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

Article abstract of DOI:10.1021/jm049252r

In this paper we describe our structure-based ligand design, synthetic strategy, and structure-activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of phenethylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl] phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates (Ranise, A.; et al. J. Med. Chem. 2003, 46, 768-781), two targeted solution-phase TC libraries were prepared by parallel synthesis. The lead optimization strategy led to para-substituted TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-4-based assays at nanomolar concentrations (EC₅₀ range: 0.04-0.01 μM). The most potent congener 50 (EC₅₀ = 0.01 μM) bears a methyl group at position 4 of the phthalimide moiety and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 μM. TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme assays. The docking model predictions were consistent with in vitro biological assays of the anti-HIV-1 activity of the TCs and related compounds synthesized.

Full text of DOI:10.1021/jm049252r

3
858
J. Med. Chem. 2005, 48, 3858-3873
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
,
§
§
§
§
§
§
Angelo Ranise,* Andrea Spallarossa, Sara Cesarini, Francesco Bondavalli, Silvia Schenone, Olga Bruno,
§
§
§
†
†
†
Giulia Menozzi, Paola Fossa, Luisa Mosti, Massimiliano La Colla, Giuseppina Sanna, Marta Murreddu,
†
†
†
†
,†
Gabriella Collu, Bernardetta Busonera, Maria Elena Marongiu, Alessandra Pani, Paolo La Colla,* and
†
Roberta Loddo
Dipartimento di Scienze Farmaceutiche, Universit a` di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy, and Dipartimento
di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche, Universit a` di
Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy
Received September 14, 2004
In this paper we describe our structure-based ligand design, synthetic strategy, and structure-
activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a
novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of pheneth-
ylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl]-
phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates
(
Ranise, A.; et al. J. Med. Chem. 2003, 46, 768-781), two targeted solution-phase TC libraries
were prepared by parallel synthesis. The lead optimization strategy led to para-substituted
TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-
4
-based assays at nanomolar concentrations (EC50 range: 0.04-0.01 µM). The most potent
congener 50 (EC50 ) 0.01 µM) bears a methyl group at position 4 of the phthalimide moiety
and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good
selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 µM.
TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but
they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase
in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme
assays. The docking model predictions were consistent with in vitro biological assays of the
anti-HIV-1 activity of the TCs and related compounds synthesized.
Introduction
Chart 1
Nucleoside (NRTIs) and non-nucleoside inhibitors
(
NNRTIs) targeting the HIV-1 encoded reverse tran-
1
scriptase (RT) have to be proved effective in treating
2
3-13
the HIV infection and AIDS. NNRTIs
bind to an
allosteric site (non-nucleoside binding site, NNBS)
largely contained within the RT p66 subunit, some 10
Å from the polymerase active site. Despite their chemi-
cal diversity, NNRTIs interact with the NNBS, showing
a similar three-dimensional arrangement, the so-called
“
butterfly-like conformation” typical of first-generation
1
4
NNRTIs, as demonstrated by X-ray crystallography
of HIV-1 RT-NNRTI complexes.15-23 However, the
relatively unconserved amino acid sequence of the
NNBS favors the rapid selection of NNRTI-resistant
24
viruses, both in vitro and in vivo. As a result of single-
2
5
point mutations in the NNBS, first-generation NNR-
TIs, e.g., nevirapine and delavirdine (Chart 1.1) show
a loss of potency of several orders of magnitude. In
contrast, second-generation NNRTIs, such as efavirenz
2
6
(
Chart 1.2) and some thiocarboxanilide27 and quinoxa-
28
line derivatives, result in minor losses of activity
against variants carrying either single or double NNRTI
resistance mutations. Nevertheless, the fact that cross-
resistance extends to the whole NNRTI class calls for
development of new agents capable of inhibiting clini-
cally relevant NNRTI-resistant mutants.
*
To whom correspondence should be addressed. For A.R.: phone,
+
39 010 353 8370; fax, +39 010 353 8358; e-mail, ranise@unige.it. For
P.L.C.: phone, +39 070 675 4148; fax, +39 070 675 4210; e-mail,
placolla@unica.it.
§
Universit a` di Genova.
Universit a` di Cagliari.
†
1
0.1021/jm049252r CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/05/2005

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3859
Chart 2. Chemical Structures of PETT, Trovirdine,
PETT-1, Lead Compound 12, O-TC 51, ATC-1, and
UC-38
tives, is an attractive research area for medicinal
chemists, as documented by recent, published papers.3
4-45
However, PETT isostere TCs have not been hitherto
reported in the literature. Notably, TCs, unlike PETT
derivatives such as PETT LY73497, PETT-1 (Chart 2A),
and trovirdine, have no internal H-bond, and therefore,
the lack of this conformational constraint makes them
more flexible than PETT derivatives. In this regard, it
was speculated that a greater conformational freedom
of inhibitors might be a useful design feature for
reducing drug resistance.4
6-49
With the aim to establish the key structural require-
ments of TCs for anti-HIV-1 activity, our lead optimiza-
tion strategy was centered on (1) substituent variation
on the N-phenyl ring (B1), (2) modification of the
benzene ring (A1) of the phthalimide moiety, (3) short-
ening, lengthening, and branching the ethyl spacer, (4)
modification of the thiocarbamate scaffold through
isosteric replacements (S/O and NH/O), and (5) opening
of the imide ring. Indeed, the last modification, planned
for future SAR strategies, was anticipated because in
the TC parallel synthesis several ring-opened analogues
(
O-TCs) (see Chemistry) were concomitantly obtained
along with their corresponding ring-closed congeners (C-
TCs). Since the overall conformation of enzyme-bound
5
1 (Chart 2A), the corresponding O-TC analogue of lead
2, approximates the X-ray structure of PETT-1 within
We have previously described a novel class of NNR-
1
TIs, i.e., O-substituted N-acyl-N-arylthiocarbamates
the NNBS (Figure 2B), the O-TCs were also tested for
HIV-1 activity. Finally, some structural variations were
performed with the aim not only to expand the SARs
but also to validate the TC docking model (for repre-
sentative examples, see derivatives 77-81 and 92-95).
2
9
(
ATCs) structurally related to N-phenethyl-N′-thiaz-
3
0,31
olylthiourea (PETT) derivatives
(Chart 2A). Among
the ATCs, the phthalimidoethyl-ATCs proved to be
potent inhibitors of the multiplication of wild-type HIV-
, significantly active against Y181C mutants but
1
ineffective against K103R mutants.
Chemistry
Figure 1 shows the relative positions and orientations
of the modeled structures of ATC-1, one of the lead
compounds of the ATC series (Chart 2B), and of its
intermediary thiocarbamate (TC) 12 (Chart 2A) super-
imposed on the X-ray structure model of PETT-1 (Chart
Combinatorial library methodologies50 play a pivotal
role in the modern drug discovery process, in particular
in lead identification and optimization efforts. Library
synthesis of small, druglike molecules is carried out by
means of manual, semiautomated, or automated syn-
thesizers. Among the combinatorial approaches, parallel
synthesis is aimed at fast preparation of a (large)
number of compounds in short times, using simple and
rapid purification methods.51
Since the new highly convergent synthesis of ATCs
was performed through a one-pot three-step proce-
dure by sequentially combining three types of building
blocks (alcohols, isothiocyanates, acyl chlorides),29 in
principle, TCs, being precursors of ATCs, could be
prepared with the above procedure by omitting the
acylation step. As previously reported,29 one of the
intermediary TCs was actually isolated by acidifying the
reaction medium, before acylating its corresponding
sodium salt. Because the synthetic protocol can be easily
adapted to parallel solution-phase synthesis (and nowa-
days the use of solution-phase techniques has been
accepted as a valuable alternative to solid-supported
2A) within the NNBS. It is apparent that the conforma-
tion of 12, unlike that of ATC-1, more closely resembles
the conformation of PETT-1. Furthermore, the thiocar-
bamate UC-38 (NSC 629243) (Chart 2C) was selected
as an anti-HIV-1 agent in the early 1990s for preclinical
3
2
development.
Starting from these preliminary remarks and taking
into account that screening of synthesis intermediates
represents one of the strategies in the search for new
lead compounds,33 12 was proposed for synthesis and
tested both in enzyme assays against HIV-1 virion RT
(
vRT) and in cell-based assays for cytotoxicity and anti-
2
9
HIV-1 activity. As anticipated by its docking model,
TC 12 proved to be able to target vRT (IC50 ) 0.6 µM)
and to selectively inhibit the HIV-1 induced cytopatho-
genicity in MT-4 cells (CC50 > 100 µM, EC50 ) 1.2 µM,
Table 3), even if it was 3-fold less potent than ATC-1
(
EC50 ) 0.4 µM).29 Therefore, to further explore the
52
potential of TCs as anti-HIV-1 agents, we designed two
small libraries of analogues of the lead compound 12.
An additional scope of our work was to compare some
relevant SARs of the TC and PETT derivatives owing
to their isosteric relationship. In the field of NNRTIs,
structure-based design of analogues of trovirdine31
chemistry approaches ), we designed and synthesized
two small, targeted TC libraries. Preliminarily, the
syntheses of alcohol building blocks 3, 4, (()-5, (()-6,
8, and 9 (Chart 3), not commercially available, were
carried out starting from anhydrides I-V and amino
alcohols (Scheme 1). Thus, according to literature
procedures,53 fusion of anhydrides I, II, IV, and V with
(Chart 2A), the most representative of PETT deriva-

3
860 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Ranise et al.
Figure 1. Stereodiagram showing superposition of TC 12, ATC-1, and PETT-1 (see Chart 1) within the NNBS. The relative
positions and conformations for TC 12 (model structure, black ball-and-stick), ATC-1 (model structure, white 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
1
6
on the basis of the amino acid residues surrounding the RT pocket, as described by Ren et al. Figure 1 indicates the different
binding mode of ATC-1 and its TC intermediate 12. Despite the lack of the internal H-bond typical of PETT derivatives, 12
assumes the butterfly-shaped bioactive conformation of NNRTIs, strongly mimicking the RT-bound PETT-1 conformation and
6
1
62
orientation. The programs MolScript and Raster3D were used for drawing the figure.
Figure 2. Stereoview showing the position and orientation of C-TC 12 (A) and its corresponding O-TC 51 (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
6
1
62
bonds are depicted as dotted lines. The drawing was realized by the programs MolScript and Raster3D.
2
9
2
-aminoethanol at 175-180 °C for 2 h gave 3, 4, 8, and
, respectively. Similarly, III condensed both with d,l-
-amino-1-propanol at 140-150 °C and with d,l-1-
sieves (library 1), or in anhydrous pyridine (library 2),
alcohols 1-7 (Chart 3A), due to their low nucleophilicity,
were transformed in the presence of a 60% NaH disper-
sion in mineral oil into their corresponding alcoholates
A1-7, which condensed in situ with appropriate isothio-
cyanates (Chart 3B) to afford either adducts B (sodium
salts of the C-TCs) or B and their ring-opened counter-
parts SS (di-sodium salts of the O-TCs), according to
the experimental results (see below). To improve the
amino-2-propanol at 160-175 °C to afford the respective
alcohols 5 and 6 as racemic mixtures. Scheme 2 sum-
marizes the general procedure for the preparation of
libraries 1 and 2. The parallel analogue synthesis was
carried out by using a manual synthesizer (Carousel
reaction station). Typically, in DMF, dried on molecular

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3861
Chart 3. Alcohols 1-10 and Isothiocyanates (Ar
Used as Synthetic Building Blocks
1
NCS)
TCs 77-81 were synthesized in parallel by reacting 10,
8 in DMF, or 9 in anhydrous THF with the proper
isothiocyanate in the presence of sodium hydride (Scheme
4
). Starting from alcohol 2, Scheme 5 illustrates the
synthesis of compounds 92-95, structurally related to
the title compounds. Derivatives 92 and 93 were pre-
pared according to a literature-reported method57 by
reacting 2, 4-tolyl chlorothionoformate, and 4-chlorophe-
nyl chlorothionoformate in the presence of DMAP.
Carbamate 94, unlike sulfur isostere 44, was synthe-
sized by heating a mixture of 1:1 pyridine/DMF of 2 and
4
-nitrophenylisocyanate at 140 °C because the proce-
dure via alcoholate was unsuccessful. Finally, 95 was
obtained by grounding a mixture of 2 and thiocarbon-
yldiimidazole in an agate mortar through a solvent-free
procedure58 until the reaction was complete (TLC
monitoring).
The formation of O-TCs and their separation from
C-TCs deserve further comment. Scheme 6a illustrates
that the SS salts can be generated either directly from
B (route b) and or indirectly from ring-opened interme-
diates 2′, 5′, 6′, and A2′,5′,6′ (route c). Clearly, these
species, derived from 2, 5, 6, and A2,5,6 (Scheme 6b), are
expected to react like the corresponding ring-closed
counterparts according to route a. Scheme 6b depicts
the common mechanism hypothesized for the imide ring
opening of B, 2, 5, 6, and A2,5,6, due to the attack of the
hydroxide ion on the carbonyls of the phthalimide
moiety. The presence of hydroxide ions in the reaction
medium can be influenced by the solvents and base
employed. Thus, DMF, dried on molecular sieves may
still contain trace water that would react with sodium
hydride to give sodium hydroxide (NaH + H2O f NaOH
yield and purity of TCs of library 2, isothiocyanates were
5
4
made more reactive by means of pyridine. Sodium
5
5
sand, currently employed in the synthesis of thiocar-
bamates, was less effective than sodium hydride (data
not shown). Upon treatment of the reaction medium
with water and ammonium chloride, C-TCs 11-22, 24-
+
H2). On the other hand, anhydrous pyridine, stored
50, and 82-91 precipitated, while ring-opened adducts
on sodium hydroxide pellets, as well as sodium hydride
dispersion in mineral oil could release alkali impurities
within the reaction medium.
SS were transformed into S (monosodium salts of the
O-TCs). Eventually, the subsequent acidification of the
reaction mother liquors with 2 M HCl caused separation
of O-TCs 51-76 as solids from the solution. Notably,
in the synthesis of C-TCs 11, 13, 24, 25, 29, 34-36, 42,
As for the easy separation of the C-TCs from the
O-TCs, it is mainly due to protolysis of basic salts B,
SS, and S, occurring under different pH conditions
(Scheme 6c). Thus, upon the weakly acidic treatment
(water and ammonium chloride) of the reaction mixture,
the ionized thiocarbamic moieties of B and SS are
protonated. This process affords their relative conjugate
acids (i.e., C-TCs and S), and sodium hydroxide as a
byproduct. Consequently, C-TCs 12, 14, 17, 18, 21, 22,
26-28, 30-33, 37-41, 44-46, 83-85 precipitate, while
salts S are kept in solution (indeed, it is not ruled out
that ammonium chloride salting-out effect contributes
to the complete separation of the C-TCs from S). In turn,
sodium hydroxide liberates ammonia from ammonium
chloride, and therefore, an NH3/NH4Cl buffer is estab-
lished, as proved by the measured pH value (about 9)
of the final mixture. The protonation of the carboxylate
group of SS cannot concomitantly occur at this pH value
because its basicity is much lower than that of the
ionized thiocarbamic moiety, as inferred indirectly from
the estimated acidity of the thiocarbamic group of, for
example, O-TC 51, which is about 8 pKa units higher
than that of the carboxylic group (see Chart 4). There-
fore, according to the Br o¨ nsted-Lowry acid-base theory,
a similar difference also has to exist in the basic
strength of their corresponding ionized forms. For
protonation of the carboxylate group of S to occur, the
4
3, 47-50, 82, 86-91 (library 1) and C-TCs 15, 16, 19,
0 (library 2), it was impossible to isolate workable
2
amounts of the corresponding O-TCs (yields were less
than 0.5-1%; data not shown). In contrast, reaction of
alcohol 2 with 3-tolyl isothiocyanate and 4-benzyloxy-
phenyl isothiocyanate afforded only O-TCs 53 and 73
(
library 2), respectively. It is noted that this anomalous
ring opening of the phthalimide moiety was also found
in hydrochlorides of basically substituted phthalimidyl
derivatives, which unexpectedly gave the corresponding
ring-opened products on careful neutralization with
5
6
alkali in cold water. In addition, in the synthesis of
C-TCs 47, 48, 49, 50 (library 1), we isolated their
corresponding O-TCs, but these derivatives have not
been fully characterized yet (the ring opening of the
asymmetric 3-methyl- and 4-methyl phthalimmide moi-
eties might give a mixture of two regioisomers). Their
elucidated structures will be reported in due course.
Purification of TCs was carried out by crystallization.
The physicochemical constants of C-TCs 11-22, 24-
5
0, 82-91 and of O-TCs 51-76 are reported Tables 1
and 2, respectively.
The dehydrative cyclization of O-TC 57 in the pres-
ence of excess P2O5 in DMF (Scheme 3) afforded C-TC
23, not directly obtainable with the above procedures.

3
862 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Ranise et al.
Scheme 1. Synthesis of Alcohols 3-6, 8, and 9a
a
Reaction conditions: (a) 2-aminoethanol, 175-180 °C, 2 h; (b) (()-2-amino-1-propanol, 140-150 °C, 2 h; (c) (()-1-amino-2-propanol,
60-175 °C, 2 h.
1
Scheme 2. General Procedure for Preparation of Libraries 1 and 2 by Parallel Analogue Synthesisa
a
Reaction conditions. Library 1: (a, b) isothiocyanate, NaH, dried DMF, 0-5 °C, then room temp for 24 h. Library 2: (a, b) isothiocyanate,
NaH, anhydrous pyridine, room temp for 15 min, then 60-65 °C for 3 h; (c) H2O, NH4Cl; (d) 2 M HCl.
pH is adjusted at low acidic values. Thus, by acidifica-
tion of the reaction mother liquors with 2 M HCl, O-TCs
Biological Results and Discussion
51-56, 58-72, and 74-76 are separated from the
The antiretroviral activity of the TCs and thionocar-
bonates 92 and 93, carbamate 94 (listed in Tables 3-7),
solution.

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3863
Table 1. Physicochemical Constants of C-TCs 11-22, 24-50, 82-91
mp (°C), in
crystallization solvent
compd
R
Ar1
yield (%)
formulak
a
11
12
13
14
15
16
17
18
19
20
21
22
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
82
83
84
85
86
87
88
89
90
91
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(phthalimido)ethyl
2-(3-methylphthalimido)ethyl
2-(3-methylphthalimido)ethyl
2-(4-methylphthalimido)ethyl
2-(4-methylphthalimido)ethyl
phthalimidomethyl
cyclohexyl
phenyl
1-naphthyl
2-tolyl
2-isopropylphenyl
2-trifluoromethylphenyl
2-metoxycarbonylphenyl
2-fluorophenyl
2-chlorophenyl
2-bromophenyl
2-nitrophenyl
2-methoxyphenyl
3-trifluoromethylphenyl
3-acetylphenyl
3-methoxycarbonylphenyl
3-chlorophenyl
3-methylsulfanylphenyl
3-nitrophenyl
3-methoxyphenyl
4-tolyl
4-ethylphenyl
132-133
40
41
24
44
37
51
53
44
40
27
27
37
46
32
15
41
43
25
38
66
11
32
18
33
26
98
32
24
36
25
29
13
45
12
26
27
35
30
62
59
44
37
16
80
24
31
39
41
55
C17H20N2O3S
C17H14N2O3S
C21H16N2O3S
C18H16N2O3S
C20H20N2O3S
C18H13F3N2O3S
C19H16N2O5S
C17H13FN2O3S
C17H13ClN2O3S
C17H13BrN2O3S
C17H13N3O5S
C18H16N2O4S
C18H13F3N2O3S
C19H16N2O4S
C19H16N2O5S
C17H13ClN2O3S
C18H16N2O3S2
C17H13N3O5S
C18H16N2O4S
C18H16N2O3S
C19H18N2O3S
C20H20N2O3S
C18H13F3N2O3S
C20H18N2O5S
C19H16N2O4S
C18H13N3O3S
C17H13FN2O3S
C17H13ClN2O3S
C17H13BrN2O3S
C17H13IN2O3S
C19H19N3O3S
C21H23N3O3S
C17H13N3O5S
C18H16N2O4S
C19H18N2O4S
C18H15ClN2O3S
C18H15N3O5S
C18H15ClN2O3S
C18H15N3O5S
C16H11N3O5S
C18H15ClN2O3S
C18H15N3O5S
C19H18N2O3S
C19H15N3O3S
C18H15ClN2O3S
C18H15BrN2O3S
C18H15N3O5S
C18H16N2O3S
C18H15N3O5S
a
118-119
b
148-150
a
128-129
c
135-136
a
128-130
a
122-124
a
135-137
d
138-140
e
123-125
a
145-146
f
134-135
a
165-167
a
116-117
c
80-81
f
159-161
a
102-104
a
190-191
a
98-100
a
133-134
b
149-151
a
4-isopropylphenyl
4-trifluoromethylphenyl
4-ethoxycarbonylphenyl
4-acetylphenyl
4-cyanophenyl
160-162
a
169-171
a
166-168
h
179-180
a
174-176
a
4-fluorophenyl
150-152
a
4-chlorophenyl
4-bromophenyl
4-iodophenyl
157-159
a
156-158
a
177-179
f
4-dimethylaminophenyl
4-diethylaminophenyl
4-nitrophenyl
4-methoxypheny
4-ethoxyphenyl
4-chlorophenyl
4-nitrophenyl
4-chlorophenyl
4-nitrophenyl
4-nitrophenyl
4-chlorophenyl
4-nitrophenyl
4-tolyl
4-cyanophenyl
194-196
h
166-168
a
196-197
125-127
a
a
114-115
f
169-171
h
215-217
i
182-184
i
202-204
187-189g
a
2-methyl-2-phthalimidoethyl
2-methyl-2-phthalimidoethyl
1-methyl-2-phthalimidoethyl
1-methyl-2-phthalimidoethyl
1-methyl-2-phthalimidoethyl
1-methyl-2-phthalimidoethyl
1-methyl-2-phthalimidoethyl
3-phthalimidopropyl
3-phthalimidopropyl
130-131
b
143-144
a
176-178
a
155-157
a
4-chlorophenyl
4-bromophenyl
4-nitrophenyl
phenyl
4-nitrophenyl
172-174
a
168-169
a
184-185
a
133-135
j
205-207
a
b
c
d
e
In dichloromethane-methanol. In diethyl ether-ethanol. In diethyl ether-methanol. In acetone-diethyl ether. In acetone-
f
g
h
i
j
methanol. In diethyl ether-dichloromethane. In dichloromethane-ethanol. In acetone-ethanol. In acetone. In dichloromethane-
ethanol-acetone. All compounds were analyzed for C, H, N, and S. Analytical results were within (0.4% of the theoretical values.
k
and imidazole-1-carbothioate 95 was evaluated in cell-
based assays by assessing the reduction of the HIV-1
induced cytopathogenicity in MT-4 cells. The results are
expressed as EC50 values. At the same time, the TC-
induced cytotoxicity was evaluated in parallel with the
antiretroviral activity in mock-infected MT-4 cells, and
the results are expressed as CC50 values. Trovirdine was
used as the reference compound.
C-TCs 31, 37-40, and 44, whose potency was confirmed
in HIV-1 infected C8166 cells (Table 9) along with 41,
were also tested against the Y181C resistant strain
(Table 10).
Table 3 summarizes the cytotoxic and antiretroviral
activities of the lead compound 12 and its analogues
modified in ring B1 (11 and 13-46). It is worth nothing
that while 12 was active in the low-micromolar concen-
tration range, PETT isosteres (ring B ) phenyl), not
having the internal H bond, were almost inactive29
likely because such derivatives cannot assume the
“high-activity rigid conformation” of PETT-1 (Chart 2A),
as illustrated in Figure 1. This evidence points out a
substantial difference between the TC and PETT series.
C-TC 41 and its O-TC 69 counterpart were also
assayed for inhibitory activity against HIV-1 recombi-
nant wild-type (wt) RT (rRT) and rRTs carrying clini-
cally relevant single and double mutations such as
5
7,58
Y181C, K103R, K103N plus Y181C.
Trovirdine and
efavirenz were used as reference compounds (Table 8).

3
864 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Ranise et al.
Table 2. Physicochemical Constants of O-TCs 51-76
mp (°C), in
crystallization solvent
compd
R1
R2
Ar1
yield (%)
formulag
a
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
H
H
phenyl
2-tolyl
2-methoxycarbonylphenyl
2-fluorophenyl
2-nitrophenyl
2-methoxyphenyl
3-tolyl
114-116
10
28
47
36
26
15
48
15
9
15
41
18
35
14
30
30
14
22
27
21
19
22
44
27
39
15
C17H16N2O4S
a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
167-169
C18H18N2O4S
C19H18N2O6S
C17H15FN2O4S
C17H15N3O6S
C18H18N2O5S
C18H18N2O4S
C19H18N2O6S
C17H15ClN2O4S
C18H18N2O5S
C18H18N2O4S2
C18H18N2O4S
C19H20N2O4S
C20H22N2O4S
C18H15N3O4S
C17H15FN2O4S
C17H15ClN2O4S
C17H15BrN2O4S
C17H15IN2O4S
C17H15N3O6S
C18H18N2O5S
C19H20N2O5S
C24H22N2O5S
C19H20N2O4S
C18H17ClN2O4S
C18H17N3O6S
b
H
149-151
c
H
154-156
d
H
159-161
d
H
150-151
d
H
162-164
e
H
3-methoxycarbonylphenyl
3-chlorophenyl
3-methoxyphenyl
3-methylsulfanylphenyl
4-tolyl
151-152
c
H
136-138
d
H
139-141
a
H
138-139
a
H
153-155
f
H
4-ethylphenyl
158-159
a
H
4-isopropylphenyl
4-cyanophenyl
4-fluorophenyl
4-chlorophenyl
4-bromophenyl
4-iodophenyl
162-163
d
H
123-125
a
H
172-173
a
H
156-158
a
H
248-250
a
H
175-177
a
H
4-nitrophenyl
167-168
b
H
4-methoxyphenyl
4-ethoxyphenyl
4-benzyloxyphenyl
4-tolyl
164-165
a
H
170-171
f
H
141-143
a
H
173-175
d
CH3
CH3
4-chlorophenyl
4-nitrophenyl
184-186
d
194-196
a
In dichloromethane-methanol. In acetone-ethanol. c In diethyl ether-ethanol. d In acetone-methanol. e In diethyl ether-acetone.
b
f
In dichloromethane-diethyl ether. g All compounds were analyzed for C, H, N, and S. Analytical results were within (0.4% of the
theoretical values.
Scheme 3. Synthesis of C-TCs 23a
Scheme 4. Parallel Synthesis of TCs 77-81a
a
Reaction conditions: (a) dried DMF, P2O5, 65 °C for 5 h,
Na2CO3.
To increase flexibility of the lead, we replaced aromatic
ring B1 with a cyclohexyl nucleus. This modification
enhanced the potency because TC 11 resulted in being
4
-fold more potent than 12. To further probe spatial
requirements, the B1 ring was replaced with the
-naphthyl nucleus, but the activity of 13 was dimin-
1
ished by about 5-fold in comparison to 12. Therefore,
the hydrophobic interactions of ring B1 could not be
improved by synthesizing an analogue where an extra
aromatic ring is fused onto the original ring of the lead.
Congeners 14-22, 23-30, and 31-46 bear ortho,
meta, and para substituents, respectively, having a wide
range of electronic (inductive and/or mesomeric), lipo-
philic, and steric properties. The ortho derivatives (14-
a
Reaction conditions: (a) dried DMF, NaH, isothiocyanate,
room temp for 20 h; (b) anhydrous THF, NaH, isothiocyanate, room
temp for 20 h.
trations (EC50 ) 20-140 nM), with the exception of
4-ethoxycarbonyl 35, 4-ethoxy 46, 4-dimethylamino 42,
and 4-diethylamino 43 (those last two compounds were
synthesized to enhance water solubility of TCs), which
were weak inhibitors. Among the most potent C-TCs,
4-methyl 31 and 4-iodo 41 (EC50 ) 0.02 µM) were
equipotent to trovirdine, followed by 4-bromo 40, 4-meth-
oxy 45 (EC50 ) 0.03 µM), 4-isopropyl 33, 4-trifluro-
methyl 34, 4-chloro 39, and 4-nitro 44 (EC50 ) 0.04 µM).
Therefore, the activity was strongly affected by the
substitution pattern of the N-phenyl ring with the
2
2) showed EC50 values in the range 3.0-65 µM.
Exceptions were 2-methoxycarbonyl 17 and 2-nitro 21,
which were inactive. Apart from 3-nitro 29 (EC50 ) 0.6
µM), most of the meta derivatives (23-30) were also
micromolar inhibitors, even if they were slightly more
active (EC50 ) 1.5-21 µM) than the ortho-substituted
C-TCs. In contrast, most of the para-substituted TCs
(31-46) proved to be active at low-nanomolar concen-

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3865
Scheme 5. Synthesis of Thionocarbonates 92, 93,
Carbamate 94, and Phthalimidoethyl
Table 4 shows the effect of the opening of the
phthalimide ring and of modification of ring B1 on
cytotoxicity and antiviral activity. Apart from 2-meth-
oxycarbonyl 53, 2-nitro 55, and 3-methoxycarbonyl 58,
which were inactive, O-TCs 51, 52, 54, 56, 57, 59-73
were selectively active against HIV-1. As in the case of
the C-TCs, the potency of ring-opened congeners de-
pends on the position of the substituent on the N-phenyl
ring. In fact, the EC50 values of ortho (52, 53, 55, and
a
Imidazole-1-carbothioate 95
5
6) and meta congeners (57, 59-61) are in the micro-
molar range (EC50 ) 65-3 µM) except 2-fluoro O-TC
5
2
6
4, which is 6 times more potent (EC50 ) 0.5 µM) than
-fluoro C-TC 18. The para-substituted O-TCs 62, 63,
5-71, and 73 were active at submicromolar concentra-
tions (EC50 ) 0.1-0.5 µM), with the exception of
-ethoxy 72 and 4-isopropyl 64, which showed EC50
4
values of 2 and 3.7 µM, respectively. Therefore, even
though the general pattern of activity of the O-TCs
mirrored that of the C-TCs (para > meta and ortho),
overall the para-substituted O-TCs were less potent
than the corresponding para-substituted C-TCs. For
example, O-TCs 4-methyl 62 and 4-iodo 69 were 20- and
6.5-fold less potent than C-TCs 4-methyl 31 and 4-iodo
a
Reaction conditions: (a) acetonitrile, DMAP, chlorothionofor-
mate, room temp for 24 h; (b) DMF/Py (1:1), p-nitrophenyl
isocyanate, 140 °C for 5 h; (c) mixture of 2 and thiocarbonyldi-
imidazole ground in a mortar for 15 min.
4
1, respectively. Inspection of Tables 3 and 4 reveals
that the potency order of the para-substituted C-TCs
4-Me ) 4-I > 4-Br ) 4-MeO > 4-Cl ) 4-NO2 > 4-CN >
-Et > 4-F) is different from that of the O-TCs bearing
the same para substituents (4-Cl ) 4-Br ) 4-CN ∼ 4-I
4-MeO ∼ 4-F > 4-NO2 ) 4-Et > 4-Me). Moreover, the
following potency trend: para . meta > ortho, as well
exemplified by positional isomers 14, 23, 31 (2-, 3-,
-tolyl), 22, 30, 45 (2-, 3-, 4-methoxy), 19, 27, 39 (2-, 3-,
-chloro), 21, 29, 44 (2-, 3-, 4-nitro), 16, 24, 34 (2-, 3-,
-trifluromethyl), 15, 33 (2-, 4-isopropyl), 18, 38 (2-,
-fluro), and 20, 40 (2-, 4-bromo). Furthermore, the data
indicate that the electronic properties of the para
substituents do not seem to affect activity; in fact, the
most potent para-substituted derivatives bear both
electron-withdrawing groups, such as nitro (44), triflu-
oromethyl (34), chloro (39), bromo (40), and iodo (41),
and electron-donating groups, such as methoxy (45),
methyl (31), and isopropyl (33).
(
4
4
4
4
4
>
activity data of the O-TCs are not affected by the
electronic nature and the steric demand of the para
substituents. Thus, 4-cyano 65 was equipotent to 4-chlo-
ro 67, 4-bromo 68, and 4-iodo 69 (EC50 ) 0.1 µM), and
4-methoxy 71 and 4-fluoro 66 were nearly equally active
(EC50 ) 0.20 and 0.25 µM, respectively). On the other
hand, 4-nitro 70 showed the same activity as 4-ethyl
63 (EC50 ) 0.30 µM), which in turn was 1.3- and 12.3-
fold more active than 4-methyl 62 and 4-isopropyl 64,
respectively. After identification of some para-substi-
tuted rings B1 as suitable moieties, all derivatives
As far as the size of the para substituens is concerned,
the relative potency of the C-TCs bearing halogens and
cognate functions (such as the cyano and trifluromethyl
groups) is in the order iodo 41 > bromo 40 > chloro 39
(except 90) reported in Tables 5-7 carry the more
promising groups, such as nitro, chloro, bromo, cyano,
and methyl at position 4 of ring B1.
)
trifluromethyl 34 > cyano 37 > fluoro 38, making
apparent that the potency correlates with the presence
of more sterically demanding groups (in this context,
the same activity shown by 34 and 39 correlates with
the comparable size of the chlorine atom and the
trifluromethyl group). It is intriguing that the p-
chloroanilino moiety of 39 is also peculiar to other potent
NNRTIs, such as TIBO (R-82913, R-86183), the PETT
derivative MSC-127, dihydroquinazolinethiones, quin-
oxalines (S-2720), oxathiin carboxanilide (NSC 615985),
To enhance the hydrophobic interactions within the
highly hydrophobic RT pocket, C-TCs 47-50 having a
methyl group at the 3- and 4-position of ring A1 were
prepared and tested (Table 5). Introduction of a methyl
group at position 3 of the phthalimide scaffold dimin-
ished the activity because 4-chloro 47 and 4-nitro 48
were 0.5- and 1.8-fold less potent than the corresponding
4-chloro 39 and 4-nitro 44, respectively. Conversely,
positional isomers 49 and 50, carrying the methyl group
at position 4 of the phthalimide moiety, were 1.6- and
7-fold more potent than 39 and 44, respectively. Con-
gener 50 was the most active C-TC (EC50 ) 0.01 µM),
being 2-fold more potent than trovirdine.
Furthermore, to evaluate the importance of the
aromatic ring A1 in eliciting hydrophobic interactions
and to probe the spatial relationship of the fused rings,
succinimido TC 77, lacking ring A1, and bicyclic TCs
78-81 were synthesized. As expected, 77 showed a 60-
fold lower activity with respect to 39 likely because of
the loss of hydrophobic aromatic interactions of ring A1.
The change of ring A1 with a cyclenic equivalent of the
3
and UC-38 (Chart 2C). Conversely, the results concern-
ing the p-dialkylamino-, p-alkyl-, and p-alkoxy-C-TCs
suggest that the activity decreases with the steric
demand of the substituents. Thus, diethylamino 43 was
2
3
.3-fold less active than dimethylamino 42, and ethyl
2 and isopropyl 33 were 4- and 2-fold less potent than
methyl 31, respectively. An even more dramatic de-
crease in activity was observed in the alkoxy series, in
which ethoxy 46 was about 233-fold less active than
methoxy 45. These differences in activity can be ratio-
nalized by the docking models of some of the above para-
substituted C-TCs (see below).

3
866 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Ranise et al.
Scheme 6. (a, b) Hypothesized Mechanism for Formation of SS (Routes b and c) and (c) Separation of the C-TCs from
Their Corresponding O-TCs for Protolysis of Salts B, SS, and S under Different pH Conditions
Chart 4. Calculated pK
a
of C-TC 44, Carbamate 94,
spacer (83-89) with the aim of evaluating both the
optimal A1-B1 distance and the effects on the overall
bioactive conformations of TCs. Shortening or extending
the spacer by only one methylene unit completely
abolished the activity or caused a significant drop in the
anti-HIV-1 potency (TC 82, the lower homologue of 44,
was inactive, whereas 90 and 91, the superior homo-
logues of 12 and 44, respectively, were inactive or poorly
active). Addition of a methyl group on the spacer carbon
atom adjacent to the thiocarbamic oxygen resulted in a
strong decrease in activity: 4-methyl 85, 4-cyano 86,
a
and O-TC 51
a
These values have been obtained through ACD Labs 7.0
software.
phenyl ring, such as the cyclohexenyl ring, or with a
more rigid bridged system, such as the norbornene
nucleus, caused a drastic decrease of activity. Thus,
4
-cloro 87, 4-bromo 88, and 4-nitro 89 were much less
4
-chloro 78 and 4-bromo 79 were about 235- and 77-
active than the corresponding unbranched C-TC deriva-
tives 31, 37, 39, 40, and 44, respectively. When the
methyl group is located on the carbon adjacent to the
imide nitrogen, a lesser decrease of activity was ob-
served, depending on the nature of the para substituent.
Thus, 4-chloro 83 and 4-nitro 84 were 1.2- and 15-fold
less potent than unbranched 4-chloro 39 and 4-nitro 44,
respectively. The same effect, due to methyl-branching,
fold less active than 39 and 40, respectively. Similarly,
4
4
-chloro 80 and 4-nitro 81 were less active than 39 and
4. The lower activity of 78-81 with respect to 39, 40,
and 44 is probably due not only to loss of aromatic
interactions but also to nonplanar spatial rearrange-
ment of the fused rings (cis and cis-endo, respectively).
Table 6 summarizes the results obtained by shortening
(82), lengthening (90, 91), and branching the ethyl

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3867
Table 3. Effects of Modification of the N-Phenyl Ring on
Table 4. Effects of the Opening of the Imide Ring and of
Modification of the N-Phenyl Ring on Cytotoxicity and
a
Cytotoxicity and Anti-HIV-1 Activity (C-TCs 11-46)
a
Anti-HIV-1 Activity (O-TCs 51-73)
CC50 b EC50 c
CC50 b EC50 c
compd
Ar1
(µM)
(µM)
SI d
compd
Ar1
(µM)
(µM)
SI d
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
1
cyclohexyl
phenyl
1-naphthyl
2-tolyl
>100
>100
>100
>100
>100
>100
0.3
1.2
6.5
3.7
43
>333
>83.3
>15.4
>27
51
phenyl
2-tolyl
>100
>100
3
>33.3
>3.7
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
52
27
53
2-methoxycarbonylphenyl >100 >100
54
2-fluorophenyl
2-nitrophenyl
2-methoxyphenyl
3-tolyl
>100
0.5
>200
2-isopropylphenyl
2-trifluoromethylphenyl
2-metoxycarbonylphenyl
2-fluorophenyl
>2.3
>1.5
55
>100 >100
65
56
>100
>100
65
5.0
>1.5
>20
45 >45
57
>100
>100
>100
3.0
17
14
>33
>5.9
>7.1
58
3-methoxycarbonylphenyl >100 >100
2-chlorophenyl
59
3-chlorophenyl
3-methoxyphenyl
3-methylsulfanylphenyl
4-tolyl
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
3.0
2.6
11
0.4
0.3
3.7
0.1 >1000
0.25 >400
0.1 >1000
0.1 >1000
0.13 >769
0.30 >333
>33.3
>38.5
>9.1
>250
>333
>27
2-bromophenyl
60
2-nitrophenyl
>100 >100
61
2-methoxyphenyl
3-tolyl
>100
>100
>100
>100
5.6
1.5
13
4.3
21
2.0
7.0
0.6
4.0
>17.9
>66.7
>7.7
>23
>4.8
>50
>14.3
>167
>25
62
63
4-ethylphenyl
3-trifluoromethylphenyl
3-acetylphenyl
3-methoxycarbonylphenyl >100
3-chlorophenyl
3-methylsulfanylphenyl
3-nitrophenyl
3-methoxyphenyl
4-tolyl
4-ethylphenyl
64
4-isopropylphenyl
4-cyanophenyl
4-fluorophenyl
4-chlorophenyl
4-bromophenyl
4-iodophenyl
65
66
>100
>100
>100
>100
>100
>100
94
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
65
67
68
69
70
4-nitrophenyl
0.02 >5000
0.08 >1250
0.04 2350
0.04 >2500
71
4-methoxyphenyl
4-ethoxyphenyl
4-benzyloxyphenyl
0.2
2.0
0.5
>500
50
>200
72
4-isopropylphenyl
4-trifluoromethylphenyl
4-ethoxycarbonylphenyl
4-acetylphenyl
73
>100
60
trovirdine
0.02 3000
23
>4.3
a
Data represent mean values for three separate experiments.
0.14 >714
0.07 >1429
0.10 >1000
0.04 >2500
0.03 >3333
0.02 >5000
0.60 >167
b
Variation among triplicate samples was less than 10%. Com-
4-cyanophenyl
pound concentration required to reduce the viability of mock-
infected cells by 50%, as determined by the MTT method.
4-fluorophenyl
4-chlorophenyl
c
Compound concentration required to achieve 50% protection of
4-bromophenyl
MT-4 cell from HIV-1 induced cytopathogenecity, as determined
by the MTT method. Selectivity index: CC50/EC50 ratio.
4-iodophenyl
d
4-dimethylaminophenyl
4-diethylaminophenyl
4-nitrophenyl
1.40
>71
0.04 1625
4-methoxyphenyl
4-ethoxyphenyl
>100
>100
60
0.03 >3333
4-methyl 92 and 4-chloro 93 thionocarbonates were
practically inactive (EC50 > 50 µM), whereas 4-nitro
carbamate 94 (EC50 ) 0.5 µM) was 12.5 less potent than
4-nitro C-TC 44. The lack of activity of 92, 93, and 95
7.0
>14.3
trovirdine
0.02 3000
a
Data represent mean values for three separate experiments.
Variation among triplicate samples was less than 10%. b Com-
pound concentration required to reduce the viability of mock-
infected cells by 50%, as determined by the MTT method.
(
data not shown), the last embodying the thiocarbamic
nitrogen into an imidazole nucleus, emphasizes the
crucial role played by the NH group and, to a lesser
extent, by the thione sulfur in binding to the RT (see
Molecular Modeling).
c
Compound concentration required to achieve 50% protection of
MT-4 cell from HIV-1 induced cytopathogenecity, as determined
by the MTT method. d Selectivity index: CC50/EC50 ratio.
C-TC 4-iodo 41 and O-TC 4-iodo 69 were also tested
in enzyme assays against recombinant wt RT (rRT) and
rRTs carrying clinically relevant NNRTI resistance
mutations (K103R, Y181C, and K103N + Y181C);
evafirenz and trovirdine were used as reference com-
pounds (Table 8). Assays against both wt and mutant
enzymes proved that TCs, like efavirenz and trovirdine,
targeted the HIV-1-RT. The IC50 values of 41 and 69
were 4.5- and 13-fold lower than the respective EC50
values (Tables 3 and 4), thus making more evident the
different potency of the test compounds in enzyme- and
cell-based assays. Perhaps the principal factor that can
account for these results is influenced by the nature of
the rRT; it would be unable to adequately mimic the
native enzyme, being generally a p66-p66 dimer.
Notably, similar differences between enzymatic and cell
culture activity have also been observed for ATC deriva-
tives.27
was found in O-TCs 4-methyl 74, 4-chloro 75, and
4
-nitro 76, even though their decrease in activity was
greater when compared to the ring-closed counterparts.
C-TCs 83-89 and O-TCs 74-76 were tested as
racemates, and no attempt was made to resolve them.
As a matter of fact, judging from the range of antiviral
potency, resolution of the racemates might not result
in a significant activity increase of the resultant enan-
tiomers. Interestingly, the activity trend due to modi-
fication of the ethyl linker of TCs is similar to the
31
activity trend obtained in the PETT series. Therefore,
the unbranched ethyl linker is optimal for the activity
of both TC and PETT series.
Table 7 indicates that isosteric replacement of the NH
group or thione sulfur of the thiocarbamic moiety with
oxygen (92, 93, and 94 are isosteres of 31, 39, and 44,
respectively) was not beneficial for activity. Thus,

3
868 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Ranise et al.
Table 5. Effects of Modification and Omission of the Phthalimide Phenyl Ring on Cytotoxicity and Anti-HIV-1 Activity (TCs 47-50,
7-81)a
7
compd
A
W
CC50 b (µM)
EC50 c (µM)
SI d
4
4
4
5
7
7
7
8
8
7
3-methylphthalimido
Cl
NO2
Cl
>100
71
>100
80
>100
>100
>100
>100
>100
60
0.05
0.07
0.025
0.01
2.4
>2000
1014
8
9
0
7
8
9
0
1
3-methylphthalimido
4-methylphthalimido
>4000
8000
4-methylphthalimido
NO2
Cl
succinimido
>42
cis-1,2,3,6-tetrahydrophthalimido
cis-1,2,3,6-tetrahydrophthalimido
cis-5-norbornene-endo-2,3-dicarboximido
cis-5-norbornene-endo-2,3-dicarboximido
Cl
9.4
>10.6
Br
2.3
68
>100
0.02
>43
>1.5
Cl
NO2
trovirdine
3000
a
b
Data represent mean values for three separate experiments. Variation among triplicate samples was less than 10%. Compound
c
concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method. Compound concentration
required to achieve 50% protection of MT-4 cell from HIV-1 induced cytopathogenecity, as determined by the MTT method. d Selectivity
index: CC50/EC50 ratio.
Table 6. Effect of Modification of the Aliphatic Spacer on Cytotoxicity and Anti-HIV-1 Activity (C-TCs 82-87, O-TCs 74-76)a
compd
B1/2
n
R1
R2
Ar1
CC50 b (µM)
EC50 c (µM)
SI d
8
8
8
8
8
8
8
8
9
9
7
7
7
2
B1
B1
B1
B1
B1
B1
B1
B1
B1
B1
B2
B2
B2
0
1
1
1
1
1
1
1
2
2
1
1
1
H
4-nitrophenyl
4-chlorophenyl
4-nitrophenyl
4-tolyl
4-cyanophenyl
4-chlorophenyl
4-bromophenyl
4-nitrophenyl
phenyl
4-nitrophenyl
4-tolyl
4-chlorophenyl
4-nitrophenyl
23
44
>23
0.05
0.6
48
e
e
e
e
e
e
e
3
4
5
6
7
8
9
0
1
4
5
6
CH3
CH3
H
H
880
H
>100
>100
>100
>100
>100
>100
>100
g100
>100
>100
>100
60
>167
CH3
CH3
CH3
CH3
CH3
H
>2.1
H
36
>2.7
>58.8
>43.5
>11.6
H
1.7
2.3
8.6
>100
19
H
H
H
H
H
g5.3
>2.4
>20
>7.7
3000
e
e
e
H
CH3
H
41
CH3
CH3
5.0
13
H
trovirdine
0.02
a
b
Data represent mean values for three separate experiments. Variation among triplicate samples was less than 10%. Compound
c
concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method. Compound concentration
d
required to achieve 50% protection of MT-4 cell from HIV-1 induced cytopathogenecity, as determined by the MTT method. Selectivity
e
index: CC50/EC50 ratio. TCs tested as racemic mixtures.
Unlike efavirenz, 41, 69, and trovirdine were inactive
against the double mutant. However, 41 showed the
same level of activity as efavirenz against the K103R
mutant (IC50 ) 2.3 vs 2.1 µM, respectively), while 69
and trovirdine were inactive (IC50 > 20 µM). Conversely,
turned out to be generally unsusceptible to the test
compounds (results not shown), with the exception of
44 that showed a weak activity against the K103N
mutant with an EC50 value of 20 µM.
6
9 was 3-fold more active than trovirdine against
Molecular Modeling
Y181C, while 41 was inactive. Probably the different
resistance profile of 41 and 69 against the above single
mutations correlates with the preservation or opening
of the imide ring.
To guide the lead optimization strategy and rational-
ize the SARs, computational studies were performed to
construct a docking model of the TCs into the HIV-1
RT NNBS according to the procedure previously de-
scribed for the ATCs.29 Briefly, low-energy conformers
of the TCs, obtained by Monte Carlo random search,
were docked (Dock 4.0) in the NNBS. The X-ray coor-
dinates of the crystal complex of PETT-1 (Chart 2A)
with HIV-1 RT (PDB code 1DTQ)16 were employed after
erasing the PETT derivative from the complex. The
resulting RT-TC complexes were energy-minimized by
simulated annealing, followed by Powell minimization.
The following features of the TC docking models are
worthy to be outlined.
The doses required to reduce HIV-1 p24 antigen levels
by 90% were determined for para-substituted C-TCs 31,
37-41, and 44 in comparison with trovirdine (Table 9).
The EC90 values of 31, 39, 40, and 44 confirmed their
highest activity. Finally, 31, 37-40, and 44 were tested
in cell-based assays against HIV-1 strains, carrying the
clinically relevant Y181C mutation in comparison with
the reference compound trovirdine (Table 10). The data
indicate that the Y181C mutant was significantly
inhibited by C-TCs 31, 37, 39, 40, 44. In contrast, the
K103R and the K103N + Y181C double mutant strains

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3869
Table 7. Effects of Isosteric Modifications (NH/O and O/S) on
As shown in Figure 2A, the RT-12 complex is
stabilized by a hydrogen bond, involving the Lys101
main chain carbonyl and the NH group. Another polar
interaction occurs between one of the imidic oxygen
atoms and the carboxylic group of Glu138 (distance of
a
Cytotoxicity and Anti-HIV-1 Activity
3
.2 Å) from the p51 subunit [thereafter Glu138 (B)]. This
interaction prompted selection of alcohol building blocks
see Chart 3A) featured by a five-membered imide ring.
(
1
6
compd
G
Z
W
CC50 b (µM) EC50 c (µM)
SI d
Moreover, ring A1, like ring A of PETT-1, establishes
hydrophobic contacts with Val106 and π-π interactions
with Tyr181, Tyr188, and Phe227, while ring B1 inter-
acts with the Leu100, Lys103, Pro236, and Tyr318 side
chains. Furthermore, the ethyl linker is involved in
hydrophobic contacts with the Val179 side chain and
the sulfur atom has van der Waals contacts with the
Lys101 backbone. The docking model of 11 explains why
the replacement of ring B1 with a cyclohexyl nucleus
caused a 4-fold increase of activity in comparison with
e
92
S
S
O
O
O
CH3
Cl
>100
>100
>100
60
>100
>100
f
9
3
4
g
9
NH NO2
0.5
0.02
>200
3000
trovirdine
a
Data represent mean values for three separate experiments.
Variation among triplicate samples was less than 10%. Com-
b
pound concentration required to reduce the viability of mock-
infected cells by 50%, as determined by the MTT method.
c
Compound concentration required to achieve 50% protection of
MT-4 cell from HIV-1 induced cytopathogenecity, as determined
d
e
by the MTT method. Selectivity index: CC50/EC50 ratio. Isostere
12. In the RT-11 complex the larger volume of the
of C-TC 31. Isostere of C-TC 39. g Isostere of C-TC 44.
f
cyclohexyl nucleus would better fill the spacious region
hosting ring B1. This ring is likely to be engaged in
extensive hydrophobic interactions with Leu100, Lys101,
Val106, His235, Pro236, and Tyr318. Moreover, the
complex is stabilized by a number of van der Waals
contacts between the thiocarbonyl sulfur atom and the
NH group of the Lys101 main chain. Another strategy
to efficiently fill the region hosting ring B1 is to
introduce substituents onto the para position of this
ring. Therefore, a greater number of para-substituted
isothiocyanates were selected as building blocks in the
TC synthesis. The models of the RT-39 and RT-44
complexes show that the 4-chloro and 4-nitro substitu-
ents strongly interact with Phe227, Leu234, His235,
Pro236, and Tyr318 and that van der Waals contacts
between the thiocarbonyl group and the Lys101 back-
bone are strengthened. Congener 4-iodo 41 was 2-fold
more potent than 4-chloro 39 probably because in the
RT-41 complex the bulkier iodine atom makes better
contacts with the above amino acid residues. Notably,
the other para substituents such as methyl, ethyl,
methoxy, and ethoxy groups cause different positions
of the inhibitor within the NNBS, due to rotation of ring
B1, relative to its orientation in the hypothetical model
of 12. Thus, in the RT-45 complex, rotation of ring B1
of about 54° provides strong interactions between the
methoxy group and Phe227 and His235, with the ether
oxygen establishing a polar interaction with the Tyr318
OH group. Conversely, in the RT-46 complex, because
the rotation of ring B1 is about 78°, the NH function is
unable to form a hydrogen bond to the Lys101 main
chain carbonyl and the 4-ethoxy group, unlike the
Table 8. IC50 of C-TCs 41 and O-TC 69 against Wild Type and
Single and Double Mutant Recombinant RTs
IC50 a (µM)
compd
wtIIIB
K103R
Y181C
K103N + Y181C
4
6
1
0.09
1.7
0.014
1.03
2.3
>20
2.1
>20
4.8
0.02
15
>20
>20
0.5
9
efavirenz
trovirdine
>20
>20
a
Compound concentration required to inhibit in enzyme assays
the HIV-1 recombinant RT activities by 50%.
Table 9. EC90 Values of Selected C-TCs against wt HIV-1
compd
31
EC90 a (µM)
0.015
0.1
0.1
0.04
0.015
0.04
0.03
0.015
3
3
3
7
8
9
40
41
44
trovirdine
a
Compound concentration required to reduce the amount of p24
by 90% in virus-infected C8166 cultures.
Table 10. EC50 Values of Selected C-TCs against the Y181C
a
Mutant in Cell-Based Assays
EC50 b (µM)
compd
3
3
1
1.8
5.9
38
7
38
3
4
4
9
0
4
2.3
2.4
6.0
1.2
trovirdine
4-methoxy group, does not interact with Phe227, His235,
a
Data represent mean values for three separate experiments.
and Tyr318. The docking models of several ortho- and
meta-substituted C-TCs, such as 17, 19, 21, 27, pre-
dicted that both the ortho and the meta groups are not
in proximity to amino acid residues suitable for ad-
ditional interactions (data not shown). The effects on
antiviral activity due to the introduction of a methyl
group at the 3- and 4-position of the phthalimide ring
are readily explained by the docking model of TCs 47-
b
Variation among triplicate samples was less than 10%. Com-
pound concentration required to achieve 50% protection of MT-4
cell from HIV-1 induced cytopathogenecity, as determined by the
MTT method.
oxygens and the Glu138(B) carboxylic group. Con-
versely, in the RT-49 and RT-50 complexes the
4-methyl group is in proximity to be engaged in hydro-
phobic interactions with the highly conserved Trp229.
Conformational analyses offered a possible explanation
for the great difference in activity between 83, 84 and
85-89, carrying a methyl group in positions 2 and 1 of
5
0. The minor potency of 47 and 48 is explained by theR
steric clash of the 3-methyl group with the Trp229 C
atom, which forces the inhibitors in an orientation that
weakens the polar interaction between one of the imidic

3
870 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Ranise et al.
the ethyl spacer, respectively. The Macromodel program
DRIVE module) predicted 83 and 87, selected as the
representatives of the above TCs, to exist in three low-
energy conformations, i.e., one anti and two gauche
mum potency was reached by introducing an additional
methyl group at the 4-position of the phthalimide
framework in the p-nitro C-TC 44. In terms of resistance
against the clinically relevant mutations, the major
molecular flexibility of the TCs with respect to PETT
derivatives did not give the eagerly awaited results.
Nevertheless, the significant activity of 41 (IC50 ) 2.3
µM) against the K103R mutant in enzyme assays and
of 31, 37, 39, 40, and 44 against the Y181C in cell-based
assays offers a stimulus for the design of new TC
analogues with better resistance profile.
(
(data not shown). Calculations indicated the anti con-
former of 83 to be about 0.5 kcal/mol lower in energy
with respect to the anti conformer of 87. There is a
correlation between the more potent 83 and the prefer-
ence for the anti conformation. The lack of activity of
9
2 and 93 is probably due to the replacement of the NH
group with oxygen, causing the loss of the H-bond
involving the NH group and the Lys101 main chain
carbonyl. The lower activity of carbamate 94 compared
with 44 can be explained by the replacement of the
sulfur with a less polarizable oxygen, which would be
expected to create less favorable dispersion interactions.
Furthermore, the energy required to desolvate a car-
bonyl will be greater than for a thione group, which
cannot form such strong interactions with water.19 On
the other hand, the NH group of 44, adjacent to a
thiocarbonyl group, is more acidic than the NH group
of 94, close to a carbonyl (the calculated pKa of 44 is
about 100 times greater than the pKa of 94; see Chart
Experimental Section
Chemistry. Starting alcohols 1, 2, 7, 10, anhydrides I-V,
and other reagents such as 60% sodium hydride dispersion in
mineral oil, 2-aminoethanol, (()-2-amino-1-propanol, (()-1-
amino-2-propanol, isothiocyanates, p-chloro thionoformates,
and p-tolyl thionoformates, phenyl isocyanate, 4-nitrophenyl
isocyanate, and thiocarbonyldiimidazole were purchased from
Chiminord and Aldrich Chemical, Milan (Italy). Solvents
(
THF, pyridine, DMF) were reagent grade. DMF was dried on
1
molecular sieves (5Å, /16 in. pellets). Organic solutions were
dried over anhydrous sodium sulfate and concentrated using
a rotatory evaporator operating at reduced pressure of about
1
5-20 Torr. TLC systems for routine monitoring of reaction
4
). Therefore, the thiocarbamic NH group is a better
mixtures and for confirming the purity of analytical samples
employed aluminum-backed silica gel plates (Merck DC-
Alufolien Kieselgel 60 F254) with chloroform or chloroform-
methanol as developing solvents. Developed plates were
visualized by UV light and iodine. The parallel solution-phase
chemistry was performed by using a Carousel Reaction Station
H-bond donor group than the carbamic NH group, and
as consequence, it will form stronger hydrogen bonds.
These observations are consistent with the H-bond
donor role proposed for the NH group to the Lys101
carbonyl (Figure 2A). Interestingly, also in the docking
model of 51 (Figure 2B), the NH group forms an H-bond
with the Lys101 carbonyl and the sulfur atom has van
der Waals contacts with the Lys101 backbone. The
N-phenyl ring makes hydrophobic interactions with
Val106, His235, Pro236, and Tyr318, while the phthalyl
moiety is in contact with Leu100, Tyr181, and Tyr188.
The carbamoyl-ethyl linker has a number of van der
Waals interactions with Val179, Tyr181, Tyr188, Val189,
and Gly190, while the carboxylic group interacts with
the Leu100 and Tyr181 side chains.
Finally, the binding model proposed for 12 also helps
to partially rationalize the activity decrease of the TCs
tested against NNRTI resistant strains. The decrease
of activity of C-TCs 31, 37-41, 44 in cell-based assays
and of O-TC 69 in an enzyme-based assay against the
Y181C mutant emphasizes the role of Tyr181 in the
binding to the TCs. This mutation causes a dramatic
change from a hydrophobic environment to a hydrophilic
environment. According to the model, one of the most
favorable hydrophobic contacts is due to the π-π
stacking interactions of Tyr181 and ring A1. Mutation
of Tyr181 to nonaromatic cysteine would abolish most
of these hydrophobic contacts. Overall, information
regarding the hypothetical TC binding mode proved to
be useful in selecting alcohol and isothiocyanate build-
ing blocks for the TC library synthesis. Moreover, the
relevant predictions of the TC models are supported by
the reported SARs.
(
Radleys Discovery Technologies, Italian distributor: StepBio,
Bologna).
Melting points were determined on a Fisher-Johns ap-
paratus and are uncorrected. Microanalyses were performed
on an EA 1110 elemental analyzer from FISON Instruments
(
Milan, Italy). The IR spectra were recorded on a Perkin-Elmer
1
398 spectrometer, using KBr disks. The H NMR spectra were
recorded in CDCl on a Varian Gemini 200 instrument.
3
Chemical shifts were reported in δ (ppm) units relative to the
internal reference tetramethylsilane, and the splitting patterns
were described as follows: s (singlet), d (doublet), t (triplet),
m (multiplet), and bs (broad singlet). The first-order values
reported for coupling constants were given in hertz. The
synthesis of trovirdine was performed according to the pub-
3
1
lished procedure. All the new compounds reported in this
paper gave satisfactory spectral and microanalysis data.
General Procedure for the Preparation of Starting
Alcohols 3, 4, 8, 9. A mixture of 2-aminoethanol (0.7 g, 12
mmol) and the proper anhydride (3-methylphthalic anhydride
I, 4-methylphthalic anhydride II, cis-1,2,3,6-tetrahydrophthal-
ic anhydride IV, or cis-5-norbornene-endo-2,3-dicarboxylic
anhydride V; 10 mmol) was heated at 175-180 °C under
stirring for 2 h. After cooling, the reaction mixture was
dissolved in CH
2
Cl
2
and washed with water (3 × 50 mL). The
organic layer was dried and evaporated under reduced pres-
sure to give the desired product, which was purified by
crystallization from diethyl ether-dichloromethane (6:1).
2-(2-Hydroxyethyl)-4-methyl-1H-isoindole-1,3(2H)-di-
one (3): 1.83 g (89%), mp 109-110 °C.
2-(2-Hydroxyethyl)-5-methyl-1H-isoindole-1,3(2H)-di-
one (4): 1.39 g (68%), mp 94-96 °C.
(
3aR,7aS)-2-(2-Hydroxyethyl)-3a,4,7,7a-tetrahydro-1H-
isoindole-1,3(2H)-dione (8): 1.19 g (61%), mp 78-80 °C.
(
3aR,4S,7S,7aS)-2-(2-Hydroxyethyl)-3a,4,7,7a-tetrahy-
Conclusion
dro-1H-4,7-methanoisoindole-1,3-dione (9): 0.97 g (47%),
mp 91-92 °C.
In this study we have described a new class of NNRTI
TC isosteres of PETT derivatives. The biological results
show that the C-TCs bearing para substituents on the
N-phenyl ring, such as methyl, iodo, chloro, bromo,
nitro, methoxy, were very potent inhibitors, but maxi-
Synthesis of (()-2-(2-Hydroxy-1-methylethyl)-1H-isoin-
dole-1,3(2H)-dione(5). A mixture of III (1.48 g, 10 mmol) and
d,l-alanilol (0.7 g, 12 mmol) was heated at 140-150 °C under
stirring for 2 h. After cooling, the mixture was dissolved in
methylene chloride and washed with water (3 × 50 mL). The

Thiocarbamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3871
organic layer was dried and evaporated under reduced pres-
O-{2-[(3aR,7aS)-1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-
isoindol-2-yl]ethyl} 4-chlorophenylthiocarbamate (78):
1.46 g (40%), mp 118-119 °C.
O-{2-[(3aR,7aS)-1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-
isoindol-2-yl]ethyl} 4-bromophenylthiocarbamate (79):
2.0 g (49%), mp 155-156 °C.
O-{2-[(3aR,4S,7S,7aS)-1,3-Dioxo-1,3,3a,4,7a-hexahydro-
2H-4,7-methanoisoindol-2-yl]ethyl} 4-chlorophenylthio-
carbamate (80): 2.56 g (68%), mp 156-158 °C.
O-{2-[(3aR,4S,7S,7aS)-1,3-Dioxo-1,3,3a,4,7a-hexahydro-
2H-4,7-methanoisoindol-2-yl]ethyl} 4-nitrophenylthio-
carbamate (81): 3.10 g (80%), mp 199-201 °C.
2 2 2
sure to yield 5, which was crystallized from Et O-CH Cl (6:
1
): 1.27 g (62%), mp 104-106 °C.
Synthesis of (()-2-(2-Hydroxypropyl)-1H-isoindole-1,3-
2H)-dione (6). A mixture of III (1.48 g, 10 mmol) and
-amino-2-propanol (0.7 g, 12 mmol) was heated at 160-170
C under stirring for 2 h. After cooling, the mixture was
dissolved in methylene chloride and washed with water (3 ×
0 mL). The organic layer was dried and evaporated under
(
1
°
5
reduced pressure to yield 6, which was crystallized from
chloroform: 1.03 g (50%), mp 90-92 °C.
General Procedure for the Preparation of Library 1.
To an ice-cooled, stirred solution of the proper alcohol (1-7,
General Procedure for the Preparation of Compounds
9
(
2 and 93. To a solution of 2 (1.91 g, 10 mmol) and DMAP
1
0 mmol) and isothiocyanate (10 mmol) in dried DMF (15 mL),
2.2 g, 18 mmol) in acetonitrile (25 mL), p-chlorophenyl
a 60% sodium hydride dispersion in mineral oil (0.45 g, ∼10
mmol) was added in a single portion. After 1 h, the mixture
was allowed to stand at room temperature and stirring was
prolonged for 24 h. On addition of water (50 mL) and
ammonium chloride (7 g) as a powder, the C-TCs precipitated
were filtered off. The crude solids were dissolved in dichlo-
romethane, and the resulting solutions were filtered through
a pad of Florisil (5 cm × 2 cm). After the solutions were
evaporated in vacuo, the residues were crystallized from
suitable solvents (Table 1) to afford TCs 11-13, 21, 24-26,
chlorothionoformate or p-tolyl chlorothionoformate (10 mmol)
was added in a single portion. The mixture was stirred at room
temperature for 24 h. Following addition of water (50 mL),
the precipitate was filtered off and dissolved in chloroform.
The organic solution was then washed with water (3 × 20 mL),
dried, and evaporated under reduced pressure. The oily residue
was crystallized from a mixture of diethyl ether and petroleum
ether to afford the desired product.
O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] O-(4-
tolyl)thiocarbonate (92): 3.11 g (91%), mp 106-108 °C.
O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl] O-(4-
chlorophenyl)thiocarbonate (93): 2.54 g (78%), mp 124-
126 °C.
Synthesis of O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-
2-yl)ethyl] 4-Nitrophenylcarbamate (94). 2 (1.0 g, 5 mmol)
and 4-nitrophenyl isocyanate (0.9 g, 5 mmol) were dissolved
in a 1:1 mixture of pyridine (15 mL) and DMF (15 mL) and
then heated at 140 °C for 5 h. Following addition of water (150
mL), the precipitate was filtered and dissolved in a hot
methanol-acetone mixture (1:3) from which 94 crystallized
as a yellow solid: 0.30 g (17%), mp 234-236 °C.
2
8, 29, 31, 32, 34-44, 46-50, 79, 82-91. Upon acidification
(
pH 0) of the reaction mother liquors with 2 M HCl (20 mL),
the O-TCs precipitated as solids or separated as oils, which
crystallized on standing. The crude solids were then filtered
off and purified by crystallization from proper solvents (Table
2) to afford O-TCs 51, 55, 58, 61-63, 65-70, 72, 74-76.
General Procedure for the Preparation of Library 2.
A 60% sodium hydride dispersion in mineral oil (0.44 g, 10
mmol) was added in a single portion to a solution of alcohol 2
(
1.91, 10 mmol) and proper isothiocyanate (10 mmol) in
anhydrous pyridine (15 mL). The reaction mixture was stirred
at room temperature for 15 min and then heated at 60-65 °C
for 3 h. After addition of water (50 mL) and NH Cl (7 g) as a
4
Synthesis of O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-
-yl)ethyl] 1H-Imidazole-1-carbothioate (95). 2 (1.52 g, 8
2
powder, the C-TCs precipitated as solids or, occasionally,
separated as oils. The solids were filtered off and dissolved in
dichloromethane, and the resulting solutions were dried
mmol) and thiocarbonyldiimidazole (1.8 g, 10 mmol) were
mixed and ground in a mortar, prolonging the manual mixing
for 15 min. Then, the paste was washed with water and
dissolved in chloroform. The solution was dried and evaporated
under reduced pressure. The oily residue was crystallized from
ethyl ether to give 95: 2.17 g (72%), mp 143-145 °C.
(
solution a). The oils were extracted with dichloromethane (30
mL × 2), and the combined extracts were washed with 2 N
HCl (20 mL × 4), dried, and filtered through a pad of Florisil
(
5 cm × 2 cm) (solution b). Then, the dichloromethane solutions
a and b were evaporated under reduced pressure, and the
resulting residues were crystallized from the suitable solvents
to afford C-TCs 14-20, 22, 27, 30, 33, 45 (Table 1). On
treatment of the reaction mother liquors with 2 N HCl (80 mL),
the products, precipitated as solids, were worked up according
to the procedure for library 1 to give O-TCs 52-54, 56, 57,
Acknowledgment. This work was supported by
MURST (Cofinanziamento Nazionale), CNR (Rome),
and Ministero Italiano della SalutesIstituto Superiore
di Sanit a` (Grant No. 40D.46). The authors thank Mr.
O. Gagliardo for the microanalyses and Mr. F. Tuberoni
and Dr. C. Rossi for the IR and NMR spectra.
5
9, 60, 64, 71, 73 (Table 2).
Synthesis of O-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-
2
-yl)ethyl] 3-Tolylthiocarbamate (23). Phosphorus pentox-
Supporting Information Available: Spectroscopic (IR
ide (2.84 g, 20 mmol) was added to a solution of 57 (3.58 g, 10
1
and H NMR) and microanalytical data for all synthesized
mmol) in dried DMF (40 mL). The resulting suspension was
compounds described in the Experimental Section and experi-
mental procedures for molecular modeling and biological
evaluation. This material is available free of charge via the
Internet at http://pubs.acs.org.
2 3
heated at 65 °C for 5 h and then treated with 1 M Na CO (50
mL). The solid precipitated was filtered, washed with water,
and crystallized from a mixture of dichloromethane and
methanol to yield 23: 0.71 g (21%), mp 102-104 °C.
General Procedure for Preparation of TCs 77-81. A
References
6
0% sodium hydride dispersion in mineral oil (0.44 g, 10 mmol)
(
1) Jonckheere, H.; Anne, J.; De Clercq, E. The HIV-1 reverse
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was added to an iced-cooled solution of 8 (1.95 g, 10 mmol) or
0 (1.43 g, 10 mmol) in dried DMF (15 mL) or of 9 (2.07 g, 10
1
mmol) in anhydrous THF (15 mL). As soon as hydrogen
evolution ceased, the proper isothiocyanate (10 mmol) was
added to the mixture, which was allowed to react at room
temperature for 20 h. After treatment with water (50 mL) and
(2) De Clercq, E. New developments in anti-HIV chemotherapy.
Farmaco 2001, 56, 3-12.
(
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