Follow on-based optimization of the biphenyl-DAPYs as HIV-1 nonnucleoside reverse transcriptase inhibitors against the wild-type and mutant strains

Article abstract of DOI:10.1016/j.bioorg.2019.102974

The present work follows our preliminary discovery of biphenyl diarylpyrimidines (DAPYs) as HIV-1 nonnucleoside reverse transcriptase inhibitors. Further structural optimization of biphenyl-DAPYs led to the identification of a new series of biphenyl-substituted thiophene[3,2-d]pyrimidine analogues by a scaffold-hopping strategy. Biological evaluation of this series showed that these compounds possessed up to single-digit nanomolar potency (EC₅₀ = 7.8–526.2 nM) and prominently low toxicity (CC₅₀ = 18.5–280.8 μM) against wild-type (WT) HIV-1-infected cells. Furthermore, the results also demonstrated that compounds 29–32 exhibited high, broad-spectrum antiviral effects against clinically observed HIV-1 mutants. Specifically, compound 30, which had the highest selectivity index (SI = 16094) and the best anti-reverse transcriptase ability (IC₅₀ = 39 nM), displayed marked inhibitory activity (EC₅₀ = 13.5, 9.4, 17.0, 52.0, and 58.2 nM) against WT, K103N, E138K, L100I, Y181C mutants and moderate activity against double mutants. This study provides important avenues for the further design of HIV-1 inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.102974

Bioorganic Chemistry 89 (2019) 102974
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Follow on-based optimization of the biphenyl-DAPYs as HIV-1
nonnucleoside reverse transcriptase inhibitors against the wild-type and
mutant strains
a,b
a,b
a,b
c
c
Yali Sang , Sheng Han , Shuwen Han , Christophe Pannecouque , Erik De Clercq ,
a,b
a,b,⁎
Chunlin Zhuang , Fener Chen
a
b
c
Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, People’s Republic of China
Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
The present work follows our preliminary discovery of biphenyl diarylpyrimidines (DAPYs) as HIV-1 non-
nucleoside reverse transcriptase inhibitors. Further structural optimization of biphenyl-DAPYs led to the iden-
tification of a new series of biphenyl-substituted thiophene[3,2-d]pyrimidine analogues by a scaffold-hopping
strategy. Biological evaluation of this series showed that these compounds possessed up to single-digit nano-
molar potency (EC50 = 7.8–526.2 nM) and prominently low toxicity (CC50 = 18.5–280.8 μM) against wild-type
HIV-1
Biphenyl
Thiophene [3,2-d] pyrimidines
DAPYs
Reverse transcriptase
(
WT) HIV-1-infected cells. Furthermore, the results also demonstrated that compounds 29–32 exhibited high,
broad-spectrum antiviral effects against clinically observed HIV-1 mutants. Specifically, compound 30, which
had the highest selectivity index (SI = 16094) and the best anti-reverse transcriptase ability (IC50 = 39 nM),
displayed marked inhibitory activity (EC50 = 13.5, 9.4, 17.0, 52.0, and 58.2 nM) against WT, K103N, E138K,
L100I, Y181C mutants and moderate activity against double mutants. This study provides important avenues for
the further design of HIV-1 inhibitors.
1
. Introduction
group G in 3 maximized the interaction with the NNRTI binding pocket
(
NNIBP) by extending the length of the π-π conjugated system to im-
In our continuous studies of novel HIV-1 nonnucleoside reverse
prove the potentially antiviral activity [8]. Liu and his colleagues syn-
thesized a series of new DAPYs 4 with longer and bulkier alkyne groups
in the left wing (e.g., aromatic ring, cycloalkane, etc.), and the newly
synthesized compounds showed significantly better antiviral profiles
because of the beneficial effects of the extensive interactions between
the extended left ring and the functional residues in the hydrophobic
tunnel [9]. Despite the above advantages of these variants, the related
toxicity and/or unfavorable selectivity index (SI) has particularly lim-
ited the potential for further drug development, and the present study
has raised concerns around these serious problems.
transcriptase inhibitors (NNRTIs) [1–5], we recently identified a series
of biphenyl-DAPY derivatives with low nanomolar antiviral activity
against HIV-1 wild-type strains and a panel of NNRTI-resistant single
and double mutant strains [6]. It was noteworthy that the inhibitory
ability of the most potent compound 1 was 1.8–5.4-fold greater against
L100I, K103N, E138K, and Y181C compared with ETV. Other variants
of biphenyl derivatives have also been explored as HIV-1 inhibitors
(
Fig. 1). Wang and coworkers described a report on a novel series of 2-
hydroxyisoquinoline-1,3-dione (HID, 2) with dual inhibitory activity,
which consistently inhibited HIV reverse transcriptase (RT)-associated
RNase H and polymerase with IC50 values in low to submicromolar
range [7]. Guillemont et al. developed DAPY analogues 3 with different
spacer groups G (e.g., 2-furyl heterocycle, alkenyl group, etc.) between
the cyano moiety and the phenyl ring that displayed substantially po-
tent inhibitory capacity against the panel of the mutant virus. The
structure-activity relationship (SAR) studies illustrated that the spacer
The previous reports from the Liu group indicated that thiophene
[3,2-d]pyrimidine could be a useful architecture for DAPY-type agents
with significant inhibitory activity toward resistance-associated HIV-1
variants [10,11]. In particular, these compounds exhibited good safety
with low toxicity and high SI values. Our previous work also demon-
strated that a fused aromatic core (e.g., benzopyrimidine ring) could
strengthen the van der Waals interaction between compounds and the
⁎
Corresponding author.
E-mail address: rfchen@fudan.edu.cn (F. Chen).
https://doi.org/10.1016/j.bioorg.2019.102974
Received 25 March 2019; Received in revised form 16 April 2019; Accepted 4 May 2019
Available online 04 May 2019
0045-2068/ © 2019 Published by Elsevier Inc.

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
a
Scheme 1. Synthesis of compounds 14–32. Reagents and conditions: (a)
PdCl
2
, K
2
CO
3
, PEG400, H
2
O, 25 °C, 5–24 h; (b) urea, 190 °C, 2.5 h; (c) POCl ,
3
DMF (cat.), 110 °C, 8 h; (d) (i) NaOH, THF, H
e) 4-Aminobenzonitrile, EtOH, N , 85 °C, 12 h; (f) POCl
CO , DMF, 80 °C, 8 h.
2
O, 50 °C, 4 h. (ii) AcOH, 35 °C, 2 h;
(
2
3
, 110 °C, 2 h; (g) 7a-7s,
K
2
3
Fig. 1. DAPY lead 1 and variants of biphenyl derivatives.
functional residues V179 and E138 to increase the specificity of these
counterparts toward HIV-1 RT.
In drug discovery, building strong interactions of the analogues with
conserved regions of the NNIBP is a widely accepted strategy to en-
hance the anti-RT potency of NNRTIs [13–15]. The Dousson group
demonstrated that a polar cyano group specially interacting with the
highly conserved residue, W229, is particularly beneficial to improve
mutant resilience [16]. Therefore, a cyano group is selected as a key
component in this framework with the hope to improve the binding
affinities of the biphenyl-DAPYs. The resistance profiles of these new
compounds against HIV-1 wild-type and prevalent drug-resistant mu-
tant variants are further evaluated in this article.
2
. Results and discussion
2.1. Chemistry
The synthetic route to the desired compounds 14–32 is outlined in
Scheme 1. First, the key intermediates, 4′-hydroxy-[1,1′-biphenyl]-4-
carbonitrile 7a-7s were generated by Suzuki-Miyaura cross-coupling
reaction of 5 and 6a-6s. Then, treatment of commercially available
methyl 3-aminothiophene-2-carboxylate 8 with urea under the solvent-
free conditions generated 1,3-dihydrothiopheno[3,2-d]pyrimidine-2,4-
dione 9, which followed by chlorination, hydrolysis and nucleophilic
substitution reactions provided the intermediate 13. Subsequent reac-
tion with the key intermediates 7a-7s yielded the final compounds
1
4–32.
2.2. Antiviral activity
The newly synthesized biphenyl compounds were evaluated for
Fig. 2. Illustration of the optimization strategy to develop new biphenyl-DAPY
analogues.
their antiviral potency and cytotoxicity in MT-4 cells infected with WT
HIV-1 strain (IIIB), HIV-2 strain (ROD) or a wide range of clinically
observed drug-resistant single and double mutant strains (L100I,
K103N, E138K, Y181C, Y188L, K103N + Y181C (RES056) and
F227L + V106A). The values of the anti-HIV activity (EC50), cytotoxi-
city (CC50), and selectivity index (SI, CC50/EC50 ratio) of the synthe-
sized NNRTI derivatives are illustrated in Tables 1 and 2. The FDA-
approved drugs, nevirapine (NVP), efavirenz (EFV), and etravirine
adjacent residues (e.g., E138, V179, etc.) in NNIBP [12]. These deri-
vatives with fused rings adjacent to the solvent-exposed tolerant region
(
the largely open region in front of K103, E138, and V179) not only
improved the anti-HIV-1 potency but also reduced the potential risk of
cytotoxicity with high SI values. In the present work, we further as-
sembled a thiophene ring into the biphenyl-DAPYs by a scaffold-hop-
ping strategy to address current disadvantages of anti-HIV drugs
(
ETV), were selected as reference compounds.
As summarized in Table 1, the biphenyl compounds with a cyano
(
Fig. 2). The present pharmacophore involves additional electrostatic
group and a thiophene attached to the pyrimidine exhibited high an-
tiviral activity against the WT HIV-1 (IIIB) strain with submicromolar to
interactions between the thiophene[3,2-d]pyrimidine structure and
2

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
Table 1
Activity and cytotoxicity against HIV-1 (IIIB) and HIV-2 (ROD) strains in MT-4 cells.
Compounds
R
EC50a
CC50 (μM)b
SI (IIIB)c
HIV-1 (IIIB) (nM)
ROD (μM)
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
H
67.4 ± 22.5
54.5 ± 10.5
58.8 ± 14.0
47.5 ± 25.9
101.5 ± 41.0
39.7 ± 7.1
229.6 ± 50.5
54.6 ± 15.6
526.2 ± 389.8
70.2 ± 14.9
76.6 ± 23.0
7.8 ± 3.7
41.1 ± 6.4
> 8.7
> 280.8
> 4151
341
2-Me
3-Me
2-F
18.5 ± 5.9
187.3 ± 45.4
> 269.9
56.4 ± 21.1
> 125.0
36.5 ± 13.4
> 16.1
3152
> 5580
> 2682
852
3-F
> 269.9
2-Cl
3-Cl
33.6 ± 16.1
> 261.0
75.8 ± 26.3
95.1 ± 43.8
> 13.6
> 1176
> 4401
50
2-CF
3
3
> 243.6
3-CF
26.5 ± 5.0
> 265.9
2-CN
3-CN
2-NO
3-NO
≥124.5
> 3744
2512
1.4 ± 0.6
> 107.2
> 34.4
192.1 ± 48.0
218.8 ± 37.3
70.2 ± 19.2
> 263.1
2
2
28,346
803
87.7 ± 30.6
35.8 ± 23.2
25.3 ± 6.8
38 ± 12.7
2-OMe
57.5 ± 11.3
> 116.9
1.4 ± 0.3
> 104.3
> 37.8
> 7289
9506
3-OMe
246.1 ± 6.5
223.8 ± 69.0
216.9 ± 6.7
78.6 ± 22.0
142.7 ± 63.4
> 15.9
2,6-diMe
2,3-diF
2,6-diF
2-Me-6-Cl
5868
13.5 ± 5.6
12.3 ± 2.1
34.5 ± 16.2
309.4 ± 57.7
7.3 ± 8.3
16,094
6423
> 70.3
4144
NVP
EFV
ETV
> 4.0
> 51
> 2.0
> 6.4
> 863
> 833
5.5 ± 4.1
> 2.0
> 4.6
a
b
c
EC50: The effective concentration required to protect the cell against viral cytopathicity by 50% in MT-4 cells.
CC50: The cytotoxic concentration of the compound that reduces the normal uninfected MT-4 cell viability by 50%.
SI: selectivity index, ratio CC50/EC50 (WT).
Table 2
the best anti-HIV-1 activity with an IC50 value of 7.8 nM. The ortho-
substitutions are favorable to the selectivity (SI, ranging from 341 to
28346), with two exceptions (15 and 19). (2) Di-substituted compounds
exhibit different anti-HIV-1 activity. The di-fluorinated derivatives (30
and 31) exhibited EC50 values of low nanomolar range. However, di-
methyl (29) and 2-methyl-6-chloride (32) had an approximately 3-fold
decrease of anti-HIV-1 compared to the di-fluorinated derivatives, in-
Inhibitory activity of the selected compounds toward WT HIV-1 RT.
IC50 (nM)a
IC50 (nM)a
Compounds
Compounds
2
2
3
5
9
0
63 ± 12
31
41 ±
9
44 ±
39 ±
2
6
32
44 ± 14
NVP
404 ± 85
a
dicating the tolerance of small substitutions on C -position. (3) Al-
6
IC50: inhibitory concentration of the test compound required to inhibit
though these analogues showed comparable or less potency against the
WT HIV-1, they had much better selectivity against HIV-1 over ROD
biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into WT HIV-1
RT by 50%.
(
EC50 > 3.0–269.9 µM), and their cytotoxicities were much lower
single-digit nanomolar EC50 values (EC50 = 7.8–526.2 nM). The SAR
analysis indicated that (1) the derivatives substituted on position 2 are
better than the substituted derivatives on position 3, with only one
exception (compound 27). Compound 25 with 2-nitro group showed
(CC50 > 18.5–280.8 µM)
(CC50 > 4.6–15.9 µM).
than
the
four
reference
drugs
Because good anti-HIV-1 activity, low cytotoxicity and/or high SI of
compounds 25 and 29–32 were observed, they were further
Table 3
Activity and selectivity index of the selected compounds against a panel of clinically relevant HIV-1 mutant strains in MT-4 cells.
Compounds
EC50 (nM)a (SI)b
L100I
K103N
Y181C
Y188L
E138K
RES056
F227L + V106A
2
2
3
3
3
5
9
0
1
2
18.2 ± 3.9
5.5 ± 0.6
55.1 ± 6.1
> 15140.0
(14)
6.5 ± 0.5
> 218821.4
(1)
≥40911.7
(11)
(
12,118)
(39,719)
(40,039)
(34,045)
23.2 ± 4.2 (8014)
52.0 ± 8.3 (4116)
47.8 ± 8.3
33.8 ± 4.2 (5318)
57.1 ± 10.6 (3197)
50.7 ± 17.0 (3531)
42.3 ± 4.2 (44,254)
152.2 ± 42.3 (1191)
253.6 ± 8.5
(
703)
9.4 ± 2.1
58.2 ± 33.3
(3753)
228.6 ± 41.6
(940)
17.0 ± 0.2
(12,725)
16.6 ± 2.1
(4724)
> 2037.1
(106)
602.8 ± 20.8
(354)
(
23,187)
8.7 ± 2.7
(9106)
120.6 ± 18.7
(654)
> 20308.0
(4)
> 78552.4
(11)
≥7233.70
(1)
(
1615)
28.4 ± 16.2
30.4 ± 6.1
(4079)
48.7 ± 14.2
(2665)
107.5 ± 42.6
(1183)
52.7 ± 6.1
(2418)
148.0 ± 20.3
(854)
405.6 ± 6.1
(314)
(
4505)
NVP
EFV
ETV
2102.3 ± 751.5 (8)
44.4 ± 9.5
> 10075.0
> 15866.7
(X1)
> 15866.7
(X1)
210.2 ± 26.3
(75)
> 15866.7
(X1)
> 15866.7
(X1)
(
X1)
101.6 ± 54.0
(63)
7.3 ± 0.6
(870)
292.0 ± 44.4
(22)
6.3 ± 0.6
(1013)
279.3 ± 98.4
(23)
273.0 ± 28.6
(23)
(
140)
7.1 ± 2.8
640)
3.2 ± 0.5
(1429)
12.0 ± 1.4
(388)
20.0 ± 7.6
(231)
6.5 ± 5.8
(721)
55.3 ± 9.2
(83)
15.2 ± 16.1
(302)
(
a
b
EC50: The effective concentration required to protect the cell against viral cytopathicity by 50% in MT-4 cells.
SI: selectivity index, ratio CC50/EC50 (mutant strains).
3

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
investigated for their inhibitory activity against WT HIV-1 RT enzymes.
NVP was chosen as the reference drug. To our delight, the five novel
NNRTI derivatives displayed great inhibitory activity against WT HIV-1
RT with IC50 values ranging from 39 nM to 63 nM, which were
position of the pyrimidine was essential for antiviral potency by in-
teracting with V106, P236, L100, L234, and Y318, as well as attributing
to the hydrogen-bonding interaction with the main chain of the residue
K101 as a hydrogen bond donor. Notably, the additional thiophene ring
further extended into the solvent-exposed tolerant region and estab-
lished nonpolar interactions with E138 and V179 residues as expected.
Another hydrogen bond between the nitrogen atom of the thiophene
[3,2-d]pyrimidine and the K101 side-chain was observed. Collectively,
these interactions are proposed to have contributed to the binding af-
finity.
6
.4–10.5-fold higher than that of NVP (IC50 = 404 nM) (Table 2).
Then, the five representative compounds were also selected to be
evaluated for their efficacy against the HIV-1 mutant strains, bearing
the L100I, K103N, Y181C, Y188L, E138K, RES056 (K103N + Y181C)
and F227L + V106A mutations that are associated with resistance to
NVP and EFV (Table 3).
Overall, they showed low EC50 values against most of the mutant
variants and much higher SI values than those observed for the positive
To further elucidate the resistance features of these compounds,
molecular models were created for the K103N and E138K variants of
the HIV-1 RT (Fig. 4). The binding orientation of compound 30 in
complexes with K103N mutant HIV-1 RT was similar to the binding
orientation in those with WT-HIV-1 RT (Fig. 3). Differently, a water-
mediated hydrogen bond (2.6, 2.2 Å) between the amino hydrogen of
N103 and the nitrogen atom in the central pyrimidine ring was formed
(Fig. 4A), indicating the contribution to the efficacy against the drug-
resistant K103N mutant virus. Consistent with the findings of the Liu
group [17], the predicted complex of the E138K RT/30 complex
showed that the Glu to Lys did not impair the electrostatic interactions
between the thiophene[3,2-d]pyrimidine structure and backbone K138
(Fig. 4B). Compound 30 inserted into the NNIBP as the 30/WT com-
plex, forming strong hydrophobic interactions with residue K138. As a
result, compound 30 retained its potency to this mutation as WT RT.
drugs. Compound 25 bearing a NO group presented prominent in-
2
hibitory efficacy (EC50 = 18, 5.5, 55.1 and 6.5 nM) against L100I,
K103N, Y181C, and E138K mutations, comparable to that of EFA and
ETV. Meanwhile, the SI values of 25 against L100I, K103N, Y181C, and
E138K mutations (SI = 12,118, 39,719, 40,039 and 34,045) were ob-
viously better than those of all of the references. However, it was in-
active to Y188L and double mutant variants RES056 and
F227L + V106A. Compound 31 showed a similar profile as compound
2
5, which was inactive against the double mutants. Compound 30 was
favorable to K103N and E138K (EC50 = 9.4 nM and 17.0 nM, respec-
tively) and showed EC50 values lower than 1 µM toward double mutant
variants RES056 and F227L + V106A. The 2,6-dimethyl-substituted 29
was the most potent inhibitor toward the dual mutant variants RES056
(
EC50 = 152 nM, SI = 1191) and F227L + V106A (EC50 = 253 nM,
SI = 703), and it demonstrated similar activity and a similar SI as the 2-
methyl-6-chloro-substituted 32, which were comparable to that of EFV,
ETV and better than NVP. Taking the chemical structure into con-
sideration, the methyl group in the di-substituted compounds (29, 32)
was positive to the potency against double mutants compared to the di-
fluoro compounds (30, 31).
3
. Conclusion
In summary, novel NNRTIs that combine structural features of 4-
cyano-1,1′-biphenyl substitutions and the thiophene[3,2-d]pyrimidine
were synthesized to overcome the high toxicity and improve the po-
tency. Current structural modification analysis indicated that (1) the
presence of a cyano group on the biphenyl ring was essential to interact
with the highly conserved W229 residue and endowed these derivatives
with promising activities and specificity in the wild-type and drug-re-
sistant mutant HIV-1 strains, (2) optimization of the substituents on the
biphenyl group could maintain or improve antiviral ability and form an
excellent combination of the biological efficiency and cytotoxicity, and
(3) the central thiophene[3,2-d]pyrimidine ring played a key role in
enhancing the specificity of these candidates targeting HIV-1 RT.
Overall, the newly designed compounds with tri-substitutions on the
biphenyl ring exhibited high broad-spectrum antiviral effects and low
cytotoxicity or high SI. This study provides a promising 4-cyano-1,1′-
biphenyl thiophene[3,2-d]pyrimidine scaffold as the novel lead tem-
plate for further investigation on anti-HIV-1 agents.
2.3. Molecular modeling analysis
Molecular docking was performed to explain the binding mode of
the representative compound 30 to HIV-1 RT, which has the best ac-
tivity at enzyme level (Fig. 3A). The docking model possessed similar
binding orientation with typical horseshoe conformational shape, con-
sistent with our earlier report on compound 1 (Fig. 3B). The biphenyl
group was inserted deeply into the aromatic-rich subpocket, which was
composed by hydrophobic amino acid residues Y181, Y188, F227, and
W229. Meanwhile, the cyano group became a powerful attachment to a
certain conserved region of W229 via extending the length of the π-π
conjugated system. The para-cyanoaniline group of 30 at the C -
2
Fig. 3. (A) Predicted binding mode of 30 with the HIV-1 WT RT crystal structure (PDB:2ZD1). (B) Superimposition of 1 and 30 with WT RT (pink, 1; yellow, 30).
Hydrogen bonds are indicated as orange dashed lines. The figure was generated using PyMol (http://pymol.sourceforge.net/). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
4

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
Fig. 4. (A) Predicted binding mode of 30 with the HIV-1 K103N RT crystal structure (PDB: 6C0K). (B) Predicted binding mode of 30 with the HIV-1 E138K RT crystal
structure (PDB: 6C0L). Hydrogen bonds are indicated as red dashed lines. The figure was generated using PyMol (http://pymol.sourceforge.net/).
4
. Experimental
.1. General
POCl was freshly distilled with an absorption apparatus for tail gas
4.3. Preparation of 9
4
Treatment of commercially available methyl 3-aminothiophene-2-
carboxylate 8 (10.0 g, 63.62 mmol, 1.0 equiv.) with urea (19.1 g,
318.09 mmol, 5.0 equiv.) at 190 °C for 2.5 h under the solvent-free
3
2
under N . EtOH was freshly distilled from Magnesium and iodine under
conditions
generated
intermediate
thieno[3,2-d]pyrimidine-
N
2
. Unless otherwise specified, all reagents and solvents were pur-
2,4(1H,3H)-dione (9). Upon cooling to about 90 °C, the reaction mix-
ture was poured into aqueous solution of sodium hydroxide (200 mL,
1 N) slowly. Then the mixture was stirred for 1 h and any insoluble
material removed by filtration. The mixture was then acidified by 15%
HCl solution until pH was adjusted to 4–5 to yield desired compound 9
chased from commercial sources and used as received. Flash column
chromatography was performed on silica gel (300–400 mesh). Thin-
layer chromatography (TLC) carried out on 0.25 mm silica gel pla-
tesvisualized with UV light (λ = 254 nm) and/or by staining with
ethanolic phosphomolybdic acid (PMA) or iodine. Proton nuclear
as a white precipitate, which was collected by filtration, washed by
1
1
magnetic resonance ( H NMR) and carbon nuclear magnetic resonance
water and dried. Yield: 92%. H NMR (400 MHz, DMSO-d
6
) δ: 11.53 (s,
1
3
(
C NMR) spectra were recorded in DMSO-d
6
or CF
3
COOD, CDCl
3
on a
1H), 11.19 (s, 1H), 8.03 (d, J = 5.0 Hz, 1H), 6.91 (d, J = 5.0 Hz, 1H).
+
Bruker AV-400 spectrometer with tetramethylsilane (TMS) as the in-
ternal standard. Chemical shifts (δ) are given in ppm relative to TMS,
coupling constants (J) in Hz. Melting points were measured on WRS-1B
digital melting-point apparatus. EI-MS were recorded on an Agilent
MS (ESI) m/z C
6
H
4
N
2
O
2
S: calcd 168.00, found 169.07 [M + 1]
.
4.4. Preparation of 10
6
890 N/5975 spectrometer and ESI-MS were recorded on a Waters
To a solution of intermediates 9 (9.0 g, 53.52 mmol) in phosphorus
oxychloride (60 mL) were added a few drops of dimethyl sulfoxide
under a nitrogen atmosphere. After the mixture was stirred for 8 h at
105 °C, TLC analysis indicated the reaction was complete. The solvent
was removed under reduced pressure and the residue was poured onto
ice-water with vigorous stirring yielding a precipitate. The precipitate
Micromass Quattro Micro spectrometer. High-resolution mass spectra
were recorded on Bruker ApeXIII 7.0 TESLA FTMS. The purities of the
compounds were analyzed by HPLC (Agilent 1260) using a C18 column
(
Eslipse XDB, 4.6 * 150 mm, 5 μm) with methanol/water as the mobile
phase at a flow rate of 1 mL/min: (a) 0–10 min, 30% MeOH; (b)
1
2
3
0–15 min, 30–60% MeOH; (c) 15–20 min, 60–95% MeOH; (d)
0–35 min, 95% MeOH; (e) 35–37 min, 95–30% MeOH; (f) 37–40 min,
0% MeOH. All final compounds exhibited purities greater than 95%.
was then collected by filtration, washed by H O and dried to give the
2
1
intermediates 10 as off-white solid. Yield: 73%. H NMR (400 MHz,
DMSO-d ) δ: 8.69 (d, J = 5.4 Hz, 1H), 7.72 (d, J = 5.4 Hz, 1H). MS
6
+
(
ESI) m/z C
6
H
2
Cl
2
N
2
S: calcd 203.93, found 204.94 [M + 1]
.
4.2. General procedure for the preparation of final compounds 7a-7s
4.5. Preparation of 11
A reaction mixture of (4-cyanophenyl)boronic acid 5 (150 mg,
.02 mmol, 1.0 equiv.), 4-bromo-2-chloro-6-methylphenol 6s (271 mg,
.22 mmol, 1.2 equiv.) and potassium carbonate (353 mg, 2.55 mmol,
10 (8.0 g, 39.01 mmol, 1.0 equiv.) was dissolved in 80 mL of a
1
1
2
mixture solvent of THF and H O (4:1), and a solution in which NaOH
2
(3.9 g, 97.53 mmol, 2.5 equiv.) was dissolved in 17 mL of H O was
2
.5 equiv.) in PEG400/H
2
0 (4 mL/4 mL) was stirred at room tempera-
added. The resulting mixture was heated to 50 °C and stirred for 4 h.
ture for 15 min, and then PdCl
2
(1.8 mg, 0.01 mmol, 0.01 equiv.) was
Then the reaction solution was cooled to 40 °C, 4.8 mL of acetic acid
was added to the reaction solution and stirred for another 2 h. The
added to it. Stirring was continued for an additional 15 h until complete
consumption of starting material as judged by TLC. Then the reaction
mixture was poured into water (20 mL) and extracted with ethyl acetate
resulting white solid was filtered, washed with H O and dried to obtain
2
1
11 in 89% yield. H NMR (400 MHz, DMSO-d ) δ: 13.47 (s, 1H), 8.17
6
(
10 mL * 4). The organic layers were washed with brine, dried over
(d, J = 5.2 Hz, 1H), 7.31 (d, J = 5.2 Hz, 1H). MS (ESI) m/z
+
anhydrous Na
2
SO
4
, filtered and condensed. The residue was then pur-
C H ClN OS: calcd 185.97, found 186.92 [M + 1]
.
6
3
2
ified via flash chromatography on silica gel, eluting with EtOAc/pet-
1
roleum ethe to 7s as white solid in 54% yield. H NMR (400 MHz,
4.6. Preparation of 12
DMSO-d ) δ: 9.50 (1H, s), 7.87–7.78 (4H, m), 7.60 (1H, s), 7.49 (1H, s),
6
2
.27 (3H, s). MS (ESI) m/z C1
4
H10ClNO: calcd 243.05, found 244.11
A solution of intermediate 11 (6.5 g, 34.83 mmol, 1.0 equiv.) and 4-
+
[
M + 1]
.
aminobenzonitrile (4.11 g, 34.83 mmol, 1.0 equiv.) in pure EtOH
The compounds 7a-7r were synthesized following the general pro-
(65 mL) was stirred at 85 °C for 12 h under N atmosphere. Then the
2
cedure, starting from 5 and phenol with different substituents 6a-6r.
solution was cooled to room temperature and stand for another15 h.
The resulting precipitate was collected by filtration, and dried to give
crude 12, which was used directly in the next step without further
5

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
+
purification. MS (ESI) m/z C13
H
8
4
N OS: calcd 268.04, found 269.13
z C27
H
17
N
5
OS: calcd 459.1154, found 460.1217 [M + H] . HPLC
+
[
M + 1]
.
analysis: retention time = 22.5 min; peak area, 95.1%.
4
.7. Preparation of 13
4.8.4. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
3′-fluoro-[1,1′-biphenyl]-4-carbonitrile (17)
1
To a solution of intermediate 12 (5.0 g, 18.64 mmol) in phosphorus
Yield: 89%. mp: 290.0–291.2 °C. H NMR (400 MHz, DMSO-d
6
) δ:
oxychloride (40 mL) were added a few drops of dimethyl sulfoxide
under a nitrogen atmosphere. After the mixture was stirred for 2 h at
10.12 (s, 1H), 8.44 (d, J = 5.3 Hz, 1H), 8.05–7.99 (m, 5H), 7.80–7.71
13
(m, 4H), 7.54–7.50 (m, 3H). C NMR (101 MHz, CF
3
COOD, CDCl ) δ:
3
1
05 °C, TLC analysis indicated the reaction was complete. The solvent
165.89, 153.94, 151.71, 150.89, 143.77, 143.14, 140.63, 140.20,
138.92, 133.48, 133.12, 127.74, 123.97, 121.73, 118.40, 117.56,
was removed under reduced pressure and the residue was poured onto
ice-water with vigorous stirring. The pH of the solution was adjusted to
116.75, 116.03, 115.84, 112.76, 110.60, 109.93, 107.18. HRMS (ESI)
+
7
by 1 N NaOH aqueous solution and the solution was stirred for an-
m/z C26
H14FN
5
OS: calcd 463.0903, found 464.0973 [M + H] . HPLC
other 2 h yielding a precipitate. The precipitate was then collected by
filtration. The collected solid was stirred in 50 mL of a mixture solvent
analysis: retention time = 22.2 min; peak area, 96.6%.
of acetone and water (4:1), and then filtered to get a white solid 13 in
4.8.5. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
1
8
0% yield. H NMR (400 MHz, DMSO-d
6
) δ: 10.56 (s, 1H), 8.47 (d,
2′-fluoro-[1,1′-biphenyl]-4-carbonitrile (18)
1
J = 5.1 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.52
Yield: 81%. mp: 288.3–290.4 °C. H NMR (400 MHz, CF
3
COOD,
(
d, J = 5.2 Hz, 1H). MS (ESI) m/z C13
H
7
4
ClN S: calcd 286.01, found
CDCl
3
) δ: 8.43 (d, J = 5.4 Hz, 1H), 7.96 (d, J = 8.2 Hz, 2H), 7.84 (d,
+
2
87.16 [M + 1]
.
J = 7.9 Hz, 2H), 7.74 (t, J = 8.5 Hz, 1H), 7.59–7.52 (m, 5H), 7.31 (t,
1
3
J = 8.1 Hz, 2H). C NMR (101 MHz, CF
159.82, 151.88, 151.77, 151.71, 150.68, 143.06, 140.19, 139.87,
33.20, 132.97, 131.56, 131.52, 129.66, 129.63, 121.84, 118.49,
3
COOD, CDCl ) δ: 166.36,
3
4.8. General procedure for the preparation of the final compounds 14–32
1
Different substituted 4′-hydroxy-[1,1′-biphenyl]-4-carbonitrile
118.15, 118.11, 117.50, 112.84, 111.60, 110.93, 110.67, 110.36,
(
0.2 mmol, 1.0 equiv.) was dissolved in DMF (3 mL) in the presence of
110.02, 107.33. HRMS (ESI) m/z C26
H
14FN OS: calcd 463.0903, found
5
+
K
2
CO
0.2 mmol, 1.0 equiv.). The reaction mixture was stirred at 85 °C for
–8 h (monitored by TLC), and then the reaction was cooled to ambient
3
(0.6 mmol, 3.0 equiv.), followed by addition of the compound 13
464.0973 [M + H] . HPLC analysis: retention time = 22.5 min; peak
area, 95.8%.
(
6
temperature and poured into 15 mL H
2
O yielding a precipitate. The
4.8.6. 3′-Chloro-4′-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-
precipitate was collected by filtration and the residue was then purified
via flash chromatography on silica gel, eluting with EtOAc/petroleum
ether (1/5, v/v) to obtain 14–32 as white solid.
yl)oxy)-[1,1′-biphenyl]-4-carbonitrile (19)
1
Yield: 68%. mp: 289.4–292.0 °C. H NMR (400 MHz, CF
3
COOD,
CDCl
3
) δ: 8.43 (d, J = 5.4 Hz, 1H), 8.14 (s, 1H), 8.04 (d, J = 10.0 Hz,
1
H), 7.98 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.61 (d,
13
4
.8.1. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
J = 8.5 Hz, 1H), 7.57 (d, J = 5.4 Hz, 1H), 7.44 (q, J = 8.9 Hz, 4H).
C
[
1,1′-biphenyl]-4-carbonitrile (14)
NMR (101 MHz, CF
3
COOD, CDCl ) δ: 165.92, 151.60, 150.92, 147.83,
3
1
Yield: 75%. mp: 274.3–275.8 °C. H NMR (400 MHz, DMSO-d
6
) δ:
143.66, 143.18, 140.24, 133.49, 133.10, 129.52, 127.86, 127.80,
127.25, 123.98, 121.66, 118.47, 117.58, 112.82, 111.25, 110.60,
110.00, 107.10. HPLC analysis: retention time = 22.4 min; peak area,
95.7%.
1
0.06 (s, 1H), 8.38 (d, J = 5.2 Hz, 1H), 7.97 (s, 4H), 7.92 (d,
J = 8.5 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.55–7.51 (m, 4H), 7.47 (d,
1
3
J = 5.3 Hz, 1H). C NMR (101 MHz, CF
3
COOD, CDCl
3
) δ: 166.76,
1
1
1
4
51.79, 151.55, 150.52, 144.88, 142.75, 140.33, 138.84, 133.38,
33.16, 129.09, 127.81, 122.34, 121.36, 118.48, 117.54, 117.10,
4.8.7. 2′-Chloro-4′-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-
16.73, 112.83, 110.00, 106.95. HRMS (ESI) m/z C26
H
15
N
5
OS: calcd
yl)oxy)-[1,1′-biphenyl]-4-carbonitrile (20)
+
1
45.0997, found 446.1068 [M + H]
.
HPLC analysis: retention
Yield: 90%. mp: 276.7–278.6 °C. H NMR (400 MHz, DMSO-d
6
) δ:
time = 22.3 min; peak area, 96.8%.
10.10 (s, 1H), 8.40 (d, J = 5.3 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.86
(
d, J = 8.4 Hz, 2H), 7.82 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.65–7.54
1
3
4
.8.2. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
(m, 4H), 7.48 (d, J = 5.3 Hz, 1H). C NMR (101 MHz, CF COOD,
3
3
′-methyl-[1,1′-biphenyl]-4-carbonitrile (15)
CDCl ) δ: 166.28, 151.74, 151.16, 150.75, 143.35, 143.05, 140.18,
3
1
Yield: 67%. mp: 261.1–262.5 °C. H NMR (400 MHz, DMSO-d
6
) δ:
133.53, 133.25, 132.71, 132.19, 130.27, 123.71, 121.96, 120.46,
118.55, 117.60, 112.90, 111.56, 110.54, 110.07, 107.44. HPLC ana-
lysis: retention time = 22.7 min; peak area, 96.0%.
1
7
0.10 (s, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.96 (s, 4H), 7.84 (s, 1H),
13
.79–7.74 (m, 3H), 7.50–7.45 (m, 4H), 2.23 (s, 3H).
C NMR
(
101 MHz, CF
3
COOD, CDCl ) δ: 166.50, 151.55, 150.73, 150.59,
3
1
1
1
4
45.13, 142.76, 140.40, 138.87, 133.33, 133.17, 131.32, 130.75,
4.8.8. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
27.79, 126.51, 122.38, 121.19, 118.53, 117.63, 112.87, 111.31,
3′-(trifluoromethyl)-[1,1′-biphenyl]-4-carbonitrile (21)
1
10.05, 109.83, 106.85, 15.67. HRMS (ESI) m/z C27
H
17
N
5
OS: calcd
Yield: 75%. mp: 245.3–247.9 °C. H NMR (400 MHz, CF
3
COOD,
+
59.1154, found 460.1215 [M + H]
.
HPLC analysis: retention
CDCl
3
) δ: 8.43 (d, J = 5.4 Hz, 1H), 8.14 (s, 1H), 8.04 (d, J = 10.0 Hz,
time = 22.6 min; peak area, 98.6%.
1H), 7.98 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.61 (d,
1
3
J = 8.5 Hz, 1H), 7.57 (d, J = 5.4 Hz, 1H), 7.44 (q, J = 8.9 Hz, 4H).
C
4
.8.3. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
NMR (101 MHz, CF
3
COOD, CDCl ) δ: 166.45, 151.69, 150.85, 148.51,
3
2
′-methyl-[1,1′-biphenyl]-4-carbonitrile (16)
143.46, 140.14, 139.43, 133.52, 133.09, 132.03, 127.83, 126.51,
126.46, 124.94, 123.99, 121.83, 117.42, 112.81, 111.55, 110.79,
1
Yield: 83%. mp: 243.9–246.6 °C. H NMR (400 MHz, DMSO-d
6
) δ:
1
0.07 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 7.95 (d, J = 7.6 Hz, 2H), 7.86
109.99, 107.25. HRMS (ESI) m/z C27
H
F
14 3
5
N OS: calcd 513.0871, found
+
(
d, J = 8.3 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H),
514.0940 [M + H] . HPLC analysis: retention time = 22.1 min; peak
7
1
1
1
1
.46 (d, J = 5.2 Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.3 Hz,
area, 97.9%.
13
H), 2.30 (s, 3H). C NMR (101 MHz, CF
3
COOD, CDCl ) δ: 166.81,
3
51.58, 151.16, 150.35, 146.17, 142.62, 140.46, 139.51, 138.20,
33.14, 132.78, 131.04, 129.97, 123.29, 121.49, 119.06, 118.47,
17.45, 112.82, 111.73, 110.00, 109.69, 106.92, 19.88. HRMS (ESI) m/
4.8.9. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
2′-(trifluoromethyl)-[1,1′-biphenyl]-4-carbonitrile (22)
Yield: 91%. mp: 244.4–246.2 °C. 1H NMR (400 MHz, CF
COOD,
3
6

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
CDCl
3
) δ: 8.39 (d, J = 5.3 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.79 (s,
127.86, 123.03, 121.20, 120.34, 118.43, 117.43, 112.79, 111.73,
1
3
1
H), 7.59–7.51 (m, 7H), 7.45 (d, J = 8.4 Hz, 2H). C NMR (101 MHz,
111.39, 110.02, 109.96, 106.72, 55.95. HRMS (ESI) m/z C27
H
17
N
5
2
O S:
+
CF
3
COOD, CDCl
3
) δ: 166.23, 151.88, 150.80, 150.68, 143.65, 143.15,
calcd 475.1103, found 476.1166 [M + H] . HPLC analysis: retention
time = 22.0 min; peak area, 97.0%.
1
1
1
5
40.02, 138.68, 133.52, 133.19, 132.42, 130.73, 130.42, 129.81,
23.46, 120.14, 120.09, 118.47, 117.58, 112.82, 111.61, 110.80,
10.00, 107.63. HRMS (ESI) m/z C27
H
14
F
3
N OS: calcd 513.0871, found
5
+
4.8.15. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
14.0943 [M + H] . HPLC analysis: retention time = 22.4 min; peak
3
′-methoxy-[1,1′-biphenyl]-4-carbonitrile (28)
area, 96.6%.
1
Yield: 71%. mp: 214.9–216.2 °C. H NMR (400 MHz, CF
3
COOD,
CDCl ) δ: 8.41 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 7.9 Hz, 2H), 7.82 (d,
3
4
.8.10. 4-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
J = 7.9 Hz, 2H), 7.64–7.47 (m, 6H), 7.09 (d, J = 8.3 Hz, 1H), 7.04 (s,
[
1,1′-biphenyl]-3,4′-dicarbonitrile (23)
1
3
1
1H), 3.89 (s, 3H). C NMR (101 MHz, CF
3
COOD, CDCl
3
) δ: 166.82,
Yield: 88%. mp: > 300 °C. H NMR (400 MHz, CF
3
COOD, CDCl ) δ:
3
1
1
57.83, 152.47, 151.57, 150.45, 142.89, 142.73, 140.39, 133.20,
8
2
.43 (d, J = 5.3 Hz, 1H), 8.11 (d, J = 12.2 Hz, 2H), 7.96 (d, J = 7.9 Hz,
32.46, 131.59, 130.20, 128.26, 121.42, 118.45, 117.42, 113.87,
H), 7.81 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.57 (d,
1
3
112.80, 111.66, 109.98, 109.26, 106.98, 105.45, 55.66. HRMS (ESI) m/
J = 5.3 Hz, 1H), 7.53–7.36 (m, 4H). C NMR (101 MHz, CF
3
COOD,
+
z C27
H
17
5
N O
2
S: calcd 475.1103, found 476.1164 [M + H] . HPLC
CDCl
3
) δ: 165.50, 152.53, 151.86, 151.57, 143.96, 142.23, 139.98,
analysis: retention time = 22.2 min; peak area, 97.9%.
1
1
34.07, 133.71, 133.15, 132.57, 127.84, 124.16, 122.18, 118.49,
17.74, 113.04, 112.84, 111.50, 111.29, 110.01, 107.69, 107.35.
HRMS (ESI) m/z C27
H
14
6
N OS: calcd 470.0950, found 469.0869 [M -
4
.8.16. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
+
H] . HPLC analysis: retention time = 22.0 min; peak area, 98.9%.
3
′,5′-dimethyl-[1,1′-biphenyl]-4-carbonitrile (29)
1
Yield: 93%. mp: 287.1–288.5 °C. H NMR (400 MHz, CF
3
COOD,
4
.8.11. 4-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
CDCl ) δ: 8.37 (d, J = 5.0 Hz, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.82 (d,
3
[
1,1′-biphenyl]-2,4′-dicarbonitrile (24)
J = 8.2 Hz, 2H), 7.55 (d, J = 5.1 Hz, 1H), 7.52 (s, 2H), 7.34 (s, 4H),
1
Yield: 76%. mp: > 300 °C. H NMR (400 MHz, CF
3
COOD, CDCl
3
) δ:
13
2
1
1
1
.28 (s, 6H). C NMR (101 MHz, CF
3
COOD, CDCl ) δ: 166.00, 151.59,
3
8
7
.43 (d, J = 5.3 Hz, 1H), 8.01 (d, J = 7.9 Hz, 2H), 7.91 (s, 1H),
50.79, 149.74, 145.24, 142.73, 140.45, 138.51, 133.28, 133.17,
31.12, 128.15, 127.77, 120.96, 118.63, 117.84, 112.97, 110.82,
13
.83–7.74 (m, 4H), 7.62–7.55 (m, 3H), 7.48 (d, J = 8.4 Hz, 2H).
COOD, CDCl ) δ: 165.89, 151.91, 150.98, 150.86,
43.50, 143.20, 141.44, 139.93, 133.36, 133.25, 131.92, 129.44,
C
NMR (101 MHz, CF
3
3
10.14, 106.98, 15.99. HRMS (ESI) m/z C28
H
19
5
N OS: calcd 473.1310,
1
1
1
4
+
found 474.1376 [M + H] . HPLC analysis: retention time = 22.8 min;
27.74, 127.25, 122.39, 118.45, 117.56, 112.81, 112.00, 111.55,
peak area, 95.0%.
09.99, 107.88. HRMS (ESI) m/z C27
H
14
6
N OS: calcd 470.0950, found
+
71.1014 [M + H] . HPLC analysis: retention time = 22.6 min; peak
4
.8.17. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
area, 96.8%.
2
′,3′-difluoro-[1,1′-biphenyl]-4-carbonitrile (30)
1
Yield: 86%. mp: 279.0–280.7 °C. H NMR (400 MHz, DMSO-d
6
) δ:
4
.8.12. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
1
0.14 (s, 1H), 8.46 (d, J = 4.9 Hz, 1H), 8.05 (d, J = 7.8 Hz, 2H),
13
3
′-nitro-[1,1′-biphenyl]-4-carbonitrile (25)
1
7.90–7.84 (m, 4H), 7.63–7.58 (m, 4H), 7.53 (d, J = 5.0 Hz, 1H).
C
Yield: 92%. mp: 297.5–298.6 °C. H NMR (400 MHz, CF
3
COOD,
NMR (101 MHz, CF
3
COOD, CDCl ) δ: 165.50, 151.50 (d, J = 89.4 Hz,
3
CDCl ) δ: 8.55 (d, J = 1.9 Hz, 1H), 8.44 (d, J = 5.4 Hz, 1H), 8.16 (dd,
3
1
1
C), 141.71, 138.94, 133.11, 133.08, 129.52, 129.49, 124.64, 122.43,
J = 8.4, 2.0 Hz, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 8.3 Hz, 2H),
18.42, 118.32, 117.49, 112.78, 110.91, 109.96, 107.65. HRMS (ESI)
7
2
1
1
1
.69 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 5.4 Hz, 1H), 7.45 (d, J = 8.6 Hz,
+
13
m/z C
26 13
H
F N OS: calcd 481.0809, found 482.0884 [M + H] . HPLC
H), 7.35 (d, J = 8.7 Hz, 2H). C NMR (101 MHz, CF
3
COOD, CDCl
3
) δ:
2
5
analysis: retention time = 22.4 min; peak area, 98.5%.
66.09, 151.78, 151.20, 144.18, 143.74, 142.23, 141.31, 140.20,
39.97, 134.07, 133.72, 133.10, 127.83, 125.92, 124.90, 122.20,
18.44, 117.54, 112.80, 111.46, 109.97, 107.53. HRMS (ESI) m/z
4
.8.18. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
+
C
26
H
14
N
6
O
3
S: calcd 490.0848, found 491.0923 [M + H] . HPLC ana-
3
′,5′-difluoro-[1,1′-biphenyl]-4-carbonitrile (31)
lysis: retention time = 21.3 min; peak area, 95.3%.
1
Yield: 82%. mp: > 300 °C. H NMR (400 MHz, DMSO-d
6
) δ: 10.17
(
s, 1H), 8.46 (d, J = 5.3 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.99 (d,
4
.8.13. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
J = 8.2 Hz, 2H), 7.90 (d, J = 9.3 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H),
2
′-nitro-[1,1′-biphenyl]-4-carbonitrile (26)
13
7
1
1
1
4
.58–7.48 (m, 3H). C NMR (101 MHz, CF
3
COOD, CDCl ) δ: 165.25,
3
1
Yield: 79%. mp: 255.8–257.7 °C. H NMR (400 MHz, DMSO-d
6
) δ:
55.04, 151.86, 151.30, 143.68, 142.81, 140.23, 140.08, 133.59,
1
0.11 (s, 1H), 8.42 (d, J = 5.3 Hz, 1H), 8.29 (s, 1H), 8.00 (d,
33.19, 133.13, 127.67, 122.08, 118.45, 117.61, 112.80, 111.49,
J = 8.2 Hz, 2H), 7.93 (d, J = 10.6 Hz, 1H), 7.84 (d, J = 8.6 Hz, 2H),
11.25, 111.04, 109.98, 107.47. HRMS (ESI) m/z C26
H
13
F
2
N OS: calcd
5
7
2
1
1
1
.78 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.6 Hz,
+
81.0809, found 482.0874 [M + H]
. HPLC analysis: retention
1
3
H), 7.50 (d, J = 5.3 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ: 165.06,
6
time = 22.5 min; peak area, 98.8%.
63.73, 157.07, 152.48, 149.25, 145.27, 141.77, 138.40, 133.63,
33.24, 133.21, 131.81, 129.55, 127.80, 123.86, 119.99, 119.24,
18.96, 118.81, 111.75, 109.71, 102.83. HRMS (ESI) m/z
4.8.19. 3′-Chloro-4′-((2-((4-cyanophenyl)amino)thieno[3,2-d]pyrimidin-
+
C
26
H
14
N
6
O
3
S: calcd 490.0848, found 491.0917 [M + H] . HPLC ana-
4-yl)oxy)-5′-methyl-[1,1′-biphenyl]-4-carbonitrile (32)
1
lysis: retention time = 22.6 min; peak area, 96.5%.
Yield: 76%. mp: > 300 °C. H NMR (400 MHz, CF
3
COOD, CDCl ) δ:
3
8
.41 (d, J = 5.4 Hz, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.2 Hz,
4
.8.14. 4′-((2-((4-Cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-
2H), 7.73 (s, 1H), 7.57 (d, J = 5.7 Hz, 2H), 7.39 (s, 4H), 2.37 (s, 3H).
1
3
2
′-methoxy-[1,1′-biphenyl]-4-carbonitrile (27)
C NMR (101 MHz, CF
3
COOD, CDCl ) δ: 165.55, 151.64, 151.02,
3
1
Yield: 80%. mp: 280.0–282.0 °C. H NMR (400 MHz, DMSO-d
6
) δ:
146.65, 143.88, 143.16, 140.28, 139.60, 133.58, 133.44, 133.12,
1
0.07 (s, 1H), 8.37 (d, J = 5.3 Hz, 1H), 8.04–7.92 (m, 5H), 7.76 (d,
13
128.78, 127.78, 126.87, 121.46, 118.48, 117.73, 112.83, 110.98,
J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.51–7.43 (m, 4H), 3.85 (s, 3H). C NMR
110.48, 110.01, 107.08, 16.13. HRMS (ESI) m/z C27
H
16ClN OS: calcd
5
+
(
101 MHz, CF
3
COOD, CDCl
3
) δ: 166.56, 151.45, 151.17, 150.43,
493.0764, found 494.0827 [M + H]
time = 22.7 min; peak area, 96.9%.
. HPLC analysis: retention
1
45.32, 142.58, 140.82, 140.53, 140.16, 133.35, 133.12, 128.12,
7

Y. Sang, et al.
Bioorganic Chemistry 89 (2019) 102974
4
.9. Antiviral activity assays
estimate the binding energy. Prior to docking, the protein was prepared
by removing water molecules, the ligand ETV, and other unnecessary
small molecules from the crystal structure of the HIV-1 RT complex
[18]. Simultaneously, 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 the 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 amino acid 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 Kollman all-
atom approach for the protein and the Gasteiger-Hückel 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 more
than 10,000 generations using a genetic algorithm, and the 20-best-
4.9.1. In vitro anti-HIV assay
The anti-HIV activity of the compounds was evaluated against WT
HIV-1 (HIV-IIIB strain), single- and double- mutant strains (L100I,
K103N, E138K, Y181C, Y188L, K103N + Y181C (RES056) and
F227L + V106A), in MT-4 cell cultures using the method of 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly,
virus stocks were titrated in MT-4 cells and expressed as the 50% cell
culture infective dose (CCID50). MT-4 cells were suspended in culture
5
medium at 1 × 10 cells/mL and infected with HIV at a multiplicity of
infection of 0.02. Immediately after viral infection,100 mL of the cell
suspension was placed in each well of a flat-bottomed micro-titer tray
containing various concentrations of the test compounds. The test
compounds were dissolved in DMSO at 50 mM or higher. After 4 days of
incubation at 37 °C, the number of viable cells was determined using
the MTT method. Compounds were tested in parallel for cytotoxic ef-
fects in uninfected MT-4 cells.
scoring ligand-protein complexes were kept for the further analyses.
2
4.9.2. HIV-1 RT inhibition assay
The -log(K
d
)
values of the 20-best-scoring complexes, which re-
Recombinant wild type p66/p51 HIV-1 RT was expressed and pur-
presented the binding affinities of the ligand with RT, encompassed a
−2 −9
ified as previously described. The RT assay was performed with the
EnzCheck Reverse Transcriptase Assay kit (Molecular Probes,
Invitrogen), as described by the Manufacturer. The assay was based on
the dsDNA quantitation reagent Pico-Green. This reagent showed a
pronounced increase in fluorescence signal upon binding to dsDNA or
RNA-DNA hetero-duplexes. Single-stranded nucleic acids generated
only minor fluorescence signal enhancement when a sufficiently high
dye: base pair ratio was applied. This condition was met in the assay.
A poly(rA) template of approximately 350 bases long and an oligo
wide scope of the functional classes (10 –10 ). Only the highest
scoring 3D structural model of the ligand-bound RT was chosen to
define the binding interaction.
Acknowledgements
We gratefully acknowledge the generous support provided by
National Natural Science Foundation of P.R. China under Grant No.
21372050, and Shanghai Municipal Natural Science Foundation under
Grant No. 13ZR1402200.
(
dT)16 primer were annealed in a molar ratio of 1:1.2 (60 min, at room
temperature). 52 ng of the RNA/DNA was brought into each well of a
6-well plate in a volume of 20 mL polymerization buffer (60 mM Trise
HCl, 60 mM KCl, 8 mM MgCl , 13 mM DTT, 100 mM dTTP, pH = 8.1).
We thank K. Erven, K. Uyttersprot, and C. Heens for technical as-
sistance with the HIV assays.
9
2
5
.0 mL of RT enzyme solution, diluted to a suitable concentration in an
Appendix A. Supplementary material
enzyme dilution buffer (50 mM Trise-HCl, 20% glycerol, 2 mM DTT,
pH = 7.6), was added. The reaction mixture was incubated at 25 °C for
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.102974.
4
0 min and then stopped by the addition of EDTA (15 mM). Hetero
duplexes were then detected by addition of Pico-Green. Signals were
read using an excitation wavelength of 490 nm and emission detection
at 523 nm using a spectrofluorometer (Safire 2, Tecan). To test the
biological activity of compounds against RT, 1.0 mL of compound in
DMSO was added to each well before the addition of RT enzyme so-
lution. Control wells without compound contained the same amount of
DMSO. Results were expressed as relative fluorescence, i.e. the fluor-
escence signal of the reaction mixed with compound divided by the
signal of the same reaction mixed without compound.
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