Synthesis and biological investigation of S-aryl-S-DABO derivatives as HIV-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.03.080

S-Aryl-S-DABO derivatives, a novel subclass of S-DABO anti-HIV-1 agents, were synthesized via Ullmann type reaction starting from the corresponding 2-thiouracils by the aid of microwave irradiation. The results of their evaluation as inhibitors of RT are

Full text of DOI:10.1016/j.bmcl.2006.03.080

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3541–3544
Synthesis and biological investigation of S-aryl-S-DABO
derivatives as HIV-1 inhibitors
a
a
b
b
´
´
Claudia Mugnaini, Fabrizio Manetti, Jose A. Este, Imma Clotet-Codina,
Giovanni Maga,c Reynel Cancio,c Maurizio Bottaa,* and Federico Corellia,*
a
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, Via De Gasperi, 2 I-53100 Siena, Italy
bRetrovirology Laboratory irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Auto´noma de Barcelona,
E-08916 Badalona, Spain
cIstituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy
Received 16 March 2006; revised 22 March 2006; accepted 24 March 2006
Available online 18 April 2006
Abstract—S-Aryl-S-DABO derivatives, a novel subclass of S-DABO anti-HIV-1 agents, were synthesized via Ullmann type reaction
starting from the corresponding 2-thiouracils by the aid of microwave irradiation. The results of their evaluation as inhibitors of RT
are reported together with their antiviral activity in cellular assays.
Ó 2006 Elsevier Ltd. All rights reserved.
The reverse transcriptase (RT) of the human immunode-
ficiency virus type 1 (HIV-1 RT) is one of the main tar-
gets of drugs used in the treatment of AIDS.1,2 Several
RT inhibitors have been developed and approved by
the FDA and are currently in clinical use. In particular,
the non-nucleoside RT inhibitors (NNRTIs)3–5 are high-
ly effective drugs with few side effects. However, RT
mutations rapidly emerge that confer resistance to all
known NNRTIs, including the clinically established
drugs, such as nevirapine and efavirenz (Chart 1), thus
reducing their effectiveness.6,7 Therefore, new NNRTIs
that are effective against the existing drug-resistant viral
strains are urgently needed.
O
N
O
CH3
R2
CH3
HN
n S
HN
X
S
N
R1
R
Cl
Cl
R3
R1
1
2
NH2
NC
Br
O
O
N
H3C
HN
F3C
N
N
H
Cl
H3C
CH3
O
N
N
N
In line with these requirements, new classes of NNRTIs
have been recently described, among which DAPY
derivatives, such as TMC125,8 show high inhibitory
potency against several clinically relevant mutant
strains. According to Arnold et al.,9 this aspect is due
to their high structural flexibility that allows for multiple
binding conformations and hence confers the capability
of targeting the RT of different mutants.
N
H
O
CN
nevirapine
efavirenz
TMC125
Chart 1.
During the course of our investigations on potential
inhibitors of RT, we became involved in the preparation
of a number of dihydro-alkylthio-benzyl-oxopyrimi-
dines (S-DABOs)10,11 of general structure 1,12 character-
ized by the presence of an arylalkylthio substituent at
position 2 which was widely varied both in terms of sub-
stitution pattern and length of the alkyl spacer. Some of
these compounds displayed potent RT inhibiting
Keywords: HIV-1; RT inhibitors; S-DABO derivatives; Pyrimidinones;
Microwave-assisted synthesis.
*
Corresponding authors. E-mail: corelli@unisi.it
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.080

3542
C. Mugnaini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3541–3544
activity and antiviral properties on cell lines infected
with either wild type or mutant HIV-1.
presence of molecular sieves. This time, compound 4
could be obtained in 23% yield after chromatographic
purification. Despite the low yield, the Ullmann type
condensation was selected as the best methodology for
a number of reasons: (i) its high reproducibility; (ii)
the availability of a large number of arylboronic acids,
much easier to handle than the diazonium salts of the
corresponding anilines; (iii) the dramatic reduction of
reaction times by the use of microwave irradiation.
As an extension of our research, we planned to investi-
gate the antiviral properties of new compounds of struc-
ture 2 which, bearing an aromatic/heteroaromatic ring
directly linked to the sulfur atom, can be regarded as hy-
brids between DAPY and S-DABO compounds.13
The target compounds 2 can, in principle, be obtained
according to two different approaches: the condensation
between an S-arylthiourea and a b-ketoester, or aryla-
tion of the appropriate 2-thiouracil. The first approach,
used on a model reaction, gave no positive results. In-
deed, when S-phenylisothiourea14 was treated with the
highly reactive 4-chloroacetoacetate in a range of differ-
ent conditions,15,16 complex reaction mixtures were al-
ways obtained, accompanied by decomposition of the
starting material in alkaline media17 (Scheme 1).
According to this result, we decided to prepare first dif-
ferently 6-substituted 2-thiouracils,12,13 and then to
introduce an aromatic or heteroaromatic substituent in
position 2.
Having chosen this procedure for the synthesis of com-
pounds of interest, we proceeded with the optimization
of the reaction conditions. The use of 1,10-phenantro-
line22 instead of TMEDA and an excess (3 equivalents)
of the arylboronic acid instead of a stoichiometric
amount resulted to be particularly fruitful. Also the
purification step, which proved to be particularly trou-
blesome because of the similar chromatographic behav-
iour of the arylboronic acids and the target compounds,
needed some adjustments.
In particular, a filtration on CeliteÒ of the reaction mix-
ture followed by treatment with an alkaline solution to
remove the excess of arylboronic acid gave compounds
2a–d in acceptable purity; conversely, for compounds
2e–i, a rapid filtration on silica gel of the reaction mix-
ture, followed by recrystallization, gave the best results.
To this aim, the arylation via diazonium salts18 was
compared to an Ullmann type reaction19–21 on a model
substrate. Commercially available 2-thio-6-methyluracil
was thus reacted in parallel with (a) the diazonium salt
of p-chloroaniline and (b) phenylboronic acid in the
presence of Cu(OAc)2ÆH2O under microwave irradiation
(Scheme 2).
In order to verify the influence of the reaction tempera-
ture and, in particular, microwave irradiation, we per-
formed the reaction between 5a (Scheme 3) and
phenylboronic acid under different conditions. When
the reaction was carried out at room temperature, com-
pound 2b was obtained in 50% yield after 24 h; working
at reflux temperature, the reaction time was reduced to
30 min with a dramatic loss in reaction yield (27%).
Thus, the irradiation with microwaves at 85 °C proved
to be the best option, providing the highest yield
(64%) of 2b in the shortest reaction time (10 min).
In the first reaction the corresponding 2-arylthiopyri-
midinone 3 was obtained in 25% yield after a laborious
chromatographic purification followed by crystalliza-
tion from EtOH. Analogous results in terms of yield
were obtained following the second approach and using
TMEDA as a base and 1,2-DCE as the solvent in the
Accordingly, compounds 2a–i were obtained in accept-
able to good yield by the use of this copper-mediated
arylation procedure23 starting from 2-thiouracils 5a,b
and arylboronic acids 6 (Scheme 3 and Table 1). In spite
of some attempts, the yield of compound 2a could not
be improved over 30%.
O
O
O
N
Cl
O
NH
NH2
HN
Cl
S
S
Scheme 1.
To the best of our knowledge, this is the first example of
the synthesis of S-arylpyrimidinones by the use of an
Ullmann type reaction. In fact, only a single example
a
O
O
N
Cl
NH2
Cl
HN
HN
B(OH)2
R3
NaNO2, HCl 50%
0 ˚C
S
N
H
CH3
S
CH3
O
O
X
R2
CH3
3
CH3
HN
HN
HS
R2
Cu(OAc)2
6
X
OH
b
N
R1
S
N
R1
O
O
N
1,10-phenantroline
molecular sieves
1,2-DCE
R1
R3
R1
B
HN
OH
HN
MW, 85 ˚C, 10 min
Cu(OAc)2.H2O, TMEDA
molecular sieves, 1,2-DCE
MW, 85 ˚C, 10 min
S
N
H
CH3
S
CH3
5a: R1 = F
5b: R1 = Cl
2a-i
4
Scheme 2.
Scheme 3.

C. Mugnaini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3541–3544
3543
Table 1.
Compound
oxyphenylboronic acid in a different solvent (CH2Cl2,
THF) proved to be unsatisfactory, leading to complex
reaction mixtures.
R1
R2
R3
X
Mpa (°C)
Yield
(%)
2a
2b
2c
2d
2e
2f
F
F
F
CH
CH
CH
CH
CH
CH
CH
CH
N
193–194
178–179
208–210
201 (dec)
220–221
208–209
223–224
233–234
222–223
30
64
88
85
49
73
59
51
76
Compounds 2a–i 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 cytotox-
icity and anti-HIV activity, in comparison with nevi-
rapine and efavirenz used as reference drugs.12 In
particular, the following mutants were used: K103N
and Y181I for enzymatic tests, K103N, Y188L,
Y181C and IRLL98 (bearing the K101Q, Y181C
and G190A mutations conferring resistance to nevi-
rapine, delavirdine and efavirenz) for tests on cell
lines. The results of these assays are reported in
Table 2.
F
H
Cl
H
H
H
H
H
F
F
F
OCH3
OCH3
H
Cl
Cl
Cl
Cl
Cl
2g
2h
2i
F
CN
OCH3
H
H
a After recrystallization from MeOH.
of C–S coupling catalyzed by Cu(OAc)2 on a 2-phenyl-
thio-1,4-dihydropyrimidine21 has been reported in the
literature so far.
As a general consideration, compounds 2a–d, charac-
terized by the presence of 2,6-difluorobenzyl moiety
at C-6, show interesting antiviral activity with an
EC50 ranging from 0.67 to 0.087 lM, while the activ-
ity of the 2,6-dichlorobenzyl-substituted derivatives
2e–i is more modest. This aspect is particularly evi-
dent if we consider the couples 2a/2g, 2b/2f and 2d/
2e: in all cases the 2,6-difluorobenzyl-substituted part-
ner is, at least, one order of magnitude more active
than the 2,6-dichloro-substituted counterpart, as far
as the activity against wt strain is concerned. No sub-
stantial difference in antiviral activity against mutant
strains can be observed within the two series of
compounds.
However, when applied to 5b and 2,6-difluoro-4-meth-
oxyphenylboronic acid, the reaction gave unexpected re-
sults (Scheme 4).
In fact, the bicyclic compound 7 was obtained instead of
the expected S-arylpyrimidinone as a result of alkylation
of the sulfur atom by DCE, used as the reaction solvent,
followed by cyclization. Attempts to obtain the S-aryl-
pyrimidinone by reacting 5b and 2,6-difluoro-4-meth-
B(OH)2
F
F
O
N
O
N
In line with the results of the cellular tests, the enzy-
matic activity of compounds 2a–i is limited to the wt
RT, regardless of the substitution pattern. Neverthe-
less, compound 2i, bearing a pyridylthio substituent
at C-2, merits some attention as the most active
compound in the enzymatic assay. Quite unexpectedly,
all compounds 2, and in particular compounds 2a–d,
proved to be more active in cellular tests than in enzy-
matic assays.
CH3
CH3
HN
HS
N
OCH3
S
Cu(OAc)2, 1,10-phenantroline,
molecular sieves 1,2-DCE
MW, 85 ˚C, 10 min
Cl
Cl
Cl
Cl
5b
7
Scheme 4.
Table 2.
a,b
ID50 (lM)
a,c
EC50 (lM)
d
Compound
CC50
wt
K103N
Y181I
NL4-3 wt
IRLL98
K103N
Y181C
Y188L
2a
2b
30
32
nae
na
na
na
na
na
na
na
na
8.0
0.4
na
na
na
na
na
na
na
na
na
20
0.1
0.31
0.09
0.10
0.67
26.5
1.80
3.36
2.49
2.72
0.052
0.001
0.003
25.8
16.7
>65.8
42.5
8.01
—
—
>65.8
>72.7
>66.1
>57.5
>61.4
>66.3
>60.5
>27.6
>61.3
>7.50
0.30
>65.8
>72.7
>66.1
57.5
2c
2d
22
na
>66.1
>57.5
>61.4
4.91
—
—
>57.5
>61.4
>66.3
>60.5
>27.6
>61.7
3.90
2e
na
>61.4
12.1
—
>61.4
>66.3
>60.5
27. 6
>61.3
>7.50
>0.32
3.70
2f
2g
36.0
10.0
23.0
3.5
0.4
44.7
>27.6
8.68
2h
2i
5.10
6.52
—
Nevirapine
Efavirenz
AZT
>7.50
0.20
0.008
0.04
0.057
0.003
—
—
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.24,25
d CC50, cytotoxic concentration 50 or needed concentration to induce 50% death of non-infected cells evaluated with the MTT method in MT-4 cells.
e na, not active at 400 lM (the highest concentration tested).

3544
C. Mugnaini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3541–3544
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are less cytotoxic than reference drugs, with compound
2b showing a selectivity index (SI = CC50/EC50wt) > 835,
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S-arylation procedure. Some of the new compounds dis-
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along with low cytotoxicity.
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Acknowledgments
This study was partially supported by grants from the
European TRIoH Consortium (LSHB-2003-503480),
´
the Fundacio La Marato de TV3 project 020930 (JAE)
and the Spanish Ministerio de Ciencia y Tecnologıa pro-
´
´
ject BFI-2003-00405 (JAE). GM was supported by the
ISS-National Research Program on AIDS (Grants
40F.78 and 40F.48).
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