4-Substituted 2-hydroxyisoquinoline-1,3(2 H,4 H)-diones as a novel class of HIV-1 integrase inhibitors

Article abstract of DOI:10.1021/ml400009t

A series of 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides featuring an N-hydroxyimide chelating functionality was evaluated for their inhibitory properties against human immunodeficiency virus type 1 integrase (HIV-1 IN). Several derivatives displayed low nanomolar IC₅₀ values comparable to that of the clinically used raltegravir. A marked effect of one compound on both primary IN-catalyzed reactions, strand transfer (ST), and 3′ processing (3′-P), emphasizes a novel IN inhibition mechanism establishing it as a potential new generation IN inhibitor. Substitution of the 2-hydroxyisoquinoline-1,3-dione scaffold at position 4 by carboxamido chains was beneficial for antiviral activity since reproducible low micromolar anti-HIV activities were obtained for the first time within this scaffold.

Full text of DOI:10.1021/ml400009t

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Letter
4-Substituted 2-Hydroxyisoquinoline-1,3(2H,4H)-
diones as a Novel Class of HIV-1 Integrase Inhibitors
Muriel Billamboz, Virginie Suchaud, Fabrice Bailly, Cedric Lion, Jonas Demeulemeester,
Christina Calmels, Marie-Line Andréola, Frauke Christ, Zeger Debyser, and Philippe Cotelle
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400009t • Publication Date (Web): 17 May 2013
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4-Substituted 2-Hydroxyisoquinoline-1,3(2H,4H)-diones as a
Novel Class of HIV-1Integrase Inhibitors
Muriel Billamboz,†,& Virginie Suchaud,†,& Fabrice Bailly,*,†, & Cedric Lion,†, & Jonas Demeulemeester,#
Christina Calmels,§ Marie-Line Andréola,§ Frauke Christ,# Zeger Debyser,# and Philippe Cotelle†,&
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†Université Lille Nord de France, F-59000 Lille, France
&Université Lille 1 Sciences & Technologies, EA 4478 Chimie Moléculaire et Formulation, F-59655 Villeneuve d'Ascq,
France
#KU Leuven, Molecular Virology and Gene Therapy (VCTB+5), Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
§UMR 5234 CNRS, Université Bordeaux Segalen, 146 Rue Léo Saignat, F-33076 Bordeaux, France
KEYWORDS HIV, antiretroviral, integrase, 3’ processing, 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamide
Supporting Information Placeholder
ABSTRACT: A series of 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides featuring a N-hydroxyimide chelating functionality
was evaluated for their inhibitory properties against human immunodeficiency virus type 1 integrase (HIV-1 IN). Several deriva-
tives displayed low nanomolar IC50 values comparable to that of the clinically used raltegravir. A marked effect of one compound
on both primary IN-catalyzed reactions, strand transfer (ST) and 3’ processing (3’-P), emphasizes a novel IN inhibition mechanism
establishing it as a potential new generation IN inhibitor. Substitution of the 2-hydroxyisoquinoline-1,3-dione scaffold at position 4
by carboxamido chains was beneficial for antiviral activity since reproducible low micromolar anti-HIV activities were obtained for
the first time within this scaffold.
HIV-1 integrase (IN) is a 32kDa protein that plays a crucial
role in HIV infection by incorporating the retrotranscribed
viral DNA into the host chromosomal DNA. IN has been
extensively studied as therapeutic target in the field of AIDS
antiretroviral therapy since it establishes irreversible infection
and has no cellular equivalent, which limits toxicity.1,2 The
integration process involves a sequence of 2 reactions which
both require the presence of metallic cofactors: in the cyto-
plasm, a DNA-IN complex is formed that catalyzes the endo-
nucleolytic cleavage of a dinucleotide at each 3’-end of the
dsDNA (the 3'-processing step,3'-P) After transport into the
nucleus, the strand transfer step (ST), catalyzed by the in-
tasome (a specific tetramer of IN and viral DNA ends) then
joins each 3'-end of this recessed DNA to a 5'-end in the host
DNA.
cally.9 Dual inhibitors against IN and reverse transcriptase
(RT) have also been investigated. Very recently, a non-
nucleoside pyrimidine-2,4-dione RT inhibitor was 3-N hy-
droxylated, leading to a N-hydroxyimide derivative capable of
inhibiting IN.10
We previously designed and studied several series of 2-
hydroxyisoquinoline-1,3-diones as potential dual inhibitors of
HIV-1 IN and RT associated RNase H activities. A few hits
displaying high selectivity for IN or the RT associated RNase
H function with submicromolar IC50 values were discov-
ered.11,12,13 Unfortunately nearly all tested compounds exhibit-
ed high cellular cytotoxicity in cell culture which limited their
applications as antiviral agents.
The first FDA-approved drug acting as a strong selective ST
inhibitor3 is raltegravir (Isentress™). Elvitegravir, also recent-
ly approved, can be given once daily when combined with a
booster (as part of the fixed-dose combination tablet Stribild),4
but cross-resistance rules out treatment of patients failing on
raltegravir therapy.5,6 Dolutegravir (S/GSK1349572)7 is cur-
rently in phase III clinical trials. Although superior to ralte-
gravir, it also exhibits significant resistance overlap.8 It can
therefore be stated that IN still remains an “orphan” in terms
of marketable drugs and an attractive and scientifically chal-
lenging target. One of the most innovative strategies so far
was the design of inhibitors targeting the Lens Epithelium
Derived Growth Factor (LEDGF)/p75 binding site on inte-
grase (LEDGINs). These small molecules hinder the interac-
tion of IN with the cellular cofactor LEDGF/p75. In addition
LEDGINs stabilize integrase dimers and inhibit IN allosteri-
Figure 1. Structures of raltegravir, elvitegravir, dolutegravir and
compound 33 pointing out the key components of the HIV-1 IN
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inhibitory pharmacophore: the magnesium chelating moiety (red)
and the hydrophobic halogenobenzyl group (green).
protected compounds 8-21.The O-benzyl protecting group was
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finally removed with boron tribromide or boron trichloride to
afford 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides 22-37.
The target compounds were obtained either as the keto form or as
a keto/enol mixture with a large preference for the keto form
(measured by 1H NMR).
Careful examination of known potent IN strand transfer in-
hibitors (INSTIs) like raltegravir and dolutegravir points out
the key presence of a magnesium chelating triad of oxygen
atoms. Studies have also revealed the crucial importance of a
hydrophobic side chain bearing a mono- or polyhalogenated
benzyl group in the pharmacophore (Figure 1).6 With the pub-
lication of their PFV intasome crystal structures, Hare et al.
recently demonstrated that this aromatic component invades a
pocket at the protein-DNA interface which is natively occu-
pied by the 3´-terminal base of viral DNA.14 This led us to
substitute our scaffold at position 4 with aromatic side chains,
as exemplified by compound 33. Not only could this enhance
the magnesium-chelating properties of our molecules by ex-
tending the co-planar arrangement of oxygen atoms, but also
their affinity for the IN catalytic center through additional
hydrophobic contacts, namely π-stacking interactions with
retroviral DNA bases.
Table 1 reports the enzyme inhibitory activities of this series.
First, it can be observed that compounds 8 and 9 are completely
devoid of inhibitory activity against HIV-1 IN. This strongly
suggests that the N-hydroxyl function participating in the chelat-
ing pharmacophore is indeed crucial to IN binding. The introduc-
tion of a phenylcarboxamido side chain (compounds 22-25) led to
strong HIV-1 IN inhibition with IC50 values in the nanomolar
range similar to that of raltegravir. The overall tendency seems to
indicate that the linker's length between the amide function and
the aromatic ring does not have a drastic influence on activity,
since compounds 31, 32 and 33 bearing benzylcarboxamido- side
chains display IC50 values similar to those of their respective
homologues 35, 36 and 34 with phenethylcarboxamido- side
chains. Even though halogen substitution of the aromatic ring
seems favorable, its m,p-disubstitution with donating oxygenated
substituents (-OH or -OMe) had a dramatic effect on the HIV-1
IN inhibition with a one-log collapse of the IC50 values (31, 32,
35, 36) when compared to the o,p-disubstituted compounds 29
and 30. This may be explained by unfavorable steric hindrance
preventing optimal π-stacking with the DNA cytosine in the hy-
drophobic cavity rather than by electronic effects. Moreover,
replacing the amide proton by a methyl group (compound 37)
provoked a 17-fold decrease of the HIV-1 IN inhibition, which
stresses the importance of the side chain orientation and of the
supposed intramolecular H-bond within the complex. Apart from
overall HIV-1 IN inhibition, careful examination of the com-
pounds' effects on HIV-1 IN 3’-P and ST activities was also very
instructive. A glance at the data of raltegravir evidences its well-
known selectivity for ST over 3'-P, measured here at 128-fold.
Close values of the IN overall and ST inhibitions were also ob-
served for compounds 25-28 and 31-36, which may infer ST
selectivity. In contrast, there was a discrepancy between overall
IN and ST inhibition for compounds 23, 24, 29 and 30. The ST
IC50 values are 10- to 15-fold higher than the overall inhibition,
suggesting either a synergistic effect of both primary IN functions
or the intervention of an unknown mechanism of IN inhibition.
The 3'-P inhibition of compounds 23, 30 and 33 was thus exam-
ined, and the calculated ratios IC50(3’ P)/IC50(ST) were measured
at 5.7, 4.1 and 0.7, respectively. Unlike raltegravir, ST selectivity
is not retained for these isoquinoline-4-carboxamides, and to the
best of our knowledge, compound 33 is the first low molecular IN
inhibitor which displays inhibition of both IN catalytic steps in the
low nanomolar range. We also tested some compounds against
HIV-1 RT associated RNase H activity (Table 1) and submi-
cromolar IC50 values were obtained for the most active com-
pounds 29-30, 32-33 and 36.
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Herein we propose further insight into the design of HIV IN
inhibitors based on the 2-hydroxyisoquinoline-1,3-dione scaffold.
The design, synthesis and pharmacological evaluation of a novel
series of 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides bear-
ing diverse substituted phenyl, benzyl or phenethyl side chains
was carried out. The carboxamide link was chosen not only for its
greater stability in physiological conditions, but also for its pro-
pensity to create an intramolecular hydrogen bond with the oxy-
gen at position 3,thus orienting the aromatic side chain towards
the desired hydrophobic cavity, as demonstrated by preliminary
molecular modeling and docking studies (data not shown/see
supplementary information). Non-hydroxylated analogues 8 and 9
were also synthesized to validate the N-hydroxyl function as a key
structural feature for magnesium complexation. The overall in
vitro HIV-1 IN inhibitory potency, the anti-HIV activity in MT-4
cells and cytotoxicity of the compounds were evaluated. Addi-
tionally, the in vitro inhibitions of HIV-1 IN 3'-P activity and RT
associated RNase H function were also measured for the most
interesting compounds.
Scheme 1. Reagents and conditions: (a) BnONH2 or CH3NH2,
BOP, NMM, CH2Cl2, rt; SOCl2 then AcOEt, NH3 g; (b) 2.5 M
KOH/MeOH, rt; (c) R4NH2, toluene, reflux; (d) BCl3 or BBr3,
CH2Cl2, rt then H2O
Finally this series was evaluated in a cell-based antiviral assay
against HIV-1 (Table 2). As expected, the non N-2 hydroxylated
compounds 8 and 9 were not active, suggesting that the antiviral
potencies may strongly be related to the metal-chelating ability
and integrase inhibition. The amide proton also proved essential
for antiviral activity (EC50> 107 µM for compound 37). Unfortu-
nately, compounds 28, 31, 32, 35 and 36 were either poorly active
or cytotoxic. Compounds 22-27, 33 and 34 however, displayed
low micromolar EC50 values ranging from 1.75 to 5.08 µM. Alt-
hough these are modest in comparison to that of the ST selective
raltegravir (EC50 = 6.0 nM), it is still a very promising result since
it is the first time that cytotoxicity is overcome on this scaffold.
Compound 33 displayed a good aqueous solubility of 190.0 ꢀM
The synthetic pathway was based upon a formerly reported
route for the synthesis of 4-alkoxycarbonylisoquinoline-1,3-
diones.15 Acid precursor 1 was obtained in two steps from com-
mercial homophthalic acid. After coupling of O-
benzylhydroxylamine, ammonia or N-methylamine, the corre-
sponding amides 2-4 were quantitatively cyclized in mild alkaline
conditions into ester precursors 5-7 (Scheme 1). Diversification of
the side chain was then obtained via addition-elimination of vari-
ous primary or secondary amines on the ester moiety to afford
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gravir and elvitegravir in time of addition experiments.24 This
and a partition coefficient, logD of 0.56. At a 10 ꢀM concentra-
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4
5
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8
attests for the cellular inhibition of integrase by this scaffold,
whereas inhibition of the RT associated RNase H function re-
mained a side activity. Half of the series showed an advantageous
window between antiviral efficacy and cellular toxicity (21- to 86-
fold).
tion, a mean permeability coefficient Papp (Caco-2 cells, pH
6.5/7.4) below 0.2 10-6 cm/s was determined and high human
plasma protein binding (mean of 99.9% protein bound) was ob-
served, which might account for the lack of strong antiviral activi-
ty. This issue is currently being addressed through further phar-
macomodulation. It must be stressed that although we previously
designed several compounds based on this scaffold, it is the first
time that a reproducible low micromolar antiviral activity is at-
tained in conjunction with a low nanomolar integrase inhibition.
Moreover, compound 33 showed a profile similar to that of ralte-
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Table 1. Inhibitory activities of compounds 8-9 and of the 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides 22-37
IC50 (µM)
Comp.
R4
3’ P/STd
Overall INa
199
STb
79
3’ Pc
RNase He
8(R2 = H, not OH)
9 (R2 = CH3, not
OH)
> 250
0.31
> 250
0.73
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23
0.020
0.010
0.277
0.110
1.58
5.7
16.6
7.31
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26
0.8
1.12
0.03
0.08
27
0.076
0.084
14.0
28
29
1.19
1.03
0.035
0.254
0.63
0.52
30
31
0.035
0.235
0.572
0.307
2.35
4.1
0.205
0.056
0.258
0.099
0.90
0.36
32
33
0.06
0.7
34
35
0.030
0.495
0.020
0.126
3.85
0.13
36
0.673
0.296
4
’
(R
CH3)
=
37
5.21
5.6
Raltegravir
0.010
0.007
0.90
128
a,b,c Concentration required to inhibit by 50% the in vitro overall, strand transfer and 3’ processing integrase activities, respectively. d IC50
3’ P / IC50 ST ratio.e Concentration required to inhibit by 50% the in vitro RNase H activity.
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Figure 2. Putative binding modes of compound 33 in the PFV IN catalytic site obtained by molecular docking using the GOLD docking
suite and the CHEMPLP fitness function. The ligand is depicted in orange, magnesium cations in green, IN in blue and viral DNA in pink.
Pose A: the three oxygens on the heterocyclic core contribute to Mg2+ chelation, allowing an intramolecular H-bond within the ligand. π-
stacking interactions occur with deoxycytosine C16 and deoxyadenosine A17. Pose B: both π-stacking interactions occur as well. The
exocyclic amide oxygen contributes to the metal chelation pharmacophore, at the expense of internal ligand torsion.
Table 2. Anti-HIV activities of compounds 8-9 and of the 2-
hydroxy-1,3-dioxoisoquinoline-4-carboxamides 22-37
iant 3'-deoxyadenosine is flipped out of the active site in the
case of elvitegravir and MK-0536, a raltegravir-derived INSTI
with improved resistance profile,17 it seems to participate in
additional π-stacking interactions with the core of dolutegravir
in the 3S3M structure. In a similar manner, the extended pla-
nar aromatic nature of our N-hydroxyisoquinoline-1,3-dione
core bearing the metal chelating pharmacophore infers a great
propensity to interact with this 3'-deoxyadenosine via π-
stacking contacts.
a
b
Comp.
EC50
CC50
TIc
(ꢀM)
(ꢀM)
8
9
22
23
24
25
26
> 250
> 250
4.95
3.34
2.47
> 250
> 250
105.5
12.3
64.0
114.5
130
21.3
3.7
25.9
65.4
41.7
> 22
6.7
1.75
3.12
As expected, two possible binding modes were obtained for
compound 33 using this model (figure 2), both of which show
similar statistical significance and high overall fitness function
scoring. Both poses involve (a) dual magnesium complexation
(b) π-stacking of the fluorobenzyl side chain with the invariant
deoxycytosine C16 (c) π-stacking of the central isoquinoline
moiety with the invariant terminal 3'-deoxyadenosine A17.
Although pose 2B involving the exocyclic oxygen in the che-
lation pharmacophore is not to be excluded, we strongly think
that pose 2A is more likely to occur in reality. A closer look at
the weighed terms of the CHEMPLP fitness function indeed
reveals that despite a slightly better metal chelation score, the
ligand conformation in pose 2B requires significant internal
torsion and close steric contacts in the carboxamide linkage.
Conversely, not only does pose 2A allow a more favorable
dihedral angle at this linkage, but it also involves an additional
intramolecular hydrogen bond between the amide proton of
the 4-(4-fluorobenzylcarboxamido) side chain with the oxygen
at position 3, which may direct and maintain the aromatic ring
towards the desired hydrophobic pocket. If this docking model
may only reflect the ST inhibition mechanics of our mole-
cules, we cannot yet provide a theoretical explanation for the
activity on 3'-processing.
27
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29
30
31
32
33
34
5.7
> 125
118.5
60.4
> 125
125
11
202
123.5
> 125
63
17.63
9.24
7.94
> 125
> 11
2.34
6.5
> 16
86.3
24
> 1.8
5.08
35
36
37
70.77
> 63
> 107.2
0.006
107.2
> 8.0
Raltegravir
> 1333
a
Effective concentration required to reduce HIV-1-induced cyto-
b
pathic effect by 50% in MT-4 cells. Cytotoxic concentration
c
required to reduce MT-4 cell viability by 50%. Therapeutic
index, defined by CC50/EC50.
In silico docking studies were also performed in order to de-
termine a possible binding mode with the target. Although our
previously reported method was originally based on the
PDB:3L2T crystallographic structure of PFV-IN intasome in
complex with raltegravir,14 we decided to adapt it to the more
recent 3S3M X-ray structure of the PFV intasome bound to
dolutegravir (see supporting information).16 Whereas the invar-
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To our knowledge, it is the first time that such cumulative and
Corresponding Author
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synergistic effects on both integrase primary functions leading to
strong integrase inhibition are observed. Little is known about 3’-
P inhibition mechanism, since only ST selective IN inhibitors
have been co-crystallized with PFV IN so far. Crystal structures
of PFV IN bound to unprocessed viral DNA prior to 3’-P were
recently reported. As stipulated by Hare et al.,18 the selectivity of
known IN inhibitors for ST may be explained by the fact that their
binding to the catalytic site in pre-3'-P configuration would re-
quire the displacement of the 3'-terminal AATtrinucleotide, in-
volving the rupture of phosphate-metal and phosphate-amide
interactions, as opposed to the displacement of only one deoxy-
adenosine at the ST stage. Considering the apparent mobility of
the unprocessed terminal 3'-dinucleotide in the catalytic site, such
a mechanism might be envisaged. However, the energetic barrier
needed for such a displacement might be difficult to reach, and a
compound would need to establish additional contacts in the pre-
3’-P complex in order to balance the energetics of binding and
maintain its potency.
* E-mail: fabrice.bailly@univ-lille1.fr. Phone: +33 320 33 72 31.
Fax: +33 320 33 63 09.
Funding Sources
This work was financially supported by grants from le Ministère
de l’Enseignement Supérieur et de la Recherche Française, le
Centre National de la Recherche Scientifique (CNRS) and
l’Agence Nationale de la Recherche contre le Sida (ANRS). Ex-
periments at KU Leuven were funded by the FP7 project
CHAARM and the IWT.
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ABBREVIATIONS
HIV-1 IN, human immunodeficiency virus type 1 integrase; ST,
strand transfer; 3’-P, 3’ processing; AIDS, acquired immune
deficiency disease; FDA, food and drug administration; LEDGF,
lens epithelium derived growth factor; LEDGIN, inhibitor of the
lens epithelium derived growth factor binding site in integrase;
RT, reverse transcriptase; INSTI, integrase strand transfer inhibi-
tor; PFV, prototype foamy virus.
Compounds inhibiting 3’-P and ST catalytic activities in the
same range are rare. They are exemplified by some conjugates of
single-stranded oligonucleotides with hydrophobic molecules
acting in the low nanomolar range19 and by low molecular com-
pounds like numerous polyphenols, salicylhydrazides,20 pyranodi-
pyrimidines,21 and styrylquinolines.22,23 The latter were very
instructive, as a few compounds inhibited HIV-1 replication at
low micromolar concentrations. After being characterized as
metal chelating inhibitors, further cellular mechanistic investiga-
tions showed that they exert their IN inhibitory activity by inter-
acting with the viral DNA or target DNA binding regions of IN
and/or by interfering with the nuclear import mechanism of IN. In
our case, the loss of the N-hydroxyl function strongly impaired
inhibitory properties, suggesting that our compounds may target
the HIV-1 IN catalytic site. However, at this stage, another mech-
anism of inhibition cannot entirely be ruled out. The alternative
would consist of an allosteric mechanism through interactions
with a specific region of IN prior to DNA binding. No effect of
compound 33 was observed on IN/LEDGF binding
(IC50IN/LEDGF>>100 ꢀM), excluding the interference with the
cellular cofactor LEDGF/p75.
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ASSOCIATED CONTENT
Supporting Information
Synthesis of 8-9 and 22-37, experimental details, docking
protocol, compound characterization (1H, 13C NMR, HRMS)
for final products, biological assay methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Pommier, Y.; Wang, Z N-3 hydroxylation of pyrimidine-2,4-
AUTHOR INFORMATION
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ACS Medicinal Chemistry Letters
Table of Contents
1
2
3
4
5
O
NHR4
O
6
7
N
OH
8
9
O
10
11
R4 =
X
12
13
14
15
16
17
18
19
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
46
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60
X ,
,
X
IC50 (HIV-1 integrase) = 0.010-1.19 ꢀM
EC50 (HIV-1 inf ected MT-4 cells) = 2.34-9.24 ꢀM
7
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