Synthesis and anti-HIV activity of 2-naphthyl substituted DAPY analogues as non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.05.036

Nine newly 6-cynao-2-naphthyl substituted diarylpyrimidines (DAPY) were synthesized as non-nucleoside reverse transcriptase inhibitors on the basis of our previous work. The antiviral and cytotoxicity evaluation indicated that these compounds displayed strong activity against wild-type HIV-1 at nanomolar concentrations with selectivity index SI greater than 23 779. The most active compounds 3c and 3e exhibited activity against the double mutant (103N+181C) strains at an EC₅₀ of 0.16 and 0.15 μM, and were more activity than that of efavirenz.

Full text of DOI:10.1016/j.bmc.2010.05.036

Bioorganic & Medicinal Chemistry 18 (2010) 4601–4605
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-HIV activity of 2-naphthyl substituted DAPY analogues
as non-nucleoside reverse transcriptase inhibitors
Yong-Hong Liang a, Qiu-Qin He a, Zhao-Sen Zeng a, Zhi-Qian Liu a, Xiao-Qing Feng a,
c
c
c
Fen-Er Chen a,b, , Jan Balzarini , Christophe Pannecouque , Erik De Clercq
*
a Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
b Institute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine newly 6-cynao-2-naphthyl substituted diarylpyrimidines (DAPY) were synthesized as non-nucleo-
side reverse transcriptase inhibitors on the basis of our previous work. The antiviral and cytotoxicity eval-
uation indicated that these compounds displayed strong activity against wild-type HIV-1 at nanomolar
concentrations with selectivity index SI greater than 23 779. The most active compounds 3c and 3e
Received 20 April 2010
Revised 13 May 2010
Accepted 14 May 2010
Available online 20 May 2010
exhibited activity against the double mutant (103N+181C) strains at an EC50 of 0.16 and 0.15
were more activity than that of efavirenz.
lM, and
Keywords:
HIV-1
Ó 2010 Elsevier Ltd. All rights reserved.
Reverse transcriptase
NNRTIs
DAPY
1. Introduction
2. Results and discussion
2.1. Chemistry
The development of new and more potent mutation-resistant
non-nucleoside reverse transcriptase inhibitors (NNRTIs) is still
an arduous task for the treatment of the HIV-1-infected patients
due to the drug compliance, adverse effects and cross-resistance.
Recently, we designed and synthesized a novel class of the naph-
thyl-substituted DAPY analogues1–3 with strong activity against
the HIV-1 LAI virus and high selectivity index, as compared to
two lead compounds TMC278 and TMC125 (Fig. 1)4–7 The struc-
ture–activity relationship (SAR) and theoretical calculated stud-
ies1,3 have revealed that the cyano group at position C-6 on the
naphthalene ring plays a vital role in the activity against the
wild-type and double mutant HIV-1 strains, and the dual-substi-
tuted compounds at positions C-1 and C-3 on the naphthalene ring
exhibit more potent activity against the mutant virus than the non-
or mono-substituted compounds.
The target compounds were synthesized via the short route de-
tailed in Scheme 1 according to our previously reported protocol.2
The 4-chloropyrimidines 1 were treated with the 6-cyano-2-naph-
thol derivatives 2a–e at 110 °C in the presence of DMF to provide
the 6-cyano-2-naphthyl substituted DAPY compounds 3a–i. The
spectroscopic data of the target compounds 3a–i were consistent
with the structures shown in Scheme 2.
The intermediate 6-cyano-2-naphthol derivatives 2a–e were
synthesized using the method as shown in Scheme 2. Following
the reported method, the intermediate 6-cyano-2-naphthol (5)
(R3)n
CN
CN
O
CN
NH
CN
NH
In continuation of our efforts in improving the anti-HIV activity
of the naphthyl-substituted DAPY analogues against the clinically
relevant HIV-1 mutant strains, nine newly 6-cyano-2-naphthyl
substituted DAPY analogues were designed and synthesized.
CN
NH
N
X
N
HN
N
N
N
R2
Br
R1
N
NH2
Naph-DAPYs
TMC125 (etravirine)
TMC278 (rilpivirine)
* Corresponding author. Tel.: +86 21 65643809; fax: +86 21 65643811.
E-mail address: rfchen@fudan.edu.cn (F.-E. Chen).
Figure 1. The chemical structures of NNRTIs.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.036

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Y.-H. Liang et al. / Bioorg. Med. Chem. 18 (2010) 4601–4605
for three representative compounds 3a–c was performed
N
(Table 1). The results strongly support all of the derivatives in this
study as NNRTIs, as they have no possibility of being phosphory-
lated for NRTI activity against RT. As shown in Table 1, the 6-cya-
no-2-naphthyl substituted DAPY derivatives displayed strong
inhibitory activity against wild-type HIV-1 at nanomolar concen-
N
R3
N
OH
R4
NC
2a-e
R3
R4
N
tration level (EC50 = 0.012–0.002
lM) and low cytotoxicity (CC50 =
Cl
N
NH
O
NH
a
118.44–331.56 M), resulting in high selectivity index (SI) values
l
N
N
of 23,779–158,228.
The mono-substituted derivative 3a, 3b and 3d exhibited an
excellent potency against wild-type virus (EC50 = 0.002 and
R1
R1
R2
1
R2
3a-i
0.006 lM), and the compounds 3a and 3d displayed a moderate
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 110 °C, 8–12 h, N2.
activity against the double mutants virus K103N+Y181C and were
more potent compared to the non-substituted compound 3j.3 How-
ever, the 3-methyoxy-substituted compound 3b displayed no activ-
ity against the double mutant form, nor did the previously reported
3-bromo-substituted compound 3l.3 Introduction of double substi-
tuted R3 and R4 groups at positions C-1 and C-3 of the naphthyl ring
was also investigated; the compounds 3c and 3e showed activity
was synthesized from 6-cyano-2-naphthol (4). Then, the treatment
of the intermediate 5 with Br2 yielded 1-bromo-6-cyano-2-naphthol
(6a). The 1,3-dibromo-6-cyano-2-naphthol (6b) was synthesized
using the reported method for 1,3,6-tribromo-2-naphthol.8,9 Reac-
tion of 6b with Sn provided 3-bromo-6-cyano-2-naphthol (6c).
Then, the methoxy-substituted 6-cyano-2-naphthol derivatives
2a–c were prepared by the methoxylation of the corresponding
bromo-substituted 6-cyano-2-naphthol compounds 2a–c. The
treatment of 5 and 2c with NCS in the presence of NaH provided
the 1-chloro-substituted naphthol compounds 2d–e.10
against wild-type HIV-1 at an EC50 value of 0.005 and 0.007 lM,
respectively. The potency against the double mutant virus
K103N+Y181C was slightly increased compared to compound 3a.
The compounds 3f and 3h with a methyl-substituted pyrimidine
ring were much less active against the double mutant virus than
the corresponding non-substituted compound 3j and 3k.
In order to investigate the binding model of the designed dual-
substituted compound with RT, a molecular docking study was
performed using the program AUTODOCK 4.0.113 according to the pre-
vious reported procedures.3 Figure 2 shows the theoretical binding
model of compounds 1u and 1cam to the non-nucleoside inhibitor
binding pocket (NNIBP) of HIV-1 RT. As the previous theoretical
binding model of compound 3j with RT,3 the dual–substituted
compound 3c adopt a horseshoe conformation in the NNIBP and
2.2. Biological activity
According to the MTT method,11,12 the newly synthesized com-
pounds 3a–i were evaluated for antiviral and cytotoxicity activity
in MT-4 cells infected with the HIV-1 (LAI strain, IIIB) wild-type virus
and HIV-1 double mutant virus K103N+Y181C (lysine replaced at
position 103 by asparagine, and tyrosine at position 181 by cys-
teine), in comparison with three FDA-approved drugs nevirapine,
delavirdine, and efavirenz used as reference compounds.
the left ‘wing’ naphthalene ring exhibits strong
p–p interactions
with five residues (Tyr181, Tyr188, Phe227, and Trp229) surround-
ing the NNIBP.
To lend support to the status of naphthyl-substituted DAPY
analogues as NNRTIs, an in vitro steady-state RT inhibition assay
Cl
OH
NC
2d
f
Br
OH
OH
Br
OH
Br
OH
a
b
d
NC
NC
NC
NC
Br
6c
6b
5
4
e
c
c
Br
OMe
OH
OH
OMe
OH
NC
NC
OMe
6a
2b
2c
c
f
OMe
Cl
OH
OH
NC
NC
OMe
2e
2a
Scheme 2. Reagents and conditions: (a) CuCN, DMF, reflux, 2 h; (b) (i) Br2, AcOH, AcONa, rt, 2 h; (ii) SnCl2(H2O)2, AcOH, 5 h; (c) MeONa, CuI, DMF, 110 °C, 3 h; (d) Sn, HCl,
AcOH, reflux, 4 h; (e) Br2, AcOH, rt, 1 h; (f) NCS, NaH, THF, reflux, 4 h.

Y.-H. Liang et al. / Bioorg. Med. Chem. 18 (2010) 4601–4605
4603
Table 1
Anti-HIV-1 activity and cytotoxicity of compounds 3a–i in MT-4 cells
R3
H
N
O
N
N
NC
R1
CN
R4
R2
M)c
SId
a
b
Compd
R1
R2
R3
R4
IC50
(lg/mL)
EC50
(l
M)
CC50(l
WT(IIIB)
103 N+181C
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
OMe
H
OMe
Cl
Cl
H
Br
H
Br
H
Br
H
H
0.9
0.52
0.04
nd
nd
nd
nd
nd
nd
3.17
1.33
0.50
0.37
nd
0.002
0.002
0.005
0.006
0.007
0.007
0.012
0.003
0.007
0.003
0.002
0.001
0.075
0.072
0.003
0.38
318. 07
0.16
0.70
0.15
331.56
0.83
331.56
0.22
6.30
0.24
>211.45
>318.07
>118.44
268.79
>190.65
>331.56
>274.12
>331.56
>274.12
>68.80
282.63
>56.53
>15.02
>3.62
P134,032
>158,228
P25,000
47,964
P25,701
>44,856
>23,779
>96,451
>40,068
20,548
181,247
50,357
>200
OMe
OMe
H
OMe
H
H
H
H
H
H
Me
Me
H
H
H
3j3
3k3
3l3
NEV
DEV
EFV
H
H
H
Br
>56.53
>15.02
>3.62
0.56
50
>2112
nd
>63.36
a
b
c
Concentration required to inhibit by 50% the in vitro RNA-dependent DNA polymerase activity of recombinant RT.
Effective concentration required to protect the cell against viral cytopathicity by 50% in MT-4 cells.
Concentration of compound that reduces normal uninfected MT-4 cell viability by 50%.
Selectivity index: ratio CC50/EC50 (wild-type).
d
4. Experimental section
4.1. Chemistry
Melting points were measured on a WRS-1 digital melting
point apparatus. 1H NMR and 13C NMR spectra on a Bruker AV
400 MHz spectrometer were recorded in DMSO-d6. Chemical shifts
are reported in d (ppm) units relative to the internal standard tet-
ramethylsilane (TMS). Mass spectra were obtained on a Agilent
MS/5975 mass spectrometer. Elemental analyses were performed
on a CARLOERBA 1106 instrument and the results of elemental
analyses for C, H, and N were within (0.4% of the theoretical val-
ues. All chemicals and solvents used were of reagent grade and
were purified and dried by standard methods before use. All air-
sensitive reactions were run 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
eluent. Flash chromatography separations were obtained on silica
gel (300–400 mesh).
4.1.1. Preparation of 6-hydroxy-2-naphthonitrile (5)
A mixture of 6-bromo-2-naphthol (32 g, 143 mmol), CuCN
(20 g, 0.225) and DMF (80 mL) was stirred at 160 °C for 4 h. To
the resulting mixture was added 10% NaOH (200 mL), and then
insoluble material was filtered and washed with water (100 mL).
The filtrate and washings were combined and filtered again. The
filtrate was acidified with 10% HCl to pH 2–3 and stirred at room
temperature for 30 h. Precipitated brown solids were collected by
filtration to give 21 g of 5. This crude brown solid was dried and
used in the next reaction without further purification.
Figure 2. Binding model of compounds 3c in the NNIBP. This figure was prepared
using the programs AUTODOCKTOOLS-1.5.0 and VIEWERLITE
.
3. Conclusion
In summary, we synthesized a novel series of 2-naphthyl
substituted DAPYs. The biological test results indicated that the
target compounds showed potent antiviral activity with EC50
values in the low-nanomolar concentration range and high SI value
of 23,779–158,228. The double substituted compounds 3c and 3e
displayed an excellent potency against the double mutant
(103N+181C) strains at an EC50 of 0.16 and 0.15 lM and were more
active compared to efavirenz. The results form a solid basis for con-
tinued exploration of the DAPY family of RT inhibitors.
4.1.2. Preparation of 5-bromo-6-hydroxy-2-naphthonitrile (6a)
6-hydroxy-2-naphthonitrile 5 (6.8 g, mmol) were dissolved in
glacial acetic acid (112 mL) with heating. The solution was then
cooled to 25 °C and bromine (33.4 g, 209.0 mmol) was added over
30 mins. The reaction was stirred for 2 h at room temperature.
Yield 94%; mp 181.4–182.7 °C; 1H NMR (400 MHz, DMSO-d6) d

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Y.-H. Liang et al. / Bioorg. Med. Chem. 18 (2010) 4601–4605
7.63–7.66 (m, 1H), 7.75 (d, 1H J = 9.2 Hz), 7.85 (d, 1H J = 9.2 Hz),
8.08 (d, 1H J = 9.2 Hz), 8.23 (s, 1H), 10.88 (s, 1H, OH).
for 4 h. Then the reaction mixture poured the reaction into 200 mL
cold water, and the resulting brown precipitate was filtered off.
The crude brown solid 2d–e was dried to be used in the next step
without further purification.
4.1.3. Preparation of 5,7-dibromo-6-hydroxy-2-naphthonitrile
(6b)
Br
Br
O
Br
OH
OH
SnCl2
HOAc
Br2
HOAc
NC
Br
NC
Br
NC
6b
5
4.1.6.1. 5-Chloro-6-hydroxy-2-naphthonitrile (2d). Yield 67%;
mp 186.4–187.8 °C; 1H NMR (400 MHz, DMSO-d6) d 7.43 (d, 1H,
J = 8.8 Hz), 7.84 (d, 1H, J = 8.8 Hz), 7.92 (d, 1H, J = 8.8 Hz), 8.12 (d,
1H, J = 8.8 Hz), 8.51 (s, 1H), 11.13 (s, 1H, OH).
Sodium acetate (15 g, 182.9 mmol) and 6-hydroxy-2-naphtho-
nitrile 5 (6.8 g, 40.2 mmol) were dissolved in glacial acetic acid
(112 mL) with heating. The solution was then cooled to 0 °C and
bromine (33.4 g, 209.0 mmol) was added in five portions over
10 min. The reaction was stirred for 2 h at room temperature. Ice
was added to the reaction causing the formation of a light yellow
precipitate, which was collected on a coarse glass frit and washed
with cold water, followed by petroleum ether and left to air dry.
A suspension of the yellow power 5,5,7-tribromo-6-oxo-5,6-dihy-
dronaphthalene-2-carbonitrile and SnCl2(H2O)2 in glacial acetic
acid (150 mL) was stirred overnight at room temperature, then
15% HCl (50) was added to the reaction mixture causing the forma-
tion of a white precipitate, which was filtered and washed with
cold water. The white crude solid was recrystallized from EtOH,
4.1.6.2. 5-Chloro-6-hydroxy-7-methoxy-2-naphthonitrile (2e).
Yield 73%; mp 183.4–184.2 °C; 1H NMR (400 MHz, DMSO-d6) d
3.98 (s, 3H, CH3), 7.49 (s, 1H), 7.69 (d, 1H, J = 8.8 Hz), 8.03 (d, 1H,
J = 8.8 Hz), 8.36 (s, 1H), 10.59 (s, 1H, OH).
4.1.7. General procedure for the preparation of 3a–i
Potassium carbonate (10 mmol) was added to a solution of b-
naphthol derivatives (2 mmol) in 20 mL of anhydrous DMF and
was stirred for 5 min. Then 4-(4-chloro-pyrimidin-2-ylamino)ben-
zonitrile 6 (2 mmol) was added. The reaction mixture was heated
to 80 °C under nitrogen atmosphere for 8–12 h. Next, the mixture
was treated with cold water (200 mL), and the resulting precipitate
was filtered off. The crude products 3a–i were recrystallized from
1,4-dioxane or EtOAc.
yield 6b as
a
yellow solid. Mp 210.8–211.5 °C; 1H NMR
(400 MHz, DMSO-d6) d 7.89 (m, 1H), 8.12 (d, 1H, J = 8.8 Hz), 8.46
(s, 1H), 8.51 (s, 1H), 10.92 (s, 1H, OH).
4.1.4. Preparation of 7-bromo-6-hydroxy-2-naphthonitrile (6c)
A suspension of 5, 7-dibromo-6-hydroxy-2-naphthonitrile (8 g,
6b) and Sn (15 g) in the mixture of 150 mL EtOH and 20 mL concd
HCl was refluxed for 4 h, then poured the reaction mixture into
cold water (250 mL) and obtained the white solid. Yield 71%; mp
237.4–238.4 °C; 1H NMR (400 MHz, DMSO-d6) d 7.36 (s, 1H),
7.63–7.66 (m, 1H), 7.87 (d, 1H, J = 8.4 Hz), 8.31 (s, 1H), 8.36 (s,
1H), 11.23 (s, 1H, OH).
4.1.7.1. 6-(2-(4-Cyanophenylamino)pyrimidin-4-yloxy)-5-meth-
oxy-2-naphthonitrile (3a). Yield 89.3%; recrystallized from
AcOEt, mp 249.2–250.1 °C; 1H NMR (400 MHz, DMSO-d6) d 3.90
(s, 3H, CH3), 6.76 (d, 1H, J = 4.0 Hz, CH), 7.25–7.55 (m, 4H, Ph),
7.64–8.50 (m, 5H, naph), 8.71 (s, 1H, CH), 10.10 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6) d 61.77, 99.70, 102.55, 108.66, 118.22,
118.99, 119.27, 123.33, 125.13, 126.97, 129.96, 131.14, 132.42,
143.19, 144.35, 146.29, 158.98, 160.42, 168.97. MS (EI) m/z:
393.1 (M+); Anal. (C23H15N6O2) C, H, N.
4.1.5. General procedure for the preparation of 2a–c
A suspension of 6a–c (4 mmol), sodium methoxide (20 mmol)
and CuI (1 mmol) in DMF (50 mL) was heated to 120 °C for 3 h,
then poured the reaction mixture into cold water (150 mL). After
filtering, the filtrate was added to 5% HCl (100 mL) and was stirred
for 30 min.
4.1.7.2. 6-(2-(4-Cyanophenylamino)pyrimidin-4-yloxy)-7-meth-
oxy-2-naphthonitrile (3b). Yield 41.8%; recrystallized from 1,4-
dioxane, mp 253.2–254.8 °C; 1H NMR (400 MHz, DMSO-d6) d 3.83
(s, 3H, OCH3), 6.69 (d, J = 5.6 Hz, 1H), 7.28–7.67 (m, 4H, Ph), 7.54–
8.52 (m, 5H, naph), 8.45 (d, J = 5.6 Hz, 1H), 10.07 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6) d 56.63, 99.94, 102.97, 108.98, 109.02,
118.66, 119.79, 119.81, 121.34, 125.39. 129.25, 130.47. 131.98,
132.98, 133.04, 144.47, 144.92, 152.33, 159.40. 160.67, 169.56. MS
(EI) m/z: 393.1 (M+); Anal. (C24H17N5O3) C, H, N.
4.1.5.1. 6-Hydroxy-5-methoxy-2-naphthonitrile (2a). Yield 74%;
mp 143.2–144.6 °C; 1H NMR (400 MHz, DMSO-d6) d 3.85 (s, 3H,
OCH3), 7.33 (d, 1H, J = 8.8 Hz), 7.64–7.67 (m, 1H), 8.02 (d, 1H,
J = 8.8 Hz), 8.41 (d, 1H, J = 1.2 Hz), 11.00 (s, 1H, OH).
4.1.7.3. 6-(2-(4-Cyanophenylamino)pyrimidin-4-yloxy)-5,7-dime-
thoxy-2-naphthonitrile (3c). Yield 85.9%; flash chromatography
separation and then recrystallized from 1,4-dioxane, 249.3–
250.4 °C; 1H NMR (400 MHz, DMSO-d6) d 3.85 (s, 3H, OCH3), 3.91
(s, 3H, OCH3), 6.78 (d, J = 4.0 Hz, 1H), 7.24–7.52 (m, 4H, Ph), 7.54–
8.54 (m, 4H, naph), 8.49 (d, J = 4.0 Hz, 1H), 10.11 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6) d 56.83, 62.23, 99.77, 102.98, 104.10,
109.61, 118.62, 119.63, 119.76, 123.76, 125.20, 125.46, 131.61,
132.93, 133.28, 135.82, 144.87, 148.00, 153.19, 160.83, 169.18. MS
(EI) m/z: 423.2 (M+); Anal. (C23H15N6O2) C, H, N.
4.1.5.2. 6-Hydroxy-5,7-dimethoxy-2-naphthonitrile (2b). Yield
64%; mp 125.5–125.9 °C; d 1H NMR (400 MHz, DMSO-d6) d 3.85 (s,
3H, CH3), 3.93 (s, 3H, CH3), 7.28 (s, 1H), 7.56 (d, J = 12 Hz, 1H), 7.95
(d, J = 12 Hz, 1H), 8.29 (s, 1H), 9.79 (s, 1H, OH).
4.1.5.3. 6-Hydroxy-7-methoxy-2-naphthonitrile (2c). Yield 72%;
mp164.2–165.4 °C;1HNMR (400 MHz, DMSO-d6)d 3.91(s, 3H, CH3),
7.24 (s, 1H), 7.41 (s, 1H), 7.49 (d, 1H, J = 1.6 Hz), 7.77 (d, 1H,
J = 1.2 Hz), 8.24 (s, 1H), 10.24 (s, 1H, OH).
4.1.6. General procedure for the preparation of 2d–e
4.1.7.4. 5-Chloro-6-(2-(4-cyanophenylamino)pyrimidin-4-yloxy)-
2-naphthonitrile (3d). Yield 71.6%; recrystallized from 1,4-diox-
ane, mp 297.5–298.7 °C; 1H NMR (400 MHz, DMSO-d6) d 6.82 (d,
Sodium hydride (10 mmol) was added to a solution of 2a or 2c
(3 mmol) in anhydrous THF (50 mL) and stirred for 5 min. Then
NCS (3 mmol) was added to the reaction mixture and was refluxed

Y.-H. Liang et al. / Bioorg. Med. Chem. 18 (2010) 4601–4605
4605
J = 4.0 Hz, 1H), 7.27–7.52 (m, 4H, Ph), 8.02–8.54 (m, 5H, naph), 8.82
4.2. Biological study
(s, 1H, CH), 10.12 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
100.18, 103.15, 109.78, 118.81, 119.09, 119.72, 123.09, 125.27,
125.55, 129.19, 130.08, 131.49, 132.81, 132.93, 135.93, 144.72,
159.33, 161.18, 168.88. MS (EI) m/z: 393.1 (M+); Anal.
(C22H12ClN5O) C, H, N.
4.2.1. Anti-HIV assay in MT-4 cells
The anti-HIV activity and cytotoxicity were evaluated against
wild-type HIV-1 strain IIIB in MT-4 cells using the 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) meth-
od.11,12 Briefly, virus stocks were titrated in MT-4 cells and
expressed as the 50% cell culture infective dose (CCID50). MT-4 cells
were suspended in culture medium at 1 Â 105 cells/mL and in-
fected with HIV at a multiplicity of infection of 0.02. Immediately
4.1.7.5. 5-Chloro-6-(2-(4-cyanophenylamino)pyrimidin-4-yloxy)-
7-methoxy-2-naphthonitrile (3e). Yield 33.4%; flash chromatog-
raphy separation and recrystallized from 1,4-dioxane, mp
268.4–269.2 °C; 1H NMR (400 MHz, DMSO-d6) d 12 13C NMR
(400 MHz, DMSO-d6) d 3.89 (s, 3H, CH3), 6.82 (d, J = 4.4 Hz, 1H),
7.25–7..49 (m, 4H, Ph), 7.82–8.63 (m, 4H, naph), 8.52 (d,
J = 4.4 Hz, 1H), 10.11 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
57.21, 99.71, 103.15, 108.27, 110.23, 118.66, 119.25, 119.72,
124.69, 125.54, 126.74, 127.57, 131.95, 132.94, 133.76, 141.45,
144.75, 152.66, 159.35, 161.13, 168.47. MS (EI) m/z: 427.1 (M+);
Anal. (C23H14ClN5O2) C, H, N.
after virus infection, 100 lL of the cell suspension was placed in
each well of a flat-bottomed microtiter tray containing various
concentrations of the test compounds. Stock solutions of the test
compounds were dissolved in DMSO at 50 mM or higher. After
4 days of incubation of virus-infected cells with the compounds
at 37 °C, the number of viable cells was determined using the
MTT method. Compounds were tested in parallel for cytotoxic ef-
fects in uninfected MT-4 cells.
4.2.2. HIV RT assay
4.1.7.6. 6-(2-(4-Cyanophenylamino)-5-methylpyrimidin-4-yloxy)-
2-naphthonitrile (3f). Yield 85.6%; recrystallized from 1,4-diox-
ane, mp 258.4–259.2 °C; 1H NMR (400 MHz, DMSO-d6) d 2.28 (s,
3H, CH3), 7.27–7.57 (m, 4H, Ph), 7.65–8.40 (m, 4H, naph), 8.72(s,
1H, CH), 9.93 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d 12.25,
102.46, 108.57, 118.40, 119.48, 119.64, 119.89, 124.61, 127.55,
129.40, 130.30, 130.95, 132.92, 134.79, 135.85, 145.16, 152.43,
157.75, 159.89, 167.90; MS (EI) m/z: 377.1 (M+); Anal.
(C23H15N6BrO2) C, H, N.
Reactions were performed under the conditions described for
the HIV-1 RT RNA-dependent DNA polymerase activity assay.
Incorporation of Biotin-dUTP into poly(rA)/oligo(dT) at different
concentrations of DNA or Biotin-dUTP was monitored in the pres-
ence of increasing fixed amounts of inhibitors. The activity of RT
was obtained through testing the amount of Biotin-dUTP by HRP-
labeled Streptavidin. For IC50 determinations, a range of inhibitor
concentrations between 0.2 IC50 and 5 IC50 was used.
Acknowledgments
4.1.7.7. 5-Bromo-6-(2-(4-cyanophenylamino)-5-methylpyrimi-
din-4-yloxy)-2-naphthonitrile (3g). Yield 83.3%; recrystallized
from 1,4-dioxane, mp 268.4–269.2 °C; 1H NMR (400 MHz, DMSO-
d6) d 2.43 (s, 3H, CH3), 6.70 (s, 1H, CH), 7.23–7.52 (m, 4H, Ph),
7.76–8.82 (m, 5H, naph), 10.09 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d 24.16, 98.81, 102.94, 109.70. 115.32, 118.69, 119.10,
119.77, 125.40, 128.19, 129.36, 130.78, 131.58, 132.89, 134.24,
135.47, 144.91, 151.15, 158.93, 169.35, 171.15; MS (EI) m/z:
457.0 (M+); Anal. (C23H15N5BrO2) C, H, N.
This work was financially supported by the National Natural
Science Foundation of China (No. 20872018 and 30672536) and
the Science and Technology Commission of Shanghai Municipality
(No. 09431902800).
References and notes
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4.1.7.8. 6-(2-(4-Cyanophenylamino)-6-methylpyrimidin-4-yloxy)-
2-naphthonitrile (3h). Yield 82.4%; recrystallized from 1,4-diox-
ane, mp 223.4–224.2 °C; 1H NMR (400 MHz, DMSO-d6) d 2.24 (s,
3H, CH3), 7.24–7.54 (m, 4H, P), 7.62–8.68 (m, 6H, naph), 8.36 (s,
1H, CH), 9.90 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d 12.25,
102.45, 108.57, 118.39 (2C), 119.48, 119.65, 119.89, 124.61,
127.54, 129.40, 130.29, 132.94 (2C), 134.78, 135.84, 145.15,
153.41, 157.73, 159.87, 167.89; MS (EI) m/z: 377.1 (M+); Anal.
(C23H15N6O2) C, H, N.
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din-4-yloxy)-2-naphthonitrile (3i). Yield 81.8%; recrystallized
from 1,4-dioxane, mp 254.4–225.2 °C; 1H NMR (400 MHz, DMSO-
d6) d 2.30 (s, 3H, CH3), 7.17–7.43 (m, 4H, Ph), 7.78–8.80 (m, 5H,
naph), 8.40 (s, 1H, CH), 9.91 (s, 1H, NH); 13C NMR (100 MHz,
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DMSO-d6)
d 12.17, 102.57, 109.22, 109.69, 115.26, 118.33,
119.10, 119.81, 125.64, 128.17, 129.35, 130.70, 131.57, 132.86,
134.22, 135.46, 145.01, 151.42, 157.67, 160.17, 167.10; MS (EI)
m/z: 457.0 (M+); Anal. (C23H15N6BrO2) C, H, N.
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