Design and optimization of tricyclic phtalimide analogues as novel inhibitors of HIV-1 integrase

Article abstract of DOI:10.1021/jm049559q

Human immunodeficiency virus type-1 integrase is an essential enzyme for effective viral replication and hence a valid target for the design of inhibitors. We report here on the design and synthesis of a novel series of phthalimide analogues as integrase

Full text of DOI:10.1021/jm049559q

1930
J. Med. Chem. 2005, 48, 1930-1940
Design and Optimization of Tricyclic Phtalimide Analogues as Novel Inhibitors
of HIV-1 Integrase
Wim G. Verschueren,*,† Inge Dierynck,† Katie I. E. Amssoms,† Lili Hu,† Paul M. J. G. Boonants,†
Geert M. E. Pille,† Frits F. D. Daeyaert,‡ Kurt Hertogs,† Dominique L. N. G. Surleraux,† and
Piet B. T. P. Wigerinck†
Tibotec BVBA, Generaal de Wittelaan L 11B 3, B-2800 Mechelen, Belgium, and Center for Molecular Design,
Johnson & Johnson Pharmaceutical Research & Development, Antwerpsesteenweg 37, B-2350 Vosselaar, Belgium
Received June 7, 2004
Human immunodeficiency virus type-1 integrase is an essential enzyme for effective viral
replication and hence a valid target for the design of inhibitors. We report here on the design
and synthesis of a novel series of phthalimide analogues as integrase inhibitors. The short
synthetic pathway enabled us to synthesize a series of analogues with a defined structure
diversity. The presence of a single carbonyl-hydroxy-aromatic nitrogen motif was shown to
be essential for the enzymatic activity and this was confirmed by molecular docking studies.
The enzymatically most active compound from this series is 7-(3,4-dichlorobenzyl)-5,9-
dihydroxypyrrolo[3,4-g]quinoxaline-6,8-dione (15l) with an IC50 value of 112 nM on the HIV-1
integrase enzyme, while ((7-(4-chlorobenzyl)-5,9-dihydroxy-pyrrolo[3,4-g]quinoxaline-6,8-dione
(15k)) showed an EC50 of 270 nM against HIV-1 in a cell-based assay.
Introduction
There is therefore a high unmet need for drugs with
new mechanisms of action, which are not limited by
existing drug resistance, to combat the HIV-1 infection.
Recently, a large interest in HIV integrase has emerged.
This enzyme is an attractive target for anti-HIV drug
discovery and development because it is essential to the
viral life cycle and is absent in the host cell.3-5
Since 1981, acquired immunodeficiency syndrome or
AIDS has become a major worldwide epidemic. AIDS
is caused by human immunodeficiency virus (HIV). By
killing or damaging cells of the immune system, HIV
progressively destroys the body’s ability to withstand
infections and certain cancers. Looking at the life cycle
of this retrovirus, three essential enzymes, encoded by
the HIV pol gene can be distinguished: (i) reverse
transcriptase, (ii) integrase, and (iii) protease.1 Reverse
transcriptase is an RNA/DNA-dependent DNA poly-
merase that transcribes the viral genomic RNA into
proviral DNA. Integrase then associates with a series
of cellular factors and the proviral DNA to form the
preintegration complex (PIC). The complex migrates to
the nucleus, where integration takes place, involving,
for example, cleavage of two nucleotides from each 3′-
terminal of the proviral DNA and the strand transfer
reaction, both catalyzed by the viral enzyme. This
process is essential for viral replication and hence a
valid target for the design of inhibitors. As of today,
whereas integrase inhibitors have been identified, which
inhibit both the viral enzyme and the virus replication
in cell-based assays as well in animal models, no
inhibitor of HIV integrase has been approved yet.2
The HIV-1 protease (PR) hydrolyzes viral polyproteins
into functional protein or peptide products that are
essential for viral assembly and subsequent replication.
Various protease and reverse transcriptase inhibitors
are currently available as approved antiretroviral drugs;
however, their suboptimal use associated with the high
mutation rate of HIV have rendered those drugs of very
limited use for a large proportion of patients, due to viral
resistance to these inhibitors.
Molecular Modeling
Structure-based drug-design efforts on HIV integrase
have been hampered by the lack of structural informa-
tion on the exact binding mode of known inhibitors. One
structure of an inhibitor cocrystallized with the core
domain of HIV integrase has been published.6 We and
others,7,8 however, believe that the observed binding
position of the inhibitor in this crystal structure is
influenced by crystal packing effects and does not reflect
the binding mode in an active biological system. Various
reported QSAR studies on different series of integrase
inhibitors using methods that do not require structural
information about the target enzyme have been pub-
lished.9-12 The picture that emerges from those efforts
is that different classes of inhibitors seem to have
different mechanisms of action.13 For a subset of these
classes, we have hypothesized, using the structural in-
formation from crystal structures of the core domain of
HIV integrase complexed to Mg2+, a binding mode based
on the presence of a metal chelating pharmacophore.14,15
HIV integrase consists of three distinct domains: the
N-terminal domain, the catalytic core domain, and the
C-terminal domain. X-ray and/or NMR structures of the
separate subdomains and of the core domains with the
N-terminal and C-terminal domains have been pub-
lished.14-24 The catalytic core domain comprises the
residues 51-212. This domain contains two aspartates
and one glutamate (Asp64, Asp116, and Glu152, the
“D,D(35),E” motif) that are essential for catalytic activ-
ity of various DNAses and are believed to bind Mg2+ or
* To whom correspondence should be addressed. Phone: +32
(0)15293178. Fax: +32 (0)15401257. E-mail: wversch1@tibbe.jnj.com.
† Tibotec BVBA.
‡ Johnson & Johnson Pharmaceutical Research & Development.
10.1021/jm049559q CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/16/2004

Phtalimides as Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1931
Figure 1. Proposed binding mode of diketo acid 128 into the
core domain of HIV-1 integrase. The Mg2+ ion is cpk rendered.
The inhibitor, the amino acid side chains of Asp64, Asp116,
Glu152, Thr66, and Cys65, and the two water molecules
completing the Mg2+ coordination are rendered in stick.
Figure 2. Various classes of compounds that can be docked
into the core domain of HIV-1 integrase using the proposed
docking protocol: 1, diketo acids;26 2, naphthyridines;29 3, 1,3-
diketones;29 4, â-keto amides.30
Mn2+. The presence of either of these divalent ions is
required for HIV integrase catalytic activity. Also, the
binding and activity of integrase inhibitors is metal
dependent.25-27 In two X-ray structures of the core
domain, a single Mg2+ ion is chelated by Asp64 and
Asp116.14,15 We propose a binding mode in which inhib-
itors bind through bidentate chelation of this ion. To
determine the orientation of a bidentate integrase inhib-
itor in the integrase enzyme, we carried out manual
docking studies using the diketo acid I (see Figure 1).
This compound was the most active in a series of
compounds where the angle formed by the lines ex-
tended from the diketo acid and benzyl groups toward
the central aromatic ring was varied by structurally
altering this central ring.28 Given the chelation of the
Mg2+ by the aspartates 64 and 116 and the octahedral
coordination of Mg2+, there are a limited number of
ways (10, see the Experimental Section) to complete this
coordination with a bidentate ligand. The configuration
with the lowest ligand-enzyme nonbond interaction
energy is show in Figure 1. In this orientation, the
benzyl group makes a turn inward to the protein, filling
a hydrophobic pocket formed in part by the flexible outer
loop of the integrase core domain. It has been shown
that this loop is important for enzymatic activity.22 The
acid group acts as a hydrogen-bond acceptor to the
hydroxy group of ThrA66, while the enol hydrogen forms
a hydrogen bond toward the carbonyl oxygen of Cys65.
Interestingly, it has been described that HIV-1 grown
in the presence of the diketo acid inhibitor L-708,906
developed resistance as the result of mutations of
ThrA66.29 Starting from the proposed integrase-inhibi-
tor complex shown in Figure 1,30 we have developed the
following protocol for docking putative HIV-1 integrase
inhibitors.
between each pair of possible coordinating atoms of the
ligand. For the diketo acid in Figure 1, these are
between the keto and enol or between the enol and
carbonyl acid oxygens. For the phthalimide compounds
these are between the carbonyl and hydroxyl and
between the hydroxyl and the pyrazine nitrogens. All
bidentate chelates are subjected to molecular mechanics
minimization.
The Mg2+-ligand chelates are then superimposed
upon the coordinates of the Mg2+ and the two removed
water molecules in the integrase active site structural
model.31 This superposition is carried out using Kabsch’
algorithm for the superposition of two vectors. As the
coordinating atoms are equivalent, this can be done in
two ways. For each pose, the nonbond interaction
between the chelate and the integrase enzyme is
calculated using a precalculated grid.
The chelate-enzyme complexes with the lowest non-
bond interaction energy calculated in the previous step
are subjected to full molecular mechanics energy mini-
mization. The complex with the lowest chelate-enzyme
interaction energy after this final minimization is
retained as the final docking result.
The force field used in the molecular mechanics
calculations is an in-house adapted version of MMFF94.32
The nonbond interaction grid is calculated using a 4-8
Lennard-Jones-type potential fitted to the original 7-14
functional form. The 4-8 functional form is used as it
has been documented that the use of softer potentials
improves existing docking algorithms.33 The fast non-
bond energy calculation using the precalculated grids
is used to select low-energy chelate-enzymes, which can
then be subjected to the computationaly much more
expensive full molecular mechanics minimization. To
reduce the computational time, only five chelate-
enzyme complexes are minimized.
First, a conformational analysis of the ligand is
carried out using a genetic algorithm (GA). The outcome
of this GA is a list of fully molecular mechanics
optimized low-energy conformers. The parameters of the
GA have been fine-tuned such that in several runs using
different initial configurations, the same low-energy
conformations are consistently generated.
With this docking protocol, various classes of HIV
integrase inhibitors can be docked in a consistent way.
Examples include the diketo acids 1, naphthyridines 2,
1-3 diketones 3, and â-keto amides 4 (see Figure 2).34,35
For each conformer, all possible bidentate complexes
with a Mg2+ ion are formed by placing a Mg2+ ion

1932 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Verschueren et al.
Scheme 2. Synthesis of 1-Substituted
Piperidin-4-ylpyrrolidine-2,5-dione Analogues 27-29a
a Reagents and conditions: (a) 10% Pd/C-H2, CH3OH, rt, 24
h; (b) Boc2O, CH2Cl2, rt, 24 h; (c) benzoyl chloride, Na2CO3, THF-
H2O (1:1), rt, 24 h.
Scheme 3. Synthesis of
1-substituted-pyrrolidine-2,5-dione Analogues 30a-da
Figure 3. Design of tricyclic phthalimide analogues 7-18.
Scheme 1. Synthesis of 1-Substituted
Pyrrolidine-2,5-dione Analogues 24a-z, 24aa-cc, 25,
and 26a-ca
a Reagents and conditions: (a) boronic acid, Pd(OAc)2, NaTM-
SPP, Et3N, THF-H2O (1:1), reflux, 0.5-24 h.
Scheme 4. Synthesis of Tricyclic Phtalimide Analogues
7-18a
a Reagents and conditions: (a) NaH, CH3OH (catalytic amount),
THF, reflux, 48 h.
20a-z, 20aa-cc in acetic acid as a solvent and a
catalytic amount of DMAP to yield the different pyrro-
lidine-2,5-dione analogues 24a-z, 24aa-cc. Hydroge-
nation of the commercially available 1-[(1R)-1-phenyl-
ethyl)pyrrole-2,5-dione (21) gave the analogue 25 as
described in method B. Method C consists of a nucleo-
philic substitution between pyrrolidine-2,5-dione (22)
and the substituted benzyl bromide analogue 23a-c in
the presence of potassium carbonate as a base.
Debenzylation of the 24c 1-(1-benzylpiperidin-4-yl)-
pyrrolidine-2,5-dione) yielded the deprotected piperidine
analogue 27 (Scheme 2). The secondary amine was
protected as a carbamate with di-tert-butyl dicarbonate
in dichloromethane to give the carbamoylated pyrroli-
dine-2,5-dione 28. In route C, the amine function of 27
was acylated with benzoyl chloride in tetrahydrofurane:
water (1:1) by applying sodium carbonate as a base.
Compounds 30a-d were achieved via a Suzuki cou-
pling, in the presence of palladium acetate, triphenyl-
phosphine-3,3′,3′′-trisulfonic acid trisodium salt (Na
TMSPP) as a water-soluble ligand, a boronic acid deriv-
ative, and triethylamine (Et3N) as a base as described
in Scheme 3.
The tricyclic phthalimide analogues 7-18 (Scheme 4)
were synthesized via a double Claisen condensation of
an appropriately N-substituted succinimide 24a-z,
24aa-cc, 25, 26a-c, 27, 28, 29, 30a-d, and 31 with
a Reagents and conditions: (a) DMAP, HOAc, reflux, 2-72 h;
(b)10% Pd/C-H2, CH3OH, rt, 24 h; (c) K2CO3, 18-crown-6, CH3CN,
reflux, 24 h.
This information, together with the diketo acid moiety
present in compound 5 and the motif present in the
azanaphthyridine analogue 6 (L-870,810), both de-
scribed by Pais et al., were used to design and synthe-
size a novel series of phthalimide analogues as candi-
date integrase inhibitors (see Figure 3).36
In this paper we report the systematic search for new,
potent, and selective integrase inhibitors. The structure-
activity relationship of several classes of phthalimide
analogues as integrase inhibitors was investigated.
Chemistry
The general synthetic route we applied for the syn-
thesis of the tricyclic phthalimide analogues 7-18 is
summarized in Schemes 1-4. In Scheme 1 we describe
three different approaches to synthesize the 1-substi-
tuted-pyrrolidine-2,5-dione analogues 24a-z, 24aa-cc,
25, and 26a-c. Method A starts from dihydrofuran-
2,5-dione (19), which reacted with different amines

Phtalimides as Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1933
Table 1. Biochemical and Biological Evaluation of Tricyclic Phtalimide Analogues 7-18

1934 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Table 1 (Continued)
Verschueren et al.

Phtalimides as Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1935
Table 1 (Continued)
a The standard deviation is given for experiments where the number of determinations is more then two. All the determinations are
quadruplet values. ND ) not determined. b The reference compounds (5, 6) have an IC50 of 0.05 and 0.01 µM, respectively, and an EC95
value of 0.10 µM.34

1936 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Verschueren et al.
tion in vitro.36,39 Several phthalimides were synthesized
in order to explore the aryl- and R-moieties. One of the
first compounds synthesized in this series was com-
pound 7 (2-(4-fluorobenzyl)-4,9-dihydroxybenzo[f]isoin-
dole-1,3-dione) with a phenyl ring on the left. The
inactivity of compound 7 showed that a hydrogen-bond
acceptor adjacent to the phenol function was essential
in comparison with the pyrazine analogue 10 (pIC50
)
6.68). The pyrazine ring was kept constant in a series
of R1-variations. The important carbonyl-hydroxy-
aromatic nitrogen motif is present twice in this mol-
ecule. In the series of benzodioxole derivatives the
spacer length was explored (n ) 1-3). The optimal chain
length is 1, as was shown by compound 13b with a pIC50
value of 6.25. This is consistent with the ability to direct
the hydrophobic substituents toward the flexible loop
of HIV-integrase. A set of mono- and dimethoxybenzyl
derivatives was synthesized 14a-f. These derivatives
showed similar results, except the 2,6-dimethoxybenzyl
analogue 14c, with a 0.5 log reduced activity on HIV-
integrase, and the 2,4-dimethoxybenzyl derivative 14e,
which in this series is clearly the least active compound
in the cell-based anti-HIV assay, having a pEC50 value
of 4.87. Some alkyl analogues, 11a-e and 12a-b, were
made but showed reduced activity on the enzyme and
no activity in cell culture except the cyclohexylmethyl
derivative 11e (pEC50 ) 5.46). The two enantiomers of
the R-methylbenzyl substituted compounds 12a-b were
toxic. The pyridine analogue 16 lost 1 log of enzymatic
activity (pIC50 ) 5.17) in comparison with the benzo-
dioxole derivative 13b and was not active in the cell-
based anti-HIV assay. A few aryl- and phenyl-substi-
tuted benzyl derivatives were made (17a-e). Compound
17e was the most interesting compound of this series:
this more lipophilic compound was less active on the
enzyme but showed a comparable activity with 13b in
the cell-based anti-HIV assay. Another series of methyl,
trifluoromethyl, trifluoromethoxy, and halogenated ben-
zyl derivatives 15a-n and 10 were synthesized. The
monohalogenated (F, Cl, Br) compounds showed a
similar enzymatic activity with a pIC50 of 6.5, while
compound 15k was clearly better in the cell-based anti-
HIV assay, where a pEC50 of 6.57 was measured. In the
dihalogenated benzyl derivatives series, the chloro-
substituted analogues showed a superior activity, which
Figure 4. Compound 15l docked into the core domain of
HIV-1 integrase.
the aromatic ortho diester 32a-d37 in tetrahydrofuran
solution and sodium hydride as base in the presence of
a catalytic amount of methanol as described by Murray
et al.38 The yields and the different types of compounds
that were synthesized are given in Table 1.
Results and Discussion
Molecular Modeling. The proposed binding hypoth-
esis, based on the presence of a metal-chelating phar-
macophore, and docking analysis procedure have been
helpful in the optimization of the present class of
tricyclic phthalimide analogues derived from compound
10. Figure 4 shows the result of the docking of com-
pound 15l, one of the more potent analogues, into the
integrase core domain using the above-described dock-
ing protocol. The carbonyl and hydroxy oxygens chelate
the Mg2+ ion in the active site. The Asp64 and Asp116
acid side chains and two water molecules complete the
octahedral coordination sphere of the ion. Important
interactions with the enzyme are hydrogen bonds (i)
between the left pyrazine nitrogen and the hydroxy
group of Thr66 and (ii) between the enol hydrogen and
the backbone carbonyl of Cys65 and (iii) the hydrophobic
interaction of the benzyl substituent with the flexible
loop formed by residues 140-148. Most phthalimide
analogues can be consistently docked in this binding
mode using the proposed docking protocol. Exceptions
are compounds with larger substituents on the phthal-
imide moiety, like 17a, 17b, and 17c. A number of SAR
observations can be rationalized with the aid of the
proposed binding model. The presence of a single
carbonyl-hydroxy-aromatic nitrogen motif is essential
for activity. However, only one such motif is necessary,
as demonstrated in the activities of compounds such as
8a and 9. Also important is the “turn” in the molecules
to direct the hydrophobic substituent on the phthalimide
nitrogen toward the flexible loop. This is illustrated by
the lower antiviral activity of compounds 11a, 11b, 11c,
and 13a.
was illustrated by the 3,4-dichlorobenzyl (15l) (pIC50
)
6.95 and pEC50 ) 6.04) and the 3,5-dichlorobenzyl (15m)
(pIC50 ) 6.73 and pEC50 ) 6.14) analogues. One of the
most active compounds was the m-bromobenzyl deriva-
tive 10, which showed a pIC50 of 6.68. This m-bro-
mobenzyl substituent was chosen to explore the aro-
matic moiety on the “left” part of the molecule. The
pyridine 8a and methylpyridine 8b showed similar
activities.
All the tricyclic phthalimide analogues demonstrated
a similar activity on the enzyme when compared with
the naphthyridine analogue 6 described by Pais et al.
but where less selective.36
Inhibitory Properties of a Series of Tricyclic
Phthalimide Analogues 7-18. All the compounds
were tested for their ability to inhibit the HIV-1 virus.
All the data are shown in Table 1. Compounds 5 and 6
described by Pais et al. are shown for comparison,
because of their known ability to inhibit HIV-1 integra-
Conclusion
The design and synthesis of tricyclic phthalimides
analogues resulted in a new class of HIV-1 integrase
inhibitors. The short synthetic pathway allowed us to
synthesize a series of phthalimide analogues with a

Phtalimides as Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1937
in dichloromethane (50 mL). The organic layer was washed
with saturated NaHCO3 solution (50 mL) and 1 N HCl
solution. The organic layer was dried over magnesium sulfate
and evaporated under reduced pressure to yield the title
compound as a white crystalline product (1.75 g, 65%).
The following products (24b-z, 24aa-cc) were prepared
in this manner; the yields and different reaction times are
mentioned for each product. All the products were identified
by LRMS, and a few representative compounds were charac-
terized by 1H NMR and 13C NMR spectra (see the Supporting
Information).
defined structure diversity. Essential structural fea-
tures, including the carbonyl-hydroxy-aromatic nitro-
gen motif, for the enzymatic activity were confirmed.
The enzymatically most active compound from this
series is 15l with an IC50 value of 112 nM on the HIV-1
integrase enzyme, while [(7-(4-chlorobenzyl)-5,9-dihy-
droxypyrrolo[3,4-g]quinoxaline-6,8-dione (15k)] showed
an EC50 of 270 nM against HIV-1 in a cell-based assay.
Further research is ongoing to increase the cell penetra-
tion and selectivity of these tricyclic phthalimide series.
1-Cyclopentylpyrrolidine-2,5-dione (24b): 69% yield.
1-(1-Benzylpiperidin-4-yl)pyrrolidine-2,5-dione (24c): 80%
yield.
Experimental Section
1-[(1S)-1-Phenylethyl)pyrrolidine-2,5-dione (24d): 86% yield,
72 h.
1-Benzo[1,3]dioxol-5-ylpyrrolidine-2,5-dione (24e): 93% yield.
1-Benzo[1,3]dioxol-5-ylmethylpyrrolidine-2,5-dione (24f): 87%
yield, 72 h.
1-(2-Benzo[1,3]dioxol-5-ylethyl)pyrrolidine-2,5-dione (24g):
100% yield.
1-(2-Methoxybenzyl)pyrrolidine-2,5-dione (24h): 83% yield.
1-(4-Methoxybenzyl)pyrrolidine-2,5-dione (24i): 74% yield,
72 h.
1-(2,6-Dimethoxybenzyl)pyrrolidine-2,5-dione (24j): 79%
yield.
1-(2,3-Dimethoxybenzyl)pyrrolidine-2,5-dione (24k): 70%
yield, 2 h.
1-(2,4-Dimethoxybenzyl)pyrrolidine-2,5-dione (24l): 75%
yield, 2 h.
1-(2,5-Dimethoxybenzyl)pyrrolidine-2,5-dione (24m): 81%
yield.
General Experimental Procedures. NMR spectra were
recorded, using standard software packages, on a Bruker
1
Avance 400 spectrometer, operating at 400 MHz for H and
100 MHz for 13C and with DMSO or CDCl3 as solvent unless
otherwise stated. In every case tetramethylsilane (TMS) was
used as internal standard. Chemical shifts are given in ppm
and J values in Hz. Multiplicity is indicated using the following
abbreviations: d for a doublet, t for a triplet, m for a multiplet,
etc. An additional 2-D measurement (HSQC) was performed
for complete structure elucidation of the tricyclic phthalimide
analogues. With this measurement, it was proven that the
benzylic CH2 for the different tricyclic phthalimide analogues
has a value of 39.9 ppm unless otherwise stated. For the sake
of brevity, we opted to completely characterize (NMR included)
one representative example of each subset of final products
7-18. Low-resolution mass spectra (LRMS) were collected on
a single quadrupole mass spectrometer (Waters ZMD), with
ion trap (ThermoFinnigan LCQ Deca) or time-of-flight (Waters
LCT) using electrospray ionization (ESI) in positive or negative
mode.
1-(4-Methylbenzyl)pyrrolidine-2,5-dione (24n): 92% yield.
1-(4-Trifluoromethoxybenzyl)pyrrolidine-2,5-dione (24o): 85%
yield.
All reagents were purchased from commercial sources
(Acros, Aldrich, ABCR, etc.) and were used as such. Column
chromatography was carried out on silica gel 60 Å, 60-200
µm (ROCC). Thin-layer chromatography was performed on
silica gel 60 F254 plates (Merck). Analytical HPLC was done
on a Waters Alliance 2690 (pump + autosampler) system
equipped with a Waters 996 photodiode array-detector. The
purity of the final compounds 7-18 was determined with three
HPLC systems using a UV-detector: In system 1, a Waters
Xterra MS C18 column (3.5 µm, 4.60 mm × 100 mm) was used
and the UV detection range was set at 230-300 nm. In system
2, a Thermo Hypersil BDS C18 column (3.5 µm, 4 mm × 100
mm) was used and a single wavelength (λmax) was set. In
system 3, an Alltech Prevail C18 column (3 µm, 4 mm × 100
mm) was used and a single wavelength (λmax) was set. The
applied gradient was the same for systems 1 and 2: 10 mM
ammonium formiate and 0.1% formic acid in water (mobile
phase A) and acetonitrile (mobile phase B), with a gradient
from 5% to 95% acetonitrile in 12 min. The applied gradient
for system 3 was 0.1% trifluoroacetic acid in water (mobile
phase A) and acetonitrile (mobile phase B), with a gradient
from 0% to 95% acetonitrile in 12 min. The flow rate was 1.0
mL/min for the three systems. The retention time of each
compound in both systems is given in minutes. Elemental
analyses were performed on a Carlo-Erba EA1110 analyzer.
The coulometric Karl Fisher method was used for water
determination.
General Reaction Procedure 1. Representative Pro-
cedures for the Synthesis of 1-Substituted Pyrrolidine-
2,5-dione Analogues 24a-z, 24aa-cc, 25, and 26a-c.
Method A: Representative Procedure for the Synthesis
of 1-Substituted Pyrrolidine-2,5-dione Analogues 24a-z
and 24aa-cc. Synthesis of 1-(3-Bromobenzyl)pyrrolidine-2,5-
dione (24a). Dihydrofuran-2,5-dione (19) (1.00 g, 0.01 mol) and
3-bromobenzylamine (20a-z, 20aa-cc) (1.86 g 0.01 mol) were
dissolved in acetic acid (25 mL), followed by the addition of a
catalytic amount of dimethylpyridin-4-ylamine (DMAP). The
solution was refluxed for 24 h. After completion of the reaction,
the solution was cooled to room temperature. The solvent was
removed under reduced pressure and the residue was dissolved
1-(3-Fluorobenzyl)pyrrolidine-2,5-dione (24p): 64% yield, 72
h.
1-(4-Fluorobenzyl)pyrrolidine-2,5-dione (24q): 91% yield.
1-(4-Bromo-2-fluorobenzyl)pyrrolidine-2,5-dione (24r): 75%
yield.
1-(2-Bromo-6-fluorobenzyl)pyrrolidine-2,5-dione (24s): 83%
yield.
1-(3-Chlorobenzyl)pyrrolidine-2,5-dione (24t): 70% yield.
1-(4-Chlorobenzyl)pyrrolidine-2,5-dione (24u): 70% yield.
1-(3,4-Dichlorobenzyl)pyrrolidine-2,5-dione (24v): 68% yield.
1-(3,5-Dichlorobenzyl)pyrrolidine-2,5-dione (24w): 55% yield.
1-(2-Bromobenzyl)pyrrolidine-2,5-dione (24x): 61% yield.
1-(4-Bromobenzyl)pyrrolidine-2,5-dione (24y): 48% yield.
1-Cyclohexylmethylpyrrolidine-2,5-dione (24z): 82% yield,
72 h.
1-Pyridin-4-ylmethylpyrrolidine-2,5-dione (24aa): 75% yield.
1-Naphthalen-2-ylmethylpyrrolidine-2,5-dione (24bb): 98%
yield.
1-[(1S)-1-Phenethyl]pyrrolidine-2,5-dione (24cc): 99% yield.
Method B. Synthesis of 1-[(1R)-1-Phenylethyl]pyrro-
lidine-2,5-dione (25). After displacing the air with N2, pal-
ladium on charcoal (10%, 0.30 g) was added followed by the
addition of a solution of 1-[(1R)-1-phenylethyl]pyrrole-2,5-dione
(1 g, 4.97 mmol) (21) in methanol (100 mL). For a few minutes
vacuum was applied and replaced by H2. After stirring for 24
h at room temperature, N2 was then introduced in the flask
to displace the remaining H2. The palladium catalyst was
removed by filtration over a bed of Celite under N2 and the
filter was washed with methanol. The filtrate was evaporated
under reduced pressure (1.01 g, 100%).
Method C. Representative Procedure for the Synthe-
sis of 1-Substituted Pyrrolidine-2,5-dione Analogues
26a-c. Synthesis of 1-(3-Trifluoromethylbenzyl)pyrro-
lidine-2,5-dione (26a). Pyrrolidine-2,5-dione (2.30 g, 23.22
mmol) (22), 1-bromomethyl-3-trifluoromethyl-benzene (5.05 g,
21.11 mmol) (23a-c), K2CO3 (6.18 g, 49.83 g), and 18-crown-6
(0.10 g) were dissolved in acetonitrile (100 mL). The solution
was refluxed for 24 h. The solution was concentrated and the
residue was purified by column chromatography on elution

1938 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Verschueren et al.
with dichloromethane (4.34 g, 80%) to yield the title compound
as a white crystalline product.
7-(3-Bromobenzyl)-5,9-dihydroxy-2-methylpyrrolo[3,4-g]quin-
oline-6,8-dione (8b): 62% yield, 120 h.
7-(3-Bromobenzyl)-5,9-dihydroxypyrrolo[3,4-g]cinnoline-6,8-
dione (9): 4% yield.
7-(3-Bromobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (10): 60% yield.
5,9-Dihydroxy-7-methylpyrrolo[3,4-g]quinoxaline-6,8-di-
one (11a): 66% yield.
7-Cyclopentyl-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-6,8-di-
one (11b): 31% yield.
5,9-Dihydroxy-7-piperidin-4-ylpyrrolo[3,4-g]quinoxaline-6,8-
dione(11c): 78% yield.
7-(1-Benzoylpiperidin-4-yl)-5,9-dihydroxypyrrolo[3,4-g]qui-
noxaline-6,8-dione (11d): 44% yield.
7-Cyclohexylmethyl-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (11e): 13% yield.
5,9-Dihydroxy-7-[(1R)-1-phenylethyl)pyrrolo[3,4-g]quinoxa-
line-6,8-dione (12a): 26% yield.
5,9-Dihydroxy-7-[(1S)-1-phenylethyl]pyrrolo[3,4-g]quinoxa-
line-6,8-dione (12b): 17% yield.
7-Benzo[1,3]dioxol-5-yl-5,9-dihydroxypyrrolo[3,4-g]quinoxa-
line-6,8-dione(13 a): 74% yield.
7-Benzo[1,3]dioxol-5-ylmethyl-5,9-dihydroxypyrrolo[3,4-g]-
quinoxaline-6,8-dione (13b): 55% yield.
7-(2-Benzo[1,3]dioxol-5-ylethyl)-5,9-dihydroxypyrrolo[3,4-g]-
quinoxaline-6,8-dione (13c): 28% yield.
5,9-Dihydroxy-7-(2-methoxybenzyl)pyrrolo[3,4-g]quinoxaline-
6,8-dione (14a): 81% yield.
5,9-Dihydroxy-7-(4-methoxybenzyl)pyrrolo[3,4-g]quinoxaline-
6,8-dione (14b): 4% yield.
The following products were prepared in the same manner:
1-(3,5-Difluorobenzyl)pyrrolidine-2,5-dione (26b) and
1-(3-chloro-4-fluorobenzyl)pyrrolidine-2,5-dione (26c).
Preparation of 1-Substituted Piperidin-4-ylpyrroli-
dine-2,5-dione Analogues (27-29). Method A: Synthesis
of 1-Piperidin-4-ylmethylpyrrolidine-2,5-dione (27). The
removal of the benzyl moiety of 1-(1-benzylpiperidin-4-yl)-
pyrrolidine-2,5-dione (24c) (12.20 g, 45.19 mmol) was done
with palladium on charcoal and hydrogen as previously
described (8.13 g, 100%).
Method B: Synthesis of 4-(2,5-Dioxopyrrolidin-1-yl-
methyl)piperidine-1-carboxylic Acid tert-Butyl Ester
(28). 1-Piperidin-4-ylmethylpyrrolidine-2,5-dione (27) (1.96 g,
10.73 mmol) and di-tert-butyl dicarbonate (2.48 g, 11.35 mmol)
were dissolved in dichloromethane (50 mL). The solution was
stirred at room temperature for 24 h. The solution was
concentrated and coevaporated with toluene (3.02 g, 100%).
Method C: Synthesis of 1-(1-Benzoylpiperidin-4-yl-
methyl)pyrrolidine-2,5-dione (29). 1-Piperidin-4-ylmeth-
ylpyrrolidine-2,5-dione (27) (1 g, 3.38 mmol), benzoyl chloride
(0.47 g, 3.38 mmol), and Na2CO3 (0.36 g, 3.38 mmol) were
dissolved in a mixture of tetrahydrofuran:water (1:1) (30 mL).
The solution was stirred at room temperature for 24 h. After
completion of the reaction, the organic layer was separated
and concentrated. The organic layer was washed with 1 N HCl
solution (2 × 40 mL), dried over magnesium sulfate, and
evaporated under reduced pressure to yield the title compound
29.
General Reaction Procedure 2. Representative Pro-
cedure for the Synthesis of 1-Substituted Pyrrolidine-
2,5-dione Analogues 30a-d. Method A: Synthesis of 1-(3-
Thiophen-2-ylbenzyl)pyrrolidine-2,5-dione (30a). 1-(3-
Bromobenzyl)pyrrolidine-2,5-dione (24a) (method A, general
reaction procedure 1) (0.54 g, 2 mmol), thiopheneboronic acid
(0.26 g, 2 mmol), palladium acetate (22.50 mg, 0.10 mmol),
triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (Na
TMSPP) (0.11 g, 0.20 mmol), and triethylamine (1.01 g, 10
mmol) were dissolved in a mixture of tetrahydrofuran:water
(1:1) (30 mL). The solution was refluxed for 30 min. The
solution was cooled to room temperature and the organic layer
was separated. The organic layer was concentrated, dissolved
in dichloromethane (30 mL), and washed with 10% citric acid
solution (30 mL). The organic layer was dried over magnesium
sulfate and evaporated under reduced pressure to yield the
title compound 30a (0.31 g, 57%).
The following products were prepared in the same manner.
1-Biphenyl-4-ylmethylpyrrolidine-2,5-dione (30b): 58% yield,
24 h.
1-(2′-Fluorobiphenyl-3-ylmethyl)pyrrolidine-2,5-dione
(30c): 76% yield, 24 h.
1-(2′-Fluorobiphenyl-4-ylmethyl)pyrrolidine-2,5-dione
(30d): 56% yield, 24 h.
1-Methylpyrrolidine-2,5-dione (31) was purchased from Al-
drich.
7-(2,6-Dimethoxybenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinox-
aline-6,8-dione (14c): 50% yield.
7-(2,3-Dimethoxybenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinox-
aline-6,8-dione (14d): 23% yield.
7-(2,4-Dimethoxybenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinox-
aline-6,8-dione (14e): 31% yield.
7-(2,5-Dimethoxybenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinox-
aline-6,8-dione (14f): 44% yield.
5,9-Dihydroxy-7-(4-methylbenzyl)pyrrolo[3,4-g]quinoxaline-
6,8-dione (15a): 41% yield.
5,9-Dihydroxy-7-(4-trifluoromethoxybenzyl)pyrrolo[3,4-g]-
quinoxaline-6,8-di one (15b): 15% yield.
5,9-Dihydroxy-7-(4-trifluoromethylbenzyl)pyrrolo[3,4-g]qui-
noxaline-6,8-dione (15c): 29% yield.
7-(3-Fluorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (15d): 11% yield.
7-(4-Fluorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (15e): 21% yield.
7-(4-Bromo-2-fluorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]qui-
noxaline-6,8-dione (15f): 57% yield.
7-(5-Bromo-2-fluorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]qui-
noxaline-6,8-dione (15g): 77% yield.
7-(3,5-Difluorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxa-
line-6,8-dione (15h): 52% yield.
7-(3-Chloro-4-fluorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]qui-
noxaline-6,8-dione (15i): 36% yield.
General Reaction Procedure 3. Representative Pro-
cedure for the Preparation of Phthalimide Analogues
(7-18).33 Method A. Synthesis of 7-(3-bromobenzyl)-5,9-
dihydroxy-pyrrolo[3,4-g]quinoline-6,8-dione (8a). Pyri-
dine-2,3-dicarboxylic acid dimethyl ester (0.20 g, 1.04 mmol)
(32a) and 1-(3-bromobenzyl)pyrrolidine-2,5-dione (0.28 g, 1.04
mmol) (24a) were dissolved in tetrahydrofuran (30 mL),
followed by the addition of sodium hydride (0.17 g, 4.16 mmol)
and methanol (catalytic). The solution was refluxed for 48 h.
The solution was cooled to room temperature and concentrated.
The residue was suspended in water. The aqueous solution
was acidified with 12 N HCl solution, followed by the addition
of ether. The resulting heterogeneous mixture was rapidly
stirred for 1 h. The crystals were filtered off, washed with
ether, and dried in vacuo for 1 h (0.31 g, 75%).
7-(3-Chlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (15j): 54% yield.
7-(4-Chlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (15k): 48% yield; LRMS (ES+) m/z 358 (M + H)+;
LRMS (ES-) m/z 356 (M - H)-; HPLC (system 1) tR 6.62 min,
98.56%; HPLC (system 2) (286 nm) tR 6.13 min, 98.58%; HPLC
(system 3) (288 nm) tR 6.38 min, 96.78%. Anal. Calcd for C17H10-
ClN3O4‚0.05H2O: C, 57.25; H, 2.85; N, 11.78. Found: C, 56.03;
H, 2.89; N, 11.33.
7-(3,4-Dichlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxa-
line-6,8-dione (15l): 58% yield; 1H NMR (DMSO) δ 4.84 (s,
2H, C6H5CH2), 7.39 (dd, 1H, J ) 2.03 and J ) 8.35, CHarom),
7.66-7.69 (m, 1H, CHarom), 7.69 (s, 1H, CHarom), 9.20 (s, 2H,
CHarom (2×)), 11.20-11.50 (brs, 2H, OH (2×)); 13C NMR
(DMSO) δ 39.9 (CH2), 109.9 (Carom), 127.8, 129.5 (CHarom), 130.0
(Carom), 130.7 (CHarom), 131.0 (Carom), 138.1, 138.7, 145.1 (Carom),
145.5 (CHarom), 165.2 (CO); LRMS (ES+) m/z 390 (M + H)+;
LRMS (ES-): m/z 388 (M - H)-; HPLC (system 1) tR 7.16
The following products were prepared in the same manner.
2-(4-Fluorobenzyl)-4,9-dihydroxybenzo[f]isoindole-1,3-di-
one (7): 34% yield.

Phtalimides as Inhibitors of HIV-1 Integrase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1939
min, 98.18%; HPLC (system 2) (293 nm) tR 6.69 min, 98.56%;
HPLC (system 3) (288 nm) tR 6.96 min, 98.09%. Anal.
(C17H9Cl2N3O4‚0.12H2O) C, H, N.
7-(3,5-Dichlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxa-
line-6,8-dione (15m): 61% yield.
In complex minimizations the CR backbone atoms are tethered
to their crystal structure positions with a harmonic potential
with a force constant of 10 kcal/mol. Å. All software is
developed in-house and is run on a Silicon Graphics Origin
2000 system.
Integrase Activity Assay. The integrase activity was
determined using an oligonucleotide-based assay in which the
DNA strand transfer by preformed complexes of integrase and
processed DNA was measured by means of an ELISA test in
microtiter plate format. Recombinant His-tagged HIV-1 inte-
grase was produced in the Escherichia coli strain BL21(DE3)
from the plasmid pINSD.His.sol (NIH) as described previ-
ously.40 HPLC-purified oligonucleotides (Proligo) were used for
the preparation of viral DNA substrate and target DNA. INb-
1C: 5′-bGTGTGGAAAATCTCTAGCAGT-3′, IN-1NC: 5′-ACT-
GCTAGAGATTTTCCACAC-3′. INT5: 5′-TGACCAAGGGCT-
AATTCACTf-3′, INT6: 5′-AGTGAATTAGCCCTTGGTCAf-3′.
INb-1C is 5′-biotinylated, and INT5 and INT6 are labeled with
FITC at the 3′-end. INb-1C and IN-1NC correspond to the U5
end of the HIV-1 LTR. The DNA substrate and target DNA
were made by annealing INb-1C and IN-1NC, and INT5 and
INT6, respectively. An equimolar mixture of both oligonucle-
otides was heated shortly at 95 °C in the presence of 100 mM
NaCl and allowed to cool slowly to room temperature.
For the integration strand transfer reactions, 20 nM bioti-
nylated DNA substrate was preincubated with 300 nM HIV-
integrase at 37 °C for 5 min. Serial dilutions of the compounds
and 50 nM target DNA were added to the reaction mix
containing 20 mM Hepes pH 7.5, 25 mM NaCl, 5 mM MnCl2,
2 mM DTT, and 50 µg/mL BSA, and the mixture incubated
for 2 h at 37 °C. The reaction mix was transferred to
streptavidine-coated plates (NoAb discoveries), which were
prewashed with 5× SSCT, and incubated for 1 h at room
temperature. Plates were washed with 2× SSCT, anti-FITC
POD-coupled antibody (Roche) was added, and the plates were
incubated for 1 h at room temperature. After a final washing
step with PBST, BM chemiluminescent POD-substrate (Roche)
was added, and luminescence was read.
Cell-Based Anti-HIV and Toxicity Assay. The antiviral
activity has been determined with a cell-based replication
assay. This assay directly measures the ongoing replication
of virus in MT4 cells via the specific interaction of HIV-tat
with LTR sequences coupled to GFP. In the toxicity assay, a
reduced expression of the GFP reporter protein serves as a
marker for the cellular toxicity of a compound. Briefly, various
concentrations of the test compounds are brought into a 384-
well microtiter plate. Subsequently, MT4 cells and HIV-1/LAI
(wild type) are added to the plate at a concentration of
respectively 150 000 cells/mL and 200 cell culture 50% infec-
tious doses (CCID50). To determine the toxicity of the test
compound, mock-infected cell cultures containing an identical
compound concentration range are incubated for 3 days (37
°C, 5% CO2) in parallel with the HIV-infected cell cultures.
On the basis of the calculated percent inhibition for each
compound concentration, dose-response curves are plotted
and EC50 and pEC50 and CC50 and pCC50 values are calculated.
7-(2-Bromobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-
6,8-dione (15n): 27% yield.
5,9-Dihydroxy-7-pyridin-4-ylmethylpyrrolo[3,4-g]quinoxaline-
6,8-dione (16): 9% yield.
5,9-Dihydroxy-7-naphthalen-2-ylmethylpyrrolo[3,4-g]qui-
noxaline-6,8-dione (17a): 8% yield.
5,9-Dihydroxy-7-(3-thiophen-2-ylbenzyl)pyrrolo[3,4-g]qui-
noxaline-6,8-dione (17b): 48% yield.
7-(2′-Fluorobiphenylyl-3-ylmethyl)-5,9-dihydroxypyrrolo[3,4-
g]quinoxaline-6,8-dione (17c): 39% yield.
7-(2′-Fluorobiphenylyl-4-ylmethyl)-5,9-dihydroxypyrrolo[3,4-
g]quinoxaline-6,8-dione(17d): 41% yield.
7-Biphenylyl-4-ylmethyl-5,9-dihydroxypyrrolo[3,4-g]quinox-
aline-6,8-dione(17e ): 19% yield.
5,9-Dihydroxy-7-phenethylpyrrolo[3,4-g]quinoxaline-6,8-di-
one (18): 26% yield.
Reaction Procedures of Heterocycles Diester Ana-
logues (32a-d). Pyridine-2,3-dicarboxylic acid dimethyl
ester (32a) was purchased from ABCR.
Synthesis of 6-Methylpyridine-2,3-dicarboxylic Acid
Dimethyl Ester (32b). 6-Methylpyridine-2,3-dicarboxylic acid
(5.10 g, 28.18 mmol) was dissolved in methanol (80 mL),
followed by the addition of 6 N HCl in 2-propanol solution (3
mL). The solution was refluxed for 72 h. The solution was
cooled to room temperature and concentrated. The residue was
coevaporated with toluene (5.81 g, 99%).
Synthesis of Pyridazine-3,4-dicarboxylic Acid Diethyl
Ester (32c). This compound was synthesized according to the
procedure described by Vors et al.32
Synthesis of Pyrazine-2,3-dicarboxylic Acid Dimethyl
Ester (32d). Pyrazine-2,3-dicarboxylic acid (21.86 g, 0.13 mol)
was dissolved in methanol (400 mL), followed by the addition
of 6 N HCl in 2-propanol solution (15 mL). The solution was
heated at reflux for 24 h. The residue was evaporated and the
residue was dissolved in dichloromethane (50 mL). The organic
layer was washed with saturated sodium bicarbonate solution
(3 × 50 mL), dried over magnesium sulfate, and evaporated
under reduced pressure to yield the title compound (25.48 g,
100%), which solidified on standing.
Molecular Modeling Methods. The A-chain of the tri-
meric crystal structure with PDB code 1BIU14 was used as the
basis for our docking protocol. The protein surface loop
comprised of residues 141-147 is not resolved in this struc-
ture. The coordinates of these residues were therefore obtained
from the PDB structure with code 1BIS14 by superimposing
the CR atoms of three residues on either side of the gap in the
1BIU structure. At the active site, we added two more water
molecules to provide for an octahedral coordination of the Mg2+
ion. Hydrogen atoms were added to the structure and a
molecular mechanics minimization was carried out with the
positions of the CR atoms tethered to their crystal structure
coordinates. The enol form of the diketo acid I (Figure 2) was
manually positioned into the binding pocket by removing two
water molecules from the coordination sphere of the Mg2+ ion
and replacing these by the keto carbonyl and hydroxy oxygen
atoms of the inhibitor. As there are four water molecules and
the diketo acid can coordinate adjacent positions of the Mg2+
coordination sphere in two ways, there are 10 possible orienta-
tions of the inhibitor, most of which severely clash with the
enzyme. The configuration shown in Figure 1 is the one with
the lowest enzyme-inhibitor interaction after molecular me-
chanics minimization of the manually constructed complex.
The diketo acid was removed from this complex, and the
coordinates of the enzyme, Mg2+ ion, and its two remaining
coordinating water molecules form the binding pocket used
in the docking protocol described in the modeling section.
Minimization of enzyme-ligand complexes is carried out until
the root-mean-square gradient is below 0.05 kcal/mol Å; for
ligands and Mg2+ chelates, this threshold is 10-6 kcal/mol Å.
Acknowledgment. The authors thank Marleen Van
Dooren for her analytical support and Marie-Pierre de
Bethune, Karen Manson, Frank Daelemans and Gem-
ma Campabadal I Monfa` for reviewing the manuscript.
Supporting Information Available: Spectroscopic data
for intermediate and final compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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