Thiocarbamates as non-nucleoside HIV-1 reverse transcriptase inhibitors. Part 1: Parallel synthesis, molecular modelling and structure-activity relationship studies on O-[2-(hetero)arylethyl]-N-phenylthiocarbamates

Article abstract of DOI:10.1016/j.bmc.2007.12.050

In order to expand the structure-activity relationship (SAR) studies on Thiocarbamates (TCs), a recently discovered class of potent non-nucleoside HIV-1 reverse transcriptase inhibitors, 38 analogues of the lead O-[2-(2-pyridyl)ethyl]-N-phenylthiocarbamate 1 were prepared by parallel solution-phase synthesis. The SAR strategy was focused on the variation (mono- and disubstitution) of the N-phenyl ring and the replacement of the 2-pyridyl with 4-pyridyl, 2-thienyl and phenyl rings. The majority of the new TCs proved to prevent the wild-type HIV-1 multiplication in MT-4 cell culture and the most potent congeners displayed an EC₅₀ value of 100 nM. Two TCs were active also at micromolar concentrations against the Y181C- and/or K103N/Y181C-resistant mutants. Docking simulations helped to rationalize the SARs.

Full text of DOI:10.1016/j.bmc.2007.12.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4160–4172
Thiocarbamates as non-nucleoside HIV-1 reverse transcriptase
inhibitors. Part 1: Parallel synthesis, molecular modelling
and structure–activity relationship studies on
O-[2-(hetero)arylethyl]-N-phenylthiocarbamates
Sara Cesarini,a,* Andrea Spallarossa,a Angelo Ranise,a Paola Fossa,a Paolo La Colla,b,*
Giuseppina Sanna,b Gabriella Collub and Roberta Loddob
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,
b
`
Km 4,500, I-09042 Monserrato (Cagliari), Italy
Received 1 August 2007; accepted 20 December 2007
Available online 25 December 2007
Abstract—In order to expand the structure–activity relationship (SAR) studies on Thiocarbamates (TCs), a recently discovered class
of potent non-nucleoside HIV-1 reverse transcriptase inhibitors, 38 analogues of the lead O-[2-(2-pyridyl)ethyl]-N-phenylthiocarba-
mate 1 were prepared by parallel solution-phase synthesis. The SAR strategy was focused on the variation (mono- and disubstitu-
tion) of the N-phenyl ring and the replacement of the 2-pyridyl with 4-pyridyl, 2-thienyl and phenyl rings. The majority of the new
TCs proved to prevent the wild-type HIV-1 multiplication in MT-4 cell culture and the most potent congeners displayed an EC50
value of 100 nM. Two TCs were active also at micromolar concentrations against the Y181C- and/or K103N/Y181C-resistant
mutants. Docking simulations helped to rationalize the SARs.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
either single or double NNRTI resistance mutations.
Despite their chemical diversity,23 most of the first gen-
eration NNRTIs bind in a superimposable ‘butterfly-
like’ mode, as assessed by X-ray crystallography.24–31
On the basis of this binding mode, Wing I, Wing II
and body-linker have been identified as modular seg-
ments susceptible of modification to afford new classes
of NNRTI.24 Wing I and Wing II generally contain aro-
matic rings that have p–p interactions with aromatic
amino acids within the NNBS (Tyr181, Tyr188,
Trp229 and Tyr318). The fact that cross-resistance ex-
tends to the whole NNRTI class calls for development
of new agents capable of inhibiting clinically relevant
NNRTI-resistant mutants.32,33
Non-nucleoside inhibitors (NNRTIs) targeting the HIV-
1-encoded reverse transcriptase (RT) have proved to be
effective in treating the HIV infection and AIDS.1,2
NNRTIs3–15 bind to the non-nucleoside binding site
(NNBS), an allosteric hydrophobic pocket located
˚
about 10 A far from the polymerase active site. First
generation NNRTIs (e.g. nevirapine, delavirdine, tivira-
pine16 and loviride17) are effective against wild-type
HIV-1 but show significantly lower potency against
common NNRTI-resistant mutants. In contrast, second
generation NNRTIs, such as efavirenz,18 some thiocar-
boxanilide,19 quinoxaline,20 imidoylthiourea (ITU),
diaryltriazine (DATA) and diarylpyrimidine (DAPY)
derivatives21,22 retain activity against variants carrying
We have previously described a novel class of potent
NNRTIs, i.e. O-(2-phthalimidoethyl)–N-arylthiocarba-
mates (C-TCs) and their corresponding ring-opened
analogues (O-TCs) (Fig. 1a shows the leads C-TC 12
and O-TC 51).34 In particular, the N-para-substituted-
phenyl C-TCs proved to be potent inhibitors of the mul-
tiplication of wild type HIV-1, significantly active
against the Y181C mutant, but ineffective against the
Keywords: Thiocarbamates; HIV-1; Non-nucleoside reverse transcrip-
tase inhibitors; Parallel synthesis.
*
Corresponding authors. Tel.: +39 010 353 8370; fax: +39 010 353
8358 (S.C.); tel.: +39 070 675 4148; fax: +39 070 675 4210 (P.L.C.);
e-mail addresses: sara.cesarini@unige.it; placolla@unica.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.050

S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
4161
a
O
COOH
S
S
H
N
N
O
N
H
O
N
H
Wing II
O
O
Wing I
C-TC 12
O-TC 51
Cl
S
S
N
O
N
O
N
COOiPr
H
H
Wing I
Wing II
Wing II
UC-38
TC 1
b
Y
N
X
S
S
N
S
N
N
NH
N
H
NH
G
NH
Br
H
H
N
N
S
Wing I
PETT
Trovirdine (X = Y = H, G = Br)
PETT-1 (X = F, Y = OC2H5, G = CN)
PEPT
OMe
OMe
S
S
N
S
S
NH
Br
N
H
NH
Br
N
N
H
NH
Br
H
N
N
PHI-236
PHI-346
PHI-443
Figure 1. Chemical structure of C-TC 12,34 O-TC 51,34 lead compound 1, UC-38, PETT, Trovirdine, PETT-1, PEPT, PHI-236, PHI-346 and PHI-
443. (a) TCs and (b) PETT derivatives.
K103R- and K103N/Y181C-resistant mutants. Thiocar-
bamates (TCs) are isosterically related to N-phenethyl-
N0-thiazolylthiourea (PETT) NNRTIs (Fig. 1b).30,35–49
In PETT derivatives, Wing I is represented by an intra-
molecular H-bonded heterocyclic ring, generally a 2-
aminothiazolyl group or a 5-bromopyridyl group
(Fig. 1b). Wing II often consists of a 2-pyridyl, 2-thienyl,
1-cycloesenyl or phenyl ring, separated from the thio-
urea function by an ethyl linker. Docking studies on
C-TCs suggested that also these inhibitors assume a
‘butterfly-like’ bioactive conformation.34 In C-TCs,
Wing I is a phenyl and Wing II is a phthalimide scaffold
separated from the thiocarbamate function by an ethyl
linker (Fig. 1a). In consequence of the isosteric NH/O
replacement, TCs have no internal H-bond, which is in-
stead essential for PETT anti-HIV activity.35 The lack of
this conformational constrain makes TCs more flexible
than thiourea NNRTIs. In this regard, it was speculated
that a greater conformational freedom of inhibitors
might be a useful design feature for reducing drug resis-
tance.21,22,50,51 In the field of NNRTIs, structure-based
design of analogues of Trovirdine and PETT is an
attractive research area for medicinal chemists, as docu-
mented by papers recently published.34,42,44–49,52 In par-
ticular, some of PETT derivatives, such as PHI-236,
PHI-346 and PHI-443 (Fig. 1b),41 are under develop-
ment as vaginal and rectal microbicides for curbing
mucosal HIV transmission via semen.46,47 In addition,
N-phenethyl-N0-5-bromopyridylthiourea (PEPT) (Fig. 1b)
analogues possess dual function as anti-HIV agents with
antioxidant properties.43 Notably, the thiocarbamate
UC-38 (Fig. 1a) was selected as an anti HIV-1 agent
in the early 1990s for preclinical development.53
In order to further explore the potential of TCs as anti-
HIV-1 agents, expand the structure–activity relationship
(SAR) studies on this class and investigate the effects on
antiretroviral activity due to the replacement of the

4162
S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
Table 2. Cytotoxicity and anti HIV-1 activity of O-[2-(2-pyri-
dyl)ethyl]N-disubstituted-phenylthiocarbamates 21–28a
phthalimide moiety in C-TCs, we wanted to prepare a
new series of TCs. Therefore, we initially designed, syn-
thesized and tested O-[2-(2-pyridyl)ethyl]-N-phen-
H
O
N
ylthiocarbamate
1
(Fig. 1a), which shares the
heteroaryl-ethyl portion with Trovirdine and the N-
phenylthiocarbamic moiety with C-TC 12. TC 1 proved
to be active with an EC50 value of 7.4 lM and was se-
lected as a lead compound. Since the lead structure
can incorporate a large number of diversity points and
a variety of substitution patterns, a simple, parallel, con-
vergent solution-phase method was set up for rapid
analogueing. We focused our SAR strategy on
structural modifications of 1 by keeping constant the
2-pyridylethyl substructure and varying the N-phenyl
portion (1–28, Tables 1 and 2). These modifications
concern the monosubstitution with various groups at
position ortho, meta and para (1–20) and the disubstitu-
tion with equal (21–25) or different (26–28) groups.
Then, we synthesized a number of analogues with the
2-pyridyl replaced by a phenyl (29–33), 2-thienyl (34),
or 4-pyridyl ring (35–38) (Table 3), and the N-phenyl
unsubstituted or monosubstituted at para position with
functional groups with various electronic, steric and
lipophilic properties (CH3, Cl, NO2 and OCH3).
X,Y
S
N
SId
b
c
Compound
X,Y
CC50
EC50
21
22
2,5-F,F
2,6-F,F
3,5-F,F
2,4-Cl,Cl
3,4-Cl,Cl
>100
>100
>100
>100
40
9.0
38
>11
>2.6
>11
>34
200
84
23
24
9.0
2.9
0.2
0.7
5.5
0.1
0.02
25
26
3-Cl,4-CH3
4-Cl,3-CF3
4-Cl,3-NO2
59
42
27
28
7.6
41
60
410
3000
Trovirdine
a,b,c,d See legend to Table 1.
Table 3. Cytotoxicity and anti HIV-1 activity of O-[2-(4-pyridyl)- or -
(2-thienyl)- or (phenyl)-ethyl]N-phenylthiocarbamates 29–38a
H
O
N
Ar
S
X
SId
b
c
Compound
Ar
X
CC50
EC50
Table 1. Cytotoxicity and anti HIV-1 activity of O-[2-(2-pyri-
dyl)ethyl]N-monosubstituted-phenylthiocarbamates 1–20a
29
30
phenyl
phenyl
H
100
>100
>100
>100
36
6.0
4.3
0.5
0.9
17
17
CH3
Cl
>23
>200
>111
2.1
31
32
phenyl
phenyl
phenyl
H
NO2
OCH3
H
O
N
33
34
X
2-thienyl
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
>100
>100
70
14
63
>7.1
>1.6
<1.0
—
S
N
35
36
CH3
Cl
SId
b
c
>70
Compound
X
CC50
EC50
37
38
NO2
OCH3
>100
59
60
>100
>59
0.02
1
H
>100
>100
>100
>100
7.4
69
>14
>1.4
>2.3
>13
P91
34
<1.0
3000
2
2-CH3
2-Cl
Trovirdine
3
43
a,b,c,d See legend to Table 1.
4
3-CH3
3-F
3-Cl
7.7
1.1
1.4
1.6
1.7
10
5
P100
47
6
7
3-NO2
4-CH3
4-C2H5
100
75
63
44
2. Chemistry
8
9
10
44
45
4.4
In the modern drug discovery process,54 and particularly
in lead identification and optimisation, parallel synthesis
plays an important role as it allows to produce a (large)
number of compounds in short times, by using simple
and rapid purification methods. Recently, solution-
phase chemistry has largely supplanted solid-phase
chemistry as the method of choice for parallel synthesis
of small organic molecules.55
4-CH(CH3)2
4-CF3
4-CN
>45
13
<1.0
3.8
11
12
50
45
0.2
225
—
13
14
4-COCH3
4-F
4-Cl
P100
>100
52
>100
1.2
0.1
>83
520
185
68
15
16
4-Br
4-I
37
34
0.2
0.5
17
18
4-N(CH3)2
4-NO2
4-OCH3
60
>60
0.7
<1.0
>143
8.6
19
20
>100
48
60
TC 1–38 were prepared by a convergent solution-phase
parallel synthesis (Scheme 1), by using ordered arrays
of spatially separated reaction vessels (Carousel reaction
stationTM). This two-step procedure combines two build-
ing blocks (alcohols and isothiocyanates, functionalised
according to the planned SARs). Starting alcohols A1–4
(Fig. 2a) were first transformed into their corresponding
salts in the presence of sodium hydride in dry THF
(emerged during the synthetic method set-up as a better
solvent than DMF and pyridine). Then, the alcoholates
5.6
0.02
Trovirdine
3000
a Data mean values for three separate experiments. Variation among
triplicate samples was less than 10%.
b Compound concentration (lM) required to reduce the viability of
mock-infected cells by 50%, as determined by the MTT method.
c Compound concentration (lM) required to achieve 50% protection of
MT-4 cell from HIV-1 induced cytopathogenicity, as determined by
the MTT method.
d Selectivity index: CC50/EC50 ratio.

S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
4163
H
N
O
N
O
(a) (b)
(c)
(Het)Ar
Ar2
(Het)Ar
Ar2
A1-4
-
Na+
S
S
B1-38
1-38
Scheme 1. Reagents and conditions: (a) NaH, dry THF, rt, 30 min; (b) Ar2AN@C@S (I1ꢀ28), (for 2 and 4, pre-heating at 60 ꢁC for 2 h), rt, 18 h; (c)
NH4Cl(aq). For the structure list of alcohols A1–4 and isothiocyanates I1–28, see Figure 2.
a
N
S
N
OH
OH
OH
OH
A1
A2
A3
A4
Ar2
Ar2
b
I1 phenyl
I2 2-tolyl
I15 4-chlorophenyl
I16 4-bromophenyl
I17 4-iodophenyl
I18 4-dimethylaminophenyl
I19 4-nitrophenyl
I3 2-chlorophenyl
I4 3-tolyl
I5 3-fluorophenyl
I6 3-chlorophenyl
I7 3-nitrophenyl
I8 4-tolyl
I20 4-methoxyphenyl
I21 2,5-difluorophenyl
I22 2,6-difluorophenyl
I23 3,5-difluorophenyl
I24 2,4-dichlorophenyl
I25 3,4-dichlorophenyl
I26 3-chloro-4-methylphenyl
I27 4-chloro-3-trifluoromethylphenyl
I28 4-chloro-3-nitrophenyl
I9 4-ethylphenyl
I10 4-i-propylphenyl
I11 4-trifluoromethylphenyl
I12 4-cyanophenyl
I13 4-acetylphenyl
I14 4-fluorophenyl
Figure 2. Building blocks used. (a) Alcohols A1ꢀ4 and (b) isothiocyanates Ar2NCS (I1ꢀ28).
(B1–38) condensed in situ with the suitable isothiocyanate
(I1–28, Fig. 2b) to give the corresponding thiocarbamate
sodium salts, which were converted into the desired
products by treatment with a solution of NH4Cl in water.
The work-up simply required filtrations or extractions
and the final products were purified by crystallization
(only 2 and 4 were purified by chromatography). The
yields (not optimized) ranged from 15% to 96%.
Table 4. Anti HIV-1 activity of 7 and 16 against Y181C and K103N/
Y181C resistant mutants
Compound
EC50 (lM)a
K103N/Y181C
Y181C
7
16
87
20
91
n.a.b
0.04
Efavirenz
0.01
a See Footnote ‘c’ of legend to Table 1.
b Not active.
3. Biological results and discussion
rigid conformation’, because of the lack of the intramo-
lecular H bond (Fig. 1b). This evidence points out a sub-
stantial difference between the TC and PETT series.
The antiretroviral activity of TC 1–38 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
compound (Tables 1–3). The most potent derivatives
were also tested against the clinically relevant K103R-,
Y181C- and K103N/Y181C-resistant mutants,56,57
employing Efavirenz as reference molecule (Table 4).
Congeners 1–20 (Table 1) bear N-phenyl ring ortho,
meta and para substituents with various electronic
(inductive and/or mesomeric), lipophilic and steric prop-
erties. The ortho derivatives 2 and 3 showed low activity
(EC50 = 69 and 43 lM, respectively). The meta ana-
logues (4–7) proved to be active in the low micromolar
concentration range (EC50 = 1.1–7.7 lM). Most of the
para-substituted TCs (8–20) showed EC50 values in the
0.1–13 lM concentration range, except for 4-isopropyl
10, 4-acetyl 13 and 4-dimethylamino 18 (the last com-
pound was synthesized to enhance water solubility of
TCs), which were inactive. The most potent derivatives
(EC50 = 0.1–0.7 lM) were 12 (4-cyano), 15 (4-chloro),
16 (4-bromo), 17 (4-iodo) and 19 (4-nitro), which were
Most of the new TCs resulted to prevent the wild-type
HIV-1 multiplication and 10 out of 38 turned out to
be submicromolar inhibitors. Notably, PETT deriva-
tives possessing a phenyl ring as Wing I were almost
inactive,35 as they could not assume the ‘high-activity

4164
S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
respectively 37-, 74-, 37-, 15- and 11-fold more active
than the lead compound 1. As previously observed for
C-TCs,34 the activity was strongly affected by the substi-
tution pattern of the N-phenyl ring with the following
potency trend: para > meta > ortho, as clearly exempli-
fied by positional isomers 2, 4, 8 (2-, 3-, 4-tolyl); 3, 6,
15 (2-, 3-, 4-chloro) and 7, 19 (3-, 4-nitro). However, dif-
ferently from C-TCs,34 also the electronic properties of
the substituents seemed to affect activity. In general,
the analogues bearing an electron-withdrawing group
were more potent than the congeners with an electron-
donating group in the same position, as it appears com-
paring 3 (2-chloro) with 2 (2-methyl); 5 (3-fluoro), 6 (3-
chloro) and 7 (3-nitro) with 4 (3-methyl); 12 (4-cyano),
14 (4-fluoro), 15 (4-chloro), 16 (4-bromo), 17 (4-iodo)
and 19 (4-nitro) with 8 (4-methyl), 9 (4-ethyl), 10 (4-iso-
propyl), 18 (4-dimethylamino) and 20 (4-methoxy).
Regarding the size of the para substituents, data in Ta-
ble 1 indicate that activity decreases with the steric de-
mand of the substituents. Thus, the potency is in the
order chloro 15 > bromo 16 > iodo 17 and methyl
8 > ethyl 9 > isopropyl 10, the last compound being
inactive like the other analogues bearing a sterically
demanding group 13 (acetyl) and 18 (dimethylamino).
Notably, the potency order of the para-halo-substituted
derivatives of 1 is inverse when compared to the corre-
sponding analogues of C-TC 12.34
the replacement of the 2-pyridyl ring with a 4-pyridyl
(35–38) caused a decrease or loss in activity (compare
35 with 8; 36 with 15; 37 with 19; 38 with 20), while
with a 2-thienyl led to the potency halving (34 vs 1).
This contrasted with the SARs of PETT derivatives,
according to which the replacement of the 2-pyridyl
with a 2-thienyl ring diminished the activity only sligh-
tly,41c and the replacement with a phenyl caused a
more significant potency decrease.35,36 Conversely, the
drop in activity due to the pyridine nitrogen shift to
the 4-position was in accordance with the PETT
SARs.35
The activity of TC 19 was confirmed also by means of
the determination of the dose required to reduce HIV-
1 p24 antigen levels by 90% in virus infected C8166 cul-
tures (EC90 = 0.8lM) in comparison with Trovirdine
(EC90 = 0.015 lM). In cell-based assays, the Y181C,
K103R and K103N + Y181C mutated strains proved
to be unsusceptible to the TCs, with the exception of 7
and 16 (Table 4), which turned out to be weakly active
against Y181C- and/or K103N + Y181C-resistant
mutants.
All compounds, except the 2-pyridyl derivatives 10 and
18 and the 4-pyridyl derivatives 36 and 38, showed val-
ues of CC50 higher than EC50 (CC50 in many cases supe-
rior to 100 lM).
Given the positive results obtained with the above-
mentioned para-halo-substituted TCs, we wanted to
evaluate the effects of the disubstitution with halogen
atoms (21–25) and the combination of halogens with
electron-donating (26) or electron-withdrawing (27
and 28) groups (Table 2). All the analogues synthesized
were active (EC50 = 0.1–38 lM). The most potent com-
pounds shared the 3,4-disubstitution pattern, and out
of them, TC 28 (4-chloro and 3-nitro) proved to be
as active as 15 (4-chloro) (EC50 = 0.1 lM), and more
potent than 25 (3,4-dichloro) and 26 (3-chloro, 4-
methyl) which displayed sub-micromolar activities
(EC50 = 0.2 and 0.7 lM, respectively). The presence
of a trifluoromethyl group at position 3 (27) or a chlo-
rine atom at position 2 (24) of the 4-chloro-phenyl
moiety caused a 55- or a 29-fold decrease in potency,
respectively. The difluoro-TCs (21–23) showed activi-
ties in the micromolar concentration range (EC50 = 9–
38 lM), and in particular 21 (2,5-difluoro) and 23
(3,5-difluoro) were only slightly less active than the
lead compound 1.
4. Molecular modeling
To rationalize the most relevant SARs, computational
studies were performed to construct a docking model
of pyridylethyl- and phenylethyl-TCs bound into the
HIV-1 RT NNBS. Briefly, TC 1, 12, 15, 19, 25, 28, 29,
31, 32 and 36 were docked (Autodock 3.05) into the
NNBS using the X-ray coordinates of RT–PETT-1 com-
plex (PDB code 1DTQ)30 as template structure. The
resulting RT–TC complexes were energy-minimized by
a combined protocol of simulated annealing and Powell
minimization. As shown in Figure 3a, the RT-1 complex
is stabilized by a hydrogen bond between the thiocarba-
mic NH group and the Lys101 main chain carbonyl (the
same hydrogen bond was present in the complexes be-
tween RT and C-TCs34 and between RT and PETT
derivatives38) and van der Waals contacts between the
thiocarbonyl sulfur atom and the Lys101 backbone
NH group. Moreover, the 2-pyridyl ring (positioned at
the ‘top’ of the NNRTI pocket) establishes p–p interac-
tions with Tyr181 and its nitrogen atom is engaged in a
polar interaction with the carboxyl group of Glu138(p51
subunit). The N-phenyl is involved in hydrophobic con-
tacts with the side chain of Leu100, Lys103, Val106,
Pro236 and Tyr318. Figure 4 shows that the bioactive
conformation of 1 resembles the ‘butterfly-like’ confor-
mation of C-TCs and PETT derivatives, with the 2-py-
ridyl and phenyl rings representing Wing 2 and Wing
1, respectively (Fig. 1a).
Successively, to evaluate the effects of the replacement
of the 2-pyridyl of the lead 1 with other (hetero)aryl
groups, TC 29–38 (Table 3) were prepared. The N-phe-
nyl was unsubstituted or monosubstituted at the para
position (according to the previous results) with groups
with various electronic, steric and lipophilic properties.
The 2-phenylethyl derivatives (29–33) were similarly or
slightly less active than the corresponding 2-(2-pyri-
dyl)ethyl TCs (compare 29 with 1, 30 with 8, 31 with
15, 32 with 19 and 33 with 20), and particularly the
congeners bearing an electron-withdrawing group (31:
4-chloro; 32: 4-nitro) showed sub-micromolar activity
(EC50 = 0.5 and 0.9 lM, respectively). On the contrary,
Docking simulations suggest that the increase in activ-
ity of the N-para-substituted-phenyl derivatives 15 and
19 compared to 1 is due to the further stabilizing effect

S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
4165
Figure 3. Stereoview showing the position and orientation of TC 1 (a) and 29 (b). The ligands are represented as black balls-and-sticks, while the
residues lining RT non-nucleoside binding site are shown as gray sticks. Hydrogen bonds are depicted as dotted lines. The drawing was realized by
the programmes MolScript62 and Raster 3D.63
Figure 4. Stereodiagram showing superposition of TC 1, C-TC 1234 and PETT-1 (Fig. 1) within the NNBS. The relative positions and conformations
for TC 1 (model structure, white ball-and-stick), C-TC 12 (model structure, black ball-and-stick) and PETT-1 (X-ray structure, gray ball-and-stick)
are shown in their respective complexes with RT. The structures were superimposed on the basis of the amino acid residues surrounding the RT
pocket, as described by Ren et al.30 Despite the lack of the internal H-bond typical of PETT derivatives, TC 1 and C-TC 12 assume the ‘butterfly-like’
bioactive conformation, strongly mimicking the RT-bound PETT-1 conformation and orientation. The programmes MolScript62 and Raster 3D63
were used to draw the Figure.
of the chlorine atom (15) or the nitro group (19), which
make extensive interactions with Leu234, His235 and
Pro236. In 19 the nitro group establishes also contacts
with Phe227 and a polar interaction with the Pro236

4166
S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
nitrogen. The cyano group of 12 compensates the lack
of interactions with Leu234, making a number of con-
tacts with Val106 and Phe227. The higher potency of
28 in comparison with 1 would be due to the formation
of a hydrogen bond between the meta-nitro group and
the Tyr318 hydroxyl, and to hydrophobic contacts
involving the para-chlorine atom, Val106 and Phe227.
In the 3,4-dichloro-derivative 25, the chlorine atoms
make extensive interactions with Val106, Lys103 and
Pro236. The shift of the polar nitrogen atom of the
pyridyl ring from position 2 to 4 (36) causes loss in
activity because the nitrogen atom can establish neither
the polar interaction with Glu138(p51) nor hydropho-
bic contacts with the ring plane of the Trp229 side-
chain.
6. Experimental
6.1. Chemistry
6.1.1. General. All chemicals were purchased by Chimi-
nord and Aldrich Chemical, Milan (Italy). Solvents were
reagent grade. THF was distilled in the presence of so-
dium. Unless otherwise stated, all commercial reagents
were used without further purification. Organic solu-
tions 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 or petroleum
ether/ethyl acetate were used as developing solvents
and detection of spots was made by UV light and/or
by iodine vapours. Merck silica gel, 230–400 mesh,
was used for chromatography.
In the modelled RT-29 complex (Fig. 3b), the phenyl
replacing the 2-pyridyl establishes hydrophobic con-
tacts with Tyr181 and Tyr188. The hydrogen bond be-
tween the thiocarbamic NH group and the Lys101
main chain carbonyl is conserved. The ethyl linker is
involved in hydrophobic contacts with the Val179
and Gly190 main chains, while the N-phenyl estab-
lishes van der Waals interactions with the Lys101 car-
bonyl oxygen and hydrophobic contacts with the side
chains of Leu100, Lys103, Val106 and Pro236. TC 29
is able to keep the same level of antiviral activity of
1 in spite of the lack of the polar interaction with
Glu138(p51) and of the p–p interactions, presumably
because these are compensated by the number of
hydrophobic contacts in which 29 is engaged. The in-
crease in activity of 31 and 32 compared to 29 would
be due to the further stabilizing effect of a number of
van der Waals contacts between the thiocarbonyl sulfur
atom and the NH group of the Lys101 backbone. In
addition, in the RT-31 complex the phenyl of the
phenylethyl moiety establishes p–p stacking interactions
with Tyr181 and the chlorine atom makes van der Waals
contacts with Phe227 and Leu234. In the RT-32 complex,
the p–p stacking are missing, but the nitro group is in-
volved in van der Waals interactions with Val106,
Phe227, Leu234, His235, Pro236 and Tyr318.
The parallel solution-phase chemistry was performed by
using a Carousel Reaction StationTM (Radleys Discovery
Technologies, Italian distributor: StepBio, Bologna).
The evaporation of solutions in parallel fashion was per-
formed with an EvaposelTM apparatus (Radleys Discov-
ery 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.
IR spectra were recorded on a Perkin-Elmer 398 spec-
trometer as KBr discs. H NMR spectra were recorded
1
in CDCl3 or DMSO-d6 on a Varian Gemini 200 instru-
ment. Chemical shifts were reported in d (ppm) units rel-
ative to the internal standard tetramethylsilane, and the
splitting patterns were described as follows: s (singlet), d
(doublet), dd (doublet of doublets), t (triplet), m (multi-
plet) and br s (broad singlet). The first order values re-
ported for coupling constants J were given in Hertz.
Elemental analyses were performed by an EA1110 Ele-
mental Analyser (Fison-Instruments, Milan) and were
within 0.4% of the theoretical values. The synthesis
of Trovirdine was accomplished according to the pub-
lished procedure.35
5. Conclusions
Our SAR expansion strategy has highlighted that the
replacement of the phthalimide with a 2-pyridyl or a
phenyl ring led to TCs with anti-HIV-1 activity in the
micro- and sub-micromolar concentration range. The
most potent TCs 15 and 28 displayed an EC50 value of
100 nM. The N-phenyl para-substitution with electron-
withdrawing groups was found to be beneficial. TC 7
and 16 also resulted weakly effective against Y181C
and/or K103N + Y181C mutant strains. However, the
easy synthetic accessibility of TCs prompted us to con-
tinue in the design and synthesis of new molecules with
the aim at identifying additional activity parameters in
the TC series and improving potency and resistance pro-
file. Further SAR studies on O-(2-phenylethyl)-N-aryl-
thiocarbamates, keeping constant the N-phenyl
substitution patterns emerged as the best in the present
work, will be the object of the following paper.
6.1.2. Parallel synthesis of O-[2-(2-pyridyl)ethyl]N-phen-
ylthiocarbamates 1–28; O-(2-phenylethyl)N-phenylthiocarba-
mates 29–33; O-[2-(2-thienyl)ethyl]N-phenylthiocarbamate
34 and O-[2-(4-pyridyl)ethyl]N-phenylthiocarbamates 35–
38. Sodium hydride (60%) dispersion in mineral oil
(0.40 g, 10 mmol) was added in a single portion at rt
to each numbered reaction tube of a 12-Carousel Reac-
tion StationTM, containing a stirred solution of the start-
ing alcohol A1ꢀ4 (10 mmol) in dry THF (30 mL). After
stirring for 30 min, the proper isothiocyanate I1–28
(10 mmol) was added to each reaction mixture, which
was then stirred for 18 h at rt (for 2 and 4, pre-heating
at 60 ꢁC for 2 h). Two different types of work-up were
carried out. Work-up (i) (1–6, 8–21, 25, 28, 29 and
34): after parallel evaporation of THF in vacuo using
an EvaposelTM apparatus, a solution of NH4Cl (1 g in

S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
4167
40 mL of water) was added into each tube. The contents
of the tubes were then transferred into a set of separat-
ing funnels. Further solution of NH4Cl (3 g in 120 mL
of water) was added into each funnel. After parallel
extraction with diethyl ether (CH2Cl2 for 13), the com-
bined extracts of each reaction were washed with water,
dried over anhydrous Na2SO4 and filtered in parallel
through pads of Florisil (diameter 5 · 2 cm) by an in-
house device. Evaporation in parallel under reduced
pressure using an EvaposelTM apparatus gave residues
which were purified by crystallization from the suitable
solvents or solvent mixtures. For 2 and 4, the residue
was purified by chromatography (eluents: petroleum
ether/ethyl acetate 1:1 for 2 and CH2Cl2/methanol 98:2
for 4). Work-up (ii) (7, 22–24, 26, 27, 30–33 and 35–
38): after parallel evaporation of THF in vacuo using
an EvaposelTM apparatus, a solution of NH4Cl (1 g in
40 mL of water) was added into each tube. The contents
of the tubes were then transferred into a set of beakers.
Further solution of NH4Cl (3 g in 120 mL of water) was
added into each beaker. The precipitates obtained were
filtered off in parallel by an in-house device and dis-
solved in CH2Cl2 (diethyl ether for 22, 24, 26 and 27).
The solutions were washed with water, dried over anhy-
drous Na2SO4 and filtered in parallel through pads of
Florisil (diameter 5 · 2 cm) by an in-house device. Evap-
oration in parallel under reduced pressure using an
EvaposelTM apparatus gave residues which were purified
by crystallization from the suitable solvents or solvent
mixtures.
3H, CH3), 3.26 (t, J = 6.6 Hz, 2H, CH2Py), 4.98 (t,
J = 6.6 Hz, 2H, CH2O), 6.50–7.83 (m, 7H, arom. H),
8.45–8.83 (m, 2H, py H + NH, exchangeable). Anal.
Calcd for C15H16N2OS: C, 66.15; H, 5.92; N, 10.29; S,
11.77. Found: C, 66.20; H, 6.04; N, 10.45; S, 11.87.
6.1.2.5. O-[2-(2-pyridyl)ethyl]N-(3-fluorophenyl)thio-
carbamate (5). Mp 87–88 ꢁC; yield: 21% from acetone/
methanol. IR (KBr) cmꢀ1: 3185, 3127. 1H NMR
(CDCl3) d: 3.08–3.27 (m, 2H, CH2Py), 4.74–5.01 (m,
2H, CH2O), 6.58–7.12 (m, 6H, arom. H), 7.44–7.62
(m, 1H, arom. H), 8.39–8.54 (m, 1H, py H), 9.16 (br s,
1H, NH, exchangeable). Anal. Calcd for C14H13FN2OS:
C, 60.85; H, 4.74; N, 10.14; S, 11.60. Found: C, 60.75;
H, 4.72; N, 10.13; S, 11.50.
6.1.2.6. O-[2-(2-pyridyl)ethyl]N-(3-chlorophenyl)thio-
carbamate (6). Mp 65–66 ꢁC; yield: 15% from diethyl
1
ether. IR (KBr) cmꢀ1: 3222, 3171. H NMR (CDCl3)
d: 3.20 (t, J = 6.4 Hz, 2H, CH2Py), 4.83–4.98 (m, 2H,
CH2O), 6.95–7.23 (m, 7H, arom. H), 7.49–7.62 (m,
1H, py H), 8.78 (br s, 1H, NH, exchangeable). Anal.
Calcd for C14H13ClN2OS: C, 57.43; H, 4.48; N, 9.57;
S, 10.95. Found: C, 57.53; H, 4.52; N, 9.29; S, 10.93.
6.1.2.7. O-[2-(2-pyridyl)ethyl]N-(3-nitrophenyl)thio-
carbamate (7). Mp 142–144 ꢁC; yield: 48% from acetone.
IR (KBr) cmꢀ1: 3177, 3123, 1526, 1351. 1H NMR
(CDCl3) d: 3.32 (t, J = 6 Hz, 2H, CH2Py), 5.04 (t,
J = 6 Hz, 2H, CH2O), 7.03–8.40 (m, 7H, arom. H),
8.52–8.72 (m, 1H, py H), 8.98 (br s, 1H, NH, exchange-
able). Anal. Calcd for C14H13N3O3S: C, 55.43; H, 4.32;
N, 13.85; S, 10.57. Found: C, 55.54; H, 4.54; N, 13.73; S,
10.51.
6.1.2.1. O-[2-(2-pyridyl)ethyl]N-phenylthiocarbamate
(1). Mp 168–170 ꢁC; yield: 68% from CH2Cl2/methanol.
IR (KBr) cmꢀ1: 3220. 1H NMR (CDCl3) d: 3.26 (t,
J = 6.6 Hz, 2H, CH2Py), 4.98 (t, J = 6.6 Hz, 2H,
CH2O), 6.86–8.77 (m, 9H, arom. H), 9.15 (br s, 1H,
NH, exchangeable). Anal. Calcd for C14H14N2OS: C,
65.09; H, 5.46; N, 10.84; S, 12.41. Found: C, 64.87; H,
5.46; N, 10.71; S, 12.12.
6.1.2.8. O-[2-(2-pyridyl)ethyl]N-(4-tolyl)thiocarbamate
(8). Mp 110–111 ꢁC; yield: 38% from diethyl ether/
methanol. IR (KBr) cmꢀ1: 3228, 3178. 1H NMR
(CDCl3) d: 2.24 (s, 3H, CH3), 3.18 (t, J = 6.6 Hz, 2H,
CH2Py), 4.79–4.96 (m, 2H, CH2O), 6.77–7.22 (m, 6H,
arom. H), 7.47–7.59 (m, 1H, arom. H), 8.40 (br s, 1H,
NH, exchangeable), 8.47–8.53 (m, 1H, py H). Anal.
Calcd for C15H16N2OS: C, 66.15; H, 5.92; N, 10.29; S,
11.77. Found: C, 66.15; H, 5.95; N, 10.28; S, 11.70.
6.1.2.2. O-[2-(2-pyridyl)ethyl]N-(2-tolyl)thiocarbamate
(2). Mp 59–61 ꢁC; yield: 29% from petroleum ether/ethyl
1
acetate. IR (KBr) cmꢀ1: 3134, 2872; H NMR (CDCl3)
d: 2.19 (s, 3H, CH3), 3.16 (t, J = 6.6 Hz, 2H, CH2Py),
4.87 (t, J = 6.6 Hz, 2H, CH2O), 6.65–7.78 (m, 7H, arom.
H), 8.45–8.83 (m, 2H, py H + NH, exchangeable).
Anal. Calcd for C15H16N2OS: C, 66.15; H, 5.92; N,
10.29; S, 11.77. Found: C, 66.24; H, 5.99; N, 10.38; S,
11.74.
6.1.2.9. O-[2-(2-pyridyl)ethyl]N-(4-ethylphenyl)thiocar-
bamate (9). Mp 71–73 ꢁC; yield: 27% from methanol. IR
1
(KBr) cmꢀ1: 3171, 2964. H NMR (CDCl3) d: 1.14 (t,
J = 7 Hz, 3H, CH3), 2.53 (q, J = 7 Hz, 2H, CH2Ph),
3.18 (t, J = 6.6 Hz, 2H, CH2Py), 4.76–4.97 (m, 2H,
CH2O), 6.81–7.22 (m, 6H, arom. H), 7.47–7.58 (m,
1H, arom. H), 8.46–8.57 (m, 1H, py H), 8.67 (br s,
1H, NH, exchangeable). Anal. Calcd for C16H18N2OS:
C, 67.10; H, 6.34; N, 9.78; S, 11.19. Found: C, 67.14;
H, 6.43; N, 9.87; S, 11.00.
6.1.2.3. O-[2-(2-pyridyl)ethyl]N-(2-chlorophenyl)thio-
carbamate (3). Mp 64–65 ꢁC; yield: 38% from acetone.
IR (KBr) cmꢀ1: 3103. 1H NMR (CDCl3) d: 3.18 (t,
J = 6.6 Hz, 2H, CH2Py), 4.88 (t, J = 6.6 Hz, 2H,
CH2O), 6.93–7.32 (m, 6H, arom. H), 7.47–7.58 (m,
1H, arom. H),8.42 (br s, 1H, NH, exchangeable),
8.48–8.55 (m, 1H, py H). Anal. Calcd for
C14H13ClN2OS: C, 57.43; H, 4.48; N, 9.57; S, 10.95.
Found: C, 57.25; H, 4.53; N, 9.52; S, 10.89.
6.1.2.10. O-[2-(2-pyridyl)ethyl]N-(4-i-propylphenyl)thio-
carbamate (10). Mp 66–67 ꢁC; yield: 48% from diethyl
1
ether/methanol. IR (KBr) cmꢀ1: 3171, 2954. H NMR
(CDCl3) d: 1.22 (d, J = 7 Hz, 6H, 2CH3), 2.70–3.07
(m, 1H, CHPh), 3.24 (t, J = 6.6 Hz, 2H, CH2Py), 4.98
(t, J = 6.6 Hz, 2H, CH2O), 6.99–7.35 (m, 6H, arom.
H), 7.46–7.70 (m, 1H, arom. H), 8.49–8.71 (m, 1H, py
6.1.2.4. O-[2-(2-pyridyl)ethyl]N-(3-tolyl)thiocarbamate
4. Mp 47–49 ꢁC; yield: 63% from CH2Cl2/methanol. IR
1
(KBr) cmꢀ1: 3124, 2947; H NMR (CDCl3) d: 2.27 (s,

4168
S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
H), 9.00 (br s, 1H, NH, exchangeable). Anal. Calcd for
C17H20N2OS: C, 67.97; H, 6.71; N, 9.32; S, 10.67.
Found: C, 67.71; H, 6.67; N, 9.31; S, 10.66.
6.1.2.17. O-[2-(2-pyridyl)ethyl]N-(4-iodophenyl)thio-
carbamate (17). Mp 108–109 ꢁC; yield: 70% from diethyl
1
ether. IR (KBr) cmꢀ1: 3206, 3158. H NMR (CDCl3) d:
3.25 (t, J = 6.6 Hz, 2H, CH2Py), 4.97 (t, J = 6.6 Hz, 2H,
CH2O), 6.77–7.76 (m, 7H, arom. H), 8.45–8.71 (m, 1H,
py H), 8.92 (br s, 1H, NH, exchangeable). Anal. Calcd
for C14H13IN2OS: C, 43.76; H, 3.41; N, 7.29; S, 8.34.
Found: C, 43.75; H, 3.46; N, 7.23; S, 8.33.
6.1.2.11. O-[2-(2-pyridyl)ethyl]N-(4-trifluoromethyl-
phenyl)thiocarbamate (11). Mp 112–114 ꢁC; yield: 19%
from diethyl ether/methanol. IR (KBr) cmꢀ1: 3180,
3116; 1H NMR (CDCl3) d: 3.21 (t, J = 6.4 Hz, 2H,
CH2Py), 4.93 (t, J = 6.4 Hz, 2H, CH2O), 7.03–7.62 (m,
7H, arom. H), 8.47–8.53 (m, 1H, py H), 8.81 (br s,
1H, NH, exchangeable). Anal. Calcd for
C15H13F3N2OS: C, 55.21; H, 4.02; N, 8.58; S, 9.82.
Found: C, 55.02; H, 3.93; N, 8.55; S, 9.72.
6.1.2.18. O-[2-(2-pyridyl)ethyl]N-(4-dimethylaminophe-
nyl)thiocarbamate (18). Mp 108–110 ꢁC; yield: 28% from
CH2Cl2/methanol. IR (KBr) cmꢀ1: 3193, 3155, 2924. 1H
NMR (DMSO-d6) d: 2.76 (s, 3H, CH3), 2.79 (s, 3H,
CH3), 3.02–3.14 (m, 2H, CH2Py), 4.64–4.78 (m, 2H,
CH2O), 6.38–6.50 (m, 1H, arom. H), 6.53–6.66 (m,
1H, arom. H), 6.77–6.88 (m, 1H, arom. H), 7.13–7.33
(m, 3H, arom. H), 7.59–7.73 (m, 1H, py H), 8.44–8.50
(m, 1H, py H), 10.73 (br s, 1H, NH, exchangeable).
Anal. Calcd for C16H19N3OS: C, 63.76; H, 6.35; N,
13.94; S, 10.64. Found: C, 63.57; H, 6.54; N, 13.91; S,
10.60.
6.1.2.12. O-[2-(2-pyridyl)ethyl]N-(4-cyanophenyl)thio-
carbamate (12). Mp 156–157 ꢁC; yield: 36% from diethyl
1
ether/methanol. IR (KBr) cmꢀ1: 3170, 2226. H NMR
(DMSO-d6) d: 3.29 (t, J = 6.6 Hz, 2H, CH2Py), 5.00 (t,
J = 6.6 Hz, 2H, CH2O), 7.05–7.80 (m, 7H, arom. H),
8.50–8.73 (m, 1H, py H), 10.61 (br s, 1H, NH, exchange-
able). Anal. Calcd for C15H13N3OS: C, 63.58; H, 4.62;
N, 14.83; S, 11.31. Found: C, 63.79; H, 4.63; N, 14.81;
S, 11.29.
6.1.2.19. O-[2-(2-pyridyl)ethyl]N-(4-nitrophenyl)thio-
carbamate (19). Mp 172–174 ꢁC; yield: 66% from
CH2Cl2/methanol. IR (KBr) cmꢀ1: 3200. 1H NMR
(DMSO-d6) d: 3.28 (t, J = 6.6 Hz, 2H, CH2Py), 4.96 (t,
J = 6.6 Hz, 2H, CH2O), 7.08–8.40 (m, 8H, arom. H),
8.65 (br s, 1H, NH, exchangeable). Anal. Calcd for
C14H13N3O3S: C, 55.43; H, 4.32; N, 13.85; S, 10.57.
Found: C, 55.70; H, 4.27; N, 13.55; S, 10.21.
6.1.2.13. O-[2-(2-pyridyl)ethyl]N-(4-acetylphenyl)thio-
carbamate (13). Mp 135–137 ꢁC; yield: 17% from diethyl
ether/methanol. IR (KBr) cmꢀ1: 3342, 3286, 3162, 1675.
1H NMR (DMSO-d6) d: 2.56 (s, 3H, CH3), 3.14–3.48
(m, 2H, CH2Py), 4.78–4.99 (m, 2H, CH2O), 7.14–8.81
(m, 8H, arom. H), 11.38 (br s, 1H, NH, exchangeable).
Anal. Calcd for C16H16N2O2S: C, 63.98; H, 5.37; N,
9.33; S, 10.67. Found: C, 64.10; H, 5.59; N, 9.10; S,
10.66.
6.1.2.20. O-[2-(2-pyridyl)ethyl]N-(4-methoxyphenyl)thio-
carbamate (20). Mp 90–91 ꢁC; yield: 58% from diethyl
1
ether/petroleum ether. IR (KBr) cmꢀ1: 3198, 2955. H
6.1.2.14. O-[2-(2-pyridyl)ethyl]N-(4-fluorophenyl)thio-
carbamate (14). Mp 95–96 ꢁC; yield: 55% from diethyl
NMR (CDCl3) d: 3.24 (t, J = 6.6 Hz, 2H, CH2Py),
3.79 (s, 3H, CH3), 4.97 (t, J = 6.6 Hz, 2H, CH2O),
6.63–7.73 (m, 7H, arom. H), 8.50–8.72 (m, 1H, py H),
9.32 (br s, 1H, NH, exchangeable). Anal. Calcd for
C15H16N2O2S: C, 62.48; H, 5.59; N, 9.71; S, 11.12.
Found: C, 62.59; H, 5.60; N, 9.66; S, 11.16.
1
ether/methanol. IR (KBr) cmꢀ1: 3179, 3126. H NMR
(CDCl3) d: 3.24 (t, J = 6.6 Hz, 2H, CH2Py), 4.96 (t,
J = 6.6 Hz, 2H, CH2O), 6.76–7.39 (m, 6H, arom. H),
7.46–7.73 (m, 1H, arom. H), 8.48–8.68 (m, 1H, py H),
9.12 (br s, 1H, NH, exchangeable). Anal. Calcd for
C14H13FN2OS: C, 60.85; H, 4.74; N, 10.14; S, 11.60.
Found: C, 60.95; H, 4.62; N, 10.03; S, 11.62.
6.1.2.21. O-[2-(2-pyridyl)ethyl]N-(2,5-difluorophenyl)thio-
carbamate (21). Mp 69–71 ꢁC; yield: 37% from diethyl
ether/ethanol. IR (KBr) cmꢀ1
:
3107. 1H NMR
6.1.2.15. O-[2-(2-pyridyl)ethyl]N-(4-chlorophenyl)thio-
carbamate (15). Mp 114–115 ꢁC; yield: 33% from diethyl
(DMSO-d6) d: 2.93–3.23 (m, 2H, CH2Py), 4.60–4.94
(m, 2H, CH2O), 6.82–7.81 (m, 6H, arom. H), 8.29–
8.60 (m, 1H, arom. H); 10.86 (s, 1H, NH, exchangeable),
8.48–8.55 (m, 1H, py H). Anal. Calcd for
C14H12F2N2OS: C, 57.13; H, 4.11; N, 9.52; S, 10.89.
Found: C, 57.20; H, 4.31; N, 9.40; S, 10.82.
1
ether. IR (KBr) cmꢀ1: 3200, 3158. H NMR (CDCl3) d:
3.27 (t, J = 6.6 Hz, 2H, CH2Py), 5.00 (t, J = 6.6 Hz, 2H,
CH2O), 7.02–7.41 (m, 6H, arom. H), 7.50–7.78 (m, 1H,
arom. H), 8.51–8.73 (m, 1H, py H), 9.08 (br s, 1H, NH,
exchangeable). Anal. Calcd for C14H13ClN2OS: C,
57.43; H, 4.48; N, 9.57; S, 10.95. Found: C, 57.76; H,
4.67; N, 9.49; S, 10.85.
6.1.2.22. O-[2-(2-pyridyl)ethyl]N-(2,6-difluorophenyl)thio-
carbamate (22). Mp 92–94 ꢁC; yield: 40% from diethyl
1
ether/ethanol. IR (KBr) cmꢀ1: 3100. H NMR (CDCl3)
6.1.2.16. O-[2-(2-pyridyl)ethyl]N-(4-bromophenyl)thio-
carbamate (16). Mp 115–117 ꢁC; yield: 37% from diethyl
ether/methanol. IR (KBr) cmꢀ1: 3154; 1H NMR
(CDCl3) d: 3.25 (t, J = 6.6 Hz, 2H, CH2Py), 4.97 (t,
J = 6.6 Hz, 2H, CH2O), 6.91–7.74 (m, 7H, arom. H),
8.47–8.74 (m, 1H, py H), 9.15 (br s, 1H, NH, exchange-
able). Anal. Calcd for C14H13BrN2OS: C, 49.86; H, 3.89;
N, 8.31; S, 9.51. Found: C, 50.11; H, 4.11; N, 8.23; S,
9.49.
d: 3.18 (t, J = 6.6 Hz, 2H, CH2Py), 4.87 (t, J = 6.6 Hz,
2H, CH2O), 6.67–7.79 (m, 6H, arom. H), 8.38–8.67
(m, 1H, py H), 8.88 (br s, 1H, NH, exchangeable). Calcd
for C14H12F2N2OS: C, 57.13; H, 4.11; N, 9.52; S, 10.89.
Found: C, 57.34; H, 4.18; N, 9.49; S, 10.88.
6.1.2.23. O-[2-(2-pyridyl)ethyl]N-(3,5-difluorophenyl)
thiocarbamate (23). Mp 122–124 ꢁC; yield: 79% from
1
CH2Cl2/diethyl ether. IR (KBr) cmꢀ1: 3197, 3118. H

S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
4169
NMR (CDCl3) d: 3.28 (t, J = 6 Hz, 2H, CH2Py), 5.01 (t,
J = 6 Hz, 2H, CH2O), 6.36–7.38 (m, 5H, arom. H),
7.48–7.78 (m, 1H, arom. H), 8.50–8.71 (m, 1H, py H),
9.36 (br s, 1H, NH, exchangeable). Anal. Calcd for
C14H12F2N2OS: C, 57.13; H, 4.11; N, 9.52; S, 10.89.
Found: C, 57.19; H, 4.19; N, 9.25; S, 11.03.
NH, exchangeable). 13C NMR (CDCl3) d: 66.19, 115.23,
121.85, 129.55, 130.13, 158.92 (C–N), 188.62 (C@S).
Anal. Calcd for C15H15NOS: C, 65.91; H, 5.53; N, 5.12;
S, 11.73. Found: C, 66.12; H, 5.60; N, 5.13; S, 11.59.
6.1.2.30. O-(2-phenylethyl)N-(4-tolyl)thiocarbamate (30).
Mp 97–99 ꢁC; yield: 91% from CH2Cl2/methanol. IR
1
6.1.2.24. O-[2-(2-pyridyl)ethyl]N-(2,4-dichlorophenyl)
thiocarbamate (24). Mp 96–97 ꢁC; yield: 41% from
(KBr) cmꢀ1: 3209, 2922. H NMR (CDCl3) d: 2.27 (s,
3H, CH3), 3.03 (t, J = 6.6 Hz, 2H, CH2Ph), 4.76 (t,
J = 6.6 Hz, 2H, CH2O), 6.74–7.57 (m, 9H, arom. H),
8.85 (br s, 1H, NH, exchangeable). Anal. Calcd for
C16H17NOS: C, 70.82; H, 6.31; N, 5.16; S, 11.81. Found:
C, 70.86; H, 6.66; N, 5.14; S, 11.75.
1
diethyl ether/ethanol. IR (KBr) cmꢀ1: 3138. H NMR
(CDCl3) d: 3.26 (t, J = 6.6 Hz, 2H, CH2Py), 4.98 (t,
J = 6.6 Hz, 2H, CH2O), 6.99–7.90 (m, 6H, arom. H),
8.42–8.76 (m, 2H, py H + NH, exchangeable). Anal.
Calcd for C14H12Cl2N2OS: C, 51.39; H, 3.7; N, 8.56;
S, 9.8. Found: C, 51.39; H, 3.79; N, 8.45; S, 9.83.
6.1.2.31. O-(2-phenylethyl)N-(4-chlorophenyl)thiocar-
bamate (31). Mp 117–118 ꢁC; yield: 82% from CH2Cl2/
1
6.1.2.25. O-[2-(2-pyridyl)ethyl]N-(3,4-dichlorophenyl)
thiocarbamate (25). Mp 106–108 ꢁC; yield: 26% from
CH2Cl2/methanol. IR (KBr) cmꢀ1: 3158. 1H NMR
(CDCl3) d: 3.27 (t, J = 6.6 Hz, 2H, CH2Py), 5.00 (t,
J = 6.6 Hz, 2H, CH2O), 6.97–7.76 (m, 6H, arom. H),
8.52–8.71 (m, 1H, py H), 8.94 (br s, 1H, NH, exchange-
able). Anal. Calcd for C14H12Cl2N2OS: C, 51.39; H,
3.70; N, 8.56; S, 9.80. Found: C, 51.41; H, 3.72; N,
8.28; S, 9.77.
methanol. IR (KBr) cmꢀ1: 3208. H NMR (CDCl3) d:
3.05 (t, J = 6.6 Hz, 2H, CH2Ph), 4.80 (t, J = 6.6 Hz,
2H, CH2O), 7.05–7.50 (m, 9H, arom. H), 9.14 (br s,
1H, NH, exchangeable). Anal. Calcd for C15H14ClNOS:
C, 61.74; H, 4.84; N, 4.80; S, 10.99. Found: C, 61.99; H,
4.85; N, 4.84; S, 11.00.
6.1.2.32. O-(2-phenylethyl)N-(4-nitrophenyl)thiocarba-
mate (32). Mp 142–143 ꢁC; yield: 92% from CH2Cl2/
1
methanol. IR (KBr) cmꢀ1: 3266, 1551, 1335. H NMR
6.1.2.26. O-[2-(2-pyridyl)ethyl]N-(3-chloro-4-methyl-
phenyl)thiocarbamate (26). Mp 84–86 ꢁC; yield: 54%
from diethyl ether/ethanol. IR (KBr) cmꢀ1: 3191,
(CDCl3) d: 3.14 (t, J = 6.6 Hz, 2H, CH2Ph), 4.39 (t,
J = 6.6 Hz, 2H, CH2O), 7.14–7.54 (m, 7H, arom. H),
7.98–8.30 (m, 2H, arom. H), 8.90 (br s, 1H, NH,
exchangeable). Anal. Calcd for C15H14N2O3S: C,
59.59; H, 4.67; N, 9.27; S, 10.60. Found: C, 59.54; H,
4.67; N, 9.23; S, 10.53.
1
3152. H NMR (CDCl3) d: 2.22 (s, 3H, CH3), 3.16 (t,
J = 6.4 Hz, 2H, CH2Py), 4.75–4.97 (m, 2H, CH2O),
6.95–7.23 (m, 5H, arom. H), 7.45–7.58 (m, 1H, py H),
8.42–8.53 (m, 1H, py H), 8.95 (br s, 1H, NH, exchange-
able). Calcd for C15H15ClN2OS: C, 58.72; H, 4.93; N,
9.13; S, 10.45. Found: C, 58.77; H, 5.01; N, 9.09; S,
10.48.
6.1.2.33. O-(2-phenylethyl)N-(4-methoxyphenyl)thio-
carbamate (33). Mp 83–85 ꢁC; yield: 96% from
CH2Cl2/methanol. IR (KBr) cmꢀ1: 3228, 2926. 1H
NMR (CDCl3) d: 3.75 (s, 3H, CH3), 4.17–4.42 (m, 2H,
CH2Ph), 4.75–5.05 (m, 2H, CH2O), 6.72–7.56 (m, 9H,
arom. H), 8.90 (br s, 1H, NH, exchangeable). Anal.
Calcd for C16H17NO2S: C, 66.87; H, 5.96; N, 4.87; S,
11.16. Found: C, 66.76; H, 6.17; N, 4.62; S, 11.13.
6.1.2.27. O-[2-(2-pyridyl)ethyl]N-(4-chloro-3-trifluoro-
methylphenyl)thiocarbamate (27). Mp 115 ꢁC; yield: 37%
1
from diethyl ether/methanol. IR (KBr) cmꢀ1: 3178. H
NMR (CDCl3) d: 3.26 (t, J = 6.6 Hz, 2H, CH2Py),
4.98 (t, J = 6.6 Hz, 2H, CH2O), 7.00–7.88 (m, 6H, arom.
H), 8.44–8.68 (m, 1H, py H), 9.42 (br s, 1H, NH,
exchangeable). Anal. Calcd for C15H12ClF3N2OS: C,
49.94; H, 3.35; N, 7.76; S, 8.89. Found: C, 49.97; H,
3.31; N, 7.68; S, 8.85.
6.1.2.34. O-[2-(2-thienyl)ethyl]N-phenylthiocarbamate
(34). Mp 93–94 ꢁC; yield: 76% from CH2Cl2/methanol.
IR (KBr) cmꢀ1: 3210. 1H NMR (CDCl3) d: 3.28 (t,
J = 6 Hz, 2H, CH2Thioph.), 4.80 (t, J = 6 Hz, 2H,
CH2O), 6.53–7.12 (m, 8H, arom. H), 8.83 (br s, 1H,
NH, exchangeable). Anal. Calcd for C13H13NOS2: C,
59.29; H, 4.98; N, 5.32; S, 24.35. Found: C, 59.51; H,
5.11; N, 5.30; S, 24.04.
6.1.2.28. O-[2-(2-pyridyl)ethyl]N-(4-chloro-3-nitro-
phenyl)thiocarbamate (28). Mp 148–150 ꢁC; yield: 24%
from CH2Cl2/methanol. IR (KBr) cmꢀ1: 3157, 1534,
1339. 1H NMR (CDCl3) d: 3.21 (t, J = 6 Hz, 2H,
CH2Py), 4.84–4.97 (m, 2H, CH2O), 7.09–7.21 (m, 3H,
arom. H), 7.28–7.38 (m, 2H, arom. H), 7.53–7.67 (m,
1H, py H), 8.47–8.53 (m, 1H, pyr H), 8.68 (br s, 1H,
NH, exchangeable). Anal. Calcd for C14H12ClN3O3S:
C, 49.78; H, 3.58; N, 12.44; S, 9.49. Found: C, 49.76;
H, 3.62; N, 12.31; S, 9.53.
6.1.2.35. O-[2-(4-pyridyl)ethyl]N-(4-tolyl)thiocarbamate
(35). Mp 130–132 ꢁC; yield: 91% from CH2Cl2/ether. IR
1
(KBr) cmꢀ1: 3117, 2956. H NMR (CDCl3) d: 2.31 (s,
3H, CH3), 3.05 (t, J = 6.6 Hz, 2H, CH2Py), 4.83 (t,
J = 6.6 Hz, 2H, CH2O), 6.93–7.34 (m, 6H, arom. H),
8.33–8.67 (m, 2H, py H), 9.57 (br s, 1H, NH, exchange-
able). Anal. Calcd for C15H16N2OS: C, 66.15; H, 5.92;
N, 10.29; S, 11.77. Found: C, 66.03; H, 6.11; N, 10.41;
S, 11.78.
6.1.2.29. O-(2-phenylethyl)N-phenylthiocarbamate (29).
Mp 91–93 ꢁC; yield: 60% from CH2Cl2/methanol. IR
(KBr) cmꢀ1: 3170. 1H NMR (CDCl3) d: 4.27 (t,
J = 5.0 Hz, 2H, CH2Ph), 4.91 (t, J = 5.0 Hz, 2H,
CH2O), 6.68–7.55 (m, 10H, arom. H), 8.82 (br s, 1H,
6.1.2.36. O-[2-(4-pyridyl)ethyl]N-(4-chlorophenyl)thio-
carbamate (36). Mp 129–131 ꢁC; yield: 79% from diethyl

4170
S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
1
ether. IR (KBr) cmꢀ1: 3164, 3101. H NMR (CDCl3) d:
3.08 (t, J = 6.6 Hz, 2H, CH2Py), 4.85 (t, J = 6.6 Hz, 2H,
CH2O), 7.05–7.48 (m, 6H, arom. H), 8.46–8.69 (m, 2H,
py H), 8.94 (br s, 1H, NH, exchangeable). Anal. Calcd
for C14H13ClN2OS: C, 57.43; H, 4.48; N, 9.57; S,
10.95. Found: C, 57.28; H, 4.51; N, 9.63; S, 10.92.
All the calculations were carried out on Silicon Graphic
Indigo2 and Origin 200 workstations. Model analyses
were performed using the CCP4 program suite.61 The
programs Molscript62and Raster3D63 were used to draw
the figures.
6.1.2.37. O-[2-(4-pyridyl)ethyl]N-(4-nitrophenyl)thio-
carbamate (37). Mp 141–144 ꢁC; yield: 93% from
CH2Cl2/methanol. IR (KBr) cmꢀ1: 3102, 1501, 1307;
1H NMR (DMSO-d6) d: 2.92–3.08 (m, 2H, CH2Py),
4.57–4.76 (m, 2H, CH2O), 7.17–7.78 (m, 4H, arom.
H), 7.95–8.12 (m, 2H, arom. H), 8.32–8.47 (m, 2H, py
H), 11.57 (br s, 1H, NH, exchangeable). Anal. Calcd
for C14H13N3O3S: C, 55.43; H, 4.32; N, 13.85; S,
10.57. Found: C, 55.44; H, 4.34; N, 13.99; S, 10.60.
Acknowledgments
This work was supported by MURST (Cofinanziamento
Nazionale), CNR (Rome) and Ministero Italiano della
Salute—Istituto Superiore di Sanita (Grant No.
40D.46). 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.
`
6.1.2.38. O-[2-(4-pyridyl)ethyl]N-(4-methoxyphenyl)
thiocarbamate (38). Mp 118–120 ꢁC; yield: 88% from
CH2Cl2/methanol. IR (KBr) cmꢀ1: 3152, 2923. 1H
NMR (CDCl3) d: 3.04 (t, J = 6.6 Hz, 2H, CH2Py),
3.79 (s, 3H, CH3), 4.81 (t, J = 6.6 Hz, 2H, CH2O),
6.69–7.53 (m, 6H, arom H), 8.41–8.66 (m, 2H, py H),
9.56 (br s, 1H, NH, exchangeable). Anal. Calcd for
C15H16N2O2S: C, 62.48; H, 5.59; N, 9.71; S, 11.12.
Found: C, 62.59; H, 5.66; N, 9.68; S, 11.01.
References and notes
1. Jonckheere, H.; Anne, J.; De Clercq, E. Med. Res. Rev.
2000, 20, 129.
2. De Clercq, E. Farmaco 2001, 56, 3.
3. Artico, M. Farmaco 1996, 51, 305.
4. Artico, M. Drugs Future 2002, 27, 159.
5. Balzarini, J. Curr. Top. Med. Chem. 2004, 4, 921.
6. De Clercq, E. Expert Opin. Invest. Drugs 1994, 3, 253.
7. De Clercq, E. J. Med. Chem. 1995, 38, 2491.
8. De Clercq, E. Clin. Microbiol. Rev. 1995, 8, 200.
9. De Clercq, E. Antiviral Res. 1998, 38, 153.
10. De Clercq, E. Collect. Czech. Chem. Commun. 1998, 63, 449.
11. De Clercq, E. Chem. Biodiversity 2004, 1, 44.
12. Pauwels, R. Curr. Opin. Pharmacol. 2004, 4, 437.
13. Pedersen, O. S.; Pedersen, E. B. Antiviral Chem. Chemo-
ther. 1999, 10, 285.
6.2. Virology: Materials and methods
The biological evaluation of the synthesized compounds
was performed according to the previously reported
procedures.34,52
14. Pedersen, O. S.; Pedersen, E. B. Synthesis 2000, 4, 479.
15. Tucker, T. J.; Lumma, W. C.; Culberson, J. C. Methods
Enzymol. 1996, 275, 440.
16. Pauwels, R.; Andries, K.; Debyser, Z.; Kukla, M. J.;
Schols, D.; Breslin, H. J.; Woestenborghs, R.; Des-
myter, J.; Janssen, M. A. C.; De Clercq, E.; Janssen,
P. A. J. Antimicrob. Agents Chemother. 1994, 38,
2863.
17. Pauwels, R.; Andries, K.; Debyser, Z.; van Daele, P.;
Schols, D.; Stoffels, P.; De Vreese, K.; Woestenborghs, R.;
Vandamme, A. M.; Janssen, C. G. M.; Anne, J.;
Cauwenberghi, G.; Desmyter, J.; Heykants, J.; Janssen,
M. A. C.; De Clercq, E.; Janssen, P. A. J. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 1711.
18. Young, S. D.; Britcher, S. F.; Tran, L. O.; Payne, L. S.;
Lumma, W. C.; Lyle, T. A.; Huff, J. R.; Anderson, P. S.;
Olsen, D. B.; Carroll, S. S.; Emini, E. Antimicrob. Agents
Chemother. 1995, 39, 2602.
19. Balzarini, J.; Pelemans, H.; Aquaro, S.; Perno, C. F.;
Witvrouw, M.; Schols, D.; De Clercq, E.; Karlsson, A.
Mol. Pharmacol. 1996, 50, 394.
20. Kleim, J. P.; Bender, R.; Billhardt, U. M.; Meichsner, C.;
Riess, G.; Rosner, M.; Winkler, I.; Paessens, A. Antimic-
rob. Agents Chemother. 1993, 37, 1659.
21. Das, K.; Clark, A. D. J.; Lewi, P. J.; Heeres, J.; De Jonge,
M. R.; Koymans, L. M.; Vinkers, H. M.; Daeyaert, F.;
Ludovici, D. W.; Kukla, M. J.; De Corte, B.; Kavash, R.
W.; Ho, C. Y.; Ye, H.; Lichtenstein, M. A.; Andries, K.;
Pauwels, R.; De Bethune, M. P.; Boyer, P. L.; Clark, P.;
Hughes, S. H.; Janssen, P. A.; Arnold, E. J. Med. Chem.
2004, 47, 2550.
6.3. Molecular modeling
The molecular structures of 1 and 29 were built by
Insight II (Builder module), parameterized according
to CVFF force field58 and their RT complexes were
calculated using Autodock 3.05.59 After the removal
of the PETT inhibitor from the crystal structure
solved by Ren et al.30 (PDB code 1DTQ), hydrogens
were added and partial charges were assigned accord-
ing to CVFF force field (Insight II, biopolymer mod-
ule). The ligand ‘root’ was defined automatically and
all bonds were allowed to freely rotate.
A
˚
50 · 50 · 50-grid (grid spacing 0.375 A) 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 con-
formers 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 A) and the best conformation of the low energy,
highest populated cluster was select as the binding
conformation. The resulting models were energy-min-
imized by CNS,60 performing a cycle of simulated
annealing (starting temperature 1000 K) followed by
120 cycles of Powell minimization.
The minimized structures of RT-1 and RT-29 complexes
were used as starting structures for the calculation of 12,
15, 19, 25, 28, 31, 32 and 36 binding modes.
22. Das, K.; Lewi, P. J.; Hughes, S. H.; Arnold, E. Prog.
Biophys. Mol. Biol. 2005, 88, 209.

S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
4171
23. Tachedjian, G.; Goff, S. P. Curr. Opin. Invest. Drugs 2003,
4, 966.
Chem. Lett. 1998, 8, 1511; (b) Ho¨gberg, M.; Sahlberg, C.;
´
Engelhardt, P.; Noreen, R.; Kangasmetsa¨, J.; Johansson,
N. G.; Oberg, B.; Vrang, L.; Zhang, H.; Sahlberg, B.-L.;
¨
24. (a) Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries,
K.; Janssen, P. A.; Hughes, S. H.; Arnold, E. Nat. Struct.
Biol. 1995, 2, 407; (b) Ding, J.; Das, K.; Tantillo, C.;
Zhang, W.; Clark, A. D. J.; Jessen, S.; Lu, V.; Hsiou, Y.;
Jacobo-Molina, A.; Andries, K.; Pauwels, R.; Moereels,
H.; Koymans, L.; Janssen, P. A. J.; Smith, R. H. J.;
Kroeger Koepke, M.; Michejda, C. J.; Hughes, S. H.;
Arnold, E. Structure 1995, 3, 365.
25. Hopkins, A. L.; Ren, J.; Esnouf, R. M.; Willcox, B. E.;
Jones, E. Y.; Ross, C.; Miyasaka, T.; Walker, R. T.;
Tanaka, H.; Stammers, D. K.; Stuart, D. I. J. Med. Chem.
1996, 39, 1589.
Unge, T.; Lo¨vgren, S.; Fridborg, K.; Ba¨ckbro, K. J. Med.
Chem. 1999, 42, 4150.
41. (a) Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.;
Uckun, F. M. Bioorg. Med. Chem. Lett. 1999, 9, 1593; (b)
Uckun, F. M.; Mao, C.; Pendergrass, S.; Maher, D.; Zhu,
D.; Tuel-Ahlgren, L.; Venkatachalam, T. K. Bioorg. Med.
Chem. Lett. 1999, 9, 2721; (c) Uckun, F. M.; Pendergrass,
S.; Maher, D.; Zhu, D.; Tuel-Ahlgren, L.; Mao, C.;
Venkatachalam, T. K. Bioorg. Med. Chem. Lett. 1999, 9,
3411; (d) Uckun, F. M.; Erbeck, D.; Tibbles, H.; Qazi, S.;
Venkatachalam, T. K. Arzneimittelforschung 2007, 57,
164.
26. Lindberg, J.; Sigurdsson, S.; Lowgren, S.; Andersson, H.
O.; Sahlberg, C.; Noreen, R.; Fridborg, K.; Zhang, H.;
Unge, T. Eur. J. Biochem. 2002, 269, 1670.
42. Ho¨gberg, M.; Engelhardt, P.; Vrang, L.; Zhang, H.
Bioorg. Med. Chem. Lett. 2000, 10, 265.
27. Ren, J.; Esnouf, R.; Garman, E.; Somers, D.; Ross, C.;
Kirby, I.; Keeling, J.; Darby, G.; Jones, Y.; Stuart, D.;
Stammers, D. Nat. Struct. Biol. 1995, 2, 293.
43. Dong, Y.; Venkatachalam, T. K.; Narla, R. K.; Trieu, V.
N.; Sudbeck, E. A.; Uckun, F. M. Bioorg. Med. Chem.
Lett. 2000, 10, 87, and references therein.
28. Ren, J.; Esnouf, R.; Hopkins, A.; Ross, C.; Jones, Y.;
Stammers, D.; Stuart, D. Structure 1995, 3, 915.
29. Ren, J.; Esnouf, R. M.; Hopkins, A. L.; Stuart, D. I.;
Stammers, D. K. J. Med. Chem. 1999, 42, 3845.
30. Ren, J.; Diprose, J.; Warren, J.; Esnouf, R. M.; Bird, L.
E.; Ikemizu, S.; Slater, M.; Milton, J.; Balzarini, J.; Stuart,
D. I.; Stammers, D. K. J. Biol. Chem. 2000, 275, 5633.
31. Ren, J.; Milton, J.; Weaver, K. L.; Short, S. A.; Stuart, D.
I.; Stammers, D. K. Structure 2000, 8, 1089.
44. Campiani, G.; Fabbrini, M.; Morelli, E.; Nacci, V.; Greco,
G.; Novellino, E.; Maga, G.; Spadari, S.; Bergamini, A.;
Faggioli, E.; Uccella, I.; Bolacchi, F.; Marini, S.; Coletta,
M.; Fracasso, C.; Caccia, S. Antiviral. Chem. Chemother.
2000, 11, 141.
45. (a) Venkatachalam, T. K.; Sudbeck, E. A.; Mao, C.;
Uckun, F. M. Bioorg. Med. Chem. Lett. 2000, 10, 2071; (b)
Venkatachalam, T. K.; Mao, C.; Uckun, F. M. Bioorg.
Med. Chem. 2004, 12, 4275.
32. Richman, D. D.; Morton, S. C.; Wrin, T.; Hellmann, N.;
Berry, S.; Shapiro, M. F.; Bozzette, S. A. AIDS 2004, 18,
1393.
33. Leigh Brown, A. J.; Frost, S. D.; Mathews, W. C.;
Dawson, K.; Hellmann, N. S.; Daar, E. S.; Richman, D.
D.; Little, S. J. J. Infect. Dis. 2003, 187, 683.
34. 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, M. E.; Pani, A.; La Colla, P.;
Loddo, R. J. Med. Chem. 2005, 48, 3858.
46. D’Cruz, O. J.; Uckun, F. M. J. Antimicrob. Chemoter.
2006, 57, 411, and references therein.
47. D’Cruz, O. J.; Uckun, F. M. Curr. HIV Res. 2006, 4, 329.
48. D’Cruz, O. J.; Uckun, F. M. J. Enzyme Inhib. Med. Chem.
2006, 21, 329.
49. Ravichandran, V.; Agrawal, R. K. Bioorg. Med. Chem.
Lett. 2007, 17, 2197.
50. Hsiou, Y.; Das, K.; Ding, J.; Clark, A. D. J.; Kleim, J. P.;
Rosner, M.; Winkler, I.; Riess, G.; Hughes, S. H.; Arnold,
E. J. Mol. Biol. 1998, 284, 313.
51. Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun,
F. M. Biochem. Pharmacol. 2000, 60, 1251.
52. Ranise, A.; Spallarossa, A.; Schenone, S.; Bruno, O.;
Bondavalli, F.; Vargiu, L.; Marceddu, T.; Mura, M.;
La Colla, P.; Pani, A. J. Med. Chem. 2003, 46,
768.
35. Bell, F. W.; Cantrell, A. S.; Hogberg, M.; Jaskunas, S. R.;
Johansson, N. G.; Jordan, C. L.; Kinnick, M. D.; Lind, P.;
¨
´
Morin, J. M., Jr.; Noreen, R.; Oberg, B.; Palkowitz, J. A.;
Parrish, C. A.; Pranc, P.; Sahlberg, C.; Ternansky, R. J.;
Vasileff, R. T.; Vrang, L.; West, S. J.; Zhang, H.; Zhou,
X.-X. J. Med. Chem. 1995, 38, 4929.
53. Strickley, R. G.; Anderson, B. D. Pharm. Res. 1993, 10,
1076, and references therein.
36. Ahgren, C.; Backro, K.; Bell, F. W.; Cantrell, A. S.;
Clemens, M.; Colacino, J. M.; Deeter, J. B.; Engelhardt,
P.; Hogberg, M.; Jaskunas, S. R.; Johansson, N. G.;
Jordan, C. L.; Kasher, J. S.; Kinnick, M. D.; Lind, P.;
54. (a) Mitscher, L.; Dutta, A., six ed.. In Burger’s Medicinal
Chemistry and Drug Discovery; Abraham, D. J., Ed.; John
Wiley and Sons, Inc., 2003; Vol. 2, pp 14–22; (b) Sanchez-
Martin, R. M.; Mittoo, S.; Bradley, M. Curr. Top. Med.
Chem. 2004, 4, 653; (c) Gupta, S. P. Comb. Chem. High
Throughput Screening 2005, 8, 375.
55. (a) An, H.; Cook, P. D. Chem. Rev. 2000, 100, 3311; (b)
Cork, D.; Hird, N. Drug Discov. Today 2002, 7, 56; (c)
Weidner, J. Drug Discov. Today 2004, 9, 375.
56. Maass, G.; Immendoerfer, U.; Koenig, B.; Leser, U.;
Mueller, B.; Goody, R.; Pfaff, E. Antimicrob. Agents
Chemother. 1993, 37, 2612.
57. Tantillo, C.; Ding, J.; Jacobo-Molina, A.; Nanni, R.
G.; Boyer, P. L.; Hughes, S. H.; Pauwels, R.; Andries,
K.; Janssen, P. A.; Arnold, E. J. Mol. Biol. 1994,
243, 369.
58. Dauber-Osguthorpe, P.; Roberts, V. A.; Osguthorpe, D.
J.; Wolff, J.; Genest, M.; Hagler, A. T. Proteins 1988,
4, 31.
´
Lopez, C.; Morin, J. M., Jr.; Muesing, M. A.; Noreen, R.;
Oberg, B.; Paget, C. J.; Palkowitz, J. A.; Parrish, C. A.;
¨
Pranc, P.; Rippy, M. K.; Rydergard, C.; Sahlberg, C.;
Swanson, S.; Ternansky, R. J.; Unge, T.; Vasileff, R. T.;
Vrang, L.; West, S. J.; Zhang, H.; Zhou, X.-X. Antimicrob.
Agents Chemother. 1995, 39, 1329.
37. Cantrell, A. S.; Engelhardt, P.; Hogberg, M.; Jaskunas, S.
R.; Johansson, N. G.; Jordan, C. L.; Kangasmetsa, J.;
Kinnick, M. D.; Lind, P.; Morin, J. M., Jr.; Muesing, M.
¨
´
A.; Noreen, R.; Oberg, B.; Pranc, P.; Sahlberg, C.;
Ternansky, R. J.; Vasileff, R. T.; Vrang, L.; West, S. J.;
Zhang, H. J. Med. Chem. 1996, 39, 4261.
38. Vig, R.; Mao, C.; Venkatachalam, T. K.; Tuel-Ahlgren,
L.; Sudbeck, E. A.; Uckun, F. M. Bioorg. Med. Chem.
1998, 6, 1789.
39. Mao, C.; Vig, R.; Venkatachalam, T. K.; Sudbeck, E. A.;
Uckun, F. M. Bioorg. Med. Chem. Lett. 1998, 8, 2213.
59. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem.
1998, 19, 1639.
´
40. (a) Sahlberg, C.; Noreen, R.; Engelhardt, P.; Ho¨gberg, M.;
Kangasmetsa¨, J.; Vrang, L.; Zhang, H. Bioorg. Med.

4172
S. Cesarini et al. / Bioorg. Med. Chem. 16 (2008) 4160–4172
60. Brunger, A. T.; Adams, P. D.; Clore, G. M.; Delano, W. L.;
Gros, P.; Grosse-Kunstleve, R. W.; Jiangj, S.; Kuszewski, J.;
Nilges, N.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.;
Warren, G. L. Acta Crystallogr. 1998, D54, 905.
61. Collaborative Computational Project, Number 4 Acta
Crystallogr. 1994, D50, 760.
62. Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946.
63. Esnouf, R. M. J. Mol. Graph. Model. 1997, 15, 132.