Discovery of piperidine-linked pyridine analogues as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1002/cmdc.201300130

In our continued efforts to discover more active and less toxic HIV-1 non-nucleoside reverse transcriptase inhibitors, we recently designed a novel series of piperidine-linked pyridine analogues on the basis of diarylpyrimidine derivatives, among which two drugs-etravirine and rilpivirine-are approved for use by the US FDA. The title compounds were evaluated for activity against wild-type and resistant mutant strains of HIV-1 as well as HIV-2 in MT-4 cells. The highly potent compound BD-c1 (EC₅₀=10nM, CC₅₀≥146μM, SI≥14126) displays lower cytotoxicity and higher selectivity than etravirine (EC₅₀=2.2nM, CC₅₀=28μM, SI=12884) against wild-type HIV-1. Compound BD-e2 (EC₅₀=5.1nM) shows greater antiviral efficacy against wild-type HIV-1 than do the four reference drugs nevirapine, delavirdine, efavirenz, and zidovudine. Many compounds were also found to be active against the frequently observed drug-resistant double mutant (K103N+Y181C) HIV-1 strain. Herein we report the design, synthesis, anti-HIV evaluation, preliminary structure-activity relationships, and molecular simulations of novel piperidine-linked pyridine analogues.

Full text of DOI:10.1002/cmdc.201300130

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DOI: 10.1002/cmdc.201300130
Discovery of Piperidine-Linked Pyridine Analogues as
Potent Non-nucleoside HIV-1 Reverse Transcriptase
Inhibitors
Xuwang Chen,[a] Yuanyuan Li,[a] Shufang Ding,[a] Jan Balzarini,[b] Christophe Pannecouque,[b]
Erik De Clercq,[b] Huiqing Liu,[c] and Xinyong Liu*[a]
In our continued efforts to discover more active and less toxic
HIV-1 non-nucleoside reverse transcriptase inhibitors, we re-
cently designed a novel series of piperidine-linked pyridine an-
alogues on the basis of diarylpyrimidine derivatives, among
which two drugs—etravirine and rilpivirine—are approved for
use by the US FDA. The title compounds were evaluated for
activity against wild-type and resistant mutant strains of HIV-
1 as well as HIV-2 in MT-4 cells. The highly potent compound
BD-c1 (EC50 =10 nm, CC50 ꢀ146 mm, SIꢀ14126) displays lower
cytotoxicity and higher selectivity than etravirine (EC50 =
2.2 nm, CC50 =28 mm, SI=12884) against wild-type HIV-1. Com-
pound BD-e2 (EC50 =5.1 nm) shows greater antiviral efficacy
against wild-type HIV-1 than do the four reference drugs nevir-
apine, delavirdine, efavirenz, and zidovudine. Many com-
pounds were also found to be active against the frequently
observed drug-resistant double mutant (K103N+Y181C) HIV-
1 strain. Herein we report the design, synthesis, anti-HIV evalu-
ation, preliminary structure–activity relationships, and molecu-
lar simulations of novel piperidine-linked pyridine analogues.
Introduction
According to statistics reported by the World Health Organiza-
tion (WHO)/UNAIDS regarding the acquired immunodeficiency
syndrome (AIDS) epidemic, there were 34 million people infect-
ed with human immunodeficiency virus (HIV) in 2012,[1] under-
scoring the importance of continued drug discovery efforts
against this virus. Although unprecedented success has been
realized by several drugs approved for the treatment of HIV in-
fections[2] and use of the highly active antiretroviral therapy
(HAART) regimen, undesirable toxic side effects along with
rapid emergence of drug resistance resulting from prolonged
HAART often compromise clinical application and effectiveness
of treatment.[3] Therefore, the discovery and development of
novel anti-HIV drugs with high potency and low toxicity is
clearly needed.
diverse structural modifications and optimizations over the
past decade.[4] Among the structural modifications, DAPY deriv-
atives with piperidine substitutions at the right wing (com-
pound 1) show excellent activity against HIV-1 replication
(wild-type EC50 =0.3 nm).[5] Encouraged by this result, we de-
signed and synthesized piperidine-linked triazine derivatives
that show high potency and low cytotoxicity (compound 2,
wild-type EC50 =4.61 nm, SI=5945, Figure 1).[6] In our contin-
ued efforts to discover more active and less toxic HIV-
1 NNRTIs, we recently designed a series of piperidine-linked
pyridine analogues (Figure 2) with the following structural
HIV-1 non-nucleoside reverse transcriptase inhibitors
(NNRTIs), with five drugs approved by the US Food and Drug
Administration (FDA), have become an indispensable compo-
nent of first-line drug regimens. Both etravirine and rilpivirine
are structural members of the diarylpyrimidine (DAPY) family
of derivatives, which have attracted considerable attention in
[a] Dr. X. Chen, Y. Li, S. Ding, Prof. X. Liu
Department of Medicinal Chemistry
Key Laboratory of Chemical Biology (Ministry of Education)
School of Pharmaceutical Sciences, Shandong University
44 West Culture Road, 250012 Ji’nan, Shandong (P.R. China)
E-mail: xinyongl@sdu.edu.cn
[b] Prof. J. Balzarini, Prof. C. Pannecouque, Prof. E. De Clercq
Rega Institute for Medical Research, KU Leuven
Minderbroedersstraat 10, 3000 Leuven (Belgium)
[c] Dr. H. Liu
Institute of Pharmacology, School of Medicine, Shandong University
44 West Culture Road, 250012 Ji’nan, Shandong (P.R. China)
Figure 1. Structures of HIV-1 NNRTIs.
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Results and Discussion
Chemistry
An expeditious synthesis of the
novel piperidine-linked pyridine
analogues was carried out in our
research group. As depicted in
Scheme 1, the intermediate S-2
was readily obtained through
nucleophilic substitution be-
Figure 2. Design strategies of new piperidine-linked pyridine compounds.
tween 4-amino-1-Boc-piperidine
and the starting material 2,6-di-
characteristics: 1) a nitro or amino group was introduced onto
pyridine ring as a potential hydrogen bond acceptor or donor
instead of the N atom of the pyrimidine or triazine ring, which
was observed similarly in diarylpyridine NNRTIs;[7] 2) the para
substituents (R1 =Me, CN) of 2,4,6-trisubstituted phenol or ani-
line, and the linker (X=O, NH) were taken into consideration
for combining the lead compounds 1 and 2. Also, the hetero-
cyclic aryl (Ar) group, and phenyl group with polar hydrophilic
substituents, were designed to orient directly into the protein–
solvent interface, as reported previously.
chloro-3-nitropyridine (S-1) at room temperature with good
yield (85%).[8] Coupling intermediate S-2 and sterically hin-
dered 2,4,6-trimethylphenol (3 equiv) with cesium carbonate
(4.5 equiv) as the base in dioxane at reflux for 7 h gave the key
intermediate S-3. The reduced product S-5 was prepared via
catalytic hydrogenation of intermediate S-3 under Pd-C/H2/
MeOH conditions at room temperature in satisfactory yield
(98%). Accordingly, intermediates S-3’ and S-5’ with R1 =CN
were obtained by following the same procedure as the prepa-
ration of S-3 and S-5. The mixture of S-2 and the liquid 2,4,6-
trimethylaniline (2 equiv) was heated at 1308C without the ad-
dition of solvent for 5 h to afford key intermediate S-7.
Intermediates S-3, S-3’, S-5, S-5’, and S-7 were dissolved in
dichloromethane in the presence of trifluoroacetic acid (TFA,
10 equiv) and stirred at room temperature to yield intermedi-
Scheme 1. Synthesis of compounds BD-a1–a4, BD-b1–b4, BD-c1–c4, BD-d1–d4 and BD-e1–e4. Reagents and conditions: a) Na2CO3, EtOH, RT, overnight;
b) Cs2CO3, dioxane, reflux, 7 h; c) TFA, CH2Cl2, RT, overnight; d) substituted benzyl chloride or 4-picolyl chloride hydrochloride, K2CO3, acetone, RT, overnight;
e) Pd-C, H2, MeOH, RT, overnight; f) D (1308C), 5 h.
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ates S-4, S-4’, S-6, S-6’, and S-8,
respectively, with yields >90%.[9]
These were alkylated at the N
atom of piperidine to afford the
title compounds BD-a1–a4, BD-
b1–b4, BD-c1–c4, BD-d1–d4,
and BD-e1–e4.
Melting point, thin-layer chro-
matography (TLC), 1H NMR, and
ESIMS analysis of all final com-
pounds, as well as HRMS data
and 13C NMR spectra for repre-
sentative compounds from each
sub-series, were determined and
are in full agreement with the
proposed structures.
Table 1. Anti-HIV activity and cytotoxicity in MT-4 cells infected with wild-type HIV-1 (IIIB) and HIV-2 (ROD).
[a]
Compd
Ar
EC50
CC50 [mm][b]
SI[c]
HIV-1 [nm]
HIV-2 [mm]
>24
>22
>19
>279
>28
>26
>26
>30
ꢀ146
>27
>27
HIV-1
1375
2600
1600
>335
241
HIV-2
<1
BD-a1
BD-a2
BD-a3
BD-a4
BD-b1
BD-b2
BD-b3
BD-b4
BD-c1
BD-c2
BD-c3
BD-c4
BD-d1
BD-d2
BD-d3
BD-d4
BD-e1
BD-e2
BD-e3
BD-e4
1[5]
4-SO2Me-Ph
4-SO2NH2-Ph
4-CONH2-Ph
pyridin-4-yl
4-SO2Me-Ph
4-SO2NH2-Ph
4-CONH2-Ph
pyridin-4-yl
4-SO2Me-Ph
4-SO2NH2-Ph
4-CONH2-Ph
pyridin-4-yl
4-SO2Me-Ph
4-SO2NH2-Ph
4-CONH2-Ph
pyridin-4-yl
4-SO2Me-Ph
4-SO2NH2-Ph
4-CONH2-Ph
pyridin-4-yl
18Æ12
8.5Æ1.6
12Æ5.3
833Æ651
116Æ55
112Æ97
58Æ5.7
84Æ9.2
10Æ2.5
19Æ6.9
31Æ7.0
39Æ10
47Æ15
53Æ23
36Æ13
56Æ11
24Æ1.8
22Æ1.9
19Æ7.9
<1
<1
>279
–
–
28Æ1.5
26Æ2.9
26Æ4.0
30Æ3.5
<1
<1
<1
<1
234
459
358
ꢀ146
ꢀ14126
1470
877
27Æ1.6
27Æ1.1
26Æ3.4
28Æ3.0
24Æ1.0
27Æ2.5
31Æ1.1
21Æ9.1
14Æ8.0
25Æ2.1
31Æ5.8
<1
<1
ꢁ4
<1
<1
<1
<1
<1
<1
<1
<1
ꢀ6.7
654
607
456
742
>28
>24
>27
>31
>21
>14
>25
>31
559
7.8Æ0.77
5.1Æ2.4
9.6Æ1.5
10Æ2.6
0.3
2662
2692
2645
2935
Anti-HIV activity in MT-4 cells
The 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide
(MTT) method, as reported previ-
ously,[6,10] was used to evaluate
the newly synthesized com-
pounds along with four refer-
ence drugs: nevirapine (NVP),
delavirdine mesylate (DLV), efa-
virenz (EFV), and zidovudine
(AZT). These compounds were
assayed for their anti-HIV activity
and cytotoxicity in MT-4 cells in-
fected with wild-type HIV-1 (IIIB),
resistant mutant strain (K103N+
2[6]
4.61Æ1.90
113Æ69
107Æ69
8.7Æ1.5
6.5Æ2.4
2.2Æ0.4
>27.39
ND[d]
ND
27.39Æ2.34
5945
>132
>338
>727
>14445
12884
<1
ND
ND
ND
NVP
DLV
EFV
AZT
ETV[,11e]
>15
>36
>6.3
>93
28Æ12
ND
0.006Æ0.001
>28
>15690
<1
[a] Compound concentration required for 50% protection of MT-4 cells against HIV-1-induced cytotoxicity; data
represent the mean ÆSD of at least two separate experiments performed in triplicate. [b] Compound concen-
tration required to decrease normal uninfected cell viability by 50%.; data represent the mean ÆSD of at least
two separate experiments performed in triplicate. [c] Selectivity index: CC50/EC50; “–” indicate incalculable
values. [d] Not determined. [e] Data were obtained from the same research group using the same method.
Y181C) of HIV-1, as well as HIV-2 (ROD strain). Results of the
biological evaluations, expressed as EC50 (50% HIV-1 replication
inhibitory concentration), CC50 (50% cytotoxic concentration),
and SI (selectivity index, given by the CC50/EC50 ratio) values,
are summarized in Tables 1 and 2, and data represent the
mean of at least two separate experiments.
a drug-resistant mutant strain of HIV-1 (K103N+Y181C), which
is a frequently encountered mutant resulting from the treat-
ment of AIDS patients with HIV-1 NNRTIs.[12] As the data in
Table 2 indicate, the compounds show activity (EC50 values) in
the low-micromolar concentration range and good relative ac-
tivity (fold resistance). Compounds BD-d1 (EC50 =0.99 mm) and
BD-d2 (EC50 =0.99 mm) show better activities than the refer-
ence drugs NVP and DLV, and the relative activity of the two
compounds (respective fold resistance: 21 and 19) is superior
to that of the reference drugs NVP, DLV, and EFV, but still inferi-
or to that of the new drug ETV (fold resistance: 16).
All the newly designed and synthesized compounds were
active against wild-type HIV-1, with EC50 values ranging from
5.1 nm to 832 nm, and most compounds exhibited stronger in-
hibitory activity than those of NVP and DLV, as listed in Table 1.
Compound BD-e2 was the most potent compound, with an
EC50 value of 5.1 nm, which proved lower than those of the
four reference drugs NVP, DLV, EFV, and AZT, but higher than
that of the newly FDA-approved drug etravirine (ETV) against
wild-type HIV-1 (EC50 =2.2 nm,[11] assayed in the same research
group using the same method). Compound BD-c1 (EC50 =
10 nm) showed significantly low cytotoxicity, with a CC50 value
>146 mm and high selectivity, with an SI value >14126,
making BD-c1 a promising lead compound similar to the new
drug ETV (CC50 =28 mm, SI=12884). Furthermore, none of the
newly synthesized compounds were active against HIV-2 (ROD)
at a subtoxic concentration in MT-4 cells.
Preliminary structure–activity relationship (SAR) information
based on the results of the antiviral assays indicates the follow-
ing: 1) by comparison with the activities between compounds
BD-a1–a4 and BD-e1–e4, compounds with an ÀNHÀ linker are
more active than those with an ÀOÀ linker; 2) reduction of the
ÀNO2 group (compounds BD-a1–a3 and BD-c1–c4) to an ÀNH2
group (compounds BD-b1–b3 and BD-d1–d4) decreased the
activity against wild-type HIV-1, but increased the activity
against the resistant mutant strain of HIV-1; 3) compounds
with a 4-cyano-2,6-dimethylphenyl group (R1 =CN, BD-d1–d4),
with the same moiety as the new drug ETV, showed better ac-
tivity against wild-type and the resistant mutant strain of HIV-
1 than compounds with a 2,4,6-trimethylphenyl group (R1 =
Notably, most of the compounds (BD-a3, BD-b1–b4, BD-c4,
and BD-d1–d4) displayed low-micromolar activities against
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hibit the activity of HIV-1 RT effectively, and therefore can be
classified as HIV-1 RT inhibitors.
Table 2. Anti-HIV activities and cytotoxicity in MT-4 cells infected with re-
sistant mutant strain (K103N+Y181C).
Compd
EC50 [mm]
FR[a]
CC50 [mm]
SI
Molecular simulation analysis
BD-a1
BD-a2
BD-a3
BD-a4
BD-b1
BD-b2
BD-b3
BD-b4
BD-c1
BD-c2
BD-c3
BD-c4
BD-d1
BD-d2
BD-d3
BD-d4
BD-e1
BD-e2
BD-e3
BD-e4
1[5]
>24
ꢀ5.7
5.8Æ0.9
>1374
ꢀ673
500
ꢀ287
48
24Æ1.8
22Æ1.9
19Æ7.8
<1
ꢁ4
3
The new compounds were designed on the basis of piperi-
dine-substituted DAPY derivatives (compound 1) and piperi-
dine-substituted triazine derivatives (compound 2). To deter-
mine the binding mode of the newly synthesized compounds,
molecular simulation studies were carried out. The most prom-
ising compounds BD-c1 and BD-e2, as well as lead compounds
1 and 2, were docked into the non-nucleoside inhibitor bind-
ing pocket (NNIBP) of wild-type HIV-1 RT (PDB code: 3M8Q)[5]
using the software package Surflex-Dock SYBYL-X 1.1. Default
parameters were used as described in the SYBYL-X 1.1 manual,
unless otherwise specified; this is illustrated in detail in the Ex-
perimental Section.
ꢀ239
>279
–
5.6Æ2.5
1.5Æ0.58
3.9Æ0.58
2.5Æ1.3
28Æ1.5
26Æ2.9
26Æ4.0
30Æ3.5
5
17
7
14
68
30
12
ꢀ32
ꢀ7.6
>27
ꢀ3045
ꢀ410
>876
198
ꢀ146
–
27Æ1.6
27Æ1.1
25Æ3.3
28Æ3.0
24Æ1.0
27Æ2.5
31Æ1.1
21Æ9.1
14Æ8.0
25Æ2.1
31Æ5.8
ꢁ4
<1
3
29
24
24
30
<1
ꢁ2
<1
<1
7.7Æ3.5
0.99Æ0.06
0.99Æ0.01
1.1Æ0.08
1.0Æ0.2
21
19
31
18
>21
>2662
ꢀ1156
>2645
>2934
67
It is apparent that the new compounds BD-c1 and BD-e2
can bind the NNIBP in a manner similar to that of lead com-
pounds 1 and 2, as observed by superimposition of the
docked conformations (Figure 3a). The Ar groups of the four
compounds were oriented directly into the protein–solvent in-
terface, and in particular, the polar hydrophilic substituents at
the para position extend to the exterior water (Figure 3b). De-
tailed analysis of the docking results revealed the following no-
table features, which were also observed for the lead com-
pounds 1 and 2 as well as other DAPY compounds:[13] 1) The
4-cyano-2,6-dimethylphenyl group of BD-c1 and the mesityl
moiety of BD-e2 fit into a sub-pocket formed by hydrophobic
aromatic residues Tyr181, Tyr188, Phe227, and Trp229. Looking
closely at this part, it is clear that the 2,4,6-position-substituted
phenyl group is parallel to the side chain phenyl group of
Tyr181 or Tyr188, exhibiting p–p interactions, and is perpendic-
ular to the ring planes of Phe227 and the highly conserved
Trp229, forming interesting CH–p interactions. 2) The nitro
group and the ÀNHÀ linker of compounds BD-c1 and BD-e2
form two hydrogen bonds with the backbone carbonyl and a-
amino group of Lys101, which are able to decrease the binding
free energy for inhibitors (Figure 3c,d). Additionally, the À
SO2Me group of BD-c1 could form a hydrogen bond with
Val106, and the ÀSO2NH2 group of BD-e2 forms hydrogen
bonds with Val106 and Lys104 (Figure 3c,d), which are located
around the protein–solvent interface and could improve stabil-
ity of RT–inhibitor complex. However, the new compounds dis-
play less potent activity against the resistant K103N+Y181C
mutant strain of HIV-1 because of the following reason accord-
ing to the analysis above. The new compounds form two hy-
drogen bonds with the Lys101 backbone, whereas lead com-
pound 1 forms another hydrogen bond with the Lys103 back-
bone through a water bridge, and this hydrogen bond interac-
tion with main chain is unlikely to be disrupted by side chain
mutations.
ꢀ5.9
>25
>31
0.020
2[6]
0.56Æ0.12
5.1Æ5.1
121.6
27.39Æ2.34
49
>3
NVP
DLV
EFV
AZT
ETV[,11b]
45
>338
58
>15
>36
>36
>6.3
>93
28Æ11
–
0.5Æ0.1
>12
ꢀ14445
817
0.0063Æ0.00003
0.034Æ0.005
1.0
15
[a] Fold resistance: ratio of EC50 value against the drug-resistant strain
over EC50 value against wild-type HIV-1 (EC50mutant/EC50wt); data represent
the mean ÆSD of at least two separate experiments performed in tripli-
cate. [b] Data were obtained from the same research group using the
same method.
Me, BD-b1–b4). The biological evaluation results and the im-
portant SAR information analyzed above will be beneficial in
the future design of new potent compounds.
Inhibition of HIV-1 RT
To directly prove that the newly synthesized compounds
target HIV-1 RT, the active compound BD-c1 was selected to
carry out recombinant HIV-1 RT inhibitory assays using the
template/primer hybrid poly(A)·oligo(dT)15 (RT kit, Roche). The
results show that BD-c1 displays good, but slightly inferior in-
hibitory activity (IC50 =1.2 mm) against HIV-1 RT relative to the
reference drug ETV (IC50 =0.94 mm) (Table 3). Thus, the newly
synthesized piperidine-linked pyridine analogues do indeed in-
Table 3. In vitro recombinant HIV-1 RT inhibitory assay of compounds
BD-c1 and ETV.
Compd
IC50 [mm][a]
Overall, the binding model analysis supports our initial
design hypothesis of introducing a nitro group as a potential
hydrogen bond receptor in place of the N atom of pyrimidine
or triazine rings. Further structural optimizations will consider
these aspects.
BD-c1
ETV
1.2
0.94
[a] Data were obtained by standard ELISA, and values are the mean of
two parallel determinations following the kit protocol.
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Figure 3. Predicted binding modes and docking poses (PDB code: 3M8Q) shown by PyMOL:[14] a) superimposition of the docked conformations of com-
pounds 1 (red), 2 (magenta), BD-c1 (blue), and BD-e2 (yellow); b) superimposition of the docked conformations in transparent surface mode; c) compound
BD-c1; d) compound BD-e2. Hydrogen bonds are indicated with dashed lines in yellow, and hydrogen atoms are omitted for clarity.
Conclusions
Experimental Section
Chemistry
In summary, we designed and synthesized a series of piperi-
dine-linked pyridine analogues to continue our research into
the discovery of more active and less toxic HIV-1 NNRTIs. From
a chemical point of view, the preparation of these compounds
is particularly simple and was carried out via an expeditious
route. The screening results in MT-4 cells indicate that most
compounds show good activity against wild-type HIV-1 in the
nanomolar concentration range and moderate activity against
the K103N+Y181C resistant mutant strain of HIV-1, with EC50
values in the low-micromolar concentration range. Compound
BD-c1 (EC50 =10 nm, CC50 ꢀ146 mm, SIꢀ14126) shows lower
cytotoxicity and higher selectivity than the newly FDA-ap-
proved drug ETV, and BD-e2 (EC50 =5.1 nm) shows better anti-
viral efficacy than the four reference drugs NVP, DLV, EFV, and
AZT. Compounds BD-c1 and BD-e2 could be further developed
as lead anti-HIV-1 agents. In addition, preliminary SAR studies
and molecular simulations were performed to analyze the in-
teractions between these analogues and HIV-1 RT. Further
studies are ongoing in our laboratories and will be reported in
due course.
All melting points were determined on a micromelting point appa-
ratus and are uncorrected. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were obtained on a Brucker AV400 NMR spec-
trometer in the indicated solvents. Chemical shifts (d) are reported
1
with TMS as internal reference for H NMR spectra and [D6]DMSO
(d=39.6 ppm) or CDCl3 (d=79.6 ppm) for 13C NMR spectra. Mass
spectra were taken on an LC Autosampler Standard G1313A instru-
ment. TLC was performed on silica gel GF254 for TLC, and spots
were visualized by iodine vapor or by irradiation with UV light
(l 254 nm). Flash column chromatography was performed on
column packed with silica gel 60 (200–300 mesh). Solvents were re-
agent grade, and when necessary, were purified and dried by stan-
dard methods. Reagents purchased from commercial suppliers
were used without further purification.
General procedure for the synthesis of title compounds
BD-a1–a4 and BD-c1–c4
(6-Chloro-3-nitropyridin-2-yl)-(1-Boc-piperidin-4-yl)amine (S-2):
Na2CO3 (0.21 g, 2.0 mmol) and 4-amino-1-Boc-piperidine (0.20 g,
1.0 mmol) were slowly added to a solution of 2,6-dichloro-3-nitro-
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pyridine (S-1, 0.19 g, 1.0 mmol) in EtOH (10 mL) at 08C. The mix-
ture was stirred at room temperature overnight, and the solvent
turned yellow. After removal of the solvent under reduced pres-
sure, H2O (20 mL) was added, and the mixture was extracted with
EtOAc (2ꢁ10 mL). Combined organic layers were washed with a sa-
turated solution of NaCl (10 mL) and dried over anhydrous Na2SO4.
Purification on silica gel gave 2 as a yellow solid. Yield: 85%; TLC Rf
(EtOAc/petroleum ether (PE) 1:8)=0.32; mp: 128–1308C; 1H NMR
(400 MHz, CDCl3): d=8.36 (d, 1H, PyH, J=8.60 Hz), 8.28 (d, 1H, J=
7.28 Hz), 6.63 (d, 1H, PyH, J=8.64 Hz), 4.30–4.37 (m, 1H), 4.07 (d,
2H, J=11.64 Hz), 3.02 (t, 2H, J=11.80 Hz), 2.06 (d, 2H, J=
11.64 Hz), 1.50–1.54 (m, 2H), 1.48 ppm (s, 9H, 3ꢁCH3); ESIMS: m/z
357.3 [M+H]+, 374.4 [M+NH4]+, 379.4 [M+Na]+, 301.4 [(MÀ56)+
H]+.
Title compounds BD-a1–a4 and BD-c1–c4: Intermediate S-4 (or S-
4’, 0.5 mmol) was dissolved in anhydrous acetone (10 mL) in the
presence of anhydrous K2CO3 (0.14 g, 1.0 mmol) at 08C, followed
by the addition of appropriately substituted benzyl chloride (or 4-
picolyl chloride hydrochloride, 0.6 mmol). The reaction mixture was
stirred at room temperature overnight. The solvent was removed
under reduced pressure, and H2O (20 mL) was added. After extrac-
tion with EtOAc (2ꢁ10 mL), the organic phase was washed with
a saturated solution of NaCl (10 mL) and dried over anhydrous
Na2SO4. The product was further purified by flash column chroma-
tography to afford title compounds BD-a1–a4 and BD-c1–c4.
6-(Mesityloxy)-N-(1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)-3-
nitropyridin-2-amine (BD-a1): Yellow solid, yield: 64%; TLC Rf
(EtOAc/PE 1:2)=0.15; mp: 195–1978C; 1H NMR (400 MHz, CDCl3):
d=8.40 (d, 1H, PyH, J=9.04 Hz), 8.27 (d, 1H, J=6.24 Hz), 7.90 (d,
2H, PhH, J=8.24 Hz), 7.53 (d, 2H, PhH, J=8.24 Hz), 6.87 (s, 2H,
PhH), 6.22 (d, 1H, PyH, J=9.00 Hz), 3.52 (s, 2H, CH2), 3.47 (brs, 1H),
3.06 (s, 3H, CH3), 2.68 (d, 2H, J=10.80 Hz), 2.30 (s, 3H, CH3), 2.04
(s, 6H, 2ꢁCH3), 1.87 (d, 2H, J=9.60 Hz), 1.74 (d, 2H, J=10.80 Hz),
1.48 ppm (d, 2H, J=9.92 Hz); ESIMS: m/z 525.5 [M+H]+.
(6-Mesityloxy-3-nitropyridin-2-yl)-(1-Boc-piperidin-4-yl)amine (S-
3): Cs2CO3 (2.9 g, 9.0 mmol) was slowly added to a solution of
2,4,6-trimethylphenol (0.82 g, 6.0 mmol) in dioxane (10 mL). The
mixture was stirred at room temperature for 15 min, and then in-
termediate S-2 (0.71 g, 2.0 mmol) was added. The reaction mixture
was held a reflux for 7 h, after which the solvent was removed
under reduced pressure. H2O (10 mL) was added to the resulting
residue, and the solution was extracted with EtOAc (2ꢁ10 mL). The
combined organic layers were washed with a saturated solution of
NaCl (10 mL) and subsequently dried over anhydrous Na2SO4. Pu-
rification by flash column chromatography afforded intermediate
S-3 as a yellow solid. Yield: 66%; TLC Rf (EtOAc/PE 1:8, run two
4-((4-(6-(Mesityloxy)-3-nitropyridin-2-ylamino)piperidin-1-yl)me-
thyl)benzenesulfonamide (BD-a2): Yellow solid, yield: 58%; TLC Rf
(EtOAc/PE 1:1)=0.27; mp: 289–2918C; 1H NMR (400 MHz,
[D6]DMSO): d=8.44 (d, 1H, PyH, J=9.04 Hz), 8.19 (d, 1H, J=
6.52 Hz), 7.79 (d, 2H, PhH, J=8.12 Hz), 7.46 (d, 2H, PhH, J=
7.96 Hz), 7.32 (s, 2H), 6.93 (s, 2H, PhH), 6.42 (d, 1H, PyH, J=
9.00 Hz), 3.45 (s, 2H, CH2), 3.26 (brs, 1H), 2.62 (d, 2H, J=10.60 Hz),
2.27 (s, 3H, CH3), 1.99 (s, 6H, 2ꢁCH3), 1.64–1.69 (m, 2H), 1.58 (d,
2H, J=10.72 Hz), 1.41–1.46 ppm (m, 2H); 13C NMR (100 MHz,
CDCl3): d=165.47, 152.00, 148.12, 143.23, 143.16, 140.05, 134.91,
130.03, 129.45, 129.30, 126.08, 122.82, 99.64, 61.94, 52.27, 49.62,
31.22, 20.84, 16.34 ppm; ESIMS: m/z 526.3 [M+H]+, 548.5 [M+
Na]+.
1
times)=0.54; mp: 74–768C; H NMR (400 MHz, CDCl3): d=8.39 (d,
1H, PyH, J=9.04 Hz), 8.23 (d, 1H, J=6.32 Hz), 6.78 (s, 2H, PhH),
6.24 (d, 1H, PyH, J=9.04 Hz), 3.91 (brs, 2H), 3.49–3.53 (m, 1H), 2.52
(m, 2H), 2.29 (s, 3H, CH3), 2.05 (s, 6H, 2ꢁCH3), 1.70–1.72 (m, 3H),
1.50–1.54 (m, 2H), 1.45 ppm (s, 9H, 3ꢁCH3); ESIMS: m/z 457.6 [M+
H]+, 401.5 [(MÀ56)+H]+.
(6-(4-Cyano-2,6-dimethylphenoxy)-3-nitropyridin-2-yl)-(1-Boc-pi-
peridin-4-yl)amine
(S-3’):
4-Hydroxy-3,5-dimethylbenzonitrile
4-((4-(6-(Mesityloxy)-3-nitropyridin-2-ylamino)piperidin-1-yl)me-
thyl)benzamide (BD-a3): Yellow solid, yield: 71%; TLC Rf (EtOAc)=
0.31; mp: 221–2238C; 1H NMR (400 MHz, CDCl3): d=8.40 (d, 1H,
PyH, J=9.00 Hz), 8.27 (d, 1H, J=6.36 Hz), 7.78 (d, 2H, PhH, J=
8.16 Hz), 7.41 (d, 2H, PhH, J=8.04 Hz), 6.86 (s, 2H, PhH), 6.22 (d,
1H, PyH, J=9.04 Hz), 6.07 (brs, 1H), 5.70 (brs, 1H), 3.50 (s, 2H,
CH2), 3.44 (brs, 1H), 2.69 (d, 2H, J=11.56 Hz), 2.30 (s, 3H, CH3), 2.04
(s, 6H, 2ꢁCH3), 1.83–1.88 (m, 2H), 1.73 (d, 2H, J=10.96 Hz), 1.43–
1.51 ppm (m, 2H); ESIMS: m/z 490.2 [M+H]+, 512.2 [M+Na]+.
(0.22 g, 1.5 mmol), Cs2CO3 (0.80 g, 2.5 mmol), and intermediate S-2
(0.17 g, 0.48 mmol) were used, following the same procedure as
described for the preparation of S-3, to give S-3’ as a yellow solid.
1
Yield: 72%; TLC Rf (EtOAc/PE 1:3)=0.30; mp: 182–1848C; H NMR
(400 MHz, CDCl3): d=8.49 (d, 1H, PyH, J=9.00 Hz), 8.28 (d, 1H, J=
6.64 Hz), 7.44 (s, 2H, PhH), 6.35 (d, 1H, PyH, J=9.00 Hz), 3.92 (d,
2H, J=12.12 Hz), 3.38–3.45 (m, 1H), 2.55 (m, 2H), 2.15 (s, 6H, 2ꢁ
CH3), 1.67 (d, 2H, J=10.20 Hz), 1.46 ppm (s, 9H, 3ꢁCH3); ESIMS: m/
z 468.5 [M+H]+, 485.7 [M+NH4]+, 412.5 [(MÀ56)+H]+, 368.4
[(MÀ100)+H]+.
6-(Mesityloxy)-3-nitro-N-(1-(pyridin-4-ylmethyl)piperidin-4-yl)-
pyridin-2-amine (BD-a4): Yellow oil, yield: 61%; TLC Rf (EtOAc/PE
3:1)=0.22; 1H NMR (400 MHz, CDCl3): d=8.55 (d, 2H, PyH, J=
5.76 Hz), 8.40 (d, 1H, PyH, J=9.00 Hz), 8.27 (d, 1H, J=6.20 Hz),
7.28 (d, 2H, PyH, J=5.76 Hz), 6.86 (s, 2H, PhH), 6.23 (d, 1H, PyH,
J=9.04 Hz), 3.49 (s, 2H, CH2), 2.69 (d, 2H, J=11.68 Hz), 2.29 (s, 3H,
CH3), 2.04 (s, 6H, 2ꢁCH3), 1.87–1.92 (m, 2H), 1.75 (d, 2H, J=
11.48 Hz), 1.45–1.54 ppm (m, 2H); ESIMS: m/z 448.6 [M+H]+.
6-(Mesityloxy)-3-nitro-N-(piperidin-4-yl)pyridin-2-amine
(S-4):
TFA (2.6 mL, 35 mmol) was added dropwise under stirring to a solu-
tion of intermediate S-3 (2.0 g, 4.4 mmol) in CH2Cl2 (10 mL) at
room temperature, and the mixture was stirred overnight. After re-
moval of the solvent under reduced pressure, H2O (10 mL) was
added, and the mixture was neutralized with 2n NaOH(aq) to pH 8.
After extraction with EtOAc (2ꢁ10 mL), the combined organic
layers were dried over anhydrous Na2SO4, filtered, and after evapo-
ration of the solvent the product S-4 was obtained as a yellow
solid. Yield: 97%; TLC Rf (MeOH/CH2Cl2 1:15, add 1 drop of Et3N)=
0.67; mp: 246–2488C; ESIMS: m/z 357.4 [M+H]+.
3,5-Dimethyl-4-(6-(1-(4-(methylsulfonyl)benzyl)piperidin-4-ylami-
no)-5-nitropyridin-2-yloxy)benzonitrile (BD-c1): Yellow solid,
1
yield: 62%; TLC Rf (EtOAc/PE 1:1)=0.22; mp: 208–2108C; H NMR
(400 MHz, CDCl3): d=8.48 (d, 1H, PyH, J=8.92 Hz), 8.25 (d, 1H, J=
6.52 Hz), 7.90 (d, 2H, PhH, J=8.32 Hz), 7.55 (d, 2H, PhH, J=
8.28 Hz), 7.42 (s, 2H, PhH), 6.35 (d, 1H, PyH, J=8.92 Hz), 3.54 (s,
2H, CH2), 3.26–3.29 (m, 1H), 3.07 (s, 3H, CH3), 2.68 (d, 2H, J=
11.76 Hz), 2.14 (s, 6H, 2ꢁCH3), 1.78–1.83 (m, 2H), 1.66 (d, 2H, J=
10.68 Hz), 1.40–1.50 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=
164.53, 154.07, 151.91, 145.19, 139.86, 139.27, 132.86, 132.15,
129.68, 127.42, 123.39, 118.57, 109.38, 98.86, 62.20, 52.06, 48.99,
6-(4-Cyano-2,6-dimethylphenoxy)-3-nitro-N-(piperidin-4-yl)pyri-
din-2-amine (S-4’): Intermediate S-3’ (0.20 g, 0.43 mmol) and TFA
(0.50 mL, 6.7 mmol) were combined, following the same procedure
as used in the preparation of S-4 to give S-4’ as a yellow solid.
Yield: 94%; TLC Rf (MeOH/CH2Cl2 1:15, add 1 drop of Et3N)=0.25;
mp: 222–2248C; ESIMS: m/z 368.4 [M+H]+.
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44.49, 31.51, 16.31 ppm; ESIMS: m/z 536.4 [M+H]+, 558.4 [M+
Na]+; HRMS (ESI): m/z [M+H]+ calcd for C27H30N5O5S: 536.1962,
found: 536.1954.
ate S-6’ as a gray solid. Yield: 96%; TLC Rf (MeOH/CH2Cl2 1:5, add
1 drop of Et3N)=0.14; mp: 200–2028C; ESIMS: m/z 338.6 [M+H]+.
Title compounds BD-b1–b4 and BD-d1–d4: Intermediate S-6 (or
S-6’, 0.5 mmol) was dissolved in anhydrous acetone (10 mL) in the
presence of anhydrous K2CO3 (0.14 g, 1.0 mmol) at 08C, followed
by addition of the appropriately substituted benzyl chloride (or 4-
picolyl chloride hydrochloride, 0.6 mmol). The reaction mixture was
stirred at room temperature overnight. The solvent was removed
under reduced pressure, and H2O (20 mL) was added. After extrac-
tion with EtOAc (2ꢁ10 mL), the organic phase was washed with
a saturated solution of NaCl (10 mL) and dried over anhydrous
Na2SO4. Further purification was done by flash column chromatog-
raphy to afford title compounds BD-b1–b4 and BD-d1–d4.
4-((4-(6-(4-Cyano-2,6-dimethylphenoxy)-3-nitropyridin-2-ylami-
no)piperidin-1-yl)methyl)benzenesulfonamide (BD-c2): Yellow
solid, yield: 55%; TLC Rf (EtOAc/PE 1:1)=0.17; mp: 225–2278C;
1H NMR (400 MHz, CDCl3): d=8.47 (d, 1H, PyH, J=8.92 Hz), 8.24 (d,
1H, J=6.60 Hz), 7.79 (d, 2H, PhH, J=8.36 Hz), 7.47 (d, 2H, PhH, J=
8.32 Hz), 7.40 (s, 2H, PhH), 6.34 (d, 1H, PyH, J=9.00 Hz), 4.93 (s,
2H), 3.54 (s, 2H, CH2), 3.19–3.26 (m, 1H), 2.68 (d, 2H, J=11.84 Hz),
2.13 (s, 6H, 2ꢁCH3), 1.76–1.81 (m, 2H), 1.65 (d, 2H, J=10.20 Hz),
1.39–1.48 ppm (m, 2H); ESIMS: m/z 537.4 [M+H]+, 559.4 [M+Na]+
.
4-((4-(6-(4-Cyano-2,6-dimethylphenoxy)-3-nitropyridin-2-ylami-
no)piperidin-1-yl)methyl)benzamide (BD-c3): Yellow solid, yield:
74%; TLC Rf (EtOAc/PE 3:1)=0.19; mp: 151–1538C; 1H NMR
(400 MHz, CDCl3): d=8.47 (d, 1H, PyH, J=8.88 Hz), 8.24 (d, 1H, J=
6.48 Hz), 7.80 (d, 2H, PhH, J=8.16 Hz), 7.40 (d, 2H, PhH, J=
8.00 Hz), 7.40 (s, 2H, PhH), 6.33 (d, 1H, PyH, J=8.92 Hz), 6.20 (brs,
1H), 5.65 (brs, 1H), 3.54 (s, 2H, CH2), 3.17–3.24 (m, 1H), 2.69 (d, 2H,
J=11.68 Hz), 2.12 (s, 6H, 2ꢁCH3), 1.74–1.79 (m, 2H), 1.65 (d, 2H,
J=10.24 Hz), 1.43–1.48 ppm (m, 2H); ESIMS: m/z 501.5 [M+H]+.
6-(Mesityloxy)-N2-(1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)pyr-
idine-2,3-diamine (BD-b1): Gray solid, yield: 43%; TLC Rf (MeOH/
CH2Cl2 1:10)=0.35; mp: 81–838C; 1H NMR (400 MHz, CDCl3): d=
7.90 (d, 2H, PhH, J=8.28 Hz), 7.57 (d, 2H, PhH, J=8.12 Hz), 6.84 (s,
2H, PhH), 6.82 (d, 1H, PyH, J=8.12 Hz), 5.68 (d, 1H, PyH, J=
7.92 Hz), 4.27 (d, 1H, J=7.00 Hz), 3.69–3.73 (m, 1H), 3.60 (s, 2H,
CH2), 3.06 (s, 3H, CH3), 2.78 (d, 4H, J=11.28 Hz), 2.27 (s, 3H, CH3),
2.14 (d, 2H, J=11.00 Hz), 2.08 (s, 6H, 2ꢁCH3), 1.97 (d, 2H, J=
10.68 Hz), 1.44–1.51 ppm (m, 2H); ESIMS: m/z 495.3 [M+H]+.
3,5-Dimethyl-4-(5-nitro-6-(1-(pyridin-4-ylmethyl)piperidin-4-yla-
mino)pyridin-2-yloxy)benzonitrile (BD-c4): Yellow solid, yield:
52%; TLC Rf (EtOAc/PE 3:1)=0.19; mp: 142–1448C; 1H NMR
(400 MHz, CDCl3): d=8.56 (dd, 2H, PyH, J1 =5.96 Hz, J2 =1.48 Hz),
8.48 (d, 1H, PyH, J=8.92 Hz), 8.27 (d, 1H, J=6.36 Hz), 7.42 (s, 2H,
PhH), 7.29 (d, 2H, PyH, J=5.76 Hz), 6.34 (d, 1H, PyH, J=9.00 Hz),
3.48 (s, 2H, CH2), 3.28–3.30 (m,1H), 2.70 (d, 2H, J=11.68 Hz), 2.13
(s, 6H, 2ꢁCH3), 1.79–1.85 (m, 2H), 1.67 (d, 2H, J=10.84 Hz), 1.44–
1.53 ppm (m, 2H); ESIMS: m/z 459.6 [M+H]+.
4-((4-(3-Amino-6-(mesityloxy)pyridin-2-ylamino)piperidin-1-yl)-
methyl)benzenesulfonamide (BD-b2): Gray solid, yield: 46%; TLC
Rf (MeOH/CH2Cl2 1:10)=0.14; mp: 225–2278C; 1H NMR (400 MHz,
CDCl3): d=7.90 (d, 2H, PhH, J=8.28 Hz), 7.56 (d, 2H, PhH, J=
8.00 Hz), 6.84 (s, 2H, PhH), 6.82 (d, 1H, PyH, J=7.84 Hz), 5.69 (d,
1H, PyH, J=7.90 Hz), 4.87 (brs, 2H), 4.31(brs, 1H), 3.73 (brs, 1H),
3.65 (s, 2H, CH2), 2.84 (brs, 2H), 2.27 (s, 3H, CH3), 2.18 (m, 2H), 2.07
(s, 6H, 2ꢁCH3), 1.99 ppm (d, 2H, J=11.64 Hz); ESIMS: m/z 496.2
[M+H]+.
4-((4-(3-Amino-6-(mesityloxy)pyridin-2-ylamino)piperidin-1-yl)-
methyl)benzamide (BD-b3): Gray solid, yield: 51%; TLC Rf (MeOH/
1
CH2Cl2 1:10)=0.12; mp: 174–1768C; H NMR (400 MHz, CDCl3): d=
General procedure for the synthesis of title compounds
BD-b1–b4 and BD-d1–d4
7.78 (d, 2H, PhH, J=8.12 Hz), 7.43 (d, 2H, PhH, J=8.12 Hz), 6.84 (s,
2H, PhH), 6.81 (d, 1H, PyH, J=7.84 Hz), 6.08 (brs, 1H), 5.70 (d, 1H,
PyH, J=7.92 Hz), 5.62 (brs, 1H), 4.27 (d, 1H, J=4.44 Hz), 3.69 (d,
1H, J=4.28 Hz), 3.61 (s, 2H, CH2), 2.83 (d, 2H, J=10.32 Hz), 2.27 (s,
3H, CH3), 2.10–2.13 (m, 2H), 2.07 (s, 6H, 2ꢁCH3), 1.97 (d, 2H, J=
10.64 Hz), 1.43–1.52 ppm (m, 2H); ESIMS: m/z 460.2 [M+H]+.
6-(Mesityloxy)-N2-(1-Boc-piperidin-4-yl)pyridine-2,3-diamine (S-
5): MeOH (10 mL) was added to a mixture of intermediate S-3
(0.15 g, 0.33 mmol) and palladium on charcoal (10%, 7.5 mg), and
this was kept under H2 atmosphere. The reaction was stirred at
room temperature overnight. Pd-C was removed by filtration, and
the solvent was removed under reduced pressure. The gray residue
(S-5) was dried over anhydrous Na2SO4. Yield: 98%; TLC Rf (EtOAc/
PE 1:1)=0.34; mp: 189–1918C; ESIMS: m/z 427.6 [M+H]+.
6-(Mesityloxy)-N2-(1-(pyridin-4-ylmethyl)piperidin-4-yl)pyridine-
2,3-diamine (BD-b4): Gray solid, yield: 47%; TLC Rf (MeOH/CH2Cl2
1
1:10)=0.12; mp: 131–1338C; H NMR (400 MHz, CDCl3): d=8.55 (d,
2H, PyH, J=4.52 Hz), 7.30 (d, 2H, PyH, J=5.48 Hz), 6.84 (s, 2H,
PhH), 6.82 (d, 1H, PyH, J=7.88 Hz), 5.70 (d, 1H, PyH, J=7.96 Hz),
4.27 (d, 1H, J=6.48 Hz), 3.67–3.71 (m, 1H), 3.52 (s, 2H, CH2), 2.78
(d, 2H, J=11.24 Hz), 2.27 (s, 3H, CH3), 2.12 (m, 2H), 2.08 (s, 6H, 2ꢁ
CH3), 1.97 (d, 2H, J=11.48 Hz), 1.45–1.53 ppm (m, 2H); ESIMS: m/z
418.6 [M+H]+, 209.9 [(M+2)/2]+.
6-(4-Cyano-2,6-dimethylphenoxy)-N2-(1-Boc-piperidin-4-yl)pyri-
dine-2,3-diamine (S-5’): Intermediate S-3’ (0.20 g, 0.43 mmol) and
Pd-C (10%, 10 mg) were used, following the procedure as de-
scribed for the preparation of intermediate S-5 to give intermedi-
ate S-5’ as a gray solid. Yield: 95%; TLC Rf (EtOAc/PE 1:1)=0.19;
mp: 182–1848C; ESIMS: m/z 438.6 [M+H]+, 382.6 [(MÀ56)+H]+.
6-(Mesityloxy)-N2-(piperidin-4-yl)pyridine-2,3-diamine (S-6): Inter-
mediate S-5 (0.20 g, 0.47 mmol) and TFA (0.50 mL, 6.7 mmol) were
used, following the procedure as described for the preparation of
intermediate S-4 to give intermediate S-6 as a gray solid. Yield:
95%; TLC Rf (MeOH/CH2Cl2 1:10, add 1 drop of Et3N)=0.67; mp:
152–1548C; ESIMS: m/z 327.6 [M+H]+.
4-(5-Amino-6-(1-(4-(methylsulfonyl)benzyl)piperidin-4-ylamino)-
pyridin-2-yloxy)-3,5-dimethylbenzonitrile (BD-d1): Gray solid,
yield: 44%; TLC Rf (MeOH/CH2Cl2 1:12)=0.16; mp: 193–1958C;
1H NMR (400 MHz, CDCl3): d=7.90 (d, 2H, PhH, J=8.28 Hz), 7.57 (d,
2H, PhH, J=7.88 Hz), 7.35 (s, 2H, PhH), 6.91 (d, 1H, PyH, J=
7.92 Hz), 5.98 (d, 1H, PyH, J=7.84 Hz), 4.20 (d, 1H, J=6.36 Hz),
3.57 (s, 2H, CH2), 3.26–3.30 (m, 1H), 3.07 (s, 3H, CH3), 2.86 (brs,
2H), 2.73 (d, 2H, J=9.76 Hz), 2.13 (s, 6H, 2ꢁCH3), 1.90 (d, 2H, J=
10.88 Hz), 1.78 (d, 2H, J=11.40 Hz), 1.38 ppm (d, 2H, J=10.88 Hz);
ESIMS: m/z 506.4 [M+H]+.
6-(4-Cyano-2,6-dimethylphenoxy)-N2-(piperidin-4-yl)pyridine-2,3-
diamine (S-6’): Intermediate S-5’ (0.60 g, 1.4 mmol) and TFA
(1.0 mL, 13.5 mmol) were used, following the procedure as de-
scribed for the preparation of intermediate S-4 to give intermedi-
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 11
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4-((4-(3-Amino-6-(4-cyano-2,6-dimethylphenoxy)pyridin-2-ylami-
no)piperidin-1-yl)methyl)benzenesulfonamide (BD-d2): Gray
solid, yield: 46%; TLC Rf (MeOH/CH2Cl2 1:10)=0.21; mp: 224–
2268C; 1H NMR (400 MHz, [D6]DMSO): d=7.79 (d, 2H, PhH, J=
8.28 Hz), 7.57 (s, 2H, PhH), 7.48 (s, 2H), 7.32 (s, 2H), 6.77 (d, 1H,
PyH, J=7.80 Hz), 5.94 (d, 1H, PyH, J=7.80 Hz), 5.42 (brs, 1H), 4.34
(brs, 2H), 3.45 (s, 2H, CH2), 3.02 (brs, 1H), 2.86 (brs, 2H), 2.62 (brs,
2H), 2.05 (s, 6H, 2ꢁCH3), 1.59–1.67 ppm (m, 4H); ESIMS: m/z 507.1
[M+H]+, 529.1 [M+Na]+.
sure, and H2O (20 mL) was added, followed by extraction with
EtOAc (2ꢁ10 mL). The organic phase was washed with saturated
NaCl (10 mL), dried over anhydrous Na2SO4, and was purified by
flash column chromatography to afford title compounds BD-e1–
e4.
N6-Mesityl-N2-(1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)-3-nitro-
pyridine-2,6-diamine (BD-e1): Yellow solid, yield: 63%; TLC Rf
(EtOAc/PE 1:1)=0.23; mp: 106–1088C; 1H NMR (400 MHz, CDCl3):
d=8.77 (brs, 1H), 8.13 (d, 1H, PyH, J=8.96 Hz), 7.91 (d, 2H, PhH,
J=8.12 Hz), 7.59 (d, 2H, PhH, J=4.28 Hz), 6.95 (s, 2H, PhH), 6.51
(brs, 1H), 5.36 (brs, 1H), 4.24 (brs, 1H), 3.66 (s, 2H, CH2), 3.06 (s,
3H, CH3), 2.83 (brs, 2H), 2.31 (s, 3H, CH3), 2.17 (s, 6H, 2ꢁCH3),
2.02–2.12 (m, 2H), 1.48–1.94 ppm (m, 4H); ESIMS: m/z 524.5 [M+
H]+, 546.3 [M+Na]+.
4-((4-(3-Amino-6-(4-cyano-2,6-dimethylphenoxy)pyridin-2-ylami-
no)piperidin-1-yl)methyl)benzamide (BD-d3): Gray solid, yield:
53%; TLC Rf (MeOH/CH2Cl2 1:10)=0.17; mp: 165–1678C; 1H NMR
(400 MHz, [D6]DMSO): d=7.92 (s, 1H), 7.83 (d, 2H, PhH, J=
7.84 Hz), 7.57 (s, 2H, PhH), 7.35 (d, 2H, PhH, J=7.20 Hz), 7.30 (s,
1H), 6.77 (d, 1H, PyH, J=7.84 Hz), 5.94 (d, 1H, PyH, J=7.80 Hz),
5.40 (d, 1H, J=5.32 Hz), 4.33 (brs, 2H), 3.42 (s, 2H, CH2), 3.02 (brs,
1H), 2.65 (d, 1H, J=8.32 Hz), 2.05 (s, 6H, 2ꢁCH3), 1.58–1.66 ppm
(m, 4H); ESIMS: m/z 471.6 [M+H]+.
4-((4-(6-(Mesitylamino)-3-nitropyridin-2-ylamino)piperidin-1-yl)-
methyl)benzenesulfonamide (BD-e2): Yellow solid, yield: 58%;
TLC Rf (EtOAc/PE 1:1)=0.11; mp: 138–1408C; 1H NMR (400 MHz,
CDCl3): d=8.75 (brs, 1H), 8.11 (d, 1H, PyH, J=9.04 Hz), 7.88 (d, 2H,
PhH, J=8.24 Hz), 7.49 (d, 2H, PhH, J=7.80 Hz), 6.95 (s, 2H, PhH),
6.59 (brs, 1H), 5.36 (brs, 1H), 5.18 (brs, 2H), 4.24 (brs, 1H), 3.60 (s,
2H, CH2), 2.79 (brs, 2H), 2.31 (s, 3H, CH3), 2.16 (s, 6H, 2ꢁCH3),
1.85–2.11 (m, 4H), 1.58–1.72 ppm (m, 2H); 13C NMR (100 MHz,
CDCl3): d=160.89, 153.50, 143.98, 140.75, 143.16, 137.57, 136.49,
131.66, 129.54, 129.39, 127.10, 126.63, 120.44, 96.65, 62.39, 51.99,
31.83, 20.98, 18.25 ppm; ESIMS: m/z 525.5 [M+H]+, 547.4 [M+
Na]+; HRMS (ESI): m/z [M+H]+ calcd for C26H33N6O4S: 525.2279,
found: 525.2269.
4-(5-Amino-6-(1-(pyridin-4-ylmethyl)piperidin-4-ylamino)pyridin-
2-yloxy)-3,5-dimethylbenzonitrile (BD-d4): Gray solid, yield: 48%;
TLC Rf (MeOH/CH2Cl2 1:10)=0.24; mp: 158–1608C; 1H NMR
(400 MHz, CDCl3): d=8.55 (dd, 2H, PyH, J1 =5.92 Hz, J2 =1.44 Hz),
7.36 (s, 2H, PhH), 7.28 (d, 2H, PyH, J=5.92 Hz), 6.91 (d, 1H, PyH,
J=7.84 Hz), 5.94 (d, 1H, PyH, J=7.88 Hz), 4.20 (d, 2H, J=6.84 Hz),
3.47 (s, 2H, CH2), 3.25–3.33 (m, 1H), 2.71 (d, 2H, J=11.84 Hz), 2.13
(s, 6H, 2ꢁCH3), 1.86–1.1.92 (m, 2H), 1.77 ppm (d, 2H, J=11.96 Hz);
ESIMS: m/z 429.5 [M+H]+.
4-((4-(6-(Mesitylamino)-3-nitropyridin-2-ylamino)piperidin-1-yl)-
methyl)benzamide (BD-e3): Yellow solid, yield: 67%; TLC Rf
(EtOAc/PE 3:1)=0.18; mp: 255–2578C; 1H NMR (400 MHz, CDCl3):
d=8.75 (brs, 1H), 8.13 (d, 1H, PyH, J=8.88 Hz), 7.78 (d, 2H, PhH,
J=8.20 Hz), 7.44 (d, 2H, PhH, J=7.64 Hz), 6.95 (s, 2H, PhH), 6.51
(brs, 1H), 6.07 (brs, 1H), 5.66 (brs, 1H), 5.35 (brs, 1H), 4.23 (brs,
1H), 3.60 (s, 2H, CH2), 2.82 (brs, 2H), 2.31 (s, 3H, CH3), 2.17 (s, 6H,
2ꢁCH3), 2.08 (brs, 2H), 1.71 ppm (brs, 2H); ESIMS: m/z 489.5 [M+
H]+, 511.6 [M+Na]+.
General procedure for the synthesis of title compounds
BD-e1–e4
N6-Mesityl-N2-(1-Boc-piperidin-4-yl)-3-nitropyridine-2,6-diamine
(S-7): A mixture of 2,4,6-trimethylaniline (0.27 g, 2.0 mmol) and in-
termediate S-2 (0.36 g, 1.0 mmol) was heated at 1308C overnight.
After addition of H2O, the reaction mixture was extracted with
EtOAc (2ꢁ10 mL). The combined organic layers were washed with
a saturated solution of NaCl (10 mL) and dried over anhydrous
Na2SO4. Purification on silica gel gave S-7 as a yellow solid. Yield:
65%; TLC Rf (EtOAc/PE 1:5)=0.41; mp: 177–1798C; 1H NMR
(400 MHz, CDCl3): d=8.72 (s, 1H, PyH), 8.14 (d, 1H, J=8.00 Hz),
6.96 (s, 2H, PhH), 6.51 (s, 1H), 5.37 (s, 1H), 4.34 (s, 1H), 4.05 (s, 2H),
3.03 (s, 2H), 2.32 (s, 3H, CH3), 2.18 (s, 6H, 2ꢁCH3), 2.06 (s, 2H),
1.50–1.54 (m, 2H), 1.48 ppm (s, 9H, 3ꢁCH3); ESIMS: m/z 456.5 [M+
H]+,478.5 [M+Na]+, 400.4 [(MÀ56)+H]+.
N6-Mesityl-3-nitro-N2-(1-(pyridin-4-ylmethyl)piperidin-4-yl)pyri-
dine-2,6-diamine (BD-e4): Yellow solid, yield: 55%; TLC Rf
1
(EtOAc)=0.18; mp: 228–2308C; H NMR (400 MHz, CDCl3): d=8.77
(brs, 1H), 8.56 (d, 2H, PyH, J=5.84 Hz), 8.13 (d, 1H, PyH, J=
8.60 Hz), 7.30 (d, 2H, PyH, J=4.04 Hz), 6.95 (s, 2H, PhH), 6.54 (brs,
1H), 5.36 (brs, 1H), 4.24 (brs, 1H), 3.56 (s, 2H, CH2), 2.82 (brs, 2H),
2.31 (s, 3H, CH3), 2.17 (s, 6H, 2ꢁCH3), 2.02–2.10 (m, 2H), 1.65–
1.89 ppm (m, 4H); ESIMS: m/z 447.5 [M+H]+.
N6-Mesityl-3-nitro-N2-(piperidin-4-yl)pyridine-2,6-diamine (S-8):
TFA (2.6 mL, 35 mmol) was added dropwise, under stirring, to a so-
lution of intermediate S-7 (2.0 g, 4.4 mmol) in CH2Cl2 (10 mL) at
room temperature and stirred overnight. After removal of the sol-
vent under reduced pressure, H2O (10 mL) and EtOAc (10 mL) were
added. An aqueous 2n HCl solution was added dropwise (to
pH 2), and the aqueous phase was separated and subsequently
neutralized with 2n NaOH(aq) to pH 8 to obtain a yellow precipi-
tate. Filtration and drying gave the product (S-8) as a yellow solid.
Yield: 95%; TLC Rf (MeOH/CH2Cl2 1:10, add 1 drop of Et3N)=0.31;
mp: 187–1898C; ESIMS: m/z 356.4 [M+H]+.
In vitro anti-HIV activity assays
The anti-HIV activity and cytotoxicity of the compounds were eval-
uated against wild-type HIV-1 strain IIIB, a double RT mutant
(K103N+Y181C) HIV-1 strain, and HIV-2 strain ROD in MT-4 cell cul-
tures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
bromide (MTT) method as previously described.[8,13] Briefly, stock
solutions (10ꢁ final concentration) of test compounds were added
in 25 mL volumes to two series of triplicate wells so as to allow si-
multaneous evaluation of their effects on mock- and HIV-infected
cells at the beginning of each experiment. Serial fivefold dilutions
of test compounds (final 200 mL volume per well) were made di-
rectly in flat-bottomed 96-well microtiter trays using a Biomek
3000 robot (Beckman Instruments). Untreated control HIV- and
mock-infected cell samples were included for each sample. HIV-
1 (IIIB)[15] or HIV-2 (ROD)[16] stock (50 mL) at 100–300 CCID50 (cell cul-
Title compounds BD-e1–e4: Intermediate S-8 (0.5 mmol) was dis-
solved in anhydrous acetone (10 mL) in the presence of anhydrous
K2CO3 (0.14 g, 1.0 mmol) at 08C, followed by addition of the appro-
priately substituted benzyl chloride (or 4-picolyl chloride hydro-
chloride, 0.6 mmol). The reaction mixture was stirred at room tem-
perature overnight. The solvent was removed under reduced pres-
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ture infectious dose) or culture medium was added to either the
infected or mock-infected wells of the microtiter tray. Mock-infect-
ed cells were used to evaluate the effect of test compounds on un-
infected cells in order to assess the cytotoxicity of the test com-
pounds. Exponentially growing MT-4 cells were centrifuged for
5 min at 220 g, and the supernatant was discarded. The MT-4 cells
were resuspended at 6ꢁ105 cellsmLÀ1, 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 spectro-
photometrically by MTT assay.
entropic, and salvation terms, was trained to estimate the dissocia-
tion constant (Kd) expressed in Àlog(Kd)2. Prior to docking, the pro-
tein was prepared by removing water molecules, the ligand, and
other unnecessary small molecules from the crystal structure com-
plex (PDB code: 3M8Q); simultaneously, polar hydrogen atoms
were added to the protein. Surflex-Dock default settings were used
for other parameters, such as the number of starting conforma-
tions per molecule, the size to expand search grid, the maximum
number of rotatable bonds per molecule, and the maximum
number of poses per ligand. During the docking procedure, all of
the single bonds in residue side chains inside the defined RT bind-
ing pocket were regarded as rotatable or flexible, and the ligand
was allowed to rotate on all single bonds and to move flexibly
within the tentative binding pocket. The atomic charges were re-
calculated using the Kollman all-atom approach for the protein
and the Gasteiger–Hꢂckel approach for the ligand. The binding in-
teraction energy was calculated to include van der Waals, electro-
static, and torsional energy terms defined in the Tripos force field.
The 20 best-scoring ligand–protein complexes were kept for fur-
ther analyses. The Àlog(Kd)2 values of the 20 best-scoring com-
plexes, which represent the binding affinities of ligand with RT,
ranged a wide scope of functional classes. Therefore, only the
highest-scoring 3D structural model of the ligand-bound RT was
chosen to define the binding interaction.
The MTT assay is based on the reduction of yellow-colored MTT
(Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase
activity 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 (Infinite
M1000, Tecan), at two wavelengths (540 and 690 nm). All data
were calculated using the median optical density (OD) values of
three wells. The 50% cytotoxic concentration (CC50) is defined as
the concentration of the test compound required to decrease 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% effec-
tive concentration (EC50).
Recombinant HIV-1 RT inhibitory assays
Acknowledgements
The HIV-RT inhibition assay was performed by using an RT assay kit
(Roche), and the procedure for assaying RT inhibition was per-
formed as described in the kit protocol. Briefly, the reaction mix-
ture consisted of template/primer complex, 2’-deoxynucleotide 5’-
triphosphates (dNTPs) and reverse transcriptase (RT) in lysis buffer
with or without inhibitors. After 1 h incubation at 378C the reac-
tion mixture was transferred to a streptavidin-coated microtiter
plate (MTP). The biotin-labeled dNTPs that are incorporated in the
template through RT activity are bound to streptavidin. The un-
bound dNTPs were washed with wash buffer, and antidigoxigenin–
peroxidase (DIG–POD) was added into the MTP. The DIG-labeled
dNTPs incorporated in the template are bound to anti-DIG–POD
antibody. The unbound anti-DIG–POD was washed, and the perox-
ide substrate (ABST) was added to the MTP. A colored reaction
product was produced during cleavage of the substrate, as cata-
lyzed by POD. The absorbance of the sample was determined at
OD405 using a microtiter plate ELISA reader. The resulting color in-
tensity is directly proportional to RT activity. The percent inhibitory
activity of RT inhibitors was calculated by comparison with
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (NSFC Nos. 81273354,
81102320, 30873133, 30772629, and 30371686), the Key Project
of NSFC for International Cooperation (No. 30910103908), the Re-
search Fund for the Doctoral Program of Higher Education of
China (Nos. 20110131130005 and 20110131120037), the Natural
Science Foundation of Shandong Province (ZR2009CM016), the
Independent Innovation Foundation of Shandong University
(IIFSDU, No. 2010GN044), the Shandong Postdoctoral Innovation
Science Research Special Program (No. 201002023), the China
Postdoctoral Science Foundation funded project (Nos.
20100481282 and 2012T50584), and KU Leuven (GOA 10/014).
We thank K. Erven, K. Uyttersprot, and C. Heens for technical as-
sistance with the anti-HIV assays. The authors declare no compet-
ing financial interest.
a sample lacking inhibitor. Percent inhibition values were calculat-
Keywords: antiviral agents · biological activity
molecular simulations · reverse transcriptase
· HIV-1 ·
+inhibitor
ed by the following:
OD405Àinhibitor)ꢁ100].
%
Inhibition=100À[(OD405
/
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Received: March 21, 2013
Published online on && &&, 0000
[13] a) P. A. Janssen, P. J. Lewi, E. Arnold, F. Daeyaert, M. de Jonge, J. Heeres,
L. Koymans, M. Vinkers, J. Guillemont, E. Pasquier, M. Kukla, D. Ludovici,
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Selective and effective! Compound
BD-c1 shows impressively low cyto-
toxicity (CC50 >146 mm), high selectivity
(SI>14,126), and good inhibitory activi-
ty toward HIV-1 reverse transcriptase.
With qualities similar to those of the
new FDA-approved drug etravirine
(CC50 =28 mm, SI=12884), BD-c1 is
a promising lead compound for the de-
velopment of even more efficacious
anti-HIV drugs.
X. Chen, Y. Li, S. Ding, J. Balzarini,
C. Pannecouque, E. De Clercq, H. Liu,
X. Liu*
&& – &&
Discovery of Piperidine-Linked
Pyridine Analogues as Potent Non-
nucleoside HIV-1 Reverse
Transcriptase Inhibitors
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