Synthesis and biological evaluation of pyridinone analogues as novel potent HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.bmc.2014.11.012

A novel 2-pyridinone scaffold was rationally designed and synthesized based on the active anti-HIV agent 1 (LAM-trans) via an efficient method. The biological results revealed that some target compounds inhibited HIV-1 reverse transcriptase in the lower m

Full text of DOI:10.1016/j.bmc.2014.11.012

Bioorganic & Medicinal Chemistry 23 (2015) 149–159
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of pyridinone analogues as novel
potent HIV-1 NNRTIs
Yuanyuan Cao a,b, Yu Zhang a, Shaotong Wu a, Quanzhi Yang a, Xuefeng Sun a, Jianxiong Zhao a, Fen Pei a,
Ying Guo a, Chao Tian a, Zhili Zhang a, Haining Wang d, Liying Ma d, Junyi Liu a,c, , Xiaowei Wang a,
⇑
⇑
a Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
b Taihe Hospital Affiliated with Hubei University of Medicine, Shiyan 442000, Hubei, China
c State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
d State Key Laboratory for Infection Disease Prevention and Control, National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease
Control and Prevention, Beijing 102206, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel 2-pyridinone scaffold was rationally designed and synthesized based on the active anti-HIV agent
Received 15 September 2014
Revised 6 November 2014
Accepted 7 November 2014
Available online 14 November 2014
1 (LAM-trans) via an efficient method. The biological results revealed that some target compounds inhib-
ited HIV-1 reverse transcriptase in the lower micromolar concentration range (IC50 0.089–0.68
Notably, the most promising compound 25b exhibited extremely potent inhibitory activity against
HIV-1 replication with an EC50 value of 0.0563 M and the viral selectivity index amounted to 3466.8.
Molecular modeling studies were performed, and some SARs were rationalized.
Ó 2014 Elsevier Ltd. All rights reserved.
lm).
l
Keywords:
Pyridinone analogues
HIV-1
NNRTIs
1. Introduction
unrelated drugs.5–7 Therefore, it is important to develop new NNR-
TIs which can effectively inhibit the existing drug-resistant viral
strains.
The reverse transcriptase (RT) of the human immunodeficiency
virus type 1 (HIV-1) has been identified as an important viral target
for the discovery and development of anti-HIV therapeutic agents.
Accordingly, two functionally distinct classes of HIV-1 RT inhibi-
tors (nucleoside and non-nucleoside) have been developed and
are being used clinically. Especially, non-nucleoside RT inhibitors
(NNRTIs), with high antiviral potency and the favorable pharmaco-
kinetic properties, have gained an important role in clinical use,
and become an indispensable component in HAART regimen.1–4
So far, five NNRTIs have gained approval for clinical use: nevirapine
(NVP), delavirdine (DLV), efavirenz (EFV), etravirine (ETR) and rilp-
ivirine (RPV). Like other types of anti-HIV drugs, the therapeutic
efficacy of NNRTIs is weakened by the emergence of mutants,
in vitro and in vivo, that show cross-resistance to other structurally
Among the structurally diverse NNRTIs, pyridinone scaffolds
demonstrated high potency against HIV-1 wild type and drug-
resistant strains.8 A contribution from our laboratories to this
effort was the development of pyridinone derivatives, especially
the compound 1 (LAM-trans) (Fig. 1) which was highly effective
against a panel of RTIs-resistant strains with single (Y181C and
K103 N) and double (A17) mutations in RT with high selectivity
index (SI), and would be identified as a promising lead compound.9
Preliminary structure and activity relationship (SAR) on this
series has revealed that the substituents on the C-3, C-4 and C-6
of the pyridinone ring were very important for antiviral activity.10
The docking results displayed that the C-6 side chain could be
located at the region similar to that of the N-1 side chain of
compound 2 (TNK-651), and form the
p-p interaction with the
Tyr318 and Pro236 residues.
Abbreviations: HIV, human immunodeficiency virus; NNRTIs, non-nucleoside
reverse transcriptase inhibitors; SARs, structure and activity relationships; HAART,
highly active antiretroviral therapy; RT, reverse transcriptase; NVP, nevirapine;
DLV, delavirdine; EFV, efavirenz; ETR, etravirine; RPV, rilpivirine; RTIs, reverse
transcriptase inhibitors; NIS, N-iodosuccinimide; NBS, N-bromosuccinimide; DMF,
N,N-dimethylformamide; NNBP, non-nucleoside binding pocket; PDB, protein
database; SI, selectivity index.
Encouraged by these promising results, we were inspired to
carry out the optimization program on this scaffold which was
focused on the C-3 and C-4 positions (Fig. 1) with an efficient syn-
thetic strategy, while keeping the above mentioned key interaction
features required for RT inhibition.
Based on our previous study, substituent with steric or lipo-
philic character on the C-3 position would be beneficial for the
⇑
Corresponding authors. Tel./fax: +86 10 82805203 (X.W.).
E-mail address: xiaoweiwang@bjmu.edu.cn (X. Wang).
http://dx.doi.org/10.1016/j.bmc.2014.11.012
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

150
Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
O
N
To explore the relationships between structure and activity of
O
the target compounds, we synthesized a series of novel pyridinone
derivatives (Fig. 1), and tested their HIV-1 RT inhibitory potency.
The active compounds were further evaluated in vitro against the
HIV-1 laboratory-adapted strain (SF33) on TZM-bl cell lines.
HN
HN
O
O
O
2. Results and discussion
2.1. Chemistry
1 (LAM-trans)
2 (TNK-651)
O
R1
3
HN
Almost all the target compounds had been synthesized from the
common intermediate 4, which was conveniently prepared
through condensation of ethyl 3-oxo-5-phenyl pentanoate 3 with
diethylmalonate in the presence of ammonia (Scheme 1).9
Chlorination with phosphorus oxychloride and benzyltriethyl-
ammonium chloride provided the requisite 4-chloro-3-carbeth-
oxy-2-pyridinone 5, which reacted with the corresponding aryl
mercaptan or amine to afford 4-thioaryl and 4-amino substituent
pyridinones respectively in a Michael addition/retro-Michael pro-
cess.12 The transformation would be beneficial with an electron
withdrawing group on the C-3 position of pyridinone ring. Subse-
quently, the 4-amino pyridinone anologues 10a–d were subjected
to 2N HCl and then halogenated with NIS or NBS to give the target
compound 12a–d and 13a–d successively. However, considering
3-carbethoxy-4-thioaryl pyridinone, the yield of hydrolysis-
decarboxylation was unacceptable (30%). Therefore, we adjusted
the sequence of reactions. The compound 5 involved decarboxyl-
ation of the C-3 ester group prior to reaction with the correspond-
ing aryl mercaptan to afford the 7a–b with improved yield (70%),
which were converted to 8a–b and 9a–b by reaction with NIS or
NBS, respectively.
R1= I, Br,CH(CH3)2, N(CH3)2
X
= O, S, NH
4
6
X
R2
R2= saturated ring, aromatic ring
target compounds
Figure 1. Structure of typical NNRTIs and the target compounds.
inhibitory activity. Therefore, according to the isosteric principle,
the isopropyl and the N,N-dimethyl as the preferred substituents
were introduced on this position, and expected to ‘trigger’ the con-
formation of the C-4 substituent with the improved anti-HIV-1 RT
activity.11 Moreover, the halogen substituent could form a halogen
bond with the carbonyl of the Tyr181 residue and act as a vehicle
to incorporate diverse functionality at C-3 of the pyridinone ring.
The optimization on C-4 position, which is surrounded by the
aromatic residues Tyr188, Phe227, and Trp229 in the hydrophobic
binding pocket, was carried out by introducing saturated and
aromatic rings to provide the opportunity to compare the influence
on anti-HIV activity. Considering the modification on linker was
little concerned. We incorporated an oxygen, sulfur or nitrogen
atom, with different electro negativity, into the bridging site to
obtain more SAR information about this category compounds.
For the preparation of the aryloxy analogue, the approach did
not appear sufficiently effective. Based on the report of Kappe
R
2 = a ph;
b cyclohexanyl;
O
c 2- methylcyclohexanyl;
I
HN
d
cyclopentanyl
NHR2
e
O
O
COOEt
NHR2
HN
c
HN
12a-d
O
NHR2
Br
e
HN
10a-d
11a-d
NHR2
13a-d
d
O
O
O
O
COOEt
OH
COOEt
Cl
HN
a
b
HN
O
3
4
5
O
I
HN
O
e
SR1
O
HN
8a-b
HN
c
d
SR1
e
O
Cl
Br
SR1
7a-b
R1 = a Ph; b p-CH3Ph
HN
6
9a-b
Scheme 1. Reagents and reaction conditions: (a) NH3/MeOH, diethyl malonate/EtONa, reflux, 5 d, yield 33%; (b) POCl3, benzyltriethylammoniumchloride, CH3CN, rt, 12 h,
yield 80%; (c) 2 N HCl, reflux, 2 d, yields 65–76%; (d) Corresponding HSR1 or H2NR2, ethanol, reflux, 2 d, yields 62–76%; (e) NIS/NBS, THF, rt, 8 h, yields 90–96%.

Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
151
and El-Mariah,13 reaction of 4-hydroxy substituted pyridinone 14
with (dichloroiodo)benzene led to rapid formation of dipole, which
was isolated and then heated in dry DMF to take rearrangement to
the 3-iodo-6-phenethyl-4-phenoxy-2-pyridinone 15. The aryloxy
and iodo substituents were introduced adjacent to each other on
the pyridinone nucleus successively, and the target compound 15
was conveniently obtained with high yield (95%) (Scheme 2).14
Introducing the N,N-dimethyl moiety on the C-3 position was
performed as illustrated in Scheme 3. Treatment of 4-hydroxy-6-
phenethyl-2-pyridinone (14) with fuming nitric acid in acetic acid
afforded the 3-nitro product 16, which reacted with methylsufonyl
chloride in triethylamine to give the bismesylated compound 17.
Subsequently, the nucleophilic substitution reaction took place
on the C-4 position selectively to afford the compound 1815 which
was treated under mild hydrolysis condition with sodium methox-
ide in CH3OH to provide the intermediate 19. Hydrogenation of the
nitro group, using Pd/C as the catalytic agent, provided the amine
20 with 90% yield, which was following converted to the target
compound 21 successfully under reductive alkylation conditions.
Further modifications on the C-3 position, with isopropyl moi-
ety, were carried out according to Scheme 4, in which 5 was used
as the starting material. Concerning the linker with different atoms
(oxygen or sulfur atom), the synthetic routes are slightly different.
4-Chloro-3-carbethoxy-2-pyridinone 5 was treated with K2CO3 in
DMF or Et3N in ethanol, and then reacted with phenol or thiophe-
nol alternatively to afford 22a–b. Conversion of 22 to 23 involved
ester reduction using MeLi leading to the expected tertiary alcohol
derivative 23, which was followed by reaction with acetic anhy-
dride or thionyl chloride to provide 24a–b.16 The 3-propylene
derivatives were hydrogenated to yield the target compounds
25a–b bearing isopropyl moiety on the C-3 position.
2.2. Biological evaluation
The target compounds were initially evaluated in enzymatic
tests for their ability to inhibit highly purified recombinant HIV-1
RT using a poly(ra)/oligo(dT)15 as homopolymer template with
nevirapine as a reference compound. The assay results are summa-
rized in Table 1, some compounds exhibited promising inhibition
with IC50 values, which are much better than NVP.
Firstly, comparison of the inhibitory activity of compounds
modified at the C-3, we found that the substituent with iodine
(8a IC50 = 20.29 lM,) was superior to bromine (9a IC50 = 57.14 lM),
as shown in Table 1. In addition, the isopropyl moiety could initiate
the ‘trigger action’ required for improved activity. As we
expected, the inhibitory activity was improved enormously (25a
O
O
O
COOEt
I+
HN
HN
a
b(i)
HN
OH
OH
O-
4
14
b(ii)
O
O
I
I+
HN
HN
_
O
O
15
Scheme 2. Reagents and reaction conditions: (a) 2N HCl, reflux, 2 d, yield 73%; (b) (i) Na2CO3, H2O, room temperature, (dichloroiodo)benzene; (ii) DMF, reflux, 2 h, yield 95%.
O
O
OMs
NO2
OH
a
b
NO2
OMs
HN
HN
N
OH
16
14
17
OMs
O
O
NO2
S
NO2
S
NH2
S
N
c
d
HN
e
HN
18
19
20
O
N
S
f
HN
21
Scheme 3. Reagents and reaction conditions: (a) nitric acid fuming, acetic acid, 80 °C, 2 min, yield 75%; (b) methanesulfonyl chloride, triethylamine, CH2Cl2, 12 h, yield 85%;
(c) thiophenol, triethylamine, CH2Cl2, 12 h, yield 72%; (d) CH3ONa/CH3OH, reflux, 1 h, yield 70%; (e) H2, Pd/C, CH2Cl2, room temperature, 2 d, yield 75%; (f) NaBH3CN, HCHO,
HOAc, CH3CN, 1 h, yield 80%.

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Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
O
O
X
O
O
O
HN
O
HN
O
b
a
HN
OH
Cl
X
5
22a-b
23a-b
O
O
c
HN
d
HN
X
X
X = a S;
b O
24a-b
25a-b
Scheme 4. Reagents and reaction conditions: (a) benzenethiol, triethylamine, ethanol, reflux, 2 d, yield 83%; or phenol, K2CO3, DMF, reflux, 8 h, yield 89%; (b) MeLi, THF,
ꢀ78 °C, 12 h, yield 70–71%; (c) SOCl2, THF, room temperature, 12 h, yield 70%; or (CH3CO)2O, reflux, 1 h, yield 80%; (d) H2, Pd/C, CH2Cl2, room temperature, 4 d, yield 80–95%.
Table 1
HIV-1 RT inhibitory activity of target compoundsa
O
R1
HN
X
R2
a
a
Compds
R1
R2
X
IC50
(l
M)
Compds
R1
R2
X
IC50 (lM)
8b
I
I
I
I
I
I
I
p-CH3Ph
S
12.03
15.22
0.42
24.01
8.14
20.29
0.29
0.089
4.65
9b
Br
Br
Br
Br
Br
Br
p-CH3Ph
S
16.89
40.26
0.68
43.34
11.35
57.14
15.21
0.093
12b
12c
12d
12a
8a
Cyclohexanyl
NH
NH
NH
NH
S
13b
13c
12d
13a
9a
Cyclohexanyl
NH
NH
NH
NH
S
2-Methylcyclohexanylc
2-Methylcyclohexanylc
Cyclopentanyl
Cyclopentanyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
15
O
O
21
25a
N,N-Dimethyl
Isopropyl
S
S
25b
Isopropyl
NVPb
a
Effective dose (lM) of the compounds required to inhibit HIV-1 RT activity by 50%. Data represent mean values for three separate experiments; variation among triplicate
samples was less than 15%.
b
Nevirapine (NVP) was used as the reference compounds.
Mixture of stereoisomers.
c
IC50 = 0.093 lM, 25b IC50 = 0.089 lM), which illustrated that the
overall steric and lipophilic characteristics were clearly beneficial
Table 2
Antiviral activity of compounds on HIV-1SF33 infection in TZM-bl cell linesa
for the inhibitory activity. However, according to the isosteric
principle, we used the N,N-dimethyl (21 IC50 = 15.21 lM) to
replace the isopropyl and found that the activity was decreased
significantly.
Compds
EC50
(
l
M)/HIV-1SF33
CC50
(
l
M)
SIc
15
0.150 0.001
2.710 0.001b
13.680 0.003b
0. 136 0.001
0.0563 0.001
0.190 0.001
180.754
50.161
130.905
135.775
195.182
204.003
1205
18.5
9.6
998.3
3466.8
1073.7
12c
13c
25a
25b
NVPd
Secondly, we turned our attention to the linker on the C-4 posi-
tion. With the oxygen bridge (15 IC50 = 0.29
activity was more 28 times and 72 times than that of nitrogen
(12a IC50 =8.14 M) and sulfur (8a IC50 = 20.90 M), respectively
lM), the inhibitory
a
Each compound was tested in triplicate, and the data were presented as the
mean SD.
Mixture of stereoisomers.
SI was calculated based on the CC50 for TZM-bl cell and EC50 for inhibiting
infection by HIV-1SF33
l
l
(Table 1). One possible explanation for this result was that the oxy-
gen atom, with its favorable electro negativity, could provide the
better interaction with the NNIBP.10 Additionally, when a methyl
moiety was introduced to the para-position of the benzene ring,
the inhibitory activity was improved.
b
c
.
d
Nevirapine (NVP) was used as the reference compound.
Finally, an investigation of the R2 substituent was performed. As
shown in Table 1, 12a–d, which contained rings ranging from phe-
nyl, cyclopentyl to cycloheptyl, were evaluated against HIV-1 RT.
The biological results displayed that the substituent with a phenyl
ring was superior to both that of cyclopentyl and cyclohexyl.
Notably, a methyl-bearing moiety on the cycle ring (12c), which
is a mixture of stereoisomers, had the inhibitory activity superior
to the other compounds. There would be differences in the confor-
mational contribution of the cyclic moiety to the binding of the
inhibitors to RT. For the cyclopentanyl substituted compound,
the decreased inhibitory activity could be a result of the unfavor-
able steric interactions with the enzyme.
Subsequently, the anti-HIV-1 activity and cytotoxicity of the
compounds 15, 12c, 13c, 25a and 25b were determined in parallel
using nevirapine as reference compound. As illustrated in Table 2,
some compounds were capable of inhibiting wild-type virus infec-
tion by HIV-1SF33 in TZM-bl cell with EC50 values in the lower
micromolar concentration range (0.0563–2.71
lM). Notably, 25b
was the most promising compound. It exhibited extremely potent

Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
153
Figure 2. Superimposed stereoview of the docked 2 (purple) and 25a (yellow) with 1RT2. The Hydrogen bonds are shown as purple dashed lines.
2
25a
Figure 3. Docking results of 2 (TNK-651) and 25a into the 1RT2 NNBP. Hydrogen bonds are shown as cyan dashed lines.
inhibitory activity against HIV-1 replication with an EC50 value of
0.0563 M and the viral selectivity amounted to 34,668, which is
l
much better than NVP.
3. Computational modeling
In addition, molecular modeling studies were carried out to elu-
cidate the effect of structural features on anti-HIV activity. The tar-
get compound 25a was flexibly docked into the binding site of HIV-
1 RT (PDB 1RT2), using the GOLD 3.0.1 docking program. The
results displayed that 25a adopted the approximate conformation
comparable to that of the 2/RT complex (Fig. 2).
Inspection of the 25a/RT complex (Fig. 3), the compound could
establish several ligand-receptor interactions as that of 2, which
may be characterized as the hydrogen bond with the backbone
NH of Lys101, the favorable
p-stacking interaction with Tyr188
and the ‘trigger action’ of the isopropyl group.
15
Recently, the existence of a halogen bond was regarded as one
of the important interactions leading to improve the anti-HIV-1
activity.17 Upon inspection of the docking result of 15/RT (Fig. 4),
the halogen bond was observed. The iodine atom of 15 is only
3.14 Å away from the carbonyl oxygen of Tyr188, which is less than
the van der waals contact distance of 3.55 Å.
Figure 4. Docking results of 15 into the 1RT2 NNBP. The Hydrogen bond is shown
as a cyan dashed line, and halogen bond is shown as a pink dashed line.
4. Conclusion
Overall, the combination of hydrophobic and
actions may favor the binding of the 2-pyridinone scaffold to RT.
p-stacking inter-
In summary, through rational design we have generated the
novel 2-pyridinone derivatives as inhibitors of HIV-1 RT in compar-

154
Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
ison with nevirapine and also explored the SAR. Interestingly, the
biological studies revealed that several 2-pyridinone derivatives
showed activity in the low micromolecular range with EC50 values
Yield 33%; white solid; mp 192–193 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 12.58 (s, 1H, OH), 11.51 (br s, 1H, NH), 7.18–
7.31 (m, 5H, ArH), 5.81 (s, 1H, pyridinone H), 4.26 (q, J = 7.2 Hz,
2H, OCH2CH3), 2.86–2.90 (m, 2H, ArCH2CH2), 2.69–2.73 (m, 2H,
ArCH2CH2), 1.26 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz,
ranging from 0.0563 to 2.71 lM. Notably 25a and 25b displayed
the best profile against HIV-1 both in enzyme and cell-based
assays. These results appear to confirm that the isopropyl moiety
on the C-3 would be beneficial to improve the antiviral activity.
It is worth mentioning that, the SI values of the derivatives for
inhibiting HIV-1SF33 infection are in the range of 18.5–3466.8.
Because of their excellent potency, the biological and the structural
data would serve as a valuable guide to the further optimization of
the anti-HIV activity of the 6-phenethyl pyridinone family.
DMSO-d6)
d (ppm) 172.82, 170.57, 161.14, 154.97, 140.68,
128.78, 126.61, 98.53, 97.52, 61.09, 34.54, 34.09, 14.58; MS (ESI):
m/z, 288.50 [M+H]+.
5.1.3. Ethyl 4-chloro-2-oxo-6-phenethyl-1,2-dihydropyridine-3-
carboxylate (5)
To a solution of compound 4 (4.6 g, 16.0 mmol) and benzyltri-
ethylammonium chloride (14.5 g, 64.0 mmol) in acetonitrile
(50 mL) was added phosphorus oxychloride (2.3 mL, 24.0 mmol).
The obtained mixture was stirred at room temperature for 24 h.
After evaporation of the solvent, 10 mL of ice water was added,
and a white precipitate formed, which was collected and recrystal-
lized with ethanol to give compound 5.
5. Experimental section
5.1. Chemistry
The structural characterization was performed with an NMR
spectrometer and a high resolution mass spectrometer (HRMS).
NMR spectra were recorded on a Bruker Avance 300 or Avance
500 with tetramethylsilane (TMS) as an internal standard, and
chemical shifts are reported in (ppm). Melting points were deter-
mined on a WBS-1B type digital melting-point apparatus and are
uncorrected. All the reactions monitoring were performed with
TLC which was performed on aluminum-backed silica gel plates
(60 F254) with spots visualized by UV detection. Silica gel H
(200–300 mesh or 500 mesh) was used for the column chromatog-
raphy and silica gel was used for TLC plates. Unless otherwise sta-
ted, all reagents were purchased from commercial companies.
When necessary, they were purified and dried by standard meth-
ods. Organic solutions were dried over anhydrous sodium sulfate.
Yield 80%; white solid; mp 147–149 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.64 (br s, 1H, NH), 7.21–7.35 (m, 5H, ArH), 6.19
(s, 1H, pyridinone H), 4.41 (q, J = 7.2 Hz, 2H, OCH2CH3), 2.98–3.03
(m, 2H, ArCH2CH2), 2.85–2.90 (m, 2H, ArCH2CH2), 1.33 (t,
J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) d (ppm)
163.98, 162.43, 150.79, 146.37, 128.59, 128.50, 126.53, 139.70,
121.66, 107.64, 61.90, 35.48, 34.99, 14.11; MS (ESI): m/z, 306.40
[M+H]+.
5.1.4. 4-Chloro-6-phenethylpyridin-2(1H)-one (6)
A solution of the compound 5 (5.0 g, 16.4 mmol) in 2 N HCl
(50 mL) was refluxed for 2 d. After the reaction was completed
(TLC), Na2CO3 was added to adjust the mixture to neutral. The
resulting white precipitate was filtered, washed with water and
dried to give the compound 6. Yield 72%; white solid; mp 147–
149 °C; 1H NMR (400 MHz, CDCl3) d (ppm) 13.34 (br s, 1H, NH),
7.23–7.34 (m, 5H, ArH), 6.49 (d, J = 1.6 Hz, 1H, pyridinone H),
6.09 (d, J = 1.6 Hz, 1H, pyridinone H), 3.00–3.05 (m, 2H, ArCH2CH2),
2.87–2.92 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, CDCl3) d (ppm)
165.28, 149.66, 148.96, 128.57, 128.49, 126.48, 139.78, 116.06,
107.43, 35.01, 34.80; MS (ESI): m/z, 234.27 [M+H]+.
5.1.1. Ethyl 3-oxo-5-phenylpentanoate (3)9
Hydrocinnamoyl chloride (11.2 mL, 75.6 mmol) was added
dropwise to a solution of Meldrum’s acid (10.9 g, 75.6 mmol) and
pyridine (12.4 mL) in CH2Cl2 (140 mL) at 0 °C. The solution was
stirred for 30 min at 0 °C and then was allowed to room tempera-
ture for 24 h. The reaction mixture was washed with 10% aqueous
HCl (2 ꢁ 50 mL) and H2O (50 mL) separately. The organic layer was
dried with MgSO4, filtered and concentrated under reduced pres-
sure. The crude residue was dissolved in EtOH (100 mL) and heated
to reflux for 24 h. The mixture was concentrated under reduced
pressure to give the dark oily residue which was purified by flash
chromatography using EtOAc/petroleum ether (1:50 V/V) as the
eluent to give required compound 3.
5.2. General procedure for the synthesis of the compounds 7a–b
To a solution of compound 6 (2.8 g, 12.0 mmol) and the corre-
sponding benzenethiol (1.3 mL, 13.0 mmol) in 20 mL of ethanol
was added triethylamine (1 mL, 7.2 mmol). The obtained mixture
was heated to reflux for 2 d. After completion of the reaction as
indicated by TLC, the mixture was cooled and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy using EtOAc/petroleum ether (1:1 V/V) as the eluent to give
the compounds 7a–b.
Yield 95%; yellow liquid; 1H NMR (400 MHz, CDCl3) d (ppm)
7.17–7.29 (m, 5H, ArH), 4.16 (q, J = 7.2 Hz, 2H, OCH2CH3), 3.41 (s,
2H, COCH2CO), 2.85–2.92 (m, 4H, ArCH2CH2), 1.25 (t, J = 7.2 Hz,
3H, OCH2CH3).
5.1.2. Ethyl 4-hydroxy-2-oxo-6-phenethyl-1,2-dihydropyridine-
3-carboxylate (4)9
5.2.1. 6-Phenethyl-4-(phenylthio)pyridin-2(1H)-one (7a)
Yield 65%; white solid; mp 180–182 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.97 (br s, 1H, NH), 7.17–7.54 (m, 10H, ArH),
5.91 (s, 1H, pyridinone H), 5.84 (s, 1H, pyridinone H), 2.94–2.98
(m, 2H, ArCH2CH2), 2.79–2.83 (m, 2H, ArCH2CH2); 13C NMR
(100 MHz, CDCl3) d (ppm) 164.77, 156.66, 147.82, 135.53, 129.89,
128.53, 128.46, 126.30, 116.05, 110.60, 140.04, 107.31, 104.38,
34.84, 34.64; MS (ESI): m/z, 308.54 [M+H]+.
A mixture of compound 3 (5 g, 24.0 mmol) and 12.8 M NH3 in
MeOH (100 mL) was heated to reflux for 8 h under N2. After cool-
ing, the mixture was concentrated to dryness to provide ethyl 3-
amino-5-phenylpent-2-enoate which was used without further
purification.
To a solution of 1.2 g (48.0 mmol) of sodium in 40 mL of ethanol
was added a solution of 7.0 mL (48.0 mmol) of diethyl malonate in
20 mL of ethanol. The resulting yellow mixture was heated to
reflux for 2 h. After cooling, another solution of 5.0 g (24.0 mmol)
of ethyl 3-amino-5-phenylpent- 2-enoate in 24 mL of ethanol
was added. The mixture was refluxed for 5 d, and then acidified
with 2 N HCl. The precipitate was filtered, washed with water
and dried to yield the compound 4.
5.2.2. 6-Phenethyl-4-(p-tolylthio)pyridin-2(1H)-one (7b)
Yield 62%; white solid; mp 104–106 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.96 (br s, 1H, NH), 7.17–7,42 (m, 10H, ArH),
5.87 (s, 1H, pyridinone H), 5.83 (s, 1H, pyridinone H), 2.93–2.97
(m, 2H, ArCH2CH2), 2.78–2.82 (m, 2H, ArCH2CH2), 2.43 (s, 3H,
ArCH3); 13C NMR (100 MHz, CDCl3) d (ppm) 164.82, 157.20,

Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
155
147.70, 140.10, 135.60, 130.67, 128.54, 128.43, 126.26, 125.00,
5.4.1. Ethyl 2-oxo-6-phenethyl-4-(phenylamino)-1,2-
dihydropyridine-3-carboxylate (10a)
140.25, 110.31, 104.18, 34.76, 34.62, 21.35; MS (ESI): m/z, 322.40
[M+H]+.
Yield 73%; white solid; mp 190–192 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 10.88 (br s, 1H, NH), 7.05–7.37 (m, 10H, ArH),
5.70 (s, 1H, pyridinone H), 4.34 (q, J = 7.2 Hz, 2H, OCH2CH3), 3.86
(s, 1H, ArNH), 2.98–3.03 (m, 2H, ArCH2CH2), 2.78–2.82 (m, 2H,
ArCH2CH2), 1.32 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz,
CDCl3) d (ppm) 170.20, 164.39, 159.43, 151.86, 140.03, 129.44,
128.54, 128.44, 126.27, 125.99, 125.37, 138.23, 125.26, 94.72,
60.46, 35.16, 34.13, 14.34; MS (ESI): m/z, 385.48 [M+Na]+.
5.3. General procedure for the synthesis of the compounds 8a–b
and 9a–9b
The compound 7a–7b (2.0 mmol) was dissolved in 10 mL anhy-
drous THF, and NIS (NBS) (2.0 mmol) was added to the mixture,
which was protected from light and stirred at room temperature
overnight. After evaporating the solvent, the residue was purified
by column chromatography using EtOAc/petroleum ether (1:1 V/
V) as the eluent to give the compounds 8a–b and 9a–b.
5.4.2. Ethyl 4-(cyclohexylamino)-2-oxo-6-phenethyl-1,2-
dihydropyridine-3-carboxylate (10b)
Yield 75%; white solid; mp 213–215 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 11.93 (br s, 1H, NH), 9.36 (br s, 1H, cyclohexyl
NH), 7.17–7.30 (m, 5H, ArH), 5.44 (s, 1H, pyridinone H), 4.28 (q,
J = 7.2 Hz, 2H, OCH2CH3), 3.26–3.28 (m, 1H, NHCH), 3.02–3.06 (m,
2H, ArCH2CH2), 2.83–2.87 (m, 2H, ArCH2CH2), 1.26–1.86 (m, 13H,
CH3 + cyclohexyl H); 13C NMR (100 MHz, CDCl3) d (ppm) 170.44,
164.40, 159.74, 151.52, 128.53, 128.40, 126.25, 140.20, 93.23,
91.54, 59.95, 51.05, 32.78, 25.50, 24.44, 35.07, 34.15, 14.38; MS
(ESI): m/z, 369.45 [M+H]+.
5.3.1. 3-Iodo-6-phenethyl-4-(phenylthio)pyridin-2(1H)-one (8a)
Yield 91%; white solid; mp 225–227 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 11.85 (br s, 1H, NH), 7.01–7.57 (m, 10H, ArH),
5.06 (s, 1H, pyridinone H), 2.66–2.68 (m, 2H, ArCH2CH2), 2.52–
2.57 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, DMSO-d6) d (ppm)
159.90, 158.29, 153.39, 140.33, 135.66, 130.73, 130.62, 130.33,
128.73, 126.57, 148.02, 107.17, 102.16, 34.12, 34.07; HRMS (ESI):
m/z, calcd. for C19H16INOS [M+H]+: 434.3059, found 434.0071.
5.4.3. Ethyl 4-((2-methylcyclohexyl)amino)-2-oxo-6-phenethyl-
1,2-dihydropyridine-3-carboxylate (10c)
5.3.2. 3-Iodo-6-phenethyl-4-(p-tolylthio)pyridin-2(1H)-one (8b)
Yield 92%; white solid; mp 216–218 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.11 (br s, 1H, NH), 7.20–7.42 (m, 10H, ArH), 5.32
(s, 1H, pyridinone H), 2.88–2.92 (m, 2H, ArCH2CH2), 2.69–2.73 (m,
2H, ArCH2CH2), 2.46 (s, 3H, ArCH3); 13C NMR (100 MHz, CDCl3) d
(ppm) 161.86, 161.35, 147.55, 140.04, 135.70, 130.78, 128.71,
128.41, 126.93, 140.60, 126.24, 103.93, 35.31, 34.99, 21.49; HRMS
(ESI): m/z, calcd. for C20H18INOS [M+H]+: 448.3325, found 448.0224.
Yield 70%; white solid; mp 177–180 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.11 (br s, 1H, NH), 9.34 (d, J = 8 Hz, 1H, cyclohexyl
NH), 7.17–7.28 (m, 5H, ArH), 5.41 (s, 1H, pyridinone H), 4.28 (q,
J = 7.2 Hz, 2H, OCH2CH3), 3.48–3.52 (m, 1H, NHCH), 3.03–3.07 (m,
2H, ArCH2CH2), 2.83–2.87 (m, 2H, ArCH2CH2), 0.87–1.85 (m, 15H,
CH3 + cyclohexyl H + cyclohexyl CH3); 13C NMR (100 MHz, CDCl3)
d (ppm) 170.55, 164.51, 160.34, 140.27, 128.55, 128.37, 126.22,
151.50, 93.18, 91.36, 59.89, 57.86, 53.00, 38.44, 33.22, 25.48,
25.18, 19.42, 35.07, 34.15, 14.37; MS (ESI): m/z, 383.46 [M+H]+.
5.3.3. 3-Bromo-6-phenethyl-4-(phenylthio)pyridin-2(1H)-one
(9a)
5.4.4. Ethyl 4-(cyclopentylamino)-2-oxo-6-phenethyl-1,2-
dihydropyridine-3-carboxylate (10d)
Yield 95%; white solid; mp 204–207 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.04 (br s, 1H, NH), 7.18–7.55 (m, 10H, ArH),
5.32 (s, 1H, pyridinone H), 2.86–2.90 (m, 2H, ArCH2CH2), 2.69–
2.73 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, CDCl3) d (ppm)
160.41, 156.30, 146.45, 135.88, 130.75, 130.22, 129.95, 129.36,
128.64, 128.53, 139.91, 126.26, 103.73, 35.38, 34.95; HRMS (ESI):
m/z, calcd. for C19H16BrNOS [M+H]+: 386.0136, found 386.0204.
Yield 76%; white solid; mp 190–192 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.04 (br s, 1H, NH), 9.36 (d, J = 6 Hz, 1H, cyclopen-
tyl NH), 7.19–7.30 (m, 5H, ArH), 5.51 (s, 1H, pyridinone H), 4.26 (q,
J = 7.2 Hz, 2H, OCH2CH3), 3.75–3.80 (m, 1H, NHCH), 3.03–3.07 (m,
2H, ArCH2CH2), 2.84–2.88 (m, 2H, ArCH2CH2), 1.48–1.97 (m, 8H,
cyclopentyl H), 1.27 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3) d (ppm) 170.41, 164.28, 160.28, 151.63, 128.51, 128.41,
126.26, 140.20, 93.71, 91.62, 59.97, 54.03, 33.56, 23.93, 35.12,
34.14, 14.36; MS (ESI): m/z, 355.70 [M+H]+.
5.3.4. 3-Bromo-6-phenethyl-4-(p-tolylthio)pyridin-2(1H)-one
(9b)
Yield 96%; white solid; mp 209–211 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.89 (br s, 1H, NH), 7.18–7.41 (m, 10H, ArH),
5.33 (s, 1H, pyridinone H), 2.86–2.90 (m, 2H, ArCH2CH2), 2.69–
2.73 (m, 2H, ArCH2CH2), 2.46 (s, 3H, ArCH3); 13C NMR (100 MHz,
CDCl3) d (ppm) 160.19, 156.98, 146.26, 139.84, 135.84, 130.77,
128.60, 128.43, 126.29, 140.68, 125.62, 103.84, 35.25, 34.87,
5.5. General procedure for the synthesis of the compounds 11a–d
A solution of the compounds 10a–d (16.4 mmol) in 2 N HCl
(30 mL) was heated to reflux for 2 d. When the reaction was com-
pleted (TLC), the mixture was extracted with CH2Cl2 (2 ꢁ 20 mL).
The combined organic layers were dried over MgSO4 and evapo-
rated under reduced pressure. The resulting residue was chromato-
graphed on a silica gel column with EtOAc/petroleum ether (1:1 V/
V) as the eluent to give the compounds 11a–d.
21.46; HRMS (ESI): m/z, calcd. for
C
20H18BrNOS [M+H]+:
400.3320, found 400.0370.
5.4. General procedure for the synthesis of the compounds 10a–d
5.5.1. 6-Phenethyl-4-(phenylamino)pyridin-2(1H)-one (11a)
Yield 76%; white solid; mp 268–270 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 10.91 (br s, 1H, NH), 8.60 (s, 1H, ArNH), 7.01–
7.34 (m, 10H, ArH), 5.70 (s, 1H, pyridinone H), 5.52 (s, 1H, pyridinone
To a solution of the compound 5 (3.9 g, 13.0 mmol) in 30 mL of
ethanol was added triethylamine (1 mL). Then, the corresponding
aniline (13.5 mmol) was added dropwise. The obtained mixture
was heated to reflux for 2 d. The mixture was monitored by TLC
to ensure the progress of the reaction. The reaction mixture was
subsequently diluted with water (50 mL), acidified with HCl and
extracted with CH2Cl2. Finally, the organic layer was washed with
water and the organic phase was concentrated to dryness, the res-
idue was purified by column chromatography using EtOAc/petro-
leum ether (1:1 V/V) as the eluent to give the compounds 10a–d
as the white solids.
H), 2.85–2.89 (m, 2H, ArCH2CH2), 2.62–2.66 (m, 2H, ArCH2CH2); 13
C
NMR (100 MHz, DMSO-d6) d (ppm) 164.75, 154.15, 148.45, 141.14,
129.65, 128.85, 128.78, 126.52, 123.28, 121.57, 140.74, 97.14, 92.50,
34.77, 34.56; MS (ESI): m/z, 291.26 [M+H]+.
5.5.2. 4-(Cyclohexylamino)-6-phenethylpyridin-2(1H)-one (11b)
Yield 72%; white solid; mp 290–292 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 10.37 (br s, 1H, NH), 7.16–7.30 (m, 5H, ArH),

156
Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
6.23 (d, J = 7.6 Hz, 1H, cyclohexyl NH), 5.48 (s, 1H, pyridinone H),
4.94 (s, 1H, pyridinone H), 3.08–3.10 (m, 1H, NHCH), 2.80–2.85
(m, 2H, ArCH2CH2), 2.52–2.57 (m, 2H, ArCH2CH2), 1.07–1.85 (m,
H), 4.82 (d, J = 8.4 Hz, 1H, cyclohexyl NH), 3.55–3.56 (m, 1H,
NHCH), 3.02–3.06 (m, 2H, ArCH2CH2), 2.86–2.92 (m, 2H, ArCH2-
CH2), 0.90–1.95 (m, 12H, cyclohexyl H + cyclohexyl CH3); 13C
NMR (100 MHz, CDCl3) d (ppm) 162.96, 156.35, 148.90, 140.66,
128.91, 128.39, 148.78, 126.13, 93.54, 67.00, 58.51, 53.48, 38.89,
25.50, 25.30, 19.44, 35.75, 35.39; HRMS (ESI): m/z, calcd. for
10H, cyclohexyl H); 13C NMR (100 MHz, DMSO-d6)
d (ppm)
156.31, 147.14, 141.22, 128.66, 126.38, 113.32, 128.72, 102.49,
96.10, 50.54, 32.56, 25.82, 24.88, 34.79, 34.59; MS (ESI): m/z,
297.25 [M+H]+.
C
20H25IN2O [M+H]+ 437.3298, found 437.1086.
5.5.3. 4-((2-Methylcyclohexyl)amino)-6-phenethylpyridin-2(1H)-
one (11c)
5.6.4. 4-(Cyclopentylamino)-3-iodo-6-phenethylpyridin-2(1H)-
one (12d)
Yield 65%; white solid; mp 233–236 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.01 (br s, 1H, NH), 7.20–7.31 (m, 5H, ArH), 5.42
(s, 1H, pyridinone H), 5.33 (s, 1H, pyridinone H), 3.96 (d,
J = 8.4 Hz, 1H, cyclohexyl NH), 3.46 (m, 1H, NHCH), 2.98–3.02 (m,
2H, ArCH2CH2), 2.77–2.81 (m, 2H, ArCH2CH2), 0.87–2.08 (m, 12H,
cyclohexyl H + cyclohexyl CH3); 13C NMR (100 MHz, CDCl3) d
(ppm) 166.48, 156.86, 147.79, 128.63, 128.38, 126.14, 140.57,
97.65, 89.71, 57.28, 52.52, 38.77, 32.98, 25.65, 25.30, 19.32,
34.92, 34.54; MS (ESI): m/z, 311.30 [M+H]+.
Yield 91%; yellow solid; mp 191–193 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.80 (br s, 1H, NH), 7.21–7.42 (m, 5H, ArH), 5.59
(s, 1H, pyridinone H), 4.95 (d, J = 6.8 Hz, 1H, cyclopentyl NH),
3.80–3.88 (m, 1H, NHCH), 3.02–3.07 (m, 2H, ArCH2CH2), 2.85–
2.89 (m, 2H, ArCH2CH2), 1.48–2.07 (m, 8H, cyclopentyl H); 13C
NMR (100 MHz, CDCl3) d (ppm) 162.96, 156.41, 148.88, 128.90,
128.41, 126.15, 140.67, 93.81, 66.97, 54.72, 33.79, 23.82, 35.84,
35.48; HRMS (ESI): m/z, calcd. for C18H21IN2O [M+H]+ 409.2766,
found 409.0764.
5.5.4. 4-(Cyclopentylamino)-6-phenethylpyridin-2(1H)-one (11d)
Yield 75%; white solid; mp 143–145 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 10.39 (br s, 1H, NH), 7.17–7.31 (m, 5H, ArH),
6.36 (d, J = 6.4 Hz, 1H, cyclopentyl NH), 5.48 (s, 1H, pyridinone
H), 4.94 (s, 1H, pyridinone H), 3.51–3.64 (m, 1H, NHCH), 2.81–
2.85 (m, 2H, ArCH2CH2), 2.53–2.58 (m, 2H, ArCH2CH2), 1.22–1.90
(m, 8H, cyclopentyl H); 13C NMR (100 MHz, DMSO-d6) d (ppm)
164.70, 156.74, 141.30, 128.77, 126.48, 146.99, 96.82, 89.07,
53.31, 32.64, 24.12, 34.69, 34.49; MS (ESI): m/z, 283.26 [M+H]+.
5.6.5. 3-Bromo-6-phenethyl-4-(phenylamino)pyridin-2(1H)-one
(13a)
Yield 95%; white solid; mp 188–190 °C; 1H NMR (400 MHz,
CDCl3)
d (ppm) 13.06 (br s, 1H, NH), 7.10–7.42 (m, 10H,
ArH),6.71 (s, 1H, ArNH), 5.84 (s, 1H, pyridinone H), 3.00–3.04 (m,
2H, ArCH2CH2), 2.81–2.85 (m, 2H, ArCH2CH2); 13C NMR
(100 MHz, CDCl3) d (ppm) 162.06, 151.83, 147.73, 140.43, 129.60,
128.83, 128.43, 126.17, 125.62, 124.28, 138.50, 95.11, 91.89,
35.73, 35.24; HRMS (ESI): m/z, calcd. for C19H17BrN2O [M+H]+
369.2551, found 369.0599.
5.6. General procedure for the synthesis of the compounds 12a–
d and 13a–d
5.6.6. 3-Bromo-4-(cyclohexylamino)-6-phenethylpyridin-2(1H)-
one (13b)
Compound 11a–d (2.0 mmol) was dissolved in 10 mL anhydrous
THF, and NIS (NBS) (2.1 mmol) was added portionwise to the mix-
ture, which was protected from light and stirred at room tempera-
ture overnight. After evaporating the solvent, the residue was
purified by column chromatography using EtOAc/petroleum ether
(1:1 V/V) as the eluent to give the compounds 12a–d and 13a–d.
Yield 92%; yellow liquid; 1H NMR (400 MHz, CDCl3) d (ppm)
12.49 (br s, 1H, NH), 7.20–7.31 (m, 5H, ArH), 5.58 (s, 1H, cyclohexyl
NH), 5.01 (s, 1H, pyridinone H), 3.24–3.26 (m, 1H, NHCH), 3.01–
3.05 (m, 2H, ArCH2CH2), 2.87–2.91 (m, 2H, ArCH2CH2), 1.21–1.92
(m, 10H, cyclohexyl H); 13C NMR (100 MHz, CDCl3) d (ppm)
160.72, 153.13, 147.74, 128.72, 128.47, 126.28, 140.21, 94.73,
89.17, 51.55, 33.18, 25.40, 24.59, 35.47, 35.07; HRMS (ESI): m/z,
calcd. for C19H23BrN2O [M+H]+ 375.3027, found 375.1068.
5.6.1. 3-Iodo-6-phenethyl-4-(phenylamino)pyridin-2(1H)-one
(12a)
Yield 92%; white solid; mp 212–214 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 11.36 (br s, 1H, NH), 7.53 (s, 1H, ArNH), 7.01–
7.33 (m, 10H, ArH), 5.56 (s, 1H, pyridinone H), 2.79–2.83 (m, 2H,
ArCH2CH2), 2.59–2.63 (m, 2H, ArCH2CH2); 13C NMR (100 MHz,
5.6.7. 3-Bromo-4-((2-methylcyclohexyl)amino)-6-
phenethylpyridin-2(1H)-one (13c)
Yield 96%; yellow liquid; 1H NMR (400 MHz, CDCl3) d (ppm)
12.73 (br s, 1H, NH), 7.20–7.38 (m, 5H, ArH), 5.54 (s, 1H, pyridinone
H), 4.83 (d, J = 8.8 Hz, 1H, cyclohexyl NH), 3.01–3.05 (m, 2H, ArCH2-
CH2), 2.84–2.88 (m, 2H, ArCH2CH2), 0.91–1.94 (m, 13H, cyclohexyl
H + cyclohexyl CH3); 13C NMR (100 MHz, CDCl3) d (ppm) 161.43,
153.47, 147.67, 128.85, 128.40, 126.15, 140.61, 93.89, 89.44,
58.14, 38.93, 34.38, 33.90, 25.52, 25.33, 19.39, 35.83, 35.33; HRMS
DMSO-d6)
d (ppm) 161.82, 154.95, 148.14, 140.18, 129.59,
128.90, 128.73, 126.49, 124.56, 123.75, 140.78, 95.26, 71.22,
34.57, 34.35; HRMS (ESI): m/z, calcd. for C19H17IN2O [M+H]+:
417.2555, found 417.0459.
5.6.2. 4-(Cyclohexylamino)-3-iodo-6-phenethylpyridin-2(1H)-
one (12b)
(ESI): m/z, calcd. for
C
20H25BrN2O [M+H]+ 389.3293, found
Yield 93%; yellow solid; mp 156–158 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.42 (s, 1H, NH), 7.20–7.37 (m, 5H, ArH), 5.50 (s,
1H, pyridinone H), 4.93 (d, J = 7.6 Hz, 1H, cyclohexyl NH), 3.28–
3.30 (m, 1H, NHCH), 2.99–3.04 (m, 2H, ArCH2CH2), 2.84–2.88 (m,
2H, ArCH2CH2), 0.86–1.93 (m, 10H, cyclohexyl H); 13C NMR
(100 MHz, CDCl3) d (ppm) 155.89, 148.68, 140.50, 128.42, 126.18,
111.32, 128.83, 93.71, 51.71, 35.63, 35.29, 33.21, 29.71, 25.47,
24.55; HRMS (ESI): m/z, calcd. for C19H23IN2O [M+H]+: 423.3032,
found 423.0928.
389.1225.
5.6.8. 3-Bromo-4-(cyclopentylamino)-6-phenethylpyridin-2(1H)-
one (13d)
Yield 91%; white solid; mp 178–180 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.80 (br s, 1H, NH), 7.20–7.39 (m, 5H, ArH), 5.63
(s, 1H, pyridinone H), 4.96 (d, J = 6.4 Hz, 1H, cyclopentyl NH),
3.77–3.83 (m, 1H, NHCH), 3.01–3.05 (m, 2H, ArCH2CH2), 2.84–
2.88 (m, 2H, ArCH2CH2), 1.46–2.05 (m, 8H, cyclopentyl H); 13C
NMR (100 MHz, CDCl3) d (ppm) 161.44, 153.47, 147.80, 128.81,
128.41, 126.16, 140.60, 94.21, 89.53, 54.41, 33.79, 23.82, 35.86,
35.38; HRMS (ESI): m/z, calcd. for C18H21BrN2O [M+H]+ 361.2761,
found 361.0903.
5.6.3. 3-Iodo-4-((2-methylcyclohexyl)amino)-6-
phenethylpyridin-2(1H)-one (12c)
Yield 95%; yellow liquid; 1H NMR (400 MHz, CDCl3) d (ppm)
12.77 (br s, 1H, NH), 7.18–7.40 (m, 5H, ArH), 5.48 (s, 1H, pyridinone

Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
157
5.6.9. 4-Hydroxy-6-phenethylpyridin-2(1H)-one (14)
164.54, 149.87, 148.94, 139.62, 128.71, 128.38, 126.56, 115.52,
A solution of the compound 4 (4.7 g, 16.4 mmol) in 2 N HCl
(10 mL) was heated to reflux for 2 d. After the reaction was com-
pleted, Na2CO3 was added to adjust the mixture to neutral. A white
precipitate formed and was filtered off. The solid was recrystallized
from ethanol to give the compound 14.
41.72, 34.62, 39.52, 39.48; MS (ESI): m/z, 417.35 [M+H]+.
5.6.13. 3-Nitro-6-phenethyl-4-(phenylthio)pyridin-2-yl
methanesulfonate (18)
To a solution of compound 17 (4.2 g, 10 mmol) in 50 mL of CH2-
Cl2 was added thiophenol (1.1 mL, 10.1 mmol) dropwise. Then
1 mL of triethylamine was added. The obtained mixture was stirred
at room temperature overnight. When the reaction was complete
(TLC), the solution was washed with 1N HCl (25 mL) and sat. aq
NaHCO3 (25 mL) successively. The organic layer was dried
(Na2SO4), filtered and concentrated under reduced pressure. The
residue was purified by flash chromatography using EtOAc/
petroleum ether (1:2 V/V) as the eluent to give the compound 18.
Yield 72%; yellow solid; mp 160–162 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 7.01–7.56 (m, 10H, ArH), 6.32 (s, 1H, pyridinone
H), 3.52 (s, 3H, CH3), 2.88–2.94 (m, 4H, ArCH2CH2); 13C NMR
(100 MHz, CDCl3) d (ppm) 161.20, 151.16, 148.94, 130.90, 130.48,
128.55, 128.33, 127.81, 126.32, 139.87, 135.63, 119.66, 41.67,
39.05, 34.53; MS (ESI): m/z, 431.39 [M+H]+.
Yield 73%; white solid; mp 272–274 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 11.23 (br s, 1H, NH), 10.52 (s, 1H, OH),
7.16–7.30 (m, 5H, ArH), 5.65 (s, 1H, pyridinone H), 5.40 (s, 1H,
pyridinone H), 2.83–2.88 (m, 2H, ArCH2CH2), 2.63–2.68 (m, 2H,
ArCH2CH2); 13C NMR (100 MHz, DMSO-d6)
d (ppm) 168.12,
165.46, 149.66, 128.81, 128.76, 126.53, 141.07, 98.44, 96.63,
34.52; MS (ESI): m/z, 214.18 [MꢀH]ꢀ.
5.6.10. 3-Iodo-6-phenethyl-4-phenoxypyridin-2(1H)-one (15)
Dichloroiodobenzene (2.7 g, 10.0 mmol) was suspended in
water (20 mL) containing sodium carbonate (1.0 g, 9.1 mmol) and
stirred for 30 min at room temperature. To this mixture, a solution
of compound 14 (2 g, 9.0 mmol) in water (20 mL) containing also
sodium carbonate (1.0 g, 9.1 mmol) was added. After stirring for
1 h at room temperature, the precipitate was filtered off, washed
with water, dried in vacuo, and suspended in DMF (20 mL). After
heating under reflux for 2 h, the solvent was removed in vacuo.
The residue was purified by column chromatography using
EtOAc/petroleum ether (1:1 V/V) as the eluent to give the com-
pound 15.
5.6.14. 3-Nitro-6-phenethyl-4-(phenylthio)pyridin-2(1H)-one
(19)
To a solution of 0.2 g (10.0 mmol) of sodium in 50 mL of meth-
anol was added compound 18 (4.3 g, 10.0 mmol). The mixture was
heated to reflux for 2 h. When the reaction was complete (TLC), the
mixture was subsequently diluted with water (30 mL), acidified
with HCl and extracted with CH2Cl2. Finally, the organic phase
was concentrated to dryness. The residue was purified by flash
chromatography using EtOAc/petroleum ether (1:1 V/V) as the elu-
ent to give the compound 19.
Yield 95%; white solid; mp 200–202 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 11.97 (br s, 1H, NH), 6.95–7.44 (m, 10H, ArH),
5.45 (s, 1H, pyridinone H), 2.77–2.81 (m, 2H, ArCH2CH2), 2.64–
2.66 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, DMSO-d6) d (ppm)
166.81, 162.80, 154.39, 150.54, 130.69, 128.84, 128.73, 126.56,
125.42, 119.96, 140.54, 97.24, 76.73, 34.57, 34.30; HRMS (ESI):
m/z, calcd. for C19H16INO2 [M+H]+ 418.2403, found 418.0307.
Yield 70%; yellow solid; mp 213–215 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.23 (br s, 1H, NH), 7.10–7.56 (m, 10H, ArH),
5.44 (s, 1H, pyridinone H), 2.85–2.89 (m, 2H, ArCH2CH2), 2.72–
2.76 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, CDCl3) d (ppm)
157.44, 156.43, 151.18, 135.77, 130.81, 130.23, 128.62, 128.48,
126.53, 139.07, 104.12, 35.54, 34.50; MS (ESI): m/z, 353.40 [M+H]+.
5.6.11. 4-Hydroxy-3-nitro-6-phenethylpyridin-2(1H)-one (16)
A suspension of the compound 14 (5.0 g, 23.2 mmol) in 40 mL of
acetic acid was stirred at 80 °C until the solution was clear. The
nitric acid fuming (1 mL, 23.3 mmol) was added dropwise. The
mixture was stirred for 5 min at 80 °C; 50 mL of ice water was
added immediately, and the yellow precipitate formed and was fil-
tered off. The solid was washed with water to give the compound
16.
5.6.15. 3-Amino-6-phenethyl-4-(phenylthio)pyridin-2(1H)-one
(20)
To a pressure reaction bottle were added compound 19 (50 mg,
0.1 mmol) and Pd/C (0.07 mmol) in 15 mL of CH2Cl2. The mixture
was placed under hydrogen (1 atm) and stirred at room temperature
for 2 d. After filtration and evaporation of the solvent, the resulting
residue was purified by column chromatography using EtOAc/
petroleum ether (1:1 V/V) as the eluent to give the compound 20.
Yield 75%; yellow solid; mp 130–133 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.50 (br s, 1H, NH), 7.19–7.32 (m, 10H, ArH),
5.93 (s, 1H, pyridinone H), 2.95–2.99 (m, 2H, ArCH2CH2), 2.77–
2.81 (m, 2H, ArCH2CH2), 1.28 (s, 2H, NH2); 13C NMR (100 MHz,
CDCl3) d (ppm) 144.41, 140.56, 133.44, 129.29, 128.48, 128.44,
126.83, 126.19, 118.57, 136.32, 133.79, 109.89, 35.31, 34.62; MS
(ESI): m/z, 323.49 [M+H]+.
Yield 75%; yellow solid; mp 244–245 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 11.91 (br s, 1H, NH), 7.18–7.32 (m, 5H, ArH),
5.82 (s, 1H, pyridinone H), 2.84–2.89 (m, 2H, ArCH2CH2), 2.69–
2.74 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, DMSO-d6) d (ppm)
161.98, 157.26, 152.50, 128.82, 126.68, 140.59, 125.98, 97.39,
34.59, 34.26; MS (ESI): m/z, 259.32 [MꢀH]ꢀ.
5.6.12. 2-(Methanesulfonyloxy)-3-nitro-6-(2-phenylethyl)
pyridin-4-yl methanesulfonate (17)
To a solution of compound 16 (2.8 g, 12.0 mmol) and methane-
sulfonyl chloride (1.5 mL, 19.0 mmol) in 40 mL of CH2Cl2 was
added triethylamine (1 mL, 7.2 mmol). The obtained mixture was
stirred at room temperature until TLC indicated that the starting
material was no longer present. The reaction mixture was diluted
with water (35 mL), and extracted with CH2Cl2. The organic layer
was washed with water and the organic phase was concentrated
to dryness. The residue was purified by column chromatography
using EtOAc/petroleum ether (1:5 V/V) as the eluent to give the
compound 17.
5.6.16. 3-(Dimethylamino)-6-phenethyl-4-(phenylthio)pyridin-
2(1H)-one (21)
NaBH3CN (40 mg, 0.6 mmol) was added portionwise at room
temperature to a solution of the compound 20 (50 mg, 0.2 mmol)
and HCHO (37% in H2O, 1.4 mmol) in CH3CN (10 mL). The mixture
was stirred at room temperature for 15 min before slow addition of
HOAc (0.5 mL). The reaction was stirred at room temperature for
1 h, then basified with 10% aqueous K2CO3 and extracted with
CH2Cl2. The combined organic layers were washed with H2O,
dried over MgSO4, and concentrated under reduced pressure. The
resulting residue was purified by column chromatography using
Yield 85%; yellow solid; mp 133–135 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 7.16–7.33 (m, 6H, ArH + pyridinone H), 3.51 (s,
3H, CH3), 3.32 (s, 3H, CH3), 3.18–3.23 (m, 2H, ArCH2CH2), 3.08–
3.12 (m, 2H, ArCH2CH2); 13C NMR (100 MHz, CDCl3) d (ppm)

158
Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
EtOAc/petroleum ether (1:1 V/V) as the eluent to give the
compound 21.
5.59 (s, 1H, pyridinone H), 5.46 (s, 1H, OH), 2.82–2.91 (m, 2H,
ArCH2CH2), 2.64–2.71 (m, 2H, ArCH2CH2), 2.06 (s, 3H, CH3), 1.81
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3) d (ppm) 151.16, 144.21,
139.81, 135.19, 134.97, 129.75, 129.60, 129.39, 128.44,128.37,
135.59, 126.30, 107.77, 60.39, 34.56, 34.37, 29.91, 21.04; MS
(ESI): m/z, 364.47 [MꢀH]ꢀ.
Yield 80%; yellow solid; mp 194–196 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 12.93 (br s, 1H, NH), 7.18–7.52 (m, 10H, ArH),
5.28 (s, 1H, pyridinone H), 2.90–2.94 (m, 2H, ArCH2CH2), 2.87 (s,
6H, NCH3CH3), 2.67–2.71 (m, 2H, ArCH2CH2); 13C NMR (100 MHz,
CDCl3) d (ppm) 162.05, 153.49, 140.40, 131.98, 131.21, 129.49,
129.24, 128.47, 128.38, 144.67, 135.76, 126.20, 102.69, 41.81,
29.71, 35.08, 34.93; HRMS (ESI): m/z, calcd. for C21H22N2OS
[M+H]+ 351.4772, found 351.1523.
5.7.2. 3-(2-Hydroxypropan-2-yl)-6-phenethyl-4-
phenoxypyridin-2(1H)-one (23b)
Yield 71%; white solid; mp 168–170 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.35 (br s, 1H, NH), 8.00 (s, 1H, OH), 6.90–7.40
(m, 10H, ArH), 5.59 (s, 1H, pyridinone H), 2.97–3.01 (m, 2H, ArCH2-
CH2), 2.80–2.84 (m, 2H, ArCH2CH2), 1.70 (s, 6H, COHCH3CH3); 13C
NMR (100 MHz, CDCl3) d (ppm) 167.62, 162.73, 154.53, 139.60,
130.08, 128.55, 128.52, 126.36, 124.60, 119.90, 147.29, 119.65,
101.02, 72.47, 34.74, 30.26; MS (ESI): m/z, 348.40 [MꢀH]ꢀ.
5.6.17. Ethyl 2-oxo-6-phenethyl-4-(phenylthio)-1,2-
dihydropyridine-3-carboxylate (22a)
A mixture of the compound 5 (3.7 g, 12.0 mmol) and benzene-
thiol (1.3 mL, 13.0 mmol) in 50 mL of ethanol was stirred until
homogeneity. 1 mL of triethylamine was added dropwise. The
obtained mixture was heated to reflux for 2 d. After cooling and
removing the solvent, the residue was purified by column chroma-
tography using EtOAc/petroleum ether (1:1 V/V) as the eluent to
give the compound 22a.
5.7.3. 6-Phenethyl-4-(phenylthio)-3-(prop-1-en-2-yl)pyridin-
2(1H)-one (24a)
Yield 83%; white solid; mp 178–180 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.23 (br s, 1H, NH), 7.10–7.49 (m, 10H, ArH),
5.45 (s, 1H, pyridinone H), 4.40 (q, J = 7.2 Hz, 2H, OCH2CH3),
2.84–2.88 (m, 2H, ArCH2CH2), 2.69–2.73 (m, 2H, ArCH2CH2), 1.33
(t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) d (ppm)
165.83, 162.07, 158.21, 149.91, 135.52, 129.98, 129.86, 129.78,
128.43, 126.29, 139.80, 115.27, 104.51, 61.41, 35.23, 34.40,
14.23; MS (ESI): m/z, 380.68 [M+H]+.
To a solution of the compound 23a (40 mg, 0.1 mmol) in 20 mL of
anhydrous THF, the thionyl chloride (0.02 mL, 0.2 mmol) was added
in one portion. The mixture was stirred at room temperature over-
night. After the evaporation of the solvent, the residue was dissolved
in 10 mL of dichloromethane. This solution was basified with a cold
sodium bicarbonate solution, washed with brine, and dried over
magnesium sulfate. After evaporation of the solvent, the residue
was purified by column chromatography using EtOAc/petroleum
ether (1:1 V/V) as the eluent to give the compound 24a.
5.6.18. Ethyl 2-oxo-6-phenethyl-4-phenoxy-1,2-
dihydropyridine-3-carboxylate (22b)
Yield 70%; white solid; mp 183–185 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 7.18–7.49 (m, 10H, ArH), 5.48 (s, 1H, pyridinone
H), 5.46 (s, 1H, C@CH2), 5.11 (s, 1H, C@CH2), 2.86–2.90 (m, 2H,
ArCH2CH2), 2.66–2.70 (m, 2H, ArCH2CH2), 2.13 (s, 3H, CH3); 13C
To a suspension of the compound 5 (0.1 g, 0.3 mmol) in 20 mL of
anhydrous DMF was added K2CO3 (70 mg, 0.5 mmol). Then, phenol
(0.04 g, 0.5 mmol) was added. The mixture was heated to reflux for
8 h carefully. After cooling, the reaction mixture was subsequently
diluted with water (20 mL), acidified with HCl and extracted with
CH2Cl2. The organic phase was concentrated to dryness. The residue
was purified by column chromatography using EtOAc/petroleum
ether (1:1 V/V) as the eluent to give the compound 22b.
Yield 89%; white solid; mp 182–184 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.40 (br s, 1H, NH), 7.02–7.42 (m, 10H, ArH),
5.53 (s, 1H, pyridinone H), 4.35 (q, J = 7.2 Hz, 2H, OCH2CH3),
2.93–2.97 (m, 2H, ArCH2CH2), 2.78–2.83 (m, 2H, ArCH2CH2), 1.28
(t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) d (ppm)
165.87, 164.66, 164.24, 153.91, 139.87, 128.55, 128.47, 126.30,
125.43, 130.00, 120.67, 97.48, 61.33, 35.72, 34.83, 14.17; MS
(ESI): m/z, 364.35 [M+H]+.
NMR (100 MHz, CDCl3)
d
(ppm) 162.12, 157.63, 151.68,
145.80,140.21, 135.22, 131.00, 129.60, 129.31, 128.53, 128.37,
140.32, 126.23, 118.47, 104.11, 35.40, 34.87, 21.56; MS (ESI):
m/z, 348.61 [M+H]+.
5.7.4. 6-Phenethyl-4-phenoxy-3-(prop-1-en-2-yl)pyridin-2(1H)-
one (24b)
The compound 23b (40 mg, 0.1 mmol) was dissolved in 10 mL of
acetic anhydride. The mixture was heated to reflux for 1 h. After
cooling, the reaction mixture was subsequently diluted with water
(10 mL), extracted with CH2Cl2. Finally, the organic layer was
washed with water and the organic phase was concentrated to
dryness. The residue was purified by column chromatography
using EtOAc/petroleum ether (1:5 V/V) as the eluent to give the
compound 24b.
5.7. General procedure for the synthesis of the compounds 23a–b
Yield 80%; colorless liquid; 1H NMR (400 MHz, CDCl3) d (ppm)
6.97–7.41 (m, 10H, ArH), 6.32 (s, 1H, pyridinone H), 5.34 (s, 1H,
C@CH2), 5.07 (s, 1H, C@CH2), 2.91–2.98 (m, 4H, ArCH2CH2), 2.07
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3) d (ppm) 171.11, 169.12,
159.92, 164.26,154.67, 136.55, 130.10, 128.47, 128.32, 125.93,
125.08, 120.30, 117.80, 109.43, 39.53, 35.52, 21.03; MS (ESI):
m/z, 332.47 [M+H]+.
The compound 22a–b (2.0 mmol) was dissolved in 10 mL of dry
THF. After cooling to ꢀ78 °C, a total of 7 mL (20 mmol) of methyl-
lithium (3 M) was added in one portion. The reaction mixture was
kept at -78 °C for 1 h and at 0 °C for 2 h, then 12 h at room temper-
ature. The reaction was ended with 2 mL of water and slowly neu-
tralized with cold 1 N HCl. The organic layer was separated, and the
aqueous phase was extracted twice with 10 mL of ethyl acetate.
The combined organic solution was subsequently washed with
sodium bicarbonate and brine, and dried over the anhydrous
sodium sulfate. After evaporation of the solvent, the residue was
purified by column chromatography using EtOAc/petroleum ether
(1:1 V/V) as the eluent to give the compounds 23a–b.
5.8. General procedure for the synthesis of the compounds 25a–b
To a pressure reaction bottle were added 24a–b (0.08 mmol)
and Pd/C (0.08 mmol) in 15 mL of CH2Cl2. The mixture was placed
under hydrogen (1 atm) and stirred at room temperature over-
night. After filtration and evaporation of the solvent, the resulting
residue was purified by column chromatography using EtOAc/
petroleum ether (1:1 V/V) as the eluent to give the target products
25a–b.
5.7.1. 3-(2-Hydroxypropan-2-yl)-6-phenethyl-4-
(phenylthio)pyridin-2(1H)-one (23a)
Yield 70%; yellow solid; mp 126–129 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.11 (br s, 1H, NH), 7.07–7.45 (m, 10H, ArH),

Y. Cao et al. / Bioorg. Med. Chem. 23 (2015) 149–159
159
5.8.1. 3-Isopropyl-6-phenethyl-4-(phenylthio)pyridin-2(1H)-
one (25a)
5.9.3. Assessment of in vitro cytotoxicity in MT-4 cells
An XTT assay, as previously described,18 was used to assess the
cytotoxicity of target compounds to MT4 cells. Briefly, a compound
at graded concentrations was added to MT4 cells (5 ꢁ 104/well),
followed by incubation at 37 °C for 3 days. Ten microliters of
CCK-8 reagent were added to the cells. After incubation at 37 °C
for 4 h to allow color development of the XTT formazan product,
the absorbance of each well was then read at 450 nm in a Victor2
1420 Multilabel Counter (Wallace-PerkinElmer Life and Analytical
Sciences Inc., Boston, MA). The percent of cytotoxicity and CC50
(concentration causing 50% cytotoxicity) were calculated as previ-
ously described.19
Yield 80%; yellow solid; mp 138–140 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 7.17–7.46 (m, 10H, ArH), 5.47 (s, 1H, pyridinone
H), 3.40–3.47 (m, 1H, CHCH3CH3), 2.89–2.93 (m, 2H, ArCH2CH2),
2.65–2.69 (m, 2H, ArCH2CH2), 1.43 (d, J = 6.8 Hz, 6H, CH3); 13C
NMR (100 MHz, CDCl3) d (ppm) 163.32, 149.45, 144.52,134.48,
131.63, 129.75, 129.52, 128.82, 128.47, 128.37, 140.43, 126.17,
104.97, 34.94, 34.86, 29.79, 19.29; HRMS (ESI): m/z, calcd. for
C
22H23NOS [M+H]+ 350.4891, found 350.1563.
5.8.2. 3-Isopropyl-6-phenethyl-4-phenoxypyridin-2(1H)-one
(25b)
Yield 95%; white solid; mp 189–191 °C; 1H NMR (400 MHz,
CDCl3) d (ppm) 13.28 (br s, 1H, NH), 6.95–7.40 (m, 10H, ArH),
5.58 (s, 1H, pyridinone H), 3.53–3.61 (m, 1H, CHCH3CH3), 2.99–
3.04 (m, 2H, ArCH2CH2), 2.78–2.82 (m, 2H, ArCH2CH2), 1.38 (d,
J = 6.8 Hz, 6H, CH3); 13C NMR (100 MHz, CDCl3) d (ppm) 166.59,
163.17, 155.54, 146.61, 129.85, 128.58, 128.40, 126.19, 123.91,
121.39, 140.52, 119.28, 99.53, 35.41, 35.19, 24.54, 20.23; HRMS
(ESI): m/z, calcd. for C22H23NO2 [M+H]+ 334.4235, found 334.1802.
5.10. Molecular modeling
The docking studies were conducted by using software GOLD
3.0.1 and the protein crystal structures of wild type RT/1 complex
(PDB ID: 1RT2). Following the default setting of Chemscore in the
software tool, top scored docked poses were visually inspected
for each ligand in the DS 2.5 based on relevant molecules. The
radius of the binding site sphere was defined by the original ligand
as 12.4 Å.
5.9. Biological testing assays
Acknowledgments
5.9.1. Assay for measuring the inhibitory activity of compounds
against HIV-1 RT
The research work was supported by the National Natural
Science Foundation of China (21172014, 20972011 and
21042009) and Grants from the Ministry of Science and Technol-
ogy of China (2009ZX09301-010) for financial support.
Oligo(dT) (TaKaRa Co., Japan) was immobilized via its 50-termi-
nal phosphate to Covalink-NH microtiter plates (NUNC Co.,
Denmark). The biotin-dUTP was incorporated by reverse transcrip-
tase (Sigma). Briefly, a serial concentration of inhibitor was added
to the mixture, which contained 1 mol/L Tris-HCl (pH 8.3), 1 mol/L
References and notes
MgCl2, 1 mol/L KCl, 0.5 mol/L DTT (D,L,-dithiothreitol), 1% BSA
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(Albumin Bovine V), 5 mg/mL poly (A), 5 mmol/L biotin-11-dUT-
P, and 0.1 mmol/L dTTP. The reaction mixture was incubated at
37 °C for 1 h and washed with a buffer containing 100 mmol/L
Tris–HCl (pH 7.5), 0.1 mol/L NaCl, 0.1 mol/L MgCl2, and 0.02%
Tween-20. After 100 lL of 1% BSA was added to each well and incu-
bated at room temperature for another 1 h, the plate was washed
with the same buffer. Before further incubation at 37 °C for 1 h,
50
lL of SA-ALP (Alkaline Phosphatase Streptavidin) solution was
added per well and washed again as above. Finally, 100
lL of PNPP
(p-nitrophenyl phosphate, disodium) (1 mg/mL, pH 9.5) was added
and incubated at 37 °C for 30 min. The reaction was stopped by
addition of 0.5 M NaOH. The inhibitory activity of the compounds
was detected and quantified using a colorimetric streptavidin-
alkaline phosphatase reporter system.
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5.9.2. Assay for measuring the inhibitory activity of compounds
on HIV-1SF33 infectious
TZM-bl cells, HIV-1SF33 were obtained from the NIH AIDS
Research and Reference Reagent Program (Germantown, MD). The
inhibitory activity of target compounds on infection by a labora-
tory-adapted HIV-1 strain SF33 were tested in TZM-bl cells. Briefly,
TZM-bl cells (4 ꢁ 104/well) were infected by addition of 200 TCID50
of HIV-1, followed by incubation for 2 h at 37 °C before addition of
compounds at serial dilutions. After further incubation at 37 °C for
7 days, p24 was measured using a commercial enzyme-linked
immunosorbent assay (ELISA) kit (Vironostika HIV-1 Microelisa sys-
tem; BioMérieux; Marcy l’Etoile, France).The concentration of a
compound for inhibiting 50% viral replication (EC50) was deter-
mined by nonlinear regression using GraphPad Prism 5.01.
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