Ruthenium arene complexes as HIV-1 integrase strand transfer inhibitors

Article abstract of DOI:10.1016/j.jinorgbio.2012.09.021

The quinolone HL¹ and the hydroxypyrimidine-carboxamide HL ² were designed and synthesized as models of the HIV integrase strand transfer inhibitors Elvitegravir and Raltegravir (brand name Isentress), with the aim to study their com

Full text of DOI:10.1016/j.jinorgbio.2012.09.021

Journal of Inorganic Biochemistry 118 (2013) 74–82
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Ruthenium arene complexes as HIV-1 integrase strand transfer inhibitors
a
a
a
a
Mauro Carcelli a, , Alessia Bacchi , Paolo Pelagatti , Gabriele Rispoli , Dominga Rogolino ,
⁎
Tino W. Sanchez c, Mario Sechi b, Nouri Neamati c
Dipartimento di Chimica, Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy
Dipartimento di Chimica e Farmacia, Università di Sassari, Via Vienna 2, 07100 Sassari, Italy
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA
a
b
c
a r t i c l e i n f o
a b s t r a c t
The quinolone HL1 and the hydroxypyrimidine-carboxamide HL2 were designed and synthesized as models of
the HIV integrase strand transfer inhibitors Elvitegravir and Raltegravir (brand name Isentress), with the aim
to study their complexing behavior and their biological activity. The Ru(arene) complexes [RuCl(η6-p-cym)L1],
[RuCl(η6-p-cym)L2] and [RuCl(hexamethylbenzene)L2] were also synthesized and spectroscopically character-
ized and their X-ray diffraction structures were discussed. The ligands and the complexes showed inhibition
potency in the sub/low-micromolar concentration range in anti-HIV-1 integrase enzymatic assays, with selectiv-
ity toward strand transfer catalytic process, without any significant cytotoxicity on cancer cells.
© 2012 Elsevier Inc. All rights reserved.
Article history:
Received 23 July 2012
Received in revised form 19 September 2012
Accepted 21 September 2012
Available online 28 September 2012
Keywords:
HIV integrase
Strand transfer inhibitor
Metal complex
Ru complex
1. Introduction
first IN inhibitor to be approved by the US FDA, in late 2007 (brand
name Isentress) [19]. The integration process promoted by IN consists
The pharmacological properties of metal-based compounds are a
topic of great interest in drug discovery, since they have found very im-
portant clinical applications [1,2]. The cytoxicity of metal complexes has
been extensively investigated [2,3], but the research on their antiviral
properties remains scarce [4]. In particular, ruthenium(II) arene com-
plexes have received much attention in the last years, because they
are promising candidates in cancer treatment [2,5,6], also thanks to
their low toxicity and the peculiar activity against platinum-resistant
tumors. On the contrary, in the literature very few studies are available
about the antiviral activity of ruthenium compounds and, in particular,
about their activity toward HIV-1 infection/replication [4,7–9]. There-
fore, in line with our recent research [10–13], we have decided to eval-
uate the activity of some ruthenium(II) η6-arene compounds in the
inhibition of the enzyme HIV-1 Integrase (IN).
IN, which catalyzes the integration of proviral cDNA into the host
cell genome [14–16], has emerged as a promising target in antiretrovi-
ral drug design, and it has been recently validated for the treatment of
HIV/AIDS. At least five IN inhibitors have been tested in HIV-infected
patients [17,18] and several others are currently under advanced pre-
clinical and long-term toxicity studies. Raltegravir (Chart 1) was the
of two distinct reactions, 3′-processing (3′-P) and strand transfer (ST).
In the first step the enzyme removes a terminal dinucleotide from the
viral DNA, generating two CA-3′-hydroxyl recessed ends, which are
the reactive intermediates required for the next step. The enzyme, still
bound to the 3′-processed viral DNA, translocates to the nucleus of
the infected cell as a part of the pre-integration complex. Here ST step
occurs, which consists of a trans-esterification reaction of the viral
DNA 3′-OH on the phosphodiester backbone of host DNA. In the active
site of IN there are two Mg2+ ions, which are fundamental for the activ-
ity of the enzyme: they are the target of the selective ST inhibitors such
as Raltegravir.
Starting from the β-diketoacid family of selective IN ST inhibitors
(for example L-731,988, Chart 1), the replacement of the keto-enolic
moiety and/or of the carboxylic group with bioisoster groups led to sev-
eral other classes of potent inhibitors, that retained selectivity for the ST
process. Among the others, there are (Chart 1) the keto-enol triazoles
(for example S-1360) [14], the naphthyridines (L-870,810) [20,21], the
hydroxypyrimidine-carboxamides (MK-0518 or Raltegravir) [22,23],
the quinolone-carboxylic acids (GS-9137 or Elvitegravir) [24,25], and
the pyrido-pyrazino oxazine derivatives (S/GSK1349572) [26]. All
these selective IN ST inhibitors share a common scaffold that is able to
chelate metal ions, in accordance with the “two-metal binding model”
[27] that emerged as an important strategy for the development of IN
inhibitors [28].
⁎
Corresponding author at: Dipartimento di Chimica, Università di Parma Campus
Universitario — V.le G.P.Usberti 17/A, 43124 Parma, Italy. Tel.: +39 0521 905427;
fax: +39 0521 905557.
Quinolones are well-known antibacterial agents, since they are
able to inhibit the bacterial DNA gyrase [29,30]. Their biological
E-mail address: mauro.carcelli@unipr.it (M. Carcelli).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.09.021

M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
75
Raltegravir
Elvitegravir
Chart 1. Representative HIV-1 IN ST inhibitors.
activities were extensively explored [31–33] and also their interac-
tions with metals [34–38], and in particular with Mg2+, that is prob-
ably involved in the mechanism of action [39]. However, few
examples of ruthenium complexes with this class of ligands are
known [40–43] and none of them has been tested for antiviral activ-
ity. As far as the coordinating behavior of the hydroxypyrimidine-
carboxamide moiety of Raltegravir is concerned, also in this case
few literature data can be found [13,44]. Moreover, the strategy to
use biologically active ligands with ruthenium arene moiety has
been already successfully exploited [45,46].
With these considerations in mind, we synthesized some new
ruthenium(II) arene compounds with ligands chosen as a model of the
quinolone Elvitegravir (HL1, 6-benzyl-1-(2-hydroxyethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid) and as a model of Raltegravir (HL2,
N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,
3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-
4-carboxamide) (Scheme 1). The model ligands are relatively easy to
prepare and yet they retain the pharmacophoric motif of the parent
HIV IN inhibitors [13].
and of the corresponding ruthenium p-cymene complexes as well as
their cytotoxic effect toward a model cancer cell line, were evaluated.
2. Experimental
2.1. Material and methods
Solvents and reagents were purchased from Sigma-Aldrich, Merck
or Carlo Erba and used without further purification. All reactions were
performed under a nitrogen atmosphere by using standard Schlenk
technique and syringes to transfer solutions. Purity of compounds
was determined by elemental analysis and verified to be ≥95% for
all synthesized molecules. Elemental analyses were performed by
using a Carlo Erba Model EA 1108 apparatus. Nuclear Magnetic Reso-
nance (NMR) spectra were recorded at 27 °C on a Bruker Avance
400 FT spectrophotometer by using SiMe4 as internal standard; the
assignment of exchangeable protons (OH and NH) was confirmed
by the addition of D2O. IR spectra were obtained with a Nicolet
5PCFT-IR spectrophotometer in the 4000–400 cm−1 range, in reflec-
tance mode on the powder. Mass spectra were obtained on an attenuat-
ed total electrospray ionization (ESI) time-of-flight Micromass 4LCZ
spectrometer (ESI/MS).
Herein we report on the synthesis and the characterization of
the organometallic compounds [RuCl(η6-p-cymene)L1] (3), [RuCl
(η6-p-cymene)L2] (4) and [RuCl(η6-hexamethylbenzene)L1] (5)
(Scheme 2), with the discussion of the crystal structure of (3) and
(4). Finally, the anti-HIV-1 IN enzymatic activity of the free ligands
6-Benzyl-4-oxo-1,4-dihydroquinolin-3-carboxylate ethyl ester (1)
(Scheme 3) and HL2 were synthesized as previously reported [13].

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M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
3N HCl solution until pH is neutral. The precipitate was filtered,
washed several times with water and dried under reduced pressure
to give a beige solid. Yield: 55%. M.p.: 195–197 °C (dec.). 1H NMR
(DMSO): δ 8.54 (s, 1H, _CH); 8.08 (s, 1H, Ar\H); 7.76 (d, 1H,
Ar\H); 7.64 (d, 1H, Ar\H); 7.28–7.21 (m, 5H, Ar\H); 5.00 (t, br,
1H, OH); 4.40 (t, 2H, CH2); 4.21 (s, 2H, CH2); 4.10 (s, 2H, CH2); 3.72
(q, 2H, CH2); 1.27 (t, 3H, CH3). Anal. Calcd. for C21H21NO4: C 71.78,
H 6.02, N 3.99. Found: C 71.60, H 6.41, N 4.02. ESI/MS (+, m/z):
352.4 [MH+].
Elvitegravir
6-Benzyl-1-(2-hydroxyethyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid, HL1
A solution of (2) (3.35 g, 10.9 mmol) and 2% NaOH (11 mL) was
refluxed for 3H and then acidified at r.t. by using 1N HCl. The precip-
itate was filtered off and recrystallized from water/ethanol giving rise
to a white powder. Yield: 97%. M.p.: 187–188 °C (dec.). IR (cm−1):
ν
OH =3312; νC_O =1697, 1610. 1H NMR (DMSO): δ 15.32 (s, br,
1H, OH); 8.86 (s, 1H, _CH); 8.23 (s, 1H, Ar\H); 8.02 (d, 1H,
Ar\H); 7.83 (d, 1H, Ar\H); 7.30–7.21 (m, 5H, Ar\H); 5.02 (t, br,
1H, OH); 4.60 (t, 2H, CH2); 4.17 (s, 2H, CH2); 3.74 (q, 2H, CH2). 13C
NMR (DMSO-d6): δ 178.2, 166.6, 144.8, 140.6, 139.9, 138.1, 135.1,
128.9, 128.7, 126.3, 124.5, 124.1, 120.0, 107.5, 40.6. Anal. Calcd. for
C
19H17NO4: C 70.58, H 5.30, N 4.33. Found: C 70.39, H 5.27, N 4.20.
ESI/MS (+, m/z): 324.3 [MH+]. Crystallization from chloroform
afforded crystals suitable for X-ray diffraction analysis.
[RuCl(η6-p-cym)L1], (3)
HL1 (100 mg, 0.3 mmol) was suspended in 15 ml of methanol,
and 12 mg (0.3 mmol) of NaOH (s) was added. The clear solution
was stirred 30 min at r.t. and then 92 mg (0.15 mmol) of
[Ru(p-cym)Cl2]2 (s) was added. The orange solution was stirred at
r.t. overnight, then the solvent was eliminated. The residue was
dissolved in dichloromethane and filtered on celite. The filtrate was
collected and the solvent was removed under vacuum. The crude
product was triturated with n-pentane to afford a yellow powder.
Yield: 54%. IR (cm−1): νOH =3733; νC_O =1614, 1587, 1479. 1H
NMR (CD2Cl2): δ 8.82 (s, 1H, _CH), 8.31 (s, 1H, Ar\H), 7.62 (s, 2H,
Ar\H), 7.33–7.26 (m, 5H, Ar\H), 5.56 (d, 2H, p-cym), 5.27 (d, br,
2H, p-cym), 4.90 (s, br, OH), 4.37 (s, 2H, CH2), 4.17 (s, 2H, CH2),
3.84 (s, 2H, CH2), 2.92 (m, 1H, CH p-cym), 2.10 (s, 3H, CH3 p-cym),
1.33–1.24 (d, 6H, (CH3)2CH p-cym). Anal. Calcd. for C29H30ClNO4
Ru·H2O: C 57.00, H 5.28, N 2.29. Found: C 57.35, H 5.60, N 2.00. ESI/
MS (+, m/z): 558.2 [M\Cl]+. Crystals suitable for X-ray diffraction
analysis were obtained by slow evaporation of a solution of (3) in
dichloromethane/n-pentane.
Raltegravir
Scheme 1. Chemical structures of the model ligands HL1 and HL2.
2.2. Synthesis
6-Benzyl-1-(2-hydroxyethyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate
ethyl ester, (2)
To a solution of (1) (0.61 g, 1.98 mmol) in anhydrous N,N-
dimethylformamide (13 mL), 60% sodium hydride in oil dispersion
(1.5 eq, 0.071 g, 2.98 mmol) and iodoethanol (1.5 eq, 0.510 g,
2.97 mmol, 0.231 mL) were added at room temperature. The
resulting mixture was stirred at 90 °C for 24 h. The reaction was
cooled and quenched with water. The mixture was acidified with a
[RuCl(η6-p-cym)L2], (4)
HL2 was deprotonated before reacting with Ru(II): the ligand was
suspended in water and 1.3 equivalents of NaOH (s) were added.
The clear solution was stirred at r.t. for 30 min, then the solvent
was eliminated under vacuum. The crude solid was dissolved in
Ar
F
Ru
H
N
O
N
Cl
Ru
O
Cl
O
O
O
N
N
O
OH
Ar=p-cymene(4)
(3)
hexamethylbenzene(5)
Scheme 2. The Ru(II)–arene complexes.

M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
77
O
N
O
O
COOH
COOEt
i)
COOEt
ii)
N
H
N
1
(1)
HL
(2)
OH
OH
Scheme 3. Preparation of the ligand HL1. Reagents and conditions: i) NaH 60%, ICH2CH2OH, dry DMF, 90 °C for 24 hours; ii) NaOH 2%, refluxing 3 h, then HCl 1N.
dichloromethane, filtered, and the solvent was then eliminated. The
residue was triturated with diethyl ether. The sodium salt of the
ligand (80 mg, 0.23 mmol) was dissolved in 10 mL of methanol and
solid [RuCl2(η6-p-cym)]2 (72 mg, 0.11 mmol) was added. The yellow
solution was stirred at r.t. overnight, the solvent was eliminated, the
residue was dissolved in dichloromethane and filtered on celite. The fil-
trate was collected, the solvent was removed under vacuum and then
the crude product was triturated with diethyl ether to afford an orange
powder. Yield: 72%. IR (cm−1): νNH=3338; νC_O=1666, 1595. 1H
NMR (CDCl3): major isomer, δ 9.54 (s, br, 1H, NH), 7.40–7.30
(m, overlapping signals, Ar\H), 7.09–7.02 (m, overlapping signals,
Ar-H), 5.52 (m, overlapping signals, CH p-cym), 5.24 (m, overlapping sig-
nals, CH p-cym), 4.90 (dd, diastereotopic CH2), 4.38 (dd, diastereotopic
CH2), 3.66 (s, 3H, CH3), 3.0–2.86 (m, overlapping signals, CH), 2.78–2.74
(m, 1H, CH), 2.18 (s, 3H, CH3 p-cym), 1.34–1.29 (dd, 12H, (CH3)CH ligand
and (CH3)2CH p-cym). Minor isomer, δ 8.29 (s, br, NH), 4.62 (d, CH2), 3.45
(s, CH3), 2.27 (s, CH3 p-cym), 1.17–1.15 (m, (CH3)2CH p-cym). 19F
{1H}-NMR (CDCl3): δ major isomer, −115.88; minor isomer, −114.97.
Anal. Calcd. for C26H31ClFN3O3Ru·0.5H2O: C 52.21, H 5.39, N 7.02.
Found: C 52.15, H 5.34, N 6.76. ESI/MS (+, m/z): 589.9 [MH]+; 553.1
[M\Cl]+; 454.8 [M-p-cym]+. Crystals suitable for X-ray diffraction
analysis were obtained by slow evaporation of a solution of (4) in
dichloromethane/n-hexane.
CH3 hexamethylbenz.), 1.36–1.27 (dd, 6H, (CH3)2CH). Minor isomer,
δ 8.32 (s, br, NH), 4.66 (d, CH2), 3.45 (s, CH3), 2.92 (m, br, 1H,
(CH3)2CH), 1.19 (dd, (CH3)2CH). 13C{1H}-NMR (CD2Cl2): major isomer,
169.6; 164.4; 163.3; 160.9; 154.8; 147.4; 135.20; 130.2, 130.1; 115.4;
115.2;88.7; 42.7; 31.0; 31.0 (overlapping signals); 20.9 (overlapping
signals); 15.5; minor isomer: 168.0; 162.1; 153.7; 147.1; 133.7; 128.7;
128.6; 115.6; 115.5; 90.3; 43.0; 14.1. 19F{1H}-NMR (CD2Cl2): δ major
isomer, −116.56; minor isomer, −116.23. Anal. Calcd. for C30H39ClFN3
O3Ru·0.5H2O: C 53.71, H 5.80, N 6.71. Found: C 53.70, H 5.89, N 6.57.
2.3. X-ray crystallography
Single crystal X-ray diffraction data were collected at T=293 K
using the MoKα radiation (λ=0.71073 Å) on a SMART APEX2 diffrac-
tometer for (3) and (4), and on a Siemens AED diffractometer equipped
with scintillation detector and CuKα radiation (λ=1.54178 Å) for HL1.
Lorentz, polarization, and absorption corrections were applied [47,48].
Structures were solved by direct methods using SIR97 [49] and refined
by full-matrix least-squares on all F2 using SHELXL97 [50] implemented
in the WinGX package [51]. Hydrogen atoms were introduced in calcu-
lated positions for (3) and (4), and were found on difference maps and
refined for HL1. Anisotropic displacement parameters were refined for
all non-hydrogen atoms. Molecule (4) is chiral on the ruthenium atom
and undergoes spontaneous resolution by crystallizing in the acentric
P21 monoclinic space group. However the crystals were all affected by
severe racemic twinning, that was refined by the TWIN/BASF instruc-
tions in Shelxl; the twinned component refined to 0.65/0.35 and resid-
uals reproducing the centrosymmetric ghost image were found in the
final Fourier difference map. Hydrogen bonds have been analyzed
with SHELXL97 [50] and PARST97 [51] and extensive use was made of
the Cambridge Crystallographic Data Centre packages [52,53] for the
[RuCl(hexamethylbenzene)L2], (5)
The same as (4), by using [RuCl2(hexamethylbenzene)]2. Orange
powder. Yield: 75%. IR (cm−1): νNH=3225; νC_O=1652, 1598. 1H
NMR (CD2Cl2): major isomer, δ 9.73 (s, br, 1H, NH), 7.44–7.36
(m, overlapping signals, Ar\H), 7.10 (m, br, overlapping signals,
Ar\H), 4.91 (dd, 2H, diastereotopic CH2), 4.35 (dd, 2H, diastereotopic
CH2), 3.65 (s, 3H, CH3), 3.0 (m, br, 1H, CH), 2.0 (s, overlapping signals,
Table 1
Crystal data and structure refinement for HL1, (3) and (4).
HL1
(4)
(3)
Empirical formula
Formula weight
C19H17NO4
323.34
C26H31ClFN3O3Ru
589.06
C29H30ClNO4Ru
593.06
Crystal system
Space group
monoclinic
P21/c
monoclinic
P21
triclinic
P-1
a/Å
b/Å
c/Å
α/°
β/°
γ/°
22.861(2)
4.9769(1)
14.0769(8)
10.3817(7)
10.1538(7)
12.7994(9)
12.662(2)
13.369(2)
18.519(3)
69.875(3)
81.063(3)
61.999(3)
2598.9(8)
4
87.102(2)
100.009(1)
Volume/Å3
Z
1599.6(2)
4
1.343
1328.7(2)
2
1.472
ρ
calc mg/mm3
1.516
0.742
μ/mm−1
0.777
0.729
F(000)
680.0
604.0
1216.0
2θ range for data collection
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R1, wR2 [I>=2σ (I)]
Final R1, wR2 [all data]
Largest ΔF max/min e Å−3
7.74–139.78°
2935
2869 [R(int)=0.0314]
2869/0/285
0.941
0.0451, 0.1054
0.0746, 0.1221
0.13/−0.18
3.24–52.74°
15965
5413 [R(int)=0.0314]
5413/17/293
1.060
0.0679, 0.1938
0.0789, 0.2137
2.21/−0.51
3.64–48.5°
19584
8335 [R(int)=0.0481]
8335/0/651
1.037
0.0570, 0.1487
0.0856, 0.1680
1.40/−0.69

78
M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
+
+
Ru
Ru
O
H2O
Ru
O
Cl-
+2HL1
O
Cl-
Cl
HO
OH
O
OH2
OH
Ru
(3)
Scheme 4. Hydrolysis of complex (3).
analysis of crystal packing. Table 1 summarizes crystal data and struc-
ture determination results. Crystallographic data (excluding structure
factors) for HL1, (4) and (3) have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication CCDC
878234–878236. Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(fax: (+44) 1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
1 h. Reactions were quenched by the addition of an equal volume
(16 μL) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025%
xylene cyanol and 0.025% bromophenol blue). An aliquot (5 μL) was
electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-
borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8 M urea).
Gels were dried, exposed in a PhosphorImager cassette, analyzed
using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences)
and quantitated using ImageQuant 5.2. Percent inhibition (% I) was cal-
culated using the following equation:
2.4. Biological materials, chemicals, and enzymes
% I ¼ 100 X ½1−ðD−CÞ=ðN−CÞꢀ
All compounds were dissolved in DMSO and the stock solutions were
stored at −20 °C. The γ[32P]-ATP was purchased from PerkinElmer. The
expression system for wild-type IN was a generous gift of Dr. Robert
Craigie, Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD.
where C, N, and D are the fractions of 21-mer substrate converted to
19-mer (3′-proc product) or ST products for DNA alone, DNA plus IN,
and DNA plus IN plus drug, respectively. The IC50 values were deter-
mined by plotting the logarithm of drug concentration versus percent
inhibition to obtain concentration that produced 50% inhibition.
2.5. Preparation of oligonucleotide substrates
The oligonucleotides 21top, 5′-GTGTGGAAAATCTCTAGCAGT-3′
and 21bot, 5′-ACTGCTAGAGATTTTCCACAC-3′ were purchased from
Norris Cancer Center Core Facility (University of Southern California)
and purified by UV shadowing on polyacrylamide gel. To analyze the
extent of 3′-P and ST using 5′-end labeled substrates, 21top was
5′-end labeled using T4 polynucleotide kinase (Epicentre, Madison,
WI) and γ [32P]-ATP (Amersham Biosciences or ICN). The kinase
was heat-inactivated and 21bot was added in 1.5-molar excess. The
mixture was heated at 95 °C, allowed to cool slowly to room temper-
ature, and run through a spin 25 mini-column (USA Scientific) to sep-
arate annealed double-stranded oligonucleotide from unincorporated
material.
2.7. Cell culture
The MCF7 breast cancer cell line was purchased from the American
Type Cell Culture. Cells were maintained in culture under 35 passages
and tested regularly for mycoplasma contamination using Plasmo
TestTM (InvivoGen). Cells were maintained as monolayer cultures in
Dulbecco's Modified Eagle Medium (DMEM) containing 10% heat-
inactivated fetal bovine serum and supplemented with 2 mmol/L L-
glutamine at 37 °C in a humidified atmosphere of 5% CO2. For subcul-
ture and experiments cells were washed with 1× DPBS, detached
using 0.025% Trypsin-EDTA (Cellgro), collected in growth media and
centrifuged. All experiments were performed in growth media using
subconfluent cells in the exponential growth phase.
2.6. Integrase assays
To determine the extent of 3′-P and ST, wild-type IN was
preincubated at a final concentration of 200 nM with the inhibitor in re-
action buffer (50 mM NaCl, 1 mM HEPES, pH 7.5, 50 μM EDTA, 50 μM
dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/ml bovine
serum albumin, 10 mM 2-mercaptoethanol, 10% DMSO, and 25 mM
MOPS, pH 7.2) at 30 °C for 30 min (HEPES, 4-(2-hydroxyethyl)-1-
piperazine ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic
acid). Then, 20 nM of the 5′-end 32P-labeled linear oligonucleotide
substrate was added, and incubation was continued for an additional
2.8. Cytotoxicity assays
Cytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as previously described
[54]. Briefly, cells were seeded in 96-well microtiter plates and
allowed to adhere overnight. Cells were subsequently treated with
continuous exposure to the corresponding drug for 72 h. A MTT solu-
tion (at a final concentration of 0.5 mg/mL) was added to each well,
and cells were incubated for 4 h at 37 °C. After removal of the
Fig. 1. Molecular structure and labeling of HL1, with thermal displacement ellipsoids drawn at the 50% probability level (left), and crystal packing with hydrogen bonds displayed as
dashed lines (right).

M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
79
medium, DMSO was added and the absorbance was read at 570 nm.
All assays were done in triplicate. The CC50 was then determined for
each drug from a plot of log (drug concentration) versus percentage
of cells killed.
presence of two species (see Experimental section). In particular, in
the 13C{1H}-NMR spectrum of (5) there are two sets of signals, but no
shift of the tertiary amide carbon is observed in comparison with the
free ligand. The same situation was found in the 13C{1H}-NMR spectrum
of an X-ray characterized Mg(II) complex, where HL2 is deprotonated
and forms a six-membered chelation ring [13]. This excludes that one
of the two species present in the solution of (5) can originate by the
involvement of the tertiary amide moiety in the coordination to the
metal center, with the formation of a five-membered chelation ring
(Scheme 2, inset).
3. Results and discussion
3.1. Chemistry
HL1 was prepared according to Scheme 3, while HL2 was synthesized
as previously reported [13]. The ruthenium(II) complexes (3), (4) and
(5) (Scheme 2) were obtained by reacting in methanol at room tem-
perature [RuCl2(η6-p-cym)]2 or [RuCl2(η6-hexamethylbenzene)]2 with
two equivalents of the ligand, that was previously deprotonated with
NaOH. The Ru(II) complexes have been isolated as yellow–orange pow-
ders in high yields and they show good solubility in chlorinated and
alcoholic solvents. Their full characterization has been obtained by spec-
troscopic methods (NMR, IR), MS, elemental analysis and by single crys-
tal X-ray diffraction analysis.
The data clearly indicate that the ligands are anionic and
bidentate, giving rise to pseudo-octahedral organometallic com-
pounds (Scheme 2). In particular, HL2 has two different compartments
that can be involved in coordination, giving rise to five- or six-
membered chelation rings (Scheme 2, inset). In the IR spectra of (4)
and (5), the carbonylic stretching absorption of the secondary amide
shifts to lower wave numbers (from 1636 cm−1 in HL2 to 1595 and
1598 cm−1 in (4) and (5), respectively), as observed in other complexes
that we have previously synthesized [13]. This is in accord with the in-
volvement of the secondary amide group in the coordination to the
metal ion. Effectively, the X-ray diffraction analysis of (4) (vide infra)
confirms that the deprotonated ligand is coordinated to Ru(II) through
the hydroxyl oxygen and the carbonyl oxygen of the secondary amide,
while the tertiary amide is excluded by the coordination sphere. The
pseudo-octahedral coordination of the metal is then completed by a
chloride ion and by the p-cymene moiety.
It seems reasonable to conclude that the two sets of signals in the
spectra of (4) and (5) are due to the fact that the rotation of the
p-fluorobenzyl amide chain is hindered by the arene bound to the ru-
thenium ion.
In the 1H-NMR spectrum of (3) only one set of signals is present
and the acidic proton is absent. In the IR spectra, the absorption of
the ν(C_O) at 1697 cm−1 of the free quinolone HL1 was replaced
by two strong bands at 1614 and 1479 cm−1, attributable to the
asymmetric and symmetric ν(O\C\O) vibrations, respectively. The
pyridone stretching vibration is shifted upon coordination from
1610 cm−1 to 1587 cm−1. The changes in the IR spectra suggest
that the mono-deprotonated ligand HL1 chelates Ru(II) through the
pyridone and one carboxylate oxygen [13]; the coordination sphere
is then completed by a chlorine ion and the p-cymene moiety, as ob-
served in other ruthenium(II) complexes with quinolone ligands
[40,41]. The structure of (3) was confirmed by X-ray analysis on sin-
gle crystal (vide infra).
The solution behavior of ruthenium(II) organometallic com-
pounds in the presence of water is very important, since it can be re-
lated to their biological activity. Compound (4) was dispersed in D2O,
where it is not completely soluble. In its 1H and 19F{1H}-NMR spectra
there are the signals of a third species containing the ligand still
complexed with Ru(II). No variations were observed in the spectra
up to 4 days. It is reasonably to suppose that the third set of signals
arises from the formation of an aqua-complex, where a water mole-
cule replaces the chlorine ion in the coordination sphere of the
metal [40,41].
Compound (3) was dispersed in D2O as well. In this case, in its
1H-NMR spectrum it is possible to observe the presence of signals that
can be attributed to a hydroxo-bridged Ru(II) dimer, that is the product
of the hydrolysis of the complex, as for analogous half-sandwich ruthe-
nium complexes (Scheme 4) [40,41]. In fact, in addition to the signals of
(3), in the p-cymene region there are two doublets at 5.27 and
5.05 ppm, a multiplet at 2.56 ppm and a doublet at 1.11 ppm. The
hydroxo-complex forms immediately, with a ratio of about 0.75:1 be-
tween (3) and the new species (about 0.55:1 after 24 h).
The 1H-NMR spectrum of (4) is rather interesting. Apart from the
absence of the OH proton and the variation of the chemical shift due
to the complexation, in (4) Ru is a chiral center, so that the benzylic
CH2 protons are diasterotopic and produce an AB quartet. Moreover,
there are two sets of signals, probably due to two different isomers.
The presence of two species is confirmed by 19F-NMR in CDCl3,
where two signals are detected (at −115.88 and −114.97 ppm for
the major and minor isomers, respectively; −114.42 ppm in the
free ligand). Crystals of (4) were dissolved in CD2Cl2 and 1H and 19F
{1H}-NMR spectra were immediately recorded: they are identical to
that obtained by dissolution of the powder. Finally, the spectra were
recorded at different temperatures and the ratio between the two iso-
mers changes from 1:3 at −30 °C to 1:2 at +50 °C.
3.2. X-ray crystallographic studies
In order to simplify the arene on the Ru(II) ion, the
hexamethylbenzene analog (5) has been synthesized. The 1H- and 19F
{1H}-NMR spectra of (5) are analogous to the ones of (4), showing the
The molecular and crystal structures of the free ligand HL1 are
shown in Fig. 1. The benzyl and the hydroxyethyl groups confer a
Fig. 2. Molecular structure and labeling of one molecule of (3), with thermal displacement ellipsoids drawn at the 50% probability level (left), and crystal packing with hydrogen
bonds displayed as dashed lines (right).

80
M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
skewed shape to the molecule, whose core is constituted by the planar
quinolone system; in fact both are almost perpendicular to the mole-
cular core (C9\C8\C11\C12=101°; C6\N1\C18\C19=99°), and
point out of the same face of the fused rings. The bond geometry is
not significantly different from the average values for similar systems
[55]. The carboxylic acid OH group is involved in a hydrogen bond,
forming an intramolecular six-membered ring (O1\H…O3=2.503
(2)Å, 153(3)°) that locks the \COOH potentially rotatable group in
the plane of quinolone. The remaining hydrogen bond hydroxyl donor
and carboxylic acceptor of HL1, are employed in an intermolecular
hydrogen bond that arranges the crystal packing in arrays along the
c axis (Fig. 1, O4\H…O2(x, 1/2−y, 1/2+z)=2.709(2)Å, 164(3)°).
The molecular structure of the corresponding ruthenium complex
(3) is shown in Fig. 2. The ligands are arranged around ruthenium in a
distorted octahedron, with the p-cymene ring occupying three posi-
tions of the polyhedron of coordination. Two other positions are occu-
pied by the chelating system of HL1, comprising the deprotonated
oxygen of the carboxylic acid and the 4-oxo group. The remaining posi-
tion is occupied by one chloride ligand. Complex (3) crystallizes with
two independent molecules in the asymmetric unit, that slightly differ
only for the orientation of the p-cymene around the bond axis between
ruthenium and the center of the p-cymene ring (Cl\Ru\C\CH3=71
and 102° in the two cases). The co-crystallization of crystallographic in-
dependent conformers differing only in the orientation of the p-cymene
ring for ruthenium half-sandwich complexes is related to the known
fluxionality of the ruthenium–arene bond in solution, and it has already
been observed in similar cases [56,57]. Similar to the intramolecular
hydrogen bond in the free ligand, here the chelation on ruthenium
locks the carboxylate group in the plane of the quinolone system,
maintaining the planarity of the molecular core. However, the chelation
of ruthenium produces a slight angular deformation of the system, since
the C12\C13\O1 bond angle in (3) widens (126.9(6) and 125.4(6)° for
the two molecules) with respect to the corresponding C2\C3\O3 in
HL1 (122.3(2)°). The six-membered chelation ring adopts an envelope
conformation in both independent molecules, forcing the two donor ox-
ygens slightly out of the molecular plane. The ruthenium atoms are 0.64
and 0.50 Å out of the chelation planes in the two cases. Regarding the
general molecular shape, the benzylic and hydroxyethyl substituents
are nearly perpendicular to the quinoline ring, but with respect to the
free ligand the hydroxyethyl group is rotated on the opposite side
(C21\C20\C23\C24=106 and 101°; C16\N1\C17\C18=−96
and −93° respectively for the two independent molecules). The
hydroxyl groups of the two independent molecules form hydrogen
bonds to the coordinated oxygen of the carboxylate groups originating
arrays along which the two independent molecules alternate in the
crystal packing (Fig. 2).
Fig. 3. Molecular structure and labeling of (4), with thermal displacement ellipsoids
drawn at the 50% probability level.
comparison of (3) and (4) shows how the different chelation sites af-
fect the overall molecular shape (Fig. 4). The most significant supra-
molecular features in the crystal packing of (4) are the NH…Cl
hydrogen bonds between the amidic NH group and the ruthenium-
bound chloride (N1\H…Cl1(1−x, 1/2+y, 1−z)=3.439(8) Å, 129°).
It is well recognized that metal bound halides are very good hydrogen
bond acceptors [58] and similar examples of hydrogen bonds to chlo-
rides in half-sandwich ruthenium units have been reported recently
[59].
3.3. Inhibition of HIV-1 IN
The ligand HL1 and the complexes (3) and (4) were tested for their
ability to inhibit 3′-P and ST catalytic activities by employing purified
enzyme (Fig. 5). All the compounds showed inhibition potency in sub
to low micromolar concentration range (Table 2); [RuCl2(p-cym)]2
was evaluated for comparison, and it was found inactive up to
500 μM. The ligand HL2 (IC50, ST=0.14 0.03 μM; 3′-P=10 8 μM)
Complex (4) crystallizes in the acentric P21 space group, due to a
spontaneous resolution of the chiral centers on the ruthenium atom.
The molecular structure is reported in Fig. 3. The coordination around
ruthenium is the same distorted octahedron observed for (3), where-
by in (4) the chelation is attained by means of the amidic and of the
4-oxo oxygen donors. Due to twinning problems discussed in the
Experimental section, the bond geometry is not accurate enough to
be compared with related compounds; however it can be noted that
the angle C14\C19\O1=132.2(8)° is broadened by the chelation,
similarly to complex (3). The more flexible nature of the HL2 ligand
with respect to HL1 results in a general higher deviation from planar-
ity of the complex. The ligand skeleton is much less planar than the
one observed for the quinolone ligand in (3), with deviations up to
0.51 Å (O3) and rms deviation of 0.20 Å compared to a rms deviation
of 0.06 Å and 0.12 Å with maximum displacements of 0.13 Å (O2)
and 0.24 Å (O5) for the two molecules of (5). The six-membered che-
lation ring has a slightly twisted envelope conformation with rutheni-
um out of the chelation plane by 0.77 Å. Similarly to HL1 and to (3),
the p-fluoro benzylic substituent is almost perpendicular to the
average molecular plane (C19\N1\C20\C21=−96°). The direct
Fig. 4. Comparison of the molecular structures of (3) (blue) and (4) (gray). Hydrogen
atoms are omitted for clarity.

M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
81
Fig. 5. Schematic representation of IN activity in vitro and representative gels showing inhibition of purified IN by HL1, (3) and (4). a) A 21-mer blunt-end oligonucleotide corre-
sponding to the U5 end of the HIV-1 LTR, 5′ end-labeled with 32P, is reacted with purified IN. The first step (3′-P) involves nucleolytic cleavage of two bases from the 3′-end,
resulting in a 19-mer oligonucleotide. Subsequently, 3′ ends are covalently joined at several sites to another identical oligonucleotide that serves as the target DNA (ST). The prod-
ucts formed by this reaction migrate slower than the original substrate on a polyacrylamide gel. b) Line named as DNA indicates DNA alone; line termed INT indicates IN and DNA
with no drug; other lines: IN, DNA and selected drug concentrations (μM) as indicated in each line.
[13] is about 20 fold more potent than the quinolone HL1 (IC50, ST=
3.1 1.1 μM; 3′-P=93 9 μM), confirming a trend that was already
observed for similar compounds [13]. (4) (IC50, ST=1.8 0.78 μM;
3′-P=44 16 μM) resulted the most active complex, even if in the ST
process it is about 13 fold less active than the corresponding free ligand
HL2. (4) retained selectivity towards ST, with a selectivity index of about
25 (71 for HL2, Table 2). Also for (3), there is a lowering of the activity in
the ST process inhibition of about 5–6 fold with respect to the corre-
sponding free ligand. While (3) and (4) are less active than the corre-
sponding free ligands, the bis-chelate complexes [ML22]·nH2O (M=
Mg(II), Mn(II), Co(II) and Zn(II)) and [ML2]·nH2O (M=Mg(II) and
Mn(II); HL: 6-benyl-4-oxo-1,4-dihydroquinolin-3-carboxylic acid, a
quinolone similar to HL1) showed an analogous or even a better inhibi-
tion profile [13].
HL1, HL2, (3) and (4) were also tested for their cytotoxicity against a
model breast cancer cell line (MCF7, Table 2): none of them showed
antiproliferative activity (CC50>20 μM) at concentration values higher
than their enzymatic inhibition activity values.
It is worth noting that (3) and (4) are, as far as we know, the first
ruthenium complexes with a significant activity against HIV-1 IN.
4. Conclusions
The new ruthenium complexes (3), (4) and (5) were synthesized,
characterized and the X-ray diffraction structures of (3) and (4) were
discussed. (3) was one of the few examples of ruthenium quinolone
complexes, a class of compounds recently studied also for their anti-
cancer properties [41]. Although (3) and (4) demonstrated good inhi-
bition against HIV IN, the Ru(II) arene moiety did not contribute to
enhance significantly the intrinsic activity of the parent ligands HL1
and HL2. However (3) and (4) resulted among the few Ru(II) com-
plexes active against HIV [7–9] and, in particular, the first ones that
were active against HIV IN. The activity of (3) was influenced by the
partial hydrolysis of the complex in aqueous solution (see NMR ex-
periments) with release of HL1. On the other hand, the activity of
(4), that was quite stable in aqueous solution, seemed to be due to
the complex, that evidently was able to influence HIV IN activity.
The metal complexes can inhibit the activity of HIV-1 IN by carry-
ing the ligand inside the active site of the enzyme, where the ligand
can interfere with the magnesium metal cofactors. Nevertheless, on
the basis of the available data, assertions about the mechanism of in-
hibition of HIV IN by the metal complexes are inevitably speculative.
Previously, it was shown that ruthenium(II) arene complexes can
show significant cytotoxicity in several cancer cell lines [2–6]. Therefore,
Table 2
Inhibition of HIV-1 IN activity and cytotoxicity of ligands and complexes.
Compound
HIV-1 integrase inhibition IC50 (μΜ)
aSI
bMCF7
cCC50 (μM)
3′-Processing
Strand transfer
HL1
HL2
(3)
(4)
93
10
>100
9
8
3.1 1.1
0.14 0.03
30
>20
>20
>20
>20
71.4
>5.9
24.4
17
7
44 16
1.8 0.78
a
SI, Selectivity Index.
MCF7, breast cancer cell line.
CC50, cytotoxic concentration 50%.
b
c

82
M. Carcelli et al. / Journal of Inorganic Biochemistry 118 (2013) 74–82
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