Hybrid diarylbenzopyrimidine non-nucleoside reverse transcriptase inhibitors as promising new leads for improved anti-HIV-1 chemotherapy.

Abstract

Molecular hybridization of the known anti-HIV-1 template DPC083 and etravirine based on docking model overlay has been generated a novel series of diarylbenzopyrimidine analogues (DABPs) (5a-z). These new hybrids were assessed for their activity against HIV in MT-4 cell cultures. Most of these compounds showed good activity against wild-type HIV-1 and mutant viruses. In particular, compound 5r showed the most potent activity against wild-type HIV-1 with an EC50 value of 1.8 nM, and with a selectivity index up to 111,954. It also proved more active against mutant L100I, K103N, Y188L, and K103N+Y181C RT HIV-1 strains than efavirenz.

Full text

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Hybrid diarylbenzopyrimidine non-nucleoside reverse transcriptase
inhibitors as promising new leads for improved anti-HIV-1 chemotherapy
Zhao-Sen Zenga, Qiu-Qin Hea, Yong-Hong Lianga, Xiao-Qing Fenga, Fen-Er Chena,b,*, Erik De Clercqc,
Jan Balzarinic, Christophe Pannecouquec
aDepartment of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
bInstitute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
cRega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
a r t i c l ei n f o
Article history:
Received 30 April 2010
Revised 28 May 2010
Accepted 28 May 2010
Available online 4 June 2010
Keywords:
HIV-1 Reverse transcriptase
NNRTIs
DABPs
Molecular hybridization
SAR
a b s t r a c t
Molecular hybridization of the known anti-HIV-1 template DPC083 and etravirine based on docking
model overlay has been generated a novel series of diarylbenzopyrimidine analogues (DABPs) (5a–z).
These new hybrids were assessed for their activity against HIV in MT-4 cell cultures. Most of these com-
pounds showed good activity against wild-type HIV-1 and mutant viruses. In particular, compound 5r
showed the most potent activity against wild-type HIV-1 with an EC50value of 1.8 nM, and with a selec-
tivity index up to 111,954. It also proved more active against mutant L100I, K103N, Y188L, and
K103N + Y181C RT HIV-1 strains than efavirenz.
? 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Diarylpyrimidine analogues (DAPYs, 1, Fig. 1), first discovered
by Dr. Paul Janssen and his co-workers in 2001,1have attracted
considerable attention due to their excellent activity and have
led to the identification of highly potent compounds against both
HIV-1 RT wild-type and mutant virus strains,2–8such as the mar-
keted etravirine (2, Fig. 1).1,9Another emerging clinical candidate
DPC083 (3, Fig. 1),10,11a more recent second-generation congener
of the licensed NNRTI drug efavirenz (4, Fig. 1), are characterized
by the improved profile of resilience to common resistance muta-
tions. However, there is still a need for exploring additional novel
chemical entities that present high potency and a broadened spec-
trum of activity toward a wide range of HIV-1 variants through
molecular hybridization12of DPC083 with etravirine.
Our docking model of overlaying the lower-energy conforma-
tions of DPC083 and etravirine in the binding pocket of HIV-1 RT
(Fig. 2) reveals that the hybrid molecules, derived from the fusion
of the aromatic ring with the pyrimidine ring of etravirine (Fig. 3),
share the features of DPC083 and etravirine. The steric and elec-
tronic similarities between DPC083 and etravirine suggested to
us that they might overlap within the binding pocket and interact
similarly with HIV-1 RT. On the basis of insights into the molecular
modeling studies of these hybrid molecules with HIV-1 RT, a series
of new diarylbenzopyrimidine hybrids 5 (DABPs) were synthesized
and evaluated as novel NNRTIs.
2. Results and discussion
2.1. Chemistry
The synthetic route for the target compounds (5a–z) is outlined
in Scheme 1. Treatment of the known intermediate 2,4-dichloro-
quinazolines 9a–b, which was prepared from 2-aminobenzoic acid
in a three-step sequence (reduction, cyclization, and chlorina-
tion),13with the corresponding phenols in the presence of anhy-
drous K2CO3at 80 ?C in EtOH provided the ethers (10a–z), which
were converted into the desired target compounds 5a–z in 32–
68% yields upon treatment with 4-cyanoaniline at 190 ?C for 2 h
under solvent-free condition.
The structures of all these hybrids were determined by mass
spectra,1H NMR, and13C NMR data. The structure of the represen-
tative compound 5o in these series was further evaluated by X-ray
analysis (Fig. 4).
2.2. Biology
The evaluation of the antiviral activity of the newly synthesized
hybrids DABPs 5a–z was based on the potency of inhibition of the
0968-0896/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.081
* Corresponding author. Tel./fax: +86 21 65643811.
E-mail address: rfchen@fudan.edu.cn (F.-E. Chen).
Bioorganic & Medicinal Chemistry 18 (2010) 5039–5047
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc

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replication of wild-type HIV-1 (IIIB) virus, the double mutant virus
K103N + Y181C RT HIV-1 and HIV-2 strain ROD, respectively. Com-
pounds 5r and 5u were further tested for their antiviral activity
against a panel of important mutant virus strains (E138K, L100I,
K103N, Y181C, and Y188L RT) and a double mutant virus strain
(F227L + V106A). The concentrations required to achieve 50% pro-
tection from HIV cytopathicity in MT-4 cells were determined by
the MTT method,14,15and are expressed as EC50(nM or lM) values.
The cytotoxic effects (CC50, lM) were assayed in parallel with the
antiretroviral activity. Three FDA approved drugs nevirapine
(NEV), delavirdine (DEV), and efavirenz (EFV) were chosen as refer-
ence drugs. All results are presented in Table 1.
As seen from the results listed in Table 1, most of the tested
compounds showed excellent activity against wild-type HIV-1
with a wide range of EC50values ranging from 1331 to 1.8 nM. It
is worth noting that compound 5r was the most potent compound,
with an EC50value of 1.8 nM against wild-type HIV-1, which is
much more effective than the reference compounds nevirapine
(by 731-fold) and delavirdine (by 266-fold). More importantly, it
also exhibited a very high selectivity index (111,954). Some other
compounds, 5n, 5t–w and 5z also showed high anti-HIV-1 potency
(EC50= 11, 8.7, 4.7, 7.5, 16, and 8.5 nM, respectively) and excellent
selectivity indices (SI = 3215, 22,005, 46,947, 2969, 6771, and
13,832, respectively). These promising results appear to validate
that the molecular hybridization of DPC083 with etravirine is ben-
eficial to reinforce the van der Waals interaction between inhibi-
tors and amino acid residues within the NNRTI binding pocket,
thus enhancing their anti-HIV potency.
The activities against the double mutant strain K103N + Y181C
of these compounds were also assessed. Compound 5r turned out
to be the most potent inhibitor with an EC50of 0.06 lM, being
about two times lower than that of efavirenz (0.11 lM). Com-
pounds 5m, 5o–p, and 5u–v also displayed high anti-HIV-1 activity
against the double mutant virus strain (EC50= 0.53, 0.35, 0.59, 0.2,
and 0.29 lM, respectively), been more potent than nevirapine or
delavirdine.
The potency of all title compounds to inhibit the replication of
the HIV-2 ROD virus in MT-4 cells shown that some compounds
also displayed micromolar activity. Compound 5p was the most
potent among these hybrids with an EC50value 4.62 lM against
HIV-2 ROD.
The results listed in Table 1 revealed some important SAR infor-
mation on the role of different substitutions in the DABPs. First of
all, the nature of the Ar in the DABPs was investigated by preparing
a series of Ar analogues of DABPs. Analogs that are mono-substi-
tuted on the Ar ring gave enhanced activity (5b–j) as compared
with the unsubstituted compound 5a, regardless of the electronic
effect and steric bulk. In contrast to the anti-HIV potency of com-
pounds 5b, 5d, and 5c demonstrated a positional preference for
ortho substitution in the Ar ring. The activity of the synthesized
ortho and para congeners (5b, 5e–f and 5d, 5g–j) revealed that
the chloro and methyl provided similar activity in inhibiting
wild-type HIV-1, whereas other substituents such as F, Br, and
MeO led to decreased activity. Clearly, di- or tri-substitution signif-
icantly increased inhibitory activity against wild-type HIV-1 as
well as double mutant virus K103N + Y181C as compared to the
corresponding mono-substitution (5n > 5m > 5b, or 5l > 5k > 5f).
Maximum activity was observed with the compound 5r with a
2,6-diMe-4-cyano phenyl group as the Ar group. Increasing the ste-
ric bulk of ortho disubstituents in the Ar ring resulted in a de-
creased antiviral activity (5r > 5t > 5w > 5y, or 5u > 5v). These
rules were in agreement with the previous results as disclosed
by our group.8It is worth noting that introducing Cl at the 6-posi-
tion of the quinazoline ring led to a slight decrease of the anti-HIV-
1 potency (5z < 5r).
Furthermore, promising compounds 5r and 5u were subse-
quentlytestedfortheirantiviralactivityagainstapanelofimportant
single mutant RT (E138K, L100I, K103N, Y181C, and Y188L) and
Figure 2. Superposition of lower-energy docking binding conformations of DPC083
(pink) and etravirine (gray) in the binding pocket of HIV-1 RT.
N
N
ONH
N
NH2
Br
CN
N
N
XNH
R1
R2
R3
1 DAPYs 2 Etravirine
N
H
NH
O
Cl
F3C
3 DPC083
N
H
O
O
Cl
F3C
4 Efavirenz
Figure 1. Chemical structures of DAPYs, etravirine, DPC083, and efavirenz.
N
N
ONH
N
N
XNH
N
N
+
NH2
Br
CN
NH
H
N
O
F3C
Cl
DPC083 Etravirine
R1
R2
5 DABPs
Figure 3. Molecular hybridization of DPC083 and etravirine to create the diary-
lbenzopyrimidines (DABPs) template 5.
5040
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double RT mutant strains (F227L + V106A and K103N + Y181C)
(Table 2). It was found that these two compounds showed excellent
activity against wild-type and mutant strains. Compound 5r and
efavirenz were equipotent inhibitors against the E138K, L100I,
F227L + V106A RT mutant virus strains. Although the compound
5r is approximately five times less potent than efavirenz against
the Y181C RT HIV-1 mutant, for the K103N and Y188L single RT
HIV-1 mutants, compound 5r was 6–20-fold more potent than the
clinically used efavirenz. Compound 5u also kept potent activity
against the K103N RT mutant virus strain. The re-assessed activities
against wild-type and K103N + Y181C were in accordance with the
above mentioned.
2.3. Molecular modeling calculations
Compound 5r, as the representative of the hybrid molecules,
was docked into the non-nucleoside binding site (NNBS) with
Molegro Virtual Docker demo version16(Fig. 5) to investigate the
binding mode of our hybrid compounds with NNBS of HIV-1 RT.
The coordinates of the NNBS were taken from the crystal structure
of the RT/4-(4-(mesitylamino)pyrimidin-2-ylamino)benzonitrile
(TMC120) complex (pdb code: 1S6Q).17The model revealed that
the analogue binds to HIV-1 RT in the horseshoe mode of action,
like most of the DAPYs compounds do.17According to this mode
of action, the NH group that is linking the quinazoline ring and
4-cyanophenyl group could create the hydrogen bonds with the
amino acid residues Lys101 and Lys103. The left Ar ring interacts
through p–p interactions with a hydrophobic pocket formed
mainly by the side chains of aromatic amino acids Tyr181,
Tyr188, Phe227, Tyr318, and Trp229. Secondly, this mode of bind-
ing of 5r allows to keep the conformational flexibility which may
compensate for the effects of resistance mutations. Finally, the fus-
ing phenyl parts of the hybrid molecule enhanced the van der
Waals interaction between 5r and the adjacent amino acid residues
(Glu138, Val179, Lys101, and Lys103) within the binding pocket of
RT. All these interactions would favor the high binding affinity and
increased activity against wild-type and mutant virus strains.
3. Conclusion
In summary, we designed and synthesized a novel series of
diarylbenzopyrimidine analogues (DABPs) and evaluated their
activity against HIV in MT-4 cells. The activities against HIV-1 indi-
cated that the designed compounds showed potent antiviral activ-
ity with EC50values in the low nanomolar range. DABPs derivatives
with 2,6-dime-4-cyano phenyl group as Ar group (5r) showed the
most potent activity against wild-type HIV-1 with an EC50value of
1.8 nM, and with a selectivity index up to 111,954. Also, compound
N
N
N
H
O
Ar
CN
N
N
N
N
OH
O
NH2
OH
O
NO2
a
b
c
d
e
RR
RR
OH
OH
Cl
Cl
N
N
Cl
O
Ar
R
R
10a-z5a-z
6a-b7a-b8a-b9a-b
6, 7, 8, 9
R
a
HCl
b
a
HC6H5
b
H2-Me-C6H4
c
H3-Me-C6H4
d
H4-Me-C6H4
e
H 2-MeO-C6H4
f
H 2-Cl-C6H4
g
H4-F-C6H4
h
H 4-Cl-C6H4
i
H 4-Br-C6H4
R Ar
j
Hp-MeO-C6H5
k
H 2,4-diCl-C6H3
l
H2,4,6-triCl-C6H2
m
H2,6-diMe-C6H3
n
H2,4,6-triMe-C6H2
o
H 2,4-diBr-6-Me-C6H2
p
H2,6-diBr-4-Me-C6H2
q
H2,4,6-triBr-C6H2
r
H4-CN-2,6-diMe-C6H2
RAr
s
H4-CN-2-MeO-C6H3
t
H4-CN-2,6-diMeO-C6H2
u
H 2-Cl-4-CN-6-MeO-C6H2
v
H2-Cl-4-CN-6-EtO-C6H2
w
H4-CN-2,6-diEtO-C6H2
x
H 4-CN-2-MeO-6-PrO-C6H2
y
H4-CN-2-EtO-6-PrO-C6H2
z
Cl4-CN-2,6-diMe-C6H2
R Ar
5, 10
5, 10 5, 10
Scheme 1. Synthetic route to target compounds 5a–z. Reagents and conditions: (a) Raney Ni, NaBH4, 20 min, 30–40 ?C; (b) urea, 180 ?C, 3 h; (c) POCl3, N,N-diethyl aniline,
reflux, 2 h; (d) K2CO3, phenol derivatives, EtOH, 5 min, then 9, 80 ?C, 12 h; (e) 4-cyanoaniline, 180–190 ?C, 2 h.
Figure 4. Crystal structure of compound 5o.
Z.-S. Zeng et al./Bioorg. Med. Chem. 18 (2010) 5039–5047
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5r showed excellent potency against the L100I, K103N, Y188L, and
K103N + Y181C mutant strains with EC50value 18, 3.6, 36, and
60 nM, respectively. The compound can serve as the basis for fur-
ther modification in searching for more effective candidates for im-
proved anti-HIV-1 chemotherapy.
4. Experimental section
4.1. Chemistry
Melting points were measured on a WRS-1 digital melting point
apparatus and are uncorrected.1H NMR and13C NMR spectra on a
Brucker AV 400 MHz spectrometer were recorded in CDCl3 or
DMSO-d6. Chemical shifts are reported in d (ppm) units relative to
the internal standard tetramethylsilane (TMS). Mass spectra were
obtained on an Agilent MS/5975 mass spectrometer. All chemicals
andsolventsusedwereofreagentgradeandwerepurifiedanddried
bystandardmethodsbeforeuse.Allair-sensitivereactionswererun
under a nitrogen atmosphere. All the reactions were monitored by
TLC on pre-coated silica gel G plates at 254 nm under a UV lamp
using ethyl acetate/hexane as eluents. Flash chromatography sepa-
rations were obtained on silica gel (300–400 mesh).
Table 1
Anti-HIV-1 activity and cytotoxicity of compounds 5a–z in MT-4 cellsa
CompdEC50b
CC50c(lM)SId
WT (IIIB) (nM) HIV-2 (lM)K103N + Y181C (lM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
NEV
EFV
DEV
1331 ± 769
57 ± 20
823 ± 340
105 ± 51
120 ± 103
37 ± 5.3
590 ± 421
190 ± 16
264 ± 72
190 ± 49
23 ± 8.8
21 ± 9.3
23 ± 13
11 ± 0.8
19 ± 10
20 ± 9.8
28 ± 12
1.8 ± 0.5
33 ± 13
8.7 ± 3.3
4.7 ± 1.9
7.5 ± 1.8
16 ± 3.5
13 ± 5.1
58 ± 9.3
8.5 ± 2.1
1316 ± 827
2.1 ± 1.3
479 ± 131
>369
>355
>355
>86
>26
>37
>351
>197
>214
>339
>31
>55
P10
>34
P14
P4.6
P8.6
32
>140
>194
>219
>21
>108
>5.2
>122
>117
—
—
—
>369
>355
>355
>86
>149
>37
>351
>197
>214
>339
>31
1.5 ± 1
0.53 ± 0.27
2.0 ± 0.9
0.35 ± 0.14
0.59 ± 0.2
0.93 ± 0.5
0.06 ± 0.02
>140
0.92 ± 0.19
0.16
0.29 ± 0.09
>108
>5.2
>122
0.7
P15
0.11 ± 0.03
>43
370
355
355
87 ± 69
150 ± 89
38 ± 2.8
>351
198 ± 9.2
215 ± 35
340
32 ± 4
56 ± 19
35 ± 2
>34 ± 1.4
150 ± 18
25 ± 2.3
58 ± 21
203 ± 35
P140
194 ± 12
220 ± 11
22 ± 4.4
108 ± 8.3
5.2 ± 0.8
123 ± 11
117 ± 6.2
P15
P6.3
>43
>279
>6211
>436
831
1265
982
>607
1037
810
>1794
1412
2626
1511
3215
7931
1281
2122
111,954
64195
22,005
46,947
2969
6771
408
2084
13,832
>114
>3082
>92
aAll data represent mean values for at least two separate experiments.
bEC50: effective concentration of compound required to protect the cell against viral cytopathogenicity by 50% in MT-4 cells.
cCC50: cytotoxic concentration of compound that reduces the normal uninfected MT-4 cell viability by 50%.
dSI: selectivity index: ratio CC50/EC50.
Table 2
Antiviral activity (nM) against wild-type HIV-1 LAI virus and a panel of single and double mutant strains of compounds 5r and 5u
CompdLAI E138K L100I K103NY181C Y188LF227L + V106A K103N + Y181C
5r
5u
NEV
DEV
EFV
2.3 ± 1
5.4 ± 1.9
102 ± 64
610 ± 174
2.2 ± 0.9
11
12 ± 1.6
162 ± 117
566 ± 457
5.4 ± 0.3
18 ± 1.5
110 ± 49
2030 ± 1241
7952 ± 9547
32
63.6
65.4
6729 ± 1128
6514 ± 8779
66 ± 19
31 ± 20
49 ± 23.3
>15,037
6253 ± 4749
5.7 ± 0.6
36 ± 28
280 ± 210
>15,037
6427 ± 458
222 ± 54
107 ± 92
467 ± 140
>15,037
9847 ± 7538
104 ± 107
56 ± 15
159
>15,037
?43,573
85
Figure 5. Model of 5r docked into the HIV-1 RT non-nucleoside binding site.
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Z.-S. Zeng et al./Bioorg. Med. Chem. 18 (2010) 5039–5047

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4.2. General procedure for the synthesis of 7a–b13a
To a solution of 2-nitrobenzoic acid 6a–b (100 mmol) in meth-
anol (200 mL) Raney nickel (590 mg, 10 mol %) was added at room
temperature. Thereafter, sodium borohydride (7.6 g, 200 mmol,
2 equiv) was slowly and carefully added in a few portions by keep-
ing the temperature between 30 and 40 ?C with external cooling.
The reaction mixture was stirred for 20 min. The catalyst was fil-
tered off, washed with methanol (30 mL), and immediately im-
mersed in distilled water to prevent spontaneous combustion.
The filtrate was evaporated to dryness and the residue was parti-
tioned between water (200 mL) and dichloromethane (200 mL).
After separation of the phases, water layer was additionally ex-
tracted with dichloromethane (3 ? 60 mL). Combined organic ex-
tracts were dried over anhydrous Na2SO4, filtered and evaporated
to dryness to afford products which could be used in the next step
without further purification.
4.3. General procedure for the synthesis of 8a–b13b
A flask containing urea (15.0 g, 0.25 mol) and 2-aminobenzoic
acid 7a–b (62.5 mmol) was heated at 180 ?C. After being stirred
for 3 h, the reaction mixture was cooled to 100 ?C and an equal vol-
ume of water was added. The obtained suspension was left to stir
for 10 min, after which it was cooled to room temperature. The
precipitate was filtered off and recrystallized from DMF to obtain
the title compounds.
4.4. General procedure for the synthesis of 9a–b13b
A mixture of 80 ml POCl3, N,N-diethylaniline (300 mmol) and
quinazoline-2,4-diol derivatives 8a–b (150 mmol) was refluxed
for 2 h. The cooling mixture poured into 250 mL ice-water and
the mixture was stirred at room temperature. The resulting precip-
itate was filtered off and washed with 50 mL water, and dried to
give products 9a–b to be used in the next step without further
purification.
4.5. General procedure for the synthesis of 10a–z18
Potassium carbonate (10 mmol) was added to a solution of phe-
nol derivatives (2 mmol) in 20 mL of anhydrous EtOH and was stir-
red for 5 min. Then, 2,4-dichloroquinazolines 9a–b (2 mmol) were
then added. The reaction mixture was refluxed for 12 h. After com-
pletion, the reaction mixture was treated with cold water
(200 mL), and the resulting precipitate was filtered off. The crude
products 10a–z were recrystallized from DMF.
4.5.1. 2-Chloro-4-phenoxyquinazoline (10a)
Yield: 87.5%. Mp 117.1–117.7 ?C;1H NMR (DMSO-d6) d (ppm)
7.35–7.56 (m, 5H, Ar00H), 7.48 (t, 1H, J = 8.0 Hz, ArH7), 7.80 (d, 1H,
J = 8.4 Hz, ArH6), 8.08 (t, 1H, J = 8.4 Hz, ArH8), 8.39 (d, 1H,
J = 8.4 Hz, ArH9); MS (ESI) m/z 257 (M++1).
4.5.2. 2-Chloro-4-(o-tolyloxy)quinazoline (10b)
Yield: 86.3%. Mp 110.9–111.3 ?C (lit.18mp 128–130 ?C).1H NMR
(DMSO-d6) d (ppm) 2.15 (s, 3H, CH3), 7.28–7.42 (m, 4H, Ar00H3,4,5,6),
7.81–7.85 (m, 1H, ArH7), 7.95–7.97 (m, 1H, ArH6), 8.08–8.12 (m,
1H, ArH8), 8,43–8.45 (m, 1H, ArH9); MS (ESI) m/z 271 (M++1).
4.5.3. 2-Chloro-4-(m-tolyloxy)quinazoline (10c)
Yield: 86.9%. Mp 134.2–134.6 ?C (lit.18mp 139–141.5 ?C).
NMR (DMSO-d6) d (ppm) 2.37 (s, 3H, CH3), 7.17–7.19 (m, 3H, Ar00H),
7.40 (t, 1H, J = 8.0 Hz, Ar00H), 7.79 (t, 1H, J = 8.0 Hz, ArH7), 7.93 (d,
1H, J = 8.4 Hz, ArH6), 8.06 (td, 1H, J = 7.2 Hz, J0= 0.8 Hz, ArH8),
8.36 (d, 1H, J = 8.0 Hz, ArH9); MS (ESI) m/z 271 (M++1).
1H
4.5.4. 2-Chloro-4-(p-tolyloxy)quinazoline (10d)
Yield: 87.5%. Mp 148.4–149.0 ?C (lit.18mp 154–156 ?C).1H NMR
(DMSO-d6) d (ppm) 2.38 (s, 3H, CH3), 7.27 (d, 2H, J = 8.4 Hz,
Ar00H3,5), 7.32 (d, 2H, J = 8.4 Hz, Ar00H2,6), 7.80 (t, 1H, J = 8.0 Hz,
ArH7), 7.93 (d, 1H, J = 8.0 Hz, ArH6), 8.08 (td, 1H, J = 8.4 Hz,
J0= 1.2 Hz, ArH8), 8.38 (d, 1H, J = 8.0 Hz, ArH9); MS (ESI) m/z 271
(M++1).
4.5.5. 2-Chloro-4-(2-methoxyphenoxy)quinazoline (10e)
Yield: 87.5%. Mp 117.1–117.7 ?C.1H NMR (DMSO-d6) d (ppm)
7.35–7.56 (m, 5H, Ar00H), 7.48 (t, 1H, J = 8.0 Hz, ArH7), 7.80 (d, 1H,
J = 8.4 Hz, ArH6), 8.08 (t, 1H, J = 8.4 Hz, ArH8), 8.39 (d, 1H,
J = 8.4 Hz, ArH9); MS (ESI) m/z 287 (M++1).
4.5.6. 2-Chloro-4-(2-chlorophenoxy)quinazoline (10f)
Yield: 68.4%. Mp 163.9–164.4 ?C (lit.18mp 166–167 ?C).1H NMR
(DMSO-d6) d (ppm) 7.42–7.71 (m, 4H, Ar00H), 7.84 (t, 1H, J = 7.6 Hz,
ArH7), 7.98 (d, 1H, J = 8.4 Hz, ArH6), 8.12 (t, 1H, J = 8.4 Hz, ArH8),
8.43 (d, 1H, J = 8.0 Hz, ArH9); MS (ESI) m/z 292 (M++1).
4.5.7. 2-Chloro-4-(4-fluorophenoxy)quinazoline (10g)
Yield: 68.4%. Mp 143.2–142.4 ?C (lit.18mp 120–122 ?C).1H NMR
(DMSO-d6) d (ppm) 7.33–7.46 (m, 4H, Ar00H), 7.79 (t, 1H, J = 8.0 Hz,
ArH7), 7.92 (d, 1H, J = 8.8 Hz, ArH6), 8.07 (t, 1H, J = 8.4 Hz, ArH8),
8.36 (d, 1H, J = 7.6 Hz, ArH9); MS (ESI) m/z 275 (M++1).
4.5.8. 2-Chloro-4-(4-chlorophenoxy)quinazoline (10h)
Yield: 76.3%. Mp 158.6–160.2 ?C (lit.18mp 145–147 ?C).1H NMR
(DMSO-d6) d (ppm) 7.47 (d, 2H, J = 6.8 Hz, Ar00H3,5), 7.59 (d, 2H,
J = 6.8 Hz, Ar00H2,6), 7.80 (t, 1H, J = 8.0 Hz, ArH6), 7.95 (d, 1H,
J = 8.4 Hz, ArH7), 8.09 (t, 1H, J = 8.0 Hz, ArH8), 8.37 (d, 1H,
J = 8.4 Hz, ArH9); MS (ESI) m/z 292 (M++1).
4.5.9. 4-(4-Bromophenoxy)-2-chloroquinazoline (10i)
Yield: 85.5%. Mp 160.1–160.2 ?C (lit.18mp 161.5–163.5 ?C).1H
NMR (DMSO-d6) d (ppm) 7.41 (d, 2H, J = 6.8 Hz, Ar00H2,6), 7.73 (d,
2H, J = 6.8 Hz, Ar00H3,5), 7.81 (t, 1H, J = 8.4 Hz, ArH7), 7.95 (d, 1H,
J = 8.8 Hz, ArH6), 8.09 (td, 2H, J = 8.4 Hz, J0= 1.2 Hz, ArH8), 8.38 (d,
1H, J = 8.4 Hz, ArH9); MS (ESI) m/z 335 (M++1).
4.5.10. 2-Chloro-4-(4-methoxyphenoxy)quinazoline (10j)
Yield: 89.0%. Mp 127.9–128.0 ?C.1H NMR (DMSO-d6) d (ppm)
3.87 (s, 3H, CH3O), 7.01 (d, 2H, J = 6.8 Hz, Ar00H3,5), 7.22 (d, 2H,
J = 6.8 Hz, Ar00H2,6), 7.65–7.69 (m, 1H, ArH7), 7.93–7.95 (m, 2H,
ArH6,8), 8.36 (d, 1H, J = 8.4 Hz, ArH9); MS (ESI) m/z 287 (M++1).
4.5.11. 2-Chloro-4-(2,4-dichlorophenoxy)quinazoline (10k)
Yield: 74.2%. Mp 172.4–172.6 ?C (lit.18mp 174.5–1175.5 ?C).1H
NMR (DMSO-d6) d (ppm) 7.59–7.88 (m, 4H, Ar00H + ArH6), 7.97 (d,
1H, J = 8.8 Hz, ArH7), 8.11 (t, 1H, J = 8.4 Hz, ArH8), 8.40 (d, 1H,
J = 8.0 Hz, ArH9); MS (ESI) m/z 326 (M++1).
4.5.12. 2-Chloro-4-(2,4,6-trichlorophenoxy)quinazoline (10l)
Yield: 88.9%. Mp 223.2–224.1 ?C (lit.18mp 228–230 ?C).1H NMR
(DMSO-d6) d (ppm) 7.94 (dt, 1H, J = 6.8 Hz, J0= 1.2 Hz, ArH7), 8.04
(s, 2H, Ar00H3,5), 8.09 (d, 1H, J = 8.4 Hz, ArH6), 7.91 (dt, 1H,
J = 6.8 Hz, J0= 1.2 Hz, ArH8), 8.36 (dd, 1H, J = 8.4 Hz, J0= 0.8 Hz,
ArH9); MS (ESI) m/z 361 (M++1).
4.5.13. 2-Chloro-4-(2,6-dimethylphenoxy)quinazoline (10m)
Yield: 87.3%. Mp 136.5–138.1 ?C (lit.18mp 140–142 ?C).1H NMR
(DMSO-d6) d (ppm) 2.12 (s, 6H, 2CH3), 7.22–7.26 (m, 3H, Ar00H),
7.87 (t, 1H, J = 8.0 Hz, ArH7), 8.00 (d, 1H, J = 8.8 Hz, ArH6), 8.13 (t,
1H, J = 7.2 Hz, ArH8), 8.50 (d, 1H, J = 8.0 Hz, ArH9); MS (ESI) m/z
285 (M++1).
Z.-S. Zeng et al./Bioorg. Med. Chem. 18 (2010) 5039–5047
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4.5.14. 2-Chloro-4-(mesityloxy)quinazoline (10n)
Yield: 66.9%. Mp 163.6–164.5 ?C.1H NMR (DMSO-d6) d (ppm)
2.04 (s, 6H, 2CH3), 2.30 (s, 3H, CH3), 7.01 (s, 2H, Ar00H3,5), 7.82 (t,
1H, J = 8.0 Hz, ArH7), 7.96 (d, 1H, J = 8.4 Hz, ArH6), 8.10 (t, 1H,
J = 8.4 Hz, ArH8), 8.46 (d, 1H, J = 8.0 Hz, ArH9); MS (ESI) m/z 299
(M++1).
4.5.15. 2-Chloro-4-(2,4-dibromo-6-methylphenoxy)quinazoline
(10o)
Yield: 81.7%. Mp 205.2–205.5 ?C.1H NMR (DMSO-d6) d (ppm)
2.21 (s, 3H, CH3), 7.72–7.73 (m, 1H, Ar00H), 7.87 (t, 1H, J = 8.4 Hz,
ArH6), 7.89–7.90 (m, 1H, Ar00H), 8.01 (d, 1H, J = 8.0 Hz, ArH7) 8.15
(td, 1H, J = 8.0 Hz, J0= 1.2 Hz, ArH8), 8.46 (d, 1H, J = 7.6 Hz, ArH9);
MS (ESI) m/z 429 (M++1).
4.5.16. 2-Chloro-4-(2,6-dibromo-4-methylphenoxy)quinazoline
(10p)
Yield: 80.7%. Mp 194.6–195.6 ?C.1H NMR (DMSO-d6) d (ppm)
2.39 (s, 3H, CH3), 7.70 (s, 2H, Ar00H), 7.70–8.19 (m, 3H, ArH6,7,8),
8.47 (dd, 1H, J = 8.0 Hz, J0= 0.8 Hz, ArH9); MS (ESI) m/z 429 (M++1).
4.5.17. 2-Chloro-4-(2,4,6-tribromophenoxy)quinazoline (10q)
Yield: 77.9%. Mp 190.3–190.9 ?C (lit.18mp 228–230 ?C).1H NMR
(DMSO-d6) d (ppm) 7.57–8.16 (m, 5H, ArH6,7,8+ Ar00H), 8.43 (d, 1H,
J = 8.0 Hz, ArH9); MS (ESI) m/z 494 (M++1).
4.5.18. 4-(2-Chloroquinazolin-4-yloxy)-3,5-dimethylbenzo-
nitrile (10r)
Yield: 85.6%. Mp 251.2–252.0 ?C.1H NMR (DMSO-d6) d (ppm)
2.20 (s, 6H, 2CH3), 7.71 (s, 2H, Ar00H3,5), 7.86 (t, 1H, J = 7.6 Hz,
ArH7), 7.96 (d, 1H, J = 8.4 Hz, ArH6), 8.12 (t, 1H, J = 8.0 Hz, ArH8),
8.51 (d, 1H, J = 8.4 Hz, ArH9); MS (ESI) m/z 310 (M++1).
4.5.19. 4-(2-Chloroquinazolin-4-yloxy)-3-methoxybenzonitrile
(10s)
Yield: 88.6%. Mp 240.1–240.8 ?C.1H NMR (DMSO-d6) d (ppm)
3.84 (s, 3H, CH3O), 7.31–8.00 (m, 6H, ArH6,7,8+ Ar00H), 8.39 (d, 1H,
J = 8.0 Hz, ArH9); MS (ESI) m/z 312 (M++1).
4.5.20. 4-(2-Chloroquinazolin-4-yloxy)-3,5-dimethoxybenzoni-
trile (10t)
Yield: 80.2%. Mp 270.2–270.7 ?C.1H NMR (DMSO-d6) d (ppm)
3.79 (s, 6H, 2CH3O), 7.43 (s, 2H, Ar00H3,5), 7.81 (t, 1H, J = 7.6 Hz,
ArH6), 7.96 (d, 1H, J = 8.4 Hz, ArH7), 8.09 (t, 1H, J = 7.6 Hz, ArH8),
8.37 (d, 1H, J = 8.4 Hz, ArH9); MS (ESI) m/z 342 (M++1).
4.5.21. 3-Chloro-4-(2-chloroquinazolin-4-yloxy)-5-methoxyben-
zonitrile (10u)
Yield: 74.4%. Mp 241.6–242.0 ?C.1H NMR (DMSO-d6) d (ppm)
3.66 (s, 3H, CH3O), 7.13–7.87 (m, 6H, ArH + Ar00H3,5); MS (ESI) m/
z 347 (M++1).
4.5.22. 3-Chloro-4-(2-chloroquinazolin-4-yloxy)-5-ethoxybenzo-
nitrile (10v)
Yield: 92.7%. Mp 185.2–187.6 ?C.1H NMR (DMSO-d6) d (ppm)
1.00 (t, 3H, J = 7.2 Hz, CH3), 4.13 (q, 2H, J = 7.2 Hz, CH2O), 7.80–
7.89 (m, 3H, ArH7+ Ar00H3,5), 7.99 (d, 1H, J = 8.4 Hz, ArH6), 8,13 (t,
1H, J = 8.4 Hz, ArH8), 8.41 (d, 1H, J = 8.0 Hz, ArH9); MS (ESI) m/z
361 (M++1).
4.5.23. 4-(2-Chloroquinazolin-4-yloxy)-3,5-diethoxybenzo-
nitrile (10w)
Yield: 76.9%. Mp 191.3–192.0 ?C.1H NMR (DMSO-d6) d (ppm)
1.06 (t, 6H, J = 7.2 Hz, 2CH3), 4.09 (q, 4H, J = 7.2 Hz, 2CH2O), 7.16
(s, 2H, Ar00H3,5), 7.82 (t, 1H, J = 8.0 Hz, ArH7), 7.97 (d, 2H,
J = 8.4 Hz ArH6), 8.11 (t, 1H, J = 8.4 Hz, ArH8), 8.38 (d, 1H,
J = 8.0 Hz, ArH9); MS (ESI) m/z 370 (M++1).
4.5.24. 4-(2-Chloroquinazolin-4-yloxy)-3-methoxy-5-propoxy-
benzonitrile (10x)
Yield: 73.9%. Mp 169.3–171.2 ?C.1H NMR (DMSO-d6) d (ppm)
0.67 (t, 3H, J = 7.2 Hz, CH3), 1.53 (m, 2H, CH2), 3.89 (s, 3H, CH3O),
4.05 (t, 2H, J = 7.2 Hz, CH2O), 7.27–7.66 (m, 2H, Ar00H), 7.82 (t, 1H,
J = 8.4 Hz, ArH7), 7.94 (d, 1H, J = 8.4 Hz, ArH6), 8.09 (t, 1H,
J = 8.4 Hz, ArH8), 8.43 (d, 1H, J = 8.4 Hz, ArH9); MS (ESI) m/z 370
(M++1).
4.5.25. 4-(2-Chloroquinazolin-4-yloxy)-3-ethoxy-5-propoxyben-
zonitrile (10y)
Yield: 82.3%. Mp 192.7–194.0 ?C.1H NMR (DMSO-d6) d (ppm)
0.54 (t, 3H, J = 7.2 Hz, CH3), 0.97–1.45 (m, 5H, CH3+ CH2), 3.96–
4.14 (m, 4H, 2CH2O), 7.39–8.38 (m, 6H, ArH + Ar00H); MS (ESI) m/z
384 (M++1).
4.5.26. 4-(2,6-Dichloroquinazolin-4-yloxy)-3,5-dimethylbenzo-
nitrile (10z)
Yield: 72.6%. Mp 267.4–269.2 ?C.1H NMR (DMSO-d6) d (ppm)
2.13 (s, 6H, 2CH3), 7.15–8.51 (m, 5H, ArH + Ar00H); MS (ESI) m/z
345 (M++1).
4.6. General procedure for the synthesis of 5a–z
Compounds 10a–z (10 mmol) and 4-cyanoaniline (35 mmol)
were thoroughly mixed and the mixture was slowly heated to
180–190 ?C and maintained at this temperature for about 2 h. After
cooling and dissolving the mixture in DMF and subsequent decol-
orization with charcoal, the product was precipitated with water.
The precipitate was filtered and further purified by silica column
chromatography (AcOEt/petroleum ether = 1:3).
4.6.1. 4-(4-Phenoxyquinazolin-2-ylamino)benzonitrile (5a)
Yield 59.6%. Mp 230.7–231.9 ?C;
7.43 (m, 3H, Ar0H2,6+ Ar00H4), 7.48 (t, 1H, J = 7.6 Hz, ArH7), 7.55–
7.59 (m, 4H, Ar00H2,3,5,6), 7.72 (d, 1H, J = 8.0 Hz, ArH6), 7.87–7.91
(m, 3H, ArH8+ Ar0H3,5), 8,23 (d, 1H, J = 8.0 Hz, ArH9), 10.04 (s, 1H,
NH);13C NMR (DMSO-d6) d 102.3, 112.0, 118.5 (2C), 119.5, 122.2
(2C), 123.7, 124.2, 125.4, 126.0, 129.9 (2C), 132.6 (2C), 134.9,
144.8, 152.4, 152.6, 155.1, 167.2; MS (ESI) m/z 337 (M+?1). Anal.
Calcd for C21H14N4O: C, 75.54; H, 4.17; N, 16.56. Found: C, 75.42;
H, 4.28; N, 16.66.
1H NMR (DMSO-d6) d 7.38–
4.6.2. 4-(4-(o-Tolyloxy)quinazolin-2-ylamino)benzonitrile (5b)
Yield 45.1%. Mp 197.3–197.4 ?C;1H NMR (DMSO-d6) d 2.16 (s,
3H, CH3), 7.30–7.44 (m, 4H, Ar00H3,4,5,6), 7.49 (td, 1H, J = 7.6 Hz,
J0= 1.2 Hz, ArH7), 7.56 (d, 2H, J = 8.8 Hz, Ar0H2,6), 7.72 (d, 1H,
J = 8.4 Hz, ArH6), 7.86 (d, 2H, J = 8.8 Hz, Ar0H3,5), 7.90 (td, 1H,
J = 7.6 Hz, J0= 1.2 Hz, ArH8), 8.27 (dd, 1H, J = 8.0 Hz, J0= 0.8 Hz,
ArH9), 10.04 (s, 1H, NH);
112.2, 118.9 (2C), 120.0, 122.9, 124.2, 124.8, 125.9, 126.7, 128.0,
130.7, 131.9, 133.1, 135.4 (2C), 145.4, 151.4, 153.1, 155.7, 167.2;
MS (ESI) m/z 353 (M++1). Anal. Calcd for C22H16N4O: C, 74.98; H,
4.58; N, 15.90. Found: C, 75.06; H, 4.43; N, 15.99.
13C NMR (DMSO-d6) d 16.4, 102.8,
4.6.3. 4-(4-(m-Tolyloxy)quinazolin-2-ylamino)benzonitrile (5c)
Yield 68.3%. Mp 219.7–220.3 ?C;1H NMR (DMSO-d6) d 2.39 (s,
3H, CH3), 7.18–7.22 (m, 3H, Ar00H2,4,6), 7.43 (d, 1H, J = 7.6 Hz, Ar00H5),
7.48 (td, 1H, J = 8.0 Hz, J0= 1.2 Hz, ArH7), 7.59 (d, 2H, J = 8.4 Hz,
Ar0H2,6), 7.72 (d, 1H, J = 8.4 Hz, ArH6), 7.88 (td, 1H, J = 7.2 Hz,
J0= 1.2 Hz, ArH8), 7.93 (d, 2H, J = 8.4 Hz, Ar0H3,5), 8.22 (dd, 1H,
J = 8.4 Hz, J0= 1.2 Hz, ArH9), 10.03 (s, 1H, NH);13C NMR (DMSO-
d6) d 20.8, 102.3, 112.0, 118.4 (2C), 119.0, 119.4, 122.4, 123.6,
5044
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124.1, 125.3, 126.5, 129.5, 132.6 (2C), 134.8, 139.6, 144.8, 152.3,
152.5, 155.0, 167.1; MS (ESI) m/z 353 (M++1). Anal. Calcd for
C22H16N4O: C, 74.98; H, 4.58; N, 15.90. Found: C, 75.18; H, 4.56;
N, 15.82.
4.6.4. 4-(4-(p-Tolyloxy)quinazolin-2-ylamino)benzonitrile (5d)
Yield 56.6%. Mp 220.6–220.8 ?C;1H NMR (DMSO-d6) d 2.39 (s,
3H, CH3), 7.26 (d, 2H, J = 8.8 Hz, Ar0H2,6), 7.33 (d, 2H, J = 8.4 Hz,
Ar00H3,5), 7.46 (t, 1H, J = 8.0 Hz, ArH7), 7.58 (d, 2H, J = 8.4 Hz,
Ar00H2,6), 7.70 (d, 1H, J = 8.4 Hz, ArH6), 7.87 (td, 1H, J = 8.0 Hz,
J0= 1.6 Hz, ArH8), 7.94 (d, 2H, J = 8.8 Hz, Ar0H3,5), 8.20 (dd, 1H,
J = 8.4 Hz, J0= 0.8 Hz, ArH9), 9.98 (s, 1H, NH);13C NMR (DMSO-d6)
d 20.5, 102.4, 112.1, 118.5 (2C), 119.5, 121.8 (2C), 123.7, 124.2,
125.4, 130.2 (2C), 132.6 (2C), 134.8, 135.1, 144.9, 150.1, 152.5,
155.1, 167.2; MS(ESI)
m/z
C22H16N4O: C, 74.98; H, 4.58; N, 15.90. Found: C, 75.11; H, 4.60;
N, 15.81.
351(M+?1). Anal.Calcdfor
4.6.5. 4-(4-(2-Methoxyphenoxy)quinazolin-2-ylamino)benzo-
nitrile (5e)
Yield 52.9%. Mp 220.0–220.5 ?C;1H NMR (DMSO-d6) d 3.73 (s,
3H, CH3O), 7.10 (td, 1H, J = 7.6 Hz, J0= 1.6 Hz, Ar00H6), 7.30 (dd, 1H,
J = 8.4 Hz, J0= 1.2 Hz, Ar00H3), 7.35–7.41 (m, 2H, Ar00H4,5), 7.47 (td,
1H, J = 8.0 Hz, J0= 0.8 Hz, ArH7), 7.56 (d, 2H, J = 8.8 Hz, Ar0H2,6),
7.71 (d, 1H, J = 8.4 Hz, ArH6), 7.84 (d, 2H, J = 8.8 Hz, Ar0H3,5), 7.89
(td, 1H, J = 8.4 Hz, J0= 1.2 Hz, ArH8), 8.22 (d, 1H, J = 8.4 Hz, ArH9),
10.06 (s, 1H, NH);
113.4, 118.3 (2C), 119.5, 121.0, 123.1, 123.8, 124.2, 125.4, 127.2,
132.6 (2C), 134.9, 140.9, 144.9, 151.1, 152.6, 155.2, 166.8; MS
(ESI) m/z 367 (M+?1). Anal. Calcd for C22H16N4O2: C, 71.73; H,
4.38; N, 15.21. Found: C, 71.77; H, 4.27; N, 15.12.
13C NMR (DMSO-d6) d 55.8, 102.3, 111.6,
4.6.6. 4-(4-(2-Chlorophenoxy)quinazolin-2-ylamino)benzo-
nitrile (5f)
Yield 51.8%. Mp 190.9–192.3 ?C;
7.61 (m, 6H, ArH7+ Ar0H2,6+ Ar00H4,5,6), 7.69–7.90 (m, 5H, ArH6,8+
Ar0H3,5+ Ar00H3), 8.24 (d, 1H, J = 8.4 Hz, ArH9), 10.08 (s, 1H, NH);
13C NMR (DMSO-d6) d 102.6, 111.5, 118.5 (2C), 119.5, 123.7,
124.5, 124.7, 125.5, 126.2, 127.9, 128.9, 130.6, 132.7 (2C), 135.3,
144.8, 148.3, 152.8, 155.0, 166.4; MS (ESI) m/z 373 (M+?1). Anal.
Calcd for C21H13N4OCl: C, 67.66; H, 3.51; N, 15.03; Cl, 9.51. Found:
C, 67.81; H, 3.44; N, 14.97; Cl, 9.58.
1H NMR (DMSO-d6) d 7.44–
4.6.7. 4-(4-(4-Fluorophenoxy)quinazolin-2-ylamino)benzo-
nitrile (5g)
Yield 55.6%. Mp 214.2–214.7 ?C;
7.39 (m, 2H, Ar0H2,6), 7.44–7.48 (m, 3H, ArH7+ Ar00H3,5), 7.60 (d,
2H, J = 8.4 Hz, Ar00H2,6), 7.70 (d, 1H, J = 8.4 Hz, ArH6), 7.85–7.93
(m, 3H, ArH8+ Ar0H3,5), 8,20 (d, 1H, J = 8.0 Hz, ArH9), 9.93 (s, 1H,
NH);13C NMR (DMSO-d6) d 102.9, 112.5, 116.8 (2C), 117.1, 119.0
(2C), 120.0, 124.2, 124.5, 124.7 (2C), 125.9, 133.2 (2C), 135.4,
145.3, 148.9, 153.1, 155.5, 167.7; MS (ESI) m/z 357 (M+?1). Anal.
Calcd for C21H13N4OF: C, 70.78; H, 3.68; N, 15.72; F, 5.33. Found:
C, 70.72; H, 3.83; N, 15.77; F, 5.21.
1H NMR (DMSO-d6) d 7.37–
4.6.8. 4-(4-(4-Chlorophenoxy)quinazolin-2-ylamino)benzo-
nitrile (5h)
Yield 55.9%. Mp 252.3–252.7 ?C;
7.48 (m, 3H, ArH7+ Ar0H2,6), 7.58–7.61 (m, 4H, Ar0H3,5+ Ar00H3,5),
7.71 (d, 1H, J = 8.4 Hz, ArH6), 7.87 (dt, 1H, J = 7.2 Hz, J0= 1.6 Hz,
ArH8), 7.93 (d, 2H, J = 8.4 Hz, Ar00H2,6), 8,19 (d, 1H, J = 8.0 Hz,
ArH9), 9.99 (s, 1H, NH);13C NMR (DMSO-d6) d 102.5, 112.0, 118.6
(2C), 119.6, 123.8, 124.3 (2C), 124.4, 125.5, 129.8 (2C), 130.2,
132.7 (2C), 135.0, 144.9, 151.2, 152.6, 154.9, 167.1; MS (ESI) m/z
373 (M+?1). Anal. Calcd for C21H13N4OCl: C, 67.66; H, 3.51; N,
15.03; Cl, 9.51. Found: C, 67.62; H, 3.49; N, 15.06; Cl, 9.62.
1H NMR (DMSO-d6) d 7.44–
4.6.9. 4-(4-(4-Bromophenoxy)quinazolin-2-ylamino)benzo-
nitrile (5i)
Yield 55.0%. Mp 267.3–267.6 ?C;1H NMR (DMSO-d6) d 7.41 (d,
2H, J = 6.8 Hz, Ar0H2,6), 7.47 (t, 1H, J = 7.2 Hz, ArH7), 7,61 (d, 2H,
J = 8.8 Hz, Ar00H2,6), 7.71–7.75 (m, 3H, ArH6+ Ar0H3,5), 7.88 (td, 1H,
J = 8.4 Hz, J0= 1.2 Hz, ArH8), 7.94 (d, 2H, J = 8.4 Hz, Ar00H3,5), 8.21
(d, 1H, J = 8.4 Hz, ArH9), 10.02 (s, 1H, NH);13C NMR (DMSO-d6) d
103.0, 112.5, 118.8 (2C), 119.0 (2C), 120.0, 124.2, 124.8, 125.2
(2C), 126.0, 133.1, 133.2 (2C), 135.5, 145.3, 152.2, 153.1, 155.4,
167.4; MS (ESI) m/z 417 (M++1). Anal. Calcd for C21H13N4OBr: C,
60.45; H, 3.14; N, 13.43; Br, 19.15. Found: C, 60.66; H, 3.02; N,
13.40; Br, 19.22.
4.6.10. 4-(4-(4-Methoxyphenoxy)quinazolin-2-ylamino)benzo-
nitrile (5j)
Yield 59.1%. Mp 218.2–218.4 ?C;1H NMR (DMSO-d6) d 3.83 (s,
3H, CH3O), 7.08 (d, 2H, J = 6.8 Hz, Ar00H3,5), 7.32 (d, 2H, J = 6.8 Hz,
Ar00H2,6), 7.47 (td, 1H, J = 8.0 Hz, J0= 0.8 Hz, ArH7), 7.59 (d, 2H,
J = 8.8 Hz, Ar0H2,6), 7.71 (d, 1H, J = 8.4 Hz, ArH6), 7.87 (td, 1H,
J = 8.4 Hz, J0= 1.2 Hz, ArH8), 7.95 (d, 2H, J = 8.4 Hz, Ar0H3,5), 8.21
(dd, 1H, J = 8.4 Hz, J0= 1.2 Hz, ArH9), 9.99 (s, 1H, NH);
(DMSO-d6) d 56.0, 102.9, 112.6, 115.3 (2C), 119.0 (2C), 120.1,
123.5 (2C), 124.2, 124.7, 125.9, 133.2, 135.3 (2C), 145.4, 146.2,
153.0, 155.6, 157.6, 167.9; MS (ESI) m/z 369 (M++1). Anal. Calcd
for C22H16N4O2: C, 71.73; H, 4.38; N, 15.21. Found: C, 71.79; H,
4.46; N, 15.03.
13C NMR
4.6.11. 4-(4-(2,4-Dichlorophenoxy)quinazolin-2-ylamino)benzo-
nitrile (5k)
Yield 56.6%. Mp 213.1–214.0 ?C;1H NMR (DMSO-d6) d 7.49 (t,
1H, J = 7.6 Hz, Ar00H7), 7.60–7.66 (m, 4H, ArH6+ Ar0H2,6+ Ar00H5),
7.73 (d, 1H, J = 8.8 Hz, ArH6), 7.88–7.91 (m, 4H, ArH8+ Ar0H3,5+
Ar00H3), 8.22 (d, 1H, J = 8.0 Hz, ArH9), 10.06 (s, 1H, NH);13C NMR
(DMSO-d6) d 103.2, 111.8, 119.0 (2C), 120.0, 124.1, 125.0, 126.0,
126.6, 128.0, 129.4, 130.5, 131.7, 133.2 (2C), 135.8, 145.1, 147.8,
153.2, 155.2, 166.6; MS (ESI) m/z 407 (M++1). Anal. Calcd for
C21H12N4OCl2: C, 61.93; H, 2.97; N, 13.76; Cl, 17.41. Found: C,
61.78; H, 3.12; N, 13.88; Cl, 17.25.
4.6.12. 4-(4-(2,4,6-Trichlorophenoxy)quinazolin-2-ylamino)-
benzonitrile (5l)
Yield 56.4%. Mp 252.4–253.1 ?C;1H NMR (DMSO-d6) d 7.34 (s,
1H, NH), 7.53–7.56 (m, 3H, ArH7+ Ar00H3,5), 7.60 (d, 2H, J = 7.2 Hz,
Ar0H2,6), 7.80 (d, 2H, J = 8.8 Hz, Ar0H3,5), 7.86 (d, 1H, J = 8.0 Hz,
ArH6), 7.91 (dt, 1H, J = 6.8 Hz, J0= 1.2 Hz, ArH8), 8.36 (dd, 1H,
J = 8.0 Hz, J0= 0.8 Hz, ArH9);
117.8 (2C), 118.8 (2C), 119.0, 123.4, 124.3, 125.7, 129.7 (2C),
131.7, 132.7 (2C), 134.7, 143.1, 144.1, 152.9, 153.9, 165.2; MS
(ESI) m/z 441 (M+?1). Anal. Calcd for C21H11N4OCl3: C, 57.10; H,
2.51; N, 12.68; Cl, 24.08. Found: C, 57.02; H, 2.78; N, 12.86; Cl,
23.89.
13C NMR (DMSO-d6) d 104.2, 111.4,
4.6.13. 4-(4-(2,6-Dimethylphenoxy)quinazolin-2-ylamino)-
benzonitrile (5m)
Yield 62.9%. Mp 159.2–160.9 ?C;1H NMR (DMSO-d6) d 2.11 (s,
6H, 2CH3), 7.21–7.24 (m, 3H, Ar0H2,6+ Ar00H4), 7.58–7.61 (m, 4H,
Ar0H3,5+ Ar00H3,5), 7.49 (t, 1H, J = 7.2 Hz, ArH7), 7.54 (d, 1H,
J = 8.8 Hz, Ar00H3,5), 7.73 (d, 1H, J = 8.4 Hz, ArH6), 7.84 (d, 2H,
J = 8.0 Hz, Ar0H3,5), 7.89 (t, 1H, J = 8.0 Hz, ArH8), 8.30 (d, 1H,
J = 7.6 Hz, ArH9), 10.08 (s, 1H, NH);
(2C), 102.5, 111.4, 118.5 (2C), 119.5, 123.8, 124.4, 125.4, 126.0,
128.9 (2C), 132.0 (2C), 132.7 (2C), 135.1, 144.9, 149.6, 152.5,
155.3,166.1;MS(ESI)
m/z
C23H18N4O: C, 75.39; H, 4.95; N, 15.29. Found: C, 75.21; H, 5.12;
N, 15.33.
13C NMR (DMSO-d6) d 16.1
367(M+?1).Anal. Calcdfor
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4.6.14. 4-(4-(Mesityloxy)quinazolin-2-ylamino)benzonitrile (5n)
Yield 50.7%. Mp 206.9–207.9 ?C;1H NMR (DMSO-d6) d 2.07 (s,
6H, 2CH3), 2.33 (s, 3H, CH3), 7.04 (s, 2H, Ar00H3,5), 7.50 (t, 1H,
J = 8.0 Hz, ArH7), 7.57 (d, 2H, J = 8.8 Hz, Ar0H2,6), 7.74 (d, 1H,
J = 8.4 Hz, ArH6), 7.88–7.93 (m, 3H, ArH8+ Ar0H3,5), 8.29 (dd, 1H,
J = 8.0 Hz, J0= 0.8 Hz, ArH9), 10.08 (s, 1H, NH);13C NMR (DMSO-
d6) d 15.4 (2C), 19.8, 102.0, 110.9, 118.1 (2C), 118.9, 123.2, 123.8,
124.7, 128.8 (2C), 129.2 (2C), 132.1 (2C), 134.4, 134.5, 144.2,
146.8, 151.9, 154.7, 165.6; MS (ESI) m/z 381 (M++1). Anal. Calcd
for C24H20N4O: C, 75.77; H, 5.30; N, 14.73. Found: C, 75.63; H,
5.45; N, 14.89.
4.6.15. 4-(4-(2,4-Dibromo-6-methylphenoxy)quinazolin-2-
ylamino)benzonitrile (5o)
Yield 49.4%. Mp 218.6–220.2 ?C;1H NMR (DMSO-d6) d 2.20 (s,
3H, CH3), 7.51 (t, 1H, J = 7.6 Hz, ArH7), 7.61 (d, 2H, J = 8.8 Hz,
Ar0H2,6), 7.73–7.76 (m, 2H, ArH6+ Ar00H5), 7.88–7.94 (m, 4H,
ArH8+ Ar0H3,5+ Ar00H3), 8.27 (d, 1H, J = 8.0 Hz, ArH9), 10.09 (s, 1H,
NH);13C NMR (DMSO-d6) d 16.3, 102.6, 111.2, 117.6, 118.5 (2C),
118.9, 119.5, 123.6, 124.5, 125.6, 132.7, 132.8 (2C), 133.3, 135.3,
135.5, 144.7, 147.6, 152.8, 154.9, 165.2; MS (ESI) m/z 511 (M++1).
Anal. Calcd for C22H14N4OBr2: C, 51.79; H, 2.77; N, 10.98; Br,
31.32. Found: C, 51.62; H, 2.95; N, 11.09; Br, 31.28.
4.6.16. 4-(4-(2,6-Dibromo-4-methylphenoxy)quinazolin-2-
ylamino)benzonitrile (5p)
Yield 50.8%. Mp 249.1–249.6 ?C;1H NMR (DMSO-d6) d 2.41 (s,
3H, CH3), 7.53 (t, 1H, J = 8.0 Hz, ArH7), 7.62 (d, 2H, J = 8.8 Hz,
Ar0H2,6), 7.72 (s, 2H, Ar00H3,5), 7.76 (d, 1H, J = 8.4 Hz, ArH6), 7.90–
7.96 (m, 3H, ArH8+ Ar0H3,5), 8.27 (dd, 1H, J = 8.0 Hz, J0= 0.8 Hz,
ArH9), 10.14 (s, 1H, NH);
110.6, 116.5 (2C), 117.9 (2C), 119.4, 123.0, 124.1, 125.0, 132.2
(2C), 132.7 (2C), 134.8, 139.0, 144.0, 144.1, 152.4, 154.3, 164.6;
MS (ESI) m/z 511 (M++1). Anal. Calcd for C22H14N4OBr2: C, 51.79;
H, 2.77; N, 10.98; Br, 31.32. Found: C, 51.77; H, 2.91; N, 10.87;
Br, 31.45.
13C NMR (DMSO-d6) d 19.3, 102.2,
4.6.17. 4-(4-(2,4,6-Tribromophenoxy)quinazolin-2-ylamino)-
benzonitrile (5q)
Yield 52.2%. Mp 261.0–261.2 ?C;1H NMR (DMSO-d6) d 7.54 (t,
1H, J = 7.2 Hz, ArH7), 7.64 (d, 2H, J = 8.4 Hz, Ar0H2,6), 7.78 (d, 1H,
J = 8.4 Hz, ArH6), 7.90–7.97 (m, 3H, ArH8+ Ar0H3,5), 8.21 (s, 2H,
Ar00H3,5), 8.27 (dd, 1H, J = 8.0 Hz, ArH9), 10.16 (s, 1H, NH);
NMR (DMSO-d6) d (ppm) 102.8, 110.9, 118.5 (2C), 118.8 (2C),
119.4, 120.0, 123.5, 124.8, 125.6, 132.7 (2C), 135.1 (2C), 135.6,
144.5, 146.7, 152.9, 154.6, 164.8; MS (ESI) m/z 573 (M++1). Anal.
Calcd for C21H11N4OBr3: C, 43.86; H, 1.93; N, 9.47; Br, 41.69. Found:
C, 43.58; H, 1.99; N, 9.87; Br, 41.72.
13C
4.6.18. 4-(2-(4-Cyanophenylamino)quinazolin-4-yloxy)-3,5-
dimethylbenzonitrile (5r)
Yield 44.7%. Mp 291.2–292.1 ?C;1H NMR (DMSO-d6) d 2.07 (s,
6H, 2CH3), 2.33 (s, 3H, CH3), 7.04 (s, 2H, Ar00H3,5), 7.50 (t, 1H,
J = 8.0 Hz, ArH7), 7.57 (d, 2H, J = 8.8 Hz, Ar0H2,6), 7.74 (d, 1H,
J = 8.4 Hz, ArH6), 7.88–7.93 (m, 3H, ArH8+ Ar0H3,5), 8.29 (dd, 1H,
J = 8.0 Hz, J0= 0.8 Hz, ArH9), 10.08 (s, 1H, NH);13C NMR (DMSO-
d6) d 15.4 (2C), 19.8, 102.0, 110.9, 118.1 (2C), 118.9, 123.2, 123.8,
124.7, 128.8 (2C), 129.2 (2C), 132.1 (2C), 134.4, 134.5, 144.2,
146.8, 151.9, 154.7, 165.6; MS (ESI) m/z 381 (M++1). Anal. Calcd
for C24H17N5O: C, 73.64; H, 4.38; N, 17.89. Found: C, 73.43; H,
4.32; N, 17.98.
4.6.19. 4-(2-(4-Cyanophenylamino)quinazolin-4-yloxy)-3-
methoxybenzonitrile (5s)
Yield 54.6%. Mp 251.7–252.4 ?C;1H NMR (DMSO-d6) d 3.80 (s,
3H, CH3O), 7.49 (t, 1H, J = 8.0 Hz, ArH7), 7.61–7.64 (m, 4H,
Ar0H2,6+ Ar00H3,5), 7.73 (d, 1H, J = 8.4 Hz, ArH6), 7.84 (s, 1H, Ar00H3),
7.86–7.90 (m, 3H, ArH8+ Ar0H3,5), 8.21 (d, 1H, J = 8.0 Hz, ArH9),
10.06 (s, 1H, NH);
111.4, 117.0, 118.4 (2C), 118.5, 118.6, 119.5, 123.7, 124.4, 125.5,
125.9, 132.7 (2C), 135.1, 144.7, 144.9, 151.7, 152.6, 154.9, 166.3;
MS (ESI) m/z 394 (M++1). Anal. Calcd for C23H15N5O2: C, 70.22; H,
3.84; N, 17.80. Found: C, 70.29; H, 3.73; N, 17.93.
13C NMR (DMSO-d6) d 56.6, 102.5, 109.8,
4.6.20. 4-(2-(4-Cyanophenylamino)quinazolin-4-yloxy)-3,5-
dimethoxybenzonitrile (5t)
Yield 55.1%. Mp 285.3–285.7 ?C;1H NMR (DMSO-d6) d 3.85 (s,
6H, 2CH3O), 7.51–7.54 (m, 3H, ArH7+ Ar00H3,5), 7.66 (d, 2H,
J = 8.8 Hz, Ar0H2,6), 7.77 (d, 1H, J = 8.8 Hz, ArH6), 7.89–7.96 (m, 3H,
ArH8+ Ar0H3,5), 8.24 (d, 1H, J = 8.0 Hz, ArH9), 10.12 (s, 1H, NH);
13C NMR (DMSO-d6) d 55.9 (2C), 102.6, 109.3, 110.0 (2C), 111.2,
118.4 (2C), 118.7, 119.6, 123.8, 124.5, 125.9, 132.8 (2C), 133.6,
135.2, 144.8, 152.7 (2C), 152.8, 155.1, 165.9; MS (ESI) m/z 424
(M+?1). Anal. Calcd for C24H17N5O3: C, 68.08; H, 4.05; N, 16.54.
Found: C, 68.19; H, 3.96; N, 16.45.
4.6.21. 3-Chloro-4-(2-(4-cyanophenylamino)quinazolin-4-
yloxy)-5-methoxybenzonitrile (5u)
Yield 42.0%. Mp 267.2–269.5 ?C;1H NMR (DMSO-d6) d 3.83 (s,
3H, CH3O), 7.51 (t, 1H, J = 7.6 Hz, ArH7), 7.64 (d, 2H, J = 8.8 Hz,
Ar0H2,6),7.75(d, 1H,
J = 8.4 Hz,
ArH8+ Ar0H3,5+ Ar00H3,5), 8.22 (d, 1H, J = 8.0 Hz, ArH9), 10.11 (s,
1H, NH);13C NMR (DMSO-d6) d 57.3, 102.8, 110.5, 110.8, 116.3,
117.4, 118.5 (2C), 119.2, 123.6, 124.8, 125.6, 125.8, 128.5, 132.8
(2C), 135.5, 142.3, 144.6, 152.9, 153.2, 154.7, 165.2; MS (ESI) m/z
428 (M+?1). Anal. Calcd for C23H14N5O2Cl: C, 64.57; H, 3.30; N,
16.37; Cl, 8.29. Found: C, 64.47; H, 3.39; N, 16.44; Cl, 8.39.
ArH6),7.88–7.94(m, 5H,
4.6.22. 3-Chloro-4-(2-(4-cyanophenylamino)quinazolin-4-yloxy)-
5-ethoxybenzonitrile (5v)
Yield 38.5%. Mp 262.2–263.7 ?C;1H NMR (DMSO-d6) d 1.02 (t,
3H, J = 7.2 Hz, CH3), 4.12 (q, 2H, J = 7.2 Hz, CH2O), 7.49 (t, 1H,
J = 7.2 Hz, ArH7), 7.62 (d, 2H, J = 8.8 Hz, Ar0H2,6), 7.74 (d, 1H,
J = 8.4 Hz, ArH6), 7.84–7.93 (m, 5H, ArH8+ Ar0H3,5+ Ar00H3,5), 8.21
(d, 1H, J = 8.0 Hz, ArH9), 10.10 (s, 1H, NH);13C NMR (DMSO-d6) d
14.5, 65.9, 103.2, 110.8, 111.3, 117.5, 117.8, 118.9 (2C), 119.6,
124.0, 125.2, 126.0, 126.2, 128.8, 133.2 (2C), 135.9, 142.4, 145.0,
152.7, 153.3, 155.2, 165.8; MS (ESI) m/z 442 (M+?1). Anal. Calcd
for C24H16N5O2Cl: C, 65.24; H, 3.65; N, 15.85; Cl, 8.02. Found: C,
65.35; H, 3.53; N, 15.76; Cl, 8.19.
4.6.23. 4-(2-(4-Cyanophenylamino)quinazolin-4-yloxy)-3,5-
diethoxybenzonitrile (5w)
Yield 50.9%. Mp 217.2–217.7 ?C;1H NMR (DMSO-d6) d 1.06 (t,
6H, J = 7.2 Hz, 2CH3), 4.08 (q, 4H, J = 7.2 Hz, 2CH2O), 7.43 (s, 2H,
Ar00H3,5), 7.48 (t, 1H, J = 8.0 Hz, ArH7), 7.60 (d, 2H, J = 8.8 Hz, Ar0H2,6),
7.76 (d, 1H, J = 8.8 Hz, ArH6), 7.85–7.91 (m, 3H, ArH8+ Ar0H3,5), 8.20
(d, 1H, J = 8.8 Hz, ArH9), 10.07 (s, 1H, NH);13C NMR (DMSO-d6) d
14.3 (2C), 65.0 (2C), 102.5, 109.1, 110.9 (2C), 111.3, 118.4 (2C),
118.7, 119.5, 123.7, 124.5, 125.5, 132.7 (2C), 134.6, 135.1, 144.8,
151.9 (2C), 152.7, 155.0, 166.1; MS (ESI) m/z 452 (M+?1). Anal.
Calcd for C26H21N5O3: C, 69.17; H, 4.69; N, 15.51. Found: C,
69.22; H, 4.85; N, 15.32.
4.6.24. 4-(2-(4-Cyanophenylamino)quinazolin-4-yloxy)-3-meth-
oxy-5-propoxybenzonitrile (5x)
Yield 32.5%. Mp 196.8–198.4 ?C;1H NMR (DMSO-d6) d 0.53 (t,
3H, J = 7.2 Hz, CH3), 1.35–1.45 (m, 2H, CH2), 3.78 (t, 2H, J = 7.2 Hz,
CH2O), 3.81 (s, 3H, CH3O), 7.44–7.49 (m, 3H, ArH7+ Ar0H2,6), 7.59
(s, 1H, Ar00H3), 7.61 (s, 1H, Ar00H5), 7.71 (d, 1H, J = 8.4 Hz, ArH6),
7.83–7.90 (m, 3H, ArH8+ Ar0H3,5), 8.19 (d, 1H, J = 8.4 Hz, ArH9),
10.07 (s, 1H, NH);
13C NMR (DMSO-d6) d 10.3, 22.1, 57.2, 71.0,
5046
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103.0, 109.7, 110.3, 111.4, 111.7, 118.8 (2C), 119.1, 120.0, 124.1,
124.9, 126.0, 133.2 (2C), 134.5, 135.6, 145.3, 152.4, 153.2, 153.3,
155.5, 166.5; MS(ESI)
m/z
C26H21N5O3: C, 69.17; H, 4.69; N, 15.51. Found: C, 69.29; H, 4.81;
N, 15.32.
452(M+?1).Anal.Calcd for
4.6.25. 4-(2-(4-Cyanophenylamino)quinazolin-4-yloxy)-3-ethoxy-
5-propoxybenzonitrile (5y)
Yield 54.5%. Mp 179.9–181.2 ?C;1H NMR (DMSO-d6) d 0.54 (t,
3H, J = 7.2 Hz, CH3), 1.08–1.12 (m, 3H, CH3), 1.39–1.43 (m, 2H,
CH2), 3.98–4.13 (m, 4H, 2CH2O), 7.39–8.20 (m, 10H, ArH6,7,8,9+
Ar0H2,3,5,6+ Ar00H3,5), 10.04 (s, 1H, NH);13C NMR (DMSO-d6) d 9.8,
14.3, 21.7, 22.1, 66.0, 71.7, 102.6, 109.1, 110.9, 111.3, 118.4 (2C),
118.9, 119.5, 123.7, 124.4, 125.5, 133.7 (2C), 134.5, 135.1, 144.8,
151.9, 152.0, 152.7, 156.1, 166.1; MS (ESI) m/z 466 (M+?1). Anal.
Calcd for C27H23N5O3: C, 69.66; H, 4.98; N, 15.04. Found: C,
69.78; H, 5.07; N, 14.87.
4.6.26. 4-(6-Chloro-2-(4-cyanophenylamino)quinazolin-4-
yloxy)-3,5-dimethylbenzonitrile (5z)
Yield 49.6%. Mp 293.5–294.9 ?C;1H NMR (DMSO-d6) d 2.15 (s,
6H, 2CH3), 7.59 (d, 2H, J = 8.4 Hz, Ar0H2,6), 7.74 (d, 1H, J = 8.4 Hz,
ArH8), 7.78 (s, 2H, Ar00H3,5), 7.82–7.91 (m, 3H, ArH8+ Ar0H3,5), 8.28
(d, 1H, J = 2.4 Hz, ArH6), 10.10 (s, 1H, NH);13C NMR (DMSO-d6) d
16.3 (2C), 103.4, 109.5, 112.4, 119.0 (2C), 119.1, 119.9, 123.0,
128.3, 128.7, 133.1 (2C), 133.2 (2C), 133.3 (2C), 136.0, 144.9,
151.9, 153.6, 155.7, 165.1; MS (ESI) m/z 426 (M+?1). Anal. Calcd
for C24H16N5OCl: C, 67.69; H, 3.79; N, 16.44; Cl, 8.32. Found: C,
67.88; H, 3.72; N, 16.32; Cl, 8.45.
4.7. Antiviral assay
The anti-HIV activity and cytotoxicity of the compounds were
evaluated against wild-type HIV-1 strain IIIB in MT-4 cell cultures
usingthe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) method.14Briefly, 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 ? 105cells/mL and infected with HIV at a multiplicity of infection
of 0.02. Immediately after viral infection, 100 lL of the cell suspen-
sion was placed in each well of a flat-bottomed microtiter tray con-
taining 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 deter-
mined using the MTT method. Compounds were tested in parallel
for cytotoxic effects in uninfected MT-4 cells. The selection and
characterization of mutant virus strains have been performed
previously.
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (No. 20872018 and 30672536), the Concerted Actions of
the K.U.Leuven (GOA-10/14) and the CHAARM microbicide project
of the European Commission for the financial support of this
research.
References and notes
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