(Hetero)aroyl esters of 2-(N-phthalimido)ethanol and analogues: Parallel synthesis, anti-HIV-1 activity and cytotoxicity

Article abstract of DOI:10.1007/s00044-009-9192-x

The structural simplification of the non-nucleoside reverse transcriptase inhibitors (NNRTIs) O-(2-phthalimidoethyl)-N-(hetero)aroyl-N-arylthiocarbamates led us to design (hetero)aroyl esters of 2-(N-phthalimido)ethanol as a potential new class of anti-HI

Full text of DOI:10.1007/s00044-009-9192-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:311–336
DOI 10.1007/s00044-009-9192-x
ORIGINAL RESEARCH
(Hetero)aroyl esters of 2-(N-phthalimido)ethanol
and analogues: parallel synthesis, anti-HIV-1
activity and cytotoxicity
Sara Cesarini Æ Andrea Spallarossa Æ Angelo Ranise Æ
Silvia Schenone Æ Paolo La Colla Æ Gabriella Collu Æ
Giuseppina Sanna Æ Roberta Loddo
Received: 7 November 2008 / Accepted: 17 March 2009 / Published online: 23 April 2009
¨
Ó Birkhauser Boston 2009
Abstract The structural simplification of the non-nucleoside reverse transcriptase
inhibitors (NNRTIs) O-(2-phthalimidoethyl)-N-(hetero)aroyl-N-arylthiocarbamates
led us to design (hetero)aroyl esters of 2-(N-phthalimido)ethanol as a potential new
class of anti-HIV-1 agents. The setup of a solution-phase parallel synthesis method
allowed the rapid preparation of a high number of analogues. In cell-based assays,
20 of 34 esters showed anti-HIV-1 activity ranging from nanomolar to micromolar
concentrations. The most potent esters had only a minor effect or were ineffective in
enzyme assay against HIV-1 reverse transcriptase. Variations on the O-(2-phtha-
limidoethyl) moiety led to compounds devoid of antiretroviral activity, but cyto-
toxic, in particular those bearing the 4-chloro-3-nitrobenzoyl moiety. The most
cytotoxic compound displayed a CC50 value of 1.6 lM.
Keywords Anti-HIV-1 activity Á Cytotoxicity Á Parallel synthesis Á
Esters
Introduction
HIV-1 reverse transcriptase (RT) catalyses the conversion of a single-stranded RNA
into a double-stranded DNA, which is then integrated into the host cell genome. The
S. Cesarini (&) Á A. Spallarossa Á A. Ranise Á S. Schenone
`
Dipartimento di Scienze Farmaceutiche, Universita di Genova,
Viale Benedetto XV 3, 16132 Genova, Italy
e-mail: sara.cesarini@unige.it
P. La Colla Á G. Collu Á G. Sanna Á R. Loddo
`
Dipartimento di Scienze e Tecnologie Biomediche, Universita di Cagliari,
Cittadella Universitaria, S.S. 554, Km 4,500, 09042 Monserrato (Cagliari), Italy

312
Med Chem Res (2010) 19:311–336
key role of RT in HIV-1 life cycle pointed out this enzyme as a privileged target for
the highly active antiretroviral therapy (HAART) (Barbaro et al. 2005; De Clercq,
2001, 2005a, b; Jonckheere et al., 2000). Non-nucleoside RT inhibitors (NNRTIs)
are chemically diverse and selective RT targeting agents that bind to an allosteric
˚
hydrophobic pocket (NNRTI binding site, NNIBS) located approximately 10 A
from the polymerase active site. Upon inhibitor interaction, the NNIBS is created
and the enzyme is locked into an inactive form by affecting the geometry of the
polymerase active site aspartyl residues (Balzarini, 2004). During the past 15 years,
more than 50 structurally diverse NNRTIs have been described (Balzarini, 2004;
Campiani et al., 2002; De Clercq, 1998, 2004; Pauwels, 2004; Pedersen and
Pedersen, 1999, 2000). The therapeutic efficacy of NNRTIs is limited by the
emergence of drug-resistant mutants (Leigh Brown et al., 2003; Richman et al.,
2004). Therefore, the need for new, selective, and potent drugs also able to inhibit
HIV-1 mutated forms is still a challenge.
In our previous studies, we reported the discovery of O-(2-phthalimidoethyl)-N-
(hetero)aroyl-N-arylthiocarbamates (ATCs) (Ranise et al., 2003; Spallarossa et al.,
2008a) and the corresponding nonacylated derivatives O-(2-phthalimidoethyl)-N-
arylthiocarbamates (C-TCs) (Ranise et al., 2005; Spallarossa et al., 2008b) (Fig. 1
shows the leads ATC 25c and C-TC 12), as classes of potent NNRTIs. C-TCs are
isosterically related to the NNRTI family of N-phenethyl-N’-thiazolylthiourea
(PETT) derivatives (Bell et al., 1995; Ren et al., 2000; Vig et al., 1998), of which
trovirdine is one of the most representative analogues (Fig. 2).
ATCs can be retrosynthetically divided into three molecular portions: a, b, and c
(Fig. 1), corresponding to the building blocks used for their synthesis (Ranise et al.,
b
O
S
N
O
N
a
O
O
ATC 25c
c
O
O
a
a
S
O
b
N
N
N
H
O
O
c
O
O
C-TC 12
1
Fig. 1 Chemical structure of ATC lead compound (ATC 25c) (Ranise et al., 2003), segmented in the
molecular portions a, b, and c, the derived C-TC lead (C-TC 12) (Ranise et al., 2005) and the lead of the
title compounds (1)

Med Chem Res (2010) 19:311–336
313
Y
N
X
S
N
S
N
NH
N
H
NH
G
H
N
S
PETT
trovirdine (X = Y = H, G = Br)
PETT-1 (X = F, Y = OC2H5, G = CN)
Fig. 2 Chemical structures of PETT, trovirdine, and PETT-1
2003; Spallarossa et al., 2008a): alcohols (a), isothiocyanates (b), and acyl chlorides
(c). Molecular simplification of bioactive molecules is one of the strategies often
successfully applied in medicinal chemistry and that has led to the discovery of new
drugs (Silverman, 2004; Thomas, 2000). Because the molecular simplification of
ATCs by the elimination of portion c had been successful (C-TCs: combination of
portions a and b), we tried to apply again this strategy—this time by combing
building blocks a and c (i.e., eliminating b)—with the goal to identify a new class
of anti-HIV-1 agents. Thus, we synthesized the ester 1 (Fig. 1) and tested it
against wild-type HIV-1 in cell-based assay. The compound showed an EC50
value of 55 lM. The easy synthetic accessibility of the structures made it an
attractive target for potential optimization. Thus, we setup a parallel synthesis
method, which allowed the rapid preparation of a high number of analogues of 1,
aimed at the structure-activity relationship (SAR) profiling. According to our SAR
strategy, we modified the lead 1 by keeping constant the O-(2-phthalimidoethyl)
substructure (portion a) and varying the benzoyl moiety (portion c) (1–34;
Tables 1 and 2). In particular, we investigated the monosubstitution with
functional groups with various electronic, steric, and lipophilic properties at
position ortho, meta, and para (1–10); the disubstitution with equal (11–18) or
different (19–28) groups; the replacement of the phenyl ring with a heteroaryl
(29–31), 1-phenylpropyl (32), styryl (33), or 2-naphthyl (34) group. Then, the
benzoyl moiety was substituted according to the substitution patterns emerged as
the most favorable in the first series, and portion a was modified (35–55; Tables 3,
4, and 5) by shortening (35, 36), branching (37–39), or lengthening (43, 44) the
ethyl linker; by introducing a methyl (40, 41) or a tert-butyl (42) at position 4 of
the phthalimide substructure; by partially (45, 46) or completely (47, 48) reducing
the phthalimide benzene ring; by replacing the phthalimide moiety with para-
bromo- (49) and para-nitro-phenoxyl (50), phenylsulphonyl (51), and 3-indolyl
(52) groups; or by replacing the 2-phthalimidoethyl portion with the 1,2-
dihydroacenaphthylen-1-yl substructure (53–55).

314
Med Chem Res (2010) 19:311–336
Table 1 Physical and chemical data, cytotoxicity, and anti-HIV-1 activity of compounds 1–28a
O
X,(Y)
N
O
O
O
Compound
X,Y
cryst. solvent(s)
mp (°C)
% Yield
CCb50
ECc50
1
H
CH2Cl2/MeOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
EtOH
115–116
73–75
57
69
71
76
69
92
74
74
95
81
69
63
65
60
69
72
95
69
60
52
60
71
57
72
66
56
61
76
[100
[100
[100
[100
[100
[100
[100
92
55
2
2-Cl
80
3
2-NO2
111–113
75–77
80
4
3-CH3
82
5
3-F
122–124
108–110
144–145
99–101
75
6
3-Cl
93
7
3-NO2
69
8
4-C(CH3)3
4-CH2CH2CH2CH2CH3
4-Br
[92
14
9
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/CHCH3
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/MeOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
64–66
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
trovirdine
a
165–167
115–117
101–103
100–102
107–109
131–133
132–134
174–175
155–156
122–124
86–87
[100
[100
[100
0.2
2,3-F2
2,4-F2
2,5-F2
2,6-F2
49
3,4-F2
[100
[100
0.7
3,5-F2
3,4-Cl2
3,5-Cl2
27
2-Cl-4-NO2
2-CH3-5-F
2-F-5-CF3
2-F-5-Cl
2-F-5-NO2
2-Cl-5-F
2-Cl-5-NO2
2-Br-5-F
4-Cl-3-NO2
4-Br-3-CH3
15
47
98–100
[100
[100
[100
[100
[30
35
129–131
136–138
107–109
134–136
108–110
164–166
162–164
[100
[100
[100
60
0.7
60
0.02
Data mean values for three independent experiments. Variation among triplicate samples was \10%
b
Compound concentration [lM] required to reduce the viability of mock-infected MT-4 cells by 50%, as
determined by the MTT method
c
Compound concentration [lM] required to achieve 50% protection of MT-4 cell from wt HIV-1
induced cytopathogenicity, as determined by the MTT method

Med Chem Res (2010) 19:311–336
315
Table 2 Physical and chemical data, cytotoxicity, and anti-HIV-1 activity of compounds 29–34a
O
N
O
R
O
O
Compound
R
cryst. solvent(s)
CH2Cl2/EtOH
mp (°C)
% Yield
36
CCb50
ECc50
29
125–127
[100
[100
[100
28
N
Cl
30
31
pyridine/EtOH
CH2Cl2/EtOH
156–158
137–139
42
34
[100
[100
Cl
N
O
32d
33
CH3COCH3
65–66
74
75
64
[100
[100
[100
60
16
50
CHCl3
115–116
148–150
34
CHCl3
[100
trovirdine
0.02
a,b,c
See legend for Table 1
d
Assayed as racemic mixture
Results and discussion
Chemistry
Esters 1–55 were prepared (Scheme 1) by solution-phase parallel synthesis, by
reacting alcohols
A1–14 (Fig. 3a) with the suitable acyl chloride (C1–34; Fig. 3b). All the building
blocks used, with the exception of alcohols A3–6, A8, and A9, were commercially

316
Med Chem Res (2010) 19:311–336
Table 3 Physical and chemical data, cytotoxicity, and anti-HIV-1 activity of compounds 35–44a
O
R1
X,Y
N
O
n
W
R2
O
O
Compound
n
R1 R2
W
X,Y
cryst. solvent(s) mp (°C)
%
Yield
CCb50 ECc50
35
0
0
1
1
1
1
1
1
–
H
H
2,5-F2
CH2Cl2/EtOH
CH2Cl2/EtOH
Et2O/EtOH
143–145 65
189–190 41
[100 [100
11 [11
36
–
H
H
4-Cl-3-NO2
2,5-F2
37d
38d
39d
40
CH3
H
H
H
78–80 54
60–62 36
136–137 35
123–125 62
146–148 61
65–67 87
[100 [100
[100 [100
34 [34
CH3
CH3
H
H
2,5-F2
Et2O/EtOH
H
H
4-Cl-3-NO2
2,5-F2
CH2Cl2/EtOH
CH2Cl2/EtOH
CH2Cl2/EtOH
H
CH3
CH3
[100 [100
81 [81
41
H
H
4-Cl-3-NO2
42
H
H
C(CH3)3 2,5-F2
CH2Cl2/
petroleum
ether
[100 [100
43
2
2
H
H
H
H
H
H
2,5-F2
CH2Cl2/EtOH
CH2Cl2/EtOH
107–109 57
152–154 50
[100 [100
[100 [100
60 0.02
44
4-Cl-3-NO2
trovirdine
a,b,c
See legend for Table 1
d
Assayed as racemic mixture
available. The synthetic procedure, previously described for the preparation of A3–6
and A8 (Ranise et al., 2005; Spallarossa et al., 2008b), and in this case successfully
applied to the synthesis of A9 (Table 6), is based on the condensation of the proper
anhydride and aminoalcohol in solvent-free conditions.
The acylation reactions (Scheme 1) were performed in parallel, in ordered arrays
of reaction vessels (Carousel-12 Reaction StationsTM), by using pyridine as solvent
and stirring at rt for 18 h. The addition of N,N,N’,N’-tetramethylethylenediamine
(TMEDA) increased the yields. The workup simply required filtrations or
extractions and the final products were purified by crystallization. The yields (not
optimized) ranged from 34–95% (Tables 1, 2, 3, 4, and 5).
Biological activity
Title compounds were evaluated for their cytotoxicity and anti-HIV-1 activity in
MT-4 cells (Tables 1, 2, 3, 4, and 5), using trovirdine as the reference compound. The
most potent derivatives were tested in enzyme assays against wt HIV-1 RT (Table 7).
All esters also were assayed against some NNRTI resistant strains (Maas et al., 1993;
Tantillo et al., 1994) (Y181C, K103N ? Y181C and K103R ? V179D ? P225H
(EFVR), using efavirenz as the reference molecule (Table 8).

Med Chem Res (2010) 19:311–336
317
Table 4 Physical and chemical data, cytotoxicity, and anti-HIV-1 activity of compounds 45–52a
X,Y
O
G
O
Compound
G
X,Y
cryst. solvent(s) mp (°C) % Yield CCb50 EC5c0
45
2,5-F2
Et2O/EtOH
76–77
99–100
68–70
60
50
42
[100 [100
O
H
H
N
O
46
47
48
49
4-Cl-3-NO2 CH2Cl2/EtOH
36 [36
O
H
H
N
O
2,5-F2
Et2O/EtOH
[100 [100
O
H
H
N
O
4-Cl-3-NO2 CH2Cl2/EtOH
117–119 51
28 [28
O
H
H
N
O
4-Cl-3-NO2 CH2Cl2/EtOH
4-Cl-3-NO2 CH2Cl2/EtOH
95–97
47
59 [59
Br
O
O
50
51
124–126 58
117–119 83
47 [47
19 [19
O2N
3,4-Cl2
CH2Cl2/EtOH
O
S
O
52
4-Cl-3-NO2 CH2Cl2/EtOH
152–154 41
6
[6
N
H
trovirdine
60 0.02
a,b,c
See legend for Table 1

318
Med Chem Res (2010) 19:311–336
Table 5 Physical and chemical data, cytotoxicity, and anti-HIV-1 activity of compounds 53–55a
X,Y
O
O
Compound
X,Y
cryst. solvent(s)
mp (°C)
% Yield
CCb50
ECc50
53d
54d
55d
3,4-Cl2
CH2Cl2/EtOH
80–82
103–106
83–85
76
53
65
42
1.6
8
[100
[100
[100
0.02
4-Cl-3-NO2
4-Br-3-CH3
CH3COCH3/EtOH
CH2Cl2/EtOH
trovirdine
60
a,b,c
See legend for Table 1
d
Assayed as racemic mixture
O
R
R'
(a)
R
OH
O
A1-14
1-55
Scheme 1 Reagents and conditions: (a) pyridine, TMEDA (1.1 eq), R’COCl (C1–34) (1.1 eq), rt, 18 h.
The structures of alcohols (A1–14) and acyl chlorides (C1–34) are listed in Fig. 3
All the 2-phthalimidoethyl derivatives with a monosubstituted or unsubstituted
benzoyl portion (1–10, Table 1), with the exception of 8 and 10, showed anti-HIV-1
activity in the micromolar concentration range (EC50 = 14–93 lM). The electronic
properties of the substituent seemed not to affect the antiretroviral activity.
Differently from the para-n-pentyl derivative (9: EC50 = 14 lM), the para-tert-
butyl analogue (8) was inactive and slightly cytotoxic (CC50 = 92 lM). Among the
disubstituted-benzoic esters (11–28; Table 1), compounds 13 (2,5-difluoro), 14 (2,6-
difluoro), 17 (3,4-dichloro), 18 (3,5-dichloro), 19 (2-chloro-4-nitro), 20 (2-methyl-
5-fluoro), 26 (2-bromo-5-fluoro), 27 (4-chloro-3-nitro), and 28 (4-bromo-3-methyl)
showed anti-HIV activity ranging from nanomolar to micromolar concentrations
(EC50 = 0.2–60 lM). In particular, 13, 17, and 27 emerged as the most active
compounds of the series, with an EC50 value of 200, 700, and 700 nM, respectively.
Unlike the other chloro-nitro congeners, 25 (2-chloro-5-nitro) was inactive, but it
showed antiproliferative activity (CC50 = 30 lM). Data reported in Table 2
highlight that the replacement of the benzoyl portion with the 2,5-dimethylfuroyl
moiety (31, EC50 = 28 lM) was beneficial (compare 31 with 1), whereas the
replacement with a 2- or 6-chloroisonicotinoyl (29, 30) or a more sterically
demanding 2-naphthoyl (34) was detrimental. Ester 1 and its vinylogue 33
displayed comparable EC50 values. In the lead structure 1, the introduction of an

Med Chem Res (2010) 19:311–336
319
a) Alcohols ROH (A1-14
)
n
W
A
R1
R2
0
1
1
1
1
1
2
-
H
H
H
H
A1
A2
A3
A4
A5
A6
A7
O
N
H
R1
OH
CH3
H
H
H
H
OH
n
CH3
H
W
R 2
O
H
CH3
C(CH3)3
H
H
H
A1-7
A14
H
H
O
O
N
O
H
H
H
N
Br
O
H
O
OH
A8
A9
A10
G
G =
A8-13
O
O2N
O
S
N
O
H
A11
A12
A13
b)Acyl chlorides R'COCl (C1-34
)
R'CO
I1 benzoyl
I2 2-chlorobenzoyl
I3 2-nitrobenzoyl
R'CO
I18 3,5-dichlorobenzoyl
I19 2-chloro-4-nitrobenzoyl
I20 2-methyl-5-fluorobenzoyl
I4 3-methylbenzoyl
I5 3-fluorobenzoyl
I6 3-chlorobenzoyl
I7 3-nitrobenzoyl
I8 4-tert-butylbenzoyl
I9 4-n-pentylbenzoyl
I10 4-bromobenzoyl
I11 2,3-difluorobenzoyl
I12 2,4-difluorobenzoyl
I13 2,5-difluorobenzoyl
I14 2,6-difluorobenzoyl
I15 3,4-difluorobenzoyl
I16 3,5-difluorobenzoyl
I17 3,4-dichlorobenzoyl
I21 2-fluoro-5-trifluoromethylbenzoyl
I22 2-fluoro-5-chlorobenzoyl
I23 2-fluoro-5-nitrobenzoyl
I24 2-chloro-5-fluorobenzoyl
I25 2-chloro-5-nitrobenzoyl
I26 2-bromo-5-fluorobenzoyl
I27 4-chloro-3-nitrobenzoyl
I28 4-bromo-3-methylbenzoyl
I29 2-chloronicotinoyl
I30 6-chloronicotinoyl
I31 2,5-dimethylfuran-3-carbonyl
I32 2-phenylbutyryl
I33 trans-cinnamoyl
I34 2-naphthoyl
Fig. 3 Alcohols and acyl chlorides used as synthetic building blocks. a Alcohols ROH (A1–14). b Acyl
chlorides R’COCl (C1–34
)

320
Med Chem Res (2010) 19:311–336
Table 6 Reagents and conditions: (a) 2-aminoethanol (1 eq), 175–180°C, 2 h. Microanalyses of com-
pounds A9 and 1–55. For each formula the theoretical and experimental values were reported in the first
and the second row, respectively
O
H
(a)
O
A9
H
O
Compd
Formula
C
H
N
S
A9
C10H15NO3
60.90
60.67
69.15
69.39
61.92
61.96
60.00
59.80
69.88
69.82
65.18
64.98
61.92
62.00
60.00
59.79
71.78
72.09
72.31
72.04
54.57
54.91
61.64
61.39
61.64
61.73
61.64
61.56
61.64
61.51
61.64
61.39
7.67
8.02
4.44
4.76
3.67
3.38
3.55
3.88
4.89
5.11
3.86
3.95
3.67
4.00
3.55
3.57
6.02
6.01
6.34
5.97
3.23
3.31
3.35
3.63
3.35
3.55
3.35
3.63
3.35
3.39
3.35
3.61
7.10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.17
4.74
4.84
4.25
4.31
8.23
8.17
4.53
4.90
4.47
4.52
4.25
4.54
8.23
8.18
3.99
3.98
3.83
4.01
3.74
3.74
4.23
4.32
4.23
4.19
4.23
4.55
4.23
4.17
4.23
4.14
1
C17H13NO4
2
C17H12ClNO4
3
C
C
17H12N2O6
18H15NO4
4
5
C17H12FNO4
6
C
C
C
C
C
C
C
C
C
17H12ClNO4
17H12N2O6
21H21NO4
7
8
9
22H23NO4
10
11
12
13
14
15
17H12BrNO4
17H11F2NO4
17H11F2NO4
17H11F2NO4
17H11F2NO4
C17H11F2NO4

Med Chem Res (2010) 19:311–336
321
Table 6 continued
Compd
Formula
C
H
N
S
16
C
C
17H11F2NO4
17H11Cl2NO4
61.64
61.45
56.07
55.97
56.07
56.11
54.49
54.62
66.05
65.89
56.70
56.45
58.72
58.99
56.99
56.77
58.72
58.57
54.49
54.50
52.06
52.04
54.49
54.40
55.69
55.43
58.11
57.98
58.11
58.16
65.17
65.33
71.20
71.02
71.02
70.66
73.04
73.09
60.58
60.68
3.35
3.54
3.04
3.03
3.04
3.05
2.96
2.98
4.31
4.46
2.91
2.85
3.19
3.42
3.09
3.15
3.19
3.18
2.96
3.07
2.83
2.70
2.96
2.98
3.63
3.71
3.35
3.68
3.35
3.03
4.83
5.05
5.68
6.07
4.71
4.82
4.38
4.35
2.86
2.55
4.23
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4.19
3.85
3.76
3.85
3.87
7.48
7.46
4.28
4.26
3.67
3.67
4.03
4.04
7.82
7.71
4.03
4.06
7.48
7.16
3.57
z3.53
7.48
7.36
3.61
3.42
8.47
8.44
8.47
8.49
4.47
4.42
4.15
4.27
4.36
4.36
4.06
4.02
4.42
4.49
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
C17H11Cl2NO4
C
C
C
C
C
C
C
C
C
17H11ClN2O6
18H14FNO4
18H11F4NO4
17H11ClFNO4
17H11FN2O6
17H11ClFNO4
17H11ClN2O6
17H11BrFNO4
17H11ClN2O6
C18H14BrNO4
C
C
C
16H11ClN2O4
16H11ClN2O4
17H15NO5
C20H19NO4
C
C
19H15NO4
21H15NO4
C16H9F2NO4

322
Med Chem Res (2010) 19:311–336
Table 6 continued
Compd
Formula
C
H
N
S
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
C
C
16H9ClN2O6
18H13F2NO4
53.28
53.41
62.61
62.59
62.61
62.72
55.61
55.86
62.61
62.47
55.61
55.86
65.11
64.92
62.61
62.61
55.61
55.55
60.90
60.61
53.91
54.28
60.53
60.52
53.62
53.67
44.97
45.14
49.13
49.38
50.15
50.52
59.23
59.40
66.49
66.29
64.51
64.66
53.72
53.36
2.51
2.48
3.79
4.16
3.79
3.79
3.37
3.30
3.79
3.85
3.37
3.30
4.94
4.94
3.79
3.83
3.37
3.28
4.51
4.59
3.99
3.72
5.08
5.27
4.50
4.24
2.77
2.81
3.02
2.94
3.37
3.27
3.80
3.71
3.52
3.46
3.42
3.53
3.38
3.68
7.77
–
7.78
4.06
4.10
4.06
4.11
7.21
7.21
4.06
4.22
7.21
7.21
3.62
3.96
4.06
4.15
7.21
7.20
4.18
4.22
7.40
7.48
4.15
4.19
7.36
7.34
3.50
3.44
7.64
7.74
–
–
–
–
C18H13F2NO4
–
–
C
C
C
C
C
C
C
C
C
18H13ClN2O6
18H13F2NO4
18H13ClN2O6
21H19F2NO4
18H13F2NO4
18H13ClN2O6
17H15F2NO4
17H15ClN2O6
17H17F2NO4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
C17H17ClN2O6
–
–
C
C
C
15H11BrClNO5
15H11ClN2O7
15H12Cl2O4S
–
–
–
–
8.93
8.81
–
–
C17H13ClN2O4
8.13
8.13
–
–
C
C
19H12Cl2O2
19H12ClNO4
–
–
–
3.96
3.91
–
–
–
C20H15BrO2
–
–
–

Med Chem Res (2010) 19:311–336
323
Table 7 Activity of compounds 13, 17, and 27 in enzyme assay against wt HIV-1 RT
Compound
ICa50
13
[5 mM
17
2
0.6 mM
27
1.1 0.2 mM
1.3 0.3 nM
efavirenz
Data mean values for three independent experiments. Standard deviations were\20%. Residual activity
was plotted versus increasing concentration of compounds. The curve was fit with Kaleidagraph (Synergy
Software) to obtain IC50 values
a
Compound concentration required to inhibit the wild-type HIV-1 RT activity by 50%
Table 8 Anti-HIV-1 activity of compound 26 against the Y181C resistant mutant
Compound
ECa50
26
90
efavirenz
0.01
Data mean values for three independent experiments. Variation among triplicate samples was \10%
a
Compound concentration [lM] required to achieve 50% protection of MT-4 cell from HIV-1 induced
cytopathogenicity, as determined by the MTT method
ethyl-substituted methylene between the carbonyl function and the benzene ring
(32, EC50 = 16 lM) caused a 3.4-fold increase in activity.
Tables 3 and 4 display that, in the structures of 13 (2,5-difluorobenzoyl) and 27
(4-chloro-3-nitrobenzoyl), the shortening (35, 36), branching (37–39), and length-
ening (43, 44) of the ethyl linker, the introduction of a methyl (40, 41), or a tert-
butyl (42) at position 4 of the phthalimide substructure, and the partial (45, 46) or
complete (47, 48) reduction of the phthalimide benzene ring led to compounds
devoid of antiretroviral activity. Interestingly, all the 4-chloro-3-nitro derivatives
(except for 44), unlike the corresponding 2,5-difluoro analogues, were endowed
with antiproliferative activity (36, 39, 41, 46, 48: CC50 = 11–81 lM).
The replacement of the phthalimide substructure with other aromatic moieties
bearing a potential hydrogen-bond acceptor group [para-bromo- (49) and para–
nitro-phenoxyl (50), phenylsulphonyl (51)] or an hydrogen-bond donor function
[3-indolyl (52)] caused the loss of the anti-HIV-1 activity (49, 50, and 52 vs. 27; 51
vs. 17), but afforded antiproliferative compounds (49–52: CC50 = 6–59 lM).
Data reported in Table 5 show that the replacement of 2-phthalimidoethyl
portion with a conformationally constrained form of the 2-(1-naphthyl)ethyl moiety
(53–55) produced the loss of the antiretroviral activity (53 vs. 17; 54 vs. 27; 28 vs.
55) and the gain in cytotoxicity (53–55: CC50 = 1.6–42 lM). In particular, the
4-chloro-3-nitrobenzoyl ester (54) turned out to be 5- and 26-fold more potent than
the 3,4-dichloro (53) and 4-bromo-3-methyl (55) analogues, respectively, and
emerged as the most cytotoxic derivative of the entire series (CC50 = 1.6 lM).
To investigate whether the molecular target of the anti HIV-1 activity of the title
compounds was the RT, the most potent esters (13, 17, and 27) were tested in

324
Med Chem Res (2010) 19:311–336
enzyme assays against the wt HIV-1 RT. Unexpectedly, derivatives 17 and 27 had
only a minor effect on the activity of the viral polymerase, whereas 13 resulted to be
ineffective (Table 7), thus suggesting a different molecular target for this new class
of anti-HIV-1 agents.
Only compound 26 (2-bromo-5-fluorobenzoyl) was able to reduce, even if
weakly, the multiplication of the Y181C resistant strain (Table 8). Also the
K103N ? Y181C and K103R ? V179D ? P225H (EFVR) mutated strains proved
to be unsusceptible to the tested compounds (data not shown).
Conclusions
In this study, a molecular simplification strategy was applied to the potent NNRTIs
ATCs, with the goal to identify a new class of anti-HIV-1 agents. The setup of a
solution-phase parallel synthesis method allowed the rapid preparation of a high
number of analogues of lead 1. In cell-based assays, 20 of 34 esters of 2-(N-
phthalimido)ethanol displayed anti-HIV-1 activity ranging from nanomolar to
micromolar concentrations and one of them (26) resulted weakly effective against
Y181C mutant strain. However, the most potent esters (13, 17, and 27) had only a
minor effect or were ineffective in enzyme assay against HIV-1 RT. Interestingly,
variations on the O-(2-phthalimidoethyl) moiety led to compounds devoid of
antiretroviral activity, but cytotoxic, in particular those bearing the 4-chloro-3-
nitrobenzoyl moiety. Among them, the ester of 1,2-dihydroacenaphthylen-1-ol (54)
resulted to be the most cytotoxic derivative of the entire series. Future study will be
focused on the identification of the molecular targets to which the anti-HIV-1 and
cytotoxic activities of the title compounds are related.
Experimental section
Chemistry
All chemicals were purchased from Sigma-Aldrich Chemical Co., Alfa Aesar, and
Lancaster and used without further purification, unless otherwise stated. Alcohols
A3–6 and A8 were prepared according to the previously reported protocol (Ranise
et al., 2005; Spallarossa et al., 2008b). The synthesis of trovirdine was
accomplished according to the published procedure (Bell et al., 1995). Solvents
were reagent grade. Organic solutions were dried over anhydrous sodium sulphate.
Thin layer chromatography (TLC) system for routine monitoring 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 were used as developing solvents and detection of spots was made by UV
light and/or by iodine vapors. The parallel solution phase chemistry was performed
by using a Carousel-12 Reaction StationTM (Radleys Discovery Technologies,
Italian distributor: StepBio, Bologna). The evaporation of solutions was performed
in parallel with an EvaposelTM apparatus (Radleys Discovery Technologies, Italian

Med Chem Res (2010) 19:311–336
325
distributor: StepBio, Bologna) operating at reduced pressure of approximately 15–
20 Torr. Yields were not optimized. Melting points were 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 at 200 MHz on a Varian Gemini 200 instrument. Chemical shifts were reported
in d (ppm) units relative to the internal standard tetramethyl silane, and the splitting
patterns were described as follows: s (singlet), d (doublet), dd (doublet of doublets),
t (triplet), m (multiplet), and bs (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.
Synthesis of (3aR,7aS)-2-(2-hydroxyethyl)hexahydro-1H-isoindole-1,3(2H)-dione
(A9)
A mixture of 2-aminoethanol (40 mmol, 2.414 ml) and (3aR,7aS)-hexahydro-2-
benzofuran-1,3-dione (40 mmol, 6.167 g) was heated at 175–180°C under stirring
for 2 h. After cooling, the reaction mixture was dissolved in DCM (dichlorometh-
ane) and washed with a 2 N HCl solution (2 9 50 ml). The organic layer was dried
over anhydrous Na2SO4 and evaporated under reduced pressure to give A9, which
was purified by distillation (210°C/0.45 mmHg). Yield: 61%.
IR (KBr) cm-1: 3457, 2946, 2863, 1773, 1710. 1H NMR (CDCl3) d: 1.24–1.41 (m,
4H, 2CH2), 1.59–1.82 (m, 4H, 2CH2), 2.49 (bs, 1H, OH, exchangeable), 2.75–2.86
(m, 2H, 2CH), 3.55–3.72 (m, 4H, CH2N ? CH2O). Anal. (C10H15NO3), C, H, N.
Parallel synthesis of compounds 1–55
The suitable acyl chloride (5.5 mmol) was added dropwise to each numbered
reaction tube of a Carousel-12 Reaction StationTM, containing a stirred solution of
the opportune alcohol (A1–14) (5 mmol) and TMEDA (5.5 mmol, 0.825 ml) in
pyridine (10 ml). The resulting mixtures were stirred for 18 h at rt. Successively, a
2N HCl solution (25 ml) was added into each tube (except for 29 and 30), the
contents of the tubes were transferred into a set of beakers and then, more 2N HCl
solution (65 ml) was added into each beaker. For 29 and 30, 2N HCl solution was
added only until the mixture reached pH = 5. Two different types of work-up were
performed. Workup (i) (1–7, 9–11, 14, 17, 18, 20–26, 28, 29, 31, 33–36, 39–42, 44,
45, 47, 51, 53, 55): the precipitates obtained were filtered off in parallel by an in-
house device and dissolved in DCM. The solutions were transferred into a set of
separating funnels, washed with brine, dried over anhydrous Na2SO4, and filtered in
parallel through pads of Florisil (diameter 5 9 2 cm) by an in-house device. Parallel
evaporating in vacuo using an EvaposelTM apparatus gave residues, which were
purified by crystallization from the suitable solvent or solvent mixture (Tables 1, 2,
3, 4, and 5). Workup (ii) (8, 12, 13, 15, 16, 19, 27, 30, 32, 37, 38, 43, 46, 48–50, 52,
54): the contents of the beakers were transferred into a set of separating funnels.
After parallel extraction with DCM, the combined extracts of each reaction were

326
Med Chem Res (2010) 19:311–336
washed with brine and then treated as for workup (i). Yields, crystallization
solvent(s) and melting points are reported in Tables 1, 2, 3, 4, and 5.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl benzoate (1)
1
IR (KBr) cm-1: 1767, 1712. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H, CH2N),
4.57 (t, J = 5.4 Hz, 2H, CH2O), 7.47–7.62 (m, 3H, arom. H), 7.71–8.2 (m, 6H,
arom. H). Anal. (C17H13NO4), C, H, N. Melting point consistent with data reported
in literature (Mizuch et al., 1957).
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-chlorobenzoate (2)
1
IR (KBr) cm-1: 1772, 1732, 1711. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H,
CH2N), 4.62 (t, J = 5.4 Hz, 2H, CH2O), 7.3–8.04 (m, 8H, arom. H). Anal.
(C17H12ClNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-nitrobenzoate (3)
IR (KBr) cm-1: 1771, 1733, 1709, 1532, 1351. 1H NMR (CDCl3) d: 4.09
(t, J = 5.4 Hz, 2H, CH2N), 4.62 (t, J = 5.4 Hz, 2H, CH2O), 7.36–8.05 (m, 8H,
arom. H). Anal. (C17H12N2O6), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3-methylbenzoate (4)
1
IR (KBr) cm-1: 1779, 1731, 1715. H NMR (CDCl3) d: 2.35 (s, 3H, CH3), 4.11
(t, J = 5.4 Hz, 2H, CH2N), 4.54 (t, J = 5.4 Hz, 2H, CH2O), 7.22–8.03 (m, 8H,
arom. H). Anal. (C18H15NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3-fluorobenzoate (5)
1
IR (KBr) cm-1: 1777, 1710. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H, CH2N),
4.58 (t, J = 5.4 Hz, 2H, CH2O), 7.19–8.0 (m, 8H, arom. H). Anal. (C17H12FNO4),
C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3-chlorobenzoate (6)
1
IR (KBr) cm-1: 1776, 1729, 1718. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.57 (t, J = 5.4 Hz, 2H, CH2O), 7.4–8.09 (m, 8H, arom. H). Anal.
(C17H12ClNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3-nitrobenzoate (7)
IR (KBr) cm-1: 1773, 1729, 1703, 1536, 1350. 1H NMR (CDCl3) d: 4.16
(t, J = 5.4 Hz, 2H, CH2N), 4.64 (t, J = 5.4 Hz, 2H, CH2O), 7.6–8.88 (m, 8H, arom.
H). Anal. (C17H12N2O6), C, H, N.

Med Chem Res (2010) 19:311–336
327
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 4-tert-butylbenzoate (8)
1
IR (KBr) cm-1: 2954, 1781, 1720. H NMR (CDCl3) d: 1.31 (s, 9H, 3CH3), 4.13
(t, J = 5.4 Hz, 2H, CH2N), 4.57 (t, J = 5.4 Hz, 2H, CH2O), 7.3–8.09 (m, 8H, arom.
H). Anal. (C21H21NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 4-pentylbenzoate (9)
1
IR (KBr) cm-1: 2966, 1771, 1732, 1713. H NMR (CDCl3) d: 0.81 (t, J = 7.1 Hz,
3H, CH3), 1.62–1.86 (m, 4H, 2CH2), 1.94–2.17 (m, 2H, CH2), 3.42 (t, J = 7.1 Hz,
2H, CH2Ph), 3.87–4.04 (m, 2H, CH2N), 4.21–4.43 (m, 2H, CH2O), 7.07–7.37 (m,
4H, arom. H), 7.65–7.95 (m, 4H, ftal. arom. H). Anal. (C22H23NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 4-bromobenzoate (10)
1
IR (KBr) cm-1: 1774, 1731, 1713. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.58 (t, J = 5.4 Hz, 2H, CH2O), 7.44–8.03 (m, 8H, arom. H). Anal.
(C17H12BrNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2,3-difluorobenzoate (11)
1
IR (KBr) cm-1: 1776, 1733, 1709. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.61 (t, J = 5.4 Hz, 2H, CH2O), 7.05–7.4 (m, 2H, arom. H), 7.53–7.97 (m,
5H, arom. H). Anal. (C17H11F2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2,4-difluorobenzoate (12)
1
IR (KBr) cm-1: 1781, 1733, 1710. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.58 (t, J = 5.4 Hz, 2H, CH2O), 6.7–7.1 (m, 2H, arom. H), 7.7–8.05 (m,
5H, arom. H). Anal. (C17H11F2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2,5-difluorobenzoate (13)
1
IR (KBr) cm-1: 1775, 1733, 1712. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H,
CH2N), 4.62 (t, J = 5.4 Hz, 2H, CH2O), 7.09–7.29 (m, 2H, arom. H), 7.6–7.95 (m,
5H, arom. H). Anal. (C17H11F2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2,6-difluorobenzoate (14)
1
IR (KBr) cm-1: 1773, 1746, 1709. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H,
CH2N), 4.66 (t, J = 5.4 Hz, 2H, CH2O), 6.58–7.98 (m, 7H, arom. H). Anal.
(C17H11F2NO4), C, H, N.

328
Med Chem Res (2010) 19:311–336
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3,4-difluorobenzoate (15)
1
IR (KBr) cm-1: 1780, 1734, 1715. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H,
CH2N), 4.58 (t, J = 5.4 Hz, 2H, CH2O), 7.09–7.43 (m, 1H, arom. H), 7.62–8.09 (m,
6H, arom. H). Anal. (C17H11F2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3,5-difluorobenzoate (16)
1
IR (KBr) cm-1: 1779, 1723. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H, CH2N),
4.59 (t, J = 5.4 Hz, 2H, CH2O), 6.87–7.2 (m, 1H, arom. H), 7.42–8.01 (m, 6H,
arom. H). Anal. (C17H11F2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3,4-dichlorobenzoate (17)
1
IR (KBr) cm-1: 1773, 1733, 1717. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.57 (t, J = 5.4 Hz, 2H, CH2O), 7.4–8.14 (m, 7H, arom. H). Anal.
(C17H11Cl2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 3,5-dichlorobenzoate (18)
1
IR (KBr) cm-1: 1777, 1735, 1716. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.57 (t, J = 5.4 Hz, 2H, CH2O), 7.46–8.0 (m, 7H, arom. H). Anal.
(C17H11Cl2NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-chloro-4-nitrobenzoate (19)
IR (KBr) cm-1: 1772, 1736, 1710, 1526, 1355. 1H NMR (CDCl3) d: 4.14 (t,
J = 5.4 Hz, 2H, CH2N), 4.69 (t, J = 5.4 Hz, 2H, CH2O), 7.72–8.36 (m, 7H, arom.
H). Anal. (C17H11ClN2O6), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl
5-fluoro-2-methylbenzoate (20)
1
IR (KBr) cm-1: 1780, 1726. H NMR (DMSO-d6) d: 2.42 (s, 3H, CH3), 3.96–4.22
(m, 2H, CH2 N), 4.38–4.65 (m, CH2O), 7.24–8.0 (m, 7H, arom. H). Anal.
(C18H14FNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-fluoro-5-
(trifluoromethyl)benzoate (21)
1
IR (KBr) cm-1: 1774, 1739, 1716. H NMR (CDCl3) d: 4.15 (t, J = 5.4 Hz, 2H,
CH2 N), 4.64 (t, J = 5.4 Hz, 2H, CH2O), 7.07–8.35 (m, 7H, arom. H). Anal.
(C18H11F4NO4), C, H, N.

Med Chem Res (2010) 19:311–336
329
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 5-chloro-
2-fluorobenzoate (22)
1
IR (KBr) cm-1: 1769, 1713. H NMR (CDCl3) d: 4.13 (t, J = 5.4 Hz, 2H, CH2N),
4.63 (t, J = 5.4 Hz, 2H, CH2O), 6.87–8.17 (m, 7H, arom. H). Anal.
(C17H11ClFNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-fluoro-5-nitrobenzoate (23)
1
IR (KBr) cm-1: 1776, 1725, 1706, 1533, 1348. H NMR (DMSO-d6) d: 4.05 (t,
J = 5.4 Hz, 2H, CH2N), 4.60 (t, J = 5.4 Hz, 2H, CH2O), 7.42–8.73 (m, 7H, arom.
H). Anal. (C17H11FN2O6), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-chloro-
5-fluorobenzoate (24)
1
IR (KBr) cm-1: 1778, 1745, 1721. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.63 (t, J = 5.4 Hz, 2H, CH2O), 7.1–7.95 (m, 7H, arom. H). Anal.
(C17H11ClFNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-chloro-5-nitrobenzoate (25)
IR (KBr) cm-1: 1774, 1743, 1713, 1529, 1355. 1H NMR (CDCl3) d: 4.16 (t,
J = 5.4 Hz, 2H, CH2N), 4.68 (t, J = 5.4 Hz, 2H, CH2O), 7.5–8.46 (m, 6H, arom.
H), 8.62–8.79 (m, 1H, arom. H). Anal. (C17H11ClN2O6), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-bromo-5-fluorobenzoate (26)
1
IR (KBr) cm-1: 1778, 1744, 1721. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.62 (t, J = 5.4 Hz, 2H, CH2O), 6.88–8.03 (m, 7H, arom. H). Anal.
(C17H11BrFNO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 4-chloro-3-nitrobenzoate (27)
1
IR (KBr) cm-1: 1773, 1737, 1711. H NMR (CDCl3) d: 4.15 (t, J = 5.4 Hz, 2H,
CH2N), 4.69 (t, J = 5.4 Hz, 2H, CH2O), 7.7–8.4 (m, 7H, arom. H). Anal.
(C17H11ClN2O6), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 4-bromo-3-methylbenzoate (28)
1
IR (KBr) cm-1: 1774, 1730. H NMR (CDCl3) d: 2.42 (s, 3H, CH3), 4.13 (t,
J = 5.4 Hz, 2H, CH2N), 4.57 (t, J = 5.4 Hz, 2H, CH2O), 7.59–7.96 (m, 7H, arom.
H). Anal. (C18H14BrNO4), C, H, N.

330
Med Chem Res (2010) 19:311–336
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-chloronicotinate (29)
1
IR (KBr) cm-1: 1771, 1735, 1711. H NMR (CDCl3) d: 4.12 (t, J = 5.4 Hz, 2H,
CH2N), 4.64 (t, J = 5.4 Hz, 2H, CH2O), 7.23–7.95 (m, 5H, arom. H), 8.10-8.63 (m,
2H, arom. H). Anal. (C16H11ClN2O4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 6-chloronicotinate (30)
1
IR (KBr) cm-1: 1774, 1713. H NMR (CDCl3) d: 4.15 (t, J = 5.4 Hz, 2H, CH2N),
4.62 (t, J = 5.4 Hz, 2H, CH2O), 7.3–7.57 (m, 1H, arom. H), 7.72–7.97 (m, 4H,
arom. H), 8.13-8.40 (m, 1H, arom. H), 8.89-9.07 (m, 1H, arom. H). Anal.
(C16H11ClN2O4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2,5-dimethyl-3-furoate (31)
1
IR (KBr) cm-1: 1774, 1714. H NMR (CDCl3) d: 2.21 (s, 3H, CH3), 2.45 (s, 3H,
CH3), 4.06 (t, J = 5.4 Hz, 2H, CH2N), 4.47 (t, J = 5.4 Hz, 2H, CH2O), 6.27 (bs,
1H, fur. H), 7.71–7.95 (m, 4H, arom. H). Anal. (C17H15NO5), C, H, N.
( ) 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-phenylbutanoate (32)
1
IR (KBr) cm-1: 1771, 1732, 1714. H NMR (CDCl3) d: 0.82 (t, J = 7.0 Hz, 3H,
CH3), 1.64-2.13 (m, 2H, CH2), 3.42 (t, J = 7.6 Hz, 1H, CHPh), 3.93 (t, J = 5.4 Hz,
2H, CH2N), 4.33 (t, J = 5.4 Hz, 2H, CH2O), 7.09–7.3 (m, 5H, arom. H), 7.68–7.89
(m, 4H, arom. H). Anal. (C20H19NO4), C, H, N.
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl (2E)-3-phenylacrylate (33)
IR (KBr) cm-1: 1777, 1722, 1709. 1H NMR (CDCl3) d: 3.92–4.22 (m, 2H, CH2N),
4.26–4.58 (m, 2H, CH2O), 6.40 (d, J = 15.8 Hz, 1H, CH=CH–CO), 7.28–7.95 (m,
10H, arom. H). Anal. (C19H15NO4), C, H, N. Consistent with data reported in
literature (Sova et al., 1993).
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl 2-naphthoate (34)
1
IR (KBr) cm-1: 1778, 1725, 1712. H NMR (CDCl3) d: 4.19 (t, J = 5.4 Hz, 2H,
CH2N), 4.64 (t, J = 5.4 Hz, 2H, CH2O), 7.49–8.06 (m, 11H, arom. H). Anal.
(C21H15NO4), C, H, N.
(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl 2,5-difluorobenzoate (35)
1
IR (KBr) cm-1: 1780, 1740, 1722. H NMR (CDCl3) d: 6.01 (s, 2H, CH2), 7.04–
8.07 (m, 7H, arom. H). Anal. (C16H9F2NO4), C, H, N.

Med Chem Res (2010) 19:311–336
331
(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl 4-chloro-3-nitrobenzoate (36)
1
IR (KBr) cm-1: 1785, 1738, 1717, 1538, 1348. H NMR (CDCl3) d: 6.03 (s, 2H,
CH2), 7.53–8.0 (m, 5H, arom. H), 8.09–8.57 (m, 2H, arom. H). Anal.
(C16H9ClN2O6), C, H, N.
( ) 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl 2,5-difluorobenzoate (37)
1
IR (KBr) cm-1: 1774, 1720, 1704. H NMR (CDCl3) d: 1.61 (d, J = 7.0 Hz, 3H,
CH3), 4.53–5.0 (m, 3H, CHN ? CH2O), 6.95–8.08 (m, 7H, arom. H). Anal.
(C18H13F2NO4), C, H, N.
( ) 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-1-methylethyl
2,5-difluorobenzoate (38)
1
IR (KBr) cm-1: 1774, 1712. H NMR (CDCl3) d: 1.47 (d, J = 6.8 Hz, 3H, CH3),
4.02 (d, J = 5.4 Hz, 2H, CH2N), 5.47–5.8 (m, 1H, CHO), 6.99–7.98 (m, 7H, arom.
H). Anal. (C18H13F2NO4), C, H, N.
( ) 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-1-methylethyl
4-chloro-3-nitrobenzoate (39)
IR (KBr) cm-1: 1774, 1714, 1535, 1351. 1H NMR (CDCl3) d: 1.48 (d, J = 6.8 Hz,
3H, CH3), 4.03 (d, J = 5.4 Hz, 2H, CH2N), 5.28–5.56 (m, 1H, CHO), 7.54–7.55 (m,
5H, arom. H), 8.06–8.56 (m, 2H, arom. H). Anal. (C18H13ClN2O6), C, H, N.
2-(5-methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl
2,5-difluorobenzoate (40)
1
IR (KBr) cm-1: 1769, 1734, 1702. H NMR (CDCl3) d: 2.51 (s, 3H, CH3), 4.09 (t,
J = 5.4 Hz, 2H, CH2N), 4.59 (t, J = 5.4 Hz, 2H, CH2O), 7.02–7.82 (m, 6H, arom.
H). Anal. (C18H13F2NO4), C, H, N.
2-(5-methyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl
4-chloro-3-nitrobenzoate (41)
1
IR (KBr) cm-1: 1771, 1730, 1708, 1533, 1349. H NMR (CDCl3) d: 2.52 (s, 3H,
CH3), 4.11 (t, J = 5.4 Hz, 2H, CH2N), 4.60 (t, J = 5.4 Hz, 2H, CH2O), 7.55–7.8
(m, 4H, arom. H), 8.04–8.56 (m, 2H, arom. H). Anal. (C18H13ClN2O6), C, H, N.
2-(5-tert-butyl-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl
2,5-difluorobenzoate (42)
IR (KBr) cm-1: 2958, 1771, 1707. 1H NMR (DMSO-d6) d: 1.24 (s, 9H, 3CH3), 3.87
(t, J = 5.4 Hz, 2H, CH2N), 4.39 (t, J = 5.4 Hz, 2H, CH2O), 7.22–7.82 (m, 6H,
arom. H). Anal. (C21H19F2NO4), C, H, N.

332
Med Chem Res (2010) 19:311–336
3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl 2,5-difluorobenzoate (43)
1
IR (KBr) cm-1: 1765, 1727, 1705. H NMR (CDCl3) d: 2.02-2.17 (m, 2H, CH2),
3.81 (t, J = 6.6 Hz, 2H, CH2N), 4.31 (t, J = 6.0 Hz, 2H, CH2O), 6.9–7.17 (m, 2H,
arom. H), 7.44–7.77 (m, 5H, arom. H). Anal. (C18H13F2NO4), C, H, N.
3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl 4-chloro-3-nitrobenzoate (44)
1
IR (KBr) cm-1: 1771, 1709, 1537, 1338. H NMR (CDCl3) d: 2.10-2.37 (m, 2H,
CH2), 3.93 (t, J = 7.2 Hz, 2H, CH2N), 4.45 (t, J = 6.6 Hz, 2H, CH2O), 7.55–7.90
(m, 5H, arom. H), 8.05–8.56 (m, 2H, arom. H). Anal. (C18H13ClN2O6), C, H, N.
2-[(3aR,7aS)-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindol-2-yl]ethyl
2,5-difluorobenzoate (45)
1
IR (KBr) cm-1: 2952, 1735, 1694. H NMR (CDCl3) d: 2.2–2.62 (m, 4H, 2CH2),
3.04–3.26 (m, 2H, 2CHCO), 3.88 (t, J = 5.4 Hz, 2H, CH2N), 4.48 (t, J = 5.4 Hz,
2H, CH2O), 5.75–5.95 (m, 2H, CH=CH), 7–7.75 (m, 3H, arom. H). Anal.
(C17H15F2NO4), C, H, N.
2-[(3aR,7aS)-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindol-2-yl]ethyl
4-chloro-3-nitrobenzoate (46)
IR (KBr) cm-1: 2960, 1772, 1717, 1703, 1537, 1340. 1H NMR (CDCl3) d: 2.2–2.63
(m, 4H, 2CH2), 3.03–3.29 (m, 2H, 2CHCO), 3.93 (t, J = 5.4 Hz, 2H, CH2N), 4.51
(t, J = 5.4 Hz, 2H, CH2O), 5.75–5.95 (m, 2H, CH=CH), 7.71 (d, J = 8.1 Hz, arom.
H5), 8.19 (dd, J = 8.1 Hz, J = 2.1 Hz, arom. H6), 8.47 (d, J = 2.1 Hz, arom. H2).
Anal. (C17H15ClN2O6), C, H, N.
2-[(3aR,7aS)-1,3-dioxooctahydro-2H-isoindol-2-yl]ethyl
2,5-difluorobenzoate (47)
1
IR (KBr) cm-1: 2949, 1772, 1730, 1707. H NMR (CDCl3) d: 1.26–2.0 (m, 8H,
4CH2), 2.77–2.98 (m, 2H, 2CHCO), 3.91 (t, J = 5.4 Hz, 2H, CH2N), 4.53 (t,
J = 5.4 Hz, 2H, CH2O), 7.02–7.37 (m, 2H, arom. H), 7.45-7.79 (m, 1H, arom. H).
Anal. (C17H17F2NO4), C, H, N.
2-[(3aR,7aS)-1,3-dioxooctahydro-2H-isoindol-2-yl]ethyl 4-chloro-
3-nitrobenzoate (48)
1
IR (KBr) cm-1: 2933, 1775, 1725, 1703, 1539, 1360. H NMR (CDCl3) d: 1.27–
1.99 (m, 8H, 4CH2), 2.79–3.05 (m, 2H, 2CHCO), 3.94 (t, J = 5.4 Hz, 2H, CH2N),
4.56 (t, J = 5.4 Hz, 2H, CH2O), 7.70 (d, J = 8.1 Hz, arom. H5), 8.20 (dd,
J = 8.1 Hz, J = 1.8 Hz, arom. H6), 8.48 (d, J = 1.8 Hz, arom. H2). Anal.
(C17H17ClN2O6), C, H, N.

Med Chem Res (2010) 19:311–336
333
2-(4-bromophenoxy)ethyl 4-chloro-3-nitrobenzoate (49)
1
IR (KBr) cm-1: 1726, 1538, 1360. H NMR (DMSO-d6) d: 4.24–4.81 (m, 4H,
2CH2), 7.00 (d, J = 9.0 Hz, 2H, arom. H), 7.50 (d, J = 9.0 Hz, 2H, arom. H), 7.81–
8.65 (m, 3H, arom. H). Anal. (C15H11BrClNO5), C, H, N.
2-(4-nitrophenoxy)ethyl 4-chloro-3-nitrobenzoate (50)
1
IR (KBr) cm-1: 1725, 1541, 1363. H NMR (CDCl3) d: 4.35–4.95 (m, 4H, 2CH2),
7.05 (d, J = 9.0 Hz, 2H, arom. H), 7.67 (d, J = 9.0 Hz, 1H, arom. H), 8.03–8.60
(m, 4H, arom. H). Anal. (C15H11ClN2O7), C, H, N.
2-(phenylsulfonyl)ethyl 3,4-dichlorobenzoate (51)
1
IR (KBr) cm-1: 1712, 1325, 1144. H NMR (CDCl3) d: 3.63 (t, J = 6.0 Hz, 2H,
CH2S), 4.71 (t, J = 6.0 Hz, 2H, CH2O), 7.43–8.11 (m, 8H, arom. H). Anal.
(C15H12Cl2O4S), C, H, S.
2-(1H-indol-3-yl)ethyl 4-chloro-3-nitrobenzoate (52)
IR (KBr) cm-1: 3368, 1715, 1527, 1354. 1H NMR (DMSO-d6) d: 3.23 (t,
J = 7.0 Hz, 2H, CH2C), 4.62 (t, J = 7.0 Hz, 2H, CH2O), 7–7.74 (m, 6H, arom. H),
8.02–8.24 (m, 1H, arom. H), 8.41–8.51 (m, 1H, arom. H), 11.11 (bs, 1H, NH,
exchangeable). Anal. (C17H13ClN2O4), C, H, N.
( ) 1,2-dihydroacenaphthylen-1-yl 3,4-dichlorobenzoate (53)
1
IR (KBr) cm-1: 1720. H NMR (DMSO-d6) d: 3.35–3.48 (m, 1H, CH2), 3.79–3.95
(m, 1H, CH2), 6.72-6.78 (m, 1H, CHO), 7.31–7.38 (m, 1H, arom. H), 7.44–8.06 (m,
8H, arom. H). Anal. (C19H12Cl2O2), C, H.
( ) 1,2-dihydroacenaphthylen-1-yl 4-chloro-3-nitrobenzoate (54)
1
IR (KBr) cm-1: 1713, 1537, 1358. H NMR (CDCl3) d: 3.47–3.63 (m, 1H, CH2),
3.7–3.92 (m, 1H, CH2), 6.73–6.98 (m, 1H, CHO), 7.25–7.85 (m, 6H, arom. H),
7.93–8.22 (m, 2H, arom. H), 8.36–8.51 (m, 1H, arom. H). Anal. (C19H12ClNO4), C,
H, N.
( ) 1,2-dihydroacenaphthylen-1-yl 4-bromo-3-methylbenzoate (55)
IR (KBr) cm-1: 1706. 1H NMR (CDCl3) d: 3.36–3.48 (m, 1H, CH2), 3.83–3.98 (m,
1H, CH2), 6.78-6.87 (m, 1H, CHO), 7.27–7.34 (m, 1H, arom. H), 7.43–7.87 (m, 8H,
arom. H). Anal. (C20H15BrO2), C, H.

334
Med Chem Res (2010) 19:311–336
Virology: materials and methods
Compounds: The compounds were solubilized in DMSO (dimethylsulfoxide) at an
initial concentration of 100 mM and then were diluted in culture medium.
Cells and viruses: MT-4, C8166, H9/IIIB, and CEM cells were grown at 37°C in
a 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% fetal calf
serum (FCS), 100 IU/ml of penicillin G and 100 lg/ml of streptomycin. Cell
cultures were checked periodically for the absence of mycoplasma contamination
with a Myco Tect (Gibco). Human immunodeficiency virus type 1 (HIV-1, IIIB
strain) was obtained from supernatants of persistently infected H9/IIIB. HIV-1 stock
solution had titer of 4.5 9 106 CCID50/ml (CCID50 = 50% cell culture infectious
dose). The Y181C mutant (NIH N119) derives from an AZT-sensitive clinical
isolate passaged initially in CEM, and then in MT-4 cells, in the presence of
nevirapine (10 lM). The double mutant K103N ? Y181C (NIH A17) derives from
the IIIB strain passaged in H9 cells in the presence of BI-RG 587 (1 lM). The triple
mutant K103R ? V179D ? P225H (EFVR) mutant was derived from an IIIB strain
passaged in MT-4 cells in the presence of efavirenz (up to 2 lM). Y181C,
K103N ? Y181C and K103R ? V179D ? P225H (EFVR) stock solutions had
titers of 1.2 9 108, 2.1 9 107, and 4.0 9 107 CCID50/ml, respectively.
HIV titration
HIV titration was performed in C8166 cells by the standard limiting dilution method
(dilution 1:2, four replica wells/dilution) in 96-well plates. The infectious virus titer
was determined by light microscope scoring of cytopathogenicity after 4 days of
incubation, and the virus titers were expressed as CCID50/ml.
Anti-HIV assays
The activity of the test compounds against multiplication of wild type HIV-1,
Y181C, K103N ? Y181C, and K103R ? V179D ? P225H (EFVR) in acutely
infected cells was based on inhibition of virus-induced cytopathogenicity in MT-4
cells. Briefly, 50 ll of culture medium containing 1 9 104 cells were added to each
well of flat-bottom microtiter trays containing 50 ll of culture medium with or
without various concentrations of the test compounds. Then, 20 ll of HIV-1
suspensions (containing the appropriate amount of CCID50 to cause complete
cytopathogenicity at day 4) were added. After incubation at 37°C, cell viability was
determined by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide
(MTT) method (Artico et al., 1996; Pauwels et al., 1988). The cytotoxicity of
compounds was evaluated in parallel with their antiviral activity. It was based on the
viability of mock-infected cells, as monitored by the MTT method.
RT assays
Assays were performed using a nonradioactive method. Purified wt HIV-1 RT was
purchased from Ambion and assayed for its RNA-dependent polymerase-associated

Med Chem Res (2010) 19:311–336
335
activity in a 20-ll volume containing 60 mM Tris–HCl (pH 8.1), 60 mM KCl,
8 mM MgCl2, 13 mM DTT, 2.5 ng/ll template:primer [poly(A)-oligo(dT)16], and
100 lM dTTP. After incubation for 30 min at 37°C, reactions were stopped by
addition of 2 ll of 200 mM EDTA. Then, 138 ll of PicoGreen Quantitation
Reagent (Molecular Probes), diluted 1/345 in TE, was added to each sample and
incubated at room temperature, protected from ambient light, for 5 min.
Fluorescence of samples in wells of a 96-well microtiter plate was determined in
a fluorescence microplate reader (VICTOR3 Multilabel Plate Reader, Perkin Elmer).
Acknowledgements This work was supported by MURST (Cofinanziamento Nazionale), CNR (Rome)
`
and Ministero Italiano della Salute - Istituto Superiore di Sanita (grant no. 40D.46). Fondazione Carige is
gratefully acknowledged for financially supporting S.C. The authors thank Mr. O. Gagliardo for the
microanalyses, Mr. F. Tuberoni, Dr. M. Anzaldi, Dr. R. Raggio, and Dr. C. Rossi, for the IR and NMR
spectra.
References
Artico M, Silvestri R, Massa S, Loi AG, Corrias S, Piras G, La Colla P (1996) 2-Sulfonyl-4-chloroanilino
moiety: a potent pharmacophore for the anti-human immunodeficiency virus type 1 activity of
pyrrolyl aryl sulfones. J Med Chem 39:522–530. doi:10.1021/jm950568w
Balzarini J (2004) Current status of the non-nucleoside reverse transcriptase inhibitors of human
immunodeficiency virus type 1. Curr Top Med Chem 4:921–944. doi:10.2174/1568026043388420
Barbaro G, Scozzafava A, Mastrolorenzo A, Supuran CT (2005) Highly active antiretroviral therapy:
current state of the art, new agents and their pharmacological interactions useful for improving
therapeutic outcome. Curr Pharm Des 11:1805–1843. doi:10.2174/1381612053764869
¨
Bell FW, Cantrell AS, Hogberg M, Jaskunas SR, Johansson NG, Jordan CL, Kinnick MD, Lind P, Morin
¨
´
JM Jr, Noreen R, Oberg B, Palkowitz JA, Parrish CA, Pranc P, Sahlberg C, Ternansky RJ, Vasileff
RT, Vrang L, West SJ, Zhang H, Zhou X (1995) Phenethylthiazolethiourea (PETT) compounds, a
new class of HIV-1 reverse transcriptase inhibitors. 1. Synthesis and basic structure-activity
relationship studies of PETT analogs. J Med Chem 38:4929–4936. doi:10.1021/jm00025a010
Campiani G, Ramunno A, Maga G, Nacci V, Fattorusso C, Catalanotti B, Morelli E, Novellino E (2002)
Non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors: past, present, and future perspectives.
Curr Pharm Des 8:615–657. doi:10.2174/1381612024607207
De Clercq E (1998) The role of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy
of HIV-1 infection. Antiviral Res 38:153–179. doi:10.1016/S0166-3542(98)00025-4
De Clercq E (2001) New developments in anti-HIV chemotherapy. Farmaco 56:3–12. doi:10.1016/
S0014-827X(01)01007-2
De Clercq E (2004) Non-nucleoside reverse transcriptase inhibitors (NNRTIs): past, present, and future.
Chem Biodivers 1:44–64. doi:10.1002/cbdv.200490012
De Clercq E (2005a) Emerging anti-HIV drugs. Expert Opin Emerg Drugs 10:241–273. doi:10.1517/
14728214.10.2.241
De Clercq E (2005b) New approaches toward anti-HIV chemotherapy. J Med Chem 48:1297–1313. doi:
10.1021/jm040158k
Jonckheere H, Anne J, De Clercq E (2000) The HIV-1 reverse transcription (RT) process as target for RT
inhibitors. Med Res Rev 20:129–154. doi:10.1002/(SICI)1098-1128(200003)20:2\129::AID-
MED2[3.0.CO;2-A
Leigh Brown AJ, Frost SD, Mathews WC, Dawson K, Hellmann NS, Daar ES, Richman DD, Little SJ
(2003) Transmission fitness of drug-resistant human immunodeficiency virus and the prevalence of
resistance in the antiretroviral-treated population. J Infect Dis 187:683–686. doi:10.1086/367989
Maass G, Immendoerfer U, Koenig B, Leser U, Mueller B, Goody R, Pfaff E (1993) Viral resistance to
the thiazolo-iso-indolinones, a new class of nonnucleoside inhibitors of human immunodeficiency
virus type 1 reverse transcriptase. Antimicrob Agents Chemother 37:2612–2617

336
Med Chem Res (2010) 19:311–336
Mizuch KG, Kasatkin NM, Gel’fer TM (1957) Reaction of acyl chlorides with some hydroxymethyl
compounds. Zh Obshch Khim 27:189–195
Pauwels R (2004) New non-nucleoside reverse transcriptase inhibitors (NNRTIs) in development for the
treatment of HIV infections. Curr Opin Pharmacol 4:437–446. doi:10.1016/j.coph.2004.07.005
Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P, Desmyter J, De Clercq E (1988)
Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds.
J Virol Methods 20:309–321. doi:10.1016/0166-0934(88)90134-6
Pedersen OS, Pedersen EB (1999) Non-nucleoside reverse transcriptase inhibitors, the NNRTI boom.
Antivir Chem Chemother 10:285–314
Pedersen OS, Pedersen EB (2000) The flourishing syntheses of non-nucleoside reverse transcriptase
inhibitors. Synthesis 4:479–495. doi:10.1055/s-2000-6357
Ranise A, Spallarossa A, Cesarini S, Bondavalli F, Schenone S, Bruno O, Menozzi G, Fossa P, Mosti L,
La Colla M, Sanna G, Murreddu M, Collu G, Busonera B, Marongiu ME, Pani A, La Colla P, Loddo
R (2005) Structure-based design, parallel synthesis, structure-activity relationship, and molecular
modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase
inhibitor isosteres of phenethylthiazolylthiourea derivatives. J Med Chem 48:3858–3873. doi:
10.1021/jm049252r
Ranise A, Spallarossa A, Schenone S, Bruno O, Bondavalli F, Vargiu L, Marceddu T, Mura M, La Colla
P, Pani A (2003) Design, synthesis, SAR, and molecular modeling studies of acylthiocarbamates: a
novel series of potent non-nucleoside HIV-1 reverse transcriptase inhibitors structurally related to
phenethylthiazolylthiourea derivatives. J Med Chem 46:768–781. doi:10.1021/jm0209984
Ren J, Diprose J, Warren J, Esnouf RM, Bird LE, Ikemizu S, Slater M, Milton J, Balzarini J, Stuart DI,
Stammers DK (2000) Phenylethylthiazolylthiourea (PETT) non-nucleoside inhibitors of HIV-1 and
HIV-2 reverse transcriptases. J Biol Chem 275:5633–5639. doi:10.1074/jbc.275.8.5633
Richman DD, Morton SC, Wrin T, Hellmann N, Berry S, Shapiro MF, Bozzette SA (2004) The
prevalence of antiretroviral drug resistance in the United States. AIDS 18:1393–1401. doi:10.1097/
01.aids.0000131310.52526.c7
Silverman RB (2004) The organic chemistry of drug design and drug action. Elsevier, Amsterdam, pp
17–20
ˇ
Sova M, Perdih A, Kotnik M, Kristan K, Rizner TL, Solmajer T, Gobec S (2006) Flavonoid and cinnamic
acid esters as inhibitors of fungal 17b-hydroxysteroid dehydrogenase: a synthesis, QSAR and
modelling study. J Biol Chem 275:5633–5639
Spallarossa A, Cesarini S, Ranise A, Bruno O, Schenone S, La Colla P, Collu G, Sanna G, Secci B, Loddo
R (2008a) 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. Eur J Med
Chem. doi:10.1016/j.ejmech.2008.09.024
Spallarossa A, Cesarini S, Ranise A, Schenone S, Bruno O, Borassi A, La Colla P, Pezzullo M, Sanna G,
Collu G, Secci B, Loddo R (2008b) Parallel synthesis, molecular modelling and further structure-
activity relationship studies of new acylthiocarbamates as potent non-nucleoside HIV-1 reverse
transcriptase inhibitors. Eur J Med Chem. doi:10.1016/j.ejmech.2008.10.032
Tantillo C, Ding J, Jacobo-Molina A, Nanni RG, Boyer PL, Hughes SH, Pauwels R, Andries K, Janssen
PA, Arnold E (1994) Locations of anti-AIDS drug binding sites and resistance mutations in the
three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug
inhibition and resistance. J Mol Biol 243:369–387. doi:10.1006/jmbi.1994.1665
Thomas G (2000) Medicinal chemistry. Wiley, New York, pp 40–42
Vig R, Mao C, Venkatachalam TK, Tuel-Ahlgren L, Sudbeck EA, Uckun FM (1998) Rational synthesis
of phenethyl–5-bromopyridyl thiourea derivatives as potent non-nucleoside inhibitors of HIV
reverse transcriptase. Bioorg Med Chem 6:1789–1797. doi:10.1016/S0968-0896(98)00108-4