,4,6-Trimethylphenyl
3,4,5-Trimethoxyphenyl
286
<1
>318
><1
><1
<1
<1
<1
<1
Ij
>318
Ik
Il
256 70
250
Im
In
Io
Ip
Iq
Ir
Is
177 11
175 6.8
179 10
198 5.4
191 7.1
>310
<1
<1
<1
><1
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Benzyl
>238
>192
>300
>192
>209
>158
>154
>161
>161
32
>238
>192
>300
>192
>209
>158
>154
>161
>161
>158
>147
>140
>254
>245
>139
>165
>33
238 25
192 5.9
300 51
192 12
209 18
158 5.9
154 5.2
161 6.4
161 10
158 15
147 9.6
140 12
254 21
>245
<1
<1
<1
<1
<1
<1
<1
<1
<1
5
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
><1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
It
Iu
4-Methylbenzyl
4-Methoxybenzyl
Phenethyl
Iv
Iw
Ix
4-methoxyphenethyl
Phenyl
IIa
IIb
IIc
IId
IIe
IIf
2-Chlorophenyl
2-Methoxyphenyl
3-Chlorophenyl
4-Fluorophenyl
4-Chlorophenyl
26
6
4-Bromophenyl
23
6
IIg
IIh
4- Cyanophenyl
≥250
>245
>139
>165
>33
≥1
><1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
4-Carbamoylphenyl
4-Sulfamoylphenyl
4-Methoxyphenyl
4-(Dimethylamino)phenyl
3,4-Difluorophenyl
3,4-Dimethylphenyl
4-Chloro-3-fluorophenyl
2,3,4-Trifluorophenyl
2,4,6-Trimethylphenyl
3,4,5-Trimethoxyphenyl
Benzyl
IIi
IIj
139 6.3
165 3.3
33 0.49
156 9.5
147 7.7
152 4.7
143 6.0
96 39
IIk
IIl
>156
>147
>152
>143
>96
>156
>147
>152
>143
>96
IIm
IIn
IIo
IIp
IIq
IIr
IIs
>204
>161
>204
>161
204 25
161 8.0
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4-Methylbenzyl
>144
>148
>141
>159
>218
>268
>195
0.24
>144
>148
>141
>159
>218
>268
>195
-
144 12
148 5.3
141 15
159 10
218 20
268 16
195 14
>15
<1
<1
<1
<1
<1
<1
<1
<1
<1
-
IIt
IIu
IIv
IIw
4-Methoxybenzyl
Phenethyl
<1
4-Methoxyphenethyl
<1
Cyclopropyl
<1
IIx
IIy
Methyl
<1
Ethyl
<1
IIz
-
-
>63
>303
NVP
DLV
0.14
-
>44
-
a R2 = H (For IIa-IIx), Methyl (For IIy), Ethyl (For IIz);
b EC50: concentration of compound required to achieve 50% protection of MT-4 cells against
HIV-1-induced cytopathic effect, as determined by the MTT method;
c CC50: concentration required to reduce the viability of mock-infected cells by 50%, as
determined by the MTT method;
d SI: selectivity index (CC50/EC50), and the SI values: >< 1 stands for ≥1 or <1 .
HIV-RT inhibitory activity evaluation
To further characterize the mechanism of action of our newely designed compounds, the
best compound of this class, IIg was evaluated as a representative compound with nevirapine
for their HIV-RT inhibitory activity using HIV-RT kit assay19. This assay is revealed (Table 2)
that compound IIg exhibit its inhibitory activity with an IC50 value of 103 μM, which is about
74 times greater than that of reference drug NVP (IC50 = 1.37μM). This difference just
corresponds to that in the data of their anti-HIV activity in MT-4 cells using the MTT assay.
Therefore, it indicates that the 1,2,6-thiadiazine-1,1-dione derivatives act as non-nucleoside
reverse transcriptase inhibitors. But its ability to bind with reverse transcriptase is insufficient,
that need to be improved by further modifications.
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Table 2. Activity of 1,2,6-thiadiazine-1,1-dione derivative IIg against HIV-1 RT
Compound
IIg
NVP
a
IC50
103μM
1.37μM
a
IC50: inhibitory concentration required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP)
incorporation into poly rA.dT by the HIV-1 RT by 50% (RT kit, Roche).
Molecular modeling study
Compound IIg, as a representative structure, was docked into the non-nucleoside
binding site (NNBS) of HIV-1 RT with the Dock module of Sybyl-X 1.1 to understand the
binding mode of our compounds. Three-dimensional coordinates of the NNBS were taken
from the crystal structure of RT/TMC125 (PDB code: 3MEC) and RT/MKC442 (PDB code:
1RT1). The modeling analysis suggested that compound IIg binds to HIV-1 RT in an U-shape
and its conformation have a close similarity to those of TMC125 and MKC442 but in two
opposite orientations 20, 21(Fig. 3). According to the first mode of action (Fig. 3a), the N atom
at 2 position of the 1, 2, 6-thiadiazine ring as well as the NH group linked to the 3-position of
the phenyl ring could make the polar contacts with Lys101. Further, results showed that the
naphthyl ring of compound IIg points to the aromatic-rich binding pocket surrounded by the
aromatic Tyr188, Phe227 and Trp229 through π–π interactions, while the 4-bromophenyl ring
parallels to Tyr318. In contrast, from the second mode (Fig. 3b), these two aromatic moieties
are just going in the opposite direction. It is worth noting that one oxygen of the sulfone
shows two polar contacts with Lys101 and Tyr181, respectively. Thus, it appears that all these
interactions of compound IIg in two modes would favor the binding affinity with the pocket.
(a)
(b)
Figure 3. Binding mode of IIg with RT: (a) Model of IIg docked into the RT non-nucleoside
binding site (PDB code: 3MEC); (b) Model of IIg docked into the RT non-nucleoside binding
site (PDB code: 1RT1)
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Physicochemical Properties study
All the compounds showed some remarkable differences in their anti-HIV activity with
nevirapine and delavirdine as shown in Table 1. Furthermore, we determined the
physicochemical properties (logP, molecular weight, topological polar surface area, molecular
volume etc.) of the most active compound IIg as a representative structure, nevirapine and
TMC125
as
control
using
free
on-line
molinspiration
software
(http://www.molinspiration.com/) for compliance to the Lipinski’s rule of five 22.
The physicochemical studies (Table 3) suggested that compound IIg, nevirapine and
TMC125 followed most closely the Lipinski’s rule of five. Although, the compound IIg has
similarity to TMC125 in terms of molecular properties, but it could not show the excellent
inhibitory action against HIV-1 replication. There would be other factors, which might be
responsible for its moderate inhibition (EC50 = 23µM) against HIV-1.
Table 3. Physicochemical propertiesa of compound IIg, nevirapine and TMC125
Compd.
Acceptable
range
IIg
EC50(µM) nViol natoms miLogP MW
nON nOHNH nrotb TPSA
MV
(< 5)
(<500
Da)
<10
<5
(<140Ǻ)
23
1
1
0
27
28
20
5.115
5.027,
1.385
442.33
435.28
266.30
5
7
5
2
3
1
4
4
1
70.56
120.65
63.58
330.203
335.95
TMC125
0.003b, 23
0.24
Nevirapine
236.618
a nViol = number of violations; natoms = no. of atoms; miLog P = molinspiration predicted Log P; MW =
molecular weight; nON - no. of hydrogen bond acceptors; nOHNH = no. of hydrogen bond donors; nrotb =
no. of rotatable bond; TPSA= topological polar surface area; MV = molar volume.
b The data were obtained from the Rega Institute for Medical Research, KU Leuven, Belgium).
Conclusion
In brief, based on the bioisosterism and molecular hybridization strategies of drug
design, the first synthesis using a convenient and efficient method, as well as biological
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evaluation of two novel series of 3,5-disubstituted 1,2,6-thiadiazine-1,1-dione derivatives as
potential HIV-1 inhibitors is described. Among them, three compounds demonstrated
moderate inhibitory activity against HIV-1 with EC50 values ranging from 23 to 32 µM.
Further, from a SAR point of view, the anti-HIV activity is strongly dependent on the nature
of the ‘wings’ of the 1,2,6-thiadiazine-1,1-dione ring. The activity order of different
substituents was as follow: naphthalen-1-ylmethyl > phenylmethyl (at C5 position); and
4-Br-phenyl > 4-Cl-phenyl > 4-F-phenyl (at 3-position amino). The results of in vitro HIV-RT
kit assay for compound IIg have shown that RT may be the target of these compounds. The
docking and physicochemical studies also provide some valuable information for the
development of novel potent NNRTIs and will encourage us for the rational design of a next
generation of suitably substituted thiadiazine derivatives.
Supporting Information
Physical properties and spectrum data of target compounds are provided.
Acknowledgments
The financial support from the National Natural Science Foundation of China (NSFC No. 81273354, No.
81102320, No. 30873133, No.30772629, No. 30371686), Key Project of NSFC for International
Cooperation (No. 30910103908), Research Fund for the Doctoral Program of Higher Education of China
(No. 20110131130005, 20110131120037), Shandong Provincial Natural Science Foundation, China (No.
ZR2009CM016), Shandong Postdoctoral Innovation Science Research Special Program (No. 201002023),
China Postdoctoral Science Foundation funded project (No.20100481282, 2012T50584) and KU Leuven
(GOA 10/014) is gratefully acknowledged. We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the HIV assays.
The authors have declared no conflict of interest.
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This article is protected by copyright. All rights reserved.