Novel modifications in the series of O-(2-phthalimidoethyl)-N-substituted thiocarbamates and their ring-opened congeners as non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.09.024

The structure-activity relationships (SARs) of N-aryl-O-(2-phthalimidoethyl)thiocarbamates (C-TCs) and their imide ring-opened congeners (O-TCs) as non-nucleoside HIV-1 reverse transcriptase inhibitors were further investigated. The SAR strategy involved

Full text of DOI:10.1016/j.ejmech.2008.09.024

European Journal of Medicinal Chemistry 44 (2009) 1650–1663
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel modifications in the series of O-(2-phthalimidoethyl)-N-substituted
thiocarbamates and their ring-opened congeners as non-nucleoside HIV-1
reverse transcriptase inhibitors
,
a
a
a
a
b,
**
Andrea Spallarossa a , Sara Cesarini , Angelo Ranise , Olga Bruno , Silvia Schenone , Paolo La Colla
,
*
Gabriella Collu b, Giuseppina Sanna b, Barbara Secci b, Roberta Loddo b
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
Dipartimento di Scienze e Tecnologie Biomediche, Universita di Cagliari, Cittadella Universitaria, S.S. 554, Km 4,500, I-09042 Monserrato (Cagliari), Italy
b
`
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2008
Received in revised form 4 September 2008
Accepted 8 September 2008
Available online 30 September 2008
The structure–activity relationships (SARs) of N-aryl-O-(2-phthalimidoethyl)thiocarbamates (C-TCs) and
their imide ring-opened congeners (O-TCs) as non-nucleoside HIV-1 reverse transcriptase inhibitors
were further investigated. The SAR strategy involved modifications of the N-phenyl ring followed by the
hybridization of the most promising N-aryl and O-(2-phthalimidoethyl) substructures. The role of
stereochemistry and tert-butyl substitution of the phthalimidoethyl moiety on activity was also inves-
tigated. Seventy-six analogues were prepared by parallel solution-phase synthesis. Twenty-two C-TCs
displayed nanomolar activity against wild-type HIV-1 and a number of analogues were effective against
the Y181C mutant. Compound 56 combined the highest activity so far identified against Y181C
Keywords:
O-(2-Phthalimidoethyl)-N-
arylthiocarbamates
HIV-1
Non-nucleoside reverse transcriptase
inhibitors
(EC50 ¼ 1.3
m
M) with good potency against the K103R mutant (EC50 ¼ 4.8
mM). Docking simulations
helped to rationalize the SARs.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Parallel synthesis
Molecular docking
1. Introduction
N-phenethyl-N0-thiazolylthiourea (PETT) derivatives (Fig. 1A)
[3,13–32]. The synthesis of thiocarbamates more structurally
similar to PETT compounds (TC-1, TC-29, Fig. 1A) [33,34] has shed
further light on the relationships between these two families of
inhibitors. Preliminary efforts [12] to optimize the lead I (Fig. 1B)
had been mainly based on independent variations of the C-TC
portions a and b (Fig. 1A) and had led to the potent derivatives II–
XIII (Fig. 1B) featured by the following substitution patterns: for
portion a: (i) methyl substitution at position 4 of the phthalimide
phenyl ring, (ii) methyl branching of the ethyl linker on the carbon
HIV-1 reverse transcriptase (RT) is a DNA-dependent poly-
merase responsible for the production of a double stranded DNA
copy of a single stranded viral RNA. RT is essential for HIV-1 life
cycle and therefore represents a privileged pharmacological target
for anti-AIDS therapy [1]. Non-nucleoside reverse transcriptase
inhibitors (NNRTIs) are a family of selective chemically diverse RT
targeting agents. Crystallographic studies have shown that all
NNRTIs bind to an allosteric binding pocket (the non-nucleoside
binding site, NNBS), located in the vicinity of the active site in the
p66 subunit [2–9]. The formation of RT–NNRTI complex results in
short- and long-range conformational changes that lock the
enzyme in an inactive form [6,10,11].
atom adjacent to the imide nitrogen (hereinafter, b-carbon Fig. 1A);
for portion b: (i) 4-monosubstitution of the phenyl ring, (ii)
replacement of the phenyl ring with a cyclohexyl.
With an aim of acquiring additional elements to maximize the
activity against wild-type and drug-resistant RT forms, a new series
of C-TCs were designed and prepared by parallel synthesis. First,
the O-(2-phthalimidoethyl) substructure was kept unmodified
while portion b was varied by embodying (cyclo)alkyl, allyl and
(hetero)arylalkyl groups (C-TCs 1–11, Table 1) or poly-substituted
phenyl moieties bearing atoms/groups endowed with different
electronic, steric and lipophilic features (F, Cl, Br, CH3, CF3, OCH3,
NO2) arranged in various patterns (C-TCs 12–39, Table 2). Notably,
O-(2-Phthalimidoethyl)-N-arylthiocarbamates (C-TCs) and their
imide ring-opened congeners (O-TCs) have been recently identified
[12] as a novel class of HIV-1 NNRTIs that are isosterically related to
* Corresponding author. Tel.: þ39 010 353 8370; fax: þ39 010 353 8358.
** Corresponding author. Tel.: þ39 070 675 4147; fax: þ39 070 675 4210.
E-mail addresses: andrea.spallarossa@unige.it (A. Spallarossa), placolla@unica.it
(P. La Colla).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.024

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1651
A
a
b
O
O
H
N
β
O
O
NH
Ar
N
H
N
Ar
α
4
S
S
COOH
3
O
C-TCs
S
O-TCs
Y
N
W
S
X
O
NH
N
H
NH
N
G
TC-1 (X = N), EC50 = 7.4 μM
Trovirdine (Y = W = H, G = Br)
TC-29 (X = CH), EC50 = 6.0 μM
PETT-1 (W = F, Y = OC2H5, G = CN)
O
O
Y
B
S
S
N
N
O
N
H
O
N
H
X
R
O
O
XIII, EC50 = 0.3 μM
X
H
H
H
H
H
H
H
H
H
H
H
R
H
H
H
H
H
H
H
H
H
H
Y
EC50 (μM)
1.2
I
H
II
CH3
C2H5
0.02
III
IV
V
0.08
COCH3 0.14
CN
F
0.07
0.10
0.04
0.03
0.02
0.03
0.05
0.025
VI
VII
VIII
IX
X
Cl
Br
I
OCH3
XI
(R/S)CH3 Cl
XII CH3
H
Cl
Fig. 1. (A) Chemical structures of thiocarbamates (C-TCs, O-TCs, TC-1, TC-29) [33,34] and PETT derivatives (Trovirdine and PETT-1). (B) Chemical structures and antiretroviral activity
of previously synthesized C-TCs I–XIII [12].
the halosubstitution of portion b was carried out also in keeping
with the paramount importance of aromatic halogen atoms
observed for the activity of other class of NNRTIs structurally
unrelated to thiocarbamates such as 2,3-diaryl-1,3-thiazolidin-4-
(thi)ones [35,36] and indolyl aryl sulphones [37]. Then, the a and
b substructures emerged as the most promising from the previous
work were hybridized for further series expansion (Tables 3 and 4).
Thus, the 4-methylphthalimidoethyl moiety was kept constant and
combined with N-cyclohexyl (40) or N-4-monosubstitutedphenyl
(CH3, C2H5, COCH3, CN, F, Br, I, OCH3) (41–48) or N-3,4-dis-
ubstitutedphenyl (Cl,Cl; Cl,CH3; Cl,CF3; Cl,NO2) (49–52) rings (Table
3). Moreover, the N-4-methyl- and N-4-chloro-phenyl moieties
were coupled with portion a bearing: (i) a tert-butyl group at
position 4 of the phthalimido phenyl ring (53 and 54, Table 3); (ii)
and aminoalcohol in solvent free conditions (Scheme 1), according
to the general procedure previously detailed [12].
Compounds 1–76 were prepared by a convergent two-step
solution-phase parallel procedure (Scheme 2) derived from our
previously reported protocols [12,33,34]. Briefly, the starting alco-
hols A1–5 (Fig. 2A) were first transformed into their corresponding
salts in the presence of sodium hydride in dry THF or DMF
(procedures A and B, respectively. See Section 6). The alcoholate
was then condensed in situ with isothiocyanates I1–49 (Fig. 2B) to
afford the mono-sodium thiocarbamates B as single adducts or in
mixture with the corresponding di-sodium ring-opened salts SS.
After treatment with an aqueous ammonium chloride solution
(pH ¼ 8), B and SS were protonated to neutral C-TCs and carbox-
ylate salts S, respectively. C-TCs were separated from the reaction
mixture and extracted in parallel with diethyl ether, whereas S
were kept in solution. Acidification of the reaction medium with
a 2 N HCl solution (pH ¼ 0) caused protonation of S, leading to O-
TCs which precipitated as free acids and were collected by filtration.
Notably, in the synthesis of C-TCs 1–10, 13, 14, 19–25, 27, 29–32, 34,
37, 38, 40–54, 58, 59 the corresponding O-TCs were isolated in trace
amounts (yields were less than 0.5–1%, data not shown). In
contrast, reaction of A1 with I44 and (S)-A2 with I13 afforded only O-
TCs 68 and 74, respectively. Details on the hypothetical mechanism
for the concomitant formation of C-TCs and O-TCs and the proce-
dures of separation have been previously reported [12]. The final
a methyl branching on the linker
57, Table 4); (iii) methyl groups on both the phthalimide position 4
and the linker -carbon (racemic 58 and 59, Table 4).
b-carbon (pure enantiomers 55–
b
Finally, O-TCs 60–76 (Table 5) allowed us to evaluate the effects
of imide ring opening concomitantly with N-phenyl ring modifi-
cations (60–72) and b-carbon methyl branching (73–76).
2. Chemistry
Preliminarily, the alcohol building blocks (R)-A2, (S)-A2, A4 and
A5 (Fig. 2A) were synthesized by condensing the proper anhydride

1652
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
Table 1
Table 2
Cytotoxicitya and anti-HIV-1 activity of C-TCs 1–11
Cytotoxicitya and anti-HIV-1 activity of C-TCs 12–39
O
O
H
N
H
O
O
N
R
N
N
X,Y
S
S
O
O
SId
Compound
R
CC50
EC50
SId
b
c
b
c
Compound
X,Y
CC50
EC50
1
2
3
4
5
6
7
8
n-Butyl
1,1,3,3-Tetramethylbutyl
Cyclopropyl
Cyclopentyl
Cyclooctyl
Cyclohexylmethyl
Allyl
Benzyl
2-Furylmethyl
2-Phenylethyl
2-(3,4-Dimethoxyphenyl)ethyl
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
60
7.2
60
>100
11
0.6
1.1
>100
26
>100
9.2
8
0.02
>13.9
>1.7
–
>9.1
>166.7
>91
–
>3.8
–
>10.9
>12.5
3000
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
2,4-Me2
2,6-Me2
3,5-Me2
2,4-F2
2,5-F2
2,6-F2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
84
6
75
4
0.5
8
7
9
30
>100
42
21
0.2
12
27
>100
23
0.8
>100
0.05
19
16.7
>1.3
>25
>200
>12.5
>14.3
>11.1
>3.3
–
>2.4
>4.8
>500
>8
>3.7
–
>4.3
>125
–
>2000
>5.3
>2.1
>28.6
10.5
>50
>9.1
>10
3,5-F2
2,3-Cl2
2,4-Cl2
2,5-Cl2
2,6-Cl2
3,4-Cl2
3,5-Cl2
4-Cl-2-Me
5-Cl-2-Me
2-Cl-6-Me
3-Cl-4-Me
2-Cl-4-NO2
4-Cl-3-NO2
4-Br-2-Me
5-Cl-2-OMe
3,4-(OMe)2
4-Cl-3-CF3
2,4,6-F3
9
10
11
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 cytopathogenecity, as determined by the MTT method.
d
Selectivity index: CC50/EC50 ratio.
48
3.5
8.0
2
11
10
9
10
0.02
products were purified by crystallization and the yields (not opti-
mized) ranged from 9% to 73% (see Section 6).
>100
>100
>100
>100
>100
60
2,3,4-Cl3
2,4,6-Cl3
4-Br-2,6-Me2
2,3,5,6-F4
38
39
Trovirdine
>11.1
>10
3000
3. Biological results and discussion
a
The antiretroviral activity of C-TCs (Tables 1–4) and O-TCs (Table
5) was evaluated in MT-4 cell-based assays by assessing the
reduction of the HIV-1 induced cytopathogenicity. The results are
expressed as EC50 values. In parallel with antiretroviral activity, the
TC-induced cytotoxicity was evaluated in mock-infected MT-4 cells
and the results are expressed as CC50 values. Trovirdine was used as
the reference drug. The most potent derivatives were also tested
against the clinically relevant K103R, Y181C and K103N/Y181C
resistant mutants [38,39], employing Efavirenz as the reference
molecule (Table 6).
Among C-TCs in which the N-phenyl ring of the lead I was
replaced by allyl, (cyclo)alkyl or (hetero)arylalkyl groups (1–10,
Table 1) only 5 (cyclooctyl) and 6 (cyclohexylmethyl) showed
improved potency in comparison with I but resulted to be less
active than the cyclohexyl derivative XIII (compare 5 and 6 with
XIII).
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 cytopathogenecity, as determined by the MTT method.
d
Selectivity index: CC50/EC50 ratio.
30 vs 29). The 3,4-pattern emerged as the most favourable; 23, 28,
30, 33 and 34 were (sub)micromolar HIV-1 inhibitors and in
particular 30 (4-Cl-3-NO2) displayed an EC50 value of 50 nM. The tri-
substituted C-TCs 35–38 and the tetrafluoro derivative 39 were
active in a narrow micromolar concentration range (EC50 range: 9–
11
mM), except for 35 that resulted to be more active.
The insertion of a methyl group at position 4 of the phthalimide
substructure led to the highly active C-TCs 40–52 (Table 3) whose
activities were influenced by the nature of portion b. Thus, the
cyclohexyl derivative 40 displayed a reduced potency in compar-
ison with the majority of its N-aryl substituted congeners. The para-
substituted phenyl analogues 41–48 showed nanomolar potencies
(EC50 range ¼ 8–70 nM) and 44 (4-CN), 46 (4-Br) and 47 (4-I)
resulted as more active than Trovirdine, while 41 (4-Me) and 42 (4-
Et) were equipotent to the reference molecule. Compound 46
turned out to be the most active C-TC so far identified. The most
potent para-substituted derivatives bear both electron-with-
drawing (F, Br, I, CN) and electron-donating (Me, Et, MeO) groups
thus confirming a marginal effect of the electronic properties of the
N-phenyl substituent on antiretroviral activity. Among the 3,4-
disubstituted analogues, 51 (4-Cl-3-NO2) emerged as the most
potent derivative (EC50 ¼ 50 nM) and 49 (diCl) and 50 (3-Cl-4-CH3)
showed submicromolar activities. In comparison with their
unsubstituted phthalimide-ring analogues, the 4-methyl
substituted C-TCs 42–47, 50, and 52 were from 2- to 7-fold more
potent whereas 40, 41, 48, 49, and 51 showed equal activity (40 vs
A complex interplay between the poly-substitution patterns and
the steric/electronic properties of the N-phenyl substituents affects
the antiretroviral properties of 12–39 (Table 2). Thus, the activity
trend of the dimethyl 12–14, difluoro 15–18 and dichloro 19–24
derivatives [2,4-: F (15) > CH3 (12) > Cl (20); 2,6-: F (17) > Cl
(22) > CH3 (13); 3,5-: CH3 (14) > F (18) > Cl (24)] suggests that the
relative positions of the N-phenyl substituents influence the anti-
retroviral activity more than how their electronic properties do.
The difluoro analogues (15–18) were similarly effective in the low
micromolar range except for 15 (2,4-diF, EC50 ¼ 0.5
mM) that turned
out to be 2.4-fold more potent than the lead I. The isosteric F/Cl
replacement decreased activity, the bulkier dichloro derivatives
being less effective than their difluoro counterparts (20 vs 15; 21 vs
16; 22 vs 17; 24 vs 18).
Moreover, except for the difluoro derivatives, the disubstituted C-
TCs bearing an ortho-substituent appeared to be less active than
their 3,4- and 3,5-positional isomers (dimethyl: 14 vs 12, 13;
dichloro: 23, 24 vs 19–22; chloro–methyl: 28 vs 25–27; chloro–nitro:

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1653
Table 3
Cytotoxicitya and anti-HIV-1 activity of C-TCs 40–54
O
S
G
N
O
N
H
X
O
SId
b
c
Compound
X
G
CC50
EC50
40
41
42
43
44
45
46
47
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cyclohexyl
4-Tolyl
>100
69
43
>100
57
100
59
89
>100
50
0.3
>333
3450
2150
>1429
5700
3333
7375
8900
>3333
250
0.02
0.02
0.07
0.01
0.03
0.008
0.01
0.03
0.2
4-Ethylphenyl
4-Acetylphenyl
4-Cyanophenyl
4-Fluorophenyl
4-Bromophenyl
4-Iodophenyl
4-Methoxyphenyl
3,4-Dichlorophenyl
48
49
CH3
CH3
50
51
52
53
CH3
CH3
CH3
C(CH3)3
C(CH3)3
3-Chloro-4-methylphenyl
4-Chloro-3-nitrophenyl
4-Chloro-3-trifluoromethylphenyl
4-Tolyl
66
>100
43
42
55
0.4
0.05
2.4
1.5
1.2
165
>2000
17.9
28
45.8
54
4-Chlorophenyl
Trovirdine
60
0.02
3000
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 cytopathogenecity, as determined by the MTT method.
d
Selectivity index: CC50/EC50 ratio.
XIII; 41 vs II; 42 vs III; 43 vs IV; 44 vs V; 45 vs VI; 46 vs VIII; 47 vs
IX; 48 vs X; 49 vs 23; 50 vs 28; 51 vs 30; 52 vs 34). The replacement
of the methyl substituent with the more sterically demanding tert-
butyl group caused a dramatic decrease in activity (53 vs 41; 54 vs
XII).
As reported in Table 4, C-TC 56 (EC50 ¼ 10 nM) was 2-fold more
active than Trovirdine and showed higher potency in comparison with
both the S isomer 57 and racemate XI (Fig. 1B) [12]. This evidence is
consistent with the data reported for PETT derivatives for which the R
isomers fit the NNRTI binding pocket much better than S enantiomers
[27,28]. The 4-tolyl analogue 55 was 7-fold less active than 56 but still
retained activity in the nanomolar concentration range. Moreover, the
comparison of 55 and 56 with their corresponding unbranched
congeners II and VII (Fig. 1B) showed opposite effects of the methyl
branching on the activity. The double methyl substitution of the linker
NNRTI binding site) led to compounds 58 and 59, active in the nano-
molarconcentration range and with a decreased activity in comparison
with their analogues bearing single variation on portion a (compare 58
with 41; 59 with XI and XII).
In accordance with the results of the previous studies [12], the
imide ring opening (O-TCs 60–76, Table 5) caused a decrease in
activity (60 vs 11; 61 vs 12; 62 vs 15; 63 vs 16; 64 vs 17; 65 vs 18; 66
vs 26; 67 vs 28; 69 vs 33; 70 vs 35; 71 vs 36; 72 vs 39; 73 vs 55; 75 vs
56; 76 vs 57). O-TCs 62, 67, 70, 73, 75 and 76 emerged as the most
Table 5
Cytotoxicitya and anti-HIV-1 activity of O-TCs 60–76
O
R
H
N
O
G
N
H
b-carbon and the phthalimide benzene ring (in an attempt to
S
strengthen the hydrophobic contacts within the highly lipophilic
COOH
SId
b
c
Compound
R
G
CC50
EC50
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
H
H
H
H
H
H
H
H
H
H
H
H
2-(3,4-Dimethoxyphenyl)ethyl
2,4-Dimethylphenyl
2,4-Difluorophenyl
2,5-Difluorophenyl
2,6-Difluorophenyl
3,5-Difluorophenyl
5-Chloro-2-methylphenyl
3-Chloro-4-methylphenyl
2-Methoxy-5-methylphenyl
3,4-Dimethoxyphenyl
2,4,6-Trifluorophenyl
2,3,4-Trichlorophenyl
2,3,5,6-Tetrafluorophenyl
4-Tolyl
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
60
63
53
3.8
51
45
62
30
4.3
88
16
7.2
44
1.6
>1.9
>26.3
>2.0
>2.2
>1.6
>3.3
>23.2
>1.1
>6.2
>13.9
>2.3
>1.4
>43.5
>7.2
>333
>43.5
3000
Table 4
Cytotoxicitya and anti-HIV-1 activity of branched C-TCs 55–59
O
Y
S
N
*
O
N
H
X
CH3
O
SId
b
c
Compound
X
Isomer
Y
CC50
EC50
55
56
H
H
H
CH3
CH3
R
R
S
R/S
R/S
CH3
Cl
Cl
CH3
Cl
76
48
84
41
34
60
0.07
0.01
0.2
0.1
0.06
0.02
1086
4800
420
410
567
H
71
57
(R)CH3
(S)CH3
(R)CH3
(S)CH3
2.3
13.8
0.3
2.3
0.02
58e
4-Tolyl
4-Chlorophenyl
4-Chlorophenyl
59e
Trovirdine
3000
76
Trovirdine
a
Data mean values for three separate experiments. Variation among triplicate
a
samples was less than 10%.
Data mean values for three separate experiments. Variation among triplicate
b
Compound concentration (
m
M) required to reduce the viability of mock-infected
samples was less than 10%.
b
cells by 50%, as determined by the MTT method.
Compound concentration (mM) required to reduce the viability of mock-infected
c
Compound concentration (
m
M) required to achieve 50% protection of MT-4 cell
cells by 50%, as determined by the MTT method.
c
from HIV-1 induced cytopathogenecity, as determined by the MTT method.
Compound concentration (mM) required to achieve 50% protection of MT-4 cell
d
Selectivity index: CC50/EC50 ratio.
Tested as a racemic mixture.
from HIV-1 induced cytopathogenecity, as determined by the MTT method.
e
d
Selectivity index: CC50/EC50 ratio.

1654
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
A
Alcohols
X
R
O
H
H
A
1
H
CH
CH
H
3
(R)-A
2
N
H
3
(S)-A
2
OH
CH
3
A3
A4
A5
X
R
C(CH
3)
3
H
CH
O
CH
3
3
B
Isothiocyanates (Ar-NCS)
Ar
Ar
n-butyl
1,1,3,3-tetramethylbutyl
2,5-difluorophenyl
2,6-difluorophenyl
3,5-difluorophenyl
2,3-dichlorophenyl
2,4-dichlorophenyl
2,5-dichlorophenyl
2,6-dichlorophenyl
3,4-dichlorophenyl
I1
I26
I27
I28
I29
I30
I31
I32
I33
I34
I35
I36
I37
I38
I39
I40
I41
I42
I43
I44
I45
I46
I2
cyclopropyl
cyclopentyl
cyclohexyl
cyclooctyl
cyclohexylmethyl
allyl
benzyl
2-furylmethyl
2-phenylethyl
2-(3,4-dimethoxyphenyl)ethyl
4-tolyl
4-ethylphenyl
4-acetylphenyl
4-cyanophenyl
4-fluorophenyl
4-chlorophenyl
4-bromophenyl
4-iodophenyl
I3
I4
I5
I6
I7
I8
I9
3,5-dichlorophenyl
I10
I11
I12
I13
I14
I15
I16
I17
I18
I19
I20
I21
3,4-dimethoxyphenyl
4-chloro-2-methylphenyl
5-chloro-2-methylphenyl
2-chloro-6-methylphenyl
3-chloro-4-methylphenyl
2-chloro-4-nitrophenyl
4-chloro-3-nitrophenyl
4-chloro3-trifluoromethylphenyl
4-bromo-2-methylphenyl
2-methoxy-5-methylphenyl
2,4,6-trifluorophenyl
2,3,4-trichlorophenyl
4-methoxyphenyl
I22 2,4-dimethylphenyl
I23 2,6-dimethylphenyl
I24 3,5-dimethylphenyl
I25 2,4-difluorophenyl
I47 2,4,6-trichlorophenyl
I48 4-bromo-2,6-dimethylphenyl
I49 2,3,5,6-tetrafluorophenyl
Fig. 2. Building blocks used.
active derivatives with EC50 values in the low micromolar or sub-
micromolar concentration range. As observed for C-TCs, the R
enantiomers 73 and 75 were from 6- to 7-fold more active than the
corresponding S isomers 74 and 76.
Y181C þ K103N resistant mutants. All compounds resulted to be
inactive against the double mutant. The 12 derivatives listed in
Table 6 were active against the Y181C variant. Compounds 30, 44,
47, 55, 56 and 59 displayed EC50 values lower than 10 mM. The R
C-TCs 1, 2, 6, 9–11, 15–18, 23, 28, 30, 33, 35–59 and O-TCs 60,
62–65, 70–76 were also screened against K103R, Y181C and
isomer 56 emerged as the most active analogue so far identified
against the Y181C strain. Differently from all the previously
prepared C-TCs, 56 showed micromolar activity against the K103R
variant in a cell-based assay (EC50 ¼ 4.8
mM). This observation
coupled with the inactivity of 57 (the S enantiomer of 56)
suggests a strong effect of stereochemistry on C-TC resistance
profile.
(a)
(R)-A2
O
O
(c)
A4
O
O
4. Molecular docking studies
O
O
(S)-A2
To rationalize the most relevant SARs, docking studies (Auto-
dock 3.05) [40] were carried out using the X-ray coordinates of RT/
C-TC complex (PDB code: 2VG5) [9] as the template structure.
In the RT/5 docking model, the ligand assumes a U-shaped
bioactive conformation consistent with the binding mode of its N-
aryl substituted analogues [9,12]. Nevertheless, the bulkiness of the
cyclooctyl ring prevents the inhibitor from forming the hydrogen
bond between the thiocarbamic NH group and Lys101 main chain
carbonyl (C(S)N–OC estimated distance: 3.3 Å). This interaction is
highly conserved in the C-TC class and in other thiocarbamic and
thiourea NNRTIs, as assessed by X-ray crystallography [3,8,9,41].
The lack of this hydrogen bond is partially compensated by the
(b)
O
(d)
A5
O
H3C
O
Scheme 1. Reagents and conditions: (a) (2R)-2-aminopropan-1-ol, 140–150 ꢀC, 2 h; (b)
(2S)-2-aminopropan-1-ol, 140–150 ꢀC, 2 h; (c) 2-aminoethanol, 175–180 ꢀC, 2 h; (d)
(ꢁ) 2-aminopropan-1-ol, 140–150 ꢀC, 2 h.

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1655
A
1-5
(a)
O
O
R
+
Ar
S
Na
O
N
-
Ar
+
N
N
H
+
-
O
N
S
X
-
+
Na
R
X
COO Na
O
B, X = H, CH3, C(CH3)3
SS (X = H, R = H, CH3)
R = H, CH3
(b)
O
O
CH3
H
S
O
N
Ar
N
H
N
Ar
O
N
H
S
X
-
+
COO Na
R
O
S
1-59
(c)
O
CH3
H
N
O
Ar
N
H
S
COOH
60-76
Scheme 2. Reagents and conditions: (a) Ar-NCS (I1–49), NaH, dry THF r.t. for 24 h (procedure A) or dried DMF 0–5 ꢀC then r.t. for 24 h; (b) aqueous NH4Cl; (c) 2 N HCl. The structures
of alcohols A1-5 and isothiocyanates I1-49 are listed in Fig. 2.
involvement of the phthalimide moiety in
p
–
p
stacking (Tyr181),
van der Waals contacts involving the sulphur atom and the Val179
side chain. The oxyethyl linker interacts with the side chains of
Leu100 and Glu138(p51), while the phthalimide moiety is involved
hydrophobic (Tyr188, Trp229) and van der Waals [Pro95 and
Glu138(p51)] interactions. Moreover, further stabilization to the
complex is provided by the hydrophobic contacts between the
cyclooctyl ring and the side chains of Lys103, Tyr318 and Val106 as
well as by van der Waals interactions between the thiocarbamic
functionality and the Lys101 backbone atoms.
in
p–p stacking interactions with the Tyr181 side chain and in
hydrophobic contacts with the Tyr188 and Trp229 residues.
Furthermore, one of the imidic oxygen interacts with the Pro95 side
chain. The N-phenyl ring hydrophobically contacts the Leu100,
Lys103, Val106 and Tyr318 side chains. The chlorine atom estab-
lishes van der Waals contacts with the His235 main chain atoms
and the nitro group is involved in a network of polar interactions
with the Gly190 and Val189 main chains thus providing further
stabilization to the complex. The impossibility of 23, 28, 33 and 34
(devoided of a 3-nitro substituent) to form polar interactions with
Gly190 and Val189 backbones would structurally rationalize the
higher potency of 30 in comparison with the other 3,4-disubsti-
tuted analogues. The lack of activity of 29 (positional isomer of 30)
would be ascribable to the ortho chlorine atom that prevents the
ligand from assuming a bioactive U-shaped conformation within
the NNBS and from establishing the hydrogen bond between the
thiocarbamic NH group and the Lys101 backbone carbonyl.
The RT/30 complex (Fig. 3A) is stabilized by a hydrogen bond
between the NH group and the main chain carbonyl of Lys101 and
Table 6
Anti-HIV-1a activity of C-TCs and O-TCs against the Y181C resistant mutant
b
Compound
EC50
30
42
44
45
47
48
51
55
2.8
16
4.5
68
9
44
30
9
1.3
12
4
18
15
0.02
Docking simulations carried out on 56 and 57 helped to ratio-
nalize the activity gap between the R and S stereoisomers. Thus, 56
would assume a bioactive conformation very similar to that
observed in the X-ray structure of its unbranched analogue VII [9]
(Fig. 3B). The RT/56 complex would be mainly stabilized by the
hydrogen bond between the NH thiocarbamic group and the Lys101
56
58c
59c
75
Trovirdine
EFV
main chain carbonyl and the
phthalimide substructure and the Tyr181 side chain. Furthermore,
van der Waals contacts would occur between the methyl on the
p–p stacking interactions between the
a
Data mean values for three separate experiments. Variation among triplicate
samples was less than 10%.
b
-
b
Compound concentration (mM) required to achieve 50% protection of MT-4 cell
carbon and the Val179, Tyr181 and Glu138(p51) side chains. To
avoid steric clashes with Val179 side chain, 57 oxyethyl linker
from HIV-1 induced cytopathogenecity, as determined by the MTT method.
c
Tested as a racemic mixture.

1656
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
Fig. 3. (A) Stereoview of the RT/30 modeled complex. The ligand is represented as black balls-and-sticks, while the amino acid residues lining the RT non-nucleoside binding site
are shown as grey sticks. (B) Stereo diagram showing superposition of C-TCs 56 (modeled structure, black ball-and-stick) and 57 (modeled structure, white ball-and-stick) using RT/
VII (grey ball-and-stick) crystallographic complex (PDB code: 2VG5) [9] as reference. Amino acid residues lining the non-nucleoside binding site are shown in sticks model, as
observed in the crystallographic structure. Hydrogen bonds are depicted as dotted lines. The programs Molscript [47] and Raster3D [48] were used for drawing the figure.
would assume a different spatial arrangement (Fig. 3B). This would
cause distortions of both the thiocarbamic functionality and the N-
phenyl ring that weaken the hydrogen bond between the NH group
and the Lys101 carbonyl thus decreasing the affinity of the inhibitor
(C(S)N–OC estimated distances: 2.7 Å and 3.0 Å for 56 and 57,
respectively).
As observed in the RT/XII model [12], in the RT/51 docking
complex the methyl group at position 4 of the phthalimide
substructure would further stabilize the complex by means of
hydrophobic interactions with the side chains of Tyr188 and
Trp229. The ability of the 51 nitro group to form polar interactions
with the Gly190 and Val189 main chains would rationalize its
higher activity in comparison with the other 3,4-disubstituted
analogues 49, 50 and 52.
the best results. The methyl substitution at position 4 of the
phthalimide of the phthalimido phenyl ring as well as the R methyl
branching of the
b-carbon atom led to potent analogues with
improved activity against wild-type HIV-1. Furthermore, some
derivatives inhibited NNRTI-resistant Y181C variant at low micro-
molar concentrations and C-TCs 56 resulted effective against both
Y181C and K103R mutants. The present study will provide the basis
for the design and the synthesis of new analogues more effective
against the clinically relevant HIV-1 mutated strains.
6. Experimental section
6.1. Chemistry
Finally, the structural comparison between RT/54 and RT/XII
modeled structures indicates that the tert-butyl group would cause
a repositioning of the inhibitor within the binding site preventing the
thiocarbamic functionality from forming the hydrogen bond with
Lys101 and minimizing the interaction of the N-phenyl ring with RT.
6.1.1. General
Alcohol A1, isothiocyanates I1–49 and the other chemicals were
purchased from Chiminord and Aldrich Chemical, Milan (Italy).
Syntheses of Trovirdine and A3 were accomplished according to the
published procedures [12,13]. Solvents were of reagent grade. THF
was distilled in the presence of sodium. DMF was dried on
molecular sieves (5 Å 1/1600 pellets). Unless otherwise stated, all
commercial reagents were used without further purification.
Organic solutions were dried over anhydrous sodium sulphate.
Thin layer chromatography (TLC) for routine monitoring of the
course of reactions and confirming the purity of analytical samples
employed aluminium-backed silica gel plates (Merck DC-Alufolien
Kieselgel 60 F254). CHCl3 or CHCl3/methanol was used as developing
5. Conclusions
Novel modifications on C-TCs and O-TCs were made to further
expand the SAR studies on this class of NNRTIs. The replacement of
the N-phenyl moiety with (aryl)alkyl portions led to weak anti-HIV-
1 inhibitors, except for the cyclooctyl derivative. Among the N-
phenyl ring poly-substituted patterns, the 3,4-arrangement gave

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1657
solvent and detection of spots was made by UV light and/or by
iodine vapours. Merck silica gel, 230–400 mesh, was used for
chromatography. The parallel solution-phase chemistry was per-
6.1.3. General procedure A for the parallel synthesis of C-TCs 1–11,
15–17, 35–39 and O-TCs 60, 62–64, 70, 72
60% sodium hydride dispersion in mineral oil (0.2 g,
A
formed by using
a
12-Carousel Reaction StationÔ (Radleys
w5 mmol) was added in single portion at r.t. to a set of reaction
tubes containing dry THF solution (15 mL) of A1 (0.45 g, 5 mmol).
After stirring for 10 min, proper isothiocyanate (5 mmol) was
added to each reaction mixture prolonging the stirring at r.t. for
24 h. The solvent of each reaction tube was evaporated by an
EvaposelÔ apparatus and the crude residue was treated with
aqueous ammonium chloride (2.5 g dissolved in 100 mL of water).
Each mixture was then transferred into a set of separating funnels
and extracted with diethyl ether (2 ꢂ 40 mL). The combined
extracts were dried, filtered through a pad of Florisil (diameter
5 ꢂ 2 cm) and evaporated in parallel by an EvaposelÔ apparatus.
The crude products were purified by crystallization to afford C-TCs.
Following acidification (pH ¼ 0) of the extracted aqueous phases
with 2 N HCl, O-TCs separated as solids. The crude solid was filtered
off and purified by crystallization.
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
determined on a Fisher–Johns apparatus and are uncorrected.
Optical rotations were measured on a Perkin Elmer 241 MC spec-
tropolarimeter; concentrations are expressed in g/100 mL and the
cell length is 1 dm. 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
d (ppm) units relative to the internal standard
tetramethylsilane, and the splitting patterns were described as
follows: s (singlet), d (doublet), t (triplet), m (multiplet) and br s
(broad singlet). The first order values reported for coupling
constants J were given in Hz. Elemental analyses were performed
by an EA1110 Elemental Analyser (Fison-Instruments, Milan) and
were within ꢁ 0.4% of the theoretical values.
6.1.3.1. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] butylth-
iocarbamate, 1. Yield 28%; mp 64–65 ꢀC (from Me2CO). IR (KBr)
3249, 2953, 2924,1772,1709 cmꢃ1; 1H NMR (CDCl3)
d 0.68–0.88 (m,
3H, CH3), 1.08–1.53 (m, 4H, CH2CH2Me), 3.05–3.18 and 3.32–3.47
(m, 4H, 2 CH2N), 3.86–4.03 (m, 2H, CH2O), 6.34 and 6.74 (br s, 1H,
NH, exchangeable), 7.58–7.82 (m, 4H, arom. H). Anal. Calcd for
C15H18N2O3S: C, 58.80; H, 5.92; N, 9.14; S, 10.47. Found: C, 58.88; H,
6.19; N, 8.97; S, 10.16.
6.1.2. General procedure for the synthesis of starting alcohols A2, A4,
and A5
A mixture of proper aminoalcohol (12 mmol) and proper
anhydride (10 mmol) was heated (for A2, A5: 145–150 ꢀC; for A4:
175–180 ꢀC) under stirring for 2 h. After cooling, the reaction
mixture was dissolved in CH2Cl2 and washed with 2 N HCl
(2 ꢂ 50 mL). The organic layer was dried and evaporated under
reduced pressure to give (R)-A2, (S)-A2, A4, A5 which were purified
by crystallization from the proper solvent mixture.
6.1.3.2. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 1,1,3,3-
tetramethylbutylthiocarbamate, 2. Yield 11%; mp 112–113 ꢀC (from
Et2O:MeOH). IR (KBr) 3232, 2951, 1775, 1716 cmꢃ1; 1H NMR (CDCl3)
d
0.81 (s, 9H, C(CH3)3), 1.24 (s, 6H, C(CH3)2), 1.48 (s, 2H, CH2), 3.98–
4.08 (m, 2H, CH2N), 4.67–4.77 (m, 2H, CH2O), 6.18 and 6.55 (br s,1H,
NH, exchangeable), 7.62–7.85 (m, 4H, arom. H). Anal. Calcd for
C19H26N2O3S: C, 62.95; H, 7.23; N, 7.73; S, 8.85. Found: C, 62.88; H,
7.23; N, 7.77; S, 8.72.
6.1.2.1. 2-[(1R)-2-Hydroxy-1-methylethyl]-1H-isoindole-1,3(2H)-
dione, (R)-A2. Yield 69%; mp 84–86 ꢀC (from CH2Cl2:Et2O), lit. [42]:
20
84–85 ꢀC (from EtOAc:petroleum ether). [
a]
¼ ꢃ14.7 (c 1.03,
D
CHCl3), lit. [39]: ꢃ14.6 (c 3.2, CHCl3). IR (KBr) 3490, 1772,
1689 cmꢃ1; 1H NMR (CDCl3)
d
1.44 (d, J ¼ 6.6 Hz, 3H, CH3), 2.67 (br s,
6.1.3.3. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] cyclo-
propylthiocarbamate, 3. Yield 49%; mp 144–145 ꢀC (from CH2Cl2
1H, OH, exchangeable), 3.86–4.10 (m, 2H, CH2), 4.21–4.74 (m, 1H,
CH), 7.61–8.01 (m, 4H, arom. H). Anal. Calcd for C11H11NO3: C, 64.38;
H, 5.40; N, 6.83. Found: C, 64.54; H, 5.16; N, 6.84.
:MeOH). IR (KBr) 3281, 1769, 1712 cmꢃ1 1H NMR (CDCl3)
; d 0.36–
0.77 (m, 4H, 2 CH2), 2.48–2.63 and 2.73–2.87 (m, 1H, CH), 3.85–4.06
(m, 2H, CH2N), 4.53–4.72 (m, 2H, CH2O), 6.47 and 6.89 (br s, 1H, NH,
exchangeable), 7.56–7.83 (m, 4H, arom. H). Anal. Calcd for
C14H14N2O3S: C, 57.92; H, 4.86; N, 9.65; S, 11.04. Found: C, 57.71; H,
4.98; N, 9.53; S, 11.35.
6.1.2.2. 2-[(1S)-2-Hydroxy-1-methylethyl]-1H-isoindole-1,3(2H)-
dione, (S)-A2. Yield 73%; mp 84–86 ꢀC (from CH2Cl2:Et2O), lit. [42]:
20
84–85 ꢀC (from EtOAc:petroleum ether). [
a]
¼ þ15.5 (c 1.05,
D
CHCl3), lit. [39]: þ16.5 (c 2.0, CHCl3). IR (KBr) 3490, 1772,
1689 cmꢃ1; 1H NMR (CDCl3)
d
1.42 (d, J ¼ 6.6 Hz, 3H, CH3), 3.16 (br s,
6.1.3.4. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] cyclo-
pentylthiocarbamate, 4. Yield 52%; mp 115–116 ꢀC (from CH2Cl2
1H, OH, exchangeable), 3.84–4.13 (m, 2H, CH2), 4.21–4.74 (m, 1H,
CH), 7.52–8.01 (m, 4H, arom. H). Anal. Calcd for C11H11NO3: C,
64.38; H, 5.40; N, 6.83. Found: C, 64.19; H, 5.50; N, 7.01.
:MeOH). IR (KBr) 3229, 2954, 2867, 1775, 1723 cmꢃ1 1H NMR
;
(CDCl3) d 1.19–2.00 (m, 8H, 4 CH2), 3.86–4.38 (m, 3H, CH and CH2N),
4.55–4.68 (m, 2H, CH2O), 6.38 and 6.68 (br s, 1H, NH, exchange-
able), 7.58–7.88 (m, 4H, arom. H). Anal. Calcd for C16H18N2O3S: C,
60.36; H, 5.70; N, 8.80; S, 10.07. Found: C, 60.42; H, 5.95; N, 8.46; S,
9.94.
6.1.2.3. 5-tert-Butyl-2-(2-hydroxyethyl)-1H-isoindole-1,3(2H)-dione,
A4. Yield 82%; mp 60–62 ꢀC (from Me2CO). IR (CHCl3) 3463, 2969,
1771,1706 cmꢃ1; 1H NMR (CDCl3)
d 1.46 (s, 9H, 3 CH3), 3.47 (br s,1H,
OH, exchangeable), 3.62–4.15 (m, 4H, 2 CH2), 7.55–8.04 (m, 3H,
arom. H). Anal. Calcd for C14H17NO3: C, 68.00; H, 6.93; N, 5.66.
Found: C, 68.26; H, 6.90; N, 5.72.
6.1.3.5. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] cyclo-
octylthiocarbamate, 5. Yield 31%; mp 93–95 ꢀC (from Et2O:MeOH).
IR (KBr) 3229, 2928, 1774, 1714 cmꢃ1 1H NMR (CDCl3)
; d 1.25–1.85
6.1.2.4. (ꢁ) 2-(2-Hydroxy-1-methylethyl)-5-methyl-1H-isoindole-1,3
(2H)-dione, A5. Yield 70%; mp 121–123 ꢀC (from CH2Cl2:Et2O), lit.
[42]: 105–107 ꢀC for the S isomer (from EtOAc:petroleum ether). IR
(m, 14H, 7 CH2), 3.70–4.17 (m, 3H, CH and CH2N), 4.48–4.67 (m, 2H,
CH2O), 6.37 and 6.83 (br s, 1H, NH, exchangeable), 7.55–7.77 (m, 4H,
arom. H). Anal. Calcd for C19H24N2O3S: C, 63.31; H, 6.71; N, 7.77; S,
8.90. Found: C, 63.11; H, 6.89; N, 7.70; S, 9.06.
(KBr) 3497, 2986, 2957, 1764, 1694 cmꢃ1; 1H NMR (CDCl3)
d 1.41 (s,
3H, CH3-Phtha), 2.94 (br s, 1H, OH, exchangeable), 3.85–4.10 (m, 2H,
CH2), 4.20–4.63 (m, 1H, CH), 7.39–7.85 (m, 4H, arom. H). Anal. Calcd
for C12H13NO3: C, 65.74; H, 5.98; N, 6.39. Found: C, 65.70; H, 5.70; N,
6.37.
6.1.3.6. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] cyclo-
hexylmethylthiocarbamate, 6. Yield 11%; mp 83–84 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3212, 2923, 1772, 1718 cmꢃ1 1H NMR
;

1658
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
(CDCl3)
d
0.73–1.22 and 1.48–1.72 (m,11H, 5 CH2 and CH), 2.93–3.02
Calcd for C17H12F2N2O3S: C, 56.35; H, 3.34; N, 7.73; S, 8.85. Found: C,
56.05; H, 3.20; N, 7.70; S, 8.37.
and 3.22–3.33 (m, 2H, CH2-cyclohexyl), 3.88–4.04 (m, 2H, CH2N),
4.58–4.68 (m, 2H, CH2O), 6.31 and 6.67 (br s,1H, NH, exchangeable),
7.59–7.83 (m, 4H, arom. H). Anal. Calcd for C18H22N2O3S: C,
62.40; H, 6.40; N, 8.09; S, 9.26. Found: C, 62.66; H, 6.47; N, 7.89; S,
8.94.
6.1.3.14. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,6-di-
fluorophenylthiocarbamate, 17. Yield 47%; mp 204–205 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3320, 1771, 1717 cmꢃ1 1H NMR (CDCl3)
;
d
3.92–4.20 (m, 2H, CH2N), 4.63–4.93 (m, 2H, CH2O), 6.62–7.10 and
6.1.3.7. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] allylth-
iocarbamate, 7. Yield 56%; mp 112–113 ꢀC (from CH2Cl2:MeOH). IR
7.52–8.02 (m, 7H, arom. H), NH, not detected. Anal. Calcd for
C17H12F2N2O3S: C, 56.35; H, 3.34; N, 7.73; S, 8.85. Found: C, 56.69; H,
3.53; N, 7.51; S, 8.55.
(KBr) 3218, 3054, 2958, 1779, 1715 cmꢃ1 1H NMR (CDCl3)
; d 3.71–
3.81 and 3.88–4.11 (m, 4H, CH2-allyl and CH2N), 4.57–4.67 (m, 2H,
CH2O), 4.88–5.22 (m, 2H, CH2]), 5.53–5.85 (m, 1H, CH]), 6.38 and
6.73 (br s, 1H, NH, exchangeable), 7.58–7.84 (m, 4H, arom. H). Anal.
Calcd for C14H14N2O3S: C, 57.92; H, 4.86; N, 9.65; S, 11.04. Found: C,
57.99; H, 5.03; N, 9.56; S, 11.07.
6.1.3.15. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,4,6-
trifluorophenylthiocarbamate, 35. Yield 44%; mp 159–161 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3239, 1773, 1709 cmꢃ1 1H NMR (CDCl3)
;
d
3.88–4.19 (m, 2H, CH2N), 4.63–4.83 (m, 2H, CH2O), 6.46–6.88 and
7.44–7.96 (m, 7H, arom. H and NH, exchangeable). Anal. Calcd for
C17H11F3N2O3S: C, 53.68; H, 2.92; N, 7.37; S, 8.43. Found: C, 53.99; H,
3.28; N, 7.15; S, 8.49.
6.1.3.8. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] benzyl-
thiocarbamate, 8. Yield 18%; mp 96–98 ꢀC (from Et2O:MeOH). IR
(KBr) 3336, 2960, 1771, 1708 cmꢃ1; 1H NMR (CDCl3)
d 3.89–4.02 (m,
2H, CH2N), 4.31–4.36 and 4.58–4.70 (m, 4H, CH2O and CH2Ph), 6.63
and 6.96 (br s, 1H, NH, exchangeable), 7.07–7.27 (m, 5H, arom. H),
7.57–7.77 (m, 4H, arom. H). Anal. Calcd for C18H16N2O3S: C, 63.51; H,
4.74; N, 8.23; S, 9.42. Found: C, 63.50; H, 4.72; N, 8.17; S, 9.27.
6.1.3.16. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,3,4-
trichlorophenylthiocarbamate, 36. Yield 42%; mp 175–178 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3213, 1776, 1722 cmꢃ1 1H NMR (CDCl3)
;
d
3.96–4.07 (m, 2H, CH2N), 4.73–4.83 (m, 2H, CH2O), 7.16–7.30 and
7.63–7.84 (m, 6H, arom. H), 8.17 (br s, 1H, NH, exchangeable). Anal.
Calcd for C17H11Cl3N2O3S: C, 47.52; H, 2.58; N, 6.52; S, 7.46. Found:
C, 47.50; H, 2.90; N, 6.81; S, 7.34.
6.1.3.9. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2-furyl-
methylthiocarbamate, 9. Yield 23%; mp 106–107 ꢀC (from Me2CO:-
MeOH). IR (KBr) 3340, 1770, 1715 cmꢃ1 1H NMR (CDCl3)
; d 3.88–
4.05 (m, 2H, CH2N), 4.27–4.36 and 4.54–4.69 (m, 4H, CH2O and CH2-
Fur), 6.08–6.13 and 6.17–6.25 (m, 2H, CH(3-fur) and CH(4-fur)), 6.74
and 6.95 (br s, 1H, NH, exchangeable), 7.06–7.09 and 7.23–7.27 (m,
1H, CH(5-fur)), 7.58–7.79 (m, 4H, arom. H). Anal. Calcd for
C16H14N2O3S: C, 58.17; H, 4.27; N, 8.48; S, 9.71. Found: C, 58.07; H,
4.46; N, 8.28; S, 9.51.
6.1.3.17. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,4,6-
trichlorophenylthiocarbamate, 37. Yield 53%; mp 201–202 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3262, 1772, 1701 cmꢃ1 1H NMR (CDCl3)
;
d
3.90–4.20 (m, 2H, CH2N), 4.59–4.91 (m, 2H, CH2O), 7.17–7.34 and
7.63–7.94 (m, 6H, arom. H), NH, not detected. Anal. Calcd for
C17H11Cl3N2O3S: C, 47.52; H, 2.58; N, 6.52; S, 7.46. Found: C, 47.80;
H, 2.88; N, 6.33; S, 7.48.
6.1.3.10. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2-phe-
nylethylthiocarbamate, 10. Yield 41%; mp 92–92 ꢀC (from CH2Cl2
6.1.3.18. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-bromo-
2,6-dimethylphenylthiocarbamate, 38. Yield 38%; mp 150–152 ꢀC
:MeOH). IR (KBr) 3226, 1774, 1716 cmꢃ1 1H NMR (CDCl3)
; d 2.60–
2.88 (m, 2H, CH2Ph), 3.33–3.47 and 3.58–3.74 (m, 2H, CH2CH2Ph),
3.84–4.03 (m, 2H, CH2N), 4.55–4.68 (m, 2H, CH2O), 6.43 and 6.83
(br s, 1H, NH, exchangeable), 7.00–7.27 (m, 5H, arom. H), 7.56–7.82
(m, 4H, arom. H). Anal. Calcd for C19H18N2O3S: C, 64.39; H, 5.12; N,
7.90; S, 9.05. Found: C, 64.27; H, 5.25; N, 7.86; S, 8.70.
(from Et2O:MeOH). IR (KBr) 3483, 3177, 1764, 1697 cmꢃ1 1H NMR
;
(CDCl3)
d 2.05 (s, 3H, CH3), 2.17 (s, 3H, CH3), 3.78–4.23 (m, 2H,
CH2N), 4.57–5.01 (m, 2H, CH2O), 6.92–7.32 and 7.65–7.95 (m, 6H,
arom. H), 8.13 (br s, 1H, NH exchangeable). Anal. Calcd for
C19H17BrN2O3S: C, 52.66; H, 3.95; N, 6.46; S, 7.40. Found: C, 52.43;
H, 4.28; N, 6.45; S, 7.30.
6.1.3.11. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2-(3,4-
dimethoxyphenyl)ethylthiocarbamate, 11. Yield 73%; mp 121–123 ꢀC
6.1.3.19. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,3,5,6-
tetrafluorophenylthiocarbamate, 39. Yield 44%; mp 150–152 ꢀC
(from Et2O:MeOH). IR (KBr) 3224,1773,1706 cmꢃ1; 1H NMR (CDCl3)
(from CH2Cl2:MeOH). IR (KBr) 3222, 1776, 1721 cmꢃ1 1H NMR
;
(CDCl3)
d 2.57–2.66 and 2.69–2.80 (m, 2H, CH2Ph), 3.31–3.43 and
3.58–3.73 (m, 2H, CH2CH2Ph), 3.77, 3.78, 3.80 (s, 6H, 2 CH3O), 3.88–
4.03 (m, 2H, CH2N), 4.57–4.66 (m, 2H, CH2O), 6.34 (br s, 1H, NH,
exchangeable), 6.57–6.74 (m, 4H, arom. H and NH, exchangeable),
7.58–7.78 (m, 4H, arom. H). Anal. Calcd for C21H22N2O5S: C, 60.85;
H, 5.35; N, 6.76; S, 7.74. Found: C, 60.79; H, 5.48; N, 6.67; S, 8.22.
d 3.88–4.01 (m, 2H, CH2N), 4.63–4.95 (m, 2H, CH2O), 6.87–7.28 and
7.69–7.96 (m, 5H, arom. H), 8.12 (br s, 1H, NH exchangeable). Anal.
Calcd for C17H10F4N2O3S: C, 51.26; H, 2.53; N, 7.03; S, 8.05. Found: C,
51.56; H, 2.94; N, 6.94; S, 7.85.
6.1.3.20. 2-[({2-[({[2-(3,4-Dimethoxyphenyl)ethyl]amino}carbonothioyl)
6.1.3.12. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,4-di-
fluorophenylthiocarbamate, 15. Yield 36%; mp 130–132 ꢀC (from
oxy]ethyl}amino)carbonyl]benzoic acid, 60. Yield 23%; mp 166–168 ꢀC
(from Me2CO:MeOH). IR (KBr) 3277, 1692, 1642 cmꢃ1 1H NMR
;
CH2Cl2:MeOH). IR (KBr) 3260, 1772, 1705 cmꢃ1
;
1H NMR (CDCl3)
(DMSO) d 2.55–2.74 (m, 2H, CH2Ph), 3.19–3.31 (m, 2H, CH2CH2Ph),
d
3.98–4.25 (m, 2H, CH2N), 4.71–4.99 (m, 2H, CH2O), 6.64–7.07 and
3.35–3.52 (m, 2H, CH2N), 3.60 and 3.63 (s, 3H, OCH3), 3.61 and 3.65
(s, 3H, OCH3), 6.56–6.83, 7.27–7.53 and 7.63–7.72 (m, 7H, arom. H),
8.40 (br s, 1H, CONH, exchangeable), 9.15 (br s, 1H, CSNH,
exchangeable), 12.76 (br s, 1H, COOH, exchangeable). Anal. Calcd for
C21H24N2O6S: C, 58.32; H, 5.59; N, 6.48; S, 7.41. Found: C, 58.08; H,
5.63; N, 6.59; S, 7.65.
7.51–8.06 (m, 7H, arom. H), 8.16 (br s, 1H, NH, exchangeable). Anal.
Calcd for C17H12F2N2O3S: C, 56.35; H, 3.34; N, 7.73; S, 8.85. Found: C,
56.54; H, 3.34; N, 7.68; S, 8.54.
6.1.3.13. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
difluorophenylthiocarbamate, 16. Yield 26%; mp 101–103 ꢀC (from
Et2O:MeOH). IR (KBr) 3225, 1773, 1710 cmꢃ1 1H NMR (CDCl3)
4.03–4.29 (m, 2H, CH2N), 4.76–5.06 (m, 2H, CH2O), 6.72–7.15 and
7.63–8.05 (m, 7H, arom. H), 8.32 (br s, 1H, NH, exchangeable). Anal.
2,5-
;
6.1.3.21. 2-({[2-({[(2,4-Difluorophenyl)amino]carbonothioyl}ox-
d
y)ethyl]amino}carbonyl)benzoic acid, 62. Yield 23%; mp 124–126 ꢀC
(from Me2CO). IR (KBr) 3298, 1678, 1635 cmꢃ1 1H NMR (DMSO)
;

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1659
d
3.13–3.62 (m, 2H, CH2N), 4.37–4.57 (m, 2H, CH2O), 6.84–7.81 (m,
6.1.4.2. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,6-di-
methylphenylthiocarbamate, 13. Yield 23%; mp 173–175 ꢀC (from
7H, arom. H), 8.24 and 8.42 (br s, 1H, CONH, exchangeable), 10.67
and 10.93 (br s, 1H, CSNH, exchangeable), 12.88 (br s, 1H, COOH,
exchangeable). Anal. Calcd for C17H14F2N2O4S: C, 53.68; H, 3.71; N,
7.36; S, 8.43. Found: C, 53.42; H, 3.79; N, 7.40; S, 8.45.
CH2Cl2:EtOH). IR (KBr) 3291, 1769, 1713 cmꢃ1 1H NMR (CDCl3)
;
d
2.10 (s, 3H, CH3), 2.21 (s, 3H, CH3), 3.96–4.22 (m, 2H, CH2N), 4.59–
5.01 (m, 2H, CH2O), 6.81–7.22 and 7.67–7.94 (m, 7H, arom. H), 8.12
(br s, 1H, NH, exchangeable). Anal. Calcd for C19H18N2O3S: C, 64.39;
H, 5.12; N, 7.90; S, 9.05. Found: C, 64.56; H, 5.04; N, 7.91; S, 8.88.
6.1.3.22. 2-({[2-({[(2,5-Difluorophenyl)amino]carbonothioyl}oxy)-
ethyl]amino}carbonyl)benzoic acid, 63. Yield 9%; mp 144–146 ꢀC
(from Me2CO:MeOH). IR (KBr) 3341, 3178, 1722 cmꢃ1
;
1H NMR
6.1.4.3. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 3,5-di-
methylphenylthiocarbamate, 14. Yield 31%; mp 153–155 ꢀC (from
(DMSO)
d 3.61–4.02 (m, 2H, CH2N), 4.15–4.39 (m, 2H, CH2O), 7.37–
8.08 (m, 7H, arom. H), 8.44 (br s,1H, CONH, exchangeable),10.92 (br
s, 1H, CSNH, exchangeable), 12.56 (br s, 1H, COOH, exchangeable).
Anal. Calcd for C17H14F2N2O4S: C, 53.68; H, 3.71; N, 7.36; S, 8.43.
Found: C, 53.79; H, 3.79; N, 7.20; S, 8.45.
CH2Cl2:EtOH). IR (KBr) 3309, 1770, 1705 cmꢃ1 1H NMR (CDCl3)
;
d
2.23 (s, 6H, 2 CH3), 4.10 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.86 (t, J ¼ 6.0 Hz,
2H, CH2O), 6.68–7.03 and 7.71–8.01 (m, 7H, arom. H), 8.39 (br s, 1H,
NH, exchangeable). Anal. Calcd for C19H18N2O3S: C, 64.39; H, 5.12;
N, 7.90; S, 9.05. Found: C, 64.36; H, 5.28; N, 7.87; S, 8.76.
6.1.3.23. 2-({[2-({[(2,6-Difluorophenyl)amino]carbonothioyl}oxy)-
ethyl]amino}carbonyl)benzoic acid, 64. Yield 30%; mp 170–172 ꢀC
6.1.4.4. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 3,5-di-
fluorophenylthiocarbamate, 18. Yield 47%; mp 156–158 ꢀC (from
(from Me2CO:MeOH). IR (KBr) 3345, 3180, 1723 cmꢃ1 1H NMR
;
(DMSO)
d
3.22–3.62 (m, 2H, CH2N), 4.34–4.57 (m, 2H, CH2O), 7.01–
Et2O:EtOH). IR (KBr) 3308, 1777,1715 cmꢃ1; 1H NMR (CDCl3)
d 3.95–
7.78 (m, 7H, arom. H), 8.25 and 8.48 (br s, 1H, CSNH, exchangeable),
10.61 and 10.91 (br s, 1H, CONH, exchangeable), 12.90 (br s, 1H,
COOH, exchangeable). Anal. Calcd for C17H14F2N2O4S: C, 53.68; H,
3.71; N, 7.36; S, 8.43. Found: C, 53.63; H, 3.51; N, 7.21; S, 8.21.
4.28 (m, 2H, CH2N), 4.73–5.02 (m, 2H, CH2O), 6.31–6.79, 6.93–7.00
and 7.62–7.99 (m, 7H, arom. H), 11.04 (br s, 1H, NH, exchangeable).
Anal. Calcd for C17H12F2N2O3S: C, 56.35; H, 3.34; N, 7.73; S, 8.85.
Found: C, 56.10; H, 3.58; N, 7.67; S, 8.93.
6.1.3.24. 2-({[2-({[(2,4,6-Trifluorophenyl)amino]carbonothioyl}oxy)-
6.1.4.5. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,3-di-
chlorophenylthiocarbamate, 19. Yield 36%; mp 119–121 ꢀC (from
ethyl]amino}carbonyl)benzoic acid, 70. Yield 22%; mp 143–145 ꢀC
(from Me2CO:MeOH). IR (KBr) 3340, 3162, 1724 cmꢃ1
;
1H NMR
CH2Cl2:MeOH). IR (KBr) 3342, 1774, 1716 cmꢃ1 1H NMR (CDCl3)
;
(DMSO)
d
3.27–3.62 (m, 2H, CH2N), 4.37–4.58 (m, 2H, CH2O), 7.10–
d
4.10 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.87 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.01–
7.88 (m, 6H, arom. H), 8.26 and 8.48 (br s, 1H, CONH, exchangeable),
10.60 and 10.83 (br s, 1H, CSNH, exchangeable), 12.95 (br s, 1H,
COOH, exchangeable). Anal. Calcd for C17H13F3N2O4S: C, 51.26; H,
3.29; N, 7.03; S, 8.05. Found: C, 51.42; H, 3.01; N, 6.93; S, 8.26.
7.32 and 7.58–7.98 (m, 7H, arom. H), 8.15 (br s, 1H, NH, exchange-
able). Anal. Calcd for C17H12Cl2N2O3S: C, 51.66; H, 3.06; N, 7.09; S,
8.11. Found: C, 51.51; H, 3.34; N, 6.95; S, 7.93.
6.1.4.6. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,4-di-
chlorophenylthiocarbamate, 20. Yield 22%; mp 154–155 ꢀC (from
6.1.3.25. 2-({[2-({[(2,3,5,6-Tetrafluorophenyl)amino]carbonothioyl}-
oxy)ethyl]amino}carbonyl)benzoic acid, 72. Yield 10%; mp 173–
CH2Cl2:MeOH). IR (KBr) 3337, 1760, 1703 cmꢃ1 1H NMR (DMSO)
;
175 ꢀC (from Me2CO). IR (KBr) 3340, 3162, 1724 cmꢃ1
;
1H NMR
d
3.94 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.73 (t, J ¼ 6.0 Hz, 2H, CH2O),
(DMSO)
d
3.18–3.62 (m, 2H, CH2N), 4.34–4.58 (m, 2H, CH2O), 7.11–
7.22–7.54 and 7.79–8.05 (m, 7H, arom. H), 10.38 (br s, 1H, NH,
exchangeable). Anal. Calcd for C17H12Cl2N2O3S: C, 51.66; H,
3.06; N, 7.09; S, 8.11. Found: C, 51.66; H, 3.07; N, 7.11; S, 8.00.
7.93 (m, 5H, arom. H), 8.38 (br s, 1H, CONH, exchangeable), 11.05 (br
s, 1H, CSNH, exchangeable), 12.86 (br s, 1H, COOH, exchangeable).
Anal. Calcd for C17H12F4N2O4S: C, 49.04; H, 2.91; N, 6.73; S, 7.70.
Found: C, 49.17; H, 3.09; N, 6.80; S, 7.62.
6.1.4.7. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,5-di-
chlorophenylthiocarbamate, 21. Yield 57%; mp 138–140 ꢀC (from
6.1.4. General procedure B for the parallel synthesis of C-TCs 12–14,
18–34, 40–59 and O-TCs 61, 65–69, 71, 73–76
CH2Cl2:EtOH). IR (KBr) 3167, 1776, 1716 cmꢃ1 1H NMR (CDCl3)
;
d
4.11 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.90 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.01–
To each ice-cooled reaction tube containing a stirred dried DMF
solution (15 mL) of the proper alcohol (5 mmol) and the proper
isothiocyanate (5 mmol), a 60% sodium hydride dispersion in
mineral oil (0.2 g, w5 mmol) was added in a single portion. Each
reaction mixture was stirred at r.t. for 24 h. Upon treatment with
aqueous ammonium chloride (2.5 g dissolved in 100 mL of water),
C-TCs separated as oils or solids. Each reaction mixture was trans-
ferred into a set of separating funnels and extracted with diethyl
ether (2 ꢂ 40 mL). The pooled organic phases were washed with
water (4 ꢂ 20 mL), dried and evaporated in parallel by an Evap-
oselÔ apparatus. The crude products were purified by crystalliza-
tion. Upon acidification (pH ¼ 0) of the reaction mother liquors
with 2 N HCl, O-TCs precipitated and were filtered off in parallel.
Then, the crude solids were purified by crystallization.
7.46 and 7.71–8.11 (m, 7H, arom. H), 8.43 (br s, 1H, NH, exchange-
able). Anal. Calcd for C17H12Cl2N2O3S: C, 51.66; H, 3.06; N, 7.09; S,
8.11. Found: C, 51.54; H, 3.35; N, 6.92; S, 7.98.
6.1.4.8. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,6-di-
chlorophenylthiocarbamate, 22. Yield 37%; mp 200–202 ꢀC (from
CH2Cl2:EtOH). IR (KBr) 3308, 1770, 1713 cmꢃ1 1H NMR (CDCl3)
;
d
3.82–4.19 (m, 2H, CH2N), 4.55–4.97 (m, 2H, CH2O), 7.03–7.49 and
7.79–7.98 (m, 7H, arom. H), 9.31 (br s, 1H, NH, exchangeable). Anal.
Calcd for C17H12Cl2N2O3S: C, 51.66; H, 3.06; N, 7.09; S, 8.11. Found: C,
51.49; H, 3.39; N, 6.79; S, 7.90.
6.1.4.9. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 3,4-di-
chlorophenylthiocarbamate, 23. Yield 49%; mp 164–166 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3380, 1778, 1710 cmꢃ1 1H NMR (DMSO)
;
6.1.4.1. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2,4-di-
methylphenylthiocarbamate, 12. Yield 15%; mp 156–158 ꢀC (from
CH2Cl2:EtOH). IR (KBr) 3337, 1770, 1717, 1702 cmꢃ1; 1H NMR (CDCl3)
d
4.09 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.89 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.07–
8.05 (m, 7H, arom. H), 10.37 (br s, 1H, NH, exchangeable). Anal.
Calcd for C17H12Cl2N2O3S: C, 51.66; H, 3.06; N, 7.09; S, 8.11. Found: C,
51.74; H, 3.14; N, 7.13; S, 8.13.
d
2.18 (s, 3H, CH3), 2.25 (s, 3H, CH3), 4.05 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.83
(t, J ¼ 6.0 Hz, 2H, CH2O), 6.81–7.16 and 7.71–7.93 (m, 7H, arom. H),
8.18 (br s, 1H, NH, exchangeable). Anal. Calcd for C19H18N2O3S: C,
64.39;H, 5.12; N, 7.90;S, 9.05. Found: C, 64.19; H, 5.32;N, 7.76; S, 8.81.
6.1.4.10. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 3,5-di-
chlorophenylthiocarbamate, 24. Yield 53%; mp 139–140 ꢀC (from

1660
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
CH2Cl2:MeOH). IR (KBr) 3180, 1775, 1710 cmꢃ1
;
1H NMR (DMSO)
(t, J ¼ 6.0 Hz, 2H, CH2O), 6.68–7.10 and 7.55–7.98 (m, 7H, arom. H),
10.12 (br s, 1H, NH, exchangeable). Anal. Calcd for C18H15ClN2O4S: C,
55.32; H, 3.87; N, 7.17; S, 8.20. Found: C, 55.10; H, 3.85; N, 7.14; S,
8.21.
d
3.97 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.76 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.03–
7.50 and 7.65–8.13 (m, 7H, arom. H), 10.32 (br s, 1H, NH,
exchangeable). Anal. Calcd for C17H12Cl2N2O3S: C, 51.66; H, 3.06; N,
7.09; S, 8.11. Found: C, 51.49; H, 3.25; N, 6.74; S, 7.87.
6.1.4.19. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 3,4-di-
methoxyphenylthiocarbamate, 33. Yield 24%; mp 99–101 ꢀC (from
6.1.4.11. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chloro-
2-methylphenylthiocarbamate, 25. Yield 38%; mp 113–115 ꢀC (from
CH2Cl2:EtOH). IR (KBr) 3337, 1771, 1705 cmꢃ1 1H NMR (CDCl3)
;
CH2Cl2:MeOH). IR (KBr) 3330, 1765, 1690 cmꢃ1
;
1H NMR (CDCl3)
d
3.86 (s, 6H, 2 OCH3), 4.08 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.84 (t,
d
2.19 (s, 3H, CH3), 4.06 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.80 (t, J ¼ 6.0 Hz,
J ¼ 6.0 Hz, 2H, CH2O), 6.63–6.96 and 7.68–7.96 (m, 7H, arom. H),
8.20 (br s, 1H, NH, exchangeable). Anal. Calcd for C19H18N2O5S: C,
59.06; H, 4.70; N, 7.25; S, 8.30. Found: C, 59.32; H, 5.06; N, 7.23; S,
8.64.
2H, CH2O), 6.88–7.39 and 7.64–8.28 (m, 7H, arom. H), 10.37 (br s,
1H, NH, exchangeable). Anal. Calcd for C18H15ClN2O3S: C, 57.68; H,
4.03; N, 7.47; S, 8.55. Found: C, 57.64; H, 4.00; N, 7.51; S, 8.28.
6.1.4.12. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 5-chloro-
2-methylphenylthiocarbamate, 26. Yield 36%; mp 125–127 ꢀC (from
6.1.4.20. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chloro-
3-trifluoromethylphenylthiocarbamate, 34. Yield 43%; mp 163–
165 ꢀC (from CH2Cl2:EtOH). IR (KBr) 3193, 1773, 1718 cmꢃ1; 1H NMR
CH2Cl2:MeOH). IR (KBr) 3175, 1775, 1712 cmꢃ1 1H NMR (DMSO)
;
d
2.07 (s, 3H, CH3), 3.95 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.71 (t, J ¼ 6.0 Hz,
(CDCl3)
d
4.11 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.89 (t, J ¼ 6.0 Hz, 2H, CH2O),
2H, CH2O), 7.03–7.32 and 7.78–8.10 (m, 7H, arom. H), 10.35 (br s, 1H,
NH, exchangeable). Anal. Calcd for C18H15ClN2O3S: C, 57.68; H, 4.03;
N, 7.47; S, 8.55. Found: C, 57.98; H, 4.37; N, 7.15; S, 8.40.
7.27–7.52 and 7.62–7.98 (m, 7H, arom. H), 8.14 (br s, 1H, NH,
exchangeable). Anal. Calcd for C18H12F3N2O3S: C, 50.42; H, 2.82; N,
6.53; S, 7.48. Found: C, 50.42; H, 3.07; N, 6.66; S, 7.58.
6.1.4.13. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2-chloro-
6-methylphenylthiocarbamate, 27. Yield 35%; mp 173–175 ꢀC (from
6.1.4.21. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)ethyl] cyclohexylthiocarbamate, 40. Yield 20%; mp 146–147 ꢀC
(from Et2O:CH2Cl2). IR (KBr) 3230, 2926, 1776, 1720 cmꢃ1; 1H NMR
CH2Cl2:EtOH). IR (KBr) 3317, 1769, 1713 cmꢃ1 1H NMR (CDCl3)
;
d
2.09–2.41 (m, 3H, CH3), 3.78–4.23 (m, 2H, CH2N), 4.59–5.01 (m,
(CDCl3) d 0.82–2.20 (m, 10H, 5 CH2-cyclohexyl), 2.52 (s, 3H, CH3),
2H, CH2O), 6.91–7.39 and 7.61–8.05 (m, 7H, arom. H), 8.34 (br s, 1H,
NH, exchangeable). Anal. Calcd for C18H15ClN2O3S: C, 57.68; H, 4.03;
N, 7.47; S, 8.55. Found: C, 57.81; H, 4.29; N, 7.21; S, 8.26.
3.21–3.31 (m, 1H, CH), 4.07 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.70 (t,
J ¼ 6.0 Hz, 2H, CH2O), 7.32 (br s, 1H, NH, exchangeable), 7.42–7.90
(m, 3H, arom. H). Anal. Calcd for C18H22N2O3S: C, 62.40; H, 6.40; N,
8.09; S, 9.25. Found: C, 62.47; H, 6.57; N, 7.97; S, 9.60.
6.1.4.14. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3-
chloro-4-methylphenylthiocarbamate, 28. Yield 12%; mp ꢃ1143–
6.1.4.22. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-
ethyl] 4-methylphenylthiocarbamate, 41. Yield 37%; mp 149–151 ꢀC
(from Et2O:CH2Cl2). IR (KBr) 3310,1767,1699 cmꢃ1; 1H NMR (CDCl3)
145 ꢀC (from CH2Cl2:MeOH). IR (KBr) 3290, 1772, 1700 cm
;
1H
NMR (CDCl3)
d
2.29 (s, 3H, CH3), 4.11 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.88
(t, J ¼ 6.0 Hz, 2H, CH2O), 6.93–8.47 (m, 7H, arom. H), 10.21 (br s, 1H,
NH, exchangeable). Anal. Calcd for C18H15ClN2O3S: C, 57.68; H, 4.03;
N, 7.47; S, 8.55. Found: C, 57.98; H, 4.18; N, 7.33; S, 8.43.
d
2.28 (s, 3H, CH3), 2.48 (s, 3H, CH3), 4.04 (t, J ¼ 6.0 Hz, 2H, CH2N),
4.85 (t, J ¼ 6.0 Hz, 2H, CH2O), 6.88–7.27 and 7.50–7.84 (m, 7H, arom.
H), 8.32 (br s, 1H, NH, exchangeable). Anal. Calcd for C19H18N2O3S:
C, 64.39; H, 5.12; N, 7.90; S, 9.05. Found: C, 64.65; H, 5.25; N, 7.86; S,
9.31.
6.1.4.15. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 2-chloro-
4-nitrophenylthiocarbamate, 29. Yield 45%; mp 142–143 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3387, 1770, 1705 cmꢃ1
;
1H NMR (DMSO)
6.1.4.23. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-ethylphenylthiocarbamate, 42. Yield 39%; mp 149–151 ꢀC (from
d
3.94 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.20 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.44–
8.27 (m, 7H, arom. H), 10.36 (br s, 1H, NH, exchangeable). Anal.
Calcd for C17H12ClN3O5S: C, 50.32; H, 2.98; N, 10.35; S, 7.90. Found:
C, 50.43; H, 2.88; N, 10.32; S, 7.97.
Et2O:CH2Cl2). IR (KBr) 3311, 1771, 1701 cmꢃ1; 1H NMR (CDCl3)
d 1.17
(t, J ¼ 6.0 Hz, 3H, CH3-ethyl), 2.37–2.75 (m, 5H, CH2 and CH3), 4.04
(t, J ¼ 6.0 Hz, 2H, CH2N), 4.85 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.07–7.86 (m,
7H, arom. H), 8.72 (br s, 1H, NH, exchangeable). Anal. Calcd for
C20H20N2O3S: C, 65.20; H, 5.47; N, 7.60; S, 8.70. Found: C, 65.24; H,
5.37; N, 7.53; S, 8.95.
6.1.4.16. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-chloro-
3-nitrophenylthiocarbamate, 30. Yield 52%; mp 205–206 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3190, 1770, 1710 cmꢃ1 1H NMR (DMSO)
;
d
4.01 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.72 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.45–
6.1.4.24. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-
ethyl] 4-acetylphenylthiocarbamate, 43. Yield 22%; mp 173–175 ꢀC
8.18 (m, 7H, arom. H),10.35 (br s,1H, NH, exchangeable). Anal. Calcd
for C17H12ClN3O5S: C, 50.32; H, 2.98; N, 10.35; S, 7.90. Found: C,
50.55; H, 2.81; N, 10.45; S, 7.55.
(from Et2O:CH2Cl2). IR (KBr) 3290, 1760, 1721, 1700 cmꢃ1 1H NMR
;
(CDCl3)
d
2.50 (s, 3H, COCH3), 2.55 (s, 3H, CH3), 4.09 (t, J ¼ 6.0 Hz,
2H, CH2N), 4.89 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.17–8.02 (m, 7H, arom. H),
9.03 (br s, 1H, NH, exchangeable). Anal. Calcd for C20H18N2O4S: C,
62.81; H, 4.74; N, 7.33; S, 8.38. Found: C, 62.53; H, 4.86; N, 7.44; S,
8.07.
6.1.4.17. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 4-bromo-
2-methylphenylthiocarbamate, 31. Yield 43%; mp 111–113 ꢀC (from
CH2Cl2:MeOH). IR (KBr) 3325, 1768, 1695 cmꢃ1 1H NMR (CDCl3)
;
d
2.50 (s, 3H, CH3), 4.41 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.84 (t, J ¼ 6.0 Hz,
2H, CH2O), 6.99–7.38 and 7.97–8.08 (m, 7H, arom. H), 9.34 (br s, 1H,
NH, exchangeable). Anal. Calcd for C18H15BrN2O3S: C, 51.56; H, 3.61;
N, 6.68; S, 7.65. Found: C, 51.27; H, 3.43; N, 6.73; S, 7.77.
6.1.4.25. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-cyanophenylthiocarbamate, 44. Yield 40%; mp 180–182 ꢀC (from
Et2O:CH2Cl2). IR (KBr) 3184, 2226, 1770, 1712, 1702 cmꢃ1 1H
;
NMR (CDCl3)
d
2.51 (s, 3H, CH3), 4.09 (t, J ¼ 6.0 Hz, 2H, CH2N),
6.1.4.18. O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] 5-chloro-
2-methoxyphenylthiocarbamate, 32. Yield 29%; mp 126–127 ꢀC
4.86 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.30–7.84 (m, 7H, arom. H), 10.32
(br s, 1H, NH, exchangeable). Anal. Calcd for C19H15N3O3S: C,
62.45; H, 4.14; N, 11.50; S, 8.77. Found: C, 62.31; H, 4.40; N, 11.32;
S, 8.57.
(from CH2Cl2:MeOH). IR (KBr) 3375, 1775, 1712 cmꢃ1 1H NMR
;
(CDCl3)
d
4.48 (s, 3H, CH3O), 4.16 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.92

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1661
6.1.4.26. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 4-fluorophenylthiocarbamate, 45. Yield 40%; mp 143–145 ꢀC
6.1.4.34. O-[2-(5-tert-butyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 4-methylphenylthiocarbamate, 53. Yield 22%; mp 118–120 ꢀC
(from Et2O:CH2Cl2). IR (KBr) 3242, 1772, 1717 cmꢃ1
;
1H NMR
(from Et2O). IR (KBr) 3232, 1763, 1700 cmꢃ1; 1H NMR (CDCl3)
d 1.35
(CDCl3)
d
2.47 (s, 3H, CH3), 4.01 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.82 (t,
(s, 9H, C(CH3)3), 2.24 (s, 3H, CH3Ph), 3.88–4.19 (m, 2H, CH2N), 4.63–
4.92 (m, 2H, CH2O), 6.89–7.27 and 7.61–7.99 (m, 7H, arom. H). Anal.
Calcd for C22H24N2O3S: C, 66.64; H, 6.10; N, 7.07; S, 8.09. Found: C,
66.72; H, 6.13; N, 7.27; S, 7.91.
J ¼ 6.0 Hz, 2H, CH2O), 6.73–7.73 (m, 7H, arom. H), 8.55 (br s, 1H, NH,
exchangeable). Anal. Calcd for C18H15FN2O3S: C, 60.33; H, 4.22; N,
7.82; S, 8.95. Found: C, 60.42; H, 4.45; N, 7.83; S, 8.75.
6.1.4.27. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 4-bromophenylthiocarbamate, 46. Yield 12%; mp 174–176 ꢀC
6.1.4.35. O-[2-(5-tert-Butyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 4-chlorophenylthiocarbamate, 54. Yield 15%; mp 101–103 ꢀC
(from Et2O:CH2Cl2). IR (KBr) 3299, 1767, 1698 cmꢃ1
;
1H NMR
(from Et2O:MeOH). IR (KBr) 3253, 2963 1771, 1718 cmꢃ1 1H NMR
;
(CDCl3)
d
2.50 (s, 3H, CH3), 3.81–4.13 (m, 2H, CH2N), 4.69–4.96 (m,
(CDCl3) d 1.31 (s, 9H, C(CH3)3), 3.94–4.04 (m, 2H, CH2N), 4.67–4.80
2H, CH2O), 7.18–7.80 (m, 7H, arom. H), 11.13 (br s, 1H, NH,
exchangeable). Anal. Calcd for C18H15BrN2O3S: C, 51.56; H, 3.61; N,
6.68; S, 7.65. Found: C, 51.47; H, 3.89; N, 6.84; S, 7.31.
(m, 2H, CH2O), 6.96–7.24, 7.63–7.67 and 7.75–7.81 (m, 7H, arom. H),
8.59 (br s, 1H, NH, exchangeable). Anal. Calcd for C21H21ClN2O3S: C,
60.50; H, 5.08; N, 6.72; S, 7.69. Found: C, 60.78; H, 5.21; N, 6.49; S,
7.44.
6.1.4.28. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
4-iodophenylthiocarbamate, 47. Yield 37%; mp 183–185 ꢀC (from
6.1.4.36. O-[(2R)-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]
Et2O:CH2Cl2). IR (KBr) 3202, 1772, 1718 cmꢃ1
;
1H NMR (CDCl3)
4-methylphenylthiocarbamate, 55. Yield 16%; mp 78–80 ꢀC (from
20
d
2.53 (s, 3H, CH3), 4.07 (t, J ¼ 6.0 Hz, 2H, CH2N), 4.85 (t, J ¼ 6.0 Hz,
EtOH).
[a
]
¼ ꢃ14.1 (c 0.97, CHCl3). IR (KBr) 3246, 1776,
D
2H, CH2O), 7.60–7.77 (m, 7H, arom. H), 8.32 (br s, 1H, NH,
exchangeable). Anal. Calcd for C18H15IN2O3S: C, 46.37; H, 3.24; N,
6.01; S, 6.88. Found: C, 46.10; H, 3.58; N, 5.95; S, 6.58.
1707 cmꢃ1
;
1H NMR (CDCl3)
d
1.44 (d, J ¼ 6.6 Hz, 3H, CH3), 2.09 (s,
3H, CH3Ph), 4.63–4.79 (m, 1H, CH), 4.81–4.94 (m, 2H, CH2O), 6.65–
6.87 (m, 4H, arom. H), 7.60–7.84 (m, 4H, arom. H), 8.23 (br s,1H, NH,
exchangeable). Anal. Calcd for C19H18N2O3S: C, 64.39; H, 5.12; N,
7.90; S, 9.05. Found: C, 64.30; H, 5.44; N, 7.56; S, 8.81.
6.1.4.29. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 4-methoxyphenylthiocarbamate, 48. Yield 39%; mp 151–
153 ꢀC (from Et2O:CH2Cl2). IR (KBr) 3311, 1768, 1701 cmꢃ1; 1H NMR
6.1.4.37. O-[(2R)-2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]
4-chlorophenylthiocarbamate, 56. Yield 38%; mp 117–119 ꢀC (from
(CDCl3)
d
2.50 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 4.05 (t, J ¼ 6.0 Hz, 2H,
20
CH2N), 4.84 (t, J ¼ 6.0 Hz, 2H, CH2O), 6.59–7.76 (m, 7H, arom. H),
8.26 (br s, 1H, NH, exchangeable). Anal. Calcd for C19H18N2O4S: C,
61.62; H, 4.90; N, 7.56; S, 8.65. Found: C, 61.87; H, 4.89; N, 7.58; S,
8.50.
Me2CO:EtOH). [
a
]
¼ ꢃ23.2 (c 0.99, CHCl3). IR (KBr) 3244, 1776,
D
1707 cmꢃ1; 1H NMR (CDCl3)
d
1.50 (d, J ¼ 6.6 Hz, 3H, CH3), 4.50–5.10
(m, 3H, CH and CH2O), 6.87–7.28 and 7.68–7.91 (m, 8H, arom. H),
8.99 (br s, 1H, NH, exchangeable). Anal. Calcd for C18H15ClN2O3S: C,
57.68; H, 4.03; N, 7.47; S, 8.55. Found: C, 57.92; H, 4.32; N, 7.20; S,
8.56.
6.1.4.30. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3,4-dichlorophenylthiocarbamate, 49. Yield 64%; mp 164–166 ꢀC
(from CH2Cl2). IR (KBr) 3316, 1773, 1715 cmꢃ1
;
1H NMR (CDCl3)
6.1.4.38. O-[(2S)-2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]
4-chlorophenylthiocarbamate, 57. Yield 22%; mp 118–120 ꢀC (from
d
2.53 (s, 3H, CH3); 3.92–4.23 (m, 2H, CH2N), 4.68–5.03 (m, 2H,
20
CH2O), 7.10–7.88 (m, 6H, arom. H), 8.12 (br s, 1H, NH, exchangeable).
Anal. Calcd for C18H14Cl2N2O3S: C, 52.82; H, 3.45; N, 6.84; S, 7.83.
Found: C, 52.74; H, 3.48; N, 6.53; S, 7.54.
Et2O:MeOH). [
a]
¼ þ22.1 (c 1.09, CHCl3). IR (KBr) 3244, 1776,
D
1707 cmꢃ1; 1H NMR (CDCl3)
(m, 3H, CH and CH2O), 6.87–7.28 and 7.68–7.91 (m, 8H, arom. H), 8.99
(br s,1H, NH, exchangeable). Anal. Calcd for C18H15ClN2O3S: C, 57.68;
H, 4.03; N, 7.47; S, 8.55. Found: C, 57.92; H, 4.32; N, 7.20; S, 8.56.
d
1.50 (d, J ¼ 6.6 Hz, 3H, CH3), 4.50–5.10
6.1.4.31. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 3-chloro-4-methylphenylthiocarbamate, 50. Yield 30%; mp
136–138 ꢀC (from CH2Cl2:MeOH). IR (KBr) 3307, 1770, 1699 cmꢃ1
;
6.1.4.39. (ꢁ) O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)propyl] 4-methylphenylthiocarbamate, 58. Yield 40%; mp 141–
143 ꢀC (from Et2O:MeOH). IR (KBr) 3330, 1763, 1695 cmꢃ1. 1H NMR
1H NMR (CDCl3)
d 2.29 (s, 3H, CH3), 2.49 (s, 3H, CH3), 3.89–4.20 (m,
2H, CH2N), 4.70–4.99 (m, 2H, CH2O), 6.81–7.85 (m, 6H, arom. H),
8.39 (br s, 1H, NH, exchangeable). Anal. Calcd for C19H17ClN2O3S: C,
58.68; H, 4.41; N, 7.20; S, 8.25. Found: C, 58.35; H, 4.68; N, 7.05; S,
8.56.
(CDCl3)
d
1.50 (d, J ¼ 6.6 Hz, 3H, CH3), 2.19 (s, 3H, CH3Ph), 2.50 (s, 3H,
CH3Phtha), 4.39–5.03 (m, 3H, CH and CH2O), 6.74–7.20 and 7.47–
7.74 (m, 7H, arom. H), 8.52 (br s, 1H, NH, exchangeable). Anal. Calcd
for C20H20N2O3S: C, 65.20; H, 5.47; N, 7.60; S, 8.70. Found: C, 65.48;
H, 5.56; N, 7.52; S, 8.52.
6.1.4.32. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
ethyl] 3-chloro-4-nitrophenylthiocarbamate, 51. Yield 49%; mp 205–
207 ꢀC (from Me2CO). IR (KBr) 3302, 1768, 1697 cmꢃ1
;
1H NMR
6.1.4.40. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
propyl] 4-chlorophenylthiocarbamate, 59. Yield 51%; mp 171–173 ꢀC
(DMSO) d 2.37 (s, 3H, CH3), 3.80–3.92 (m, 2H, CH2N), 4.64–4.76 (m,
2H, CH2O), 7.42–7.68 (m, 6H, arom. H), 11.44 (br s, 1H, NH,
exchangeable). Anal. Calcd for C18H14ClN3O5S: C, 51.49; H, 3.36; N,
10.01; S, 7.64. Found: C, 51.72; H, 3.35; N, 9.98; S, 7.95.
(from Me2CO:CH2Cl2). IR (KBr) 3312, 1763, 1694 cmꢃ1 1H NMR
;
(DMSO)
d
1.43 (d, J ¼ 6.6 Hz, 3H, CH3), 2.50 (s, 3H, CH3Phtha), 4.29–
4.94 (m, 3H, CH and CH2O), 6.93–7.36 and 7.61–7.80 (m, 7H, arom.
H), 11.31 (br s, 1H, NH, exchangeable). Anal. Calcd for
C19H17ClN2O3S: C, 58.68; H, 4.41; N, 7.20; S, 8.25. Found: C, 58.65; H,
4.25; N, 7.08; S, 8.46.
6.1.4.33. O-[2-(5-Methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]
3-chloro-4-(trifluoromethyl)phenylthiocarbamate, 52. Yield 33%; mp
133–135 ꢀC (from Et2O:MeOH). IR (KBr) 3307, 1772, 1698 cmꢃ1
;
1H NMR (CDCl3)
d
2.51 (s, 3H, CH3), 3.93–4.24 (m, 2H, CH2N),
6.1.4.41. 2-({[2-({[(2,4-Dimethylphenyl)amino]carbonothioyl}oxy)
ethyl]amino}carbonyl)benzoic acid, 61. Yield 16%; mp 145–147 ꢀC
(from Et2O:CH2Cl2). IR (KBr) 3310, 3190, 1702, 1654 cmꢃ1; 1H NMR
3.72–4.98 (m, 2H, CH2O), 7.23–7.85 (m, 6H, arom. H), 8.51 (br s,
1H, NH, exchangeable). Anal. Calcd for C19H14ClF3N2O3S: C,
51.53; H, 3.19; N, 6.33; S, 7.24. Found: C, 51.18; H, 3.27; N, 6.27; S,
7.44.
(DMSO)
d 2.13 (s, 3H, CH3), 2.27 (s, 3H, CH3), 3.30–3.78 (m, 2H,
CH2N), 4.38–4.76 (m, 2H, CH2O), 6.98–7.19 and 7.37–7.96 (m, 7H,

1662
A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
arom. H), 8.40 (br s,1H, CSNH, exchangeable), 10.43 (br s, 1H, CONH,
exchangeable), 12.81 (br s,1H, COOH, exchangeable). Anal. Calcd for
C19H20N2O4S: C, 61.27; H, 5.41; N, 7.52; S, 8.61. Found: C, 61.33; H,
5.41; N, 7.50; S, 8.68.
CONH, exchangeable), 10.93 (br s, 1H, CSNH, exchangeable), 12.88
(br s, 1H, COOH, exchangeable). Anal. Calcd for C19H20N2O4S: C,
61.27; H, 5.41; N, 7.52; S, 8.61. Found: C, 61.05; H, 5.16; N, 7.51; S,
8.71.
6.1.4.42. 2-({[2-({[(3,5-Difluorophenyl)amino]carbonothioyl}oxy)
6.1.4.49. 2-({[(1S)-1-Methyl-2-({[(4-methylphenyl)amino]carbono-
ethyl]amino}carbonyl)benzoic acid, 65. Yield 16%; mp 167–169 ꢀC
thioyl}oxy)ethyl]amino}carbonyl)benzoic acid, 74. Yield 26%; mp
20
(from Me2CO:MeOH). IR (KBr) 3331, 1698, 1650 cmꢃ1
;
1H NMR
163–165 ꢀC (from Me2CO:Et2O). [
a
]
;
¼ þ11.3 (c 0.97, Me2CO). IR
D
(DMSO)
d
3.46–3.84 (m, 2H, CH2N), 4.50–4.84 (m, 2H, CH2O), 6.74–
(KBr) 3322, 3237, 1696, 1650 cmꢃ1
1H NMR (DMSO)
d
0.99–1.19
7.98 (m, 8H, 7 arom. H and CONH), 8.57 (br s, 1H, CSNH,
exchangeable), 11.62 (br s, 1H, COOH, exchangeable). Anal. Calcd for
C17H14F2N2O4S: C, 53.68; H, 3.71; N, 7.36; S, 8.43. Found: C, 53.62; H,
4.05; N, 7.48; S, 8.41.
(m, 3H, CH3), 2.16 (s, 3H, CH3Ph), 4.13–4.50 (m, 3H, CH2O and CH),
6.97–7.23, 7.35–7.52 and 7.68–7.75 (m, 8H, arom. H), 8.26 (br s, 1H,
CONH, exchangeable), 10.93 (br s, 1H, CSNH, exchangeable), 12.88
(br s, 1H, COOH, exchangeable). Anal. Calcd for C19H20N2O4S: C,
61.27; H, 5.41; N, 7.52; S, 8.61. Found: C, 61.05; H, 5.16; N, 7.51; S,
8.71.
6.1.4.43. 2-({[2-({[(5-Chloro-2-methylphenyl)amino]carbonothioyl}
oxy)ethyl]amino}carbonyl)benzoic acid, 66. Yield 16%; mpꢃ1147–
148 ꢀC (from CH2Cl2:MeOH). IR (KBr) 3342, 3257, 1701 cm
;
1H
6.1.4.50. 2-({[(1R)-2-({[(4-Chlorophenyl)amino]carbonothioyl}oxy)-
NMR (DMSO)
d
2.20 (s, 3H, CH3), 3.35–3.80 (m, 2H, CH2N), 4.50 (t,
1-methylethyl]amino}carbonyl)benzoic acid, 75. Yield 24%; mp 158–
20
J ¼ 6.0 Hz, 2H, CH2O), 6.95–7.78 (m, 8H, 7 arom. H and CONH), 9.65
(br s, 1H, CSNH, exchangeable), 11.30 (br s, 1H, COOH, exchange-
able). Anal. Calcd for C18H17ClN2O4S: C,55.03; H, 4.36; N, 7.13; S,
8.16. Found: C, 54.90; H, 4.41; N, 7.15; S, 7.92.
160 ꢀC (from Me2CO:Et2O). [
a
]
¼ ꢃ21.1 (c 0.98, Me2CO). IR (KBr)
D
3285, 1702, 1637 cmꢃ1 1H NMR (DMSO)
; d 0.95–1.17 (m, 3H, CH3),
4.10–4.52 (m, 3H, CH2O and CH), 6.91–7.79 (m, 8H, arom. H), 8.29
(br s, 1H, CONH, exchangeable), 11.17 (br s, 1H, CSNH, exchange-
able), 12.90 (br s, 1H, COOH, exchangeable). Anal. Calcd for
C18H18ClN2O4S: C, 55.03; H, 4.36; N, 7.13; S, 8.16. Found: C, 55.19; H,
4.65; N, 7.43; S, 8.26.
6.1.4.44. 2-({[2-({[(3-Chloro-4-methylphenyl)amino]carbonothioyl}
oxy)ethyl]amino}carbonyl)benzoic acid, 67. Yield 61%; mp 166–
168 ꢀC (from CH2Cl2:MeOH). IR (KBr) 3340, 3250, 1688, 1581,
1530 cmꢃ1; 1H NMR (DMSO)
d
2.30 (s, 3H, CH3), 3.40–3.80 (m, 2H,
6.1.4.51. 2-({[(1S)-2-({[(4-Chlorophenyl)amino]carbonothioyl}oxy)-
CH2N), 4.63 (t, J ¼ 6.0 Hz, 2H, CH2O), 7.05–7.95 (m, 8H, 7 arom. H
and CONH), 8.78 (br s, 1H, CSNH, exchangeable), 11.80 (br s, 1H,
COOH, exchangeable). Anal. Calcd for C18H17ClN2O4S: C, 55.03; H,
4.36; N, 7.13; S, 8.16. Found: C, 55.04; H, 4.36; N, 7.10; S, 8.14.
1-methylethyl]amino}carbonyl)benzoic acid, 76. Yield 21%; mp 147–
20
149 ꢀC (from Me2CO). [
a
]
¼ þ20.1 (c 1.39, Me2CO). IR (KBr) 3285,
D
1702, 1637 cmꢃ1 1H NMR (DMSO)
; d 0.95–1.17 (m, 3H, CH3), 4.10–
4.52 (m, 3H, CH2O and CH), 6.91–7.79 (m, 8H, arom. H), 8.29 (br s,
1H, CONH, exchangeable), 11.17 (br s, 1H, CSNH, exchangeable),
12.90 (br s, 1H, COOH, exchangeable). Anal. Calcd for
C18H18ClN2O4S: C, 55.03; H, 4.36; N, 7.13; S, 8.16. Found: C, 55.19; H,
4.65; N, 7.43; S, 8.26.
6.1.4.45. 2-({[2-({[(2-Methoxy-5-methylphenyl)amino]carbonothioyl}
oxy)ethyl]amino}carbonyl)benzoic acid, 68. Yield 28%; mp 143–
145 ꢀC (from Et2O:CH2Cl2). IR (KBr) 3338, 3300, 1689, 1666 cmꢃ1; 1H
NMR (DMSO) d 2.20 (s, 3H, CH3), 3.29–3.87 (m, 5H, CH2N and CH3O),
4.32–4.73 (m, 2H, CH2O), 6.83–7.98 (m, 7H, arom. H), 8.47 (br s, 1H,
CSNH, exchangeable), 10.39 (br s, 1H, CONH, exchangeable), 13.07
(br s, 1H, COOH, exchangeable). Anal. Calcd for C19H20N2O5S: C,
58.75; H, 5.19; N, 7.21; S, 8.25. Found: C, 58.77; H, 5.20; N, 7.24; S,
8.02.
6.2. Virology: materials and methods
The biological evaluation of the synthesized compounds was
performed according to the previously reported procedures [12,43].
6.1.4.46. 2-({[2-({[(3,4-Dimethoxyphenyl)amino]carbonothioyl}oxy)
6.3. Molecular modelling
ethyl]amino}carbonyl)benzoic acid, 69. Yield 31%; mp 147–149 ꢀC
(from Et2O:CH2Cl2). IR (KBr) 3363, 3287, 1695, 1637 cmꢃ1
;
1H
The molecular structures of 5, 23, 28–30, 33, 34, 49–52, 54, 56
and 57 were built by Insight II (Builder module), parameterized
according to CVFF force field [44] and their RT complexes were
calculated using Autodock 3.05 [40]. After the removal of the
inhibitor from the crystal structure of RT/VII complex (PDB code
2VG5) [9], hydrogen atoms were added and partial charges were
assigned according to CVFF force field (Insight II, biopolymer
module). The ligand ‘‘root’’ was defined automatically and all bonds
were allowed to freely rotate. A 50 ꢂ 50 ꢂ 50 grid (grid spacing
0.375 Å) was centred on the NNBS and electrostatic and affinity
maps for each atom type of the ligand were calculated. The docking
search was performed over 256 conformers using the Genetic
Algorithm Local Search protocol as implemented in Autodock
(population size: 100; rate of gene mutation: 0.02; rate of cross-
over: 0.8). The docking poses were clustered (rmsd: 2.0 Å) and the
best conformation of the low energy, highest populated cluster was
selected as the binding conformation. The resulting models were
energy-minimized by CNS [45], performing a cycle of simulated
annealing (starting temperature 1000 K) followed by 120 cycles of
Powell minimization. Model analyses were performed using the
CCP4 program suite [46]. All the calculations were carried out on
Silicon Graphic Indigo2 and Origin 200 workstations.
NMR (DMSO)
d 3.48–4.01 (m, 8H, CH2N and 2 CH3O), 4.34–4.80
(m, 2H, CH2O), 6.65–7.96 (m, 7H, arom. H), 8.53 (br s, 1H, CSNH,
exchangeable), 10.25 (br s, 1H, CONH, exchangeable), 13.01 (br s,
1H, COOH, exchangeable). Anal. Calcd for C19H20N2O6S: C, 56.43;
H, 4.98; N, 6.93; S, 7.93. Found: C, 56.67; H, 4.98; N, 6.91; S, 8.13.
6.1.4.47. 2-({[2-({[(2,3,4-Trichlorophenyl)amino]carbonothioyl}oxy)
ethyl]amino}carbonyl)benzoic acid, 71. Yield 35%; mp 81–82 ꢀC
(from Me2CO). IR (KBr) 3142, 1712 cmꢃ1 1H NMR (DMSO)
; d 3.32–
3.78 (m, 2H, CH2N), 4.43–4.75 (m, 2H, CH2O), 7.08–7.98 (m, 6H,
arom. H), 8.44 (br s, 1H, CONH, exchangeable), 11.24 (br s, 1H, CSNH,
exchangeable), 13.05 (br s, 1H, COOH, exchangeable). Anal. Calcd for
C17H13Cl3N2O4S: C, 45.60; H, 2.93; N, 6.26; S, 7.16. Found: C, 45.59;
H, 3.02; N, 6.46; S, 7.41.
6.1.4.48. 2-({[(1R)-1-Methyl-2-({[(4-methylphenyl)amino]carbono-
thioyl}oxy)ethyl]amino}carbonyl)benzoic acid, 73. Yield 16%; mp
20
157–159 ꢀC (from Me2CO:Et2O). [
a
]
;
¼ ꢃ11.9 (c 0.91, Me2CO). IR
D
(KBr) 3322, 3237, 1696, 1650 cmꢃ1
1H NMR (DMSO)
d 0.99–1.19
(m, 3H, CH3), 2.16 (s, 3H, CH3Ph), 4.13–4.50 (m, 3H, CH2O and CH),
6.97–7.23, 7.35–7.52 and 7.68–7.75 (m, 8H, arom. H), 8.26 (br s, 1H,

A. Spallarossa et al. / European Journal of Medicinal Chemistry 44 (2009) 1650–1663
1663
[18] C. Mao, E.A. Sudbeck, T.K. Venkatachalam, F.M. Uckun, Bioorg. Med. Chem. Lett.
9 (1999) 1593–1598.
Acknowledgements
[19] C. Sahlberg, R. Nore´en, P. Engelhardt, M. Ho¨gberg, J. Kangasmetsa¨, L. Vrang,
H. Zhang, Bioorg. Med. Chem. Lett. 8 (1998) 1511–1516.
This work was supported by MURST (Cofinanziamento Nazio-
nale), CNR (Rome) and Ministero Italiano della Salute – Istituto
[20] F.M. Uckun, C. Mao, S. Pendergrass, D. Maher, D. Zhu, L. Tuel-Ahlgren,
T.K. Venkatachalam, Bioorg. Med. Chem. Lett. 9 (1999) 2721–2726.
[21] F.M. Uckun, S. Pendergrass, D. Maher, D. Zhu, L. Tuel-Ahlgren, C. Mao,
T.K. Venkatachalam, Bioorg. Med. Chem. Lett. 9 (1999) 3411–3416.
[22] F.M. Uckun, D. Erbeck, H. Tibbles, S. Qazi, T.K. Venkatachalam, Arznei-
mittelforschung 57 (2007) 164–170.
`
Superiore di Sanita (grant no 40D.46). 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.
[23] M. Ho¨gberg, C. Sahlberg, P. Engelhardt, R. Nore´en, J. Kangasmetsa¨,
¨
N.G. Johansson, B. Oberg, L. Vrang, H. Zhang, B.-L. Sahlberg, T. Unge, S. Lo¨vgren,
K. Fridborg, K. Ba¨ckbro, J. Med. Chem. 42 (1999) 4150–4160.
[24] M. Ho¨gberg, P. Engelhardt, L. Vrang, H. Zhang, Bioorg. Med. Chem. Lett. 10
(2000) 265–268.
References
[25] 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.
[26] G. Campiani, M. Fabbrini, E. Morelli, V. Nacci, G. Greco, E. Novellino, G. Maga,
S. Spadari, A. Bergamini, E. Faggioli, I. Uccella, F. Bolacchi, S. Marini, M. Coletta,
C. Fracasso, S. Caccia, Antiviral Chem. Chemother. 11 (2000) 141–155.
[27] T.K. Venkatachalam, E.A. Sudbeck, C. Mao, F.M. Uckun, Bioorg. Med. Chem.
Lett. 10 (2000) 2071–2074.
[1] H.C. Castro, N.I. Loureiro, M. Pujol-Luz, A.M. Souza, M.G. Albuquerque,
D.O. Santos, L.M. Cabral, I.C. Frugulhetti, C.R. Rodrigues, Curr. Med. Chem. 13
(2006) 313–324 and references therein.
[2] L.A. Kohlstaedt, J. Wang, J.M. Friedman, P.A. Rice, T.A. Steitz, Science 256 (1992)
1783–1790.
[3] 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) 5633–5639.
[4] J. Ren, C. Nichols, L.E. Bird, T. Fujiwara, H. Sugimoto, D.I. Stuart, D.K. Stammers,
J. Biol. Chem. 275 (2000) 14316–14320.
[28] T.K. Venkatachalam, C. Mao, F.M. Uckun, Bioorg. Med. Chem. 12 (2004)
4275–4284.
[29] O.J. D’Cruz, F.M. Uckun, Curr. HIV Res. 4 (2006) 329–345.
[30] O.J. D’Cruz, F.M. Uckun, J. Antimicrob. Chemother. 57 (2006) 411–423 and
references therein.
[31] O.J. D’Cruz, F.M. Uckun, J. Enzyme Inhib. Med. Chem. 21 (2006) 329–350.
[32] V. Ravichandran, R.K. Agrawal, Bioorg. Med. Chem. Lett. 17 (2007) 2197–2202.
[33] S. Cesarini, A. Spallarossa, A. Ranise, P. Fossa, P. La Colla, G. Sanna, G. Collu,
R. Loddo, Bioorg. Med. Chem. 16 (2008) 4160–4172.
[5] J. Lindberg, S. Sigurdsson, S. Lowgren, H.O. Andersson, C. Sahlberg, R. Noreen,
K. Fridborg, H. Zhang, T. Unge, Eur. J. Biochem. 269 (2002) 1670–1677.
[6] R. Esnouf, J. Ren, C. Ross, Y. Jones, D. Stammers, D. Stuart, Nat. Struct. Biol. 2
(1995) 303–308.
[7] R.M. Esnouf, J. Ren, A.L. Hopkins, C.K. Ross, E.Y. Jones, D.K. Stammers,
D.I. Stuart, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3984–3989.
[8] K. Das, A.D.J. Clark, P.J. Lewi, J. Heeres, M.R. De Jonge, L.M. Koymans, H.M. Vinkers,
F. Daeyaert, D.W. Ludovici, M.J. Kukla, B. De Corte, R.W. Kavash, C.Y. Ho, H. Ye,
[34] S. Cesarini, A. Spallarossa, A. Ranise, O. Bruno, P. La Colla, B. Secci, G. Collu,
R. Loddo, Bioorg. Med. Chem. 16 (2008) 4173–4185.
´
M.A. Lichtenstein, K. Andries, R. Pauwels, M.P. De Bethune, P.L. Boyer, P. Clark,
[35] A. Rao, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte, P. Monforte,
C. Pannecouque, M. Zappala, Il Farmaco 57 (2002) 747–751.
S.H. Hughes, P.A. Janssen, E. Arnold, J. Med. Chem. 47 (2004) 2550–2560.
[9] A. Spallarossa, S. Cesarini, A. Ranise, M. Ponassi, U. Unge, M. Bolognesi, Bio-
chem. Biophys. Res. Commun. 365 (2008) 764–770.
[10] C.A. Shaw-Reid, B. Feuston, V. Munshi, K. Getty, J. Krueger, D.J. Hazuda,
M.A. Parniak, M.D. Miller, D. Lewis, Biochemistry 44 (2005) 1595–1606.
[11] N. Sluis-Cremer, N.A. Temiz, I. Bahar, Curr. HIV Res. 2 (2004) 323–332.
[12] 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.
[13] 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.
[14] C. 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.
[36]
A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte,
P. Monforte, C. Pannecouque, M. Zappala, Il Farmaco 59 (2004) 33–39.
[37] G. La Regina, A. Coluccia, F. Piscitelli, A. Bergamini, A. Sinistro, A. Cavazza,
G. Maga, A. Samuele, S. Zanoli, E. Novellino, M. Artico, R. Silvestri, J. Med.
Chem. 50 (2007) 5034–5038.
[38] G. Maass, U. Immendoerfer, B. Koenig, U. Leser, B. Mueller, R. Goody, E. Pfaff,
Antimicrob. Agents Chemother. 37 (1993) 2612–2617.
[39] 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.
[40] 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.
[41] J. Ren, R.M. Esnouf, A.L. Hopkins, J. Warren, J. Balzarini, D.I. Stuart,
D.K. Stammers, Biochemistry 37 (1998) 14394–14403.
[42] A. Carocci, A. Catalano, F. Corbo, A. Duranti, R. Amoroso, C. Franchini,
G. Lentini, V. Tortorella, Tetrahedron: Asymmetry 11 (2000) 3619–3634.
[43] 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.
[44] P. Dauber-Osguthorpe, V.A. Roberts, D.J. Osguthorpe, J. Wolff, M. Genest,
A.T. Hagler, Proteins 4 (1988) 31–47.
[45] 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.
[46] Collaborative Computational Project, Number 4, Acta Crystallogr. D50 (1994)
760–763.
[15] 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.
[16] R. Vig, C. Mao, T.K. Venkatachalam, L. Tuel-Ahlgren, E.A. Sudbeck, F.M. Uckun,
Bioorg. Med. Chem. 6 (1998) 1789–1797.
[47] P.J. Kraulis, J. Appl. Crystallogr. 24 (1991) 946–950.
[48] R.M. Esnouf, J. Mol. Graph. Model. 15 (1997) 132–134.
[17] C. Mao, R. Vig, T.K. Venkatachalam, E.A. Sudbeck, F.M. Uckun, Bioorg. Med.
Chem. Lett. 8 (1998) 2213–2218.