Synthesis and biological evaluation of piperidine-substituted triazine derivatives as HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.02.019

A novel series of piperidine-substituted triazine derivatives have been synthesized and evaluated for anti-HIV activities in MT-4 cells. Most compounds displayed extremely promising activity against wild-type HIV-1 with EC ₅₀ values in low nanomolar concentration, better than that of Nevirapine, Delavirdine, Zidovudine and Dideoxycitidine, and higher potency towards the resistant mutant strain K103N/Y181C than that of Nevirapine and Delavirdine. Selected compounds were also assayed against reverse transcriptase with lower IC₅₀ values than that of Nevirapine. The structure-activity relationship (SAR) of these novel structural congeners was also discussed.

Full text of DOI:10.1016/j.ejmech.2012.02.019

European Journal of Medicinal Chemistry 51 (2012) 60e66
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of piperidine-substituted triazine derivatives
as HIV-1 non-nucleoside reverse transcriptase inhibitors
,
Xuwang Chen a, Peng Zhan a, Christophe Pannecouque b, Jan Balzarini b, Erik De Clercq b, 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 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-substituted triazine derivatives have been synthesized and evaluated for
anti-HIV activities in MT-4 cells. Most compounds displayed extremely promising activity against wild-
type HIV-1 with EC50 values in low nanomolar concentration, better than that of Nevirapine, Delavirdine,
Zidovudine and Dideoxycitidine, and higher potency towards the resistant mutant strain K103N/Y181C
than that of Nevirapine and Delavirdine. Selected compounds were also assayed against reverse tran-
scriptase with lower IC50 values than that of Nevirapine. The structure-activity relationship (SAR) of
these novel structural congeners was also discussed.
Received 21 September 2011
Received in revised form
3 February 2012
Accepted 8 February 2012
Available online 15 February 2012
Keywords:
HIV-1
Ó 2012 Elsevier Masson SAS. All rights reserved.
NNRTIs
Piperidine
Triazine
AIDS
SAR
1. Introduction
2011 respectively (Fig. 1) [3,4]. And also, their analogue TMC120 is
still under development as a vaginal HIV microbicide [5,6].
According to the statistic data of AIDS epidemic in 2010 by WHO,
there were 33.3 million people living with HIV, 2.6 million new HIV
infections and 1.8 million AIDS-related deaths in 2009, therefore,
AIDS is still one of the leading pandemic diseases worldwide [1].
Highly active antiretroviral therapy (HAART) provided an effective
way to treat AIDS patients by dramatically decreasing the morbidity
and mortality from the infection of HIV-1. However, the rapid
emergence of drug resistance, serious side effects and poor patient
compliance during the long-term in which the drugs are used
partly compromise the clinical application of HAART. Therefore, it is
an urgent need to design and develop new anti-AIDS drugs with
improved potency to halt the spread of HIV.
However, the pharmacokinetic profiles of most DAPYs are not
satisfactory due to the low water solubility [7e9]. Recently, in order
to enhance oral bioavailability, a series of piperidine-linked ami-
nopyrimidine derivatives were reported with good potency against
wild-type and several important resistant mutant strains of HIV-1
in both enzymic and cellular assays (Fig. 1) [10,11]. Inspired by
their good potency and bioavailability profiles, we designed a new
series of piperidine-linked triazine derivatives through incorpora-
tion of the triazine ring of DATA, a common moiety in HIV-1 NNRTIs
[12,13]. Herein, we report the synthesis, anti-HIV evaluation and
preliminary structure-activity relationship (SAR) of these hybrid
compounds.
HIV-1 non-nucleoside reverse transcriptase inhibitors (HIV-1
NNRTIs) with high antiviral potency, high specificity and low
cytotoxicity have become an indispensable component in HAART
regimen [2]. Among the NNRTIs, diaryltriazine (DATA) and diary-
lpyrimidine (DAPY) derivatives with superior activity profiles
against HIV-1 have attracted considerable attention over the past
few years. Etravirine (ETV, TMC125) and Rilpivirine, the represen-
tatives of DAPYs, were approved by FDA in January 2008 and May
2. Chemistry
The synthetic route to the target compounds 6a1e7, 6b1e7,
6a01e4 and 6b01e4 is depicted in Scheme 1. 4,6-dichloro-N-mesi-
tyl-1,3,5-triazin-2-amine (2) was obtained by substitution reaction
of commercially available 2,4,6-trichloro-1,3,5-triazine (1) with
2,4,6-trimethylaniline in ice bath [14], which was reacted with 4-
amino-1-Boc-piperidine to give 6-chloro-N2-mesityl-N4-(1-Boc-
piperidin-4-yl)- 1,3,5-triazine-2,4-diamine (3) [15]. The chlorine
atom of intermediate 3 was transformed to methylamino (4a) and
* Corresponding author. Tel.: þ86 531 88380270; fax: þ86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.019

X. Chen et al. / European Journal of Medicinal Chemistry 51 (2012) 60e66
61
Fig. 1. The structures of DAPYs, DATA and piperidine-linked aminopyrimidine derivative.
methoxyl group (4b) under the conditions of NH2CH3 (aq)/NaHCO3/
THF and Na/CH3OH respectively [16]. By removing the Boc group of
4a and 4b in the presence of trifluoroacetic acid (TFA) at room
temperature, compounds 5a and 5b were prepared in good yield,
which were then reacted with substituted benzyl chloride
(bromine) or substituted benzoic acid, achieving the final
compounds 6a1e7, 6b1e7, 6a01e4 and 6b01e4 respectively.
using the MTT method [17e20]. Nevirapine (NVP), Delavirdine
Mesylate (DLV), Efavirenz (EFV), zidovudine (azidothymidine, AZT),
dideoxycitidine (DDC) and ETV were used as reference drugs
(Table 1). The results demonstrated that compounds 6a1e7 and
6b1e7 exhibited higher inhibition activity against HIV-1 than that
of NVP, DLV, AZT and DDC, with low EC50 values ranging from 7.0 to
19.2 nM, and high selectivity indices (SI ¼ CC50/EC50) ranging from
1188 to 3240. Compounds 6a01e4 and 6b01e4 showed moderate
activity against HIV-1, of which 6a02 and 6a03 were more potent
than DDC. However, the new synthesized compounds are less
potent than ETV against wild-type HIV-1, which showed EC50
value as low as 3.0 nM. Further studies of piperidine-substituted
triazine derivatives will assist the design of the next generations
of compounds.
3. Results and discussion
3.1. Anti-HIV activity evaluation
The newly synthesized piperidine-substituted triazine deriva-
tives were evaluated for anti-HIV-1 (IIIB) activity in MT-4 cells
Scheme 1. Reagents and conditions i: K2CO3, THF; ii: NaHCO3 (5% aq), THF; iii: (a) NH2CH3 (aq), NaHCO3, THF; (b) Na/CH3OH; iv: TFA, CH2Cl2; v: substituted benzyl chloride
(bromine), K2CO3, DMF; or substituted benzoic acid, N,N’-Carbonyldiimidazole (CDI),DMF.

62
X. Chen et al. / European Journal of Medicinal Chemistry 51 (2012) 60e66
Table 1
Table 2
Activity against wild-type HIV-1 (IIIB) in MT-4 cells by MTT method.
Activity against K103N/Y181C resistant mutant strain of HIV-1 in MT-4 cells by MTT
method.
Compd
EC50
(
m
M)a
Compd
EC50 (mM)
6a1
6a2
6a3
6a4
6a5
6a6
6a7
1.3 ꢀ 0.1
5.0 ꢀ 0.1
ꢁ2.6
6b1
6b2
6b3
6b4
6b5
6b6
6b7
6b’1
6b’2
6b’3
6b’4
AZT
EFV
1.6 ꢀ 0.1
4.8 ꢀ 0.02
ꢁ6.1
4.9 ꢀ 0.4
5.8 ꢀ 0.7
4.6 ꢀ 0.5
4.6 ꢀ 0.2
>255.3
6.5 ꢀ 1.0
7.4 ꢀ 0.5
4.6 ꢀ 0.1
6.7 ꢀ 1.6
>149.2
6a01
6a02
6a03
6a04
NVP
DLV
ETV [21]
>55.4
>30.9
>33.2
>121.3
>28.8
>25.2
11.2 ꢀ 2.2
0.0086 ꢀ 0.0015
>36.2
0.57 ꢀ 0.06
0.026 ꢀ 0.004
a
EC50: concentration of compound required to achieve 50% protection of MT-4
cell against K103N/Y181C resistant mutant strain of HIV-1, as determined by the
MTT method.
Compd
R
Y
EC50 (nM)a
8.5 ꢀ 1.1
CC50
(
m
M)b
SIc
1188
6a1
6a2
6a3
6a4
6a5
6a6
6a7
6b1
6b2
6b3
6b4
6b5
6b6
6b7
6a01
6a02
6a03
6a04
6b01
6b02
6b03
6b04
NVP
DLV
EFV
AZT
DDC
ETVe [21]
4eCN
H
4eNO2
4-F
4-OMe
3eCN
2eCN
4eCN
H
4eNO2
4-F
4-OMe
3eCN
2eCN
NHMe
NHMe
NHMe
NHMe
NHMe
NHMe
NHMe
OMe
OMe
OMe
OMe
OMe
10.0 ꢀ 5.6
28.4 ꢀ 2.5
10.2 ꢀ 3.8
25.6 ꢀ 1.7
22.1 ꢀ 3.2
23.0 ꢀ 2.4
24.7 ꢀ 2.7
14.6 ꢀ 3.8
28.6 ꢀ 1.8
15.4 ꢀ 2.1
24.7 ꢀ 2.0
19.8 ꢀ 3.2
23.5 ꢀ 2.1
28.0 ꢀ 1.9
ꢁ255.3
8.8 ꢀ 0.9
8.2 ꢀ 3.1
10.2 ꢀ 0.4
9.5 ꢀ 0.2
7.0 ꢀ 2.2
9.9 ꢀ 1.3
9.2 ꢀ 2.2
11.3 ꢀ 2.8
9.4 ꢀ 2.1
11.5 ꢀ 0. 4
9.7 ꢀ 1.3
9.8 ꢀ 2.2
19.2 ꢀ 7.4
3187
1230
2884
2310
3240
2501
1598
2512
1642
2140
2043
2382
1460
>orX2d
60
Table 3
Activity of selected piperidine-substituted triazine derivatives against wild-type
HIV-1 RT.
Compd
6a2
6a6
6b1
NVP
ETV
IC50
(
m
M)a
2.46
1.97
1.77
2.74
0.55
a
IC50: Inhibitory concentration required to inhibit biotin deoxyuridine triphos-
phate (biotin-dUTP) incorporation into the HIV-1 RT by 50% (RT kit, Roche).
OMe
OMe
which means the piperidine-substituted triazine derivatives
belonged to anti-HIV-1 agents.
4eCOOH NHMe ꢁ127050.2
4eOH
H
4eNO2
NHMe
NHMe
NHMe
931.6 ꢀ 216.7
224.4 ꢀ 22.4
55.4 ꢀ 2.2
30.9 ꢀ 1.8
33.2 ꢀ 4.5
Rapid emergence of drug resistance compromise the anti-AIDS
therapy clinically, thus, the new analogues were also assayed
against the K103N/Y181C resistant mutant strain of HIV-1, which is
a frequently encountered double mutant in clinical. The results
were illustrated in Table 2 together with NVP, DLV, EFV, AZT and
ETV as reference standards. As the data indicated, most N-benzyl
analogues (6a1e7, 6b1e7) retained higher activity against the
double mutant virus than NVP and DLV, but less than AZT, EFV and
132
18
1793.9 ꢀ 224.2
4eCOOH OMe
4eOH
H
36183.9 ꢀ 2160.8 ꢁ149.2
ꢁ4
OMe
OMe
OMe
1664.7 ꢀ 108.1
1791.6 ꢀ 447.9
4374.0 ꢀ 915.5
90.1 ꢀ 30.0
32.6 ꢀ 3.6
121.3 ꢀ 30.1
73
28.8 ꢀ 1.4
ꢁ25.2
16
ꢁ6
4eNO2
>15.0
>168
>1096
>1187
>36.2
>6.3
5.4 ꢀ 0.3
20.6 ꢀ 23.6
1041.6 ꢀ 426.0
3.0 ꢀ 0.2
249.5 ꢀ 12.6 12,221
ETV. Among the N-benzyl analogues, 6a1 (EC50 ¼ 1.3
(EC50 ¼ 1.6 M) were the most potent analogues, however, ETV
M) against the
mM) and 6b1
>94.7
>4.6
>93
>1537
m
displayed very promising activity (EC50 ¼ 0.026
m
a
EC50: concentration of compound required to achieve 50% protection of MT-4
cell against HIV-1-induced cytotoxicity, as determined by the MTT method.
resistant mutant strain of HIV-1. All the N-benzoyl analogues
(6a01e4 and 6b01e4) lost their activities against K103N/Y181C
resistant mutant strain.
b
CC50: concentration required to reduce the viability of mock-infected cells by
50%, as determined by the MTT method.
c
SI: selectivity index (CC50/EC50).
d
X2: stands for ꢁ2 or <2.
e
3.2. Inhibition of HIV-1 RT
ETV: Etravirine. The data were obtained from the same laboratory (Prof. Erik De
Clercq, Rega Institute for Medical Research, K.U. Leuven, Belgium).
Meanwhile, for identification of the binding target, some active
compounds (6a2, 6a6 and 6b1) were selected to carry out an HIV-1
RT inhibitory assay, using poly(rA)-oligo(dT) as template
primer (Table 3) [20,22]. The results indicated that the tested
compounds displayed higher inhibitory activity than that of NVP
Based on the results of antiviral assay (Table 1), some important
SAR information was summarized as the following: (1) Substituents
at the C6 position of triazine ring influenced the activity: N-benzyl
analogues with Y ¼ NHMe (6a1e7) and Y ¼ OMe (6b1e7) showed
activities in the similar order of magnitude, but all the compounds
with Y ¼ NHMe (6a1e7) were slightly more potent than the cor-
responding compounds with Y ¼ OMe (6b1e7). (2) N-benzyl
analogues (6a1-7, 6b1-7) showed superior activity to the N-benzoyl
analogues (6a’1-4, 6b’1-4). (3) In the N-benzoyl analogues, the
effect of different substituents on the activity was in a clear order: H
(6a03, 6b03) > (or z) 4-hydroxy (6a02, 6b02) > 4-nitro (6a04,
6b04) > 4-carboxyl (6a01, 6b01).
(IC50 ¼ 2.74
m
M). Though the new compounds were less potent than
ETV (IC50 ¼ 0.55
m
M), it suggests that the newly synthesized
piperidine-substituted triazine derivatives bind to HIV-1 RT with
good affinity, and belong to HIV-1 NNRTIs.
4. Conclusion
In summary, a series of novel piperidine-substituted triazine
derivatives were synthesized via an expeditious route, and evaluated
for their antiviral activity against of HIV-1 (IIIB strain and K103N/
Y181C resistant mutant strains) and HIV-2 (ROD strain) in MT-4 cells,
as well as HIV-1 RT inhibitory activity (some selected compounds).
In addition, the compounds 6a1e7, 6b1e7, 6a01e4 and 6b01e4
were also tested their ability to inhibit HIV-2 (ROD) replication in
MT-4 cells. But none of them were active at subtoxic concentration,

X. Chen et al. / European Journal of Medicinal Chemistry 51 (2012) 60e66
63
Among them, compound 6a6 was identified as the most promising
candidate (EC50 ¼ 7.0 nM, SI ¼ 3240), which may contribute to the
discovery of potential anti-HIV-1 agents. Further studies are ongoing
in our laboratories and will be reported in due course.
MeOH (50 mL) 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 ꢂC for 4 h. After removal of
the solvent under reduced pressure, water (20 mL) was added and
extracted with ethyl acetate (2 ꢃ 20 mL). Combined organic phase
was dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to give the product (4b) as white solid. Yield: 94%,
mp: 190e192 ꢂC, ESI-MS: m/z 443.6 (M þ 1), 444.6 (M þ 2).
N2-mesityl-N4-methyl-N6-(piperidin-4-yl)-1,3,5-triazine-2,4,6-
triamine (5a): TFA (1 mL, 13.5 mmol) was added dropwise under
stirring to a solution of intermediate 4a (0.3 g, 0.67 mmol) in CH2Cl2
(10 mL) at room temperature and stirred overnight. The reaction
mixture was neutralized with 2N NaOH to pH8, and the organic
layer was separated and dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to give the product (5a) as
white solid. Yield: 95%, mp: 139e141 ꢂC, ESI-MS: m/z 342.4 (M þ 1),
171.8 ((M þ 2)/2).
5. Experimental Section
5.1. Chemistry
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
with
obtained on a Brucker Avance-600 NMR-spectrometer in the indi-
cated solvents. Chemical shifts are expressed in units and TMS as
a
Nexus 470FT-IR Spectrometer. 1H-NMR spectra were
d
internal reference. Mass spectra were taken on a LC Autosampler
Device: Standard G1313A instrument. TLC was performed on Silica
Gel GF254 for TLC (Merck) and spots were visualized by iodine
vapours or by irradiation with UV light (254 nm). Flash column
chromatography was performed on column packed with Silica Gel
60 (230e400 mesh). Solvents were reagent grade and, when
necessary, were purified and dried by standard methods. Concen-
tration of the reaction solutions involved the use of rotary evapo-
rator at reduced pressure.
N2-mesityl-6-methoxy-N4-(piperidin-4-yl)-1,3,5-triazine-2,4-
diamine (5b): Using intermediate 4b (0.2 g, 0.45 mmol) and TFA
(0.5 mL, 6.7 mmol) and following the procedure as in the prep-
aration of 5a to give 5b as a white solid. Yield: 95%, mp:
225e227 ꢂC, ESI-MS: m/z 343.5 (M þ 1), 172.4 ((M þ 2)/2).
5.1.2. General procedure for the synthesis of target compounds
6a1e7 and 6b1e7
5.1.1. General procedure for the synthesis of N2-mesityl-N4-methyl-
N6-(piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine (5a) and N2-mesityl-
6-methoxy-N4-(piperidin-4-yl)-1,3,5-triazine-2,4-diamine (5b)
Compounds 5a (or 5b) was dissolved in anhydrous DMF (10 mL)
in the presence of anhydrous K2CO3 (2 eq) at 0 ꢂC, followed by
addition of appropriate substituted benzyl chloride (bromine) (1.1
eq). The reaction mixture was stirred at room temperature over-
night. The solvent was removed under reduced pressure, and water
(20 mL) was added. Extracted with ethyl acetate (2 ꢃ 10 mL), and
the organic phase was washed with saturated sodium chloride
(10 mL), and dried over anhydrous Na2SO4 to give the corre-
sponding crude product, which was purified by flash column
chromatography to afford compounds 6a1e7 (or 6b1e7).
4,6-dichloro-N-mesityl-1,3,5-triazin-2-amine (2): To a solution
of cyanuric chloride (1, 0.37 g, 2 mmol) in THF (15 mL) at 0 ꢂC were
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 ꢂC for 3 h
(monitored by TLC). After removal of the solvent under reduced
pressure, water (20 mL) was added and extracted with CH2Cl2
(2 ꢃ 10 mL). Combined organic phase was washed with brine, dried
over anhydrous Na2SO4, filtered and concentrated. Purification on
silica gel gave 2 as an off-white solid. Yield: 91%, mp: 197e199 ꢂC.
1H-NMR (DMSO-d6, ppm)
d: 10.45 (s, 1H, NH), 6.94 (s, 2H, PhH),
5.1.3. 4-((4-(4-(mesitylamino)-6-(methylamino)-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzonitrile (6a1)
2.25 (s, 3H, CH3), 2.08 (s, 6H, 2 ꢃ CH3). ESI-MS: m/z 283.3 (M þ 1),
285.3 (M þ 3), 287.3 (M þ 5).
White powder, yield: 68.4%. Mp: 121e123 ꢂC. 1H-NMR (DMSO-
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/acetone/H2O (4:1:1, 30 mL) was treated with 4-
amino-1-Boc-piperidine (0.4 g, 2 mmol) and 5% w/v aqueous
NaHCO3 (8 mL). The mixture was stirred at 30 ꢂC for 8 h. THF/
acetone was evaporated under reduced pressure and extracted
with ethyl acetate (2 ꢃ 10 mL). Combined extracts were washed
with saturated sodium chloride (10 mL), dried over anhydrous
Na2SO4, and evaporated in vacuo to give the crude product.
Purification on silica gel gave 3 as an off-white solid. Yield: 92%,
d6, ppm) d: 7.78 (s, 2H, PhH), 7.49 (d, 2H, PhH), 6.82 (s, 2H, PhH),
6.45 (m, 2H), 3.53 (d, 2H, CH2), 2.71 (s, 3H, NeCH3), 2.61 (s, 2H,
CH2), 2.20 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3), 1.96 (m, 2H, CH2), 1.78
(m, 2H, CH2), 1.45 (m, 2H, CH2). IR (KBr, cmꢄ1): 3416 (yNH), 2943
(
yasCH3), 2228 (
yC^N), 1567, 1513 (yC]N), 812 (uC]N). ESI-MS: m/z
457.5 (M þ 1), 229.4 ((M þ 2)/2). C26H32N8 (456.27).
5.1.4. N2-(1-Benzylpiperidin-4-yl)-N4-mesityl-N6-methyl-1,3,5-
triazine-2,4,6-triamine (6a2)
White powder, yield: 53.6%. Mp: 111e113 ꢂC. 1H-NMR (DMSO-
mp: 203e205 ꢂC. 1H-NMR (DMSO-d6, ppm)
d: 9.13 (m, 1H), 7.77
d6, ppm) d: 7.27 (m, 5H, PhH), 6.81 (s, 2H, PhH), 6.53 (m, 2H), 3.43
(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 ꢃ CH3), 1.37 (s, 9H). ESI-MS: m/z 447.5
(M þ 1), 448.5 (M þ 2), 449.5 (M þ 3).
(d, 2H, CH2), 2.73 (s, 3H, NeCH3), 2.61 (s, 2H, CH2), 2.20 (s, 3H, CH3),
2.07 (s, 6H, 2 ꢃ CH3), 1.94 (m, 2H, CH2), 1.71 (m, 2H, CH2), 1.44 (m,
2H, CH2). IR (KBr, cmꢄ1): 3420 (yNH), 2941 (yasCH3), 1563, 1513
N2-mesityl-N4-methyl-N6-(1-Boc-piperidin-4-yl)-1,3,5-triazine-
2,4,6-triamine (4a): Intermediate 3 (0.22 g, 0.5 mmol) was dis-
solved in THF, followed by addition of saturated NaHCO3 (2 mL) and
40% w/v aqueous methylamine (0.2 mL). The reaction mixture was
stirred at 50 ꢂC for 5 h. THF was removed under reduced pressure
and extracted with ethyl acetate (2 ꢃ 10 mL). Combined organic
phase was dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure to give the product (4a) as white solid.
Yield: 96%, mp: 131e133 ꢂC, ESI-MS: m/z 442.5 (M þ 1), 443.5
(M þ 2).
(
y
C]N), 812 (
u
C]N). ESI-MS: m/z 432.5 (M þ 1), 216.9 ((M þ 2)/2).
C25H33N7 (431.28).
5.1.5. N2-mesityl-N4-methyl-N6-(1-(4-nitrobenzyl)piperidin-4-yl)-
1,3,5-triazine-2,4,6-triamine (6a3)
White powder, yield: 65.2%. Mp: 127e129 ꢂC. 1H-NMR (DMSO-
d6, ppm)
d
: 8.32 (d, 2H, J ¼ 9.6 Hz, PhH), 7.57 (d, 2H, J ¼ 9.6 Hz, PhH),
6.82 (s, 2H, PhH), 6.52 (m, 2H), 3.58 (d, 2H, CH2), 2.74 (s, 3H,
NeCH3), 2.61 (s, 2H, CH2), 2.20 (s, 3H, CH3), 2.01 (s, 6H, 2 ꢃ CH3),
1.79 (m, 2H, CH2),1.53 (m, 2H, CH2). IR (KBr, cmꢄ1): 3415 (yNH), 2943
N2-mesityl-6-methoxy-N4-(1-Boc-piperidin-4-yl)-1,3,5-triazine-
2,4-diamine (4b): Na (0.35 g, 15 mmol) was dissolved in absolute
(
yasCH3), 1567 (
y
C]N), 1518 (yasNO2), 812 (
uC]N). ESI-MS: m/z 477.3
(M þ 1), 478.3 (M þ 2). C25H32N8O2 (476.26).

64
X. Chen et al. / European Journal of Medicinal Chemistry 51 (2012) 60e66
5.1.6. N2-(1-(4-fluorobenzyl)piperidin-4-yl)-N4-mesityl-N6-methyl-
1,3,5-triazine-2,4,6-triamine (6a4)
1H), 2.74 (m,1H), 2.22 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3), 2.00 (m,1H),
1.81 (m, 1H), 1.71 (m, 1H), 1.55 (m, 1H), 1.43 (m, 1H). IR (KBr, cmꢄ1):
White powder, yield: 55.7%. Mp: 118e120 ꢂC. 1H-NMR (DMSO-
3394 (yNH), 2946 (yasCH3), 1575, 1520 ( C]N), 1498 (yasNO2), 1371
y
d6, ppm)
d: 7.29 (s, 2H, PhH), 7.13 (d, 2H, PhH), 6.81 (s, 2H, PhH),
(
yas
]
CeOeC), 815 (
u
C]N). ESI-MS: m/z 478.3 (M þ 1), 479.3 (M þ 2).
6.44 (m, 2H), 3.41 (d, 2H, CH2), 2.74 (s, 3H, NeCH3), 2.60 (s, 2H, CH2),
2.20 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3),1.91 (m, 2H, CH2),1.71 (m, 2H,
CH2), 1.43 (m, 2H, CH2). IR (KBr, cmꢄ1): 3422 (yNH), 2943 (yasCH3),
C25H31N7O3 (477.25).
5.1.13. N2-(1-(4-fluorobenzyl)piperidin-4-yl)-N4-mesityl-6-
methoxy-1,3,5-triazine-2,4-diamine (6b4)
1570, 1509(
(M þ 2). C25H32FN7 (449.27).
y
C]N), 812 (
u
C]N). ESI-MS: m/z 450.3 (M þ 1), 451.3
White powder, yield: 54.4%. Mp: 123e125 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 7.30 (m, 2H, PhH), 7.12 (m, 2H, PhH), 7.17 (m, 1H, PhH),
5.1.7. N2-mesityl-N4-(1-(4-methoxybenzyl)piperidin-4-yl)-N6-
methyl-1,3,5-triazine-2,4,6-triamine (6a5)
6.84 (s, 2H, PhH), 3.79 (s, 3H, OeCH3), 3.59 (s, 2H, CH2), 3.44 (s, 1H),
3.39 (s, 1H), 2.78 (m, 1H), 2.72 (m, 1H), 2.22 (s, 3H, CH3), 2.07 (s, 6H,
2 ꢃ CH3), 2.94 (m, 2H), 1.80 (m, 1H), 1.69 (m, 1H), 1.52 (m, 1H), 1.40
(m, 1H). IR (KBr, cmꢄ1): 3391 (yNH), 2948 (yasCH3), 1575, 1507
White powder, yield: 52.4%. Mp: 155e157 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 7.18 (d, 2H, PhH), 6.86 (s, 2H, PhH), 6.81 (s, 2H, PhH),
6.43 (m, 2H), 3.73 (s, 3H, OeCH3), 3.37 (s, 1H), 2.74 (s, 3H, NeCH3),
2.60 (d, 2H, CH2), 2.20 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3), 1.90 (m, 2H,
CH2),1.70 (m, 2H, CH2),1.42 (m, 2H, CH2). IR (KBr, cmꢄ1): 3416 (yNH),
(
y
C]N), 1371 (yas
]
CeOeC), 815 (
u
C]N). ESI-MS: m/z 451.3 (M þ 1),
452.3 (M þ 2). C25H31FN6O (450.25).
2938 (yasCH3), 1567, 1511 (
y
C]N), 1246 (yas
]CeOeC), 812 (
u
C]N).
5.1.14. N2-mesityl-6-methoxy-N4-(1-(4-methoxybenzyl)piperidin-
4-yl)-1,3,5-triazine-2,4-diamine (6b5)
ESI-MS: m/z 462.3 (M þ 1), 463.3 (M þ 2) C26H35N7O (461.29).
White powder, yield: 50.2%. Mp: 141e143 ꢂC. 1H-NMR (DMSO-
5.1.8. 3-((4-(4-(mesitylamino)-6-(methylamino)-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzonitrile (6a6)
d6, ppm)
d
: 7.20 (m, 2H, PhH), 6.88 (d, 2H, J ¼ 8.4 Hz, PhH), 6.84 (s,
2H, PhH), 3.79 (s, 3H, OeCH3), 3.73 (d, 3H), 3.59 (s, 2H, CH2), 3.38 (s,
1H), 2.78e2.61 (m, 2H), 2.22 (d, 3H), 2.07 (s, 6H, 2 ꢃ CH3), 1.87e1.96
(m, 2H), 1.78 (m, 1H), 1.68 (m, 1H), 1.49 (m, 1H), 1.37 (m, 1H). IR (KBr,
White powder, yield: 64.8%. Mp: 112e114 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 7.72 (m, 2H, PhH), 7.63 (s, 1H, PhH), 7.55 (m, 1H, PhH),
6.81 (s, 2H, PhH), 6.45 (m, 2H), 3.50 (d, 2H, CH2), 3.17 (d, 1H), 2.74 (s,
3H, NeCH3), 2.61 (s, 2H, CH2), 2.20 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3),
1.72 (m, 2H, CH2), 1.46 (m, 2H, CH2). IR (KBr, cmꢄ1): 3404 (yNH),
cmꢄ1): 3364
yas¼CeOeC), 815 (
C26H34N6O2 (462.27).
(yNH), 2944
( (yC]N), 1371
yasCH3), 1574, 1511
(
u
C]N). ESI-MS: m/z 463.3 (M þ 1), 464.3 (M þ 2).
2941 (yasCH3), 2229 (
yC^N), 1571, 1511 (yC]N), 812 (uC]N). ESI-MS:
m/z 457.3 (M þ 1), 458.3 (M þ 2). C26H32N8 (456.27).
5.1.15. 3-((4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzonitrile (6b6)
5.1.9. 2-((4-(4-(mesitylamino)-6-(methylamino)-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzonitrile (6a7)
White powder, yield: 59.7%. Mp: 192e194 ꢂC. 1H-NMR (DMSO-
d6, ppm)
d
: 7.52e7.73 (m, 3H, PhH), 7.15 (d, 1H, J ¼ 7.8Hz, PhH), 6.86
White powder, yield: 67.1%. Mp: 129e131 ꢂC. 1H-NMR (DMSO-
(s, 2H, PhH), 3.77 (s, 3H, OeCH3), 3.59 (s, 2H, CH2), 3.38 (s, 1H),
2.71e2.78 (m, 2H), 2.22 (d, 3H), 2.07 (s, 6H, 2 ꢃ CH3), 1.95e2.02 (m,
2H), 1.81 (m, 1H), 1.70 (m, 1H), 1.54 (m, 1H), 1.41 (m, 1H). IR (KBr,
d6, ppm) d: 7.80 (s, H, PhH), 7.67 (s, 1H, PhH), 7.55 (s, 1H, PhH), 7.46
(s, 1H, PhH), 6.82 (s, 2H, PhH), 6.46 (m, 2H), 3.61 (d, 2H, CH2), 2.75
(s, 3H, NeCH3), 2.61 (s, 2H, CH2), 2.20 (s, 3H, CH3), 2.08 (s, 6H,
2 ꢃ CH3), 1.74 (m, 2H, CH2), 1.45 (m, 4H). IR (KBr, cmꢄ1): 3414 (yNH),
cmꢄ1): 3386 (yNH), 2947 (yasCH3), 2229 (
yC^N), 1576, 1498 (
yC]N),
1371 (yas¼CeOeC), 815 (
u
C]N). ESI-MS: m/z 458.3 (M þ 1), 459.3
2943 (yasCH3), 2224 (
y
C^N), 1562, 1513 (
y
C]N), 812 (
u
C]N). ESI-MS:
(M þ 2). C26H31N7O (457.26).
m/z 457.3 (M þ 1), 458.3 (M þ 2). C26H32N8 (456.27).
5.1.16. 2-((4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzonitrile (6b7)
5.1.10. 4-((4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-
ylamino)piperidin-1-yl)methyl)benzonitrile (6b1)
White powder, yield: 61.2%. Mp: 211e213 ꢂC. 1H-NMR (DMSO-d6,
White powder, yield: 61.2%. Mp: 106e108 ꢂC. 1H-NMR (DMSO-
ppm) d: 7.44e7.82 (m, 4H, PhH), 7.16e7.24 (m, 1H), 6.85 (s, 2H, PhH),
d6, ppm)
d
: 7.80 (s, H, PhH), 7.67 (s,1H, PhH), 7.55 (s,1H, PhH), 7.46 (s,
3.80 (s, 3H, OeCH3), 3.63 (s, 1H), 3.59 (s, 2H, CH2), 2.74e2.83 (m, 2H),
2.21 (d, 3H), 2.12 (m, 1H), 2.07 (s, 6H, 2 ꢃ CH3), 1.79 (m, 1H), 1.71 (m,
1H),1.54 (m,1H),1.41 (m,1H). IR (KBr, cmꢄ1): 3391 (yNH), 2939 (yasCH3),
2223 (
MS: m/z 458.3 (M þ 1), 459.3 (M þ 2). C26H31N7O (457.26).
1H, PhH), 6.82 (s, 2H, PhH), 6.46 (m, 2H), 3.61 (d, 2H, CH2), 2.75 (s,
3H, NeCH3), 2.61 (s, 2H, CH2), 2.20 (s, 3H, CH3), 2.08 (s, 6H, 2 ꢃ CH3),
1.74 (m, 2H, CH2), 1.45 (m, 4H). IR (KBr, cmꢄ1): 3377 (yNH), 2948
yC^N), 1581, 1473 (y ]CeOeC), 812 (uC]N). ESI-
C]N), 1371 (yas
(
yasCH3), 2227 (
yC^N), 1575, 1498 (yC]N), 815 (uC]N). ESI-MS: m/z
458.5 (Mþ1), 459.5 (M þ 2), 480.4 (M þ Na). C26H31N7O (457.26).
5.1.17. General procedure for the synthesis of target compounds
6a01e4 and 6b01e4
5.1.11. N2-(1-benzylpiperidin-4-yl)-N4-mesityl-6-methoxy-1,3,5-
triazine-2,4-diamine (6b2)
N,N’-Carbonyldiimidazole (CDI, 1 eq) and appropriate substituted
benzoic acid (1 eq) were added to anhydrous DMF at 0 ꢂC succes-
sively, and then stirred for 30 min. Compounds 5a (or 5b) (1.1 eq)
was added to the mixture slowly and stirred at room temperature
overnight. The solvent was removed under reduced pressure, and
water (20 mL) was added. Extracted with ethyl acetate (2 ꢃ 10 mL),
and the organic phase was dried over anhydrous Na2SO4 to give the
corresponding crude product, which was purified by flash column
chromatography to afford compounds 6a01e4 (or 6b01e4).
White powder, yield: 55.7%. Mp: 104e106 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 7.20e7.34 (m, 5H, PhH), 7.13 (d, 1H), 6.84 (s, 2H, PhH),
3.81 (s, 3H, OeCH3), 3.62 (s, 2H, CH2), 3.46 (s, 1H), 3.41 (s, 1H), 2.80
(m,1H), 2.74 (m, 1H), 2.20 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3),1.68 (m,
2H, CH2), 1.39 (m, 2H). IR (KBr, cmꢄ1): 3393 (yNH), 2946 (yasCH3),
1574, 1497 (y ]CeOeC), 815 (uC]N). ESI-MS: m/z
C]N), 1370 (yas
433.6 (M þ 1), 434.5 (M þ 2). C25H32N6O (432.26).
5.1.12. N2-mesityl-6-methoxy-N4-(1-(4-nitrobenzyl)piperidin-4-
yl)-1,3,5-triazine-2,4-diamine (6b3)
5.1.18. 4-(4-(4-(mesitylamino)-6-(methylamino)-1,3,5-triazin-2-
ylamino)piperidine-1-carbonyl)benzoic acid (6a01)
White powder, yield: 63.9%. Mp: 119e121 ꢂC. 1H-NMR (DMSO-
d6, ppm)
d
: 8.20 (m, 2H, PhH), 7.60 (m, 2H, PhH), 7.17 (m,1H), 6.85 (s,
White powder, yield: 42.3%. Mp: 187e189 ꢂC.1H-NMR (DMSO-d6,
2H, PhH), 3.80 (s, 3H, OeCH3), 3.60 (d, 2H, CH2), 3.56 (s,1H), 2.81 (m,
ppm) d: 7.99 (s, 2H, PhH), 7.46 (d, 2H, PhH), 6.82 (s, 2H, PhH),

X. Chen et al. / European Journal of Medicinal Chemistry 51 (2012) 60e66
65
6.52e6.70 (m, 2H), 4.36 (s,1H), 3.92e4.10 (m,1H), 2,95e3.12 (m, 2H),
5.1.25. (4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-ylamino)
2.76 (s,1H), 2.61 (s,1H), 2.20 (s, 3H, CH3), 2.08 (s, 6H, 2 ꢃ CH3),1.91 (s,
piperidin-1-yl)(4-nitrophenyl)methanone (6b04)
2H),1.70e1.80 (m, 2H),1.29e1.49 (m, 2H). IR (KBr, cmꢄ1): 3401 (yNH),
White powder, yield: 47.2%. Mp: 123e125 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 8.29 (m, 2H, PhH), 7.63 (m, 2H, PhH), 7.19e7.34 (m, 1H),
2924 (yasCH3), 1629 (
yC]O), 1571, 1507 (yC]N), 812 (uC]N). ESI-MS:
m/z 490.4 (M þ 1), 491.4 (M þ 2). C26H31N7O3 (489.25).
6.85 (s, 2H, PhH), 4.35e4.43 (m, 1H), 3.79e3.81 (d, 2H), 3.60 (s, 1H),
3.39e3.48 (m, 1H), 3.13 (m, 1H), 2.98 (m, 1H), 2.20 (s, 3H, CH3), 2.07
(s, 6H, 2 ꢃ CH3), 1.86 (m, 1H), 1.24e1.53 (m, 4H). IR (KBr, cmꢄ1):
5.1.19. (4-hydroxyphenyl)(4-(4-(mesitylamino)-6-(methylamino)-
1,3,5-triazin-2-ylamino)piperidin-1-yl)methanone (6a02)
3385 (yNH), 2952 (yasCH3), 1633 (
yC]O), 1575, 1497 (yC]N), 1371
White powder, yield: 44.5%. Mp: 157e159 ꢂC. 1H-NMR (DMSO-
(
yas
]CeOeC), 815 (
uC]N). ESI-MS: m/z 492.4 (M þ 1), 493.4 (M þ 2).
d6, ppm)
d
: 7.21 (d, 2H, PhH), 6.82 (s, 2H, PhH), 6.78 (s, 2H, PhH),
C25H29N7O4 (491.23).
2.75 (s, 2H), 2.61 (s, 1H), 2.20 (s, 3H, CH3), 2.08 (s, 6H, 2 ꢃ CH3), 1.91
(s, 2H), 1.33 (m, 3H). IR (KBr, cmꢄ1): 3398 (yNH), 2924 (yasCH3), 1626
5.2. Biological activity
(y
C]O), 1574, 1514 (
y
C]N), 811 (
u
C]N). ESI-MS: m/z 462.3 (M þ 1),
463.3 (M þ 2). C25H31N7O2 (461.25).
5.2.1. In vitro anti-HIV activity assays
Evaluation of the antiviral activity of the compounds against
HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4 cells was per-
formed using the MTT assay as previously described [18]. Stock
solutions (10 ꢃ final concentration) of test compounds were added
5.1.20. (4-(4-(mesitylamino)-6-(methylamino)-1,3,5-triazin-2-
ylamino)piperidin-1-yl)(phenyl)methanone (6a03)
White powder, yield: 49.4%. Mp: 142e144 ꢂC. 1H-NMR (DMSO-d6,
ppm) d: 7.44 (s, 2H, PhH), 7.35 (d, 2H, PhH), 6.82 (s, 2H, PhH), 6.51 (m,
in 25-mL volumes to two series of triplicate wells so as to allow
2H), 4.10 (m,1H), 3.17 (s, 2H), 2.75 (s,1H), 2.61 (s,1H), 2.20 (s, 3H, CH3),
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
microtiter trays using a Biomek 3000 robot (Beckman instru-
ments, Fullerton, CA). Untreated control HIV- and mock-infected
cell samples were included for each sample.
2.08 (s, 6H, 2 ꢃ CH3), 1.80 (m, 2H), 1.32 (m, 2H). IR (KBr, cmꢄ1): 3385
(
yNH), 2921 (yasCH3),1636 (
yC]O),1571,1496 (yC]N), 812 (uC]N). ESI-
MS: m/z 446.5 (M þ 1), 447.4 (M þ 2). C25H31N7O (445.26).
5.1.21. (4-(4-(mesitylamino)-6-(methylamino)-1,3,5-triazin-2-
ylamino)piperidin-1-yl)(4-nitrophenyl)methanone (6a04)
White powder, yield: 50.8%. Mp: 152e154 ꢂC. 1H-NMR (DMSO-
HIV-1(IIIB) [23] or HIV-2 (ROD) [24] stock (50 mL) at 100e300
CCID50 (cell culture infectious dose) or culture medium was
added to either the infected or mock-infected wells of the
microtiter tray. Mock-infected cells were used to evaluate the
effect of test compound on uninfected cells in order to assess the
cytotoxicity of the test compound. Exponentially growing MT-4
cells [25] were centrifuged for 5 min at 1000 rpm and the
supernatant was discarded. The MT-4 cells were resuspended at
d6, ppm) d: 8.29 (d, 2H, PhH), 7.63 (d, 2H, PhH), 6.82 (s, 2H, PhH),
6.51e6.69 (m, 2H), 4.36 (s, 1H), 3.93e4.10 (m, 1H), 3.40e3.47 (m,
1H), 2.89e3.17 (m, 2H), 2.75 (s, 1H), 2.62 (s, 1H), 2.20 (s, 3H, CH3),
2.08 (s, 6H, 2 ꢃ CH3), 1.82 (m, 2H),1.24e1.51 (m, 3H). IR (KBr, cmꢄ1):
3408 (yNH), 2945 (yasCH3), 1633 (
yC]O), 1579, 1520 (yC]N), 1437
(
yasNO2), 812 (
u
C]N). ESI-MS: m/z 491.5 (M þ 1), 492.4 (M þ 2).
C25H30N8O3 (490.24).
6 ꢃ 105 cells/mL, and 50-
mL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of
mock- and HIV-infected cells was examined spectrophotometri-
cally by the MTT assay.
5.1.22. 4-(4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-
ylamino)piperidine-1-carbonyl)benzoic acid (6b01)
White powder, yield: 40.9%. Mp: 147e149 ꢂC. 1H-NMR (DMSO-
The MTT assay is based on the reduction of yellow coloured 3-
d6, ppm) d: 8.00 (m, 2H, PhH), 7.46 (m, 2H, PhH), 7.16e7.33 (m, 1H),
(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
6.85 (s, 2H, PhH), 4.39 (m, 1H), 3.93e4.23 (m, 1H), 3.81 (s, 1H), 3.62
(s, 1H), 3.11 (m, 1H), 2.91e2.97 (m, 1H), 2.21 (s, 3H, CH3), 2.08 (s, 6H,
2 ꢃ CH3),1.91 (s,1H),1.84 (m,1H),1.72 (m,1H),1.36e1.52 (m, 2H). IR
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
genase of metabolically active cells to a blue-purple formazan that
can be measured spectrophotometrically. The absorbances were
read in an eight-channel computer-controlled photometer (Multi-
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-
infected control sample by 50%. The concentration achieving 50%
protection from the cytopathic effect of the virus in infected cells
was defined as the 50% effective concentration (EC50).
(KBr, cmꢄ1): 2924 (yasCH3), 1696 (
yC]O), 1578, 1505 (yC]N), 1372
(
yas
]
CeOeC), 813 (
u
C]N). ESI-MS: m/z 491.4 (M þ 1), 492.4 (M þ 2).
C26H30N6O4 (490.23).
5.1.23. (4-hydroxyphenyl)(4-(4-(mesitylamino)-6-methoxy-1,3,5-
triazin-2-ylamino)piperidin-1-yl)methanone (6b02)
White powder, yield: 42.8%. Mp: 154e156 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 7.18e7.25(m, 2H, PhH), 6.85 (s, 2H, PhH), 6.76e6.81 (m,
2H, PhH), 3.79e3.81 (s, 1H), 3.60 (s, 1H), 2.21 (s, 3H, CH3), 2.08 (s,
6H, 2 ꢃ CH3), 1.87 (s, 1H), 1.76 (s, 1H), 1.44 (m, 1H), 1.24e1.32 (m,
2H). IR (KBr, cmꢄ1): 3388 (yNH), 2952 (yasCH3), 1578, 1501 (
yC]N),
5.2.2. HIV-1 RT inhibition assay
1371 (yas
(M þ 2). C25H30N6O3 (462.24).
]
CeOeC), 814 (
u
C]N). ESI-MS: m/z 463.3 (M þ 1), 464.3
Inhibition of HIV-1 RT assay was performed by using homo-
polymer template/primer linked to microtiter plate, biotin-dUTP
and RT with detection of ELISA for quantifying expression [22].
The incorporated quantities of the biotin-dUTP into the template
represented the activity of HIV-1 RT. IC50 values corresponded to
the concentration of the piperidine-substituted triazine derivatives
required to inhibit biotin-dUTP incorporation by 50%.
5.1.24. (4-(4-(mesitylamino)-6-methoxy-1,3,5-triazin-2-ylamino)
piperidin-1-yl)(phenyl)methanone (6b03)
White powder, yield: 45.5%. Mp: 106e108 ꢂC. 1H-NMR (DMSO-
d6, ppm) d: 7.30e7.46 (m, 5H, PhH), 6.85 (s, 2H, PhH), 4.36e4.43 (m,
1H), 3.93e4.01 (m, 1H), 3.79e3.84 (d, 2H), 3.60 (s, 1H), 3.10 (s, 1H),
2.91 (m, 1H), 2.21 (s, 3H, CH3), 2.07 (s, 6H, 2 ꢃ CH3), 1.71e1.84 (m,
2H), 1.24e1.52 (m, 3H). IR (KBr, cmꢄ1): 2950 (yasCH3), 1575, 1497
Conflict of interest
(
y
C]N), 1370 (yas
]
CeOeC), 815 (
u
C]N). ESI-MS: 447.6 (M þ 1),
448.5 (M þ 2). C25H30N6O2 (446.24).
None.

66
X. Chen et al. / European Journal of Medicinal Chemistry 51 (2012) 60e66
[10] D.J. Kertesz, C. Brotherton-Pleiss, M.M. Yang, Z.G. Wang, X.F. Lin, Z.X. Qiu,
Acknowledgment
D.R. Hirschfeld, S. Gleason, T. Mirzadegan, P.W. Dunten, S.F. Harris,
A.G. Villasenor, J.Q. Hang, G.M. Heilek, K. Klumpp, Bioorg. Med. Chem. Lett. 20
(2010) 4215e4218.
The financial support from the National Natural Science Foun-
dation of China (NSFC No. 81102320, No. 30873133, No. 30772629
and 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 and No. 070422083), Independent Innovation
Foundation of Shandong University (IIFSDU, No. 2010GN044),
Shandong Postdoctoral Innovation Science Research Special
Program (No. 201002023) and China Postdoctoral Science
Foundation funded project (No. 20100481282) is gratefully
acknowledged.
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