Discovery of biphenyl-substituted diarylpyrimidines as non-nucleoside reverse transcriptase inhibitors with high potency against wild-type and mutant HIV-1

Article abstract of DOI:10.1016/j.ejmech.2018.01.016

A novel series of diarylpyrimidine (DAPY) derivatives bearing the biphenyl motif with multiple substituted groups was synthesized as human immunodeficiency virus (HIV)-1 non-nucleoside reverse transcriptase inhibitors. All of the target compounds were evaluated for their in vitro activity against HIV in MT-4 cells. Most of the compounds exhibited excellent activity with low nanomolar EC₅₀ values against wild-type, single and double mutant HIV-1 strains. Compound 4b displayed an EC₅₀ value of 1 nM against HIV-1 IIIB, 1.3 nM against L100I, 0.84 nM against K103 N, 1.5 nM against Y181C, 11 nM against Y188L, 2 nM against E138K, 10 nM against K103 N + Y181C, and almost 110 nM against F227L + V106. The improvement in the selectivity and potency of the target molecules against the wild-type and mutant HIV-1 strains validated our hypothesis. The biphenyl ring in the DAPY derivatives could strengthen the π-π stacking effect between the target molecule and the non-nucleoside inhibitor-binding pocket in the reverse transcriptase by extending the conjugating systems. This research represented a significant step toward the discovery of novel therapeutic DAPYs for treating acquired immunodeficiency syndrome in patients infected with HIV-1.

Full text of DOI:10.1016/j.ejmech.2018.01.016

Accepted Manuscript
Discovery of biphenyl-substituted diarylpyrimidines as non-nucleoside reverse
transcriptase inhibitors with high potency against wild-type and mutant HIV-1
KaiJun Jin, Hong Yin, Erik De Clercq, Christophe Pannecouque, Ge Meng, FenEr
Chen
PII:
S0223-5234(18)30016-3
10.1016/j.ejmech.2018.01.016
EJMECH 10093
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 November 2017
Revised Date: 28 December 2017
Accepted Date: 5 January 2018
Please cite this article as: K. Jin, H. Yin, E. De Clercq, C. Pannecouque, G. Meng, F. Chen, Discovery
of biphenyl-substituted diarylpyrimidines as non-nucleoside reverse transcriptase inhibitors with high
potency against wild-type and mutant HIV-1, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.01.016.
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Discovery of Biphenyl-substituted Diarylpyrimidines as
Non-nucleoside Reverse Transcriptase Inhibitors with High Potency against Wild-Type and Mutant HIV-1
KaiJun Jin a, b, §, Hong Yin a, §, Erik De Clercqc, Christophe Pannecouque c, Ge Menga, b, FenEr Chen a, b*
a Engineering Center of Catalysis and Synthesis for Chiral molecules, Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic
of China
b Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, People’s Republic of China
c Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
§ KaiJun Jin and Hong Yin contributed equally.
* Corresponding authors.
1

ACCEPTED MANUSCRIPT
2

ACCEPTED MANUSCRIPT
Discovery of Biphenyl-substituted Diarylpyrimidines as Non-nucleoside Reverse Transcriptase Inhibitors
with High Potency against Wild-Type and Mutant HIV-1
KaiJun Jin a, b, §, Hong Yin a, §, Erik De Clercqc, Christophe Pannecouque c, Ge Menga, b, FenEr Chen a, b*
a Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of
China
b Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, People’s Republic of China
c Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
§ KaiJun Jin and Hong Yin contributed equally.
* Corresponding authors.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel series of diarylpyrimidine (DAPY) derivatives bearing the biphenyl motif with multiple
substituted groups was synthesized as human immunodeficiency virus (HIV)-1 non-nucleoside
reverse transcriptase inhibitors. All of the target compounds were evaluated for their in vitro
activity against HIV in MT-4 cells. Most of the compounds exhibited excellent activity with low
nanomolar EC50 values against wild-type, single and double mutant HIV-1 strains. Compound
4b displayed an EC50 value of 1 nM against HIV-1 IIIB, 1.3 nM against L100I, 0.84 nM against
K103N, 1.5 nM against Y181C, 11 nM against Y188L, 2 nM against E138K, 10 nM against
K103N+Y181C, and almost 110 nM against F227L+V106. The improvement in the selectivity
and potency of the target molecules against the wild-type and mutant HIV-1 strains validated our
hypothesis. The biphenyl ring in the DAPY derivatives could strengthen the π-π stacking effect
between the target molecule and the non-nucleoside inhibitor-binding pocket in the reverse
transcriptase by extending the conjugating systems. This research represented a significant step
toward the discovery of novel therapeutic DAPYs for treating acquired immunodeficiency
syndrome in patients infected with HIV-1.
Available online
Keywords:
Biphenyl;
Diarylpyrimidines;
HIV-1;
Reverse transcriptase;
Dock;
SAR

NNRTIs[13].
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1. Introduction
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
have played an important role in the highly active
antiretroviral therapy for acquired immunodeficiency
The crystal structure of the DAPY analogue complex
with HIV-1 reverse transcriptase (RT) and our 3D-QSAR
studies revealed that the left wing of DAPYs interact with the
hydrophobic/aromatic site of HIV-1 RT including, among
others, amino acid residues including Tyr181, Tyr188, Trp229
and Phe227, as shown in Fig. 2[14, 15].
syndrome
(AIDS)
patients
infected
with human
immunodeficiency virus (HIV)[1-4]. The diarylpyrimidine
analogues (DAPYs, 1) are the most efficacious NNRTIs
developed as the therapeutic agents against HIV-1, they are
interacting in a noncompetitive manner targeted at the
hydrophobic allosteric binding site (NNRTIs binding pocket,
NNIBP) like other NNRTIs, which is located about 10 Å
distant from the catalytic site of RT[5].
Fig. 2. The binding model of ETV (2, blue) and RPV (3, red) with
HIV-1 RT (PDB code: 2ZD1).
Thus, extending the length of the π-π conjugated system
in the left wing of DAPYs was expected to enhance their
aromaticity to strengthen the π-π stacking interactions between
the target molecules and amino acid residues Tyr 181, Tyr 188
and Trp229 within the active binding pocket of RT. We
hypothesized that introducing a biphenyl group with the large
π-π conjugated system might provide the reasonable
replacement for the phenyl moiety on the left wing in DAPY
molecules to protrude further into the hydrophobic/aromatic
pocket, significantly contributing toward the improved
biological activity against HIV-1 strains (Fig. 3).
Fig. 1. Chemical structure of DAPYs
DAPYs are quite unique for their sufficient flexibility
through ‘‘wiggling’’ and “jiggling” in their conformational
transformation process, which is quite different from the
first-generation NNRTIs with the highly rigid structural
features[6]. The innate flexibility of DAPYs allow them to
adopt the suitable conformations in NNIBP showing different
size for mutation, explaining their potent activity against the
wild type and drug-resistant mutant strains of HIV-1[7-9].
Recently, good examples have been set in identification of
new DAPY NNRTIs based on their crystal structures with RT,
SAR analysis and molecular modeling studies, offering the
great value to design new DAPYs as the promising new
anti-HIV NNRTIs drug candidates. For example, Etravirine (2,
ETV) and Rilpivirine (3, RPV), two DAPY drugs with low
drug resistance that are approved by the FDA (Fig. 1),
exhibited a broad spectrum of activities against wild-type (WT)
and clinically relevant HIV-1 mutant strains with nanomolar
EC50 values[10, 11].
Although the emergence of drug resistance of the above two
drugs was delayed, mutations did arise following the clinical
administration[12], the novel potential DAPY analogues with
the good safety properties and the pharmacokinetic profiles are
still urgently needed to overcome the drug resistance. The
superior pharmacological features and the characteristic
molecular binding modes of DAPYs with NNIBP could
encourage the medicinal chemists to find more potent HIV-1
Fig. 3. The strategy for designing the target molecules
As part of our continuing research program to develop
new DAPYs as HIV-1 NNRTIs[16-20], a series of biphenyl
substituted DAPY analogues (BP-DAPYs, 4) was synthesized
and evaluated for their antiviral biological activities (Scheme
2

1). The preliminary structure–activity relationships of these
Treating 7 with 4-bromo-2,6-dimethylaniline 8 at 170°C for 3
h under the solvent-free conditions generated diphenyl amino
pyrimidine 9[21], which was converted into the desired target
molecules 4a–v at a 40%–60% yield via Suzuki-Miyaura cross
coupling with arylboronic acids catalyzed by Pd(dppf)Cl2
under reaction conditions that involved boiling in a mixed
solvent of dioxane and water[22].
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new DAPY analogues are discussed below.
2. Results and discussion
2.1. Chemistry
The synthesis of target molecules 4a–v is depicted in
Scheme 1. The intermediate chloropyrimidine 7 was obtained
from the commercially available methylthio pyrimidine 5 in
two steps following our previously reported procedure[16-18].
Scheme 1. Reagents and conditions: (a): (i) 4-aminobenzonitrile (6), 180–190°C, 8 h, 90%; (ii) POCl3, refluxing, 1 h, 82%; (b):
4-bromo-2,6-dimethylaniline (8), 160–170°C, 3 h, 80%; (c): ArB(OH)2 (10), Pd(dppf)Cl2, dioxane, H2O, N2, refluxing, 5–10 h, 40%–60%.
2.2. Antiviral activity
The newly synthesized compounds were evaluated for
their biological activities against WT HIV-1 strain IIIB and
HIV-2 strain ROD, respectively. The FDA approved drugs
Nevirapine (NVP), Efavirenz (EFV), Etravirine (ETV) and
azidothymidine (AZT), were included as the reference drugs.
The biological results are represented as EC50 values (anti-HIV
activity), CC50 values (cytotoxicity) and SI values (selectivity
index, given by the CC50/EC50 ratio) (Table 1 and Table 2)
Table 1
Anti-HIV activity and cytotoxicity of compounds 4a–v in MT-4 cellsa
EC50(nM)b
Compds
CC50(nM)c
SI( B)ds
HIV-1IIIB
2.8 ± 1.1
1 ± 0.6
ROD
>4170
>2080
>920
4170 ±1630
2080 ± 720
920 ± 270
1516
2059
601
4a
4b
4c
4d
4e
4f
1.5 ± 0.1
3.7 ± 2..7
5.3 ±3
>4190
>2160
>3990
>3990
>4120
>2120
>5570
>3670
4190 ± 1600
2160 ± 540
3990 ± 1160
4170 ± 1640
4120 ± 1440
2120 ± 640
5570 ± 1450
3670 ± 1390
1129
409
2.6 ± 1.7
1.4 ± 0.6
1.7 ± 0.9
1.4 ± 0.6
3.6 ± 1.4
5.9 ± 2.2
1151
2953
2377
1542
1543
622
4g
4h
4i
4j
4k
3

2.4 ± 1.4
2.4 ± 1.4
2.9 ± 1.8
3 ± 1.9
>3820
3820 ± 1260
1579
1205
1050
316
4l
4m
4n
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>2890
>2990
>940
2890 ± 490
2990 ± 930
940 ± 330
4160 ± 1580
4030 ± 1450
4210 ± 1530
2590 ± 340
4140 ±1500
4290 ± 1600
3910 ±1630
>4000
4o
10 ± 5
>4160
>4030
>4210
>2590
>4140
>4290
>3910
418
4p
8.4 ± 3.9
6.9 ± 3.1
7.1 ± 5.6
23 ± 12
34 ± 14
33 ± 14
24 ± 6
477
4q
606
4r
364
4s
177
4t
125
4u
120
4v
>168
>4624
>1320
>971
NVP
EFV
ETV
AZT
0.43 ± 0
1.5 ± 0.1
2.1 ± 0.3
>2000
>2000
>2000
a All data represent the mean values of at least three separate experiments.
b EC50: The effective concentration required to protect MT-4 cells against viral cytopathicity by 50%.
c CC50: The cytotoxic concentration of the compound that reduces the normal uninfected MT-4 cell viability by 50%.
d SI: selectivity index, ratio CC50/EC50 (WT).
Table 1 revealed that all of the newly synthesized
compounds exhibited good anti-HIV activity with the low
nanomolar EC50 values against the WT HIV-1 strain ranging
from 34 nM to 1 nM showing the SI values ranging from 120
to 2953. Overall, most of the compounds were more potent
than NVP (EC50 (IIIB) = 24 nM), which have almost reached the
a fluorine atom on the diphenyl ring (4g–i) had superior
potency over other compounds without the fluorine
substitution. The activity of the synthesized congeners (4m–o)
revealed that the methoxyl group exhibited similar activity to
that of 4a. By contrast, some other substituents in this region
led to the deleterious effects on the anti-HIV potency, such as
the alkyl groups (in 4j–l), and the halogen-derivative groups in
(4d–f and 4p–r). Interestingly, all of the di-substituted
compounds including 4s, 4t, 4u and 4v exhibited low
biological activities, which were less potent than both the
mono-substituted compounds and the non-substituted
compounds (Table 1). This result indicated that increasing the
steric hindrance of the biphenyl ring might decrease the
biological activity.
level of AZT (EC50
= 2.1 nM). It worth noting that
(IIIB)
compounds 4b, 4c, 4g, 4h and 4i exhibited the potent activity
against WT HIV replication (EC50(IIIB) values of 1, 1.5, 1.4, 1.7
and 1.4 nM, respectively), with similar potency to ETV
(EC50(IIIB) = 1.5 nM). Furthermore, these new compounds
showed low micromolar activity against the HIV-2 ROD virus
in MT-4 cells, among which compound 4c was the most potent
with an EC50 value of 0.92 µM.
The active BP-DAPYs described above (4b, 4c, 4g and 4i)
were also tested in the MT-4 cell line for their ability of inhibit
several single and double resistant virus strains (L100I,
K103N, E138K, Y181C, Y188L, K103N/Y181C (RES056)
and F227L/V106A) (Table 2). All retained high inhibitory
activity with EC50 values ranging from the low-micromolar
concentration level to the single-digit nanomolar level.
Notably, compound 4b showed the low nanomolar EC50 values
toward all of the single mutant strains and the double mutant
strain RES056 (≤11 nM), especially for the mutant strains of
K103N (EC50 = 0.84 nM) and Y181C (EC50 = 1.5 nM), which
were more active than the reference drugs NVP and EFV, with
Concerning the variation in the diphenyl ring in the left
wing, the unsubstituted compound 4a showed the significantly
higher inhibitory activity than other compounds, which was 10
times higher than that of NVP. Compounds with a cyano
group were clearly superior to the other alternatives (4b vs 4d,
g, j, m, o, p and 4c vs 4e, f, k, n, q, Table 1). A cyano group at
the C-4 position of the diphenyl ring produced the best results
(4b vs 4c), as expected from our published results on the
DAPY derivatives as the inhibitors of HIV-1 RT[23]. In
general, the attachment positions of the substituents on the
biphenyl ring almost exerted no effect on the anti-HIV potency
(4d–f, 4g–i, 4j–l, 4m–o and 4p–r). In addition, analogues with
4

similar activity to ETV. In the inhibition of double mutant
EFV (EC =50 nM), but more potent than NVP (EC ≥4000
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50
50
strain F227L+V106A, 4b (EC50≥110 nM) was less potent than
nM).
Table 2
The biological activity of the active compounds (4b, 4c, 4g and 4i) on the mutant HIV-1virus strains
EC50 a(nM)
Compds
L100I
1.3 ± 0.1
17 ± 1
K103N
0.84 ± 066
2.3 ± 0.6
2.3 ± 0.2
3.2 ± 1.4
1850 ± 40
19 ± 0.0
Y181C
1.5 ± 0.0
11 ± 1
Y188L
11 ± 1
E138K
2 ± 0.6
RES056b
10 ± 2
F227L+V106Ab
≥110
4b
4c
>920
8.1 ± 0.3
8.1 ± 0.4
7.8 ± 0.0
58 ± 0.0
1.6 ± 0.1
7.3 ± 06
2.2 ± 0.1
270 ± 30
190 ± 10
230 ± 30
>4000
>920
10 ± 1
8.3 ± 0.9
8 ± 0.6
160 ± 0.0
130 ± 0.0
>4000
320 ± 100
≥390
4g
12 ±3
4i
400 ± 90
1.0 ± 2
2.7 ± 0.0
1.3 ± 0.0
>4000
>4000
NVP
EFV
ETV
AZT
1.5 ± 00
8.1 ± 0.0
1.7 ± 0.2
59 ± 12
7.9 ± 0.2
1.6 ± 0.0
40 ± 2
50 ± 11
7 ± 0.4
1.5 ± 0.0
10 ± 1
1.9 ± 0.1
1.5 ± 0
1.0 ± 0.1
a EC50: The effective concentration required to protect MT-4 cells against viral cytopathicity by 50%.
b The double mutation strain (RES056 referred to K103N/Y181C).
2.3. HIV-1 RT inhibition assay
low-micromolar RT inhibitory activity (0.116–0.009 µM)
superior to that of NVP (IC50 = 0.176 µM). Compound 4i
showed excellent affinity to HIV-1 RT (IC50 = 0.009 µM),
being nearly equipotent to that of EFV (IC50 = 0.002 µM). The
newly synthesized representative derivatives therefore showed
high affinity to WT HIV-1 RT, indicating that they are
potential HIV-1 NNRTIs.
To confirm the binding target, an enzymatic assay against
WT HIV-1 RT was conducted for all of the compounds using
poly[A]•oligo[dT]15 as the template primer (RT kit, Roche).
NVP and EFV were used as the reference drugs in this assay.
As shown in Table 3, all of the target compounds displayed
Table 3
The inhibitory activity of the compounds 4a–x against WT HIV-1 RT a
Compds
4a
IC50b (µM)
Comps
4m
4n
IC50 (µM)
0.016 ± 0.003
0.014 ± 0.005
0.011 ± 0.004
0.026 ± 0.001
0.020 ± 0.002
0.020 ± 0.000
0.017 ± 0.001
0.012 ± 0.002
0.009 ± 0.002
0.019 ± 0.000
0.030 ± 0.005
0.018 ± 0.010
0.027 ± 0.005
0.027 ± 0.010
0.021 ± 0.012
0.067 ± 0.009
0.058 ± 0.001
0.046 ± 0.004
0.037 ± 0.001
0.116 ± 0.020
0.046 ± 0.004
0.052 ± 0.016
0.176 ± 0.062
0.002 ± 0.001
4b
4c
4o
4d
4e
4p
4q
4f
4r
4g
4s
4h
4i
4t
4u
4j
4v
4k
NVP
EFV
4l
a Data represent the mean values of at least two separate experiments.
b IC50: inhibitory concentration of test compound required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into WT
HIV-1 RT by 50%.
2.4. Molecular modeling analysis
To investigate the binding models and rationalize the
structure–activity relationship (SAR) results of the newly
5

synthesized compounds, 4b and 4u were docked into the
theoretical binding models of these compounds are shown in
Fig. 4.
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non-nucleoside inhibitor-binding pocket (NNIBP) of HIV-1
RT using the software surflex-Docking Sybyle-X 1.2. The
Fig. 4. The predicted binding models of the compounds 4b (B) and 4u (C) with RT WT (PDB ID: 2ZD1); the binding model of 4b (D)
with the double-mutant F227L+V106A (mutated by 2ZD1 using Sybyl). The whole HIV-1 enzyme is shown as the colored ribbon (A).
The results showed that both of the molecules bind to the
NNIBP in a “U” shape, similarly to most other DAPY
compounds. The NH group linking the pyrimidine ring to
4-cyanophenyl formed two hydrogen bonds with the amino
acid residues Lys101 and Lys103. The biphenyl group fitted
well into the aromatic-rich subpocket consisting of amino acid
residues Tyr181, Tyr188, Phe227 and Trp229, and exhibited
the positive face-to-face π-π stacking interactions with these
aromatic amino acid residues.
achieved by the free rotation of the σ-bond between the two
phenyl rings. Furthermore, one of the chlorine atoms in
compound 4u protruded toward the phenyl ring of Phe227
resulting in a dipole–dipole interaction with NNIBP induced
by the strong electronegativity of the chloride atoms. All of
these factors decreased the biological activity of the
dichlorine-substituted DAPY analogue.
The interaction model between compound 4b and the
double mutant of HIV-1 RT (V106A and F227L) derived from
WT HIV-1 (2ZD1) indicated that the aromatic interaction
between the biphenyl group of 4b and the double mutant
HIV-1 RT NNBP was lost due to the lack of the phenyl ring of
L227 (the red line in D versus that in B and C in Fig. 4). The
According to this model, the bulky steric effects of the
double chlorine atoms in 4u might constrain the flexibility of
the two phenyl rings connected by a single bond. A suitable
pharmacological conformation for NNIBP could not be
6

distance between 4b and A106 in the NNIBP increased
All of these effects contributed toward enhancing the π-π
stacking effect between the target molecules and the aromatic
amino acid residues in NNBP that are necessary to maintain
the desired biological activity. Further optimization of these
biphenyl-substituted diarylpyrimidine derivatives to improve
their drug resistance profiles and poor solubility is an ongoing
part of our research.
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significantly, resulting in the loss of the intermolecular
interaction between 4b and the mutated amino acid residue
(the blue line in D versus the green line B and C in Fig. 4). All
of these observations at the molecular level might explain the
significant decrease in biological activity of compound 4b
against the HIV-1 double mutant (V106A and F227L).
Based on SAR analysis and molecular modelling, it might
also be concluded that introducing a bulky group to the
biphenyl ring might be detrimental for the aromatic interaction
with the target enzyme, thus decreasing the biological activity.
The cyano group at the para-position of phenyl ring IV also
extended the length of the conjugation system of the target
molecule, thus enhancing the aromatic interaction with the
active binding pocket of this enzyme. Both the SAR summary
and the results of docking analysis supported each other,
providing the useful insight that will aid the future design of
novel HIV-1 NNRTIs.
4. Experiment section
4.1. Chemistry
Chemical reagents and solvents, purchased from
commercial sources, were of analytical grade and were used
without further purification. All air-sensitive reactions were
run under a nitrogen atmosphere. All the reactions were
monitored by TLC on the re-coated silica gel G plates at 254
nm under a UV lamp using ethyl acetate/n-hexane as the
eluent. Flash column chromatography was performed on glass
column packed with silica gel (200-300 mesh) using ethyl
acetate/n-hexane as the eluent. Melting points were measured
In general, the results of the modelling study supported
our original designing hypothesis and deepened our
understanding of the interactions between these compounds
and the HIV-1 RT NNIBP.
1
on a SGW X-1 microscopic melting point apparatus. H NMR
and 13C NMR spectra were recorded on a Bruker AV400 MHz
spectrometer in DMSO-d6. Chemical shifts were reported in δ
(ppm) units relative to the internal standard tetramethylsilane
(TMS). Mass spectra and HRMS were obtained on a Waters
Quattro Micromass instrument and Bruker Compact
instrument, respectively, using electrospray ionization (ESI)
techniques. The purities of the target compounds were ≥ 95%,
measured by HPLC, performed on an Agilent 1200 HPLC
system with UV detector and Agilent Eclipse Plus C18 column
(150 x 4.6 mm, 5 mm), eluting with a mixture of solvents H2O
(A) and CH3CN (B) from VA:VB=90:10 to 10:90. Peaks were
detected at λ 254 nm with a flow rate of 1.0 mL/min.
4.1.1 General procedure to prepare the intermediates of 7, 8
and 10.
3. Conclusion
In summary, 22 new HIV-1 RT inhibitors with a
pyrimidine and biphenyl group skeleton were synthesized and
evaluated for their biological activity. Exploiting the
aromatic-rich region in the NNIBP via introducing a biphenyl
group to the left wing of DAPYs was shown to be a successful
strategy for the design of potential HIV-1 RT inhibitors, which
was further supported by molecular modelling analysis. Most
of the new target compounds showed excellent activity against
WT HIV-1 with EC50 values in the single-digit nanomolar
concentration range. Some of the compounds exhibited good
activity with low nanomolar EC50 values against NNRTI
resistant mutant virus in MT-4 cells. Five of the 22 compounds
exhibited high potency with EC50 values of less than 2 nM.
Two compounds showed equivalent potency to ETV, while
three compounds were more potent than ETV. The compound
with the highest activity showed low nanomolar values against
most single and double mutant strains. Preliminary SAR
analysis of this series of DAPYs showed that besides retaining
the 2,6-dimethyl groups on the left ring of DAPYs,
introducing a para-substituted cyano group on the phenyl ring
(IV) might be crucial for enhancing the biological activity
against both the WT and mutant HIV-1. Furthermore,
2,6-dimethyl groups could enhance the electron density while
contributing to the conjugation system of the whole molecule.
The intermediates including 7 and 9 were obtained easily
according to the improved procedure published previously
[16-18, 21].
4.1.2 General procedure to prepare 4a-v
.
A mixture of 4-((4-((4-bromo-2,6-dimethylphenyl)amino)
pyrimidin-2-yl)amino)benzonitrile (10) (0.394 g, 1.00 mmol),
Pd(dppf)Cl2 (0.036 g, 0.05 mmol), K2CO3 (0.27 g, 2.00 mmol)
and one of the various kinds of arylboronic acid (1.00 mmol)
in an aqueous solution of dioxane (15 mL, dioxane : H2O, v:
v=10:1) was heated to 110 °C under N2 atmosphere for 6-8 h.
The reaction was monitored by TLC analysis until its
completion. The reaction mixture was then poured into brine
(50 mL), before being extracted with ethyl acetate (40 mL×3).
7

The organic layer was separated before being dried with
3277, 3180, 2227, 1582, 1528, 1423; HRMS calcd for
C24H20N6 [M+H]+: 417.1822, found: 417.1815.
4.1.2.4.
ACCEPTED MANUSCRIPT
anhydrous Na2SO4. The mixture was then concentrated under
the reduced pressure. The crude product was purified by
column chromatography to afford the target compound, with
the yield of 40-60%, respectively.
4-((4-((4'-chloro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)pyri
midin-2-yl)amino)benzonitrile (4d)
4.1.2.1.
4-((4-((3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)
The title product was synthesized following the general
procedure, starting from 9 and 4-chlorophenylboronic acid
pyrimidin-2-yl)amino)benzonitrile (4a)
1
The title product was synthesized following the general
(10d). Yield 51%, white solid, mp 271-273 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H, NH), 8.88 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.50-7.76 (m, 10H, ArH), 6.37 (s, 1H,
pyrimidine-H), 2.23 (s, 6H, CH3×2); 13C NMR (101 MHz,
DMSO-d6) δ: 162.47, 159.77, 156.14, 146.10, 139.13,
137.11,137.03, 132.89, 132.68, 129.33, 128.77, 126.69, 126.63,
120.17, 118.43, 101.77, 99.24, 18.90; IR: υ 3280, 3185, 2221,
1586, 1521, 1423 cm-1; HRMS calcd for C25H20ClN5 [M+H]+:
426.1480, found: 426.1480.
procedure, starting from 9 and phenylboronic acid (10a). Yield
1
43%, white solid, m.p. 227-230 °C (EA/PE); H NMR (400
MHz, DMSO-d6) δ: 9.56 (s, 1H, NH), 8.88 (s, 1H，NH), 8.02
(s, 2H, ArH), 7.69-7.71 (m, 4H, ArH), 7.48-7.50 (m, 4H, ArH),
7.35-7.39 (m, 2H, ArH), 6.38 (s, 1H, pyrimidine-H), 2.24 (s,
6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ: 162.49,
159.77, 156.12, 146.11, 140.39, 138.63, 136.92, 132.86,
129.39, 127.80, 127.04, 126.71, 126.68, 120.16, 118.45,
101.69, 99.26, 18.92; IR: υ 3297, 3179, 2214, 1586, 1523,
1426 cm-1; HRMS calcd for C24H20N6 [M+H]+:392.1870,
found: 393.1860.
4.1.2.5.
4-((4-((3'-chloro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)pyri
midin-2-yl)amino)benzonitrile (4e)
4.1.2.2.
The title product was synthesized following the general
procedure, starting from 9 and 3-chlorophenylboronic acid
4'-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)amino)-3',5'-dim
ethyl-(1,1'-biphenyl)-4-carbonitrile (4b)
1
(10e). Yield 60%, white solid, mp 245-248 °C (EA/PE); H
The title product was synthesized following the general
procedure, starting from 9 and 4-cyanophenylboronic acid
NMR (400 MHz, DMSO-d6) δ: 9.56 (s, 1H, NH), 8.89 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.78-7.69 (m, 4H, ArH), 7.54-7.49 (m,
3H, ArH), 7.41-7.45 (m, 2H, ArH), 6.36 (s, 1H, pyrimidine-H),
2.24 (s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ:
162.46, 159.77, 156.17, 146.09, 142.52, 137.12, 137.10,
134.25, 132.93, 132.91, 132.85, 131.20, 127.62, 126.77,
125.69 , 120.15, 118.44, 118.43, 101.75, 99.27, 18.78; IR: υ
3294, 3176, 2218, 1582, 1524, 1422 cm-1; HRMS calcd for
C25H20ClN5 [M+H]+: 426.1480, found: 426.1470.
1
(10b). Yield 54%, white solid, m.p. 284-286 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.58 (s, 1H, NH), 8.91 (s, 1H,
NH), 7.98 (m, 6H, ArH), 7.65 (m, 4H, ArH), 7.39 (s, 1H,
pyrimidine-H), 6.38 (s, 1H, pyrimidine-H), 2.25 (s, 6H, CH3×
2); 13C NMR (101 MHz, DMSO-d6) δ: 162.41, 159.77 , 156.34,
146.07, 144.80, 137.28,137.25, 133.30, 132.97, 132.93, 127.87,
127.12, 120.17, 119.39, 118.43, 110.36, 101.81, 99.32, 18.89;
IR: υ 3371, 3185, 2221, 1581, 1525, 1413 cm-1; HRMS calcd
for C24H20N6 [M+H]+:417.1822, found:417.1812.
4.1.2.6.
4-((4-((2'-chloro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)pyri
midin-2-yl)amino)benzonitrile (4f)
4.1.2.3.
4'-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)amino)-3',5'-dim
ethyl-(1,1'-biphenyl)-3-carbonitrile (4c)
The title product was synthesized following the general
procedure, starting from 9 and 2-chlorophenylboronic acid
The title product was synthesized following the general
procedure, starting from 9 and 3-cyanophenylboronic acid
1
(10f). Yield 55%, white solid, mp 230-232°C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.61 (s, 1H, NH), 8.87 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.71-7.59 (m, 3H, ArH), 7.44-7.40 (m,
4H, ArH), 7.25 (s, 2H, ArH), 6.37 (s, 1H, pyrimidine-H), 2.22
(s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ: 162.45,
159.80, 156.51, 146.09, 140.13, 132.93, 131.93, 131.77,
130.35, 129.56, 127.93, 120.18, 118.41, 101.56, 99.06, 18.96;
IR: υ 3301, 3179, 2214, 1586, 1524, 1427 cm-1; HRMS calcd
for C25H20ClN5 [M+H]+: 426.1480, found: 426.1478.
1
(10c). Yield 49%, white solid, mp>300 °C (EA/PE); H NMR
(400 MHz, DMSO-d6) δ: 9.57 (s, 1H, NH), 8.89 (s, 1H, NH),
8.22 (s, 1H, ArH), 8.09-8.07 (m, 3H, ArH), 7.84-7.60 (m, 6H,
ArH), 7.40 (s, 1H, pyrimidine-H), 6.37 (s, 1H, pyrimidine-H),
2.25 (s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ:
162.44, 159.77, 156.08, 146.08, 141.38, 137.21, 132.96,
132.88, 131.74, 131.36, 130.59, 126.97, 126.91, 126.87,
120.16, 119.31, 118.42, 112.62, 101.78, 99.34, 18.86; IR: υ
8

4.1.2.7
The title product was synthesized following the general
ACCEPTED MANUSCRIPT
4-((4-((4'-fluoro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)pyri
procedure, starting from 9 and 4-methylbenzeneboronic acid
1
midin-2-yl)amino)benzonitrile (4g)
(10j). Yield 45%, white solid, mp 208-210 °C (EA/PE); H
The title product was synthesized following the general
procedure, starting from 9 and 4-fluorophenylboronic acid
NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H, NH), 8.86 (s, 1H,
NH), 8.01 (s, 2H, ArH), 7.70-7.60 (m, 4H, ArH), 7.46-7.28 (m,
6H, ArH), 6.36 (s, 1H, pyrimidine-H), 2.36 (s, 3H, CH3), 2.22
(s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ: 162.51,
159.77, 156.16, 146.12, 138.44, 137.07, 136.79, 132.82 ,
130.00, 126.85, 126.39,126.36, 120.16, 118.42, 103.97, 101.65,
99.37, 21.15, 18.92; IR: υ 3286, 3195, 2219, 1585, 1525, 1424
cm-1; HRMS calcd for C26H23N5 [M+H]+: 406.2026, found:
406.2019.
1
(10g). Yield 46%, white solid, mp 246-248 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H, NH), 8.87 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.70-7.77 (m, 4H, ArH), 7.47 (s, 2H,
ArH), 7.28-7.33 (m, 3H, ArH), 6.36 (s, 1H, pyrimidine-H),
2.23 (s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ:
163.51, 162.49, 161.08, 159.77, 156.09, 146.11, 136.94,
132.87, 129.02, 128.94, 126.62, 120.17 (s), 118.43, 116.26,
116.04, 101.68, 99.26, 18.91; IR: υ 3278, 3181, 2216, 1587,
1520, 1423 cm-1; HRMS calcd for C25H20FN5 [M+H]+:
410.1776, found: 410.1765.
4.1.2.11. 4-((4-((3,3',5-trimethyl-(1,1'-biphenyl)-4-yl)amino)
pyrimidin-2-yl)amino)benzonitrile (4k)
The title product was synthesized following the general
4.1.2.8.
procedure, starting from 9 and 3-methylbenzeneboronic acid
1
4-((4-((3'-fluoro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)pyri
midin-2-yl)amino)benzonitrile (4h)
(10k). Yield 40%, white solid, mp 191-194 °C (EA/PE); H
NMR (400 MHz, Acetone-d6) δ: 8.75 (s, 1H, NH), 8.05 (m, 3H,
ArH), 7.80 (s, 1H, NH), 7.56-7.50 (m, 5H, ArH), 7.38 (m, 2H,
ArH), 7.21 (d, J = 7.4 Hz, 1H, ArH), 6.43 (s, 1H,
pyrimidine-H), 2.44 (s, 3H, CH3), 2.31 (s, 6H, CH3×2); 13C
NMR (101 MHz, Acetone-d6) δ: 162.70, 159.77, 156.56,
145.60, 140.62, 138.32, 137.00, 132.54, 128.76, 128.00,
127.57, 126.73, 126.65, 126.59, 123.93, 119.23, 118.19,
102.71, 98.77, 20.68, 17.85; IR: υ 3284, 3195, 2222, 1584,
1524, 1418 cm-1; HRMS calcd for C26H23N5 [M+H]+:
406.2026, found: 406.2014.
The title product was synthesized following the general
procedure, starting from 9 and 3-fluorophenylboronic acid
1
(10h). Yield 47%, white solid, mp 249-251 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.57 (s, 1H, NH), 8.89 (s, 1H,
NH), 7.99 (s, 2H, ArH), 7.73-7.51 (m, 7H, ArH), 7.22 (s, 1H,
ArH), 7.19 (dd, J = 13.2, 5.1 Hz, 1H, ArH), 6.37 (s, 1H,
pyrimidine-H), 2.24 (s, 6H, CH3×2); 13C NMR (101 MHz,
DMSO-d6) δ: 164.43, 162.47, 162.01, 159.77, 156.15, 146.10,
142.85, 137.04, 132.89, 131.32, 131.23, 126.83, 123.06,
120.16, 118.43, 114.58, 114.37, 113.81, 113.58, 101.76, 99.28,
18.90; IR: υ 3294, 3179, 2219, 1582, 1525, 1424 cm-1;HRMS
calcd for C25H20FN5 [M+H]+: 410.1776, found: 410.1777.
4.1.2.9.
4.1.2.12. 4-((4-((2',3,5-trimethyl-(1,1'-biphenyl)-4-yl)amino)
pyrimidin-2-yl)amino)benzonitrile (4l)
The title product was synthesized following the general
procedure, starting from 9 and 2-methylbenzeneboronic acid
1
4-((4-((2'-fluoro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)pyri
midin-2-yl)amino)benzonitrile (4i)
(10l). Yield 57%, white solid, mp 241-243 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.60 (s, 1H, NH), 8.84 (s, 1H,
NH), 8.02 (s, 1H, ArH), 7.70 (s, 2H, ArH), 7.41-7.24 (m, 6H,
ArH), 7.15 (s, 2H, ArH), 6.37 (s, 1H, pyrimidine-H), 2.30 (s,
3H, CH3), 2.22 (s, 6H, CH3 ×2); 13C NMR (101 MHz,
DMSO-d6) δ: 162.47, 159.80, 156.19, 146.13, 141.56, 139.81,
136.08, 135.13, 132.85, 130.80, 129.88, 128.97, 127.71,
126.35, 120.18, 118.40, 101.62, 99.14, 20.72, 18.87; IR: υ
3304, 3179, 2213, 1588, 1524, 1428 cm-1; HRMS calcd for
C26H23N5 [M+H]+: 406.2026, found: 406.2024.
The title product was synthesized following the general
procedure, starting from 9 and 2-fluorophenylboronic acid
1
(10i). Yield 50%, white solid, mp 227-229 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.59 (s, 1H, NH), 8.90 (s, 1H,
NH), 8.02 (s, 1H, ArH), 7.56-7.54 (m, 2H, ArH), 7.42 (t, J =
7.9 Hz, 1H, ArH), 7.36-7.30 (m, 7H, ArH), 6.37 (s, 1H,
pyrimidine-H), 2.22 (s, 6H, CH3×2); 13C NMR (101 MHz,
DMSO-d6) δ: 162.47, 160.84, 159.77, 158.39, 156.16, 146.09,
136.60, 132.87, 131.24, 131.21, 129.87, 128.83, 125.40,125.36,
120.18, 118.42, 116.70, 116.47, 101.70, 99.30, 18.85; IR: υ
3282, 3186, 2222, 1588, 1521, 1423 cm-1; HRMS calcd for
C25H20FN5 [M+H]+: 410.1776, found: 410.1769.
4.1.2.13.
4-((4-((4'-methoxy-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)py
rimidin-2-yl)amino)benzonitrile (4m)
The title product was synthesized following the general
4.1.2.10. 4-((4-((3,4',5-trimethyl-(1,1'-biphenyl)-4-yl)amino)
pyrimidin-2-yl)amino)benzonitrile (4j)
procedure, starting from 9 and 4-methoxyphenylboronic acid
1
(10m). Yield 52%, white solid, mp 202-204 °C (EA/PE); H
9

NMR (400 MHz, DMSO-d ) δ: 9.55 (s, 1H, NH), 8.85 (s, 1H,
(EA/PE); 1H NMR (400 MHz, DMSO-d ) δ: 9.58 (s, 1H, NH),
ACCEPTED MANUSCRIPT
6
6
NH), 8.07-8.02 (m, 2H, ArH), 7.71-7.64 (m, 4H, ArH),
8.92 (s, 1H, NH), 8.03-7.92 (m, 4H, ArH), 7.82-7.72 (m, 4H,
ArH), 7.56 (s, 2H, ArH), 7.37 (s, 1H, pyrimidine-H), 6.39 (s,
1H, pyrimidine-H), 2.25 (s, 6H, CH3×2); 13C NMR (101 MHz,
DMSO-d6) δ: 162.43, 159.76, 156.25, 146.07, 144.27, 137.24,
137.22, 132.90, 132.87, 128.03, 127.74, 127.02, 127.00,
126.22,126,18, 123.52, 120.14, 118.44, 101.79, 99.30, 18.87;
IR: υ 3280, 3195, 2222, 1585, 1523, 1422 cm-1; HRMS calcd
for C26H20F3N5 [M+H]+:460.1744, found: 460.1737.
4.1.2.17.
7.37-7.43 (m, 3H, ArH), 7.03-7.05 (m, 2H, ArH), 6.36 (s, 1H,
pyrimidine-H), 3.81 (s, 3H, OCH3), 2.22 (s, 6H, CH3×2); 13C
NMR (101 MHz, DMSO-d6) δ: 162.55, 159.78, 159.31, 155.98,
146.14, 138.24, 136.78, 136.75, 132.86, 132.81, 128.11,
126.14, 120.18, 118.43, 114.82, 101.64, 99.13, 55.64, 18.95;
IR: υ 3288, 3181, 2219, 1586, 1516, 1423 cm-1; HRMS calcd
for C26H23N5O [M+H]+: 422.1975, found: 422.1971.
4.1.2.14.
4-((4-((3'-methoxy-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)py
rimidin-2-yl)amino)benzonitrile (4n)
4-((4-((3,5-dimethyl-3'-(trifluoromethyl)-(1,1'-biphenyl)-4-yl)a
mino)pyrimidin-2-yl)amino)benzonitrile (4q)
The title product was synthesized following the general
The title product was synthesized following the general
procedure, starting from 9 and 3-(trifluoromethyl)phenyl
boronic acid (10q). Yield 56%, white solid, mp 180-182 °C
(EA/PE); 1H NMR (400 MHz, DMSO-d6) δ: 9.58 (s, 1H, NH),
8.92 (s, 1H, NH), 8.02 (s, 3H, ArH), 7.72 (s, 4H, ArH), 7.59 (s,
2H, ArH), 7.37 (s, 2H, ArH), 6.39 (s, 1H, pyrimidine-H), 2.26
(s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ: 162.46,
159.78, 156.24, 146.09, 141.44, 137.23, 132.88, 131.07,
130.78 , 130.47, 130.16, 129.84, 128.80, 126.96, 126.09,
124.35, 123.46, 123.39, 120.06, 118.42, 101.79, 99.28, 18.83;
IR: υ 3300, 3175, 2221, 1584, 1524, 1424 cm-1; HRMS calcd
for C26H20F3N5 [M+H]+: 460.1744, found: 460.1727.
4.1.2.18.
procedure, starting from 9 and 3-methoxyphenylboronic acid
1
(10n). Yield 48%, white solid, mp 239-242 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.57 (s, 1H, NH), 8.89 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.70 (s, 2H, ArH), 7.50 (s, 2H, ArH),
7.39-7.37 (m, 2H, ArH), 7.28-7.24 (m, 2H, ArH), 6.95 (d, J =
8.0 Hz, 1H, ArH), 6.37 (s, 1H, pyrimidine-H), 3.86 (s, 3H,
OCH3), 2.24 (s, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6)
δ: 162.47, 160.24, 159.77, 156.16, 146.13, 141.99, 138.60,
136.82, 132.81, 130.41, 126.79, 120.18, 119.41, 118.43,
118.41, 113.53, 112.44, 101.65, 99.19, 55.61, 18.93; IR: υ
3302, 3175, 2219, 1582, 1522, 1422 cm-1; HRMS calcd for
C26H23N5O [M+H]+: 422.1975, found: 422.1968.
4.1.2.15.
4-((4-((3,5-dimethyl-2'-(trifluoromethyl)-(1,1'-biphenyl)-4-yl)a
mino)pyrimidin-2-yl)amino)benzonitrile (4r)
4-((4-((2'-methoxy-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)py
rimidin-2-yl)amino)benzonitrile (4o)
The title product was synthesized following the general
procedure, starting from 9 and 2-(trifluoromethyl)phenyl
boronic acid (10r). Yield 51%, white solid, mp 228-231 °C
(EA/PE); 1H NMR (400 MHz, DMSO-d6) δ: 9.63 (s, 1H, NH),
8.86 (s, 1H, NH), 8.02 (s, 2H, NH), 7.85 (d, J = 7.7 Hz, 1H,
ArH), 7.73-7.62 (m, 4H, ArH), 7.44-7.42 (m, 2H, ArH), 7.14 (s,
2H, ArH), 6.37 (s, 1H, pyrimidine-H), 2.21 (s, 6H, CH3×2);
13C NMR (101 MHz, DMSO-d6) δ: 162.37, 159.82, 156.16,
146.07, 142.71, 141.02, 137.93, 135.81, 132.89, 132.66,
132.58, 128.78, 128.43, 127.45, 127.16, 126.53, 126.12,
123.40, 120.15, 118.29, 101.72, 99.20, 18.87; IR: υ 3232, 2960,
2222, 1580, 1527, 1412 cm-1; HRMS calcd for C26H20F3N5
[M+H]+: 460.1744, found: 460.1737.
The title product was synthesized following the general
procedure, starting from 9 and 2-methoxyphenylboronic acid
1
(10o). Yield 42%, white solid, mp 270-272 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.60 (s, 1H, NH), 8.85 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.70 (s, 2H, ArH), 7.38-7.27 (m, 5H,
ArH), 7.15 (d, J = 7.6 Hz, 1H, ArH), 7.05 (t, J = 7.3 Hz, 1H,
ArH), 6.36 (s, 1H, pyrimidine-H), 3.83 (s, 3H, OCH3), 2.20 (s,
6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ: 162.52,
159.79, 156.63, 156.15, 146.14, 136.84, 136.78, 132.87,
130.91, 130.10, 129.24, 121.21, 120.22, 118.42, 112.17,
101.59, 99.04, 55.96, 18.90; IR: υ 3384, 3188, 2220, 1579,
1526, 1411 cm-1; HRMS calcd for C26H23N5O [M+H]+:
422.1975, found: 422.1976.
4.1.2.19.
4.1.2.16.
4-((4-((2',3,5,6'-tetramethyl-(1,1'-biphenyl)-4-yl)amino)pyrimi
din-2-yl)amino)benzonitrile (4s)
4-((4-((3,5-dimethyl-4'-(trifluoromethyl)-(1,1'-biphenyl)-4-yl)a
mino)pyrimidin-2-yl)amino)benzonitrile (4p)
The title product was synthesized following the general
procedure, starting from 9 and 2, 6-xylylboronic acid (10s).
The title product was synthesized following the general
procedure, starting from 9 and 4-(trifluoromethyl)phenyl
boronic acid (10p). Yield 49%, white solid, mp 203-205 °C
1
Yield 50%, white solid, mp 189-192 °C (EA/PE); H NMR
(400 MHz, DMSO-d6) δ: 9.63 (s, 1H, NH), 8.83 (s, 1H, NH),
10

8.07-8.01 (m, 2H, ArH), 7.69 (s, 2H, ArH), 7.39 (s, 1H,
pyrimidine-H), 7.12-7.14 (m, 3H, ArH), 6.96 (s, 2H, ArH),
6.36 (s, 1H, pyrimidine-H), 2.21 (s, 6H, CH3×2), 2.05 (d, J =
25.0 Hz, 6H, CH3×2); 13C NMR (101 MHz, DMSO-d6) δ:
162.47, 159.83, 156.26, 146.11, 141.59, 136.26, 135.77,
135.55, 132.91, 128.79, 127.69, 127.39, 120.12, 118.54,
118.14, 101.57, 99.01, 21.06, 18.91; IR: υ 3293, 3184, 2222,
1582, 1521, 1420 cm-1; HRMS calcd for C27H25N5 [M+H]+:
420.2183, found: 420.2165.
pyrimidine-H), 6.37 (s, 1H, pyrimidine-H), 2.23 (s, 6H, CH ×
2); 13C NMR (101 MHz, DMSO-d6) δ: 162.39, 159.75, 156.50,
ACCEPTED MANUSCRIPT
3
146.04, 143.86, 143.82, 142.15, 137.33, 137.26, 135.17,
132.98, 132.93, 127.13, 126.99, 125.65, 120.12, 118.42,
101.78, 99.14, 18.52; IR: υ 3264, 3177, 2222, 1582, 1524
1423 cm-1; HRMS calcd for C25H19F2N5 [M+H]+: 428.1681,
found: 428.1675.
4.2. In vitro anti-HIV assay
4.1.2.20.
The anti-HIV activity and cytotoxicity of all the
compounds were evaluated against WT HIV-1 (HIV-IIIB
strain), single RT mutant strains (L100I, K103N, Y181C,
Y188L, and E138K), double RT mutant strains
(F227L+V106A and K103N+Y181C), and HIV-2 (strain
ROD) in MT-4 cell cultures in MT-4 cell cultures using the
4-((4-((3,3',5,5'-tetramethyl-(1,1'-biphenyl)-4-yl)amino)pyrimi
din-2-yl)amino)benzonitrile (4t)
The title product was synthesized following the general
procedure, starting from 9 and 3, 5-xylylboronic acid (10t).
1
Yield 58%, white solid, mp 223-225 °C (EA/PE); H NMR
(400 MHz, DMSO-d6) δ: 9.56 (s, 1H, NH), 8.88 (s, 1H, NH),
8.01 (s, 2H, ArH), 7.66 (s, 2H, ArH), 7.45 (s, 2H, ArH), 7.31 (s,
3H, ArH), 7.01 (s, 1H, ArH), 6.36 (s, 1H, pyrimidine-H), 2.36
(s, 6H, CH3×2), 2.22 (s, 6H, CH3×2); 13C NMR (101 MHz,
DMSO-d6) δ: 162.50, 159.74, 156.18 , 146.10, 140.46, 138.37,
136.84, 132.82, 129.22, 126.68, 124.93, 120.16, 120.12,
118.44, 118.42, 101.62, 99.14, 21.48, 18.86; IR: υ 3288, 3174,
2220, 1583, 1524, 1423 cm-1; HRMS calcd for C27H25N5
[M+H]+: 420.2183, found: 420.2164.
method
of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT)[24].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 medium at 1×105
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 effects in
uninfected MT-4 cells.
4.1.2.21.
4-((4-((3',5'-dichloro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)
pyrimidin-2-yl)amino)benzonitrile (4u)
The title product was synthesized following the general
procedure, starting from 9 and 3, 5-dichlorophenylboronic
acid (10u). Yield 48%, white solid, mp 256-259 °C (EA/PE);
1H NMR (400 MHz, DMSO-d6) δ: 9.54 (s, 1H, NH), 8.90 (s,
1H, NH), 8.02 (s, 2H, ArH), 7.75-7.66 (m, 4H, ArH),
7.56-7.51 (m, 3H, ArH), 7.36 (s, 1H, pyrimidine-H), 6.37 (s,
1H, pyrimidine-H), 2.23 (s, 6H, CH3×2); 13C NMR (101 MHz,
DMSO-d6) δ: 162.41, 159.75, 156.24, 146.03, 143.83, 137.26,
137.23, 135.16, 132.93, 127.10, 126.94, 126.35, 125.63,
120.13, 118.42, 101.80, 99.18, 18.78; IR: υ 3262, 3177, 2221,
1582, 1523, 1423 cm-1; HRMS calcd for C25H19Cl2N5 [M+H]+:
460.1090, found: 460.1078.
4.3. HIV-1 RT inhibition assay
Recombinant wild type p66/p51 HIV-1 RT was expressed
and purified as previously described[25]. 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[26]. This condition was met in the assay.
4.1.2.22.
4-((4-((3',5'-difluoro-3,5-dimethyl-(1,1'-biphenyl)-4-yl)amino)
pyrimidin-2-yl)amino)benzonitrile (4v)
A poly(rA) template of approximately 350 bases long and
anoligo(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 96-well plate in a volume of 20 mL
polymerization buffer (60 mM Trise HCl, 60 mM KCl, 8 mM
The title product was synthesized following the general
procedure, starting from 9 and 3, 5-difluorophenylboronic acid
1
(10v). Yield 47%, white solid, mp 265-268 °C (EA/PE); H
NMR (400 MHz, DMSO-d6) δ: 9.56 (s, 1H, NH), 8.90 (s, 1H,
NH), 8.02 (s, 2H, ArH), 7.78-7.56 (m, 7H, ArH), 7.38 (s, 1H,
11

MgCl , 13 mM DTT, 100 mM dTTP, pH = 8.1). 5.0 mL of RT
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-scoring ligand-protein complexes were kept for the
further analyses. The -log(Kd)2 values of the 20-best-scoring
complexes, which represented the binding affinities of the
ligand with RT, encompassed a wide scope of the functional
classes (10-2 - 10-9). Only the highest scoring 3D structural
model of the ligand-bound RT was chosen to define the
binding interaction[30, 31].
ACCEPTED MANUSCRIPT
2
enzyme solution, diluted to a suitable concentration in an
enzyme dilution buffer (50 mM Trise-HCl, 20% glycerol,
2mM DTT, pH = 7.6), was added. The reaction mixture was
incubated at 25℃ for 40 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 spectrofluo-rometer (Safire 2, Tecan). To test the biologcial
activity of compounds against RT, 1.0 mL of compound in
DMSO was added to each well before the addition of RT
enzyme solution. Control wells without compound contained
the same amount of DMSO. Results were expressed as relative
fluorescence, i.e. the fluorescence signal of the reaction mixed
with compound divided by the signal of the same reaction
mixed without compound.
4.4. Molecular docking
Molecular modelling research work was performed with
the Tripos molecular modelling software packages (Sybyl-X
1.2). All the molecules for docking analysis were built using
the standard bond lengths and angles from Sybyl-X 1.2/base
Builder before being optimized using the Tripos force field for
10000 generations two times or more, until the minimized
conformers of the ligand were the same. The flexible docking
method, called Surflex-Dock[27], docks the ligand
automatically into the ligand binding site of the receptor by
using a protocol-based approach and an empirically-derived
Acknowledgement
This research was financially supported by National
Natural Science Foundation of P. R. China under Grant No.
21372050, Shanghai Municipal Natural Science Foundation
under Grant No.13ZR1402200.
Supplementary Material
Supplementary data related to this article can be found in
the online version at the web site.
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ACCEPTED MANUSCRIPT
Highlights
1. Twenty-two new compounds were designed、synthesized and evaluated for their in vitro anti-HIV
activity as well as their anti-drug resistant activity.
2. Five out 22 compounds exhibited high potency with EC50(ⅢB) values of less than 2 nM (4b, 4c, 4g,
4h, and 4i).
3. Compound 4b also showed low nanomolar against most single-mutant and double-mutant strains
(≤11nM) except the double-mutant F227L+V106A (≥110nM).
4. The preliminary SAR of this series of compounds and docking analysis between the typical
compound 4b and 4u with wild type and mutant type of HIV RT were also investigated.
5.The interaction model indicated that the aromatic interaction between the biphenyl group of 4b
and the double mutant (F227L+V106A) disappeared for lacking the phenyl ring of L227.