Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent non-nucleoside reverse transcriptase inhibitors of wild-type and mutant HIV-1 strains

Article abstract of DOI:10.1016/j.ejmech.2019.05.011

A novel series of dihydroquinazolin-2-amine derivatives were synthesized and evaluated for their anti-HIV-1 activity in MT-4 cell cultures. All of the molecules were active against wild-type HIV-1 with EC₅₀ values ranging from 0.61 μM to 0.84 nM. The most potent inhibitor, compound 4b, had an EC₅₀ value of 0.84 nM against HIV-1 strain IIIB, and thus was more active than the reference drugs efavirenz and etravirine. Moreover, most of the compounds maintained high activity (low-micromolar EC₅₀ values)against strains bearing the reverse transcriptase (RT)E138K mutation. Compound 4b had EC₅₀ values of 3.5 nM and 66 nM against non-nucleoside reverse transcriptase inhibitor-resistant strains bearing the RT E138K and RES056 mutations. In enzyme activity assays, compound 4b exhibited an IC₅₀ value of 10 nM against HIV-1 RT. Preliminary SARs and molecular docking studies provide valuable insights for further optimization.

Full text of DOI:10.1016/j.ejmech.2019.05.011

Accepted Manuscript
Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent non-
nucleoside reverse transcriptase inhibitors of wild-type and mutant HIV-1 strains
KaiJun Jin, YaLi Sang, Sheng Han, Erik De Clercq, Christophe Pannecouque, Ge
Meng, FenEr Chen
PII:
S0223-5234(19)30420-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.011
EJMECH 11321
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 February 2018
Revised Date: 5 May 2019
Accepted Date: 6 May 2019
Please cite this article as: K. Jin, Y. Sang, S. Han, E. De Clercq, C. Pannecouque, G. Meng, F. Chen,
Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent non-nucleoside reverse
transcriptase inhibitors of wild-type and mutant HIV-1 strains, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.011.
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Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent
non-nucleoside reverse transcriptase inhibitors of wild-type and mutant HIV-1
strains
KaiJun Jin a, b, YaLi Sang a, b, Sheng Han a, b, 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
* Corresponding authors.
1

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2

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent non-nucleoside reverse
transcriptase inhibitors of wild-type and mutant HIV-1 strains
KaiJun Jin a, b, YaLi Sang a, b, Sheng Han a, b, Erik De Clercqc, Christophe Pannecouquec, Ge Menga, b*, FenEr
Chen a, b*
a Engineering Center ofCatalysis and SynthesisforChiral 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
* Corresponding authors.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel series of dihydroquinazolin-2-amine derivatives were
synthesized and evaluated for their anti-HIV-1 activity in MT-4 cell
cultures. All of the molecules were active against wild-type HIV-1
with EC50 values ranging from 0.61 µM to 0.84 nM. The most
potent inhibitor, compound 4b, had an EC50 value of 0.84 nM
against HIV-1 strain IIIB, and thus was more active than the
reference drugs efavirenz and etravirine. Moreover, most of the
compounds maintained high activity (low-micromolar EC50 values)
against strains bearing the reverse transcriptase (RT) E138K
mutation. Compound 4b had EC50 values of 3.5 nM and 66 nM
against non-nucleoside reverse transcriptase inhibitor-resistant
strains bearing the RT E138K and RES056 mutations. In enzyme
activity assays, compound 4b exhibited an IC50 value of 10 nM
against HIV-1 RT. Preliminary SARs and molecular docking
studies provide valuable insights for further optimization.
Available online
Keywords:
Anti-HIV-1 activity;
Dihydroquinazolines;
DAPYs;
Reverse transcriptase;
Efavirenz;
Etravirine.

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1. Introduction
The life cycle of human immunodeficiency
1-3.
virus type 1 (HIV-1) involves reverse transcription
of its RNA genome to cDNA followed by
integration of DNA into the host cell genome. The
HIV-1 reverse transcriptase (RT) is critical for this
process and is a major target for antiretroviral
therapies [1]. RT inhibitors fall into two main
classes: nucleoside reverse transcriptase inhibitors
(NRTIs) and non-nucleoside reverse transcriptase
inhibitors (NNRTIs) [2, 3]. Despite their high
chemical diversity, most NNRTIs interact with RT
To varying degrees, use of NNRTIs results in
the emergence of resistance mutations, which has
required the continuous development of novel
agents active against resistant HIV-1 strains.
Therefore, the design and synthesis of NNRTIs
with high efficiency using novel skeletons has
become an urgent task in drug design.
Molecular hybridization (MH) is an important
method in the efficient design of new drugs, and
might be of benefit for discovering novel active site
or allosteric inhibitors of RT with distinct
mechanisms[8, 9]. In our previous modifications of
the above NNRTI structures, we used MH
successfully to develop several series of NNRTIs,
some of which showed potency against the WT
HIV-1 strain IIIB at low nanomolar concentrations
(1.8nM)[10-13]. Inspired by these results, we made
further attempts using this strategy to develop new
NNRTIs with novel chemical structures [12, 14,
15].
in
a
noncompetitive manner at the same
hydrophobic pocket (the NNRTI binding pocket or
NNIBP) located about 10 Å from the catalytic site
of RT. Due to their high specificity and low toxicity,
NNRTIs are key components of highly active
antiretroviral therapy（HAART） used to treat HIV
infection[4].
Efavirenz
(1,
EFV),
the
first-generation NNRTI, is widely used in the clinic
due to its favorable pharmacological properties and
high potency. The activity of EFV relies heavily on
formation of ring-stacking interactions with RT
K103, and its efficacy is strongly affected by the
most common NNRTI resistance-associated
mutation, K103N[5]. Etravirine (2, ETV) and
rilpivirne (3, RPV) are second-generation NNRTIs
(Figure 1); both are diarylpyrimidine analogues
(DAPYs). Although ETV and RPV showed a broad
spectrum of activities against wild-type (WT) and
mutant HIV-1 strains, resistance mutations still
emerged and some adverse effects have been
reported[6, 7].
Analyses of structure-activity relationships
(SARs) showed that the cyclopropylacetylene and
trifluoromethyl groups are the most important
pharmacophores in EFV (1), while the 4-cyano
aniline group is essential for the biological
activities of ETV (2) and RPV (3) [16-18].
Molecular modeling of EFV and ETV in the
binding pocket of HIV-1 RT (Fig. 2) suggested that
their binding sites overlap and that they might
interact with HIV-1 RT in a similar fashion.
Therefore, a new series of dihydroquinazoline
derivatives (3a-3z) sharing the structure features of
EFV (1) and DAPYs was designed and synthesized
by fusing the basic skeletons of both 1 and 2, in the
hope that this approach would furnish analogues
with high potency and resilience to RT mutations
(Fig.3). Preliminary SARs and molecular modeling
results were used to understand the biological
activities of these novel NNRTIs.
Figure 1.Chemical structures of FDA-approved NNRTIs

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Figure 2. Superposition of lower-energy docking conformations of EFV (blue) and ETV (red) in the binding pocket of HIV-1
reverse transcriptase.
Figure 3. Molecular design strategy of dihydroquinazoline derivatives using molecular hybridization.
2. Results and discussion
2.1. Chemistry
The target compounds designed in the present
study all had the general structure shown in
Scheme 1. The synthetic route for compounds 4a-x
is also depicted in Scheme 1. Briefly,
for 8 h to give compound 8. Compound 8 was
reacted with cyclopropylacetylene in
tetrahydrofuran under n-BuLi at -50°C for 1h to
give intermediate 9, then deprotected using ceric
ammonium nitrate at room temperature for 4h to
give compound 10. Compound 10 was refluxed in
POCl3 for 9 h to give the key intermediate
11[19-21]. Finally, intermediate11was treated with
the indicated anilines 12 under reflux in n-butanol
for 5–8 h to give the desired target compounds 4a-x
with 65–80% yield [22].
1-(2-amino-5-chlorophenyl)-2,
2-trifluoroethan-1 -one (5) was converted to
intermediate by reaction with
2,
6
4-methoxy-benzenemethanol at 60 °C for 8h.
Subsequently, intermediate 6 was reacted with
potassium cyanate at 60°C for 5h to give
intermediate 7, which was then refluxed in xylene

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CF3
O
O
F3C OH
Cl
Cl
Cl
Cl
NH
N
CF3
CF3
NH2
a
c
b
N
O
N
O
NHPMB
PMB
Cl
PMB
6
5
7
8
F3C
F3C
F3C
Cl
Cl
e
d
f
d
NH
NH
NH
Cl
N
O
N
O
N
H
PMB
11
10
9
4
Ar
Ph
4
Ar
4
Ar
a
b
c
d
e
f
i
j
k
3-Br-Ph
2-Br-Ph
4-F-Ph
3-F-Ph
2-F-Ph
4-OMe-Ph
3-OMe-Ph
2-OMe-Ph
q
r
s
t
u
v
w
x
4-Me-Ph
3-Me-Ph
2-Me-Ph
3,4-DiMe-Ph
3,5-DiMe-Ph
3,4-DiCl-Ph
3,4-DiF-Ph
3-Cl-4-F-Ph
4-CN-Ph
4-CONH2-Ph
3-CN-Ph
3-NO2-Ph
3-CF3-Ph
3-Cl-Ph
F3C
Cl
d
NH
l
m
n
o
p
N
NHAr
g
h
4-Br-Ph
4a-x
Scheme 1. Reagents and conditions: (a) 4-methoxy-benzenemethanol, p-Toluenesulfonic acid, acetonitrile, 60°C, 8 h, 80%; (b)
Potassium cyanate, AcOH, H2O, 60°C, 5 h, 68%; (c) Xylene, reflux, 8h, 60%; (d) Cyclopropylacetylene, n-BuLi, tetrahydrofuran,
-50°C, 1 h, 65%; (e) ceric ammonium nitrate, acetonitrile, H2O, room temperature, 4 h, 45%; (f) POCl3, reflux, 9 h, 40%; (g)
ArNH2 (12), n-BuOH, reflux, 5–8 h, 65–80%.
compound 4v (EC50 = 30 nM), and the most active
2.2. In vitro anti-HIV-1 activity and cytotoxicity
derivative, compound 4b (EC50 = 0.84 nM).
Compound 4b (EC50 = 0.84 nM) was about nine
The newly synthesized compounds were
times more active than ETV (EC50 = 6.4 nM)
evaluated for their anti-HIV-1 activities in MT-4
andtwice as active as EFV (EC50 = 1.6 nM).
cell cultures infected with one of three HIV-1
Interestingly, all of the hybrid derivatives showed
strains: (i) the WT strain IIIB; (ii) the single RT
high activity against the single RT mutantE138K,
mutant E138K; or (iii) the double RT mutant
with EC50 values in the low-micromolar to
K103N + Y181C (RES056).The cytotoxicity of the
single-digit nanomolar range (3.4µM to 3.5 nM).
compounds was also assessed using the MTT
Notably, compound 4b displayed outstanding
method. The results were expressed as EC50 values
potency against the E138K mutant strain (EC50
=
(anti-HIV-1 activity), CC50 values (cytotoxicity)
and SI values (selectivity index, given by the
CC50/EC50 ratio). The FDA-approved drugs
nevirapine (NVP), delavirdine (DLV), EFV and
ETV were used as references (Table 1).
3.5 nM); its potency was about twice that of ETV
(EC50 = 6.3 nM) and nearly reached that of EFV
(EC50 =2 nM). This finding might be useful as a
guide for drug discovery to overcome drug
resistance associated with the E138K mutation.
As shown in Table 1, the newly synthesized
compounds 4a-x exhibited moderate to excellent
potency against WT HIV-1 with EC50 values
ranging from 0.61 to 8.4 × 10-4 µM. Five
Unfortunately, except for compound 4b (EC50
=
66nM), none of the new hybrid compounds were
active at sub-cytotoxic concentrations against the
double mutant strain RES056.
compounds showed
a low nanomolar EC50:
compound 4f (EC50 = 51 nM), compound 4j (EC50
= 88 nM), compound 4o (EC50 = 63 nM),

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Table 1
Anti-HIV-1 activity and cytotoxicity of the derivative compounds 4a-x in MT-4 cellsa.
EC50(µM)b
Compounds
CC50(µM)c
SI ( B)d
21
B
E138K
RES056
> 2.4
4a
4b
0.11 ± 0.05
0.38 ± 0.03
2.4
1.9
8.4× 10-4
±
3.5×10-3
±
6.6× 10-2 ±
4.0 × 10-4
> 2.9
2304
6.0× 10-5
0.22 ± 0.11
0.44 ± 0.06
0.12 ± 0.06
1.0 × 10-4
0.16 ± 0.05
0.65 ± 0.03
0.36 ± 0.05
4c
4d
4e
12
2.2
1.6
58
5
> 2.2
> 1.6
14
5.1 × 10-2
±
4f
0.35 ± 0.02
> 3.2
3.3
64
2.1× 10-2
0.21 ± 0.10
0.45 ± 0.04
0.15 ± 0.06
4g
4h
4i
0.48 ± 0.09
>3.4
> 2.0
> 3.4
> 2.1
2.0
>3.4
2.1
9
8
0.42 ± 0.03
14
8.8× 10-2
±
4j
0.16 ± 0.01
1.8 ± 0.28
11
133
2.4× 10-2
0.25 ± 0.04
0.16 ± 0.06
0.10 ± 0.04
0.26 ± 0.09
4k
4l
0.46 ± 0.06
0.34 ± 0.02
0.14 ± 0.04
0.41 ± 0.01
> 2.3
> 2.0
> 3.1
> 8.4
2.4
2.0
3.1
8.4
9
12
31
32
4m
4n
6.3× 10-2
±
4o
0.29 ± 0.00
> 2.6
2.7
44
2.3× 10-2
0.15 ± 0.05
0.25 ± 0.13
0.21 ± 0.10
0.14 ± 0.03
0.61 ± 0.12
0.36 ± 0.03
4p
4q
4r
4s
4t
0.19 ± 0.02
0.40 ± 0.06
0.36 ± 0.05
0.40 ± 0.13
0.56 ± 0.06
0.60 ± 0.14
> 8.6
> 4.9
> 2.1
> 4.1
> 5.8
> 2.1
8.6
5.0
2.1
4.1
5.9
2.1
58
20
10
30
8
4u
6
3.0 × 10-2
±
4v
0.17 ± 0.01
>2.1
2.1
70
1.9× 10-2
0.36 ± 0.03
0.19 ± 0.10
1.6× 10-3±
6.0 ×10-4
4w
4x
>1.8
> 1.8
> 1.8
1.8
1.9
5
0.46 ± 0.01
2.0 × 10-3±
6.0 ×10-4
10
5.5×10-2
±
EFV
ETV
>2.0
>2.0
>1269
>923
2.0× 10-2
1.5 ×10-2 ±
9.0 × 10-4
2.2 × 10-3±
5.0 × 10-4
6.3× 10-3
4.3× 10-3
±
a All data represent the mean values from three independent experiments.
bEC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity,
presented as the mean ± standard deviation (SD), determined by the MTT method.
c CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
d SI: selectivity index, ratio of CC50/EC50 (WT).
Preliminary SAR analyses revealed that the
hybrids compounds 4 plays an important role in
nature and position of aniline substitution of the
modulating their anti-HIV-1activity.Comparedwith

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the unsubstituted compound 4a (EC50 = 0.11 µM),
HIV-1 RT. Poly(A)•oligo(dT)15 was used as the
template primer for reverse transcription (RT kit,
Roche), and NVP and EFV were used as the
reference drugs in this assay (Table 2). All five
compounds displayed inhibitory activity against
RT at low micromolar concentrations (0.448–0.010
µM), and compounds 4b (IC50 = 10 nM) and 4o
(IC50 = 0.11 µM) showed superior inhibition
compared with NVP (IC50 = 0.19 µM). Notably,
compound4a showed the excellent inhibition of
HIV-1 RT (IC50 = 10 nM) and was nearly
equipotent to EFV (IC50 = 6nM). These results
suggested that the target compounds could potently
inhibit the activity of WT HIV-1 RT, and belong to
a new group of HIV-1 NNRTIs.
it is more favorable when introducing bromine
atom in the ortho position of the benzene ring (4j
vs 4m, 4p, 4s); In the meta position of the benzene
ring, trifluoromethyl is more favorable than other
substitutions (4f vs 4d, 4e, 4g, 4i, 4l, 4o and 4r).
Surprisingly, a nitrile group in the para-position of
the benzene ring to obtain the most active
compound 4b, indicating that the scaffold
of4-cyano-aniline is very important for enhancing
the potency of NNRTIs; this result is consistent
with the previous studies of NNRTIs. In the case of
di-substitution vs mono-substitution of the phenyl
ring,
compound 4t (EC50
3,5-dimethyl-substituted compound 4u (EC50
0.36µM) showed no enhanced in activity compared
withthe4-Me-substituted compound 4q (EC50
although
the
3,4-dimethylsubstituted
=
0.61 µM) and
Table
2
Inhibitory
activity
of
representative
=
dihydroquinazoline-2-amines against WT HIV-1 reverse
transcriptasea.
=
0.25 µM), the 3-Me-substituted compound 4r (EC50
= 0.21 µM) or the 2-Me-substituted compound 4s
Compounds
IC50b(µM)
4b
4f
1.0× 10-2 ± 2.0 × 10-3
0.45 ± 1.3 × 10-2
0.34 ± 5.1 × 10-2
0.11 ± 2.5 × 10-2
0.37 ± 7.1 × 10-2
0.19 ± 4.2 × 10-2
6.0 × 10-3 ± 2.0 × 10-3
(EC50
=
0.14µM).
However,
the
=
3,4-dichlorosubstituted compound 4v (EC50
4j
30nM) was more potent than the 3-Cl-substituted
4o
compound 4g (EC50 = 21 µM). It appeared that the
4v
potencies
of
ortho-substituted
derivatives
NVP
EFV
(compounds 4j, 4m and 4s) were higher than
meta-substituted derivatives (4i, 4l and 4r) and the
para-substituted congeners (4h, 4k and 4q). In
addition, compounds 4f (EC50 = 51 nM) and 4o
(EC50= 63nM) had opposite electrical properties,
but their activities were almost the same, this
shows that liposolubility and water solubility of the
substituents had a great influence on the activity
besides steric hindrance and electrical properties.
These results and SAR conclusions are consistent
with our original hypothesis in designing the target
molecules.
a
Data represent the mean values of two independent
experiments.
b
IC50: inhibitory concentration of test compound required to
inhibit biotin deoxyuridine triphosphate (biotin-dUTP)
incorporation intoHIV-1 (WT) RT by 50%, presented as the
mean ± standard deviation (SD).
2.4. Molecular modeling analysis
To better understand the binding modes of the
newly synthesized hybrid compounds and account
for the SAR conclusions, compound 4a was docked
with the NNIBP of WT HIV-1 RT (PDB code:
3MEC) using Surflex-Docking Sybyl-X 2.0[23].
The theoretical binding models are shown in Fig.4
2.3. HIV-1 RT inhibition assay
In order to evaluate their effects on HIV-1 RT,
compounds 4b,4f, 4j, 4o and 4v were tested in
enzyme activity assays using purified recombinant

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Figure 4. A): The predicted binding modes of compounds 4b (A), 4m (B) and 4v (C) with WT HIV-1 RT (PDB ID: 3MEC).
The model indicated that the representative
compound 4b adopted a similar binding orientation
and horseshoe conformation within the NNIBP
compared with ETV. The trifluoromethyl and
cyclopropyl acetylene groups were accommodated
within the aromatic-rich sub-pocket consisting of
residues Y181, Y188, F226 and W229, forming
weak π-π interaction with these residues. The
mutation. The backbone nitrogen of the
dihydroquinazoline ring and the NH linker between
the central dihydroquinazoline ring and the
benzonitrile ring formed two hydrogen bonds with
the backbone carbonyl group of K101, improving
the binding affinity between the analogue and RT.
Compounds 4m and 4v showed similar
conformations within the NNIBP as compound 4b.
The chlorine group in compound 4v also formed
dipole-dipole interaction with RT residue L234,
while such kind of interaction did not occur in
compound 4m. On the other hand, compound 4m
only formed one hydrogen bonds with Lys101,
which might explain the higher activities of
compounds 4b and 4v compared to compound 4m.
In summary, the docking analyses revealed
benzonitrile
solvent-exposed region which was surrounded by
residues V106, P236 and Y318; the
para-substituted cyan group extended towards
L234, forming dipole-dipole interaction.
moiety
extended
into
a
a
Moreover, the fused central dihydroquinazoline
ring targeted the entrance channel, forming Van der
Waals interactions with residues E138 and V179;
this might explain the high activity of this
compound against strains bearing the E138K
the binding modes of the EFV-ETV hybrid
compounds to WT HIV-1 RT and provided critical

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insights for further optimization of these new lead
3. Conclusion
In conclusion, to extend the range of NNRTI
chemicalstructures and overcomethe issue of
resistance, we designed series of
drugs.
Quattro Micromass instrument and Bruker solari
X-70 FT-MS 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 ×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.
a
dihydroquinazolin-2-amine derivatives as potent
new HIV-1 NNRTIs using MH of the lead
compounds EFV and ETV. The results showed that
all of the target compounds exhibited high potency
against WT HIV-1. Five compounds (4b, 4f, 4j, 4o
and 4v) showed excellent activity in the low
nanomolar concentration range. Furthermore, most
of the compounds maintained high activity against
HIV-1 strains bearing the RT mutation E138K. On
this basis, compound 4b might represent a potential
drug candidate for further development to address
the issue of drug resistance. Additionally,
preliminary SARs were analyzed in detail based on
the anti-HIV-1 activities of the lead molecules.
Molecular modeling studies were carried out to
investigate the structure-function relationships
between the hybrid compounds and HIV-1 RT.
Further optimization of this novel chemical
skeleton to improve its biological activity is
ongoing in our lab.
4.1.1 General procedure to prepare the
intermediates of 6, 7, 8, 9 and 10.
The intermediates including 6, 7, 8, 9 and 10
were obtained easily according to the improved
procedure published previously [19-21].
4.1.2
Synthesis
of
2,6-dichloro-4-(cyclopropylethynyl)-4-(trifluorome
thyl)-1,4-dihydroquinazoline (11).
A
solution
of
6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl
)-3,4-dihydroquinazolin-2(1H)-one (10) (3.14 g, 10
mmol) in POCl3 (10 mL) was refluxed for 10 h.
The reaction process was monitored by TLC
analysis until its completion. The reaction mixture
was then poured into brine (50 mL) after being
cooled and stirred for approximately 15 min,
filtered and the filter cake was diluted with ethyl
acetate (30 mL×3). The organic layer was then
dried with anhydrous Na2SO4. The mixture was
then concentrated under the reduced pressure. The
crude product was purified by column
chromatography to afford the target compound 11
with the yield of 45%[22].
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 on a SGW
4.1.3 General procedure for 4a-x.
To a solution of 11 (1.0eq) in n-BuOH (5 mL)
was added ArNH2 (1.5eq). The resulting mixture
was heated at 110 °C for 6-8 h and was then cooled
to r.t. and concentrated in vacuo. The crude product
was purified by column chromatography to afford
the target compound 4a-x, with the yield of
60-80%, respectively.
1
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
4.1.3.1.6-chloro-4-(cyclopropylethynyl)-N-phenyl-

ACCEPTED MANUSCRIPT
4-(trifluoromethyl)-1,4-dihydroquinazolin-2-amine
The title product was synthesized following
the general procedure, starting from 11 and
4-aminobenzamide (12c). Yield 60%, white solid,
(4a)
The title product was synthesized following the
general procedure, starting from 11 and aniline
(12a). Yield 77%, white solid, m.p. 131-133 °C
(EA(ethyl acetate)/PE (petroleum ether));1H NMR
(400 MHz, DMSO-d6) δ: 11.12 (s, 2H, NH), 7.63
(d, J = 8.6 Hz, 1H), 7.56 (s, 1H, PhH), 7.49 (m, 2H,
PhH), 7.40-7.33 (m, 4H, PhH), 1.60-1.58 (m, 1H,
CH), 0.95-0.93 (m, 2H, CH2), 0.79 (m, 2H, CH2);
1
m.p. 178-179 °C (EA/PE); H NMR (400 MHz,
DMSO-d6) δ: 11.06 (s, 2H, NH), 8.05–7.97 (m, 3H,
PhH), 7.64-7.56 (m, 2H, PhH), 7.48–7.41 (m, 3H,
PhH), 7.33 (s, 1H, PhH), 1.59 (m, 1H, CH), 0.95
(m, 2H, CH2), 0.79 (m, 2H, CH2); 13C NMR (101
MHz, DMSO-d6) δ: 167.47 (C=O), 149.00
(N=C-(NH)2), 132.14 (ArC), 129.50 (ArC), 127.71
(ArC), 117.53 (ArC), 8.89 (CH2), 8.84 (CH2), -0.70
(CC≡C); IR: υ 3172, 2971, 2249, 1651, 1487, 1170
cm-1; HRMS calcd for C21H16ClF3N4O [M+H]+:
433.1037, found: 433.1033.
13C
NMR
(101
MHz,
DMSO-d6)
δ:
149.32(N=C-(NH)2), 135.31 (ArC), 132.51
(ArC),132.20 (ArC), 130.27 (ArC, 4C), 129.58
(ArC), 127.67 (ArC), 124.57 (ArC), 123.75 (d, JCF
= 287 Hz), 119.89 (ArC), 117.35 (ArC), 94.46
(C≡C), 66.64 (C≡C), 57.48 (d, JCCF= 34 Hz), 8.90
(CH2), 8.85 (CH2), -0.71 (CC≡C); IR: υ 2842, 2250,
1639, 1546, 1483, 1168 cm-1; HRMS calcd for
C20H15ClF3N3 [M+H]+: 390.0979, found: 390.0973.
4.1.3.2.
4.1.3.4.
3-((6-chloro-4-(cyclopropylethynyl)-4-(trifluorome
thyl)-1,4-dihydroquinazolin-2-yl)
amino)benzonitrile (4d)
The title product was synthesized following
the general procedure, starting from 11 and
3-aminobenzonitrile (12d). Yield 75%, white solid,
m.p. 183-185 °C (EA/PE);1H NMR (400 MHz,
DMSO-d6) δ:11.06 (s, 2H, NH), 7.96 (s, 1H, PhH),
7.76-7.73 (m, 2H, PhH), 7.65 (m,, PhH), 7.55 (s,
1H, PhH), 7.35 (d, J = 8.4 Hz, 1H, PhH), 1.60–1.58
(m, 1H, CH), 0.95 (m, 2H, CH2), 0.80 (m, 2H,
CH2); 13C NMR (101 MHz, DMSO-d6) δ:
149.10(N=C-(NH)2), 137.20 (ArC), 132.95(ArC),
132.10(ArC), 131.46 (ArC), 130.24 (ArC),
129.53(ArC), 128.88(ArC), 127.67 (ArC), 127.45
(ArC), 123.81 (d, JCF = 286 Hz), 119.97 (ArC),
118.76 (ArC), 117.50 (ArC), 112.86 (ArC), 94.23
(C≡C), 67.02 (C≡C), 57.78 (d, JCCF= 32 Hz), 8.88
(CH2), 8.83 (CH2), -0.71(CC≡C); IR: υ 3368, 2804,
2253, 1612, 1576, 1164 cm-1; HRMS calcd for
C21H14ClF3N4 [M+H]+: 415.0932, found: 435.0925.
4.1.3.5.6-chloro-4-(cyclopropylethynyl)-N-(3-nitro
phenyl)-4-(trifluoromethyl)-1,4-dihydroquinazolin-
2-amine (4e)
4-((6-chloro-4-(cyclopropylethynyl)-4-(trifluorome
thyl)-1,4-dihydroquinazolin-2-yl)
amino)benzonitrile (4b)
The title product was synthesized following
the general procedure, starting from 11 and
4-cyanoaniline (12b). Yield 65%, white solid, m.p.
180-182°C (EA/PE);1H NMR (400 MHz,
DMSO-d6) δ: 9.16 (s, 2H, NH), 7.73 (s, 4H, PhH),
7.42-7.37(m, 2H, PhH), 7.01 (d, J = 8.2 Hz, 1H,
PhH), 1.49 (m, 1H, CH), 0.87 (m, 2H, CH2), 0.69
(m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6) δ:
151.19(N=C-(NH)2), 137.22 (ArC), 133.61 (ArC),
131.41 (ArC, 2C), 127.58 (ArC), 125.52 (ArC),
123.73 (d, JCF = 286 Hz), 119.91 (ArC), 118.93
(ArC), 118.86 (ArC), 116.78 (2C), 115.43 (ArC),
91.96 (C≡C), 68.68 (C≡C), 58.79 (d, JCCF= 32 Hz),
8.75 (CH2), 8.70 (CH2),-0.70 (CC≡C); IR: υ 2971,
2226, 1646, 1588, 1485, 1243 cm-1; HRMS calcd
for
C21H14ClF3N4
[M+H]+:
415.0932,
found:415.0934.
4.1.3.3.
The title product was synthesized following
the general procedure, starting from 11 and
3-nitroaniline (12e). Yield 66%, white solid, m.p.
183-185 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 11.11 (s, 2H, NH), 8.40 (s, 1H, PhH),
4-((6-chloro-4-(cyclopropylethynyl)-4-(trifluorome
thyl)-1,4-dihydroquinazolin-2-yl)
amino)benzamide (4c)

ACCEPTED MANUSCRIPT
8.11 (d, J = 7.6 Hz, 1H, PhH), 7.84 (d, J = 7.9 Hz,
m-chloroaniline (12g). Yield 72%, white solid, m.p.
196-198 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 11.18 (s, 2H, NH), 7.63 (d, J = 8.6
Hz, 1H, PhH), 7.56 (s, 2H, PhH), 7.49 (t, J = 8.0
Hz, 1H, PhH), 7.37 (s, 3H, PhH), 1.60 (dd, J = 8.4,
3.7 Hz, 1H, CH), 0.95 (m, 2H, CH2), 0.79 (m, 2H,
CH2); 13C NMR (101 MHz, DMSO-d6) δ: 149.12
(N=C-(NH)2), 137.47 (ArC), 134.23 (ArC), 132.79
(ArC), 132.15 (ArC), 131.73 (ArC), 129.57 (ArC),
127.68 (ArC), 126.72 (ArC), 124.04 (ArC), 123.78
(d, JCF= 286 Hz), 122.77 (ArC), 119.88 (ArC),
117.49 (ArC), 94.32 (C≡C), 66.86 (C≡C), 57.69 (d,
1H, PhH), 7.74 (t, J = 8.1 Hz, 1H, PhH), 7.62 (d, J
= 8.6 Hz, 1H, PhH), 7.55 (s, 1H, PhH), 7.33 (d, J =
7.5 Hz, 1H, PhH), 1.59 (m, 1H, CH), 0.94 (m, 2H,
CH2), 0.79 (m, 2H, CH2); 13C NMR (101 MHz,
DMSO-d6) δ: 149.09(N=C-(NH)2), 148.87(CNO2),
147.16(ArC),
137.84
(ArC),
132.04(ArC),
131.40(ArC), 129.25 (ArC), 127.70 (ArC), 123.86
(d, JCF = 286 Hz), 122.38 (ArC), 120.77 (ArC),
119.90 (ArC), 118.43 (ArC), 117.59 (ArC), 93.94
(C≡C), 67.35 (C≡C), 57.73 (q,JCCF = 21 Hz), 8.87
(CH2), 8.81(CH2), -0.69 (CC≡C); IR: υ 3357, 2906,
2246, 1658, 1538, 1171 cm-1; HRMS calcd for
C20H14ClF3N4O2 [M+H]+: 435.0830, found:
435.0830.
JCCF=
33 Hz), 8.88 (CH2), 8.83 (CH2),
-0.71(CC≡C); IR: υ 2809, 2243, 1647, 1589, 1474,
1170 cm-1; HRMS calcd for C20H14Cl2F3N3 [M+H]+:
424.0590, found: 424.0588.
4.1.3.6.
6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl
)-N-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinaz
olin-2-amine (4f)
4.1.3.8.
N-(4-bromophenyl)-6-chloro-4-(cyclopropylethyny
l)-4-(trifluoromethyl)-1,4-dihydroquinazolin-2-ami
ne (4h)
The title product was synthesized following
the general procedure, starting from 11 and
3-(trifluoromethyl)aniline (12f). Yield 68%, white
solid, m.p. 123-125°C (EA/PE); 1H NMR (400
MHz, DMSO-d6) δ: 10.62 (s, 2H, NH), 7.75 (d, J=
8.2 Hz, 2H, PhH), 7.69 (m, 2H, PhH), 7.57-7.46
(m, 2H, PhH), 7.17 (s, 1H, PhH), 1.61-1.50 (m, 1H,
CH), 0.92 (m, 2H, CH2), 0.75 (m, 2H, CH2); 13C
The title product was synthesized following
the general procedure, starting from 11 and
4-bromoanilines (12h). Yield 75%, white solid, m.p.
133-135 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 11.13 (s, 2H, NH), 7.69-7.62 (m, 2H,
PhH), 7.52 (m, 2H, PhH), 7.45-7.34 (m, 3H, PhH),
1.59 (m, 1H, CH), 0.95 (m, 2H, CH2), 0.80 (m,
2H,CH2); 13C NMR (101 MHz, DMSO-d6) δ:
148.96(N=C-(NH)2), 135.85 (ArC), 133.65 (ArC),
132.86 (ArC, 3C), 132.45(ArC), 131.93(ArC),
129.00 (ArC), 127.67(ArC), 125.81(ArC), 123.93
(d, JCF= 288 Hz), 119.82 (ArC), 117.40 (ArC),
93.67 (C≡C), 67.55 (C≡C), 57.89 (d, JCCF= 38 Hz),
8.85 (CH2), 8.80 (CH2),-0.70 (CC≡C); IR: υ 2790,
2246, 1645, 1484, 1239, 1167 cm-1; HRMS calcd
for C20H14BrClF3N3 [M+H]+: 468.0084, found:
468.0082.
NMR
(101
MHz,
DMSO-d6)
δ:
148.15(N=C-(NH)2), 141.55(ArC), 134.85(ArC),
131.58(ArC), 128.49(ArC), 127.63(ArC), 126.90
(ArC, 2C), 126.03(ArC), 124.16(d, JCF = 286 Hz),
123.88(d, JCF
121.86(ArC),
=
232 Hz), 122.72(ArC),
119.94(ArC), 117.51(ArC),
92.75(C≡C), 68.71(C≡C), 58.47(d, JCCF= 33 Hz),
8.73(CH2), 8.68(CH2), -0.81(CC≡C); IR: υ 2953,
2248, 1651, 1486, 1321, 1164 cm-1; HRMS calcd
for C25H20ClN5 [M+H]+: 458.0853, found:
458.0852.
4.1.3.9.
4.1.3.7
N-(3-bromophenyl)-6-chloro-4-(cyclopropylethyny
l)-4-(trifluoromethyl)-1,4-dihydroquinazolin-2-ami
ne (4i)
6-chloro-N-(3-chlorophenyl)-4-(cyclopropylethyny
l)-4-(trifluoromethyl)-1,4-dihydro
quinazolin-2-amine (4g)
The title product was synthesized following
the general procedure, starting from 11 and
3-bromoaniline (12i). Yield 70%, white solid, m.p.
The title product was synthesized following
the general procedure, starting from 11 and

ACCEPTED MANUSCRIPT
193-195 °C (EA/PE); 1H NMR (400 MHz,
Hz, 1H, PhH), 7.49 (m, 3H, PhH), 7.31 (m, 3H,
PhH), 1.59-1.57 (m, 1H, CH), 0.94 (m, 2H, CH2),
0.78 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6)
δ: 160.74 (d, J = 242 Hz), 149.39 (N=C-(NH)2),
133.55 (ArC), 132.22 (ArC), 131.96 (ArC), 128.97
(ArC), 127.65 (ArC), 126.54 (ArC), 123.91 (d,
JCF= 287 Hz), 119.81 (ArC), 117.29 (ArC), 116.96
(ArC), 116.73 (ArC), 93.8 (C≡C), 67.55 (C≡C),
57.74 (d, JCCF= 32 Hz), 55.39 (CH2), 8.86 (CH2),
8.81 (CH2), -0.71 (CC≡C); IR: υ 2891, 2249, 1654,
1505, 1482, 1182 cm-1; HRMS calcd for
C20H14ClF4N3 [M+H]+: 408.0885, found: 408.0882.
4.1.3.12.
DMSO-d6) δ: 11.12 (s, 2H, NH), 7.62 (m, 4H,
PhH), 7.37 (m, 3H, PhH), 1.58 (m, 1H, CH), 0.94
(m, 2H, CH2), 0.79 (m, 2H, CH2); 13C NMR (101
MHz, DMSO-d6) δ: 149.14 (N=C-(NH)2), 148.96
(ArC), 137.57 (ArC), 132.77 (ArC), 132.07 (ArC,
2C), 129.57 (ArC), 127.68 (ArC), 126.87 (ArC),
123.76 (d, JCF= 287 Hz), 123.19 (ArC), 122.45
(ArC), 119.88 (ArC), 117.47 (ArC), 94.33 (C≡C),
66.83 (C≡C), 57.67 (d, JCCF= 32 Hz), 8.89 (CH2),
8.83 (CH2), -0.71 (CC≡C); IR: υ 2807, 2243, 1648,
1587, 1473, 1184 cm-1; HRMS calcd for
C20H14BrClF3N3 [M+H]+: 468.0084, found:
468.0079.
6-chloro-4-(cyclopropylethynyl)-N-(3-fluorophenyl
4.1.3.10.
)-4-(trifluoromethyl)-1,
4-dihydro
N-(2-bromophenyl)-6-chloro-4-(cyclopropylethyny
l)-4-(trifluoromethyl)-1,
quinazolin-2-amine (4l)
The title product was synthesized following
the general procedure, starting from 11 and
3-fluoroaniline (12l). Yield 76%, white solid, m.p.
145-147 °C (EA/PE);1H NMR (400 MHz,
DMSO-d6) δ: 11.24 (s, 2H, NH), 7.63 (d, J = 8.3
Hz, 1H, PhH), 7.51 (m, 2H, PhH), 7.48-7.35 (m,
2H, PhH), 7.19 (m, 2H, PhH), 1.59 (m, 1H, CH),
0.95 (m, 2H, CH2), 0.80 (m, 2H, CH2); 13C NMR
(101 MHz, DMSO-d6) δ:162.85 (d, J = 242 Hz),
148.48 (N=C-(NH)2), 131.58-131.29 (ArC, 4),
128.28 (ArC), 127.95 (ArC), 127.63 (ArC), 124.19
(ArC, d, JCF= 287 Hz), 119.89 (ArC), 118.07
(ArC), 117.45 (ArC), 92.85 (C≡C), 68.87 (C≡C),
58.40 (d, JCCF= 32 Hz), 8.78 (CH2), 8.72 (CH2),
-0.72 (CC≡C); IR: υ 2815, 2245, 1651, 1603, 1481,
1170 cm-1; HRMS calcd for C20H14ClF4N3 [M+H]+:
408.0885, found: 408.0879.
4-dihydroquinazolin-2-amine (4j)
The title product was synthesized following
the general procedure, starting from 11 and
4-methylbenzeneboronic acid (12j). Yield 74%,
1
white solid, m.p. 215-217 °C (EA/PE); H NMR
(400 MHz, DMSO-d6) δ: 10.85 (s, 2H, NH), 7.81
(s, 1H, PhH), 7.58 (m, 4H, PhH), 7.39 (s, 1H,
PhH), 7.31 (s, 1H, PhH), 1.58 (m, 1H, CH), 0.96
(m, 2H, CH2), 0.80 (m, 2H, CH2); 13C NMR (101
MHz, DMSO-d6) δ: 149.85 (N=C-(NH)2), 145.38
(ArC), 134.03 (ArC), 133.35 (ArC), 132.64 (ArC),
132.31 (ArC), 129.70 (ArC), 128.77 (ArC), 127.75
(ArC), 123.67 (d, JCF= 287 Hz), 119.61 (ArC),
118.53 (ArC), 117.00 (ArC), 116.50 (ArC), 108.50
(C≡C), 66.76 (C≡C), 57.53 (d, JCCF= 34 Hz), 8.93
(CH2), 8.89 (CH2), -0.70 (CC≡C); IR: υ 2747, 2249,
1659, 1581, 1474, 1167 cm-1; HRMS calcd for
C20H14BrClF3N3 [M+H]+: 468.0084, found:
468.0085.
4.1.3.13.6-chloro-4-(cyclopropylethynyl)-N-(2-fluo
rophenyl)-4-(trifluoromethyl)-1,
4-dihydroquinazolin-2-amine (4m)
4.1.3.11.6-chloro-4-(cyclopropylethynyl)-N-(4-fluo
The title product was synthesized following
the general procedure, starting from 11 and
2-fluoroaniline (12m). Yield 70%, white solid, m.p.
206-208 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 11.02 (s, 2H, NH), 7.64 (d, J = 8.5
Hz, 1H, PhH), 7.56 (s, 2H, PhH), 7.42-7.34 (m, 3H,
PhH), 7.32 (t, J = 7.3 Hz, 1H, PhH), 1.59-1.57 (m,
1H, CH), 0.94 (m, 2H, CH2), 0.80 (m, 2H, CH2);
rophenyl)-4-(trifluoromethyl)-1,
4-dihydro
quinazolin-2-amine (4k)
The title product was synthesized following
the general procedure, starting from 11 and
4-fluoroaniline (12k). Yield 69%, white solid, m.p.
141-143 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 10.91 (s, 2H, NH), 7.59 (d, J = 8.5

ACCEPTED MANUSCRIPT
13C NMR (101 MHz, DMSO-d6) δ: 156.74 (d, JCF=
13C NMR (101 MHz, DMSO-d6) δ: 160.62 (ArCO),
149.21 (N=C-(NH)2), 136.51 (ArC), 132.65 (ArC),
132.17 (ArC), 131.02 (ArC), 129.50 (ArC), 127.66
(ArC), 123.76 (d, JCF= 287 Hz), 119.89 (ArC),
117.35 (ArC), 116.40 (ArC), 113.11 (ArC), 110.01
248 Hz), 149.89 (N=C-(NH)2), 132.32 (ArC, 2C),
129.68 (ArC), 128.43 (ArC), 127.76 (ArC), 125.87
(ArC), 125.63 (d, JCF= 287 Hz), 122.59 (ArC),
119.64 (ArC), 117.34 (ArC), 117.15 (ArC, 2C),
94.56 (C≡C), 66.63 (C≡C), 57.57 (d, JCCF= 33 Hz),
8.90 (CH2), 8.85 (CH2), -0.70 (CC≡C); IR: υ 2723,
2248, 1648, 1488, 1182, 1166 cm-1; HRMS calcd
for C20H14ClF4N3 [M+H]+: 408.0885, found:
408.0881.
(ArC), 94.38 (C≡C), 66.67 (C≡C), 57.53 (q, JCCF
=
34 Hz), 55.78 (OCH3), 8.89 (CH2), 8.84 (CH2),
-0.71 (CC≡C); IR: υ 2829, 2248, 1652, 1607, 1488,
1157 cm-1; HRMS calcd for C21H17ClF3N3O
[M+H]+: 420.1085, found: 420.1090.
4.1.3.14.
4.1.3.16.6-chloro-4-(cyclopropylethynyl)-N-(2-met
hoxyphenyl)-4-(trifluoromethyl)-1,
6-chloro-4-(cyclopropylethynyl)-N-(4-methoxyphe
nyl)-4-(trifluoromethyl)-1,
4-dihydroquinazolin-2-amine (4p)
4-dihydroquinazolin-2-amine (4n)
The title product was synthesized following
the general procedure, starting from 11 and
2-methoxyaniline (12p). Yield 72%, white solid,
m.p. 225-227 °C (EA/PE);1H NMR (400 MHz,
DMSO-d6) δ: 11.81 (s, 1H, NH), 10.74 (s, 2H,
PhH), 7.63 (d, J = 8.5 Hz, 1H, PhH), 7.55-7.50 (m,
1H, PhH), 7.40 (m, 3H, PhH), 7.21 (d, J = 8.2 Hz,
1H, PhH), 7.06 (t, J = 7.5 Hz, 1H, PhH), 3.80 (s,
3H, OCH3), 1.58 (m, 1H, CH), 0.94 (m, 2H, CH2),
0.79 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6)
δ: 154.10 (ArCO), 149.89 (N=C-(NH)2), 132.33
(ArC), 129.91 (ArC), 129.38 (ArC), 127.75 (ArC),
123.65 (d, JCF= 286 Hz), 122.40 (ArC), 121.42
(ArC), 119.36 (ArC), 116.91 (ArC), 113.15 (ArC),
94.43 (C≡C), 66.71 (C≡C), 57.42 (q, JCCF= 29 Hz),
56.29 (OCH3), 8.90 (CH2), 8.85 (CH2), -0.71
(CC≡C); IR: υ 2790, 2249, 1666, 1491, 1182, 1167
cm-1; HRMS calcd for C21H17ClF3N3O [M+H]+:
420.1085, found: 420.1087.
The title product was synthesized following
the general procedure, starting from 11 and
4-aminoanisole (12n). Yield 78%, white solid, m.p.
239-242 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 10.11 (s, 2H, NH), 7.49-7.40 (m, 4H,
PhH), 7.12 (d, J = 8.0 Hz, 1H, PhH), 6.97 (m, 2H,
PhH), 3.77 (s, 3H, OCH3), 1.55-1.53 (m, 1H, CH),
0.99-0.85 (m, 2H, CH2), 0.74 (m, 2H, CH2); 13C
NMR (101 MHz, DMSO-d6) δ: 157.01 (ArCO),
148.81 (N=C-(NH)2), 135.76 (ArC), 131.38 (ArC),
130.16 (ArC), 127.63 (ArC), 127.53 (ArC), 124.44
(ArC), 124.29 (d, JCF= 287 Hz), 122.59 (ArC),
119.85 (ArC), 117.33 (ArC), 114.91 (ArC, 2C),
92.28 (C≡C), 69.15 (C≡C), 58.37 (d, JCCF=32 Hz),
55.75 (OCH3), 8.72 (CH2), 8.66 (CH2), -0.79
(CC≡C); IR: υ 2968, 2244, 1655, 1509, 1245, 1166
cm-1; HRMS calcd for C21H17ClF3N3O [M+H]+:
420.1085, found: 420.1086.
4.1.3.15.
4.1.3.17.
6-chloro-4-(cyclopropylethynyl)-N-(3-methoxyphe
nyl)-4-(trifluoromethyl)-1,
6-chloro-4-(cyclopropylethynyl)-N-(p-tolyl)-4-(trifl
uoromethyl)-1,4-dihydroquinazolin-2-amine (4q)
The title product was synthesized following
the general procedure, starting from 11 and
4-toluidine (12q). Yield 76%, white solid, m.p.
173-175 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 10.88 (s, 2H, NH), 7.62 (d, J = 8.6
Hz, 1H, PhH), 7.55 (s, 1H, PhH), 7.34-7.26 (m, 5H,
PhH), 2.35 (s, 3H, CH3), 1.60-1.58 (m, 1H, CH),
0.95 (m, 2H, CH2), 0.80 (m, 2H, CH2); 13C NMR
(101 MHz, DMSO-d6) δ: 149.22 (N=C-(NH)2),
4-dihydroquinazolin-2-amine (4o)
The title product was synthesized following
the general procedure, starting from 11 and
3-methoxyaniline (12o). Yield 69%, white solid,
1
m.p. 142-144 °C (EA/PE); H NMR (400 MHz,
DMSO-d6) δ: 11.05 (s, 2H, NH), 7.64-7.56 (m, 2H,
PhH), 7.38 (m, 2H,PhH), 7.03 (s, 1H, PhH), 6.93
(m, 2H, PhH), 3.79 (s, 3H, OCH3), 1.58 (m, 1H,
CH), 0.99-0.89 (m, 2H, CH2), 0.79 (m, 2H, CH2);

ACCEPTED MANUSCRIPT
137.21 (ArC), 132.39 (ArC), 132.22 (ArC), 130.78
119.64 (ArC), 116.94 (ArC), 94.45 (C≡C), 66.67
(C≡C), 57.38 (q, JCCF = 33 Hz), 17.86 (CH3), 8.93
(CH2), 8.88 (CH2), -0.71 (CC≡C); IR: υ 2716, 2251,
1647, 1488, 1181, 1166 cm-1; HRMS calcd for
C21H17ClF3N3 [M+H]+: 404.1136, found: 404.1132.
4.1.3.20.
(ArC, 3C), 129.65 (ArC), 127.66 (ArC), 124.85
(ArC, 2C), 123.72 (d, JCF= 286 Hz), 119.76 (ArC),
117.39 (ArC), 94.63 (C≡C), 66.43 (C≡C), 57.48 (q,
JCCF= 33 Hz), 21.01 (CH3), 8.87 (CH2), 8.81 (CH2),
-0.79 (CC≡C); IR: υ 2830, 2248, 1654, 1490, 1186,
1166 cm-1; HRMS calcd for C21H17ClF3N3 [M+H]+:
404.1136, found: 404.1139.
6-chloro-4-(cyclopropylethynyl)-N-(3,4-dimethylp
henyl)-4-(trifluoromethyl)-1,4-dihydroquinazolin-2
-amine (4t)
4.1.3.18.
6-chloro-4-(cyclopropylethynyl)-N-(m-tolyl)-4-(trif
luoromethyl)-1,4-dihydroquinazolin-2-amine (4r)
The title product was synthesized following
the general procedure, starting from 11 and
m-toluidine (12r). Yield 73%, white solid, m.p.
182-184 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 11.08 (s, 2H, NH), 7.63 (dd, J = 8.6,
1.9 Hz, 1H, PhH), 7.56 (s, 1H, PhH), 7.38 (m, 2H,
PhH), 7.19 (d, J= 6.6 Hz, 3H, PhH), 2.35 (s, 3H,
CH3), 1.60-1.58 (m, 1H, CH), 0.95 (m, 2H, CH2),
0.80 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6)
The title product was synthesized following
the general procedure, starting from 11 and
3,4-xylidine (12t). Yield 77%, white solid, m.p.
161-163 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 10.99 (s, 2H, NH), 7.63 (d, J = 8.7
Hz, 1H, PhH), 7.55 (s, 1H, PhH), 7.36 (d, J = 8.6
Hz, 1H, PhH), 7.25 (d, J = 8.0 Hz, 1H, PhH), 7.16
(s, 1H, PhH), 7.10 (d, J = 7.9 Hz, 1H, PhH), 2.26
(s, 6H, 2 × CH3), 1.59 (m, 1H, CH), 0.96-0.94 (m,
2H, CH2), 0.80 (m, 2H, CH2); 13C NMR (101 MHz,
DMSO-d6) δ: 149.51 (N=C-(NH)2), 138.35 (ArC),
135.96 (ArC), 132.47 (ArC), 132.40 (ArC), 132.22
(ArC), 131.15 (ArC, 2C), 129.47 (ArC), 127.66
(ArC), 126.11 (ArC), 123.83 (d, JCF= 269 Hz),
119.85 (ArC), 117.17 (ArC), 94.42 (C≡C), 66.66
(C≡C), 57.37 (q, JCCF= 32 Hz), 19.89 (CH3), 19.40
(CH3), 8.91 (CH2), 8.86 (CH2), -0.72 (CC≡C); IR: υ
2830, 2247, 1654, 1489, 1185, 1168 cm-1; HRMS
calcd for C22H19ClF3N3 [M+H]+: 418.1292, found:
418.1290.
δ:
149.35(N=C-(NH)2),
139.81
(ArC),
135.11(ArC), 132.51 (ArC), 132.21 (ArC), 130.10
(ArC, 2C), 129.54 (ArC), 128.09 (ArC), 127.67
(ArC), 123.75 (d, JCF= 286 Hz), 121.71 (ArC),
119.90 (ArC), 117.30 (ArC), 94.44 (C≡C), 66.67
(C≡C), 57.44 (q, JCCF= 33 Hz), 21.41 (CH3), 8.91
(CH2), 8.86 (CH2), -0.71 (CC≡C); IR: υ 2818, 2245,
1652, 1479, 1184, 1166 cm-1; HRMS calcd for
C21H17ClF3N3 [M+H]+: 404.1136, found: 404.1130.
4.1.3.19.
4.1.3.21.
6-chloro-4-(cyclopropylethynyl)-N-(o-tolyl)-4-(trifl
uoromethyl)-1,4-dihydroquinazolin-2-amine (4s)
The title product was synthesized following
the general procedure, starting from 11 and
2-toluidine (12s). Yield 80%, white solid, m.p.
169-171 °C (EA/PE); 1H NMR (400 MHz,
DMSO-d6) δ: 11.27 (m, 3H, NH), 7.63-7.56(m, 2H,
PhH), 7.37 (m, 5H, PhH), 2.27 (s, 3H, CH3),
1.61-1.59 (m, 1H, CH), 0.96-0.94 (m, 2H, CH2),
0.80 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6)
δ: 150.07 (N=C-(NH)2), 135.14 (ArC), 135.10
(ArC), 132.92 (ArC), 132.28 (ArC), 131.72 (ArC),
129.45 (ArC), 128.87 (ArC), 127.86 (ArC), 127.72
(ArC), 127.36 (ArC), 123.74 (d, JCF= 287 Hz),
6-chloro-4-(cyclopropylethynyl)-N-(3,5-dimethylp
henyl)-4-(trifluoromethyl)-1,
4-dihydroquinazolin-2-amine (4u)
The title product was synthesized following
the general procedure, starting from 11 and
3,5-Dimethylaniline (12u). Yield 76%, white solid,
1
m.p. 225-227 °C (EA/PE); H NMR (400 MHz,
DMSO-d6) δ: 11.06 (s, 2H, NH), 7.63 (d, J = 8.6
Hz, 1H, PhH), 7.56 (s, 1H, PhH), 7.38 (d, J = 8.6
Hz, 1H, PhH), 6.99 (s, 3H, PhH), 2.31 (s, 6H, 2 ×
CH3), 1.61-1.58(m, 1H, CH), 0.95 (m, 2H, CH2),
0.80 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6)
δ: 149.05(N=C-(NH)2), 139.89 (ArC, 2C), 134.78
(ArC), 132.23 (ArC), 132.13 (ArC), 129.90 (ArC),

ACCEPTED MANUSCRIPT
129.15 (ArC), 127.68 (ArC), 125.09 (d, JCF= 286
Hz), 122.13 (ArC,2C), 119.64 (ArC), 117.48
(ArC), 94.86 (C≡C), 66.15 (C≡C), 57.48 (q, JCCF
(d, JCF= 287 Hz), 119.89 (ArC), 118.77 (ArC, 2C),
117.35 (ArC), 94.27 (C≡C), 66.94 (C≡C), 57.64 (q,
=
JCCF= 34 Hz), 8.88 (CH2), 8.83 (CH2), -0.72
33 Hz), 21.19 (CH3, 2C), 8.83 (CH2), 8.77 (CH2),
-0.88 (CC≡C). IR: υ 3230, 3101, 2250, 1694, 1500,
1168 cm-1; HRMS calcd for C22H19ClF3N3 [M+H]+:
418.1292, found: 418.1290.
(CC≡C); IR: υ 2892, 2248, 1654, 1482, 1239, 1173
cm-1; HRMS calcd for C20H13ClF5N3 [M+H]+:
427.0791, found: 427.0796.
4.1.3.24.
4.1.3.22.6-chloro-4-(cyclopropylethynyl)-N-(3,4-di
chlorophenyl)-4-(trifluoromethyl)-1,
6-chloro-N-(3-chloro-4-fluorophenyl)-4-(cycloprop
ylethynyl)-4-(trifluoromethyl)-1,4-dihydroquinazol
in-2-amine (4x)
4-dihydroquinazolin-2-amine (4v)
The title product was synthesized following
the general procedure, starting from 11 and
3,4-dichloroaniline (12v). Yield 69%, white solid,
The title product was synthesized following
the general procedure, starting from 11 and
3-Chloro-4-fluoroaniline (12x). Yield 72%, white
1
1
m.p. 136-138 °C (EA/PE); H NMR (400 MHz,
solid, m.p.197-198 °C (EA/PE); H NMR (400
DMSO-d6) δ: 10.84 (s, 2H, NH), 7.86 (s, 1H, PhH),
7.66 (d,J = 8.4 Hz, 1H, PhH), 7.60-7.47 (m, 2H,
PhH), 7.43 (d, J= 8.5 Hz, 1H, PhH), 7.24 (d, J =
7.0 Hz, 1H, PhH), 1.56 (m, 1H, CH), 0.92 (m, 2H,
CH2), 0.77 (m, 2H, CH2); 13C NMR (101 MHz,
DMSO-d6) δ: 148.74 (N=C-(NH)2), 137.42 (ArC),
131.97 (ArC), 131.75 (ArC), 131.60 (ArC, 2C),
128.71 (ArC), 128.22 (ArC), 127.66 (ArC, 2C),
124.07 (d, JCF= 286 Hz), 123.25 (ArC), 119.79
(ArC), 117.42 (ArC), 93.20 (C≡C), 68.13 (C≡C),
58.40 (q, JCCF = 33 Hz), 8.83 (CH2), 8.77 (CH2),
-0.69 (CC≡C); IR: υ 2929, 2250, 1637, 1473, 1242,
1167 cm-1; HRMS calcd for C20H13Cl3F3N3 [M+H]+:
458.0200, found: 458.0192.
MHz, DMSO-d6) δ: 11.03 (s, 2H, NH), 7.76 (d, J =
6.3 Hz, 1H, NH), 7.62 (d, J = 8.6 Hz, 1H, NH),
7.55-7.51 (m, 2H, PhH), 7.45-7.42 (m, 1H, PhH),
7.36 (d, J = 8.6 Hz, 1H, PhH), 1.61-1.58 (m, 1H,
CH), 0.95 (m, 2H, CH2), 0.80 (m, 2H, CH2); 13C
NMR (101 MHz, DMSO-d6) δ: 156.36 (d, J =245
Hz), 149.40 (N=C-(NH)2), 132.65 (ArC), 132.17
(ArC), 129.59 (ArC), 127.66 (ArC), 127.37 (ArC),
125.90 (ArC), 123.72 (d, JCCF = 287 Hz), 120.79
(ArC), 120.60 (ArC), 119.77 (ArC), 118.38 (ArC),
118.16 (ArC), 117.35 (ArC), 94.41 (C≡C), 66.77
(C≡C), 57.46 (d, JCCF = 33 Hz), 8.87 (CH2), 8.82
(CH2), -0.74 (CC≡C); IR: υ 2819, 2244, 2220, 1497,
1254, 1169 cm-1; HRMS calcd for C20H13Cl2F4N3
[M+H]+: 442.0495, found: 442.0493.
4.1.3.23.
6-chloro-4-(cyclopropylethynyl)-N-(3,4-difluoroph
enyl)-4-(trifluoromethyl)-1,
4.2. In vitro anti-HIV assay
The anti-HIV activity and cytotoxicity of the
newly synthesized compounds were evaluated with
WT HIV-1 (strain HIV-IIIB), double RT mutant
strains of HIV-1 IIIB (RES056 and F227L/V106A),
five single RT mutant strains of HIV-1 IIIB (L100I,
K103N, E138K, Y181C, Y188L), and HIV-2
(strain ROD) in MT-4 cell cultures using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu
4-dihydroquinazolin-2-amine (4w)
The title product was synthesized following
the general procedure, starting from 11 and
3,4-difluoroaniline (12w). Yield 74%, white solid,
m.p. 200-202 °C (EA/PE);1H NMR (400 MHz,
DMSO-d6) δ: 10.94 (s, 2H, NH), 7.69-7.52 (m, 5H,
PhH), 7.35 (d,J = 7.8 Hz, 1H, PhH), 7.27 (d, J =
7.9 Hz, 1H, PhH), 1.61-1.59 (m, 1H, CH), 0.95 (m,
2H, CH2), 0.80 (m, 2H, CH2); 13C NMR (101 MHz,
DMSO-d6) δ: 150.05 (d, J =244 Hz), 149.91 (d, J
=245 Hz), 149.39 (N=C-(NH)2), 148.83 (ArC),
148.69 (ArC, 2C), 132.98 (ArC), 132.48 (ArC),
132.11 (ArC), 129.43 (ArC), 127.65 (ArC), 123.76
m
bromide (MTT) method as described
previously[24, 25].At the beginning of each
experiment, stock solutions (10× final
concentration) of test compounds were added in 25
µL volumes to two series of triplicate wells in order
to allow simultaneous evaluation of their effects on

ACCEPTED MANUSCRIPT
mock- and HIV-infected cells. Using a Biomek
contained in the kit and the procedure for
assayingHIV-1 RT inhibition (recombinant WT
and K103N/Y181C double mutant RT enzymes)
was performed as described in the kit protocol[26].
Briefly, the reaction mixture containing HIV-1
RT enzyme, reconstituted template, and viral
3000 robot (Beckman Instruments, Fullerton, CA),
serial 5-fold dilutions of the test compounds (final
200 µL volume per well) were made directly in
flat-bottomed 96-well microtiter trays, including
untreated control HIV-1 and mock-infected cell
samples for each sample. HIV-1 (IIIB) and mutant
HIV-1 strains (RES056, F227L/V106A, L100I,
K103N, E138K, Y181C, and Y188L) or HIV-2
(ROD) stock (50 µL at 100−300 CCID50) (50%
cell culture infectious dose) or culture medium was
added to either the infected or mock-infected wells
of the microtiter tray. Mock-infected cells were
used to evaluate the effect of test compounds on
uninfected cells in order to assess the cytotoxicity
of the compounds. Exponentially growing MT-4
cells were centrifuged for 5 min at 1000 rpm, and
the supernatant was discarded. The MT-4 cells
were resuspended at 6 × 105 cells/mL, and 50 µL
aliquots were transferred to the microtiter tray
wells. At 5 days after infection, the viability of
mock- and HIV-infected cells was determined
spectro-photometrically by means of the MTT
assay.
nucleotides
[digoxigenin
(DIG)-dUTP,
biotin-dUTP, and dTTP] in the incubation buffer
with or without inhibitors was incubated for 1 h at
37 °C. Then the reaction mixture was transferred to
a streptavidin-coated microtiter plate (MTP) and
incubated for another
1 h at 37 °C. The
biotin-labeled dNTPs that were incorporated into
the cDNA chain in the presence of RT were bound
to streptavidin. The unbound dNTPs were washed
with washing buffer, and anti-DIG-POD was added
to the MTPs.
After incubation for 1 h at 37 °C, the
DIG-labeled dNTPs incorporated in cDNA were
bound to the anti-DIG-POD antibody. The
unbound anti-DIG-PODs were washed out, and the
peroxide substrate (ABST) solution was added to
the MTPs. The reaction mixture was incubated at
25 °C until the green color was sufficiently
developed for detection. The absorbance of the
sample was determined at OD405 nm using a
microtiter plate ELISA reader. The percentage
inhibitory activity of RT inhibitors was calculated
according to the following formula: % inhibition =
[O.D. value with RT but without inhibitors −O.D.
value with RT and inhibitors]/[O.D. value with RT
and inhibitors− O.D. value without RT and
inhibitors]. TheIC50 values correspond to the
concentrations of the inhibitors required to inhibit
biotin-dUTP incorporation by 50%.
The MTT assay is based on the reduction of
yellow-colored MTT (Acros Organics, Geel,
Belgium) by mitochondrial dehydrogenase of
metabolically active cells to form a blue-purple
formazan
that
can
be
measured
spectro-photometrically. The absorbances were
read in an eight-channel computer-controlled
photometer (Multiscan Ascent Reader, Lab systems,
Helsinki, Finland) at the wavelengths of 540 and
690 nm. All data were calculated using the median
optical density (OD) value of three wells. The 50%
effective antiviral concentration (EC50) led to 50%
protection from viral cytopathogenicity. The 50%
cytotoxic concentration (CC50) was defined as the
compound concentration that reduced the
absorbance (OD540) of mock-infected cells by 50%.
4.3. HIV-1 RT inhibition assay
4.4. Molecular docking
Molecular modeling studies 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 HIV-1 RT inhibition assay was conducted
by using an RT assay kit produced by Roche. All
the reagents for performing the RT reaction were

ACCEPTED MANUSCRIPT
the same. The flexible docking method, called
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].
Acknowledgement
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 scoring function[28]. The
protocol is a computational representation of a
putative ligand that binds to the intended binding
site and is a unique and essential element of the
docking algorithm[29]. The scoring function in
Surflex-Dock, which contains hydrophobic, polar,
repulsive, entropic, and solvation terms, was
trained to estimate the dissociation constant (Kd)
expressed in -log (Kd)2. The scoring function in
Surflex-Dock, which contains hydrophobic, polar,
repulsive, entropic and solvation terms, was trained
to 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 (PDB code:3MEC)[23];
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-scoring ligand-protein complexes were
kept for the further analyses. The -log(Kd)2 values
of the 20-best-scoring complexes, which
This research was financially supported by
National Natural Science Foundation of P. R. China
under Grant No. 21871055 and 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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Highlights
1. Twenty-four new compounds were synthesized as new HIV-1 NNRTIs.
2. Five out 24 compounds exhibited EC50 in the low nanomolar range.
3. Compound 4b showed the best result against the single and double mutant strain of
HIV-1.
4. The preliminary SAR and docking studies with HIV-1 RT were also investigated.