Parallel synthesis, molecular modelling and further structure-activity relationship studies of new acylthiocarbamates as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.10.032

The structure-activity relationships (SARs) of acylthiocarbamates (ATCs), a new class of non-nucleoside HIV-1 reverse transcriptase inhibitors, have been expanded. Sixty-six new analogues were prepared by parallel solution-phase synthesis. In general, the

Full text of DOI:10.1016/j.ejmech.2008.10.032

European Journal of Medicinal Chemistry 44 (2009) 2190–2201
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Parallel synthesis, molecular modelling and further structure–activity
relationship studies of new acylthiocarbamates as potent non-nucleoside
HIV-1 reverse transcriptase inhibitors
,
a
a
a
a
b
Andrea Spallarossa a , Sara Cesarini , Angelo Ranise , Silvia Schenone , Olga Bruno , Alberto Borassi ,
*
Paolo La Colla c, Margherita Pezzullo c, Giuseppina Sanna c, Gabriella Collu c, Barbara Secci c,
Roberta Loddo c
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
b Dipartimento di Chimica e Chimica Industriale, Universita’ degli Studi di Genova, Via Dodecaneso 31, I-16146 Genova, Italy
c
`
Dipartimento di Scienze e Tecnologie Biomediche, Universita di Cagliari, Cittadella Universitaria, S.S. 554, Km 4,500, I-09042 Monserrato (Cagliari), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationships (SARs) of acylthiocarbamates (ATCs), a new class of non-nucleoside
HIV-1 reverse transcriptase inhibitors, have been expanded. Sixty-six new analogues were prepared by
parallel solution-phase synthesis. In general, the potency of new ATCs was better than that of the first
series and O-[2-phthalimidoethyl] 4-chlorophenyl(3-nitrobenzoyl) thiocarbamate turned out to be the
most potent ATC so far synthesized (EC50 ¼ 1.5 nM). Several ATCs were active at micromolar concen-
trations against HIV-1 strains carrying the RT Y181C mutation and one of them was also moderately
active against the K103R variant. Docking simulations were carried out to rationalize the most relevant
SARs.
Received 9 July 2008
Received in revised form
22 October 2008
Accepted 30 October 2008
Available online 5 November 2008
Keywords:
Acylthiocarbamates
HIV-1
Ó 2008 Elsevier Masson SAS. All rights reserved.
Non-nucleoside reverse transcriptase
inhibitors
Parallel synthesis
1. Introduction
[10], mainly based on modifications of the b substructure of lead I,
had identified the N-phenyl para substitution as a key feature to
HIV-1 reverse transcriptase (RT) catalyses the conversion of
a single-stranded RNA into a double-stranded DNA that is then
integrated into the host cells’ genome. Due to its essential role in
the HIV-1 life-cycle, RT is a primary target for the highly active
antiretroviral therapy (HAART). Non-nucleoside inhibitors
(NNRTIs) [1–9] are chemically diverse and selective RT targeting
agents that lock this enzyme in an inactive form [4] by binding to an
allosteric hydrophobic pocket (namely, non–nucleoside inhibitor
binding site, NNBS) located about 10 Å far from the polymerase
active site.
gain potent inhibitors (Fig. 1B).
With the aim at acquiring additional key elements to maximize
the activity against wild-type RT and some clinically relevant
RT mutations, a new series of ATCs was designed. Initially, the
SAR strategy was focused on the variation of the substructure c
[mono- (1–16), di-substituted (hetero)aroyl (19–47) and bicyclic
(hetero)aroyl (48–50) groups] by keeping constant portion
a (2-phthalimidoethyl) and the para-substitution pattern (halide,
methyl) of portion b. Analogues 17 and 18 were synthesized to
evaluate the effect of portion c polysubstitution on the activity of
N-unsubstituted phenyl ATCs. Then, some of the most favourable
N-phenyl(4-chlorophenyl, 4-nitrophenyl) and acyl(3-nitrobenzoyl,
4-chlorobenzoyl, 2-thenoyl, 2-chloronicotinoyl) substructures were
assembled with portion a carrying modifications on the ethyl linker
(51–54) and on the phthalimide phenyl ring (55–60). Finally, we
evaluated the influence of the N-phenyl ring di- and tri-chlor-
osubstitution on the anti-HIV-1 activity by keeping constant
portion a (2-phthalimidoethyl) and embedding two of the most
promising acyl moieties (2-furoyl, 3-nitrobenzoyl) (61–66).
O-(2-Phthalimidoethyl)-N-aryl-N-acylthiocarbamates
(ATCs)
have been recently identified [10] as a new class of HIV-1 NNRTIs,
structurally related to N-phenethyl-N0-thiazolylthiourea (PETT)
[11–19] and thiocarbamate (TC) [20–23] derivatives (Fig. 1). ATCs
can be retrosynthetically fragmented into three portions (namely,
a–c. Fig. 1B). Previous structure–activity relationship (SAR) studies
* Corresponding author. Tel.: þ39 010 353 8364; fax: þ39 010 353 8358.
E-mail address: andrea.spallarossa@unige.it (A. Spallarossa).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.032

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
2191
O
A
N
Y
W
R
S
N
O
R = H, CH3
O
R2
HN
N
H
N
H
N
O
Ar
A
N
S
R1
H
COOH
G
R1, R2 = H, CH3
TC s
Trovirdine (Y = W = H, G = Br)
PETT-1 (W = F, Y = OC2H5, G = CN)
X
X = CH, N
B
X
(Het)Ar-CO
benzoyl
EC
0.4
0.1
(µM)
50
O
a
b
X
H
Cl
Cl
Cl
Cl
Cl
Cl
I
S
benzoyl
II
III
N
O
4-chlorobenzoyl
2-furoyl
0.025
0.007
0.008
0.005
0.006
O
N
IV
V
2-thenoyl
O
(Het)Ar
2-chloronicotinoyl
6-chloronicotinoyl
VI
VII
c
Fig. 1. A] PETT and TC [20–23] derivatives structurally related to ATCs. B] Molecular portions, chemical structure and antiretroviral activity of previously prepared ATCs I–VII [10].
2. Chemistry
and times were employed to increase the product yields. The
overall yields for ATCs 1–66 ranged from 12 to 88% (see Section 6).
The most relevant features of this parallel methodology are: i)
no intermediate needs to be isolated; ii) high atom economy is
realized, as only one molecule of HCl is formally lost in the whole
process (this allows the incorporation into the products of all the
chemical features of the commercially available or readily acces-
sible building blocks used); iii) minimal sample handling is
required during the purification protocol; iv) the modular character
of the synthesis allows the independent variation of portions a–c.
The synthesis of ATCs 1–66 was carried out by the paralleliza-
tion of the previously reported highly convergent three-step one-
pot solution-phase protocol [10]. As shown in Scheme 1, starting
alcohols A1–8 (Fig. 2A) were transformed into the corresponding
alcoholates Aꢀ in the presence of sodium hydride in anhydrous
aprotic solvents (DMF or pyridine) and condensed in situ with the
proper isothiocyanate B (Fig. 2B). The sodium thiocarbamate
adducts ABꢀ were subsequently coupled with acyl chlorides C
(Fig. 2C) in the presence of N,N,N0,N0-tetramethylethylenediamine
(TMEDA) to afford the desired products. Two synthetic variants
(namely, procedures P1 and P2, see Section 6) were adopted to
obviate the different reactivity of key intermediates Aꢀ and ABꢀ vs
building blocks B and C. Thus, ATCs 1–49, 52–57 and 59 were
prepared in anhydrous pyridine stirring the mixtures overnight at
rt (procedure P1). For derivatives 50, 51, 58, 60–66 (procedure P2),
dry DMF was used as reaction medium and different temperatures
3. Biological results and discussion
ATCs 1–66 were evaluated for their cytotoxicity and anti-HIV-1
activity in MT-4 cells using Trovirdine as the reference molecule
(Tables 1–5). Selected analogues were screened in enzymatic assays
against HIV-1 wt RT, using Efavirenz as control compound (Table 6).
The most potent derivatives were also tested against the clinically
S
+
(a)
(b)
S
(c)
Na
N
-
+
Ar
Ar
R
R
OH
R
O
2
Ar
Na
R
1
O
N
O
O
1
-
A
AB-
1-66
A
1-8
1-8
Scheme 1. General procedure for the preparation of ATCs 1–66 by parallel, convergent, one-pot solution-phase synthesis. Reaction conditions: (a) NaH, dry DMF or dry pyridine, 0 ꢁ
or rt; (b) Ar1–NCS (B1–12), 0 ꢁC or rt; (c) TMEDA then Ar2COCl (C1–33), rt or heating. The structures of alcohols A1–8, isothiocyanates B1–12 and acyl chlorides C1–33 are listed in Fig. 2.
C

2192
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
A] ROH A1-A8
n
0
1
1
1
1
1
2
X
H
H
H
Y
R1
R2
H
O
R1
()
A
A
A
A
A
A
A
H
-
O
1
2
3
4
5
6
7
H
CH3
H
H
OH
H
H
N
N
n
OH
CH3 H
H
H
X
H
H
H
H
H
H
CH3
H
H
A8
R2
O
O
CH3
H
Y
H
B] Ar1-NCS B1-B12
Ar1
C] Ar2-CO-Cl C1-C33
Ar2-CO
Ar2-CO
2-toluoyl
2-nitrobenzoyl
3-toluoyl
3-nitrobenzoyl
4-toluoyl
4-trifluoromethylbenzoyl
4-chlorobenzoyl
4-bromobenzoyl
4-pentyloxybenzoyl
4-nitrobenzoyl
2,3-difluorobenzoyl
2,4-difluorobenzoyl
2,5-difluorobenzoyl
2,6-difluorobenzoyl
3,4-difluorobenzoyl
3,5-difluorobenzoyl
2,3-dichlorobenzoyl
2,4-dichlorobenzoyl
2,6-dichlorobenzoyl
3,4-dichlorobenzoyl
3,5-dichlorobenzoyl
2,6-dimethoxybenzoyl
2-chloro-4-nitrobenzoyl
2-chloro-5-nitrobenzoyl
4-chloro-3-nitrobenzoyl
4-bromo-3-methylbenzoyl
1-naphthoyl
2-naphthoyl
2-furoyl
2,5-dimethyl-3-furoyl
2-thenoyl
2-chloronicotinoyl
thianaphthene-2-carbonyl
B1 phenyl
B2 4-tolyl
C1
C2
C3
C4
C5
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
B3 4-fluorophenyl
B4 4-chlorophenyl
B5 4-bromophenyl
B6 4-nitrophenyl
B7 2,3-dichlorophenyl
B8 2,4-dichlorophenyl
B9 2,6-dichlorophenyl
B10 3,4-dichlorophenyl
B11 3,5-dichlorophenyl
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
B12 2,4,6-trichlorophenyl
Fig. 2. Building blocks used for the ATC parallel synthesis.
relevant K103R, Y181C (Table 7) and K103 N/Y181C resistant
mutants [24,25].
Table 1
Cytotoxicity and anti-HIV-1 activity of ATCs 1–16.a
With the exception of 1, ATCs bearing ortho-, meta- and para-
monosubstituted (hetero)aroyl moieties (1–16, Table 1) emerged as
potent NNRTIs, active in the sub-micromolar or nanomolar
concentration range. In particular, 4, 5 and 7 were 2-, 13- and 4-fold
more active than Trovirdine, respectively, whereas 14 and 15 were
equipotent to the reference compound. Analogue 5 turned out to be
the most potent ATC so far synthesized (EC50 ¼ 1.5 nM). The
substitution pattern as well as the electronic properties of the acyl
substituent significantly affected the activity. Thus, the meta- and
para-substituted ATCs showed higher potency than the corre-
sponding ortho congeners (4 and 6 vs 2; 5 and 8 vs 3; 13 and 15 vs
11; 14 and 16 vs 12). The toluoyl derivatives were more active than
the nitrobenzoyl analogues among the ortho and para series (2 vs 3;
6 vs 8; 11 vs 12; 15 vs 16); this trend was reversed within the meta-
substituted ATCs (4 vs 5; 13 vs 14). Furthermore, the ATCs’ potency
is concomitantly influenced by the acyl substitution pattern and the
electronic nature of the portion b substituents. Thus, among the
ortho- and meta-substituted aroyl ATCs, the N-4-chlorophenyl
analogues were more active than the N-4-tolyl congeners (2 vs 11; 3
vs 12; 4 vs 13; 5 vs 14), whereas the para derivatives showed an
opposite trend (compare 6 with 15; 8 with 16).
O
Acyl
O
N
N
S
X
O
SId
b
c
Cpd
X
Acyl
CC50
EC50
1
2
F
Cl
4-pentyloxybenzoyl
2-toluoyl
>100
21
21
58
18
>100
10
>100
73
>100
>100
100
>100
100
83
8.5
>11.8
525
300
0.04
0.07
0.01
0.0015
0.05
0.005
0.06
0.035
0.2
0.20
0.40
0.04
0.02
0.02
0.03
0.02
3
4
Cl
Cl
2-nitrobenzoyl
3-toluoyl
5800
12,000
>2000
2000
1667
2086
>500
>500
250
>2500
5000
4150
1933
3000
5
6
Cl
Cl
3-nitrobenzoyl
4-toluoyl
7
8
9
10
11
12
13
14
15
16
Cl
Cl
Br
Br
CH3
CH3
CH3
CH3
CH3
CH3
4-trifluoromethylbenzoyl
4-nitrobenzoyl
4-bromobenzoyl
2-chloronicotinoyl
2-toluoyl
2-nitrobenzoyl
3-toluoyl
3-nitrobenzoyl
4-toluoyl
4-nitrobenzoyl
58
60
Trovirdine
The activities of ATCs featured by di-substituted (hetero)aroyl
groups (17–47) are shown in Table 2. The most potent derivatives
shared the N-4-chlorophenyl substructure and showed EC50 values
lower than (19, 20, 23, 24, 28, 29, 31, 34) or equal to (21, 22, 33, 35)
that of Trovirdine. The N-phenyl para substitution as well as the
electronic properties of the substituent on portion b proved to play
a pivotal role on anti-HIV-1 activity. Thus, the N-unsubstituted
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
b
Compound concentration [mM] required to reduce the viability of mock-infected
cells by 50%, as determined by the MTT method.
c
Compound concentration [mM] required to achieve 50% protection of MT-4 cell
from HIV-1 induced cytopathogenicity, as determined by the MTT method.
d
Selectivity index: CC50/EC50 ratio.

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
2193
Table 2
Table 3
Cytotoxicity and anti-HIV-1 activity of ATCs 17–47.a
Cytotoxicity and anti-HIV-1 activity of ATCs 48–50.a
O
O
Acyl
Acyl
O
N
O
N
N
N
S
S
X
Cl
EC50
O
O
SId
b
c
Cpd
X
Acyl
CC50
EC50
SId
b
c
Cpd
Acyl
CC50
48
49
50
1-naphthoyl
2-naphthoyl
Thianaphthene-2-carbonyl
6
37
56
60
0.03
0.02
0.2
200
1850
280
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3,4-dichlorobenzoyl
52
51
58
52
54
39
52
59
54
18
20
35
32
69
19
45
48
18
40
20
62
65
54
35
49
22
17
36
17
47
0.5
104
146
4-chloro-3-nitrobenzoyl
2,3-difluorobenzoyl
2,4-difluorobenzoyl
2,5-difluorobenzoyl
2,6-difluorobenzoyl
3,4-difluorobenzoyl
3,5-difluorobenzoyl
2,3-dichlorobenzoyl
2,4-dichlorobenzoyl
2,6-dichlorobenzoyl
3,4-dichlorobenzoyl
3,5-dichlorobenzoyl
2,6-dimethoxybenzoyl
2-chloro-4-nitrobenzoyl
2-chloro-5-nitrobenzoyl
4-chloro-3-nitrobenzoyl
0.35
0.008
0.01
0.02
0.02
0.008
0.007
0.1
0.03
5.6
0.009
0.008
0.4
0.008
0.07
0.02
0.007
0.02
0.03
0.04
1.0
7250
5200
2700
1950
6500
8429
540
Trovirdine
0.02
3000
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
b
Compound concentration [mM] required to reduce the viability of mock-infected
cells by 50%, as determined by the MTT method.
c
Compound concentration [mM] required to achieve 50% protection of MT-4 cell
600
3.6
from HIV-1 induced cytopathogenicity, as determined by the MTT method.
d
Selectivity index: CC50/EC50 ratio.
3889
4000
172
2375
643
2400
2571
2000
667
1,550
65
1080
175
163
2.4
340
1.6
189
783
phenyl derivatives were less active than their corresponding N-
para-substituted phenyl analogues (compare 17 with 28 and 43; 18
with 33 and 46) and the N-4-chlorophenyl ATCs (bearing an
electron-withdrawing substituent on portion b) resulted from 1.6-
to 55-fold more active than those embedding the electron-
donating methyl group (20 vs 36, 21 vs 37, 22 vs 38, 24 vs 39, 25 vs
40, 26 vs 41, 27 vs 42, 28 vs 43, 30 vs 44, 31 vs 45, 33 vs 46, 34 vs
47). The difluorobenzoyl derivatives 19–24 were highly active in
a narrow concentration range (EC50 range: 7–20 nM), thus sug-
gesting that activity does not correlate with a specific position of
the two aromatic fluorine atoms. The dichlorobenzoyl derivatives
were less active than their difluoro isosters (25 vs 19; 26 vs 20; 27
vs 22; 28 vs 23; 29 vs 24; 41 vs 36; 42 vs 38) and showed a strong
dependence of the antiretroviral activity on the acyl moiety
substitution pattern. In particular, the 3,4- and 3,5-dichloro
analogues were significantly more active than their 2,3-, 2,4- and
2,6-dichloro positional isomers (compare 28 and 29 with 25–27;
43 with 40–42). Noteworthy, 27, 38, 42 and 44 (sharing a 2,6-di-
substituted acyl moiety) emerged as the weakest derivatives of the
series indicating this pattern as particularly unfavourable for ATC
antiviral activity. The chloro-nitrobenzoyl ATCs 31–33, 45, 46
Cl
Cl
Cl
4-bromo-3-methylbenzoyl
2,5-dimethyl-3-furoyl
2,4-difluorobenzoyl
2,5-difluorobenzoyl
2,6-difluorobenzoyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3,5-difluorobenzoyl
0.05
0.2
0.3
9
0.05
2,3-dichlorobenzoyl
2,4-dichlorobenzoyl
2,6-dichlorobenzoyl
3,4-dichlorobenzoyl
2,6-dimethoxybenzoyl
2-chloro-4-nitrobenzoyl
4-chloro-3-nitrobenzoyl
4-bromo-3-methylbenzoyl
22
0.09
0.06
0.05
0.02
>100
60
>2000
3000
Trovirdine
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
b
Compound concentration [mM] required to reduce the viability of mock-infected
cells by 50%, as determined by the MTT method.
c
Compound concentration [mM] required to achieve 50% protection of MT-4 cell
from HIV-1 induced cytopathogenicity, as determined by the MTT method.
d
Selectivity index: CC50/EC50 ratio.
Table 4
Cytotoxicity and anti-HIV-1 activity of ATCs 51–60.a
R1
Acyl
N
O
( )
A
n
S
R2
X
b
c
Cpd
A
n
R1
R2
Acyl
X
CC50
EC50
>100
0.05
0.7
29
SId
–
1540
44.3
1.8
1467
>2000
>5000
817
51
N-phthalimido
N-phthalimido
N-phthalimido
N-phthalimido
N-(3-methyl)phthalimido
N-(4-methyl)phthalimido
N-(4-methyl)phthalimido
N-(4-methyl)phthalimido
N-(4-methyl)phthalimido
N-(cis-1,2,3,6-tetrahydrophthalimido)
0
1
1
2
1
1
1
1
1
1
–
CH3
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
3-nitrobenzoyl
3-nitrobenzoyl
3-nitrobenzoyl
3-nitrobenzoyl
3-nitrobenzoyl
3-nitrobenzoyl
2-thenoyl
2-chloronicotinoyl
4-chlorobenzoyl
4-chlorobenzoyl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
>100
77
31
53
44
>100
>100
49
>100
>100
60
52e
53e
54
55
56
57
58
59
60
0.03
0.05
0.02
0.06
0.04
NO2
Cl
>2500
>1.2
3000
H
H
83
0.02
Trovirdine
a
Data mean values for three separate experiments. Variation among triplicate samples was less than 10%.
b
c
Compound concentration [
Compound concentration [
m
m
M] required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
M] required to achieve 50% protection of MT-4 cell from HIV-1 induced cytopathogenicity, as determined by the MTT method.
d
e
Selectivity index: CC50/EC50 ratio.
Tested as racemic mixture.

2194
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
Table 5
Table 7
Cytotoxicity and anti-HIV-1 activity of ATCs 61–66.a
Activity of selected ATCs against Y181C resistant mutant.a
O
b
Acyl
Cpd
EC50
wt
X1
X2
O
N
Y181C
Ratioc
N
4
6
8
10
19
20
21
23
25
29
33
34
35
49
50
52
0.01
0.05
0.06
0.2
6.0
8.5
26
2.3
6.0
5.4
5.7
56
4.2
8.2
9.0
10.9
7.5
11
600
170
433
11
750
540
285
7000
42
1025
450
1557
375
550
60
S
O
X3
CC50
SId
b
c
Cpd
Acyl
X1
X2
X3
EC50
>100
2.6
17
0.69
0.008
0.01
0.02
0.008
0.1
0.008
0.02
0.007
0.02
0.02
0.2
0.05
0.03
0.06
0.02
n.d.d
61
62
63
64
65
66
2-furoyl
2-furoyl
3-nitrobenzoyl
2-furoyl
2-furoyl
2-Cl
2-Cl
2-Cl
3-Cl
3-Cl
2-Cl
3-Cl
4-Cl
6-Cl
4-Cl
5-Cl
4-Cl
H
H
H
H
100
>100
58
100
>100
20
–
>38
3.4
145
–
H
>100
>20
3-nitrobenzoyl
6-Cl
–
Trovirdine
60
0.02
3000
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
12
b
Compound concentration [mM] required to reduce the viability of mock-infected
cells by 50%, as determined by the MTT method.
Compound concentration [mM] required to achieve 50% protection of MT-4 cell
from HIV-1 induced cytopathogenicity, as determined by the MTT method.
5.0
4.2
6.7
1.2
0.01
100
140
112
60
55
58
c
Trovirdine
Efavirenz
d
Selectivity index: CC50/EC50 ratio.
n.d.d
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
b
proved to be potent derivatives within both the N-4-chlorophenyl
and the N-4-tolyl series.
Compound concentration [mM] required to achieve 50% protection of MT-4 cell
from HIV-1 induced cytopathogenicity, as determined by the MTT method.
c
EC50 (Y181C)/EC50(wt) ratio.
Not determined.
To further probe the spatial requirements of portion c, the
bicyclic (hetero)aroyl compounds 48–50 were tested (Table 3). The
naphthoyl derivatives 48 and 49 showed an EC50 comparable with
that of Trovirdine, being more active than their less hindered
benzoyl analogue II. Surprisingly, 50 showed a reduced potency in
comparison with both its naphthoyl isosters and its thenoyl
analogue (compare 50 with 48, 49 and V).
d
also for TCs [21–23] and the previously reported analogues [10])
could be ascribed to the possibility that ATCs are slow binding
kinetics inhibitors [26].
A number of ATCs [sharing the N-4-(chloro or bromo)phenyl
moiety] showed moderate activity against the Y181C strain (EC50
The EC50 values of ATCs 51–60 (Table 4), embodying some of the
most favourable b and c portions combined with variations of
portion a, indicate that: i) the ethyl linker shortening or length-
ening caused loss or drop in activity, respectively (51 and 54 vs 5);
ii) the methyl branching of the ethyl linker caused a reduction of
potency (compare 52 and 53 with 5); however, when the methyl
group was located on the C-atom adjacent to the phthalimide
nitrogen, the activity was kept high, differently from the methyla-
tion on the carbon atom next to the thiocarbamic functionality
(compare 52 with 53); iii) the methyl substitution of the phthali-
mide system was well tolerated (compare 55 and 56 with 5; 57 with
V; 58 with VI). The partial saturation of the phthalimide benzene
ring and the consequent planarity disruption of the bicyclic system
caused a dramatic decrease in activity (compare 60 with III).
The N-dichloro- and N-trichloro-substituted phenyl ATCs (61–
66, Table 5) were poorly active or inactive, differently from the
corresponding para mono-chloro-substituted analogues (compare
63 and 66 with 5; 61, 62, 64 and 65 with IV).
range ¼ 2.3–56
m
M, Table 7). The most interesting compounds
(EC50 range 2.3–5.7
mM) bear the 2,4-, 2,5- and 3,5-difluorobenzoyl
(20, 21 and 24, respectively), 2,3-dichlorobenzoyl (25) or 2-chlor-
onicotinoyl (10) moieties. Moreover, the methyl-branched 52 and
3-methyl phthalimidyl 55 (sharing the 3-nitrobenzoyl moiety)
were active in the same micromolar concentration range. In addi-
tion, ATC 10 showed a modest activity also against the K103R
mutation (EC50: 58 mM), differently from all ATCs so far synthesized
[10]. No activity against the K103 N/Y181C mutation was detected
(data not shown).
4. Modelling studies
To rationalize the most relevant SARs, docking studies (Auto-
dock 3.05) [27] were carried out using the X-ray coordinates of the
RT/PETT complex (PDB code: 1DTQ) [28] as the template structure.
The docking model built for the most potent derivative 5 sug-
gested for the inhibitor an extended conformation superimposable
with that predicted for I [10]. The RT/5 complex is mainly stabilized
by two hydrogen bonds: the former involving the thiocarbonyl
sulphur atom and the Lys101 amidic nitrogen (S–NH distance:
2.9 Å), the latter between one of the imidic carbonyls and the
Lys103 side chain amino group (Fig. 3). The higher potency of 5, in
comparison with that of the lead I, would be explained by the
hydrophobic contacts of the chlorine atom with Val179, Ile180,
Tyr181 and Tyr188 and by the polar interactions of the 3-nitro
group with the Phe227, Leu234, His235 and Pro236 main chain
atoms. According to this model, the chlorine atom would be at
halogen bond distance (Cl–O distance: 3.1 Å) [29] with the carbonyl
oxygen of Val179 main chain, thus rationalizing the higher potency
of the N-4-chlorophenyl derivatives in comparison with that of
their N-4-tolyl analogues. It should be noted that Leu234 and
To determine whether the title compounds targeted HIV-1 RT,
ATCs 10 and 59 were tested by enzyme assay (Table 6). Both
derivatives showed good affinity for wt HIV-1 wt RT with IC50 values
in the sub-micromolar concentration range. The considerable
difference of potency in enzyme- and cell-based assays (observed
Table 6
Activity of selected ATCs in enzyme assays against the wt HIV-1 wt RT.a
b
Cpd
IC50
10
59
Efavirenz
0.7 ꢂ 0.08
0.3 ꢂ 0.05
0.0013 ꢂ 0.0003
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
b
IC50
[
mM] ꢂ standard deviation.

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
2195
Fig. 3. Stereodiagram showing the position and orientation of 5 modelled structures inside the NNBS. Amino acid residues lining the NNBS are represented by lines and labelled
according to the one-letter code. The ligand is displayed in ball-and-sticks model. The hydrogen bonds are shown as dotted lines.
Pro236 (along with Trp229) are highly conserved amino acids in
the NNBS and therefore recognized of strategic relevance for the
design of new NNRTIs more resilient to the effect of RT mutations in
this site [6]. Interestingly, X-ray studies carried out on 14 (a close
analogue of 5) as free ligand [30] showed the acylthiocarbamic
group in an E,E0 conformation, whereas docking calculation pre-
dicted a Z,Z0 arrangement. As a consequence, the modelled and X-
ray structures show different torsional angles along the C(S)–N and
C(O)–N bonds with an inversion of the positions of the N-phenyl
and N-acyl moieties (Fig. 4).
The docking models of RT/38, RT/42 and RT/44 complexes
rationalize the poor activity of this 2,6-di-substituted derivatives.
Thus, the 2,6 pattern induces a conformation change responsible
for the loss of one (for 38) or both hydrogen bonds (for 42 and 44)
relative to the RT/5 docking model.
The docking models of 49 and 50 showed the two inhibitors in
an extended conformation with the naphthoyl moieties in contact
with Val106, Gly190, Trp229, Leu234. In addition, the 1-naphthyl is
in contact with Tyr188, Tyr318 and the 2-naphthyl interacts with
Leu100 and Lys103. The 1-naphthyl and the 2-naphthyl would be
rotated of about 129ꢁ and 135ꢁ relative to the 3-nitrophenyl frag-
ment of 5, respectively.
found in the first ATC series. These results encourage further efforts
to improve ATC resilience to the most important drug resistant RT
mutations.
6. Experimental section
6.1. Chemistry
6.1.1. General
N-(Hydroxymethyl)phthalimide, N-(2-hydroxyethyl)phthalimide,
N-(3-hydroxypropyl)phthalimide, isothiocyanates, acyl chlorides
and reagents (60% sodium hydride dispersion in mineral oil,
TMEDA) were purchased from Chiminord and Aldrich Chemical,
Milan (Italy). Alcohols A3–6 and A8 were prepared according to the
previously reported protocol [20]. The synthesis of Trovirdine was
accomplished according to the published procedure [31]. Solvents
were of reagent grade (DMF, pyridine). DMF was dried on molecular
sieves (5 Å 1/1600 inch pellets). Unless otherwise stated, all
commercial reagents were used without further purification.
Organic solutions were dried over anhydrous sodium sulphate.
Thin layer chromatography (TLC) system for routine monitoring the
course of parallel reactions and confirming the purity of analytical
samples employed aluminium-packed silica gel plates (Merck DC-
Alufolien Kieselgel 60 F254). CHCl3 or CHCl3/methanol was used as
a developing solvent and detection of spots was made by UV light
and/or by iodine vapours. The parallel solution-phase chemistry
was performed by using a 12-Carousel Reaction StationÔ (Radleys
Discovery Technologies, Italian distributor: StepBio, Bologna). The
evaporation of solutions in parallel fashion was performed with an
EvaposelÔ apparatus (Radleys Discovery Technologies, Italian
distributor: StepBio, Bologna) operating at reduced pressure of
about 15–20 Torr. Yields were not optimized. Melting points were
5. Conclusion
The parallel synthesis of sixty-six ATCs complemented our SAR
strategy centred on molecular modifications of lead I. The new ATCs
were, in general, more active than those previously described. In
particular, 5 emerged as the most potent ATC so far synthesized.
Besides the high potency against wild-type HIV-1, several ATCs
inhibited the Y181C variant at micromolar concentrations and one
of them showed a moderate activity against the K103R mutant, not
Fig. 4. Stereodiagram showing the superposition of 14 X-ray structure [30] (grey) and 5 docked conformation (black). The superposition has been obtained by overlying the N–C(S)–
O atoms in the two structures.

2196
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
determined on a Fisher–Johns apparatus and are uncorrected. IR
spectra were recorded on a Perkin Elmer 398 spectrometer as KBr
discs. 1H NMR spectra were recorded in CDCl3 or DMSO-d6 on
a Varian Gemini 200 instrument; chemical shifts were reported in
8.76 (m, 12H, arom H). Calcd for C24H16ClN3O6S: C, 56.53; H, 3.16; N,
8.24; S, 6.29. Found: C, 56.42; H, 3.35; N, 8.26; S, 6.30.
6.1.2.6. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(4-methylbenzoyl)thiocarbamate 6. Mp 175–176 ꢁC (from
CH2Cl2/Et2O); yield: 71%. IR (KBr) cmꢀ1 1777, 1712. 1H NMR (CDCl3)
d
(ppm) units relative to the internal reference tetramethylsilane,
and the splitting patterns were described as follows: s (singlet),
d (doublet), t (triplet) and m (multiplet). The first order values
reported for coupling constants J were given in Hz. Elemental
analyses were performed by an EA1110 Analyser, Fison Instruments
(Milan).
d
2.32 (s, 3H, CH3), 3.86 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.63 (t, J ¼ 6.0 Hz,
2H, CH2O), 6.99–7.39 and 7.62–7.95 (m, 12H, arom H). Calcd for
C25H19ClN2O4S: C, 62.69; H, 4.00; N, 5.85; S, 6.69. Found: C, 62.54;
H, 4.11; N, 5.84; S, 6.58.
6.1.2. General parallel synthetic procedure P1 for the preparation of
ATCs 1–49, 52–57, 59
6.1.2.7. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(4-trifluorometylbenzoyl)thiocarbamate 7. Mp 166–168 ꢁC
(from CH2Cl2/EtOH); yield: 18%. IR (KBr) cmꢀ1 1777, 1712. 1H NMR
Alcohols A2–7 (10 mmol) and isothiocyanates B1–6 (10 mmol)
were added to each reaction tube of a Carusel Reaction StationÔ
and dissolved in anhydrous pyridine (25 mL) under stirring at rt.
60% sodium hydride dispersion in mineral oil (0.44 g, w10 mmol)
was poured in a single portion into each tube; as soon as the
hydrogen evolution ceased, TMEDA (1.28 g, 11 mmol; for 10:
15 mmol; and for 35: 12 mmol) and the proper acyl chloride
(11 mmol; for 10 and 35: 12 mmol) were added. Each reaction
mixture was stirred for 12 h at rt (for 35: 60 ꢁC for 2 h). Each
reaction mixture was transferred into a set of separating funnels,
diluted with water (150 mL) and extracted with dichloromethane
(30 mL ꢃ 2). The combined extracts were washed with water
(30 mL ꢃ 2), 1 N HCl (30 mL ꢃ 2, except for 10), dried and filtered in
parallel through pads of Florisil (diameter 5 ꢃ 2 cm). Evaporating in
vacuo gave a residue that was purified by crystallization from the
suitable solvent or solvent mixture.
(CDCl3)
d
3.88 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.63 (t, J ¼ 6.0 Hz, 2H,
CH2O), 7.07–8.14 (m, 12H, arom H). Calcd for C25H16ClF3N2O4S: C,
56.35; H, 3.03; N, 5.26; S, 6.02. Found: C, 56.25; H, 3.18; N, 5.19; S,
6.13.
6.1.2.8. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(4-nitrobenzoyl)thiocarbamate 8. Mp 190–192 ꢁC (from
CH2Cl2/EtOH); yield: 39%. IR (KBr) cmꢀ1 1775, 1711. 1H NMR
(CDCl3)
d
3.89 (t, J ¼ 6.0 Hz, CH2N), 4.65 (t, J ¼ 6.0 Hz, CH2O), 7.15–
7.38 and 7.60–8.40 (m, 12H, arom H). Calcd for C24H16ClN3O6S: C,
56.53; H, 3.16; N, 8.24; S, 6.29. Found: C, 56.82; H, 3.08; N, 7.94; S,
6.15.
6.1.2.9. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-bro-
mobenzoyl(4-bromophenyl)thiocarbamate 9. Mp 185–186 ꢁC (from
CH2Cl2/EtOH); yield: 57%. IR (KBr) cmꢀ1 1775, 1709. 1H NMR (CDCl3)
6.1.2.1. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-fluo-
rophenyl[4-(pentyloxy)benzoyl]thiocarbamate 1. Mp 166–167 ꢁC
(from CH2Cl2/Et2O); yield: 67%. IR (KBr) cmꢀ1: 2938, 1778, 1713,
d
3.90 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.66 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.02–
7.97 (m, 12H, arom H). Calcd for C24H16Br2N2O4S: C, 49.00; H, 2.74;
N, 4.76; S, 5.45. Found: C, 48.84; H, 3.04; N, 4.53; S, 5.52.
1693. 1H NMR (DMSO)
d 0.75–1.09 (m, 3H, CH3), 3.67–4.21 (m, 6H,
3CH2/pentyl), 3.67–4.21 (m, 4H, CH2N and CH2O/pentyl), 4.36–4.73
(m, 2H, CH2O), 6.78–8.07 (m,12H, arom H). Calcd for C29H27FN2O5S:
C, 65.15; H, 5.09; N, 5.24; S, 6.00. Found: C, 65.28; H, 5.23; N, 5.29; S,
5.77.
6.1.2.10. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-bro-
mophenyl[(2-chloropyridin-3-yl)carbonyl]thiocarbamate
165–167 ꢁC (from MeOH); yield: 67%. IR (KBr) cmꢀ1 1778, 1710,
1668. 1H NMR (CDCl3)
10. Mp
d
3.80 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.59 (t,
J ¼ 6.0 Hz, 2H, CH2O), 7.16–7.29 and 7.76–7.89 (m, 8H, arom H),
7.46–7.50 and 8.41–8.44 (m, 3H, py H). Calcd for C23H15BrClN3O4S:
C, 50.71; H, 2.78; N, 7.71; S, 5.89. Found: C, 50.36; H, 2.90; N, 7.57; S,
5.69.
6.1.2.2. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(2-methylbenzoyl)thiocarbamate 2. Mp 114–116 ꢁC (from
CH2Cl2/Et2O); yield: 60%. IR (KBr) cmꢀ1 1776, 1708. 1H NMR (CDCl3)
d
2.41 (s, 3H, CH3), 3.75 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.49 (t, J ¼ 6.0 Hz,
2H, CH2O), 6.94–7.97 (m, 12H, arom H). Calcd for C25H19ClN2O4S: C,
62.69; H, 4.00; N, 5.85; S, 6.69. Found: C, 62.43; H, 4.22; N, 5.66; S,
6.82.
6.1.2.11. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2-
methylbenzoyl(4-methylphenyl)thiocarbamate 11. Mp 135–137 ꢁC
(from CH2Cl2/EtOH); yield: 27%. IR (KBr) cmꢀ1 1775, 1715, 1682. 1H
NMR (CDCl3)
d 2.32 (s, 3H, CH3), 2.42 (s, 3H, CH3/acyl), 3.71 (t,
6.1.2.3. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(2-nitrobenzoyl)thiocarbamate 3. Mp 178–179 ꢁC (from
CH2Cl2/EtOH); yield: 69%. IR (KBr) cmꢀ1 1768, 1717. 1H NMR (CDCl3)
J ¼ 6.0 Hz, 2H, CH2N), 4.48 (t, J ¼ 6.0 Hz, 2H, CH2O), 6.98–8.02 (m,
12H, arom H). Calcd for C26H22N2O4S: C, 68.11; H, 4.84; N, 6.11; S,
6.99. Found: C, 68.36; H, 4.86; N, 6.14; S, 6.79.
d
3.77 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.53 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.28–
8.38 (m, 12H, arom H). Calcd for C24H16ClN3O6S: C, 56.53; H, 3.16; N,
8.24; S, 6.29. Found: C, 56.24; H, 3.14; N, 8.21; S, 6.19.
6.1.2.12. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
methylphenyl(2-nitrobenzoyl)thiocarbamate 12. Mp 179–181 ꢁC
(from CH2Cl2/EtOH); yield: 43%. IR (KBr) cmꢀ1 1768, 1717. 1H NMR
6.1.2.4. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(3-methylbenzoyl)thiocarbamate 4. Mp 134–136 ꢁC (from
CH2Cl2/EtOH); yield: 88%. IR (KBr) cmꢀ1 1777, 1709. 1H NMR (CDCl3)
(CDCl3)
d
2.33 (s, 3H, CH3), 3.76 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.54 (t,
J ¼ 6.0 Hz, 2H, CH2O), 7.00–8.34 (m, 12H, arom H). Calcd for
C25H19N3O6S: C, 61.34; H, 3.91; N, 8.58; S, 6.55. Found: C, 61.41; H,
3.89; N, 8.62; S, 6.50.
d
2.36 (s, 3H, CH3), 3.83 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.63 (t, J ¼ 6.0 Hz,
2H, CH2O), 7.08–7.94 (m, 12H, arom H). Calcd for C25H19ClN2O4S: C,
62.69; H, 4.00; N, 5.85; S, 6.69. Found: C, 62.54; H, 4.03; N, 5.76; S,
6.42.
6.1.2.13. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3-
methylbenzoyl(4-methylphenyl)thiocarbamate 13. Mp 137–139 ꢁC
(from CH2Cl2/EtOH); yield: 42%. IR (KBr) cmꢀ1 1775, 1717. 1H NMR
6.1.2.5. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chlor-
ophenyl(3-nitrobenzoyl)thiocarbamate 5. Mp 167–169 ꢁC (from
CH2Cl2/EtOH); yield: 83%. IR (KBr) cmꢀ1 1777, 1711. 1H NMR (CDCl3)
(CDCl3)
d
2.20–2.47 (m, 6H, 2CH3), 3.77 (t, J ¼ 6.0 Hz, 2H, CH2N),
4.61 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.11–8.19 (m, 12H, arom H). Calcd for
C26H22N2O4S: C, 68.11; H, 4.84; N, 6.11; S, 6.99. Found: C, 68.12; H,
4.82; N, 6.10; S, 6.68.
d
4.00 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.68 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.21–

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
2197
6.1.2.14. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
57.55; H, 3.02; N, 5.59; S, 6.40. Found: C, 57.20; H, 3.13; N, 5.55; S,
6.18.
methylphenyl(3-nitrobenzoyl)thiocarbamate 14. Mp 178–179 ꢁC
(from CH2Cl2/EtOH); yield: 40%. IR (KBr) cmꢀ1 1771, 1712, 1688. 1H
NMR (CDCl3)
d
2.30 (s, 3H, CH3), 3.87 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.67
6.1.2.23. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
chlorophenyl(3,4-difluorobenzoyl)thiocarbamate 23. Mp
4-
188–
(t, J ¼ 6.0 Hz, 2H, CH2O), 7.07–8.77 (m, 12H, arom H). Calcd for
C25H19N3O6S: C, 61.34; H, 3.91; N, 8.58; S, 6.55. Found: C, 61.31; H,
3.91; N, 8.52; S, 6.55.
190 ꢁC (from CH2Cl2/EtOH); yield: 45%. IR (KBr) cmꢀ1 1777, 1710.
1H NMR (CDCl3)
d 3.78–4.08 (m, 2H, CH2N), 4.50–4.81 (m, 2H,
CH2O), 7.05–8.06 (m, 11H, arom H). Calcd for C24H15ClF2N2O4S: C,
57.55; H, 3.02; N, 5.59; S, 6.40. Found: C, 57.84; H, 2.90; N, 5.57;
S, 6.38.
6.1.2.15. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
methylbenzoyl(4-methylphenyl)thiocarbamate 15. Mp 142–144 ꢁC
(from CH2Cl2/EtOH); yield: 48%. IR (KBr) cmꢀ1 1773, 1710. 1H NMR
(CDCl3)
d
2.29 (s, 6H, 2CH3), 3.86 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.64 (t,
6.1.2.24. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
J ¼ 6.0 Hz, 2H, CH2O), 6.97–7.97 (m, 12H, arom H). Calcd for
C26H22N2O4S: C, 68.11; H, 4.84; N, 6.11; S, 6.99. Found: C, 68.18; H,
4.91; N, 6.16; S, 6.77.
chlorophenyl(3,5-difluorobenzoyl)thiocarbamate 24. Mp 148–149 ꢁC
(from CH2Cl2/EtOH); yield: 65%. IR (KBr) cmꢀ1 1776, 1715, 1695. 1H
NMR (CDCl3)
d
3.85 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.65 (t, J ¼ 6.0 Hz, 2H,
CH2O), 6.91–8.01 (m, 11H, arom H). Calcd for C24H15ClF2N2O4S: C,
57.55; H, 3.02; N, 5.59; S, 6.40. Found: C, 57.50; H, 3.19; N, 5.51; S,
6.32.
6.1.2.16. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
methylphenyl(4-nitrobenzoyl)thiocarbamate 16. Mp 170–172 ꢁC
(from CH2Cl2/EtOH); yield: 25%. IR (KBr) cmꢀ1 1776, 1709. 1H NMR
(CDCl3)
d
2.30 (s, 3H, CH3), 3.90 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.70 (t,
6.1.2.25. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
J ¼ 6.0 Hz, 2H, CH2O), 7.08–8.39 (m, 12H, arom H). Calcd for
C25H19N3O6S: C, 61.34; H, 3.91; N, 8.58; S, 6.55. Found: C, 61.34; H,
3.91; N, 8.56; S, 6.47.
chlorophenyl(2,3-dichlorobenzoyl)thiocarbamate 25. Mp 189–191 ꢁC
(from CH2Cl2/EtOH); yield: 37%. IR (KBr) cmꢀ1 1773, 1706. 1H NMR
(CDCl3)
d
3.80 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.57 (t, J ¼ 6.0 Hz, 2H, CH2O),
6.78–8.05 (m, 11H, arom H). Calcd for C24H15Cl3N2O4S: C, 54.00; H,
2.83; N, 5.25; S, 6.01. Found: C, 53.93; H, 2.84; N, 5.18; S, 6.21.
6.1.2.17. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3,4-
dichlorobenzoyl(phenyl)thiocarbamate 17. Mp 127–129 ꢁC (from
CH2Cl2/Et2O); yield: 57%. IR (KBr) cmꢀ1 3088, 3071, 1777, 1715. 1H
6.1.2.26. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
chlorophenyl(2,4-dichlorobenzoyl)thiocarbamate 26. Mp
4-
123–
NMR (DMSO)
d
3.85 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.61 (t, J ¼ 6.0 Hz, 2H,
CH2O), 7.20–8.10 (m, 12H, arom H). Calcd for C24H16Cl2N2O4S: C,
57.73; H, 3.23; N, 5.61; S, 6.42. Found: C, 57.64; H, 3.10; N, 5.32; S,
6.12.
125 ꢁC (from CH2Cl2/EtOH); yield: 37%. IR (KBr) cmꢀ1 1769, 1713,
1698. 1H NMR (CDCl3)
J ¼ 6.0 Hz, 2H, CH2O), 7.00–8.10 (m, 11H, arom H). Calcd for
C24H15Cl3N2O4S: C, 54.00; H, 2.83; N, 5.25; S, 6.01. Found: C, 53.78;
H, 3.00; N, 5.11; S, 5.88.
d
3.81 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.60 (t,
6.1.2.18. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chloro-3-nitrobenzoyl(phenyl)thiocarbamate 18. Mp 120–121 ꢁC
(from CH2Cl2/Et2O); yield: 12%. IR (KBr) cmꢀ1 1775, 1717. 1H NMR
6.1.2.27. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
(DMSO)
d
3.82 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.61 (t, J ¼ 6.0 Hz, 2H, CH2O),
chlorophenyl(2,6-dichlorobenzoyl)thiocarbamate 27. Mp 154–156 ꢁC
7.08–8.48 (m, 12H, arom H). Calcd for C24H16ClN3O6S: C, 56.53; H,
3.16; N, 8.24; S, 6.29. Found: C, 56.39; H, 3.00; N, 8.28; S, 6.10.
(from CH2Cl2/EtOH); yield: 43%. IR (KBr) cmꢀ1 1767, 1706. 1H NMR
(CDCl3)
d
3.84 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.68 (t, J ¼ 6.0 Hz, 2H, CH2O),
7.00–8.20 (m, 11H, arom H). Calcd for C24H15Cl3N2O4S: C, 54.00; H,
2.83; N, 5.25; S, 6.01. Found: C, 53.78; H, 2.92; N, 5.18; S, 5.82.
6.1.2.19. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chlorophenyl(2,3-difluorobenzoyl)thiocarbamate 19. Mp 149–150 ꢁC
(from CH2Cl2/EtOH); yield: 82%. IR (KBr) cmꢀ1 1776, 1708. 1H NMR
6.1.2.28. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
(CDCl3)
d
3.86 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.65 (t, J ¼ 6.0 Hz, 2H, CH2O),
chlorophenyl(3,4-dichlorobenzoyl)thiocarbamate 28. Mp 162–164 ꢁC
6.81–8.02 (m, 11H, arom H). Calcd for C24H15ClF2N2O4S: C, 57.55; H,
3.02; N, 5.59; S, 6.40. Found: C, 57.35; H, 2.98; N, 5.54; S, 6.62.
(from CH2Cl2/EtOH); yield: 70%. IR (KBr) cmꢀ1 1777, 1710. 1H NMR
(CDCl3)
d
3.97 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.65 (t, J ¼ 6.0 Hz, 2H, CH2O),
7.01–8.21 (m, 11H, arom H). Calcd for C24H15Cl3N2O4S: C, 54.00; H,
2.83; N, 5.25; S, 6.01. Found: C, 54.14; H, 2.65; N, 5.13; S, 6.06.
6.1.2.20. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chlorophenyl(2,4-difluorobenzoyl)thiocarbamate 20. Mp 121–123 ꢁC
(from Et2O/EtOH); yield: 70%. IR (KBr) cmꢀ1 1777, 1713. 1H NMR
6.1.2.29. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
(CDCl3)
d
3.87 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.58 (t, J ¼ 6.0 Hz, 2H, CH2O),
chlorophenyl(3,5-dichlorobenzoyl)thiocarbamate 29. Mp 145–147 ꢁC
7.13–8.01 (m, 11H, arom H). Calcd for C24H15ClF2N2O4S: C, 57.55; H,
3.02; N, 5.59; S, 6.40. Found: C, 57.72; H, 3.27; N, 5.45; S, 6.29.
(from CH2Cl2/EtOH); yield: 73%. IR (KBr) cmꢀ1 1778, 1715. 1H NMR
(CDCl3)
d
3.94 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.61 (t, J ¼ 6.0 Hz, 2H, CH2O),
7.18–8.08 (m, 11H, arom H). Calcd for C24H15Cl3N2O4S: C, 54.00; H,
2.83; N, 5.25; S, 6.01. Found: C, 54.28; H, 2.96; N, 5.02; S, 6.18.
6.1.2.21. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chlorophenyl(2,5-difluorobenzoyl)thiocarbamate 21. Mp 148–149 ꢁC
(from CH2Cl2/EtOH); yield: 56%. IR (KBr) cmꢀ1 1778, 1711, 1683. 1H
6.1.2.30. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
NMR (CDCl3)
d
3.84 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.65 (t, J ¼ 6.0 Hz, 2H,
chlorophenyl(2,6-dimethoxybenzoyl)thiocarbamate 30. Mp 158–
CH2O), 6.89–8.10 (m, 11H, arom H). Calcd for C24H15ClF2N2O4S: C,
57.55; H, 3.02; N, 5.59; S, 6.40. Found: C, 57.18; H, 3.07; N, 5.52; S,
6.37.
159 ꢁC (from CH2Cl2/Et2O); yield: 45%. IR (KBr) cmꢀ1 1777, 1716,
1597. 1H NMR (CDCl3)
d 3.50 (m, 8H, CH2N and 2CH3O), 4.51 (t,
J ¼ 6.0 Hz, 2H, CH2O), 6.31–6.68 and 7.01–7.98 (m, 11H, arom H).
Calcd for C26H21ClN2O6S: C, 59.49; H, 4.03; N, 5.34; S, 6.11. Found: C,
59.48; H, 4.23; N, 5.52; S, 5.96.
6.1.2.22. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chlorophenyl(2,6-difluorobenzoyl)thiocarbamate 22. Mp 135–137 ꢁC
(from CH2Cl2/EtOH); yield: 48%. IR (KBr) cmꢀ1 1775, 1709, 1692. 1H
6.1.2.31. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2-
NMR (CDCl3)
d
3.87 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.63 (t, J ¼ 6.0 Hz, 2H,
chloro-4-nitrobenzoyl(4-chlorophenyl)thiocarbamate 31. Mp 168–
CH2O), 6.61–8.02 (m, 11H, arom H). Calcd for C24H15ClF2N2O4S: C,
170 ꢁC (from CH2Cl2/EtOH); yield: 50%. IR (KBr) cmꢀ1 1772, 1715,

2198
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
1695. 1H NMR (CDCl3)
J ¼ 6.0 Hz, 2H, CH2O), 7.10–8.32 (m, 11H, arom H). Calcd for
C24H15Cl2N3O6S: C, 52.95; H, 2.78; N, 7.72; S, 5.89. Found: C, 52.61;
H, 2.83; N, 7.65; S, 5.74.
d
3.62 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.60 (t,
C
25H18F2N2O4S: C, 62.49; H, 3.78; N, 5.83; S, 6.67. Found: C, 62.22;
H, 3.80; N, 5.70; S, 6.50.
6.1.2.40. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,3-
dichlorobenzoyl(4-methylphenyl)thiocarbamate 40. Mp 114–116 ꢁC
6.1.2.32. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2-
(from CH2Cl2/EtOH); yield: 32%. IR (KBr) cmꢀ1 1773, 1715, 1685. 1H
chloro-5-nitrobenzoyl(4-chlorophenyl)thiocarbamate 32. Mp 109–
NMR (CDCl3)
d
2.31 (s, 3H, CH3), 3.78 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.54
110 ꢁC (from CH2Cl2/EtOH); yield: 30%. IR (KBr) cmꢀ1 1776, 1710. 1H
(t, J ¼ 6.0 Hz, 2H, CH2O), 7.09–8.01 (m, 11H, arom H). Calcd for
C25H18Cl2N2O4S: C, 58.49; H, 3.53; N, 5.46; S, 6.24. Found: C, 58.69;
H, 3.62; N, 5.38; S, 6.26.
NMR (CDCl3)
d 3.68–4.00 (m, 2H, CH2N), 4.41–4.79 (m, 2H, CH2O),
7.21–8.48 (m, 11H, arom H). Calcd for C24H15Cl2N3O6S: C, 52.95; H,
2.78; N, 7.72; S, 5.89. Found: C, 53.16; H, 2.88; N, 7.50; S, 5.84.
6.1.2.41. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,4-
6.1.2.33. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
dichlorobenzoyl(4-methylphenyl)thiocarbamate 41. Mp 112–114 ꢁC
chloro-3-nitrobenzoyl(4-chlorophenyl)thiocarbamate 33. Mp 119–
(from CH2Cl2/EtOH); yield: 47%. IR (KBr) cmꢀ1 1777, 1711. 1H NMR
121 ꢁC (from CH2Cl2/EtOH); yield: 41%. IR (KBr) cmꢀ1 1777, 1712. 1H
(CDCl3)
d
2.31 (s, 3H, CH3), 3.81 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.59 (t,
NMR (CDCl3)
d
3.96 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.71 (t, J ¼ 6.0 Hz, 2H,
J ¼ 6.0 Hz, 2H, CH2O), 7.07–7.96 (m, 11H, arom H). Calcd for
C25H18Cl2N2O4S: C, 58.49; H, 3.53; N, 5.46; S, 6.24. Found: C, 58.39;
H, 3.64; N, 5.38; S, 6.15.
CH2O), 7.01–8.50 (m, 11H, arom H). Calcd for C24H15Cl2N3O6S: C,
52.95; H, 2.78; N, 7.72; S, 5.89. Found: C, 52.77; H, 3.08; N, 7.54; S,
5.72.
6.1.2.42. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,6-
6.1.2.34. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
dichlorobenzoyl(4-methylphenyl)thiocarbamate 42. Mp 162–164 ꢁC
bromo-3-methylbenzoyl(4-chlorophenyl) thiocarbamate 34. Mp
(from CH2Cl2/EtOH); yield: 20%. IR (KBr) cmꢀ1 1771, 1713. 1H NMR
138–140 ꢁC (from CH2Cl2/Et2O); yield: 44%. IR (KBr) cmꢀ1 1776,
(CDCl3)
d
2.36 (s, 3H, CH3), 3.82 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.68 (t,
1710. 1H NMR (CDCl3)
d
2.41 (s, 3H, CH3), 3.88 (t, J ¼ 6.0 Hz, 2H,
J ¼ 6.0 Hz, 2H, CH2O), 7.01–8.04 (m, 11H, arom H). Calcd for
C25H18Cl2N2O4S: C, 58.49; H, 3.53; N, 5.46; S, 6.24. Found: C, 58.34;
H, 3.55; N, 5.40; S, 6.01.
CH2N), 4.67 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.20–7.94 (m, 11H, arom H).
Calcd for C25H18BrClN2O4S: C, 53.83; H, 3.25; N, 5.02; S, 5.75. Found:
C, 53.64; H, 3.23; N, 4.98; S, 5.85.
6.1.2.43. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3,4-
6.1.2.35. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
dichlorobenzoyl(4-methylphenyl)thiocarbamate 43. Mp 126–128 ꢁC
chlorophenyl(2,5-dimethyl-3-furoyl)thiocarbamate 35. Mp 118–
(from CH2Cl2/EtOH); yield: 58%. IR (KBr) cmꢀ1 1774, 1709. 1H NMR
119 ꢁC (from Et2O); yield: 39%. IR (KBr) cmꢀ1 1775, 1714. 1H NMR
(CDCl3)
d
2.30 (s, 3H, CH3), 3.91 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.70 (t,
(CDCl3)
d
1.99 (s, 3H, CH3), 2.37 (s, 3H, CH3), 3.90 (t, J ¼ 5.6 Hz,
J ¼ 6.0 Hz, 2H, CH2O), 7.09–8.08 (m, 11H, arom H). Calcd for
C25H18Cl2N2O4S: C, 58.49; H, 3.53; N, 5.46; S, 6.24. Found: C, 58.17;
H, 3.72; N, 5.17; S, 6.21.
CH2N), 4.63 (t, J ¼ 5.6 Hz, CH2O), 5.94 (s, 1H, CH fur.), 7.09–7.24 (m,
4H, arom H), 7.66–7.79 (m, 4 H, phtha. arom H). Calcd for
C24H19ClN2O5S: C, 59.69; H, 3.97; N, 5.80; S, 6.64. Found: C, 59.35;
H, 4.21; N, 5.86; S, 6.60.
6.1.2.44. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,6-
dimethoxybenzoyl(4-methylphenyl)thiocarbamate 44. Mp 115–
6.1.2.36. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,4-
117 ꢁC (from CH2Cl2/EtOH); yield: 18%. IR (KBr) cmꢀ1 1770, 1709. 1H
NMR (CDCl3) d 2.30 (s, 3H, CH3), 3.54–3.91 (m, 8H, CH2N and
2CH3O), 4.50 (t, J ¼ 6.0 Hz, 2H, CH2O), 6.36–6.63 and 7.01–7.97 (m,
11H, arom H). Calcd for C27H24N2O6S: C, 64.24; H, 4.79; N, 5.55; S,
6.35. Found: C, 64.12; H, 4.87; N, 5.58; S, 6.15.
difluorobenzoyl(4-methylphenyl)thiocarbamate 36. Mp 135–137 ꢁC
(from Et2O/EtOH); yield: 27%. IR (KBr) cmꢀ1 1773, 1715, 1691. 1H
NMR (CDCl3)
d
2.32 (s, 3H, CH3), 3.84 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.68
(t, J ¼ 6.0 Hz, 2H, CH2O), 6.61–7.26 and 7.75–7.96 (m, 11H, arom H).
Calcd for C25H18F2N2O4S: C, 62.49; H, 3.78; N, 5.83; S, 6.67. Found: C,
62.21; H, 3.77; N, 5.55; S, 6.50.
6.1.2.45. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2-
chloro-4-nitrobenzoyl(4-methylphenyl)thiocarbamate 45. Mp 141–
6.1.2.37. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,5-
143 ꢁC (from CH2Cl2/EtOH); yield: 39%. IR (KBr) cmꢀ1 1772, 1715,
difluorobenzoyl(4-methylphenyl)thiocarbamate 37. Mp 144–146 ꢁC
1691. 1H NMR (CDCl3)
d
2.31 (s, 3H, CH3), 3.87 (t, J ¼ 6.0 Hz, 2H,
(from CH2Cl2/EtOH); yield: 40%. IR (KBr) cmꢀ1 1772, 1715, 1681. 1H
CH2N), 4.66 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.11–7.29 and 7.72–8.31 (m,
11H, arom H). Calcd for C25H18ClN3O6S: C, 57.31; H, 3.46; N, 8.02; S,
6.12. Found: C, 57.32; H, 3.26; N, 7.90; S, 6.23.
NMR (CDCl3)
d
2.29 (s, 3H, CH3), 3.82 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.66
(t, J ¼ 6.0 Hz, 2H, CH2O), 6.85–8.00 (m, 11H, arom H). Calcd for
C25H18F2N2O4S: C, 62.49; H, 3.78; N, 5.83; S, 6.67. Found: C, 62.35;
H, 3.74; N, 5.82; S, 6.55.
6.1.2.46. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chloro-3-nitrobenzoyl(4-methylphenyl)thiocarbamate 46. Mp 135–
6.1.2.38. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,6-
137 ꢁC (from CH2Cl2/EtOH); yield: 26%. IR (KBr) cmꢀ1 1772, 1717. 1H
difluorobenzoyl(4-methylphenyl)thiocarbamate 38. Mp 146–148 ꢁC
NMR (CDCl3)
d
2.30 (s, 3H, CH3), 3.98 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.76
(from CH2Cl2/Et2O); yield: 32%. IR (KBr) cmꢀ1 1766, 1711. 1H NMR
(t, J ¼ 6.0 Hz, 2H, CH2O), 7.08–7.28 and 7.68–8.52 (m, 11H, arom H).
Calcd for C25H18ClN3O6S: C, 57.31; H, 3.46; N, 8.02; S, 6.12. Found: C,
57.34; H, 3.39; N, 7.82; S, 6.15.
(CDCl3)
d
2.29 (s, 3H, CH3), 3.84 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.63 (t,
J ¼ 6.0 Hz, 2H, CH2O), 6.65–8.01 (m, 11H, arom H). Calcd for
C25H18F2N2O4S: C, 62.49; H, 3.78; N, 5.83; S, 6.67. Found: C, 62.21;
H, 3.63; N, 5.74; S, 6.94.
6.1.2.47. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
bromo-3-methylbenzoyl(4-methylphenyl)thiocarbamate
ꢀ417. Mp
6.1.2.39. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3,5-
174–176 ꢁC (from CH2Cl2/EtOH); yield: 40%. IR (KBr) cm 1772,
difluorobenzoyl(4-methylphenyl)thiocarbamate 39. Mp 132–134 ꢁC
1710. 1H NMR (CDCl3)
d 2.30 (s, 3H, CH3), 2.40 (s, 3H, CH3/acyl), 3.87
(from CH2Cl2/EtOH); yield: 45%. IR (KBr) cmꢀ1 1779, 1713. 1H NMR
(t, J ¼ 6.0 Hz, 2H, CH2N), 4.68 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.05–7.97 (m,
12H, arom H). Calcd for C26H21BrN2O4S: C, 58.11; H, 3.94; N, 5.21; S,
5.97. Found: C, 58.38; H, 4.05; N, 5.19; S, 6.07.
(CDCl3)
d
2.31 (s, 3H, CH3), 3.90 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.69 (t,
J ¼ 6.0 Hz, 2H, CH2O), 7.03–7.92 (m, 12H, arom H). Calcd for

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
2199
6.1.2.48. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
(t, J ¼ 6.0 Hz, 2H, CH2O), 7.16–8.41 (m, 11H, arom H). Calcd for
C25H18ClN3O6S: C, 57.31; H, 3.46; N, 8.02; S, 6.12. Found: C, 57.00; H,
3.24; N, 7.99; S, 5.96.
chlorophenyl(1-naphthoyl)thiocarbamate 48. Mp 139–140 ꢁC (from
CH2Cl2/EtOH); yield: 41%. IR (KBr) cmꢀ1 1776, 1710. 1H NMR (CDCl3)
d
3.60 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.36 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.27–
8.42 (m,15H, arom H). Calcd for C28H19ClN2O4S: C, 65.30; H, 3.72; N,
5.44; S, 6.23. Found: C, 63.05; H, 3.75; N, 5.40; S, 6.34.
6.1.3. General parallel synthetic procedure P2 for the preparation of
ATCs 50, 51, 58, 60–66
60% sodium hydride dispersion in mineral oil (0.44 g,
w10 mmol) was poured in a single portion into each of the 12-
Carousel Reaction StationÔ tubes containing a stirred, dry DMF
(25 mL), ice-cooled solution of starting alcohols A1, A2, A4, A8
(10 mmol) and isothiocyanates B4, B7–12 (10 mmol). Each reaction
mixture was allowed to react for 1 h under stirring and then TMEDA
(1.28 g, 11 mmol) and the proper acyl chloride (11 mmol) were
added. The resulting mixtures were stirred at rt for 12 h [for 58: 6 h.
In the case of 60–62, 64 and 65 the reaction mixtures were heated
at 65–70 ꢁC for 1.5 h and then kept under stirring at room
temperature for 12 h. For 50 an extra aliquot of acyl chloride
(11 mmol) and TMEDA (11 mmol) was added and then the mixture
was heated at 75 ꢁC for 15 min]. Each reaction mixture was trans-
ferred into a set of separating funnels, diluted with water (150 mL)
and extracted with dichloromethane (30 mL ꢃ 2). The combined
extracts were washed with water (20 mL ꢃ 4) and 1 M HCl
(20 mL ꢃ 2) (except for 50 and 58), dried, filtered through a plug of
Florisil (diameter 5 ꢃ 2 cm) and evaporated under reduced pres-
sure to give a residue that was purified by crystallization from the
suitable solvent or solvent mixture.
6.1.2.49. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-
chlorophenyl(2-naphthoyl)thiocarbamate 49. Mp 163–165 ꢁC (from
CH2Cl2/EtOH); yield: 48%. IR (KBr) cmꢀ1 1777, 1709. 1H NMR (CDCl3)
d
3.84 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.66 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.28–
8.16 (m, 15H, arom H). Calcd for C28H19ClN2O4S: C, 65.30; H, 3.72; N,
5.44; S, 6.23. Found: C, 65.05; H, 3.93; N, 5.40; S, 6.37.
6.1.2.50. (ꢂ)O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]
4-chlorophenyl(3-nitrobenzoyl)thiocarbamate 52. Mp 173–175 ꢁC
(from CH2Cl2/EtOH); yield: 39%. IR (KBr) cmꢀ1 1777, 1708. 1H NMR
(CDCl3)
d
1.37 (d, J ¼ 6.0 Hz, 3H, CH3), 4.56–5.01 (m, 3H, CH and
CH2O), 7.06–7.90 (m, 12H, arom H). Calcd for C25H18ClN3O6S: C,
57.31; H, 3.46; N, 8.02; S, 6.12. Found: C, 57.64; H, 3.59; N, 7.93; S,
6.04.
6.1.2.51. (ꢂ)O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-1-meth-
ylethyl] 4-chlorophenyl(3-nitrobenzoyl)thiocarbamate 53. Mp 159–
161 ꢁC (from CH2Cl2/Et2O); yield: 13%. IR (KBr) cmꢀ1 1780, 1713. 1H
NMR (CDCl3)
d
1.05 (d, J ¼ 6.0 Hz, 3H, CH3), 3.65–4.00 (m, 3H, CH
and CH2N), 7.20–8.74 (m, 12H, arom H). Calcd for C25H18ClN3O6S: C,
57.31; H, 3.46; N, 8.02; S, 6.12. Found: C, 57.02; H, 3.78; N, 7.93; S,
6.32.
6.1.3.1. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 1-ben-
zothien-2-ylcarbonyl(4-chlorophenyl)thiocarbamate 50. Mp 145–
147 ꢁC (from Et2O); yield: 61%. IR (KBr) cmꢀ1 1777, 1713. 1H NMR
6.1.2.52. O-[3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]
4-
(CDCl3)
d
3.80 (t, J ¼ 4.8 Hz, 2H, CH2N), 4.77 (t, J ¼ 4.8 Hz, 2H, CH2O),
chlorophenyl(3-nitrobenzoyl)thiocarbamate 54. Mp 144–146 ꢁC
7.20–7.50 and 7.60–8.08 (m, 13H, arom H). Calcd for
C26H17ClN2O4S2: C, 59.94; H, 3.29; N, 5.38; S, 12.31. Found: C, 60.32;
H, 3.28; N, 5.26; S, 12.05.
(from CH2Cl2/Et2O); yield: 16%. IR (KBr) cmꢀ1 1772, 1718, 1699. 1H
NMR (CDCl3)
d 1.53–2.17 (m, 2H, C–CH2–C), 3.25–3.73 (m, 2H,
CH2N), 4.12–4.54 (m, 2H, CH2O), 7.07–8.75 (m, 12H, arom H). Calcd
for C25H18ClN3O6S: C, 57.31; H, 3.46; N, 8.02; S, 6.12. Found: C,
57.68; H, 3.56; N, 8.08; S, 6.25.
6.1.3.2. O-[(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl] 4-chlor-
ophenyl(3-nitrobenzoyl)thiocarbamate 51. Mp 135–137 ꢁC (from
CH2Cl2/EtOH); yield: 18%. IR (KBr) cmꢀ1 1782, 1722. 1H NMR (CDCl3)
6.1.2.53. O-[2-(4-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-chlorophenyl(3-nitrobenzoyl)thiocarbamate 55. Mp 140–142 ꢁC
(from CH2Cl2/EtOH); yield: 36%. IR (KBr) cmꢀ1 1766, 1707, 1691. 1H
d 5.82 (s, 2H, CH2), 7.17–8.47 (m, 12H, arom H). Calcd for
C23H14ClN3O6S: C, 55.71; H, 2.85; N, 8.47; S, 6.47. Found: C, 55.91; H,
3.03; N, 8.40; S, 6.44.
NMR (CDCl3)
d
2.70 (s, 3H, CH3), 3.88 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.69 (t,
J ¼ 6.0 Hz, 2H, CH2O), 7.02–8.68 (m, 11H, arom H). Calcd for
C25H18ClN3O6S: C, 57.31; H, 3.46; N, 8.02; S, 6.12. Found: C, 57.18; H,
3.69; N, 7.89; S, 6.30.
6.1.3.3. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-chlorophenyl[(2-chloropyridin-3-yl)carbonyl]thiocarbamate 58. Mp
156–158 ꢁC (from CH2Cl2/EtOH); yield: 68%. IR (KBr) cmꢀ11773, 1710.
1H NMR (CDCl3)
d
2.56 (s, 3H, CH3), 3.79 (t, J ¼ 5.2 Hz, 2H, CH2N), 4.58
6.1.2.54. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-chlorophenyl(3-nitrobenzoyl)thiocarbamate 56. Mp 169–170 ꢁC
(from CH2Cl2/EtOH); yield: 47%. IR (KBr) cmꢀ1 1773, 1712. 1H NMR
(t, J ¼ 5.2 Hz, 2H, CH2O), 7.22–7.35 (m, 5H, arom H), 7.56–7.87 (m, 4H,
arom H), 8.44 (m,1H, arom H). Calcd for C24H17Cl2N3O4S: C, 54.99; H,
3.41; N, 8.36; S, 6.38. Found: C, 54.90; H, 3.39; N, 7.99; S, 6.16.
(CDCl3)
d
2.66 (s, 3H, CH3), 3.88 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.62 (t,
J ¼ 6.0 Hz, 2H, CH2O), 7.10–7.80 (m, 11H, arom H). Calcd for
C25H18ClN3O6S: C, 57.31; H, 3.46; N, 8.02; S, 6.12. Found: C, 57.13; H,
3.52; N, 8.00; S, 6.25.
6.1.3.4. O-{2-[(3aR,7aS)-1,3-Dioxo-1S,3,3a,4,7,7a-hexahydro-2H-iso-
indol-2-yl]ethyl}
60. Mp 155–157 ꢁC (from CH2Cl2/EtOH); yield: 48%. IR (KBr) cmꢀ1
2950, 1787, 1695. 1H NMR (CDCl3)
1.94–2.58 (m, 4H, 2CH2), 2.88–
4-chlorobenzoyl(4-chlorophenyl)thiocarbamate
d
6.1.2.55. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-chlorophenyl(thien-2-ylcarbonyl)thiocarbamate 57. Mp 142–143 ꢁC
(from CH2Cl2/EtOH); yield: 46%. IR (KBr) cmꢀ1 1775, 1716. 1H NMR
3.17 (m, 2H, 2CH), 3.68 (t, J ¼ 6 Hz, 2H, CH2N), 4.54 (t, J ¼ 6 Hz, 2H,
CH2O), 5.80–6.01 (m, 2H, 2CH ¼ C), 7.08–8.29 (m, 8H, arom H). Calcd
for C24H20Cl2N2O4S: C, 57.25; H, 4.00; N, 5.56; S, 6.37. Found: C,
56.98; H, 3.90; N, 5.34; S, 6.05.
(DMSO)
d
2.51 (s, 3H, CH3), 3.89 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.79
(t, J ¼ 6.0 Hz, 2H, CH2O), 6.98–8.09 (m, 10H, 7 arom H and 3H thioph).
Calcd for C23H17ClN2O4S2: C, 56.96; H, 3.53; N, 5.78; S, 13.22. Found:
C, 57.10; H, 3.31; N, 5.75; S, 13.00.
6.1.3.5. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,3-
dichlorophenyl(2-furoyl)thiocarbamate 61. Mp 163–165 ꢁC (from
CH2Cl2/EtOH); yield: 49%. IR (KBr) cmꢀ1 3128, 3068, 1776, 1716,
6.1.2.56. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-chlorobenzoyl(4-nitrophenyl)thiocarbamate 59. Mp 194–197 ꢁC
(from CH2Cl2/EtOH); yield: 34%. IR (KBr) cmꢀ1 1770, 1714. 1H NMR
1684. 1H NMR (CDCl3)
d
3.92 (t, J ¼ 6 Hz, 2H, CH2N), 4.78 (t, J ¼ 6 Hz,
2H, CH2O), 6.30–6.45 (m, 1H, H-4, fur), 7.02–8.09 (m, 9H, 7 arom H
and H-3 and H-5 fur). Calcd for C22H14Cl2N2O5S: C, 54.00; H, 2.88;
N, 5.72; S, 6.55. Found: C, 54.15; H, 2.87; N, 5.71; S, 6.25.
(CDCl3)
d
2.56 (s, 3H, CH3), 3.88 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.66

2200
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
6.1.3.6. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,4-
RT/PETT complex solved by Ren et al. [28] (PDB code: 1DTQ) was
used as starting structure. The ligands were built by Insight II
(Builder module) and parameterized according to the CVFF force
field [32]. The docking calculations were carried out as previously
reported [21–23]. The RT–ATC complexes were energy-minimized
by CNS [33], performing a cycle of simulated annealing (starting
temperature 1000 K) followed by 120 cycles of Powell minimiza-
tion. All the calculations were performed on Silicon Graphic Indigo2
and Origin 200 workstations. Model analyses were carried out
using CCP4 (Collaborative Computational Project Number 4)
program suite [34]. The programs Molscript [35] and Raster3D [36]
were used for drawing the figures.
dichlorophenyl(2-furoyl)thiocarbamate 62. Mp 130–132 ꢁC (from
CH2Cl2/EtOH); yield: 74%. IR (KBr) cmꢀ1 1771, 1716, 1688. 1H NMR
(CDCl3)
d
3.95 (t, J ¼ 6 Hz, 2H, CH2N), 4.70 (t, J ¼ 6 Hz, 2H, CH2O),
6.31–6.48 (m, 1H, H-4, fur), 7.05–7.98 (m, 9H, 7 arom H and H-3 and
H-5 fur). Calcd for C22H14Cl2N2O5S: C, 54.00; H, 2.88; N, 5.72; S,
6.55. Found: C, 53.88; H, 2.77; N, 5.70; S, 6.20.
6.1.3.7. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
2,6-
dichlorophenyl(3-nitrobenzoyl)thiocarbamate 63. Mp 185–187 ꢁC
(from CH2Cl2/EtOH); yield: 45%. IR (KBr) cmꢀ11774,1713,1690.1H NMR
(CDCl3)
d
3.77 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.63 (t, J ¼ 6.0 Hz, 2H, CH2O),
7.25–8.92 (m,11H, arom H). Calcd for C25H15Cl2N3O6S: C, 52.95; H, 2.78;
N, 7.72; S, 5.89. Found: C, 52.72; H, 2.79; N, 7.62; S, 5.72.
Acknowledgements
6.1.3.8. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3,4-
This work was supported by MURST (Cofinanziamento Nazio-
nale 2004). Fondazione Banca Carige is gratefully acknowledged for
financially supporting SC. The authors thank Mr. O. Gagliardo for
the microanalyses, Mr. F. Tuberoni, Dr. C. Rossi and Dr. R. Raggio for
the IR and NMR spectra.
dichlorophenyl(2-furoyl)thiocarbamate 64. Mp 150–152 ꢁC (from
CH2Cl2/EtOH); yield: 61%. IR (KBr) cmꢀ1 3091, 1772, 1712. 1H NMR
(CDCl3)
d
3.97 (t, J ¼ 6 Hz, 2H, CH2N), 4.75 (t, J ¼ 6 Hz, 2H, CH2O),
6.37–6.60 (m, 1H, H-4, fur), 7.03–7.94 (m, 9H, 7 arom H and H-3 and
H-5 fur). Calcd for C22H14Cl2N2O5S: C, 54.00; H, 2.88; N, 5.72; S,
6.55. Found: C, 53.93; H, 2.78; N, 5.68; S, 6.35.
References
6.1.3.9. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3,5-
[1] M. Artico, Farmaco 51 (1996) 305–331.
[2] M. Artico, Drugs Future 27 (2002) 159–175.
[3] J. Balzarini, Curr. Top. Med. Chem. 4 (2004) 921–944.
[4] E. De Clercq, Chem. Biodivers. 1 (2004) 44–64.
[5] E. De Clercq, J. Med. Chem. 46 (2005) 1297–1313.
[6] R. Pauwels, Curr. Opin. Pharmacol. 4 (2004) 437–446.
[7] O.S. Pedersen, E.B. Pedersen, Antivir. Chem. Chemother. 10 (1999) 285–314.
[8] O.S. Pedersen, E.B. Pedersen, Synthesis 4 (2000) 479–495.
[9] T.J. Tucker, W.C. Lumma, J.C. Culberson, Methods Enzymol. 275 (1996)
440–472.
dichlorophenyl(2-furoyl)thiocarbamate 65. Mp 144–146 ꢁC (from
CH2Cl2/EtOH); yield: 71%. IR (KBr) cmꢀ1 3093, 1778, 1710. 1H NMR
(CDCl3)
d
3.96 (t, J ¼ 6 Hz, 2H, CH2N), 4.74 (t, J ¼ 6 Hz, 2H, CH2O),
6.47–6.51 (m, 1H, H-4, fur), 7.20–7.96 (m, 9H, 7 arom H and H-3 and
H-5 fur). Calcd for C22H14Cl2N2O5S: C, 54.00; H, 2.88; N, 5.72; S,
6.55. Found: C, 54.06; H, 2.83; N, 5.74; S, 6.35.
[10] A. Ranise, A. Spallarossa, S. Schenone, O. Bruno, F. Bondavalli, L. Vargiu,
T. Marceddu, M. Mura, P. La Colla, A. Pani, J. Med. Chem. 46 (2003) 768–781.
[11] A. Ahgren, K. Backro, F.W. Bell, A.S. Cantrell, M. Clemens, J.M. Colacino,
J.B. Deeter, P. Engelhardt, M. Hogberg, S.R. Jaskunas, N.G. Johansson,
C.L. Jordan, J.S. Kasher, M.D. Kinnick, P. Lind, C. Lopez, J.M. Morin Jr.,
M.A. Muesing, R. Noreen, B. Oberg, C.J. Paget, J.A. Palkowitz, C.A. Parrish,
P. Pranc, M.K. Rippy, C. Rydergard, C. Sahlberg, S. Swanson, R.J. Ternansky,
T. Unge, R.T. Vasileff, L. Vrang, S.J. West, H. Zhang, X.-X. Zhou, Antimicrob.
Agents Chemother. 39 (1995) 1329–1335.
[12] A.S. Cantrell, P. Engelhardt, M. Hogberg, S.R. Jaskunas, N.G. Johansson,
C.L. Jordan, J. Kangasmetsa, M.D. Kinnick, P. Lind, J.M. Morin Jr., M.A. Muesing,
R. Noreen, B. Oberg, P. Pranc, C. Sahlberg, R.J. Ternansky, R.T. Vasileff, L. Vrang,
S.J. West, H. Zhang, J. Med. Chem. 39 (1996) 4261–4274.
6.1.3.10. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
nitrobenzoyl(2,4,6-trichlorophenyl)thiocarbamate 66. Mp 185–
187 ꢁC (from CH2Cl2/EtOH); yield: 45%. IR (KBr) cmꢀ1 1774, 1718,
1694. 1H NMR (CDCl3)
J ¼ 6.0 Hz, 2H, CH2O), 7.29–8.77 (m, 10H, arom H). Calcd for
C24H14Cl3N3O6S: C, 49.80; H, 2.44; N, 7.26; S, 5.54. Found: C, 49.93;
H, 2.47; N, 7.24; S, 5.33.
3-
d
3.76 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.61 (t,
6.2. Virology
[13] O.J. D’Cruz, F.M. Uckun, J. Antimicrob. Chemother. 57 (2006) 411–423 and
references therein.
[14] Y. Dong, T.K. Venkatachalam, R.K. Narla, V.N. Trieu, E.A. Sudbeck, F.M. Uckun,
Bioorg. Med. Chem. Lett. 10 (2000) 87–90 and references therein.
6.2.1. Cell-based assays
The biological evaluation of the ATCs 1–66 was performed
according to the previously reported procedures [10].
´
¨
[15] M. Ho¨gberg, C. Sahlberg, P. Engelhardt, R. Noreen, J. Kangasmetsa,
¨
N.G. Johansson, B. Oberg, L. Vrang, H. Zhang, B.-L. Sahlberg, T. Unge, S. Lo¨vgren,
6.2.2. Enzyme assays
RT assays were carried out using a non-radioactive method.
Purified HIV-RT was purchased from Ambion and assayed for its
K. Fridborg, K. Ba¨ckbro, J. Med. Chem. 42 (1999) 4150–4160.
[16] V. Ravichandran, R.K. Agrawal, Bioorg. Med. Chem. Lett. 17 (2007) 2197–2202.
[17] F.M. Uckun, D. Erbeck, H. Tibbles, S. Qazi, T.K. Venkatachalam, Arznei-
mittelforschung 57 (2007) 164–170.
[18] T.K. Venkatachalam, E.A. Sudbeck, C. Mao, F.M. Uckun, Bioorg. Med. Chem. Lett.
10 (2000) 2071–2074.
[19] T.K. Venkatachalam, C. Mao, F.M. Uckun, Bioorg. Med. Chem.12 (2004) 4275–4284.
[20] A. Ranise, A. Spallarossa, S. Cesarini, F. Bondavalli, S. Schenone, O. Bruno,
G. Menozzi, P. Fossa, L. Mosti, M. La Colla, G. Sanna, M. Murreddu, G. Collu,
B. Busonera, M.E. Matongiu, A. Pani, P. La Colla, R. Loddo, J. Med. Chem. 48
(2005) 3858–3873.
[21] S. Cesarini, A. Spallarossa, A. Ranise, P. Fossa, P. La Colla, G. Sanna, G. Collu,
R. Loddo, Bioorg. Med. Chem. 16 (2008) 4160–4172.
[22] S. Cesarini, A. Spallarossa, A. Ranise, O. Bruno, P. La Colla, B. Secci, G. Collu,
R. Loddo, Bioorg. Med. Chem. 16 (2008) 4173–4185.
[23] A. Spallarossa, S. Cesarini, A. Ranise, O. Bruno, S. Schenone, P. La Colla, G. Collu,
G. Sanna, B. Secci, R. Loddo, Eur. J. Med. Chem. (2008). doi:10.1016/
j.ejmech.2008.09.024.
[24] C. Tantillo, J. Ding, A. Jacobo-Molina, R.G. Nanni, P.L. Boyer, S.H. Hughes,
R. Pauwels, K. Andries, P.A. Janssen, E. Arnold, J. Mol. Biol. 243 (1994) 369–387.
[25] G. Maass, U. Immendoerfer, B. Koenig, U. Leser, B. Mueller, R. Goody, E. Pfaff,
Antimicrob. Agents Chemother. 37 (1993) 2612–2617.
RNA-dependent polymerase-associated activity in a 20
containing 60 mM Tris–HCl (pH ¼ 8.1), 60 mM KCl, 8 mM MgCl2,
13 mM DTT, 2.5 ng
ꢀ1 template: primer [poly(A)-oligo(dT)16] and
100
M dTTP. After incubation for 30 min at 37 ꢁC, reactions were
stopped by addiction of 2 L of 200 mM EDTA. 138 L of PicoGreen
mL volume
mL
m
m
m
Quantitation Reagent (Molecular Probes), diluted 1/345 in TE, was
added to each sample and incubated at room temperature, pro-
tected from ambient light, for 5 min. Sample fluorescence from
each well of a 96-well microtiter plate was measured using
a
microplate VICTOR3 Multilabel Plate Reader (PerkinElmer).
Percent residual activity was plotted vs increasing concentration of
compounds. The curve was fit with Kaleidagraph (Synergy Soft-
ware) to obtain IC50 values.
[26] D. Motakis, M.A. Parniak, Antimicrob. Agents Chemother. 46 (2002) 1851–1856.
[27] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew,
A.J. Olson, J. Comput. Chem. 19 (1998) 1639–1662.
[28] J. Ren, J. Diprose, J. Warren, R.M. Esnouf, L.E. Bird, S. Ikemizu, M. Slater,
J. Milton, J. Balzarini, D.I. Stuart, D.K. Stammers, J. Biol. Chem. 275 (2000) 5633.
6.3. Molecular modelling
The docking models of analogues 5, 38, 42, 44, 49 and 50
were calculated using Autodock 3.0.5 [27]. The crystal structure of

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 2190–2201
2201
[29] P. Auffinger, F.A. Hays, E. Westhof, P.S. Ho, Proc. Natl. Acad. Sci. U S A 101
(2004) 16789–16794.
[32] P. Dauber-Osguthorpe, V.A. Roberts, D.J. Osguthorpe, J. Wolff, M. Genest,
A.T. Hagler, Proteins 4 (1988) 31–47.
[30] A. Mugnoli, A. Borassi, A. Spallarossa, S. Cesarini, Acta Crystallogr. C62 (2006)
315–317.
[33] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. Delano, P. Gros, R.W. Grosse-Kuns-
tleve, S. Jiangj, J. Kuszewski, N. Nilges, N.S. Pannu, R.J. Read, L.M. Rice,
T. Simonson, G.L. Warren, Acta Crystallogr. D54 (1998) 905–921.
[34] Collaborative Computational Project, Number 4, Acta Crystallogr. D50 (1994)
760–763.
[31] F.W. Bell, A.S. Cantrell, M. Hogberg, S.R. Jaskunas, N.G. Johansson,
C.L. Jordan, M.D. Kinnick, P. Lind, J.M. Morin Jr., R. Noreen, B. Oberg,
J.A. Palkowitz, C.A. Parrish, P. Pranc, C. Sahlberg, R.J. Ternansky,
R.T. Vasileff, L. Vrang, S.J. West, H. Zhang, X.-X. Zhou, J. Med. Chem. 38
(1995) 4929–4936.
[35] P.J. Kraulis, J. Appl. Crystallogr. 24 (1991) 946–950.
[36] R.M. Esnouf, J. Mol. Graph. Model. 15 (1997) 132–134.