Design, synthesis and anti-HIV evaluation of novel diarylnicotinamide derivatives (DANAs) targeting the entrance channel of the NNRTI binding pocket through structure-guided molecular hybridization

Article abstract of DOI:10.1016/j.ejmech.2014.09.054

Through a structure-based molecular hybridization approach, a novel series of diarylnicotinamide derivatives (DANAs) targeting the entrance channel of HIV-1 NNRTIs binding pocket (NNIBP) were rationally designed, synthesized and evaluated for their anti-H

Full text of DOI:10.1016/j.ejmech.2014.09.054

European Journal of Medicinal Chemistry 87 (2014) 52e62
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and anti-HIV evaluation of novel diarylnicotinamide
derivatives (DANAs) targeting the entrance channel of the NNRTI
binding pocket through structure-guided molecular hybridization
Zhaoqiang Liu , Wenmin Chen , Peng Zhan *, Erik De Clercq b,
a
a
a,
b
a, *
Christophe Pannecouque , Xinyong Liu
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University,
4, West Culture Road, 250012 Jinan, Shandong, PR China
a
4
b
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2014
Received in revised form
Through a structure-based molecular hybridization approach, a novel series of diarylnicotinamide de-
rivatives (DANAs) targeting the entrance channel of HIV-1 NNRTIs binding pocket (NNIBP) were ratio-
nally designed, synthesized and evaluated for their anti-HIV activities in MT-4 cells together with the
inhibition against the reverse transcriptase (RT) in an enzymatic assay. Encouragingly, most of the new
12 September 2014
Accepted 15 September 2014
Available online
DANAs were found to be active against wild-type HIV-1 with an EC50 in the range of 0.027e4.54
Among them, compound 6b11 (EC50 ¼ 0.027 M, SI > 12518) and 6b5 (EC50 ¼ 0.029 M, SI ¼ 2471) were
identified as the most potent inhibitors, which were more potent than the reference drugs nevirapine
M). Some DANAs were also active at micromolar con-
mM.
m
m
Keywords:
HIV-1
NNRTIs
DANAs
Entrance channel
Molecular hybridization
Drug design
(
EC50 ¼ 0.31
m
M) and delavirdine (EC50 ¼ 0.66
m
centrations against the K103N þ Y181C resistant mutant. Compound 6b11 exhibited the highest enzy-
matic inhibition activity (IC50 ¼ 20 nM), which is equal to that of efavirenz (EC50 ¼ 20 nM) and 31 times
higher than that of nevirapine (EC50 ¼ 0.62
mM). Preliminary structure-activity relationships (SARs) and
molecular modeling of these new DANAs have been discussed.
©
2014 Elsevier Masson SAS. All rights reserved.
1
. Introduction
superior activity profiles have attracted considerable attention over
the past few years [5]. Up to now, two DAPY derivatives, etravirine
(TMC125, ETV) and rilpivirine (TMC278, RPV), have been launched
in the marketplace by FDA for the treatment of HIV infection in
2008 and 2011 respectively (Fig. 1). Besides, indolylarylsulfone
(IAS) derivatives (lead compound L-737,126 and 7e) represent
another series of new generation NNRTIs with broad spectrum of
activity against drug-resistant HIV strains [4,6,7]. With an attempt
to improve their anti-HIV-1 activity, most work on the modification
of IASs was focused on introducing different substituents into the
2-carboxamide nitrogen [8e12].
Although DAPYs and IASs belong to two structurally different
scaffolds, these two types of NNRTIs share a similar pharmaco-
phoric feature and binding conformation. As shown in Fig. 2, a
crystallographic overlay of TMC125 [13] and 7e [7] within their
NNRTIs binding site (NNIBS) revealed that most fragments in their
structures can overlap well with each other. The pyrrolidine ring of
Acquired immune deficiency syndrome (AIDS), mainly caused
by human immunodeficiency virus type-1 (HIV-1) is still a preva-
lent disease worldwide. The most common and effective treatment
of AIDS is the highly active antiretroviral therapy (HAART). Non-
nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are
important components of HAART with high antiviral potency, high
specificity and low cytotoxicity [1]. Even so, a major problem is that
the long-term efficacy of NNRTIs is limited by the rapid emergence
of drug-resistant variants of HIV-1. Hence, the development of
novel chemical entities with high affinity for the mutated RT has
been a very active research field in recent years [2e4].
Among the more than 50 different series of NNRTIs that have
been reported so far, diarylpyrimidine (DAPY) derivatives with
7
e and the 2,6-dimethyl-4-cyanophenoxyl group of TMC125 both
*
Corresponding authors.
E-mail addresses: zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn,
participate in hydrophobic interactions with residues Tyr188 and
xinyongllab@163.com (X. Liu).
http://dx.doi.org/10.1016/j.ejmech.2014.09.054
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
53
Fig. 1. The structures of representative DAPYs (TMC125 and TMC278), IASs (L-737,126 and 7e), pDAPY and MDec5.
Tyr188. The phenyl ring of the indolyl group of 7e can overlap with
the right aryl ring of TMC125, pointing toward the solvent exposed
region. The 2-carbamoyl-pyrrole moiety functioning as the py-
rimidine ring of TMC125 developed critical hydrogen bonding with
diarylnicotinamide derivatives (DANAs) were synthesized, and
their anti-HIV activities against wild-type HIV-1 and HIV-2, as well
as their activities against the double mutant HIV-1 strain
(K103N þ Y181C) were evaluated. Preliminary structure-activity
relationships (SARs) and molecular modeling results of these new
compounds were also discussed.
2
the backbone of Lys101. In addition, the NH and Br groups on the
central pyrimidine ring of TMC125 pointed to the entrance channel
which was surrounded by amino acids Leu100, Lys101, Glu138 and
Val179 [13], the substituents at the 2-carboxamide nitrogen may
also fit into the entrance channel in the docking study of IAS de-
rivatives [10,14,15].
2. Chemistry
The expeditious and straightforward synthetic route is depicted
in Scheme 1. The synthetic work started with the commercially
available diethyl 1,3-acetonedicarboxylate (1). Reaction of 1 with
triethyl orthoformate in acetic anhydride, followed by cyclisation
with ammonia, resulted in formation of ethyl 4,6-
dihydroxynicotinate (2). Treatment of 2 with phosphorus oxy-
chloride gave ethyl 4,6-dichloronicotinate (3), which underwent
nucleophilic substitution reaction with different substituted phe-
The entrance channel was first discovered in the structural
optimization and molecular modeling study of IAS series [14,15].
More studies suggested that the introduction of nitrogen-
containing aromatic heterocycles, amino acids, oligopeptides and
so on at the 2-position of the indole ring of IAS will form the double
hydrogen bonding with Lys101 and Glu138 of the channel, thus
enhancing the potency against the wild-type or mutant virus
strains [8e12]. According to the SAR studies of the DAPY de-
rivatives, the substituents on the 5,6-position of the central py-
rimidine ring was changeable [16]. The piperidinylamino-
diarylpyrimidine (pDAPY) derivatives were designed through a
molecular hybridization strategy in our previous studies [17].
Among the pDAPY derivatives, compound MDec5 was identified as
the most potent inhibitor against wild-type and K103N þ Y181C
2 3
nols under conditions of DMF/K CO , generating intermediate ethyl
6-chloro-4-(4-cyano-2,6-dimethylphenoxy)nicotinate (4a) and
ethyl 6-chloro-4-(mesityloxy)nicotinate (4b). Then ethyl 4-(4-
cyano-2,6-dimethylphenoxy)-6-(4-cyanophenylamino)nicotinate
(5a) and ethyl 6-(4-cyanophenylamino)-4-(mesityloxy)nicotinate
(5b) were obtained from 4a and 4b by BuchwaldeHartwig reaction
with 4-aminobenzonitrile. The final derivatives 6a1-11 and 6b1-11
were obtained by ammonolysis of 5a and 5b using trimethylalu-
minium [18]. The synthesized compounds were characterized by
drug-resistant mutant HIV-1 strain with an EC50 value of 0.038
mM
and 0.95 M respectively (Fig. 1), which confirmed the design ra-
m
1
13
tionality of new multi-sites binding NNRTIs especially with the
entrance channel targeting property.
physicochemical and spectral means. The MS and H NMR, C NMR
spectral data were found in agreement with the assigned molecular
structures.
Prompted by the analyses of the crystallographic overlay of
TMC125 and 7e, the novel diarylnicotinamide scaffold was created
using crystallographic overlays based molecular hybridization. As
shown in Fig. 3, the preferred combination of the trisubstituted
phenoxy ring (A-ring), the para-cyanoaniline moiety (B-ring), and
the NH group linking the B ring and pyrimidine core (C-ring) of
DAPYs were preserved. Substituents at the 2-carboxamide nitrogen
in the IASs series were introduced to further improve the interac-
tion with the entrance channel. These newly designed
3. Results and discussion
3.1. Anti-HIV activity evaluation
The newly synthesized DANAs were evaluated for their activity
against wild-type HIV-1 strain (III
HIV-1 strain (RES056) and HIV-2 ROD strain in MT-4 cells using the
B
), K103N þ Y181C double mutant

54
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
Fig. 2. Crystallographic overlay of the RT coecrystal structures of TMC125 (green) (PDB code: 3MEC, resolution: 2.3 Å) and 7e (pink) (PDB code: 2RF2, resolution: 2.4 Å). This figure
was generated using PyMOL (www.pymol.org). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
MTT method [19e21]. Nevirapine (NVP), lamivudine (3 TC), zido-
vudine (azidothymidine, AZT), didanosine (DDI), efavirenz (EFV),
delavirdine msylate (DLV) and etravirine (ETV) were used as
reference drugs. The cytotoxicity of the compounds was deter-
mined in parallel. The results, expressed as EC50, CC50 and SI, are
illustrated in Table 1.
conjunction with previous studies suggest that the C4-position on
the 2,6-dimethylphenyl fragment (A ring) is a major determinant
for the anti-HIV-1 activity of DAPY analogues.
Obviously, contrary to our expectations, most of the N-
substituted amides resulted in a reduced anti-HIV potency when
compared to compound 6a11 or 6b11. Among the N-substituted
amides, the optimum activities were found in compounds with
As shown in Table 1, except for compound 6a1, all the novel
synthesized NNRTIs exhibited moderate (compound 6a6:
substituents containing a
p-electron character. Prop-2-yn-1-yl
EC50 ¼ 4.54
m
M; 6a8: EC50 ¼ 3.1
m
M; 6a3: EC50 ¼ 1.74
m
M) to
(6a5, 6b5) appeared to be the most favorable group, closely fol-
lowed by N-substituents in the sequence prop-2-yn-1-yl > allyl, 2-
cyanoethyl. However, bulky substitutions such as 2-(dimethyla-
mino)ethyl (6a1, 6b1) or 2,2-dimethoxyethyl (6a6, 6b6) at this
position led to significant loss of antiviral potency compared to
other analogues in each series. Besides, it is worth noting that the
introduction of a hydrophobic ring group may impair the activity,
which can be clearly seen from the contrast of compounds 6a4 and
6a2, 6b4 and 6b2. In conclusion, SAR investigation revealed that
bulky substitutions at the amide position is probably detrimental to
excellent potency (EC50 from 0.027 to 0.85
m
M) against wild-type
HIV-1. Totally, 14 compounds exhibited highly potent inhibitory
activity against HIV-1 replication, which were much better than
those of NVP, 3 TC, DDI and DLV. Among them, compounds 6b11
(
EC50 ¼ 0.027
m
M, SI > 12,518) and 6b5 (EC50 ¼ 0.029 M, SI ¼ 2471)
m
were the most active derivatives. Especially, 6b11 was about 11
times more active than NVP and 24 times more active than DLV, but
still inferior to EFV and ETV. Even so, DANAs merit further modi-
fication as potential antiviral agents. Our results unambiguously
further validates the molecular hybridization strategy based on
crystallographic overlay as an effective strategy for the discovery of
novel NNRTIs. None of these compounds showed activity against
HIV-2, which means the newly designed DANAs most likely belong
to the genuine HIV-1 NNRTIs.
activity, while
p-electron containing linear substitutions can be
tolerated at this position.
Moreover, it cannot be ignored that the replacement of the
nitrile in 6a series by a methyl moiety resulted in 6b series with
significantly exacerbated cytotoxicity (as highlighted by the CC50).
In each series, the compounds bearing a 2-(dimethylamino)ethyl
group (6a1 and 6b1) always exhibited the highest cytotoxicity. It
was also noted that the inhibitors containing an unsubstituted
amide (6a11, 6b11) revealed a remarkable reduced cytotoxicity
The preliminary SARs analysis indicated that the anti-HIV-1
(
III
B
) activity is strongly dependent on the nature of the sub-
and R . The detailed discussion of the SAR data for
stituents at R
1
2
these DANAs will be presented in due course.
Firstly, the methyl group was more favorable to their anti-HIV-1
with the higher CC50 value of >325.95 mM and >335.66 mM,
activities than the nitrile group at the R
1
position which can be
respectively. No wonder the selectivity index of 6a11 and 6b11 were
clearly observed within 6a and 6b series. These results in
the highest. From what has been discussed above, we can draw the

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
55
Fig. 3. Design of diarylnicotinamide derivatives (DANAs) based on crystallographic overlay-guided molecular hybridization.
conclusion that the cytotoxicity of DANAs was also directly related
to the nature of the substituents on R and R position.
3.2. HIV-1 RT inhibition assay
1
2
The activity of the compounds against K103N þ Y181C double
RT mutant strain (RES056), which confers resistance towards most
of the classical NNRTIs, was also tested due to the rapid drug
resistance emergence in the clinical treatment of AIDS. The results
are illustrated in Table 1. Unfortunately, most of the tested com-
pounds were inactive against the RES056 strain except for com-
pounds 6b3, 6b5 and 6b9, which showed moderate activities
against the RES056 strain with EC50 values of 7.8, 6.1 and 10.1 mM
respectively. In particular, compound 6b5 was inferior to the
reference drugs EFV and ETV, but more potent than the reference
All the compounds were tested in enzymatic assays against HIV-
1 RT (WT) using poly(rA)-oligo(dT) as template/primer and
digoxigenin-/biotin-labeled dUTP as nucleotides [22,23] in order to
confirm the binding target. Nevirapine (NVP) and efavirenz (EFV)
were used as reference drugs. As shown in Table 2, on the whole,
the enzymatic activity and SARs of these compounds is in line with
the results of the cellular tests. It is reasonable that the newly
synthesized compounds can specifically target HIV-1 RT. Among
them, eight compounds showed much better RT inhibitory activity
than that of NVP. Three compounds (6a11, 6b5, 6b11) demonstrated
extremely high potency compared to that of EFV. Especially, com-
pound 6b11 exhibited the highest enzymatic inhibition activity
(IC50 ¼ 20 nM), which is equal to that of EFV (EC50 ¼ 20 nM) and 31
drugs NVP and DLV. The results indicated that the existing of
p-
electron (prop-2-yn-1-yl and allyl) is a key chemical feature
responsible for the exceptional biological activities of the com-
pounds against the mutant HIV-1 strain, which can be seen from
times higher than that of NVP (EC50 ¼ 0.62
mM).
6
2
b5 and 6b9. Even small modifications in the R position of DANAs
can impair or restore the activity of the new resulting compounds
against the resistant HIV-1 strain, suggesting that these DANAs
have the potential to be further optimized as new derivatives with
improved antiviral efficacy and favorable drug resistance profiles.
3
.3. Molecular modeling analysis
By means of SYBYL-X 1.1 (Tripos, St. Louis, MO, USA), the most
potent compound 6b11 was docked into the NNRTIs binding pocket
Scheme 1. Reagents and conditions: i: (a) Acetic anhydride, triethyl orthoformate, 120 C; (b) NH.
ꢀ
H O, rt; ii: POCl , CH CN, triethylamine, 80 C iii: ArOH, K CO , DMF, 60 C; iv: 4-
ꢀ
ꢀ
3
2
3
3
2
3
ꢀ
ꢀ
aminobenzonitrile, Pd(OAc) , Xantphos, Cs CO , 90 C; v: Amine, trimethylaluminium, toluene, 80 C.
2 2 3

56
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
Table 1
Activity and cytotoxicity against HIV-1 (III
B
and RES056) and HIV-2 (ROD) strains in MT-4 cells.
Compds
R
1
R
2
EC50
III
(
m
M)a
CC50
(
m
M)b
SI(III
B
)c
B
ROD
RES056
6a1
CN
ꢁ8.8
ꢁ7.5
>23.2
23.19 ± 0.97
ꢂ3
6a2
6a3
6a4
6a5
6a6
CN
CN
CN
CN
CN
0.54 ± 0.05
1.74 ± 0.25
0.85 ± 0.13
0.24 ± 0.03
4.54 ± 0.57
>169.7
>61.0
>169.7
>61.0
169.68 ± 27.95
61.04 ± 5.49
ꢁ146.29
321
35
>146.5
>166.7
>90.6
>146.5
>166.7
>90.6
ꢁ175
703
20
166.67 ± 13.02
90.56 ± 25.43
6
a7
a8
CN
CN
0.31 ± 0.06
3.10 ± 0.2
>231.4
>141.8
>231.4
>142.5
231.40 ± 23.50
142.52 ± 22.63
784
46
6
6
6
a9
CN
CN
0.24 ± 0.06
0.69 ± 0.06
>207.1
>65.8
>207.1
>65.8
207.08 ± 25.79
65.82 ± 6.6
625
97
a10
6
a11
b1
CN
Me
0.05 ± 0.013
0.63 ± 0.12
>325.9
>16.7
>325.9
>16.69
>325.95
>6195
6
4.67 ± 0.37
7
6b2
6b3
6b4
6b5
6b6
Me
Me
Me
Me
Me
0.055 ± 0.002
0.067 ± 0.006
0.11 ± 0.02
ꢁ11.1
>24.0
>30.6
>73.0
>23.2
ꢁ11.15
18.89 ± 3.84
24.04 ± 2.00
30.57 ± 10.48
72.96 ± 26.85
23.19 ± 2.96
335
359
264
2471
83
7.8 ± 0.76
>30.57
0.029 ± 0.005
0.28 ± 0.04
6.1 ± 0.54
>23.19
6
6
b7
b8
Me
Me
0.24 ± 0.02
>25.7
>25.75
25.75 ± 1.77
>273.8
103
0.18 ± 0.049
>273.8
>273.82
>1492
6
6
6
b9
Me
Me
Me
0.058 ± 0.002
0.033 ± 0.002
0.027 ± 0.014
ꢁ21.5
>9.9
10.1 ± 0.26
>9.89
21.33 ± 4.20
9.89 ± 1.59
>335.66
364
310
b10
b11
>335.7
>335.66
>12,518

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
57
Table 1 (continued )
Compds
R
1
R
2
EC50
(
m
M)a
CC50
(
m
M)b
SI(III
B
)c
III
B
ROD
RES056
NVP
0.31 ± 0.015
2.22 ± 0.19
0.0071 ± 0.0008
23.20 ± 1.79
0.0063 ± 0.0005
0.66 ± 0.28
e
ꢁ7.58
>15.02
>87.24
>93.55
>211.66
>6.34
48
3
TC
8.81 ± 0.67
0.0067 ± 0.0004
44.91 ± 4.59
e
e
e
e
>39
>13,144
>9
>1014
>67
AZT
DDI
EFV
DLV
ETVd
0.010 ± 0.0025
e
0.16 ± 0.005
>43.81
0.025 ± 0.001
>43.81
>4.59
0.0041 ± 0.0001
>1127
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method.
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
b
c
SI: selectivity index, the ratio of CC50/EC50
.
d
ETV: Etravirine. The data were obtained from the same laboratory (Rega Institute for Medical Research, KU Leuven, Belgium).
(
PDB code: 3MEC). The theoretical binding mode of 6b11 to the
with the entrance channel of the NNIBP. Taking full advantage of
the valuable information from SARs analysis, further optimization
of this series of compounds aimed at improving drug resistance
profiles and exploring the salient features controlling the activity
are ongoing in our lab and will be reported in due course.
NNIBP is shown in Fig. 4. The left phenyl ring fitted into the
important hydrophobic sub-pocket, which is formed by aromatic
amino acid residues Tyr181, Tyr188, Phe227 and Trp229, exhibiting
p-p interaction with these residues. The right aniline ring extended
to the surface of solvent and protein contacting side chain of amino
acid Leu234 and Pro236. Three hydrogen bonds were formed with
backbone carbonyl and backbone amino of residue Lys101 and
Glu138, improving the affinity between RT and inhibitors. The
amide group pointed to the entrance channel forming a hydrogen
bond with Glu138. Taken together, the docking study was able to
support our original intention and is also in accordance with the
biological activity results.
5. Experimental section
5.1. Chemistry
All melting points were determined on a micromelting point
1
13
apparatus and are uncorrected. H NMR and C NMR spectra were
recorded on a Bruker AV-400 spectrometer using DMSO-d6 as
solvent and tetramethylsilane (TMS) as internal standard. Chemical
4
. Conclusion
shifts are reported in parts per million (d), and signals are expressed
as s (singlet), d (doublet), t (triplet), q (quartet) or m (multiplet).
Mass spectra were taken on an LC Autosampler Device: Standard
G1313A instrument. TLC was performed on Silica Gel GF254 for TLC
(Merck) and spots were visualized by iodine vapors or by irradia-
tion with UV light (254 nm). Flash column chromatography was
performed on column packed with Silica Gel 60 (200e300 mesh).
Solvents were reagent grade and, when necessary, were purified
In summary, a series of novel DANAs targeting the entrance
channel of NNIBP were rationally designed by using a crystallo-
graphic overlay-guided hybridization strategy, synthesized and
evaluated for their bioactivities against HIV-1 (III
B
strain and
K103N þ Y181C double-mutated strains) and HIV-2 (ROD) in MT-4
cells, as well as against HIV-1 RT activity. These efforts culminated
in the discovery of compounds 6b11 (EC50 ¼ 0.027
mM, SI > 12,518)
and 6b5 (EC50 ¼ 0.029 M, SI ¼ 2471), which showed extremely
m
promising activities against WT HIV-1 with EC50 values at double-
digit nanomolar level, much better than four clinically-used drugs
(
NVP, 3 TC, DDI and DLV), and 6b5 also possessed higher potency
towards the drug-resistant mutant RES056 strain than NVP and
DLV. Besides, the enzymatic activity order of these compounds is
essentially in line with the results of the cellular tests. Furthermore,
the detailed SARs were discussed. The molecular modeling analysis
supported our design hypothesis that the DANAs could interact
Table 2
Inhibitory activity against HIV-1 RT (WT).
Compds
IC50
(m
M)a
Compds
IC50(mM)
6
6
6
6
6
6
6
6
6
6
6
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
a11
25.77 ± 1.385
4.14 ± 0.125
11.27 ± 0.246
4.25 ± 0.076
1.29 ± 0.025
11.37 ± 1.601
3.37 ± 0.358
10.92 ± 0.430
2.85 ± 0.070
3.94 ± 0.214
0.08 ± 0.003
0.62 ± 0.038
6b1
6b2
6b3
6b4
6b5
6b6
6b7
6b8
6b9
6b10
6b11
EFV
3.45 ± 0.198
0.32 ± 0.019
0.52 ± 0.025
0.68 ± 0.011
0.07 ± 0.006
1.89 ± 0.291
0.23 ± 0.008
1.20 ± 0.105
0.17 ± 0.016
0.19 ± 0.008
0.02 ± 0
NVP
0.02 ± 0.002
a
IC50: Inhibitory concentration of tested compounds required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.
Fig. 4. Predicted binding modes of 6b11 in the NNIBP of wild-type HIV-1 RT (PDB
code: 3MEC) displayed by PyMOL.

58
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
and dried by standard methods. Concentration of the reaction so-
lutions involved the use of rotary evaporator at reduced pressure.
4b was synthesized as for 4a but using 2,4,6-trimethylphenol.
White crystals, yield: 46.4%. Mp: 74e77 C. H NMR (400 MHz,
ꢀ
1
DMSO-d
6
, ppm)
d
: 8.74 (s, 1H, pyridine-H), 7.04 (s, 2H, PhH), 6.36 (s,
), 2.29 (s, 3H, CH ), 2.03 (s,
6
). C NMR (100 MHz, DMSO-d ,
1
H, pyridine-H), 4.37 (q, 2H, J ¼ 7.1, CH
2
3
5.1.1. General procedure for the synthesis of ethyl 4,6-
13
6
H, 2 ꢃ CH
3
), 1.33 (t, 3H, J ¼ 7.1, CH
3
dihydroxynicotinate (2)
Diethyl 3-oxopentanedioate (1) is commercially available. 1
ppm)
d: 164.50,163.52,155.30,153.03,147.10,136.22,130.46 (2 ꢃ C),
H
129.94, 116.52, 109.02, 61.80, 20.83, 15.85 (2 ꢃ C), 14.47. ESI-MS: m/z
NMR (400 MHz, DMSO-d
s, 4H, 2 ꢃ CH ), 1.20 (t, 6H, J ¼ 7.1, 2 ꢃ CH
DMSO-d , ppm)
: 197.34,167.11 (2 ꢃ C), 61.07 (2 ꢃ C), 49.23 (2 ꢃ C),
4.31 (2 ꢃ C).
Diethyl 3-oxopentanedioate (1, 10.00 g, 49.45 mmol), triethyl
6
, ppm)
d
: 4.10 (q, 4H, J ¼ 7.1, 2 ꢃ CH
2
), 3.69
), C NMR (100 MHz,
13
320.3 (M þ 1), 322.4 (M þ 3). C17 18ClNO (319.10).
H
3
(
2
3
6
d
1
5
.1.4. General procedure for the synthesis of ethyl 4-(4-cyano-2,6-
dimethylphenoxy)-6-((4-cyanophenyl)amino)nicotinate (5a) and
ethyl 6-((4-cyanophenyl)amino)-4-(mesityloxy)nicotinate (5b)
Palladium acetate (0.01543 g, 0.069 mmol) and Xantphos
orthoformate (7.33 g, 49.45 mmol) and acetic anhydride (10.10 g,
ꢀ
9
8.93 mmol) were combined and stirred at 120 C for 2 h then
allowed to cool to ambient temperature. The volatiles were
removed under vacuum. The residue was then cooled in an ice bath
and aqueous ammonia (25 mL) was added in portions with stirring.
(
0.0796 g, 0.138 mmol) were dissolved in 15 mL dioxane and stirred
at ambient temperature for 15 min. 4-aminobenzonitrile (0.1625 g,
.38 mmol), 4a (0.50 g, 1.52 mmol) and Cs CO (0.673 g, 2.07 mmol)
1
2
3
ꢀ
The reaction mixture was stirred at 0 C for 1 h and was then
were added, and the flask was evacuated and backfilled with ni-
trogen for three times. The resulting mixture was stirred at 90 C for
acidified with 2N hydrochloric acid to pH < 5. The precipitate was
collected by filtration and allowed to dry to provide 2 as yellow
ꢀ
12 h. The mixture was filtrated and the filtrate was collected and
ꢀ
1
solid. Yield: 81.6%. Mp: 207e210 C. H NMR (400 MHz, DMSO-d
ppm) : 11.78 (s, br, 1H), 10.74 (s, br, 1H), 8.02 (s, 1H), 5.61 (s, 1H),
.27 (q, 2H, J ¼ 7.1), 1.29 (t, 3H, J ¼ 7.1). C NMR (100 MHz, DMSO-
, ppm) : 166.57, 166.40, 164.00, 142.97, 100.44, 98.96, 61.35,
6
,
concentrated. The crude residue was purified by silica gel chro-
matography to provide the title product 5a as white crystals. Yield:
7
1
2
2
d
13
4
ꢀ
1
7%. Mp: 243e245 C. H NMR (400 MHz, DMSO-d , ppm) d: 9.81 (s,
6
d
6
d
H, NH), 8.72 (s, 1H, pyridine-H), 7.84 (d, 2H, J ¼ 7.3 PhH), 7.83 (s,
H, PhH), 7.71 (d, 2H, J ¼ 8.8, PhH), 5.88 (s, 1H, pyridine-H), 4.32 (q,
14.46.
1
3
H, J ¼ 7.1, CH
2
), 2.15 (s, 6H, 2 ꢃ CH
3
), 1.32 (t, 3H, J ¼ 7.1, CH
3
).
C
5.1.2. General procedure for the synthesis of ethyl 4,6-
NMR (100 MHz, DMSO-d
6
, ppm) d: 163.89, 162.88, 159.39, 153.60,
dichloronicotinate (3)
153.37, 145.15, 133.79, 133.61, 133.09, 119.91, 118.85, 118.79, 109.43,
The reaction mixture of ethyl 4,6-dihydroxynicotinate (2, 5.6 g,
0.57 mmol), phosphorus oxychloride (23.44 g, 152.87 mmol) and
109.26, 103.08, 94.97, 60.90, 15.83 (2 ꢃ C), 14.63. ESI-MS: m/z 413.5
3
(M þ 1), 435.5 (M þ 23). C24
20 4 3
H N O (412.15).
triethylamine (7.73 g, 76.39 mmol) in 40 mL CH
3
CN was stirred at
5b was synthesized as for 5a but using 4b.White powder, yield:
ꢀ
ꢀ
1
8
0 C for 5 h. After removal of the solvent under reduced pressure,
53.8%. Mp: 243e245 C. H NMR (400 MHz, DMSO-d , ppm) d: 9.83
6
ꢀ
water (20 mL) was added at 0 C. The reaction mixture was alkal-
ized with K CO to pH > 7 and extracted with ethyl acetate
3 ꢃ 10 mL). The combined organic phase was dried over anhydrous
Na SO , filtered and concentrated in vacuum. The residue was
(s,1H, NH), 8.69 (s, 1H, pyridine-H), 7.86 (d, 2H, J ¼ 7.1, PhH), 7.69 (d,
2H, J ¼ 8.0, PhH), 7.04 (s, 2H, PhH), 5.94 (s, 1H, pyridine-H), 4.32 (q,
2
3
(
2H, J ¼ 7.1, CH
2
), 2.30 (s, 3H, CH
). C NMR (100 MHz, DMSO-d
3
), 2.21 (s, 6H, 2 ꢃ CH
3
), 1.32 (t, 3H,
d: 164.16, 164.11,
1
3
2
4
J ¼ 7.1, CH
3
6
, ppm)
purified by silica gel using ethyl acetate-petroleum ether. Pure
159.39, 153.04, 147.62, 145.41, 135.53, 133.51, 130.28, 130.26, 119.96,
fractions were collected and concentrated, giving the desired
compounds 3 as colorless oil. Yield: 85.1%. H NMR (400 MHz,
118.73, 109.52, 102.84, 94.88, 60.73, 20.82, 16.01 (2 ꢃ C), 14.63. ESI-
1
MS: m/z 402.5 (M þ 1), 424.5 (M þ 23). C24
23 3 3
H N O (401.17).
DMSO-d
t, 3H, J ¼ 7.1). C NMR (100 MHz, DMSO-d
52.15, 145.00, 126.30, 125.77, 62.45, 14.36. ESI-MS: m/z 220.2
M þ 1), 222.2 (M þ 3). C Cl NO (218.99).
6
, ppm)
d
: 8.83 (s, 1H), 7.97 (s, 1H), 4.38 (q, 2H, J ¼ 7.1), 1.35
1
3
(
1
6
, ppm) d: 163.12, 153.97,
5.1.5. General procedure for the synthesis of 6a1-11 and 6b1-11
Trimethylaluminium (1.6 M in toluene) (1.75 mL, 2.8 mmol) was
(
H
8 7
2
2
ꢀ
added dropwise at 0 C to a solution of different amine (2.8 mmol)
in dry toluene (10 mL) under nitrogen atmosphere and the reaction
allowed to stir for 1 h. Then 5a or 5b (0.70 mmol) was added and
5.1.3. General procedure for the synthesis of ethyl 6-chloro-4-(4-
cyano-2,6-dimethylphenoxy)nicotinate (4a) and ethyl 6-chloro-4-
ꢀ
the reaction was stirred at 70 C for 12 h. Water (20 mL) was added
(
mesityloxy)nicotinate (4b)
and extracted with ethyl acetate (3 ꢃ 10 mL). The combined organic
The reaction mixture of 4-hydroxy-3,5-dimethylbenzonitrile
2 4
phase was dried over anhydrous Na SO , filtered and concentrated
(
2 3
1.12 g, 7.61 mmol) and K CO (2.63 g, 19.06 mmol) in 30 mL DMF
in vacuum. The residue was purified by silica gel using ethyl
acetate-petroleum ether to provide the title product 6a1-10 and
was stirred at ambient temperature for 15 min. Ethyl 4,6-
dichloronicotinate (3, 2.0 g, 9.09 mmol) was added and the
6b1-10, 6a11 and 6b11 were obtained by reaction in ammonia gas
ꢀ
resulting mixture was stirred at 50 C for 24 h. After removal of the
atmosphere.
solvent under reduced pressure, water (20 mL) was added and
extracted with ethyl acetate (3 ꢃ 10 mL). The combined organic
phase was dried over anhydrous Na
2
SO
4
, filtered and concentrated
5.1.6. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
in vacuum. The residue was purified by silica gel using ethyl
acetate-petroleum ether. Pure fractions were collected and
concentrated, giving the desired compounds 4a as white crystals.
amino)-N-(2-(dimethylamino)ethyl)nicotinamide (6a1)
White powder, yield: 46.3%. Mp: 238e240 C. 1H NMR
ꢀ
6
(400 MHz, DMSO-d , ppm) d: 9.69 (s, 1H, NH), 8.70 (s, 1H, pyridine-
ꢀ
1
Yield: 50.1%. Mp: 95e100 C. H NMR (400 MHz, DMSO-d
6
, ppm)
d:
H), 8.18 (t, 1H, J ¼ 5.2, NH), 7.85 (s, 2H, PhH), 7.84 (d, 2H, J ¼ 9.2,
8
.78 (s, 1H, pyridine-H), 7.78 (s, 2H, PhH), 6.58 (s, 1H, pyridine-H),
PhH), 7.69 (d, 2H, J ¼ 8.8, PhH), 5.87 (s, 1H, pyridine-H), 3.41 (q, 2H,
4
CH
.37 (q, 2H, J ¼ 7.1, CH
2
), 2.12 (s, 6H, 2 ꢃ CH
3
), 1.33 (t, 3H, J ¼ 7.1,
J ¼ 5.6, CH
2
), 2.42 (t, 2H, J ¼ 6.5, CH
2
), 2.17 (s, 6H, 2 ꢃ CH
3
), 2.14 (s,
d: 163.06, 161.42,
13
13
3
). C NMR (100 MHz, DMSO-d , ppm) : 163.32, 163.29, 155.60,
6
d
6H, 2 ꢃ CH
3
). C NMR (100 MHz, DMSO-d
6
, ppm)
1
1
(
53.13, 152.88, 133.99 (2 ꢃ C), 132.71 (2 ꢃ C), 118.82, 116.46, 109.93,
09.49, 61.92, 15.78 (2 ꢃ C), 14.47. ESI-MS: m/z 331.4 (M þ 1), 333.5
M þ 3). C17 15ClN (330.08).
158.46, 153.21, 152.31, 145.46, 133.75, 133.60, 133.31, 120.00, 118.80,
118.43,112.36,109.61,102.61, 94.43, 58.14, 45.44 (2 ꢃ C), 37.57,15.86
H
2
O
3
26 6 2
(2 ꢃ C). ESI-MS: m/z 455.5 (M þ 1). C26H N O (454.21).

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
59
5
.1.7. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
amino)-N-propylnicotinamide (6a2)
White powder, yield: 48.5%. Mp: 278e281 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.66 (s, 1H, NH), 8.64 (s, 1H, pyridine-
5.1.12. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
amino)-N-cyclopropylnicotinamide(6a7)
White powder, yield: 74.0%. Mp: 286e290 C. 1H NMR
(400 MHz, DMSO-d , ppm) : 9.65 (s, 1H, NH), 8.50 (s, 1H, pyridine-
ꢀ
ꢀ
(
6
d
6
d
H), 8.18 (t, 1H, J ¼ 5.6, NH), 7.84 (d, 2H, J ¼ 8.6, PhH), 7.83 (s, 2H,
H), 8.18 (d, 1H, J ¼ 4.1, NH), 7.83 (d, 2H, J ¼ 9.2, PhH), 7.82 (s, 2H,
PhH), 7.68 (d, 2H, J ¼ 8.7, PhH), 5.88 (s, 1H, pyridine-H), 3.33e3.27
PhH), 7.68 (d, 2H, J ¼ 8.8, PhH), 5.86 (s, 1H, pyridine-H), 2.89e2.84
(
m, 2H, CH
H, J ¼ 7.4, CH
61.39, 158.24, 153.42, 151.70, 145.55, 133.70, 133.57, 133.38, 120.00,
18.82, 118.36, 113.33, 109.53, 102.52, 94.56, 41.29, 22.91, 15.94
2 ꢃ C), 11.77. ESI-MS: m/z 426.5 (M þ 1), 448.5 (M þ 23).
(425.19).
2
), 2.18 (s, 6H, 2 ꢃ CH
3
), 1.56 (q, 2H, J ¼ 7.2, CH
2
), 0.90 (t,
(m,1H, CH), 2.16 (s, 6H, 2 ꢃ CH
3
), 0.74e0.70 (m, 2H, CH
2
), 0.60e0.59
d: 164.97, 161.36,
13
13
3
3
). C NMR (100 MHz, DMSO-d
6
, ppm)
d: 163.34,
(m, 2H, CH
2
). C NMR (100 MHz, DMSO-d
6
, ppm)
1
1
(
158.05, 153.51, 150.79, 145.57, 133.69, 133.58, 120.01, 118.84, 118.29,
114.15, 109.44, 102.46, 94.75, 23.41, 15.95 (2 ꢃ C), 6.61 (2 ꢃ C). ESI-
MS: m/z 424.5 (M þ 1), 446.4(M þ 23). C25
21 5 2
H N O (423.17).
25 23 5 2
C H N O
5.1.13. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
5
.1.8. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
amino)-N-(2-methoxyethyl)nicotinamide (6a3)
White powder, yield: 56.1%. Mp: 242e244 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.69 (s, 1H, NH), 8.69 (s, 1H, pyridine-
amino)-N-((tetrahydrofuran-2-yl)methyl)nicotinamide (6a8)
ꢀ
1
White powder, yield: 74.1%. Mp: 258e259 C.
H NMR
ꢀ
(400 MHz, DMSO-d , ppm) : 9.68 (s, 1H, NH), 8.67 (s, 1H, pyridine-
6
d
(
6
d
H), 8.13 (t, 1H, J ¼ 5.7, NH), 7.84 (s, 2H, PhH), 7.83 (d, J ¼ 8.8, 2H,
PhH), 7.68 (d, 2H, J ¼ 8.8, PhH), 5.87 (s, 1H, pyridine-H), 4.00 (q, 1H,
H), 8.18 (s, 1H, NH), 7.84 (s, 2H, PhH), 7.84 (d, 2H, J ¼ 7.8, PhH), 7.69
(
2
d, 2H, J ¼ 8.8, PhH), 5.88 (s, 1H, pyridine-H), 3.51e3.49 (m, 4H,
J ¼ 6.1, CH), 3.74e3.70 (m, 1H, CH
2
), 3.63e3.57 (m, 1H, CH
2
), 3.40
13
ꢃ CH
2
), 3.26 (s, 3H, CH
, ppm) : 163.36, 161.42, 158.45, 153.29, 152.19, 145.47,
33.74, 133.59, 133.38, 119.99, 118.81, 118.43, 112.49, 109.61, 102.62,
3
), 2.17 (s, 6H, 2 ꢃ CH
3
). C NMR (100 MHz,
(td, 2H, J
CH ), 1.85e1.76 (m, 2H, CH
(100 MHz, DMSO-d , ppm) : 163.44, 161.39, 158.43, 153.22, 152.12,
1
¼5.8, J
2
¼ 1.9, CH
2
), 2.17 (s, 6H, 2 ꢃ CH
3
),1.93e1.87 (m,1H,
13
DMSO-d
6
d
2
2
), 1.65e1.58 (m, 1H, CH ). C NMR
2
1
9
4
6
d
4.51, 70.95, 58.41, 39.26, 15.90 (2 ꢃ C). ESI-MS: m/z 442.5 (M þ 1),
145.47, 133.78, 133.60, 133.34, 119.99, 118.79, 118.43, 112.58, 109.66,
94.51, 77.45, 67.71, 43.34, 28.78, 25.70, 15.90. ESI-MS: m/z 468.4
64.4 (M þ 23). C25 (441.18).
H
23
N
5
O
3
(M þ 1), 490.5 (M þ 23). C27
25 5 3
H N O (467.20).
5.1.9. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
amino)-N-(cyclopropylmethyl)nicotinamide (6a4)
White powder, yield: 84.9%. Mp: 258e260 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.68 (s, 1H, NH), 8.66 (s, 1H, pyridine-
5.1.14. N-allyl-4-(4-cyano-2,6-dimethylphenoxy)-6-((4-
ꢀ
cyanophenyl)amino)nicotinamide(6a9)
(
6
d
White powder, yield: 89.6%. Mp: 163e165 C. 1H NMR
ꢀ
H), 8.24 (t, 1H, J ¼ 5.7, NH), 7.84 (d, 2H, J ¼ 8.8, PhH), 7.83 (s, 2H,
6
(400 MHz, DMSO-d , ppm) d: 9.69 (s, 1H, NH), 8.67 (s, 1H, pyridine-
PhH), 7.69 (d, 2H, J ¼ 8.8, PhH), 5.88 (s, 1H, pyridine-H), 3.22 (t, 2H,
H), 8.36 (t, 1H, J ¼ 5.8, NH), 7.84 (s, 2H, PhH), 7.83 (d, J ¼ 8.8, 2H,
J ¼ 6.2, CH
2
), 2.19 (s, 6H, 2 ꢃ CH
), 0.26e0.24 (m, 2H, CH
: 163.31, 161.40,158.30, 153.42, 151.89, 145.53, 133.72,133.59,
3
), 1.10e1.06 (m, 1H, CH), 0.43e0.42
PhH), 7.69 (d, 2H, J ¼ 8.8, PhH), 5.98e5.89 (m, 1H, C]CH), 5.88 (s, 1H,
13
(
m, 2H, CH
2
2
). C NMR (100 MHz, DMSO-d
6
,
pyridine-H), 5.19 (dd, 1H, J
1
¼ 17.2, J
2
¼ 1.7, C]CH
2
), 5.09 (dd, 1H,
), 2.18 (s, 6H,
d: 163.25, 161.43,
ppm)
d
J
1
¼ 10.3, J
2
¼ 1.6, C]CH
2
), 3.97e3.95 (m, 2H, CH
). C NMR (100 MHz, DMSO-d , ppm)
2
1
3
1
1
(
33.39, 120.00, 118.83, 118.38, 113.06, 109.54, 102.54, 94.56, 43.56,
5.96 (2 ꢃ C), 11.48, 3.47 (2 ꢃ C). ESI-MS: m/z 438.5 (M þ 1), 460.5
M þ 23). C26 (437.19).
2 ꢃ CH
3
6
158.36, 153.37, 151.92, 145.51, 136.03, 133.71, 133.59, 133.40, 120.00,
118.82, 118.40, 115.08, 112.94, 109.55, 102.57, 94.51, 41.81, 15.97
23 5 2
H N O
(
(
2 ꢃ C). ESI-MS: m/z 424.5 (M þ 1), 446.4 (M þ 23). C25
21 5 2
H N O
423.17).
5
.1.10. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
amino)-N-(prop-2-yn-1-yl)nicotinamide (6a5)
White powder, yield: 81.6%. Mp: 258e259 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.72 (s, 1H, NH), 8.69 (s, 1H, pyridine-
ꢀ
5.1.15. 4-(4-Cyano-2,6-dimethylphenoxy)-N-(2-cyanoethyl)-6-((4-
(
6
d
cyanophenyl)amino)nicotinamide (6a10)
White powder, yield: 75.6%. Mp: 235e238 C. 1H NMR
ꢀ
H), 8.58 (t, 1H, J ¼ 5.6, NH), 7.85 (s, 2H, PhH), 7.84 (s, 2H, J ¼ 7.0,
PhH), 7.69 (d, 2H, J ¼ 8.8, PhH), 5.88 (s,1H, pyridine-H), 4.10 (dd, 2H,
6
(400 MHz, DMSO-d , ppm) d: 9.73 (s, 1H, NH), 8.74 (s, 1H, pyridine-
J
1
¼ 2.3, J
2
¼ 5.6, CH
2
), 3.10 (t, 1H, J ¼ 2.4, CH), 2.18 (s, 6H, 2 ꢃ CH
3
).
H), 8.54 (t, 1H, J ¼ 5.5, NH), 7.85 (s, 2H, PhH), 7.84 (d, J ¼ 7.5, 2H,
1
3
C NMR (100 MHz, DMSO-d
6
, ppm) d: 163.1, 161.47, 158.60, 153.37,
PhH), 7.70 (d, 2H, J ¼ 8.8, PhH), 5.89 (s, 1H, pyridine-H), 3.58 (q, 2H,
13
152.31, 145.42, 133.71, 133.60, 133.42, 119.99, 118.83, 118.49, 112.11,
J ¼ 6.1, CH
(100 MHz, DMSO-d
145.40, 133.69, 133.60, 133.49, 119.98, 119.69, 118.83, 118.51, 111.76,
09.59, 102.72, 94.36, 35.79, 18.06, 15.97 (2 ꢃ C). ESI-MS: m/z 437.5
M þ 1), 459.5 (M þ 23). C25 (436.16).
2
), 2.83 (t, 2H, J ¼ 6.4, CH
2
), 2.19 (s, 6H, 2 ꢃ CH
3
). C NMR
1
4
09.57, 102.69, 94.50, 82.08, 72.93, 29.12, 15.99 (2 ꢃ C). ESI-MS: m/z
6
, ppm)
d: 163.58, 161.53, 158.70, 153.34, 152.55,
22.4 (M þ 1), 444.5 (M þ 23). C25
19 5 2
H N O (421.15).
1
(
20 6 2
H N O
5.1.11. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
amino)-N-(2,2-dimethoxyethyl)nicotinamide (6a6)
White powder, yield: 70.0%. Mp: 246e248 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.70 (s, 1H, NH), 8.69 (s, 1H, pyridine-
ꢀ
5.1.16. 4-(4-Cyano-2,6-dimethylphenoxy)-6-((4-cyanophenyl)
(
6
d
amino)nicotinamide (6a11)
ꢀ
1
H), 8.14 (t, 1H, J ¼ 5.7, NH), 7.85 (s, 2H, PhH), 7.84 (d, 2H, J ¼ 8.2,
White powder, yield: 57.6%. Mp: >300 C. H NMR (400 MHz,
DMSO-d , ppm) : 9.68 (s, 1H, NH), 8.71 (s, 1H, pyridine-H), 7.84 (d,
2H, J ¼ 8.4, PhH), 7.83 (s, 2H, PhH), 7.69 (d, 2H, J ¼ 8.8, PhH), 7.61 (s,
PhH), 7.69 (d, 2H, J ¼ 8.9, PhH), 5.88 (s, 1H, pyridine-H), 4.57 (t, 1H,
6
d
J ¼ 5.6, CH), 3.45 (t, 2H, J ¼ 5.6, CH
2
), 3.30 (s, 6H, 2 ꢃ CH
, ppm)
58.54, 153.24, 152.27, 145.44, 133.76, 133.59, 133.36, 119.98, 118.79,
18.47, 112.25, 109.65, 102.67, 102.38, 94.49, 53.87 (2 ꢃ C), 41.39,
5.87 (2 ꢃ C). ESI-MS: m/z 472.4 (M þ 1), 494.4 (M þ 23).
(471.19).
3
), 2.18 (s,
: 163.42, 161.45,
13
13
6
H, 2 ꢃ CH
3
). C NMR (100 MHz, DMSO-d
6
d
2H, NH
(100 MHz, DMSO-d
2
), 5.86 (s, 1H, pyridine-H), 2.17 (s, 6H, 2 ꢃ CH
3
). C NMR
1
1
1
6
, ppm) : 164.82, 161.64, 158.58, 153.36, 152.55,
d
145.49, 133.70, 133.58, 133.37, 119.99, 118.83, 118.44, 112.51, 109.53,
102.60, 94.44, 15.96 (2 ꢃ C). ESI-MS: m/z 384.4 (M þ 1), 406.5
C
H
26 25
N
5
O
4
(M þ 23). C22
17 5 2
H N O (383.14).

60
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
5
(
.1.17. 6-((4-Cyanophenyl)amino)-N-(2-(dimethylamino)ethyl)-4-
mesityloxy)nicotinamide (6b1)
White powder, yield: 69.4%. Mp: 208e210 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.75 (s, 1H, NH), 8.73 (s, 1H, pyridine-
5.1.22. 6-((4-Cyanophenyl)amino)-N-(2,2-dimethoxyethyl)-4-
(mesityloxy)nicotinamide(6b6)
White powder, yield: 62.3%. Mp: 222e224 C. 1H NMR
(400 MHz, DMSO-d , ppm) : 9.74 (s, 1H, NH), 8.72 (s, 1H, pyridine-
ꢀ
ꢀ
(
6
d
6
d
H), 8.20 (t, 1H, J ¼ 5.1, NH), 7.86 (d, 2H, J ¼ 8.8, PhH), 7.70 (d, 2H,
H), 8.09 (t, 1H, J ¼ 5.7, NH), 7.86 (d, 2H, J ¼ 8.8, PhH), 7.68 (d, 2H,
J ¼ 8.8, PhH), 7.06 (s, 2H, PhH), 5.94 (s, 1H, pyridine-H), 3.43 (q, 2H,
J ¼ 8.8, PhH), 7.07 (s, 2H, PhH), 5.95 (s, 1H, pyridine-H), 4.56 (t, 1H,
J ¼ 5.5, CH
ꢃ CH
: 163.27, 162.63, 158.59, 152.34, 147.15, 145.66, 135.86, 133.52,
30.46, 130.31, 120.04, 118.42, 112.05, 102.43, 94.32, 58.12, 45.39
2 ꢃ C), 37.79, 20.83, 16.01 (2 ꢃ C). ESI-MS: m/z 44.7 (M þ 1).
(443.23).
2
), 2.42 (t, 2H, J ¼ 6.4, CH
2
), 2.31 (s, 3H, CH
). C NMR (100 MHz, DMSO-d
3
), 2.13 (s, 6H,
J ¼ 5.5, CH), 3.48 (t, 2H, J ¼ 5.6, CH
2
), 3.30 (s, 6H, 2 ꢃ CH
3
), 2.31 (s,
, ppm)
d: 163.59, 162.67, 158.70, 152.38, 147.15, 145.62, 135.95, 133.52,
13
13
2
d
3
), 2.09 (s, 6H, 2 ꢃ CH
3
6
, ppm)
3H, CH
3
), 2.09 (s, 6H, 2 ꢃ CH
3 6
). C NMR (100 MHz, DMSO-d
1
(
130.51, 130.33, 120.02, 118.48, 111.83, 102.52, 102.48, 94.38, 53.96
(2 ꢃ C), 41.31, 20.83, 16.02 (2 ꢃ C). ESI-MS: m/z 461.5 (M þ 1),
C
H
26 29
N
5
O
2
483.5(M þ 23). C26
28 4 4
H N O (460.21).
5
.1.23. 6-((4-Cyanophenyl)amino)-N-cyclopropyl-4-(mesityloxy)
nicotinamide (6b7)
White powder, yield: 81.1%. Mp: 253e254 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.68 (s, 1H, NH), 8.52 (s, 1H, pyridine-
5
.1.18. 6-((4-Cyanophenyl)amino)-4-(mesityloxy)-N-
propylnicotinamide (6b2)
White powder, yield: 69.7%. Mp: 224e225 C. 1H NMR
ꢀ
ꢀ
(
6
d
(
400 MHz, DMSO-d , ppm) : 9.71 (s, 1H, NH), 8.64 (s, 1H, pyridine-
6
d
H), 8.09 (d, 1H, J ¼ 3.9, NH), 7.84 (d, 2H, J ¼ 8.8, PhH), 7.67 (d, 2H,
H), 8.12 (t, 1H, J ¼ 5.2, NH), 7.84 (d, 2H, J ¼ 8.8, PhH), 7.67 (d, 2H,
J ¼ 8.8, PhH), 7.04 (s, 2H, PhH), 5.91 (s, 1H, pyridine-H), 2.90e2.85
J ¼ 8.8, PhH), 7.06 (s, 2H, PhH), 5.92 (s, 1H, pyridine-H), 3.29 (q, 2H,
(m, 1H, CH), 2.30 (s, 3H, CH
3
), 2.06 (s, 6H, 2 ꢃ CH
3
), 0.75e0.71 (m,
J ¼ 6.3, CH
J ¼ 6.2, CH
ppm) : 163.53, 162.57, 158.36, 151.73, 147.31, 145.73, 135.81, 133.54,
30.54, 130.28, 120.07, 118.33, 112.97, 102.32, 41.20, 22.92, 20.84,
6.12 (2 ꢃ C), 11.76. ESI-MS: m/z 415.6 (M þ 1), 437.6 (M þ 23).
(414.21).
2
), 2.30 (s, 3H, CH
), 0.89 (t, 3H, J ¼ 7.4, CH
3
), 2.08 (s, 6H, 2 ꢃ CH
3
), 1.54 (q, 2H,
13
13
2 2 6
2H, CH ), 0.59e0.57 (m, 2H, CH ). C NMR (100 MHz, DMSO-d ,
2
3
). C NMR (100 MHz, DMSO-d
6
,
ppm)
30.51, 130.27, 120.03, 118.32, 113.77, 102.34, 94.65, 23.35, 20.81,
6.10, 6.69. ESI-MS: m/z 413.6(M þ 1), 435.6(M þ 23). C25
412.19).
d: 165.13,162.56,158.23,150.90, 147.40,145.77,135.72,133.50,
d
1
1
(
1
1
24 4 2
H N O
25 26 4 2
C H N O
5
(
.1.24. 6-((4-Cyanophenyl)amino)-4-(mesityloxy)-N-
(tetrahydrofuran-2-yl)methyl)nicotinamide(6b8)
White powder, yield: 75.4%. Mp: 226e228 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.75 (s, 1H, NH), 8.71 (s, 1H, pyridine-
5
.1.19. 6-((4-Cyanophenyl)amino)-4-(mesityloxy)-N-(2-
methoxyethyl)nicotinamide (6b3)
White powder, yield: 80.0%. Mp: 220e224 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.75 (s, 1H, NH), 8.72 (s, 1H, pyridine-
ꢀ
ꢀ
(
6
d
(
6
d
H), 8.09 (t, 1H, J ¼ 5.7, NH), 7.86 (d, J ¼ 8.9, 2H, PhH), 7.68 (d, 2H,
H), 8.16 (t, 1H, J ¼ 5.4, NH), 7.86 (d, 2H, J ¼ 8.9, PhH), 7.68 (d, 2H,
J ¼ 8.8, PhH), 7.07 (s, 2H, PhH), 5.94 (s, 1H, pyridine-H), 4.00 (q, 1H,
J ¼ 8.8, PhH), 7.07 (s, 2H, PhH), 5.95 (s, 1H, pyridine-H), 3.53e3.48
J ¼ 6.0, CH), 3.70 (m, 1H, CH
2
), 3.60 (m, 1H, CH
2
), 3.44 (t, 2H, J ¼ 5.6,
),
). C NMR (100 MHz,
d: 163.59, 162.59, 158.62, 152.30, 147.11, 145.64,
(
m, 4H, CH
2
), 3.26 (s, 3H, CH
). C NMR (100 MHz, DMSO-d
3
), 2.31 (s, 3H, CH
3
), 2.09 (s, 6H,
d: 163.43, 162.62,
CH
.83e1.76 (m, 2H, CH
DMSO-d , ppm)
2
), 2.31 (s, 3H, CH
3
), 2.09 (s, 6H, 2 ꢃ CH
3 2
), 1.93e1.86 (m, 1H, CH
1
3
2
ꢃ CH
3
6
, ppm)
13
1
2
), 1.63e1.58 (m, 1H, CH
2
1
58.62, 152.31, 147.20, 145.65, 135.92, 133.53, 130.53, 130.31, 120.04,
18.43,112.05,102.45, 94.40, 71.02, 58.41, 39.19, 20.83,16.05 (2 ꢃ C).
ESI-MS: m/z 431.6 (M þ 1), 453.5(M þ 23). C25 (430.20).
6
1
135.97, 133.54, 130.49, 130.36, 120.04, 118.43, 112.04, 102.46, 94.38,
26 4 3
H N O
7
7.46, 67.77, 43.17, 28.69, 25.77, 20.83, 16.05. ESI-MS: m/z 457.6
(456.22).
(M þ 1), 479.5(M þ 23). C27
28 4 3
H N O
5
(
.1.20. 6-((4-Cyanophenyl)amino)-N-(cyclopropylmethyl)-4-
mesityloxy)nicotinamide (6b4)
White powder, yield: 70.6%. Mp: 228e230 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.73 (s, 1H, NH), 8.68 (s, 1H, pyridine-
5
.1.25. N-allyl-6-((4-cyanophenyl)amino)-4-(mesityloxy)
ꢀ
nicotinamide (6b9)
White powder, yield: 74.0%. Mp: 207e210 C. H NMR (400 MHz,
DMSO-d , ppm) : 9.73 (s, 1H, NH), 8.68 (s, 1H, pyridine-H), 8.27 (t,
H, J ¼ 5.8, NH), 7.86 (d, J ¼ 8.9, 2H, PhH), 7.68 (d, 2H, J ¼ 8.8, PhH),
.06 (s, 2H, PhH), 5.93 (s, 1H, pyridine-H), 5,97e5.90 (m, 1H, C]CH),
.20 (dd, 1H, J ¼ 1.7, C]CH ) 5.09 (dd, 1H, J ¼12.0, J ¼ 1.5,
¼18.8, J
C]CH ), 4.00e3.97 (m, 2H, CH ), 2.30 (s, 3H, CH ), 2.09 (s, 6H,
ꢃ CH ). C NMR (100 MHz, DMSO-d , ppm) : 163.47, 162.63,
58.49, 151.96, 147.27, 145.70, 136.08, 135.84, 133.53, 130.56, 130.29,
20.05, 118.39, 115.01, 112.62, 102.39, 94.42, 42.73, 20.83, 16.14
(
6
d
ꢀ
1
H), 8.17 (t, 1H, J ¼ 5.7, NH), 7.85 (d, 2H, J ¼ 8.8, PhH), 7.68 (d, 2H,
6
d
J ¼ 8.8, PhH), 7.06 (s, 2H, PhH), 5.94 (s, 1H, pyridine-H), 3.24 (t, 2H,
1
7
5
J ¼ 6.2, CH
2
), 2.31 (s, 3H, CH
3
), 2.09 (s, 6H, 2 ꢃ CH
3
), 1.08e1.06 (m,
), 0.26e0.23 (m, 2H, CH ). C NMR
2 2
13
1
(
1
9
4
H, CH), 0.43e0.39 (m, 2H, CH
100 MHz, DMSO-d , ppm) : 163.50, 162.59, 158.45, 151.99, 147.32,
45.71, 135.85, 133.54, 130.56, 130.30, 120.06, 118.37, 112.64, 102.36,
1
2
2
1
2
6
d
2
2
3
13
2
1
1
3
6
d
4.45, 43.50, 20.84, 16.13 (2 ꢃ C), 11.47, 3.44 (2 ꢃ C). ESI-MS: m/z
27.5 (M þ 1), 449.5 (M þ 23). C26 (426.21).
26 4 2
H N O
(
(
2 ꢃ C). ESI-MS: m/z 413.6 (M þ 1), 435.5 (M þ 23). C25
24 4 2
H N O
412.19).
5.1.21. 6-((4-Cyanophenyl)amino)-4-(mesityloxy)-N-(prop-2-yn-1-
yl)nicotinamide (6b5)
White powder, yield: 62.9%. Mp: 250e252 C. 1H NMR
400 MHz, DMSO-d , ppm) : 9.76 (s, 1H, NH), 8.72 (s, 1H, pyridine-
ꢀ
5.1.26. N-(2-cyanoethyl)-6-((4-cyanophenyl)amino)-4-
(
6
d
(mesityloxy)nicotinamide (6b10)
H), 8.51 (t, 1H, J ¼ 5.6, NH), 7.87 (d, 2H, J ¼ 8.8, PhH), 7.69 (d, 2H,
White powder, yield: 67.4%. Mp: 204e206 C. 1H NMR
ꢀ
J ¼ 8.8, PhH), 7.06 (s, 2H, PhH), 5.95 (s, 1H, pyridine-H), 4.13 (dd, 2H,
6
(400 MHz, DMSO-d , ppm) d: 9.77 (s, 1H, NH), 8.74 (s, 1H, pyridine-
J
(
1
1
(
(
1
¼ 4.6, J
s, 6H, 2 ꢃ CH
62.69, 158.73, 152.33, 147.33, 145.62, 135.86, 133.53, 130.60, 130.26,
20.03, 118.48, 111.87, 102.52, 94.42, 82.13, 72.89, 29.11, 20.84, 16.17
2 ꢃ C). ESI-MS: m/z 411.5 (M þ 1), 433.6 (M þ 23). C25
410.17).
2
¼ 2.4, CH
2
), 3.09 (t, 1H, J ¼ 2.4, CH), 2.31 (s, 3H, CH
3
), 2.10
H), 8.51 (t, 1H, J ¼ 5.8, NH), 7.86 (d, J ¼ 8.8, 2H, PhH), 7.68 (d, 2H,
1
3
3
). C NMR (100 MHz, DMSO-d , ppm) d: 163.40,
6
J ¼ 8.8, PhH), 7.07 (s, 2H, PhH), 5.94 (s, 1H, pyridine-H), 3.60 (q, 2H,
J ¼ 6.3, CH
2
), 2.82 (t, 2H, J ¼ 6.4, CH
2
), 2.31 (s, 3H, CH
3
) 2.10 (s, 6H,
d: 163.83, 162.76,
1
3
2 ꢃ CH
3
). C NMR (100 MHz, DMSO-d
6
, ppm)
H
22
N
4
O
2
158.80, 152.52, 147.30, 145.60, 135.87, 133.54, 130.67, 130.23, 120.04,
119.70, 118.49, 111.55, 102.52, 94.29, 35.75, 20.84, 18.08, 16.16

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 52e62
61
(
(
2 ꢃ C). ESI-MS: m/z 426.5 (M þ 1), 448.5(M þ 23). C25
H
23
N
5
O
2
DIG-labeled dNTPs incorporated in the template was bound to anti-
DIG-POD antibody. The unbound anti-DIG-POD was washed and
the peroxide substrate (ABST) was added to the MTP. A colored
reaction product was produced during the cleavage of the substrate
catalyzed y a peroxide enzyme. The absorbance of the sample was
determined at OD 405 nM using microtiter plate ELISA reader. The
percentage inhibitory activity of RT inhibitors was calculated by
comparison to a sample that did not contain an inhibitor. The
percentage inhibition was calculated by formula as given below: %
inhibition ¼ 100 ꢄ (OD 405 nm without inhibitor ꢄ OD405 nm with
425.19).
5
.1.27. 6-((4-Cyanophenyl)amino)-4-(mesityloxy)nicotinamide
6b11)
White powder, yield: 64.7%. Mp: >300 C. H NMR (400 MHz,
DMSO-d , ppm) : 9.73 (s, 1H, NH), 8.70 (s, 1H, pyridine-H), 7.85 (d,
H, J ¼ 8.7, PhH), 7.68 (d, 2H, J ¼ 8.7, PhH), 7.61 (s, 1H, NH), 7.55 (s,
H, NH), 7.06 (s, 2H, PhH), 5.91 (s, 1H, pyridine-H), 2.30 (s, 3H, CH ),
: 165.08,
62.81, 158.69, 152.50, 147.23, 145.67, 135.79, 133.55, 130.51, 130.30,
20.07, 118.40, 112.29,102.38, 94.34, 20.84, 16.13 (2 ꢃ C). ESI-MS: m/
z 373.3 (M þ 1), 395.4(M þ 23). C22 (372.16).
(
ꢀ
1
6
d
2
1
2
1
1
3
13
.08 (s, 6H, 2 ꢃ CH
3 6
). C NMR (100 MHz, DMSO-d , ppm) d
inhibitor)/(OD405 nm without inhibitor
ꢄ
OD405 nm
background) ꢃ 100. IC50 values corresponded to the concentration
of the tested compounds required to inhibit biotin-dUTP incorpo-
ration into the HIV-1 RT by 50%.
20 4 2
H N O
5.2. Biological activity
Conflict of interest
5.2.1. In vitro anti-HIV assay
The methodology of the anti-HIV assay has been previously
described [19,20]. Stock solutions (10 ꢃ final concentration) of test
compounds were added in 25 L volumes to two series of triplicate
The authors declare no conflict of interest.
Acknowledgment
m
wells to allow simultaneous evaluation of their effects on mock-
and HIV-infected cells at the beginning of each experiment. Serial
fivefold dilutions of test compounds were made directly in flat-
bottomed 96-well microtiter trays using a Biomek 3000 robot
The financial support from the National Natural Science Foun-
dation of China (NSFC No. 81273354, No. 81102320, No. 30873133,
No. 30772629, No. 30371686), Key Project of NSFC for International
Cooperation (No. 81420108027; 30910103908), Research Fund for
the Doctoral Program of Higher Education of China (Nos.
20110131130005, 20110131120037), Natural Science Foundation of
Shandong Province (ZR2009CM016) and KU Leuven (GOA 10/014).
(
Beckman instruments, Fullerton, CA). Untreated control HIV- and
mock-infected cell samples were included for each sample.
HIV-1 [24] (III , RES056) or HIV-2 [25] (ROD) stock (50
00e300 CCID50 (cell culture infectious dose) or culture medium
B
mL) at
1
was added to either the infected or mock-infected wells of the
microtiter tray. Mock-infected cells were used to evaluate the effect
of test compound on uninfected cells in order to assess the cyto-
toxicity of the tested compound. Exponentially growing MT-4 cells
were centrifuged for 5 min at 220 g and the supernatant was dis-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.054.
5
carded. The MT-4 cells were resuspended at 6 ꢃ 10 cells/mL, and
5
0
mL volumes were transferred to the microtiter tray wells. Five
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