Synthesis of potential HIV integrase inhibitors inspired by natural polyphenol structures

Article abstract of DOI:10.1080/14786419.2017.1354191

Drawing inspiration from the structural features of some natural polyphenols, the synthesis of two different model compounds as potential inhibitors of HIV integrase (IN) has been described. The former was characterised by a diketo acid (DKA) bioisostere, such as a β-hydroxycarbonyl moiety, between two fragments containing aromatic groups, while in the latter an epoxide linked two polyoxygenated aromatic residues. The moieties present in the structures are thought to function by chelating divalent metal ions on the enzyme catalytic site. Overall, 10 compounds were prepared and some of that submitted to molecular modelling studies (to investigate their interactions with the active site of IN), to metal titration studies (to detect their chelating capability) and to biological assays.

Full text of DOI:10.1080/14786419.2017.1354191

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis of potential HIV integrase inhibitors
inspired by natural polyphenol structures
Giuliana Righi, Romina Pelagalli, Valerio Isoni, Ilaria Tirotta, Martina Marini,
Matteo Palagri, Roberto Dallocchio, Alessandro Dessì, Beatrice Macchi,
Caterina Frezza, Gianpiero Forte, Antonella Dalla Cort, Gustavo Portalone &
Paolo Bovicelli
To cite this article: Giuliana Righi, Romina Pelagalli, Valerio Isoni, Ilaria Tirotta, Martina Marini,
Matteo Palagri, Roberto Dallocchio, Alessandro Dessì, Beatrice Macchi, Caterina Frezza, Gianpiero
Forte, Antonella Dalla Cort, Gustavo Portalone & Paolo Bovicelli (2017): Synthesis of potential
HIV integrase inhibitors inspired by natural polyphenol structures, Natural Product Research, DOI:
10.1080/14786419.2017.1354191
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Date: 27 July 2017, At: 23:08

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1354191
Synthesis of potential HIV integrase inhibitors inspired by
natural polyphenol structures
Giuliana Righia, Romina Pelagallib , Valerio Isonib, Ilaria Tirottab, Martina Marinib,
Matteo Palagrib, Roberto Dallocchioc, Alessandro Dessìc, Beatrice Macchid,
Caterina Frezzad, Gianpiero Forteb, Antonella Dalla Cortb, Gustavo Portaloneb and
Paolo Bovicellia
b
adepartment of chemistry, cNr-IBPM, “sapienza” universy of rome, rome, Italy; department of chemistry,
“sapienzauniversy of rome, roma, Italy; ccNr-IcB uos sassari, sassari, Italy; ddepartmentof system Medicine,
“tor Vergata”university, rome, Italy
ABSTRACT
ARTICLE HISTORY
received 19 May 2017
accepted 6 July 2017
Drawing inspiration from the structural features of some natural
polyphenols, the synthesis of two different model compounds
as potential inhibitors of HIV integrase (IN) has been described.
The former was characterised by a diketo acid (DKA) bioisostere,
such as a β-hydroxycarbonyl moiety, between two fragments
containing aromatic groups, while in the latter an epoxide linked
two polyoxygenated aromatic residues. The moieties present in the
structures are thought to function by chelating divalent metal ions on
the enzyme catalytic site. Overall, 10 compounds were prepared and
some of that submitted to molecular modelling studies (to investigate
their interactions with the active site of IN), to metal titration studies
(to detect their chelating capability) and to biological assays.
KEYWORDS
Inhibitors; hIV integrase; dKa
inhibitors; polyphenols
OCH3
O
O
H3CO
O
O
HO
CONTACT Giuliana righi
giuliana.righi@cnr.it
this author contributed equally to this work.
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1354191.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
G. RIGHI ET AL.
1. Introduction
HIV integrase is essential for viral replication because its primary function is to catalyse the
insertion of the double-stranded viral DNA generated by reverse transcription into host
chromosomes.
The 32 kDa IN enzyme contains 288 amino acids divided into three structural and func-
tional domains: the N-terminal domain (NTD), the catalytic core domain (CCD) and the
C-terminal domain (CTD) (Chiu and Davies 2004). The catalytic core domain (residues 50–212)
of HIV-1 integrase (IN) contains a canonical three amino acid (so-called DDE motif), formed
by two aspartates (D64 and D116) and one glutamate (E152). Mutation of any of these three
acidic residues removes integrase’s enzymatic activities and viral replication (Esposito et al.
1999). The two D64 and D116 residues form a coordination complex with a divalent metal
(Mg2+ or Mn2+), the role of which is not only limited to catalysis, but also important for specific
binding of IN to DNA, conformational change, multimerisation and assembly. Therefore, the
metal ion binding sites are highly conserved and essential for enzymatic activity as well as
for efficient viral replication. Over the past years, a large number of natural product IN inhib-
itors have been identified by screening of microbial, plant and marine organism extracts
(Koehn and Carter 2005); examples of biologically active compounds include CAPE (Fesen
et al. 1993), curcumins (Mazumder et al. 1995, 1997) flavones and flavonoids (Fesen et al.
1994), caffeoylquinic acids (Neamati et al. 1997), chicoric acid (Lin et al. 1999; Charvat et al.
2006), coumarins (Mazumder et al. 1996; Zhao et al. 1997), as well as conformationally
restricted cinnamoyl compounds (Figure 1) (Artico et al. 1998). Catechol moiety, a common
structural feature, is reported to be an efficient metal chelating agent, but also displays
remarkable cytotoxic and relatively non-selective activity. However, compounds having two
catechol units separated by a linker are in general more potent inhibitors, and an increased
hydroxylation of the phenyl ring even increases potency against IN.
Another structural feature frequently present in synthetic HIV-IN inhibitors (Zhan et al.
2016), first of which was the 5-CITEP, is the beta-diketoacid (DKA) moiety (Ingale and Bhatia
2011; Cuzzucoli Crucitti et al. 2015; Nair and Okello 2015; Corona et al. 2016). Unfortunately,
similar to polyhydroxylated compounds, DKA functionality is not only responsible for antiviral
activity, but also contributes to cytotoxicity.
Figure 1. examples of dKa and polyphenol IN inhibitors.

NATURAL PRODUCT RESEARCH
3
In order to reduce the cytotoxicity while maintaining the biological activity, we decided
to create a small library of novel potential HIV-IN inhibitors, characterised by two aromatic
fragments linked together via a β-hydroxycarbonyl DKA bioisostere (Figure 2a). Considering
that many recognised HIV-IN inhibitors contain a structural motif that coordinates the two
magnesium ions in the enzyme’s active site , we envisioned that, besides the well-know
cathecol moiety, also the β-hydroxycarbonyl core could interact with the metal ions in the
catalytic site of HIV-IN (Figure 2b). Coordination with the metal ions through the formation
of a five and/or six-membered chelate should result in the inhibition of the biological prop-
erties of the enzyme and ultimately the replication of the HIV virus.
2. Results and discussions
2.1. Chemistry
Starting from commercially available cis-buten-1,4-diol 1, epoxy acid 6 was readily prepared
in five steps (Scheme 1). Treatment of diol 1 with TBDMSCl (tert-butyldimethylsilylchloride)
(Trost et al. 2002) afforded the monosilyl ether 2, which was subsequently converted to the
corresponding epoxy alcohol 3 (Yoshimura et al. 2007) and, by an etherification with PMBBr
(4-methoxybenzyl bromide) ((König et al. 2007) followed by a desilylation, to the alcohol 5.
Finally, the oxidation of alcohol 5 with TEMPO (2,2,6,6-tetramethylpiperidinyloxy)/NaClO
in the presence of quaternary ammonium catalyst Aliquat 336 (Wolf and Spenser 1995) gave
the corresponding epoxy acid 6 in 70% yield. Epoxy acid 6 was subsequently coupled with
benzyl alcholols, phenols and phenethyl alcohols to yield the first nine derivatives. The
esterification step was carried out under different conditions depending on the alcoholic
partner utilised: CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine)/NMM (N-methylmorpholine)
Figure 2. (a) library of dKa bioisosteres based on a β-hydroxycarbonyl core and two aromatic fragments.
(b) Possible interaction of the β-hydroxycarbonyl core with metal ions.
Scheme 1. (a) Nah, tBdMscl, thF, 0 °c, 81%; (b) McPBa (m-chloroperbenzoic acid), ch2cl2, rt, 98%; (c)
Nah, PMBBr (4-methoxybenzyl bromide), thF, 0 °c, 85%; (d) tBaF (tetrabutylammonium floride), thF, rt,
96%; (e) Naclo, teMPo, KBr, Nahco3, aliquat 336, ch2cl2, 0 °c, 70%.

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G. RIGHI ET AL.
Scheme 2. (a) cdMt/NMM, ch2cl2, rt, 60–67%; (b) cdMt/NMM, ch2cl2, rt, 28–34%; (c) Phcocl, tea,
dMaP, thF, 0 °c, 82–96%.
Scheme 3. (a) NaBr, amb15, acetone, −20 °c, 88–96%; (b)ttMss(tris(trimethylsilyl)silane)/aIBN
(azobisisobutyronitrile), toluene, 70 °c, 72–83%.
system (Kamiński et al. 1998) for benzyl alcohols and phenols, and BzCl/DMAP (4-dimethyl-
amino-pyridine)/TEA (triethylamine) system (Inanaga et al. 1979; Dhimitruka and SantaLucia
2006) for phenethyl alcohols (Scheme 2).
In order to achieve the β-hydroxycarbonyl derivatives 9a-i, compounds 7a-i underwent
a two-step reaction sequence (Righi et al. 2001), consisting of a regioselective ring opening
of oxirane by NaBr/Amb15 and subsequent radical debromination (Chatgilialoglu 1988)
(Scheme 3).
Besides the synthetic work, molecular modelling and metal titration studies were carried
out to investigate the chelating ability of structures 9a-i. Unfortunately, preliminary studies
showed the inability of compounds 9a-i to chelate Mg++; consequently, a low inhibitor activ-
ity towards the enzyme’s catalytic site could be expected.
Therefore, we decided to explore another type of derivative having a conformationally
constrained linker, such as an epoxy ring, between two polyoxygenated aromatic residues,
one of which was a monoprotected catechol moiety, able to chelate the cations.
To verify this hypothesis, we prepared the model compound 13, as potential lead of future
library. Starting from the monoprotected diol 2, we prepared the epoxy alcohol 12, that,
coupled with the vanillic acid (Kang et al. 2003; Yoshida et al. 2009) efficiently provided the
target ester 13 (Scheme 4).

NATURAL PRODUCT RESEARCH
5
The molecular structure of crystalline compound 13 was studied by X-ray diffraction.
Results are summarised in the supplementary material (Figure S1 and Table S1).
2.2. Molecular docking
All compounds 9a-i were docked and the best docking results come from analogue 9b.
Molecule 9b showed two possible arrangements in the HIV-IN active site core domain.
One of them showed (Figure 3a) a hydrogen bond with the amino acid residue Asn155 and
an interaction with Mg2+. The mean binding energy (MBE) was −3.00 kcal/mol, the estimate
free energy of binding (EFEB, ΔGbind) −3.14 kcal/mol and the estimated inhibition constant
(EIG, Ki) 5.01 mM. The latter showed (Figure 3b) an interaction with Mg2+, an MBE value of
−2.59 kcal/mol, a ΔGbind value of −2.64 kcal/mol and a Ki value of 11.59 mM. Thus, according
to these docking results, 9b is binding to the active site of IN.
Although the values found by the interactions docking of 9b structure are not particularly
strong; the study of this compounds is anyway interesting and goes in the right direction.
Also the docking results of 13 showed two possible arrangements. In one of those
arrangements, compound 13 seemed to be able to interact with the active site via hydrogen
bonding with the amino acid residue Asn64. Also Mg2+ seemed to form a coordination bridge
between the ester and epoxide groups; one of them shows (Figure 4a) that this compound
interacts with the active site by hydrogen bonding to the amino acid residue Asn64 and in
this bound orientation, Mg2+ ion forms coordination bridge between the epoxide ring and
the ester group. The mean binding energy (MBE) is −5.19 kcal/mol, the estimated free energy
of binding (EFEB, ΔGbind) is −6.55 kcal/mol, while the estimated inhibition constant (EIC, Ki)
is 15.72 µM. The second arrangement shows that the compound 13 interacts with two amino
acid residues in the active site, Asp 64 and Glu 152 (Figure 4b). The mean binding energy
Scheme 4. (a) dIad, tPP, thF, rt, 75%; (b) tBaF, thF, rt, 85%; (c) m-cPBa, ch2cl2, rt, 85%; (d) 4-hydroxy-3-
methoxybenzoyc acid, dIad, tPP, thF, rt, 48%.
Figure 3. Possible arrangements in the hIV-IN active site core domain of 9b.

6
G. RIGHI ET AL.
(MBE) is −4.53 kcal/mol, the estimated free energy of binding (EFEB, ΔGbind) is −4.88 kcal/
mol, while the estimated inhibition constant (EIC, Ki) is 262.79 µM. These results indicated
good interactions and tight binding with the active site of IN.
2.3. Metal titration studies
To investigate the interaction of the synthesised derivatives with divalent metal cations such
as Mg+2, preliminary UV–vis titrations were undertaken. Due to the poor solubility in water
of the compounds, the experiments were carried out in DMSO. Unfortunately, the spectra
of compounds 9a-i did not change upon addition of increasing amounts of Mg(OAc)2, sug-
gesting negligible interaction between the two partners. Therefore, we focused our attention
on derivative 13. UV–vis spectra recorded in the presence of increasing amounts of magne-
sium acetate in DMSO (Figure 5a) show strong variations in the absorption.
As the observed changes in the absorption spectra of 13 can be also attributed to the
deprotonation of the phenolic group by the acetate ion (Sarmini and Kenndler 1999), we
recorded the spectra of compound 13 adding increasing amounts of magnesium chloride
(Figure 5b). The hyperchromic effect observed upon Mg2+ addition together with a slight
bathochromic shift (c.a. 2 nm) and the appearance of a shoulder at 320 nm indicate the
Figure 4. Possible arrangements in the hIV-IN active site core domain of 13.
Figure 5. (a) absorption changes of compound 13 upon addition of Mg(oac)2. the dark curve indicates
the spectrum of 13. the arrows indicate how the absorption curve shift depends on the concentration of
the metal ion. (b) absorption changes of compound 13 upon addition of Mgcl2. the dark curve indicates
the spectrum of 13. Mg2+concentrations are the same as in the experiment with acetate salt.

NATURAL PRODUCT RESEARCH
% of inhibition HIV infection
7
Table 1. Inhibitory activity of the compound 13 on hIV infection.
Compound
CC50a Molt-3 (µM)
13 125 µM
241
25
6
1
87
80
70
13 75 µM
13 5 µM
raltegravir 125 µM
raltegravir 75 µM
raltegravir 5 µM
850
acc50 is the cytotoxic concentration 50% required to inhibit 50% metabolic activity evaluated in cell line Molt-3 by Mts.
occurrence of interactions between 13 and the metal ion. However, due to the presence on
the molecule of a number of different potential coordinating sites, a quantitative analysis
was prevented.
2.4. Biological results
In consideration of the results obtained in the metal titration studies, at the moment the
potential activity to inhibit HIV infection was tested only on compound 13. Raltegravir, the
first FDA-approved HIV-integrase inhibitor (Summa et al. 2008), was used as internal positive
control in the assay.
The results reported in Table 1 showed that compound 13 was endowed with cytotoxic
activity towards MOLT-3 cells, 3.5 times higher compared to Raltegravir. Dose effect studies
reveal that compound 13 at 125 μM was able to inhibit 25% of HIV infection, although the
IC50 (inhibitory concentration 50%) on HIV infection could not be tested by rising the con-
centration owing to its cytotoxic effect.
3. Conclusions
In this study, we have designed and synthesised two different model structures as potential
inhibitors of HIV integrase. The former was characterised by a DKA bioisostere, such as a
β-hydroxycarbonyl moiety, between two fragments containing aromatic groups, while in
the latter an epoxide linked two polyoxygenated aromatic residues. The moieties present
in the structures are thought to function by chelating divalent metal ions on the enzyme
catalytic site.
Although the preliminary molecular modelling studies indicated favourable interactions
and tight binding with the active site of IN for both structures, only the second showed a
promising ability to chelate the metal cations.
Preliminary biological assays on compound 13 showed that it was able to partially inhibit
HIV infection at a concentration in the micromolar range, although significantly less effi-
ciently than Raltegravir.
Disclosure statement
No potential conflict of interest was reported by the authors.

8
G. RIGHI ET AL.
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