Structure-based design, synthesis, and biological evaluation of conformationally restricted novel 2-alkylthio-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4 (3H)-ones as non-nucleoside inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1021/jm010853h

5-Alkyl-2-(alkylthio)-6-(2,6-difluorobenzyl)-3,4-dihydropyrimidin-4(3H)-ones (S-DABOs 2) have been recently described as a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) active at nan

Full text of DOI:10.1021/jm010853h

2544
J . Med. Chem. 2001, 44, 2544-2554
Str u ctu r e-Ba sed Design , Syn th esis, a n d Biologica l Eva lu a tion of
Con for m a tion a lly Restr icted Novel 2-Alk ylth io-6-[1-(2,6-d iflu or op h en yl)a lk yl]-
3,4-d ih yd r o-5-a lk ylp yr im id in -4(3H)-on es a s Non -n u cleosid e In h ibitor s of HIV-1
Rever se Tr a n scr ip ta se
Antonello Mai,† Gianluca Sbardella,† Marino Artico,*,† Rino Ragno,‡ Silvio Massa,§ Ettore Novellino,|
Giovanni Greco,| Antonio Lavecchia,| Chiara Musiu, Massimiliano La Colla,# Chiara Murgioni,#
Paolo La Colla,*,# and Roberta Loddo#
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Universita` degli Studi di Roma
“La Sapienza”, P.le A. Moro 5, I-00185 Roma, Italy, Dipartimento di Studi di Chimica e Tecnologie delle Sostanze
Biologicamente Attive, 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, via Aldo Moro, I-53100 Siena, Italy,
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` degli Studi di Napoli “Federico II”, Via D. Montesano 49,
I-80131 Napoli, Italy, Novirio Pharmaceuticals, Inc., Boston, and Dipartimento di Biologia Sperimentale, Sezione di
Microbiologia, Universita` degli Studi di Cagliari, Cittadella Universitaria SS 554, I-09042 Monserrato (Cagliari), Italy
Received February 26, 2001
5-Alkyl-2-(alkylthio)-6-(2,6-difluorobenzyl)-3,4-dihydropyrimidin-4(3H)-ones (S-DABOs, 2) have
been recently described as a new class of human immunodeficiency virus type 1 (HIV-1) non-
nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) active at nanomolar concentrations
(Mai, A. et al. J . Med. Chem. 1999, 42, 619-627). In pursuing our lead optimization efforts,
we designed novel conformationally restricted S-DABOs, 3, featuring a methyl at the benzylic
carbon (Y ) Me) and at the pyrimidine 5-position (R ) Me). Conformational analyses and
docking simulations suggested that the presence of both methyls would significantly reduce
conformational flexibility without compromising, in the R enantiomers, the capability of fitting
into the RT non-nucleoside binding pocket. To develop structure-activity relationships, we
prepared several congeners of type 3 belonging to the thymine (R ) Me) and uracil (R ) H)
series, featuring various 2-alkylthio side chains (X ) Me, i-Pr, n-Bu, i-Bu, s-Bu, c-pentyl, and
c-hexyl) and aryl moieties different from the 2,6-difluorophenyl (Ar ) phenyl, 2,6-dichlorophenyl,
1-naphthyl). Moreover, R-ethyl derivatives (Y ) Et) were included in the synthetic project in
addition to R-methyl derivatives (Y ) Me). All of the new compounds were evaluated for their
cytotoxicity and anti-HIV-1 activity in MT-4 cells, and some of them were assayed against
highly purified recombinant wild-type HIV-1 RT using homopolymeric template primers. The
results were expressed as CC50 (cytotoxicity), EC50 (anti-HIV-1 activity), SI (selectivity, given
by the CC50/EC50 ratio), and IC50 (RT inhibitory activity) values. In the 2,6-difluorobenzyl-
thymine (R ) Me) series, methylation of the benzylic carbon improved anti-HIV-1 and RT
inhibitory activities together with selectivity. Compound 3w (Ar ) 2,6-F2-Ph, R ) Y ) Me, X
) c-pentyl) turned out the most potent and selective among the S-DABOs reported to date
(CC50 > 200 µM, EC50 ) 6 nM, IC50 ) 5 nM, and SI > 33 333). Assays performed on the pure
enantiomer (+)-3w , much more active than (-)-3w , yielded the following results: CC50 > 200
µM, EC50 ) 2 nM, IC50 ) 8 nM, and SI > 100 000, under conditions wherein MKC-442 was
less active and selective (CC50 > 200 µM, EC50 ) 30 nM, IC50 ) 40 nM, SI > 6666). The 2,6-
difluorophenylethylthymines (R ) Me) were generally endowed with higher potency compared
with the uracil counterparts (R ) H). In the 2,6-difluorophenyl series the best and the least
performant 2-alkylthio side chains were the 2-c-pentylthio and the 2-methylthio, respectively.
When the methyl at the benzylic carbon was replaced by an ethyl, activity was retained or
decreased slightly, thus suggesting that the dimensions of the cavity within the RT hosting
this substituent would not be compatible with groups larger than ethyl. Aryl moieties different
from the 2,6-difluorophenyl (phenyl, 1-naphthyl, 2,6-dichlorophenyl) were generally detrimental
to activity, consistent with a favorable electronic effect exerted by the 2,6-fluorines on a putative
charge-transfer interaction between the aromatic moieties of the inhibitor and Tyr188.
In tr od u ction
responsible for the conversion of single-stranded RNA
viral genome to double-stranded DNA.1-3 Two distinct
The human immunodeficiency virus type 1 (HIV-1)
reverse transcriptase (RT) is a multifunctional enzyme
‡ Dipartimento di Studi di Chimica e Tecnologie delle Sostanze
Biologicamente Attive, Universita` degli Studi di Roma “La Sapienza”.
§ Universita` degli Studi di Siena.
* To whom correspondence should be addressed. For M.A.: phone
and fax, +39064462731; e-mail, marino.artico@uniroma1.it. For P.L.C.:
phone, +390706754147; fax, +390706754210; e-mail, placolla@unica.it.
† Dipartimento di Studi Farmaceutici, Universita` degli Studi di
Roma “La Sapienza”.
| Universita` degli Studi di Napoli “Federico II”.
Novirio Pharmaceuticals, Inc.
#
Universita` degli Studi di Cagliari.
10.1021/jm010853h CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/10/2001

Non-nucleoside Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2545
Ch a r t 1. Structures of Well-Known RT Inhibitors
classes of RT inhibitors are currently being used in the
treatment of AIDS: the nucleoside (NRTIs) and non-
nucleoside (NNRTIs) inhibitors (Chart 1). The NRTIs,
whose prototype is zidovudine,4 are phosphorylated and
act competitively at the catalytic site of the enzyme as
DNA chain-terminating analogues of the natural deoxy-
nucleoside triphosphates.5 However, their therapeutic
effectiveness is limited by toxicity, unfavorable phar-
macokinetics, and emergence of resistant viral strains.6-8
NNRTIs comprise many structurally diverse com-
pounds such as nevirapine,9 TIBO,10 BHAP,11 R-APA,12
PETT,13 HEPT,14 and TNK-651.15 Crystal structures of
several RT/inhibitor complexes15-23 clearly show that
NNRTIs share a common mode of action, binding to an
allosteric site located about 10 Å from the catalytic
site. Although these agents generally exhibit low toxicity
and favorable pharmacokinetic properties, resistant
viral populations rapidly develop following their admin-
istration.24,25 Therefore, there is still a need to identify
new NNRTIs endowed with high inhibitory activity
against wild-type RT as well as its clinically relevant
mutants.26
In pursuing our lead optimization efforts, we have
recently investigated novel S-DABOs of general form-
ula 3 as conformationally restricted analogues of the
2,6-difluorobenzyl-S-DABOs 2. The present paper de-
scribes the design, the synthesis, biological evaluation,
and the structure-activity relationships (SARs) of
compounds 3.
Dihydroalkoxybenzyloxopyrimidines (DABOs, 1), struc-
turally related to HEPT, were disclosed as NNRTIs by
our group in 1993.27-29 Since then, we have been
performing many structural modifications on DABO
lead compounds with the aim of improving potency.30,31
The most fruitful result of these efforts was the discov-
ery of 2,6-difluorobenzyl-S-DABO derivatives (2)32 active
at nanomolar concentrations in cell-based and enzy-
matic assays. Compounds 2 were designed prompted by
computational studies suggesting that 2,6-dihalogena-
tion of the S-DABO phenyl moiety would enhance a
charge-transfer interaction between the π-stacking aro-
matic rings of the ligand and Tyr188.
Com p u ter -Aid ed Design
A typical strategy to improve potency of a lead
compound is to synthesise conformationally restricted
analogues with at least one conformation, among a few
energetically allowed conformations, recognized by the
target. Structural modifications of previously reported
2,6-difluorobenzyl-S-DABOs 232 were therefore sought
to reduce flexibility without producing any steric misfit
between the ligands and the RT non-nucleoside binding
cleft. This problem was approached using computational
tools as herein summarized (procedures are detailed in
the Experimental Section).

2546 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Mai et al.
recognized by RT, the S-DABO (R)-3w (Ar ) 2,6-F2-Ph,
R ) Y ) (R)-Me, X ) c-pentyl) was docked into the non-
nucleoside binding pocket of the target enzyme. The
docking calculations differed in some methodological
aspects from those described in a previous paper.32
Particularly, the structure of RT that cocrystallized with
TNK-65115 (unavailable at the time we designed the
first 2,6-difluorobenzyl-S-DABOs) was selected to take
into account a conformational change induced onto the
Tyr181 side chain by highly potent inhibitors. Our
previous models were in fact based on the coordinates
of the RT/HEPT complex16 wherein the Tyr181 side
chain is not “switched on”. Moreover, a more rigorous
computational procedure was applied on the basis of the
Amber force field parametrization35,36 and molecular
dynamics (MD) calculations.
Throughout the 500 ps MD simulation of the RT/(R)-
3w complex, τ1 and τ2 fluctuated within g- and g+
ranges centered on mean values of -51° and 87°. Figure
3 shows the model obtained by energy-minimizing the
structure of the complex averaged over the last 100 ps.
The RT-bound conformation of the ligand is character-
ized by a “propeller-like” disposition of the thymine and
the 2,6-difluorophenyl rings with τ1 ) -47° and τ2 )
88°. These values correspond to a family of conforma-
tions stabilized by the (R)-R-methyl group (highlighted
by an arrow in Figure 2). The pyrimidine NH-CdO
moiety is engaged in two hydrogen bonds with the
Lys101 CdO and NH, respectively. The 2,6-difluorophen-
yl ring contacts favorably the Tyr181 side chain, giving
rise to a π-stacking interaction. The 2-c-pentylthio
substituent fits into the Pro236 hairpin pocket.15 Fi-
nally, the R-methyl group is well accommodated in a
small cavity made up of Val179 side chain and Tyr181
plus Tyr188 main chains.
The results of the conformational analyses and dock-
ing simulations gave support to the preparation and
biological evaluation of conformationally restricted
R-methyl-2,6-difluorobenzyl-S-DABOs such as com-
pound 3w . To develop SARs, we planned the synthesis
of various congeners of general formula 3 belonging to
either the thymine (R ) Me) and uracil (R ) H) series,
featuring various 2-alkylthio side chains (X ) Me, i-Pr,
n-Bu, i-Bu, s-Bu, c-pentyl, and c-hexyl) and aryl moieties
different from the 2,6-difluorophenyl (Ar ) phenyl, 2,6-
dichlorophenyl, 1-naphthyl). To probe the dimensions
of the cavity within RT hosting the substituent on the
benzylic carbon, R-ethyl derivatives (Y ) Et) were
included in the synthetic project in addition to R-methyl
derivatives (Y ) Me).
F igu r e 1. Torsion angles τ1 and τ2 are defined by the atoms
in bold (top). Effects of R-methylation and chirality on stability
of the [g-,g+] conformation in the thymine series (R ) Me) of
2,6-difluorobenzyl-S-DABOs are shown (bottom).
A variety of different conformations are accessible to
the 2,6-difluorobenzyl side chain of compounds 2 be-
cause of rotation around the two bonds connecting the
pyrimidine and the 2,6-difluorophenyl rings. Each con-
formation is univocally identified through the torsion
angles τ1 and τ2 defined in Figure 1. Docking studies
performed in our laboratories32,33 suggested that the RT-
bound conformation of these molecules is characterized
by a gauche-, gauche+ (g-, g+) arrangement about τ1 and
τ2, respectively (Figure 1). Insertion of a methyl group
on the benzylic carbon of 2,6-difluorobenzyl-5-methyl-
S-DABOs (thymine series) selects or destabilizes con-
formations of the [g-,g+] family depending on whether
the configuration is R or S, respectively. In fact, while
in the R enantiomer the R-methyl points outward from
thymine 5-methyl, in the S enantiomer the two methyl
groups are eclipsed and sterically repel each other
(Figure 1).
Conformational analyses were performed on four 2,6-
difluorobenzyl-S-DABO models34 differing in the pres-
ence or absence of a methyl at the pyrimidine 5-position
and the benzylic carbon with R configuration. The
results are summarized in Figure 2 as scatter plots in
the plane of τ1 and τ 2. In these diagrams, a conformation
is labeled with a circle (“low energy”) or a cross depend-
ing on whether its strain energy (Estrain) is within or
above 2 kcal/mol. Estrain was calculated as the difference
in energy with respect to the global minimum confor-
mation. Expectedly, the number of low-energy confor-
mations reaches a minimum when both methyls are
present: only seven low-energy conformations of [g-,g+]
type out of the 180 generated. A single methyl decreases
flexibility to a lesser extent compared with the desm-
ethyl counterpart: 39 or 41 vs 72 low-energy conforma-
tions.
Ch em istr y
The synthesis of the title compounds was accom-
plished by two alternative routes: (i) preparation of 6-(1-
arylethyl)- and 6-(1-arylpropyl)-3,4-dihydro-2-thioxopy-
rimidin-4(3H)-ones (6) followed by S-alkylation in dry
DMF with the appropriate haloalkane in the presence
of potassium carbonate to afford 3a -j, s, u -x, a ′-j′
(Scheme 1) and (ii) direct lithiation of 2-alkylthio-6-(2,6-
difluorobenzyl)-3,4-dihydropyrimidin-4(3H)-ones at 0 °C
with lithium diisopropylamide (LDA) in dry THF and
hexamethylphosphoramide (HMPA) followed by treat-
ment with the proper alkyl halide (Scheme 2).
To verify whether, among the conformation of [g-,g+]
type selected by the (R)-R-methyl group, there was any
Thiouracil and thiothymine derivatives 6 were ob-
tained by condensation of thiourea in alkaline medium

Non-nucleoside Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2547
F igu r e 2. Distribution of the strain energies (Estrain) among the conformations of 2,6-difluorobenzyl-S-DABO models differing in
the presence or absence of a methyl group at the pyrimidine 5-position and the benzylic carbon with R configuration. Torsion
angles τ1 and τ2 are defined in Figure 1. Circle and cross labels denote Estrain values within and above 2 kcal/mol, respectively.
F igu r e 3. Model of (R)-3w docked into the RT non-nucleoside binding site. Putative intermolecular hydrogen bonds are highlighted
by dashed lines. Only residues within 3.5 Å from the ligand are shown. For sake of clarity, Glu138, Val106, and the side chain
of Lys101 are not displayed.
with ethyl 4-aryl-3-oxopentanoates and ethyl 4-aryl-3-
oxohexanoates (5), respectively. Preparation of the latter
â-oxoesters was performed by reacting potassium mo-
noethylmalonate or 2-methylmalonate with the proper
acylimidazolides in the presence of the magnesium
dichloride/triethylamine system.32 The required 2-aryl-
propionic or 2-arylbutyric acids 4 were obtained from
the related 2-arylacetic acids by lithiation (with LDA
in THF and HMPA) and subsequent alkylation.
Chemical and physical data of the newly synthesized
derivatives are reported in Tables 1 and 2.
Resu lts a n d Discu ssion
The novel S-DABOs 3 were evaluated for their cyto-
toxicity and anti-HIV-1 activity in MT-4 cells (Table 3)
in comparison with nevirapine,9 MKC-442,15 and six
previously reported S-DABOs (2a -f).32 Selected com-
pounds were assayed against highly purified recombi-

2548 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Mai et al.
Sch em e 1a
freedom of R-methylthymines to only a few low-energy
conformations, including the one bound to RT.
The nature of the 2-alkylthio substituent modulates
antiviral RT inhibiting activity. The methylthio is the
least performant in both uracil and thymine series
probably because the alkylthio side chain must fulfill
minimal requirements of lipophilicity. Steric properties
of the 2-alkylthio substituent also affect activity as
examplified by the 3- and 5-fold drop of activity passing
from the c-pentylthio derivatives 3w and 3p to the
corresponding c-hexylthio homologues 3x and 3q.
When the methyl at the benzylic carbon is replaced
by an ethyl, activity is retained or decreases slightly (up
to 5-fold passing from 3p to 3l′) with the exception of
the pairs 3q, 3m ′ and 3h , 3h ′ wherein the R-ethyl
derivatives are 3-fold more active. These results suggest
that the dimensions of the cavity within the RT hosting
the R-substituent would not be compatible with groups
larger than ethyl.
Aryl moieties different from the 2,6-difluorophenyl
(phenyl, 1-naphthyl, 2,6-dichlorophenyl) are generally
detrimental to activity, the only exception being the
1-naphthyl derivative 3g, which is slightly more active
than the corresponding 2,6-difluorophenyl derivative 3p .
The above findings are in accordance with a favorable
electronic effect exerted by the 2,6-fluorines on a puta-
tive charge-transfer interaction between the aromatic
moieties of the inhibitor and Tyr188.
The doses required to reduce HIV-1 p24 antigen levels
by 90%, 99%, and 99.9% were determined for a few
representative 2,6-difluorobenzyl-S-DABOs 2 and 3 in
comparison with MKC-442 (Table 4). In these secondary
tests, compound 3w confirms its highest activity (EC90
) 6 nM, EC99 ) 15 nM, EC99.9 ) 33 nM).
Three compounds (2f, 3r , and 3t) among those most
active were submitted to a preliminary test against the
Y181C HIV-1 mutant strain using nevirapine and
efavirenz38 as reference drugs. As shown by prelimi-
nary data on Y181C inhibition (Table 5), the presence
of substituents at both the C-5 pyrimidine ring and
the CH2 linker plays an important role. In fact, the
thymine derivative 2f, which does not bear substitu-
ents at the methylene linker, is less potent than the
linker-substituted thymine derivative 3t. However, the
nature and length of the S-substituent play a determi-
nant role for inhibitory potency against Y181C mu-
tant as proved by comparing the potent sec-butylthio
derivative 3t with the scarcely active methylthio coun-
terpart 3r .
a
R ) H, Me; Y ) Me, Et; Ar ) phenyl, 1-naphthyl, 2,6-F2-
phenyl, 2,6-Cl2-phenyl; X ) alkyl, cycloalkyl. (a) (1) LDA, THF,
and HMPA, 0 °C; (2) YI, H+. (b) (1) CDI, CH3CN, room temp; (2)
EtOCOCHRCOOK, (C2H5)3N, MgCl2, CH3CN, room temp; (3) 13%
HCl. (c) (1) C2H5ONa, NH2CSNH2, C2H5OH, reflux; (2) 2 N HCl.
(d) X-halide, K2CO3, DMF, room temp. (e) C6H11I, K2CO3, DMF,
80 °C.
Sch em e 2a
a
R ) H, Me; X ) alkyl, cycloalkyl; Y ) Me, Et. (a) (1) LDA,
THF, and HMPA, 0 °C; (2) alkyl halide, H+.
nant wild-type HIV-1 RT using homopolymeric template
primers. The results, expressed as CC50 (cytotoxicity),
EC50 (anti-HIV-1 activity), SI (selectivity, given by the
CC50/EC50 ratio), and IC50 (RT inhibitory activity)
values, are summarized in Table 3.
In the 2,6-difluorobenzylthymine (R ) Me) series,
methylation of the benzylic carbon improves anti-HIV-1
and RT inhibitory activities (compare 3s, 3t, and 3w vs
the R-desmethyl counterparts 2d , 2e, and 2f) together
with selectivity. Compound 3w is the most potent and
selective among the newly disclosed S-DABOs (CC50
>
200 µM, EC50 ) 6 nM, IC50 ) 5 nM, and SI > 33 333),
inhibiting HIV-1 replication in MT-4 cells more ef-
fectively than MKC-442 (by 5-fold), nevirapine (by 50-
fold), and its R-desmethyl homologue 2f (by 13-fold).
Assays performed on pure enantiomers of 3w , obtained
by resolution of the racemic mixture by chiral HPLC,37
showed that configuration is a crucial determinant for
activity since (+)-3w (CC50 > 200 µM, EC50 ) 2 nM,
IC50 ) 8 nM, and SI > 100 000) is 350 and 125 times
more active than (-)-3w in the cell-based and enzymatic
tests, respectively. All of the 2,6-difluorobenzyluracils
are generally less active by 4- to 9-fold than the
corresponding thymines (except 3o and 3t, which are
equipotent). In contrast, uracil and thymine derivatives
of type 2 exhibited similar anti-HIV and anti-RT activi-
Studies are in progress to confirm that, having as
untouchable basic structure the 6-[1-(2,6-difluorophen-
yl)ethyl]-5-methyl-3,4-dihydropyrimidin-4-one moiety,
the activity of F2-S-DABOs against Y181C mutant
strain could be properly modulated by introducing
appropriate alkyl (or cycloalkyl)thio substituents in the
C-2 position.
Con clu sion s
32
ties (compare 2a , 2b, and 2c with 2d , 2e, and 2f). In
other words, methylation of the benzylic carbon en-
hances activity provided that an additional methyl
group is in place at the pyrimidine 5-position, as though
the two methyls exerted a sort of synergistic effect on
potency. These findings are consistent with our hypoth-
esis of the two methyls restricting the conformational
Conformationally restricted S-DABOs 3, bearing a
methyl or ethyl group on the benzylic carbon, were
designed as novel NNRTIs using computational meth-
ods. Biological evaluation of the newly synthesized
compounds identified the 2,6-difluorobenzyl-R-meth-
ylthymine derivatives as those most active in reducing

Non-nucleoside Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2549
Ta ble 1. Physical and Chemical Data of Compounds 3
compd
Ar
R
Y
X
mp, °C
recryst solvent
synth schemea
% yield
formulab
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
i-Pr
s-Bu
118-119
97-98
n-hexane
n-hexane
n-hexane
n-hexane
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
88
69
74
62
70
58
46
69
63
49
41
60
56
54
55
54
32
49
69
55
58
62
60
48
82
62
75
58
67
52
44
64
58
62
58
59
54
C15H18N2Os
C
C
C
C
C
C
16H20N2OS
17H20N2OS
18H22N2OS
19H20N2OS
21H22N2OS
22H24N2OS
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
Me
i-Pr
n-Bu
i-Bu
s-Bu
cyclopentyl
cyclohexyl
Me
i-Pr
s-Bu
n-Bu
i-Bu
cyclopentyl
cyclohexyl
i-Pr
95-96
142-143
161-163
oil
1-naphthyl
1-naphthyl
1-naphthyl
2,6-Cl2-Ph
2,6-Cl2-Ph
2,6-Cl2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
Ph
n-hexane/cyclohex
177-178
168-170
181-182
211-212
179-180
155-156
149-150
159-160
133-134
165.5-166.5
206-207
202-203
177-178
135-136
122-123
152-153
196-197
208-209
144-145
138-139
168-169
175.5-176.5
163-164
oil
n-hexane
diethyl ether
cyclohexane
benzene/cyclohex
benzene
cyclohexane
cyclohexane
cyclohexane
n-hexane
cyclohexane
benzene
benzene/cyclohex
n-hexane/cyclohex
cyclohexane
n-hexane
cyclohexane
cyclohexane
n-hexane/cyclohex
cyclohexane
cyclohexane
cyclohexane
cyclohexane
cyclohexane
C15H16Cl2N2OS
C17H18Cl2N2OS
C18H20Cl2N2OS
C13H12F2N2OS
C15H16F2N2OS
C16H18F2N2OS
C16H18F2N2OS
C16H18F2N2OS
C17H18F2N2OS
C18H20F2N2OS
C14H14F2N2OS
C16H18F2N2OS
C17H20F2N2OS
C17H20F2N2OS
C17H20F2N2OS
C18H20F2N2OS
C19H22F2N2OS
H
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
3s
3t
3u
3v
3w
3x
3a ′
3b′
3c′
3d ′
3e′
3f′
3 g′
3h ′
3i′
3j′
3k ′
3l′
3m ′
C
C
C
C
C
C
16H20N2OS
17H22N2OS
18H22N2OS
19H24N2OS
20H22N2OS
22H24N2OS
Ph
Ph
Ph
s-Bu
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
1-naphthyl
1-naphthyl
1-naphthyl
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
126-127
166-168
168-169
198-199
149-150
141-143
154-155
n-hexane
C23H26N2OS
diethyl ether
diethyl ether
cyclohexane
cyclohexane
cyclohexane
cyclohexane
C16H18Cl2N2OS
C18H20Cl2N2OS
C19H22Cl2N2OS
C16H18F2N2OS
C18H20F2N2OS
C19H22F2N2OS
a
b
See Schemes 1 and 2. All compounds were analyzed for C, H, N, S and, when required, Cl and F. Analytical results were within
(0.4% of the theoretical values.
Ta ble 2. Physical and Chemical Data of Compounds 4-6
compd
Ar
R
Y
mp, °C
recryst solvent
synth schemea
% yield
formulab
C13H12O2
C9H8Cl2O2
C9H8F2O2
C14H14O2
C10H10Cl2O2
C10H10F2O2
C13H16O3
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
1-naphthyl
2,6-Cl2-Ph
2,6-F2-Ph
1-naphthyl
2,6-Cl2-Ph
2,6-F2-Ph
Ph
1-naphthyl
2,6-Cl2-Ph
2,6-F2-Ph
2,6-F2-Ph
Ph
1-naphthyl
2,6-Cl2-Ph
2,6-F2-Ph
Ph
1-naphthyl
2,6-Cl2-Ph
2,6-F2-Ph
2,6-F2-Ph
Ph
Me
Me
Me
Et
Et
Et
Me
Me
Me
Me
Me
Et
Et
Et
Et
Me
Me
Me
Me
Me
Et
142-143
124-125
oil
86-87
131-132
oil
oil
oil
oil
oil
cyclohexane
cyclohexane
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
c
c
c
c
c
c
c
c
c
86
68
65
100
59
47
88
98
86
90
74
94
94
89
87
64
41
54
59
40
48
42
61
42
n-hexane
cyclohexane
H
H
H
H
Me
H
H
H
H
H
H
H
H
Me
H
H
H
H
C17H18O3
C13H14Cl2O3
C13H14F2O3
C14H16F2O3
C14H18O3
oil
oil
oil
oil
C18H20O3
C14H16Cl2O3
C14H16F2O3
C12H12N2OS
oil
205-206
229-230
252-253
267-268
267-269
242-243
244-246
222-223
202-203
ethanol
acetonitrile
ethanol
ethanol
acetonitrile
ethanol
ethanol/water
ethanol
diethyl ether
C16H14N2OS
C12H10Cl2N2OS
C12H10F2N2OS
C13H12F2N2OS
C
C
13H14N2OS
17H16N2OS
6g
6h
6i
1-naphthyl
2,6-Cl2-Ph
2,6-F2-Ph
Et
Et
Et
C13H12Cl2N2OS
C13H12F2N2OS
a
b
See schemes 1. All compounds were analyzed for C, H, and, when required, N, S, Cl, and F. Analytical results were within (0.4%
of the theoretical values.
HIV-1-induced cytopathicity in MT-4 cells and inhibiting
wild-type RT, thus confirming the rationale leading to
their synthesis. The significant improvement of potency
associated with two methyl groups, one at the benzylic
carbon and the other at the pyrimidine 5-position, was
related to an intramolecular steric effect; in the R
enantiomer the conformational space is restricted to a
few low-energy conformations including the one recog-

2550 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Ta ble 3. Cytotoxicity and Anti-HIV-1 Activity of Compounds 3a
Mai et al.
[µM]
b
c
d
compd
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
Ar
R
Y
X
CC50
EC50
IC50
SIe
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
i-Pr
s-Bu
>200f
200
159
149
119
93
0.9
0.23
0.6
0.6
1.1
0.5
0.14
1.2
1.0
2.9
0.18
0.05
0.07
0.05
0.03
0.03
0.16
0.04
2.3
>222
870
333
248
108
186
321.4
97.5
78.3
>69
115
3340
2857
NDg
2.1
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
Me
ND
5.8
0.4
1-naphthyl
1-naphthyl
1-naphthyl
2,6-Cl2-Ph
2,6-Cl2-Ph
2,6-Cl2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
Ph
45
117
2.6
ND
ND
ND
0.2
ND
ND
ND
ND
0.05
ND
0.02
78.3
>200
20.8
i-Pr
n-Bu
i-Bu
s-Bu
167
>200
>200
>200
>200
>200
>200
200
>4000
>6666
>6666
>1250
>5000
>28570
6666
14000
>20000
>33333
>100000
>286
>11111
250
cyclopentyl
cyclohexyl
Me
3q
3r
3s
3t
3u
3v
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
i-Pr
s-Bu
n-Bu
i-Bu
0.007
0.03
0.008
0.01
0.006
0.002
0.07
0.018
0.8
0.019
0.02
ND
>200
112
>200
>200
>200
200
>200
>200
>200
50
ND
3w
[+]-3w h
[-]-3w h
3x
3a ′
3b′
3c′
3d ′
3e′
3f′
3g′
3h ′
3i′
3j′
3k ′
3l′
cyclopentyl
cyclopentyl
cyclopentyl
cyclohexyl
i-Pr
0.005
0.008
1.0
ND
2.3
ND
>10
3.7
0.8
ND
1.4
ND
ND
ND
ND
ND
ND
0.05
0.05
0.08
0.05
0.05
0.08
0.04
0.3
Ph
Ph
Ph
s-Bu
0.4
1.0
1.3
1.5
3.0
0.18
0.4
1.0
3.6
0.08
0.15
0.05
0.05
0.04
0.08
0.05
0.05
0.08
0.03
0.3
>500
>200
>154
33.3
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
cyclopentyl
cyclohexyl
i-Pr
51
1-naphthyl
1-naphthyl
1-naphthyl
2,6-Cl2-Ph
2,6-Cl2-Ph
2,6-Cl2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
16.9
>200
23.4
17
94
>200
>500
70
23.4
200
130
>55.5
875
1333
>200
>200
>200
>200
>200
>200
200
>200
200
>200
3m ′
2a i
2b
2c
2d
2600
>4000
>5000
>2500
>4000
>4000
>2500
6666
s-Bu
cyclopentyl
i-Pr
2e
2f
s-Bu
cyclopentyl
MKC-422
nevirapine
>666
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 detemined 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 for crystallization of compounds in the culture medium
Not
g
h
i
determined. [+] and [-] are referred to CDC values recorded at 244 nm (ref 37). Reference 32.
Ta ble 4. Anti-HIV-1 Activity of F2-S-DABOs 2 and 3 as
Determined by Reduction of p24 Levels
Ta ble 5. Activity of Selected F2-S-DABOs 2 and 3 against
Y181C HIV-1 Mutant Strain
[µM]
EC50, µMa
(Y181C/
b
a
a
a
compd
R
Y
X
WTIIIB Y181C WTIIIB)
compd
2a
2c
2d
2f
3l
3p
R
Y
X
EC90
0.042
cyclopentyl 0.080
i-Pr 0.026
cyclopentyl 0.040
Me i-Pr 0.042
Me cyclopentyl 0.015
EC99
EC99.9
2f
3r
3t
Me
cyclopentyl 0.04
1.2
g10
0.3
>30
0.025
(30)
(g270)
(15)
(>81)
(6)
H
H
Me
Me
H
i-Pr
0.083
0.150
0.065
0.075
0.083
0.035
0.015
0.035
0.150
0.041
0.190
0.300
0.150
0.150
0.190
0.090
0.033
0.090
0.300
0.082
Me Me Me
Me Me s-Bu
0.037
0.02
0.37
nevirapine
efavirenz
0.004
H
a
Compound dose required to achieve 50% protection of MT-4
cells for wild-type and Y181C HIV-1-induced cytopathogenicity,
as determined by the MTT method. Fold resistance.
3w
3k ′
3l′
MKC-442
Me Me cyclopentyl 0.006
H
H
Et
Et
i-Pr
0.015
b
cyclopentyl 0.080
0.014
tiomer (-)-3w , exhibited the highest activity and se-
lectivity among the so-far reported S-DABOs (CC50
200 µM, EC50 ) 2 nM, IC50 ) 8 nM, SI > 100 000) under
conditions wherein the MKC-442 was less active and
selective (CC50 > 200 µM, EC50 ) 30 nM, IC50 ) 40 nM,
a
Compound dose required to reduce p24 antigen levels by 90%
(EC90), 99% (EC99), and 99.9% (EC99.9).
>
nized by RT. Compound (+)-3w , significantly more
active and less cytotoxic than the corresponding enan-

Non-nucleoside Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2551
3H, OCH2CH3), 1.79-1.97 (m, 1H, CHCH2CH3), 2.19-2.36 (m,
1H, CHCH2CH3), 3.24-3.27 (d, 2H, COCH2CO), 3.97-4.07 (q,
2H, OCH2CH3), 4.43-4.50 (t, 1H, CHCH2CH3), 7.24-7.34 (m,
1H, C-2 naphthyl-H), 7.39-7.58 (m, 3H, C-3,6,7 naphthyl-H),
7.76-7.89 (m, 2H, C-4,8 naphthyl-H), 8.05-8.09 (m, 1H, C-5
naphthyl-H). IR: 1725, 1695 cm-1. Anal. C, H.
SI > 6666). These results make compound (+)-3w an
interesting candidate for further biological and phar-
macological studies aimed at evaluating its effectiveness
as an anti-AIDS agent.
Exp er im en ta l Section
Syn th esis of 6. Exa m p le: 6-[1-(2,6-Dich lor op h en yl)-
eth yl]-3,4-d ih yd r o-2-th ioxop yr im id in -4(3H)-on e (6c). So-
dium metal (0.60 g, 26.2 mmol) was dissolved in 50 mL of
absolute ethanol, and thiourea (1.39 g, 18.2 mmol) and ethyl
4-(2,6-dichlorophenyl)-3-oxopentanoate (5c) (3.53 g, 13.1 mmol)
were added to the clear solution. The mixture was heated at
reflux for 5 h. After the mixture was cooled, the solvent was
distilled in vacuo at 40-50 °C until dry and the residue was
dissolved in a little water (20 mL) and made acidic with 2 N
HCl. The resulting precipitate was filtered under reduced
pressure, washed with diethyl ether, and vacuum-dried at 80
°C for 12 h to give 2.2 g of 6c as a pure solid. 1H NMR (DMSO-
d6): δ 1.46-1.50 (d, 2H, CH3), 4.54-4.57 (q, 1H, CH-Ar), 5.71
(s, 1H, C-5 H), 7.28-7.35 (m, 1H, C-4 Ar-H), 7.42-7.47 (m,
2H, C-3,5 Ar-H), 12.17 (s, 1H, NH exchanged with D2O), 12.36
(s, 1H, NH exchanged with D2O). IR: 3100, 1630 cm-1. Anal.
C, H, Cl, N, S.
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 310 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 standards meth-
ods. The concentrations of solutions after reactions and extrac-
tions 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. Arylacetic acids, N,N′-
carbonyldiimidazole, iodomethane, 2-iodopropane, 1-iodo-2-
methylpropane, 2-iodobutane, 1-iodobutane, cyclopentyl bro-
mide, and cyclohexyl iodide were purchased from Aldrich
Chimica, Milan (Italy), or Lancaster Synthesis GmbH, Milan
(Italy).
Syn th esis of 3 fr om 6. Exa m p le 1: 2-Cyclop en tylth io-
3,4-d ih yd r o-6-(1-p h en yl)eth ylp yr im id in -4(3H)-on e (3c). A
mixture of 3,4-dihydro-6-(1-phenylethyl)-2-thioxopyrimidin-
4(3H)-one (6a ) (0.15 g, 0.65 mmol), cyclopentyl bromide (0.11
g, 0.08 mL, 0.71 mmol), and potassium carbonate (0.09 g, 0.65
mmol) in 1 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 organic layers were collected, washed with brine (3 × 50
mL), dried, and evaporated to furnish crude 3c, which was
purified by chromatography on silica gel column (eluent:
Syn th eses. The specific examples presented below il-
lustrate general synthetic methods. As a rule, samples pre-
pared for physical (Tables 1 and 2) and biological studies
(Tables 3-5) were dried in high vacuum over P2O5 for 20 h at
temperatures ranging from 25 to 110 °C, depending on the
sample melting point.
1
n-hexane/ethyl acetate/methanol 12/3/1). H NMR (CDCl3): δ
Syn th esis of 4. Exa m p le: 2-(2,6-Diflu or op h en yl)p r o-
p ion ic Acid (4c). 2-(2,6-Difluorophenyl)acetic acid (3.00 g,
17.43 mmol) was added portionwise to a cooled (0 °C) 2.0 M
solution of LDA in heptane/THF/ethylbenzene (26.5 mL, 52.29
mmol), and then HMPA (4.57 mL, 26.14 mmol) and THF (10
mL) were added to the mixture. The resulting solution was
stirred at room temperature for 30 min, then it was recooled
(0 °C), treated with methyl iodide (3.72 g, 1.63 mL, 26.14
mmol), and stirred at room temperature for 5 min. After being
stirred, the clear solution was poured into cold water (200 mL)
and 2 N HCl (20 mL) and extracted with ethyl acetate (3 × 50
mL). The organic layers were collected, washed with brine (100
mL), dried, and evaporated to furnish a residue that was
purified by column chromatography (silica gel, ethyl acetate/
1.56-1.59 (d, 3H, CHCH3), 1.79-1.71 (m, 6H, C-2eq,5eq, C-3,
C-4 cyclopentane-H overlapped signal), 2.07-2.09 (m, 2H,
C-2ax,5ax cyclopentane-H), 3.82-3.93 (q, 1H, CHCH3), 3.93-
4.02 (m, 1H, SCH overlapped signal), 6.02 (s, 1H, C-5 H), 7.25-
7.27 (m, 5H, Ph), 13.00 (broad s, 1H, NH exchanged with D2O).
IR: 2950, 1630 cm-1. Anal. C, H, N, S.
Syn th esis of 3 fr om 6. Exa m p le 2: 2-Cycloh exylth io-
3,4-d ih yd r o-6-[1-(1-n a p h t h yl)p r op yl]p yr im id in -4(3H )-
on e (3g′). A mixture of 3,4-dihydro-6-[1-(1-naphthylpropyl)]-
2-thioxopyrimidin-4(3H)-one (6g) (0.50 g, 1.69 mmol), cyclohexyl
iodide (0.39 g, 0.24 mL, 1.86 mmol), and potassium carbonate
(0.23 g, 1.69 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 3g′, which
1
chloroform 1/1). H NMR (CDCl3): δ 1.49-1.52 (d, 3H, CH3),
4.07-4.18 (q, 1H, CH), 6.82-6.90 (m, 2H, C-3,5 Ar-H), 7.15-
7.24 (m, 1H, C-4 Ar-H), 7.95 (s, 1H, OH exchangeable with
D2O). IR: 3060, 1700 cm-1. Anal. C, H, F.
was purified by chromatography on
a silica gel column
(eluent: n-hexane/ethyl acetate/methanol, 12/3/1). 1H NMR
(CDCl3): δ 0.93-1.00 (t, 3H, CHCH2CH3), 1.28-1.63 (m, 6H,
C-3,4,5 cyclohexane-H), 1.63-1.70 (m, 2H, C-2eq,6eq cyclo-
hexane-H overlapped signal), 1.91-1.96 (m, 2H, C-2ax,6ax
cyclohexane-H), 2.06-2.24 (m, 1H, CHCH2CH3), 2.24-2.34 (m,
1H, CHCH2CH3), 3.72-3.76 (m, 1H, SCH), 4.39-4.46 (t, 1H,
CHCH2CH3), 6.11 (s, 1H, C-5 H), 7.42-7.50 (m, 4H, C-2,3,6,7
naphthyl-H), 7.69-7.73 (m, 1H, C-4 naphthyl-H), 7.79-7.84
(m, 1H, C-8 naphthyl-H), 8.07-8.11 (m, 1H, C-5 naphthyl-H),
Syn th esis of 5. Exa m p le: Eth yl 4-(1-Na p h th yl)-3-oxo-
h exa n oa te (5g). Triethylamine (11.6 mL, 83.2 mmol) and
magnesium dichloride (6.33 g, 66.5 mmol) were added to a
stirred suspension of potassium ethylmalonate (9.51 g, 55.8
mmol) in dry acetonitrile (100 mL), and stirring was continued
at room temperature for 2 h. Then, a solution of the 2-(1-
naphthyl)butanoic imidazolide in the same solvent (50 mL),
prepared 15 min before by reaction between 2-(1-naphthyl)-
butanoic acid (4d ) (5.70 g, 26.6 mmol) and N,N′-carbonyldi-
imidazole (5.17 g, 32.0 mmol) in acetonitrile (50 mL), was
added. The reaction mixture was stirred overnight at room
temperature and then was completed by heating at reflux for
2 h. After the mixture was cooled, 13% HCl (100 mL) was
cautiously added while the temperature was kept below 25
°C, and the resulting clear mixture was stirred for a further
15 min. The organic layer was separated and evaporated, and
the residue was treated with ethyl acetate (100 mL). The
aqueous layer was extracted with ethyl acetate (2 × 50 mL),
and the organic phases were collected, washed with sodium
hydrogen carbonate saturated solution (2 × 100 mL) and brine
(100 mL), dried, and concentrated to give pure 5g (7.08 g). 1H
NMR (CDCl3): δ 0.85-0.93 (t, 3H, CHCH2CH3), 1.10-1.17 (t,
12.59 (s, 1H, NH exchanged with D2O). IR: 2900, 1645 cm-1
Anal. C, H, N, S.
.
Syn th esis of 3 fr om 2. Exa m p le: 2-Cyclop en tylth io-6-
[1-(2,6-d iflu or op h e n yl)p r op yl]-3,4-d ih yd r op yr im id in -
4(3H)-on e (3l′). 2-Cyclopentylthio-6-(2,6-difluorobenzyl)-3,4-
dihydropyrimidin-4(3H)-one (2c)32 (0.70 g, 2.08 mmol) was
added portionwise to a cooled (0 °C) 2.0 M solution of LDA in
heptane/THF/ethylbenzene (6.3 mL, 12.48 mmol), and then
HMPA (0.55 mL, 3.12 mmol) and THF (10 mL) were added to
the mixture. The resulting solution was stirred at room
temperature for 30 min, then it was recooled (0 °C), rapidly
treated with ethyl iodide (0.48 g, 0.25 mL, 3.12 mmol), and
stirred at room temperature overnight. After the mixture

2552 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Mai et al.
was stirred, the clear solution was poured into cold water (200
mL) and 2 N HCl (20 mL) and extracted with ethyl acetate (3
× 50 mL). The organic layers were collected, washed with brine
(100 mL), dried, and evaporated to furnish a residue that was
purified by column chromatography (silica gel, ethyl acetate/
chloroform 1/1). 1H NMR (CDCl3): δ 0.87-0.94 (t, 3H,
CHCH2CH3), 1.25-1.84 (m, 8H, C-2eq,6eq,3,4,5 cyclohexane-
H), 1.95-2.10 (m, 3H, C-2ax,6ax cyclohexane-H and CHCH2-
CH3), 2.16-2.26 (m, 1H, CHCH2CH3), 3.63-3.67 (m, 1H, SCH),
4.06-4.14 (dd, 1H, CHCH2CH3), 6.13 (s, 1H, C-5 H), 6.77-
6.85 (t, 2H, C-3,5 Ar-H), 7.11-7.18 (m, 1H, C-4 Ar-H), 12.80
(s, 1H, NH exchanged with D2O). IR: 2800, 1645 cm-1. Anal.
C, H, F, N, S.
cally varied through 20° steps: τ1 within the 0-340° range
and τ2 within the 0-160° range owing to the equivalence of
the 2 and 6 positions of the 2,6-difluorophenyl ring. Each of
the generated conformations was energy-minimized without
perturbing the scanned torsion angles for a maximum of 15
iterations. The scatter plots in Figure 2 were obtained by
calculating the strain energy (Estrain) of each conformation as
the difference in energy with respect to the global minimum
conformation.
The structure of the non-nucleoside binding site (NNBS) of
RT was taken from the 2.5 Å resolution of the HIV-1 RT/TNK-
651 complex15 filed in the Brookhaven Protein Data Bank43
with the entry code 1RT2. The NNBS consisted of a set of 68
amino acids located within 15 Å from the non-hydrogen atoms
of the bound inhibitor. Water molecules were removed. Hy-
drogens were added to the unfilled valences of the protein. Lys,
Asp, and Glu side chains were modeled in their ionized forms.
The tautomeric states of His residues were assigned by visual
inspection. The starting geometry of the NNBS/(R)-3w complex
was modeled on the reported NNBS/2f complex.44 The NNBS
extracted from RT/TNK-651 was first aligned on the NNBS/
2f complex about the backbone atoms and then filled with (R)-
3w obtained by mutation of 2f.
Energy minimizations (EM) and molecular dynamics (MD)
calculations were based on the all-atom Cornell et al. force
field45 available within the Amber package, which was modi-
fied to include additional parameters for (R)-3w . Most of these
parameters were either adapted by analogy from others
included in the Amber force field or derived from the MM2
force field.46 Parameters related to the bond distances, bond
angles, and torsion angles involving the sulfur atom in the
ligand were taken from a parametrization of sulfur-containing
compounds.47,48 Partial atomic charges for the ligand was
calculated with the semiempirical quantum mechanics method
AM1,49 available in the Mopac program.50
An tivir a l Assa y P r oced u r es. Com p ou n d s. Compounds
were solubilized in DMSO at 200 mM and then diluted into a
culture medium.
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.
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.
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.
The geometry of the NNBS/(R)-3w complex was prelimi-
narly optimized by energy minimization with the Sander/
Amber module (50 000 steps; distance-dependent dielectric
function of ꢀ ) 4r), applying an energy penalty force constant
of 5 kcal/mol to the protein backbone atoms. The geometry-
optimized structures were then used as the starting point for
subsequent 500 ps MD simulation, during which the protein
backbone atoms were constrained as done in the previous step.
A time step of 1 fs and a nonbonded pairlist updated every 25
fs were used for the MD simulations. The temperature was
mantained at 300 K using the Berendsen algorithm51 with a
0.2 ps coupling constant. Four snapshots, extracted every 25
ps from the last 100 ps of MD simulation, were very similar
to one another in terms of root-mean-square deviation. An
average structure was calculated from the last 100 ps trajec-
tory and energy-minimized using the steepest descent and
conjugate gradient methods available within the Sander/
Amber to yield the NNBS/(R)-3w complex shown in Figure 3.
RT Assa ys. Assays were performed as previously de-
scribed.32
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.
Com p u ta tion a l Stu d ies. Computations were performed
using the software packages Sybyl40 and Amber35,36 running
on a Silicon Graphics R10000 workstation. Models of S-DABOs
were available from previous studies,32 directly or after
modifications according to the standard parameters of the
molecular mechanics Tripos force field.41 In each structure the
methyl group of the 2-methylthio side chain and the pyrimi-
dine N1 were oriented in a syn disposition. Geometry optimi-
zations were realized with the Sybyl/Maximin2 minimizer
selecting the BFGS (Broyden, Fletcher, Goldfarb, and Shanno)
algorithm42 and a root-mean-square gradient of the forces
acting on each atom of 0.05 kcal mol-1 Å-1 as the convergence
criterion. Conformational analyses on the S-DABOs in Figure
2 were carried out using the Sybyl/Gridsearch routine. The
torsion angles τ1 and τ2 (defined in Figure 2) were systemati-
Ack n ow led gm en t. The authors thank Italian Min-
istero della Sanita`sIstituto Superiore di Sanita`sIX
Progetto AIDS 1996 (Grants 40A.0.06 and 9403-59) for
financial aid. Acknowledgments are due to Italian
MURST (40% funds) and Regione Autonoma Sardegna
(Assessorato Sanita`) for partial support.
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