A Versatile and Practical Synthesis toward the Development of Novel HIV-1 Integrase Inhibitors

Article abstract of DOI:10.1002/cmdc.201000510

As a continuation of our previous work, which resulted in the identification of a new hit compound as an HIV-1 integrase inhibitor, three novel series of salicylic acid derivatives were synthesized using three versatile and practical synthetic strategies and were assayed for their capacity to inhibit the catalytic activity of HIV-1 integrase. Biological evaluations revealed that some of the synthesized compounds possess good inhibitory potency in enzymatic assays and are able to inhibit viral replication in MT-4 cells at low micromolar concentrations. Finally, docking studies were conducted to analyze the binding mode of the synthesized compounds within the DNA binding site of integrase in order to refine their structure-activity relationships. Easy as A, B, C: Three new series of salicylic acid derivatives were designed and synthesized to investigate their activity toward HIV-1 integrase. Some of these compounds were obtained with microwave-assisted procedures developed and optimized in our research group, which allowed us to rapidly generate several final compounds of high purity.

Full text of DOI:10.1002/cmdc.201000510

DOI: 10.1002/cmdc.201000510
A Versatile and Practical Synthesis toward the
Development of Novel HIV-1 Integrase Inhibitors
Marta Rinaldi,[a] Cristina Tintori,[a] Luigi Franchi,[a] Giulia Vignaroli,[a] Anna Innitzer,[a]
Silvio Massa,[a] Josꢀ A. Estꢀ,[d] Encarna Gonzalo,[d] Frauke Christ,[b] Zeger Debyser,[b] and
Maurizio Botta*[a, c]
As a continuation of our previous work, which resulted in the
identification of a new hit compound as an HIV-1 integrase in-
hibitor, three novel series of salicylic acid derivatives were syn-
thesized using three versatile and practical synthetic strategies
and were assayed for their capacity to inhibit the catalytic ac-
tivity of HIV-1 integrase. Biological evaluations revealed that
some of the synthesized compounds possess good inhibitory
potency in enzymatic assays and are able to inhibit viral repli-
cation in MT-4 cells at low micromolar concentrations. Finally,
docking studies were conducted to analyze the binding mode
of the synthesized compounds within the DNA binding site of
integrase in order to refine their structure–activity relation-
ships.
Introduction
Acquired immunodeficiency syndrome (AIDS), caused by the
human immunodeficiency virus (HIV), has become a major epi-
demic, infecting more than 39 million people worldwide.
State-of-the-art highly active antiretroviral therapy (HAART)
against this lethal disease is based on cocktails of different
drug classes that target various steps of the HIV replication
cycle. Before late 2007, four different drug classes had been
approved by the US Food and Drug Administration (FDA) for
the treatment of AIDS: 1) nucleoside reverse transcriptase in-
hibitors, 2) non-nucleoside reverse transcriptase inhibitors,
3) protease inhibitors, and 4) fusion inhibitors.[1,2] These drugs
have proven successful for the treatment of HIV-1, but consid-
ering their adverse side effects and the emergence of resistant
mutant strains, new therapies against the HIV virus are urgent-
ly required.[3,4] In October 2007, the FDA approved raltegravir
(MK-0518), the first example of a drug active against HIV-1 inte-
grase (IN), validating IN as a new target in the field of anti-HIV
drug research.[5–7]
virtual screening protocol[14,15] that allowed us to identify a
new hit (compound 17),[16] which proved to be active against
both the integration activity of IN and HIV-1 replication at the
micromolar level. Furthermore, the synthesis and biological
evaluation of a small family of structurally related compounds
were performed.[17]
We found that some of the synthesized compounds exhibit
inhibitory potency against IN and antiviral activity in cellular
assays at micromolar concentrations. The biological results in-
IN is an essential enzyme for the integration of viral DNA
into the host cell genome. The process occurs in two spatially
and temporally different steps known as 3’ processing and
strand transfer. The 3’ processing event occurs in the cyto-
plasm, where IN binds the viral DNA and then removes a dinu-
cleotide from each strand at the 3’ end adjacent to a con-
served CA sequence. The formed complex is then transported
into the nucleus, where the strand transfer reaction takes
place, and the 3’ ends of the viral DNA are covalently linked to
the 5’ ends of the host cell DNA.[8–10] The essential multifunc-
tional role of IN in the viral replication cycle and the absence
of a counterpart in host cells make this enzyme an interesting
target for the development of new anti-HIV drugs. During the
past few years, many studies have been focused on the discov-
ery of novel potential integrase inhibitors.[11–13] In this context,
we recently disclosed our own work in the field, presenting a
[a] Dr. M. Rinaldi, Dr. C. Tintori, Dr. L. Franchi, Dr. G. Vignaroli, Dr. A. Innitzer,
Prof. S. Massa, Prof. M. Botta
Dipartimento Farmaco Chimico Tecnologico
Universitꢀ degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena (Italy)
Fax: (+39)0577234333
E-mail: botta@unisi.it
[b] Dr. F. Christ, Prof. Z. Debyser
Molecular Medicine, Katholieke Universiteit Leuven
Kapucijnenvoer 33, 3000 Leuven, Flanders (Belgium)
[c] Prof. M. Botta
Biotechnology, College of Science and Technology
Temple University, Biolife Science Building
Suite 333, 1900 N. 12thStreet, Philadelphia, PA 19122 (USA)
[d] Prof. J. A. Estꢁ, Dr. E. Gonzalo
Laboratori de Retrovirologia irsiCaixa
Hospital Universitari Germans Trias i Pujol
Universitat Autꢂnoma de Barcelona
Ctra. Del Canyet s/n, 08916 Badalona (Spain)
ChemMedChem 2011, 6, 343 – 352
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
343

M. Botta et al.
MED
dicate that: 1) the acidic moiety is fundamental for the activity;
2) the potency of these compounds could be improved by the
introduction of hydroxy groups on the phenyl moiety; 3) the
introduction of substituents at position 3 of the furan ring
leads to compounds with inferior activity; and 4) the thiobarbi-
turic acid group can be substituted with other heterocyclic
moieties, such as rhodanine or thiohydantoin, without loss of
activity.
5 of the furan ring, keeping the 2-salicylic furan moiety fixed.
The new compounds A, B, and C allowed us to explore wheth-
er molecules in these series engage in stronger interactions
with the 140–149 loop than the previously reported hit com-
pound 17. Concerning series A derivatives, we replaced the
heterocycle of compound 17 with a benzyl moiety linked di-
rectly to the furan ring (general structure A, Figure 2), keeping
the salicylic biaryl portion of the molecule fixed, which was
suggested to be crucial for activity.[15] This modification was at-
tempted to better investigate two aspects: 1) the importance
of the hydrogen bond interaction established by the hit 17
with the backbone carbonyl group of Gln148 (determining
whether it is essential for the inhibition of IN activity); and
2) the influence of more extensive hydrophobic contacts with
the 140–149 loop on receptor binding . Furthermore, we de-
signed two series of 2-salicylic furan derivatives containing a
substituted rhodanine moiety at position 5 of the furan ring
(general structures B and C, Figure 2). The aim of this modifica-
tion was to obtain elongated molecules capable of establish-
ing more extensive interactions within the binding site, espe-
cially with Tyr143, which was shown to be an important resi-
due for IN binding with DNA. Various substituents were intro-
duced at two different positions of the rhodanine ring to ex-
plore the space around the heterocycle. Biological results
showed that among the benzyl derivatives, only one com-
pound possesses weak activity against IN. In contrast, the ap-
proach to the substituted rhodanines was more successful; in
Docking studies with hit compound 17 (Figure 1) suggest
that the salicylic ring is located in the region usually occupied
by the phosphate groups of viral DNA, where it establishes
Figure 1. Schematic representation of the binding mode of compound 17.
strong electrostatic interactions with Lys159. Interestingly,
Lys159 is involved in the specific recognition of viral DNA by
integrase, and alteration of this contact has been proposed as
the mechanism of action of 3’-processing inhibitors.[13] On the
other hand, the thiobarbituric moiety is directed toward a very
flexible loop containing amino acid residues 140–149, and its
NH group interacts with the backbone carbonyl group of
Gln148. The 140–149 loop has been proposed to be involved
fact, most of the compounds with general structure
C
(Figure 2) showed good activity results in the IN inhibition
assay. Moreover, docking studies with the new synthesized
compounds are in agreement with biological data and help to
refine the structure–activity relationship analysis.
in a conformational modification of the enzyme after binding Results and Discussion
with DNA, and consequently, stabilization of the viral DNA–IN
complex.[13,18] Through mutagenesis and photo-crosslinking
Chemistry
studies, several amino acids that are involved in the DNA bind-
ing of IN, in particular Tyr143, have been identified, although
these do not belong directly to the catalytic site of the
enzyme.[19,20]
The final compounds with general structure A were obtained
by starting from the commercially available methyl-4-iodosaly-
cilate 1, which was used in a Suzuki coupling with the 2-furan-
boronic acid 2 (Scheme 1). The coupled product 3 was then
hydrolyzed with an aqueous solution of sodium hydroxide to
afford the free acid analogue 4, which was protected with
TIPSCl. The protected compound 5 was treated with various
Grignard reagents: 4-fluorophenylmagnesium bromide 6a, 3-
fluorophenylmagnesium bromide 6b, and 3-phenylphenylmag-
nesium bromide 6c. In the case of the 4-fluorobenzyl deriva-
tive 7a, the protecting groups were removed directly with
TBAF to yield the final com-
In this work, pursuing our goal to identify novel HIV-1 IN in-
hibitors capable of blocking the IN–viral DNA interaction, and
taking into account structure–activity relationship studies per-
formed on the previous set of compounds,[17] we synthesized
three new series of second-generation salicylic acid derivatives
(A, B, and C in Figure 2). These compounds were designed to
investigate the influence of synthetic derivatizations at position
pound 8a; in the other cases,
compounds 7a–c were simulta-
neously reduced and deprotect-
ed with TMSCl and NaI to obtain
the final compounds 9a–c. In
most cases, synthetic steps pro-
ceeded with acceptable yield.
Figure 2. General structures of the three novel series of compounds.
However, an optimized proce-
344
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 343 – 352

HIV-1 Integrase Inhibitors
by changing only the amine employed. We used morpholine,
thiomorpholine, and N-methylpiperazine as secondary amines,
while benzylamine was used as the primary amine (Table 1).
After dissolving all reagents in ethanol, heating at 1508C for
20 min by microwave irradiation is required to effect comple-
tion. After acidic workup, the desired products are collected by
filtration from ethanol.
The other series of substituted rhodanine derivatives with
general structure C was obtained by exploiting a very practical
and rapid procedure that was recently developed by our re-
search group (Scheme 3).[26] This methodology consists of a se-
quential, one-pot, two-step process under microwave-assisted
irradiation and allows the formation of desired final com-
pounds in just a few minutes with high purity after a simple
precipitation under acidic conditions.
Following the optimized protocol, bis(carboxymethyl)trithio-
carbonate and the suitable amine were dissolved in DME in
the presence of triethylamine, and the resulting mixture was
heated at 908C under microwave irradiation for 10 min. After
this time, the 5-aryl-2-furaldehyde 4 was added, and the reac-
tion mixture was heated again at 1108C under microwave irra-
diation for 5 min. The desired compounds were obtained by
filtration from methanol under acidic conditions.
Scheme 1. Synthesis of benzyl derivatives 8a and 9a–c: a) Pd(PPh3)2Cl2,
Na2CO3, DMF/EtOH, RT, 1.5 h; b) 1n NaOH, CH3OH, reflux, overnight;
c) TIPSCl, imidazole, dry DMF, 0!608C, 16 h; d) Grignard reagent, dry THF,
ꢀ788C, 1 h; ea) TBAF, dry THF, 08C!RT, 30 min; eb) TMSCl, NaI, dry CH3CN,
30 min.
This methodology allowed us to obtain, in a very short time,
a collection of final compounds for testing against HIV-1 IN ac-
tivity. Considering the versatility and efficiency of the protocol,
dure for the direct and regiose-
lective arylation of 2-acylfurans
was recently reported.[21] All
compounds belonging to this
series are listed in Table 1.
To generate compounds with
general structure B, several pro-
cedures have been report-
ed;[22–25] we adapted a recently
Scheme 3. Synthesis of substituted rhodanine derivatives 16a–n: a) DME, Et3N, 908C, MW, 10 min; b) DME, Et3N,
110 8C, MW, 5 min.
published synthetic strategy that describes a one-pot, multi-
component, microwave-assisted reaction that allows the de-
sired final compounds to be obtained directly (Scheme 2).[22]
The methodology involves 5-aryl-2-furaldehyde 4 (synthesized
as reported above), rhodanine, and several secondary or pri-
mary amines (11 a–d) that catalyze a Knoevenagel condensa-
tion between the aldehyde and rhodanine, and then act as nu-
cleophiles in the displacement of the thiocarbonyl sulfur. This
microwave-assisted procedure allows one to obtain, in very
short reaction times and easy workup, several final compounds
we employed both aromatic and aliphatic primary amines to
better investigate how the nature of the substituents can
affect the interactions with the IN binding site. The final com-
pounds 16a–n are listed in Table 1.
Compounds 12a–d and 16a–n are present as potential E
1
and Z isomers: in all cases, in the H and 13C NMR spectra of
compounds 12a–d and 16a–n, only one isomer was observed,
3
and the Z configuration was assigned by the JC4–H6 coupling
constant measurement. In fact, the experimental J values of
these derivatives are in agreement with those previously re-
ported for analogous 4-thiazolidinones.[22–25,27–30]
Biological activity and docking studies
All the synthesized compounds were tested with an
ELISA-based IN activity assay, and the biological re-
sults are listed in Table 1.[31,32] Concerning benzyl de-
rivatives, the results show that only compound 8a
possesses weak activity against the HIV-1 virus. For
these derivatives, docking studies suggest a binding
Scheme 2. Synthesis of substituted rhodanine derivatives 12a–d: a) EtOH, 1508C, MW,
20 min.
ChemMedChem 2011, 6, 343 – 352
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
345

M. Botta et al.
MED
activity. Interestingly, the difference between the
fairly active compound 8a and the reduced analogue
9a is represented by the hydroxy group formed after
allowing the Grignard reagent to react with the alde-
hyde. Therefore, such a hydroxy group is responsible
for the moderate activity exhibited by compound 8a.
In agreement with this observation, the docking cal-
culation for compound 8a highlighted that such a
hydroxy group could positively interact with Asp116
through a hydrogen bond contact when the chiral
center is the S configuration (Figure 4).
Table 1. Anti-IN activity of compounds 8a, 9a–c, 12a–d, and 16a–n.
Compd
R
R1
IC50 [mm][a]
43.0ꢁ15.6
8a
9a
9b
9c
4-F
4-F
OH
H
>250
3-F
3-Ph
H
H
>250
>250
Regarding the biological results of the substituted
rhodanine derivatives, the three compounds 12a,
12b, and 12d showed moderate activity in the IN in-
hibition assays, whereas compounds 16a–n gave
more satisfactory results. In the series of substituted
rhodanine derivatives 16a–n, a substituent-depen-
dent effect was observed: the best results were ob-
tained when aromatic moieties were introduced on
the rhodanine ring, while the substitution with ali-
phatic groups at the same position of this ring de-
creased the inhibitory potency against HIV-1 IN.
12a
12b
134.0ꢁ64.0
93.0ꢁ5.0
12c
>250
12d
16a
16b
130.0ꢁ39.0
6.9ꢁ1.2
Considering these results, we aimed to determine
a comprehensive structure–activity relationship. It
was observed that the first attempt at molecular
complication, which led to the study of compounds
12a–d, was unsuccessful. Docking studies with these
compounds did not converge to a highly populated
cluster, suggesting that such compounds may be
unable to find a stable pose in the binding site. A dif-
ferent behavior was observed in the docking studies
with derivatives 16a–n. All these compounds were
found to bind the DNA binding site of IN with an ori-
entation similar to that of the hit compound 17, dis-
cussed above. Specifically, the salicylic ring is engag-
ed in hydrogen bonding with polar amino acid resi-
dues: hydrogen bond contacts between the acidic
group of the inhibitor and the side chain of Lys159
as well as with the backbone NH group of His67 con-
tribute significantly to the stabilization of the com-
plex. In addition, the hydroxy group is involved in a
hydrogen bond with Glu152. On the other hand, the
rhodanine moiety is directed toward the 140–149
loop, and positive van der Waals contacts involving
the side chains of Ile151 and Ile141 were found.
Moreover, another hydrophobic interaction was ob-
served between the rhodanine substituent and
Tyr143 for compounds substituted with aromatic
groups such as 16a–d, 16h, and 16l–n. This addi-
tional hydrophobic contact could explain the im-
proved activity observed if aromatic moieties are in-
3.7ꢁ1.1
16c
32.8ꢁ11.5
16d
202.3ꢁ67.0
16e
16 f
114.9ꢁ3.7
86.1ꢁ5.4
16g
140.3ꢁ8.7
16h
16i
16l
17.3ꢁ3.8
CH3
>125
20.4ꢁ2.3
16m
16n
24.1ꢁ5.8
33.8ꢁ10.0
17
MK-0518
8.9ꢁ0.01
0.08ꢁ0.012
[a] IC50: concentration required to inhibit overall in vitro integrase activity by 50%;
values represent the mean ꢁSD of three experiments.
mode similar to that of the hit compound 17. As an example,
Figure 3 shows the predicted binding mode for compound 9a.
Despite the fact that the benzyl moiety establishes positive
van der Waals contacts within the binding site, the loss of the
hydrogen bond interaction established between the heterocy-
cle of compound 17 and Gln148 appears to be detrimental for
troduced on the rhodanine ring (16a–c, 16h, and 16l–n)
rather than aliphatic groups (16e–g, 16i). For compounds
16a–c, 16h, and 16l–n, the loss of a hydrogen bond with
Gln148 seems to be compensated by the van der Waals inter-
action with Tyr143. For example, Figure 5 shows the predicted
binding mode of compound 16a.
346
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 343 – 352

HIV-1 Integrase Inhibitors
Figure 3. Graphical representation of the binding mode of compound 9a.
For clarity, only a few residues are labeled; nonpolar hydrogen atoms are
omitted, and hydrogen bond interactions are represented by black dashed
lines.
Figure 5. Graphical representation of the binding mode of compound 16a.
For clarity, only a few residues are labeled; nonpolar hydrogen atoms are
omitted, and hydrogen bond interactions are represented by black dashed
lines.
Table 2. Cytotoxicity and antiviral activity of compounds 16a, 16b, 16d,
and 12a.
Compd
EC50 [mm][a]
CC50 [mm][b]
SI [CC50/EC50]
16a
16b
1.7
8.2
31
27
18
3
16d
12a
17
MK-0518
>15
>50
15
15
–
–
>9
>50
>140
>2
0.0008
>2600
[a] Effective concentration required to decrease HIV-1-induced cytopathic
effect by 50% in MT-4 cells. [b] Cytotoxic concentration required to de-
crease MT-4 cell viability by 50%. Values represent the mean of two ex-
periments.
Conclusions
Figure 4. Graphical representation of the binding mode of compound 8a.
For clarity, only a few residues are labeled; nonpolar hydrogen atoms are
omitted, and hydrogen bond interactions are represented by black dashed
lines.
We designed and synthesized three novel series of salicylic
acid derivatives 8a, 9a–c, 12a–d, and 16a–n to investigate
their activity against HIV-1 IN. In particular, compounds 12a–d
and 16a–n were obtained by using microwave-assisted proce-
dures that were developed and optimized in our research
group, which allowed us to obtain several final compounds of
high purity in a short time.
Some of the more interesting compounds belonging to this
series were also tested in 3’-processing and ST assays, but no
great difference in terms of activity was observed between the
two steps.
Biological evaluation revealed that some of the synthesized
compounds possess good inhibitory potency in enzymatic
assays. Docking studies of the new molecules were successfully
used to rationalize their structure–activity relationship. The re-
sults of these simulations show that the hydrogen bond be-
tween our previously reported hit compounds and Gln148 is a
crucial interaction for determining the affinity toward IN. In
fact, the ligands that cannot form the same hydrogen bond
contact are characterized by loss of activity. The IN inhibitory
activity could be restored for compounds that are able to es-
tablish extensive van der Waals interactions with Tyr143. Over-
all, these results emphasized Gln148 and Tyr143 as two impor-
tant residues to be targeted for the design of new IN inhibi-
The most active compounds in terms of overall integration
activity, 16a and 16b as well as the inactive compounds 12a
and 16d, were selected to evaluate their ability to inhibit the
HIV-induced cytopathic effect in a human lymphocyte MT-4
cell culture infected with HIV-1 NL4-3 (wt) using the MT-4/MTT
assay.[33,34] Remarkably, compounds 16a and 16b showed low
micromolar activity (EC50 =1.7 and 8.2 mm, respectively), where-
as compounds 12a and 16d were inactive at subtoxic concen-
trations (Table 2). Further modifications of compounds 16a–b
aiming at improving these results are in progress.
ChemMedChem 2011, 6, 343 – 352
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
347

M. Botta et al.
MED
pletion (TLC) and was quenched with H2O and 2n HCl, then EtOAc
was added, and the mixture was stirred until the two layers
became clear. The aqueous layer was extracted three times with
EtOAc, then the organic layers were washed with H2O and brine,
dried over Na2SO4, filtered, and evaporated under reduced pres-
sure. The crude product was purified by flash chromatography
using PE/EtOAc=7:3 as eluent to yield the desired product 3 as a
light-orange solid (yield: 99%); mp=1508C (decomposition);
1H NMR (CDCl3, 400 MHz): d=10.84 (s, 1H), 9.69 (s, 1H), 7.91–7.88
(d, 1H, J=12 Hz), 7.40–7.39 (d, 1H, J=4 Hz), 7.35–7.32 (m, 3H),
6.94–6.93 (d, 1H, J=4 Hz), 3.97 (s, 3H); 13C NMR (CDCl3, 100 MHz):
d=177.47, 161.69, 157.37, 152.53, 135.17, 130.58, 122.56, 115.74,
113.73, 112.82, 109.84, 52.40, 29.59; MS (ES): m/z 245.0 [Mꢀ1]ꢀ;
Anal. (C13H10O5) C, H, N.
tors. Compounds 16a and 16b were also able to inhibit HIV
replication in MT-4 cells at low micromolar concentrations with
an appreciable selectivity index (SI), indicating that aromatic-
substituted rhodanines are also good scaffolds for further de-
velopment of IN inhibitors. Further modifications of these new
hit compounds, with the aim of improving activity, are current-
ly ongoing in our laboratories.
Experimental Section
Chemistry
General information: All commercially available chemicals were
used as purchased. Anhydrous reactions were run under a positive
pressure of dry N2. Thin-layer chromatography (TLC) was carried
out using Merck TLC plates: silica gel 60 F254. Chromatographic pu-
rifications were performed on columns packed with Merck 60 silica
4-(5-Formylfuran-2-yl)-2-hydroxybenzoic acid (4): Compound 3
(1.73 mmol) was dissolved in 30 mL CH3OH, then a solution of
NaOH (1m, 5.00 mmol) was added dropwise and at a rate of
1 mLhꢀ1 to the reaction mixture. After the first addition of NaOH
solution, the reaction mixture was heated at reflux, and the follow-
ing additions were performed at this temperature. The reaction
mixture was stirred overnight until completion (TLC). Organic sol-
vent was removed under reduced pressure, then some H2O
(~10 mL) was added, and the aqueous layer was extracted three
times with Et2O; the aqueous layer was then acidified to pH 1 with
HCl (6n) and a precipitate appeared. The desired product 4 was
obtained as a brown–red solid (yield: 93%); mp=2308C (decom-
1
gel, 23–400 mesh, for the flash technique. H and 13C NMR spectra
were recorded at 400 MHz on a Bruker Avance DPX400 spectrom-
eter. Chemical shifts are reported relative to (CH3)4Si at d=
0.00 ppm. Melting points were measured using a Gallenkamp melt-
ing point apparatus and are uncorrected. Elemental analyses were
performed on a PerkinElmer PE 2004 elemental analyzer, and the
data for C, H, and N are within 0.4% of theoretical values.
HPLC and MS analysis: The purity of the tested compounds was
assessed by reversed-phase liquid chromatography and a mass
spectrometer (Agilent series 1100 LC/MSD) equipped with a UV de-
tector (l=254 nm) and an electrospray ionization (ESI) source. The
LC elution method (using a Zorbax Eclipse XDB, 4.6ꢂ150 mm,
5 mm C8 column) involved the following: (compounds 8a and 9a–
c) T=258C, mobile phase composed of A) 60% CH3OH and B) 40%
H2O at a flow rate of 0.8 mLminꢀ1; (compounds 12a–d and 16a–n)
T=258C, mobile phase composed of A) 70% CH3CN and B) 30%
H2O with 0.5% formic acid at a flow rate of 1.0 mLminꢀ1; (all sol-
vents were HPLC grade, Sigma Aldrich). All analyzed compounds
meet ꢂ95% purity criteria. MS data were obtained using an Agi-
lent 1100 LC/MSD VL system (G1946C) with a 0.4 mLminꢀ1 flow
rate using a binary solvent system of 95:5 CH3OH/H2O. UV detec-
tion was monitored at l=254 nm. Mass spectra were acquired in
negative mode scanning over the mass range of 50–1500 Da. The
following ion source parameters were used: drying gas flow,
9 mLminꢀ1; nebulizer pressure, 40 psig; drying gas temperature,
3508C.
1
position); H NMR (DMSO, 400 MHz): d=9.63 (s, 1H), 7.88–7.86 (d,
1H, J=8 Hz), 7.66–7.65 (d, 1H, J=4 Hz), 7.45–7.39 (m, 3H);
13C NMR (DMSO, 100 MHz): d=178.72, 171.68, 161.69, 156.82,
152.63, 135.11, 131.72, 125.14, 116.02, 114.06, 113.29, 111.53; MS
(ES): m/z 231.0 [Mꢀ1]ꢀ; Anal. (C12H8O5) C, H, N.
Triisopropylsilyl
4-(5-formylfuran-2-yl)-2-(triisopropylsilyloxy)-
benzoate (5): Compound 4 (0.23 mmol) was dissolved in 1.5 mL
dry DMF, then imidazole was added (1.16 mmol), and the reaction
mixture was cooled to 08C before adding triisopropylsilyl chloride
(1.07 mmol). The reaction mixture was heated at 608C for 17 h.
H2O was added, and the aqueous layer was extracted three times
with EtOAc; the organic layers were then washed with brine, dried
over Na2SO4, filtered, and evaporated under reduced pressure. The
crude product was purified by flash chromatography using PE/
EtOAc=95:5 as eluent and then with a kugelrohr apparatus to
yield the desired product as a yellow oil (yield: 35%). 1H NMR
(CDCl3, 400 MHz): d=9.69 (s, 1H), 7.85–7.83 (d, 1H, J=8 Hz), 7.39–
7.37 (d, 1H, J=8 Hz), 7.32–7.31 (m, 2H), 6.85–6.84 (d, 1H, J=4 Hz),
1.43–1.33 (m, 6H), 1.15–1.13 (m, 36H); 13C NMR (CDCl3, 100 MHz):
d=177.34, 163.81, 157.85, 156.91, 152.57, 132.92, 132.41, 124.41,
122.49, 117.00, 109.06, 18.01, 17.90, 13.11, 12.08; MS (ES): m/z 387.1
[Mꢀ157]ꢀ, 566.8 [M+23]+; Anal. (C30H48O5Si2) C, H, N.
Microwave irradiation experiments: Microwave reactions were
conducted using a CEM Discover Synthesis Unit (CEM Corp., Mat-
thews, NC, USA). The instrument consists of a continuous focused
microwave power delivery system with operator-selectable power
output from 0 to 300 W. The temperature of the contents of the
vessel was monitored with a calibrated IR temperature control
mounted under the reaction vessel. All experiments were per-
formed using a stirring option, whereby the contents of the vessel
are stirred by a rotating magnetic plate located below the floor of
the microwave cavity and a Teflon-coated magnetic stir bar in the
vessel.
General procedure for the synthesis of compounds 7a–c: Com-
pound 5 (0.30 mmol) was dissolved in 10 mL dry THF; then the so-
lution was cooled to ꢀ788C and Grignard reagent solution in THF
(0.48 mmol) was added dropwise. The reaction mixture was stirred
at ꢀ788C for 1 h until completion (TLC). A saturated solution of
NH4Cl was added to the reaction mixture, then the aqueous layer
was extracted three times with EtOAc, then the organic layers
were washed with brine, dried over Na2SO4, filtered and evaporat-
ed under reduced pressure. The crude products were purified by
flash chromatography using PE/EtOAc=9:1 as eluent to yield de-
sired products as colorless oils.
Methyl 4-(5-formylfuran-2-yl)-2-hydroxybenzoate (3): Methyl-4-
iodosalycilate 1 (1.00 mmol) and 5-formyl-2-furan boronic acid 2
(1.30 mmol) were dissolved in 10 mL DMF and 15 mL EtOH. The re-
action mixture was stirred for 10 min under N2, then Pd(PPh3)2Cl2
(0.10 mmol) and a solution of Na2CO3 (2m, 6 mmol) were added to
the reaction mixture, and the light-orange suspension was stirred
under N2 at room temperature. After 1 h the reaction went to com-
Triisopropylsilyl 4-(5-((4-fluorophenyl)(hydroxy)methyl)furan-2-
yl)-2-(triisopropylsilyloxy)benzoate (7a): (yield: 73%); 1H NMR
348
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 343 – 352

HIV-1 Integrase Inhibitors
(CDCl3, 400 MHz): d=7.79–7.77 (d, 1H, J=8 Hz), 7.47–7.44 (m,
2H), 7.18–1.16 (d, 1H, J=8 Hz), 7.12 (s, 1H), 7.09–7.04 (m, 2H),
6.61–6.60 (d, 1H, J=4 Hz), 6.27–6.26 (d, 1H, J=4 Hz), 5.85 (s, 1H),
1.42–1.29 (m, 6H), 1.15–1.12 (m, 36H); 13C NMR (CDCl3, 100 MHz):
d=164.01, 163.69, 156.79, 156.03, 152.79, 136.40, 134.40, 132.19,
128.31, 128.23, 122.16, 115.38, 115.15, 109.42, 107.26, 69.58, 17.91,
17.82, 12.97, 11.98; MS (ES): m/z 483.1 [Mꢀ157]ꢀ; Anal.
(C36H53FO5Si2) C, H, N.
4-(5-(4-Fluorobenzyl)furan-2-yl)-2-hydroxybenzoic acid (9a):
(yield 83%); mp=2028C (decomposition); 1H NMR ((CD3)2CO,
400 MHz): d=7.94–7.92 (d, 1H, J=8 Hz), 7.46–7.43 (m, 2H), 7.30–
7.28 (d, 1H, J=8 Hz), 7.25 (s, 1H), 7.17–7.15 (m, 2H), 7.03–7.02 (d,
1H, J=4 Hz), 6.32–6.31 (d, 1H, J=4 Hz), 4.15 (s, 2H); 13C NMR
((CD3)2CO, 100 MHz): d=171.42, 162.77, 162.41, 156.03, 151.36,
137.53, 134.02, 130.87, 130.44, 130.36, 115.12, 114.90, 114.03,
110.71, 110.45, 109.37, 108.90, 33.11; MS (ES): m/z 311.0 [Mꢀ1]ꢀ;
Anal. (C18H13FO4) C, H, N.
Triisopropylsilyl 4-(5-((3-fluorophenyl)(hydroxy)methyl)furan-2-
yl)-2-(triisopropylsilyloxy)benzoate (7b): (yield: 63%); 1H NMR
(CDCl3, 400 MHz): d=7.79–7.77 (d, J=8 Hz, 1H), 7.33 (m, 1H), 7.24
(m, 1H), 7.18–7.16 (d, J=8 Hz, 1H), 7.13 (s, 1H), 7.02 (m, 1H), 6.61–
6.60 (d, J=4 Hz, 1H), 6.30–6.29 (d, J=4 Hz, 1H), 5.86 (s, 1H), 2.62
(s, 1H), 1.35 (m, 6H), 1.10 (m, 36H); 13C NMR (CDCl3, 100 MHz): d=
164.2, 157.1, 155.8, 153.5, 153.2, 134.6, 132.4, 130.2, 130.1, 123.1,
122.5, 115.7, 115.2, 113.9, 113.6, 109.9, 107.5, 69.8, 18.2, 18.1, 13.2,
12.2; MS (ES): m/z 483.1 [Mꢀ157]ꢀ; Anal. (C36H53FO5Si2) C, H, N.
4-(5-((3-Fluorophenyl)(hydroxy)methyl)furan-2-yl)-2-hydroxyben-
zoic acid (9b): (yield: 41%); mp=1778C (decomposition); H NMR
1
(MeOD, 400 MHz): d=7.83–7.81 (d, 1H, J=8 Hz), 7.31 (m, 1H),
7.12 (m, 3H), 7.02–7.00 (d, 1H, J=8 Hz), 6.94 (m, 1H), 6.82–6.81 (d,
1H, J=4 Hz), 6.20–6.19 (d, 1H, J=4 Hz), 4.05 (s, 2H); 13C NMR
(MeOD, 100 MHz): d=164.10, 162.00, 161.67, 154.96, 151.69,
140.57, 136.92, 130.64, 129.81, 124.14, 115.07, 113.62, 112.95,
112.74, 110.35, 108.66, 108.37, 33.42; MS (ES)=m/z 311.0 [Mꢀ1]ꢀ;
Anal. (C18H13FO5) C, H, N.
Triisopropylsilyl 4-(5-(biphenyl-3-yl(hydroxy)methyl)furan-2-yl)-
2-(triisopropylsilyloxy)benzoate (7c): (yield: 66%); H NMR (CDCl3,
4-(5-(Biphenyl-3-ylmethyl)furan-2-yl)-2-hydroxybenzoic acid (9c):
(yield: 50%); mp=1898C (decomposition); 1H NMR (MeOD,
400 MHz): d=7.82–7.80 (d, 1H, J=8 Hz), 7.58–7.56 (d, 1H, J=
8 Hz), 7.53 (s, 1H), 7.48–7.46 (d, 1H, J=8 Hz), 7.42–7.36 (m, 2H),
7.32–7.30 (d, 1H, J=8 Hz), 7.27–7.25 (d, 1H, J=8 Hz), 7.14–7.12 (m,
2H), 6.79–6.78 (d, 1H, J=4 Hz), 6.18–6.17 (d, 1H, J=4 Hz), 4.09 (s,
2H); 13C NMR (DMSO, 100 MHz): d=172.02, 162.08, 156.06, 151.21,
140.84, 140.44, 138.91, 136.71, 131.37, 129.55, 129.32, 128.12,
127.87, 127.48, 127.07, 125.34, 114.14, 112.37, 110.78, 110.10,
109.63, 34.15; MS (ES): m/z 369.1 [Mꢀ1]ꢀ; Anal. (C24H18O4) C, H, N.
1
400 MHz): d=7.80–7.78 (d, 1H, J=8 Hz), 7.71 (s, 1H), 7.61–7.57
(m, 3H), 7.48–7.42 (m, 4H), 7.37–7.35 (d, 1H, J=8 Hz), 7.20–7.18 (d,
1H, J=8 Hz), 7.14 (s, 1H), 6.62–6.61 (d, 1H, J=4 Hz), 6.31–6.30 (d,
1H, J=4 Hz), 5.96 (s, 1H), 1.44–1.27 (m, 6H), 1.15–1.10 (m, 36H);
13C NMR (CDCl3, 100 MHz): d=163.99, 156.82, 152.78, 141.49,
141.11, 140.80, 134.51, 132.21, 128.90, 128.71, 127.36, 127.11,
126.89, 125.44, 125.34, 122.12, 115.48, 109.58, 107.35, 70.33, 17.93,
17.85, 12.97, 12.00; MS (ES): m/z 541.2 [Mꢀ157]ꢀ, 721.3 [M+23]+;
Anal. (C42H58O5Si2) C, H, N.
General procedure for the synthesis of compounds 12a–d: To a
suspension of rhodanine (0.13 mmol) in EtOH, 5-aryl-2-furaldehyde
4 (0.13 mmol) and amine (0.39 mmol) were added. The reaction
mixture was heated by microwave irradiation at 1508C for 20 min.
The reaction vessel was allowed to cool to room temperature and
a drop of HCl (2n) was added. The precipitated solid was filtered
off, washed with H2O and EtOH, and dried under vacuum.
4-(5-((4-Fluorophenyl)(hydroxy)methyl)furan-2-yl)-2-hydroxyben-
zoic acid (8a): Compound 7a (0.12 mmol) was dissolved in 2 mL
dry THF, then the solution was cooled to 08C. A solution of TBAF
(1m in THF, 0.31 mmol) was added dropwise to the reaction mix-
ture at 08C. The reaction mixture was allowed to warm to room
temperature and stirred for 30 min. EtOAc was added and the or-
ganic layer was washed three times with a saturated solution of
NH4Cl, then with brine, dried over Na2SO4 and evaporated under
reduced pressure. The crude product was dissolved in a solution of
NaOH (1n) and the aqueous layer was extracted three times with
Et2O. The aqueous layer was acidified until pH 1 with HCl (6n) and
extracted three times with EtOAc. The organic layers were washed
with brine, dried over Na2SO4, filtered and evaporated under re-
duced pressure. The crude product was purified by flash chroma-
tography using EtOAc with some drops of acetic acid as eluent to
yield the desired product as a white solid (yield: 51%); mp=1628C
(Z)-2-Hydroxy-4-(5-((2-morpholino-4-oxothiazol-5(4H)-ylidene)-
methyl)furan-2-yl)benzoic acid (12a): (yield: 75%); Yellow solid;
1
mp=2678C (decomposition); H NMR (DMSO, 400 MHz): d=7.91–
7.89 (d, 1H, J=8 Hz), 7.50 (s, 1H), 7.43–7.36 (m, 3H), 7.16–7.15 (d,
1H, J=4 Hz), 3.93–3.91 (m, 2H), 3.80–3.78 (m, 2H), 3.74–3.71 (m,
4H); 13C NMR (DMSO, 100 MHz): d=179.10, 175.06, 171.69, 161.74,
154.74, 150.67, 135.53, 131.77, 127.02, 119.42, 116.47, 115.35,
113.06, 112.47, 112.12, 66.06, 65.89, 48.82, 48.68; MS (ES): m/z 399.0
[Mꢀ1]ꢀ; Anal. (C19H16N2O6S) C, H, N.
1
(decomposition); H NMR (MeOD, 400 MHz): d=7.84–7.82 (d, 1H,
(Z)-2-Hydroxy-4-(5-((4-oxo-2-thiomorpholinothiazol-5(4H)-ylide-
ne)methyl)furan-2-yl)benzoic acid (12b): (yield: 31%); Yellow
J=8 Hz), 7.51–7.48 (m, 2H), 7.19–7.16 (m, 2H), 7.12–7.08 (m, 2H),
6.84–6.83 (d, 1H, J=4 Hz), 6.25–6.24 (d, 1H, J=4 Hz), 5.82 (s, 1H);
MS (ES): m/z 327.0 [Mꢀ1]ꢀ; Anal. (C18H13FO5) C, H, N.
1
solid; mp=2178C (decomposition); H NMR (DMSO, 400 MHz): d=
7.92–7.90 (d, 1H, J=8 Hz), 7.50 (s, 1H), 7.43–7.36 (m, 3H), 7.16–
7.15 (d, 1H, J=4 Hz), 4.19–4.17 (m, 2H), 3.96–3.94 (m, 2H), 2.87–
2.85 (m, 2H), 2.79–2.77 (m, 2H); 13C NMR (DMSO, 100 MHz): d=
179.18, 175.01, 171.71, 161.76, 154.78, 150.67, 135.53, 131.77,
127.02, 119.43, 116.53, 115.35, 113.08, 112.47, 112.13, 51.85, 51.22,
27.40, 26.87; MS (ES): m/z 415.0 [Mꢀ1]ꢀ; Anal. (C19H16N2O5S2) C, H,
N.
General procedure for the synthesis of compounds 9a–c: NaI
(0.69 mmol) was dissolved in 1 mL dry CH3CN and TMSCl
(0.69 mmol) was added, a white precipitate formed. Compound
7a–c (0.12 mmol) was dissolved in 3 mL dry CH3CN and this solu-
tion was added with a double needle to the reaction mixture at
room temperature. The reaction mixture was stirred 30 min at
room temperature. H2O and EtOAc were added, layers were sepa-
rated, and the aqueous layer was extracted three times with
EtOAc, then organic layers were washed with brine, dried over
Na2SO4, filtered and evaporated under reduced pressure. The crude
products were purified by flash chromatography using PE/EtOAc=
9:1 as eluent to obtain the desired products as white solids.
(Z)-2-Hydroxy-4-(5-((2-(4-methylpiperazin-1-yl)-4-oxothiazol-
5(4H)-ylidene)methyl)furan-2-yl)benzoic acid (12c): (yield: 76%);
Yellow solid; mp=2658C (decomposition); 1H NMR (DMSO,
400 MHz): d=7.90–7.88 (d, 1H, J=8 Hz), 7.52 (s, 1H), 7.39–7.35
(m, 3H), 7.16–7.15 (d, 1H, J=4 Hz), 4.20–4.18 (m, 2H), 4.02–4.00
(m, 2H), 3.39–3.33 (m, 4H) 2.78 (s, 3H); 13C NMR (DMSO, 100 MHz):
ChemMedChem 2011, 6, 343 – 352
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
349

M. Botta et al.
MED
d=179.33, 175.71, 171.82, 161.84, 155.16, 150.29, 134.94, 131.76,
126.67, 120.08, 117.14, 115.11, 114.06, 112.35, 112.11, 51.94, 51.70,
45.48, 44.92, 42.54; MS (ES): m/z 412.0 [Mꢀ1]ꢀ; Anal. (C20H19N3O5S)
C, H, N.
161.89, 156.87, 150.45, 147.69, 147.21, 138.05, 134.73, 131.75,
129.01, 122.31, 121.56, 119.90, 113.90, 109.35, 109.01, 108.52,
108.20, 101.49, 101.09, 47.46; MS (ES): m/z 480.0 [Mꢀ1]ꢀ; Anal.
(C23H15NO7S2) C, H, N.
(Z)-4-(5-((2-(Benzylamino)-4-oxothiazol-5(4H)-ylidene)methyl)fur-
an-2-yl)-2-hydroxybenzoic acid (12d): (yield: 73%); Yellow solid;
mp=2798C (decomposition); H NMR (DMSO, 400 MHz): d=7.84–
7.82 (d, 1H, J=8 Hz), 7.46 (s, 1H), 7.39–7.30 (m, 8H), 7.12–7.11 (d,
1H, J=4 Hz), 4.73 (s, 2H); 13C NMR (DMSO, 100 MHz): d=179.12,
174.24, 171.74, 161.92, 154.46, 150.83, 137.44, 135.62, 131.44,
128.95, 128.12, 127.94, 127.38, 119.12, 115.91, 115.32, 112.85,
112.49, 111.83, 48.26; MS (ES): m/z 419.0 [Mꢀ1]ꢀ; Anal.
(C22H16N2O5S) C, H, N.
(Z)-2-Hydroxy-4-(5-((4-oxo-3-propyl-2-thioxothiazolidin-5-ylide-
ne)methyl)furan-2-yl)benzoic acid (16e): (yield: 46%); Yellow-
brown solid; mp=2758C (decomposition); 1H NMR (DMSO,
400 MHz): d=7.86–7.84 (d, 1H, J=8 Hz), 7.55 (s, 1H), 7.39–7.38 (d,
1H, J=4 Hz), 7.30–7.26 (m, 3H), 3.95–3.92 (t, 2H, J=7.6 Hz), 1.66–
1.61 (m, 2H), 0.89–0.85 (t, 3H, J=7.2 Hz); 13C NMR (DMSO,
100 MHz): d=194.23, 171.77, 166.90, 161.78, 156.60, 150.45,
134.89, 131.66, 123.00, 120.07, 118.28, 115.48, 113.41, 112.98,
112.36, 46.03, 20.28, 11.44; MS (ES): m/z 388.0 [Mꢀ1]ꢀ; Anal.
(C18H15NO5S2) C, H, N.
1
General procedure for the synthesis of compounds 16a–n: To a
solution of bis(carboxymethyl)trithiocarbonate 13 (0.22 mmol) in
DME (1.0 mL) were added Et3N (0.22 mmol) and the opportune
amine (R1NH2, 0.22 mmol). The reaction mixture was heated at
908C for 10 min under microwave irradiation. After this time, the
5-aryl-2-furaldehyde 4 (0.22 mmol) was added, and the mixture
was heated at 1108C for 5 min under microwave irradiation. The
reaction mixture was evaporated to dryness, and the residue was
resuspended with CH3OH (~2 mL) and a drop of HCl 2n; the final
rhodanine derivatives were obtained as a pure precipitate, isolated
by filtration, washed with H2O and hexane, and finally dried under
high vacuum.
(Z)-4-(5-((3-(Cyclopropylmethyl)-4-oxo-2-thioxothiazolidin-5-yli-
dene)methyl)furan-2-yl)-2-hydroxybenzoic acid (16 f): (yield:
50%); Yellow solid; mp=2768C (decomposition); H NMR (DMSO,
1
400 MHz): d=7.87–7.85 (d, 1H, J=8.4 Hz), 7.58 (s, 1H), 7.40–7.39
(d, 1H, J=3.2 Hz), 7.32–7.28 (m, 3H), 3.89–3.87 (d, 2H, J=7.2 Hz),
1.20–1.30 (m, 1H), 0.49–0.47 (d, 2H, J=7.2 Hz), 0.40–0.39 (d, 2H,
J=3.6 Hz); 13C NMR (DMSO, 100 MHz): d=194.50, 171.77, 167.13,
161.79, 156.68, 150.43, 134.86, 131.80, 131.53, 123.12, 120.03,
115.50, 113.46, 113.03, 112.39, 48.88, 9.58, 4.03; MS (ES): m/z 400.0
[Mꢀ1]ꢀ; Anal. (C19H15NO5S2) C, H, N.
(Z)-2-Hydroxy-4-(5-((4-oxo-3-(prop-2-ynyl)-2-thioxothiazolidin-5-
ylidene)methyl)furan-2-yl)benzoic acid (16g): (yield: 15%); Yellow
(Z)-4-(5-((3-(4-Fluorobenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene)-
methyl)furan-2-yl)-2-hydroxybenzoic acid (16a): (yield: 35%);
Yellow solid; mp=2608C (decomposition); 1H NMR (DMSO,
400 MHz): d=7.86–7.84 (d, 1H, J=8 Hz), 7.69 (s, 1H), 7.39–7.37
(m, 4H), 7.27–7.25 (m, 2H), 7.18–7.14 (m, 2H), 5.21 (s, 2H); 13C NMR
(DMSO, 100 MHz): d=194.23, 171.67, 166.97, 162.16, 157.19,
150.33, 134.30, 131.72, 131.63, 130.53, 130.41, 125.94, 123.61,
119.68, 119.01, 118.79, 116.01, 115.75, 115.57, 115.21, 114.98, 112.88,
112.66, 46.81; MS (ES): m/z 454.0 [Mꢀ1]ꢀ; Anal. (C22H14FNO5S2) C, H,
N.
1
solid; mp=2698C (decomposition); H NMR (DMSO, 400 MHz): d=
7.94–7.92 (d, 1H, J=8 Hz), 7.75 (s, 1H), 7.50–7.49 (d, 1H, J=4 Hz),
7.41–7.38 (m, 3H), 4.78 (s, 2H), 3.31 (s, 1H); 13C NMR (DMSO,
100 MHz): d=193.13, 171.70, 165.95, 161.84, 156.95, 150.43,
134.83, 131.88, 123.65, 119.59, 119.16, 115.61, 113.84, 113.17,
112.52, 77.08, 75.05, 33.87; MS (ES): m/z 384.0 [Mꢀ1]ꢀ; Anal.
(C18H15NO5S2) C, H, N.
(Z)-4-(5-((3-(4-Chlorobenzyl)-4-oxo-2-thioxothiazolidin-5-ylide-
ne)methyl)furan-2-yl)-2-hydroxybenzoic acid (16h): (yield: 40%);
Yellow solid; mp=2758C (decomposition); 1H NMR (DMSO,
400 MHz): d=7.94–7.92 (d, 1H, J=8 Hz), 7.73 (s, 1H), 7.50–7.34
(m, 8H), 5.22 (s, 2H); 13C NMR (DMSO, 100 MHz): d=194.18,
171.55, 166.92, 161.82, 156.96, 150.50, 134.87, 134.34, 132.79,
131.79, 130.00, 128.89, 123.26, 119.99, 118.86, 115.56, 113.91,
112.97, 112.55, 46.92; MS (ES): m/z 469.9 [Mꢀ1]ꢀ; Anal.
(C22H14ClNO5S2) C, H, N.
(Z)-4-(5-((3-Benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)-
furan-2-yl)-2-hydroxybenzoic acid (16b): (yield: 75%); Yellow
1
solid; mp=2758C (decomposition); H NMR (DMSO, 400 MHz): d=
7.90–7.88 (d, 1H, J=8 Hz), 7.68 (s, 1H), 7.45–7.44 (d, 1H, J=4 Hz),
7.36–7.27 (m, 8H), 5.22 (s, 2H); 13C NMR (DMSO, 100 MHz): d=
194.21, 171.71, 166.96, 161.90, 156.90, 150.43, 135.34, 134.72,
131.80, 128.94, 128.02, 123.48, 119.85, 118.92, 118.78, 115.51,
113.98, 113.11, 112.94, 112.50, 112.36, 47.49; MS (ES): m/z 435.9.0
[Mꢀ1]ꢀ; Anal. (C22H15NO5S2) C, H, N.
(Z)-2-Hydroxy-4-(5-((3-methyl-4-oxo-2-thioxothiazolidin-5-ylide-
ne)methyl)furan-2-yl)benzoic acid (16i): (yield: 38%); Yellow
1
(Z)-2-Hydroxy-4-(5-((3-(3-methoxybenzyl)-4-oxo-2-thioxothiazoli-
din-5-ylidene)methyl)furan-2-yl)benzoic acid (16c): (yield: 41%);
Yellow solid; mp=1778C (decomposition); 1H NMR (DMSO,
400 MHz): d=7.93–7.91 (d, 1H, J=8 Hz), 7.71 (s, 1H), 7.48–7.47 (d,
1H, J=4 Hz), 7.40–7.37 (m, 3H), 7.26–7.22 (m, 1H), 6.88–6.84 (s,
2H), 3.72 (s, 3H); 13C NMR (DMSO, 100 MHz): d=194.29, 171.71,
168.10, 166.98, 161.90, 159.72, 156.83, 150.51, 136.82, 134.87,
131.85, 130.09, 123.40, 119.92, 118.89, 115.56, 113.75, 113.30,
113.14, 112.47, 55.43, 47.41; MS (ES): m/z 466.0 [Mꢀ1]ꢀ; Anal.
(C23H17NO6S2) C, H, N.
solid; mp>3008C (decomposition); H NMR (DMSO, 400 MHz): d=
7.86–7.84 (d, 1H, J=8 Hz), 7.55 (s, 1H), 7.38–7.37 (d, 1H, J=4 Hz),
7.30–7.25 (m, 3H), 3.35 (s, 3H); 13C NMR (DMSO, 100 MHz): d=
194.31, 171.74, 166.88, 161.77, 156.54, 150.42, 134.86, 131.64,
122.93, 120.39, 118.19, 115.46, 113.40, 112.95, 112.33, 31.43; MS
(ES): m/z 359.9 [Mꢀ1]ꢀ; Anal. (C16H11NO5S2) C, H, N.
(Z)-2-Hydroxy-4-(5-((3-(4-methoxybenzyl)-4-oxo-2-thioxothiazoli-
din-5-ylidene)methyl)furan-2-yl)benzoic acid (16l): (yield: 37%);
Orange solid; mp=2738C (decomposition); 1H NMR (DMSO,
400 MHz): d=7.88–7.86 (d, 1H, J=8.4 Hz), 7.63 (s, 1H), 7.42–7.41
(d, 1H, J=2.8 Hz), 7.33–7.27 (m, 5H), 6.88–6.86 (d, 2H, J=8 Hz),
5.13 (s, 2H), 3.70 (s, 3H); 13C NMR (DMSO, 100 MHz): d=194.07,
171.75, 166.94, 161.80, 159.22, 156.77, 150.45, 134.84, 131.72,
130.23, 129.90, 127.35, 126.91, 123.29, 119.90, 118.73, 115.52,
114.29, 113.55, 113.04, 112.42, 55.44, 46.98; MS (ES): m/z 466.0
[Mꢀ1]ꢀ; Anal. (C23H17NO6S2) C, H, N.
(Z)-4-(5-((3-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-oxo-2-thioxothia-
zolidin-5-ylidene)methyl)furan-2-yl)-2-hydroxybenzoic
acid
(16d): (yield: 49%); Orange solid; mp=2838C (decomposition);
1H NMR (DMSO, 400 MHz): d=7.92–7.90 (d, 1H, J=8 Hz), 7.67 (s,
1H), 7.43–7.34 (m, 4H), 6.91 (s, 1H), 6.84 (s, 2H), 5.97 (s, 2H), 5.14
(s, 2H); 13C NMR (DMSO, 100 MHz): d=194.18, 171.72, 166.97,
350
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 343 – 352

HIV-1 Integrase Inhibitors
(Z)-4-(5-((3-(2-(1H-Indol-3-yl)ethyl)-4-oxo-2-thioxothiazolidin-5-
ylidene)methyl)furan-2-yl)-2-hydroxybenzoic acid (16m): (yield:
55%); Yellow-brown solid; mp=2548C (decomposition); 1H NMR:
((CD3)2CO, 400 MHz) d=8.00–7.98 (d, 1H, J=8.4 Hz), 7.76–7.74 (d,
1H, J=8 Hz), 7.64 (s, 1H), 7.47–7.33 (m, 5H), 7.22 (s, 1H), 7.11–7.02
(m, 2H) 4.41–4.37 (m, 2H), 3.19–3.15 (m, 2H); 13C NMR (DMSO,
100 MHz): d=194.15, 171.77, 166.77, 161.81, 156.64, 150.49,
136.64, 134.91, 131.77, 127.45, 123.59, 123.10, 121.47, 120.25,
118.86, 118.53, 118.33, 115.53, 113.52, 113.09, 112.40, 111.90, 110.38,
45.23, 22.82; MS (ES): m/z 489.0 [Mꢀ1]ꢀ; Anal. (C25H18N2O5S2) C, H,
N.
1.6 mm) and 4 mL annealed oligonucleotides (7 nm) were added in
a final reaction volume of 40 mL containing 10 mm MgCl2, 5 mm di-
thiothreitol, 20 mm HEPES (pH 7.5), 5% poly(ethylene glycol), and
15% DMSO. The final concentration of IN in this assay was 160 nm.
The reaction was carried out for 1 h at 378C. Reaction products
were denatured with 30 mm NaOH and detected by ELISA on
avidin-coated plates.
Anti-HIV activity in lymphoid cells: The biological activity of the
compounds was tested in the lymphoid MT-4 cell line (received
from the NIH AIDS Reagent Program) against the wild-type HIV-1
NL4-3 strain. In brief, MT-4 cells were infected with the appropriate
HIV-1 strain (or mock-infected to determine cytotoxicity) in the
presence of various drug concentrations. At day five post-infection,
a tetrazolium-based colorimetric method (MTT method) was used
to evaluate the number of viable cells.[33,34]
(Z)-4-(5-((3-(3-Chlorobenzyl)-4-oxo-2-thioxothiazolidin-5-ylide-
ne)methyl)furan-2-yl)-2-hydroxybenzoic acid (16n): (yield: 51%);
1
Yellow-brown solid; mp=2898C (decomposition); H NMR: (DMSO,
400 MHz) d=7.91–7.89 (d, 1H, J=8.4 Hz), 7.69 (s, 1H), 7.47–7.46
(d, 1H, J=3.6 Hz), 7.39–7.35 (m, 6H), 7.27–7.26 (d, 1H, J=6.4 Hz),
5.22 (s, 2H); 13C NMR (DMSO, 100 MHz): d=194.32, 171.72, 166.96,
161.81, 156.84, 150.49, 138.07, 137.76, 134.86, 133.54, 131.85,
128.01, 126.53, 119.97, 119.03, 115.67, 115.49, 113.66, 113.23,
112.70, 46.92; MS (ES): m/z 470.0 [Mꢀ1]ꢀ; Anal. (C22H14ClNO5S2) C,
H, N.
Abbreviations
DME, 1,2-dimethoxyethane; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; PE, petroleum ether; SD, standard deviation;
TBAF, tetra-n-butylammonium fluoride; THF, tetrahydrofuran;
TIPSCl, triisopropylsilyl chloride; TMSCl, trimethylsilyl chloride.
Docking studies
Acknowledgements
Docking studies of all the synthesized compounds were performed
within the DNA binding domain of IN using the software package
Gold 4.1.[35–37] GoldScore was chosen as the fitness function. The
genetic algorithm parameter settings of Gold were employed
using the Search efficiency set at 100%. Thirty runs were carried
out for each ligand. Results differing by <1.5 ꢃ in ligand all-atom
root mean square deviation (RMSD) were clustered together. For
each inhibitor, the first-ranked solution as well as the lowest-
energy conformation of the most populated cluster were analyzed.
This work was supported by the European Union collaborative
projects: “THINC” (grant no. HEALTH-2007-2.3.2-1) and “CHAARM”
(grant no. HEALTH-F3-2009-242135). It was also supported by the
Italian Ministero dell’Istruzione, dell’Universitꢀ e della Ricerca,
Prin 2008 research project (grant no. 2008CE75SA_004). We
thank Barbara van Remoortel and Martine Michiels for excellent
technical assistance. F.C. is supported by a grant from the Indus-
trial Research Fund (IOF) from the Katholieke Universiteit Leuven
(Flanders, Belgium).
Three structures of the protein, characterized by different loop
conformations, were used for docking calculations, as previously
described.[14] The Mg2+-bound water molecules were kept during
the docking calculations by using the option toggle (water mole-
cules can be displaced during the docking at the cost of a penalty).
Structures of inhibitors were built using Maestro 3D-sketcher, mini-
mized with the OPLS_2005 force field, using the Polak–Ribiere con-
jugated gradient method (0.001 kJꢃꢀ1 molꢀ1 convergence), and fi-
nally, docked in the protein.[38,39] Among the three studied confor-
mations of the protein, in the structure PDB ID: 1BL3,[40] all the
studied inhibitors showed a high score value. Therefore, structure
1BL3 was chosen for the analysis of the ligand–protein complexes.
Finally, post-docking analysis was performed, and Figures 3, 4, and
5 were created using the program PyMOL (version 1.2).[41]
Keywords: antiviral agents · HIV · inhibitors · integrase
[1] D. C. Meadows, J. Gervay-Hague, ChemMedChem 2006, 1, 16–29.
[2] E. De Clercq, J. Med. Chem. 2005, 48, 1297–1313.
[3] P. Yin, D. Das, H. Mitsuya, Cell. Mol. Life Sci. 2006, 63, 1706–1724.
[4] T. Imamichi, Curr. Pharm. Des. 2004, 10, 4039–4053.
[5] R. Dayam, R. Gundla, L. Q. Al-Mawsawi, N. Neamati, Med. Res. Rev. 2008,
28, 118–154.
[6] T. H. Evering, M. Markowitz, Drugs Today 2007, 43, 865–877.
[7] V. Summa, A. Petrocchi, F. Bonelli, B. Crescenzi, M. Donghi, M. Ferrara, F.
Fiore, C. Gardelli, O. G. Paz, D. J. Hazuda, P. Jones, O. Kinzel, R. Laufer, E.
Monteagudo, E. Muraglia, E. Nizi, F. Orvieto, P. Pace, G. Pescatore, R.
Scarpelli, K. Stillmock, M. V. Witmer, M. Rowley, J. Med. Chem. 2008, 51,
5843–5855.
Biology
[8] D. Esposito, R. Craigie, Adv. Virus Res. 1999, 52, 319–333.
[9] Y. Goldgur, R. Craigie, G. H. Cohen, T. Fujiwara, T. Yoshinaga, T. Fujishita,
H. Sugimoto, T. Endo, H. Murai, D. R. Davies, Proc. Natl. Acad. Sci. USA
1999, 96, 13040–13043.
[10] F. Christ, W. Thys, J. De Rijck, R. Gijsbers, A. Albanese, D. Arosio, S. Emi-
liani, J. C. Rain, R. Benarous, A. Cereseto, Z. Debyser, Curr. Biol. 2008, 18,
1192–1202.
[11] D. J. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler, A.
Espeseth, L. Gabryelski, W. Schleif, C. Blau, M. D. Miller, Science 2000,
287, 646–650.
[12] Y. Pommier, A. A. Johnson, C. Marchand, Nat. Rev. Drug Discovery 2005,
4, 236–248.
Integrase assays: The enzymatic integration reactions were carried
out with minor modifications as described previously.[32] To deter-
mine the susceptibility of the HIV-1 IN enzyme to different com-
pounds, we used an enzyme-linked immunosorbent assay
(ELISA).[42] The overall integration assay uses an oligonucleotide
substrate for which one oligonucleotide (5’-ACT GCT AGA GAT TTT
CCA CAC TGA CTA AAA GGG TC-3’) is labeled with biotin at the 3’
end and the other oligonucleotide (5’-GAC CCT TTT AGT CAG TGT
GGA AAA TCT CTA GCA GT-3’) is labeled with digoxigenin at the 5’
end. The IN enzyme was diluted in 750 mm NaCl, 10 mm Tris
(pH 7.6), 10% glycerol, and 1 mm b-mercaptoethanol. To perform
the reaction, 4 mL diluted IN (corresponding to a concentration of
[13] S. Hare, S. S. Gupta, E. Valkov, A. Engelman, P. Cherepanov, Nature 2010,
464, 232–237.
ChemMedChem 2011, 6, 343 – 352
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
351

M. Botta et al.
MED
[14] C. Mugnaini, S. Rajamaki, C. Tintori, F. Corelli, S. Massa, M. Witvrouw, Z.
Debyser, V. Veljkovic, M. Botta, Bioorg. Med. Chem. Lett. 2007, 17, 5370–
5373.
[15] C. Tintori, F. Manetti, N. Veljkovic, V. Perovic, J. Vercammen, S. Hayes, S.
Massa, M. Witvrouw, Z. Debyser, V. Veljkovic, M. Botta, J. Chem. Inf.
Model. 2007, 47, 1536–1544.
[30] P. Vicini, A. Geronikaki, K. Anastasia, M. Incerti, F. Zani, Bioorg. Med.
Chem. 2006, 14, 3859–3864.
[31] M. Dubois, F. Bailly, G. Mbemba, J. F. Mouscadet, Z. Debyser, M. Witv-
rouw, P. Cotelle, J. Med. Chem. 2008, 51, 2575–2579.
[32] Z. Debyser, P. Cherepanov, W. Pluymers, E. De Clercq, Methods Mol. Biol.
2001, 160, 139–155.
[16] C. Tintori, V. Corradi, M. Magnani, F. Manetti, M. Botta, J. Chem. Inf.
Model. 2008, 48, 2166–2179.
[33] G. Moncunill, M. Armand-Ugꢄn, E. Pauls, B. Clotet, J. A. Estꢀ, AIDS 2008,
22, 23–31.
[17] S. Rajamaki, A. Innitzer, C. Falciani, C. Tintori, F. Christ, M. Witvrouw, Z.
Debyser, S. Massa, M. Botta, Bioorg. Med. Chem. Lett. 2009, 19, 3615–
3618.
[34] G. Moncunill, M. Armand-Ugꢄn, I. Clotet-Codina, E. Pauls, E. Ballana, A.
Llano, B. Romagnoli, J. W. Vrijbloed, F. O. Gombert, B. Clotet, S. De Mar-
co, J. A. Estꢀ, Mol. Pharmacol. 2008, 73, 1264.
[18] F. Dyda, A. B. Hickman, T. M. Jenkins, A. Engelman, R. Craigie, D. R.
Davies, Science 1994, 266, 1981–1986.
[19] D. Esposito, R. Craigie, EMBO J. 1998, 17, 5832–5843.
[20] A. A. Johnson, C. Marchand, S. S. Patil, R. Costi, S. R. Di, T. R., Jr. Burke, Y.
Pommier, Mol. Pharmacol. 2007, 71, 893–901.
[35] G. Jones, P. Willett, R. C. Glen, J. Mol. Biol. 1995, 245, 43–53.
[36] G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J. Mol. Biol. 1997,
267, 727–748.
[37] M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray, R. D. Taylor, Pro-
teins Struct. Funct. Bioinf. 2003, 52, 609–623.
[21] L. Franchi, M. Rinaldi, G. Vignaroli, A. Innitzer, M. Radi, M. Botta, Synthe-
sis 2010, 3927–3933.
[22] M. Anderluh, M. Jukic, R. Petric, Tetrahedron 2009, 65, 344–350.
[23] K. Bourahla, A. Derdour, M. Rahmouni, F. Carreaux, J. P. Bazureau, Tetra-
hedron Lett. 2007, 48, 5785–5789.
[38] Maestro, ver. 8.5, Schrçdinger, New York, NY (USA), 2008.
[39] W. L. Jorgensen, D. S. Maxwell, J. Tirado-Rives, J. Am. Chem. Soc. 1996,
118, 11225–11236.
[40] S. Maignan, J.-P. Guilloteau, Q. Zhou-Liu, C. Clꢀment-Mella, V. Mikol, J.
Mol. Biol. 1998, 282, 359–368.
[24] B. Hu, M. Malamas, J. Ellingboe, E. Largis, S. Han, R. Mulvey, J. Tillett,
Bioorg. Med. Chem. Lett. 2001, 11, 981–984.
[41] W. L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific
LLC, San Carlos, CA (USA): http://www.pymol.org.
[25] M. Pulici, F. Quartieri, Tetrahedron Lett. 2005, 46, 2387–2391.
[26] M. Radi, L. Botta, G. Casaluce, M. Bernardini, M. Botta, J. Comb. Chem.
2010, 12, 200–205.
[42] Y. Hwang, D. Rhodes, F. Bushman, Nucleic Acids Res. 2000, 28, 4884–
4892.
[27] P. Fresneau, M. Cussac, J. M. Morand, B. Szymonski, D. Tranqui, G. Le-
clerc, J. Med. Chem. 1998, 41, 4706–4715.
[28] P. Fresneau, J. M. Morand, F. Thomasson, M. Cussac, Spectrochim. Acta,
Part A 1999, 55, 2893–2898.
[29] P. C. Unangst, D. T. Connor, W. A. Cetenko, R. J. Sorenson, C. R. Kostlan,
J. C. Sircar, C. D. Wright, D. J. Schrier, R. D. Dyer, J. Med. Chem. 1994, 37,
322–328.
Received: November 25, 2010
Revised: December 22, 2010
Published online on January 18, 2011
352
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 343 – 352