Parallel solution-phase and microwave-assisted synthesis of new S-DABO derivatives endowed with subnanomolar anti-HIV-1 activity

Article abstract of DOI:10.1021/jm050744t

A simple and efficient methodology for the parallel solution-phase synthesis has been set up to obtain a series of thiouracils, in turn selectively S-benzylated under microwave irradiation to give new S-DABOs. Biological screening led to the identificatio

Full text of DOI:10.1021/jm050744t

8000
J. Med. Chem. 2005, 48, 8000-8008
Parallel Solution-Phase and Microwave-Assisted Synthesis of New S-DABO
Derivatives Endowed with Subnanomolar Anti-HIV-1 Activity
Fabrizio Manetti,† Jose´ A. Este´,‡ Imma Clotet-Codina,‡ Mercedes Armand-Ugo´n,‡ Giovanni Maga,§
Emmanuele Crespan,§ Reynel Cancio,§ Claudia Mugnaini,† Cesare Bernardini,† Andrea Togninelli,†
Caterina Carmi,| Maddalena Alongi,† Elena Petricci,† Silvio Massa,† Federico Corelli,† and Maurizio Botta*,†
Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy,
Retrovirology Laboratory irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Auto´noma de Barcelona,
E-08916 Badalona, Spain, Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy, and
Dipartimento Farmaceutico, Universita` degli Studi di Parma, Parco Area delle Scienze 27/A, I-43100 Parma, Italy
Received July 29, 2005
A simple and efficient methodology for the parallel solution-phase synthesis has been set up
to obtain a series of thiouracils, in turn selectively S-benzylated under microwave irradiation
to give new S-DABOs. Biological screening led to the identification of compounds with
nanomolar activity toward both the highly purified recombinant human immunodeficiency virus
type 1 (HIV-1) reverse transcriptase (RT) enzyme (wild-type and mutants) and wild-type (wt)
and mutant HIV-1 strains. In particular, 20 was found to be the most potent S-DABO reported
so far (ID50 ) 26 nM toward the isolated wt enzyme) with subnanomolar activity toward both
the wt and the pluriresistant virus (IRLL98) HIV-1 strain (EC50 < 0.14 nM and EC50 ) 0.22
nM, respectively). Molecular modeling calculations were also performed to investigate the
binding mode of such compounds onto the non-nucleoside reverse transcriptase inhibitor binding
site and to rationalize the relationships between their chemical structure and activity values
toward wt RT.
Introduction
portion of it is formed by amino acid residues belonging
to the p51 subunit.8,14-21 During the process of inhibitor
binding, significant conformational changes occur in the
orientation of the side chains of some residues (particu-
larly Tyr181 and Tyr188), leading to the formation of
the NNIBP accommodating the inhibitors. It is evident
from a comparison of the various RT structures that the
NNIBP has a very flexible structure and this charac-
teristic allows the enzyme to accommodate structurally
diverse inhibitors that have different shapes and sizes.22
Although NNRTIs generally exhibit low toxicity and
favorable pharmacokinetic properties, the rapid replica-
tion of HIV and its inherent variability often lead to the
generation of drug-resistant variants responsible for the
clinical NNRTI treatment failure.23-24
The current therapy against the human immunode-
ficiency virus type 1 (HIV-1), the causative agent of
acquired immunodeficiency sindrome (AIDS), is based
on four classes of drugs: nucleoside reverse tran-
scriptase inhibitors (NRTIs), non-nucleoside reverse
transcriptase inhibitors (NNRTIs), protease inhibitors
(PIs), and fusion inhibitor enfuvirtide. Although NRTIs
are equally active against HIV-1 and HIV-2 reverse
transcriptase (RT), acting at the catalytic site as DNA
chain terminators,1 NNRTIs are highly specific for
HIV-1 and include more than 30 structurally different
classes of molecules, such as nevirapine,2 TIBO,3 BHAP,4
R-APA,5 PETT,6 HEPT,7 TNK-651,8 ITU,9 DATA,10 and
DAPY11-13 (Chart 1).
Among NNRTIs, dihydro-alkoxy-benzyl-oxopyrim-
idines (DABOs) are an interesting class of compounds
active at nanomolar concentration; they were first
disclosed in 199225 and further developed during the
following years into S-DABOs (Chart 1) and related
analogues. Recently, we identified compounds 1 and 2
as byproducts of cleavage of thiopyrimidinones from
Wang resin (Chart 1).26 Considering their structural
similarity to known S-DABOs, we tested these com-
pounds as possible NNRTIs, and despite their moderate
activity (IC50 ) 400 µM), we became interested in
investigating the influence of the arylalkyl moiety on
anti-HIV activity because only scattered examples of
S-DABOs having this kind of substitution at position 2
had been reported in the literature. Herein, we describe
the microwave-assisted synthesis of new S-DABO de-
rivatives 3-30, endowed with anti-HIV activity at
nanomolar or picomolar concentrations on cell lines
infected with either wild-type or mutated HIV-1.
The HIV-1 RT is a multifunctional enzyme, consisting
of subunits p66 and p51, responsible for the conversion
of the single-stranded RNA viral genome into double-
stranded DNA. Lacking a biological counterpart in the
eukaryotic systems, RT is an attractive target for the
development of selective inhibitors.
Studies carried out on crystal structures of different
RT/NNRTI complexes suggest that NNRTIs share a
common mode of action, binding the RT enzyme at an
allosteric site corresponding to a hydrophobic pocket in
p66, called the non-nucleoside inhibitor binding pocket
(NNIBP). The NNIBP is located about 10 Å from the
catalytic site in the p66 subunit, and only a small
* Author to whom correspondence should be addressed. Phone: +39
0577 234306. Fax: +39 0577 2343330. E-mail: botta@unisi.it.
† Universita` degli Studi di Siena.
‡ Universitat Auto´noma de Barcelona.
§ Istituto di Genetica Molecolare.
| On leave from the Universita` degli Studi di Parma.
10.1021/jm050744t CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/11/2005

S-DABO Derivatives with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8001
Chart 1. Structure of Known NNRTIs
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) a. MgCl2, Et3 N, CH3CN, rt, 360
rpm, 2 h; b. substituted phenylacetic acid, N,N′-carbonyldiimida-
zole, rt, 360 rpm, overnight then reflux, 360 rpm, 2 h; c. 13% HCl,
rt, 360 rpm, 10 min; (ii) thiourea, EtONa, reflux, 360 rpm,
overnight.
In particular, starting from our lead compounds 1 and
2, we planned to design a number of S-DABO deriva-
tives characterized by the presence of (i) an arylalkylthio
substituent at position 2 that represents the focus of
our investigations. To expand the structure-activity
relationships (SARs) of S-DABOs, the substitution
pattern on the phenyl ring was broadly varied, as well
as the length of the alkyl spacer, which was increased
from one to three carbon atoms, (ii) a 5-methyl group,
which was kept fixed in all the molecules, as a confor-
mational constraint with the aim of enhancing the
affinity for the enzyme, (iii) a halogenated benzyl group
at the 6 position, which 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 NNBP of the
enzyme.27
a Reagents and conditions: (i) substituted benzyl halide, DMF,
MW, 130 °C, 5 min (method A) or substituted benzyl alcohol,
trimethylphosphine, DIAD, DMF, MW, 40 °C, 10 min (method B);
(ii) MCPBA, CH2Cl2, rt, 360 rpm, overnight.
tion vessels and reacted with three substituted pheny-
lacetyl imidazolides in the presence of a magnesium
dichloride/triethylamine system in acetonitrile according
to the Clay procedure29 to give, after a simple liquid-
phase extractive purification, the â-keto esters 35-37.
Subsequent condensation of 35-37 with thiourea in the
presence of sodium ethoxide in refluxing ethanol af-
forded 6-benzyl-2,3-dihydro-5-methyl-2-thioxopyrimidin-
4(1H)-ones (31-33).
Exploitation of this procedure might allow the rapid
synthesis of a large number of S-DABO derivatives
variously substituted at positions 2, 5, and 6 for further
SAR studies in this class of compounds.
5,6-Disubstituted thiouracils 31-33 were selectively
S-benzylated under microwave irradiation with the
appropriate substituted benzyl halide in dry DMF in the
presence or not of potassium carbonate (Method A)
(Scheme 2).30 Alternatively, 31-33 were S-benzylated
using the appropriate benzyl alcohol via a microwave-
assisted Mitsunobu reaction in the presence of trimeth-
ylphosphine and diisopropylazodicarboxylate (DIAD) in
Chemistry
As an extension of our ongoing efforts toward the
development of new methodologies for the synthesis of
pyrimidine and pyrimidinone derivatives28 and in view
of a more extensive functionalization of the positions 5
and 6 of the thiopyrimidinone scaffold, we have set up
a simple and efficient methodology for the parallel
solution-phase synthesis of thiouracils 31-33 using a
Bu¨chi Syncore synthesizer (Scheme 1). Potassium ethyl
2-methylmalonate (34) was partitioned into three reac-

8002 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Table 1. Chemical and Physical Data of Derivatives 3-30 and 38-40
Manetti et al.
cmpd
R1
R2
n
mp (°C)
recryst solvent
yield (%)
method of synthesis
3
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diF
2,6-diF
2,6-diF
2,6-diF
2,6-diF
4-F
H
4-F
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
1
1
1
1
1
1
1
1
1
1
1
1
236-237
205-206
214
CH3CN
CH3CN
50
50
65
67
64
72
73
79
60
78
55
50
51
77
72
65
50
53
67
52
49
57
40
59
56
63
58
69
71
55
50
A
A
A
A
B
A
B
B
A
A
A
A
A
A
B
B
A
B
B
A
A
A
A
A
A
A
B
B
4
5
4-Cl
CH3CN
6
4-Br
4-OH
219
CH3CN
7
204-206
197-199
218-221
201-205
221-223
232-235
231-232
238-239
232
CH3CN
8
4-OCH3
4-OCH2CH3
4-OBu
4-iPr
CH3CN
9
CH3CN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38
39
40
CH3CN
CH3CN
4-CN
CH3CN
4-NO2
CH3CN
3-F
CH3CN
3-Cl
CH3CN
3-CN
203
CH3CN
2,4-diOCH3
3,4-diOCH3
3,5-diOCH3
4-OCH3
4-OCH3
4-OCH3
4-iPr
217-219
195-196
246-247
182-183
180-182
170-171
179-181
247-248
248-250
228-230
246
CH3OH/CHCl3
CH3OH/CHCl3
CH2Cl2
CH3CN
CH3CN
CH3CN
CH3OH/CH3CN
CH3CN
4-CN
4-NO2
3,5-diOCH3
4-NO2
4-OCH3
CH3CN
CH3CN
CH3CN
4-F
190
192-194
>250
171-174
175-180
241-244
CH3CN
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
CH3CN/CH2Cl2
CH3OH/CHCl3
CH3CN
4-OCH3
4-NO2
4-Br
CH3CN
CH3CN
dry DMF (Method B). In both cases, the use of micro-
waves allowed us to obtain, in a few minutes, the
desired products in high yield and good purity.
Finally, sulfides 6, 8, and 13 were oxidized to sulfones
38-40 with m-chloroperbenzoic acid in dichloromethane
at room temperature.31
All of the compounds were obtained in more than 95%
purity, as shown by HPLC-MS analysis. Chemical and
physical data of the new compounds are reported in
Table 1.
activity of a 2,6-difluorobenzyl substituent at position
6,32 in our case this type of substitution proved to be
less profitable with respect to the corresponding 2,6-
dichlorobenzyl group. In fact, although compound 8 was
highly active both in enzymatic and cell tests, showing
appreciable activity also against IRLL98, compound 22
retained activity only against the wt RT. Moreover, a
striking difference in activity can be observed between
that of 13 (displaying a full-range activity at low
concentrations so as to emerge as one of the most
interesting compounds of the series) and that of 25,
whose activity was limited to wt virus and unrelated to
RT inhibiting properties. In line with these observa-
tions, the presence of a 6-(4-fluorobenzyl) group led to
the substantially inactive compounds 27 and 28.
On the basis of these results, we kept the 6-(2,6-
dichlorobenzyl) substituent fixed and systematically
modified the C-2 position. The 4-hydroxybenzyl group,
found in our lead compounds 1 and 2 and also present
in compound 7, did not positively contribute to the
antiviral activity. Replacement of the OH group with
H, F, Cl, Br, iPr, and CN improved the general biological
profile (compounds 3-6, and 12), with activity ranging
from 0.39 to 103 µM in enzymatic tests, and from 0.11
to 0.83 µM on cells infected with wt virus. No interesting
activity was observed on mutant strains, with the
exception of 12, which retained activity on clinically
relevant mutants at a micromolar or submicromolar
concentration. Moving the substituent (F, Cl, CN) from
a para to a meta position on the aromatic ring did not
modify the biological response to the molecules (14-
16). On the other hand, replacement of the OH group
with NO2 led to the very interesting compound 13 (vide
supra), whereas substitution of the OH group with
alkoxy groups gave compounds 8-10, whose activities
were strongly dependent on the length of the alkyl
Results and Discussion
Title compounds 3-30 and 38-40, a new family of
S-DABOs characterized by the presence of an arylalky-
lthio substitution at C-2, a methyl group at C-5, and a
halogenated benzyl group at C-6 of the pyrimidinone
nucleus, were prepared in a straightforward fashion by
alkylation of three different 6-substituted 2-thiouracils
(31-33) and subsequent oxidation of the sulfur atom
(38-40).
The new compounds were evaluated in enzymatic
tests for their ability to inhibit either wild-type (wt) or
mutated RTs as well as on MT-4 cells for cytotoxicity
and anti-HIV-activity, in comparison with nevirapine
and efavirenz, used as reference drugs. In particular,
the following mutants were used: K103N and Y181I for
enzymatic tests, K103N, Y188L, and pluriresistant virus
(IRLL98) (bearing the K101Q, Y181C, and G190A
mutations conferring resistance to nevirapine, delavir-
dine, and efavirenz, respectively) for tests on cell lines.
The results of these assays are reported in Table 2.
Moreover, Table 3 reports fold-resistance values of
selected compounds against a panel of mutant strains,
in comparison to those of AZT, nevirapine, and efavirenz.
Although previous findings obtained with other S-
DABO series highlighted the importance for optimal

S-DABO Derivatives with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8003
Table 2. Anti HIV-1 Activity and Cytotoxicity of Compounds 3-30 and 38-40
ID50 (µM)a,b
EC50 (µM)a,c
IRLL98
f
cmpd
wt
K103N
Y181I
NL4-3 wt
K103N
Y188L
CC50
3
2.5
16
350
na
na
9
na
102
na
na
na
na
0.12
35
400
na
na
na
na
3.2
0.5
3.5
na
na
na
na
na
na
na
na
10
nad
na
na
na
na
150
na
na
na
na
0.22
na
400
na
na
na
na
1.5
0.3
na
na
na
na
na
na
na
na
na
27
0.83
0.11
>64
2.7
>64
27
>59
>53
>41
4.7
>58
>3.1
>58
0.10
11
>61
>59
>60
>55
10
>55
0.87
>1.0
0.69
33% at 62
>65
>62
>60
>65
>8.9
5.3
>48
>2.8
>53
>50
3.9
>64
>61
>59
>53
>41
>32
>58
>3.1
>58
>60
31%
>61
>59
>60
>55
>55
>55
6.9
>64
>61
>59
13
4
5
20
0.47
2.1
6
0.39
na
0.004
2
2.8
na
0.79
>41
0.007
0.25
>53
>41
0.047
1.9
7
41
8
32
>58
3.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38
39
40
>3.1
35% at 2.3
0.17
34% at 11e
>57
1.0
45% at 11
>60
>58
>61
>59
>60
>55
>55
>55
>57
1.0
>64
>62
>65
>62
>60
>65
8.9
>57
>48
2.8
>53
>50
>7.5
>0.32
3.7
103
0.009
2.0
0.79
3.3
0.15
2.2
2.3
0.31
4.8
9.5
0.89
5.0
25
1.33
>55
0.93
>55
0.00022
0.11
0.69
16
1.2
0.20
256
0.026
0.037
0.007
3
33
<0.00014
0.00044
0.026
0.58
>1.0
10
>62
>65
>62
>60
>65
>8.9
33
0.76
na
0.052
0.074
18
>65
>62
>60
>65
>8.9
0.51
>48
>2.8
>53
>50
>7.5
0.2
na
na
>65
na
>8.9
0.12
0.5
3.5
13
>48
>2.8
>53
>50
>7.5
0.3
0.006
na
>2.8
>53
na
na
8
na
na
20
na
>50
nevirapine
efavirenz
AZT
0.4
0.052
0.001
0.003
0.04
0.4
0.1
0.057
0.003
0.008
0.003
a Data represent mean values of at least two experiments. b ID50: Inhibiting dose 50 or needed dose to inhibit 50% of enzyme. c EC50
:
Effective concentration 50 or needed concentration to inhibit 50% HIV-induced cell death, evaluated with MTT method in MT-4 cells.
d na: Not active at 400 µM (the highest concentration tested). e Percent inhibition of HIV-induced cell death at the reported micromolar
concentration. f CC50: Cytotoxic concentration 50 or needed concentration to induce 50% death of noninfected cells evaluated with the
MTT method in MT-4 cells.
Table 3. Fold Resistance Values of Selected Compounds
Against a Panel of Mutant Strains, in Comparison to Those of
AZT, Nevirapine, and Efavirenz
antiviral activity was the presence of a bulky group on
the aryl moiety, as in compounds 11, 23, and 30.
A very marked improvement of the biological profile
was obtained by increasing the length of the linker
connecting the aromatic ring to the sulfur atom. Com-
pounds 20 and 21 (n ) 2 and 3, respectively) proved to
be the most interesting among the new derivatives. In
particular, 20 exhibited anti-HIV activity on cells
infected with either wild-type or pluriresistant virus
(IRLL98) at subnanomolar concentration, together with
low cytotoxicity (SI > 410 000). On the whole, this
compound showed an anti-HIV profile superior to that
of nevirapine and comparable to that of efavirenz, thus
emerging as the most active S-DABO analogue reported
so far. Further lengthening of the linker led to com-
pound 21 that, though retaining very good anti-HIV
activity, was endowed with higher cytotoxicity. Finally,
basically inactive compounds (38-40) were obtained by
oxidation of the sulfur atom.
Biological data showed some contradictions between
ID50 and EC50 values for several compounds, probably
due to the fact that cellular tests reflect the interference
of a compound in viral steps not shown in the pure RT
enzymatic tests (i.e., enzymatic tests account only for
the DNA-RNA-dependent synthesis inhibition, whereas
cellular test results represent the inhibition of all viral
replication phases). Moreover, we cannot exclude the
possibility that the low cellular activity or the inactivity
fold resistance
EC50 (µM)
toward NL4-3 wt
cmpd
IRLL98
K103N
Y188L
8
0.007
0.17
7
6
3.31
5
4.6
20.08
4.4
3
144
300
670
0.5
3000
>60.1
31%
>67.5
>276
302.71
283.5
1
12
13
0.79
10.64
>67.5
51.2
20.63
45.5
1
16
0.89
0.20
0.026
0.12
0.003
0.052
0.001
18
22
29
AZT
NVP
EVZ
70
80
144
400
moiety. Thus, whereas 9 and 10 (p-ethoxy and p-butoxy
groups, respectively) were only moderately active on the
wt, compound 8 (p-methoxy) was endowed with very
significant activity in the nanomolar range toward both
wt and IRLL98 strains, as well as with low toxicity (SI
> 4600). The contemporary presence on the aromatic
ring of more than one methoxy group negatively affected
the biological activity. In particular, although a modest
effect on wt RT can still be ascribed to compounds 17,
18, and 29 (having a substituent at the para position),
no interesting activity was demonstrated by 19 and 26
(R ) 3,5-dimethoxyphenyl). Even more detrimental to

8004 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Manetti et al.
Figure 1. Stereographic (A, the inhibitor shown in ball-and-stick notation) and schematic (B, the inhibitor drawn with thick
lines) representations of the orientation mode of compound 8 into the NNIBD. The terminal methyl group of the side chain at
position 2 contacts both Pro225 and Pro236 at the surface of RT. Moreover, the 3-NH group of the pyrimidinone ring is involved
in a hydrogen-bond interaction with the carbonyl oxygen of Lys101. Finally, the benzyl chain at position 6 is accommodated
within a large pocket mainly defined by the hydrophobic side chains of Leu100, Trp229, Phe227, Tyr181, Tyr183, and Tyr188.
For sake of clarity, only representative amino acids are shown.
of some of our compounds could be the consequence of
poor adsorption through the T-cell membrane, in addi-
tion to a poor affinity for the corresponding receptor
counterpart.
toward the solvent-accessible surface defined by Pro225
and Pro236. In further detail, although the sulfur atom
of 8 was located at a distance of about 3.7 Å from the
backbone-NH group of Lys101, the benzyl moiety was
accommodated into a large pocket mainly defined by
Val106, Pro225, Pro236, and Tyr318 (Figure 1). Good
profitable hydrophobic interactions were found between
the alkyl side chain of Val106 and the phenyl ring of
the inhibitor as well as between the terminal methyl
group with both Pro225 and Pro236. Finally, the benzyl
substituent at position 6 of 8 was embedded into an
extended hydrophobic region defined by the aromatic
side chains of Tyr181, Tyr188, Phe227, and Trp229 as
well as by Leu100 and Leu234. The major difference
between the experimental and calculated complexes
involved the interaction between the 6-benzyl side chain
of the inhibitors and the side chain of Trp229. In fact,
although a T-tilted interaction was shown in the com-
plex with MKC-442, a π-π interaction was found in the
complex with 8. This was mainly due to the fact that a
Molecular Modeling Calculations
To investigate the binding mode of the new RT
inhibitors, molecular-docking simulations were per-
formed by means of the software Autodock. Results
showed 8 assuming an orientation very similar to that
of the cocrystallized inhibitor (MKC-442, emivirine). In
particular, the pyrimidinone ring of 8 was superposable
to that of MKC-442, allowing for a hydrogen-bond
contact between its NH group at position 3 with the
carbonyl moiety of Lys101 (1.9 Å), suggested to be a
crucial interaction key for inhibitors belonging to the
DABO and S-DABO classes of compounds.32 Moreover,
the side chains of both Lys103 and Val106 were also
found at close contact with the pyrimidinone nucleus.
The extended side chains at position 2 of both 8 and
MKC-442 were lined up in a parallel way and pointed

S-DABO Derivatives with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8005
significant conformational rearrangement occurred within
the aromatic cage accommodating the substituent at
position 6. In fact, the aromatic side chains of Tyr183,
Tyr188, and Trp229 approached the inhibitor, leading
to a structural system where the indole nucleus was
sandwiched between the 6-benzyl group of the inhibitor
and the phenyl ring of Tyr183 (allowing π-π interac-
tions) and interacted with a T-tilted contact with the
aromatic moiety of Tyr188. On the other hand, although
Phe227 retained its original position, a rotation of the
Tyr181 side chain occurred that pushed it about 1.8 Å
away from the NNIBP.
Similar results were found for the nitro derivative 13,
with a major difference involving the benzylthio side
chain that was located in a region of space comprised
between the corresponding side chain of 8 and the N1
side chain of the crystallized inhibitor. In particular, due
to a rotation of about 20° around the C2-S bond, the
nitro group was inserted between Pro225 and the side
chain of Phe227, and one of its electron-rich oxygen
atoms was found at the proper distance (1.6 Å) to make
a hydrogen-bond contact with the backbone-NH group
of Phe227.
Lengthening the benzyl moiety of 8 to a phenylethyl
(20) and phenylpropyl (21) chain led their p-methoxy
group to go beyond the opening defined by Pro225 and
Pro236 and, thus, to be exposed to the solvent. More-
over, such a shift of the phenyl ring caused the lack of
its hydrophobic interactions with the side chain of
Val106. The pyrimidinone nucleus of 21 maintained an
orientation comparable to that of 8, including the
hydrogen-bond contact with Lys101 as well as the
hydrophobic interactions with a part of the Val106 side
chain. Similarly, no substantial difference was found for
the location of the benzyl substituent at position 6, in
comparison to that of 8 and the crystallized inhibitor.
On the other hand, the 6-benzylpyrimidinone system
of 20 underwent a significant conformational rearrange-
ment, allowing for a hydrogen-bond contact involving
the carbonyl oxygen and the backbone-NH group of
Lys101, in addition to the hydrogen-bond contact be-
tween the 3-NH group of the inhibitor and the carbonyl
of the same amino acid. Moreover, due to the fact that
the benzyl group at the position 6 was characterized by
an alternative orientation into the hydrophobic cage
previously described, the profitable aromatic-aromatic
interaction involving the side chain of Trp229 was lost.
In summary, docking calculations suggested that the
p-methoxybenzylthio group at position 2 of 8 seems to
be characterized by the optimal length to fulfill the
tunnel delimited, at the surface of the RT structure, by
Pro225 and Pro236. Moreover, the lower activity of 20
and 21 toward the wt RT, with respect to that of their
shorter analogue 8, was in part due to reduced hydro-
phobic interactions mainly involving the benzyl sub-
stituents at position 2 as well as to unfavorable contacts
between the terminal methyl substituent and the sol-
vent. In a similar way, analogues of 8, bearing at the
para position of the 2-benzyl substituent a hydrophobic
group larger than a methoxy moiety (exceeding the
surface of the RT and contacting the solvent), showed
lower activity than 8. In fact, the ethoxy, butoxy, and
isopropyl derivatives (9, 10, and 11) showed activity in
the micromolar range or lower.
Conclusions
Parallel solution-phase and microwave-assisted syn-
thesis was applied to the synthesis of new S-DABO
derivatives having a 2-phenylalkylthio side chain of
variable length, characterized by different substituents
and substitution patterns on the phenyl ring. Results
from anti-HIV reverse transcriptase assays and anti-
HIV activity in lymphoid cells showed activity values
in the nanomolar and subnanomolar range, respectively.
Moreover, one of the new compounds (20) was charac-
terized by an anti-HIV profile better than that of
nevirapine and comparable to that of efavirenz, thus
emerging as the most active S-DABO analogue reported
so far.
Experimental Section
Chemistry. Reagents were obtained from commercial sup-
pliers and used without further purifications. According to
standard procedures, CH2Cl2 was dried over calcium hydride,
MeOH and EtOH were dried over Mg/I2 prior to use, and DMF
was bought already anhydrous. Anhydrous reactions were run
under a positive pressure of dry N2 or argon. IR spectra were
recorded on a Perkin-Elmer BX FT-IR system using CHCl3 as
the solvent. TLC was carried out using Merck TLC plates
Kieselgel 60 F254. Chromatographic purifications were per-
formed on columns packed with Merck 60 silica gel, 23-400
mesh, for flash technique. Melting points were taken using a
Gallenkamp melting point apparatus and are uncorrected. 1H
NMR spectra were recorded with a Bruker AC200F spectrom-
eter at 200 MHz, and chemical shifts are reported in δ values,
relative to TMS at δ 0.00 ppm. CD3OD, DMSO-d6, and CD3Cl
were used as solvents. Buchi Syncore polyvap was used for
parallel synthesis, filtration, and evaporation.
HPLC and MS Analysis. The purity of the compounds was
assessed by reversed-phase liquid chromatography and mass
spectrometry (Agilent series 1100 LC/MSD) with a UV detector
at λ ) 254 nm and an electrospray ionization source (ESI).
The LC elution method (using Hypersil ODS, 4.6 mm × 200
mm, 5 µm C18 column; Zorbax Eclipse XDB, 4.6 mm × 150
mm, 5 µm C8 column) was the following: 20 min method at
25 °C, mobile phase composed of different MeOH/H2O or CH3-
CN/H2O mixtures at a flow rate of 1 mL/min (all solvents were
HPLC grade, Fluka). Mass spectral (MS) data were obtained
using an Agilent 1100 LC/MSD VL system (G1946C) with a
0.4 mL/min flow rate using a binary solvent system of 95:5
MeOH/water. UV detection was monitored at 254 nm. Mass
spectra were acquired in positive mode scanning over the mass
range of 50-1500. The following ion source parameters were
used: drying gas flow, 9 mL/min; nebulizer pressure, 40 psig;
drying gas temperature, 350 °C.
Microwave Irradiation Experiments. Microwave reac-
tions were conducted using a CEM Discover Synthesis Unit
(CEM Corp., Matthews, NC). The machine consists of a
continuous focused microwave-power delivery system with
operator-selectable power output from 0 to 300 W. The
temperature of the contents of the vessel was monitored using
a calibrated infrared temperature control mounted under the
reaction vessel. All experiments were performed using a
stirring option whereby the contents of the vessel are stirred
by means of a rotating magnetic plate located below the floor
of the microwave cavity and a Teflon-coated magnetic stir bar
in the vessel.
Synthesis. Parallel Synthesis of Ethyl 2-Methyl-4-
substituted Phenyl-3-oxobutanoates (35-37). General
Procedure. Potassium ethyl 2-methylmalonate33 (34) (0.929
g, 5.04 mmol), placed in three different vessels of the Buchi
Syncore, was suspended in dry acetonitrile (6 mL). Triethy-
lamine (1.07 mL, 7.67 mmol) and magnesium chloride (0.571
g, 6 mmol) were added to the stirred suspensions and the
mixtures stirred (360 rpm) at room temperature for 2 h. Then
were added the solutions of (substituted phenylacetyl)imida-

8006 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Manetti et al.
zolides in dry acetonitrile, prepared 15 min before by reaction
between appropriate phenylacetic acid (2.4 mmol) and N,N′-
carbonyldiimidazole (0.427 g, 2.67 mmol) in acetonitrile (3 mL).
The reaction mixtures were stirred (360 rpm) overnight at
room temperature and then refluxed for 2 h. Six milliliters of
13% HCl was added dropwise to the cooled mixtures, and the
resulting clear mixtures were stirred (360 rpm) for an ad-
ditional 10 min. Finally, the organic layers were separated in
parallel with a specific filtration unit. The aqueous phases
were extracted once with ethyl acetate (10 mL), and the
organic phases were combined and then evaporated to dryness
in parallel with the same apparatus to give 35-37 as colorless
oils to be used in the next step without further purification.
Spectroscopic and analytical data for compounds 35-37 are
in agreement with those reported in the literature.27
Parallel Synthesis of Substituted 6-Benzyl-5-methyl-
3,4-dihydro-2-thioxopyrimidin-4(3H)-ones (31-33). Gen-
eral Procedure. Sodium (0.105 g, 4.55 mmol) was placed in
three different vessels of the Buchi Syncore and dissolved in
8 mL of anhydrous ethanol under a positive pressure of dry
N2. Thiourea (0.241 g, 3.178 mmol) and 35-37 (2.27 mmol)
were added in different vessels, and the resulting mixtures
were shaken overnight at reflux temperature at 360 rpm.
Then, solvent was evaporated in parallel, and the residues
were redissolved in the least amount of water (3 mL). After
neutralization with 0.5 N acetic acid, the products were
extracted in parallel with ethyl acetate (3 × 10 mL). The
organic phases, separated with a specific filtration unit, were
combined and evaporated to dryness with the same apparatus.
Finally, the residues were washed twice with petroleum ether
to give compounds 31-33, pure enough to be used in the next
step without purification. Spectroscopic and analytical data
for compounds 31-33 are in agreement with those reported
in the literature.27
residues were purified on silica gel by flash chromatography
and recrystallized by using a suitable solvent, to give com-
pounds 7, 9, 10, 17, 18, 20, 21, 29, and 30 in very high purity.
Method B. Examples. 6-(2,6-Dichlorobenzyl)-2-(2-(4-
methoxyphenyl)ethylthio)-5-methylpyrimidin-4(3H)-
one (20). The product was purified by flash chromatography
(eluent: CH2Cl2/MeOH, 97/3) to give a white solid (yield 53%),
which was recrystallized from acetonitrile. Mp 182-183°C. IR
1
(CHCl3) (ν, cm-1): 1539, 1659, 3022. H NMR (DMSO-d6): δ
2.01 (s, 3H), 2.43 (t, 2H J ) 7.27), 2.89 (t, 2H J ) 7.26), 3.69
(s, 3H), 4.15 (s, 2H), 6.77-6.89 (m, 4H), 7.17-7.40 (m, 3H),
12.47 (br s, 1H). MS (ESI) m/z: 435 [M + H]+, 457 [M + Na]+.
HPLC (C8 column; MeOH/H2O, 80/20) tR 7.59 min. Anal.
(C21H20Cl2N2O2S) C, H, S; N.
6-(2,6-Dichlorobenzyl)-2-(3-(4-methoxyphenyl)propy-
lthio)-5-methylpyrimidin-4(3H)-one (21). The product was
purified by flash chromatography (eluent: CH2Cl2/MeOH, 97/
3) to give a white solid (yield 67%), which was recrystallized
from acetonitrile. Mp 180-182°C. IR (CHCl3) (ν, cm-1): 1540,
1
1644, 3018. H NMR (DMSO-d6): δ 1.45 (quint, 2H J ) 7.1),
2.00 (s, 3H), 2.29 (t, 2H J ) 7.2), 2.62 (t, 2H J ) 6.96), 3.68 (s,
3H), 4.12 (s, 2H), 6.78-6.98 (m, 4H), 7.18-7.41 (m, 3H), 12.50
(br s, 1H). MS (ESI) m/z: 449 [M + H]+, 471 [M + Na]+. HPLC
(C8 column; MeOH/H2O, 80/20) tR 9.55 min. Anal. (C22H22-
Cl2N2O2S) C, H, S; N.
Parallel Synthesis of Substituted 6-Benzyl-2-(benzyl-
sulfonyl)-5-methylpyrimidin-4(3H)-ones (38-40). Gen-
eral Procedure. Compounds 6, 8, and 13 (0.2 mmol) were
placed in four different vessels of the Buchi Syncore and
dissolved in 6 mL of dry CH2Cl2. A solution of m-chloroper-
benzoic acid (0.103 g, 0.6 mmol) in dry CH2Cl2 (6 mL) was
added dropwise in each vessel, and the resulting solutions were
shaken overnight at room temperature at 360 rpm. The
reaction mixtures were evaporated to dryness in parallel to
obtain crude materials that were purified by flash chroma-
tography to afford the final products.
Method A. General Procedure. Microwave-Assisted
Synthesis of Substituted 6-Benzyl-2-(benzylthio)-5-me-
thylpyrimidin-4(3H)-ones 3-6, 8, 11-16, 19, and 22-28.
Compounds 31-33 (0.3 mmol) and appropriate halobenzyl
derivatives (0.3 mmol) were suspended in the least amount of
dry DMF (0.7 mL) in sealed vessels in the presence (31, 33)
or not (32) of K2CO3 (0.041 g, 0.3 mmol), and the mixtures
were irradiated at 130 °C for 5 min. The reaction mixtures
were then diluted with water (2 mL) and extracted with ethyl
acetate (3 × 10 mL). Finally, the organic phases were dried
over anhydrous Na2SO4 and evaporated to dryness. The
residues were purified by crystallization to give compounds
3-6, 8, 11-16, 19, and 22-28 in good yields and high purity.
Examples. 6-(2,6-Dichlorobenzyl)-2-(4-methoxybenzyl-
sulfonyl)-5-methylpyrimidin-4(3H)-one (38). The product
was purified by flash chromatography (eluent: AcOEt/AcOH,
99/1) to give a white solid (yield 71%), which was recrystallized
1
from acetonitrile. Mp 171-174 °C. H NMR (CDCl3): δ 2.31
(s, 3H), 3.73 (s, 3H), 4.32 (s, 2H), 4.41 (s, 2H), 6.65-6.77 (m,
4H), 7.09-7.41 (m, 3H) (m, 7H). MS (ESI) m/z: 475 [M + Na]+,
491 [M + K]+. HPLC (C18 column; CH3CN/H2O, 70/30) tR 4.36
min. Anal. (C20H18Cl2N2O4S) C, H, S; N.
6-(2,6-Dichlorobenzyl)-2-(4-bromobenzylsulfonyl)-5-
methylpyrimidin-4(3H)-one (39). The product was purified
by flash chromatography (eluent: AcOEt/petroleum ether/
AcOH, 30/10/0.6) to give a white solid (yield 55%). Mp 241-
244 °C. 1H NMR (DMSO-d6): δ 1.85 (s, 3H), 4.10 (s, 2H), 4.23
(s, 2H), 6.79-7.60 (m, 7H). MS (ESI) m/z: 500 [M - H]-. HPLC
(C18 column; CH3OH/H2O, 75/25) tR 2.67 min. Anal. (C19H15-
Cl2N3O5S) C, H, S; N.
6-(2,6-Dichlorobenzyl)-2-(4-nitrobenzylsulfonyl)-5-me-
thylpyrimidin-4(3H)-one (40). The product was purified by
flash chromatography (eluent: AcOEt/petroleum ether/AcOH,
30/10/0.6) to give a white solid (yield 50%). Mp 175-180 °C.
1H NMR (DMSO-d6): δ 1.96 (s, 3H), 4.11 (s, 2H), 4.53 (s, 2H),
7.14-8.00 (m, 7H). MS (ESI) m/z: 466 [M - H]-. HPLC (C18
column; CH3OH/H2O, 75/25) tR 2.62 min. Anal. (C19H15-
BrCl2N2O3S) C, H, S; N.
Biology. Anti-HIV Reverse Transcriptase Assays. Re-
combinant RT wt, Lys103Asn, and Tyr181Ile were expressed
and purified to >95% purity (as judged by SDS-PAGE), as
described,34 and had specific activities on poly(rA)/oligo(dT) (see
below) of 76 570 units/mg (wt), (K103N), (Y181I); 1 unit of
DNA polymerase activity corresponds to the incorporation of
1 nmol of dNMP into acid-precipitatable material in 60 min
at 37 °C.
RNA-dependent DNA polymerase activity was assayed as
follows: a final volume of 25 µL contained buffer A (50 mM
Tris-HCl pH 7.5, 1 mM DTT, 0.2 mg/mL BSA, 4% glycerol),
10 mM MgCl2, 0.5 µg of poly(rA)/oligo(dT)10:1 (0.3 µM 3′-OH
ends), 10 µM [3H]-dTTP (1 Ci/mmol), and 5-10 nM RT.
Method A. Examples. 6-(2,6-Dichlorobenzyl)-2-(4-meth-
oxybenzylthio)-5-methylpyrimidin-4(3H)-one (8). Yield
72%. Mp 197-199 °C. IR (CHCl3) (ν, cm-1): 841, 920, 1512,
1
1645, 3018. H NMR (DMSO-d6): δ 2.02 (s, 3H), 3.66 (s, 3H),
3.91 (s, 2H), 4.17 (s, 2H), 6.62-6.77 (m, 4H), 7.21-7.47 (m,
3H), 12.52 ppm (br, 1H). MS (ESI) m/z: 443 [M + Na]+. HPLC
(C8 column; CH3CN/H2O, 50/50) tR 18.08 min. Anal. (C20H18-
Cl2N2O2S) C, H, S; N.
6-(2,6-Dichlorobenzyl)-2-(4-nitrobenzylthio)-5-meth-
ylpyrimidin-4(3H)-one (13). Yield 55%. Mp 231-232 °C. IR
1
(CHCl3) (ν, cm-1): 1348, 1523, 1647, 3007. H NMR (CDCl3):
δ 2.17 (s, 3H), 4.14 (s, 2H), 4.23 (s, 2H), 6.99-7.99 (m, 7H),
11.93 ppm (br, 1H). MS (ESI) m/z: 434 [M - H]-. HPLC (C18
column; CH3OH/H2O, 90/10) tR 3.58 min. Anal. (C19H15-
Cl2N3O3S) C, H, S; N.
Method B. General Procedure. Microwave-Assisted
Synthesis of Substituted 6-Benzyl-2-(benzylthio)-5-me-
thylpyrimidin-4(3H)-ones 7, 9, 10, 17, 18, 20, 21, 29, and
30. Compounds 31-33 (0.3 mmol) and appropriate benzyl
alcohol derivatives (0.3 mmol) were suspended in the least
amount of dry DMF (1 mL) in the presence of trimethylphos-
phine (0.3 mL) (1 M solution in toluene). The reaction mixtures
were cooled in ice bath, and DIAD was added (0.3 mmol). The
mixtures were irradiated at 40° C for 10 min and then were
diluted with water (2 mL) and extracted with diethyl ether (5
× 10 mL). Finally, the combined organic phases were dried
over anhydrous Na2SO4 and evaporated to dryness. The

S-DABO Derivatives with Anti-HIV-1 Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8007
Reactions were incubated for 10 min at 37 °C. Aliquots (20
µL) were then spotted on glass fiber filters GF/C, which were
immediately immersed in 5% ice-cold TCA. Filters were
washed twice in 5% ice-cold TCA and once in ethanol for 5
min. Incorporation of radioactive dTTP into poly(rA)/oligo(dT)
at different concentrations of DNA or dNTP was monitored in
the presence of increasing amounts of inhibitor.
Anti-HIV Activity in Lymphoid Cells. Biological activity
of the compounds was tested in the lymphoid MT-4 cell line
(received from the NIH AIDS Reagent Program) against the
wt HIV-1 NL4-3 strain and three different HIV-1 strains, as
described before.35 Briefly, MT-4 cells were infected with the
appropriate HIV-1 strain (or mock-infected to determine
cytotoxicity) in the presence of different drug concentrations.
At day five post-infection, a tetrazolim-based colorimetric
method (MTT method) was used to evaluate the number of
viable cells. The IRLL98 HIV-1 strain contains the following
mutations in the RT coding sequence:36 M41L, D67N, Y181C,
M184V, R211K, T215Y (conferring resistance to NRTI) and
mutations K101Q, Y181C, G190A (conferring resistance to
NNRTI). The HIV-1 strains containing the multi-NNRTI
mutation, K103N, or the Y188L mutant were received from
the Medical Research Council Centralised Facility for AIDS
Reagents, Herfordshire, UK.
Supporting Information Available: Details of synthesis
and elemental analysis data. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Three-dimensional coordinates of the HIV-1 RT/MKC-442
(emivirine) complex (Brookhaven Protein Data Bank entry
1RT1) were used as the input structure for docking calcula-
tions. For this aim, all cocrystallized water molecules were
deleted, and all polar hydrogens were added using the ap-
propriate tool in the builder module of Maestro. The structures
of the new S-DABO derivatives were built using the Maestro
3D-sketcher and fully minimized (Polak-Ribiere conjugate
gradient of 0.05 kJ/Å mol convergence). Atom charges assigned
to compounds during the minimization step were retained for
the following docking calculation.
For molecular docking purposes, a box of 64 × 50 × 62 points
was set that comprised all residues constituting the NNRTI
binding pocket. Starting structures of the selected compounds
were randomly defined to obtain totally unbiased results. The
genetic algorithm-local search (GA-LS) method was used with
the default settings and retrieved 100 docked conformations
from each compound. Results from Autodock calculations were
clustered using an RMSD tolerance of 2 Å and the lowest
energy conformer of the most populated cluster (the lowest
energy cluster in most cases) was selected as the most probable
binding conformer. HIV-1 RT/ligand complexes were submitted
to a full minimization of the whole structure to a 0.1 kJ/Å mol
gradient.
Acknowledgment. This study was partially sup-
ported by grants from the European TRIoH Consortium
(LSHB-2003-503480), the Fundacio´ La Marato´ de TV3
project 020930 (J.A.E.), and the Spanish Ministerio de
Ciencia y Tecnolog´ıa project BFI-2003-00405 (J.A.E.).
G.M. was supported by the ISS-National Research
Program on AIDS (Grants 40F.78 and 40F.48). C.C.
thanks the Dipartimento Farmaceutico, Universita` degli
Studi di Parma, for a leave to work at the Dipartimento
Farmaco Chimico Tecnologico, Universita` degli Studi di
Siena. M.B. thanks the Merk Research Laboratories
(2004 Academic Development Program Chemistry
Award). F.M. thanks the Divisione di Chimica Farma-
ceutica della Societa` Chimica Italiana and Farmindus-
tria for the “Premio Farmindustria 2004” award.
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