Design, synthesis, and antiviral evaluation of novel hydrazone-substituted thiophene[3,2-d]pyrimidine derivatives as potent human immunodeficiency virus-1 inhibitors

Article abstract of DOI:10.1111/cbdd.13373

In the previous studies of our laboratory, the thiophene[3,2-d]pyrimidine was identified as a promising scaffold for seeking highly potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, we designed, synthesized, and biologi

Full text of DOI:10.1111/cbdd.13373

DR ZHAO WANG (Orcid ID : 0000-0001-5431-1785)
DR PENG ZHAN (Orcid ID : 0000-0002-9675-6026)
PROFESSOR XINYONG LIU (Orcid ID : 0000-0002-5833-3807)
Article type
: Research Article
Design, Synthesis, and Antiviral Evaluation of Novel Hydrazone-substituted
Thiophene[3,2-d]pyrimidine Derivatives as Potent HIV-1 Inhibitors
Zhao Wang,† Dongwei Kang,† Meng Chen, | Gaochan Wu,† Da Feng,† Tong Zhao,† Zhongxia
Zhou,† Zhipeng Huo,† Lanlan Jing,† Xiaofang Zuo,† Dirk Daelemans,§ Erik De Clercq,§
Christophe Pannecouque, § Peng Zhan,†,* and Xinyong Liu†,*
† Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
250012 Jinan, Shandong, PR China
| Shandong Center for Disease Control and Prevention, 250014 Jinan, Shandong, PR China
§ Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy,
K.U.Leuven, Herestraat 49 Postbus 1043 (09.A097), B-3000 Leuven, Belgium.
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13373
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ABSTRACT
In the previous studies of our lab, the thiophene[3,2-d]pyrimidine was identified as a
promising scaffold for seeking highly potent HIV-1 non-nucleoside reverse transcriptase
inhibitors (NNRTIs). In the present study, we designed, synthesized, and biologically
evaluated a series of thiophene[3,2-d]pyrimidine derivatives with changed linker between the
thiophenepyrimidine core and the right wing. Some of the synthesized compounds exhibited
excellent HIV-1 inhibitory potency with low (double-digit) nanomolar EC50 values. Among
them, compound 13a exhibited the most potent anti HIV-1 activity (EC50 = 21.2 nM), which
was 10-fold greater than that of NVP (EC50 = 281 nM). Moreover, 13a showed much lower
cytotoxicity (CC50 = 183 μM) and higher SI (SI = 8632) than NVP, ETV and AZT. Besides,
some physicochemical properties and water solubility were calculated or measured. The
preliminary structure-activity relationships and molecular simulation studies of these
compounds were also discussed comprehensively to provide valuable direction for further
design and optimization.
KEYWORDS: HIV-1, NNRTIs, DAPY, thiophene[3,2-d]pyrimidine, backbone-binding,
molecular simulation
INTRODUCTION
Acquired immune deficiency syndrome (AIDS), which mainly caused by human
immunodeficiency virus type-1 (HIV-1), is one of the world's most serious infectious
diseases which threaten human's health.1 HIV-1 reverse transcriptase (RT) is a key target for
anti-AIDS drug design for its well-known mechanism and well-solved three dimensional
structure.2 Non-nucleoside RT inhibitors (NNRTIs) bind to the allosteric binding site
(NNRTIs binding pocket, NNIBP) about 10 Å distantly from the polymerase active site of
RT, leading to catalytic region's conformational change and contribute to the inhibition of its
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DNA polymerase activity.1,3 Compared with other inhibitors, NNRTIs have gained an
essential place in the highly active antiretroviral therapy (HAART) regimens to treat HIV
infection, owing to their unique antiviral potency, relatively low toxicity and high
selectivity.4 So far, five NNRTIs have been approved for clinical use by US FDA, including
first-generation drugs nevirapine (1, NVP), delavirdine (2, DLV), and efavirenz (3, EFV),
and second-generation drugs etravirine (4, ETV) and rilpivirine (5, RPV) (Figure 1).5,6
However, in view of the emergence of drug-resistance and the mutation of virus (especially
K103N/Y181C), the therapeutic efficacies of the first generation of NNRTIs were
dramatically downplayed. The second-generation drugs ETV and RPV belong to the
diarylpyrimidine (DAPY) family, which comprises the most successful NNRTIs, exhibited
broad spectrum of activities against drug-resistant HIV-1 strains, but their low solubility and
poor bioavailability limit their clinical use.7,8 Therefore, there still remains an urgent need to
conduct further modifications based on the DAPY skeleton and develop novel DAPY-based
NNRTIs with improved antiviral efficacy and favorable pharmacokinetic profiles.9
Figure 1. Chemical structures of NNRTIs approved by the US FDA
According to the traditional drug design strategy, a promising NNRTI should enter the
NNIBP effectively and occupy the binding pocket maximally, therefore interacting
effectively with the amino acids around the binding site, particularly to promote a robust
network of hydrogen bonding interactions with backbone atoms of amino acid residues in the
NNIBP.10 Previous research in our lab demonstrated that the piperidine-substituted
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thiophene[3,2-d]pyrimidine is a promising scaffold of DAPY NNRTIs.11−14 It is worth noting
that the most potent compound 6 (K-5a2) exhibited a significantly improved drug resistance
profile compared to ETV. Molecular docking results showed that 6 could form considerably
more direct or water-mediated hydrogen bonding interactions between their polar groups (the
thiophene sulfur, piperidine nitrogen and solvent-exposed sulfonamide) and the main chains
of amino acid residues throughout the NNIBP.11 This extensive network of main chain
hydrogen bonds are less susceptible to side chain mutations in the pocket, which contributes
mostly to its improved drug resistance profiles. The research work on 6 further illustrates that
the backbone-binding concept is an effective strategy to overcome drug resistance.14
Therefore, we have continued to apply the backbone-binding design strategy resulting in the
design and synthesis of a variety of thiophene[3,2-d]pyrimidine derivatives with intriguing
structural features.
Figure 2. Further structure optimization of thiophene[3,2-d]pyrimidine 6
With 6 as lead compound, we conducted the further structure modification from the
following two parts: 1) replacement the NH linker connecting the central pyridine ring and
the right ring with a hydrazone linker, with the hope that the newly increased N atom could
develop additional hydrogen bonds with the main chain of Lys103; 2) benzonitrile was
introduced to the right wing according to bioisosterism strategy. For the purpose of
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developing more hydrogen bonds with the main chain of Lys103, the fluorine atom was
introduced to the C2 and C3 position of the benzonitrile. In addition, we also introduced the
cyano vinyl group in the left wing, anticipating that it could form stronger hydrophobic
interactions with highly conserved amino acid residues (Figure 2). Herein we report the
synthesis and biological evaluation of these novel compounds. Moreover, preliminary SARs
and molecular simulation studies were also discussed to gain further insights into this series
of analogues.
CHEMISTRY
The synthetic routes of the newly designed compounds are outlined in Schemes 1-3.15,16
In Scheme 1, the commercially available 2,4-dichlorothiophene[3,2-d]pyrimidine (7) was
selected as starting material, which engaged in nucleophilic substitution reaction with
3,5-dimethyl-4-hydroxybenzonitrile in the presence of potassium carbonate to afford the
intermediate 8. Then the intermediate 9 was obtained via the hydrazinolysis of 8 in the
presence of hydrazine hydrate. 9 was reacted with
1-(tert-butoxycarbonyl)-4-formylpiperidine to afford the intermediate 10, which was directly
deprotected with trifluoroacetic acid to yield 11. Subsequently, treatment of the intermediate
11 with various substituted aromatic groups in the presence of potassium carbonate yielded
the target compounds 12a-m. The target compounds 13a-c were prepared by treating 9 with
4-cyanobenzaldehyde or fluorine substituted 4-cyanobenzaldehyde in ethanol.
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Scheme 1. Reagents and Conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (ii) 80%
hydrazine hydrate, ethanol, reflux; (iii) 1-(tert-Butoxycarbonyl)-4-formylpiperidine, ethanol, reflux; (iv) TFA,
DCM, r.t.; (v) substituted benzyl chloride (or bromine) or 2-picolyl chloride hydrochloride or 4-picolyl chloride
hydrochloride, DMF, K2CO3, r.t.
Scheme 2. Reagents and Conditions: (i) substituted 4-cyanobenzaldehyde or 4-cyanobenzaldehyde, ethanol,
reflux.
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Scheme 3. Reagents and Conditions: (i) 3,5-dimethyl-4-hydroxybenzaldehyde, DMF, K2CO3, r.t.; (ii)
(EtO)2P(O)CH2CN, t-BuOK, THF, 0°C to r.t.; (iii) 80% hydrazine hydrate, ethanol, reflux; (iv)
4-cyanobenzaldehyde, ethanol, reflux.
Similarly, treatment of 7 with 3,5-dimethyl-4-hydroxybenzaldehyde yielded 14, which
was reacted with diethyl cyanomethylphosphonate to give intermediate 15 via the
Wittig-Horner reaction. Then the target compound 17 was obtained via hydrazinolysis and
nucleophilic addition reaction, successively.
All the novel synthesized compounds were completely characterized by the spectral
means including proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic
resonance (13C NMR), and mass spectrometry (MS). The purity of all final compounds was
>95% as determined by analytical HPLC.
RESULTS AND DISCUSSION
The target compounds were evaluated for their antiviral potency in MT-4 cell cultures
infected with WT HIV-1 strain (IIIB) and double mutant strain K103N+Y181C (RES056).
Nevirapine (NVP), etravirine (ETV) and zidovudine (AZT) were selected as reference drugs.
The biological evaluation results were interpreted as EC50 (anti-HIV potency), CC50
(cytotoxicity), and SI (selectivity index, CC50/EC50 ratio), which was summarized in Table 1.
Table 1. Activity and cytotoxicity against HIV-1 (IIIB and RES056) strains in MT-4 Cells
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EC50 (μM)
SI
Compd
R
CC50 (μM)
Ar
IIIB
>3.60
>4.62
>4.65
>5.52
>13.4
>3.82
RES056
IIIB
<1
<1
<1
<1
<1
<1
RES056
12a
12b
12c
12d
12e
12f
-
-
4-SO2NH2-Ph
4-SO2CH3-Ph
4-CONH2-Ph
pyridine-4-yl
4-NO2-Ph
-
-
-
-
-
-
3.60±0.510
4.62±0.271
4.65±0.086
5.52±0.340
13.4±11.8
-
-
-
-
-
-
-
-
-
-
4-SO2NHCH3-Ph
3.82±0.329
>4.03
>4.68
-
4.03±0.470
<1
-
12g
-
12h
12i
12j
3-CONH2-Ph
4-CN-Ph
4-F-Ph
-
>23.6
-
4.68±0.147
23.6±6.54
5.48±2.50
<1
4
-
<1
-
-
-
-
5.85±2.15
>5.48
<1
>14.5
>4.42
-
-
14.5±8.43
<1
<1
-
-
12k
12l
-
-
4.42±0.701
pyridine-2-yl
>4.18
-
4.18±0.951
183±0.706
182±9.61
215±1.37
96.6±83.4
>15.0
<1
8632
4407
8366
1023
>53
-
X1
12m
13a
-
H
2-F
3-F
-
-
-
-
-
0.0212±0.005
0.0413±0.011
0.0257±0.002
0.0944±0.055
0.281±0.038
0.004±0.0003
0.007±0.001
>58.9
>56.5
X1
13b
13c
>56.5
X1
>96.6
<1
17
NVP
ETV
AZT
-
>15.0
X1
-
0.035±0.013
0.017±0.006
2.16±0.107
>7.48
540
62
-
>1069
>440
a EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by
the MTT method.
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b CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
c SI: selectivity index, the ratio of CC50/EC50
.
As depicted in the Table 1, some of the designed compounds exhibited prominent
potency against the WT HIV-1 strain with double-digit nanomolar EC50 values ranging from
21.2 nM to 94.4 nM. Especially, 13a (EC50 = 0.0212 μM) and 13c (EC50 = 0.0257 μM)
exhibited the most potent activity, being superior to that of NVP (EC50 = 0.281 μM) and
comparable to that of AZT (EC50 = 0.007 μM) and ETV (EC50 = 0.004 μM). Besides, 13a
(CC50 = 183 μM, SI = 8632) and 13c (CC50 = 215 μM, SI = 8366) displayed much lower
cytotoxicity and higher selectivity index than ETV (CC50 = 2.16 μM, SI = 540). However, all
of the synthesized compounds were inactive to the double mutant strain RES056.
Preliminary structure-activity relationships (SARs) can be concluded from the biological
results. First of all, we paid attention to the compounds 12a-m. We held the view that the
newly introduced hydrazone linker could form additional hydrogen bond with Lys103 of the
NNIBP. However, almost all the compounds exhibited no activity toward the HIV-1 WT and
RES056 at the tested concentration. Only, 12i demonstrated moderate potency with EC50
value of 5.85 μM. We supposed that the hydrazone linker might change the binding mode of
the inhibitor with NNIBP and resulted in the loss of the hydrogen bonding force with Lys103,
Lys104 and Val106, indicating that the length of linker have great influence on the antiviral
activity.
Furthermore, the replacement of the aminopiperidine with benzene ring resulted in a
significantly improved antiviral activity toward WT HIV-1 strains. The activity of 13a was
10-fold more potent than the reference drug NVP. Introduction of a fluorine atom at the C2
and C3 position of the benzonitrile of 13a yielded 13b and 13c, with an EC50 values of 0.0413
and 0.0257 μM respectively, which displayed no obvious advantages over 13a. In addition,
the replacement of the cyano group in the left wing of 13a with the cyano vinyl group gave
compound 17 with slightly reduced activity and increased cytotoxicity (EC50 = 0.0944 μM,
CC50 = 96.6 μM).
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MOLECULAR MODELING STUDIES
In order to obtain deeper insight into the binding mode of the novel designed compounds
in the NNIBP and prove the rationality of our explanation toward the activity, we conducted
molecular docking studies of the representative compounds 12a, 13a, 13b, and 13c with the
WT HIV-1 RT crystal structure by utilizing the SurflexeDock SYBYL-X 2.0 software. The
co-crystal structure of the reverse transcriptase was selected from the co-crystallization of the
small molecule ligand containing aminopiperidine (PDB code: 3M8Q17) or p-cyanoaniline
(PDB code: 3MEC18) on the right wing. Our docking results were visualized by PyMOL. And
the docking protocol was illustrated in the computational section.
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Figure 3. (a) Predicted binding modes of 13a with the HIV-1 WT RT crystal structure (PDB: 3MEC); (b)
Superimposition of 13b (yellow) and 13c (magenta) with HIV-1 WT RT (PDB: 3MEC); (c) Superimposition of
12a (orange) and 6 (white) with HIV-1 WT RT (PDB: 3M8Q). Hydrogen bonds are shown as dashed lines
(yellow). Nonpolar hydrogen atoms are hid for clarity.
It is noteworthy that the most potent compound 13a showed classical horseshoe
conformational in the NNIBP similar to other familiar NNRTIs (Figure 3a). 13a has the
following characteristic interaction modes: (i) the benzene ring of left wing is stabilized by
π-π stacking interactions with aromatic side chains of amino acid residues Tyr181, Tyr188,
Phe227 and Trp229; (ii) the thiophene ring points to the NNIBP entrance channel containing
Glu138 and Val179 and the sulfur atom forms an electrostatic interaction with Val179. (iii)
Remarkably, the N atom of the pyrimidine ring and hydrazone linker displays double
hydrogen bonding with the main-chain backbone of Lys101, which plays a crucial role in
maintaining effective antiviral activity. As shown in Figure 3b, the C3-substituted fluorine
atom of 13c displays direct hydrogen bonding with the conserved amino acid residue Tyr318,
which accounts for the more active potency of 13c than 13b. However, the hydrogen-bonding
interaction between the N atom of pyrimidine ring and Lys101 disappeared, which may
provide a rational explanation of the slightly reduced activity of 13b and 13c compared to
13a.
Furthermore, we chose the representative compound 12a to dock into the WT RT
binding pocket (Figure 3c). Compared with the lead compound 6, 12a displays only one
hydrogen bonding with Lys103, while 6 forms extensive network of main chain hydrogen
bonds with Lys103, Lys104 and Val106. The lack of these main chain hydrogen bonds
provides a rational explanation of the inactive potency of 12a-m.
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PHYSICOCHEMICAL PROPERTIES AND MEASURED WATER
SOLUBILITY
Because water solubility and hydrophilicity/lipophilicity are the important factors to
evaluate the newly designed compounds, we used free online software
(http://www.molinspiration.com/) and HPLC to investigate the physicochemical properties
and solubility of the potent compounds 13a, 13b, 13c and 17. As shown in the Table 2, the
results of physicochemical properties indicated that these four compounds were all
conformed well to the Lipinski's rule of five. Furthermore, we also investigated the water
solubility and LogP of 13a-c and 17 by using HPLC. The experimental results demonstrated
that 13a-c have well solubility and favorable LogP value, which can be considered as the
promising lead compounds for further optimization.
Table 2. Physicochemical properties and water solubility of 13a, 13b, 13c and 17
pH = 7.0 pH = 7.4 pH = 2.0
(μg/mL)c (μg/mL)d (μg/mL)d
nOHNH
compd
EC50(nM)
MW nONa
nrotba TPSAa LogPb
a
Accepted
range
-
<500
<10
<5
≤10
<140
<5
-
-
-
21.2
41.3
25.7
94.4
424.11
442.10
442.10
450.13
7
7
7
7
1
1
1
1
5
5
5
6
106.99
106.99
106.99
106.99
1.49
1.53
1.53
1.42
3.91
<<1
0.39
<<1
1.10
3.36
9.76
2.08
5.94
9.36
12.97
<<1
13a
13b
13c
17
a Using free on-line software (http://www.molinspiration.com/); MW = molecular weight; nON = no. of hydrogen bond acceptors; nOHNH = no. of
hydrogen bond donors; nrotb = no. of rotatable bonds; TPSA = topological polar surface area.
b Values were measured at pH 7 by HPLC method in duplicate; LogP = logarithm of partition coefficient.
c Measured in water at pH 7 by HPLC method in duplicate.
d Measured in phosphate buffer by HPLC method in duplicate.
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CONCLUSION
To sum up, taking our previously reported K-5a2 (compound 6) as lead compound, a
novel series of hydrazone-substituted thiophene [3,2-d] pyrimidine derivatives was designed,
synthesized and biologically evaluated. Encouragingly, four compounds exhibited remarkable
inhibitory activity against WT HIV-1 strain with EC50 values ranging from 21.2 nM to 94.4
nM. Compound 13a proved to be the most promising inhibitor with low nanomolar EC50
values of 21.2 nM. Moreover, 13a showed much lower cytotoxicity (CC50 = 183 μM) and
higher SI (SI = 8632) than NVP, ETV and AZT. The detailed molecular modeling studies
revealed the interactions between the inhibitors and WT HIV-1 RT and rationalized the
preliminary SARs. Furthermore, some physicochemical properties were predicted and water
solubility were also measured. The current results can provide a useful insight for the
development of HIV-1 NNRTIs with improved antiviral activity. Further optimizations of
this series of compounds are currently ongoing and will be reported in due course.
EXPERIMENTAL SECTION
Chemistry. All melting points were determined on a micromelting point apparatus and
are uncorrected. 1H NMR and 13C NMR spectra were obtained on a Bruker AV-400
spectrometer using DMSO-d6 as the solvent and tetramethylsilane (TMS) as the internal
standard. Chemical shifts are given inδunits (ppm) and J values were reported in hertz (Hz).
Mass spectrometry was recorded on an LC Autosampler device of Standard G1313A
instrument. TLC was completed on Silica Gel GF254 for TLC and spots were visualized by
irradiation with UV light (λ = 254 nm). Flash column chromatography was performed on
columns packed with Silica Gel 60 (200-300 mesh). Solvents were of reagent grade and were
purified and dried by standard methods if necessary. Rotary evaporator was used in the
concentration of the reaction solutions under reduced pressure. Sample purity was analyzed
on a Shimadzu SPD-20A/20AV HPLC system with a Inertsil ODS-SP, 5 μm C18 column
(150 mm × 4.6 mm). Purity of all tested compounds was >95%.
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4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (8). A mixture of
4-hydroxy-3,5-dimethylbenzonitrile (0.79 g, 5.36 mmol) with potassium carbonate (0.81 g,
5.85 mmol) in DMF (15 mL) was reacted in room temperature for 0.5 h; then added
2,4-dichlorothieno[3,2-d]pyrimidine (1.0 g, 4.88 mmol) to it. The reaction mixture continued
to react for 2 h (monitored by TLC), then the mixture was poured into ice water and left to
stand for 1 h. The precipitated white solid was collected by filtration, washed with saturated
salt solution, and recrystallized in DMF/H2O to obtain the desired product 8 as a white solid
in 88.3% yield, mp 258-260 °C. ESI-MS: m/z 316.3 (M + 1). C15H10ClN3OS (315.02).
4-((2-hydrazinylthieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (9). A
solution of 8 (1.0 g, 3.17 mmol) in 50 mL of ethanol, and then added 3 mL 80% hydrazine
hydrate to it. The mixture was refluxed at 90 °C for 5 h (monitored by TLC). The solution
was cooled to room temperature, and 50 mL of ice water was added to it. The resulting
precipitate was collected by filtration and dried to give crude product, which was purified by
flash column chromatography and recrystallized from ethyl acetate/petroleum ether to afford
the intermediate 9 as a white solid in 75.8% yield, mp 219-221 °C. 1H NMR (400 MHz,
DMSO-d6): δ 8.24 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 8.06 (s, 1H, NH), 7.73 (s, 2H,
C3,C5-Ph'-H), 7.32 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 4.09 (s, 2H, NH2), 2.12 (s,
6H, CH3×2). 13C NMR (100 MHz, DMSO-d6): δ 165.50, 163.60, 162.35, 153.24, 137.00,
133.21, 133.06, 123.70, 119.01, 109.16, 105.90, 16.25. ESI-MS: m/z 312.1 (M + 1).
C15H13N5OS (311.08).
(E)-3,5-dimethyl-4-((2-(2-(piperidin-4-ylmethylene)hydrazinyl)thieno[3,2-d]pyrimidin-4
-yl)oxy)benzonitrile (11). A solution of 9 (1.0 g, 3.21 mmol) in 50 mL of ethanol, and then
tert-butyl 4-formylpiperidine-1-carboxylate (0.82 g, 3.84 mmol) was added to it. The mixture
was refluxed at 90 °C for 4 h (monitored by TLC). The solution was cooled to room
temperature, and the resulting precipitate was collected by filtration and dried to give crude
product 10, which was used directly in the next step without further purification. Then a
solution of 10 (1.0 g, 1.97 mmol) in 5 mL of DCM was added trifluoroacetic acid (TFA)
(2.22 mL, 30 mmol) at room temperature, and the solution was stirred for 3 h (monitored by
TLC). Then the reaction mixture was alkalized to pH 9 with saturated sodium bicarbonate
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solution and washed with 20 mL of saturated salt water. The aqueous phase was extracted
with DCM (3 × 5 mL). The combined organic phase was dried, filtered, and concentrated
under vacuum to give 11 as a white solid in 64.5% yield, mp 274-276 °C. 1H NMR (400
MHz, DMSO-d6): δ 10.63 (s, 1H, NH), 8.96 (s, 2H), 8.30 (d, J = 5.3 Hz, 1H,
C6-thienopyrimidine-H), 7.75 (s, 2H, C3,C5-Ph'-H), 7.35 (dd, J = 33.6, 4.9 Hz, 2H), 3.31–2.81
(m, 4H), 2.13 (s, 6H, CH3×2), 1.94–1.53 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 165.76,
158.52, 153.26, 147.24, 137.49, 133.27, 133.10, 124.09, 119.00, 109.22, 107.48, 42.80,
36.09, 26.19, 16.28. ESI-MS: m/z 407.3 (M + 1). C21H22N6OS (406.16).
General Procedure for the Preparation of Final Compounds 12a-m. Compound 11 was
dissolved in 5 mL of anhydrous DMF, followed by addition of the anhydrous K2CO3 (1.2
equiv) and appropriate substituted benzyl chloride (bromide) or picolyl chloride
hydrochloride (1.1 equiv). The reaction mixture was stirred at room temperature for 6 h
(monitored by TLC). The solvent was removed under vacuum and the residue was taken up in
20 mL of water and extracted with ethyl acetate (3 × 10 mL). Then the organic phase was
washed with saturated sodium chloride (2 × 10 mL), dried, filtered, and purified by flash
column chromatography. Recrystallized from ethyl acetate/petroleum ether to afford the
target compounds 12a-m.
(E)-4-((4-((2-(4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hydrazono)
methyl)piperidin-1-yl)methyl)benzenesulfonamide (12a). Recrystallized from ethyl
acetate/petroleum ether as a white solid, 53.1% yield, mp 193-195 °C. 1H NMR (400 MHz,
DMSO-d6): δ 10.44 (s, 1H, NH), 8.21 (d, J = 5.4 Hz, 1H, N=CH), 7.80–7.60 (m, 4H), 7.42 (d,
J = 8.0 Hz, 2H, C3,C5-Ph''-H), 7.31 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.24 (d, J =
9.0 Hz, 3H), 3.45 (s, 2H, N-CH2), 2.70 (d, J = 11.5 Hz, 2H), 2.06 (s, 7H), 2.00–1.88 (m, 2H),
1.65–1.53 (m, 2H), 1.42―1.29 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 165.81, 162.52,
158.56, 153.27, 149.35, 143.36, 143.11, 137.36, 133.28, 133.09, 129.41, 126.05, 124.07,
109.23, 107.28, 62.15, 52.98 , 38.72, 29.90, 16.27. ESI-MS: m/z 576.3 (M + 1).
C28H29N7O3S2 (575.18). HPLC purity: 96.67%.
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(E)-3,5-dimethyl-4-((2-(2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)methylene)hydraz
inyl)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12b). Recrystallized from ethyl
acetate/petroleum ether as a white solid, 61.2% yield, mp 216-218 °C. 1H NMR (400 MHz,
DMSO-d6): δ 10.45 (s, 1H, NH), 8.21 (d, J = 5.4 Hz, 1H, N=CH), 7.81 (d, J = 8.0 Hz, 2H,
C3,C5-Ph''-H), 7.66 (s, 2H, C3,C5-Ph'-H), 7.51 (d, J = 8.1 Hz, 2H, C2,C6-Ph''-H), 7.31 (d, J =
5.4 Hz, 1H, C6-thienopyrimidine-H), 7.24 (d, J = 5.3 Hz, 1H, C7-thienopyrimidine-H), 3.49
(s, 2H, N-CH2), 3.14 (s, 3H, SO2CH3), 2.70 (d, J = 11.5 Hz, 2H), 2.24–1.84 (m, 9H), 1.60
(dd, J = 13.5, 3.8 Hz, 2H), 1.47–1.27 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 158.58,
153.27, 149.26, 145.53, 139.76, 137.36, 133.28, 133.08, 129.73, 127.39, 124.07, 118.97,
109.21, 62.09, 53.03, 44.07, 38.68, 29.88, 16.28. ESI-MS: m/z 575.5 (M + 1). C29H30N6O3S2
(574.18). HPLC purity: 99.15%.
(E)-4-((4-((2-(4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hydrazono)
methyl)piperidin-1-yl)methyl)benzamide (12c). Recrystallized from ethyl acetate/petroleum
ether as a white solid, 54.5% yield, mp 247-249 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.51
(s, 1H, NH), 8.28 (d, J = 5.3 Hz, 1H, N=CH), 7.93 (s, 1H), 7.83 (d, J = 7.9 Hz, 2H,
C3,C5-Ph''-H), 7.74 (s, 2H, CONH2), 7.46–7.21 (m, 5H), 3.50 (s, 2H, N-CH2), 2.77 (d, J =
11.3 Hz, 2H), 2.13 (s, 7H), 2.06–1.94 (m, 2H), 1.66 (dd, J = 13.2, 3.8 Hz, 2H), 1.51–1.34 (m,
2H). 13C NMR (100 MHz, DMSO-d6): δ 168.24, 165.81, 162.52, 158.57, 153.27, 149.37,
142.51, 137.34, 133.37, 133.28, 133.08, 128.83, 127.87, 124.07, 109.23, 62.42, 53.00, 38.77,
29.91, 16.27. ESI-MS: m/z 540.6 (M + 1). C29H29N7O2S (539.21). HPLC purity: 98.71%.
(E)-3,5-dimethyl-4-((2-(2-((1-(pyridin-4-ylmethyl)piperidin-4-yl)methylene)hydrazinyl)t
hieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12d). Recrystallized from ethyl
acetate/petroleum ether as a white solid, 43.8% yield, mp 214-216 °C. 1H NMR (400 MHz,
DMSO-d6): δ 10.53 (s, 1H, NH), 8.58–8.44 (m, 2H), 8.29 (d, J = 5.3 Hz, 1H,
C6-thienopyrimidine-H), 7.74 (s, 2H, C3,C5-Ph'-H), 7.38 (d, J = 5.4 Hz, 1H,
C7-thienopyrimidine-H), 7.35–7.28 (m, 3H), 3.49 (s, 2H, N-CH2), 2.77 (d, J = 11.4 Hz, 2H),
2.13 (s, 8H), 1.67 (dd, J = 13.5, 3.8 Hz, 2H), 1.54–1.30 (m, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 165.80, 162.52, 158.57, 153.27, 149.96, 149.25, 148.28, 137.36, 133.28,
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133.08, 124.08, 118.98, 109.22, 61.43, 53.04, 38.64, 29.85, 16.28. ESI-MS: m/z 499.4 (M +
1). C27H27N7OS (497.20). HPLC purity: 99.91%.
(E)-3,5-dimethyl-4-((2-(2-((1-(4-nitrobenzyl)piperidin-4-yl)methylene)hydrazinyl)thieno
[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12e). Recrystallized from ethyl acetate/petroleum
ether as a white solid, 46.9% yield, mp 220-222 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.53
(s, 1H, NH), 8.28 (d, J = 5.3 Hz, 1H, N=CH), 8.24–8.14 (m, 2H, C3,C5-Ph''-H), 7.73 (s, 2H,
C3,C5-Ph'-H), 7.59 (d, J = 8.4 Hz, 2H, C2,C6-Ph''-H), 7.38 (d, J = 5.4 Hz, 1H,
C6-thienopyrimidine-H), 7.31 (d, J = 5.3 Hz, 1H, C7-thienopyrimidine-H), 3.59 (s, 2H,
N-CH2), 2.77 (dt, J = 11.4, 3.4 Hz, 2H), 2.30–1.90 (m, 9H), 1.67 (dd, J = 13.1, 3.8 Hz, 2H),
1.53–1.33 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 165.80, 162.52, 158.58, 153.27,
149.22, 147.65, 146.94, 137.37, 133.27, 133.08, 129.99, 124.07, 123.82, 118.97, 109.21,
61.83, 53.02, 38.63, 29.87, 16.28. ESI-MS: m/z 542.5 (M + 1). C28H27N7O3S (541.19). HPLC
purity: 98.91%.
(E)-4-((4-((2-(4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hydrazono)
methyl)piperidin-1-yl)methyl)-N-methylbenzenesulfonamide (12f). Recrystallized from ethyl
acetate/petroleum ether as a white solid, 56.5% yield, mp 143-145 °C. 1H NMR (400 MHz,
DMSO-d6): δ 10.44 (s, 1H, NH), 8.21 (d, J = 5.3 Hz, 1H, N=CH), 7.66 (t, J = 4.1 Hz, 4H),
7.46 (d, J = 8.0 Hz, 2H, C2,C6-Ph''-H), 7.32 (dd, J = 11.7, 5.2 Hz, 2H), 7.24 (d, J = 5.4 Hz,
1H, C7-thienopyrimidine-H), 3.47 (s, 2H, N-CH2), 2.80–2.62 (m, 2H), 2.34 (d, J = 5.0 Hz,
3H, NHCH3), 2.06 (s, 7H), 1.95 (t, J = 11.1 Hz, 2H), 1.60 (dd, J = 13.2, 3.8 Hz, 2H),
1.45–1.27 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 165.81, 158.58, 153.27, 149.30,
144.13, 138.15, 137.35, 133.28, 133.08, 129.61, 127.11, 124.07, 118.97, 109.23, 62.13,
53.02, 38.69, 29.88, 29.15, 16.27. ESI-MS: m/z 590.5 (M + 1). C29H31N7O3S2 (589.19).
HPLC purity: 98.95%.
(E)-4-((4-((2-(4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hydrazono)
methyl)piperidin-1-yl)methyl)-N-cyclopropylbenzenesulfonamide (12g). Recrystallized from
ethyl acetate/petroleum ether as a white solid, 50.8% yield, mp 129-131 °C. 1H NMR (400
MHz, DMSO-d6): δ 10.44 (s, 1H, NH), 8.21 (d, J = 5.3 Hz, 1H, N=CH), 7.80 (d, J = 2.6 Hz,
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1H, SO2NH), 7.75–7.60 (m, 4H), 7.46 (d, J = 8.0 Hz, 2H, C2,C6-Ph''-H), 7.31 (d, J = 5.3 Hz,
1H, C6-thienopyrimidine-H), 7.24 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 3.48 (s, 2H,
N-CH2), 2.86–2.61 (m, 2H), 2.26–1.85 (m, 10H), 1.60 (dd, J = 12.9, 3.9 Hz, 2H), 1.46–1.25
(m, 2H), 0.51–0.23 (m, 4H, CH2CH2). 13C NMR (100 MHz, DMSO-d6): δ 165.81, 153.27,
149.30, 144.15, 139.15, 137.35, 133.28, 133.08, 129.56, 127.23, 124.07, 118.97, 109.22,
62.13, 53.00, 38.70, 29.88, 24.56, 16.27, 5.59. ESI-MS: m/z 616.5 (M + 1). C31H33N7O3S2
(615.21). HPLC purity: 98.57%.
(E)-3-((4-((2-(4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hydrazono)
methyl)piperidin-1-yl)methyl)benzamide (12h). Recrystallized from ethyl acetate/petroleum
ether as a white solid, 51.6% yield, mp 136-138 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.44
(s, 1H, NH), 8.21 (d, J = 5.3 Hz, 1H, N=CH), 7.91 (s, 1H), 7.83–7.59 (m, 4H), 7.46–7.17 (m,
5H), 3.43 (s, 2H, N-CH2), 2.87–2.63 (m, 2H), 2.06 (s, 8H), 1.69–1.51 (m, 2H), 1.34 (d, J =
18.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 168.41, 165.81, 162.52, 158.57, 153.27,
149.37, 137.33, 134.66, 133.27, 133.08, 132.07, 128.48, 126.47, 124.08, 118.97, 109.22,
107.27, 62.64, 52.94, 38.78, 29.85, 16.27. ESI-MS: m/z 540.2 (M + 1). C29H29N7O2S
(539.21). HPLC purity: 97.02%.
(E)-4-((2-(2-((1-(4-cyanobenzyl)piperidin-4-yl)methylene)hydrazinyl)thieno[3,2-d]pyrim
idin-4-yl)oxy)-3,5-dimethylbenzonitrile (12i). Recrystallized from ethyl acetate/petroleum
ether as a white solid, 46.2% yield, mp 248-250 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.52
(s, 1H, NH), 8.29 (d, J = 5.4 Hz, 1H, N=CH), 7.80 (d, J = 8.0 Hz, 2H, C3,C5-Ph''-H), 7.74 (s,
2H, C3,C5-Ph'-H), 7.52 (d, J = 7.9 Hz, 2H, C2,C6-Ph''-H), 7.38 (d, J = 5.4 Hz, 1H,
C6-thienopyrimidine-H), 7.31 (d, J = 5.3 Hz, 1H, C7-thienopyrimidine-H), 3.55 (s, 2H,
N-CH2), 2.89 (s, 1H), 2.75 (d, J = 15.5 Hz, 3H), 2.13 (s, 6H, CH3×2), 1.74–1.57 (m, 2H),
1.55–1.28 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 165.80, 162.52, 158.57, 153.27,
137.37, 133.28, 133.11, 133.08, 132.61, 130.18, 129.88, 124.07, 119.42, 118.98, 109.21,
107.26, 52.98, 29.86, 16.28. ESI-MS: m/z 522.4 (M + 1). C29H27N7OS (521.20). HPLC
purity: 97.07%.
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(E)-4-((2-(2-((1-(4-fluorobenzyl)piperidin-4-yl)methylene)hydrazinyl)thieno[3,2-d]pyri
midin-4-yl)oxy)-3,5-dimethylbenzonitrile (12j). Recrystallized from ethyl acetate/petroleum
ether as a white solid, 58.7% yield, mp 227-229 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.50
(s, 1H, NH), 8.28 (d, J = 5.4 Hz, 1H, N=CH), 7.73 (s, 2H, C3,C5-Ph'-H), 7.44–7.24 (m, 4H),
7.13 (t, J = 8.7 Hz, 2H), 3.43 (s, 2H, N-CH2), 2.81–2.71 (m, 2H), 2.12 (s, 6H, CH3×2), 1.98
(d, J = 6.9 Hz, 3H), 1.71–1.59 (m, 2H), 1.47–1.32 (m, 2H). 13C NMR (100 MHz, DMSO-d6):
δ 165.81, 162.52, 158.58, 153.27, 149.35, 137.35, 133.28, 133.08, 130.99, 130.91, 124.07,
118.97, 115.40, 115.19, 109.22, 107.26, 61.91, 52.85, 38.80, 29.89, 16.27, 14.56. ESI-MS:
m/z 515.4 (M + 1). C28H27FN6OS (514.20). HPLC purity: 95.19%.
(E)-3,5-dimethyl-4-((2-(2-((1-(4-(morpholinosulfonyl)benzyl)piperidin-4-yl)methylene)hydraz
inyl)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12k). Recrystallized from ethyl
acetate/petroleum ether as a white solid, 63.1% yield, mp 139-141 °C. 1H NMR (400 MHz,
DMSO-d6): δ 10.45 (s, 1H, NH), 8.21 (d, J = 5.3 Hz, 1H, N=CH), 7.64 (d, J = 18.2 Hz, 4H),
7.53 (d, J = 8.0 Hz, 2H, C2,C6-Ph''-H), 7.31 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.25
(d, J = 5.3 Hz, 1H, C7-thienopyrimidine-H), 3.71–3.40 (m, 6H), 2.91–2.63 (m, 7H), 2.06 (s,
8H), 1.60 (d, J = 11.2 Hz, 2H), 1.49–1.21 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ
165.81, 158.58, 153.27, 137.36, 133.28, 133.07, 129.76, 128.15, 124.06, 109.22, 65.72,
62.06, 53.04, 46.37, 38.68, 29.86, 16.27. ESI-MS: m/z 646.3 (M + 1). C32H35N7O4S2
(645.22). HPLC purity: 96.91%.
(E)-4-((4-((2-(4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hydrazono)
methyl)piperidin-1-yl)methyl)-N-(tetrahydro-2H-pyran-4-yl)benzenesulfonamide (12l).
Recrystallized from ethyl acetate/petroleum ether as a white solid, 56.2% yield, mp 140-142
°C. 1H NMR (400 MHz, DMSO-d6): δ 10.44 (s, 1H, NH), 8.21 (d, J = 5.4 Hz, 1H, N=CH),
7.80–7.60 (m, 5H), 7.43 (d, J = 8.0 Hz, 2H, C2,C6-Ph''-H), 7.31 (d, J = 5.4 Hz, 1H,
C6-thienopyrimidine-H), 7.24 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 3.75–3.56 (m,
2H), 3.47 (s, 2H), 3.14 (td, J = 11.4, 2.4 Hz, 3H), 2.70 (d, J = 10.9 Hz, 2H), 2.06 (s, 6H,
CH3×2), 1.95 (t, J = 11.0 Hz, 2H), 1.68–1.53 (m, 2H), 1.50–1.17 (m, 7H). 13C NMR (100
MHz, DMSO-d6): δ 165.81, 162.52, 158.58, 153.27, 149.31, 143.92, 141.00, 137.35, 133.28,
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133.08, 129.60, 126.71, 124.07, 109.22, 65.97, 62.09, 53.01, 49.68, 39.65, 38.70, 33.75,
29.88, 16.27. ESI-MS: m/z 661.3 (M + 1). C33H37N7O4S2 (659.23). HPLC purity: 99.79%.
(E)-3,5-dimethyl-4-((2-(2-((1-(pyridin-2-ylmethyl)piperidin-4-yl)methylene)hydrazinyl)t
hieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12m). Recrystallized from ethyl
acetate/petroleum ether as a white solid, 49.5% yield, mp 116-118 °C. 1H NMR (400 MHz,
DMSO-d6): δ 10.45 (s, 1H, NH), 8.41 (dd, J = 5.0, 1.7 Hz, 1H), 8.21 (d, J = 5.4 Hz, 1H,
N=CH), 7.78–7.54 (m, 3H), 7.37 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 5.3 Hz, 1H,
C6-thienopyrimidine-H), 7.24 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 7.21–7.14 (m,
1H), 3.51 (s, 2H), 2.74 (d, J = 11.2 Hz, 2H), 2.06 (s, 9H), 1.60 (dd, J = 13.4, 3.8 Hz, 2H),
1.46–1.20 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 165.81, 158.58, 153.27, 149.31,
149.18, 137.35, 136.92, 133.28, 133.07, 124.07, 123.01, 122.52, 118.97, 109.22, 64.59,
53.17, 38.65, 29.90, 16.27. ESI-MS: m/z 498.6 (M + 1). C27H27N7OS (497.20). HPLC purity:
95.10%.
4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzaldehyde (14). A mixture
of 4-hydroxy-3,5-dimethylbenzaldehyde (0.88 g, 5.85 mmol) and potassium carbonate (1.35
g, 9.75 mmol) in 15 mL of DMF was stirred at 25 °C for 15 min, and then added
2,4-dichlorothiopheno[3,2-d]pyrimidine (1.0 g, 4.88 mmol) to it. The mixture was stirred for
another 1.5 h (monitored by TLC), then poured into 30 mL of ice water and left to stand for 1
h. The precipitated white solid was collected by filtration, washed with cold water, and
recrystallized from DMF/H2O to provide 14 as a white solid in 91.3% yield, mp 285-287 °C.
ESI-MS: m/z 319.1 (M + 1). C15H11ClN2O2S (318.02).
(E)-3-(4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-
dimethylphenyl)acrylonitrile (15). A mixture of (EtO)2P(O)CH2CN (0.67 g, 3.76 mmol) and
t-BuOK (0.71 g, 6.27 mmol) in 10 mL of THF was stirred for 1 h at 0 °C, and then a solution
of 14 (1.0 g, 3.14 mmol) in 10 mL of THF was slowly added to it. The mixture was stirred for
another 1 h (monitored by TLC). The mixture was poured into 20 mL of ice water, and the
precipitate was collected, washed with cold water, and dried to afford 15 as a white solid in
85.7% yield, mp 225-227 °C. ESI-MS: m/z 342.0 (M + 1). C17H12ClN3OS (341.04).
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(E)-3-(4-((2-hydrazinylthieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitril
e (16). The synthetic method was similar to that of 9 and starting with material 15 (0.34g, 1.0
mmol). White solid, 81.6% yield, mp 193-195 °C. ESI-MS: m/z 338.4 (M + 1). C17H15N5OS
(337.10).
General Procedure for the Preparation of Final Compounds 13a-c and 17. Compounds 9
or 16 were added to anhydrous ethanol (10 mL), followed by addition of appropriate
substituted or not substituted 4-cyanobenzaldehyde (1.1 equiv). The reaction mixture was
refluxed at 90 °C for 4 h (monitored by TLC). The solvent was removed under vacuum and
recrystallized from ethanol to afford the target compounds 13a-c and 17.
(E)-4-((2-(2-(4-cyanobenzylidene)hydrazinyl)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimet
hylbenzonitrile (13a). Recrystallized from ethanol as a white solid, 70.4% yield, mp 280-282
°C. 1H NMR (400 MHz, DMSO-d6): δ 11.42 (s, 1H, NH), 8.37 (d, J = 5.3 Hz, 1H,
C6-thienopyrimidine-H), 8.10 (s, 1H, N=CH), 7.85 (d, J = 8.2 Hz, 2H, C2,C6-Ph''-H), 7.79 (s,
2H, C3,C5-Ph'-H), 7.75 (d, J = 8.1 Hz, 2H, C3,C5-Ph''-H), 7.50 (d, J = 5.3 Hz, 1H,
C7-thienopyrimidine-H), 2.17 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6): δ 165.67,
162.70, 158.11, 153.27, 140.25, 139.75, 137.93, 133.34, 133.12, 133.04, 127.16, 124.22,
119.33, 119.01, 110.98, 109.32, 108.58, 16.32. ESI-MS: m/z 425.4 (M + 1). C23H16N6OS
(424.11). HPLC purity: 99.75%.
(E)-4-((2-(2-(4-cyano-2-fluorobenzylidene)hydrazinyl)thieno[3,2-d]pyrimidin-4-yl)oxy)-
3,5-dimethylbenzonitrile (13b). Recrystallized from ethanol as a white solid, 74.5% yield, mp
265-267 °C. 1H NMR (400 MHz, DMSO-d6): δ 11.56 (s, 1H, NH), 8.39 (d, J = 5.4 Hz, 1H,
C6-thienopyrimidine-H), 8.26 (s, 1H, N=CH), 8.08–7.85 (m, 2H), 7.79 (s, 2H, C3,C5-Ph'-H),
7.71 (dd, J = 8.1, 1.5 Hz, 1H), 7.51 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 2.16 (s, 6H,
CH3×2). 13C NMR (100 MHz, DMSO-d6): δ 165.62, 162.74, 157.89, 153.24, 138.07, 133.33,
133.13, 132.35, 129.13, 128.54, 128.44, 126.95, 124.24, 120.68, 120.43, 118.99, 118.22,
112.09, 109.36, 108.90, 16.30. ESI-MS: m/z 443.5 (M + 1). C23H15FN6OS (442.10). HPLC
purity: 99.69%.
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(E)-4-((2-(2-(4-cyano-3-fluorobenzylidene)hydrazinyl)thieno[3,2-d]pyrimidin-4-yl)oxy)-
3,5-dimethylbenzonitrile (13c). Recrystallized from ethanol as a white solid, 71.2% yield, mp
286-288 °C. 1H NMR (400 MHz, DMSO-d6): δ 11.59 (s, 1H, NH), 8.39 (d, J = 5.3 Hz, 1H,
C6-thienopyrimidine-H), 8.07 (s, 1H, N=CH), 7.92 (t, J = 7.4 Hz, 1H), 7.79 (s, 2H,
C3,C5-Ph'-H), 7.58 (d, J = 8.7 Hz, 2H, C2,C6-Ph''-H), 7.50 (d, J = 5.3 Hz, 1H,
C7-thienopyrimidine-H), 2.16 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6): δ 165.59,
162.74, 157.99, 153.24, 143.60, 143.52, 138.26, 138.06, 134.64, 133.34, 133.07, 124.20,
123.38, 123.35, 118.94, 114.65, 113.21, 113.01, 109.37, 108.84, 99.46, 39.65, 16.32.
ESI-MS: m/z 443.5 (M + 1). C23H15FN6OS (442.10). HPLC purity: 99.92%.
4-((E)-(2-(4-(4-((E)-2-cyanovinyl)-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)hy
drazono)methyl)benzonitrile (17). Recrystallized from ethanol as a white solid, 67.3% yield,
mp 262-264 °C. 1H NMR (400 MHz, DMSO-d6): δ 11.41 (s, 1H, NH), 8.34 (d, J = 5.4 Hz,
1H, C6-thienopyrimidine-H), 8.12 (s, 1H, N=CH), 7.84 (d, J = 8.2 Hz, 2H, C2,C6-Ph''-H),
7.75 (d, J = 8.2 Hz, 2H, C3,C5-Ph''-H), 7.66 (d, J = 16.8 Hz, 1H, ArCH=), 7.54 (s, 2H,
C3,C5-Ph'-H), 7.49 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.48 (d, J = 16.7 Hz, 1H,
=CHCN), 2.14 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6): δ 165.52, 163.10, 158.21,
151.57, 150.37, 140.28, 139.72, 137.73, 133.06, 132.18, 131.95, 128.71, 127.16, 124.20,
119.34, 110.96, 108.69, 97.12, 16.58. ESI-MS: m/z 451.4 (M + 1). C25H18N6OS (450.13).
HPLC purity: 99.89%.
In Vitro Anti-HIV Assay. Antiviral activity and cytotoxicity of the synthesized
compounds were evaluated against WT HIV-1 strain (IIIB) and double RT mutant strain
(K103N+Y181C) in MT-4 cell cultures utilizing the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method as described
previously.19,20 Stock solutions (10× final concentration) of test compounds were added in 25
μL volumes to two series of triplicate wells in order to allow simultaneous evaluation of the
effects on mock- and HIV-infected cells. Using a Biomek 3000 robot (Beckman Instruments,
Fullerton, CA) to dilute the test compounds to 5-fold serially (final 200 μL volume per well)
in flat-bottomed 96-well microtiter trays, including untreated control HIV-1 and
mock-infected cell samples for each sample. HIV-1 (IIIB) and double mutant HIV-1 strain
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(K103N+Y181C) stock (50 μL at 100-300 CCID50) (50% cell culture infectious dose) or
culture medium was added to either the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test compounds on uninfected cells in
order to assess the cytotoxicity of compounds. Exponentially growing MT-4 cells were
centrifuged for 5 min at 1000 rpm and the supernatant was discarded. The MT-4 cells were
resuspended at 6 × 105 cells/mL, and 50 μL volumes were transferred to the microtiter tray
wells. After 5 days of infection, the viability of mock- and HIV-infected cells was examined
spectrophotometrically by using the MTT method. The 50% cytotoxic concentration (CC50)
was defined as the concentration of compound that reduced the absorbance (OD540) of
mock-infected MT-4 cells by 50%. The 50% effective concentration (EC50) was defined as
the concentration of compound that achieved 50% protection from the cytopathic effect of the
virus in infected cells.
Molecular Simulations. Molecular simulations was performed with the Tripos
molecular modeling package Sybyl-X 2.0. All the molecules for docking were built using
standard bond lengths and angles from Sybyl-X 2.0/Base Builder. The molecules (12a, 13a,
13b, 13c and 6) were optimized by using the Tripos force field for 1000 generations two
times or more until the minimized conformers of the ligand were the same. The protein was
prepared by removing the water molecules and ligand and adding polar hydrogen atoms. The
published crystal structures of the liganded HIV-1 RT complex (PDB codes: 3MEC and
3M8Q) were retrieved from the Protein Data Bank. The flexible docking method
(Surflex-Dock) docks the ligand automatically into the ligand-binding site of the receptor by
using a protocol-based approach and an empirically-derived scoring function. The protocol is
a computational representation of a putative ligand that binds to the intended binding site and
is a unique and essential element of the docking algorithm. The scoring function in
Surflex-Dock, containing hydrophobic, polar, repulsive, entropic, and solvation terms, was
trained to estimate the dissociation constant (Kd) expressed in −log(Kd)2. Surflex-Dock
default settings were used for other parameters, such as 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
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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 Kollman all-atom approach for the protein and the
GasteigereHückel approach for the ligand. The binding interaction energy was calculated,
including van der Waals, electrostatic, and torsional energy terms defined in the Tripos force
field. The structure optimization was performed for 10,000 generations using a genetic
algorithm, and the 20-best-scoring ligand-protein complexes were kept for further analysis.
The −log(Kd)2 values of the 20-best-scoring complexes, representing the binding affinities of
ligand with RT, encompassed a wide range of functional classes (10−2-10−9). Hence, only the
highest-scoring 3D structural model of ligand-bound RT was chosen to define the binding
interaction.
Measurements of water solubility and LogP. Water solubility was measured in water
at pH 7.0 and phosphate buffer at pH 7.4 and 2.0 by using an HPLC-UV method.21
Compounds were initially dissolved in DMSO at 10 mg/mL 10 μL of this stock solution was
spiked into purified water (1 mL) with the final DMSO concentration being 1%. The mixture
was ultrasonic for 2 h at room temperature and restored at room temperature overnight. The
saturated solution was filtrated through a filter membrane (pore size = 0.22 μm) and
transferred to other Eppendorf tubes for analysis by HPLC-UV. The sample was performed in
duplicate. For quantification, a model LC-20AT HPLC-UV (SHIMADZU) system was used
with an Inertsil ® ODS-SP-C18 column (150 mm × 4.6 mm, 5 μm) and MeOH/water as
eluant. The flow rate was 1.0 mL/min, and injection volume was 20 μL. Aqueous
concentration was determined by comparison of the peak area of the saturated solution with a
standard curve plotted peak area versus known concentrations, which were prepared by
solutions in MeOH at 200, 100, 50, 25, and 12.5 μg/mL.
DMSO stock solution 1 mg/mL was prepared, and then 20 μL of this solution was added
into n-octanol (1 mL) and H2O (1 mL). The mixture was ultrasonic for 2 h at room
temperature and left to sit overnight. Each solution (~0.4 mL) was transferred from two
phases, respectively, into other eppendorf tubes for HPLC analysis. The instrument and
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conditions were the same as those for solubility determination. The P (partition coefficient)
value was calculated by the peak area ratios in n-octanol and in H2O.
AUTHOR INFORMATION
Corresponding Authors
*P.Z.: e-mail, zhanpeng1982@sdu.edu.cn; phone, 086-531-88382005.
*X.L.: e-mail, xinyongl@sdu.edu.cn; phone, 086-531-88380270.
Funding
Financial support from the National Natural Science Foundation of China (NSFC Grants
81273354, 81573347), Key Project of NSFC for International Cooperation (Grant
81420108027), Young Scholars Program of Shandong University (YSPSDU Grant
2016WLJH32), the Fundamental Research Funds of Shandong University (Grant
2017JC006), Key Research and Development Project of Shandong Province (Grant
2017CXGC1401), and KU Leuven (Grant GOA 10/014) is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank K. Erven, K. Uyttersprot, and C. Heens for technical assistance with the anti-HIV
assays.
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ABBREVIATIONS
AIDS, acquired immunodeficiency syndrome; EC50, the concentration causing 50%
inhibition of antiviral activity; CC50, 50% cytotoxicity concentration; DAPY,
diarylpyrimidine; NNRTI, non-nucleoside reverse transcriptase inhibitor; NNIBP, NNRTI
binding pocket; HAART, highly active antiretroviral therapy; HIV, human
immunodeficiency virus; RT, reverse transcriptase; SAR, structure-activity relationship; SI,
selection index; WT, wild-type; NVP, nevirapine; DLV, delavirdine; EFV, efavirenz; ETV,
etravirine; RPV, rilpivirine; AZT, azidothymidine.
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