Design, synthesis, anti-HIV evaluation and molecular modeling of piperidine-linked amino-triazine derivatives as potent non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.04.030

A novel series of piperidine-linked amino-triazine derivatives were designed, synthesized and evaluated for in vitro anti-HIV activity as non-nucleoside reverse transcriptase inhibitors on the basis of our previous work. Screening results indicated that most compounds showed excellent activity against wild-type HIV-1 with EC₅₀ values in low nanomolar concentration range (especially compound 6b3, EC₅₀ = 4.61 nM, SI = 5945) and high activity against K103N/Y181C resistant mutant strain of HIV-1 with EC₅₀ values in low micromolar concentration range. In addition, preliminary structure-activity relationship and molecular modeling of these new analogs were detailed in this manuscript.

Full text of DOI:10.1016/j.bmc.2012.04.030

Bioorganic & Medicinal Chemistry 20 (2012) 3856–3864
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, anti-HIV evaluation and molecular modeling
of piperidine-linked amino-triazine derivatives as potent non-nucleoside
reverse transcriptase inhibitors
Xuwang Chen a, Peng Zhan a, Xin Liu a, Ziheng Cheng b, Caicai Meng a, Siyuan Shao a,
Christophe Pannecouque c, Erik De Clercq c, Xinyong Liu a,
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012 Ji’nan, Shandong, PR China
b Marine College, Shandong University, Weihai Wenhua West Road, No. 180, Weihai 264209, Shandong, PR China
c Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of piperidine-linked amino-triazine derivatives were designed, synthesized and evaluated
for in vitro anti-HIV activity as non-nucleoside reverse transcriptase inhibitors on the basis of our previ-
ous work. Screening results indicated that most compounds showed excellent activity against wild-type
HIV-1 with EC50 values in low nanomolar concentration range (especially compound 6b3, EC50 = 4.61 nM,
SI = 5945) and high activity against K103N/Y181C resistant mutant strain of HIV-1 with EC50 values in
low micromolar concentration range. In addition, preliminary structure–activity relationship and molec-
ular modeling of these new analogs were detailed in this manuscript.
Received 5 March 2012
Revised 11 April 2012
Accepted 16 April 2012
Available online 21 April 2012
Keywords:
HIV-1
NNRTIs
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Piperidine
Triazine
Antiviral activity
SAR
Molecular modeling
1. Introduction
Among these structurally modified analogs, piperidine-linked ami-
nopyrimidine derivatives (Fig. 2) displayed promising potency
Acquired immune deficiency syndrome (AIDS) mainly caused by
human immunodeficiency virus type-1 (HIV-1), is one of the lead-
ing pandemic diseases worldwide. Because of the lack of vaccine,
the highly active antiretroviral therapy (HAART) regimen plays a
crucial role in the treatment of HIV-1 infection, however, drug resis-
tance and side effects often compromise clinical treatment.1 HIV-1
non-nucleoside reverse transcriptase inhibitors (HIV-1 NNRTIs)
have become an indispensable component of HAART, with unique
antiviral potency, high specificity and low cytotoxicity.2 Especially,
diarylpyrimidine (DAPY) derivatives, such as Dapivirine (TMC120),
Etravirine (TMC125, ETV) and Rilpivirine (TMC278) showed highly
potent activity against wild-type and NNRTI-resistant strains of
HIV-1 (Fig. 1),3,4 of which, TMC125 and TMC278 have been
approved by FDA, providing an additional treatment option for
HAART regimen. Further research of DAPY derivatives was focused
on diverse structural modifications, including the left wing,5–7 the
linker between the left benzene ring and the central pyrimidine
ring,8–10 the central pyrimidine core11–14 and the right wing.15,16
against wild-type and several important resistant mutant strains
of HIV-1, as well as enhanced water solubility to potentially melio-
rate the pharmacokinetic profile, which make it extensive concern
in DAPY analogs.
Recently, we designed and synthesized a novel series of DAPY
analogs with piperidine-substituted triazine moiety, which are
structurally similar to the piperidine-linked aminopyrimidine
derivatives. From biological assay, we had discovered some new
lead compounds to be potent and selective HIV-1 NNRTIs with
IC50 values at nanomoles ranges.17 Encouraged by this result and
continued to pursue our studies, we undertook a new round of
structural modification of piperidine-substituted triazine deriva-
tives, in which, amino group was introduced to the triazine ring,
similar to that observed in the structure of Etravirine (Fig. 2),
which may improve hydrogen bond interaction with amino acid
residues inside the binding pocket of HIV-1 NNRT.18 In addition,
polar hydrophilic substituents and heterocycle were introduced
to the right wing (Fig. 2) that is oriented directly into the exterior
water through a rather small opening window in the protein/sol-
vent interface, which is thought to be playing an important role
in the capacity to inhibit HIV-1 replication and improve pharmaco-
⇑
Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0968-0896/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.030

X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
3857
CN
CN
CN
NH
CN
NH
CN
NH
O
N
HN
N
HN
N
N
Br
N
N
NH2
Etravirine (TMC125,ETV)
Dapivirine (TMC120)
Rilpivirine (TMC278)
Figure 1. The structures of DAPY derivatives.
R2
CN
O
N
Protein/solvent
interface
Introducing polar hydrophilic
substituents or heterocycle
Ar
N
NH
N
Protein/solvent
interface
N
R1
Piperidine-linkedaminopyrimidines
HN
N
NH
CN
CN
N
N
Hydrogen bond
interaction domain
NH2
Newly designed compounds
O
N
NH
N
Br
Hydrogen bond
interaction domain
NH2
Approved drug by FDA
Figure 2. The design of piperidine-linked amino-triazine derivatives.
kinetic properties.19 Herein, we will detailedly report the synthesis,
2.2. Biological activity
anti-HIV evaluation, preliminary structure–activity relationship
(SAR) and molecular modeling of the newly designed piperidine-
linked amino-triazine analogs.
The newly synthesized piperidine-linked amino-triazine deriva-
tives were tested for their anti-HIV activity and cytotoxicity in MT-
4 cells infected by wild-type HIV-1 (IIIB) and the double mutant
strains RES056 (K103N+Y181C) along with HIV-2 (ROD) according
to the MTT method reported previously.17,23–25 FDA-approved
drugs: Nevirapine (NVP), Delavirdine Mesylate (DLV), Efavirenz
(EFV), Zidovudine (Azidothymidine, AZT), Dideoxycitidine (DDC)
and Etravirine (ETV)26 were used as reference drugs. The biological
evaluation results were interpreted in EC50 (anti-HIV activity), CC50
2. Results and discussion
2.1. Chemistry
The synthesis route of the target compounds 6a1–6a8, 6b1–6b5
and 6c1–6c5 was shown in Scheme 1. The intermediate 6-chloro-
N2-mesityl-N4-(1-Boc-piperidin-4-yl)-1,3,5-triazine-2,4-diamine (3)
was prepared from structurally symmetrical 2,4,6-trichloro-1,3,
5-triazine (1) by substitution reaction with 2,4,6-trimethylaniline
and 4-amino-1-Boc-piperidine successively in mild reaction condi-
tions.20 Treatment of intermediate 3 with ammonia under high
temperature in sealed reactor,21 aqueous methylamine and sodium
methoxide gave the intermediate 4a, 4b and 4c, respectively.22 Boc
group was removed in the presence of trifluoroacetic acid (TFA) at
room temperature to afford 5a, 5b and 5c, which were alkylated
at the N atom of piperidine, achieving the final compounds
6a1–6a8, 6b1–6b5 and 6c1–6c5, respectively. Both analytical and
spectral data of all the compounds are in full agreement with the
proposed structures.
values (cytotoxicity) and SI (selectivity index, given by the CC50
EC50 ratio) as shown in Tables 1 and 2.
/
As listed in Table 1, most compounds showed high activity
against wild-type HIV-1 with EC50 values in the range of 4.61–
20.43 nM and high SI values ranging from 297 to 5945. Compound
6b3 was the most potent compound (EC50 = 4.61 nM, SI = 5945),
superior to the reference drugs NVP, DLV, EFV, AZT and DDC, but
a little inferior to ETV against wild-type HIV-1. Introduction of –
NH2 at the triazine ring (6a1–6a5) improved the activity compared
to the –NHMe series (6b1–6b5) and –OMe series (6c1–6c5) except
the compound with 4-carbamoylphenyl group (6a3). Compared to
compound 6a6, compounds 6a4, 6a5, 6a7 and 6a8 showed more
potent activity due to the presences of polar hydrophilic substitu-

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X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
Boc
N
Boc
N
Cl
N
Cl
HN
N
R
NH
HN
N
Cl
b
HN
N
NH
c
a
N
N
N
N
N
N
N
N
Cl
Cl
2
Cl
3
1
4a
, R = NH2
4b, R = NHMe
4c, R = OMe
Ar
H
N
N
e
d
HN
N
R
NH
HN
N
NH
N
N
N
N
R
5a,
6a1-6a8,
R = NH2
R = NH2
5b, R = NHMe
5c, R = OMe
6b1-6b5, R = NHMe
6c1-6c5, R = OMe
Scheme 1. Reagents and conditions (a) K2CO3/THF; (b) NaHCO3 (5%aq)/THF; (c) (c1) NH3 (aq)/THF; (c2) NH2Me(aq)/NaHCO3/THF; (c3) Na/MeOH; (d) TFA/CH2Cl2; (e) K2CO3/
DMF.
ents at the para phenyl, such as sulfamoyl, methylsulfonyl, cyano
and nitro groups. Additionally, substitution of phenyl group (6a6)
with polar hydrophilic pyridyl group (6a1) was confirmed contrib-
uting to the anti-HIV-1 activity. Among the tested compounds,
compounds 6a2, 6b2 and 6c2 with 4-COOEt-Ph displayed reduced
potency against wild-type HIV-1 and increased cytotoxicity with
lower SI values, because the ethyl group is hydrophobic and could
not extend into the exterior water of the opening RT pocket.
In summary, introduction of –NH2 at the triazine ring and polar
hydrophilic groups at the right wing were favorable to improve-
ment of activity against wild-type HIV-1 indeed. Furthermore,
none of the newly synthesized compounds were active at subtoxic
concentration against HIV-2 (ROD) in MT-4 cells.
In the treatment of HIV-1 infection, drug resistance is still a key
cause of failure for their antiviral efficacy. There are many resistant
RT mutations of HIV-1, such as L100I, K101E, K103N, V106A, V179F,
Y181C, Y188L, G190A, F227C, L234I and Y318F. Among them,
K103N and Y181C resistant mutant strains are the most often
encountered mutations in the treatment of AIDS with HIV-1 NNRTIs
clinically.27 With the aim to define the resistance profiles of these
compounds, they were also assayed against the K103N/Y181C resis-
tant mutant strain of HIV-1 with the results illustrated in Table 2. As
the data indicated, both the absolute activity against the HIV-1 mu-
tants (EC50 values) and the relative activity (fold resistance) needed
to be taken into consideration of the potency. Most compounds dis-
played high activity against K103N/Y181C resistant mutant strain
of HIV-1 with EC50 values in low-micromolar concentration ranges.
6b2 and 6c2) displayed no activity against resistant mutant
strain of HIV-1.
2.3. Molecular simulation analysis
In view of piperidine-substituted triazine derivatives and piper-
idine-linked aminopyrimidine derivatives targeting at the site of
RT,15–17 we decided to investigate the binding mode of the newly
synthesized NNRTIs. Potent compounds 6a1 and 6b3 were docked
into non-nucleoside inhibitor binding pocket (NNIBP) of wild-type
HIV-1 RT (PDB code: 3M8Q) using the software Surflex-Dock SYB-
YL-X 1.1 and the docking results were showed by PyMOL 0.99. De-
fault parameters were used as described in the SYBYL-X 1.1
manual unless otherwise specified.
As shown in Figure 3, the new compounds 6a1 and 6b3 could
bind into the NNIBP similar with piperidine-linked aminopyrimi-
dine derivatives.15 Detailed analysis of the docking results re-
vealed the following notable features that may clarify the
mechanism of action of piperidine-substituted triazine deriva-
tives. (1) Mesityl moiety of compounds 6a1 and 6b3 fits into
the important hydrophobic sub-pocket, which is formed by aro-
matic amino acid residues Y181, Y188, F227 and W229, exhibit-
ing
compounds form two hydrogen bonds with backbone carbonyl
and backbone -amino of residue K101, similar to that observed
p
-p
interaction with these residues. (2) The new
a
in DAPY compounds. Additionally, the –NH2 and –NHMe groups
can form a hydrogen bond with amino acid E138 that may im-
prove stability of RT-inhibitor complex and the affinity between
RT and inhibitor. (3) The substituted piperidine sits between ami-
no acid Y318 and V106, and the polar hydrophilic substituents
(compound 6a1) and heterocycle (compound 6b3) is located to-
ward a solvent exposure region, contacting either side chain of
amino acid L234 or P236 (Fig. 3c). This binding model supports
our initial design hypothesis that introduction of –NH2 group at
the triazine ring may improve hydrogen bond interaction with
amino acid residues of the binding pocket, and polar hydrophilic
substituents and heterocycle point directly into the protein/sol-
vent interface.
Compounds 6b1 (EC50 = 0.55
and 6b4 (EC50 = 0.51 M with a 72.7-fold resistance ratio) were
more active than reference drugs NVP, DLV and EFV, however, they
were less potent than AZT and ETV (EC50 = 0.034 M with a
lM with a 76.4-fold resistance ratio)
l
l
15.5-fold resistance ratio). It is interesting to note that, compounds
with 4-SO2NH2-Ph group (6a4, 6b4 and 6c4) showed better potency
against resistant mutant strain of HIV-1 among each series (–NH2
series, –NHMe series and –OMe series), which could also be ob-
served in NNRTIs of other structural type, such as benzophenone
(GW678248)28 and triazolylthioacetanilide (VRX-480773)29
NNRTIs. However, compounds with 4-COOEt-Ph group (6a2,

X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
3859
Table 1
Activity and cytotoxicity against HIV-1 (wild-type, IIIB) and HIV-2 (ROD) strains in MT-4 cells
Ar
N
N
R
HN
NH
N
N
6a1-6a8, R = NH2
6b1-6b5,
R = NHMe
6c1-6c5, R = OMe
SIc
a
b
Compds
R
Ar
EC50
CC50 (lM)
HIV-1 IIIB (nM)
HIV-2 ROD (lM)
6a1
6a2
6a3
6a4
6a5
6a6
6a7
6a8
6b1
6b2
6b3
6b4
6b5
6c1
6c2
6c3
6c4
6c5
NVP
DLV
EFV
AZT
DDC
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHMe
NHMe
NHMe
NHMe
NHMe
OMe
OMe
OMe
OMe
OMe
Pyridin-4-yl
4-COOEt-Ph
4-CONH2-Ph
4-SO2NH2-Ph
4-SO2Me-Ph
Phenyl
4.90 2.40
11.55 0.51
20.43 19.73
5.75 2.64
5.55 1.48
10.25 0.98
7.87 1.41
5.66 3.13
7.25 1.19
16. 66 7.94
4.61 1.90
6.99 1.09
6.06 2.04
13.32 2.88
14.71 7.04
8.64 1.49
10.62 2.58
11.90 4.54
200.40 105.31
35.01 4.73
6.57 0.87
6.00 0.57
1394.28 50.22
2.2 0.4
>20.12
>5.91
>74.36
>19.85
>5.06
>23.59
>3.46
>3.26
>25.80
>4.95
>27.39
>14.83
>9.64
>30.91
>8.48
>33.43
>19.63
>13.45
20.12 8.10
5.91 1.37
74.36 18.94
19.85 4.52
5.06 2.49
23.59 3.40
3.46 1.58
3.26 1.18
25.80 1.15
4.95 0.17
27.39 2.34
14.83 2.49
9.64 3.12
30.91 2.23
8.48 2.03
33.43 8.98
19.63 2.67
13.45 3.48
>15.02
4108
512
3640
3451
911
2302
439
576
4-NO2-Ph
4-CN-Ph
Pyridin-4-yl
4-COOEt-Ph
4-CONH2-Ph
4-SO2NH2-Ph
4-SO2Me-Ph
Pyridin-4-yl
4-COOEt-Ph
4-CONH2-Ph
4-SO2NH2-Ph
4-SO2Me-Ph
3558
297
5945
2122
1590
2320
577
3869
1849
1130
>75
>1034
>964
P8295
>68
>36.19
>6.34
P49.77
>94.69
0.007 0.0008
1.67 0.25
>27.78
d26
ETV
27.78 11.53
12884
a
b
c
EC50: Concentration of compound required to achieve 50% protection of MT-4 cell against HIV-1-induced cytotoxicity, as determined by the MTT method.
CC50: Concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
SI: Selectivity index, the ratio of CC50/EC5H0IVÀ1IIIB
.
d
ETV: Etravirine. The data were obtained from the same laboratory (Rega Institute for Medical Research, K.U. Leuven, Belgium).
Overall, all these analyses were in accordance with the biologi-
MT-4 cells along with preliminary SAR analysis. Screening results
indicated that most compounds showed excellent activity against
wild-type HIV-1 with EC50 values in low nanomolar concentration
range (6b3, EC50 = 4.61 nM, SI = 5945) and high activity against
K103N/Y181C resistant mutant strain of HIV-1 with EC50 values
cal data. Apparently, the new compounds showed excellent activ-
ity against wild-type HIV-1 as they can bind into the NNIBP of
wild-type HIV-1 RT and exhibit interactions with NNIBP. However,
the new compounds displayed less potent activity against K103N/
Y181C resistant mutant strain of HIV-1 because of the following
reasons according to the docking studies. First, the new com-
pounds form hydrogen bonds with amino acid K101 backbone,
whereas piperidine-linked aminopyrimidine derivatives form an-
other hydrogen bond with K103 backbone through a water
bridge,15 and this hydrogen bond interaction with main chain is
unlikely to be disrupted by side chain mutations. Second, mesityl
moiety which fits into the hydrophobic sub-pocket keeps parallel
in low micromolar concentration range (6b1, EC50 = 0.55
lM with
a 76.4-fold resistance ratio; 6b4 EC50 = 0.51 M with a 72.7-fold
l
resistance ratio). In addition, we also built the molecular modes
to analyze the interactions between the analogs and wild-type
HIV-1 RT. Further studies are ongoing in our laboratories and will
be reported in due course.
4. Experimental section
4.1. Chemistry
to the side chain phenyl of amino acid Y181 and forms strong
interaction, when resistant mutation at Y181 side chain emerged
the dependent interaction lost. Further structural optimiza-
p–
p
p–p
tion will consider these aspects analyzed above.
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
with a Nexus 470FT-IR Spectrometer. 1H NMR spectra were ob-
tained on a Brucker Avance-600 NMR-spectrometer in the indi-
cated solvents. Chemical shifts are expressed in d units and TMS
as internal reference. Mass spectra were taken on a LC Autosampler
3. Conclusion
In the present paper, a novel series of piperidine substituted
triazine derivatives were designed, synthesized and evaluated in

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X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
Table 2
(DMSO-d6, ppm) d: 9.13 (m, 1H), 7.77 (m, 1H), 6.87 (s, 2H, PhH),
3.91 (m, 3H), 2.81 (s, 2H), 2.22 (d, 3H, CH3), 2.07 (d, 6H, 2 Â CH3),
1.37 (s, 9H). ESI-MS: m/z 447.5 (M+1), 448.5 (M+2), 449.5 (M+3).
Activity against K103N+Y181C mutant strain of HIV-1 (RES056) in MT-4 cells
Compds
EC50
(l
M)
Fold-res.a
CC50
(
lM)
SI
C
22H31ClN6O2 [446.22].
6a1
6a2
6a3
6a4
6a5
6a6
6a7
6a8
6b1
6b2
6b3
6b4
6b5
6c1
6c2
6c3
6c4
6c5
NVP
DLV
EFV
AZT
DDC
0.97 0.07
>5.91
198.8
>511.6
162.8
105.7
887.5
481.3
>439.1
>575.8
76.4
>297.0
121.6
72.7
549.8
111.1
>576.6
128.3
88.2
20.12 8.10
5.91 1.37
74.36 18.94
19.85 4.52
5.06 2.49
23.59 3.40
3.46 1.58
3.26 1.18
25.80 1.15
4.95 0.17
27.39 2.34
14.83 2.49
9.64 3.12
30.91 2.23
8.48 2.03
33.43 8.98
19.63 2.67
13.45 3.48
>15.02
21
<1
22
32
1
4.1.3. General procedure for the synthesis of N2-mesityl-N4-(1-
Boc-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine (4a), N2-
mesityl-N4-methyl-N6-(1-Boc-piperidin-4-yl)-1,3,5-triazine-
2,4,6-triamine (4b) and N2-mesityl-6-methoxy-N4-(1-Boc-
piperidin-4-yl)-1,3,5-triazine-2,4-diamine (4c)
To a solution of solid 3 (7.1 g, 16 mmol) in THF (20 mL) was
added ammonia (28% w/v, 100 mL). Then, the mixture was trans-
ferred to the reactor, sealed and placed at 100 °C for 5 h with a
mechanical stirrer. The reaction solution was left to cool overnight
and extracted with ethyl acetate (3 Â 30 mL). Combined extracts
were washed with 2 N HCl and brine, dried over anhydrous
Na2SO4, filtered and concentrated to give the crude product. Puri-
fication on silica gel gave 4a as an off-white solid. Yield: 82%,
mp: 144–146 °C. ESI-MS: m/z 428.3 (M+1), 429.3 (M+2).
3.33 3.19
0.61 0.01
4.92 4.79
4.93 0.03
>3.46
>3.26
0.55 0.22
>4.95
0.56 0.12
0.51 0.27
3.33 3.33
1.48 0.31
>8.48
1.11 0.17
0.94 0.04
P1.59
5
<1
<1
47
<1
49
29
3
21
<1
30
21
68
X1b
X1
>12
P4397
P133.3
>75.0
>1033.6
83.6
>15.02
>36.19
0.55 0.06
0.01 0.009
>36.19
>6.34
P49.77
>94.69
C22H33N7O2 [427.27].
Intermediate 3 (0.22 g, 0.5 mmol) was dissolved in THF, fol-
1.9
lowed by addition of saturated NaHCO3 (2 mL) and 40% w/v aque-
ous methylamine (0.2 mL). The reaction mixture was stirred at
50 °C for 5 h. THF was removed under reduced pressure and ex-
tracted with ethyl acetate (2 Â 10 mL). Combined organic phase
was dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to give the product 4b as white solid. Yield: 96%,
mp: 131–133 °C, ESI-MS: m/z 442.5 (M+1), 443.5 (M+2).
c26
ETV
0.034 0.0046
15.5
27.78 11.53
817
a
Fold-res: Fold resistance, the ratio of EC50 value against drug-resistant strain
and EC50 of the wild-type HIV-1 IIIB (ECm50utant strains/EC
.
wt
50
b
X1: stands for P1 or <1.
ETV: Etravirine. The data were obtained from the same laboratory (Rega Insti-
tute for Medical Research, K.U. Leuven, Belgium).
c
C23H35N7O2 [441.29].
Na (0.35 g, 15 mmol) was dissolved in absolute MeOH (50 mL)
Device: Standard G1313A instrument. TLC was performed on Silica
Gel GF254 for TLC (Merck) and spots were visualized by iodine va-
pors or by irradiation with UV light (254 nm). Flash column chro-
matography was performed on column packed with Silica Gel 60
(200–300 mesh). Solvents were reagent grade and, when neces-
sary, were purified and dried by standard methods. Concentration
of the reaction solutions involved the use of rotary evaporator at
reduced pressure.
followed by addition of 6-chloro-N2-mesityl-N4-(1-Boc-piperidin-
4-yl)-1,3,5-triazine-2,4-diamine (3) (4.5 g, 10 mmol). The reaction
mixture was stirred at 60 °C for 4 h. After removal of the solvent
under reduced pressure, water (20 mL) was added and extracted
with ethyl acetate (2 Â 20 mL). Combined organic phase was dried
over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give the product (4c) as white solid. Yield: 94%, mp:
190–192 °C, ESI-MS: m/z 443.6 (M+1), 444.6 (M+2). C23H34N6O3
[442.27].
4.1.1. General procedure for the synthesis of 4,6-dichloro-N-
mesityl-1,3,5-triazin-2-amine (2)
4.1.4. General procedure for the synthesis of N2-mesityl-N4-
(piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine (5a), N2-mesityl-
N4-methyl-N6-(piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine (5b)
and N2-mesityl-6-methoxy-N4-(piperidin-4-yl)-1,3,5-triazine-
2,4-diamine (5c)
TFA (4 mL, 54 mmol) was added dropwise under stirring to a
solution of intermediate 4a (3.3 g, 7.7 mmol) in CH2Cl2 (20 mL) at
room temperature and stirred for 5 h. To the reaction mixture
was added water and neutralized with 2 N NaOH to pH8, and the
organic layer was separated and dried over anhydrous Na2SO4, fil-
tered and concentrated under reduced pressure to give the product
(5a) as white solid. Yield: 91%, mp: 236–238 °C, ESI-MS: m/z 328.2
(M+1), 164.7 ((M+2)/2). C17H25N7 [327.22].
To a solution of cyanuric chloride (1, 0.37 g, 2 mmol) in THF
(15 mL) at 0 °C was slowly added K2CO3 (0.55 g, 4 mmol) and
2,4,6-trimethylaniline (0.28 g, 2 mmol). The resulting mixture
was stirred at 0 °C for 3 h (monitored by TLC). The solvent was re-
moved under reduced pressure and water (20 mL) was added and
extracted with CH2Cl2 (2 Â 10 mL). Combined organic phase was
washed with brine, dried over anhydrous Na2SO4, filtered and con-
centrated. Purification on silica gel gave 2 as an off-white solid.
Yield: 91%, mp: 197–199 °C. 1H NMR (DMSO-d6, ppm) d: 10.45 (s,
1H, NH), 6.94 (s, 2H, PhH), 2.25 (s, 3H, CH3), 2.08 (s, 6H,
2 Â CH3). ESI-MS: m/z 283.3 (M+1), 285.3 (M+3), 287.3 (M+5).
C12H12Cl2N4 [282.04].
Using intermediate 4b (0.3 g, 0.67 mmol) and TFA (1 mL,
13.5 mmol) and following the procedure as in the preparation of
5a to give 5b as a white solid. Yield: 95%, mp: 139–141 °C, ESI-
MS: m/z 342.4 (M+1), 171.8 ((M+2)/2). C18H27N7 [341.45].
Using intermediate 4c (0.2 g, 0.45 mmol) and TFA (0.5 mL,
6.7 mmol) and following the procedure as in the preparation of
5a to give 5c as a white solid. Yield: 95%, mp: 225–227 °C, ESI-
MS: m/z 343.5 (M+1), 172.4 ((M+2)/2). C18H26N6O [342.44].
4.1.2. General procedure for the synthesis of 6-chloro-N2-
mesityl-N4-(1-Boc-piperidin-4-yl)-1,3,5-triazine-2,4-diamine
(3)
A solution of crude intermediate 2 (0.56 g, 2 mmol) in THF/ace-
tone/H2O (4:1:1, 30 mL) was treated with 4-amino-1-Boc-piperi-
dine (0.4 g, 2 mmol) and 5% w/v aqueous NaHCO3 (8 mL). The
mixture was stirred at 30 °C for 8 h, after that, THF/acetone was
evaporated under reduced pressure and extracted with ethyl ace-
tate (2 Â 10 mL). Combined extracts were washed with saturated
sodium chloride (10 mL), dried over anhydrous Na2SO4, and evap-
orated in vacuo to give the crude product. Purification on silica gel
gave 3 as an off-white solid. Yield: 92%, mp: 203–205 °C. 1H NMR
4.1.5. General procedure for the synthesis of target compounds
(6a1–6a8, 6b1–6b5 and 6c1–6c5)
Compounds 5a (5b or 5c) was dissolved in anhydrous DMF
(10 mL) in the presence of anhydrous K2CO3 (2 equiv) at 0 °C, fol-

X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
3861
Figure 3. Predicted binding modes between the new compounds and NNIBP of wild-type HIV-1 RT (PDB code: 3M8Q): (a) Compound 6a1; (b) Compound 6b3; (c)
Superimposition of compound 6a1 (white) and compound 6b3 (purple).
lowed by addition of appropriate substituted benzyl chloride (or 4-
picolyl chloride hydrochloride) (1.1 equiv). The reaction mixture
was stirred at room temperature overnight. The solvent was re-
moved under reduced pressure, and water (20 mL) was added. Ex-
tracted with ethyl acetate (2 Â 10 mL), and the organic phase was
washed with saturated sodium chloride (10 mL), and dried over
anhydrous Na2SO4 to give the corresponding crude products,
which were purified by flash column chromatography to afford
the target compounds 6a1–6a8 (6b1–6b5 or 6c1–6c5),
respectively.
1.94 (s, 3H), 1.44 (m, 2H), 1.39 (t, 3H, CH3). IR (KBr, cmÀ1): 3386
maNsH), 3316 ( NH), 2939 (mCasH3), 1718 (
C@O), 1560, 1494 ( C@N),
1276 (maCs—O—C), 1106 (msC—O—C), 813 (
C@N). ESI-MS: m/z 490.9
(
t
t
x
t
(M+1), 246.0 ((M+2)/2). C27H35N7O2 [489.61].
4.1.5.3. 4-((4-(4-amino-6-(mesitylamino)-1,3,5-triazin-2-ylami-
no)piperidin-1-yl)methyl)benzamide (6a3).
White powder,
yield: 71.2%. Decomposed at 266 °C. 1H NMR (DMSO-d6, ppm) d:
7.82 (s, 2H, PhH), 7.35 (s, 2H, PhH), 6.82 (s, 2H, PhH), 3.48 (d, 2H,
J = 24 Hz), 2.76 (s, 2H), 2.20 (s, 3H, CH3), 2.08 (s, 6H, 2 Â CH3),
1.88–1.99 (m, 2H), 1.66–1.76 (m, 2H), 1.39–1.49 (m, 2H). 13C
NMR (100 MHz, DMSO-d6, ppm) d: 18.79, 20.94, 32.31, 46.81,
52.72, 65.49, 127.88, 128.56, 129.01, 133.50, 134.50, 135.09,
136.38, 142.23, 165.95, 167.48, 168.27. IR (KBr, cmÀ1): 3397
4.1.5.1. N2-mesityl-N4-(1-(pyridin-4-ylmethyl)piperidin-4-yl)-
1,3,5-triazine-2,4,6-triamine (6a1).
Pale yellow powder,
yield: 67.6%. Decomposed at 233 °C. 1H NMR (CDCl3, ppm) d:
8.59 (s, 2H, PhH), 6.97 (s, 2H, PhH), 4.85 (m, 4H), 3.95 (m, 1H),
3.52 (s, 2H, CH2), 2.81 (s, 2H), 2.35 (s, 3H, CH3), 2.25 (s, 6H,
2 Â CH3), 2.05 (s, 4H), 1.51 (s, 2H). 13C NMR (100 MHz, CDCl3,
ppm) d: 18.59, 20.97, 32.33, 46.91, 52.37, 61.83, 123.75, 128.79,
132.46, 136.59, 147.92, 149.80, 165.72, 167.23. IR (KBr, cmÀ1):
(
maNsH), 2924 (mCasH3), 1671 (
813 ( C@N). ESI-MS: m/z 461.6 (M+1). C25H32N8O [460.57].
t C@O), 1612 (bNH), 1568, 1494 (tC@N),
x
4.1.5.4. 4-((4-(4-amino-6-(mesitylamino)-1,3,5-triazin-2-ylami-
no)piperidin-1-yl)methyl)benzenesulfonamide
3460 (maNsH), 3399 (
t
NH), 2936 (maCsH3), 1559, 1487 (
tC@N), 812 (
x
C@N).
(6a4).
White powder, yield: 70.4%. Decomposed at 242 °C. 1H
ESI-MS: m/z 419.4 (M+1), 210.3 ((M+2)/2). C23H30N8 [418.54].
NMR (DMSO-d6, ppm) d: 7.77 (s, 2H, PhH), 7.47 (d, 2H, J = 9.6 Hz,
PhH), 7.31 (s, 2H), 6.82 (s, 2H, PhH), 6.11 (d, 1H, J = 3.0 Hz), 3.51
(d, 2H, J = 24.6 Hz), 2.76 (s, 2H), 2.20 (s, 3H, CH3), 2.08 (s, 6H,
2 Â CH3), 1.83–2.05 (m, 2H), 1.62–1.80 (m, 2H), 1.45–1.55 (m,
4.1.5.2. ethyl 4-((4-(4-amino-6-(mesitylamino)-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzoate
(6a2).
White
powder, yield: 69.4%. Mp: 141–143 °C. 1H NMR (CDCl3, ppm) d:
7.99 (d, 2H, J = 7.8 Hz, PhH), 7.39 (s, 2H, PhH), 6.91 (s, 2H, PhH),
4.80 (m, 3H), 4.37 (q, 2H, J = 7.2 Hz, CH2), 3.80 (m, 1H), 3.55 (s,
2H, CH2), 2.77 (s, 2H), 2.29 (s, 3H, CH3), 2.19 (s, 6H, 2 Â CH3),
2H). IR (KBr, cmÀ1): 3457 (mNasH), 3420 (
t
NH), 2946 (maCsH3), 1556,
S@O), 813
C@N). ESI-MS: m/z 497.6 (M+1), 249.5 ((M+2)/2). C24H32N8O2S
[496.63].
1486 (
t
C@N), 1369 (mas
), 1164 (t O@S@O), 1097 (t
O@S@O
(x

3862
X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
4.1.5.5. N2-mesityl-N4-(1-(4-(methylsulfonyl)benzyl)piperidin-
J = 23.4 Hz), 6.82 (s, 2H, PhH), 6.53 (m, 1H), 3.48 (d, 2H, J = 25.2 Hz),
2.74 (s, 3H, NCH3), 2.60 (d, 2H, J = 9.0 Hz), 2.20 (s, 3H, CH3), 2.07 (s,
6H, 2 Â CH3), 1.91–1.99 (m, 2H), 1.66–1.77 (m, 2H), 1.40–1.50 (m,
2H). 13C NMR (100 MHz, DMSO-d6, ppm) d: 18.81, 20.94, 27.59,
32.23, 47.31, 52.81, 62.23, 127.86, 128.51, 128.91, 133.43, 134.59,
135.01, 136.33, 142.52, 165.66, 166.90, 168.31. IR (KBr, cmÀ1):
4-yl)-1,3,5-triazine-2,4,6-triamine (6a5).
White powder,
yield: 66.8%. Decomposed at 242 °C. 1H NMR (CDCl3, ppm) d:
7.89 (d, 2H, J = 6.6 Hz, PhH), 7.53 (s, 2H, PhH), 6.91 (s, 2H, PhH),
4.83 (m, 3H), 3.80 (m, 1H), 3.57 (s, 2H, CH2), 3.06 (s, 3H, CH3),
2.75 (s, 2H), 2.29 (s, 3H, CH3), 2.19 (s, 6H, 2 Â CH3), 1.94 (s, 2H),
1.43 (s, 2H). IR (KBr, cmÀ1): 3422 (
t
NH), 2943 (maCsH3), 1560, 1513
3402 (
1556, 1516
C26H34N8O [474.60].
tNH), 3271 (
t
NH), 2923 (maCsH3), 1674 (
C@N). ESI-MS: m/z 475.5 (M+1),
t C@O), 1613 (bNH),
(t
C@N), 1372 (mas
), 1150 (
t
O@S@O), 1090 (
t
S@O), 812 (
x
C@N).
(
tC@N), 812 (x
O@S O
ESI-MS: m/z 496.6 @(M+1). C25H33N7O2S [495.64].
4.1.5.6. N2-(1-benzylpiperidin-4-yl)-N4-mesityl-1,3,5-triazine-
4.1.5.12. 4-((4-(4-(mesitylamino)-6-(methylamino)-1,3,5-tria-
zin-2-ylamino)piperidin-1-yl)methyl)benzenesulfonamide
(6b4).
2,4,6-triamine (6a6).
White powder, yield: 68.5%. Mp: 253–
255 °C. 1H NMR (CDCl3, ppm) d: 7.31 (s, 4H, PhH), 6.90 (s, 2H,
PhH), 4.75 (m, 4H), 3.90 (m, 1H), 3.52 (s, 2H, CH2), 2.81 (s, 2H),
2.29 (s, 3H, CH3), 2.19 (s, 6H, 2 Â CH3), 1.96 (s, 4H), 1.46 (s, 2H).
White powder, yield: 68.3%. Mp: 170–172 °C. 1H NMR
(CDCl3, ppm) d: 7.79 (d, 2H, J = 7.2 Hz, PhH), 7.50 (d, 2H,
J = 7.8 Hz, PhH), 6.79 (s, 2H, PhH), 5.15 (s, 2H, NH2), 4.92 (s, 2H),
3.88 (m, 1H), 3.55 (s, 2H, CH2), 2.91 (s, 3H, NCH3), 2.76 (s, 2H),
2.25 (s, 3H, CH3), 2.12 (s, 6H, 2 Â CH3), 1.82–2.04 (m, 5H), 1.48
IR (KBr, cmÀ1): 3463 (maNsH), 3402 ( NH), 2934 (mCasH3),
tNH), 3309 (t
1557, 1486 (tC@N), 811 (xC@N). ESI-MS: m/z 418.4 (M+1), 209.9
((M+2)/2). C24H31N7 [417.55].
(m, 2H). IR (KBr, cmÀ1): 3382 (
t
NH), 2942 (maCsH3), 1571, 1494 (
t
C@N),
1371 (mas
), 1160 (
tO@S@O), 1097 (
tS@O), 811 (xC@N). ESI-MS: m/
S@O
4.1.5.7. N2-mesityl-N4-(1-(4-nitrobenzyl)piperidin-4-yl)-1,3,5-
z 511.7 O(@M+1). C25H34N8O2S [510.65].
triazine-2,4,6-triamine (6a7).
White powder, yield: 69.1%.
Mp: 248–250 °C. 1H NMR (CDCl3, ppm) d: 8.17 (s, 2H, PhH), 7.50
(s, 2H, PhH), 6.91 (s, 2H, PhH), 4.84 (m, 4H), 3.80 (m, 1H), 3.58 (s,
2H, CH2), 2.74 (s, 2H), 2.29 (s, 3H, CH3), 2.19 (s, 6H, 2 Â CH3),
4.1.5.13.
N2-mesityl-N4-methyl-N6-(1-(4-(methylsulfo-
nyl)benzyl)piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine
(6b5).
White powder, yield: 71.4%. Mp: 125–127 °C. 1H NMR
1.94 (s, 2H), 1.43 (s, 2H). IR (KBr, cmÀ1): 3456 (mNasH), 3411 (
t
NH),
(CDCl3, ppm) d: 7.88 (d, 2H, J = 7.8 Hz, PhH), 7.53 (d, 2H,
J = 7.2 Hz, PhH), 6.89 (s, 2H, PhH), 4.86 (s, 2H), 3.90 (m, 1H), 3.57
(s, 2H, CH2), 3.05 (s, 3H, CH3), 2.94 (s, 3H, NCH3), 2.76 (s, 2H),
2.28 (s, 3H, CH3), 2.19 (s, 6H, 2 Â CH3), 1.90–2.09 (m, 3H), 1.50
3309 (t t x
NH), 2920 (maCsH3), 1557, 1483 ( C@N), 1518 (maNsO2), 813 (
C@N).
ESI-MS: m/z 463.4 (M+1), 232.3 ((M+2)/2). C24H30N8O2 [462.55].
4.1.5.8. 4-((4-(4-amino-6-(mesitylamino)-1,3,5-triazin-2-ylami-
(m, 2H). IR (KBr, cmÀ1): 3394 (
t
NH), 3263 (
), 1150 ( O@S@O), 1089 (
xC@N). ESI-MS: m/z 510.6 (M+1). C26H35N7O2S [509.67].
t
NH), 2922 (maCsH3),
S@O),
no)piperidin-1-yl)methyl)benzonitrile (6a8).
White pow-
1570, 1513 (
812 (
t
C@N), 1371 (mas
t
t
O@S@O
der, yield: 71.0%. Decomposed at 228 °C. 1H NMR (CDCl3, ppm) d:
7.60 (d, 2H, J = 7.2 Hz, PhH), 7.44 (s, 2H, PhH), 6.91 (s, 2H, PhH),
4.84 (m, 3H), 3.54 (s, 2H, CH2), 2.74 (s, 2H), 2.29 (s, 3H, CH3),
2.19 (s, 6H, 2 Â CH3), 1.96 (s, 2H), 1.45 (s, 2H), 1.26 (s, 2H). IR
4.1.5.14.
N2-mesityl-6-methoxy-N4-(1-(pyridin-4-ylmethyl)
piperidin-4-yl)-1,3,5-triazine-2,4-diamine (6c1). Pale yel-
(KBr, cmÀ1): 3397 (
C@N), 813 (
25H30N8 [442.56].
t
NH), 2923 (maCsH3), 2228 (
t
C„N), 1564, 1497
low powder, yield: 66.9%. Mp: 122–124 °C. 1H NMR (CDCl3, ppm)
d: 8.54 (s, 2H, PhH), 7.53 (s, 2H, PhH), 6.90 (d, 2H, J = 24 Hz,
PhH), 6.23 (m, 1H), 5.06 (m, 1H), 3.93 (s, 3H, OCH3), 3.71 (s, 1H),
3.50 (s, 2H, CH2), 2.78 (s, 2H), 2.29 (s, 3H, CH3), 2.19 (s, 6H,
2 Â CH3), 1.98 (m, 2H), 1.80 (m, 2H), 1.49 (m, 2H). 13C NMR
(100 MHz, DMSO-d6, ppm) d: 18.10, 20.41, 31.30, 47.30, 52.24,
53.09, 60.69, 123.55, 128.00, 128.15, 133.29, 135.57, 147.68,
(
C
t
xC@N). ESI-MS: m/z 443.6 (M+1), 222.3 ((M+2)/2).
4.1.5.9. N2-mesityl-N4-methyl-N6-(1-(pyridin-4-ylmethyl)piper-
idin-4-yl)-1,3,5-triazine-2,4,6-triamine (6b1). Pale yellow
powder, yield: 67.7%. Mp: 134–136 °C. 1H NMR (CDCl3, ppm) d:
8.54 (d, 2H, J = 3.6 Hz, PhH), 6.90 (s, 2H, PhH), 6.23 (m, 1H), 4.95
(s, 2H), 3.49 (s, 2H, CH2), 2.94, (s, 3H, NCH3), 2.77 (s, 2H), 2.29 (s,
3H, CH3), 2.20 (s, 6H, 2 Â CH3), 2.04 (m, 2H), 1.89 (m, 2H), 1.50
(m, 2H). 13C NMR (100 MHz, CDCl3, ppm) d: 18.59, 20.99, 27.52,
32.28, 52.38, 61.58, 123.79, 128.67, 136.12, 147.88, 149.79. IR
149.40, 166.03, 166.44, 170.58. IR (KBr, cmÀ1): 3392 (
t
NH), 3249
), 815
(
(
t t
NH), 2948 (maCsH3), 1577, 1499 ( C@N), 1371 (mas
x
C@N). ESI-MS: m/z 434.7 (M+1), 218.1 ((M+2)@/2). C24H31N7O
CAOAC
[433.55].
(KBr, cmÀ1): 3423 (
812 ( C@N). ESI-MS: m/z 433.7 (M+1), 217.6 ((M+2)/2). C24H32N8
t
NH), 3261 (
t
NH), 2943 (maCsH3), 1560, 1513 (
t
C@N),
4.1.5.15. ethyl 4-((4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-
2-ylamino)piperidin-1-yl)methyl)benzoate (6c2). White
x
[432.56].
powder, yield: 69.8%. Mp: 142–144 °C. 1H NMR (CDCl3, ppm) d:
8.00 (s, 2H, PhH), 7.40 (s, 2H, PhH), 6.90 (d, 2H, J = 22.8 Hz, PhH),
6.29 (m, 1H), 5.05 (m, 1H), 4.37 (q, 2H, J = 6.6 Hz, CH2), 3.92 (s,
3H, OCH3), 3.71 (s, 1H), 3.56 (s, 2H, CH2), 2.79 (s, 2H), 2.29 (s, 3H,
CH3), 2.19 (s, 6H, 2 Â CH3), 1.97 (m, 3H), 1.43 (m, 2H), 1.39 (t,
4.1.5.10. ethyl 4-((4-(4-(mesitylamino)-6-(methylamino)-1,3,5-
triazin-2-ylamino)piperidin-1-yl)methyl)benzoate
(6b2).
White powder, yield: 70.4%. Mp: 127–129 °C. 1H NMR
(CDCl3, ppm) d: 7.99 (d, 2H, J = 8.4 Hz, PhH), 7.38 (d, 2H,
J = 8.4 Hz, PhH), 6.89 (s, 2H, PhH), 4.84 (s, 2H), 4.37 (q, 2H,
J = 7.2 Hz, CH2), 3.54 (s, 2H, CH2), 2.91 (s, 3H, NCH3), 2.76 (s, 2H),
2.28 (s, 3H, CH3), 2.19 (s, 6H, 2 Â CH3), 1.97 (m, 2H), 1.46 (m,
3H, J = 6.0 Hz, CH3). IR (KBr, cmÀ1): 3371 (
t
NH), 3253 (
t
NH), 2949
), 1275
(
(
maCsH3), 1719 (
t C@O), 1575, 1499 (t
C@N), 1369 (mas
@CAOAC
mCasAOAC), 1106 (mCs AOAC), 815 (
x
C@N). ESI-MS: m/z 505.6 (M+1).
C28H36N6O3 [504.62].
2H), 1.39 (t, 3H, J = 7.2 Hz, CH3). IR (KBr, cmÀ1): 3400 (
t
NH), 3263
C@N), 1275 (maCsAOAC),
C@N). ESI-MS: m/z 504.6 (M+1), C28H37N7O2
(t
NH), 2941 (maCsH3), 1718 (
t
C@O), 1560, 1513 (
t
4.1.5.16.
4-((4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-
1106 (maCsAOAC), 812 (
x
ylamino)piperidin-1-yl)methyl)benzamide (6c3). White
[503.64].
powder, yield: 72.4%. Mp: 155–157 °C. 1H NMR (CDCl3, ppm) d:
7.73 (d, 2H, J = 7.8 Hz, PhH), 7.39 (d, 2H, J = 6.6 Hz, PhH), 6.90 (d,
2H, J = 29.4 Hz, PhH), 5.95 (s, 2H, NH2), 3.92 (s, 3H, OCH3), 3.71
(s, 1H), 3.53 (s, 2H, CH2), 2.78 (s, 2H), 2.30 (s, 3H, CH3), 2.20 (s,
6H, 2 Â CH3), 1.96 (m, 2H), 1.77 (s, 2H), 1.48 (m, 2H). 13C NMR
(100 MHz, DMSO-d6, ppm) d: 18.10, 20.41, 31.34, 47.42, 52.21,
4.1.5.11. 4-((4-(4-(mesitylamino)-6-(methylamino)-1,3,5-tria-
zin-2-ylamino)piperidin-1-yl)methyl)benzamide
(6b3).
NMR (DMSO-d6, ppm) d: 7.92 (s, 2H, PhH), 7.82 (s, 2H), 7.33 (d, 3H,
White powder, yield: 66.7%. Decomposed at 237 °C. 1H

X. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3856–3864
3863
53.09, 61.65, 127.29, 128.00, 128.15, 128.31, 132.87, 133.29,
135.57, 141.95, 166.03, 166.43, 167.69, 170.58. IR (KBr, cmÀ1):
protection from the cytopathic effect of the virus in infected cells
was defined as the 50% effective concentration (EC50).
3255 (
t
NH), 2923 (maCsH3), 1672 (
), 814 (
t
C@O), 1575, 1498 (
tC@N), 1373
(
mas
x
C@N). ESI-MS: m/z 476.4 (M+1). C26H33N7O2
Acknowledgments
[4@75.59].
CAOAC
The financial support from the National Natural Science Foun-
dation of China (NSFC Nos. 81102320, 30873133, 30772629,
30371686), Key Project of NSFC for International Cooperation
(No. 30910103908), Research Fund for the Doctoral Program of
4.1.5.17.
4-((4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzenesulfonamide
(6c4).
White powder, yield: 71.1%. Mp: 160–162 °C. 1H NMR
(CDCl3, ppm) d: 7.81 (d, 2H, J = 7.2 Hz, PhH), 7.46 (d, 2H,
J = 7.2 Hz, PhH), 6.84 (d, 2H, J = 34.8 Hz, PhH), 6.35 (m, 1H), 5.16
(s, 2H, NH2), 3.91 (s, 3H, OCH3), 3.71 (s, 1H), 3.55 (s, 2H, CH2),
2.78 (s, 2H), 2.25 (s, 3H, CH3), 2.13 (s, 6H, 2 Â CH3), 1.97 (m, 2H),
Higher
Education
of
China
(Nos.
20110131130005,
20110131120037), Independent Innovation Foundation of Shan-
dong University (IIFSDU, No. 2010GN044), Shandong Postdoctoral
Innovation Science Research Special Program (No.201002023), Chi-
1.84 (m, 2H), 1.49 (m, 2H). IR (KBr, cmÀ1): 3350 (
t
NH), 2924
O@S@O), 1098
(x
C@N). ESI-MS: m/zO@5@12.7 (M+1). C25H33N7O3S
na
Postdoctoral
Science
Foundation
funded
project
(
(
maCsH3), 1580, 1400 (
S@O), 814
t
C@N), 1385 (mas
), 1161 (
t
(No.20100481282) 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.
S O
t
[511.64].
4.1.5.18.
N2-mesityl-6-methoxy-N4-(1-(4-(methylsulfonyl)
References and notes
benzyl)piperidin-4-yl)-1,3,5-triazine-2,4-diamine
(6c5).
White powder, yield: 72.0%. Mp: 121–123 °C. 1H NMR
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(CDCl3, ppm) d: 7.88 (d, 2H, J = 7.2 Hz, PhH), 7.53 (d, 2H,
J = 7.2 Hz, PhH), 6.90 (d, 2H, J = 22.8 Hz, PhH), 3.93 (s, 3H, OCH3),
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t
NH), 3257 (
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C@N). ESI-MS: m/z 511.6 (M+1). C26H34N6O3S [510.65].
t
NH), 2923 (maCsH3),
S@O),
1578, 1498 (
815 (
t
C@N), 1384 (mas
t
t
O@S@O
x
4.2. In vitro anti-HIV activity assays
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25-lL volumes to two series of triplicate wells so as to allow simul-
taneous 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 microti-
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were included for each sample.
HIV-1(IIIB)30 or HIV-2 (ROD)31 stock (50
lL) 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.
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pound on uninfected cells in order to assess the cytotoxicity of
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lL
volumes were transferred to the microtiter tray wells. Five days
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bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
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scan Ascent Reader, Labsystems, Helsinki, Finland), at two wave-
lengths (540 and 690 nm). All data were calculated using the
median OD (optical density) value of tree wells. 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%
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