Non-nucleoside HIV reverse transcriptase inhibitors, Part 6[1]: Synthesis and anti-HIV activity of novel 2-[(arylcarbonylmethyl)thio]-6-arylthio DABO analogues

Article abstract of DOI:10.1002/ardp.200400961

2-(Arylcarbonylmethyl)thio-6a-naphthylmethyl derivatives of dihydro-alkoxy-benzyl-oxopyrimidines (DABO) were newly found to exhibit activity against both HIV-1 and HIV-2. To further explore their structure-activity relationship, the modified S-DABO analog

Full text of DOI:10.1002/ardp.200400961

Arch. Pharm. Chem. Life Sci. 2005, 338, 457−461
DOI 10.1002/ardp.200400961
457
Non-nucleoside HIV Reverse Transcriptase
Inhibitors, Part 6[1]: Synthesis and Anti-HIV Activity
of Novel 2-[(Arylcarbonylmethyl)thio]-6-arylthio
DABO Analogues
Guang-Fu Suna, Yun-Yan Kuanga, Fen-Er Chena, Erik De Clercqb, Jan Balzarinib,
Christophe Pannecouqueb
a Department of Chemistry, Fudan University, Shanghai, People’s Republic of China
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium
2-(Arylcarbonylmethyl)thio-6α-naphthylmethyl derivatives of dihydro-alkoxy-benzyl-oxopyrimidines
(DABO) were newly found to exhibit activity against both HIV-1 and HIV-2. To further explore their
structure-activity relationship, the modified S-DABO analogues (5aϪg and 6eϪf) with a 1-naphthyl-
thio or phenylthio group at the C-6 position were synthesized. S-Alkylation of 5-ethyl-2-thiouracil
with substituted 2-bromo-acetophenones provided crude 2-[(arylcarbonylmethyl)thio]-5-ethyl-(3H)-
uracil 2aϪe, which was directly subjected to toluenesulfonylation with TsCl to afford disulfonate 4aϪe.
Substitution of 4aϪe with arylthiol afforded the desired S-DABO analogues 5aϪg and 6eϪf. The
compounds were evaluated for their in vitro anti-HIV activity in MT-4 cells. The IC50 values for anti-
HIV-1 activity fall into the range 0.37Ϫ29.50 µM, and the IC50 values for anti-HIV-2 activity fall into
the range 23.11Ϫ181.07 µM. The results indicated that these compounds are moderately active against
HIV-1 and HIV-2.
Keywords: NNRTI; S-DABO analogues; Antiviral activity; HIV-1; HIV-2
Received: November 15, 2004; Accepted: July 1, 2005
Introduction
Dihydro-alkoxy-benzyl-oxopyrimidine (DABO) (Figure 1)
analogues are known as a typical class of NNRTI with simi-
lar structures related to HEPT (Figure 1). A series of DABO
derivatives has been found to have excellent anti-HIV-1 ac-
tivities [7, 8]. Very recently, when introducing a β-carbonyl
group into the C-2 side chain of 6α-naphthylmethyl S-
DABO, we found that the resulting 2-(arylcarbonylmethyl)-
thio-6α-naphthylmethyl S-DABO derivatives (Figure 1) ex-
hibited marked activity against both HIV-1 and HIV-2 [9].
Among them, the candidates bearing an ethyl group at the
C-5 position of the pyrimidin-4-one ring were more effective
against HIV-2 than those bearing a methyl or isopropyl
group at the same position. These interesting results
prompted us to further explore the structure-activity re-
lationship of SϪDABO. Herein, a series of 6-arylthio-sub-
stituted S-DABO derivatives were designed, synthesized and
evaluated for their anti-HIV activity.
Since the discovery of Zidovudine (AZT), reverse tran-
scriptase (RT) inhibitors of human immunodeficiency virus
(HIV) have played an important role in the treatment of
AIDS in the past two decades [1, 2]. As we know, RT inhibi-
tors are divided into two functionally distinct classes [3Ϫ5]:
(A) nucleoside RT inhibitors (NRTI) such as AZT, which
bind to the polymerase site and thereby act as competitive
inhibitors and exhibit equal activity against HIV-1 and
HIV-2; (B) non-nucleoside RT inhibitors (NNRTI) as exem-
plified by Nevirapine, Efavirenz, HEPT and others, which
bind to a specific allosteric site close to the polymerase site
of HIV-1 RT, lead to a non-competitive inhibition mecha-
nism and generally are highly potent against HIV-1. To date,
14 RT inhibitors have been successfully approved as drugs
for the treatment of AIDS, but their clinical use is limited
because of the severe adverse side effects or drug resistance
[2, 6]. Therefore, researchers in the RT inhibitor field now
focus on finding novel compounds with different mecha-
nisms of action and strong inhibition of the resistant mu-
tants.
Results and discussion
Chemistry
The synthetic route to the target compounds 5aϪf and 6eϪf
is outlined in Scheme 1. According to the previously re-
ported procedure [10], 5-ethyl-2-thiouracil 1 was easily pre-
pared by the condensation of diethyl ethylmalonate with
Correspondence: Fen-Er Chen, Department of Chemistry,
Fudan University, Shanghai 200433, People’s Republic of China.
Phone: ϩ86 21 65642021, Fax: ϩ86 21 65642021, e-mail:
rfchen@fudan.edu.cn
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Chen et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 457−461
Figure 1. Chemical structures of DABO derivatives and HEPT.
Scheme 1. Synthesis of the DABO analogues 5aϪg and 6eϪf. Reagents and conditions: (a) NH2CSNH2, NaOCH3/CH3OH,
reflux, 6 h; (b) RCOCH2Br, K2CO3/DMF, r. t., 12 h; (c) TsCl, Et3N/CH2Cl2, 0°C, 6 h; (d) ArSH, EtOH/0.5N NaOH-EtOH, r. t.,
12 h, then 10% NaOH-H2O, 3 h.
thiourea in the presence of CH3ONa/CH3OH with 83%
yield. S-Alkylation of 1 with various substituted 2-bromo-
acetophenones in the presence of K2CO3 in anhydrous
DMF solution provided a mixture of 2-[(arylcarbonyl-
methyl)thio]-5-ethyl-(3H)-uracils 2aϪe and 2-[(arylcarbon-
ylmethyl)thio]-5-ethyl-(1H)-uracils 3a؊e with 81Ϫ97%
yields. Without separation, the mixture was directly sub-
jected to toluenesulfonylation with TsCl in the Et3N/CH2Cl2
system to afford disulfonates 4aϪe with 37Ϫ70% yields,
which reacted with 1-naphthalenethiol or benzenethiol un-
der alkaline conditions to afford 5aϪg with 39Ϫ58% yields,
along with their isomers 6eϪf with 8Ϫ24% yields. The
structures of two isomers were determined by comparing
the spectral data with similar compounds [8, 11].
MT-4 cells using the MTT method [12]. The used virus
strains are HIV-1 strain IIIB and HIV-2 strain ROD. The
results were summarized in Table 1.
As can be seen from the anti-HIV-1 activity data listed in
Table 1, the compounds (5c, 5e) with a 6-(1-naphthylthio)
substituent were 1.1-fold and 14.9-fold, respectively, more
active than their counterparts with a 6-benzenethiol sub-
stituent (5f, 5g), and these results suggested that the 6-(1-
naphthylthio) group contributed more to improve the bio-
logical activity than the 6-benzenethiol group. Moreover,
the pyrimidinone derivatives 5aϪg inhibited HIV-1 repli-
cation in a range of micromolar concentrations (IC50
ϭ
0.37Ϫ6.72 µM), and compounds 6eϪf showed less activity
(IC50 ϭ 16.8 and 29.5 µM, respectively). It has been de-
duced that the 3-NH function enhances anti-HIV activity
by donation of a hydrogen bond to the carbonyl oxygen of
Lys101 in RT [8]; our results further confirmed that the 3-
NH function of DABO was essential for the inhibition of
HIV RT.
Biological properties
The novel S-DABO derivatives (5aϪg and 6eϪf) were
evaluated for their cytotoxicity and anti-HIV activity in
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Arch. Pharm. Chem. Life Sci. 2005, 338, 457−461
Novel 2-[(Arylcarbonylmethyl)thio]-6-arylthio DABO Analogues
459
Table 1. Biological activities of compounds 5aϪh and 6eϪf against HIV-1 and HIV-2.
Compound
Ar
R
IC50 µM
CC50 µM
SI (HIV-1)
HIV-1
HIV-2
5a
5b
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
Phenyl
Phenyl
1-Naphthyl
Phenyl
Ph
4-Cl-Ph
4-F-Ph
2,4-F2-Ph
4-H3CO-Ph
4-F-Ph
4-H3CO-Ph
4-H3CO-Ph
4-F-Ph
0.37
5.94
3.33
0.38
0.45
3.53
6.72
16.77
29.50
5.06
5.37
> 26.39
> 25.5
> 23.11
> 31.20
> 157.58
> 37.5
> 181.07
36.15
> 27.50
> 405.8
2.71
36.74
22.44
26.04
41.03
160.6
36.5
181.48
74.09
29.58
399
101
4
8
5c
5d
107
359
10
27
4
< 1
79
98
5e
5f
5g
6e
6f
HEPT
DDI
529
General procedure for the synthesis of 4aϪe
It was also found that all compounds showed less activity
against HIV-2 in comparison with that against HIV-1, and
the replacement of the 6α-naphthylthio group by the 6-phe-
nylthio group or the replacement of the 3-NH function by
the 1-NH function in target compounds had no influence
on the anti-HIV-2 activity. Moreover, the compounds with
a 4-methoxyphenyl group at the C-2 side chain (5e, 5g and
6e) showed less cytotoxicity.
To a stirred mixture of crude 5-ethyl-2-[(arylcarbonylmethyl)thio]-
uracils 3aϪe (3.0 mmol), Et3N (5.0 mL) and CH2Cl2 (20 mL) was
added dropwise a solution of TsCl (1.2 g, 6.2 mmol) in CH2Cl2
(10 mL) at 0Ϫ5°C. The reaction mixture was stirred at room tem-
perature for an additional 6 h; then H2O (20 mL) was added drop-
wise and the organic phase was separated and the aqueous phase
was extracted with CH2Cl2 (2 ϫ 15 mL). The combined organic
phases were washed with H2O (2 ϫ 20 mL) and dried over anhy-
drous Na2SO4. The solvent was evaporated in vacuo, and the residue
was purified by column chromatography on silica gel (eluent
AcOEt/petroleum ether, 1 : 2 vol/vol, 60Ϫ90°C) to afford 4a؊e with
37Ϫ70% yields.
In conclusion, the novel 2-[(arylcarbonylmethyl)thio]-6-
arylthio S-DABO were synthesized and evaluated for their
anti-HIV activity. The results revealed that the target com-
pounds had moderate activities against HIV-1 and HIV-2,
but were less active in comparison with the previously re-
ported 2-(arylcarbonylmethyl)thio S-DABO derivatives [9].
5-Ethyl-2-(phenacylmethyl)thio-4,6-di(p-tolylsulfonyloxy)primidine
(4a)
Yield 1.14 g (64%) as a yellow powder. mp 91Ϫ93°C. 1H-NMR
(CDCl3): δ ϭ 1.01 (t, 3H, J ϭ 7.7 Hz, CH3), 2.40 (s, 6H, ϫ 2 CH3),
2.45 (q, 2H, J ϭ 7.7 Hz, CH2), 4.45 (s, 2H, SCH2), 6.95Ϫ7.96 (m,
13H, Harom).
Experimental
General
5-Ethyl-2-[(4Ј-chlorophenacylmethyl)thio]-4,6-di(p-tolylsulfonyl-
oxy)primidine (4b)
Melting points (mp) were measured on a WRS-1B digital melting
point apparatus and are uncorrected. IR spectra were recorded on
Yield 1.32 g (70%) as a yellow powder. mp 127Ϫ129°C. 1H-NMR
(CDCl3): δ ϭ 1.09 (t, 3H, J ϭ 7.4 Hz, CH3), 2.46 (s, 6H, ϫ 2 CH3),
2.53 (q, 2H, J ϭ 7.4 Hz, CH2), 4.55 (s, 2H, SCH2), 7.36 (d, 4H, J ϭ
8.2 Hz, Harom), 7.52 (d, 2H, J ϭ 8.8 Hz, Harom), 7.93 (d, 4H, J ϭ
8.2 Hz, Harom), 7.97 (d, 2H, J ϭ 8.8 Hz, Harom).
an Avvatar 360 FT-IR instrument. H- and 13C-NMR spectra were
1
recorded with a JEOL EX-400 (400 MHz) spectrometer. Chemical
shifts (δ) were expressed in ppm with the protonated solvent as ref-
erence. Mass spectra (MS) were recorded on MAT95 and for the
electronic impacts (EI) at 70 eV. Column chromatography was per-
formed with silica gel G (300Ϫ400 mesh); TLC was performed on
plates coated with silica gel GF254. Solvents were purified using
standard procedures [13].
5-Ethyl-2-[(4Ј-methoxyphenacylmethyl)thio]-4,6-di(p-tolylsulfonyl-
oxy)primidine (4c)
Yield 1.17 g (62%) as a yellow powder. mp 123Ϫ124°C. 1H-NMR
(CDCl3): δ ϭ 1.10 (t, 3H, J ϭ 7.7 Hz, CH3), 2.41 (s, 6H, ϫ 2 CH3),
2.54 (q, 2H, J ϭ 7.7 Hz, CH2), 3.89 (s, 3H, OCH3), 4.46 (s, 2H,
SCH2), 6.98 (d, 2H, J ϭ 9.2 Hz, Harom), 7.32 (d, 4H, J ϭ 8.3 Hz,
Harom), 7.92 (d, 4H, J ϭ 8.3 Hz, Harom), 7.96 (d, 2H, J ϭ 9.2 Hz,
Chemistry
General procedure for the synthesis of 2aϪe and 3a؊e
A mixture of 5-ethyl-2-thiouracil (1) (0.624 g, 3.6 mmol), 2-bromo-
acetophenone or substituted 2-bromo-acetophenone (4.0 mmol)
and anhydrous K2CO3 (0.556 g, 4 mmol) in anhydrous DMF (10
mL) was stirred at room temperature for 12 h, then was poured into
cold H2O (40 mL) and neutralized with 10% aq. HCl to pH 5Ϫ6.
The white precipitate was collected by filtration, successively washed
with cold H2O (3 ϫ 15 mL) and ether (2 ϫ 20 mL), and dried in
vacuo to afford the mixture of 2aϪe and 3aϪe with 84Ϫ97% yields,
which were directly used in the next step without separation.
H
arom).
5-Ethyl-2-[(4Ј-fluorophenacylmethyl)thio]-4,6-di(p-tolylsulfonyl-
oxy)primidine (4d)
Yield 0.99 g (54%) as a yellow powder. mp 128Ϫ131°C. 1H-NMR
(CDCl3): δ ϭ 1.06 (t, 3H, J ϭ 7.8 Hz, CH3), 2.41 (s, 6H, ϫ 2 CH3),
2.47 (q, 2H, J ϭ 7.8 Hz, CH2), 4.51 (s, 2H, SCH2), 7.16 (m, 2H,
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Chen et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 457−461
Harom), 7.32 (d, 4H, J ϭ 8.2 Hz, Harom), 7.89 (d, 4H, J ϭ 8.2 Hz,
Harom), 8.03 (m, 2H, Harom).
Hz, CH2), 4.53 (s, 2H, SCH2), 6.92Ϫ7.99 (m, 10H, Harom), 9.42 (s,
1H, NH). 13C-NMR (CDCl3): δ ϭ 14.1, 21.9, 38.6, 106.1, 113.3,
123.3, 123.9, 125.0, 126.2, 126.7, 126.9, 127.1, 127.5, 128.3, 129.4,
129.6, 138.4, 143.4, 152.6, 154.2, 165.6, 166.1, 168.4, 191.9. MS: m/z
(%): 468 (20.3), 327 (43.7), 313 (32), 141 (100).
5-Ethyl-2-[(2Ј,4Ј-difluorophenacylmethyl)thio]-4,6-di(p-tolyl-
sulfonyloxy)primidine (4e)
Yield 0.71 g (37%) as a yellow powder. mp 136Ϫ138°C. 1H-NMR
(CDCl3): δ ϭ 1.07 (t, 3H, J ϭ 7.8 Hz, CH3), 2.41 (s, 6H, ϫ 2 CH3),
2.46 (q, 2H, J ϭ 7.8 Hz, CH2), 4.61 (s, 2H, SCH2), 6.97 (m, 2H,
Harom), 7.27 (d, 4H, J ϭ 8.2 Hz, Harom), 7.52 (m, 1H, Harom), 7.87
(d, 4H, J ϭ 8.2 Hz, Harom).
5-Ethyl-2-[(4Ј-methoxyphenacylmethyl)thio]-6-1-naphthylthio-
pyrimidin-4(3H)-one (5e)
Yield: 0.12 g (52%) as a white powder. mp 197Ϫ198°C. 1H-NMR
(CDCl3): δ ϭ 1.04 (t, 3H, J ϭ 7.3 Hz, CH3), 2.43 (q, 2H, J ϭ 7.3
Hz, CH2), 3.90 (s, 3H, OCH3), 4.51 (s, 2H, SCH2), 6.97Ϫ7.99 (m,
11H, Harom), 10.01 (s, 1H, NH). 13C-NMR (CDCl3): δ ϭ 13.4, 21.8,
37.7, 55.6, 114.2, 121.5, 123.9, 124.3, 124.6, 125.6, 125.9, 126.4,
127.5, 127.8, 128.4, 129.8, 130.4, 134.0, 158.2, 161.7, 163.1, 163.4,
192.8. MS: m/z (%): 462 (16.2), 327 (28.3), 313 (36.8), 135 (100).
General procedure for the synthesis of 5a؊g and 6eϪf
To
a stirred mixture of 1-naphthalenethiol or benzenethiol
(0.5 mmol) and 0.5 M ethanolic NaOH solution (1.0 mL) in EtOH
(4 mL) under a nitrogen atmosphere was added 4aϪe (0.5 mmol)
in one portion; the resulting mixture was stirred overnight at room
temperature until 4aϪe had disappeared (monitored by TLC).
Then, 10% NaOH solution (2 mL) was added and the mixture was
continuously stirred for an additional 3 h at room temperature. The
solvent was evaporated in vacuo, and the residue was suspended in
H2O (5 mL), acidified to pH 5Ϫ6 with 1 M aq. HCl and extracted
with CH2Cl2 (2 ϫ 10 mL). The combined organic phases were dried
over anhydrous Na2SO4, filtered and evaporated to dryness; the resi-
due was purified by column chromatography on silica gel (eluent
AcOEt/petroleum ether, 1 : 1 vol/vol, 60Ϫ90°C) to afford 5aϪg
and 6eϪf.
2-[(4Ј-Fluorophenacylmethyl)thio]-5-ethyl-6-phenylthio pyrimidin-
4(3H)-one (5f)
Yield: 0.094 g (47%) as a white solid. mp 171Ϫ174°C. 1H-NMR
(CDCl3): δ ϭ 1.07 (t, 3H, J ϭ 7.3 Hz, CH3), 2.61 (q, 2H, J ϭ 7.3
Hz, CH2), 4.52 (s, 2H, SCH2), 6.92Ϫ7.45 (m, 9H, Harom), 9.81 (s,
1H, NH). 13C-NMR (CDCl3): δ ϭ 13.7, 21.8, 38.1, 118.3 (d), 124.6,
125.6, 127.6, 128.8, 129.6, 132.8, 134.1, 152.9, 153.5, 165.7, 166.9,
193.2. MS: m/z (%): 400 (23.4), 277 (68.1), 263 (52.3), 123 (100).
2-[(4Ј-Methoxyphenacylmethyl)thio]-5-ethyl-6-phenylthio pyrimi-
din-4(3H)-one (5g)
2-[(Phenacylmethyl)thio]-5-ethyl-6α-naphthylthio pyrimidin-4(3H)-
one (5a)
Yield: 0.081 g (39%) as a yellow solid. mp 153Ϫ155°C. 1H-NMR
(CDCl3): δ ϭ 1.05 (t, 3H, J ϭ 7.3 Hz, CH3), 2.58 (q, 2H, J ϭ 7.3
Hz, CH2), 3.89 (s, 3H, OCH3), 4.53 (s, 2H, SCH2), 6.89Ϫ7.38 (m,
11H, Harom). 10.12 (br, 1H, NH), 13C-NMR (CDCl3): δ ϭ 13.3,
21.2, 36.8, 55.3, 114.3 (d), 116.7, 123.9, 127.5, 128.6, 129.8, 132.6,
133.5, 152.2, 153.1, 162.7, 165.3, 192.7. MS: m/z (%): 412 (26.7),
277 (100), 263 (49.2), 135 (83.2).
Yield: 0.10 g (46%) as a white powder. mp 178Ϫ181°C. 1H-NMR
(CDCl3): δ ϭ 1.01 (t, 3H, J ϭ 7.3 Hz, CH3), 2.38 (q, 2H, J ϭ 7.3
Hz, CH2), 4.53 (s, 2H, SCH2), 7.09Ϫ7.98 (m, 12H, Harom), 8.67 (s,
1H, NH). 13C-NMR (CDCl3): δ ϭ 13.8, 18.4, 37.6, 122.7, 124.4 (d)
124.8, 125.1, 125.3, 126.3, 126.8, 127.0, 127.4, 127.7, 127.9, 128.8,
130.6, 134.1 (d), 136.1, 151.2, 159.9, 163.0, 192.8. MS: m/z (%): 432
(24.6), 327 (74.1), 313 (62.7), 105 (100).
5-Ethyl-2-[(4Ј-methoxyphenacylmethyl)thio]-6-1-naphthylthio-
pyrimidin-4(1H)-one (6e)
2-[(4Ј-Chlorophenacylmethyl)thio]-5-ethyl-6α-naphthylthio pyrimi-
din-4(3H)-one (5b)
Yield: 0.075 g (24%) as a yellow powder. mp 105Ϫ108°C. 1H-NMR
(CDCl3): δ ϭ 0.89 (t, 3H, J ϭ 7.3 Hz, CH3), 2.38 (q, 2H, J ϭ 7.3
Hz, CH2), 3.49 (s, 1H, NH), 3.84 (s, 3H, OCH3), 4.41 (s, 2H, SCH2),
6.84Ϫ8.02 (m, 11H, Harom). 13C-NMR (CDCl3): δ ϭ 13.1, 20.7,
36.5, 55.3, 114.2 (d), 118.3, 122.3, 123.7, 123.9, 124.5, 125.8, 126.1,
126.6, 127.8, 128.1, 128.3, 129.9, 133.6, 151.7, 153.4, 162.8, 167.3,
191.3. MS: m/z (%): 462 (5.1), 327 (63.6), 154 (23.1), 135 (100).
Yield: 0.18 g (58%) as a white solid. mp 182Ϫ184°C. 1H-NMR
(CDCl3): δ ϭ 0.94 (t, 3H, J ϭ 7.4 Hz, CH3), 2.42 (q, 2H, J ϭ 7.4
Hz, CH2), 4.45 (s, 2H, SCH2), 7.15Ϫ8.05 (m, 11H, Harom), 9.73 (s,
1H, NH). 13C-NMR (CDCl3): δ ϭ 13.7, 22.3, 37.9, 122.4, 123.4 (d),
125.1, 125.6, 126.3, 126.8, 127.3, 127.9, 128.4, 128.7, 129.3, 129.7,
131.8, 134.4, 137.7, 154.2, 158.1, 165.3, 192.6. MS: m/z (%): 466
(6.0), 327 (21.3), 313 (24.5), 139(100).
2-[(4Ј-Fluorophenacylmethyl)thio]-5-ethyl-6-phenylthio pyrimidin-
4(1H)-one (6f)
2-[(4Ј-Fluorophenacylmethyl)thio]-5-ethyl-6α-naphthylthio pyrimi-
din-4(3H)-one (5c)
Yield: 0.042 g (21%) as a yellow solid. mp 93Ϫ95°C. 1H-NMR
(CDCl3): δ ϭ 0.86 (t, 3H, J ϭ 7.3 Hz, CH3), 2.41 (q, 2H, J ϭ 7.3
Hz, CH2), 3.51 (s, 1H, NH), 4.40 (s, 2H, SCH2), 7.09Ϫ7.99 (m,
11H, Harom). 13C-NMR (CDCl3): δ ϭ 13.3, 20.9, 37.1, 115.2 (d),
118.7, 124.3, 125.4, 126.9, 127.5, 131.9, 133.7, 148.3, 149.1, 165.2,
168.5, 191.7. MS: m/z (%): 400 (4.9), 291 (35.3), 277 (54.8), 123
(100).
Yield: 0.15 g (48%) as a white solid. mp 175Ϫ177°C. 1H-NMR
(CDCl3): δ ϭ 0.98 (t, 3H, J ϭ 7.4 Hz, CH3), 2.41 (q, 2H, J ϭ 7.4
Hz, CH2), 4.50 (s, 2H, SCH2), 7.10Ϫ7.94 (m, 11H, Harom), 9.16 (s,
1H, NH). 13C-NMR (CDCl3): δ ϭ 13.5, 21.7, 37.9, 116.8 (d), 123.1,
125.0, 126.1, 126.5, 127.1, 127.4, 127.7, 127.9, 128.7, 129.3, 129.7,
130.8, 134.1, 151.3, 153.1, 165.3, 166.7, 192.5. MS: m/z (%): 450
(17.7), 327 (100), 313 (78.6), 123 (80.1).
Anti-HIV-1 activity assays
The activity of the compounds against HIV-1 (HTLV-IIIB strain)
and the Y181CϩK103N mutant strain was based on the inhibition
of the virus-induced cytopathic effect on MT-4 cells using the MTT
method. Briefly, virus stocks were titrated in MT-4 cells and ex-
pressed as 50% cell culture-infective dose (CCID50). MT-4 cells
2-[(2Ј,4Ј-Difluorophenacylmethyl)thio]-5-ethyl-6α-naphthylthio pyr-
imidin-4(3H)-one (5d)
Yield: 0.13 g (42%) as a yellow solid. mp 166Ϫ168°C. 1H-NMR
(CDCl3): δ ϭ 0.99 (t, 3H, J ϭ 7.3 Hz, CH3), 2.45 (q, 2H, J ϭ 7.3
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Arch. Pharm. Chem. Life Sci. 2005, 338, 457−461
Novel 2-[(Arylcarbonylmethyl)thio]-6-arylthio DABO Analogues
461
were suspended in culture medium at 1 ϫ 105 cells/mL and infected
with HIV at a multiplicity of infection of 0.02. Immediately after
virus infection, 100 µL of the cell suspension was brought into each
well of a flat-bottom microtiter tray containing various concen-
trations of the test compounds. The test compounds were dissolved
in dimethyl sulfoxide at 50 mM or higher. After a 4-day incubation
at 37°C, the number of viable cells was determined by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method. Compounds were tested in parallel for their cytotoxic effect
on uninfected MT-4 cells.
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