New chloro,fluorobenzylindole derivatives as integrase strand-transfer inhibitors (INSTIs) and their mode of action

Article abstract of DOI:10.1016/j.bmc.2010.06.063

The life cycle of HIV-1 requires extensive assistance from the integrase (IN) enzyme which therefore constitutes an attractive therapeutic target for the development of anti-AIDS agents. We herein report the synthesis and biological evaluation of new HIV

Full text of DOI:10.1016/j.bmc.2010.06.063

Bioorganic & Medicinal Chemistry 18 (2010) 5510–5518
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New chloro,fluorobenzylindole derivatives as integrase strand-transfer
inhibitors (INSTIs) and their mode of action
b
a
c
c
Stefania Ferro a, Laura De Luca a, , Maria Letizia Barreca , Sara De Grazia , Frauke Christ , Zeger Debyser ,
*
Alba Chimirri a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, I-98168 Messina, Italy
b Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Via del Liceo, 1, I-06123 Perugia, Italy
c Molecular Virology and Gene Therapy, Molecular Medicine, Katholieke Universiteit Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The life cycle of HIV-1 requires extensive assistance from the integrase (IN) enzyme which therefore con-
stitutes an attractive therapeutic target for the development of anti-AIDS agents. We herein report the
synthesis and biological evaluation of new HIV integrase strand-transfer inhibitors (INSTIs) which proved
to be also potent anti-HIV agents. The binding mode of the most representative molecules were also stud-
ied by induced-fit docking (IFD). The obtained IFD results were consistent with the mechanism of action
proposed for this class of IN inhibitors, that is metal chelating/binding agents.
Received 11 February 2010
Revised 11 June 2010
Accepted 16 June 2010
Available online 22 June 2010
Keywords:
HIV-1 integrase
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Induced-fit docking
Integrase strand-transfer inhibitors
Microwave-assisted organic synthesis
1. Introduction
mediated by HIV-1-Integrase (IN) and also that there is no known
human counterpart,3 this essential enzyme might constitute a
AIDS (Acquired immune deficiency syndrome), caused by Hu-
man Immodeficiency Virus (HIV), is a severe infectious disease
responsible for a large number of deaths every year. Studies in
HIV biology have provided important information about the main
steps of the virus life cycle which consists of viral entry, reverse
transcription, integration, gene expression, virion assembly, bud-
ding and maturation. The officially approved drugs belong to the
class of reverse transcription and protease inhibitors and, recently,
viral entry and integrase inhibitors.
Despite the successes with such treatments as HAART (highly
active antiretroviral therapy) combination regimens, the perma-
nent use of anti-AIDS drugs induces drug-resistant viral variants
and emergence of unwanted metabolic side effects.1
In addition, some data have shown that HIV can survive in ex-
tremely long-living cells and be reactivated even after years of
HAART regimens.2 Therefore, the discovery and the development
of new more potent and less toxic anti-HIV agents capable of sup-
pressing drug-resistant HIV strains and/or targeting different
stages in the virus life cycle are still warranted.
therapeutically highly advantageous target.4
IN catalyzes the insertion of HIV-1 DNA into the genome of the
host cell through a multistep process which includes the following
different biochemical steps: (i) assembly of a stable complex with
specific DNA sequences at the end of the HIV-1 long terminal re-
peat (LTR) regions, (ii) endonucleolytic processing of the viral
DNA to remove the terminal dinucleotide from each 30 end (30P),
and (iii) strand-transfer (ST) in which the viral DNA 30 ends are
covalently legated to the host chromosomal DNA. No energy source
(e.g., ATP) is needed for the integration reaction, and only divalent
cations are required for catalytic activity.5,6
In particular, the two metal cofactors of HIV IN are thought to
both be Mg2+ ions under physiological conditions. Even if the exact
mechanism of action of IN inhibitors has not been completely elu-
cidated, it is believed that the inhibitors of the IN stand transfer
(INSTIs) step bind to the acceptor DNA site of the enzyme and
act by sequestering the metal ions bound in the IN active site to
form a ligand–M2+–IN complex.7–9
Moreover, it has been demonstrated that INSTIs bind at the
IN–DNA interface rather than to IN alone,10,11 thus acting as inter-
facial inhibitors of protein–nucleic acid interactions.12,13
Research has focused on the molecular binding of INSTIs to
integrase complexes because of the increasing importance of selec-
tive INSTIs as antiviral compounds and of their unique mechanism
of action.12
Considering that successful completion of the HIV-1 viral life
cycle depends in part on the integration of complementary DNA
* Corresponding author. Tel.: +39 0906766464; fax: +39 0906766402.
E-mail address: ldeluca@unime.it (L.D. Luca).
0968-0896/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.063

S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
5511
Unfortunately, a full understanding of the binding mode of INS-
TIs has been hampered by the lack of detailed information about
the structure of the three-domain protein and its interaction with
the DNA substrates (viral and target DNA), metal cofactors and
inhibitors. In the last decade of antiviral research, the discovery
of the keto-enol acid class, often referred as diketoacid (DKA) class,
and their analogues and bioisosters as IN inhibitors was a major
advance in the validation of IN as a therapeutically viable antiret-
roviral drug target.12,14–18
In particular a DKA-analogue MK-0518 (1), known as raltegra-
vir, is the first and only integrase inhibitor (INI) approved by the
Food and Drug Administration (FDA) for the treatment of AIDS act-
ing as INSTI.19–21
Moreover other HIV-1 IN inhibitors that have been clinically
evaluated, such as GS-9137 (2), MK-2048 (3), GS-9160, GS-9224,
GSK-364735, and BMS-707035 belong to the same chemical class
of DKA-like derivatives which have been shown to specifically in-
hibit the DNA ST step of integration and represent the major leads
in the development of anti-HIV-1 IN drugs.22
Previously, our molecular modeling studies had suggested to us
the rational design and synthesis of new potential DKA-INIs char-
acterized by the presence of a 1H-benzylindole skeleton.23,24 The
biological results showed that all derivatives selectively inhibited
the ST step at nanomolar concentration and in particular allowed
us to identify a new ‘lead compound’ named CHI-1043 (4) charac-
terized by the presence of a methoxy group at C-4 of the indole
system and bearing a fluorine atom on the para position of the ben-
zyl moiety (Fig. 1).25
stall leading to a rapid decline in the viral load and then of the
infection. For these reason, intensive research over the last two
decades have been addressed to the discovery and development
of small molecules inhibitors of the INST. Several previous publica-
tions have described our intense work on anti-HIV agents.23,24,27–34
In particular, we generated a 3D pharmacophore model by Catalyst
program for DKA-like derivatives acting as INSTIs25 consisting of
seven features: four hydrogen-bond acceptors (A1–A4), two hydro-
phobic aliphatic regions (Z1 and Z2) and one aromatic feature (Y).
The superimposition of our lead compound CHI-1043 on the model
is shown in Figure 2A: the b-diketo acid moiety maps the three
hydrogen-bond acceptors (A1–A3), the methoxy group occupies
aliphatic region Z1 and the p-fluorobenzyl ring overlaps the aro-
matic feature Y. For the purpose of lead optimization, the effect
of introducing substituents on the benzene-fused ring of an indole
system has already been examined25 and the biological results
showed that the methoxy derivatives exhibited the best biological
profile inhibiting HIV-1 IN at a nanomolar to low micromolar
range. Encouraged by these results and in order to extend our
understanding of the structure–activity relationships of this class
of INIs and explore the chemical features required for the IN inhi-
bition, we decided to keep unchanged the indole part of the mole-
cule and to investigate the effects of different substituents on the
benzyl group at N1.
Although INSTIs are structurally diverse and encompass a vari-
ety of pharmacophores, all appear to have features in common,
reflecting a likely similar mode of action. In particular, the presence
of a metal-binding moiety, in our compounds represented by the
hydroxy acid group, and an enzyme-binding moiety appears spe-
cially important. This last portion contains one or more aromatic
hydrophobic residues needed to anchor the INSTIs to a hydropho-
bic pocket that forms upon binding of viral DNA.35 In particular a
halogen-substituted aromatic functionality linked to the chelating
portion of the inhibitors has been hypothesized may bind in one or
more hydrophobic pockets of IN–DNA complex.
On these bases, and also taking into account some structural
characteristics of potent IN inhibitors in clinical trials (i.e.,
GS-9137 elvitegravir (2), MK-2048 (3) etc.) we herein report the
rational design, synthesis and structure–activity relationships
(SAR) of new chloro–fluoro-benzylindoles CHI-1043 analogues
with the aim of improving the activity of this class of compounds
as well as their selective index. Finally, docking results are herein
reported to further clarify their mechanism of action.
Furthermore, several reports highlighted the importance of the
halogen substituents effects on IN inhibitory potency. In particular
we observed that some new HIV-1 IN inhibitors in clinical phase,
including compounds such as elvitegravir (2) and MK-2048 (3),
were charactherized by a chloro,fluorobenzyl substitution as com-
mon feature.
2. Results and discussion
2.1. Rational design
The superimposition of these compounds on our pharmaco-
phore hypothesis showed that the chloro,fluorobenzyl moiety of
these ligands occupied the hydrophobic feature Y of our model
(Fig. 2B–C). This observation suggested to us some chemical mod-
The IN strand-transfer reaction occurs inside the nucleus and
produces a functional integrated proviral DNA which forms the
template for transcription of new viral RNA needed for new viri-
ons.26 Thus without successful integration, viral replication would
O
OH
OH
O
N
O
N
O
H
N
N
O
F
N
N
O
Cl
O
HN
F
OH
1 (MK-0518, raltegravir)
2 (GS-9137, elvitegravir)
F
H
N
O
N
F
N
N
O
N
N
COOH
OMe
Cl
O
OH
HO
O
3 (MK-2048)
4 (CHI-1043 )
Figure 1. Chemical structures of integrase strand-transfer inhibitors.

5512
S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
Figure 2. The top scoring HipHop pharmacophore Hypo1 is mapped to IN inhibitors 4 (A), 2 (B) 3 (C) (A1–A4, hydrogen-bond acceptor, green; Z1–Z2, hydrophobic aliphatic,
blue; Y, hydrophobic aromatic, cyan).
ifications on this part of our lead compound CHI-1043. With the
aim of studying the effect on biological activity of the nature and
position of substituents on the benzyl moiety we replaced the flu-
oro to chloro group and also examined the influence of introducing
two or more substituents in different positions.
A small library of novel chloro,fluorobenzyl derivatives (20–45)
was then designed and tested as INIs and anti-HIV agents (see Sec-
tions 2.2 and 2.3).
sorbent assays as described previously.38 However, their inhibitory
effect on the HIV-induced cytopathic effect (CPE) in human lympho-
cyte MT-4 cell culture was determined by the MT-4/MTT-assay.39
Also in this new series of molecules the diketo acids (33–35 and
37–45) inhibited the enzyme at nanomolar concentration and
exhibit a higher potency than that of the corresponding esters
(20–22) and (24–32), thus suggesting the importance of ionizable
carboxylic acid as metal ion binding motif. Surprendently deriva-
tive 36, showed a different behavior limitedly to the enzymatic
activity.
2.2. Chemistry
The biological results suggested that compounds bearing only a
chlorine atom on 2- (33) or 3-position (34) were more potent than
4-substituted derivative (35) at inhibiting both over-all and
strand-transfer steps as well as HIV replication in MT-4 cells.
Moreover, out of the disubstituted derivatives, compounds
37–43 showed a potent inhibition of strand-transfer and in over-
all steps thus indicating the importance to maintain a chloro
substitution at 2- or 3-position of the benzyl moiety. Moreover
derivative 39 with the chloro substituent at the C-2 and a fluoro
group at C-6 displayed a good antiviral activity and the highest
selectivity index in the current series of molecules (Table 1).
With these findings our study may contribute to extend the
understanding of the structure–activity parameters proving that
the diversity of halogen-substituted benzyl groups influences the
inhibition potency and the selectivity of these INIs.
With the aim of obtaining further information about the struc-
ture–activity relationship (SAR) of the benzylindole DKA deriva-
tives 20–45 we planned the synthesis of the designed compounds.
Previously25,36 we described the preparation of 4-[1-benzyl-
4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acids in
which several steps of the synthetic approach were optimized by
the application of microwave-assisted technology, thereby increas-
ing the yields and reducing the reaction times as well as having
cleaner reactions and using less solvent.
We report herein an alternative way to perform the 3-acetyla-
tion of 4-OCH3-1H-indole (5) through a similar Vilsmeier–Haack
reaction using N,N-dimethylacetamide and phosphoryl chloride
according to the procedure reported by Murakami et al.37 This ap-
proach led to a significant improvement in reaction conditions giv-
ing a cleaner reaction and increasing the yield of the intermediate
6. (Scheme 1).
2.4. Molecular modeling studies
Successively, by application of microwave assisted synthesis it
was N-alkylated by treatment with the appropriate substituted-
benzyl bromide to give intermediates 7–19.
Coupling with diethyl oxalate easily provided diketo esters 20–
32 which were finally converted into the corresponding benzylin-
dolediketo acids (33–45) in basic medium.
In order to better analyze their possible accommodation into
the active site, we also performed docking studies on this new ser-
ies of compounds starting from our previous results which pro-
vided new insights into the possible mechanism of action and
binding mode of INSTIs.40
The structural characteristics of obtained compounds were
determined by means of analytic and spectroscopic methods.
In particular, a ternary HIV-1 IN–Mg–DNA model was built and
used by us as target for induced-fit docking (IFD) studies. Our in
silico results were consistent with the available resistance muta-
tion data and revealed that all the inhibitors bound the same
region of the protein and share a similar mode of action at the IN
active site. More specifically, the compounds: (a) have a pharmaco-
phore that can establish interactions with the Mg2+ ions;
(b) showed close interactions with the three catalytic residues
(D64, D116 and E152); (c) interacted with the viral donor DNA
and (d) were able to occupy a deep region defined by the catalytic
loop and the viral DNA through their substituted aromatic tail.
The same IFD protocol has now been applied to the most active
compounds herein reported in order to explore their possible bind-
ing conformation.
2.3. Biological activity
All the synthesized derivatives, diketo esters (20–32) and the
corresponding diketo acids (33–45), which were designed to merge
their features with those of our previously reported benzylindole
derivatives (i.e., CHI-1043), were tested. Their inhibitory effect on
IN enzymatic activity and against HIV-1 replication (Table 1) was
compared with those of some our already published substituted
fluorobenzyl derivatives.36
To determine the susceptibility of the HIV-1 integrase enzyme
towards synthesized compounds we used enzyme-linked immuno-

S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
5513
RR'
H
N
H
N
N
i
ii
CH3
7, 20, 33, R = 2Cl, R' = H,
8, 21, 34, R = 3Cl, R' = H,
9, 22, 35, R = 4Cl, R' = H,
10, 23, 36, R = 2Cl, R' = 3F,
11, 24, 37, R = 2Cl, R' = 4F,
12, 25, 38, R = 2Cl, R' = 5F,
13, 26, 39, R = 2Cl, R' = 6F,
14, 27, 40, R = 3Cl, R' = 2F,
15, 28, 41, R = 3Cl, R' = 4F,
16, 29, 42, R = 3Cl, R' = 5F,
17, 30, 43, R = 3Cl, R' = 6F,
18, 31, 44 R = 4Cl, R' = 2F,
19, 32, 45 R = 4Cl, R' = 3F
CH3
OMe
OMe
OMe
7-19
O
O
5
6
iii
RR'
RR'
N
N
iv
COOEt
OH
COOH
OMe
OMe
33-45
O
O
HO
20-32
Scheme 1. Reagents and conditions: (i) POCl3, CH3CON(CH3)2, rt,12 h; (ii) ArCH2Br, NaH, DMF, 50 °C, 5 min, 100 Watt; (iii) diethyl oxalate, dry CH3ONa, THF, two separate
steps under the same conditions: 50 °C, 2 min, 250 Watt; (iv) 2 N NaOH, MeOH, rt, 1.5 h.
Table 1
Inhibition of HIV-1 integrase enzymatic activity, replication of HIV-1 (IIIB), and cytotoxicity in MT-4 cells
Compound
IN enzymatic activity
STb
IC50
Activity in MT-4 cells
Cytotoxicityd
Over-alla
IC50 M)
HIV-1c
EC50
SIe
(
l
(
lM)
(
lM)
EC50 (lM)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
CHI-1043
MK-518
GS-9137
13.1 0.4
4.78 1.56
5.78 3.85
1.39 0.78
1.74 0.79
2.7 0.25
2.86 1.07
1.37 0.14
0.36 0.1
1.30 0.09
1.61 1.37
1.15 0.22
5.05 2.62
0.71 0.24
0.13 0.09
1.02 0.94
5.24 1.23
0.54 0.14
0.42 0.16
0.53 0.38
0.74 0.21
0.04 0.00
0.24 0.01
0.32 0.08
2.53 0.39
3.33 0.89
0.08 0.003
0.009 0.0002
0.004 0.003
0.73 0.18
2.89 0.86
8.26 4.32
9.2 6.79
2.56 1.17
2.94 1.88
1.2 0.11
2.51 0.08
3.25 0.86
41.32 0.00
2.52 0.26
4.06 1.56
16.39 0.6
4.66 0.00
2.31 0.02
1.87 0.19
4.27 0.68
18.13 0.00
>17.00
34.51 4.64
52.89 3.76
41.32 0.00
18.5 3.5
51.43 28.35
39.63 5.67
25.36 0.00
46.9 0.00
49.3 3.6
14
17
1
7
13
3
5
0.74 0.26
0.07 0.04
3.17 0.70
0.89 0.53
3.32 1.31
20.35 5.52
0.52 0.35
0.03 0.01
0.59 0.06
12.74 1.79
0.06 0.04
0.27 0.07
0.13 0.03
0.03 0.02
0.12 0.01
0.25 0.01
0.09 0.02
5.52 1.58
4.58 0.19
0.14 0.11
0.007 0.0005
0.015 0.002
20
26
14
1
<1
<1
64
47
2
18
11
8
111
35
74
62
3
58.2 3.9
18.13 0.00
17.00 0.00
63.70
>63.68
0.42 0.00
0.64 0.00
20.58 0.00
0.36 0.05
4.3 2.1
27.0
30.15 1.85
33.47 5.27
6.5 0.5
47.16 4.9
26.26 4.13
61.97 22.84
14.95 3.65
31.65 18.25
32.15 3.05
10.75 2.95
23.00 0.5
23.31
3.3 0.86
0.56 0.003
0.43 0.00
0.43 0.005
0.52 0.05
4.17
>22.52
>23.31
0.59 0.38
0.013 0.0005
0.0008 0.00009
<1
<1
70
>1387
>2650
41.1 16.7
>18
2.12 0.36
a
b
c
Concentration required to inhibit the in vitro overall integrase activity by 50%.
Concentration required to inhibit the in vitro strand-transfer step by 50%.
Effective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells.
Cytotoxic concentration to reduce MT-4 cell viability by 50%.
d
e
Selectivity index: ratio CC50/EC50. All data represent average results SD.
Figure 3 reports the binding mode of derivative 39, selected for
The results of the IFD study showed that compound 39 is able to
interact with the Mg2+ ions and also presents close interactions
with both viral DNA strands (Fig. 3).
The benzyl group points toward the active site loop (residues
140–149), making strong hydrophobic interactions with I141; no
its interesting biological profile (Table 1): it is active at nanomolar
concentration as INST inhibitor and shows potent antiviral activity
against HIV-1 in MT-4 cells as well as the highest selectivity index
of the series.

5514
S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
Figure 3. (A) Induced-fit docking results for 39. IN/DNA residues lying within a distance of 5 Å from the docked inhibitor are shown in yellow. G24 and C25 are 50-DNA
nucleotide bases, while A20 is the terminal adenine of the 30-processed viral DNA. The divalent metal ions are shown as magenta spheres, the active site loop is highlighted in
cyan, and the viral DNA backbone is depicted in blue. B) Binding mode for 39. Molecular surfaces are shown for IN (gray), catalytic loop (residues 140–149; cyan), metal ions
(magenta), 30-DNA strand (green), and 50-DNA strand (yellow). IFD conformations for 39 and elvitegravir (2) are colored in white and orange, respectively. This figure was
prepared using PyMol.46
hydrogen-bond was observed between our compound and the
macromolecular target (Fig. 3A). A comparison between the IFD
conformations obtained for elvitegravir (2) and compound 39
highlighted that their substituted aromatic tail occupy a similar re-
gion of the macromolecular target: the fluoro,chlorobenzyl group
of both molecules was in fact directed toward the active site loop
residues and deeply occupies a region defined by the loop itself
and the donor DNA (Fig. 3B).
4.2. Induced-fit methodology
The IFD protocol43 developed by Schrçdinger44 was employed in
this study. Briefly, IFD methodology uses the docking program
Glide to account for ligand flexibility and the Refinement module
in the Prime program to account for receptor flexibility. The Schrç-
dinger IFD protocol models induced-fit docking of one or more li-
gands using the following steps (the description below is from
the IFD manual):
3. Conclusion
1. Constrained minimization of the receptor (Glide protein prepa-
ration, refinement only) with an RMSD cutoff of 0.18.
2. Initial Glide docking of each ligand using a softened potential
(van der Waals radii scaling). By default, a maximum 20 poses
per ligand are retained, and by default, poses to be retained
must have a Coulomb–vdW score <100 and an H-bond score
<ꢀ0.05.
We herein report design, synthesis and biological evaluation of
a new series of potent INIs which allowed us to further clarify the
SAR of our benzylindolediketo acids. In this study we extended the
investigation of the hydrophobic portion that is of the enzyme-
binding moiety of INSTIs. As a result some new derivatives more
potent and with a selectivity index higher than that of our lead
compound CHI-1043 were obtained. In some cases the new mole-
cules (34, 37 and 40), showed an IN strand-transfer inhibition com-
parable to that of elvitegravir (2).
Finally, the IFD results highlighted that our derivatives present
a binding mode similar to that of elvitegravir (2) and confirmed the
importance of lipophilic groups able to occupy the hydrophobic
cavity in the INSTI binding site. Considering that the diketo acid
moiety seems to negatively influence the selectivity of the mole-
cules, additional modifications in this direction are in progress to
further optimize the biological profile of so far obtained com-
pounds and in an attempt to throw useful structural information
to construct ideal INSTIs.
3. One round of Prime side chain prediction for each protein–
ligand complex, on residues within a given distance of any
ligand pose (6 Å in our study).
4. Prime minimization of the same set of residues and the ligand
for each protein–ligand complex pose. The receptor structure
in each pose now reflects an induced-fit to the ligand structure
and conformation.
5. Glide re-docking of each protein–ligand complex structure
within
a specified energy of the lowest-energy structure
(default: 30 kcalmol ꢀ1). The ligand is now rigorously docked,
using default Glide settings, into the induced-fit receptor
structure.
6. Estimation of the binding energy (IFDScore) for each output
pose.
4. Experimental section
In our study, all docking calculations were run in the ‘Standard
Precision’ mode of Glide, and the center of the grid box was defined
by the manually selected Mg ions. All of the docked structures
were automatically ranked according to the IFD score. RMSDs were
computed by all atoms except non polar hydrogen atoms.
4.1. Preparation of IN–DNA–Mg complex and ligands for the IFD
protocol
Ligand structures were constructed using the Schrçdinger
Maestro41 and were then submitted to Polak–Ribiere conjugate
gradient minimization (0.0005 kJÅꢀ1 molꢀ1 convergence). The
compounds were presented in their enolic tautomeric form, since
it has been clearly established that DKAs mainly exist in this form
in solution, the carboxylic moiety was considered as carboxylate,
and the enolic oxygen was considered as enolate given the influ-
ence of the two metal ions in the binding site.42
4.3. Chemistry
All microwave-assisted reactions were carried out in a CEM Fo-
cused Microwave Synthesis System, Model Discover working at the
potency necessary for refluxing under atmospheric conditions (i.e.,
250–300 W). Melting points were determined on a BUCHI Melting

S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
5515
Point B-545 apparatus and are uncorrected. Elemental analyses (C,
H and N) were carried out on a Carlo Erba Model 1106 Elemental
Analyzer and the results are within 0.4% of the theoretical values.
Merck Silica Gel 60 F254 plates were used for analytical TLC; col-
umn chromatography was performed on Merck silica gel 60
(230–400 mesh) and Flash Chromatography (FC) on a Biotage SP1
EXP. 1H NMR spectra were recorded in CDCl3 with TMS as internal
standard or DMSO on a Varian Gemini-300 spectrometer. Chemical
shifts are expressed in d (ppm) and coupling constants (J) in
hertz.
4.3.8. 3-Acetyl-1-(2-chloro-6-fluorobenzyl)-4-methoxy-1H-indole
(13)
Mp 108–110 °C, yield 46%; 1H NMR (d) 2.65 (s, 3H, CH3), 3.96 (s,
3H, OCH3), 5.41 (s, 2H, CH2), 6.69–7.67 (m, 7H, ArH). Anal. Calcd for
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.02; H, 4.64;
N, 4.35.
4.3.9. 3-Acetyl-1-(3-chloro-2-fluorobenzyl)-4-methoxy-1H-indole
(14)
Mp 121–123 °C, yield 71%; 1H NMR (d) 2.70 (s, 3H, CH3), 3.98 (s,
3H, OCH3), 5.36 (s, 2H, CH2), 6.69–7.75 (m, 7H, ArH). Anal. Calcd for
4.3.1. Synthesis of 3-acetyl-4-methoxy-1H-indole (6)
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.23; H, 4.71;
Phosphoryl chloride (0.92 ml, 10 mmol) was added to ice cold
dimethylacetamide (2.79 ml, 30 mmol) with stirring and cooling
in ice. 4-Methoxy-1H-indole 5 (147.18 mg, 1 mmol) was added
and reaction mixture was stirred at room temperature for 12 h,
then poured over ice and basified with 4 N aqueous sodium
hydroxide solution. The mixture was extracted with ethyl acetate
and dried over Na2SO4. After removal of the solvent under reduced
pressure, the residue was powdered by treatment with diethyl
ether and recrystallized from dichloromethane. Mp 113–115 °C,
yield 89%. 1H NMR (d) 2.70 (s, 3H, CH3), 3.97 (s, 3H, OCH3), 6.68–
7.79 (m, 4H, ArH), 8.69 (br s, 1H, NH). Anal. Calcd for C11H11NO2:
C, 69.83; H, 5.86; N, 7.40. Found: C, 69.62; H, 5.63; N, 7.51.
3-Acetyl-4-methoxy-1-benzyl-1H-indoles (7–19) were pre-
pared following previously reported procedure.36
N, 4.11.
4.3.10. 3-Acetyl-1-(3-chloro-4-fluorobenzyl)-4-methoxy-1H-indole
(15)
Mp 103–105 °C, yield 61%; 1H NMR (d) 2.70 (s, 3H, CH3), 3.97 (s,
3H, OCH3), 5.24 (s, 2H, CH2), 6.69–7.71 (m, 7H, ArH). Anal. Calcd for
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.02; H, 4.44;
N, 4.57.
4.3.11. 3-Acetyl-1-(3-chloro-5-fluorobenzyl)-4-methoxy-1H-indole
(16)
Mp 133–135 °C, yield 66%; 1H NMR (d) 2.71 (s, 3H, CH3), 3.98 (s,
3H, OCH3), 5.26 (s, 2H, CH2), 6.69–7.71 (m, 7H, ArH). Anal. Calcd for
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.27; H, 4.71;
N, 4.09.
4.3.2. 3-Acetyl-1-(2-chlorobenzyl)-4-methoxy-1H-indole (7)
Mp 131–133 °C, yield 40%; 1H NMR d 2.50 (s, 3H, CH3), 3.87 (s,
3H, OCH3), 5.54 (s, 2H, CH2), 6.72–8.07 (m, 8H, ArH). Anal. Calcd for
4.3.12. 3-Acetyl-1-(3-chloro-6-fluorobenzyl)-4-methoxy-1H-indole
(17)
C18H16ClNO2: C, 68.90; H, 5.14; N, 4.46. Found: C, 69.07; H, 5.34; N,
Mp 123–125 °C, yield 68%; 1H NMR (d) 2.70 (s, 3H, CH3), 3.98 (s,
3H, OCH3), 5.30 (s, 2H, CH2), 6.70–7.74 (m, 7H, ArH). Anal. Calcd for
4.13.
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.33; H, 4.62;
4.3.3. 3-Acetyl-1-(3-chlorobenzyl)-4-methoxy-1H-indole (8)
Mp 115–117 °C, yield 63%;1H NMR (d) 2.62 (s, 3H, CH3), 3.94 (s,
3H, OCH3), 5.37 (s, 2H, CH2), 6.69–7.84 (m, 8H, ArH). Anal. Calcd for
N, 4.13.
4.3.13. 3-Acetyl-1-(4-chloro-2-fluorobenzyl)-4-methoxy-1H-indole
(18)
C18H16ClNO2: C, 68.90; H, 5.14; N, 4.46. Found: C, 68.72; H, 5.42; N,
4.64.
Mp 140–142 °C, yield 66%; 1H NMR (d) 2.69 (s, 3H, CH3), 3.97 (s,
3H, OCH3), 5.30 (s, 2H, CH2), 6.68–7.72 (m, 7H, ArH). Anal. Calcd for
4.3.4. 3-Acetyl-1-(4-chlorobenzyl)-4-methoxy-1H-indole (9)
Mp 129–131 °C, yield 60%; 1H NMR (d) 2.53 (s, 3H, CH3), 3.84 (s,
3H, OCH3), 5.44 (s, 2H, CH2), 6.69–8.19 (m, 8H, ArH). Anal. Calcd for
C18H15Cl2NO2: C, 68.90; H, 5.14; N, 4.46. Found: C, 69.12; H, 5.06;
N, 4.24.
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.38; H, 4.27;
N, 4.51.
4.3.14. 3-Acetyl-1-(4-chloro-3-fluorobenzyl)-4-methoxy-1H-indole
(19)
Mp 158–160, yield 72%; 1NMR (d) 2.71 (s, 3H, CH3), 3.98 (s, 3H,
OCH3), 5.27 (s, 2H, CH2), 6.69–7.71 (m, 7H, ArH). Anal. Calcd for
4.3.5. 3-Acetyl-1-(2-chloro-3-fluorobenzyl)-4-methoxy-1H-indole
(10)
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.32; H,
Mp 165–167 °C, yield 68%; 1H NMR (d) 2.70 (s, 3H, CH3), 3.99 (s,
3H, OCH3), 5.41 (s, 2H, CH2), 6.46–7.71 (m, 7H, ArH). Anal. Calcd for
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.32; H, 4.28;
N, 4.06.
4.24; N, 4.31.
Ethyl 4-[1-benzyl-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxo-
but-2-enoates (20–32) were prepared following previously re-
ported procedure.36
4.3.6. 3-Acetyl-1-(2-chloro-4-fluorobenzyl)-4-methoxy-1H-indole
(11)
4.3.15. Ethyl 4-[1-(2-chlorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (20)
Mp 151–153 °C, yield 67%; 1H NMR (d) 2.70 (s, 3H, CH3), 3.99 (s,
3H, OCH3), 5.35 (s, 2H, CH2), 6.67–7.70 (m, 7H, ArH). Anal. Calcd for
Mp 205 °C dec, yield 93%; 1H NMR (d) 1.23 (t, J = 7.1, 3H, CH3),
3.84 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.47 (s, 2H, CH2),
6.56–8.52 (m, 9H, 8ArH and CH). Anal. Calcd for C22H20ClNO5: C,
63.85; H, 4.87; N, 3.38. Found: C, 63.56; H, 4.41; N, 3.52.
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.42; H, 4.37;
N, 4.31.
4.3.7. 3-Acetyl-1-(2-chloro-5-fluorobenzyl)-4-methoxy-1H-
indole (12)
4.3.16. Ethyl 4-[1-(3-chlorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (21)
Mp 132–134 °C, yield 42%; 1H NMR (d) 2.54 (s, 3H, CH3), 3.87 (s,
3H, OCH3), 5.53 (s, 2H, CH2), 6.52–8.09 (m, 7H, ArH). Anal. Calcd for
Mp 180 °C dec, yield 87%; 1H NMR (d) 1.23 (t, J = 7.1, 3H, CH3),
3.82 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.41 (s, 2H, CH2),
6.56–8.51 (m, 9H, 8ArH and CH). Anal. Calcd for C22H20ClNO5: C,
63.85; H, 4.87; N, 3.38. Found: C, 63.71; H, 4.94 N, 3.25.
C18H15ClFNO2: C, 65.16; H, 4.56; N, 4.22. Found: C, 65.27; H, 4.73;
N, 4.08.

5516
S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
4.3.17. Ethyl 4-[1-(4-chlorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (22)
4.3.26. Ethyl 4-[1-(4-chloro-2-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (31)
Mp 215 °C dec, yield 91%; 1H NMR (d) 1.22 (t, J = 7.1, 3H, CH3),
3.82 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.39 (s, 2H, CH2),
6.55–8.51 (m, 9H, 8ArH and CH). Anal. Calcd for C22H20ClNO5: C,
63.85; H, 4.87; N, 3.38. Found: C, 63.61; H, 4.95 N, 3.54.
Mp 228–230 °C dec, yield 89%; 1H NMR (d) 1.28 (t, J = 7.1, 3H,
CH3), 3.89 (s, 3H, OCH3), 4.23 (q, J = 7.1, 2H, CH2), 5.53 (s, 2H,
CH2), 6.76–8.33 (m, 8H, 7ArH and CH). Anal. Calcd for
C22H19ClFNO5: C, 61.19; H, 4.43; N, 3.24. Found: C, 61.32; H,
4.68; N, 3.39.
4.3.18. Ethyl 4-[1-(2-chloro-3-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (23)
4.3.27. Ethyl 4-[1-(4-chloro-3-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (32)
Mp 242 °C dec, yield 96%; 1H NMR (d) 1.22 (t, J = 7.1, 3H, CH3),
3.84 (s, 3H, OCH3), 4.09 (q, J = 7.1, 2H, CH2), 5.53 (s, 2H, CH2),
6.52–7.60 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C,
61.19; H, 4.43; N, 3.24. Found: C, 61.29; H, 4.63; N, 3.53.
Mp 186–188 °C, yield 81%; 1H NMR (d) 1.22 (t, J = 7.1, 3H, CH3),
3.82 (s, 3H, OCH3), 4.09 (q, J = 7.1, 2H, CH2), 5.41 (s, 2H, CH2), 6.53–
8.50 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C, 61.19;
H, 4.43; N, 3.24. Found: C, 61.44; H, 4.28; N, 3.42.
4-[1-Benzyl-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-
enoic acid (33–45) were prepared following previously reported
procedure.36
4.3.19. Ethyl 4-[1-(2-chloro-4-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (24)
Mp 250 °C dec, yield 89%; 1H NMR (d) 1.22 (t, J = 7.1, 3H, CH3),
3.83 (s, 3H, OCH3), 4.08 (q, J = 7.1, 2H, CH2), 5.44 (s, 2H, CH2),
6.58–8.49 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C,
61.19; H, 4.43; N, 3.24. Found: C, 61.44; H, 4.72; N, 3.51.
4.3.28. 4-[1-(2-Chlorobenzyl)-4-methoxy-1H-indol-3-yl]-2-hydr-
oxy-4-oxobut-2-enoic acid (33)
Mp 160–162 °C, yield 65%; 1H NMR (d) 3.91 (s, 3H, OCH3), 5.60
(s, 2H, CH2), 6.78–8.36 (m, 9H, 8ArH and CH). Anal. Calcd for
C20H16ClNO5: C, 62.27; H, 4.18; N, 3.63. Found: C, 62.48; H, 4.61;
N, 3.31.
4.3.20. Ethyl 4-[1-(2-chloro-5-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (25)
Mp 190 °C dec, yield 88%; 1H NMR (d) 1.23 (t, J = 7.1, 3H, CH3),
3.84 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.47 (s, 2H, CH2),
6.48–8.49 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C,
61.19; H, 4.43; N, 3.24. Found: C, 61.32; H, 4.26; N, 3.52.
4.3.29. 4-[1-(3-Chlorobenzyl)-4-methoxy-1H-indol-3-yl]-2-hydr-
oxy-4-oxobut-2-enoic acid (34)
Mp 160–162 °C, yield 65%; 1H NMR (d) 3.90 (s, 3H, OCH3), 5.52
(s, 2H, CH2), 6.79–8.56 (m, 9H, 8ArH and CH). Anal. Calcd for
C
20H16ClNO5: C, 62.27; H, 4.18; N, 3.63. Found: C, 62.38; H, 4.29;
4.3.21. Ethyl 4-[1-(2-chloro-6-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (26)
N, 3.47.
Mp 245 °C dec, yield 91%; 1H NMR (d) 1.22 (t, J = 7.1, 3H, CH3),
3.83 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.46 (s, 2H, CH2),
6.63–8.48 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C,
61.19; H, 4.43; N, 3.24. Found: C, 61.44; H, 4.72; N, 3.09.
4.3.30. 4-[1-(4-Chlorobenzyl)-4-methoxy-1H-indol-3-yl]-2-hydr-
oxy-4-oxobut-2-enoic acid (35)
Mp 170–172 °C, yield 48%; 1H NMR (d) 3.89 (s, 3H, OCH3), 5.51
(s, 2H, CH2), 6.78–8.53 (m, 9H, 8ArH and CH). Anal. Calcd for
C
20H16ClNO5: C, 62.27; H, 4.18; N, 3.63. Found: C, 62.41; H, 4.35;
4.3.22. Ethyl 4-[1-(3-chloro-2-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (27)
N, 3.44.
Mp 198–200 °C, yield 88%; 1H NMR (d) 1.23 (t, J = 7.1, 3H, CH3),
3.81 (s, 3H, OCH3), 4.11 (q, J = 7.1, 2H, CH2), 5.41 (s, 2H, CH2), 6.56–
8.49 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C, 61.19;
H, 4.43; N, 3.24. Found: C, 61.24; H, 4.56; N, 3.37.
4.3.31. 4-[1-(2-Chloro-3-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (36)
Mp 160–162 °C, yield 35%; 1H NMR (d) 3.92 (s, 3H, OCH3), 5.66
(s, 2H, CH2), 6.56–8.42 (m, 8H, 7ArH and CH). Anal. Calcd for
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.38; H,
3.61; N, 3.55.
4.3.23. Ethyl 4-[1-(3-chloro-4-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (28)
Mp 207 °C dec, yield 79%; 1H NMR (d) 1.22 (t, J = 7.1, 3H, CH3),
3.81 (s, 3H, OCH3), 4.08 (q, J = 7.1, 2H, CH2), 5.38 (s, 2H, CH2),
6.50–8.52 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19ClFNO5: C,
61.19; H, 4.43; N, 3.24. Found: C, 61.53; H, 4.62; N, 3.58.
4.3.32. 4-[1-(2-Chloro-4-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (37)
Mp 266–288 °C dec, yield 58%; 1H NMR (d) 3.85 (s, 3H, OCH3),
5.51 (s, 2H, CH2), 6.69–8.05 (m, 8H, 7ArH and CH). Anal. Calcd for
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.76; H,
3.97; N, 3.58.
4.3.24. Ethyl 4-[1-(3-chloro-5-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (29)
4.3.33. 4-[1-(2-Chloro-5-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (38)
Mp 223–225 °C, yield 82%; 1H NMR (d) 1.21 (t, J = 7.1, 3H, CH3),
3.81 (s, 3H, OCH3), 4.12 (q, J = 7.1, 2H, CH2), 5.41 (s, 2H, CH2), 6.62–
8.49 (m, 8H, 7ArH and CH). Anal. Calcd for C22H19FNO5: C, 61.19; H,
4.43; N, 3.24. Found: C, 61.32; H, 4.28; N, 3.48.
Mp 164–166 °C, yield 41%; 1H NMR (d) 3.92 (s, 3H, OCH3), 5.60
(s, 2H, CH2), 6.64–8.40 (m, 8H, 7ArH and CH). Anal. Calcd for
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.21; H,
3.59; N, 3.62.
4.3.25. Ethyl 4-[1-(3-chloro-6-fluorobenzyl)-4-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (30)
4.3.34. 4-[1-(2-Chloro-6-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (39)
Mp 198–200 °C dec, yield 86%; 1H NMR (d) 1.59 (t, J = 7.1, 3H,
CH3), 3.82 (s, 3H, OCH3), 4.09 (q, J = 7.1, 2H, CH2), 5.43 (s, 2H,
CH2), 6.53–8.51 (m, 8H, 7ArH and CH). Anal. Calcd for
Mp 198–200 °C, yield 59%; 1H NMR (d) 3.90 (s, 3H, OCH3), 5.62
(s, 2H, CH2), 6.81–8.13 (m, 8H, 7ArH and CH). Anal. Calcd for
C22H19ClFNO5: C, 61.19; H, 4.43; N, 3.24. Found: C, 61.34; H,
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.36; H,
4.27; N, 3.51.
3.61; N, 3.58.

S. Ferro et al. / Bioorg. Med. Chem. 18 (2010) 5510–5518
5517
4.3.35. 4-[1-(3-Chloro-2-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (40)
reduction of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) by mitochondrial dehydroge-
nase of metabolically active cells to a blue formazan derivative,
which can be measured spectrophotometrically. The 50% cell cul-
ture infective dose (CCID50) of the HIV(IIIB) strain was determined
by titration of the virus stock using MT-4 cells. For the drug-sus-
ceptibility assays, MT-4 cells were infected with 100–300 CCID50
of the virus stock in the presence of fivefold serial dilutions of
the antiviral drugs. The concentration of the various compounds
that achieved 50% protection against the CPE of the different HIV
strains, which is defined as the EC50, was determined. In parallel
the 50% cytotoxic concentration (CC50) was determined.
Mp 268 °C dec, yield 63%; 1H NMR (d) 3.85 (s, 3H, OCH3), 5.57 (s,
2H, CH2), 6.70–8.14 (m, 8H, 7ArH and CH). Anal. Calcd for
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.58; H,
3.52; N, 3.71.
4.3.36. 4-[1-(3-Chloro-4-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (41)
Mp 218–220 °C dec, yield 79%;1H NMR (d) 3.89 (s, 3H, OCH3),
5.50 (s, 2H, CH2), 6.78–8.55 (m, 8H, 7ArH and CH). Anal. Calcd for
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.61; H,
3.59; N, 3.31.
Acknowledgment
4.3.37. 4-[1-(3-Chloro-5-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (42)
This work was supported by THINC project (European
commission HEALTH-F3-2008-201032).
Mp 165–167 °C, yield 71%; 1H NMR (d) 3.89 (s, 3H, OCH3), 5.52
(s, 2H, CH2), 6.81–8.56 (m, 8H, 7ArH and CH). C20H15ClFNO5: C,
59.49; H, 3.74; N, 3.47. Found: C, 59.35; H, 3.51; N, 3.63.
References and notes
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E.; Schleif, W. A.; Emini, E. A. J. Virol. 1992, 66, 7414.
4.3.38. 4-[1-(3-Chloro-6-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (43)
Mp 200–202 °C, yield 67%; 1H NMR (d) 3.84 (s, 3H, OCH3), 5.49
(s, 2H, CH2), 6.69–8.14 (m, 8H, 7ArH and CH). Anal. Calcd for
4. Anthony, N. J. Curr. Top. Med. Chem. 2004, 4, 979.
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.38; H,
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6. Lubkowski, J.; Dauter, Z.; Yang, F.; Alexandratos, J.; Merkel, G.; Skalka, A. M.;
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3.43; N, 3.51.
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Med. Chem. 2006, 14, 8430.
8. Marchand, C.; Johnson, A. A.; Karki, R. G.; Pais, G. C.; Zhang, X.; Cowansage, K.;
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A.; Egbertson, M.; Bourgeois, M.; Melamed, J.; Wai, J. S.; Young, S.; Vacca, J.;
Hazuda, D. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6661.
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4.3.39. 4-[1-(4-Chloro-2-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (44)
Mp 168–170 °C dec, yield 72%; 1H NMR (d) 3.90 (s, 3H, OCH3),
5.57 (s, 2H, CH2), 6.80–8.44 (m, 8H, 7ArH and CH). Anal. Calcd for
C20H15ClFNO5: C, 59.49; H, 3.74; N, 3.47. Found: C, 59.72; H,
3.68; N, 3.59.
4.3.40. 4-[1-(4-Chloro-3-fluorobenzyl)-4-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (45)
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Mp 156–158 °C, yield 68%; 1H NMR (d) 3.90 (s, 3H, OCH3), 5.53
(s, 2H, CH2), 6.79–8.56 (m, 7H, 6ArH and CH). Anal. Calcd for
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To determine the susceptibility of the HIV-1 integrase enzyme
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anol. To perform the reaction 4
to a concentration of 1.6 M) and 4
(7 nM) was added in a final reaction volume of 40
l
l diluted integrase (corresponding
l of annealed oligonucleotides
l containing
l
l
l
10 mM MgCl2, 5 mM DTT, 20 mM HEPES pH 7.5, 5% PEG and 15%
DMSO. The reaction was carried out for 1 h at 37 °C. Reaction prod-
ucts were denatured with 30 mM NaOH and detected by an immu-
nosorbent assay on avidin coated plates.45
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