5-alkyl-2-(alkylthio)-6-(2,6-dihalophenylmethyl)-3,4-dihydropyrimidin- 4(3H)-ones: Novel potent and selective dihydro-alkoxy-benzyl-oxopyrimidine derivatives

Article abstract of DOI:10.1021/jm980260f

Molecular modeling analysis of compounds belonging to the recently published series of dihydro-alkoxy-benzyl-oxopyrimidines (DABOs), such as S- DABOs and DATNOs, gave support to the design of new 2,6-disubstituted benzyl- DABO derivatives as highly potent

Full text of DOI:10.1021/jm980260f

J . Med. Chem. 1999, 42, 619-627
619
5-Alk yl-2-(a lk ylth io)-6-(2,6-d ih a lop h en ylm eth yl)-3,4-d ih yd r op yr im id in -4(3H)-on es:
Novel P oten t a n d Selective Dih yd r o-a lk oxy-ben zyl-oxop yr im id in e Der iva tives
Antonello Mai,§ Marino Artico,*,§ Gianluca Sbardella,§ Silvio Massa,† Ettore Novellino,‡ Giovanni Greco,∇
Anna Giulia Loi,| Enzo Tramontano,| Maria Elena Marongiu,| and Paolo La Colla*,|
Dipartimento di Studi Farmaceutici, Istituto Pasteur-Fondazione Cenci Bolognetti, Universita` degli Studi di Roma “La
Sapienza”, P.le A. Moro 5, I-00185 Roma, Italy, Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Siena,
Banchi di Sotto 55, I-53100 Siena, Italy, Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno, P.za
Vittorio Emanuele 9, I-84084 Penta (Salerno), Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` degli
Studi di Napoli “Federico II”, via D. Montesano 49, I-80731 Napoli, Italy, and Dipartimento di Biologia Sperimentale, Sezione
di Microbiologia, Universita` degli Studi di Cagliari, V.le Regina Margherita 45, I-09124 Cagliari, Italy
Received April 30, 1998
Molecular modeling analysis of compounds belonging to the recently published series of dihydro-
alkoxy-benzyl-oxopyrimidines (DABOs), such as S-DABOs and DATNOs, gave support to the
design of new 2,6-disubstituted benzyl-DABO derivatives as highly potent and specific inhibitors
of the HIV-1 reverse transcriptase (RT). To follow up on the novel DABO derivatives, we decided
to investigate the effect of electron-withdrawing substituents in the benzyl unit of the S-DABO
skeleton versus their anti-HIV-1 activity. Such chemical modifications impacted the inhibitory
activity, especially when two halogen units were introduced at positions 2 and 6 in the phenyl
portion of the benzyl group bound to C-6 of the pyrimidine ring. Various 5-alkyl-2-(alkyl(or
cycloalkyl)thio)-6-(2,6-dichloro(or 2,6-difluoro)phenylmethyl)-3,4-dihydropyrimidin-4(3H)-ones
were then synthesized and tested as anti-HIV-1 agents in both cell-based and enzyme
(recombinant reverse transcriptase, rRT) assays. Among the various mono- and disubstituted
phenyl derivatives, the most potent were those containing a 6-(2,6-difluorophenylmethyl)
substituent (F-DABOs), which showed EC50’s ranging between 40 and 90 nM and selectivity
indexes up to g5000. An excellent correlation was found between EC50 and IC50 values which
confirmed that these compounds act as inhibitors of the HIV-1 RT. The structure-activity
relationships of the newly synthesized pyrimidinones are presented herein.
Ch a r t 1. Dihydro-alkoxy-benzyl-oxopyrimidines
(DABOs) and Related Derivatives
Currently available drugs for AIDS therapy are
divided into two groups: those that prevent infection
of target cells (nucleoside (NRTIs) and nonnucleoside
reverse transcriptase inhibitors (NNRTIs)) and those
that prevent HIV-1-infected cells from yielding infec-
tious viruses (protease inhibitors).1 Monotherapy with
antiretroviral agents has shown limited effects, very
likely due to the interplay of phenomena such as: (1)
high viral loads and multiplication rates of HIV-1, (2)
incomplete inhibition of viral replication, and (3) emer-
gence of drug-resistant mutants.2 For this reason,
combination therapies with two or more drugs have
been proposed for a more effective treatment of AIDS.3
Potent suppression of HIV-1 replication over prolonged
periods has been accomplished with regimens including
reverse transcriptase (RT) and protease inhibitors,4
although on stopping therapies viremia has rapidly
reappeared. In the attempt to obtain better results,
research is now focused on exploiting new targets and
enhancements of “old” drugs. Among the latter, new
NNRTIs possibly endowed with better pharmacokinetic
profiles, such as capability to inhibit clinically relevant
mutants and, hopefully, to minimize the HIV-1 multi-
plication,5 are being pursued.
Numerous classes of NNRTIs have been discovered
with dihydro-alkyloxy-benzyl-oxopyrimidines (DABOs)
being one of them. The lead compound, an isotrimetho-
prim derivative (1, Chart 1), has been synthesized as a
possible dihydrofolate reductase inhibitor, and when
* To whom correspondence should be sent.
§ Universita` degli Studi di Roma “La Sapienza”.
† Universita` degli Studi di Siena.
‡ Universita` degli Studi di Salerno.
∇ Universita` degli Studi di Napoli “Federico II”.
| Universita` degli Studi di Cagliari.
10.1021/jm980260f CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/05/1999

620 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Mai et al.
Sch em e 1a
tested against HIV-1 because of its structural similarity
with HEPT,6,7 it was a selective, although not very
potent, HIV-1 inhibitor.8
On the basis of these findings, various oxopyrimidines
(2, 3) have been synthesized and found to be specific
HIV-1 inhibitors targeted at the reverse transcriptase.9-12
Structure-activity relationship (SAR) studies have
shown that the presence of the C-2 alkoxy (DABO)/
alkylthio (S-DABO) side chain is a structural determi-
nant for the antiviral activity of these compounds,
whereas the length and type of substituent at C-2 (C1-6
alkyl-, isoalkyl-, or cycloalkylthio), the substituent at
C-5 (H, Me, or Et), and the presence of a monomethyl
(3) or dimethyl (3,5) substitution in the phenyl portion
of the benzyl moiety have modulatory effects on po-
tency.12 However, (1) modifications of the pyrimidine
base such as N-3 alkylations, (2) introduction of a
variety of substituents at C-4, (3) shift of the benzyl
group from C-6 to C-5, (4) decrease or increase of the
distance between the pyrimidine and the phenyl ring,
and (5) replacement of the C-6 benzyl with smaller
(propyl) substituents have led to substantial loss of
activity. The only exception has been the replacement
of the C-6 benzyl with a bulkier substituent such as
1-naphthylmethyl (DATNOs, 4).13
a
(a) (1) (C2H5)3N, MgCl2, CH3CN, rt then reflux, (2) 13% HCl;
(b) (1) C2H5ONa, NH2CSNH2, C2H5OH, reflux, (2) 2 N HCl; (c)
CH3I, DMF, rt; (d) R6 halide, K2CO3, DMF, rt; (e) C6H11I, K2CO3,
DMF, 80 °C.
Sch em e 2a
The possible binding mode of the above S-DABOs and
DATNOs to the nonnucleoside binding site (NNBS) of
the HIV-1 RT was recently examined in our laboratory
through molecular modeling methods based on the
crystal structure of the RT/HEPT complex reported by
Ren et al.14 The results of these modeling studies
prompted the synthesis and biological evaluation of
novel DABOs 5 bearing fluorine or chlorine atoms at
the 2- and 6-positions of the phenyl ring. A priori, the
2,6-dihalogenation was expected to improve a putative
π-stacking interaction between the electron-deficient
benzene ring of the ligand and the electron-rich benzene
ring of Tyr188 located in the nonnucleoside binding cleft
of RT. To better define the effects of electron-withdraw-
ing substituents on the inhibitory activity, additional
DABOs were designed featuring a fluorine, chlorine, or
nitro group at various positions of the phenyl ring. In
the herein series, the pyrimidine has C-2 alkylthio
substituents (e.g., methyl, isopropyl, isobutyl, n-butyl,
sec-butyl, cyclopentyl, and cyclohexyl) and C-5 substit-
uents of hydrogen (uracil) or methyl (thymine).
a
(f) HNO3, H2SO4, 0 °C then rt.
of the magnesium dichloride-triethylamine system.
When compared to our previously reported procedure
for â-oxoester preparation,13 this new method afforded
almost quantitative yields of ethyl arylacetylacetates 7,
which were sufficiently pure to be used for subsequent
reactions without further purification.
Condensation of the above â-oxoesters with thio-
urea12,23 afforded the related uracil and thymine deriva-
tives 8, which were S-alkylated in dry DMF with
iodomethane (2 equiv) or with the appropriate halo-
alkane (or halocycloalkane, 1 equiv) in the presence of
potassium carbonate to yield the required 5-alkyl-2-
(alkyl(or cycloalkyl)thio)-3,4-dihydro-6-(substituted phen-
ylmethyl)pyrimidine-4(3H)-ones 5 (Scheme 1).
Ch em istr y
The ethyl arylacetylacetates, needed for the pyrimi-
dine syntheses, were prepared by several methods. One
method involved Claisen condensation of ethyl arylac-
etates or aroyl chlorides with ethyl acetate15 or, alter-
natively, acylation of diethyl malonate followed by
partial hydrolysis and decarboxylation16,17 (classic meth-
ods). The second approaches are based on the use of
ethylmalonic acid,18 tert-butyl ethyl malonate,19 or Mel-
drum’s acid and subsequent ethanolysis.20-22 However,
these methods can lead to undesired byproducts or
retro-condensation reactions, and they afford the re-
quired â-oxoesters in moderate or low yields. To obviate
these disadvantages, we prepared the ethyl arylacetyl-
acetates 7 by a simple and high-yielding method by the
reaction of potassium monoethyl malonate or methyl
malonate with (arylacetyl)imidazolides 6 in the presence
2-(sec-Butylthio)-3,4-dihydro-6-(4-nitrophenylmethyl)-
pyrimidin-4(3H)-one derivatives 5i,q were obtained by
direct nitration of 2-(sec-butylthio)-3,4-dihydro-6-(phen-
ylmethyl)pyrimidin-4(3H)-one and its 5-methyl deriva-
tive12 (Scheme 2). Chemical and physical data for
derivatives 5, 7, and 8 are reported in Tables 1 and 2.
Resu lts a n d Discu ssion
Com p u ter -Assisted Design of Novel DABOs. The
computational work was carried out by molecular
mechanics (Tripos force field26), conformational analysis,
docking, and graphics routines available within the
SYBYL program.27 Details of these procedures are
described in the Experimental Section, whereas the
results are summarized below.

Novel Potent and Selective DABO Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 621
Ta ble 1. Physical and Chemical Data of Compound 5
substituent
recryst
method of
synthesis
compd
R1
R2
Cl
H
H
F
R3
R4
R5
R6
mp, °C
solventa
yield, %
formulab
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
120-121
98-99
a
b
b
a
b
c
d
d
e
b
b
b
b
b
b
b
b
f
D
D
D
D
D
D
D
D
F
D
D
D
D
D
D
D
F
C
D
D
D
D
D
E
C
D
D
D
D
D
E
C
D
D
D
D
D
E
C
D
D
D
D
D
E
58
92
74
68
67
94
68
54
100
68
75
79
65
72
69
88
100
98
81
62
56
55
54
49
95
74
46
49
48
45
40
93
78
52
63
53
49
48
92
76
65
59
49
54
49
C15H17ClN2OS
Cl
H
H
F
H
H
NO2
H
H
Cl
H
H
F
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
F
H
H
NO2
H
H
Cl
H
H
F
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
C
C
15H17ClN2OS
15H17ClN2OS
15H17FN2OS
C15H17FN2OS
C15H17FN2OS
C15H17N3O3S
C15H17N3O3S
C15H17N3O3S
16H19ClN2OS
16H19ClN2OS
16H19ClN2OS
16H19FN2OS
16H19FN2OS
16H19FN2OS
125-126
106-107
96-97
H
H
NO2
H
H
Cl
H
H
F
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
98-99
148.0-148.5
127-128
128-130
170-171
145-146
163-165
120.5-121.5
146-147
154-155
163-164
178-180
237-238
230-231
153-154
143.5-144.5
183-184
185-186
200-201
197-198
174-175
126-127
136-137
149-150
168-169
164-165
260-261
241-242
179-180
208-209
204-205
252-253
237-238
218.5-219.5
164-165
178-179
161-162
128-129
192-193
191-192
H
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
C
C
C
C
C
C
C16H19N3O3S
C16H19N3O3S
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
12H10Cl2N2OS
14H14Cl2N2OS
15H16Cl2N2OS
15H16Cl2N2OS
15H16Cl2N2OS
16H16Cl2N2OS
17H18Cl2N2OS
12H10F2N2OS
14H14F2N2OS
15H16F2N2OS
15H16F2N2OS
15H16F2N2OS
16H16F2N2OS
17H18F2N2OS
13H12Cl2N2OS
15H16Cl2N2OS
16H18Cl2N2OS
16H18Cl2N2OS
16H18Cl2N2OS
17H18Cl2N2OS
18H20Cl2N2OS
13H12F2N2OS
15H16F2N2OS
16H18F2N2OS
16H18F2N2OS
16H18F2N2OS
17H18F2N2OS
18H20F2N2OS
5s
5t
f
b
b
d
b
d
f
b
b
b
a
b
f
d
b
b
b
b
d
b
f
b
b
b
c
b
b
5u
5v
5v
5x
5y
5z
5a ′
5b′
5c′
5d ′
5e′
5f′
5g′
5h ′
5i′
5j′
5k ′
5l′
5m ′
5n ′
5o′
5p ′
5q′
5r ′
5s′
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
F
F
F
F
a
Code: a, n-hexane/cyclohexane; b, cyclohexane; c, n-hexane; d, benzene/cyclohexane; e, diethyl ether/petroleum ether; f, benzene.
All compounds were analyzed for C, H, N, S and, when required, Cl and F; analytical results were within (0.4% of theoretical values.
b
Ch a r t 2. Previously Reported S-DABOs12 Used as
References in SAR Studies and Molecular Modeling
methyl derivative, such as 10b, was based on two
considerations: (i) the cyclopentyl group is fairly rigid
and lacks chiral centers, and its medium size makes it
representative of both small (methyl) and large (sec-
butyl) groups of the 2-alkylthio side chain; (ii) it was
soon realized that any conformation energetically al-
lowed to a 5-methyl derivative could also be attained
by the less encumbered 5-desmethyl counterpart.
The model of the RT/10b complex, like those later
developed for other DABOs of general formula 5, was
derived through the following steps: (i) manual align-
ment of the 2-(cyclopentylthio)thymine moiety within
the NNBS using the enzyme-bound conformation of
HEPT as template; (ii) systematic search of the ligand
conformation in which the 6-arylmethyl chain fills the
largest volume within the binding cleft; (iii) geometry
optimization of the resulting complex by keeping the
protein backbone atoms fixed.
The design of 2,6-dihalogenated DABOs was prompted
by inspection of a previously derived model of compound
10b (Chart 2) docked into the NNBS of HIV-1 RT
(unpublished results). Coordinates of the NNBS were
taken from the crystal structure of the RT/HEPT
complex reported at 3.0 Å resolution by Ren et al.14 The
high degree of similarity between HEPT and DABOs
justified the selection of such a complex among those
currently available28 from the Brookhaven Protein Data
Bank.29 The choice of modeling a 2-(cyclopentylthio)-5-
Figure 1 shows the theoretical binding mode of 5r ′ to
the NNBS. The following features of the model are
worthy to be outlined. The thymine and 2,6-difluorophen-

622 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Mai et al.
Ta ble 2. Physical and Chemical Data of Compounds 7 and 8
substituent
compd
R1
R2
Cl
H
H
F
H
H
NO2
H
Cl
H
H
F
H
H
H
Cl
F
Cl
F
Cl
H
H
F
H
H
NO2
H
Cl
H
H
F
H
H
H
Cl
F
Cl
F
R3
R4
R5
mp, °C
recryst solventa
yield, %
formulaa
b
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
H
H
Cl
H
H
F
H
H
NO2
H
Cl
H
H
F
H
NO2
H
H
H
H
H
Cl
H
H
F
H
H
NO2
H
Cl
H
H
F
H
NO2
H
H
H
H
Cl
H
H
F
H
H
H
H
Cl
H
H
F
H
H
H
H
H
H
H
Cl
H
H
F
H
H
H
H
Cl
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
oil
oil
oil
oil
oil
oil
85
94
90
85
94
92
98
90
90
98
100
89
88
100
82
99
87
94
87
43
52
75
85
60
62
18
83
56
59
64
52
55
62
46
78
84
40
56
C12H13ClO3
C12H13ClO3
C12H13ClO3
C12H13FO3
C12H13FO3
C12H13FO3
c
56-57
59-60
oil
diethyl ether/petroleum ether
benzene/cyclohexane
C12H13NO5
C12H13NO5
d
C13H15ClO3
C13H15ClO3
C13H15ClO3
C13H15FO3
C13H15FO3
C13H15FO3
C13H15NO5
C12H12Cl2O3
C12H12F2O3
C13H14Cl2O3
C13H14F2O3
C11H9ClN2OS
7j
7k
7l
oil
oil
oil
oil
oil
oil
7m
7n
7o
7p
7q
7r
7s
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
75-76
37-38
98-99
oil
n-hexane
n-hexane
n-hexane
H
Me
Me
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
240-242
239-240
242-243
239-241
220-221
225-227
248-249
246-248
243-245
229-230
262-263 dec
246-247
251-252
255-256
268-269
>280
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
ethanol
C
C
11H9ClN2OS
11H9ClN2OSe
C11H9FN2OS
11H9FN2OS
11H9FN2OS
C11H9N3O3S
C11H9N3O3S
12H11ClN2OS
12H11ClN2OS
12H11ClN2OS
12H11FN2OS
12H11FN2OS
12H11FN2OS
C12H11N3O3S
C11H8Cl2N2OS
11H8F2N2OS
12H10Cl2N2OS
12H10F2N2OS
C
C
f
H
Me
Me
Me
Me
Me
Me
Me
Me
H
C
C
C
C
C
C
8m
8n
8o
8p
8q
8r
H
H
H
H
H
>280
258-260
278-280 dec
C
C
C
Me
Me
H
H
Cl
F
8s
a
All compounds were analyzed for C, H, N, S and, when required, Cl and F; analytical results were within (0.4% of theoretical values.
Reference 24. c Reference 20. Reference 21. e Reference 19. f Reference 25.
b
d
the “butterfly” orientation of the planar rings of HEPT
and nevirapine, respectively, in their enzyme-bound
conformations.14,30 The thymine moieties of 5r ′ and
HEPT occupy the same position in the NNBS, the only
difference being a rotation by ca. 30° about one another
within the same plane. A hydrogen bond is donated by
the 3-NH function of 5r ′ to the carbonyl oxygen of
Lys101 (N‚‚‚O distance 3.4 Å) which is analogous to that
observed for the RT/HEPT complex (N‚‚‚O distance 3.1
Å). The 2-cyclopentylthio group fits into a pocket
reported to exhibit great flexibility about the Pro236
hairpin31 which is also occupied by the 1-(2-hydroxy-
ethoxy)methyl substituent of HEPT. The 2,6-difluo-
robenzyl moiety is surrounded by the side chains of
Pro95, Tyr181, Tyr188, Phe227, Trp229, Leu100, and
Leu234. Particularly, the electron-rich benzene ring of
Tyr188 appears to be optimally oriented for a favorable
π-stacking interaction with that of the ligand (made
electron-deficient by the two fluorines): the planes of
the two aromatic rings are fairly parallel and separated
by a distance of 3.8 Å.
F igu r e 1. 2,6-Difluoro-DABO (5r ′) (yellow) docked into the
nonnucleoside binding site of RT (only 17 residues are dis-
played). The Tyr188 side chain, hypothesized to give rise to a
favorable π-stacking interaction with the 2,6-difluorophenyl
moiety of the ligand, is red.
Given the many approximations implicit in our dock-
ing calculations and the known limitations of molecular
mechanics in describing polarization effects and charge-
transfer interactions, the 2,6-difluoro- and 2,6-dichloro-
DABOs were predicted to be more potent than their
corresponding parent compounds on qualitative bases.
yl parts of the ligand exhibit a “propeller-like” disposi-
tion which is intermediate between the “skewed” and

Novel Potent and Selective DABO Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 623
Ta ble 3. Cytotoxicity and Anti-HIV-1 Activity of Compound 5a
substituent
compd
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5v
5x
R1
R2
Cl
H
H
F
R3
R4
R5
R6
CC50, µMb
EC50, µMc
IC50, µMd
SIe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
Me
i-propyl
n-butyl
i-butyl
s-butyl
c-pentyl
c-hexyl
Me
i-propyl
n-butyl
i-butyl
s-butyl
c-pentyl
c-hexyl
Me
i-propyl
n-butyl
i-butyl
s-butyl
c-pentyl
c-hexyl
Me
i-propyl
n-butyl
i-butyl
s-butyl
c-pentyl
c-hexyl
200
116
120
200
1.0
2.0
5.0
0.26
0.7
8.7
0.25
0.4
1.5
0.27
0.96
9.5
0.41
1.2
11
0.35
2
3.2
1.9
0.44
0.45
0.14
0.4
1.6
1.9
4.8
0.3
0.6
7.4
0.3
0.3
1.2
0.4
0.9
8.5
0.4
1.5
13
0.4
1.8
3.0
1.5
0.5
0.6
0.1
0.4
0.4
0.8
0.05
0.20
0.20
0.05
0.08
0.09
NDg
1.15
1.15
1.10
0.06
1.7
200
58
24
Cl
H
H
F
H
H
NO2
H
H
Cl
H
H
F
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
F
H
H
NO2
H
H
Cl
H
H
F
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
769
f
H
H
NO2
H
H
Cl
H
H
F
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
>200
>286
g200
>200
157
23
>800
392
101
>741
>208
>20
341
H
151
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
>200
>200
>200
140
>200
105
>200
>200
>200
>200
>200
>200
>200
>200
>200
g200
g200
162
>166
9.5
>571
>100
>62
>105
>454
>444
>1428
>500
>333
g247
g4000
900
0.6
5y
5z
0.81
0.05
0.18
0.14
0.04
0.08
0.08
38
1.3
1.1
1.2
0.05
1.8
5a ′
5b′
5c′
5d ′
5e′
5f′
5g′
5h ′
5i′
182
1300
g200
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
>200
>200
164
g200
>200
>200
150
g5000
>2500
>2500
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
5.2
>154
>171
>166
>4000
>111
>9
1053
>4000
>2500
1640
g4000
>2500
>2222
125
5j′
5k ′
5l′
22
4.9
0.2
5m ′
5n ′
5o′
5p ′
5q′
5r ′
5s′
9a
0.19
0.05
0.08
0.1
0.05
0.08
0.09
1.2
0.05
0.09
0.09
0.05
0.08
0.07
1.00
0.5
F
F
F
F
9b
86
0.6
140
10a
10b
nevirapine
MKC-442
147
1.7
0.6
0.25
0.03
1.6
0.5
0.3
0.04
86
>200
>200
200
>333
>800
6666
a
b
Data represent mean values of at least two separate experiments. Compound dose required to reduce the viability of mock-infected
cells by 50%, as determined by the MTT method. c Compound dose required to achieve 50% protection of MT-4 cells from HIV-1-induced
cytopathogenicity,as determined by the MTT method. Compound dose required to inhibit the HIV-1 rRT activity by 50%. e Selectivity
d
index, CC50/EC50 ratio. f Higher concentrations could not be achieved because of crystallization of compounds in the culture medium.
g
Not determined.
Analogues bearing a single electron-attracting group (F,
Cl, or NO2) at the 2-, 3-, or 4-position were also
suggested for the synthesis in order to enrich the
amount of information achievable from SAR data. The
above structures were predicted by molecular modeling
analyses to attain low-energy conformations fitting into
the NNBS cavity with sufficient shape complementarity.
Cytotoxicity, An tivir a l Activity, a n d SAR Stu d -
ies. On the basis of the above considerations, we
synthesized the uracil and thymine derivatives 5a -s′
listed in Table 3. Compounds 5a -q are characterized
by chlorine, fluorine, or nitro substituents at either the
ortho, meta, or para positions of the phenyl ring, and
all possess a sec-butyl side chain at position 2 of the
pyrimidine. Compounds 5r -s′ are 2,6-dichloro and 2,6-
difluoro derivatives and have different alkylthio side
chains.
Title compounds were evaluated for cytotoxicity and
anti-HIV-1 activity in MT-4 cells (Table 3) in comparison
with MKC-44232 and nevirapine,33,34 used as reference
drugs. The majority of derivatives are noncytotoxic for
MT-4 cells at doses as high as 200 µM, whereas the
remaining nine compounds show CC50 values ranging
from 105 to 182 µM.
Several ortho- and meta-monosubstituted DABOs
(5d ,g,h ,j,p ) are up to 3 times more active than their
parent compounds 9a ,b, whereas other monosubstituted
derivatives are equally active (5a ,e,m ) or slightly less

624 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Mai et al.
acids, N,N-carbonyldiimidazole, iodomethane, 2-iodopropane,
1-iodo-2-methylpropane, 2-iodobutane, 1-iodobutane, cyclo-
pentyl bromide, and cyclohexyl iodide were purchased from
Aldrich Chimica, Milan (Italy), and Lancaster Synthesis
GmbH, Milan (Italy).
Syn th eses. The specific examples presented below il-
lustrate general synthetic methods A-F. As a rule, samples
prepared for physical (Tables 1 and 2) and biological (Table 3)
studies were dried in high vacuum over P2O5 for 20 h at
temperatures ranging from 25 to 110 °C, depending on the
sample melting point.
Meth od A. Exa m p le: Eth yl 4-(2,6-Dich lor op h en yl)-
a cetyla ceta te (7p ). Triethylamine (8.7 mL, 62.4 mmol) and
magnesium chloride (4.64 g, 48.73 mmol) were added to a
stirred suspension of potassium ethyl malonate (6.97 g, 40.95
mmol) in dry acetonitrile (50 mL), and stirring was continued
at room temperature for 2 h. Then, a solution of the (2,6-
dichlorophenylacetyl)imidazolide in dry acetonitrile (50 mL),
prepared 15 min before by reaction between 2,6-dichlorophe-
nylacetic acid (4.00 g, 19.49 mmol) and N,N′-carbonyldiimi-
dazole (3.47 g, 21.40 mmol) in acetonitrile (20 mL), was added.
The reaction was allowed to stir overnight at room tempera-
ture and then heated at reflux for 2 h. After the mixture had
cooled, 13% HCl (50 mL) was cautiously added while keeping
the temperature below 25 °C, and the resulting clear mixture
was stirred for a further 15 min. The organic layer was
separated and evaporated; then the residue was treated with
ethyl acetate (50 mL). The aqueous layer was extracted with
ethyl acetate (2 × 50 mL), and the organic phases were
combined, washed with saturated sodium bicarbonate solution
(2 × 100 mL) and brine (100 mL), dried, and concentrated to
give pure 7p (5.3 g): 1H NMR (CDCl3) δ 1.25-1.32 (t, 3H,
CH2CH3), 3.56 (s, 2H, COCH2CO), 4.17-4.24 (m, 2H, CH2CH3),
4.24 (s, 2H, COCH2Ar), 7.16-7.20 (m, 1H, 4-H Ar), 7.30-7.34
(m, 2H, 3,5-H Ar); IR ν 1725, 1700 cm-1. Anal. C, H, Cl.
active (5b,k ,n ). As a rule, the shift of substituents from
the ortho to the meta or para position is accompanied
by loss of activity. The difference in potency between
ortho- and para-substituted derivatives ranges between
5-fold (5c vs 5a ) and 33-fold (5f vs 5d ) and is likely due
to the slightly unfavorable steric interaction between
the 4-substituent and the Trp229 side chain of the
NNBS.
In the dihalo-substituted series (compounds 5r -s′),
difluoro derivatives are usually more active than the
dichloro counterparts. The sole exception is represented
by 5j′ which, together with its 5c′,q′ sec-butyl difluoro
homologues, is active at nanomolar concentrations and
shows a potency significantly higher than that of
nevirapine and comparable to that of MKC-442. Inter-
estingly, also in dihalo-substituted DABOs the alkylthio
substituents modulate the anti-HIV-1 potency. How-
ever, a peculiar feature of the latter, and in particular
of thymine derivatives, is the very small differences in
potency among compounds carrying different alkyl
substituents in the alkylthio chain.
The above results appear to confirm our hypothesis
about the favorable effect of 2,6-dihalogenation related
to an attractive π-stacking interaction taking place
between the benzene rings of Tyr188 and DABOs. Also
consistent with the hypothesis is the fact that the
presence of a single electron-withdrawing group at the
ortho or meta position of the phenyl ring of DABOs
reduces the beneficial electronic effect on the antiviral
activity produced by 2,6-dihalogenation.
Since previous DABO derivatives targeted the HIV-1
RT, the title compounds were also tested in enzyme
assays against highly purified recombinant HIV-1 RT
using homopolymeric template primers, and their in-
hibitory activity was expressed as IC50. An excellent
correlation was found between the EC50 and IC50 values
indicating that the in vitro antiproliferative activity does
entirely reflect the capability of the tested compounds
to inhibit the enzyme. This conclusion is strengthened
by the fact that, besides being specific inhibitors of the
HIV-1 acute infection, DABOs inhibit neither the mul-
tiplication of HIV-2 nor that of HIV-1 in chronically
infected H9/IIIB cells (results not shown).
Meth od B. Exa m p le: 6-(2-Ch lor op h en ylm eth yl)-3,4-
d ih yd r o-5-m eth yl-2-th iop yr im id in -4(3H)-on e (8i). Sodium
metal (0.42 g, 0,018 g-atom) was dissolved in 50 mL of absolute
ethanol, and thiourea (0.97 g, 12.74 mmol) and ethyl 2-(2-
chlorophenylacetyl)propionate (7i) (2.34 g, 9.19 mmol) were
added to the clear solution. The mixture was heated at reflux
for 5 h. After cooling, the solvent was removed, and the residue
was dissolved in water (20 mL) and acidified with 2 N HCl.
The resulting precipitate was filtered, washed with diethyl
ether, and dried in vacuo at 80 °C for 12 h to give 8i (1.37 g)
as a pure solid: 1H NMR (DMSO-d6) δ 1.63 (s, 3H, C5-CH3),
3.90 (s, 2H, CH2Ar), 7.04-7.09 (m, 1H, 4-H Ar), 7.26-7.30 (m,
2H, 5,6-H Ar), 7.45-7.50 (m, 1H, 3-H Ar), 12.22 (s, 1H, NH
exchangeable with D2O), 12.43 (s, 1H, NH exchangeable with
D2O); IR ν 3200, 2940, 1635 cm-1. Anal. C, H, Cl, N, S.
In conclusion, the new dihalogen-substituted DABOs
emerge as noncytotoxic, anti-HIV-1 agents whose po-
tency is comparable to that of MKC-442, the most
important of the structurally related HEPT class of
NNRTIs.
Meth od C. Exa m p le: 6-(2,6-Diflu or op h en ylm eth yl)-3,4-
d ih yd r o-5-m e t h yl-2-(m e t h ylt h io)p yr im id in -4(3H )-on e
(5m ′). A mixture of 6-(2,6-difluorophenylmethyl)-3,4-dihydro-
5-methyl-2-thiopyrimidin-4(3H)-one (8s) (0.30 g, 1.12 mmol)
and iodomethane (0.32 g, 0.14 mL, 2.24 mmol) in 2 mL of
anhydrous DMF was stirred at room temperature for 24 h.
The suspension was then diluted with cold water (100 mL)
and extracted with ethyl acetate (3 × 50 mL). The combined
organic extract was washed with brine (3 × 50 mL), dried,
and evaporated to furnish crude 5m ′ (0.30 g), which was
recrystallized: 1H NMR (DMSO-d6) δ 2.02 (s, 3H, C5-CH3), 2.15
(s, 3H, SCH3), 3.89 (s, 2H, CH2Ar), 7.00-7.07 (m, 2H, 3,5-H
Ar), 7.26-7.41 (m, 1H, 4-H Ar), 12.53 (broad s, 1H, NH
exchangeable with D2O); IR ν 2950, 1640 cm-1. Anal. C, H, F,
N, S.
Meth od D. Exa m p le: 6-(2,6-Diflu or op h en ylm eth yl)-
3,4-d ih yd r o-2-(1-m e t h ylp r op ylt h io)p yr im id in -4(3H )-
on e (5c′). A mixture of 6-(2,6-difluorophenylmethyl)-3,4-
dihydro-2-thiopyrimidin-4(3H)-one (8q) (0.50 g, 2.03 mmol),
2-iodobutane (0.40 g, 0.25 mL, 2.23 mmol), potassium carbon-
ate (0.27 g, 1.95 mmol), and 2 mL of anhydrous DMF was
stirred at room temperature for 24 h. After treatment with
cold water (200 mL), the solution was extracted with ethyl
acetate (3 × 50 mL). The combined organic extract was washed
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Bu¨chi 530
melting point apparatus and are uncorrected. Infrared (IR)
spectra (KBr) were recorded on a Perkin-Elmer 297 instru-
ment. 1H NMR spectra were recorded at 200 MHz on a Bruker
AC 200 spectrometer; chemical shifts are reported in δ (ppm)
units relative to the internal reference tetramethylsilane (Me4-
Si). All compounds were routinely checked by TLC and 1H
NMR. TLC was performed on aluminum-backed silica gel
plates (Merck DC-Alufolien Kieselgel 60 F254) with spots
visualized by UV light. All solvents were reagent grade and,
when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions
involved the use of a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Organic solutions were dried over
anhydrous sodium sulfate. Analytical results are within
(0.40% of the theoretical values. Substituted phenylacetic

Novel Potent and Selective DABO Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 625
with brine (3 × 50 mL), dried, and evaporated to furnish crude
5c′, which was purified by chromatography over silica gel
column (eluent: chloroform): 1H NMR (CDCl3) δ 0.89-0.96
(t, 3H, CH2CH3), 1.26-1.30 (d, 3H, CHCH3), 1.57-1.67 (m, 2H,
CH2CH3), 3.74-3.86 (m, 1H, CHCH3), 3.86 (s, 2H, CH2Ar), 5.95
(s, 1H, C-5 H), 6.82-6.89 (m, 2H, 3,5-H Ar), 7.19-7.24 (m,
1H, 4-H Ar), 13.02 (broad s, 1H, NH exchangeable with D2O);
IR ν 2900, 1640 cm-1. Anal. C, H, F, N, S.
F igu r e 2.
Meth od E. Exa m p le: 2-(Cycloh exylth io)-6-(2,6-d ich lo-
r op h en ylm eth yl)-3,4-d ih yd r op yr im id in -4(3H)-on e (5x). A
mixture of 6-(2,6-dichlorophenylmethyl)-3,4-dihydro-2-thio-
pyrimidin-4(3H)-one (8p ) (0.50 g, 1.74 mmol), cyclohexyl iodide
(0.40 g, 0.25 mL, 1.91 mmol), and potassium carbonate (0.24
g, 1.74 mmol) in 2 mL of anhydrous DMF was heated at 80 °C
for 15 h. After cooling, the reaction mixture was treated with
cold water (100 mL) and extracted with ethyl acetate (3 × 50
mL). The organic layers were collected, washed with brine (3
× 50 mL), dried, and evaporated to furnish crude 5x, which
was purified by chromatography on silica gel column (eluent:
n-hexane/ethyl acetate/methanol, 12/3/1): 1H NMR (CDCl3) δ
1.26-1.48 (m, 6H, C3,C4,C5 cyclohexane-H), 1.56-1.67 (m, 2H,
relaxation for each conformation. The number of output
conformations to be examined was reduced by setting the
“energy window” (energy difference between the generated
conformation and the current minimum) to 2.0 kcal/mol. The
resulting conformations were classified in the grid space of
torsional angles into a smaller number of “families” using the
FAMILY option of the SYBYL/MOLECULAR SPREADSHEET
routine. The lowest energy conformer of each family was fully
energy-minimized, and among the resulting structures we
identified the global minimum energy conformer.
Molecular superimpositions were performed by minimizing
the root-mean-square distance between selected pairs of atoms
using the SYBYL/FIT and SYBYL/MATCH commands.
The geometry of the nonnucleoside binding site (NNBS) of
RT was derived from a 3.0 Å resolution structure of the HIV-1
RT/HEPT complex14 filed in the Brookhaven Protein Data
Bank28,29 (entry code 1RTI). The NNBS consisted of a set of
69 amino acids located within a distance of 10 Å from any non-
hydrogen atom of the bound inhibitor. Hydrogens were added
to the unfilled valences of HEPT and of the amino acids
according to standard geometries of the Tripos force field.
Charges of the NNBS atoms were calculated by the Gasteiger-
Hu¨ckel method (Lys, Asp, and Glu side chains were modeled
in their ionized forms).
Docking of the 2,6-difluoro-DABO (5r ′) into the NNBS was
carried out in two steps. First, the 2-(cyclopentylthio)-5-
methyldihydropyrimidinone (CPMP) moiety was superimposed
on the thymine ring of HEPT with overlap of the common
NHCO fragments. HEPT was removed from the site. Using
the SYBYL/DOCK routine, the CPMP moiety was rotated and
translated manually within the plane of the pyrimidine ring
by monitoring the ligand site interaction energy made up by
steric and electrostatic contributions. During this maneuver,
the atoms of the 2,6-difluorophenylmethyl substituent of the
ligand were ignored by the force field, the hydrogen bond
distance between the hydrogen of the pyrimidine 3-NH group
and the carbonyl oxygen of Lys101 was monitored, and the
torsional angles τ3 and τ4 determining the orientation of the
2-cyclopentylthio substituent were adjusted manually. The
process, constrained by requirements of (i) coplanarity of the
pyrimidine and thymine rings of 5r ′ and HEPT and (ii)
preservation of the above-mentioned hydrogen bond, ended up
until no improvement of the interaction energy was apparently
possible.
The docking continued by seeking the optimal position of
the previously neglected 2,6-difluorobenzyl group. This was
realized using the SYBYL/SEARCH routine. Torsional angles
τ1 and τ2 were rotated by 10° increments in the 0-350° range.
A van der Waals scaling factor of 0.7 was applied to both ligand
and site atoms. A limited number of output conformations,
very similar to one another, were obtained. A trial active
conformation was chosen as that with the lowest volume in
common with the protein atoms (this volume was computed
using the SYBYL/MVOLUME command).
C
2eq,C6eq cyclohexane-H), 1.91-1.96 (m, 2H, C2ax,C6ax cyclohex-
ane-H), 3.69-3.74 (m, 1H, SCH), 4.17 (s, 2H, CH2Ar), 5.89 (s,
1H, C5-H), 7.08-7.16 (m, 1H, 4-H Ar), 7.24-7.32 (m, 2H, 3,5-H
Ar), 12.62 (broad s, 1H, NH exchangeable with D2O); IR ν 2900,
1645 cm-1. Anal. C, H, Cl, N, S.
Meth od F . Exa m p le: 3,4-Dih yd r o-2-(1-m eth ylp r op yl-
th io)-6-(4-n itr op h en ylm eth yl)p yr im id in -4(3H)-on e (5i). A
65% solution of nitric acid (0.05 mL, 1.09 mmol) was cautiously
added to an ice-cooled mixture of 3,4-dihydro-2-(1-methylpro-
pylthio)-6-(phenylmethyl)pyrimidin-4(3H)-one (9a )12 (0.30 g,
1.09 mmol) and 98% sulfuric acid (5 mL), and the resulting
mixture was stirred at room temperature for 1 h. After
treatment with cold water (100 mL), the solution was extracted
with ethyl acetate (3 × 50 mL) and the combined organic
extract was washed with brine (3 × 50 mL), dried, and
evaporated to furnish crude 5i (0.34 g) that was homogeneous
by TLC analysis (SiO2/n-hexane-acetone, 3:1): 1H NMR
(CDCl3) δ 0.90-0.97 (t, 3H, CH2CH3), 1.29-1.32 (d, 3H,
CHCH3), 1.59-1.68 (m, 2H, CH2CH3), 3.77-3.87 (m, 1H,
CHCH3), 3.87 (s, 2H, CH2Ar), 6.02 (s, 1H, C5-H), 7.39-7.43
(d, 2H, 2,6-H Ar), 8.13-8.18 (d, 2H, 3,5-H Ar), 12.82 (broad s,
1H, NH exchangeable with D2O); IR ν 2950, 1640, 1510, 1335
cm-1. Anal. C, H, N, S.
Molecu la r Mod elin g. Molecular modeling studies were
performed with the software package SYBYL27 running on a
Silicon Graphics Iris Indigo XS24 workstation. Herein we
describe the computational procedure applied to the 2,6-
difluoro-DABO (5r ′); analogues of 5r ′ were modeled following
a similar approach.
A model of 5r ′, based on the crystal structure of 6-(3-
methylphenylmethyl)-2-(2-methylpropylthio)-S-DABO,35 was
created using the SYBYL fragment library. The 2-cyclopent-
ylthio substituent was consistently modeled as a half-chair
equatorially substituted conformation.
Conformational energies were calculated with the Tripos
force field protocol26 including the electrostatic contribution.
Partial atomic charges were assigned according to the
Gasteiger-Hu¨ckel method.36,37 Geometry optimizations were
performed with the SYBYL/MAXIMIN2 minimizer by applying
the Broyden-Fletcher-Goldfarb-Shanno algorithm38 and set-
ting a root-mean-square gradient of the forces acting on each
atom of 0.05 kcal/mol Å as convergence criterion. Under these
conditions, energy minimization reproduced satisfactorily the
solid-state geometry of 6-(3-methylphenylmethyl)-2-(2-meth-
ylpropylthio)-S-DABO35 about the thymine and phenylmethyl
moieties.
Systematic conformational analyses were carried out using
the SYBYL/SEARCH routine to identify low-energy conforma-
tions of 5r ′. Torsional angles τ1-τ4 (defined in Figure 2) were
scanned by 10° increments throughout a 0-350° range. A van
der Waals scaling factor of 0.7 was applied to “soften” steric
contacts. Such scaling was aimed to minimize the typical
limitations of systematic “rigid” search consisting of lack of
Geometric optimization of the RT/5r ′ complex was per-
formed to relieve sterically undesirable contacts between
ligand and NNBS. The protein backbone atoms were kept
fixed. The complex was minimized to an attractive interaction
energy of -10 kcal/mol.
The putative active conformation of 5r ′, characterized by
an energy of 1.7 kcal/mol above that of the corresponding
global minimum conformer, was associated with the following
torsional angles: τ1 ) -52°, τ2 ) -93°, τ3 ) -27°, τ4 ) -56°.
An t ivir a l Assa y P r oced u r es. 1. Com p ou n d s. Com-

626 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Mai et al.
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pounds were solubilized in DMSO at 200 mM and then diluted
into culture medium.
2. Cells a n d Vir u ses. MT-4, C8166, H9/IIIB, and CEM cells
were grown at 37 °C in a 5% CO2 atmosphere in RPMI 1640
medium, supplemented with 10% fetal calf serum (FCS), 100
IU/mL penicillin G, and 100 µg/mL streptomycin. Cell cultures
were checked periodically for the absence of mycoplasma
contamination with a MycoTect Kit (Gibco). Human immuno-
deficiency viruses type-1 (HIV-1, IIIB strain) and type-2 (HIV-
2, ROD strain; kindly provided by Dr. L. Montagnier, Paris)
were obtained from supernatants of persistently infected H9/
IIIB and CEM cells, respectively. HIV-1 and HIV-2 stock
solutions had titers of 4.5 × 106 and 1.4 × 105 50% cell culture
infectious dose (CCID50)/mL, respectively.
3. HIV Titr a tion . Titration of HIV was performed in C8166
cells by the standard limiting dilution method (dilution 1:2,
four replica wells/dilution) in 96-well plates. The infectious
virus titer was determined by light microscope scoring of
cytopathicity after 4 days of incubation, and the virus titers
were expressed as CCID50/mL.
4. An ti-HIV Assa ys. Activity of the compounds against
HIV-1 and HIV-2 multiplication in acutely infected cells was
based on the inhibition of virus-induced cytopathicity in MT-4
and C8166 cells, respectively. Briefly, 50 µL of culture medium
containing 1 × 104 cells was added to each well of flat-bottom
microtiter trays containing 50 µL of culture medium with or
without various concentrations of the test compounds. Then
20 µL of an HIV suspension containing 100 (HIV-1) or 1000
(HIV-2) CCID50 was added. After a 4-day incubation (5 days
for HIV-2) at 37 °C, the number of viable cells was determined
by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) method.39 Cytotoxicity of the compounds was
evaluated in parallel with their antiviral activity. It was based
on the viability of mock-infected cells, as monitored by the
MTT method.
5. RT Assa ys. Assays were performed as previously de-
scribed.40 Briefly, purified rRT was assayed for its RNA-
dependent polymerase-associated activity in a 50-µL volume
containing: 50 mM Tris-HCl (pH 7.8), 80 mM KCl, 6 mM
MgCl2, 1 mM DTT, 0.1 mg mL-1 BSA, 0.5 OD260 unit mL-1
template:primer [poly(rC)-oligo(dG)12-18], and 10 mM [3H]-
dGTP (1 Ci mmol-1). After incubation for 30 min at 37 °C, the
samples were spotted on glass fiber filters (Whatman GF/A),
and the acid-insoluble radioactivity was determined.
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Ack n ow led gm en t. The authors thank Italian Min-
istero della Sanita` - Istituto Superiore di Sanita` - IX
Progetto AIDS 1996 (Grants 40A.0.06 and 9403-59) for
financial aid. Thanks are also due to Italian MURST
(40% funds) and Regione Autonoma Sardegna (Asses-
sorato Sanita`) for partial support.
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