2-hydroxyisoquinoline-1,3(2H,4H)-diones (HIDs) as human immunodeficiency virus type 1 integrase inhibitors: Influence of the alkylcarboxamide substitution of position 4

Article abstract of DOI:10.1016/j.ejmech.2016.03.083

Herein, we report further insight into the biological activities displayed by the 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) scaffold. Previous studies have evidenced the marked fruitful effect of substitution of this two-metal binding pharmacophore at

Full text of DOI:10.1016/j.ejmech.2016.03.083

European Journal of Medicinal Chemistry 117 (2016) 256e268
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
2-hydroxyisoquinoline-1,3(2H,4H)-diones (HIDs) as human
immunodeficiency virus type 1 integrase inhibitors: Influence of the
alkylcarboxamide substitution of position 4
,
,
,
b
,
, c
Muriel Billamboz a b, Virginie Suchaud a b, Fabrice Bailly a *, Cedric Lion a
,
d
e
e
a, b
ꢀ
Marie-Line Andreola , Frauke Christ , Zeger Debyser , Philippe Cotelle
Universite Lille Nord de France, F-59000 Lille, France
a
ꢀ
b
ꢀ
Centre de Recherches Jean-Pierre Aubert UMR-S1172, Faculte des Sciences Pharmaceutiques et Biologiques, 3 rue du Professeur Laguesse, BP83, F-59006
Lille, France
c
ꢀ
UMR CNRS 8576, Unite de Glycobiologie Structurale et Fonctionnelle, F-59655 Villeneuve d'Ascq, France
d
ꢀ
ꢀ
Laboratoire MFP, UMR 5234 CNRS, Universite Bordeaux Segalen, FR Transbiomed, 146 Rue Leo Saignat, F-33076 Bordeaux, France
e Molecular Medicine, K.U. 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:
Herein, we report further insight into the biological activities displayed by the 2-hydroxyisoquinoline-
1,3(2H,4H)-dione (HID) scaffold. Previous studies have evidenced the marked fruitful effect of substi-
tution of this two-metal binding pharmacophore at position 4 by phenyl and benzyl carboxamido chains.
Strong human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors in the low nanomolar range
with micromolar (even down to low nanomolar) anti-HIV activities were obtained. Keeping this essential
4-carboxamido function, we investigated the influence of the replacement of phenyl and benzyl groups
by various alkyl chains. This study shows that the recurrent halogenobenzyl pharmacophore found in the
INSTIs can be efficiently replaced by an n-alkyl group. With an optimal length of six carbons, we observed
a biological profile and a high barrier to resistance equivalent to those of a previously reported hit
compound bearing a 4-fluorobenzyl group.
Received 6 November 2015
Received in revised form
25 March 2016
Accepted 26 March 2016
Available online 28 March 2016
Keywords:
HIV-1 integrase
Two-metal binding pharmacophore
Antiretroviral
© 2016 Elsevier Masson SAS. All rights reserved.
2-hydroxyisoquinoline-1,3(2H,4H)-diones
1. Introduction
a preferred first line agent as part of the highly active antiretroviral
therapy (HAART) treatment guidelines since 2009 [5]. Indeed it has
Over the last two decades, intense efforts have been devoted to
the development of HIV-1 integrase (IN) inhibitors and the pyr-
imidone scaffold was intensively investigated [1], leading to the
release of Raltegravir (Fig. 1), the first HIV-1 IN inhibitor approved
by the Food and Drug Administration (FDA) in 2007 [2], which
opened up a new class of antiretrovirals agents [3,4]. It has become
a favorable long-term efficacy and safety profile in integrase-in-
hibitor-naïve patients with triple-class resistant HIV in whom an-
tiretroviral therapy is failing. However, after several years of clinical
use, resistance to this integrase strand transfer inhibitor (INSTI)
involving mutations at IN amino acids Y143, Q148, and N155 has
quickly emerged [6e9]. Elvitegravir, the second FDA-approved
INSTI, needs to be boosted by the pharmacoenhancer termed
cobicistat and is prescribed as a potent once-daily single tablet also
including two potent nucleos(t)ide reverse transcriptase inhibitors
(NRTIs), emtricitabine and tenofovir [10e12]. Although this novel
anti-HIV tablet could offer new perspective for patients failing
existing antiviral regimes, cross resistance with raltegravir rules out
any treatment option for patients failing on raltegravir therapy
[13,14]. Dolutegravir, is a second generation integrase inhibitor that
provides distinct advantages compared with first generation inte-
grase inhibitors. Unlike raltegravir, dolutegravir in coformulation
with abacavir and lamivudine can be given once daily for patients
Abbreviations: AZT, azidothymidine; CPE, cytopathic effect; CYP, cytochrome
P450; FC, fold-change in EC50 relative to wild-type strain of HIV-1; FDA, food and
drug administration; LEDGF, lens epithelium-derived growth factor; HAART, highly
active antiretroviral therapy; HID, 2-hydroxyisoquinoline-1,3(2H,4H)-dione; HIV-1,
human immunodeficiency virus type 1; IN, integrase; INSTI, integrase strand
transfer inhibitor; 30-P, 30-processing; PFV, prototype foamy virus; RT, reverse
transcriptase; ST, strand transfer; TI, therapeutic index; TOA, time of addition.
* Corresponding author. Centre de Recherches Jean-Pierre Aubert UMR-S1172,
ꢀ
Faculte des Sciences Pharmaceutiques et Biologiques, 3 rue du Professeur Laguesse,
BP83, F-59006 Lille, France.
E-mail address: fabrice.bailly@univ-lille1.fr (F. Bailly).
http://dx.doi.org/10.1016/j.ejmech.2016.03.083
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
257
hydrophobic aminoacid residues), we investigated whether the N-
benzylated chain at position 4 of the HID scaffold could be replaced
by alkyl chains that would occupy this cavity through pure van der
Waals interactions.
2. Results and discussion
2.1. Chemistry
The target 2-hydroxyisoquinoline-1,3(2H,4H)-diones were syn-
thesized according to our previously reported method [28]. The key
ester precursor 1, methyl 2-(benzyloxy)-1,3-dioxo-1,2,3,4-
tetrahydroisoquinoline-4-carboxylate was converted to carbox-
amides 2e16 by addition-elimination of various amines. Finally, the
O-benzyl protecting group was removed by action of boron tri-
chloride or hydrogenation at room temperature over 5% Pd/C
(Scheme 1). A series of primary carboxamides with increasing
linear alkyl chains (3e9 carbons, 17, 19, 23e27) were synthesized
(Table 1), together with representative secondary amides (com-
pounds 20, 21 and 31). The influence of the ramification (com-
pound 18, R4 ¼ isopropyl; compound 22, R4 ¼ tertbutyl) of the alkyl
chain and of the bulkiness (compound 28e30, R4 ¼ cyclopropyl,
cyclopentyl, cyclohexyl) of the cycloalkyl chain was also briefly
investigated.
Fig. 1. Structures of the INSTI raltegravir, the hit compound 32, the lead compound 33
and of herein studied N-alkyl carboxamides pointing out the key components of the
HIV-1 IN inhibitory pharmacophore: the magnesium chelating moiety (red) and the
hydrophobic halogenobenzyl or alkyl group (green). (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this
article.)
who are antiretroviral treatment naïve without the requirement of
a pharmacokinetic booster which minimizes the drugedrug inter-
action potential of dolutegravir [15e17]. Moreover, dolutegravir
showed a more robust resistance profile than raltegravir and elvi-
tegravir although some viruses containing E138K, G140S, or R148H
mutations had lower susceptibility and may also diminish the
likehood of long-term clinical success [18,19]. All these INSTIs were
shown to inhibit HIV-1 IN by chelating the two magnesium ions of
the catalytic core through a triad of oxygen atoms [20,21]. Thus, in
the current clinical context, integrase inhibitors should have a high
genetic barrier to existing INSTIs to be novel serious candidates. For
this purpose, alternative targets like the cellular cofactor lens
epithelium-derived growth factor (LEDGF/p75) are promising tar-
gets [22,23] or continuous modulation of valuable scaffolds like
naphthyridine is still relevant [24].
2.2. Integrase and ribonuclease H inhibitory properties
Table 1 shows the biological properties of this series of N-alkyl-
2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides. As far as the
compounds (17, 19, 23e27) with increasing linear chains are con-
cerned, compounds 17 (R4 ¼ propyl), 26 (R4 ¼ octyl) and 27
(R4 ¼ nonyl) displayed weakened HIV-1 IN inhibition when
compared with our reference hit and lead compounds 32 and 33,
with IC50 values of 1.82, 9.05 and 6.65 mM, respectively. Compounds
19, 23, and 25 (R4 ¼ butyl, pentyl, and heptyl) inhibit HIV-1 IN in the
submicromolar IC50 range, with values from 0.65
mM to 0.86
mM.
Compound 24 bearing an hexyl side-chain (IC50 0.085
mM) is the
only representative in this series that displayed excellent integrase
inhibitory activity in the same nanomolar range as lead 33 (IC50
Following early pioneer studies on the 2-hydroxyisoquinoline-
1,3(2H,4H)-dione (HID) scaffold [25,26], we recently reported the
interesting biological properties of some derivatives substituted at
position 4 by carboxamide chains [27e29]. These compounds dis-
played strong IN inhibitory potencies comparable to that of the
clinically used raltegravir. One hit compound 32 (Fig. 1) potently
inhibited ST and 30 P IN catalytic activities while it kept activity
against a panel of raltegravir-resistant HIV-1 variants and did not
induce any resistance selection in cell culture [27]. A crystal
structure of this compound bound to the wild-type prototype
foamy virus (PFV) intasome revealed that the compact heterocyclic
scaffold displaying all three Mg2þ chelating oxygen atoms from a
single ring showed an overall binding mode similar to previous
INSTIs [27]. Further substitution of this hit compound at position 7
by electron-withdrawing groups and particularly the nitro function
led to the discovery of the lead compound 33 (Fig. 1) with low
nanomolar anti-HIV potency [29].
0.010 mM). Fig. 2 shows the variation of the integrase inhibitory
activity according to the length of the linear alkyl chain, clearly
evidencing an optimal length of six carbons since compound 24
(R4 ¼ hexyl, IC50 ¼ 0.085
mM) is 20- to 105-fold more active than
compounds 17 (R4 ¼ propyl, IC50 ¼ 1.82
m
M), 26 (R4 ¼ octyl,
IC50 ¼ 9.05
m
M) and 27 (R4 ¼ nonyl, IC50 ¼ 6.65
mM) whereas there
is only one-log difference with the close compounds 19 (R4 ¼ butyl,
IC50 ¼ 0.66
m
M), 23 (R4 ¼ pentyl, IC50 ¼ 0.65
mM) and 25
(R4 ¼ heptyl, IC50 ¼ 0.86
mM).
The ramification of the propyl chain did not have a great impact
on the integrase inhibition with close IC50 values of 1.82 and
4.43
mM for the linear and ramified counterparts 17 and 18,
Herein we elaborated and studied
a novel series of 2-
hydroxyisoquinoline-1,3(2H,4H)-diones, in which we replaced the
p-fluorobenzyl side chain by various alkyl chains. These synthetic
modulations, aimed at generating more hydrophobic molecules,
were performed in order to assess the influence of the nature of the
side chain on the enzymatic inhibitory and antiviral properties
within this scaffold. Owing to the nature of the hydrophobic pocket
occupied by this substituent at the catalytic site (not only due to
aromatic DNA bases pairs but also containing non-aromatic
Scheme 1. Synthesis of target compounds 17e31. Reagents and conditions: (i) 2.0 eq.
R4NH2, toluene, Dean Stark apparatus, reflux, 15 h (45e83%); (ii) BCl3, 5.0 or 6.0 eq,
CH2Cl2, 15 min, rt then H2O, 5 min, rt (49e88%) or H2, 5% Pd/C, EtOAc/MeOH, rt, 4 h
(52e90%).

258
M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
Table 1
Inhibition of HIV-1 in, antiviral activity, and cytotoxicity of substituted 2-hydroxyisoquinoline-1,3(2H,4H)-diones 17e34, and raltegravir.
0
00
Comp.
R7
R4
R4
IC50
(
m
M)
IC50
ST
(m
M)
IC50
(
m
M)
EC50
(
m
M)d
CC50
(m
M)e
TIf
Overall
RNase Hc
INa
INb
Raltegravir26
17 (Keto)g
18 (Keto)g
19 (Keto)g
20 (Keto)g
21 (Keto)g
22 (Keto)g
23 (Keto)g
24 (Keto)g
25 (Keto)g
26 (Keto)g
27 (Keto)g
28 (Keto)g
0.010
1.82
4.43
0.66
8.66
123
4.45
0.65
0.085
0.86
9.05
6.65
2.13
0.007
1.99
5.05
0.48
8.70
0.006
28.0
30.2
9.20
>63.8
>28
>8
131
162.5
128
63.8
28
142
>1333
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eCH3
eCH2CH3
eH
eH
eH
eH
eH
eH
eH
e(CH2)2CH3
eCH(CH3)2
e(CH2)3CH3
e(CH2)3CH3
eCH2CH3
4.7
5.4
14
15.1
206
eC(CH3)3
4.46
0.53
0.195
1.05
9.13
6.67
4.00
>142
10.7
e(CH2)4CH3
e(CH2)5CH3
e(CH2)6CH3
e(CH2)7CH3
e(CH2)8CH3
130
12.1
21.2
3.4
3.5
3.0
5.35
23.2
2.70
57.7
109.5
98.5
88.5
32.5
28.0
29.0
42.7
>250
>6
29 (Keto)g
30 (Keto)g
eH
eH
eH
eH
1.36
0.7
3.63
3.36
61.9
80.2
>250
>250
>4
>3
31 (Keto)g
eH
eH
8.12
9.10
>120
2.34
120
202
3228 (Keto)g
eH
eH
eH
0.056
0.099
0.36
86.3
3329 (Enol)g
3429 (Enol)g
eNO2
eNO2
0.010
0.060
0.015
0.010
0.11
4.21
121
1100
e(CH2)5CH3
>250
>59
a,b,c
Concentration required to inhibit by 50% the in vitro overall integrase, strand transfer integrase and RNase H activities, respectively.
Effective concentration required to reduce HIV-1 induced cytopathic effect by 50% in MT-4 cells.
Cytotoxic concentration required to reduce by 50% MT-4 cell viability.
Therapeutic index defined by CC50/EC50 ratio.
Major tautomeric form present in DMSO solution.
d
e
f
g
respectively. On the contrary, the homologous compound 19
cyclic analogues 28 (R4 ¼ cyclopropyl, IC50 ¼ 2.13
m
M) and 29
(R4 ¼ butyl, IC50 ¼ 0.66
m
M) did not retain its submicromolar in-
(R4 ¼ cyclopentyl, IC50 ¼ 1.36
mM). In contrast, the cyclic C6 com-
hibition upon ramification (compound 22, R4
¼
tert-butyl,
pound 30 (R4 ¼ cyclohexyl, IC50 ¼ 0.7
mM) was 8.2-fold less active
IC50 ¼ 4.45
m
M). Cyclization of the linear alkyl chain did not
than its acyclic counterpart 24 (R4 ¼ hexyl, IC50 ¼ 0.085
mM). The
strongly modify the antiintegrase activities of the C3 and C5 de-
extension of the cycle size was slightly favorable since the cyclic C6
rivatives since compounds 17 (R4 ¼ propyl, IC50 ¼ 1.82
m
M) and 23
compound 30 (R4 ¼ cyclohexyl, IC50 ¼ 0.7
mM) was 3.0-fold more
(R4 ¼ pentyl, IC50 ¼ 0.65
m
M) were as active as their respective
active than the cyclic C3 compound 28 (R4
¼
cyclopropyl,
IC50 ¼ 2.13
mM) but integrase inhibition in the nanomolar range was
not reached within this small cyclic series 28e30. Secondary car-
boxamides 20, 21 and 31 were revealed as modest integrase in-
hibitors with IC50 values in the large micromolar range. The
negative effect of a second alkyl group on the nitrogen atom of the
carboxamide function is well pinpointed by comparison of the
properties of the primary carboxamide 19 (IC50 ¼ 0.66
mM) and its
methylated secondary derivative 20 (IC50 ¼ 8.66 M). This evolu-
m
tion is corroborated by the study of our previous series of carbox-
amides [27,28]. We observed that replacing the amide proton by a
methyl group provoked a strong decrease of the HIV-1 IN inhibi-
tion, which stressed the importance of an intramolecular hydrogen
bond between the exocyclic amide hydrogen and the oxygen at
position 3. Preliminary docking studies [28,29] subsequently fol-
lowed by crystal structure of 32 in complex with the PFV IN inta-
some (PDB: 4IKF) [27] were in perfect accordance to support the
hypothesis that this intramolecular hydrogen bond allows
Fig. 2. Effect of the length of the linear alkyl side chain at position 4 on HIV-1 IN
inhibitory activity of HIDs 17, 19, 23e27.

M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
259
convenient orientation of the side chain towards the desired hy-
drophobic pocket near the IN catalytic site. As a whole, the inte-
grase inhibitory potencies of these alkyl carboxamides were weak
with IC50 values in the large micromolar or submicromolar ranges.
Replacing the phenyl and benzyl carboxamides at position 4 of the
scaffold by alkyl carboxamides gave modest to good HIV-1 IN in-
hibitors. We previously obtained a reproducible low nanomolar
antiintegrase potency within the phenyl and benzyl carboxamides
series [27-29]. Herein, 24 reaches this level with an IC50 value
selectivity. This behavior was previously reported for similar iso-
quinoline-4-carboxamides [28]. We also tested some compounds
against HIV-1 Reverse Transciptase (RT) associated RNase H activity
(Table 1) since this scaffold was originally developed for the design
of dual inhibitors of HIV-1 IN and RT associated RNase H domain
[25]. A micromolar IC50 value (5.35
pound 24 whereas compounds 19 and 25 were less inhibitory with
IC50 values of 15.1 and 23.2 M, respectively. In this series of
mM) was obtained for com-
m
alkylcarboxamides, the RNase H inhibition did not reach the sub-
micromolar level obtained previously for some benzylcarbox-
amides [28].
(IC50 ¼ 0.085
(IC50 0.056
(IC50 ¼ 0.010 M). Crystal structure of 32 in complex with the PFV
mM) close to those of our hit compound, 32
¼
mM) and the clinically used INSTI, raltegravir
m
IN intasome (PDB:4IKF) showed that the extending p-fluorobenzyl
group makes extensive van der Waals interactions with conserved
residues P214 and Q215 (HIV equivalents P145, Q146), the invariant
CA dinucleotide, and the guanosine 4 base from the nontransferred
strand and stacks its aromatic ring against the 30 penultimate
cytosine base [27]. The absence of an exocyclic aromatic ring
2.3. Docking studies
The tendency observed for integrase inhibition was confirmed
by in silico molecular docking studies on this series of HIDs bearing
a linear alkyl side-chain 17, 19, 23e27 (Fig. 3 and see Supporting
Information), applying our previously reported protocol [29].
In our previous work on compound 32 and other benzylated
HIDs, we reported an optimized docking protocol based on the
PDB:3S3M structure of the prototype foamy virus (PFV) co-
crystallized with dolutegravir. We then confirmed the binding
mode obtained with this procedure for 32 by experimental X-ray
co-crystal structure in the PFV intasome [27e29]. These results
evidenced that the dual chelation of the two magnesium co-factors
within the catalytic site allows the N-benzylamido side chain to
occupy a known hydrophobic pocket and interact with a cytosine
making pep stacking had to be compensed by optimized Van der
Waals interactions. This was achieved with 24: its optimal linear
hexyl chain seems to interact as efficiently as 32 with HIV-1 cata-
lytic core. Compounds' effects on HIV-1 ST activity was also
examined (Table 1). Close values of the IN overall and ST inhibitions
were observed for all the series, which may infer ST selectivity as it
is well-known for the pioneer INSTI, raltegravir. However, the
calculated ratio IC50(30-P)/IC50(ST) was measured at 6.0 for the hit
compound 24 (IC50 (30-P) ¼ 0.51
mM), revealing a moderate ST
Fig. 3. Proposed binding mode of compound 24 as obtained by molecular docking in the PFV IN catalytic site (PDB:3S3M).Viral DNA is depicted in pink, PFV IN in blue, magnesium
cations in green and the ligand in gold. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

260
M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
via
nature of
p
-stacking, in a similar way to dolutegravir. Although the real
-stacking is still debated in the literature nowadays [30],
p
the main energetic contribution to these interactions are Van Der
Waals dispersion and electrostatics. Owing to the fact that the
hydrophobic nature of the pocket created at the interface between
integrase and the nucleic acid chain at the catalytic site is not only
due to DNA bases but also to non-aromatic hydrophobic residues of
the protein, it seemed natural to investigate whether or not
replacing the benzylated side chain at position 4 by alkyl sub-
stituents could be beneficial.
As expected, the outcome of molecular docking for all com-
pounds except for 17 was similar to that of our recently published
4-benzylcarboxamido derivatives. No satisfying docking pose could
be obtained for propyl derivative 17: the short 3-carbon side-chain
is not long enough to occupy the hydrophobic pocket formed at the
protein-DNA interface, natively occupied by the 3’-terminal base of
viral DNA. Therefore compound 17 can efficiently chelate the two
magnesium cofactors within the catalytic site, but there is no
additional stabilizing interaction coming from the side-chain,
which confirms the essential nature of this hydrophobic substitu-
ent. All the other compounds exhibited a clear docking pose similar
to that obtained for our p-fluorobenzylated hit compounds [28,29]:
the binding mode previously identified for 32 and 33 is retained
with regard to chelation of both metallic cofactors by the phar-
macophore and to the intramolecular H-bond within the ligand,
stabilizing the side-chain in an ideal position for occupation of the
Fig. 4. Time-of-addition experiments. After infection of MT-4 cells with HIV-1 IIIB,
inhibitors (50- and 100-fold EC50) were added at indicated time points spanning from
1 to 25 h postinfection. Virus replication was determined by p24 antigen determina-
tion in the supernatant at 30 h postinfection. The comparative profiles of RT inhibitor,
AZT, HDIs 24 and 32, INSTI raltegravir (Ral) and protease inhibitor ritonavir (Rit) are
presented.
25 h post-infection for RT inhibitor AZT, INSTI raltegravir and
protease inhibitor ritonavir, respectively (Fig. 4). Compound 24
matched the profile observed for the INSTI raltegravir and our hit
compound 32, attesting for its cellular inhibition of HIV-1 inte-
grase (Fig. 4).
hydrophobic pocket. Interestingly, the lack of
p-stacking between
Table 1 shows that HIV-1 IN inhibitions and antiviral activities
can be reasonably correlated. Our new compounds were moder-
ately active in vitro with EC50 values ranging from 9.2 to 51.8 mM
and four compounds (20e22, 31) displayed limiting cellular cyto-
toxicity. Interestingly, compound 24 with the best antiintegrase
activity of the series displayed also the lowest EC50 value of
the inhibitor and the penultimate cytosine of the DNA seems well
compensated by hydrophobic interactions, the alkyl side-chain
being ideally placed in the cavity, as observed in the different en-
ergetic terms of the obtained scores for the CHEMPLP function of
the Gold docking suite, most notably the attractive hydro-
phobicehydrophobic term (data not shown). This correlates with
the fact that these alkyl derivatives maintain quite a good inhibitory
activity on HIV-1 IN when compared to previously reported series
that do not bear carboxamido groups in position 4 [26]. Because
molecular docking is not quantitative and this docking protocol
does not allow for flexibility of the DNA structure, it would seem
hazardous to go further than a qualitative assessment here. How-
ever, this cavity being quite short in length, it appears that it cannot
accommodate longer linear chains in their most stable conforma-
tion, thus requiring less favorable eclipsed conformations of the
sp3-hybridized carbon atoms: the longer the hydrocarbon chain,
the higher the internal torsion of the ligand. Additionally, in the
identified poses, the termini of octyl and nonyl chains in com-
pounds 26 and 27 is directly exposed to the aqueous medium and
does not fit into the cavity. The fact that compound 24 exhibits the
best activity in this series can be directly correlated to its optimal
occupancy of the hydrophobic pocket (when compared to propyl
17, butyl 19 and pentyl 23) while minimizing conformational strain
and water exposure of the side-chain (when compared to heptyl 25,
octyl 26 and nonyl 27) (Fig. 2; see Supporting Information, super-
imposition of the best ligand docking poses).
2.70
mM, roughly equal to that of hit compound 32
(EC50 ¼ 2.34
mM). An explanation for the lack of significant anti-
viral activities could be an unfavorable tautomeric equilibrium
within this series of HIDs, whose major tautomeric form present in
DMSO solution is the keto one (Table 1; see 1H and 13C NMR
spectra of compound 23 in Supporting Information). In contrast,
we previously evidenced a good relationship between the enoli-
zation ability of HIDs and low submicromolar antiviral activities
[29]. We found that the substitution by electron-withdrawing
functions like cyano and, particularly nitro moieties led to the
displacement of the keto-enol equilibrium towards enol com-
pounds, with higher lipophilicities and improved anti-HIV activ-
ities [29]. Furthermore, careful comparison of the antiviral
activities of the HIDs 24, 32, 33 and 34 (Table 1) evidences the
cumulative positive effect of a p-fluorobenzyl group at position 4
and of a nitro function at position 7. Compound 34 bearing a hexyl
chain at position 4 and a nitro function at position 7 was therefore
synthesized. Even though its HIV-1 IN inhibitory activity was
slightly improved when compared to its counterpart 24
(IC50 ¼ 0.060
mM vs. 0.085 mM, respectively), the improvement
upon introduction of this nitro substituent did not translate in
terms of in vivo HIV-1 replication (EC50 ¼ 4.21
mM) as it did for p-
2.4. Antiviral properties
To unambiguously attribute the antiviral activities of our HID
compounds to a specific cell inhibition of HIV-1 IN, time-of-
addition (TOA) experiments were first performed before doing
the evaluation in a cell-based antiviral assay against HIV-1.
Compound 24 was tested together with a set of known HIV
replication inhibitors. The compounds were added at different
time points after infection of MT-4 cells with HIV-1 IIIB, and p24
antigen production in the supernatants was measured at 30 h
postinfection. The loss of activity was delayed to 4e5 h, 10e11 h,
Table 2
Cross resistance of HIDs 24, 32, dolutegravir (Dol.) with INSTI raltegravir (Ral.). Fold-
change (FC) in EC50 relative to wild-type strain of HIV-1.
Resistance
Ral.
Virus strain
Ral.27
Dol.
24
3227
Q148H
N155H
G140S/Q148H
9.0
5.0
486
n.d.a
n.d.a
5.0
1.0
1.0
1.0
1.0
1.0
1.0
a
Not determined.

M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
261
fluorobenzylated derivatives 32 (EC50
¼
2.34
m
M) and 33
CYP2C19 and CYP2D6). The largest inhibition (42.0%) was
observed with CYP3A4, but it did not reach the marked unfavor-
able effect of 33 (100%). In addition, an extended study on the
toxicity of this compound was performed (Table 4). The effects on
cell number, intracellular free calcium, nuclear size, membrane
permeability, and mitochondrial potential were measured. Com-
pound 24 like 32 and 33 did not display any severe toxicity in
(EC50 ¼ 0.11 M). Therefore, with this low nanomolar potency,
m
derivative 33 remains the lead compound in this series.
Regarding the therapeutic index, the hit compound 24
(TI ¼ 21.2; Table 1) was the best HID of the series. This encouraging
result has to be improved to reach the level of compound 32
(TI ¼ 86.3).
As mentioned before, the current primary objective for the
development of novel marketable antiintegrase drugs is to discover
compounds with minimal toxicity that retain good effectiveness
against known resistant mutants [8,9,24]. This led us to test our hit
compound 24 against the relevant Y143R, N155H mutants and also
the double mutant, Q148H/G140S. Table 2 shows the fold changes
in the EC50 values of compounds 24, 32 and dolutegravir tested
against raltegravir-resistant strains. Like the previous hit 32, com-
pound 24 retained completely its activity against N155H, Q148H
mutants and the G140S/Q148H double mutant. For this double
mutant, the HID compounds are even better than dolutegravir,
these tests, even at a concentration of 100 mM. In contrast, the
reference compound, cerivastatin, displayed submicromolar IC50
values for each test. Finally compound 24 was tested in a hERG
(human Ether-a-go-go-Related Gene potassium channel 1;
Table 5) inhibition assay. Inhibition of this voltage gated potassium
ion channel, a transmembrane protein encoded by the hERG gene,
is known to be undesirable due to the possibility of QT prolon-
gation, which can lead to fatal cardiac arrhythmia. Compound 24
like 32 and 33 did not significantly inhibited the tail current with
5.8, 12.2 and 19.4% inhibitions at the concentrations of 0.1
mM,
1.0 M and 10 M, respectively. In the same conditions, the control
m
m
which displayed a 5-fold change in EC50
.
compound Eꢁ4031 induced hERG-mediated cardiac toxicity with
an IC50 value of 24 nM.
2.5. Preliminary ADMETox evaluation of 24
3. Conclusion
As reported for our previous lead compound 33, we performed
a preliminary ADMETox evaluation of 24 [29]. This hit compound
displayed a good aqueous solubility of 160.3 mM (200.0 mM for 33)
and a partition coefficient, logD of 1.26 (1.24 for 33). As for the
compounds 32 and 33 [28,29], compound 24 presents a high
human plasma protein binding (mean of 99.9% protein bound;
mean of 93.2% recovery). However, regarding A-B permeability
(Caco-2 cells, pH 6.5/7.4), a mean permeability coefficient Papp of
Herein, we investigated the antiintegrase and antiviral proper-
ties of a novel series of HIDs substituted at position 4 by various
alkylcarboxamides. With the objective to replace the recurrent
halobenzyl pharmacophore by a simple alkul group, we found one
hit compound 24 displaying integrase inhibition in the low nano-
molar range. Its EC50 value on HIV-1 replication, is equivalent to
that of compound 32. This study evidenced an optimal length of six
carbons for the alkyl side chain that can be correlated with the
optimal fitting within the hydrophobic pocket created by
displacement of the 3‘-adenosine (A17). The importance of a pri-
mary carboxamido side function to allow for an intramolecular
hydrogen bond between the amide proton and the carbonyl oxygen
at position 3 was confirmed. On the whole, the preliminary
ADMETox assessment of compound 24 evidenced a pharmacoki-
netic profile similar to that of our previous hit 32. High plasma
protein binding is still certainly responsible for the lack of strong
nanomolar antiviral activity. But, without any severe toxicity,
compound 24 is the first HID displaying a largely positive mean
permeability coefficient Papp of 24.6 ꢀ 10ꢁ6 cm/s with satisfactory
features for uptake after oral administration. Moreover, in contrast
to dolutegravir, compound 24 retained completely its antiviral ac-
tivity against the relevant mutants Q148H, N155H and the double
mutant G140S/Q148H. To our knowledge, these HID compounds
are the unique HIV-1 IN inhibitors able to display such properties
against resistant IN mutants. The advantageous permeability co-
efficient of compound 24 and its high barrier to resistance will
24.6 ꢀ 10ꢁ6 cm/s was determined at a 10
mM concentration while
negligible values below 0.1 and 0.2 ꢀ 10ꢁ6 cm/s were obtained for
32 [28] and 33 [29], respectively. This represents a large advantage
of 24 over 32 and 33. This hit compound has satisfactory features
for uptake after oral administration since a desired Papp value for a
candidate drug should be > 15.0 ꢀ 10ꢁ6 cm/s. P-gp is an important
transporter protein found in cells throughout the body, such as
those lining the intestine and bloodebrain barrier. It is believed to
play an important role in defining the extent of distribution of
drug molecules and limiting their oral and brain exposures. Up to
a concentration of 100.0
mM, compound 24 like 32 and 33,
inhibited the P-gp efflux at a minor extent (Table 3) while the
reference compound, verapamil, reduced by 50% this efflux at a
concentration of 8.0
mM. The cytochrome P450 (CYP) is a large
superfamily of enzymes involved in drug metabolism via oxidation
of organic substances. Changes in the activity or expression levels
of CYPs are the major source of adverse effects of drugs. As shown
in Table 3, 24 like 32 at a concentration of 10
mM only faintly
inhibited up to 25.0% the different CYPs tested (CYP1A2, CYP2C9,
Table 3
Inhibition of different members of the cytochrome P450 (CYP450) mixed-function oxidase system and of P-gp efflux by HIDs 24, 32, 33 and reference compounds.
Comp.
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
P-gp inhibition (MDR1-MDCKII)
24
12.7a
5.2a
1.4a
1.4f
Nd
Nd
Nd
Nd
25.3a
6.4a
76.5a
Nd
9.3a
18.0a
11.4a
Nd
24.6
6.6
42.0ꢂ
35.9ꢂ
106ꢂ
Nd
Nd
Nd
ꢁ5.5c
ꢁ5.4c
ꢁ5.1c
2.0d
5.3e
3227
ꢁ4.0d
ꢁ3.7e
3329
Ndb
Nd
ꢁ3.2d
7.6e
Furafyline
Sulphaphenazole
Tranylcypromine
Quinidine
Ketoconazole
0.18f
Nd
Nd
Nd
3.1f
Nd
Nd
Nd
Nd
0.017f
Nd
Nd
Nd
0.28f
c,d,e
P-gp inhibition at 1.0
IC50 values in M.
Percentage inhibition of control values at a 10.0
Not determined.
m
M, 30.0
m
M and 100.0
m
M.
f
m
a
mM dose.
b

262
M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
Table 4
Detailed analysis of in vitro toxicity of HIDs 24, 32, 33 and cerivastatin.
Assay
Comp.
1.0
m
M
30.0
m
M
100.0
m
M
IC50 (m
M)f
Cytotoxicity (cell number)a
24
ꢁ10.0
ꢁ22.8
ꢁ20.1
Ndg
ꢁ1.5
ꢁ1.7
ꢁ1.3
Nd
ꢁ7.0
ꢁ1.0
2.0
Nd
0.1
4.9
25.3
16.0
ꢁ7.4
Nd
32.3
29.9
18.3
Nd
26.8
18.7
22.1
Nd
23.6
41.2
6.7
Nd
Nd
Nd
0.96
Nd
Nd
Nd
0.34
Nd
Nd
Nd
0.13
Nd
Nd
Nd
0.80
Nd
Nd
Nd
0.60
3227
25.7
12.6
Nd
5.8
9.6
18.8
Nd
12.1
13.1
23.8
Nd
3329
Cerivastatin
24
Intracellular free calciumb
Nuclear sizec
3227
3329
Cerivastatin
24
3227
3329
Cerivastatin
24
Membrane permeabilityd
Mitochondrial membrane potentiale
7.0
3227
ꢁ0.5
20.3
23.9
Nd
3329
0
Cerivastatin
24
Nd
5.7
11.8
8.4
Nd
Nd
33.9
6.3
22.4
Nd
5.1
3227
ꢁ21.2
ꢁ23.8
Nd
3329
Cerivastatin
a
Percentage reduction relative to untreated control.
Percentage increase relative to untreated control.
Percentage reduction relative to untreated control.
Percentage increase relative to untreated control.
Percentage reduction relative to untreated control.
Concentration required to inhibit aee.
Not determined.
b
c
d
e
f
g
Table 5
uncorrected. Mass spectra (ElectroSpray Ionization, ESI) were
recorded on a Micromass Quattro II spectrometer. HRMS mea-
surements were made on an Apex Qe 9.4 T Bruker Daltonics
spectrometer. Analytes dissolved in methanol (3 mM solutions)
were diluted with a water/methanol/formic acid solution (50/50/
Analysis of cardiac toxicity of HIDs 24, 32, 33 and the reference compound E-4031
(hERG automated patch-clamp).
Comp.
0.1
m
M
1.0
m
M
10.0
m
M
IC50 (mM)
24
5.8a
3.4
12.2
10.6
5.1
19.4
13.3
3.9
Nd
Nd
Nd
3227
3329
E-4031
0.1, % v/v) to afford 3
mM solutions and infused into the mass
2.6
spectrometer nano ESI source in positive mode at a rate of 1
mL/min.
Ndb
Nd
Nd
0.024
The purity of the tested compounds was established by combustion
analysis, confirming a purity ꢃ95%. Elemental analyses (C, H, N)
were performed by CNRS laboratories (Vernaison, France); the
analytical results were within 0.4% of the theoretical values.
a
Percentage inhibition of tail current.
Not determined.
b
encourage us to further pharmacomodulate this scaffold and reach
antiviral activities in the nanomolar range. In order to overcome
high plasma protein binding and improve the biological properties
within this series, alkyl side chains containing heteroatoms may be
inserted at position 4 of compound 24 or on its unsubstituted ar-
omatic benzenic ring.
4.1.2. Synthesis of the carboxamides 2e16
4 . 1. 2 . 1. N - P r o p y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (2). Ester
1
(0.32 g,
1.0 mmol) and propylamine (0.12 g, 2.0 mmol) were dissolved in
toluene (100 mL). The mixture was refluxed for 15 h using a Dean
Stark apparatus. After cooling, the solution was concentrated in
vacuo. The residue was dissolved in EtOAc. The organic layer was
washed with 2.0 M HCl and dried over Na2SO4. After concentration
in vacuo, the residue was triturated in ether and the precipitate was
filtered and dried at room temperature giving 2 as a white solid
(80%); mp 166 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
4. Experimental section
4.1. Chemistry
3
3
d
¼ 0.86 (t, 3 H, CH3, J ¼ 7.3 Hz), 1.45 (sext, 2 H, CH2, J ¼ 7.1 Hz),
4.1.1. General details
3.06 (q, 2 H, NHeCH2, 3J ¼ 6.3 Hz), 4.98 (d, 1 H, OCH2, 2J ¼ 9.8 Hz),
All reagents and solvents were purchased from Aldrich-Chimie
(Saint-Quentin-Fallavier, France) of ACS reagent grade and were
used as provided. Thin layer chromatography analyses were per-
formed on plastic sheets precoated with silica gel 60F254 (Merck).
5.03 (d, 1 H, OCH2, 2J ¼ 9.8 Hz), 5.12 (s, 1 H, CH), 7.38e7.45 (m, 4 H,
H
Ar), 8.10 (d, 1 H, HAr, 3J ¼ 7.8 Hz), 8.80 (t, 1 H, NH, 3J ¼ 5.4 Hz); 13
C
NMR (75 MHz, DMSO-d6):
d
¼ 11.3 (CH3), 22.2 (CH2), 40.9 (CH2),
55.8 (CH), 77.4 (OCH2), 125.3 (CIV), 126.6 (CH), 128.1 (CH), 128.3
(CH), 128.4 (2 CH), 128.9 (CH), 129.4 (2 CH), 134.2 (CH), 134.5 (CIV),
133.9 (CIV), 161.1 (CO), 164.6 (CO), 167.0 (CO); ESI-MS: m/z ¼ 353
(M þ H)þ.
SiO2, 40e63 mesh (Merck) or 30 mm HP-Silprepacked SNAP col-
umns (Biotage) were used for flash column chromatography. NMR
spectra were obtained on an AC 300 Bruker spectrometer in the
appropriate solvent with TMS as internal reference. Chemical shifts
are reported in
d units (ppm) and are assigned as singlets (s),
doublets (d), doublets of doublets (dd), triplets (t), quartets (q),
quintets (quin), sextuplets (sext), multiplets (m), and broad signals
(br) … Melting points were obtained on a Reichert Thermopan
melting point apparatus, equipped with a microscope and are
4 .1. 2 . 2 . N - I s o p r o p yl - 2 - b e n z yl o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (3). White solid (74%); mp
158 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.07 (d,
3 H, CH3, 3J ¼ 6.4 Hz), 1.14 (d, 3 H, CH3, 3J ¼ 6.4 Hz), 3.76 (sext, 1 H,

M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
263
CH, 3J ¼ 6.6 Hz), 4.99 (d, 1 H, OCH2, 2J ¼ 9.6 Hz), 5.04 (d, 1 H, OCH2,
2J ¼ 9.6 Hz), 5.08 (s, 1 H, CH), 7.42e7.45 (m, 4 H, HAr), 7.57e7.60 (m,
3 H, HAr), 7.74 (t, 1 H, HAr, 3J ¼ 7.2 Hz), 8.10 (d, 1 H, H8, 3J ¼ 7.7 Hz),
8.74 (dapp, 1 H, NH, 3J ¼ 7.4 Hz); 13C NMR (75 MHz, DMSO-d6):
3J ¼ 7.0 Hz), 4.79 (s, 1 H, CH), 5.11 (d, 1 H, OCH2, 2J ¼ 9.0 Hz), 5.15 (d,
1 H, OCH2, 2J ¼ 9.0 Hz), 6.50 (s, 1 H, NH), 7.34e7.66 (m, 8 H, HAr),
8.24 (d, 1 H, H8, 3J ¼ 7.3 Hz); 13C NMR (75 MHz, CDCl3):
d
¼ 13.9
(CH3), 22.2 (CH2), 28.9 (2 CH2), 40.5 (CH2), 55.7 (CH), 78.5 (OCH2),
125.3 (CIV), 127.8 (CH), 128.5 (2 CH), 128.6 (CH), 128.9 (CH), 129.1
(CH), 129.9 (2 CH), 133.1 (CIV), 133.8 (CIV), 134.0 (CH), 161.1 (CO),
164.9 (CO), 165.0 (CO); ESI-MS: m/z ¼ 381 (M þ H)þ.
d
¼ 22.0 (2 CH3), 41.3 (CH), 55.7 (CH), 77.3 (OCH2), 125.2 (CH), 128.0
(CH), 128.3 (3 CH), 128.8 (CH), 129.4 (2 CH), 134.1 (CH), 134.4 (CIV),
134.9 (CIV), 161.1 (CO), 164.5 (CO), 165.0 (CO); ESI-MS: m/z ¼ 353
(M þ H)þ.
4 . 1. 2 . 8 . N - H e x y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
4 . 1. 2 . 3 . N - B u t y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (4). White solid (83%); mp
tetrahydroisoquinoline-4-carboxamide (9). White solid (83%); mp
139e140 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.88 (t,
166 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.85 (t, 3 H,
3 H, CH3, 3J ¼ 7.0 Hz), 1.29 (m, 6 H, CH2), 1.51 (quin, 2 H, CH2,
3J ¼ 7.0 Hz), 3.25 (t, 2 H, CH2, 3J ¼ 7.0 Hz), 4.80 (s, 1 H, CH), 5.12 (d,
1 H, OCH2, 2J ¼ 9.2 Hz), 5.16 (d, 1 H, OCH2, 2J ¼ 9.2 Hz), 6.51 (t, 1 H,
NH, 3J ¼ 6.0 Hz), 7.28 (dd, 1 H, H5, 3J ¼ 8.0 Hz, 4J ¼ 1.5 Hz), 7.30e7.40
(m, 3 H, HAr), 7.51e7.68 (m, 4 H, HAr), 8.23 (dd, 1 H, H8, 3J ¼ 8.0 Hz,
CH3, 3J ¼ 7.0 Hz), 1.12e1.40 (m, 4 H, 2 CH2), 3.28 (q, 2 H, NHeCH2,
3J ¼ 6.9 Hz), 4.80 (s, 1 H, CH), 5.14 (s, 2 H, OCH2), 6.47 (t, 1 H, NH,
3J ¼ 7.0 Hz), 7.40 (d,1 H, HAr, 3J ¼ 7.7 Hz), 7.52 (t,1 H, HAr, 3J ¼ 7.6 Hz),
7.69 (t, 1 H, HAr, 3J ¼ 7.4 Hz), 8.07 (d, 1 H, H8, 3J ¼ 6.8 Hz); 13C NMR
(75 MHz, CDCl3):
d
¼ 13.7 (CH3), 20.0 (CH2), 31.3 (CH2), 40.3 (CH2),
4J ¼ 1.5 Hz); 13C NMR (75 MHz, CDCl3):
d
¼ 14.0 (CH3), 22.5 (CH2),
55.7 (CH), 78.5 (OCH2), 125.3 (CIV), 128.1 (CH), 128.5 (2 CH), 128.7
(CH), 129.0 (CH), 129.2 (CH), 130.0 (2 CH), 133.0 (CIV), 133.8 (CIV),
134.0 (CH), 161.1 (CO), 164.6 (CO), 165.1 (CO); ESI-MS: m/z ¼ 367
(M þ H)þ.
26.4 (CH2), 29.2 (CH2), 31.4 (CH2), 40.6 (CH2), 55.5 (CH), 78.6
(OCH2), 125.2 (CIV), 128.5 (2 CH), 128.6 (CH), 128.8 (CH), 129.0 (CH),
129.2 (CH), 130.0 (2 CH), 132.7 (CIV), 133.7 (CIV), 133.9 (CH), 160.5
(CO), 164.0 (CO), 165.1 (CO); ESI-MS: m/z ¼ 395 (M þ H)þ.
4.1.2.4. N-Butyl-N-methyl-2-benzyloxy-1,3-dioxo-1,2,3,4-
4 . 1. 2 . 9 . N - H e p t y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (5). White solid (69%); mp
tetrahydroisoquinoline-4-carboxamide (10). White solid (48%); mp
117e120 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.83 (t,
131e134 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.80 (t,
3 H, CH3, 3J ¼ 8.4 Hz), 1.20e1.59 (m, 4 H, 2 CH2), 2.88 (s, 3 H,
NHeCH3), 3.07 (s, 3 H, NHeCH3), 3.30 (t, 2 H, NHeCH2, 3J ¼ 6.7 Hz),
3.56 (t, 2 H, NHeCH2, 3J ¼ 6.7 Hz), 4.99 (s, 1 H, CH), 5.23 (s, 1 H, CH),
7.06 (m, 1 H, HAr), 7.23e7.25 (m, 3 H, HAr), 7.36e7.47 (m, 4 H, HAr),
3 H, CH3, 3J ¼ 7.0 Hz), 1.19e1.21 (m, 8 H, 4 CH2), 1.43e1.45 (m, 2 H,
CH2), 3.14 (q, 2 H, NHeCH2, 3J ¼ 7.1 Hz), 4.71 (s, 1 H, CH), 5.05 (m,
2 H, OCH2), 6.40 (m,1 H, NH), 7.19 (d, 1 H, HAr, 3J ¼ 7.2 Hz), 7.26e7.30
(m, 3 H, HAr), 7.44e7.56 (m, 4 H, HAr), 8.16 (d, 1 H, H8, 3J ¼ 8.3 Hz);
8.10 (d, 1 H, H8, 3J ¼ 7.2 Hz); 13C NMR (75 MHz, CDCl3):
d
¼ 13.8
13C NMR (75 MHz, CDCl3):
d
¼ 14.1 (CH3), 22.6 (CH2), 26.8 (CH2),
(CH3), 19.9 (CH2), 20.0 (CH2), 29.0 (CH2), 30.9 (CH2), 34.6 (NHCH3),
36.5 (NHCH3), 48.6 (NHCH2), 51.0 (NHCH2), 51.7 (CH), 52.5 (CH),
78.4 (OCH2), 125.9 (CIV), 126.1 (CIV), 126.4 (CH), 128.4 (2 CH), 128.6
(CH), 129.0 (CH), 129.3 (CH), 129.9 (2 CH), 133.9 (CIV), 134.2 (CH),
161.1 (CO), 164.2 (CO), 165.9 (CO); ESI-MS: m/z ¼ 381 (M þ H)þ.
28.9 (CH2), 29.3 (CH2), 31.7 (CH2), 40.5 (CH2), 55.7 (CH), 78.5
(OCH2), 125.3 (CIV), 127.8 (CH), 128.5 (3 CH), 129.0 (CH), 129.2 (CH),
129.9 (2 CH),133.2 (CIV), 133.8 (CIV), 134.0 (CH), 161.2 (CO), 165.0
(CO), 165.1 (CO); ESI-MS: m/z ¼ 409 (M þ H)þ.
4 . 1. 2 . 1 0 . N - O c t y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
4 .1. 2 . 5 . N , N - D i e t h yl - 2 - b e n z yl o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (6). White solid (67%); mp
tetrahydroisoquinoline-4-carboxamide (11). White solid (49%); mp
137e139 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.80 (t,
114 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.11 (t,
3 H, CH3, 3J ¼ 7.3 Hz), 1.18e1.22 (m, 10 H, 5 CH2), 1.43e1.45 (m, 2 H,
CH2), 3.18 (q, 2 H, NHeCH2, 3J ¼ 7.5 Hz), 4.71 (s, 1 H, CH), 5.07 (m,
2 H, OCH2), 6.37 (m,1 H, NH), 7.20 (d,1 H, HAr, 3J ¼ 8.0 Hz), 7.31e7.33
(m, 3 H, HAr), 7.44e7.59 (m, 4 H, HAr), 8.16 (d, 1 H, H8, 3J ¼ 7.7 Hz);
3 H, CH3, 3J ¼ 6.7 Hz), 1.29 (t, 3 H, CH3, 3J ¼ 6.5 Hz), 3.24e3.43 (m,
2 H, NHeCH2), 3.61e3.76 (m, 2 H, NHeCH2), 5.00 (s, 2 H, OCH2),
5.80 (s, 1 H, CH), 6.50 (s, 1 H, NH), 7.28 (d, 1 H, HAr
,
3J ¼ 7.7 Hz),
7.30e7.40 (m, 3 H, HAr), 7.56e7.61 (m, 3 H, HAr), 7.74 (t, 1 H, HAr
,
13C NMR (75 MHz, CDCl3):
d
¼ 14.1 (CH3), 22.6 (CH2), 26.8 (CH2),
3J ¼ 7.2 Hz), 8.11 (d, 1 H, H8, 3J ¼ 7.6 Hz); 13C NMR (75 MHz, DMSO-
29.2 (3 CH2), 31.8 (CH2), 40.5 (CH2), 55.7 (CH), 78.5 (OCH2), 125.3
(CIV), 127.8 (CH), 128.5 (3 CH), 128.9 (CH), 129.2 (CH), 129.9 (2
CH),133.2 (CIV), 133.8 (CIV), 134.0 (CH), 161.1 (CO), 165.0 (CO), 165.1
(CO); ESI-MS: m/z ¼ 423 (M þ H)þ.
d6):
d
¼ 12.7 (CH3), 14.7 (CH3), 40.5 (CH2), 42.8 (CH2), 51.1 (CH), 77.4
(OCH2), 125.5 (CIV), 126.6 (CH), 128.1 (CH), 128.2 (CH), 128.4 (2 CH),
128.9 (CH), 129.4 (2 CH), 134.3 (CH), 135.3 (2 CIV), 160.9 (CO), 164.5
(CO), 165.7 (CO); ESI-MS: m/z ¼ 367 (M þ H)þ.
4 . 1. 2 . 11. N - N o n y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
4 .1. 2 . 6 . N -Te r t - b u t yl - 2 - b e n z yl o x y- 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (7). Beige solid (70%); mp
tetrahydroisoquinoline-4-carboxamide (12). White solid (65%); mp
146 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.89 (t, 3 H,
143 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.31 (s,
CH3, 3J ¼ 7.0 Hz), 1.28e1.60 (m, 14 H, 7 CH2), 3.28 (q, 2 H, NHeCH2,
3J ¼ 6.0 Hz), 4.78 (s, 1 H, CH), 5.14 (m, 2 H, OCH2), 6.43 (m, 1 H, NH),
7.30e7.68 (m, 8 H, HAr), 8.25 (d, 1 H, H8, 3J ¼ 7.6 Hz); 13C NMR
9 H, 3 CH3), 5.04 (d, 1 H, OCH2, 2J ¼ 9.3 Hz), 5.08 (d, 1 H, OCH2,
2J ¼ 9.3 Hz), 5.19 (s, 1 H, CH), 7.48 (m, 3 H, HAr), 7.51 (d, 1 H, HAr
,
3J ¼ 7.7 Hz), 7.60e7.65 (m, 3 H, HAr), 7.80 (t,1 H, HAr, 3J ¼ 7.7 Hz), 8.15
(75 MHz, CDCl3):
d
¼ 14.1 (CH3), 22.6 (CH2), 26.8 (2 CH2), 29.2 (3
(d, 1 H, H8, 3J ¼ 7.7 Hz), 8.55 (s, 1 H, NH); 13C NMR (75 MHz, DMSO-
CH2), 29.5 (CH2), 31.8 (CH2), 40.5 (CH2), 55.7 (CH), 78.5 (OCH2),
125.3 (CIV), 127.9 (CH), 128.5 (2 CH), 128.6 (CH), 128.9 (CH), 129.1
(CH), 129.9 (2 CH), 133.1 (CIV), 133.8 (CIV), 134.0 (CH), 161.1 (CO),
164.8 (CO), 165.0 (CO); ESI-MS: m/z ¼ 437 (M þ H)þ.
d6):
d
¼ 28.1 (3 CH3), 50.9 (CIV), 56.2 (CH), 77.3 (OCH2), 125.2 (CIV),
126.4 (CH), 128.0 (CH), 128.3 (2 CH), 128.8 (CH), 129.3 (2 CH), 134.0
(CH), 134.5 (CIV), 135.1 (CIV), 161.1 (CO), 164.6 (CO), 165.2 (CO); ESI-
MS: m/z ¼ 367 (M þ H)þ.
4.1.2.12. N-Cyclopropyl-2-benzyloxy-1,3-dioxo-1,2,3,4-
tetrahydroisoquinoline-4-carboxamide (13). White solid (71%); mp
183 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
4 . 1. 2 . 7 . N - P e n t y l - 2 - b e n z y l o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (8). White solid (45%); mp
134 ꢂC; 100% keto form; 1H NMR (300 MHz, CDCl3):
d
¼ 0.90 (t, 3 H,
d
¼ 0.37e0.52 (m, 2 H, 2 CH), 0.62e0.75 (m, 2 H, 2 CH), 2.63e2.72
CH3, 3J ¼ 6.4 Hz), 1.32e1.65 (m, 6 H, 3 CH2), 3.25 (q, 2 H, NHeCH2,
(m, 1 H, CH), 5.00 (d, 1 H, OCH2, 2J ¼ 10.0 Hz), 5.04 (d, 1 H, OCH2,

264
M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
2J ¼ 10.0 Hz), 5.00 (s, 1 H, CH), 7.34e7.50 (m, 4 H, HAr), 7.51e7.65 (m,
3 H, HAr), 7.74 (t, 1 H, HAr, 3J ¼ 7.4 Hz), 8.10 (d, 1 H, H8, 3J ¼ 7.4 Hz),
5.39, N, 10.66.
8.90 (d, 1 H, NH, 3J ¼ 3.1 Hz); 13C NMR (75 MHz, DMSO-d6):
d
¼ 6.6
4 .1. 3 . 2 . N - I s o p r o p y l - 2 - h y d r o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (18). Intermediate 3 (0.35 g,
1.0 mmol) was dissolved in a minimum of CH2Cl2 and boron tri-
chloride (1.0 M solution in CH2Cl2, 6.0 mL, 6.0 mmol) was added
dropwise at room temperature. The solution was stirred for 15 min
and water (20 mL) was slowly added. After 5 min stirring, the
precipitate was filtered and the aqueous layer was extracted with
ethyle acetate. Organic layers were dried over Na2SO4 and
concentrated in vacuo. Organic residues and precipitate were
gathered and triturated with ether. Insoluble materials were
filtered and dried at room temperature giving compound 16 as a
beige solid (56%); mp 190 ꢂC; 100% keto form; 1H NMR (300 MHz,
(2 CH2), 23.7 (CH2), 56.4 (CH), 55.6 (CH), 78.2 (OCH2), 120.0 (CIV),
126.1 (CIV), 127.4 (CH), 128.9 (CH), 129.1 (2 CH), 129.2 (2 CH), 129.7
(CH), 130.2 (2 CH), 135.0 (CH), 135.5 (CIV), 161.9 (CO), 165.2 (CO),
167.8 (CO); ESI-MS: m/z ¼ 351 (M þ H)þ.
4.1.2.13. N-Cyclopentyl-2-benzyloxy-1,3-dioxo-1,2,3,4-
tetrahydroisoquinoline-4-carboxamide (14). White solid (77%); mp
188 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.40e1.90 (m, 8 H, 4 CH2), 3.95 (sext, 1 H, CH, 3J ¼ 6.3 Hz), 4.99
(d, 1 H, OCH2, 2J ¼ 9.6 Hz), 5.04 (d, 1 H, OCH2, 2J ¼ 9.6 Hz), 5.10 (s,
1 H, CH), 7.40e7.43 (m, 4 H, HAr), 7.55e7.59 (m, 3 H, HAr), 7.74 (t,1 H,
HAr
,
3J ¼ 7.6 Hz), 8.09 (d, 1 H, H8, 3J ¼ 7.9 Hz), 8.79 (dapp, 1 H, NH,
DMSO-d6):
d
¼ 1.12 (d, 3 H, CH3, 3J ¼ 6.5 Hz), 1.19 (d, 3 H, CH3,
3J ¼ 7.3 Hz); 13C NMR (75 MHz, DMSO-d6):
d
¼ 23.4 (CH2), 23.5
3J ¼ 6.5 Hz), 3.80 (sext, 1 H, CH, 3J ¼ 6.5 Hz), 5.07 (s, 1 H, CH), 7.45 (d,
(CH2), 32.1 (CH2), 32.2 (CH2), 50.9 (CH), 55.6 (CH), 77.3 (OCH2),
125.2 (CIV), 126.4 (CH), 128.0 (CH), 128.2 (CH), 128.3 (2 CH), 128.8
(CH), 129.3 (2 CH), 134.1 (CH), 134.4 (CIV), 134.9 (CIV), 161.0 (CO),
164.5 (CO), 165.3 (CO); ESI-MS: m/z ¼ 379 (M þ H)þ.
1 H, HAr, 3J ¼ 7.7 Hz), 7.60 (t, 1 H, HAr, 3J ¼ 7.7 Hz), 7.77 (t, 1 H, HAr
,
3J ¼ 7.3 Hz), 8.12 (d, 1 H, H8, 3J ¼ 7.5 Hz), 8.75 (dapp, 1 H, NH,
3J ¼ 7.3 Hz); 13C NMR (75 MHz, DMSO-d6):
d
¼ 22.1 (2 CH3), 41.3
(CH), 55.3 (CH), 125.4 (CIV), 126.4 (CH), 127.9 (CH), 128.2 (CH), 133.8
(CH), 134.7 (CIV), 161.6 (CO), 164.8 (CO), 165.1 (CO); ESI-MS: m/
z ¼ 263 (M þ H)þ; HRMS calcd for C13H14N2O4 262.09536; found:
262.09605; Anal. Calc. For C13H14N2O4: C, 59.54, H, 5.38, N, 10.68;
Found: C, 59.45, H, 5.37, N, 10.67.
4.1.2.14. N-Cyclohexyl-2-benzyloxy-1,3-dioxo-1,2,3,4-
tetrahydroisoquinoline-4-carboxamide (15). White solid (85%); mp
192 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.15e1.35 (m, 6 H, 3 CH2), 1.47e1.86 (m, 4 H, 2 CH2), 3.46 (m,1 H,
CH), 4.98 (d, 1 H, OCH2, 2J ¼ 9.6 Hz), 5.03 (d, 1 H, OCH2, 2J ¼ 9.6 Hz),
4.1.3.3. N-Butyl-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-
4-carboxamide (19). Hydrogenation over Pd/C 5%. White solid
(53%); mp 180 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
5.11 (s, 1 H, CH), 7.39e7.48 (m, 4 H, HAr), 7.52e7.62 (m, 3 H, HAr),
7.73 (td, 1 H, HAr
,
3J ¼ 7.6 Hz, 4J ¼ 1.3 Hz), 8.09 (dd, 1 H, H8,
3J ¼ 7.8 Hz, 4J ¼ 1.0 Hz), 8.69 (dapp, 1 H, NH, 3J ¼ 7.7 Hz); 13C NMR
d
¼ 0.92 (t, 3 H, CH3, 3J ¼ 7.0 Hz), 1.15e1.36 (m, 4 H, 2 CH2), 3.25 (q,
(75 MHz, DMSO-d6):
d
¼ 24.2 (CH2), 25.1 (2 CH2), 32.0 (2 CH2), 48.2
2 H, NHeCH2, 3J ¼ 7.0 Hz), 5.00 (s, 1 H, CH), 7.40 (d, 1 H, HAr
,
(CH), 55.6 (CH), 77.7 (OCH2), 125.3 (CIV), 126.4 (CH), 128.0 (CH),
128.2 (CH), 128.4 (2 CH), 128.8 (CH), 129.4 (2 CH), 134.1 (CH), 134.5
(CIV), 134.9 (CIV), 161.0 (CO), 164.5 (CO), 165.0 (CO); ESI-MS: m/
z ¼ 393 (M þ H)þ.
3J ¼ 7.7 Hz), 7.69 (t, 1 H, HAr, 3J ¼ 7.4 Hz), 7.69 (t, 1 H, HAr, 3J ¼ 7.4 Hz),
8.15 (d,1 H, H8, 3J ¼ 8.1 Hz), 8.72 (dapp,1 H, NH, 3J ¼ 7.0 Hz),10.45 (s,
1 H, OH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 14.0 (CH3), 19.8 (CH2),
31.3 (CH2), 39.2 (CH2), 55.8 (CH), 125.8 (CIV), 127.0 (CH), 128.3 (CH),
128.6 (CH),135.1 (CIV),162.0 (CO),165.2 (CO),166.4 (CO); ESI-MS: m/
z ¼ 277 (M þ H)þ; HRMS calcd for C14H16N2O4 276.11101; found:
276.11213; Anal. Calc. For C14H16N2O4: C, 60.86, H, 5.84, N, 10.14;
Found: C, 60.98, H, 5.83, N, 10.11.
4.1.2.15. (2-Benzyloxy-1,3-dioxoisoquinolin-4-yl)(piperidin-1-yl)
methanone (16). White solid (67%); mp 132 ꢂC; 100% keto form; 1H
NMR (300 MHz, DMSO-d6):
d
¼ 1.48e1.62 (m, 6 H, 3 CH2), 3.35 (t,
2 H, NeCH2, 3J ¼ 5.1 Hz), 3.66 (t, 2 H, NeCH2, 3J ¼ 5.1 Hz), 5.00 (s,
2 H, OCH2), 5.93 (s, 1 H, CH), 7.30 (d, 1 H, HAr, 3J ¼ 7.2 Hz), 7.42e7.56
4.1.3.4. N-Butyl-N-methyl-2-hydroxy-1,3-dioxo-1,2,3,4-
tetrahydroisoquinoline-4-carboxamide (20). Hydrogenation over
Pd/C 5%. Brown solid (62%); mp 120e124 ꢂC; 100% keto form; 1H
(m, 6 H, HAr), 7.74 (t, 1 H, HAr
,
3J ¼ 7.2 Hz), 8.12 (d, 1 H, H8,
3J ¼ 7.7 Hz); 13C NMR (75 MHz, DMSO-d6):
d
¼ 23.8 (CH3), 25.3
(CH2), 26.1 (CH2), 43.2 (CH2), 47.5 (CH2), 51.0 (CH), 77.5 (OCH2),
125.4 (CIV), 126.9 (CH), 128.1 (CH), 128.3 (CH), 128.4 (2 CH), 128.9
(CH), 129.4 (2 CH), 134.2 (CH), 134.3 (CIV), 135.1 (CIV), 160.9 (CO),
164.4 (CO), 164.6 (CO); ESI-MS: m/z ¼ 379 (M þ H)þ.
NMR (300 MHz, DMSO-d6):
d
¼ 0.92 (t, 3 H, CH3, 3J ¼ 8.4 Hz),
1.27e1.70 (m, 4 H, 2 CH2), 2.95 (s, 3 H, NHeCH3), 3.34 (m, 2 H,
NHeCH2), 5.90 (s, 1 H, CH), 7.32 (d, 1 H, HAr, 3J ¼ 7.5 Hz), 7.61 (t, 1 H,
HAr
,
3J ¼ 7.5 Hz), 7.76 (d, 1 H, HAr
,
3J ¼ 7.5 Hz), 8.12 (d, 1 H, H8,
3J ¼ 7.2 Hz); 13C NMR (75 MHz, CDCl3):
d
¼ 14.2 (CH3), 19.8 (CH2),
4.1.3. Deprotection of the precursors 2e16
4 . 1 . 3 . 1 . N - P r o p y l - 2 - h y d r o x y - 1 , 3 - d i o x o - 1 , 2 , 3 , 4 -
29.0 (CH2), 31.0 (CH2), 34.2 (NHCH3), 36.7 (NHCH3), 47.7 (NHCH2),
50.7 (NHCH2), 51.0 (CH), 51.5 (CH), 126.0 (CIV), 127.3 (CH), 128.3
(CH), 128.6 (CH), 134.3 (CH), 135.6 (CIV), 161.9 (CO), 165.2 (CO), 167.3
(CO); ESI-MS: m/z ¼ 291 (M þ H)þ; HRMS calcd for C15H18N2O4
290.12666; found: 290.12567; Anal. Calc. For C15H18N2O4: C, 62.06,
H, 6.25, N, 9.65; Found: C, 62.21, H, 6.23, N, 9.66.
tetrahydroisoquinoline-4-carboxamide
(17). Intermediate
2
(0.265 g, 0.74 mmol) was dissolved in a mixture of ethyle acetate
(10 mL) and methanol (5 mL) and hydrogenated for 4 h at room
temperature over Pd/C 5% (20 mg). The catalyst was filtered and the
solvent was removed in vacuo. Trituration of the residue in ether
afforded the deprotected compound 15 as a white solid (90%); mp
4 .1. 3 . 5 . N , N - D i e t h y l - 2 - h y d r o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (21). Hydrogenation over
Pd/C 5%. White solid (52%); mp 178 ꢂC; 100% keto form; 1H NMR
180 ꢂC; 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 0.92 (t,
3 H, CH3, 3J ¼ 7.1 Hz), 1.51 (sext, 2 H, NHeCH2eCH2, 3J ¼ 7.0 Hz), 3.11
(dapp, 2 H, NHeCH2, 3J ¼ 5.6 Hz), 5.13 (s, 1 H, CH), 7.46 (d, 1 H, HAr
,
(300 MHz, DMSO-d6):
d
¼ 1.21 (t, 3 H, CH3, 3J ¼ 6.4 Hz), 1.28 (t, 3 H,
3J ¼ 7.7 Hz), 7.60 (t, 1 H, HAr, 3J ¼ 7.2 Hz), 7.76 (t, 1 H, HAr, 3J ¼ 7.2 Hz),
CH3, 3J ¼ 6.6 Hz), 3.21e3.41 (m, 2 H, NHeCH2), 3.60e3.73 (m, 2 H,
NHeCH2), 5.77 (s, 1 H, CH), 7.22 (d, 1 H, HAr, 3J ¼ 7.7 Hz), 7.55 (t, 1 H,
8.11 (d, 1 H, H8, 3J ¼ 7.7 Hz), 8.82 (m, 1 H, NH), 10.80 (s, 1 H, OH); 13
C
NMR (75 MHz, DMSO-d6):
d
¼ 11.3 (CH3), 22.1 (CH2), 40.9 (CH2),
HAr
,
3J ¼ 7.6 Hz), 7.70 (t, 1 H, HAr
,
3J ¼ 7.5 Hz), 8.08 (d, 1 H, H8,
55.4 (CH), 125.4 (CIV), 126.6 (CH), 127.9 (CH), 133.8 (CH), 134.7 (CIV),
161.6 (CO), 164.8 (CO), 166.1 (CO); ESI-MS: m/z ¼ 263 (M þ H)þ;
HRMS calcd for C13H14N2O4 262.09536; found: 262.09355; Anal.
Calc. For C13H14N2O4: C, 59.54, H, 5.38, N, 10.68; Found: C, 59.67, H,
3J ¼ 7.7 Hz), 10.50 (s, 1 H, OH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 12.7 (CH3), 14.8 (CH3), 40.4 (CH2), 42.8 (CH2), 50.6 (CH), 125.5
(CIV), 126.5 (CH), 127.9 (CH), 128.0 (CH), 133.9 (CH), 135.3 (CIV), 161.5
(CO), 164.8 (CO), 166.0 (CO); ESI-MS: m/z ¼ 277 (M þ H)þ; HRMS

M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
265
calcd for C14H16N2O4 276.11101; found: 276.11170; Anal. Calc. For
14H16N2O4: C, 60.86, H, 5.84, N, 10.14; Found: C, 60.69, H, 5.86, N,
Found: C, 63.99, H, 6.95, N, 8.82.
C
10.11.
4 . 1 . 3 . 1 0 . N - O c t y l - 2 - h y d r o x y - 1 , 3 - d i o x o - 1 , 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (26). Hydrogenation over
4 .1. 3 . 6 . N -Te r t - b u t y l - 2 - h y d r o x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (22). Treatment with boron
trichloride. White solid (49%); mp 174 ꢂC; 100% keto form; 1H NMR
Pd/C 5%. Beige solid (67%); mp 92e96 ꢂC; 100% keto form; 1H NMR
3
(300 MHz, DMSO-d6):
d
¼ 0.92 (t, 3 H, CH3, J ¼ 7.3 Hz), 1.29e1.31
(m, 10 H, 5 CH2), 1.47e1.49 (m, 2 H, CH2), 5.11 (s, 1 H, CH), 7.44 (d,
(300 MHz, DMSO-d6):
d
¼ 1.25 (s, 9 H, 3 CH3), 5.09 (s, 1 H, CH), 7.42
1 H, HAr, 3J ¼ 7.3 Hz), 7.60 (t, 1 H, HAr, 3J ¼ 7.0 Hz), 7.75 (t, 1 H, HAr
,
(d, 1 H, HAr, 3J ¼ 7.3 Hz), 7.53 (t, 1 H, HAr, 3J ¼ 7.7 Hz), 7.70 (t, 1 H, HAr
,
3J ¼ 7.0 Hz), 8.11 (d, 1 H, H8, 3J ¼ 7.3 Hz), 8.80 (s, 1 H, NH), 10.63 (s,
3J ¼ 7.7 Hz), 8.05 (d, 1 H, H8, 3J ¼ 7.7 Hz), 8.47 (s, 1 H, NH); 13C NMR
1 H, OH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 13.9 (CH3), 22.0 (CH2),
(75 MHz, DMSO-d6):
d
¼ 28.1 (3 CH3), 50.9 (CIV), 55.7 (CH), 125.3
26.2 (CH2), 28.6 (2 CH2), 28.7 (CH2), 31.1 (CH2), 39.0 (CH2), 55.3
(CH), 125.3 (CIV), 126.5 (CH), 127.8 (CH), 128.1 (CH), 133.6 (CH), 134.6
(CIV), 161.5 (CO), 164.7 (CO), 165.9 (CO); ESI-MS: m/z ¼ 333
(CIV), 126.3 (CH), 127.8 (CH), 128.0 (CH), 133.7 (CH), 134.9 (CIV), 161.6
(CO), 164.9 (CO), 165.3 (CO); ESI-MS: m/z ¼ 277 (M þ H)þ; HRMS
calcd for C14H16N2O4 276.11101; found: 276.10935; Anal. Calc. For
(M
þ
H)þ; HRMS calcd for C18H24N2O4 332.17361; found:
C
14H16N2O4: C, 60.86, H, 5.84, N, 10.14; Found: C, 61.05, H, 5.86, N,
332.17889; Anal. Calc. For C18H24N2O4: C, 65.04, H, 7.28, N, 8.43;
Found: C, 65.14, H, 7.27, N, 8.42.
10.15.
4 . 1 . 3 . 7 . N - P e n t y l - 2 - h y d r o x y - 1 , 3 - d i o x o - 1 , 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (23). Hydrogenation over
Pd/C 5%. White solid (52%); mp 130 ꢂC (dec); 100% keto form; 1H
4 . 1. 3 . 11. N - N o n y l - 2 - h y d r o x y - 1, 3 - d i o x o - 1 , 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (27). Hydrogenation over
Pd/C 5%. White solid (54%); mp 164 ꢂC; 100% keto form; 1H NMR
3
NMR (300 MHz, DMSO-d6):
d
¼ 0.80 (t, 3 H, CH3, 3J ¼ 6.6 Hz),
(300 MHz, DMSO-d6):
d
¼ 0.70 (t, 3 H, CH3, J ¼ 7.0 Hz), 1.10e1.40
1.21e1.22 (m, 4 H, 2 CH2), 1.37e1.39 (m, 2 H, CH2), 3.00 (m, 2 H,
NHeCH2), 4.99 (s, 1 H, CH), 7.33 (d, 1 H, HAr, 3J ¼ 7.0 Hz), 7.48 (t, 1 H,
(m, 14 H, 7 CH2), 3.05 (m, 2 H, NHeCH2), 5.00 (s, 1 H, CH), 7.35 (d,
1 H, HAr, 3J ¼ 7.0 Hz), 7.42 (t, 1 H, HAr, 3J ¼ 7.0 Hz), 7.59 (t, 1 H, HAr
,
HAr
,
3J ¼ 7.0 Hz), 7.63 (t, 1 H, HAr
,
3J ¼ 7.0 Hz), 8.00 (d, 1 H, H8,
3J ¼ 7.0 Hz), 8.04 (d, 1 H, H8, 3J ¼ 7.5 Hz), 8.72 (m, 1 H, NH), 10.56 (s,
3J ¼ 7.5 Hz), 8.68 (m, 1 H, NH), 10.51 (s, 1 H, OH); 13C NMR (75 MHz,
1 H, OH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 13.9 (CH3), 22.0 (CH2),
DMSO-d6):
d
¼ 14.3 (CH3), 22.2 (CH2), 28.9 (2 CH2), 39.5 (CH2), 55.8
26.2 (CH2), 28.6 (CH2), 28.7 (CH2), 28.9 (CH2), 29.5 (CH2), 31.2 (CH2),
39.0 (CH2), 55.3 (CH), 125.3 (CIV), 126.5 (CH), 127.8 (CH), 128.1 (CH),
133.6 (CH),134.6 (CIV),161.5 (CO),164.7 (CO),165.9 (CO); ESI-MS: m/
z ¼ 347 (M þ H)þ; HRMS calcd for C19H26N2O4 346.18926; found:
346.18787; Anal. Calc. For C19H26N2O4: C, 65.88, H, 7.57, N, 8.09;
Found: C, 65.80, H, 7.55, N, 8.10.
(CH), 125.2 (CIV), 127.0 (CH), 128.3 (CH), 128.6 (CH), 134.1 (CH), 135.3
(CIV), 162.0 (CO), 165.2 (CO), 166.4 (CO); ESI-MS: m/z ¼ 291
(M
þ
H)þ; HRMS calcd for C15H18N2O4 290.12666; found:
290.12559; Anal. Calc. For C15H18N2O4: C, 62.06, H, 6.25, N, 9.65;
Found: C, 62.15, H, 6.23, N, 9.68.
4 . 1 . 3 . 8 . N - H e x y l - 2 - h y d r o x y - 1 , 3 - d i o x o - 1 , 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (24). Treatment with boron
trichloride. Light brown solid (79%); mp 137e138 ꢂC; 90% keto
form; 10% enol form; Keto form; 1H NMR (300 MHz, DMSO-d6):
4.1. 3.12. N-Cyclopropyl-2-hydroxy-1, 3-dioxo-1, 2, 3, 4-
tetrahydroisoquinoline-4-carboxamide (28). Treatment with boron
trichloride. Brown solid (68%); mp 185e190 ꢂC; 100% keto form; 1H
NMR (300 MHz, DMSO-d6):
d
¼ 0.38e0.52 (m, 2 H, 2 CH), 0.61e0.74
d
¼ 0.89 (t, 3 H, CH3, 3J ¼ 7.0 Hz), 1.24 (m, 6 H, CH2), 1.40 (quin, 2 H,
(m, 2 H, 2 CH), 2.64 (m, 1 H, CH), 4.96 (s, 1 H, CH), 7.35 (d, 1 H, HAr,
CH2, 3J ¼ 7.0 Hz), 3.05 (m, 2 H, CH2), 5.05 (s, 1 H, CH), 7.38 (dd, 1 H,
3J ¼ 7.4 Hz), 7.55 (t, 1 H, HAr, 3J ¼ 7.3 Hz), 7.70 (t, 1 H, HAr, 3J ¼ 7.3 Hz),
H5, 3J ¼ 8.0 Hz, 4J ¼ 1.5 Hz), 7.54 (td, 1 H, HAr, 3J ¼ 8.0 Hz, 4J ¼ 1.5 Hz),
8.10 (d, 1 H, H8, J ¼ 7.6 Hz), 8.87 (m, 1 H, NH); 13C NMR (75 MHz,
3
7.69 (td, 1 H, HAr
,
3J ¼ 8.0 Hz, 4J ¼ 1.5 Hz), 8.05 (dd, 1 H, H8,
DMSO-d6):
d
¼ 6.3 (CH2), 22.9 (CH), 55.1 (CH), 125.5 (CIV), 126.5
3J ¼ 8.0 Hz, 4J ¼ 1.5 Hz), 8.75 (t, 1 H, NH, 3J ¼ 6.5 Hz); 13C NMR
(CH), 127.9 (CH), 128.2 (CH), 133.8 (CH), 161.6 (CO), 165.0 (CO), 167.1
(CO); ESI-MS: m/z ¼ 261 (M þ H)þ; HRMS calcd for C13H12N2O4
260.07971; found: 260.07992; Anal. Calc. For C13H12N2O4: C, 60.00,
H, 4.65, N, 10.76; Found: C, 59.91, H, 4.60, N, 10.74.
(75 MHz, DMSO-d6):
(CH2), 30.8 (CH2), 40.1 (CH2), 55.3 (CH), 125.3 (CIV), 126.4 (CH), 127.8
(CH), 128.1 (CH), 133.7 (CH), 134.6 (CIV), 161.5 (CO), 164.7 (CO), 165.9
d
¼ 13.9 (CH3), 22.0 (CH2), 25.8 (CH2), 28.7
1
(CO); Enol form; only several peaks could be attributed on the H
NMR spectrum; 1H NMR (300 MHz, DMSO-d6):
d
¼ 0.73 (t, 3 H, CH3,
4.1. 3.13. N-Cyclopentyl-2-hydroxy-1, 3-dioxo-1, 2, 3, 4-
tetrahydroisoquinoline-4-carboxamide (29). Hydrogenation over
Pd/C 5%. Brown solid (61%); mp 160 ꢂC; 100% keto form; 1H NMR
3J ¼ 7.0 Hz), 3.24 (t, 2 H, CH2, 3J ¼ 7.0 Hz), 7.32 (td, 1 H, HAr
,
3J ¼ 7.5 Hz, 4J ¼ 1.2 Hz), 8.15 (dd,1 H, H8, 3J ¼ 8.0 Hz, 4J ¼ 1.2 Hz); ESI-
MS: m/z ¼ 305 (M þ H)þ; HRMS calcd for C16H20N2O4 304.14231;
found: 304.14043; Anal. Calc. For C16H20N2O4: C, 63.14, H, 6.62, N,
9.20; Found: C, 63.25, H, 6.61, N, 9.17.
(300 MHz, DMSO-d6):
d
¼ 1.40e1.80 (m, 8 H, 4 CH2), 3.97 (m, 1 H,
CH), 5.09 (s, 1 H, CH), 7.42 (d, 1 H, HAr, 3J ¼ 7.0 Hz), 7.57 (t, 1 H, HAr
,
3J ¼ 7.2 Hz), 7.72 (t, 1 H, HAr, 3J ¼ 7.3 Hz), 8.10 (d, 1 H, H8, 3J ¼ 7.6 Hz),
8.81 (dapp,1 H, NH, 3J ¼ 6.1 Hz),10.63 (s,1 H, OH); 13C NMR (75 MHz,
4 . 1 . 3 . 9 . N - H e p t y l - 2 - h y d r o x y - 1, 3 - d i o x o - 1 , 2 , 3 , 4 -
DMSO-d6):
d
¼ 23.41 (CH2), 23.45 (CH2), 32.1 (CH2), 32.2 (CH2), 50.9
tetrahydroisoquinoline-4-carboxamide (25). Hydrogenation over
(CH), 55.1 (CH), 125.3 (CIV), 126.3 (CH), 127.8 (CH), 128.1 (CH), 133.7
(CH), 134.7 (CIV), 161.5 (CO), 164.7 (CO), 165.4 (CO); ESI-MS: m/
z ¼ 289 (M þ H)þ; HRMS calcd for C15H16N2O4 288.11101; found:
288.11012; Anal. Calc. For C15H16N2O4: C, 62.49, H, 5.59, N, 9.72;
Found: C, 62.56, H, 5.58, N, 9.70.
Pd/C 5%. Grey solid (74%); mp 120e130 ꢂC; 100% keto form; 1H NMR
3
(300 MHz, DMSO-d6):
d
¼ 0.90 (t, 3 H, CH3, J ¼ 7.0 Hz), 1.23e1.25
(m, 8 H, 4 CH2), 1.40e1.42 (m, 2 H, CH2), 3.07 (q, 2 H, NHeCH2,
3J ¼ 7.1 Hz), 5.08 (s, 1 H, CH), 7.40 (d, 1 H, HAr, 3J ¼ 7.2 Hz)), 7.55 (t,
1 H, HAr, 3J ¼ 7.5 Hz), 7.70 (t, 1 H, HAr, J ¼ 7.5 Hz), 8.11 (d, 1 H, H8,
3
3J ¼ 7.5 Hz), 8.82 (s, 1 H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 13.9
4 .1. 3 .14 . N - C yc l o h e x yl - 2 - hy d ro x y - 1, 3 - d i o x o - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-4-carboxamide (30). Treatment with boron
trichloride. Grey solid (76%); mp 165e170 ꢂC; 100% keto form; 1H
(CH3), 22.0 (CH2), 26.1 (CH2), 28.3 (CH2), 28.8 (CH2), 31.2 (CH2), 38.7
(CH2), 55.3 (CH), 125.3 (CIV), 126.4 (CH), 127.8 (CH), 128.1 (CH), 133.6
(CH), 134.6 (CIV), 161.5 (CO), 164.7 (CO), 165.9 (CO); ESI-MS: m/
z ¼ 319 (M þ H)þ; HRMS calcd for C17H22N2O4 318.15796; found:
318.15884; Anal. Calc. For C17H22N2O4: C, 64.13, H, 6.97, N, 8.80;
NMR (300 MHz, DMSO-d6):
d
¼ 1.14e1.42 (m, 6 H, 3 CH2), 1.46e1.94
(m, 4 H, 2 CH2), 3.81 (m, 1 H, CH), 5.07 (s, 1 H, CH), 7.42 (d, 1 H, HAr
,
3J ¼ 7.6 Hz), 7.50 (t, 1 H, HAr, 3J ¼ 7.4 Hz), 7.68 (t, 1 H, HAr, 3J ¼ 7.0 Hz),

266
M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
8.06 (d, 1 H, H8, 3J ¼ 7.6 Hz), 8.67 (dapp, 1 H, NH, 3J ¼ 7.2 Hz), 10.57 (s,
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by
mitochondrial dehydrogenase of metabolically active cells to a blue
formazan derivative, which can be measured spectrophotometri-
cally. The 50% cell culture infective dose (CCID50) of the HIV-1 (IIIB)
strain was determined by titration of the virus stock using MT-
4 cells. The origin of HIV-1 strain IIIB has been described previously
[32]. For the drug susceptibility assays, MT-4 cells were infected
with 100e300 CCID50 of the virus stock in the presence of 5-fold
serial dilutions of the antiviral drugs. The concentration of
various compounds achieving 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.
1 H, OH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 24.3 (2 CH2), 25.3 (CH2),
32.5 (2 CH2), 47.1 (CH), 55.2 (CH), 120.4 (CIV), 122.1 (CH), 124.2 (CH),
126.0 (CH),130.8 (CH), 135.3 (CIV), 158.9 (CO),161.6 (CO),164.8 (CO);
ESI-MS: m/z ¼ 303 (M þ H)þ; HRMS calcd for C16H18N2O4
302.12666; found: 302.12654; Anal. Calc. For C16H18N2O4: C, 63.56,
H, 6.00, N, 9.27; Found: C, 63.60, H, 6.02, N, 9.25.
4.1.3.15. (2-Hydroxy-1,3-dioxoisoquinolin-4-yl)(piperidin-1-yl)meth-
anone (31). Hydrogenation over Pd/C 5%. Black solid (64%);
mp > 128 ꢂC (dec); 100% keto form; 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.49e1.64 (m, 6 H, 3 CH2), 3.35 (t, 2 H, NeCH2, 3J ¼ 5.1 Hz), 3.66
(t, 2 H, NeCH2, 3J ¼ 5.1 Hz), 5.97 (s, 1 H, CH), 7.30 (d, 1 H, HAr
,
3J ¼ 7.7 Hz), 7.61 (t, 1 H, HAr, 3J ¼ 7.7 Hz), 7.76 (t, 1 H, HAr, 3J ¼ 7.7 Hz),
4.2.4. Reverse transcriptase RNase H assay
8.12 (d, 1 H, H8, 3J ¼ 7.7 Hz), 10.8 (s, 1 H, OH); 13C NMR (75 MHz,
The substrate for RNase H activity was prepared as previously
described [33]. E. coli RNA polymerase used single-stranded calf
thymus DNA as a template to synthesize complementary 3H-
labeled RNA. For RNase H activity, recombinant HIV-1 RT (4.5 pmol)
[34] was incubated with the appropriate compound for 10 min at
DMSO-d6):
d
¼ 23.7 (CH3), 25.1 (CH2), 26.0 (CH2), 43.0 (CH2), 47.4
(CH2), 50.3 (CH), 125.3 (CIV), 126.7 (CH), 127.7 (CH), 127.9 (CH), 133.7
(CH), 135.0 (CIV), 161.3 (CO), 164.6 (CO), 164.7 (CO); ESI-MS: m/
z ¼ 289 (M þ H)þ; HRMS calcd for C15H16N2O4 288.11101; found:
288.10986; Anal. Calc. For C15H16N2O4: C, 62.49, H, 5.59, N, 9.72;
Found: C, 62.47, H, 5.60, N, 9.73.
37 ꢂC in 20
mL. The components of the incubation mixture were
added to reach a final concentration of 50 mM TriseHCl (pH 8.0),
10 mM dithiothreitol, 6 mM MgCl2, 80 mM KCl, and the labeled
4.2. Biological assays
nucleic acid duplex (20,000 cpm) in a final volume of 50 mL. After
incubation for 10 min at 37 ꢂC, the reaction was stopped by addition
of 1 mL of cold 10% TCA containing 0.1 M sodium pyrophosphate,
the acid-precipitable material was collected on nitrocellulose filters
and washed, the radioactivity was determined and the radioactivity
released from the hybrid was determined by subtraction from the
undigested hybrid control.
4.2.1. Integrase inhibition
To determine the susceptibility of the HIV-1 integrase enzyme to
different compounds, an enzyme-linked immunosorbent assay was
used. This assay uses an oligonucleotide substrate in which one
oligo (50-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC-30) is
labeled with biotin on the 30 end and in which the other oligo is
labeled with digoxigenin at the 50 end. For the overall integration
assay, the second 50-digoxigenin-labeled oligo is 50-GACCCTTT-
TAGTCAGTGTGGAAAATCTCTAGCAGT-30. For the strand transfer
assay, a pre-cleaved oligonucleotide substrate (the second oligo-
nucleotide lacks GT [underlined] at the 30 end) was used. The
integrase was diluted in 750 mM NaCl, 10 mM Tris (pH 7.6), 10%
4.2.5. ADMETox studies
Aqueous solubility (PBS, pH 7.4; Cerep catalogue reference
0435), partition coefficient (log D, n-octanol/PBS, pH 7.4; Cerep
catalogue reference 0417), human plasma protein binding (Cerep
catalogue reference 2194), A-B permeability coefficient (Papp, Caco-
2 cells, pH 6.5/7.4; Cerep catalogue reference 3318), inhibition of P-
gp efflux (MDR1-MDCKII, calcein AM substrate; Cerep catalogue
reference 1324), inhibition of CYP1A2 (recombinant, CEC substrate,
Cerep catalogue reference 0389), inhibition of CYP2C9 (recombi-
nant, MFC substrate, Cerep catalogue reference 0412), inhibition of
CYP2C19 (recombinant, CEC substrate, Cerep catalogue reference
0390), inhibition of CYP2D6 (recombinant, MFC substrate, Cerep
catalogue reference 1338), inhibition of CYP3A4 (recombinant, BFC
substrate, Cerep catalogue reference 0391), cardiac toxicity (hErg,
automated patch clamp, Cerep catalogue reference 2245), in vitro
cytotoxicity panel (cell number, intracellular free calcium, nuclear
size, membrane permeability, mitochondrial membrane potential,
http://www.cerep.fr/cerep/users/pages/downloads/Documents/
Marketing/Pharmacology%20&%20ADME/OTP/CytotoxicityPanel.
pdf) were determined in standard assays by Cerep, France (www.
cerep.fr).
glycerol, 1 mM
albumin. To perform the reaction, 4
sponds to a concentration of WT integrase of 1.6
annealed oligos (7 nM) were added in a final reaction volume of
40 L containing 10 mM MgCl2, 5 mM dithiothreitol, 20 mM HEPES
b
-mercaptoethanol, and 0.1 mg/mL bovine serum
L of diluted integrase (corre-
M) and 4 L of
m
m
m
m
(pH 7.5), 0.5% polyethylene glycol, and 15% DMSO. As such, the final
concentration of integrase in this assay was 160 nM. The reaction
was carried out for 1 h at 37 ꢂC. The reaction products were de-
natured with 30 mM NaOH and detected by ELISA on avidin-coated
plates.
4.2.2. Time of addition experiments
100,000 MT-4 cells per well in a 96-well microtiter plate were
infected with HIV-IIIB at a multiplicity of infection of 0.7. Com-
pounds were added at different time points after infection (1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 24 and 25h) as described previously [31]. Viral
p24 antigen production was determined 30 h postinfection by
ELISA (Innogenetics, Belgium). Compounds were added at 50 and
100 times their EC50 as determined by the drug susceptibility assay
(MTT/MT-4).
4.3. Docking procedure
We previously reported that the 2-hydroxyisoquinoline-1,3-
(2H,4H)-dione scaffold complexes magnesium as the enol or eno-
late form, as shown by NMR studies [25]. Predictions of the ioni-
zation state by use of the SPARC online calculator [35] indicate that
such enols are deprotonated in aqueous media at physiological pH
in the presence of magnesium cations; hence ligands were modeled
as the dianionic enolate form. Compounds 17, 19, 23e27 were thus
created and their geometry minimized at the HF/3-21G level by use
of the Gaussian 09 package [36]. The minimized ligands were then
submitted to our previously reported docking protocol using the
PDB 3S3M crystallographic structure, which was validated for this
4.2.3. In vitro anti-HIV and drug susceptibility assays
The inhibitory effect of antiviral drugs on the HIV-1-induced
cytopathic effect (CPE) in human lymphocyte MT-4 cell culture
was determined by the MT-4/MTT assay. MT-4 cells were obtained
through the AIDS Research and Reference Reagent Program, Divi-
sion of AIDs, NIH. The cells were grown in RPMI 1640 supplemented
with 10% FCS and 20
assay is based on the reduction of the yellow colored 3-(4,5-
mg/mL gentamicin (RPMI-complete). This

M. Billamboz et al. / European Journal of Medicinal Chemistry 117 (2016) 256e268
267
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