Rational Design, Synthesis and Evaluation of Coumarin Derivatives as Protein-protein Interaction Inhibitors

Article abstract of DOI:10.1002/minf.201501034

Herein we describe the design and synthesis of a new series of coumarin derivatives searching for novel HIV-1 integrase (IN) allosteric inhibitors. All new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 IN enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75. A combined approach of docking and molecular dynamic simulations has been applied to clarify the activity of the new compounds. Specifically, the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA) was calculated, whereas hydrogen bond occupancies were monitored throughout simulations methods.

Full text of DOI:10.1002/minf.201501034

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DOI: 10.1002/minf.201501034
Rational design, synthesis and evaluation of coumarin
derivatives as protein-protein interaction inhibitors
Laura De Luca,*[a] Fatima E. Agharbaoui,*[a] Rosaria Gitto,[a] Maria Rosa Buemi,[a] Frauke Christ,[b] Zeger Debyser,[b]
and Stefania Ferro[a]
Abstract: Herein we describe the design and synthesis of
a new series of coumarin derivatives searching for novel
HIV-1 integrase (IN) allosteric inhibitors. All new obtained
compounds were tested in order to evaluate their ability to
inhibit the interaction between the HIV-1 IN enzyme and
the nuclear protein lens epithelium growth factor LEDGF/
p75. A combined approach of docking and molecular dy-
namic simulations has been applied to clarify the activity of
the new compounds. Specifically, the binding free energies
by using the method of molecular mechanics-generalized
Born surface area (MM-GBSA) was calculated, whereas hy-
drogen bond occupancies were monitored throughout sim-
ulations methods.
Keywords: HIV-1 · IN · LEDGF/p75 · Docking · Molecular Dynamics
1 Introduction
Since the discovery of HIV-1, the etiological agent of the ac-
quired immune deficiency syndrome (AIDS), 26 antiretrovi-
ral compounds have been approved by the US Food and
Drug Administration (FDA). Based on “Global AIDS Re-
sponse Progress Reporting 2015”[1] there are nearly 15 mil-
lion of people receiving the therapy.[2] The current most ef-
fective AIDS treatment, referred as combinatorial AntiRetro-
viral Therapy (cART), includes reverse transcriptase inhibi-
tors (RTIs), protease inhibitors (PIs), fusion inhibitors (FIs),
coreceptor inhibitors (CRIs) and integrase inhibitors (INIs),
used in combination regimens.[3–6]
gration of viral DNA into the host genome, an highly or-
ganized multistep process that relies on several cellular
proteins for completion. So an alternative approach to
block the HIV-1 integration step, instead of targeting direct-
ly the catalytic activity of IN, is to interfere with the interac-
tion between the enzyme and the cellular cofactors, stop-
ping integration in allosteric way.
Among several identified IN cofactors, lens epithelium-
derived growth factor or transcriptional co-activator p75
(LEDGF/p75) is emerged as a target of great interest for the
development of a novel generation of integration inhibi-
tors.[11–14]
LEDGF/p75 binds to the interface of IN dimer and pro-
motes IN tetramerization of enzyme resulting in the func-
tional form of IN required for concerted integration.[15] So,
after the initial identification and validation of the interac-
tion between LEDGF/p75 and the enzyme[16–17] a crucial
role for this cofactor in HIV-1 replication was evidenced
through different mutagenesis, transdominant and knock-
out studies.[18–22] In the last decade some compounds have
been discovered as promising LEDGF/p75 inhibitors.
Figure 1 displays the most representative compounds from
different chemical classes.[12,23–29]
As consequence of these medical advances the AIDS-re-
lated mortality has dropped sharply reducing in incidence,
and the syndrome has gradually become a controllable
chronic disease.
However, it was recently demonstrated that low-level of
viremia (LLV) of HIV-1 in the plasma is relatively common
among patients on cART regimen.[7–10] This phenomenon is
often associated with a greater risk of virologic failure as
emergence of drug resistance and immune activation.
Moreover the need for lifelong treatment, the frequently
associated toxic effects, anatomical barriers and also the ex-
istence of virus-reservoirs are some of the obstacles in the
severely hurt patients compliance, a decisive factor in ach-
ieving a successful response to cART in HIV-1 infection.
Thus, the development of safer and potentially promising
antiretroviral agents is eagerly needed, and considerable ef-
forts have been made to identify new molecules capable of
suppressing drug-resistant HIV-1 strains and/or targeting
different stages in the virus life cycle.
[a] L. De Luca, F. E. Agharbaoui, R. Gitto, M. R. Buemi, S. Ferro
Dipartimento di Scienze Chimiche, Biologiche
Farmaceutiche ed Ambientali, Universitꢀ degli Studi di Messina
Viale Annunziata, I-98168, Messina, Italy
*e-mail: ldeluca@unime.it
fagharbaoui@unime.it
[b] F. Christ, Z. Debyser
It is now widely known that the stability of HIV-1 infection
critically depends on the HIV-1 Integrase (IN) mediated inte-
Molecular Virology and Gene Therapy KU Leuven and IRC KULAK
Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
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Considering that Natural Products (NPs) have historically
represented a remarkably source for new medicines as well
as the origin of lead compounds,[30–31] we have recently re-
ported the application of a virtual screening strategy to
access novel drug-like natural compounds as potential pro-
tein-protein interaction inhibitors (PPIIs). As results, new hit
structures from natural sources proved to be active as PPIIs
of the IN-LEDGF/p75 complex. Among them, we selected
the 8-methyl-2-oxo-4-phenyl-2H-chromen-7-yl)oxy](phenyl)-
acetic acid (CR) (Figure 1) structurally characterized by cou-
marin nucleus. It is well-known that coumarins are a versa-
tile class of heterocycles displaying a wide range of thera-
peutic activities. Particularly, coumarin derivatives have
been shown to be pharmacologically useful as anti-coagu-
lants, anti-oxidants, antitumorals, free radical scavengers
and also anti-HIV agents.[32–38]
It is interesting to note that the 4-phenylcoumarin core
of hit compound CR resembles the heterocyclic systems of
inhibitors CX5016, BI1001 and CX14442 depicted in
Figure 1. Moreover, CR and other LEDGF/p75 inhibitors
share the carboxylate function, for which has been demon-
strated a pivotal role in the inhibition of IN-LEDGF/p75 in-
teraction.[14,23]
Figure 1. Chemical structures of well-known LEDGF/p75 inhibitors.
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1
In the current work several structural modifications, com-
putational studies and biological screening of new 4-phe-
nylcoumarin derivatives are presented with the aim of ach-
ieving further information about the mechanism of inhibi-
tion of IN-LEDGF/p75 interaction.
Mp: 240–2428, yield 70%. H NMR (DMSO-d6): 2.18 (s, 3H,
CH3), 6.15 (s, 1H, CH), 6.65 (d, J=8.8, 1H, ArH), 6.77 (d, J=
8.8, 1H, ArH), 7.41–7.65 (m, 4H, ArH), 10.56 (bs, 1H, OH).
Anal. ( C16H11ClO3).
4-(3-Chlorophenyl)-7-hydroxy-8-methyl-2H-chromen-2-
one (10)
1
Mp: 315–3178, yield 79%. H NMR (DMSO-d6): 2.18 (s, 3H,
2 Experimental Section
CH3), 6.18 (s, 1H, CH), 6.83 (d, J=8.5, 1H, ArH), 7.04 (d, J=
9.0, 1H, ArH), 7.44 (d, J=8.5, 1H, ArH), 7.53–7.59 (m, 3H,
ArH), 10.59 (bs, 1H, OH). Anal. (C16H11ClO3).
4-(4-Chlorophenyl)-7-hydroxy-8-methyl-2H-chromen-2-
one (11)
2.1 Chemistry
All starting materials and reagents commercially available
(Sigma-Aldrich Milan Italy and Alfa Aesar Karlsruhe Germa-
ny) were used without further purification. Microwave-as-
sisted reactions were carried out in a CEM Focused Micro-
wave Synthesis System Model Discover working at the
power necessary for refluxing under atmospheric condi-
tions. Melting points were determined on a BUCHI Melting
Point B-545 apparatus and are uncorrected.
1
Mp: 253–2558, yield 87%. H NMR (DMSO-d6): 2.19 (s, 3H,
CH3), 6.15 (s, 1H, CH), 6.83 (d, J=9.0, 1H, ArH), 7.08 (d, J=
9.0, 1H, ArH), 7.50 (d, J=9.0, 2H, ArH), 7.61 (d, J=8.5, 2H,
ArH), 10.55 (bs, 1H, OH). Anal. (C16H11ClO3).
7-Hydroxy-8-methyl-4-(2-methylphenyl)-2H-chromen-
2-one (12)
1
Elemental analysis (C, H, N) were carried out on a Carlo
Erba Model 1106 Elemental Analyzer and the results are
within Æ0.4% of the theoretical values. Merck silica gel 60
F254 plates were used for analytical TLC; column chromatog-
raphy was performed on Merck silica gel 60 (230–
400 mesh). Flash Chromatography (FC) was carried out on
Mp: 108–1108, yield 26%. H NMR (DMSO-d6): 2.07 (s, 3H,
CH3), 2.18 (s, 3H, CH3), 6.06 (s, 1H, CH), 6.64 (d, J=8.2, 1H,
ArH), 6.76 (d, J=8.8, 1H, ArH), 7.19 (d, J=7.6, 1H, ArH),
7.31–7.40 (m, 3H, ArH), 10.53 (bs, 1H, OH). Anal. (C17H14O3).
7-Hydroxy-8-methyl-4-(3-methylphenyl)-2H-chromen-
2-one (13)
1
1
a Biotage SP1 EXP. H-NMR spectra were recorded in CDCl3
Mp: 181–1838, yield 88%. H NMR (DMSO-d6): 2.19 (s, 3H,
with TMS as internal standard or [D6]DMSO on a Varian
Gemini-300 spectrometer. Chemical shifts were expressed
in d (ppm) and coupling constants (J) in Hertz (Hz). All ex-
changeable protons were confirmed by addition of D2O.
Mass spectrometry analysis were realized on Bruker MicrO-
TOF (ESI) equipped with an Agilent 1200 LC.
CH3), 2.38 (s, 3H, CH3), 6.10 (s, 1H, CH), 6.82 (d, J=8.8, 1H,
ArH), 7.12 (d, J=8.8, 1H, ArH), 7.27–7.43 (m, 4H, ArH), 10.56
(bs, 1H, OH). Anal. (C17H14O3).
7-Hydroxy-8-methyl-4-(4-methylphenyl)-2H-chromen-
2-one (14)
1
Mp: 259–2618, yield 63%. H NMR (DMSO-d6): 2.19 (s, 3H,
General procedures for the synthesis of 7-hydroxy-2H-
chromen-2-ones. (4 and 7–14)
CH3), 2.38 (s, 3H, CH3), 6.09 (s, 1H, CH), 6.82 (d, J=8.5, 1H,
ArH), 7.13 (d, J=8.5, 1H, ArH), 7.35–7.37 (m, 4H, ArH), 10.53
(bs, 1H, OH). Anal. (C17H14O3).
Compounds 4 and 7–14 were prepared by reaction of re-
sorcinol (1), 2-chlororesorcinol (2) or 2-methylresorcinol (3)
(1 mmol) with malic acid or with the appropriate ethyl ben-
zoylacetate (1.2 mmol), in aqueous 70% sulfuric acid
(2 mL). The resulting solution was stirred at room tempera-
ture for 3 hours and then was poured into ice. The ob-
tained precipitate was filtered, washed with water, dried
and crystallized with ethanol.
General procedures for the synthesis of ethyl [(2-oxo-
4-phenyl-2H-chromen-7-yl)oxy]acetates (5 and 15–22)
To a well-stirred solution of 7-hydroxy-2H-chromen-2-one
(4, 7–14) (1 mmol) and potassium carbonate (5 mmol), in
absolute acetone (3 mL), the appropriate ethyl bromophen-
yl acetate (1.2 mmol) was added dropwise. The reaction
mixture was stirred and refluxed for 5 h at 1108. After that
time the mixture was cooled to room temperature and fil-
tered to remove the inorganic material. The solution was
evaporated in vacuo to give the crude product. The residue
was purified by crystallization with ethanol.
7-Hydroxy-8-methyl-2H-chromen-2-one (4)
1
Mp: 195–1978, yield 49%. H NMR (DMSO-d6): 2.15 (s, 3H,
CH3), 6.22 (d, 1H, J=8.8, CH), 6.82 (d, 1H, J=8.2, CH), 7.90
(d, 1H, J=9.4, ArH), 8.00 (d, 1H, J=9.3, ArH), 10.48 (bs, 1H,
OH). Anal. (C10H8O3).
Ethyl [(8-methyl-2-oxo-2H-chromen-7-yl)oxy](phenyl)-
acetate (5)
7-Hydroxy-4-phenyl-2H-chromen-2-one (7)
1
1
Mp: 195–1978, yield 53%. H NMR (DMSO-d6): 6.14 (s, 1H,
Mp: 125–1268, yield 73%. H NMR (CDCl3): 1.19 (t, J=7.1,
CH), 6.76–6.80 (m, 2H, ArH), 7.27 (d, J=8.2, 1H, ArH), 7.49–
7.56 (m, 5H, ArH), 10.65 (bs, 1H, OH). Anal. (C15H10O3).
8-Chloro-7-hydroxy-4-phenyl-2H-chromen-2-one (8)
3H, CH3), 2.45 (s, 3H, CH3),4.18 (q, J=7.1, 2H, CH2), 5.71 (s,
1H, CH), 6.26 (d, J=9.3, 1H, CH), 6.72 (d, J=8.2, 1H, CH),
7.21–7.62 (m, 7H, ArH). Anal. (C20H18O5).
1
Mp: 214–2158, yield 41%. H NMR (CDCl3): 5.89 (s, 1H,
Ethyl [(2-oxo-4-phenyl-2H-chromen-7-yl)oxy](phenyl)-
acetate (15)
CH), 6.66 (d, J=8.8, 1H, ArH), 6.95 (d, J=8.8, 1H, ArH),
7.15–7.25 (m, 5H, ArH). Anal. (C15H9ClO3).
4-(2-Chlorophenyl)-7-hydroxy-8-methyl-2H-chromen-2-
one (9)
1
Mp: 102–1048, yield 92%. H NMR (DMSO-d6): 1.11 (t, J=
7.1, 3H, CH3), 4.15 (q, J=7.1, 2H, CH2), 6.23 (s, 1H, CH), 6.25
(s, 1H, CH), 7.00 (d, J=8.8, 1H, ArH), 7.10 (s, 1H, ArH), 7.36
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(d, J=8.8, 1H, ArH), 7.41–7.58 (m, 10H, ArH). Anal.
(C25H20O5).
that was collected and crystallized with ethanol to give the
carboxylic acid derivatives 6 and 15–22.
[(8-Methyl-2-oxo-2H-chromen-7-yl)oxy](phenyl)acetic
acid (6)
Ethyl [(8-chloro-2-oxo-4-phenyl-2H-chromen-7-yl)oxy]-
(phenyl)acetate (16)
1
1
Mp: 175–1768, yield 54%. H NMR (DMSO-d6): 1.11 (t, J=
Mp: Dec, yield 89%. H NMR (CDCl3): 2.33 (s, 3H, CH3),
7.1, 3H, CH3), 4.14 (q, J=7.1, 2H, CH2), 6.24 (s, 1H, CH), 6.26
(s, 1H, CH), 6.92 (d, J=8.9, 1H, ArH), 7.35 (d, J=8.9, 1H,
ArH), 7.40–7.60 (m, 10H, ArH). Anal. (C25H19ClO5).
Ethyl {[8-methyl-4-(2-chlorophenyl)-2-oxo-2H-chromen-
7-yl]oxy}(phenyl)acetate (17)
5.63 (s, 1H, CH), 5.68 (s, 1H, CH), 6.46 (s, 1H, CH), 6.96–7.60
(m, 7H, ArH). Anal. Calcd for C18H14O5. ESI(À), CH3OH, HR-
MS :ion [MÀH]À, m/z 310, C18H14O5, m/z theory 310,09, m/z
found 309,08.
[(2-Oxo-4-phenyl-2H-chromen-7-yl)oxy](phenyl)acetic
acid (23)
1
Mp: 188–1908C, yield 96%. H NMR (DMSO-d6): 1.23 (t,
1
J=7.4, 3H, CH3), 2.33 (s, 3H, CH3), 4.10 (q, J=7.4, 2H, CH2),
6.18 (s, 1H, CH), 6.29 (s, 1H, CH), 6.76 (t, J=8.8, 1H, ArH),
6.93 (d, J=8.8, 1H, ArH), 7.39–7.65 (m, 9H, ArH). Anal.
(C26H21O5).
Mp: 240–2428, yield 62%. H NMR (DMSO-d6): 6.06 (s, 1H,
CH), 6.24 (s, 1H, CH), 7.00 (dd, J=8.8, J=9.3, 1H, ArH), 7.08
(d, 1H, J=2.3, ArH), 7.35 (d, J=8.8, 1H, ArH), 7.39–7.58 (m,
10H, ArH). Anal. Calcd for C23H16O5. ESI(À), CH3OH, HR-
MS :ion [MÀH]À, m/z 372, C23H16O5, m/z theory 372,10, m/z
found 371,09.
Ethyl {[8-methyl-4-(3-chlorophenyl)-2-oxo-2H-chromen-
7-yl]oxy}(phenyl)acetate (18)
1
Mp: 187–1898, yield 77%. H NMR (DMSO-d6): 1.21 (t, J=
[(8-Chloro-2-oxo-4-phenyl-2H-chromen-7-yl)oxy](phe-
nyl)acetic acid (24)
7.3, 3H, CH3), 2.34 (s, 3H, CH3), 4.10 (q, J=7.3, 2H, CH2), 6.21
(s, H, CH), 6.31 (s, 1H, CH), 6.99 (d, J=9.4, 1H, ArH), 7.15 (d,
J=8.8, 1H, ArH), 7.41–7.47 (m, 4H, ArH), 7.57–7.60 (m, 5H,
ArH). Anal. (C26H21O5).
1
Mp: 220–2228, yield 98%. H NMR (DMSO-d6): 6.16 (s, 1H,
CH), 6.33 (s, 1H, CH), 7.13 (d, J=9.4, 1H, ArH), 7.34–7.61 (m,
10H, ArH), 7.71 (d, J=8.7, 1H, ArH). Anal. Calcd for
C23H15ClO5. ESI(À), CH3OH, HR-MS: ion [MÀH]À, m/z 406,
C23H15ClO5, m/z theory 406,06, m/z found 405,05.
{[4-(2-Chlorophenyl)-8-methyl-2-oxo-2H-chromen-7-
yl]oxy}(phenyl) acetic acid (25)
Ethyl {[8-methyl-4-(4-chlorophenyl)-2-oxo-2H-chromen-
7-yl]oxy}(phenyl)acetate (19)
1
Mp: 194–1968, yield 98%. H NMR (DMSO–d6): 1.11 (t, J=
7.5, 3H, CH3), 2.34 (s, 3H, CH3), 4.02 (q, J=7.5, 2H, CH2), 6.22
(s, 1H, CH), 6.28 (s, 1H, CH), 6.99 (d, J=9.4, 1H, ArH), 7.19
(d, J=8.8, 1H, ArH), 7.41–7.47 (m, 3H, ArH), 7.44 (d, J=8.8,
2H, ArH), 7.57–7.63 (m, 4H, ArH). Anal. (C26H21O5).
1
Mp: 262–2648, yield 99%. H NMR (DMSO-d6): 2.34 (s, 3H,
CH3), 6.03 (s, 1H, CH), 6.29 (s, 1H, CH), 6.79 (d, J=9.0 1H,
ArH), 6.94 (d, J=8.8, 1H, ArH), 7.40–7.66 (m, 9H, ArH). Anal.
Calcd for C24H17ClO5. ESI(À), CH3OH, HR-MS: ion [MÀH]À,
m/z 420, C24H17ClO5, m/z theory 420,08, m/z found 419,07.
{[4-(3-Chlorophenyl)-8-methyl-2-oxo-2H-chromen-7-
yl]oxy}(phenyl) acetic acid (26)
Ethyl
{[8-methyl-4-(2-methylphenyl)-2-oxo-2H-chro-
men-7-yl]oxy}(phenyl)acetate (20)
1
Mp: 167–1698, yield 55%. H NMR (DMSO-d6): 1.07 (t, J=
7.0, 3H, CH3), 2.09 (s, 3H, CH3), 2.34 (s, 3H, CH3), 4.10 (q, 2H,
J=7.0, CH2), 6.13 (s, 1H, CH), 6.20 (s, 1H, CH), 6.75 (d, J=
8.8, 1H, ArH), 6.93 (d, J=8.8, 1H, ArH), 7.17–7.45 (m, 7H,
ArH), 7.55–7.59 (m, 2H, ArH). Anal. (C27H24O5).
1
Mp: 2958 dec., yield 99%. H NMR (DMSO-d6): 2.34 (s, 3H,
CH3), 6.05 (s, 1H, CH), 6.30 (s, 1H, CH), 6.99 (d, J=9.4 1H,
ArH), 7.16 (d, J=8.8, 1H, ArH), 7.40–7.46 (m, 4H, ArH), 7.57–
7.61 (m, 5H, ArH). Anal. Calcd for C24H17ClO5. ESI(À), CH3OH,
HR-MS :ion [MÀH]À, m/z 420, C24H17ClO5, m/z theory
420,08, m/z found 419,07.
Ethyl
{[8-methyl-4-(3-methylphenyl)-2-oxo-2H-chro-
men-7-yl]oxy}(phenyl)acetate (21)
1
Mp: 1818 dec., yield 98%. H NMR (DMSO-d6): 1.09 (t, J=
7.0, 3H, CH3), 2.34 (s, 3H, CH3), 2.38 (s, 3H, CH3), 4.10 (q, J=
7.0, 2H, CH2), 6.17 (s, 1H, CH), 6.22 (s, 1H, CH), 6.99 (d, J=
9.3, 1H, ArH), 7.21–7.46 (m, 8H, ArH), 7.58 (d, J=9.4, 2H,
ArH). Anal. (C27H24O5).
{[4-(4-Chlorophenyl)-8-methyl-2-oxo-2H-chromen-7-
yl]oxy}(phenyl) acetic acid (27)
Mp: 246–2488, yield 99%. H NMR (DMSO-d6): 2.33 (s, 3H,
CH3), 6.05 (s, 1H, CH), 6.26 (s, 1H, CH), 6.97 (d, J=9.1, 1H,
ArH), 7.18 (d, J=8.5, 1H, ArH), 7.37–7.53 (m, 5H, ArH), 7.57–
7.61 (m, 4H, ArH). Anal. Calcd for C24H17ClO5. ESI(À), CH3OH,
HR-MS :ion [MÀH]À, m/z 420, C24H17ClO5, m/z theory
420,08, m/z found 419,07.
1
Ethyl
{[8-methyl-4-(4-methylphenyl)-2-oxo-2H-chro-
men-7-yl]oxy}(phenyl)acetate (22)
1
Mp: 148–1508, yield 56%. H NMR (DMSO-d6): 1.09 (t, J=
7.0, 3H, CH3), 2.34 (s, 3H, CH3), 2.39 (s, 3H, CH3), 4.10 (q, J=
7.0, 2H, CH2), 6.17 (s, 1H, CH), 6.21 (s, 1H, CH), 6.99 (d, J=
9.4, 1H, ArH), 7.24 (d, J=8.8, 1H, ArH), 7.34–7.60 (m, 9H,
ArH). Anal. (C27H24O5).
{[8-Methyl-4-(2-methylphenyl)-2-oxo-2H-chromen-7-
yl]oxy}(phenyl) acetic acid (28)
1
Mp: 114–1178, yield 61%. H NMR (DMSO-d6): 2.07 (s, 3H,
General procedures for the synthesis of [(2-oxo-4-
phenyl-2H-chromen-7-yl)oxy]acetic acids. (6 and 23–30)
Derivatives 5, 15–22 (1 mmol) were solubilized in ethanol
(5 mL), treated with a water solution of NaOH (5N, 2 mL)
and stirred at room temperature for 1.5 h. Then the reac-
tion mixture was acidified with conc. HCl to afford a solid
CH3), 2.34 (s, 3H, CH3), 6.00 ( s, 1H, CH), 6.19 (s, 1H, CH),
6.76 (d, J=8.8 1H, ArH), 6.92 (d, J=8.2, 1H, ArH), 7.20–7.45
(m, 7H, ArH), 7.44–7.58 (m, 2H, ArH). Anal. Calcd for
C25H20O5. ESI(À), CH3OH, HR-MS :ion [MÀH]À, m/z 400,
C25H20O5, m/z theory 400,13, m/z found 399,12.
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{[8-Methyl-4-(3-methylphenyl)-2-oxo-2H-chromen-7-
Mp: 121–1238, yield 81%. H NMR (DMSO-d6): 1.14 (t, J=
yl]oxy}(phenyl) acetic acid (29)
Mp: 107–1098, yield 32%. H NMR (DMSO-d6): 2.34 (s, 3H,
7.1, 3H, CH3), 1.58 (s, 6H, 2CH3), 2.26 (s, 3H, CH3), 4.15 (q,
J=7.1, 2H, CH2), 6.25 (s, 1H, CH), 6.64 (d, J=9.4, 1H, ArH),
7.21 (d, J=8.8, 1H, ArH), 7.49–7.57 (m, 5H, ArH). Anal.
(C22H22O5).
General procedures for the synthesis of [(8-methyl-2-
oxo-4-phenyl-2H-chromen-7-yl)oxy]acetic acid (35) and
2-[(8-methyl-2-oxo-4-phenyl-2H-chromen-7-yl)oxy]propa-
noic acids (35–37)
1
CH3), 2.38 (s, 3H, CH3), 5.95 ( s, 1H, CH), 6.21 (s, 1H, CH),
6.97 (d, J=8.8, 1H, ArH), 7.20–7.43 (m, 8H, ArH), 7.59 (d, J=
8.2, 2H, ArH). Anal. Calcd for C25H20O5. ESI(À), CH3OH, HR-
MS :ion [MÀH]À, m/z 400, C25H20O5, m/z theory 400,13, m/z
found 399,12.
{[8-Methyl-4-(4-methylphenyl)-2-oxo-2H-chromen-7-
yl]oxy}(phenyl) acetic acid (30)
The intermediates (33–34) obtained in the previous step
were deprotected according to procedure previously de-
scribed for compounds 6 and 23–30.
1
Mp: 2638 dec., yield 97%. H NMR (DMSO-d6): 2.33 (s, 3H,
CH3), 2.38 (s, 3H, CH3), 6.03 ( s, 1H, CH), 6.19 (s, 1H, CH),
6.99 (d, J=9.3, 1H, ArH), 7.25 (d, J=8.8, 1H, ArH), 7.36–7.43
(m, 7H, ArH), 7.60 (m, 2H, ArH). Anal. Calcd for C25H20O5.
ESI(À), CH3OH, HR-MS :ion [MÀH]À, m/z 400, C25H20O5, m/z
theory 400,13, m/z found 399,12.
[(8-Methyl-2-oxo-4-phenyl-2H-chromen-7-yl)oxy]acetic
acid (35)
1
Mp: 233–2358, yield 99%. H NMR (DMSO-d6): 2.27 (s, 3H,
CH3), 4.83 ( s, 2H, CH2), 6.23 (s, 1H, CH), 6.93 (d, J=9.0 1H,
ArH), 7.21 (d, J=9.1, 1H, ArH), 7.45–7.56 (m, 5H, ArH). Anal.
Calcd for C18H14O5. ESI(À), CH3OH, HR-MS :ion [MÀH]À, m/z
310, C18H14O5, m/z theory 310,09, m/z found 309,08.
2-[(8-Methyl-2-oxo-4-phenyl-2H-chromen-7-yl)oxy]pro-
panoic acid (36)
Synthesis of 7-hydroxy-8-methyl-4-phenyl-2H-chro-
men-2-one (31)
Ethyl benzoylacetate (1.2 mmol) was added to a solution
of 2-methylresorcinol (1 mmol) in aqueous 70% sulfuric
acid solution (2 mL). The resulting mixture was stirred at
room temperature for 3 hours and then was poured into
ice. The obtained precipitate was filtered, washed with
water, dried and crystallized with ethanol.
1
Mp: 236–2388, yield 98%. H NMR (DMSO-d6): 1.54 (d, J=
6.9, 3H, CH3), 2.26 (s, 3H, CH3), 5.01 (q, J=6.9, 1H, CH), 6.22
(s, 1H, CH), 6.86 (d, J=9.0, 1H, ArH), 7.20 (d, J=9.0, 1H,
ArH), 7.46–7.55 (m, 5H, ArH). Anal. Calcd for C19H16O5.
ESI(À), CH3OH, HR-MS :ion [MÀH]À, m/z 324, C19H16O5, m/z
theory 324,10, m/z found 323,09.
1
Mp: 223–2258, yield 93 %. H NMR (DMSO-d6): 2.19 (s, 3H,
CH3), 6.12 (s, 1H, CH), 6.82 (d, J=8.8, 1H, ArH), 7.11 (d, J=
8.5, 1H, ArH), 7.46–7.55 (m, 5H, ArH), 10.54 (bs, 1H, OH).
Anal. (C16H12O3).
General procedures for the synthesis of ethyl [(8-
methyl-2-oxo-4-phenyl-2H-chromen-7-yl)oxy]acetate (32)
and ethyl 2-[(8-methyl-2-oxo-4-phenyl-2H-chromen-7-
yl)oxy]propanoates (33–34)
2-Methyl-2-[(8-methyl-2-oxo-4-phenyl-2H-chromen-7-
yl)oxy]propanoic acid (37)
1
Mp: 122–1248, yield 71%. H NMR (DMSO-d6): 1.57 (s, 6H,
2CH3), 2.25 (s, 3H, CH3), 6.23 (s, 1H, CH), 6.71 (d, J=8.8, 1H,
ArH), 7.20 (d, J=8.8, 1H, ArH), 7.50–7.56 (m, 5H, ArH). Anal.
Calcd for C20H18O5. ESI(À), CH3OH, HR-MS :ion [MÀH]À, m/z
338, C20H18O5, m/z theory 324,10, m/z found 337,10.
To a solution of 7-hydroxy-8-methyl-4-phenyl-2H-chro-
men-2-one (31) (1 mmol) in absolute acetone (3 mL), potas-
sium carbonate (5 mmol) and the appropriate ethyl bromo-
phenyl acetate (1.2 mmol) were added. The mixture was
heated to reflux for 5 h, until TLC indicated the disappear-
ance of the starting material. Then, it was cooled to 08C
and filtered to remove the inorganic material. The solution
was evaporated in vacuo to give the crude product. The
residue was purified by crystallization with ethanol.
Ethyl [(8-methyl-2-oxo-4-phenyl-2H-chromen-7-yl)oxy]-
acetate (32)
2.2 Biological Assays
2.2.1 LEDGF/p75-HIV-1 Integrase Interaction Screening
The AlphaScreen assay was performed as previously de-
scribed.[46] Reactions were performed in 25 ml final volume
in 384-well Optiwellꢁ microtiter plates (PerkinÀElmer). The
reaction buffer contained 25 mM TrisÀHCl (pH 7.4), 150 mM
NaCl, 1 mM MgCl2, 0.01% (v/v) Tween-20 and 0.1% (w/v)
bovine serum albumin. His6-tagged integrase (300 nM final
concentration) was incubated with the compounds at 48
for 30 min. The compounds were added in varying concen-
trations from 1 up to 100 nM. Afterwards 100 nM of re-
combinant flag-LEDGF/p75 was added and incubation was
extended by another hour at 48. Subsequently, 5 ml of Ni-
chelate-coated acceptor beads and 5 ml of anti-flag donor
beads were added to a final concentration of 20 mg/ml of
both beads. Proteins and beads were incubated at 308 for
1 h in order to allow association to occur. Exposure of the
reaction to direct light was prevented as much as possible
and the emission of light from the acceptor beads was
1
Mp: 143–1458, yield 54%. H NMR (DMSO-d6): 1.19 (t, J=
7.2, 3H, CH3), 2.28 (s, 3H, CH3), 4.15 (q, J=7.2, 2H, CH2), 4.94
(s, 2H, CH2), 6.24 (s, 1H, CH), 6.93 (d, J=9.1, 1H, ArH), 7.21
(d, J=9.0, 1H, ArH), 7.49–7.57 (m, 5H, ArH). Anal. (C20H18O5).
Ethyl
2-[(8-methyl-2-oxo-4-phenyl-2H-chromen-7-yl)-
oxy]propanoate (33)
1
Mp: 136–1388, yield 51%. H NMR (DMSO-d6): 1.15 (t, J=
6.9, 3H, CH3), 1.55 (d, J=6.9, 3H, CH3), 2.27 (s, 3H, CH3), 4.11
(q, J=6.9, 2H, CH2), 5.10 (q, J=6.9, 1H, CH), 6.24 (s, 1H, CH),
6.87 (d, J=9.0, 1H, ArH), 7.20 (d, J=9.1, 1H, ArH), 7.48–7.56
(m, 5H, ArH). Anal. (C21H20O5).
Ethyl 2-methyl-2-[(8-methyl-2-oxo-4-phenyl-2H-chro-
men-7-yl)oxy]propanoate (34)
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measured in the EnVision plate reader (PerkinÀElmer, Bene-
scoring function is successful. These values were small
enough and supported the hypothesis that experimental
binding modes could be reproduced with accuracy using
this protocol. The standard default settings were used in all
calculations.
lux) and analyzed using the EnVision manager software.
2.2.2 In vitro Anti-HIV and Drug Susceptibility Assays
The inhibitory effect of antiviral drugs on the HIV-induced
cytopathic effect (CPE) in human lymphocyte MT-4 cell cul-
ture was determined by the MT-4/MTT-assay.[53] This assay is
based on the reduction of the yellow coloured 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by
mitochondrial dehydrogenase of metabolically active cells
to a blue formazan derivative, which can be measured
spectrophotometrically. The 50% cell culture infective dose
(CCID50) of the HIV(IIIB) strain was determined by titration of
the virus stock using MT-4 cells. For the drug-susceptibility
assays, MT-4 cells were infected with 100–300 CCID50 of the
virus stock in the presence of five-fold serial dilutions of
the antiviral drugs. The concentration of the various com-
pounds that achieved 50% protection against the CPE of
the different HIV strains, which is defined as the EC50, was
determined. In parallel the 50% cytotoxic concentration
(CC50) was determined.
*
Docking studies
Docking studies were performed using the genetic opti-
mization for ligand docking (GOLD) software package ver-
sion 4.1.1 from the Cambridge Crystallographic Data
Centre (CCDC)[50] as described in our previous paper.[42]
For the prediction of ligand binding positions GoldScore
fitness function was used. For each ligand 100 independent
runs and a maximum of 15000 genetic operations were
performed using the default operator weights and a popula-
tion size of 100 chromosomes.
Default cutoff values of 2.5 ꢂ for hydrogen bonds and
4.0 ꢂ for van der Waals interactions were employed. Auto-
matic bond settings were used, allowing the torsion angles
of all acyclic, rotatable bonds in the ligand to vary except
for amide bonds. Results differing by less than 0.75 ꢂ in
ligand-all-atom RMSD were clustered together.
Results differing by less than 1.00 ꢂ in ligand-all atom
RMSD were clustered together. A 20.0 ꢂ radius active site
was drawn on the original position of the LEDGF/p75 IBD
dipeptide Ile365-Asp366 and automated cavity detection
was used. Two hydrogen bond constraints were used to
specify that two protein atoms should be hydrogen-
bonded to the ligand, namely NH backbone of Glu170 and
His171 with a constraint weight of 5.
2.3 Computational Studies
2.3.1 Docking Simulations
*
Ligand preparation
3D structure of each ligand was constructed using Dis-
covery Studio2.5.5 and minimized using CHARMmforcefield
followed by Smart Minimizer algorithm performing 1000
steps of Steepest Descent with a root mean square (RMS)
gradient tolerance of 3, followed by Conjugate Gradient
minimization, until the RMS gradient for potential energy
was less than 0.05 kcal/mol/ꢂ.
Binding energy of the minimized complex was calculated
using the MM-GBSA method[51] implemented in the AMBER
program.
2.3.2 Molecular Dynamics Simulations
*
Protein preparation
*
Model Preparation
The protein was prepared using Discovery Studio 2.5.5.[54]
For our docking simulations the crystal structure of the
dimeric CCD of HIV-1 IN complexed with the IBD of LEDGF/
p75 was retrieved from RCSB Protein Data Bank
(PDB :2B4J).[47] First, the LEDGF/p75 structure and the water
molecules solved by X-ray crystallography were removed,
then the missing hydrogens were added to the pattern.
The starting model for simulations of IN-LEDGF/p75 was
prepared as described in our previous paper.[42] In brief,
from the X-ray structure 2B4J of IN CCD (chains A and B) in
complex with the LEDGFIBD (chains C and D)[47] was used.
First, chain D and water molecules were removed from the
structure. Then, the missing residues of the INCCD were
added by superimposing chain C of the HIV-1 IN 1BL3[55]
structure and energy-minimized using Maestro[56] with
a RMSD of 0.30 ꢂ. From the resulting complex, the chain C
of LEDGFIBD was castoff in order to simulate IN-PPIIs com-
plexes.
*
Validation of docking protocol
The validation of the docking protocol was performed by
docking the native co-crystallized ligands of the two crystal
structures with the PDB codes 3LPT and 3LPU, into LEDGF/
p75 binding site. The comparison of docking results with
the co-crystallized form showed success rates with the
docked ligand strictly superimposed with the crystallized
conformation with RMSD=1.01 ꢂ indicating that the used
*
Molecular Dynamics
MD simulations were carried out using the sander
module of AMBER 11[52] and parm 99.dat and frcmod.ff03
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parameter files.[57–58] These parameters were assigned to
the designed ligands, while partial charges were calculated
using the AM1-BCC method as implemented in the Ante-
chamber suite of AMBER 11.
All obtained information has been used in a virtual
screening strategy that led to a selection of nine in silico
hits that were tested in AlphaScreen assay[46] to evaluate
their ability to prevent IN-LEDGF/p75 interaction. Eight of
them have exhibited inhibitory effects at 100 mM ranging
from 30% to 88% and for six of them the IC50 value was
determined.[27]
The geometry of the system was minimized in order to
remove any bad contact using the steepest descent algo-
rithm for the first 250 steps before switching to the conju-
gate gradient algorithm for the remaining 250 steps.
Solvent effects were taken into account by using the
generalized Born implicit solvent model. The minimized
structure was the input for MD runs using constant-temper-
ature Langevin dynamics at 300 K for 100 ps with a time
step of 1fs and a distance cutoff of 12.0 ꢂ for the nonbond-
ed interactions.
These compounds have been considered as starting
point for further hit-to-lead optimization, for which com-
pound CR (IC50 =37.25 mM) has represented an encourag-
ing “hit compound”; the Figure 2 shows its plausible bind-
ing mode into the allosteric site of integrase catalytic core
domain (CCD).
As result of this study, the carboxylic function seems to
form hydrogen bonds with the IN backbone nitrogen
atoms of Glu170 and His171 residues, the same interactions
mediated by LEDGF/p75 hotspot residue of Asp366.[47] Ad-
ditionally, a hydrogen bond with the hydroxyl group of the
IN side chain of the Thr174 residue is highlighted. The re-
maining portion of the molecule is housed within the
dimer interface cleft made up by IN chain A residues
Thr174, Gln168, Ala169 and Met178 and IN chain B residues
Leu102, Ala 128, Ala129 and Trp131. Thereby it is able to
establish hydrophobic contacts with both the CCD subu-
nits. The interactions between the IN CCD and compound
CR were examined using PyMOL[48] and LIGPLUS.[49]
*
Analysis of MD Trajectories and Free Energy
Snapshots of the complexes during the simulations and
the average structures were obtained with the Ptraj
module of the AMBER 11 suite.[52]
The hydrogen bonds were detected when the acceptor-
donor atom distance was lower than 3.5 ꢂ and the accept-
or-H-donor angle was more than 1208. The MM-GBSA
method[51] implemented in the AMBER program was used
to evaluate the ligand-protein interaction free energies of
the minimized complex and the 100 snapshots extracted at
1 ps intervals.
Considering these docking results, further efforts can be
made to perform structural modifications on the different
moieties of CR compound, in order to study the structural
requirements for PPIIs and to design new coumarin deriva-
tives. Thus, keeping unchanged both the coumarin “core”
For MM-GBSA analysis, snapshots at 40 ps intervals were
extracted from the last 4 ns of the MD trajectory, and the
binding free energies were averaged over the ensemble of
conformers produced (100 snapshots for each trajectory).
3 Results and Discussion
3.1 Rational Design
As a continuation of our previous studies on anti-HIV
agents and in particular on HIV-IN enzyme inhibi-
tors,[11–12,27,39–45] herein we report the results of a research on
PPIIs.
It is well known that the identification of small molecules
able to interfere with IN-LEDGF/p75 interaction could pro-
vide an enormous impetus in the field of antiretroviral re-
search.
Thus, in order to obtain useful insights for the develop-
ment of new small molecules that specifically disrupt this
PPI, we have previously investigated the most important
contacts between some PPI inhibitors and the IN-LEDGF/
p75 complex.[27]
By means of a combination of docking and ultrashort
MD, we have generated a weighted ensemble of protein-
ligand configurations and estimated the binding affinity
averaged over snapshots taken from the MD trajectories,
together with the presence of fundamental hydrogen
bonds.[42]
Figure 2. Binding mode of reference compound CR in complex
with IN CCD. Key residues of the pocket are presented. Hydrogen
bonds are shown by dotted lines as well as their occupancies
during MD simulations as percentage. The figure was created
using PyMOL software.
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rivatives 35–37 were generated by saponification with
NaOH aqueous solution.
3.3 Biological Results
The synthesized coumarin derivatives were tested in Al-
phaScreen assay in order to evaluate their inhibitory effects
on the IN-LEDGF/p75 protein interaction, and the biological
results were compared with the reference compound CR.
As reported in Table 1, we found that all new derivatives
inhibited IN-LEDGF/p75 interaction, displaying a percentage
of inhibition ranging from 31% to 88%, at fixed-dose of
100 mM. On the basis of structural modifications summar-
ized in Figure 3, we can collect the following SAR consider-
ations. Removal of ring A (6) and ring B (35), or replace-
ment of the latter with small alkyl groups (36,37), negative-
ly affects the inhibition of the IN-LEDGF/p75 interaction.
Similarly substitution of 8-methyl group of the coumarin
scaffold with chlorine atom was unfavorable for the PPI in-
hibitory effects.
Figure 3. Design strategy for achieving new coumarin derivatives.
On the contrary the best results were observed for deriv-
atives 27 (IC50 =60.80 mM), 28 (IC50 =60.42 mM) and 29
(IC50 =32.10 mM) in which, keeping unchanged both the
methyl group on the benzene fused ring and the unsubsti-
tuted ring B, a methyl group or a chlorine atom on the
ring A have been inserted.
and carboxylic group, we decided to explore the role of
the two phenyl rings (A and B) and 8-methyl group. The
designed modifications are displayed in Figure 3.
Specifically, we focused on the substitutions at the
phenyl ring A to explore the interaction within the hydro-
phobic cavity formed by Ala128, Trp131, Trp132 and
Met178 residues. The structural modifications on phenyl
ring A have been suggested by the pattern of substituents
of other discovered LEDGF/p75 inhibitors (see Figure 1).
Then, we replaced the 8-methyl group with a chlorine
atom to investigate the role of hydrophobic interaction as
well as the electronic properties of the methyl substituent.
We also studied the 8-unsubstituted analogs. To obtain
more information about the hydrophobic area neighboring
carboxyl moiety, the phenyl ring B has been removed and
it has been replaced with one or two methyl groups.
Table 1. Inhibition of IN-LEDGF/p75 interaction of compounds 6,
23–30 and 35–37
Cpd
% [a]
IC50(mM) [b]
R
R’
R’’
R’’’
6
36
75
33
47
73
55
71
88
78
29
31
73
87
>100
>100
>100
>100
CH3
H
Cl
C6H5
H
H
2-Cl
3-Cl
4-Cl
2-CH3
3-CH3
4-CH3
H
—
—
H
H
H
H
H
H
H
H
H
H
CH3
H
23
24
25
26
27
28
29
30
35
36
37
CR
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
>100
60.80Æ2.2
60.42Æ2.4
32.10Æ2.8
>100
ND
ND
3.2 Synthesis
As shown in Scheme 1, the synthesis of designed coumarin
derivatives 6, 23–30 has been carried out through a classic
Pechmann condensation. The unsubstituted (1) or substi-
tuted (2,3) resorcinol reacted with malic acid or the suita-
ble ethyl benzoylacetate to give intermediates 4 and 7–14,
which were alkylated by treatment with ethyl bromophenyl
acetate and potassium carbonate in acetone. Finally, the
obtained derivatives 5 and 15–22 were converted into the
target compounds 6 and 23–30 by hydrolysis at room tem-
perature in basic medium.
H
H
H
CH3
CH3
C6H5
100Æ4.5
37.25
[a] % inhibition at 100 mM; [b] C50: Concentration required to inhib-
it the HIV-1 IN-LEDGF/p75 interaction by 50%. ND: Not Deter-
mined.
3.4 Computational Studies
Following a similar procedure, the synthesis of com-
pounds 35–37 was performed (Scheme 1). Specifically, the
reaction between methyl resorcinol (3) and ethyl benzoyla-
cetate furnished coumarine scaffold 31. It was treated with
the suitable ethyl bromo derivatives to give the ethyl ester
intermediates 32–34. Finally, the desired carboxylic acid de-
In order to rationalize the obtained results, the binding
mode of the designed compounds was studied. The dock-
ing calculation into the LEDGF/p75-IBD binding pocket in
the CCD of IN (PDB ID: 2B4J) was realized using GOLD (Ge-
netic Optimization for Ligand Docking) software package.[50]
So as to take into account the flexible side chain of residue
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Scheme 1. Reagents and conditions: (a) H2SO4, rt, 3 h; (b) ethyl bromophenyl acetate, dry K2CO3, acetone, 1108C, 5 h; (c) EtOH, NaOH, rt,
1,5 h. (d) suitable ethyl bromoacetate, dry K2CO3, acetone, 1108C, 5 h; (e) EtOH, NaOH, rt, 1,5 h.
Gln95, two different conformations of IN CCD were used.
Two clusters were taken for additional analysis.
To eliminate bad contacts, the geometry of the systems
was minimized using the steepest descent algorithm fol-
lowed by a conjugate gradient. The solvent effects were
considered through the generalized Born implicit solvent
model.
with hot-spot residues were calculated by AMBER 11 con-
sidering all steps of the MD simulation and are shown in
Table 3; when the occupancies were more than 20% in the
investigated time period; distances between involved
atoms are also indicated.
Table 2. Binding free energy estimation
The output complex was used to estimate ligand binding
MM-GBSA[51]
method
compound
DGbind (complex)
DGbind (snapshots average)
free
energy
using
the
kcal/mol
kcal/mol
([DGbind(complex), Table 2]) followed by additional analysis
applying ultrashort Molecular Dynamics simulations using
Sander module of AMBER 11.[52] The resulting system was
used to estimate the binding affinity averaged over snap-
shots taken from the MD trajectories using the MM-GBSA
method ([DGbind(snapshots average), Table 2]). This proce-
dure allowed us to illustrate the behavior of the complex
IN-ligands.
Specifically we observed that the ranking of the predict-
ed binding free energies are in good agreement with the
experimental IC50 except for low active inhibitor 25.
With the aim of highlighting stable and unstable hydro-
gen bonding of the starting structure, H-bond interactions
6
À20.9
À24.2
À27.7
À33.4
À26.7
À29.0
À29.1
À29.5
À26.1
À25.7
À26.2
À25.7
À31.8
À22.6
À26.7
À28.7
À31.9
À24.1
À30.2
À32.1
À32.9
À31.2
À21.9
À22.7
À25.2
À29.0
23
24
25
26
27
28
29
30
35
36
37
CR
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Table 3. Hydrogen bonds analysis from the results of MD simulation for IN in complex with compound CR and the designed compounds.
IN complex with
donor
acceptor
Occupancy (%) [a]
Distance (ꢂ) [b]
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
His171(A) NH
Thr174(A) OH
Glu170(A) NH
Glu170(A) NH
His171(A) NH
His171(A) NH
O3
O3
O4
O3
O4
O3
O3
O4
O3
O4
O3
O3
O4
O3
O4
O3
O3
O4
O3
O3
O3
O4
O3
O3
O3
O4
O3
O3
O3
O4
O3
O3
O3
O4
O3
O3
O3
O4
O3
O3
O3
O4
O3
O4
O3
O4
O3
O3
O3
O4
O3
O3
O4
O3
O4
O3
O3
O4
72.4
89.4
18.9
16.5
32.6
46.1
64.9
46.9
41.4
10.3
40.1
58.2
56.7
36.4
12.5
83.2
90.7
87.3
70.9
7.4
2.935 (0.24)
2.863 (0.14)
3.036 (0.22)
3.199 (0.20)
3.054 (0.18)
2.834 (0.22)
2.981 (0.20)
2.950 (0.19)
3.075 (0.18)
3.064 (0.20)
2.917 (0.19)
2.934 (0.22)
2.977 (0.20)
3.175 (0.18)
3.085 (0.21)
2.901 (0.23)
2.945 (0.18)
3.038 (0.19)
3.130 (0.18)
2.943 (0.24)
2.933 (0.18)
3.018 (0.23)
3.183 (0.16)
2.879 (0.23)
2.958 (0.19)
3.011 (0.20)
3.064 (0.19)
2.885 (0.23)
2.942 (0.17)
3.095 (0.18)
3.052 (0.17)
2.926 (0.25)
2.976 (0.18)
3.021 (0.20)
3.052 (0.17)
2.909 (0.24)
2.973 (0.20)
3.002 (0.19)
3.064 (0.18)
2.946 (0.27)
2.917 (0.20)
2.946 (0.27)
3.182 (0.19)
3.222 (0.20)
2.948 (0.25)
3.012 (0.22)
2.942 (0.19)
3.242 (0.18)
2.982 (0.25)
2.902 (0.18)
3.067 (0.21)
3.182 (0.20)
3.144 (0.19)
2.871 (0.24)
2.873 (0.15)
3.123 (0.20)
3.182 (0.18)
3.087 (0.21)
6
23
24
25
87.4
14.7
6.4
26
27
78.8
86.0
84.2
59.3
83.7
91.7
81.0
58.6
82.2
87.0
97.7
95.6
75.4
89.5
75.0
93.1
29.6
58.4
46.9
23.0
10.8
54.4
78.1
70.9
34.5
59.9
80.7
52.8
34.5
13.4
88.6
97.3
42.7
47.8
19.4
28
29
30
35
36
37
CR
[a] The listed donor and acceptor pairs satisfy the criteria for the hydrogen bond over 20.0% occupancy during the entire simulation.
[b] The average distance between the hydrogen-acceptor atom and hydrogen-donor atom in the investigated time period with standard
error (SE=standard deviation/N1/2) in parentheses.
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3.5 Results Analysis
The insertion of substituents on the ring A (23–30) showed
a significant influence on the inhibitory profile. With the
aim of explaining the best activity of {[8-methyl-4-(3-meth-
ylphenyl)-2-oxo-2H-chromen-7-yl]oxy}phenyl)acetic
acid
(29), we analyzed its binding pose as shown in Figure 4.
To predict the impact of performed structural modifica-
tions on binding affinity, we used ultrashort molecular dy-
namics. As results the binding energy over snapshots aver-
age of compound 29 (Table 2) is better both than the refer-
ence compound CR and the other compounds displaying
substitution on ring A (25–28 and 30).
Additionally, hydrogen bond analysis revealed that the
carboxylate forms the same contacts with residues Glu170,
His171 and Thr174 of IN, and the lipophilic group is well
lodged in the hydrophobic pocket formed by residues
Ala128, Trp131, Trp132 and Met178 of IN. Moreover, com-
pound 29 showed a 97% occupancy with Glu170 and the
contact between the carboxylic acid and backbone NH of
the His171 was increased by 50% corresponding to the ref-
erence compound CR.
On the contrary, the removal of ring A (6) from the struc-
ture unfavorably affects the biological results (36% of in-
hibition) highlighting the importance of this ring. Figure 5
displays the binding mode of compound 6 (blue) into the
IN allosteric binding site and the alignment with “reference
Figure 5. Binding mode of compound 6 (blue) and the reference
compound CR (magenta) in complex with IN CCD. Key residues of
the pocket are presented. The figure was created using PyMOL
software.
compound” CR (magenta). The binding mode of 6 suggest-
ed the relevant role of the ring A for the hydrophobic con-
tacts. Moreover, the binding energy analysis and hydrogen
bond occupancies (Table 2) also confirmed that the dele-
tion of this ring generates a molecule with a low affinity
(Table 1).
Concerning the ring B, the removal (i.e. compound 35) or
replacement with small alkyl groups (36, 37) determined
detrimental effects on the binding IN-LEDGF/p75 inhibition
thus highlighting the significant role of this portion for the
biological profile for this class of compounds. In figure 6,
the exploration of binding mode of compounds 35–37 is
reported. There was a slightly different binding mode in
comparison with to the reference compound CR (Figure 6)
due to shift of the carboxylic acid that was revealed by the
distance measurement (Table 3). This movement has affect-
ed the stability of the complex, which was illustrated also
by the decrease of binding energy and the hydrogen bond
occupancies, lower than CR compound especially for the
Thr174 and the His171.
Similarly, the removal of the 8-methyl group (e.g. 23) and
its replacement by chlorine atom (24) was not favorable for
the PPI inhibitory effects. It was observed that these modifi-
cations faintly affected the binding mode of compounds
23 and 24 in comparison with CR (Figure 7). These findings
were highlighted by the measurement of the distances be-
Figure 4. Binding mode of {[8-methyl-4-(3-methylphenyl)-2-oxo-
2H-chromen-7-yl]oxy}(phenyl)acetic acid (29) in complex with IN
CCD. Key residues of the pocket are presented. Hydrogen bonds
are shown by dotted lines as well as their occupancies during MD
simulations as percentage. The figure was created using PyMOL
software.
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Figure 7. Binding mode of compound 23 (cyan), 24 (yellow) and
the reference compound CR (magenta) in complex with IN CCD.
Key residues of the pocket are presented. The figure was created
using PyMOL software.
Figure 6. Binding mode of compound 35 (cyan), 36 (green), 37
(yellow) and the reference compound CR (magenta) in complex
with IN CCD. Key residues of the pocket are presented. The figure
was created using PyMOL software.
tween carboxylic acids in regards to the reference com-
pound CR. Furthermore, the analysis of hydrogen bond oc-
cupancies (Table 3) showed a decreasing by 20 to 55%
with Glu170, His171 and Thr174 for the 23 and 24 deriva-
tives.
To obtain more information about the interaction within
the IN-LEDGF interface, we compared the docking-predict-
ed binding mode of 29 with the crystallized position of
more potent inhibitors CX0516 and KF116 (Figure 8). As re-
sults, we found the overlapping of the carboxylic group for
all analysed compounds. Whereas a different orientation
has been found in the occupancy of the hydrophobic area
lined by Ala128 and Trp131. These findings could furnish
new suggestion for the design on new analogues.
Figure 8. Docking-predicted binding mode of 29 compared to
crystallized position of HIV-1 IN CCD in complex with CX0516 (ma-
genta, pdb code: 3LPU) and KF116 (yellow, pdb code: 4O5B). The
figure was created using PyMOL software.
4 Conclusions
To aim of improving inhibitory activity of our reference
molecule CR and achieving new information about the
mechanism of action, a series of modifications on the cou-
marin scaffold was planned and carried out. The new syn-
thesized compounds 6, 23–37 were evaluated to establish
their ability to block the IN-LEDGF/p75 interaction. The
best biological results were obtained for derivatives 27–29,
which were able to inhibit the PPI at micromolar concentra-
tion ranging from 60.42 to 32.10 mM. Specifically, for com-
pound 29 we found that the IC50 value was very similar to
reference compound CR. An extensive SAR has been dis-
cussed considering docking and molecular dynamic simula-
tions.
Conflict of Interest
None declared.
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Acknowledgements
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Received: December 14, 2015
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Published online: && &&, 0000
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FULL PAPERS
L. De Luca,* F. E. Agharbaoui,* R. Gitto,
M. R. Buemi, F. Christ, Z. Debyser,
S. Ferro
&& – &&
Rational design, synthesis and
evaluation of coumarin derivatives
as protein-protein interaction
inhibitors
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