Pharmacophore-based design of HIV-1 integrase strand-transfer inhibitors

Article abstract of DOI:10.1021/jm050549e

Using a training set of diketo-like acid HIV-1 integrase (IN) strand-transfer inhibitors, a 3D pharmacophore model was derived having quantitative predictive ability in terms of activity. The best statistical hypothesis consisted of four features (one hydrophobic aromatic region, two hydrogen-bond acceptors, and one hydrogen-bond donor) with r of 0.96. The resulting pharmacophore model guided the rational design of benzylindoles as new potent IN inhibitors, whose microwave-assisted synthesis and biological evaluation are reported.

Full text of DOI:10.1021/jm050549e

7084
J. Med. Chem. 2005, 48, 7084
Pharmacophore-Based Design of HIV-1 Integrase Strand-Transfer Inhibitors
Maria Letizia Barreca,†,* Stefania Ferro,†,* Angela Rao,† Laura De Luca,† Maria Zappala`,†
Anna-Maria Monforte,† Zeger Debyser,§ Myriam Witvrouw,§ and Alba Chimirri†
Dipartimento Farmaco-Chimico, Universita` di Messina, Viale Annunziata, 98168 Messina, Italy, and Molecular Medicine,
Katholieke Universiteit Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
Received June 11, 2005
Using a training set of diketo-like acid HIV-1 integrase (IN) strand-transfer inhibitors, a 3D
pharmacophore model was derived having quantitative predictive ability in terms of activity.
The best statistical hypothesis consisted of four features (one hydrophobic aromatic region,
two hydrogen-bond acceptors, and one hydrogen-bond donor) with r of 0.96. The resulting
pharmacophore model guided the rational design of benzylindoles as new potent IN inhibitors,
whose microwave-assisted synthesis and biological evaluation are reported.
Introduction
for virtual screening, we have found several compounds
that contain the specified 3D patterns of chemical
functions. Biological testing showed that our strategy
was successful in searching for new structural leads as
HIV-1 IN inhibitors.6
We have now extended our investigation to the
development of 3D QSAR models for the same family
of IN inhibitors. In fact, even if several HIV IN phar-
macophores have already been published,7-12 to the best
of our knowledge no attempts to build quantitative
models for DKA-like strand-transfer-selective inhibitors
have as yet been reported. In fact, the only two previ-
ously reported pharmacophores for DKA-like derivatives
are “qualitative” hypotheses 6,12 (generated without the
use of activity data), whereas we have now developed a
more sophisticated “quantitative predictive” model,
since the biological activities were taken into account.
Human immunodeficiency virus type 1 (HIV-1) en-
codes three enzymes which are required for viral
replication: reverse transcriptase, protease, and inte-
grase (IN). Although drugs targeting reverse tran-
scriptase and protease are widely used and have shown
effectiveness particularly when employed in combina-
tion, toxicity and development of resistant strains have
limited their usefulness.1 Therefore, there is ever-
increasing impetus for the discovery of new agents
directed against alternative sites in the viral life cycle.
IN has thus emerged as an attractive target for anti-
HIV therapy, both because it is necessary for stable
infection and because known functional analogues are
lacking in the human host.2 Integrase inserts a double-
stranded DNA copy of the viral RNA genome into the
chromosomes of an infected cell through two separate
reactions; in the ‘‘3′-processing” step, IN removes two
nucleotides from each 3′ end of viral cDNA, while in the
“strand transfer” reaction, the two newly processed 3′-
viral DNA ends are inserted into the host cell DNA. For
the integration reaction, no source of energy (e.g. no
ATP) is needed and only divalent cations such as Mn2+
or Mg2+ are required for the catalytic activity.
On the basis of the best hypothesis, we designed and
synthesized a novel potential class of â-diketo acid-
containing inhibitors of IN, and the evaluation of the
IN inhibitory activity confirmed the strength of our
rational approach.
Results and Discussion
6,13,14
Although a wide variety of compounds have been
reported as IN inhibitors,3 drugs active against this
enzyme have not as yet been approved by the FDA. One
of the major leads in the development of anti-HIV-1 IN
drugs is represented by â-diketo acids4 (DKAs) and their
derivatives3 which have been shown to selectively
inhibit the strand transfer step by sequestering the
divalent cations bound in the active site of the enzyme,5
and to block HIV-1 replication in infected cells.
Considering that the only two IN inhibitors so far in
clinical trials,3 Merck’s L-870,810 and Shionogi/Glaxo-
SmithKline’s S-1360 (Figure 1, 1 and 19), belong to the
â-diketo-like acid family, we have recently constructed
a three-dimensional pharmacophore model for the bind-
ing of DKAs to the enzyme.6 Using this model as a query
Continuing our work in this research field
and
in an attempt to identify new and potent IN inhibitors,
we tried to generate the simplest hypothesis that could
correctly predict the activity of compounds belonging to
the DKA class. The 3D QSAR hypotheses obtained
should give important guidance for the design of novel
HIV-1 integrase inhibitors.
Pharmacophore Hypothesis Generation and Ra-
tional Design of New DKA Analogues. The algo-
rithm HypoGen15 implemented in the Catalyst pack-
age16 was used to derive automated SAR pharmacophore
models, called “hypotheses”, from a data set consisting
of 33 molecules acting as IN strand-transfer-selective
inhibitors and including the most representative DKAs
and DKA-like derivatives reported in the litera-
ture.3,5,17-20 Among them, 17 compounds were selected
as the training set (TS), and the other 16 were used as
the prediction set (PS). The structures and IN inhibitory
activity values of TS (1-17) and PS molecules (18-33)
are reported in Figure 1.
* To whom correspondence should be addressed. Phone: (++39)
090-6766464. Fax:
(++39) 090-355613. E-mail:
barreca@
pharma.unime.it; sferro@pharma.unime.it.
† Universita` di Messina.
§ Katholieke Universiteit Leuven and IRC KULAK.
10.1021/jm050549e CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/06/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7085
Figure 1. Chemical structures of the 33 data set molecules (TS, 1-17 and PS, 18-33) with their experimental (exp IC50) and
estimated (est IC50) IN strand transfer inhibitory activities, both expressed in µM.
The range of in vitro IN inhibitory activity, expressed
as IC50 for strand transfer inhibition, spanned 5 orders
of magnitude (0.01-100 µM), making this a good data
set for HypoGen module.
It has been suggested that the DKAs act by coordi-
nating the two magnesium ions required for IN activity;
in particular, the carboxylic group (or its bioisosteric
equivalent) interacts with one Mg2+, while the ketoenol
moiety binds the other one.5
In the pharmacophore-based investigation of DKA-
like derivatives involving Catalyst, on the basis of the
chemical features of the TS compounds and their
proposed mechanism of action, the following chemical
functions were selected in the feature dictionary: hy-
drogen bond acceptor (HBA), hydrogen bond donor
(HBD), hydrophobic (HY), hydrophobic aliphatic (HYAl),
and hydrophobic aromatic (HYAr) groups. In fact, even
if the metal-binding function is not included in the
Catalyst feature dictionary, it can be replaced by a
standard hydrogen-bond acceptor/donor function.15
Using the selected TS molecules, HypoGen algorithm
constructed the 10 simplest hypotheses that showed the
best correlation between estimated and measured ac-
tivities, and the results of statistical significance and
predictive ability are presented in Table 1.
The quality of the generated pharmacophore hypoth-
eses was evaluated by considering the cost functions
calculated by the HypoGen module during hypothesis
generation.15 The Fixed cost of the 10 top-scored hy-
potheses was 67.0 bits, well separated from the Null

7086 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Table 1. Summary of the HypoGen Runs for the DKA Dataset
Brief Articles
our hypothesis of the mapping of the hydrogen bond
sites described by the 7-carbonyl-8-hydroxy-[1,6]-naph-
thyridine moiety. The hydrophobic aromatic feature was
instead located at the centroid of the benzyl ring. The
lowest-cost hypothesis Hypo1 fitted the input data well
(Figure 1 and Table 1 in the Supporting Information).
We also used Hypo1 to perform a regression analysis
with the PS of 16 compounds (Figure 1, 18-33) in order
to check the predictive power of this model. Linear
regression of the predicted activities for PS IN inhibitors
versus the experimental ones gave a fairly good cor-
relation coefficient of 0.85, confirming the validity of the
most statistically significant HypoGen hypothesis in
predicting the IN inhibitory activity of DKAs and DKA-
like derivatives.
total
cost
cost
correl.
(r)
Hypoa
diffb RMS
featuresc
Hypo1
Hypo2
Hypo3
Hypo4
Hypo5
Hypo6
Hypo7
79.382 89.897 0.819 0.959 HBA,HBA,HBD, HYAr
79.573 89.573 0.956 0.961 HBA,HBA,HBD, HYAr
85.041 84.238 1.008 0.936 HBA,HBA,HBD, HY
86.067 83.238 1.347 0.935 HBA,HBA,HBA,HYAr
87.036 82.243 1.455 0.923 HBA,HBA, HBA,HY
87.439 81.840 1.453 0.923 HBA,HBA, HBD,HY
91.453 77.826 1.541 0.914 HBA,HBA, HBA,HY
Hypo8 105.035 63.825 2.106 0.828 HBA, HBA,HYAr,HYAr
Hypo9 105.454 63.825 2.087 0.832 HBA,HBA,HBD, HYAr
Hypo10 109.082 60.197 2.225 0.805 HBA, HBA,HBA, HYAr
a Numbers for the hypothesis are consistent with the numera-
tion as obtained by the hypothesis generation. b Difference between
the Null hypothesis and the cost of each returned hypothesis.
c HYar: hydrophobic aromatic; HY: hydrophobic; HBA: hydrogen
bond acceptor. HBD: hydrogen bond donor.
It is also worth noting that the type and number of
features encoded in the automatically generated hy-
pothesis were in full agreement with our previously
manually developed ligand-based pharmacophore model.6
However, the spatial location of HYAr differed a little
in the two hypotheses. In particular, the HypoGen runs
pointed out that to have compounds with very high IN
inhibitory activity, it is necessary to have one hydro-
phobic feature well separated by the DKA motif; the
distance between HYAr and HBA1, and HBD and
HBA2, were found to be 6.20 ( 1 Å, 8.82 ( 1 Å and
10.91 ( 1 Å, respectively.
The most active compounds in the data set assumed
a conformation that allowed proper mapping of the
HYAr feature of the generated hypothesis, whereas
most of the less active compounds (10-15, 29-33) were
unable to map this feature, as shown by the presence
of an asterisk in the mapping column of the output file
of Catalyst run (see Supporting Information).
Among the compounds that failed to map the HYAr
feature, compound 10 drew our attention; the alignment
of this IN inhibitor on Hypo1 (Figure 3A) clearly showed
that its IN inhibitory potency (IC50 ) 1.4) might be
improved by further analogue synthesis.
In fact, since 10 mapped to only three of the four
features of the lowest-cost Catalyst-generated DKA
hypothesis, we thought that the introduction of func-
tionality in this ligand, which might interact with the
fourth feature of the hypothesis (i.e. HYAr), could
provide enhanced IN inhibitory activity to the com-
pound. This idea was also supported by comparison of
the activity data of compound 15 (IC50 ) 24.2) and its
benzyl derivative 18 (IC50 ) 0.01). The designed N-
benzyl derivative of compound 10 (compound 40, Scheme
1) and the corresponding conformational models were
thus edited within Catalyst as described in the Sup-
porting Information. For the estimation of 40 we used
Hypo1 and the BestEst option of the ViewHypothesis
Workbench in Catalyst. The predicted IC50 value for 40
was 0.02 µM, and its mapping onto the top-ranked
hypothesis is represented in Figure 3B.
Figure 2. The top scoring HypoGen pharmacophore Hypo1
is mapped to the most active compound in the training set (1,
L-870,810) (HBD, hydrogen bond donor; HBA, hydrogen bond
acceptor; HYAr, hydrophobic aromatic region).
hypothesis cost score of 169.3 bits; the difference
between these two theoretical parameters is higher than
70 (102.3) and the Configuration cost was less than 17
bits, thus suggesting a robust correlation.
The top ranked pharmacophore model (Hypo1) had
the best predictive power and statistical significance and
was characterized by the highest cost difference (89.897),
the lowest rms (0.81947), and the best correlation
coefficient (0.959). These values indicated a great pre-
dictability of the 3D-QSAR pharmacophore and con-
firmed that it did not come about by chance; Hypo1 was
thus retained for further analysis.
The selected 3D hypothesis consisted of one hydro-
phobic aromatic region (HYAr), two hydrogen-bond
acceptor (HBA1 and HBA2), and one hydrogen-bond
donor (HBD) sites in a specific three-dimensional ori-
entation. Using the most active molecule (1) of the TS,
which is also one of the two IN-DKA-like inhibitors in
clinical development, a flexible fit of this molecule to
Hypo1 is shown in Figure 2.
Compound 1 and its analogues (3, 20, and 21) can be
still considered DKA-like derivatives. In fact, it has been
reported that 8-hydroxy-[1,6]-naphthyridine and 8-hy-
droxyquinoline were a suitable replacement for the 1,3-
diketoacid motif, and the molecules obtained act analo-
gously to diketo acids (that is, selective inhibition of
strand transfer).17 Considering the proposed mechanism
of action for DKAs, the interaction between 1 and the
metal ions in the IN active site was accounted for by
The promising molecular modeling results prompted
us to plan the synthesis of a series of new 1H-indole
derivatives 34-45 (Scheme 1) bearing a benzyl or
4-fluorobenzyl substituent at N-1, seeing that an over-
view of the most active DKA analogues highlighted the
presence of a fluorine atom on the benzyl moiety.
Furthermore, a chlorine atom or a methoxy group was
introduced on the benzene-fused ring of some of the
newly designed benzylindole derivatives (i.e. 36-39, 42-

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7087
Table 2. Inhibition of Integration and Anti-HIV-1 Activity, Both Expressed as µM
b
c
compd
overalla
3′-Pa
>286.2
STa
EC50
CC50
SId
34
35
36
37
38
39
40
41
42
43
44
45
1
0.76 ( 0.10
1.20 ( 0.0
5.40 ( 4.16
1.50 ( 0.57
14.30 ( 2.33
9.25 ( 3.81
4.50 ( 1.00
3.42 ( 2.23
6.07( 0.0
14.34 ( 7.14
19.92 ( 15.25
5.31 ( 0.88
4.78 ( 0.98
2.78 ( 0.39
5.32 ( 0.24
5.81 ( 1.93
0.0047 ( 0.0007
119.7 ( 66.1
27.2 ( 5.4
34.00 ( 6.78
26.53 ( 4.24
29.04 ( 0.0
127.45 ( 26.5
>78
8
>272.20
10.19 ( 5.83
3
8
0.22 ( 0.04
0.03 ( 0.01
0.39 ( 0.14
11.47 ( 2.5
162.5 ( 2.4
130.7 ( 0.0
8.28 ( 2.21
5.3 ( 1.25
0.97 ( 0.18
1.59 ( 1.08
2.16 ( 0.68
2.71( 0.0
0.07 ( 0.05
8
0.15 ( 0.05
0.68 ( 0.42
5
0.22 ( 0.08
0.80 ( 0.7
9
0.02 ( 0.0006
0.002 ( 0.001
0.010 ( 0.003
0.20 ( 0.08
0.03(0.0006
0.015 ( 0.003
0.10 ( 0.01
>4
>14
8
>74
37.24 ( 1.54
53.42 ( 6.69
86.38 ( 52.79
63.54 ( 39.53
2.15 ( 0.15
0.01 ( 0.001
0.021 ( 0.007
0.004 ( 0.001
0.0025 ( 0.0007
19
16
11
457
0.017 ( 0.006
0.019 ( 0.008
0.0005 ( 0.0003
0.12 ( 0.03
a Concentration required to inhibit by 50% the in vitro integrase activity assays as IC50 (µM). b Effective concentration required to
reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells. c Cytotoxic concentration to reduce MT-4 cell viability by 50%. d Selectivity
index: ratio CC50/EC50
.
Scheme 1a
a Reagents and conditions: (i) AcCl, Et2AlCl, CH2Cl2, 0 °C, 2 h;
(ii) benzyl or 4-fluorobenzyl bromide, NaH, DMF, 0 °C, 30 min;
(iii) diethyl oxalate, dry C2H5ONa, THF, two separated steps under
the same conditions: 50 °C, 2 min, 250 W, 300 psi; (iv) 2 N NaOH,
MeOH, rt, 1.5 h.
Figure 3. Compound 10 (A) and 40 (B) aligned on the lowest
cost pharmacophore model generated for DKA-like strand
transfer inhibitors (HBD, hydrogen bond donor; HBA, hydro-
gen bond acceptor; HYAr, hydrophobic aromatic region).
The best activity was displayed when a p-fluorine
atom was present on the benzyl moiety; in fact com-
pound 41 was 2-fold more potent compared to the
unsubstituted parent 40, and fluoro derivatives 43 and
45 were the most active compounds of the series with
IC50 ) 0.01 µM and IC50 ) 0.004 µM, respectively.
In particular the contemporary presence of a fluorine
atom on the benzyl moiety and a methoxy group on the
indole system increseased the IN inhibitor activity, and
compound 45 showed potency comparable to that of
L-870,810, one of the two IN inhibitors in clinical trials.
The relevance of our modeling assumptions is worth
noting as documented by the measured strand stransfer
inhibitory activity of 40 (IC50 ) 0.03 µM), which
compared well with the predicted value of 0.02 µM.
45) based on the observation that chloro-substituted
compound 28 (IC50 ) 0.52) was 2-fold more potent than
10 (IC50 ) 1.4), and that a methoxy group was present
in potent IN inhibitors such as 5, 22, and 23.
Inhibition of HIV-1 IN Activity. All the synthesized
compounds were tested in IN inhibition assays which
have been recently reviewed.22,23 The results showed
that the diketo acid derivates were generally more
potent than the corresponding esters and that most of
the tested compounds showed 50-100 fold selectivity
toward inhibition of strand transfer in comparison to
3′-processing (Table 2).
Our lead compound (40) had a strand stransfer
inhibitory activity of 0.03 µM; the introduction of a
chlorine atom on the indolic ring (42) led to a significant
decrease of anti-IN activity, while the presence of a
methoxy group in the same position (44) did not influ-
ence the potency.
The better inhibition of strand transfer activity by
compound 40 with respect to that reported by Sechi et
al.21 (IC50 ) 1 µM) is probably due to the fact that we
determined strand transfer activity independent of DNA
binding and 3′-processing.

7088 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Brief Articles
In Vitro Anti-HIV Assay and Drug Susceptibility
Assay. The antiviral activity of our compounds on the
HIV-induced CPE in human lymphocyte MT-4 cell
culture was determined by the MT-4/MTT-assay.24 All
compounds proved to be effective inhibitors of HIV-1
replication at micromolar concentration (Table 2), with-
out correlation between anti-HIV and anti-IN activities.
This behavior might be due to the physical-chemical
properties of our molecules. Extensive further modifica-
tions are in progress aiming at modifying the solubility
and decreasing the toxicity in this series of IN strand-
transfer-selective inhibitors.
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Experimental Section
Chemistry. The synthesis of 1H-indole derivatives 34-45
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in Scheme 1. The appropriate 1H-indole (46-48) was 3-acety-
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chloride as catalyst and then N-alkylated by treatment with
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model for DKA-like derivatives acting as inhibitors of
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design new DKAs containing a benzylindole skeleton.
Synthesized molecules proved to be potent IN-inhibitors
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Acknowledgment. We thank A. Nijs, M. Michiels,
and B. Van Remoortel (KULeuven) for excellent techni-
cal assistance with anti-IN and anti-HIV assays. Fi-
nancial support for this research by Fondo Ateneo di
Ricerca (2002, Messina, Italy), MIUR (COFIN2004,
Roma, Italy), and the TRIoH project (LSHB-CT-2003-
503480)) is gratefully acknowledged.
Note Added after ASAP Publication. Reference 21
in the version of the paper posted October 6, 2005, has
been corrected in the new version posted October 18,
2005.
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Supporting Information Available: Additional experi-
mental data are available free of charge via Internet at http://
pubs.acs.org
JM050549E