Hybrid chemistry. Part 4: Discovery of etravirine-VRX-480773 hybrids as potent HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.06.048

A novel series of etravirine-VRX-480773 hybrids were designed using structure-guided molecular hybridization strategy and fusing the pharmacophore templates of etravirine and VRX-480773. The anti-HIV-1 activity and cytotoxicity was evaluated in MT-4 cell

Full text of DOI:10.1016/j.bmc.2015.06.048

Accepted Manuscript
Hybrid chemistry: part 4. Discovery of etravirine–VRX-480773 hybrids as po-
tent HIV-1 non-nucleoside reverse transcriptase inhibitors
Zheng-Yong Wan, Yuan Tao, Ya-Feng Wang, Tian-Qi Mao, Hong Yin, Fen-Er
Chen, Hu-Ri Piao, Erik De Clercq, Dirk Daelemans, Christophe Pannecouque
PII:
S0968-0896(15)00542-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.048
BMC 12404
Reference:
Bioorganic & Medicinal Chemistry
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16 June 2015
17 June 2015
Please cite this article as: Wan, Z-Y., Tao, Y., Wang, Y-F., Mao, T-Q., Yin, H., Chen, F-E., Piao, H-R., Clercq,
E.D., Daelemans, D., Pannecouque, C., Hybrid chemistry: part 4. Discovery of etravirine–VRX-480773 hybrids as
potent HIV-1 non-nucleoside reverse transcriptase inhibitors, Bioorganic & Medicinal Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bmc.2015.06.048
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Graphical Abstract
Hybrid chemistry: part 4. Discovery of
etravirine–VRX-480773 hybrids as potent
HIV-1 non-nucleoside reverse transcriptase
inhibitors
Leave this area blank for abstract info.
Zheng-Yong Wana, Yuan Taoa, Ya-Feng Wanga, Tian-Qi Maoa,b, Hong Yina, Fen-Er Chena,b, Hu-Ri Piaoc, Erik
De Clercqd, Dirk Daelemansd, Christophe Pannecouqued
aDepartment of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
bInstitute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
c Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry
of Education, Yanbian University, Yanji 133000, People’s Republic of China
dRega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000
Leuven, Belgium

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Hybrid chemistry: part 4. Discovery of etravirine–VRX-480773 hybrids as potent
HIV-1 non-nucleoside reverse transcriptase inhibitors
Zheng-Yong Wana, Yuan Taoa, Ya-Feng Wanga, Tian-Qi Maoa,b, Hong Yina, Fen-Er Chena,b,∗, Hu-Ri
Piaoc,∗, Erik De Clercqd, Dirk Daelemansd, Christophe Pannecouqued
aDepartment of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
bInstitute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
cKey Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, Yanbian University, Yanji 133000,
People’s Republic of China
dRega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel series of etravirine–VRX-480773 hybrids were designed using structure-guided
molecular hybridization strategy and fusing the pharmacophore templates of etravirine and
VRX-480773. The anti-HIV-1 activity and cytotoxicty was evaluated in MT-4 cell cultures. The
most active hybrid compound in this series, N-(2-chlorophenyl)-2-((4-(4-cyano-2,6-
dimethylphenoxy)pyrimidin-2-yl)thio)acetamide 3d (EC50 = 0.24 µΜ, SI > 1225), was more
potent than delavirdine (EC50 = 0.66 µΜ, SI > 67) in the anti-HIV-1 in vitro cellular assay.
Studies of structure-activity relationships established a correlation between anti-HIV activity
and the substitution pattern of the acetanilide group.
Available online
Keywords:
Antiviral agent
Biological activity
Etravirine
2009 Elsevier Ltd. All rights reserved.
Molecular hybridization
VRX-480773
1. Introduction
chemical entities with improved affinity and efficacy profiles and
higher genetic barriers to resistance.
The discovery of etravirine 1 (TMC125, Fig. 1) is a
comprehensive result of the coordinated multidisciplinary
endeavor lasting for more than 20 years of research on non-
nucleoside reverse transcriptase inhibitors (NNRTIs).1 This
promising NNRTI was approved by the FDA in 2008 for AIDS
therapy owing to potency against both wild-type (WT) and
mutant virus strains. Another clinical candidate NNRTI, VRX-
480773 22 (Fig. 1) originated from high-throughput screening
(HTS) of compound libraries as a second generation NNRTI,
showed resilience to most NNRTI-resistant clinical HIV-1
isolates. However, there is still a great need for additional new
Figure 2. Superposition of lower-energy docking binding
conformations of etravirine (purple) and VRX-480773 (blue) in the
binding pocket of HIV-1 RT (protein removed for clarity).
The present work is an extension of our ongoing efforts
towards developing effective NNRTIs through
a hybrid
F—ig—ure—1. Structures of etravirine and VRX-480773.
pharmacophore approach3-5, and one that uses etravirine and
∗ Corresponding author. Tel.: +86 21 65643809; fax: +86 21 65643811; e-mail: rfchen@fudan.edu.cn
∗ Corresponding author. Tel.: +86 43 32435003; fax: +86 43 32435004; e-mail: piaohuri@aliyun.com

Figure 3. Design of etravirine–VRX-480773 hybrids 3 based on structure-guided molecular hybridization.
Scheme 1. Synthetic route of target compounds 3a-u. Reagents and conditions: (a) 4-hydroxy-3,5-dimethylbenzonitrile, NaH, THF, 40 oC, 6 h;
(b) thiourea, EtOH, reflux, 4.5 h; (c) substituted α-bromoacetamide 7a-u, KOt-Bu, DMF, r.t., 1 h.
Table 1
Biological activities of compounds 3a-u in MT-4 cellsa
Compds
R
EC50b (µΜ)
K103N+Y181C
CC50c (µΜ)
SId
pKae
Log Pe
MRe (cm3)
PSAf (Å2)
HIV-2
Ⅲ
>320
8.1 0.5
8.0 2.4
0.24 0.05
8.0 2.3
B
H
3-F
4-F
2-Cl
3-Cl
>320
>306
>306
≥10.9
>294
>294
>272
>89
>320
>306
>306
>294
>294
>294
>272
>89
>320
>306
>306
>294
>294
>294
>272
89 7
≥41
24 3.7
>287
>287
>287
>301
>288
>266
>309
>309
46 12
≥69
1
>38
>38
0.6 0.8 5.62 0.52 108.36 0.4
0.6 0.9 6.15 0.59 108.47 0.4
0.6 0.8 6.01 0.57 108.47 0.4
87.91
87.91
87.91
87.91
87.91
87.91
87.91
87.91
87.91
3a
3b
3c
3d
>1225 0.5 0.9 5.82 0.54 113.19 0.4
>37
>75
>227
17
>3.7
17
0.6 0.9 6.34 0.54 113.19 0.4
0.6 0.9 6.59 0.54 113.19 0.4
0.5 0.9 6.80 0.55 118.01 0.4
0.6 0.9 7.18 0.56 118.01 0.4
0.5 0.9 7.08 0.56 118.01 0.4
0.6 0.9 6.83 0.57 116.08 0.4
0.4 0.9 5.54 0.54 114.39 0.4
0.5 0.9 6.01 0.54 114.39 0.4
0.6 0.9 6.20 0.55 114.39 0.4
0.6 0.9 5.90 0.58 112.93 0.4
0.5 0.9 5.48 0.71 113.04 0.4
0.6 0.9 4.54 0.57 120.80 0.4
0.6 0.8 6.08 0.52 112.98 0.4
0.6 0.8 6.08 0.52 112.98 0.4
0.6 0.9 5.00 0.57 125.42 0.4
0.8 0.7 5.63 0.53 114.72 0.4
0.9 0.6 4.87 0.53 109.89 0.4
3e
3f
3g
4-Cl
3.9 1.9
1.2 0.6
5.3 1.4
10.8 2.8
1.4 0.3
0.34 0.04
41 7
1.4 0.3
4.3 0.7
6.0 1.3
2,4-DiCl
3,4-DiCl
3,5-DiCl
4-Br
2-NO2
3-NO2
4-NO2
4-CN
3h
3i
>41
>24
>41
>24
87.91
3j
3k
>287
>287
>287
>301
>288
>266
>309
>309
>46
>287
>287
>287
>301
>288
>266
>309
>309
>46
>844
>7
133.73
133.73
133.73
111.70
111.70
148.07
87.91
87.91
148.07
97.14
3l
3m
3n
3o
>205
>70
>48
>34
>52
>61
57
≥9
9
>67
>1122
41571
2-F-4-CN
4-SO2NH2
2-Me
7.9 1.9
5.9 1.7
3p
3q
4-Me
5.1 1.0
3r
3s
3t
2-Me-4-SO2NH2
4-MeO
4-OH
0.81 0.19
7.3 1.3
8.3 1.3
>69
>71
>44
>69
>71
71 17
>44
108.14
110.43
120.65
119.98
3u
DEV
0.66 0.61
0.0041 0.0002
NDg
NDg
NDg
----
----
124.34 0.4
106.87 0.4
0.025 0.002
>4.6
<39
>59
ETV
VRX-480773
0.00014 0.0000322 0.00023 0.0000252
5.8210
1.7 0.8 6.81 1.01 141.40 0.5
a All data represent mean values for at least three separate experiments.
b EC50: effective concentration required to protect the cell against viral cytopathicity by 50% in MT-4 cells.
c CC50: cytotoxic concentration of compound that reduces the normal uninfected MT-4 cell viability by 50%.
d SI: selectivity index, ratio CC50/EC50 (WT).
e Predicted by using software ACD.
f Predicted by using software ChemAxon.
g ND: not detected.
VRX-480773 as a starting point. Our docking model of
overlaying the lower-energy conformations of etravirine and
2. Chemistry
VRX-480773 templates within the NNRTIs binding sites
The synthetic route for the target compounds 3a-u is
depicted in Scheme 1. Biaryl ether was obtained by
regioselective coupling of 2,6-dimethyl-4-cyanophenol to
the 4-position of 2,4-dichloropyrimidine (
).6 Treatment of
with thiourea yielded 2-
.7 Further condensation with known
(NNIBS) reveals that most fragments in the structures can
superpose well with each other (Fig. 2). They exhibit similar
binding patterns with HIV-1 RT (Fig. 2). In this paper, a
5
4
series of etravirine–VRX-480773 hybrids
3 (Fig. 3),
2-chloropyrimidine
mercaptopyrimidine
5
characterized by a 2,6-dimethyl-4-cyanophenoxyl group, a
central pyrimidine ring and thioacetanilide moieties, were
6
synthesized and evaluated as novel NNRTIs. Preliminary intermediates α-bromoacetamide 7a-u8 created the desired
structure-activity relationships (SARs) and molecular
modeling for these hybrids are also discussed.
target compounds with yields of 48–86%.
3. Results and discussion
3.1. Biological activity

The newly synthesized etravirine–VRX-480773 hybrids were 3t). Derivatives with hydrophobic groups (3n and 3r) were
evaluated for their anti-HIV activity in MT-4 cell cultures more potent than analogs with hydrophilic substituents (3p and
infected with a WT HIV-1 strain (ⅢB), a double mutant HIV-1 3u).
strain RES056 (K103N + Y181C) or an HIV-2 strain (ROD).
Druglike physicochemical property (Table 1) of these
The results were expressed as cytotoxicity (CC50), anti-HIV hybrids were predicted by using software ACD11 or
activity (EC50) and selective index (SI = CC50/EC50). The SI ChemAxon12, respectively. As for pKa, log P and molar
indicates the specificity of the antiviral effect. The results are refractivity (MR), the patterns of hybrids were similar to the
illustrated in Table 1, and include those of the two FDA parent etravirine and VRX-480773. Furthermore, molecular
approved drugs, delavirdine (DEV) and etravirine (ETV), as the polar surface area (PSA) values were calculated by using
reference standards.
ChemAxon software as a prediction of oral bioavailability. The
As listed in Table 1, these hybrids (except for compound 3a) values for all compounds (except for compounds 3p and 3s)
were found to exhibit anti-HIV-1 ⅢB activity in the lower
micromolar range (EC50 = 0.24–41 µΜ) with SI values ranging
from 1 to > 1225. Compound 3d was the most potent inhibitor
less than 140 Å2.13
With the aim to verify their binding target, three selected title
compounds (3d, 3k, and 3s) were assessed in enzyme
with an EC50 value of 0.24 µΜ against WT HIV-1. This activity assays14,15 against highly purified recombinant HIV-1 RT using
was superior to that of the reference compound DEV (EC50
=
poly(rA)/oligo(dT)16 as template and primer, nevirapine (NVP)
0.66 µΜ, SI > 67), but inferior to that of ETV (EC50 = 0.0041 and efavirenz (EFV) as reference (Table 2). All the tested
µΜ) and VRX-480773 (EC50 = 0.00014 µΜ2). It is worth compounds showed moderate inhibitory activity against WT
noting that analogues 3k (EC50 = 0.34 µΜ, SI > 844) and 3s HIV-1 RT. The results confirmed the SAR information
(EC50 = 0.81 µΜ, SI = 57) possessed the same inhibitory obtained in antiviral evaluation in MT-4 cells. As an example,
activity as DEV against WT HIV-1. However, these hybrids compound 3d still displayed the highest inhibitory activity
lack potency against the double mutant strain RES056 and against WT HIV-1 RT with IC50 value of 0.473 µM, which was
HIV-2. The preliminary SARs analyses indicated that the higher than nevirapine (NVP, IC50 = 0.807 µM), but lower than
substitution pattern on the right wing of the hybrids might play efavirenz (EFV, IC50 = 0.022 µM). The regression analysis
a determinant role in their anti-HIV-1 ( ⅢB) activity. All
(Fig. 4) of pEC50 and pIC50 (most of data removed for clarity)
phenyl-substituted hybrids were more potent than the non- with a correlation factor of 0.87 indicated that these hybrids
substituted parent compound 3a, regardless of the electronic bind to HIV-1 RT.
effect and steric bulk. In the case of di-substitution over mono-
substitution of the phenyl ring, 2,4-dichloro-substituted 3g
(EC50 = 1.2 µΜ) showed no marked improvement on the ability
to inhibit WT virus compared with 2-chloro (3d) and 4-chloro
(3f) analogues (EC50 = 0.24 and 3.9 µΜ, respectively), with the
exception of a 10-fold boost in activity achieved when a second
methyl group was introduced in compound 3s. The inhibitory
potency was higher for the ortho-substituted derivatives (3d
and 3k) than for the para-substituted analogues (3f and 3m)
that in turn was more active than the meta-substituted
congeners (3e and 3l). For the compounds with a mono-
substituent at the C-4 position of the phenyl ring, the electron-
withdrawing groups (3j, 3m, 3f and 3n) seem to be more
advantageous than the electron-donating substituents (3r and
Table 2
Inhibitory activity of representative hybrids against WT HIV-1 RTa
Compounds
IC50b (µM)
0.473 0.155
1.169 0.216
2.775 0.117
0.807 0.326
0.022 0.006
3d
3k
3s
NVP
EFV
a Data represent the mean values of at least two separate experiments.
b IC50: inhibitory concentration required to inhibit biotin deoxyuridine
triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.
Figure 4. Regression analysis pEC50 and pIC50
Furthermore, we also performed time-of-addition with WT HIV-1. Reference compounds with known mode of
experiments in MT-4 cells in which the test compounds were action were included such as NRTI AZT that inhibits HIV RT
added at different time points after infection of the cell cultures (0-3 h post infection), NNRTIs NVP and ETV that inhibits HIV

RT (3-5 h post infection), the diketo acid EVG that inhibits the compound 3d displayed the highest activity with an EC50 value
strand transfer step of the integration (7-8 h post-infection) and of 0.24 µΜ and SI > 1225. Taking full use of the information
WP7-5 that is an inhibitor of transcription (10-16 h post- from SAR analyses and molecular modeling calculations,
infection). Addition of the etravirine–VRX-480773 hybrids, 3d, further optimization of this series of hybrids to improve their
3k and 3s, all can be postponed for 3-5 h post-infection,
coinciding with the process of reverse transcription, which is
also confirmed by a similar behavior of the NNRTIs NVP and
ETV in the experiments.
drug resistance profiles are ongoing and will be reported in due
course.
5. Material and methods
5.1. Chemistry
3.2. Molecular simulation
To investigate the HIV-1 inhibitory potencies of the newly
synthesized etravirine–VRX-480773 hybrids at a molecular
level, molecular simulation was carried out. This was done by
docking the representative compound 3a into the NNIBS of
WT HIV-1 RT (PDB code: 3C6T)16 using a Base-Builder
program and Surflex-Dock program interfaced with SYBYL-X
1.2. The results are shown in Fig. 5. The hybrid 3a was
predicted to bind with NNIBS in a “U” mode. Notable features
include: (1) the left phenyl ring fitted into the aromatic-rich
binding pocket, which was surrounded by the aromatic side
chains of amino acid residues Tyr181, Tyr188 and Trp229, and
exhibited π–π stacking interaction with these residues; (2) the
right aromatic ring extended to the enzyme/water interfaces and
made lipophilic interactions with the amino acids Phe227,
Pro236 and Tyr318; (3) a hydrogen bond was formed between
the NH group of the C2 side chain of hybrid 3a and the
backbone C=O of Lys103, improving the affinity between RT
and inhibitors.
Chemical reagents and solvents, purchased from commercial
sources, were of analytical grade and were used without further
purification. All air-sensitive reactions were run under a
nitrogen atmosphere. All the reactions were monitored by TLC
on pre-coated silica gel G plates at 254 nm under a UV lamp
using ethyl acetate/hexane as eluent. Column chromatography
separations were performed with silica gel (300–400 mesh).
Melting points were measured on a SGW X-1 microscopic
1
melting point apparatus. H NMR and 13C NMR spectra were
recorded on a Bruker AV400 MHz spectrometer in DMSO-d6.
Chemical shifts were reported in δ (ppm) units relative to the
internal standard tetramethylsilane (TMS). IR analysis were
carried out on a JASCO FT/IR-4200 series. Mass spectra and
HRMS were obtained on
a Waters Quattro Micromass
instrument and Bruker solari X-70 FT-MS instrument,
respectively, using electrospray ionization (ESI) techniques.
The purities of target compounds were ≥ 95%, measured by
HPLC, performed on an Agilent 1200 HPLC system with UV
detector and Agilent Eclipse Plus C18 column (150 × 4.6 mm, 5
µm), eluting with a mixture of solvents H2O (A) and CH3CN
(B) from VA: VB = 90:10 to 10:90. Peaks were detected at λ 254
nm with a flow rate of 1.0 mL/min.
5.2. Preparation of 4-((2-chloropyrimidin-4-yl)oxy)-3,5-
dimethylbenzonitrile (5)
Sodium hydride (2.20 g, 55 mmoL) was added in portions to
a solution of 4-hydroxy-3,5-dimethylbenzonitrile (7.35 g, 50
mmoL) in anhydrous THF (100 mL) at room temperature.6 The
resulting solution was stirred for 10 min, then 2,4-
dichloropyrimidine (4) (7.40 g, 50 mmoL) was added. The
o
reaction mixture was heated to 40 C and maintained for 6 h.
The progress of the reaction was monitored by TLC. After
completion of the reaction, the reaction mixture was cooled to
RT, poured into vigorously stirred ice water (800 mL), and
filtered; the precipitated solid was washed with chilled water,
o
and dried at 50 C under vacuum. The corresponding crude
product was recrystallized from DMF to afford pure 5 (71%
o
1
Figure 5. Predicted binding mode of the representative hybrid 3a
in NNIBS of WT HIV-1 RT (PDB code: 3C6T).
yield) as white crystals. Mp 230.3–230.9 C; H NMR (400
MHz, DMSO-d6) δ = 2.08 (s, 6H, 2CH3), 7.28 (d, J = 5.6 Hz,
1H, pyrimidine H5), 7.71 (s, 2H, PhH3,5), 8.67 (d, J = 5.6 Hz,
1H, pyrimidine H6); 13C NMR (100 MHz, DMSO-d6) δ = 15.79,
107.14, 109.27, 118.50, 132.49, 132.98, 152.34, 159.29,
162.13, 168.63; MS (ESI+) m/z 260 (M+H)+; HRMS calcd for
C13H10ClN3O [M+Na]+: 282.0410, found: 282.0409.
In summary, the computational modeling study was
helpful to better understand the interactions between the
hybrids and HIV-1 RT. The molecular binding model of
compounds 3a provided critical insights for further
development of new hybrid molecules.
5.3. Preparation of 4-((2-mercaptopyrimidin-4-yl)oxy)-3,5-
dimethylbenzonitrile (6)
4. Conclusion
4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (5)
(4.26 g, 16.4 mmoL), thiourea (1.88 g, 24.6 mmoL) and ethanol
(75 mL) were boiled under reflux for 4.5 h.7 After the mixture
was cooled, the precipitate was collected by filtration, washed
Based on
a structure-guided molecular hybridization
approach, we designed and synthesized a novel series of
etravirine–VRX-480773 hybrids as potent HIV-1 NNRTIs.
These hybrids were assessed for their anti-HIV-1 activity in
MT-4 cell cultures. Most compounds (except for compound 3a)
exhibited significant potency against WT HIV-1 strain (IIIB) at
micromolar concentrations (EC50 = 0.24–41 µΜ). Among them,
o
with EtOH (2 × 10 mL) and dried at 50 C under vacuum to
give
compound
4-((2-mercaptopyrimidin-4-yl)oxy)-3,5-
dimethylbenzonitrile (6) as a white solid. Yield: 77%. Mp
o
1
290.2–290.5 C; H NMR (400 MHz, DMSO-d6) δ = 2.09 (s,

6H, 2CH3), 6.61 (d, J = 6.8 Hz, 1H, pyrimidine H5), 7.69 (s, dimethylphenoxy)pyrimidin-2-yl)thio)acetamide (3d)
o
1
2H, PhH3,5), 7.99 (d, J = 6.8 Hz, 1H, pyrimidine H6); 13C NMR
Yield 84%; white solid, mp 170.0–170.8 C; H NMR (400
(100 MHz, DMSO-d6) δ = 15.94, 98.03, 108.89, 118.64, MHz, DMSO-d6) δ = 2.02 (s, 6H, 2CH3), 3.93 (s, 2H, CH2),
132.49, 132.73, 149.25, 152.71, 166.13, 182.38; MS (ESI+) m/z 6.95 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.17 (t, J = 7.6 Hz, 1H,
258 (M+H)+; HRMS calcd for C13H11N3OS [M+H]+: 258.0701, Ph’H4), 7.33 (t, J = 7.8 Hz, 1H, Ph’H5), 7.47 (d, J = 8.0 Hz, 1H,
found: 258.0698.
5.4. General procedure for the preparation of 3a-u
Ph’H3), 7.56 (s, 2H, PhH3,5), 7.82 (d, J = 8.4 Hz, 1H, Ph’H6),
8.58 (d, J = 5.6 Hz, 1H, pyrimidine H6), 9.48 (s, 1H, NH); 13C
Potassium tert-butoxide (0.0589 g, 0.525 mmoL) was added NMR (100 MHz, DMSO-d6) δ = 15.72, 34.91, 103.54, 108.81,
solution of 4-((2-mercaptopyrimidin-4-yl)oxy)-3,5- 118.48, 124.32, 124.84, 125.87, 127.52, 129.47, 132.25,
to
a
dimethylbenzonitrile (6) (0.1287 g, 0.50 mmoL) in 1.5 mL of
132.66, 134.69, 152.41, 160.02, 166.24, 167.02, 170.31; MS
anhydrous DMF. The mixture was stirred for 30 min before (ESI+) m/z 458 (M+Na)+; HRMS calcd for C21H17ClN4O2S
0.50 mmoL of a known intermediate α-bromoacetamide 7a-u8 [M+Na]+: 447.0658, found: 447.0658; HPLC: tR = 10.31 min,
was added dropwise as a solution in 0.6 mL of anhydrous DMF 96.3%.
and the reaction was stirred at room temperature for 1 h. The 5.4.5.
N-(3-Chlorophenyl)-2-((4-(4-cyano-2,6-
reaction was followed by TLC until its completion and water
(21 mL) was added. The mixture was extracted with ethyl
dimethylphenoxy)pyrimidin-2-yl)thio)acetamide (3e)
1
o
Yield 78%; white solid, mp 200.0–200.8 C; H NMR (400
acetate (3 × 20 mL), washed with water (3 × 10 mL) and brine MHz, DMSO-d6) δ = 1.97 (s, 6H, 2CH3), 3.77 (s, 2H, CH2),
(3 × 10 mL), and dried over Na2SO4 to give the corresponding 6.93 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.12 (d, J = 7.2 Hz,
crude product, which was purified by flash column 1H, Ph’H4), 7.34-7.36 (m, 4H, PhH3,5 + Ph’H5,6), 7.76 (s, 1H,
chromatography to afford compounds 3a-u.
5.4.1. 2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-phenylacetamide (3a)
Ph’H2), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6), 10.09 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.73, 35.19,
103.20, 108.75, 117.15, 118.22, 118.51, 122.90, 130.45,
o
1
Yield 76%; white solid, mp 211.2–211.6 C; H NMR (400 132.09, 132.45, 133.13, 140.65, 152.32, 159.92, 165.63,
MHz, DMSO-d6) δ = 1.98 (s, 6H, 2CH3), 3.78 (s, 2H, CH2), 166.93, 170.61; MS (ESI+) m/z 425 (M+H)+; HRMS calcd for
6.92 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.07 (m, 1H, Ph’H4), C21H17ClN4O2S [M+Na]+: 447.0658, found: 447.0646; HPLC:
7.31 (m, 4H, PhH3,5 + Ph’H3,5), 7.54 (d, J = 6.4 Hz, 2H, tR = 9.47 min, 97.4%.
Ph’H2,6), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6), 9.91 (s, 1H, 5.4.6.
N-(4-Chlorophenyl)-2-((4-(4-cyano-2,6-
NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.74, 35.21, dimethylphenoxy)pyrimidin-2-yl)thio)acetamide (3f)
o
1
103.18, 108.78, 118.59, 118.76, 123.21, 128.75, 132.14,
132.51, 139.27, 152.36, 159.90, 165.26, 166.95, 170.75; IR MHz, DMSO-d6) δ = 1.98 (s, 6H, 2CH3), 3.77 (s, 2H, CH2),
(KBr): ν
= 3387 (s; ν(N-H)), 2219 (s; ν(C≡N)), 1709 cm-1 (s; 6.92 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.35-7.37 (m, 4H,
ν(C=O)); MS (ESI+) m/z 391 (M+H)+; HRMS calcd for PhH3,5 + Ph’H3,5), 7.55-7.57 (d, J = 8.8 Hz, 2H, Ph’H2,6), 8.53
C21H18N4O2S [M+Na]+: 413.1048, found: 413.1042; HPLC: tR (d, J = 5.6 Hz, 1H, pyrimidine H6), 10.05 (s, 1H, NH); 13C
Yield 84%; white solid, mp 176.0–176.4 C; H NMR (400
̃
= 8.21 min, 99.2%.
NMR (100 MHz, DMSO-d6) δ = 15.77, 35.21, 103.26, 108.78,
118.63, 120.35, 126.77, 128.70, 132.20, 132.54, 138.23,
152.41, 159.96, 165.48, 167.00, 170.68; MS (ESI+) m/z 447
5.4.2.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(3-fluorophenyl)acetamide (3b)
Yield 75%; white solid, mp 204.6–205.4 C; H NMR (400 (M+Na)+; HRMS calcd for C21H17ClN4O2S [M+H]+: 425.0839,
o
1
MHz, DMSO-d6) δ = 1.97 (s, 6H, 2CH3), 3.78 (s, 2H, CH2), found: 425.0838; HPLC: tR = 10.81 min, 96.0%.
6.88-6.94 (m, 2H, pyrimidine H5 + Ph’H4), 7.24 (m, 1H, Ph’H5), 5.4.7.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
7.34-7.38 (m, 3H, PhH3,5 + Ph’H6), 7.51 (d, J = 11.2 Hz, 1H, yl)thio)-N-(2,4-dichlorophenyl)acetamide (3g)
Ph’H2), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6), 10.11 (s, 1H,
Yield 77%; white solid, mp 188.8–189.5 C; H NMR (400
o
1
NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.72, 35.20, MHz, DMSO-d6) δ = 2.02 (s, 6H, 2CH3), 3.93 (s, 2H, CH2),
103.20, 105.40 (d, JC-C-F = 26.3 Hz), 108.77, 109.54 (d, JC-C-F
21.0 Hz), 114.52 (d, JC4-F = 2.3 Hz), 118.54, 130.43 (d, JC3-F
=
=
6.94 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.41 (d, J = 8.4 Hz,
1H, Ph’H5), 7.56 (s, 2H, PhH3,5), 7.64 (s, 1H, Ph’H3), 7.83 (d, J
9.3 Hz), 132.10, 132.45, 140.90 (d, JC3-F = 10.9 Hz), 152.33, = 8.8 Hz, 1H, Ph’H6), 8.57 (d, J = 5.6 Hz, 1H, pyrimidine H6),
159.92, 163.35 (d, JC-F = 239.9 Hz), 165.61, 166.95, 170.63; 9.56 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.73,
MS (ESI+) m/z 456 (M+Na)+; HRMS calcd for C21H17FN4O2S 34.90, 103.53, 108.78, 118.46, 125.34, 125.72, 127.62, 128.86,
[M+H]+: 409.1134, found: 409.1128; HPLC: tR = 10.25 min, 128.92, 132.26, 132.65, 133.93, 152.42, 160.01, 166.43,
96.9%.
5.4.3.
167.01, 170.31; MS (ESI+) m/z 481 (M+Na)+; HRMS calcd for
C21H16Cl2N4O2S [M+Na]+: 481.0269, found: 481.0265; HPLC:
tR = 12.21 min, 97.6%.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(4-fluorophenyl)acetamide (3c)
o
1
Yield 83%; white solid, mp 206.1–206.5 C; H NMR (400 5.4.8
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
MHz, DMSO-d6) δ = 1.99 (s, 6H, 2CH3), 3.78 (s, 2H, CH2), yl)thio)-N-(3,4-dichlorophenyl)acetamide (3h)
o
1
6.92 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.15 (t, J = 8.6 Hz, 2H,
Yield 85%; white solid, mp 203.6–204.3 C; H NMR (400
Ph’H3,5), 7.40 (s, 2H, PhH3,5), 7.54-7.57 (m, 2H, Ph’H2,6), 8.54 MHz, DMSO-d6) δ = 1.98 (s, 6H, 2CH3), 3.77 (s, 2H, CH2),
(d, J = 5.6 Hz, 1H, pyrimidine H6), 9.97 (s, 1H, NH); 13C NMR 6.94 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.36 (s, 2H, PhH3,5),
(100 MHz, DMSO-d6) δ = 15.71, 35.09, 103.18, 108.73, 115.40 7.42 (d, J = 8.8 Hz, 1H, Ph’H5), 7.56 (d, J = 8.8 Hz, 1H, Ph’H6),
(d, JC-C-F = 22.1 Hz), 118.56, 120.44 (d, JC3-F = 7.7 Hz), 132.14, 7.90 (s, 1H, Ph’H2), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6),
132.50, 135.65 (d, JC4-F = 2.4 Hz), 152.36, 156.70 (d, JC-F
=
10.19 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.71,
237.8 Hz), 159.89, 165.16, 166.94, 170.69; MS (ESI+) m/z 409 35.18, 103.23, 108.73, 118.49, 118.79, 119.95, 124.62, 130.71,
(M+H)+; HRMS calcd for C21H17FN4O2S [M+H]+: 409.1134, 131.06, 132.12, 132.44, 139.25, 152.34, 159.93, 165.78,
found: 409.1135; HPLC: tR = 10.09 min, 97.7%.
5.4.4. N-(2-Chlorophenyl)-2-((4-(4-cyano-2,6-
166.94, 170.53; MS (ESI+) m/z 481 (M+Na)+; HRMS calcd for
C21H16Cl2N4O2S [M+Na]+: 481.0269, found: 481.0262; HPLC:

tR = 11.46 min, 97.5%.
5.4.9. 2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(3,5-dichlorophenyl)acetamide (3i)
97.2%.
5.4.14.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(4-cyanophenyl)acetamide (3n)
o
1
o
1
Yield 84%; white solid, mp 226.9–227.7 C; H NMR (400
Yield 82%; white solid, mp 209.0–209.4 C; H NMR (400
MHz, DMSO-d6) δ = 1.97 (s, 6H, 2CH3), 3.75 (s, 2H, CH2), MHz, DMSO-d6) δ = 1.96 (s, 6H, 2CH3), 3.79 (s, 2H, CH2),
6.94 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.29-7.33 (m, 3H, 6.93 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.31 (s, 2H, PhH3,5),
PhH3,5 + Ph’H4), 7.58 (s, 2H, Ph’H2,6), 8.54 (d, J = 5.6 Hz, 1H, 7.68 (d, J = 8.4 Hz, 2H, Ph’H3,5), 7.77 (d, J = 8.4 Hz, 2H,
pyrimidine H6), 10.22 (s, 1H, NH); 13C NMR (100 MHz,
Ph’H2,6), 8.53 (d, J = 5.6 Hz, 1H, pyrimidine H6), 10.32 (s, 1H,
DMSO-d6) δ = 15.71, 35.22, 103.25, 108.73, 116.90, 118.40, NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.77, 35.38,
122.49, 132.09, 132.40, 134.19, 141.44, 152.31, 159.96, 103.33, 105.02, 108.76, 118.63, 118.88, 119.24, 132.18,
165.97, 166.94, 170.49; MS (ESI+) m/z 481 (M+Na)+; HRMS 132.51, 133.42, 143.41, 152.41, 160.01, 166.13, 167.04,
calcd for C21H16Cl2N4O2S [M+H]+: 459.0449, found: 459.0434; 170.59; MS (ESI+) m/z 438 (M+Na)+; HRMS calcd for
HPLC: tR = 11.78 min, 98.1%.
C22H17N5O2S [M+H]+: 416.1181, found: 416.1170; HPLC: tR =
9.57 min, 98.3%.
5.4.10.
dimethylphenoxy)pyrimidin-2-yl)thio)acetamide (3j)
N-(4-Bromophenyl)-2-((4-(4-cyano-2,6-
5.4.15.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
o
1
Yield 86%; white solid, mp 160.7–161.5 C; H NMR (400 yl)thio)-N-(4-cyano-2-fluorophenyl)acetamide (3o)
o
1
MHz, DMSO-d6) δ = 1.98 (s, 6H, 2CH3), 3.78 (s, 2H, CH2),
Yield 85%; white solid, mp 217.7–218.2 C; H NMR (400
6.92 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.38 (s, 2H, PhH3,5 ), MHz, DMSO-d6) δ = 1.98 (s, 6H, 2CH3), 3.88 (s, 2H, CH2),
7.48-7.53 (m, 4H, Ph’H2,3,5,6), 8.53 (d, J = 5.6 Hz, 1H, 6.94 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.35 (s, 2H, PhH3,5),
pyrimidine H6), 10.06 (s, 1H, NH); 13C NMR (100 MHz,
7.66 (d, J = 8.4 Hz, 1H, Ph’H3), 7.87 (d, J = 11.2 Hz, 1H,
DMSO-d6) δ = 15.75, 35.22, 103.22, 108.76, 114.73, 118.60, Ph’H5), 8.26 (t, J = 8.2 Hz, 1H, Ph’H6), 8.53 (d, J = 5.6 Hz, 1H,
120.69, 131.58, 132.17, 132.51, 138.62, 152.38, 159.93, pyrimidine H6), 10.10 (s, 1H, NH); 13C NMR (100 MHz,
165.48, 166.96, 170.65; MS (ESI+) m/z 469 (M+H)+; HRMS DMSO-d6) δ = 15.81, 35.35, 103.41, 105.67 (d, JC3-F = 9.5 Hz),
calcd for C21H17BrN4O2S [M+H]+: 469.0334, found: 469.0338; 108.64, 118.09, 118.47, 119.44 (d, JC-C-F = 23.2 Hz), 121.90 (d,
HPLC: tR = 11.05 min, 97.1%.
JC-C-F = 22.0 Hz), 129.60 (d, JC4-F = 2.9 Hz), 131.68 (d, JC3-F =
5.4.11.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(2-nitrophenyl)acetamide (3k)
11.0 Hz), 132.24, 132.51, 149.92 (d, JC-F = 246.0 Hz), 152.46,
159.99, 166.83, 167.05, 170.59; MS (ESI+) m/z 456 (M+Na)+;
Yield 85%; yellow solid, mp 180.9–181.5 oC; H NMR (400 HRMS calcd for C22H16FN5O2S [M+H]+: 434.1087, found:
1
MHz, DMSO-d6) δ = 1.99 (s, 6H, 2CH3), 3.89 (s, 2H, CH2), 434.1071; HPLC: tR = 10.15 min, 97.0%.
6.96 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.36 (t, J = 7.0 Hz, 1H, 5.4.16.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
Ph’H4), 7.53 (s, 2H, PhH3,5), 7.75 (t, J = 6.4 Hz, 1H, Ph’H5), yl)thio)-N-(4-sulfamoylphenyl)acetamide (3p)
7.97-8.05 (m, 2H, Ph’H3,6), 8.57 (d, J = 5.6 Hz, 1H, pyrimidine
Yield 77%; white solid, mp 250.9–251.5 C; H NMR (400
o
1
H6), 10.52 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = MHz, DMSO-d6) δ = 1.97 (s, 6H, 2CH3), 3.82 (s, 2H, CH2),
15.69, 35.14, 103.71, 108.81, 118.53, 123.79, 124.84, 125.36, 6.93 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.28 (s, 2H, NH2), 7.38
132.10, 132.29, 132.65, 134.84, 139.83, 152.47, 160.07, (s, 2H, PhH3,5), 7.68 (d, J = 8.8 Hz, 2H, Ph’H2,6), 7.77 (d, J =
166.43, 167.10, 169.92; MS (ESI+) m/z 458 (M+Na)+; HRMS 8.8 Hz 2H, Ph’H3,5), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6),
calcd for C21H17N5O4S [M+Na]+: 458.0899, found: 458.0887; 10.28 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.74,
HPLC: tR = 10.85 min, 97.3%.
35.27, 103.23, 108.80, 118.35, 118.59, 126.70, 132.15, 132.52,
138.35, 142.07, 152.35, 159.94, 165.92, 166.93, 170.56; MS
(ESI+) m/z 492 (M+Na)+; HRMS calcd for C21H19N5O4S2
5.4.12.
yl)thio)-N-(3-nitrophenyl)acetamide (3l)
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
Yield 84%; light yellow solid, mp 192.8–193.5 oC; 1H NMR [M+H]+: 470.0957, found: 470.0954; HPLC: tR = 7.92 min,
(400 MHz, DMSO-d6) δ = 1.96 (s, 6H, 2CH3), 3.79 (s, 2H, 97.6%.
CH2), 6.94 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.28 (s, 2H, 5.4.17.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
PhH3,5), 7.63 (t, J = 8.0 Hz, 1H, Ph’H5), 7.82 (d, J = 7.6 Hz, 1H, yl)thio)-N-(o-tolyl)acetamide (3q)
Ph’H6), 7.93 (d, J = 8.0 Hz, 1H, Ph’H4), 8.54-8.55 (m, 2H,
Yield 84%; white solid, mp 167.6–168.4 C; H NMR (400
o
1
pyrimidine H6 + Ph’H2), 10.36 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = 2.05 (s, 6H, 2CH3), 2.14 (s, 3H, CH3),
MHz, DMSO-d6) δ = 15.70, 35.20, 103.25, 108.63, 112.80, 3.92 (s, 2H, CH2), 6.91 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.07
117.78, 118.47, 124.66, 130.25, 132.11, 132.41, 140.33, (t, J = 7.0 Hz, 1H, Ph’H4), 7.13-7.20 (m, 2H, Ph’H5,6), 7.40 (d, J
148.01, 152.37, 159.95, 165.97, 166.98, 170.58; MS (ESI+) m/z = 7.6 Hz, 1H, Ph’H3), 7.60 (s, 2H, PhH3,5), 8.56 (d, J = 5.6 Hz,
436 (M+H)+; HRMS calcd for C21H17N5O4S [M+H]+: 436.1079, 1H, pyrimidine H6), 9.33 (s, 1H, NH); 13C NMR (100 MHz,
found: 436.1070; HPLC: tR = 9.98 min, 96.4%.
5.4.13. 2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(4-nitrophenyl)acetamide (3m)
DMSO-d6) δ = 15.79, 17.79, 34.85, 103.36, 108.81, 118.53,
124.38, 125.15, 125.99, 130.38, 131.07, 132.38, 132.74,
136.20, 152.49, 159.97, 165.94, 166.99, 170.68; MS (ESI+) m/z
o
1
Yield 85%; white solid, mp 208.7–209.5 C; H NMR (400 405 (M+H)+; HRMS calcd for C22H20N4O2S [M+H]+: 405.1385,
MHz, DMSO-d6) δ = 1.97 (s, 6H, 2CH3), 3.82 (s, 2H, CH2), found: 405.1374; HPLC: tR = 10.46 min, 97.2%.
6.94 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.34 (s, 2H, PhH3,5), 5.4.18.
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
7.74 (d, J = 8.8 Hz, 2H, Ph’H2,6), 8.23 (d, J = 9.2 Hz, 2H, yl)thio)-N-(p-tolyl)acetamide (3r)
Ph’H3,5), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6), 10.50 (s, 1H,
Yield 85%; white solid, mp 195.8–196.3 C; H NMR (400
o
1
NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.69, 35.37, MHz, DMSO-d6) δ = 1.98 (s, 6H, 2CH3), 2.26 (s, 3H, CH3),
103.26, 108.69, 118.44, 118.54, 125.05, 132.08, 132.47, 3.77 (s, 2H, CH2), 6.91 (d, J = 5.2 Hz, 1H, pyrimidine H5), 7.10
142.16, 145.29, 152.32, 159.93, 166.20, 166.96, 170.49; MS (d, J = 7.6 Hz, 2H, Ph’H3,5), 7.39 (s, 2H, PhH3,5), 7.42 (d, J =
(ESI+) m/z 458 (M+Na)+; HRMS calcd for C21H17N5O4S 8.0 Hz, 2H, Ph’H2,6), 8.53 (d, J = 5.6 Hz, 1H, pyrimidine H6),
[M+H]+: 436.1079, found: 436.1061; HPLC: tR = 10.04 min, 9.84 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.76,

20.52, 35.18, 103.20, 108.79, 118.62, 118.80, 129.13, 132.11, and purified as previously described.14 The RT assay was
132.19, 132.56, 136.78, 152.39, 159.91, 165.04, 166.96, performed with the EnzCheck Reverse Transcriptase Assay kit
170.78; MS (ESI+) m/z 405 (M+H)+; HRMS calcd for
(Molecular Probes, Invitrogen), as described by the
C22H20N4O2S [M+Na]+: 427.1205, found: 427.1200; HPLC: tR Manufacturer. The assay was based on the dsDNA quantitation
= 10.62 min, 97.8%.
5.4.19. 2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(2-methyl-4-sulfamoylphenyl)acetamide (3s)
reagent PicoGreen. This reagent showed a pronounced increase
in fluorescence signal upon binding to dsDNA or RNA-DNA
heteroduplexes. Single-stranded nucleic acids generated only
o
1
Yield 84%; white solid, mp 222.8–223.6 C; H NMR (400 minor fluorescence signal enhancement when a sufficiently
MHz, DMSO-d6) δ = 2.04 (s, 6H, 2CH3), 2.25 (s, 3H, CH3), high dye:base pair ratio was applied.15 This condition was met
3.97 (s, 2H, CH2), 6.92 (d, J = 5.6 Hz, 1H, pyrimidine H5), 7.27 in the assay.
(s, 2H, NH2), 7.59 (s, 2H, PhH3,5), 7.61-7.72 (m, 3H, Ph’H3,5,6),
A poly(rA) template of approximately 350 bases long, and an
oligo(dT)16 primer, were annealed in a molar ratio of 1:1.2 (60
8.56 (d, J = 5.6 Hz, 1H, pyrimidine H6), 9.50 (s, 1H, NH); 13
C
NMR (100 MHz, DMSO-d6) δ = 15.78, 17.93, 34.96, 103.37, min, at room temperature). 52 ng of the RNA/DNA was
108.78, 118.50, 123.39, 123.71, 127.69, 130.64, 132.32, brought into each well of a 96-well plate in a volume of 20 mL
132.70, 139.34, 139.87, 152.45, 159.97, 166.33, 166.97, polymerization buffer (60 mM Tris-HCl, 60 mM KCl, 8 mM
170.56; MS (ESI+) m/z 409 (M+H)+; HRMS calcd for
MgCl2, 13 mM DTT, 100 mM dTTP, pH = 8.1). 5.0 mL of RT
C22H21N5O4S2 [M+H]+: 484.1113, found: 484.1115; HPLC: tR = enzyme solution, diluted to a suitable concentration in enzyme
8.45 min, 98.4%.
5.4.20. 2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(4-methoxyphenyl)acetamide (3t)
dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT,
pH = 7.6), was added. The reaction mixture was incubated at 25
oC for 40 min and then stopped by the addition of EDTA (15
o
1
Yield 86%; white solid, mp 184.5–185.3 C; H NMR (400 mM). Heteroduplexes were then detected by addition of
MHz, DMSO-d6) δ = 1.99 (s, 6H, 2CH3), 3.73 (s, 3H, CH3), PicoGreen. Signals were read using an excitation wavelength of
3.77 (s, 2H, CH2), 6.87 (d, J = 8.8 Hz, 2H, Ph’H3,5), 6.91 (d, J = 490 nm and emission detection at 523 nm using
a
5.6 Hz, 1H, pyrimidine H5), 7.42-7.47 (m, 4H, PhH3,5
+
spectrofluorometer (Safire2, Tecan). To test the activity of
Ph’H2,6), 8.54 (d, J = 5.6 Hz, 1H, pyrimidine H6), 9.80 (s, 1H, compounds against RT, 1.0 mL of compound in DMSO was
NH); 13C NMR (100 MHz, DMSO-d6) δ = 15.74, 35.07, 55.19, added to each well before the addition of RT enzyme solution.
103.17, 108.76, 113.85, 118.60, 120.25, 132.18, 132.47, Control wells without compound contained the same amount of
132.55, 152.39, 155.14, 159.89, 164.76, 166.95, 170.78; MS DMSO. Results were expressed as relative fluorescence, i. e.
(ESI+) m/z 421 (M+H)+; HRMS calcd for C22H20N4O3S the fluorescence signal of the reaction mixed with compound
[M+H]+: 421.1334, found: 421.1332; HPLC: tR = 9.74 min, divided by the signal of the same reaction mixed without
96.9%.
5.4.21.
compound.
5.7. Time-of-addition experiments
2-((4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-2-
yl)thio)-N-(4-hydroxyphenyl)acetamide (3u)
Time-of-addition experiments were adapted from Pauwels
Yield 80%; white solid, mp 199.4–200.2 C; H NMR (400 and19 Daelemans20. Briefly, MT-4 cells were infected with WT
MHz, DMSO-d6) δ = 1.99 (s, 6H, 2CH3), 3.75 (s, 2H, CH2), HIV-1 at an m.o.i. of 0.5. Following a 1 h adsorption period
6.69 (d, J = 8.4 Hz, 2H, Ph’H3,5), 6.91 (d, J = 5.6 Hz, 1H, cells were distributed in a 96-well tray at 45,000 cells/well and
pyrimidine H5), 7.32 (d, J = 8.4 Hz, 2H, Ph’H2,6), 7.43 (s, 2H, incubated at 37°C. Test compounds were added at different
PhH3,5), 8.53 (d, J = 5.6 Hz, 1H, pyrimidine H6), 9.21 (s, 1H, times (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 24, and 25 h) after infection.
OH), 9.68 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ = HIV-1 production was determined at 31 h postinfection via a
15.75, 35.05, 103.15, 108.77, 115.06, 118.61, 120.47, 131.02, p24 enzyme-linked immunosorbent assay (Perkin Elmer,
132.19, 132.58, 152.39, 153.25, 159.88, 164.54, 166.94, Brussels, Belgium). Dextran sulfate was used at 12.5 µM, AZT
o
1
170.81; MS (ESI+) m/z 407 (M+H)+; HRMS calcd for
C21H18N4O3S [M+H]+: 407.1178, found: 407.1167; HPLC: tR = aminoquinolone derivative WP7-5 at 0.25 µM and the
at 1.9 µM, ETV at 0.19 µM, EVG at 0.31 µM, the 6-
8.18 min, 97.9%.
5.5. Anti-HIV activity assay
etravirine–VRX-480773 hybrids were added at their 100-fold
their EC50 concentration obtained in the MT-4/MTT assay.
The anti-HIV activity and cytotoxicity of the compounds 3a- 5.8. Molecular docking
u were evaluated against wild-type HIV-1 strain IIIB, a double
Molecular modeling was performed with the Tripos
RT mutant (K103N + Y181C) HIV-1 strain and HIV-2 strain molecular modeling packages Sybyl-X 1.2. All the molecules
ROD in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2- for docking were built using standard bond lengths and angles
yl)-2,5-diphenyltetrazolium bromide (MTT) method.17,18 from Sybyl-X 1.2/base Builder and were then optimized using
Briefly, virus stocks were titrated in MT-4 cells and expressed the Tripos force field for 2000 generations two times or more,
as the 50% cell culture infective dose (CCID50). MT-4 cells until the minimized conformers of the ligand were the same.
were suspended in culture medium at 1 × 105 cells/mL and The flexible docking method, called Surflex-Dock21, docks the
infected with HIV at a multiplicity of infection of 0.02. ligand automatically into the ligand binding site of the receptor
Immediately after viral infection, 100 µL of the cell suspension by using a protocol-based approach and an empirically-derived
was placed in each well of a flat-bottomed microtiter tray scoring function22. The protocol is
a
computational
containing various concentrations of the test compounds. The representation of a putative ligand that binds to the intended
test compounds were dissolved in DMSO at 50 mM or higher. binding site and is a unique and essential element of the
After 4 days of incubation at 37 °C, the number of viable cells docking algorithm23. The scoring function in Surflex-Dock,
was determined using the MTT method. Compounds were which contains hydrophobic, polar, repulsive, entropic, and
tested in parallel for cytotoxic effects in uninfected MT-4 cells.
5.6. HIV-1 RT assay
solvation terms, was trained to estimate the dissociation
constant (Kd) expressed in -log (Kd)2. Prior to docking, the
Recombinant wild type p66/p51 HIV-1 RT was expressed protein was prepared by removing water molecules, the ligand,

and other unnecessary small molecules from the crystal
structure of the ligand–HIV-1 RT complex (PDB code:
3C6T)16; simultaneously, polar hydrogen atoms were added to
the protein. Surflex-Dock default settings were used for other
parameters, such as the number of starting conformations per
molecule (set to 0), the size to expand search grid (set to 8 Å),
the maximum number of rotatable bonds per molecule (set to
100), and the maximum number of poses per ligand (set to 20).
During the docking procedure, all of the single bonds in residue
side-chains inside the defined RT binding pocket were regarded
as rotatable or flexible, and the ligand was allowed to rotate at
all single bonds and move flexibly within the tentative binding
pocket. The atomic charges were recalculated using the
A. G.; Hang, J. Q.; Heilek, G. M.; Klumpp, K. Bioorg.
Med. Chem. Lett 2010 20, 4215-4218.
Brown, D. J.; Mori, K. Aust. J. Chem. 1985
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A.; Chan, J. H.; Boone, L. R. J. Med. Chem. 2006, 49,
727-739.
Weuts, I.; Dycke, F. V.; Voorspoels, J.; Cort, S. D.;
Stokbroekx, S.; Leemans, R.; Brewster, M. E.; Xu, D.;
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7
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9
Sci. 2011, 100, 260-274.
Kollman all-atom approach for the protein and the Gasteiger- 10 Jochmans, D.; Anders, M.; Keuleers, I.; Smeulders, L.;
Hückel approach for the ligand. The binding interaction energy
Kräusslich H.; Kraus, G.; Müller, B. Retrovirology 2010
,
was calculated, including van der Waals, electrostatic, and
7
, 89-102.
torsional energy terms defined in the Tripos force field. The 11 ACD/ilab2, version 12.01, Advanced Chemistry
structure optimization was performed for 20,000 generations
using a genetic algorithm, and the 20-best-scoring ligand-
Development,
www.acdlabs.com, 2014
Inc.,
Toronto,
ON,
Canada,
.
protein complexes were kept for further analyses. The -log 12 http://www.chemicalize.org/
(Kd)2 values of the 20-best-scoring complexes, which 13 Veber, D. F.; Johnson, S. R.; Cheng, H.; Smith, B. R.;
represented the binding affinities of ligand with RT,
encompassed a wide scope of functional classes (10-2–10-9).
Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45,
2615-2623.
Therefore, only the highest-scoring 3D structural model of the 14 Auwerx, J.; North, T. W.; Preston, B. D.; Klarmann, G.
ligand-bound RT was chosen to define the binding interaction24.
Acknowledgements
J.; De Clercq, E.; Balzarini, J. Mol. Pharmacol. 2002, 61,
400-406.
This research was financially supported by National Natural 15 Singer, V. L.; Jones, L. J.; Yue, S. T.; Haugland, R. P.
Science Foundation of China under Grant No. 81172918,
Shanghai Municipal Natural Science Foundation under Grant
No. 13ZR1402200, Chinese National Science and Technology
Major Project under Grant No. 2012ZX09103101-068 and
Open Foundation of Key Laboratory of Natural Resources of
Anal. Biochem. 1997, 249, 228-238.
16 Tucker, T. J.; Saggar, S.; Sisko, J. T.; Tynebor, R. M.;
Williams, T. M.; Felock, P. J.; Flynn, J. A.; Lai, M.;
Liang, Y.; McGaughey, G.; Liu, M.; Miller, M.; Moyer,
G.; Munshi, V.; Perlow-Poehnelt, R.; Prasad, S.;
Sanchez, R.; Torrent, M.; Vacca, J. P.; Wan, B.; Yan, Y.
Changbai Mountain
&
Functional Molecules (Yanbian
University), Ministry of Education.
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