Design, synthesis and preliminary SAR studies of novel N-arylmethyl substituted piperidine-linked aniline derivatives as potent HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.bmc.2013.10.033

A series of novel N-arylmethyl substituted piperidine-linked aniline derivatives were designed, synthesized and evaluated for their anti-HIV activity in MT-4 cells. All the new compounds showed moderate to potent activities against wild-type (wt) HIV-1 wi

Full text of DOI:10.1016/j.bmc.2013.10.033

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and preliminary SAR studies of novel N-arylmethyl
substituted piperidine-linked aniline derivatives as potent HIV-1
NNRTIs
a
a
a
a
a
a
c
Lingzi Zhang , Peng Zhan , Xuwang Chen , Zhenyu Li , Zhoumeng Xie , Tong Zhao , Huiqing Liu ,
b
b
b
a,
⇑
Erik De Clercq , Christophe Pannecouque , Jan Balzarini , Xinyong Liu
a
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, PR China
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
b
c
Institute of Pharmacology, School of Medicine, Shandong University, 44, West Culture Road, 250012 Ji’nan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-arylmethyl substituted piperidine-linked aniline derivatives were designed, synthe-
Received 26 September 2013
Revised 21 October 2013
Accepted 22 October 2013
Available online xxxx
sized and evaluated for their anti-HIV activity in MT-4 cells. All the new compounds showed moderate
to potent activities against wild-type (wt) HIV-1 with an EC50 range from 0.022 to 2.1
lM. Among them,
compound 5a6 (EC50 = 0.022 ± 0.0091 M, SI >10,770) was confirmed to be the most potent and selective
l
inhibitor, which proved more potent than DDI and DLV in a cell-based assay against wt HIV-1, and more
efficient than NVP in an RT (reverse transcriptase) assay. Besides, it is worth noting that compound 7a1
Keywords:
HIV-1
AIDS
NNRTIs
Synthesis
Anti-HIV-1 activity
retained moderate inhibitory activity (EC50 = 4.8 ± 0.95
lM) against the HIV-1 double RT mutant strain
(K103N/Y181C). The preliminary structure–activity relationship was discussed and rationalized by
molecular simulation.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
The number of people living with human immunodeficiency
of diarylpyrimidine (DAPY) derivatives (Fig. 1), such as the clini-
cally approved drugs, etravirine (TMC125) and rilpivirine
(TMC278), which retain activity against a large panel of HIV-1 mu-
5
,6
virus (HIV) has been rising steadily in the past two decades.
According to the latest statistic data provided by WHO, 34.0 mil-
tant strains. Whereas a defect in bioavailability of most DAPY
derivatives resulted from their low water solubility has provided
much of the motivation for further structural optimization based
1
lion people were living with HIV and 1.7 million people died from
AIDS in 2011, globally. Fortunately, the current coverage of highly
active anti-retroviral therapy (HAART) has distinctly deferred peo-
ple dying from AIDS-related causes. In the combinatory regimens
of HAART, nonnucleoside reverse transcriptase (RT) inhibitors
7
on this chemical skeleton. Subsequent modification using the
principles of molecular hybridization and bioisosteric replacement
led to the discovery of piperidine-linked aminopyrimidine deriva-
tives and diarylaniline derivatives, respectively, which displayed
broad spectrum against both wild-type (wt) and drug-resistant vir-
(
NNRTIs), identified as a chemically diverse class of compounds
8
,9
7,10
targeting at an allosteric binding pocket on HIV-1 RT have gained
a definitive place because of their high potency, low toxicity and
impressive selectivity against HIV-1 replication.2 However, the
high mutation rate of RT has promoted the selection of drug-resis-
tant virus which have significantly hindered the continued suc-
cessful antiretroviral therapy.3 Therefore, the development of
novel NNRTIs with an improved activity profile against drug-resis-
tant HIV mutants is continuously needed.
al strains. In our previous work,
a series of piperidine-linked
triazine derivatives were designed, synthesized and evaluated for
anti-HIV activities, and several compounds revealed excellent
potencies with EC50 values in low nanomolar concentration range
against the wt HIV-1 strain and in low micromolar concentration
range against the frequently encountered HIV-1 double mutant
strain K103N/Y181C.
Promoted by these results and in continuation of our study, we
designed a novel series of N-arylmethyl substituted piperidine-
linked aniline derivatives by utilizing the molecular hybridization
principle. In the newly designed compounds (Fig. 2), the 4-cyano/
methyl-2,6-dimethylphenoxy, which was assumed to be a prepon-
derant group in the left wing of TMC120 or TMC125, was adopted
,4
Investigations focused on the resistance profiles of NNRTIs for
almost two decades gave rise to the discovery and development
⇑
Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.033
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033

2
L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
CN
condition provided 4a and 4b, which were sufficiently pure for
CN
O
CN
NH
1
0,14–16
the following reactions without further purification.
Subse-
CN
NH
quently, N-alkylation reactions of 4a or 4b with appropriately
substituted arylmethyl halides achieved the final compounds
1
0
5
a1–5a10 and 5b1–5b10. Reduction of the nitrobenzene com-
N
pound where Ar was 4-sulfanilylphenyl (5a8) gave the intermedi-
ate 6a1, which afforded the desired compound 7a1 after
HN
N
N
Br
N
17
acidification. In another route, 3a was reduced by hydrogenation,
NH2
etravirine (TMC125)
rilpivirine (TMC278)
and then acetylated to give the intermediate 9, which was followed
by deprotection, affording the intermediate 10. The target product
Figure 1. Structures of clinically used DAPY derivatives.
1
1 was finally obtained by an alkylation reaction of compound 10
with 4-(bromomethyl)benzenesulfonamide. In addition, the
substituted arylmethyl halides were commercially available or
prepared according to similar methods reported in the litera-
to accommodate the hydrophobic sub-pocket of RT. And a nitro-,
amino- or amide substituted benzene, which was confirmed to
be an acceptable bioisostere of pyrimidine ring, was used as the
core ring and was expected to generate a hydrogen bond with
the amino acid of RT. Moreover, inspired by the drug design strat-
egy that forming additional protein–inhibitor interactions, espe-
cially a bidentate hydrogen-bond between the inhibitor and the
1
8–22
tures.
Both analytical and spectral data of all the compounds
are in full agreement with the proposed structures.
2.2. Biological activities
2.2.1. In vitro anti-HIV assay
1
1
amino acid main chain may prevent resistance development,
The compounds were evaluated for their in vitro anti-HIV activ-
2
3
the N-arylmethyl substituted piperidine in the right wing, previ-
ously reported to make multiple hydrogen bonds with the back-
bone NH of K103 through a water bridge,8 was employed for
improving the resistance profile. Herein, we report the synthesis,
anti-HIV activities of these newly designed compounds, as well
as their preliminary structure–activity relationship (SAR) analysis.
ity in MT-4 cells infected by wt HIV-1 strain III
B
,
commonly
encountered double RT mutant HIV-1 strain RES056
(K103N + Y181C), and HIV-2 strain ROD. The cytotoxicity of the
compounds was also tested in parallel in HIV-uninfected MT-4
cells. The results, interpreted as EC50, CC50, SI (selectivity index)
and fold resistance, are summarized in Table 1 with the marketed
drugs zidovudine (azidothymidine, AZT), dideoxyinosine (DDI),
DMP266 (efavirenz, EFV) and PNU-90152T (delavirdine, DLV) as
2
2
. Results and discussion
.1. Chemistry
2
4,25
the reference standards.
As shown in Table 1, it was gratifying to see that all the synthe-
sized compounds were superior to the reference drug DDI against
B
wt HIV-1(III ) with the exception of 11, which was totally inactive.
Half of them were more potent than the positive control drug DLV.
The active derivatives exhibited moderate to high activity against
A modified synthetic route to afford the target products
a1–5a10, 5b1–5b10, 7a1 and 11 is shown in Scheme 1. Treatment
5
of the initial material 4-chloro-2-fluoro-1-nitrobenzene (1) with
tert-butyl 4-aminopiperidine-1-carboxylate gave intermediate
the wt HIV-1 strain III
B
with EC50 values ranging from 0.022 to
1
2,13
2.1 M and SI values in the variable range of 4 to >10,770. Of all
l
2,
which was reacted with corresponding substituted phenols
the evaluated derivatives, compound 5a6 emerged as the most po-
tent and selective inhibitor against wt HIV-1, possessing a low EC50
in the catalysis of KI and PTC (phase transfer catalyst) at a high
temperature to afford the key intermediates 3a and 3b in excellent
yields. Deprotections of 3a and 3b in an easy, rapid and mild
value of 0.022 ± 0.0091
lM and a high SI of >10,770, which
Ar
N
R1
left wing
right wing
N
R1
CN
NH
molecular
O
hybridization
hetero
cycle
Ar
left wing
right wing
core ring
R1
N
previously reported piperidine-
linked DAPY derivatives
X
N
left wing
molecular
hybridization
right wing
N
R2
O
NH
core ring
R3
R1
O
CN
core ring
R2
TMC120:
1
R =Me, R ,R =H,X=NH
2
3
left wing
right wing
the present designed piperidine-
linked aniline derivatives
bioisosteric
TMC125:
1
replacement
R =CN, R =Br, R =NH , X=O
NH
2
3
2
R2
core ring
previously reported diarylaniline
derivatives
R =NO , NH
2
2
2
Figure 2. Newly designed piperidine-linked aniline derivatives.
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L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
SO
2
NH
2
2 2
SO NH
Ar
R
R
O
CN
O
CN
H
N
Cl
F
N
N
N
NO
2
1
O
NH
NH
NO
NH
NH
O
NH
HCl
Boc
N
R
O
Boc
N
NO2
NH2
2
2
4
a: R=CN
b: R=Me
5a1-5a10: R=CN
5b1-5b10: R=Me
6a1
7a1
4
2 2
SO NH
Cl
NH
NH
CN
O
Boc
N
CN
O
Boc
N
CN
O
CN
NO
2
NO
a: R=CN
2
H
N
N
3
3
2
b: R=Me
NH
NH
NH
NH
O
NH
O
O
O
2
N
H
N
H
N
H
8
9
10
11
Scheme 1. The synthetic route of target compounds. Reagents and conditions: (i) tert-butyl 4-aminopiperidine-1-carboxylate, Na
phenol, K CO , TBAB, KI, DMF, 120–130 °C, 12 h; (iii) TFA, DCM, rt, 20 min; (iv) K CO , DMF, rt; (v) Pd/C, H , THF, EtOH, rt, 36 h; (vi) con. hydrochloric acid, DCM, 0 °C; (vii) Pd/
C, H , THF, EtOH, rt, overnight; (viii) acetyl chloride, TEA, DCM, 0 °C, 1 h.
2 3
CO , DMF, 80 °C, 1 h; (ii) substituted
2
3
2
3
2
2
attained the same order of magnitude as those obtained for the ref-
erence drugs AZT and EFV.
It was especially interesting to notice that the wt HIV-1 inhibi-
tory activity was markedly enhanced when the unsaturated car-
boxyl group at the benzyl moiety was replaced by a saturated
hydroxymethyl group (5a5 versus 5a4, 5b5 versus 5b4), while the
negative impact on the cytotoxicity was further corroborated with
this replacement. This result indicated that, the unsaturated or
conjugate substituent at the para-position proved beneficial for
decreasing the cytotoxicity of the tested compound.
These analogues were also assayed against the frequently
encountered HIV-1 double mutant strain K103N/Y181C (RES056).
Both the absolute activity against the HIV-1 mutant (EC50 value)
and the relative activity (fold-resistance) were used to define the
resistance profile of the tested compounds. As shown in Table 1,
compound 7a1 was identified as the unique active compound
which was more potent than the reference drug DLV against the
Furthermore, as the data demonstrated, compounds 5a1 and
5b1 with a hydrophobic substituent nitro at the para-tail possessed
the lowest inhibitory activity against wt HIV-1 among both the ser-
ies of compounds 5a and 5b. In the meantime, all the compounds
with various hydrophilic para-substituents (5a2–5a8, 5b2–5b8)
revealed better potency in inhibiting wt HIV-1 compared with
the corresponding no-para-substituent compounds 5a10 and
5b10, which were endowed with the lowest selectivity indices.
Practically, compound 5a7/5b7 with a hydrophilic para-carbamoyl
exhibited much better antiviral activity and higher cytotoxicity
than compound 5a9/5b9 with a hydrophobic para-methoxycar-
bonyl. Furthermore, the introduction of a pyridylmethyl moiety
at the benzyl domain led to a substantial increase in the potency
and selectivity against wt HIV-1, for instance, 5a2 versus 5a10
and 5b2 versus 5b10. Thus, the results led us to hypothesize that
a para substituent at the benzyl moiety is crucial for the selectivity
index of the tested compound and a hydrophilic group accommo-
dates the chemical environment well in this region of RT, which is
grossly in accordance with results of the previously reported piper-
resistant mutant strain with an EC50 value of 4.8 ± 0.95
lM (versus
>
36 M, DLV) and a fold resistance ratio of 114 (versus >277, DLV).
l
In addition, all the title compounds were screened for their inhibi-
tion against the replication of the HIV-2 strain (ROD), but none of
them exhibited inhibitory activity at sub-toxic concentrations,
indicating that the novel series of N-arylmethyl-substituted piper-
idine-linked aniline derivatives were specific to HIV-1.
First, we focused our attention on the SAR of the substituent R
1
at the left wing. When the core ring and the right wing of each
compound were mutual coincide, cyan proved to be the favored
group for wt HIV-1 strain inhibition compared with methyl in most
instances. For example, the series 5a (with a cyano group) showed
better potency against wt HIV-1 than the series 5b (with a methyl
group), except for compounds 5a3, 5a4 and 5a7 (versus 5b3, 5b4
and 5b7, respectively).
Next, the SAR of the substituted Ar of the right wing was ex-
plored. The diverse structures of this moiety produced distinct im-
pact on the activities against the wt HIV-1. It was interesting to
notice that the antiviral efficiency and selectivity index increased
dramatically when the carbonyl at the para position of the benzyl
moiety (5a3, 5b3, 5a7 and 5b7) was replaced with sulfuryl (5a6,
7
,10
idine-linked triazine series.
We also investigated the SAR of the substituent R
ring. Obviously, the results revealed that the anti-HIV activity of
compounds 5a1–5a10 and 5b1–5b10 bearing a NO substituent
is limited to the wt HIV-1, irregardless the substitution pattern.
Nevertheless, compound 7a1 (R = NH ) retained high activity
2
at the core
5
b6, 5a8 and 5b8), especially for compounds 5a3 and 5b3 versus
compounds 5a6 and 5b6, respectively. The antiviral activity of
compound 5a8/5b8 (with-SO NH ) and the counterpart 5a6/5b6
with-SO Me) was almost at the same level. Meanwhile, the com-
2
2
2
2
2
(
2
against the wt HIV-1 and exhibited considerable inhibitory activity
against a resistant mutant RT strain of HIV-1 (K103N/Y181C) with-
out augmenting the cytotoxicity compared to compound 5a8
pounds with the carbonyl in the para substituent displayed consid-
erable antiviral potency, which decreased steadily in the order 5a7/
5
b7 (–CONH
2
) >5a3/5b3 (–COMe) >5a5/5b5 (–COOH). These re-
(R
at the NH
ity against both wt HIV-1 and HIV-2 strains. It can be concluded
that the NH at the core ring was probably beneficial for the bind-
ing affinity (potentially hydrogen bonding interaction) between
2
= NO
2
). However, compound 11, which introduced an acetyl
10
sults in conjunction with previous studies suggested that the sul-
furyl/carbonyl group included in the para-substituent played a
dominant role, when the substituent possessed appropriate
hydrophilicity.
2
group in the case of compound 7a1, displayed no activ-
2
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4
L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 1
Anti-HIV-1 activity and cytotoxicity of the newly synthesized congeners
Ar
R1
O
N
NH
R2
No.
R
1
R
2
Ar
EC50a
(
lM)
CC50b
(l
M)
SIc
Fold res.d
HIV-1
HIV-1
IIIB
HIV-2
ROD
HIV-1
RES056
>182
IIIB
RES056 RES056
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
1
7
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
b1
b2
b3
b4
b5
b6
b7
b8
b9
b10
1
CN NO
CN NO
CN NO
CN NO
CN NO
CN NO
CN NO
CN NO
CN NO
CN NO
Me NO
Me NO
Me NO
Me NO
Me NO
Me NO
Me NO
Me NO
Me NO
Me NO
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4-Nitrophenyl
Pyridin-4-yl
4-Acetylphenyl
4-(Hydroxymethyl) phenyl
4-Carboxylphenyl
4-(Methylsulfonyl) phenyl
4-Aminoacylphenyl
4-Sulfanilylphenyl
4-Methoxycarbonylphenyl
Phenyl
4-Nitrophenyl
Pyridin-4-yl
4-Acetylphenyl
4-(Hydroxymethyl) phenyl 0.096 ± 0.028
4-Carboxylphenyl
4-(Methylsulfonyl) phenyl
4-Aminoacylphenyl
4-Sulfanilylphenyl
4-Methoxycarbonylphenyl
Phenyl
0.67 ± 0.28
>182
>19
>251
>11
>30
>234
>27
182 ± 18
19 ± 3.1 336
>251 >1630
11 ± 5.9 80
30 ± 1.9 159
>234
27 ± 2.6 430
23 ± 1.7 782
269
<1
<1
X1
<1
<1
>269
>336
>1,630
>80
>159
>10,770
>430
>782
>1208
>4.4
0.056 ± 0.0033 >19
0.15 ± 0.051
0.14 ± 0.076
0.19 ± 0.10
>251
>11
>30
0.022 ± 0.0091 >234
>10,770 X1
0.062 ± 0.015
0.029 ± 0.014
0.13 ± 0.041
0.63 ± 0.32
2.1 ± 0.83
0.082 ± 0.048
0.11 ± 0.032
>27
>23
<1
<1
>23
>160
>2.8
>147
>21
>138
>25
>35
>158
>27
>160
>2.8
>147
>21
>138
>25
>35
>158
>27
>22
>248
>25
>228
>22
P160
P1208 <orX1
2.8 ± 1.6 4.4
147 ± 16 70
21 ± 3.6 258
138 ± 66 1312
25 ± 1.4 255
35 ± 8.8 161
158 ± 55
27 ± 2.1 457
22 ± 2.4 615
>248
25 ± 1.9 37
>228 X1
22 ± 0.61 505
<1
<1
<1
<1
<1
<1
<1
<1
<1
X1
<1
>70
>258
>1312
>255
>161
>5895
>457
>615
>246
>37
0.22 ± 0.049
0.027 ± 0.014
0.059 ± 0.012
0.036 ± 0.014
1.0 ± 0.054
0.66 ± 0.36
>228
5895
>22
>248
>25
>246
CN NHCOCH
a1 (Hydrochloride) CN NH
3
4-Sulfanilylphenyl
4-Sulfanilylphenyl
2
0.043 ± 0.0020 4.8 ± 0.95
4.4
114
AZT
DDI
EFV
DLV
0.0072 ± 0.00029 0.0074 ± 0.00087 0.0057 ± 0.00050 >94
>13,066 >12,594 1.0
19 ± 2.6
18 ± 1.8
>212
>6.3
>36
>11
>855
>277
>12
>12
X1
0.0074 ± 0.0026 0.52 ± 0.022
70
>277
0.13 ± 0.043
>36
All data were calculated using the median value of two or three parallel assays.
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell against HIV-induced cytotoxicity, as determined by the MTT method.
CC50: concentration required to decrease the viability of mock-infected cells by 50%, as determined by the MTT method.
SI: selectivity index (CC50/EC50). The SI values: X1 stand for P1or<1.
b
c
d
mut
wt
Fold res.: ratio of EC50 value against drug-resistant strain and EC50 of wt HIV-1(III
B
) ðEC50 =EC Þ.
50
the inhibitor and the active binding site of the double mutant
strain (K103N/Y181C), and the conjecture is also in agreement
with those reported in the literatures.9,26 Further in-depth analysis
of this issue will be presented in the following section concerning
molecular simulation.
In general, the SAR analysis above can be summarized as fol-
lows: (1) Substituted phenyl is an acceptable isosteric replace-
ment for the anti-HIV-1 activity instead of heterocycles in the
2.2.2. HIV-1 RT inhibition assay
We hypothesize the newly synthesized N-arylmethyl substi-
tuted piperidine-linked aniline derivatives as HIV-1 NNRTIs based
on the similarity with the lead compounds. To further confirm
their binding target, we selected the most active compound 5a6
for the RT enzyme assay, utilizing poly(A) ꢀ oligo(dT)15 as the tem-
plate primer. As the data indicated (Table 2), compound 5a6 was
more potent than the typical NNRTI NVP (IC50 = 0.67
IC50 value of 0.36 M, while inferior to that of ETV
(IC50 = 0.071 M). The result manifested that these newly synthe-
lM) with an
core ring domain. It may be noted that substitution at the R
2
po-
l
sition of the piperidine-linked aniline skeleton deserves further
consideration towards improving the drug-resistance profile. (2)
The potency and selectivity index of the compounds are strongly
dependent on the nature of the substituent at the para position
of the benzyl. (3) Appropriate hydrophilicity of the aforemen-
tioned substituent plays a significant role in the bioactivity of
the compounds. (4) An unsaturated or conjugate substituent
coupling directly with the para-benzyl is essential for decreasing
the cytotoxicity, and the sulfuryl is confirmed to be a privileged
group. The comprehensive SAR concluded above provides helpful
information for further design and investigation of novel anti-
HIV-1 analogues.
l
sized derivatives represented by compound 5a6 targeted the
HIV-1 RT, thus acting as classical NNRTIs.
Table 2
a
Inhibitory activity of compound 5a6 against wt HIV-1 RT
Compd
IC50b
5a6
NVP
0.67
ETV
(
lM)
0.36
0.071
a
The RT kit was commercially available and supplied by Roche.
IC50 means the inhibitory concentration of the tested compounds required to
b
inhibit biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1
RT by 50%.
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L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
2
.3. Molecular modeling
Compound 7a1 was also simulated in a K103N/Y181C mutant
RT binding pocket (PDB code: 3BGR). The result revealed that the
potent antiviral compound retained the hydrogen bond between
NH and Lys101. It is worth noting that a lose of a water-mediated
hydrogen bond with the Lys103 backbone in wt binding mode was
Molecular modeling was carried out to give further insight in
the probable binding modes at the allosteric binding site of RT
and rationalize some SAR conclusions. More specifically, the repre-
sentative compounds 5a8, 7a1 and 11 were chosen to be docked
into the nonnucleoside inhibitor binding pocket (NNIBP) of HIV-1
RT (PDB code: 3M8Q and 3BGR) in terms of Autodock Vina 1.1.2
remedied by the hydrogen bond formed between the aniline NH
2
and the Asn103 side chain CO. Thus, the bidentate hydrogen bond
was preserved in the mutant binding mode to meet the drug-resis-
tance design strategy proposed in the design and explaining why
compound 7a1 was active against the K103N/Y181C HIV-1 mutant
strain.
[
http://vina.scripps.edu] (Fig. 3). Default parameters were used as
described in the Autodock Vina 1.1.2 manual unless otherwise
specified.
As illustrated in Figure 3, compounds 5a8 and 7a1 docked into
the wt RT binding pocket (PDB code: 3M8Q) displayed a similar
binding mode as the lead piperidine-linked aminopyrimidine
In summary, the molecular simulation analysis conformed to
the original design, rationalized the SAR conclusion above and
strongly supported the bioactivity data of the newly designed
and synthesized NNRTIs. Further investigations on the structural
basis will take these aspects into account.
8
,10
derivatives.
Specifically, the pattern can be characterized as fol-
lows: (a) The left wing of compounds 5a8 and 7a1 fit into a hydro-
phobic sub-pocket mainly defined by the side chains of aromatic
amino acid residues Tyr181, Tyr188 and Trp229 and generated a
3
. Conclusion
positive p–p stacking interaction. (b) Compounds 5a8 and 7a1 re-
served features of both Lys101 and Lys103 main-chain binding
types, wherein the hydrogen bond between the N atom of the
piperidine and the backbone of residue Lys103 was bridged by a
water molecule. (c) The hydrophilic benzsulfamide tail resided be-
tween Val106 and Pro236 and was oriented to the solvent exposed
region, which can explain the SAR conclusion that a hydrophilic
substituent at the tail of the right wing was favored. However,
the position of the left wing of compound 11 revealed a deflection
in the hydrophobic sub-pocket due to the substitution of an aceta-
mino at the core ring, and directly resulted in a decreased interac-
tion between the inhibitor and the side chain of the Tyr181 residue
as well as the lose of a water-bridged hydrogen bond. In addition,
compound 11 displayed the worst overlap with the ligand, and the
docking results could account for why compounds 5a8 and 7a1
exhibited similar excellent potency, whereas compound 11
showed a sharp decrease in antiviral activity.
In summary, we have designed and synthesized a series of novel
N-arylmethyl substituted piperidine-linked aniline derivatives, and
evaluated their biological activity against HIV-1 (wt strain III and
B
double mutant strain K103N/Y181C) and HIV-2 (strain ROD). It was
gratifying to see that the majority of the title compounds exhibited
high potency against HIV-1. Among the newly synthesized congen-
ers, the most potent and selective compound against wt HIV-1 was
5
a6, which was more active than the reference drugs DDI and DLV,
with an EC50 value of 0.022 ± 0.0091 M and a SI value of >10,770.
Besides, compound 7a1 retained moderate inhibitory activity
l
(
(
EC50 = 4.8 ± 0.95
lM) against the HIV-1 double mutant strain
K103N/Y181C). These piperidine-linked anilines could be defined
as orthodox NNRTIs based on the enzyme assay and the fact that
none of the congeners was shown to be an anti-HIV-2 agent. The
preliminary SAR and molecular simulation were also studied, pro-
Figure 3. (A) The predicted binding mode of 5a8 (red) into wt NNIBP (PDB code: 3M8Q) and the overlap with the ligand (yellow). (B) The predicted binding mode of 7a1
gray) into wt NNIBP (PDB code: 3M8Q) and the overlap with the ligand (yellow). (C) The predicted binding mode of 11 (green) into wt NNIBP (PDB code: 3M8Q) and the
(
overlap with the ligand (yellow). (D) The predicted binding mode of 7a1 into double mutant (K103N/Y181C) NNIBP (PDB code: 3BGR). All the figures were generated using
PyMol (www.pymol.org).
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033

6
L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
viding helpful guidance for further rational design of novel anti-
HIV-1 NNRTIs.
for 6 h. The mixture was poured into water. The aqueous was ex-
tracted with ethyl acetate for 3 times. The combined organic phase
was washed with brine, dried over anhydrous sodium sulfate and
filtered. The filtrate was concentrated and the crude reaction prod-
uct was purified by column chromatography on silica using petro-
leum ether/ethyl acetate (10:1) as eluent to give an orange oil, and
then recrystallized to obtain the product 3b (63.5 mg, yield: 99.2%)
as a yellow solid. ESI-MS: m/z 456.5 (M+1), 473.3 (M+18), 478.4
(M+23). C25H33N O [455.24].
4
4
. Experimental section
.1. Chemistry
All melting points were determined on a micro melting point
apparatus and are uncorrected. 1H NMR spectra were recorded
on a Bruker Avance 400 spectrometer in the appropriate solvents
and tetramethylsilane (TMS) as internal standard. 13C NMR spec-
tra were run in the same instrument at 100 MHz. Chemical shifts
are expressed in d units and TMS as internal reference. Mass
3
5
4.1.4. General procedure for the synthesis of 3,5-dimethyl-4-(4-
nitro-3-(piperidin-4-ylamino)phenoxy)benzonitrile (4a) and N-
(5-(mesityloxy)-2-nitrophenyl)piperidin-4-amine (4b)
spectra were taken on
a
LC Autosampler Device: standard
To a solution of tert-butyl 4-(5-(4-cyano-2,6-dimethylphen-
oxy)-2-nitrophenylamino)piperidine-1-carboxylate (3a, 4.00 g,
8.58 mmol) in dichloromethane (13 ml) was added trifluoroacetic
acid (13 ml) dropwise for 0.5 h in a water bath at 15 °C. The reac-
tion mixture was stirred for another 0.5 h, the water bath was re-
placed by an ice-bath and the saturated sodium carbonate (90 ml)
was added in drops. The precipitate was filtered and dried in vac-
uum to give the intermediate 4a (3.09 g, yield: 98.4%) as an orange
G1313A instrument (Agilent Technologies, Company). TLC was
performed on Silica Gel GF254 for TLC (Merck) and spots were
visualized by iodine vapors or by irradiation with UV light
(
254 nm). Flash column chromatography was performed on col-
umn packed with Silica Gel 60 (200–300 mesh). Solvents were
reagent grade and, when necessary, were purified and dried by
standard methods. Concentration of the reaction solutions in-
volved the use of a rotary evaporator at reduced pressure.
solid. ESI-MS: m/z 367.3 (M+1), 389.4 (M+23). C20H22N O3
4
[
366.17].
The intermediate 4b was obtained as a yellow solid (yield:
4
.1.1. General procedure for the synthesis of tert-butyl 4-(5-
chloro-2-nitrophenylamino)piperidine-1-carboxylate (2)
mixture of 4-chloro-2-fluoro-1-nitrobenzene (1, 0.09 g,
99.5%) following the procedure in the preparation of 4a. Mp:
152–154 °C. ESI-MS: m/z 356.4 (M+1), 378.4 (M+23). C20H25N O3
[355.19].
A
3
0
.5 mmol), tert-butyl 4-aminopiperidine-1-carboxylate (0.1 g,
0
.5 mmol), and sodium carbonate (0.15 g, 1.4 mmol) in DMF
(
5 ml) was stirred under 80 °C for 1 h. Then DMF was evaporated
4.1.5. General procedure for the synthesis of target compounds
(5a1–5a10 and 5b1–5b10)
and the crude reaction product was dissolved in 10 ml dichloro-
methane (DCM) and 10 ml brine. The aqueous solution was ex-
tracted with DCM (10 mlꢀ3). The combined organic layer was
A mixture of 4a or 4b (1 equiv), corresponding substituent
(0.99–1.56 equiv) and potassium carbonate (2.11–3.22 equiv) in
DMF was stirred at room temperature for 1.5–12 h. The reaction
mixture was poured into an excess of water and filtered over cel-
atom. The residue was dissolved with dichloromethane and dried
over anhydrous sodium sulfate. The filtrate was concentrated and
purified by column chromatography on silica to afford the target
compounds (5a1–5a10 and 5b1–5b10), respectively.
washed with brine (10 mlꢀ3) and dried over anhydrous Na
2 4
SO .
After filtration, the solvent was removed by evaporation and the
residue was purified by column chromatography on silica to give
the compound 2 (0.18 g, yield: 100%) as a yellow solid. Mp: 93–
9
C
5 °C. ESI-MS: m/z 373.3 (M+18), 375.4 (M+2+18), 378.4 (M+23).
22ClN [355.13].
16
H
3 4
O
4
.1.2. General procedure for the synthesis of tert-butyl 4-(5-(4-
4.1.5.1. 3,5-Dimethyl-4-(4-nitro-3-(1-(4-nitrobenzyl)piperidin-
cyano-2,6-dimethylphenoxy)-2-nitrophenylamino)piperidine-
4-ylamino)phenoxy)benzonitrile (5a1).
Elution with petro-
1
-carboxylate (3a)
A mixture of tert-butyl 4-(5-chloro-2-nitrophenylamino)piper-
leum ether/DCM/ethyl acetate (1:1:0.1). Orange solid, yield:
1
100%, mp: 208–210 °C. H NMR (400 MHz, CDCl , ppm) d: 8.28
3
0
idine-1-carboxylate (2, 0.1 g, 0.28 mmol), 4-cyan-3,5-dimethyl-
phenol (0.04 g, 0.27 mmol), potassium carbonate (0.08 g,
(d, 1H, J = 7.1 Hz, NH), 8.19 (d, 2H, J = 8.6 Hz, C ,C -Ph -H), 8.14
3
5
0
6 2 6
(d, 1H, J = 9.5 Hz, C -Ph-H), 7.53 (d, 2H, J = 8.5 Hz, C ,C -Ph -H),
7.46 (s, 2H, C ,C -Ph -H), 6.13 (d, 1H, J = 2.4 Hz, C -Ph-H), 5.97
3 5 3
0
0
0
.58 mmol), tetrabutyl ammonium bromide (0.02 g, 0.062 mmol),
potassium iodide (0.01 g, 0.060 mmol) and DMF (5 ml) as the
solvent was heated to 120–130 °C and stirred for 12 h. The sol-
vent was removed under reduced pressure and redissolved in
1 2 5 2
(dd, 1H, J = 2.5 Hz, J = 9.5 Hz, C -Ph-H), 3.63 (s, 2H, CH ), 3.36
(m, 1H, C -Pi-H), 2.78 (t, 2H, Pi-CH ), 2.25 (t, 2H, Pi-CH ), 2.17 (s,
1
2
2
13
6H, 2ꢀ CH ), 2.00 (m, 2H, Pi-CH ), 1.68 (m, 2H, Pi-CH ). C NMR
3
2
2
00
1
0 ml DCM and 10 ml brine. The liquid was separated and the
3 4 1
(100 MHz, CDCl , ppm) d: 162.84 (Ph-C ), 153.77 (Ph -C ),
water phase was extracted with DCM (10 mlꢀ3), the organic lay-
ers were combined, washed with brine (10 mlꢀ3) and dried over
anhydrous sodium sulfate. After filtration, the solvent was re-
moved by evaporation and the residue was purified by column
chromatography on silica to give the compound 3a (0.11 g, yield:
147.21, 146.49, 146.43, 133.05, 133.01, 130.22, 129.35, 127.77,
123.59, 118.39 (C„N), 109.79, 103.13, 97.66 (Ph-C ), 62.07 (Pi-
3
CH -Py), 51.61, 48.96, 31.52, 16.16 (2ꢀ CH ). ESI-MS: m/z 502.3
2
3
(M+1), 524.4 (M+23), 540.4 (M+39). C
2
7
H
27
N O [501.20].
5 5
8
4.6%) as a yellow solid. Mp: 125–127 °C. ESI-MS: m/z 467.5
4.1.5.2.
ylmethyl)piperidin-4-ylamino)phenoxy)benzonitrile
5a2). Elution with petroleum ether/ethyl acetate/triethyl-
amine (2:1:0.2). Yellow solid, yield: 71.0%, mp: 169–171 °C.
NMR (400 MHz, CDCl ppm) d: 8.56 (dd, 2H, = 1.4 Hz,
3,5-Dimethyl-4-(4-nitro-3-(1-(pyridin-4-
(
M+1), 484.5 (M+18), 489.5 (M+23). C25 [466.22].
30 4 5
H N O
(
4
.1.3. General procedure for the synthesis of tert-butyl 4-(5-
1
H
(
(
mesityloxy)-2-nitrophenylamino)piperidine-1-carboxylate
3b)
3
,
J
1
J
2
= 4.5 Hz, C
J = 9.5 Hz,
J = 5.7 Hz, C
3
,C
5
-Py-H), 8.28 (d, J = 7.1 Hz, 1H, NH), 8.15 (d, 1H,
tert-Butyl
boxylate (2, 0.05 g, 0.14 mmol), 2,4,6-trimethylphenol (0.04 g,
.29 mmol), potassium carbonate (0.08 g, 0.58 mmol), tetrabutyl
ammonium bromide (0.01 g, 0.03 mmol), potassium iodide
0.005 g, 0.03 mmol) in DMF (1.5 ml) was stirred at 120–130 °C
4-(5-chloro-2-nitrophenylamino)piperidine-1-car-
0
C
6
-Ph-H), 7.46 (s, 2H,
,C
= 2.5 Hz, J
-Pi-H), 2.79 (t, 2H, Pi-CH
ꢀ CH ), 2.01 (m, 2H, Pi-CH ), 1.69 (m, 2H, Pi-CH
C
3
5
,C -Ph -H), 7.30 (d, 2H,
2
6
-Py-H), 6.12 (d, 1H, J = 2.4 Hz, C
= 9.5 Hz, C -Ph-H), 3.55 (s, 2H, CH
), 2.25 (t, 2H, Pi-CH ), 2.17 (s, 6H,
3
-Ph-H), 5.97 (dd,
0
1
1
2
H, J
H, C
1
2
5
2
), 3.36 (m,
1
2
2
(
13
3
2
2
). C NMR
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033

L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
0
0
(
100 MHz, CDCl
3
, ppm) d: 162.84 (Ph-C
46.49, 133.04, 133.01, 130.24, 127.76, 123.76, 118.39 (C„N),
09.79, 103.11, 97.65 (Ph-C ), 61.63 (Pi-CH -Py), 51.57, 31.41,
6.18 (2ꢀ CH ). ESI-MS: m/z 458.5 (M+1). C26 [457.21].
4
), 153.75 (Ph -C
1
), 149.88,
129.67, 127.77, 127.49, 118.39 (C„N), 109.79, 103.15, 97.66 (Ph-
1
1
1
C
3
), 62.15 (Pi-CH
2
-Py), 51.54, 44.53 (SO
2
–CH
3
), 31.42, 16.18 (2ꢀ
3
2
CH ). ESI-MS: m/z 535.3 (M+1), 557.2 (M+23). C28H N O S
3
30 4 5
3
H
27
N
5
O
3
[534.19].
4
.1.5.3. 4-(3-(1-(4-Acetylbenzyl)piperidin-4-ylamino)-4-nitro-
Elution with
petroleum ether/ethyl acetate/triethylamine (5:1:0.2). Yellow so-
4.1.5.7.
4-((4-(5-(4-Cyano-2,6-dimethylphenoxy)-2-nitrophe-
Elution
with petroleum ether/ethyl acetate/triethylamine (2:1:0.15). Yel-
phenoxy)-3,5-dimethylbenzonitrile (5a3).
nylamino)piperidin-1-yl)methyl)benzamide (5a7).
1
1
lid, yield: 66.2%. mp: 110–112 °C. H NMR (400 MHz, CDCl
3
,
low solid, yield: 88.2%, mp: 198–200 °C. H NMR (400 MHz, CDCl
3
,
ppm) d: 8.28 (d, 1H, J = 7.2 Hz, NH), 8.14 (d, 1H, J = 9.4 Hz, C
6
00
-Ph-
ppm) d: 8.26 (d, 1H, J = 7.1 Hz, NH), 8.14 (d, 1H, J = 9.4 Hz, C -Ph-H),
6
0
0
00
H), 7.93 (d, 2H, J = 8.2 Hz, C
.44 (d, 2H, J = 9.3 Hz, C ,C
H), 5.96 (dd, 1H, J
3
,C
5
0
-Ph -H), 7.45 (s, 2H, C
3
,C
5
-Ph -H),
-Ph-
-Ph-H), 3.59 (s, 2H,
-Pi-H), 2.79 (t, 2H, Pi-CH ), 2.61 (s, 3H, CO–
), 2.22 (t, 2H, Pi-CH ), 1.99 (m, 2H, Pi-
), 2.17 (s, 6H, 2ꢀ CH
), 1.68 (m, 2H, Pi-CH ). C NMR (100 MHz, CDCl , ppm) d:
), 146.53, 144.11,
36.17, 133.04, 133.00, 130.21, 128.96, 128.44, 127.72, 118.40
), 62.53 (Pi-CH -Py), 51.58,
), 16.17 (2ꢀ CH ). ESI-MS: m/z 499.4
[498.23].
7.80 (d, 2H, J = 8.1 Hz, C
(d, 2H, J = 8.3 Hz, C ,C
J = 2.4 Hz, C
3.61 (s, 2H, CH
(t, 2H, Pi-CH
(m, 2H, Pi-CH
(C@O), 162.83 (Ph-C
3
,C
5
0
-Ph -H), 7.45 (s, 2H, C
3
,C
), 6.10 (d, 1H,
= 9.5 Hz, C -Ph-H),
-Pi-H), 2.80 (t, 2H, Pi-CH ), 2.24
), 1.99 (m, 2H, Pi-CH ), 1.67
, ppm) d: 169.22
5
-Ph -H), 7.43
7
2
6
-Ph -H), 6.12 (d, 1H, J = 2.5 Hz, C
= 2.5 Hz, J
3
2
6
-Ph -H), 6.10 (br, 2H, NH
-Ph-H), 5.98 (dd, 1H, J
), 3.31 (m, 1H, C
2
1
2
= 9.5 Hz, C
5
3
1
= 2.4 Hz, J
2
5
CH
CH
CH
2
3
2
), 3.34 (m, 1H, C
1
2
2
1
2
2
3
2
), 2.16 (s, 6H, 2ꢀ CH
3
2
1
3
13
2
3
2
).
C NMR (100 MHz, CDCl
3
00
00
1
1
97.86 (C@O), 162.81 (Ph-C
4
), 153.76 (Ph -C
1
4
), 153.77 (Ph -C
1
), 146.50, 133.05, 133.01,
132.33, 130.23, 129.20, 127.73, 127.49, 118.41 (C„N), 109.75,
103.16, 97.61 (Ph-C ), 62.37 (Pi-CH
-Py), 51.39, 31.39, 16.17 (2ꢀ
CH ). ESI-MS: m/z 500.3 (M+1), 522.4 (M+23).
(
C„N), 109.77, 103.05, 97.66 (Ph-C
3
2
3
2
4
9.10, 31.54, 26.65 (CO-CH
3
3
3
28 29 5 4
C H N O
(
M+1), 521.4 (M+23). C29
H
30
N
4
O
4
[499.22].
4
.1.5.4. 4-(3-(1-(4-(Hydroxymethyl)benzyl)piperidin-4-ylami-
Elu-
4.1.5.8.
4-((4-(5-(4-Cyano-2,6-dimethylphenoxy)-2-nitrophe-
no)-4-nitrophenoxy)-3,5-dimethylbenzonitrile (5a4).
tion with petroleum ether/ethyl acetate/triethylamine (3:1:0.2).
Orange solid, yield: 38.5%, mp: 163–165 °C. H NMR (400 MHz,
nylamino)piperidin-1-yl)methyl)benzenesulfonamide
(5a8). Elution with petroleum ether/ethyl acetate/triethyl-
amine (1:1:0.2). Yellow solid, yield: 65.6%, mp: 147–149 °C.
1
1
H
CDCl
3
, ppm) d: 8.25 (d, 1H, J = 7.2 Hz, NH), 8.13 (d, 1H, J = 9.4 Hz,
NMR (400 MHz, CDCl
3
, ppm) d: 8.26 (d, 1H, J = 7.0 Hz, NH), 8.14
00
0
0
C
6
6
-Ph-H), 7.45 (s, 2H, C
.10(d, 1H, J = 2.5 Hz,
3
,C
5
-Ph -H), 7.34 (d, 4H, C
2
,C
3
,C
5
,C
6
-Ph -H),
= 2.5 Hz,
-O), 3.56 (s, 2H, Pi-CH
(d, 1H, J = 9.5 Hz, C
6
-Ph-H), 7.90 (d, 2H, J = 8.0 Hz, C
3
,C
5
-Ph -H),
0
00
C
3
-Ph-H), 5.96 (dd, 1H,
J
1
7.50 (d, 2H, J = 8.0 Hz, C
(d, 1H, C
5.20 (s, 2H, NH
2H, Pi-CH ), 2.24 (t, 2H, Pi-CH
2H, Pi-CH ), 1.67 (m, 2H, Pi-CH
ppm) d: 162.87 (Ph-C ), 153.78 (Ph -C
133.03, 130.23, 129.54, 127.70, 126.49, 118.43 (C„N), 109.73,
103.25, 97.62 (Ph-C ), 62.15 (Pi-CH
-Py), 51.35, 31.37, 16.17 (2ꢀ
CH ). ESI-MS: m/z 536.4 (M+1), 558.4 (M+23). C27
535.19].
2
,C
6
-Ph -H), 7.46 (s, 2H, C
3
,C
5
-Ph -H), 6.10
0
J
2
= 9.5 Hz, C
5
-Ph-H), 4.69 (s, 2H, Ph -CH
-Pi-H), 2.81 (t, 2H, Pi-CH
), 2.16 (s, 6H, 2ꢀ CH ), 1.99 (t, 2H, Pi-CH
). C NMR (100 MHz, CDCl
2
2
-
3
-Ph-H), 5.99 (dd, 1H, J
), 3.62 (s, 2H, CH
), 2.16 (s, 6H, 2ꢀ CH
1
= 1.6 Hz, J
), 3.35 (m, 1H, C
2
= 9.4 Hz, C
-Pi-H), 2.78 (t,
), 1.99 (m,
). C NMR (100 MHz, CDCl
5
-Ph-H),
0
Ph ), 3.32 (m, 1H, C
CH
CH
1
2
), 2.23 (m, 2H, Pi-
), 1.67 (m, 2H, Pi-
), 153.79
), 146.51, 140.08, 133.04, 132.99, 130.18, 129.38, 127.80,
27.03, 118.33 (C„N), 109.81, 103.10, 97.68 (Ph-C ), 65.06 (CH ),
2.52 (CH ). ESI-MS: m/z
), 51.24, 49.08, 31.36, 16.12 (2ꢀ CH
87.5 (M+1), 509.4 (M+23). C28 [486.23].
2
2
1
2
3
2
2
2
3
1
3
13
2
3
, ppm) d: 162.81 (Ph-C
4
2
2
3
,
0
0
00
(
Ph -C
1
4
1
), 146.51, 140.86, 133.05,
1
6
4
3
2
2
3
3
2
H
30
N
4
O
4
3
29 5 5
H N O S
[
4
.1.5.5.
4-((4-(5-(4-Cyano-2,6-dimethylphenoxy)-2-nitrophe-
Elu-
tion with ethyl acetate/methanol/acetic acid (12:1:1). Yellow
nylamino)piperidin-1-yl)methyl)benzoic acid (5a5).
4.1.5.9.
nitrophenylamino)piperidin-1-yl)methyl)benzoate
(5a9). Elution with petroleum ether/ethyl acetate (4:1). Yel-
low solid, yield: 71.4%, mp: 182–184 °C. H NMR (400 MHz, CDCl
ppm) d: 8.27 (d, 1H, J = 7.2 Hz, NH), 8.14 (d, 1H, J = 9.5 Hz, C -Ph-H),
-Ph -H), 7.42
-Ph-H),
-Ph-H), 3.92 (s, 3H, COO–
Methyl
4-((4-(5-(4-cyano-2,6-dimethylphenoxy)-2-
1
3
solid, yield: 39.2%, mp: 230–232 °C. H NMR (400 MHz, CDCl ,
1
ppm) d: 8.29 (d, 1H, J = 6.2 Hz, NH), 8.14 (d, 1H, J = 9.4 Hz, C
6
0
-Ph-
3
,
0
H), 8.05 (d, 2H, J = 7.7 Hz, C
3
,C
5
-Ph -H),7.47 (d, 2H, C
2
,C
6
-Ph -H),
6
00
0
00
7
.45 (s, 2H, C
dd, 1H, J = 1.8 Hz, J
m, 1H, C -Pi-H), 2.97 (t, 2H, Pi-CH
H, 2ꢀ CH ), 2.09 (m, 2H, Pi-CH ), 1.79 (m, 2H, Pi-CH
100 MHz, CDCl ppm) d: 162.87 (Ph-C ), 153.72 (Ph -C
46.41, 133.03, 130.25, 130.16, 129.62, 127.83, 118.37 (C„N),
09.82, 103.17, 97.70 (Ph-C ), 62.00 (Pi-CH -Py), 53.44, 51.07,
0.56, 16.18 (2ꢀ CH ). ESI-MS: m/z 499.5 (Mꢁ1). C28
3
,C
5
-Ph -H), 6.13 (d, 1H, J = 1.3 Hz, C
= 9.4 Hz, C -Ph-H), 3.77 (s, 2H, CH
), 2.49 (t, 2H, Pi-CH ), 2.15 (s,
3
-Ph-H), 5.96
8.00 (d, 2H, J = 8.3 Hz, C
(d, 2H, J = 8.1 Hz, C ,C
5.96 (dd, 1H, J = 2.5 Hz, J
CH ), 3.59 (s, 2H, CH ), 3.34 (m, 1H, C
2.22 (t, 2H, Pi-CH
), 2.16 (s, 6H, 2ꢀ CH
1.67 (m, 2H, Pi-CH
(C@O), 162.82 (Ph-C
130.22, 129.66, 129.11, 128.84, 127.74, 118.40 (C„N), 109.78,
103.04, 97.70 (Ph-C ), 62.53 (Pi-CH -Py), 52.09 (O–CH ), 51.54,
). ESI-MS: m/z 515.5 (M+1), 537.4 (M+23).
3
,C
5
0
-Ph -H),7.45 (s, 2H, C
,C
3 5
(
(
6
1
2
5
2
), 3.43
2
6
-Ph -H), 6.13 (d, 1H, J = 2.5 Hz, C
3
1
2
2
1
2 5
= 9.5 Hz, C
1
3
3
2
2
). C NMR
3
2
1
-Pi-H), 2.79 (t, 2H, Pi-CH
), 1.99 (m, 2H, Pi-CH
). C NMR (100 MHz, CDCl
2
),
),
0
0
(
3
,
4
1
),
2
3
2
1
3
1
1
3
2
3
, ppm) d: 167.01
4 1
), 153.77 (Ph -C ), 146.53, 133.04, 133.01,
0
0
3
2
3
28 4 5
H N O
[
500.21].
3
2
3
3
C
1.47, 16.18 (2ꢀ CH
3
4
.1.5.6. 3,5-Dimethyl-4-(3-(1-(4-(methylsulfonyl)benzyl)piperi-
Elution
with petroleum ether/ethyl acetate/triethylamine (5:1:0.1). Yellow
29 30 4 5
H N O
[514.22].
din-4-ylamino)-4-nitrophenoxy)benzonitrile (5a6).
4.1.5.10. 4-(3-(1-Benzylpiperidin-4-ylamino)-4-nitrophenoxy)-
3,5-dimethylbenzonitrile (5a10). Elution with petroleum
ether/ethyl acetate (5:1). Yellow solid, yield: 72.0%, mp: 156–
1
3
solid, yield: 78.1%, mp: 164–166 °C. H NMR (400 MHz, CDCl ,
ppm) d: 8.28 (d, 1H, J = 7.0 Hz, NH), 8.14(d, 1H, J = 9.5 Hz, C
6
-Ph-
0
1
H), 7.91 (d, 2H, J = 8.2 Hz, C
3
,C
5
-Ph -H), 7.58 (d, 2H, J = 7.2 Hz,
158 °C. H NMR (400 MHz, CDCl
3
, ppm) d: 8.26 (d, 1H, J = 7.2 Hz,
0
00
00
C ,C
2
6
-Ph -H), 7.46 (s, 2H, C
3
,C
= 2.5 Hz, J
-Pi-H), 3.07 (s, 3H, SO
), 2.26 (t, 2H, Pi-CH
), 1.70 (m, 2H, Pi-CH
), 153.75 (Ph -C
5
-Ph -H), 6.13 (d, 1H, J = 2.4 Hz, C
3
-
NH), 8.14 (d, 1H, J = 9.5 Hz, C
6
-Ph-H), 7.45 (s, 2H, C
-Ph -H), 7.26 (m, 1H, C
3
,C
5
-Ph -H),
0
0
Ph-H), 5.97 (dd, 1H, J
1
2
= 9.5 Hz, C
5
-Ph-H), 3.64 (s, 2H,
7.33 (m, 4H, C
1H, J = 2.5 Hz, C
Ph-H), 3.53 (s, 2H, CH
,C ,C
2 3 5
,C
6
4
-Ph -H), 6.12 (d,
CH
CH
CH
2
2
2
), 3.37 (m, 1H, C
1
2
–CH
3
), 2.79 (t, 2H, Pi-
3
-Ph-H), 5.95 (dd, 1H, J
), 3.32 (m, 1H, C
), 2.16 (s, 6H, 2ꢀ CH
1
= 2.5 Hz, J
-Pi-H), 2.80 (t, 2H, Pi-
), 1.97 (m, 2H, Pi-
). C NMR (100 MHz, CDCl , ppm) d:
2 5
= 9.5 Hz, C -
2
), 2.17 (s, 6H, 2ꢀ CH
3
), 2.00 (m, 2H, Pi-
). C NMR (100 MHz, CDCl , ppm) d:
), 146.47, 133.04, 133.02, 130.24,
2
1
1
3
2
3
CH
CH
2
), 2.20 (t, 2H, Pi-CH
), 1.65(m, 2H, Pi-CH
2
3
00
13
1
62.85 (Ph-C
4
1
2
2
3
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033

8
L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
0
0
). 13C NMR (100 MHz, CDCl
, ppm) d: 164.40 (Ph-
2 3
1
1
1
1
62.80 (Ph-C
30.21, 129.07, 128.30, 127.70, 127.14, 118.42 (C„N), 109.77,
03.00, 97.70 (Ph-C ), 63.00 (Pi-CH -Py), 51.50, 49.30, 31.59,
). ESI-MS: m/z 457.5 (M+1). C27 [456.22].
4
), 153.79 (Ph -C
1
), 146.59, 138.21, 133.05, 133.01,
(m, 2H, Pi-CH
), 147.93, 146.68, 139.99, 135.36, 130.47, 129.87, 129.70,
129.41, 127.16, 127.06, 103.79 (Ph-C ), 97.50 (Ph-C ), 65.12
(CH ), 62.61 (CH ), 51.34, 31.60, 31.29, 20.80 (Ph -C ), 16.09
2ꢀ CH ). ESI-MS: m/z 476.3 (M+1), 498.4 (M+23). C28
[475.25].
C
4
3
2
5
3
0
0
6.19 (2ꢀ CH
3
H
28
N
O
4 3
2
2
4 3
-CH
(
3
33 3 4
H N O
4
.1.5.11.
N-(5-(Mesityloxy)-2-nitrophenyl)-1-(4-nitroben-
Elution with petroleum ether/
ethyl acetate (6:1). Orange solid, yield: 98.6%, mp: 178–180 °C.
zyl)piperidin-4-amine (5b1).
4.1.5.15. 4-((4-(5-(Mesityloxy)-2-nitrophenylamino)piperidin-
1-yl)methyl)benzoic acid (5b5). Yellow solid, yield: 78.3%,
mp: 133–135 °C. H NMR (400 MHz, CDCl , ppm) d: 8.28 (d, 1H,
1
H NMR (400 MHz, CDCl
.19 (d, 2H, J = 8.6 Hz, C ,C
H), 7.52 (d, 2H, J = 8.6 Hz, C
.15 (d, 1H, J = 2.4 Hz,
= 9.5 Hz, C -Ph-H), 3.61 (s, 2H, CH
t, 2H, Pi-CH
3
, ppm) d: 8.28 (d, 1H, J = 7.2 Hz, NH),
0
1
8
3
5
-Ph -H), 8.11 (d, 1H, J = 9.5 Hz, C
6
00
-Ph-
3
0
,C
2 6
-Ph -H), 6.91 (s, 2H, C
3
,C
5
-Ph -H),
= 2.4 Hz,
-Pi-H), 2.76
), 2.25 (t, 2H, Pi-CH ), 2.07
), 2.00 (m, 2H, Pi-CH ), 1.67 (m, 2H, Pi-CH ).
C NMR (100 MHz, CDCl , ppm) d: 164.41 (Ph-C ), 147.93,
47.20, 146.64, 146.49, 135.36, 130.47, 129.89, 129.70, 129.34,
27.21, 123.59, 103.75 (Ph-C ), 97.53 (Ph-C ), 62.12 (Pi-CH -Py),
), 16.09 (2ꢀ CH ). ESI-MS:
[490.22].
J = 6.2 Hz, NH), 8.10 (d, 1H, J = 9.5 Hz, C
6
-Ph-H), 8.04 (d, 2H,
0
0
6
C
3
-Ph-H), 6.01 (dd, 1H,
), 3.38 (m, 1H, C
-Ph -CH
J
1
J = 7.5 Hz, C
(s, 2H, ,C
= 1.4 Hz, J
3
,C
5
-Ph -H), 7.44 (d, 2H, J = 7.7 Hz, C
,C
2 6
-Ph -H), 6.90
0
0
J
2
5
2
1
C
3
5
-Ph -H), 6.11 (d, 1H,
C
3
-Ph-H), 6.03 (dd, 1H,
), 3.44 (m, 1H,
00
(
(
2
), 2.31 (s, 3H, C
4
3
2
J
1
2
= 9.5 Hz, C -Ph-H), 3.84 (s, 2H, CH
5
2
00
s, 6H, C
,C
2 6
-Ph -CH
3
2
2
C
1
-Pi-H), 3.01 (t, 2H, Pi-CH
2
), 2.56 (t, 2H, Pi-CH
), 2.06 (s, 6H, C
). C NMR (100 MHz, DMSO-d
(COOH), 164.30 (Ph-C ), 147.91, 146.72, 143.62, 135.44, 131.98,
131.00, 130.39, 130.09, 129.68, 129.07, 128.92, 126.91, 103.96
(Ph-C ), 98.26 (Ph-C ), 62.02 (Pi-CH -Py), 51.50, 31.43, 30.47,
0.81 (Ph -C ). ESI-MS: m/z 490.4 (M+1),
), 16.12 (2ꢀ CH
512.5 (M+23). C28 [489.23].
2
), 2.30 (s, 3H, C
4
-
1
3
00
00
3
4
Ph -CH
((m, 2H, Pi-CH
3
), 2.06 (m, 2H, Pi-CH
2
2
,C
6
-Ph -CH
3
), 1.80
1
3
1
1
5
2
6
, ppm) d: 167.97
5
0
3
2
4
0
1.61, 48.78, 31.48, 20.80 (Ph -C
4
-CH
3
3
m/z 491.4 (M+1), 513.4 (M+23). C27
H
30
N
4
O
5
5
3
2
0
0
2
4
-CH
3
3
4
.1.5.12.
ylmethyl)piperidin-4-amine (5b2).
ether/ethyl acetate/triethylamine (1:1:0.1). Yellow solid, yield:
N-(5-(Mesityloxy)-2-nitrophenyl)-1-(pyridin-4-
31 3 5
H N O
Elution with petroleum
4.1.5.16. N-(5-(Mesityloxy)-2-nitrophenyl)-1-(4-(methylsulfo-
nyl)benzyl)piperidin-4-amine (5b6). Elution with petro-
leum ether/ethyl acetate/triethylamine (5:1:0.1). Yellow solid,
1
4
7.6%, mp: 105–107 °C. H NMR (400 MHz, CDCl
= 4.6 Hz, C
NH), 8.11 (d, 1H, J = 9.5 Hz, C
3
, ppm) d: 8.56
(
dd, 2H, J
1
= 1.4 Hz, J
2
3
5
,C -Py-H), 8.28 (d, 1H, J = 7.0 Hz,
1
6
-Ph-H), 7.30 (d, 2H, J = 5.5 Hz,
3
yield: 74.0%, mp: 136–138 °C. H NMR (400 MHz, CDCl , ppm) d:
0
C
,C
2 6
-Py-H), 6.91 (s, 2H, C
= 2.4 Hz, J
-Pi-H), 2.78 (t, 2H, Pi-CH
), 2.28 (t, 2H, Pi-CH ), 2.07 (s, 6H, C ,C
), 1.69 (m, 2H, Pi-CH
ppm) d: 164.42 (Ph-C ), 149.88, 147.91, 146.62, 135.37, 130.45,
29.89, 129.70, 127.21, 123.80, 103.84 (Ph-C ), 97.48 (Ph-C ),
-CH
), 16.09 (2ꢀ
). ESI-MS: m/z 447.5 (M+1), 469.5 (M+23).
446.23].
3
,C
5
-Ph -H), 6.13 (d, 1H, J = 2.3 Hz, C
3
-
8.28 (d, 1H, J = 7.0 Hz, NH), 8.10 (d, 1H, J = 9.5 Hz, C
6
-Ph-H), 7.90
0
0
Ph-H), 6.02 (dd, 1H, J
CH
Ph -CH
2
1
2
= 9.5 Hz, C
5
-Ph-H), 3.55 (s, 2H,
(d, 2H, J = 8.2 Hz, C
H), 6.91 (s, 2H, C ,C
6.01 (dd, 1H, J = 2.0 Hz, J
3.38 (m, 1H, C -Pi-H), 3.07 (s, 3H,SO
2.31 (s, 3H, C -Ph -CH
), 2.00 (m, 2H, Pi-CH
(100 MHz, CDCl
135.37, 130.46, 129.90, 129.71, 129.63, 127.46, 127.19, 103.78
(Ph-C ), 97.51 (Ph-C ), 62.23 (Pi-CH -Py), 51.52, 44.55 (SO –CH ),
1.42, 20.82 (Ph -C ). ESI-MS: m/z
), 16.10 (2ꢀ CH
524.5(M+1), 541.5 (M+18), 546.4 (M+23). C28 S [523.21].
3
,C
5
-Ph -H), 7.56 (d, 2H, J = 7.9 Hz, C
2 6
,C -Ph -
0
0
2
), 3.38 (m, 1H, C
1
2
), 2.31 (s, 3H, C
4
-
3
5 3
-Ph -H), 6.14 (d, 1H, J = 1.9 Hz, C -Ph-H),
0
0
3
2
2
6
3
-Ph -CH ), 2.03 (m,
1
2
5
= 9.4 Hz, C -Ph-H), 3.61 (s, 2H, CH
2
),
),
13
H, Pi-CH
2
2
). C NMR (100 MHz, CDCl
3
,
1
2
3
-CH ), 2.77 (t, 2H, Pi-CH
2
0
0
4
4
3
), 2.24 (t, 2H, Pi-CH
2
), 2.07 (s, 6H, C
2
,C
6
-
0
0
13
1
6
CH
5
3
Ph -CH
3
2
), 1.67 (m, 2H, Pi-CH
2
). C NMR
00
1.61 (Pi-CH
2
-Py), 51.50, 31.27, 20.80 (Ph -C
4
3
3 4
, ppm) d: 164.42 (Ph-C ), 147.91, 146.64, 139.32,
3
26 30 4 3
C H N O
[
5
3
2
2
3
0
0
3
4
-CH
3
3
4
.1.5.13. 1-(4-((4-(5-(Mesityloxy)-2-nitrophenylamino)piperi-
(5b3). Elution with
petroleum ether/ethyl acetate/triethylamine (5:1:0.1). Yellow so-
33 3 5
H N O
din-1-yl)methyl)phenyl)ethanone
4.1.5.17. 4-((4-(5-(Mesityloxy)-2-nitrophenylamino)piperidin-
1-yl)methyl)benzamide (5b7). Elution with petroleum
ether/ethyl acetate/triethylamine (3:1:0.27). Yellow solid, yield:
1
3
lid, yield: 87.2%, mp: 138–140 °C. H NMR (400 MHz, CDCl ,
ppm) d: 8.27 (d, 1H, J = 6.9 Hz, NH), 8.10 (d, 1H, J = 9.5 Hz, C
H), 7.93 (d, 2H, J = 8.1 Hz, C
6
-Ph-
0
1
3
,C
5
-Ph -H), 7.44 (d, 2H, J = 7.8 Hz,
3
92.8%, mp: 92–94 °C. H NMR (400 MHz, CDCl , ppm) d: 8.27 (d,
0
00
C
,C
2 6
-Ph -H), 6.91 (s, 2H, C
3
,C
= 2.0 Hz, J
-Pi-H), 2.78 (t, 2H, Pi-CH
5
-Ph -H), 6.13 (d, 1H, J = 1.9 Hz, C
= 9.4 Hz, C -Ph-H), 3.59 (s, 2H,
), 2.60 (s, 3H, CO-
), 2.23 (t, 2H, Pi-CH ), 2.07 (s, 6H,
), 2.00 (m, 2H, Pi-CH ), 1.67 (m, 2H, Pi-CH
NMR (100 MHz, CDCl , ppm) d: 197.80 (C@O), 164.40 (Ph-C
3
-
1H, J = 6.8 Hz, NH), 8.10 (d, 1H, J = 9.5 Hz, C
6
-Ph-H), 7.79 (d, 2H,
0
0
Ph-H), 6.02 (dd, 1H, J
CH
CH
1
2
5
J = 8.0 Hz, C
(s, 2H, C ,C
3
,C
5
-Ph -H), 7.43 (d, 2H, J = 7.9 Hz, C
2
6
,C -Ph -H), 6.91
0
0
2
), 3.36 (m, 1H, C
), 2.31 (s, 3H, C
1
2
3
5
-Ph -H), 6.13 (d, 1H, C
= 1.6 Hz, J = 9.4 Hz, C
-Pi-H), 2.77 (t, 2H, Pi-CH
), 2.23 (t, 2H, Pi-CH ), 2.07 (s, 6H, C
2H, Pi-CH ), 1.66 (m, 2H, Pi-CH
ppm) d: 169.25 (C@O), 164.42 (Ph-C
132.25, 130.46, 129.88, 129.70, 129.13, 127.46, 127.15, 103.78
(Ph-C ), 97.51 (Ph-C ), 62.47 (Pi-CH -Py), 51.49, 31.60, 31.40,
20.81 (Ph -C ). ESI-MS: m/z 489.6 (M+1),
), 16.10 (2ꢀ CH
11.5 (M+23). C28 [488.24].
3
-Ph-H), 6.06 (br, 2H, NH
2
),
00
3
4
-Ph -CH
3
2
6.01 (dd, 1H, J
3.36 (m, 1H, C
CH
1
2
5
-Ph-H), 3.58 (s, 2H, CH
2
00
),
00
13
C
2
,C
6
-Ph -CH
3
2
2
).
C
),
1
2
), 2.31 (s, 3H, C
4
-Ph -
), 2.01 (m,
). C NMR (100 MHz, CDCl
), 147.91, 146.68, 135.36,
0
0
3
4
3
2
2 6 3
,C -Ph -CH
1
3
1
1
5
1
C
47.94, 146.65, 136.26, 135.35, 130.46, 129.87, 129.69, 129.03,
28.45, 127.21, 103.79 (Ph-C ), 97.50 (Ph-C ), 62.50 (Pi-CH -Py),
1.52, 48.88, 31.58, 31.41, 26.62 (CO–CH ), 20.79 (Ph -C -CH ),
6.07 (2ꢀ CH ). ESI-MS: m/z 488.4 (M+1), 510.4 (M+23).
[487.25].
2
2
3
,
5
3
2
4
00
3
4
3
3
5
3
2
0
0
29
H
33
N
3
O
4
4
-CH
3
3
5
32 4 4
H N O
4
1
.1.5.14. (4-((4-(5-(Mesityloxy)-2-nitrophenylamino)piperidin-
-yl)methyl)phenyl)methanol (5b4). Elution with petro-
4.1.5.18. 4-((4-(5-(Mesityloxy)-2-nitrophenylamino)piperidin-
1-yl)methyl)benzenesulfonamide (5b8). Elution with petro-
leum ether/ethyl acetate/triethylamine (1:1:0.2). Yellow solid,
leum ether/ethyl acetate/triethylamine (4:1:0.2). Yellow solid,
1
yield: 62.7%, mp: 68–70 °C. H NMR (400 MHz, CDCl
.26 (d, 1H, J = 6.9 Hz, NH), 8.10 (d, 1H, J = 9.5 Hz, C -Ph-H), 7.33
-Ph -H), 6.12 (d, 1H,
= 2.1 Hz, J = 9.5 Hz, C -Ph-H),
-Ph ), 3.34 (m, 1H, C
3
, ppm) d:
1
8
6
3
yield: 75.3%, mp: 128–130 °C. H NMR (400 MHz, CDCl , ppm) d:
0
00
(
d, 4H, C
,C
2 3
,C ,C
5 6
-Ph -H), 6.91 (s, 2H, C
,C
3 5
8.27 (d, 1H, J = 6.4 Hz, NH), 8.10 (d, 1H, J = 9.4 Hz, C
6
-Ph-H), 7.89
0
0
J = 1.9 Hz, C
3
-Ph-H), 6.01 (dd, 1H, J
1
2
5
(d, 2H, J = 7.8 Hz, C
H), 6.91 (s, 2H, C ,C
J = 9.2 Hz, C -Ph-H), 5.14 (s, 2H, NH
1H, C -Pi-H), 2.74 (t, 2H, Pi-CH
3
,C
5
-Ph -H), 7.49 (d, 2H, J = 7.8 Hz, C
2 6
,C -Ph -
0
0
00
4
.69 (s, 2H, Ph -CH
2
–O), 3.54 (s, 2H, Pi-CH
), 2.31 (s, 3H, C
2
1
-
3
5 3
-Ph -H), 6.14 (d, 1H, C -Ph-H), 6.01 (dd, 1H,
0
0
Pi-H), 2.78 (t, 2H, Pi-CH
Pi-CH ), 2.07 (s, 6H, C ,C
2
4
-Ph -CH
3
), 2.22 (t, 2H,
), 1.66
5
2
), 3.59(s, 2H, CH
2
), 3.37 (m,
), 2.24 (t,
00
00
2
2
6
-Ph -CH
3
), 2.01 (m, 2H, Pi-CH
2
1
2
), 2.31 (s, 3H, C
4
-Ph -CH
3
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033

L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
0
0
2
H, Pi-CH
2
), 2.07 (s, 6H, C
2
,C
). C NMR (100 MHz, CDCl
47.90, 146.66, 140.83, 135.38, 130.45, 129.90, 129.71, 129.55,
27.16, 126.51, 103.84 (Ph-C ), 97.52 (Ph-C ), 62.18 (Pi-CH -Py),
), 16.10 (2ꢀ CH ). ESI-MS:
S [524.21].
6
-Ph -CH
3
), 1.98 (m, 2H, Pi-CH
2
), 1.66
4.1.7. General procedure for the synthesis of tert-butyl 4-(2-
acetamido-5-(4-cyano-2,6-
dimethylphenoxy)phenylamino)piperidine-1-carboxylate (9)
1
3
(
m, 2H, Pi-CH
2
3
, ppm) d: 164.45 (Ph-C
4
),
1
1
6
5
0
3
2
To palladium–carbon catalyst (0.05 g, 10%) was added absolute
ethyl alcohol (8 ml) gently, then the suspension was added to 3a
(0.5 g, 1.07 mmol) in THF (2 ml). The reaction mixture was exposed
to hydrogen at 30 °C and stirred for 6 h. The suspension was fil-
tered, the filtrate was concentrated and the solvent was replaced
with dichloromethane. Then triethylamine (0.16 g, 1.6 mmol) and
acetylchloride (0.1 g, 1.28 mmol) was added successively in an
ice-bath and stirred for 1 h. The reaction mixture was washed with
water for 3 times, and then the water layer was extracted with
dichloromethane for 3 times. The combined organic layers were
dried over anhydrous sodium sulfate, purified by column chroma-
tography on silica and recrystallized from diethyl ether to afford
the intermediate 9 as a creamy white solid. Yield: 80.39%. ESI-
0
0.44, 51.31, 31.27, 20.81 (Ph -C
m/z 525.6(M+1), 547.2 (M+23). C27H N O
32 4 5
4
-CH
3
3
4
.1.5.19.
no)piperidin-1-yl)methyl)benzoate (5b9).
petroleum ether/ethyl acetate (7:1). Yellow solid, yield: 81.5%,
Methyl
4-((4-(5-(mesityloxy)-2-nitrophenylami-
Elution with
1
3
mp: 168–170 °C. H NMR (400 MHz, CDCl , ppm) d: 8.27 (d, 1H,
J = 7.0 Hz, NH), 8.10 (d, 1H, J = 9.5 Hz, C
6
-Ph-H), 8.00 (d, 2H,
0
0
J = 8.2 Hz, C
s, 2H, C ,C
H, J
s, 2H, CH
3
,C
5
-Ph -H), 7.42 (d, 2H, J = 7.9 Hz, C
,C
2 6
-Ph -H), 6.91
00
(
3
5
-Ph -H), 6.13 (d, 1H, J = 2.1 Hz, C
= 9.5 Hz, C -Ph-H), 3.92 (s, 3H, COO–CH
), 3.35 (m, 1H, C
3
-Ph-H), 6.01 (dd,
1
1
= 2.2 Hz, J
2
5
3
), 3.58
(
3
2
1
-Pi-H), 2.77 (t, 2H, Pi-CH
), 2.23 (t, 2H, Pi-CH ), 2.07 (s, 6H, C ,C
.01 (m, 2H, Pi-CH ), 1.66(m, 2H, Pi-CH
, ppm) d: 167.02 (C@O), 164.40 (Ph-C
35.36, 130.47, 129.89, 129.70, 129.67, 129.09, 128.88, 127.16,
03.77 (Ph-C ), 97.50 (Ph-C ), 62.56 (Pi-CH -Py), 52.10 (O–CH ),
1.57, 31.42, 20.82 (Ph -C -CH ). ESI-MS: m/z
), 16.10 (2ꢀ CH
04.4 (M+1), 526.4 (M+23). C29 [503.24].
2
), 2.31 (s,
00
00
4
H, C -Ph -CH
3
2
2
6
-Ph -CH
3
),
34 4 4
MS: m/z 479.4 (M+1), 501.4 (M+23). C27H N O [478.26].
1
3
2
CDCl
1
1
5
5
2
2
). C NMR (100 MHz,
), 147.92, 146.67,
3
4
4.1.8. General procedure for the synthesis of N-(4-(4-cyano-2,6-
dimethylphenoxy)-2-(piperidin-4-ylamino)phenyl)acetamide
(10)
Follow the procedure in the preparation of 4a and 4b to give the
compound 10 as a white solid. Yield: 96.9%.
5
3
2
3
00
4
3
3
33 3 5
H N O
4
.1.5.20. 1-Benzyl-N-(5-(mesityloxy)-2-nitrophenyl)piperidin-4-
Elution with petroleum ether/ethyl acetate
4.1.9. General procedure for the synthesis of N-(4-(4-cyano-2,6-
dimethylphenoxy)-2-(1-(4-sulfamoylbenzyl)piperidin-4-
ylamino)phenyl)acetamide (11)
amine (5b10).
1
(
(
16:1). Yellow solid, yield: 84.0%, mp: 95–97 °C.
400 MHz, CDCl
H
NMR
3
, ppm) d: 8.27 (d, 1H, J = 7.0 Hz, NH), 8.10 (d, 1H,
Follow the procedure in the preparation of 5a and 5b using
EtOAc/methanol/triethylamine (10/1/0.2) as eluant to afford the
0
J = 9.5 Hz, C
C
6
-Ph-H), 7.32 (m, 4H, C
,C
2 3
,C ,C
5 6
-Ph -H), 7.25 (m, 1H,
-Ph-
-Ph-H), 3.53 (s, 2H,
0
00
1
4
-Ph -H), 6.91 (s, 2H, C
3
,C
5
-Ph -H), 6.12 (d, 1H, J = 2.4 Hz, C
3
product as colorless syrup. Yield: 62.2%. H NMR (400 MHz,
H), 6.01 (dd, 1H, J
CH ), 3.33 (m, 1H, C
-Ph -CH
m, 2H, Pi-CH
ppm) d: 164.38 (Ph-C
1
= 2.4 Hz, J
-Pi-H), 2.79 (t, 2H, Pi-CH
), 2.19 (t, 2H, Pi-CH ), 2.07 (s, 6H, C ,C
), 1.64 (m, 2H, Pi-CH
), 147.95, 146.73, 138.21, 135.35, 130.49,
29.87, 129.70, 129.10, 128.30, 127.16, 127.14, 103.74 (Ph-C ),
2
= 9.5 Hz, C
5
DMSO-d
6
0
, ppm) d: 9.00(s, 1H, Ph-NH-CO), 7.80 (d, 2H, J = 8.2 Hz,
00
2
1
2
), 2.31 (s, 3H,
C
C
3
,C
,C
5
-Ph -H), 7.67 (s, 2H, C
3
5
,C -Ph -H), 7.50 (d, 2H, J = 8.2 Hz,
0
0
00
0
C
(
4
3
2
2
6
3
-Ph -CH ), 1.98
2
6
-Ph -H), 7.32 (s, 2H, NH
2
), 6.97 (d, 1H, J = 8.6 Hz, C
-Ph-H), 5.71 (dd, 1H, = 2.4 Hz,
-Ph-H), 4.80(d, 1H, J = 7.5 Hz, Pi-NH), 3.55 (s, 2H,
), 3.41 (t, 2H, Pi-CH ), 3.17 (m, 1H, C -Pi-H), 2.75 (t, 2H, Pi-
), 2.11 (s, 6H, 2ꢀ CH ), 2.07 (s, 3H, CO–CH ), 1.87 (m, 2H, Pi-
), 1.44 (m, 2H, Pi-CH ). C NMR (100 MHz, DMSO-d , ppm) d:
6
-Ph-H),
13
2
2
). C NMR (100 MHz, CDCl
3
,
6.20 (d, 1H, J = 2.3 Hz,
= 8.5 Hz, C
C
3
J
1
4
J
2
5
1
9
C
5
CH
CH
CH
2
2
2
2
1
0
0
7.51 (Ph-C
3
), 63.01 (Pi-CH
2
-Py), 51.50, 49.32, 31.51, 20.82 (Ph -
3
3
1
3
4
-CH
3
), 16.11 (2ꢀ CH
3
). ESI-MS: m/z 446.5 (M+1). C27
H
31
N
3
O
3
2
6
[
445.24].
169.21 (C@O), 155.81, 155.24, 143.66, 143.17, 133.52, 133.36,
29.38, 127.97, 126.08, 119.14, 118.89 (C„N), 108.18, 100.59,
98.63 (Ph-C ), 61.85 (Pi-CH -Py), 52.22, 49.44, 31.97, 23.67
(CO–CH ). ESI-MS: m/z 548.5 (M+1). C29
), 16.14 (2ꢀ CH
[547.23].
1
4
.1.6. General procedure for the synthesis of 4-((4-(2-amino-5-
3
2
(
4-cyano-2,6-dimethylphenoxy)phenylamino)piperidin-1-
3
3
33 5 4
H N O S
yl)methyl)benzenesulfonamide hydrochloride (7a1)
To palladium–carbon catalyst (0.014 g, 10%) was added abso-
lute ethyl alcohol (1 ml) gently, then the suspension was added
to 5a8 (0.07 g) in THF (0.5 ml). The reaction mixture was exposed
to hydrogen at 30 °C and stirred for 36 h. The suspension was fil-
tered and the filtrate was purified to give 6a1 as slight pink syrup.
Subsequently, 6a1 was redissolved in DCM (0.5 ml) immediately
and concentrated hydrochloric acid (4 drops) was added to this
solution in an ice-bath. The mixture continued stirring for 0.5 h,
then the solvent was removed under reduced pressure and the res-
idue was recrystallized from ethanol to give the compound 7a1 as
4.2. In vitro anti-HIV assay
Evaluation of the antiviral activity of the compounds was per-
formed using the MTT assay as previously described.2
solutions (10ꢀ final concentration) of test compounds were added
in 25 l volumes to two series of triplicate wells so as to allow
3,27
Stock
l
simultaneous evaluation of their effects on mock-and HIV-infected
cells at the beginning of each experiment. Serial fivefold dilutions
of the 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
1
a white solid. Yield: 56.3%, mp: 207–209 °C. H NMR (400 MHz,
0
D
2
O, ppm) d: 7.94 (d, 2H, J = 8.0 Hz, C
3
,C
5
-Ph -H), 7.66 (d, 2H,
0
00
J = 8.1 Hz, C
2
,C
6
-Ph -H), 7.47 (s, 2H, C
3
,C
5
-Ph -H), 7.09 (d, 1H,
cell samples were included for each sample. HIV-1 (III
B
), HIV-1
2
8,29
J = 8.7 Hz, C
J = 8.6 Hz, C
6
-Ph-H), 6.40 (d, 1H, J = 2.1 Hz, C
-Ph-H), 4.37 (s, 2H, CH ), 3.53 (t, 2H, Pi-CH
-Pi-H), 3.08 (m, 2H, Pi-CH ), 2.21 (t, 2H, Pi-CH
), 1.65 (m, 2H, Pi-CH
3
-Ph-H), 6.01 (d, 1H,
), 3.31
), 2.01
(RES056) or HIV-2 (ROD)
stock (50 ll) at 100–300 CCID50
5
2
2
(50% 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 com-
pounds on uninfected cells in order to assess its cytotoxicity. Expo-
(
(
m, 1H, C
s, 6H, 2ꢀ CH
1
2
2
13
3
2
). C NMR (100 MHz, D
2
O,
00
0
ppm) d: 158.15 (Ph -C
1
), 154.62 (Ph-C
4
), 143.01 (Ph -C
4
), 141.70
0
30
(
Ph -C
1
), 133.81, 133.63, 133.52, 133.33, 132.38, 132.20, 126.78,
nentially growing MT-4 cells were centrifuged for 5 min at
1
CH
25.75, 119.72 (C„N), 111.65, 108.08, 103.92, 101.17, 59.52 (Pi-
1000 rpm and the supernatant was discarded. The MT-4 cells were
5
2 3
-Py), 51.69, 47.50, 28.89, 15.34 (2ꢀ CH ). ESI-MS: m/z 506.4
resuspended at 6 ꢀ 10 cells/ml, and 50
ll volumes were trans-
(
M+1), 528.4 (M+23). C27
H
31
N
5
O
3
S [505.21].
ferred to the microtiter tray wells. Five days after infection, the via-
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033

1
0
L. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
bility of mock-and HIV-infected cells was examined spectrophoto-
metrically by the MTT assay.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.033.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.033