Investigating the role of metal chelation in HIV-1 integrase strand transfer inhibitors

Article abstract of DOI:10.1021/jm200851g

HIV-1 integrase (IN) has been validated as an attractive target for the treatment of HIV/AIDS. Several studies have confirmed that the metal binding function is a crucial feature in many of the reported IN inhibitors. To provide new insights on the metal

Full text of DOI:10.1021/jm200851g

Article
pubs.acs.org/jmc
Investigating the Role of Metal Chelation in HIV-1 Integrase Strand
Transfer Inhibitors
Alessia Bacchi,† Mauro Carcelli,† Carlotta Compari,‡ Emilia Fisicaro,‡ Nicolino Pala,§ Gabriele Rispoli,†
Dominga Rogolino,*,† Tino W. Sanchez,∥ Mario Sechi,§ Valentina Sinisi,† and Nouri Neamati∥
‡
†Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica and Dipartimento di Scienze Farmacologiche,
Biologiche e Chimiche Applicate, Universita
̀
di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy
§Dipartimento di Scienze del Farmaco, Universita
̀
di Sassari, Via Muroni 23/A, 07100 Sassari, Italy
∥Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal
Avenue, Los Angeles, California 90089, United States
S
* Supporting Information
ABSTRACT: HIV-1 integrase (IN) has been validated as an attractive target for the treatment of HIV/AIDS. Several studies
have confirmed that the metal binding function is a crucial feature in many of the reported IN inhibitors. To provide new insights
on the metal chelating mechanism of IN inhibitors, we prepared a series of metal complexes of two ligands (HL1 and HL2),
designed as representative models of the clinically used compounds raltegravir and elvitegravir. Potentiometric measurements
were conducted for HL2 in the presence of Mg(II), Mn(II), Co(II), and Zn(II) in order to delineate a metal speciation model.
We also determined the X-ray structures of both of the ligands and of three representative metal complexes. Our results support
the hypothesis that several selective strand transfer inhibitors preferentially chelate one cation in solution and that the metal
complexes can interact with the active site of the enzyme.
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 preintegration
complex. Here the strand transfer step occurs, which consists of
a trans-esterification reaction of the viral DNA 3′-OH on the
phosphodiester backbone of the host DNA.
Magnesium ion is an essential cofactor in numerous enzymes
such as polymerases, exonucleases, ribonucleases, transposases,
and integrases and in many processes involving formation and
modification of phosphate chains.9 Understanding the mech-
anism of action of the inhibitors that act by chelating
magnesium is therefore of great importance in order to develop
new drugs not only against IN but also against other viral
metalloenzymes (i.e., hepatitis C virus polymerase and
influenza endonuclease).9,10
INTRODUCTION
■
HIV-1 integrase (IN) is one of the three virally encoded
enzymes responsible for the retroviral life cycle, together with
protease and reverse transcriptase. IN catalyzes the integration
of proviral cDNA into the host cell genome, a crucial step for
viral replication.1−3 Thus far, at least five IN inhibitors have
been tested in HIV-infected patients4,5 (Chart 1), and several
others are under advanced preclinical and long-term toxicity
studies. Raltegravir is the first IN inhibitor to be approved by
the U.S. FDA.6
IN is composed of three domains: the C-terminal domain,
responsible for nonspecific interaction with DNA; the N-
terminal domain, containing a “zinc-binding motif” that is
responsible for multimerization of the protein; the catalytic
core domain. The last one is highly conserved among
polymerases and polynucleotidyl transferases and comprises
the so-called “D,D(35)E” motif, a triad of amino acids that
coordinates two divalent metal ions,7 probably Mg2+ ions.8 The
integration process promoted by IN consists of two distinct
reactions: 3′-processing and strand transfer. In the first step the
Received: June 30, 2011
Published: November 8, 2011
© 2011 American Chemical Society
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Chart 1. Selected DKA-Based and Representative HIV-1 Integrase Inhibitors
Previously, several potent IN inhibitors based on the β-diketo
acids (DKAs) scaffold have been identified.11−13 The general
features of these inhibitors include a β-diketo moiety, an
aromatic or heteroaromatic portion, and a carboxylic
functionality (Chart 1). Several studies14−16 have pointed out
the importance of the hydrophobic part for activity, probably
because it allows the correct accommodation of the molecule
within the active site through a series of interactions with the
protein side chain. The replacement of the keto−enolic moiety
and/or of the carboxylic group with their bioisosteres led to
other classes of inhibitors, such as the keto−enol triazoles (S-
1360)1−3,17,18 or the more recent naphthyridinecarboxamide
(L-870,810),19,20 hydroxypyrimidinecarboxamide (HPCA, MK-
0518),21−24 quinolone carboxylic acid (QCA, GS-9137),25−28
and the pyridopyrazinooxazine derivatives (S/GSK1349572)29
(Chart 1). Selective IN strand transfer inhibitors (SSTI) share a
common scaffold that is able to chelate divalent metal ions, in
accordance with the “two-metal binding model”30 that emerged
as an important strategy for the development of new IN
inhibitors.13,31 According to this model, the DKA-based
pharmacophore is involved in the sequestration of the
magnesium cofactors, preventing the interaction between the
viral protein and the DNA substrate and thus blocking the
enzymatic action at the strand transfer stage. The X-ray crystal
structure of the full-length prototype foamy virus enzyme in
complex with the viral DNA, in the absence and in the presence
of inhibitors (raltegravir and elvitegravir), supports these
considerations, since the drugs are located within the catalytic
core with their chelating moieties oriented toward the metal
ions.32 The capability of the classical diketo acid motif to
chelate two divalent metal ions at physiological conditions were
elucidated by our33,34 and other studies.35 In particular, these
studies suggested that DKA could act when complexed with a
metal ion and not only as a free ligand.
are able to form complexes with two metal ions as DKAs or if
they preferentially form coordination compounds with other
stoichiometries. The answer is obviously important to clarify
the metal chelating mechanism and the activity of these potent
IN inhibitors.
Quinolones have been widely used as antibiotics because
they are able to selectively inhibit the bacterial DNA
gyrase.36−38 Their interaction with metals was investigated by
crystallographic and spectroscopic methods39−43 as well as by
computational tools,44,45 and their biological activities were
extensively explored.39,46−50 They can form different metal−
quinolone complexes, depending on metal to ligand ratio,
coordination mode, or the pH conditions. However, most of
the complexes present a 1:2 metal to ligand ratio, where the
ligand chelates the metal ion through the carboxylate and the
ring carbonyl oxygen atoms,36−38,49−51 whereas only few
examples of 1:1 species are available in the literature.52−54
Little is known about the coordination behavior of the
HPCA backbone.55 In particular, studies on the biological
activity of isolated metal complexes of both classes of inhibitors
are surprisingly scarce.
Therefore, to determine which type of metal complexes are
formed in solution and what structural features are important
for activity, we synthesized a model of the quinolone drug
elvitegravir (HL1, 6-benyl-4-oxo-1,4-dihydroquinolin-3-carbox-
ylic acid) and 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-car-
boxamide) (Chart 2). These simplified models are easy to
prepare and yet maintain the pharmacophoric backbone of the
known inhibitors. In particular, we wanted to elucidate the role
of the different pharmachophoric groups in the interaction with
the metal ion.
Herein, we report on the synthesis and characterization of
HL1, HL2, and a series of their metal complexes. The crystal
structures of HL1, HL2, [MnL12·2H2O]·2DMSO,
[MgL2 ·2H2O], and [CoL2 ·2H2O] are discussed, together
In the more recent and active examples of SSTI, the DKA
moiety was replaced by the hydroxypyrimidone ring conjugated
to a carboxamide side chain (raltegravir) and by the 4-
quinolone-3-carboxylic group (elvitegravir). Looking at these
bioisosteric scaffolds, we were interested to determine if they
2
2
with a potentiometric investigation on the solution behavior of
HL2. Finally, the anti-HIV-1 IN activities of the free ligands and
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Chart 2. Chemical Structures of the Model Ligands HL1 and
HL2
hydroxyl to provide the intermediate 6, followed by base-
promoted N-methylation in position 1 of the pyrimidone ring.
Both ligands were fully characterized by the usual spectroscopic
tools and by X-ray diffraction analysis, and the acidic constant
of HL2 was evaluated by potentiometric measurements (see
below).
Complexes 8 and 9 were synthesized by reacting the
deprotonated ligand HL1 with Mg(OH)2 and Mn(CH3COO)2,
respectively, in a 2:1 ligand to metal ratio (Scheme 3). The use
of a base is necessary to ensure deprotonation of the carboxylic
moiety and subsequent coordination to the metal. In the IR
spectra, the absorption of the ν(CO) at 1712 cm−1 of the
free quinolone was replaced by two strong characteristic bands
at 1597, 1420 cm−1 (8) and 1584, 1407 cm−1 (9), attributable
to the asymmetric and symmetric ν(O−C−O) vibrations,
respectively. The Δ[ν(CO2)asym−ν(CO2)sym] of 177 cm−1 is
indicative of a monodentate coordination mode of the
carboxylate moiety.56,57 The pyridone stretching vibration is
shifted upon coordination from 1618 to 1560 cm−1 (8) and
1569 cm−1 (9). The changes in the IR spectra suggest that the
ligand coordinates to the metal through the pyridone and one
of carboxylate oxygens.36−40 1H NMR, mass spectra, and
elemental analysis confirmed the expected stoichiometry, as
well as the X-ray diffraction analysis on the manganese complex
(see X-ray analysis section). With the aim to prepare 2:2 metal
to ligand species,52 the conditions of the reactions were varied
by changing pH, metal/ligand ratio, and metal salt. However,
only the monometallic 1:2 metal to ligand species (8 and 9)
were isolated with no significant amount of 2:2 metal to ligand
complexes. For similar reactions, the use of hydrothermal
conditions was previously reported39,52−54 to yield only the 1:2
species.
of the metal complexes were evaluated in enzymatic assays, and
implications between their structural features and the complex-
ing abilities, as well as a mechanistic hypothesis, are detailed.
RESULTS AND DISCUSSION
■
Chemistry. Ligands HL1 and HL2 were obtained as
outlined in Schemes 1 and 2, respectively, according to slightly
modified literature procedures.23,27 HL1 was obtained from the
ester 2 by hydrolysis with 2% NaOH (Scheme 1). 2 was
prepared by heating the 4-benzylaniline with diethyl ethox-
ymethylenemalonate at 130 °C and subsequent thermal
cyclization of the aminoacrylate 1 in Dowtherm A for 3 h at
reflux conditions (40% yield, method A). Direct condensation
and ring closure in Dowtherm A for 5 h provided 2 in modest
yield (∼27%, method B).
Complexes 10−13 were obtained by reacting HL2 with the
acetate salt of the metal in the presence of NaOH. In this case
the use of a base was strictly necessary to deprotonate the
ligand, since the pKa of the hydroxyl moiety was high (see
potentiometric section). HL2 has different coordinating groups
that can be involved in coordination, giving rise to five- or six-
membered chelating rings (Scheme 3, inset). Therefore,
bimetallic species could be present or only one metal could
be coordinated, and in this case a 1:2 metal to ligand complex is
expected. For compounds 10−13, coordination to the metal
HL2 was prepared by reaction of the ester 7 with the 4-
fluorobenzylamine as described in Scheme 2. The synthesis of
the methyl 5,6-dihydroxypyrimidine-4-carboxylate core was
achieved by conversion of isobutyronitrile into the amidoxime
3, followed by reaction with dimethyl acetylenedicarboxylate, to
give 4 in quantitative yield. Subsequent cyclization with xylene
at reflux afforded the desired intermediate 5. Finally, 7 was
prepared from 5 via regioselective benzoylation of the 5-
1
ions can be inferred by spectroscopic analysis. In the H NMR
spectra of both the magnesium and the zinc complexes a
downfield shift of the signals of about 0.2 ppm was observed
and the resonance of the OH proton, detectable in the free
a
Scheme 1. Preparation of Ligand HL1
a
Reagents and conditions: (i) 130 °C for 3 h; (A) Dowtherm A, reflux for 3 h; (B) Dowtherm A, reflux for 5 h; (ii) 2% NaOH, reflux for 3 h.
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a
Scheme 2. Preparation of Ligand HL2
a
Reagents and conditions: (i) KOH, CH3OH, room temp, 30 min, then reflux for 24 h; (ii) DMAD, anhyd CHCl3, reflux 24 h; (iii) o-xylene, reflux
for 48 h; (iv) benzoic anhydride, pyridine, room temp for 48 h; (v) Cs2CO3, MeI, anhyd THF, room temp for 24 h; (vi) 4-fluorobenzylamine, anhyd
CH3OH, sealed tube, 70 °C for 3.5 h, then 1 M NaOH, room temp for 30 min.
Scheme 3. Synthesis and Structural Representation of Complexes 8 and 9 (a) and 10−13 (b)
ligand, disappeared. A comparison of the 13C NMR spectra of
HL2 and of 10 suggests that only two of the three coordinating
groups (the hydroxyl, the secondary, and the tertiary amides)
are involved in the complexation of the metal ion. In fact, with
the exception of the one bounded to the isopropyl group, all
the resonances of the quaternary carbons were broadened upon
coordination, but only the signals attributable to the
heterocyclic carbon bonded to the hydroxyl moiety and the
carbonyl of the secondary amide are shifted from 168.4 and
157.9 to 166.9 and 165.4 ppm, respectively, indicating the
formation of the six-membered ring. In the IR spectra of the
complexes, it is possible to observe the shift of the carbonylic
stretching absorptions to lower wavenumbers, as well as the
upper shift of the NH stretching mode of about 20−40 cm−1 vs
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Figure 1. Molecular structures of HL1 (top left) and HL2 (top right) and of their complexes with manganese and magnesium, 9 (bottom left) and 10
(bottom right). Thermal ellipsoids are at the 50% probability level.
the free ligand. This is in accord with the involvement of the
secondary amide in coordination because the NH group that
forms in an intramolecular hydrogen bond in the free ligand is
not engaged in any type of interaction after coordination (see
X-ray analysis section). In order to exclude the formation of 1:1
species, also with HL2 several attempts were realized by varying
the reaction conditions. All the data (1H NMR, 13C NMR, IR,
mass spectrometry, and elemental analysis) confirmed the
isolation of the mononuclear, bis-chelated species 10−13.
These findings were further supported by potentiometric
measurements and calculations.
X-ray Crystallography Studies. To elucidate the phar-
macophoric features of the ligands and the coordination
geometries of the complexes, a series of X-ray crystallographic
studies were carried out. A Mogul58 analysis of the structure of
the two ligands shows that their bonding geometries do not
present any unusual feature. HL1 crystallizes with two
independent molecules in the asymmetric unit (only one is
shown in Figure 1) that differ only in the conformation of the
benzyl substituent around the bond C8−C11 (C9−C8−C11−
C12 = 75°, C26−C25−C28−C29 = −111°). Both molecules
form an intramolecular hydrogen bond between the carboxylic
acid and the quinolone oxygen (O2···O3 = 2.475(6) Å, O−
H···O = 159(6)°, O5···O6 = 2.466(6) Å, O−H···O = 144(6)°),
while the intermolecular hydrogen bonds between the −NH
group and the carboxylic CO acceptor link the two
independent molecules in homomeric chains (N1···O1-
(−x,y−1/2,5/2−z) = 2.798(7) Å, N−H···O = 177(6)°;
N2···O4(−x−1,y−1/2,5/2−z) = 2.853(7) Å, N−H···O =
177(6)°) (see Supporting Information). Similarly, HL2 presents
an intramolecular hydrogen bond between the −OH group and
the amidic oxygen (O2···O3 = 2.604(3) Å, O−H···O = 144(3)
°), while neighboring molecules are linked in chains by
intermolecular hydrogen bonds between the −NH donor and
the −OH acting as an acceptor (N3···O2(x,y+1/2,1/2−z) =
3.036(3) Å, N−H···O = 125(2)°) (see Supporting Informa-
tion). The quinolone oxygen is only involved in weak CH···O
contacts.
The crystal structures of 9, 10, and 12 are constituted by six-
coordinated metal centers binding two bis-chelated deproto-
nated ligands in the equatorial plane and two water molecules
at the apexes of a distorted octahedron (Figure 1; complexes 10
and 12 are isostructural, and only 10 is shown). Compound 9
was obtained from a DMSO solution and crystallized as
9·2DMSO. All the complexes are centrosymmetric. In the
isostructural complexes 10 and 12, the coordination geometry
consists of two six-membered chelation rings that are practically
planar, with M−O distances varying between 1.988(5) and
2.080(6) Å and two apical waters at longer distances (2.136(3)
Å for Mg and 2.197(6) Å for Co). The rest of the ligand
molecules are perfectly coplanar with the equatorial coordina-
tion plane, apart form the p-fluorobenzyl substituents that stick
perpendicularly out of this plane at opposite sides. The crystal
packing is described in the Supporting Information.
The coordination geometry in 9 is similar to the one
observed for 10 and 12, with four shorter M−O bonds in the
equatorial plane (2.084(7), 2.120(8)Å) and two longer apical
metal···water bonds (2.246(8) Å). The difference between
corresponding M−O bond lengths in 9, 10, and 12 reflects the
larger ionic radius of high-spin six-coordinated Mn(II) (0.97 Å)
relative to those of high-spin six-coordinated Co(II) (0.88 Å)
and six-coordinated Mg(II) (0.86 Å). The benzyl substituents
stick out of the average equatorial plane at the opposite sides.
Similar to 10 and 12, in 9 the coordination consists of two
six-membered chelation rings that in this case are puckered in
an envelope conformation with the metal at the flap deviating
by 0.45 Å from the rest of the ring. Because of the molecular
centrosymmetry, the overall effect is that the ligand skeletons
are skewed with respect to the apical axis defined by the
metal···water coordination bonds. Therefore, the overall
molecular shape of 9 is significantly different from 10 and 12
because the ligand planarity is remarkably perturbed. A similar
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Table 1. Logarithms of Formation Constants (βpqr = [MpLqHr]/{[M]p[L]q[H]r}) in Methanol/Water = 9:1 v/v, I = 0.1 M KCl at
a
25 °C for HL2 with Mg(II), Mn(II), Co(II), and Zn(II)
p
q
r
Mg(II)
Mn(II)
Co(II)
Zn(II)
1
1
1
0
1
2
0
1
0
5.11(0.23)
9.08(0.22)
5.93(0.07)
6.91(0.03)
4.87(0.20)
9.29(0.11)
−19.90(0.22)
0
10.18(0.07)
−19.88(0.13)
11.67(0.06)
−18.91(0.10)
8.639(0.002)
−2
1
L + H = LH
a
Standard deviations are given in parentheses. Charges are omitted for simplicity.
Figure 2. Distribution diagrams for the systems under investigation at L/M = 4:1 (the concentration of the metal ion is 0.75 mM for Mg(II),
Mn(II), Co(II) and 0.37 mM for Zn(II)): (a) M = Mg(II); (b) M = Mn(II); (c) M = Co(II); (d) M = Zn(II).
ligand bending is observed in the solvate forms of diaqua-
bis(ciprofloxacin)magnesium(II) dinitrate,42 rac-diaqua-bis-
(ofloxacin)magnesium(II), and diaqua-bis((S)-levofloxacin)-
magnesium(II),41 with the metal displaced by 0.63, 0.56, and
0.43 Å from the chelation plane, respectively. The deformation
observed for the ligands skeletons in 9 and in the above cited
magnesium complexes may be ascribed to a supramolecular
effect, since in 9 the complex crystallizes with one DMSO
molecule in the asymmetric unit, which is involved in a
extensive network of hydrogen bonds. The steric crowding
required for the establishment of this tight network of contacts
may consequently force the ligands to bend (Supporting
Information, Figure S4). Another interesting observation is that
molecular volume and van der Waals surface area of 9 (volume
= 494 Å3, area = 569 Å2) are significantly smaller than those of
10 and 12 (volume = 548 and 551 Å3, area = 659 and 654 Å2).
Potentiometric Measurements and Calculations. A
potentiometric study was carried out with Mg2+, Mn2+, Co2+,
Zn2+, and HL2 in order to obtain more information about the
type and the stability of the species present at physiological pH.
Unfortunately, the poor solubility of HL1 prevented the same
study on the QCA system. The titrations were performed in
methanol/water = 9/1 v/v to reduce solubility problems and to
obtain results comparable with our previous works.33,34,59
HL2 is a monoprotic acid with a pKa of 8.639 ± 0.002. A pKa
of 6.48 was reported for a similar ligand based on 6-
carboxamido-5,4-hydroxypyrimidinones (6-carboxamido-5,4-
HOPY) in aqueous solution at the same ionic strength.55
The different value we found for HL2 can be explained by the
use of mixed solvents and by the different functionalizations of
the two ligands.
The stoichiometry of the complexes with Mg2+, Mn2+, Co2+,
and Zn2+ were determined by using metal to ligand ratios from
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1:1 to 1:5. Titrations with Zn2+ must be performed at halved
concentrations because of poor solubility. Results of the best fit
of the experimental titration curves by the Hyperquad
software60 are reported in Table 1, and the corresponding
distribution diagrams are shown in Figure 2 for a metal/ligand
ratio of 1/4.
observed, thus demonstrating that both the metals and organic
scaffold are important for the activity.
Different behavior was found for compounds bearing the
HMPCA-based pharmacophoric motif. In fact, the strand
transfer inhibition values for complexes 10−13 ranged from
0.09 to 0.41 μM, thus sharing a potency similar to that of the
free ligand HL2 (Figure 3). As already observed in our previous
study on triazole derivatives,59 the Co(II) complex 12 proved
to be the most active tested compound, with an IC50 value of
0.09 ± 0.02 for strand transfer. Therefore, an interesting metal-
dependent behavior was evidenced also for HL2 and its metal
complexes. The activity of the complexes toward strand
transfer, even if with slight differences, follows the trend Mg
< Zn < Mn < Co [IC50, strand transfer = 0.41 ± 0.2, 0.23 ±
0.10, 0.18 ± 0.1, 0.09 ± 0.02 μM for Mg(II), Zn(II), Mn(II),
Co(II), respectively].
The activity of the metal complexes enforced the observation
that they are quite stable in solution (see also their log β values)
and that the complexes, not necessarily only the free ligand,
might be involved in the inhibition mechanism (Figure 4).
Another point that would support this hypothesis regards the
Co(II) complex. The reaction buffer for the assay contains
Mn2+ in millimolar concentration, to ensure enzymatic activity,
and other ligands (EDTA, DTT). When the metal complex is
dissolved, equilibria between the different metal species in
solution arise, according to the different formation constants
and concentrations. Interestingly, the Co(II) complex, which
has the higher stability constant, also shows the higher anti-IN
activity.
Monometallic Species in Strand Transfer Inhibition.
The analysis of the coordinating ability of both the HPCA and
the QCA pharmachophoric models indicates that in solution
the formation of monometallic species is more plausible than
the concurrent chelation of two metal centers. On the other
hand, the possibility of formation, in proximity of the chelation
region, of other types of interactions like hydrogen bonds
would also exert a crucial role for binding into the active site
(for example, by mediating the interaction with the protein side
chain). Effectively, a tight network of supramolecular hydrogen
bonds is observed in the crystal packing of the complexes,
involving the noncoordinated carboxylic oxygen (complex 9) or
the pyrimidone oxygen (10).
At this stage, it can be proposed that in the presence of metal
ions, the HMPCA- and QCA-based chemical pharmacophores
form complexes that could bind directly to IN, after keeping
and/or scavenging the metal, interacting as single static entity
(Figure 4). ML and ML2 could also bind to IN and engage the
second ion cofactor that is present on the active site. Either
way, the values of the stability constants of the complexes, the
concentration of divalent cation in the assays conditions, and
the similar inhibition data for the free ligand and the
corresponding complexes support the hypothesis of the
complexes as putative active forms of the IN inhibitors,
indicating that stable species present in solution are involved in
several ways in the inhibitory action. This concept was also
suggested in our previous studies.33,34,59
The species found in solution were [ML]+ and ML2 for all
the studied metal ions, with formation constants that increase
in the order Zn2+ < Mg2+ < Mn2+ < Co2+ for the ML complex
and in the order Mg2+ ∼ Zn2+ < Mn2+ < Co2+ for the ML2
complex, according to the Irving−Williams sequence.61
This model is statistically significant, and we observe a
reasonable correlation between experimental and computed
titration curves. For Co2+ and Zn2+, convergence can be
achieved only by considering the hydrolysis of the cation, by
adding the formation constant of the hydroxide (Table 1).
Interestingly, at physiological conditions, both species (ML and
ML2) are present, and Co(II) is the only ion almost completely
in the form of the ML2 complex (Figure 2).
Therefore, HL2 behaves in a different way with respect to
DKA ligands:33,34 whereas at physiologic pH the parent DKAs
are almost completely in the form M2L2 with a metal/ligand =
1/1, this ratio is less favored with HL2 (in particular the species
M2L2 cannot be found in solution) and the formation constants
of ML2 complexes are much greater than for the other species
in solution.
Inhibition of HIV-1 IN. The ligands HL1 and HL2 and
their complexes 8−13 were tested for their ability to inhibit 3′-
processing and strand transfer catalytic activities by employing
purified enzyme (Table 2). Both series of compounds showed
Table 2. Inhibition of HIV-1 IN Catalytic Activities of
Ligands and Complexes
IC50 (μM)
a
b
compd
M
3′-processing
strand transfer
SI
HL1
>100
12 ± 5
4 ± 2
2 ± 0.1
0.14 ± 0.03
0.41 ± 0.2
0.18 ± 0.1
0.09 ± 0.02
0.23 ± 0.10
>8.3
22.2
>125
71.4
19.5
38.9
21.1
11.3
8
Mg2+
Mn2+
89 ± 16
>250
9
HL2
10
11
12
13
10 ± 8
8 ± 5
7 ± 4
1.9 ± 1.1
2.6 ± 1.0
Mg2+
Mn2+
Co2+
Zn2+
b
a
M, metal ion. SI, selectivity index.
inhibition potency in the low nanomolar/micromolar concen-
tration range, with significant selectivity toward strand transfer.
In particular, HL2 (IC50, strand transfer = 0.14 ± 0.03 μM; 3′-
processing = 10 ± 8 μM) was about 100-fold more potent than
the quinolone HL1 (IC50, strand transfer = 12 ± 5 μM; 3′-
processing > 100 μM). The complexes 8−13 retained an
activity profile analogous to the corresponding free ligands, as
already observed in our previous work on DKAs and triazolic
bioisosters33,34,59 (Table 2; for a representative gel, see Figure
3).
The magnesium and manganese complexes of 8 and 9
resulted in a 4-fold and 6-fold increase in activities with respect
to the parent ligand (IC50, strand transfer of 4 ± 2 and 2 ± 0.1
μM for 8 and 9, respectively, vs 12 ± 5 μM for HL1), and 9 was
2-fold more potent than 8 (Figure 3b). Interestingly, when HL1
was involved in coordination, an enhancement in potency was
An “interfacial-type mechanism” of action for metal
complexes, where the chelation of the Mg2+ ion in the active
site of IN is important for the activity of the inhibitor, is also
possible. The inhibitors could interact with IN after 3′-
processing or in the later stage of this process. After binding
to the enzyme as preformed complexes, a DNA-induced
conformational change occurs, displacing the reactive viral
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Figure 3. Schematic representation of IN activity in vitro and representative gel showing inhibition of purified IN by HL1, HL2, and 8−13. (a) A 21-
mer blunt-end oligonucleotide corresponding 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 products formed by this reaction migrate more slowly than
the original substrate on a polyacrylamide gel. (b) Line labeled as DNA indicates DNA alone. Line labeled INT indicates IN and DNA with no drug.
Other lines are as follows: IN, DNA, and selected drug concentrations (μM) as indicated in each line.
DNA end from the active site and thus disarming the viral
nucleoprotein complex. It is also possible to speculate that
inhibitors bind to a transient IN-DNA structure formed prior to
or upon 3′-OH processing within the cytosolic preintegration
complex. This can explain why, among the identified IN
inhibitors that specifically target the strand transfer reaction
alone, several of them are highly selective for strand transfer
while others also show some activity against the 3′-processing
reaction. Therefore, the reactive species could be the metal
complex that specifically interacts with the protein and prevents
the interaction between the enzyme and its DNA (viral)
substrate.
Recently, a crystal structure of the quinolonic drug
moxifloxacin in complex with Acinetobacter baumannii top-
oisomerase IV revealed that a magnesium ion mediates the
interaction between the drug and the protein, suggesting that
an “interfacial model” could also be at the base of the mode of
action of quinolone antibacterial drugs.62
giving stable, isolable species. We have also observed that the
complexes themselves inhibit the HIV-1 IN catalytic activities
and, moreover, that their activity is metal dependent. This
means that the metal complexes (in particular the Mg(II)
complex at physiological conditions) could be involved in
different stages of the inhibition process. Another relevant
point is that the complexes that we have characterized are
monometallic and as such can participate in the inhibition
mechanism by interacting with the enzyme in many ways (i.e.,
exchange of the metals, coordination of a second metal, second
coordination sphere interactions). Molecular modeling studies
that consider this hypothesis are in progress in our laboratory.
We anticipate that the results presented here can contribute to
the mechanistic aspect of HIV-IN inhibitors action and can
expedite the design of other metal-based complexes for
different targets.
EXPERIMENTAL SECTION
■
Materials and Methods. Chemistry. Anhydrous solvents and all
reagents were purchased from Sigma-Aldrich, Merck, or Carlo Erba
and used without further purification. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere using oven-dried glassware 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 and were within ±0.4% of the theoretical values.
CONCLUSIONS
■
The studies on HIV-1 IN inhibitors and on the biology of metal
cofactors have confirmed the important role of the metals in
the inhibition process and consequently that the metal binding
function is a critical factor in the development of IN inhibitors.
The compounds related to the diketo acids family, such as
raltegravir and elvitegravir, are well suited to explore this
chemical space, and they represent milestones for medicinal
chemistry discovery and development programs. These
molecules contain the basic structural features that established
what is known as the “two-metal chelation” motif. Effectively,
we have confirmed that these inhibitors can chelate metal ions,
Melting points (mp) were determined using an Electrothermal
melting point or a Kofler apparatus and are uncorrected. Analytical
̈
thin-layer chromatography (TLC) was carried out on Merck silica gel
F-254 plates. Flash chromatography purifications were performed on
Merck silica gel 60 (230−400 mesh ASTM) as the stationary phase.
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Figure 4. Mechanism of proviral DNA integration and proposed mechanistic model for SSTI and related complexes. The integration process of the
HIV lifecycle requires two steps mediated by the IN enzyme, 3′-end processing (a, b) and strand transfer reaction (c, d). Stage a to b: IN removes
the last dinucleotide in 3′ position to the conserved CA of viral DNA, thanks to the catalytic action of the metal cofactors. The recognized adenosine
is exposed as the new 3′-end, still bound to the protein as PIC (preintegration complex). From stage c to d, the PIC nonspecifically binds to host
DNA and the two metals catalyze a five staggered base pair cut into host DNA, linking both viral 3′ ends and achieving integration of viral DNA into
the host genome. In the inhibition mechanism, inhibitors can bind as free ligands (X) or as preformed ML (Y) or ML2 (Z) complexes to the active
site within or after the 3′-processing. Complexes would bind directly to IN, after keeping and/or scavenging the metal, to form a ternary ligand−
M2+−IN complex. This would represent the first step of the inhibition process. Next, after accommodation within the active site, ligands or
complexes can interact with a second ion by interfering with the viral DNA substrate. In this manner the inhibitors cause a displacement of the
reactive viral DNA end from the active site, blocking DNA binding.
NMR spectra were recorded at 27 °C on Bruker Avance 300 FT
and Varian XL-200 spectrophotometers 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 reflectance mode on the powder. The ESI(+)-MS spectra were
collected by using a quadrupole-time-of-flight Micromass spectrometer
(Micromass, Manchester, U.K.) equipped with a pneumatically
assisted ESI interface. The system was controlled by Masslynx
software, version 4.0 (Micromass). The nebulizing gas (nitrogen,
99.999% purity) and the desolvation gas (nitrogen, 99.998% purity)
were delivered at a flow rate of 10 and 600 L/h, respectively.
Continuum mode full-scan mass spectra were acquired using an
acquisition time of 1 s and an interscan delay of 0.1 s. QqTOF external
calibration was performed using a 0.1% phosphoric acid solution, and a
fifth-order nonlinear calibration curve was usually adopted.
Synthesis of Ethyl 2-((4-Benzylphenylamino)methylen)-
malonate (1). A mixture of 4-benzylaniline (5 g, 27.3 mmol) and
diethyl ethoxymethylenemalonate (5.9 g, 27.3 mmol) was stirred at
130 °C for 3 h. On elimination of the volatile components, a brown oil
was obtained, which was used without further purification.
1
Quantitative yield. IR (Nujol, cm−1): νCO = 1752, 1645. H NMR
(CDCl3): δ 11.01 (d, 1H, J = 14 Hz, NH); 8.50 (d, 1H, J = 14 Hz,
CH); 7.34−7.17 (m, 5H, Ar-H); 7.06 (d, 2H, Ar-H, 3JHH = 8 Hz); 4.30
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3
3
(q, 2H, OCH2, JHH = 6.2 Hz); 4.27 (q, 2H, OCH2, JHH = 6.5 Hz);
3.97 (s, 2H, CH2); 1.38 (t, 3H, CH3, 3JHH = 6.3 Hz); 1.32 (t, 3H, CH3,
3JHH = 6.6 Hz).
solution was quenched with 1 N HCl and extracted with 30 mL of
ethyl acetate. The organic layer was washed three times with 1 N HCl
and twice with brine, then dried with Na2SO4. On elimination of the
solvent, a white solid was obtained, which was treated with diethyl
ether, filtered off, and dried in air. Yield: 85%. IR (cm−1): νNH = 3172;
Synthesis of Ethyl 6-Benzyl-4-oxo-1,4-dihydroquinolin-3-
carboxylate (2). Method A. A solution of 1 (9.6 g, 27.3 mmol) in
Dowterm A (30 mL) was stirred at reflux for 3 h and then cooled at
room temperature. By addition of petroleum ether, a beige powder was
obtained, which was filtered off and washed several times with
petroleum ether. Yield: 40%.
1
νCO = 1740, 1681. H NMR (DMSO-d6): δ 13.29 (s, br, 1H, NH);
8.07 (d, 2H, Ar-H, 3JHH = 7.2 Hz); 7.78 (t, 1H, Ar-H, 3JHH = 7.5 Hz);
3
7.63 (t, 2H, Ar-H, JHH = 7.6 Hz); 3.75 (s, 3H, CH3); 2.91 (m, 1H,
3
3
CH, JHH = 6.9 Hz); 1.23 (dd, 6H, CH3 JHH = 6.3 Hz). ESI/MS (+,
Method B. 4-Benzylaniline and diethyl ethoxymethylenemalonate in
equimolar amount were stirred at 130 °C for 5 h. Workup was
analogous to method A. Yield: 27%.
m/z): 316 [M+].
Synthesis of Methyl 5-Benzyloxy-2-isopropyl-1-methyl-6-
oxo-1,6-dihydroxypyrimidine-4-carboxylate (7). An amount of
3 g (9 mmol) of 6 was dissolved in 100 mL of distilled THF under
anhydrous conditions, and to the mixture was added 2 equiv of
Cs2CO3. After the mixture was stirred for 20 min at room temperature,
an amount of 26 mmol (3.7 g) of CH3I was added dropwise and the
reaction mixture was stirred for 24 h. The solution was diluted with 1
N HCl and then extracted with ethyl acetate. The organic layer was
washed with brine and then dried with Na2SO4. On elimination of the
solvent, a brown, hygroscopic solid was obtained, which was used
without further purification. Yield: 97%. IR (cm−1): νCO = 1737,
1687. 1H NMR (CDCl3): δ 8.20 (d, 2H, Ar-H, 3JHH = 7.2 Hz); 7.64 (t,
IR (cm−1): νCO = 1744, 1631. H NMR (DMSO-d6): δ 12.33 (s,
1
br, 1H, NH); 8.51 (s, 1H, CH); 7.97 (s, 1H, Ar-H); 7.56 (m, 2H, Ar-
3
H); 7.30−7.19 (m, 5H, Ar-H); 4.23 (q, 2H, OCH2, JHH = 6.4 Hz);
4.07 (s, 2H, CH2); 1.27 (t, 3H, CH3, 3JHH = 6.6 Hz). ESI/MS (+, m/
z): 307 [MH+].
Synthesis of 6-Benyl-4-oxo-1,4-dihydroquinolin-3-carbox-
ylic Acid (HL1). A solution of 2 (3.35 g, 10.9 mmol) and 2%
NaOH (11 mL) was refluxed for 3 h and then acidified at room
temperature by using 1 N HCl. The precipitate was filtered off and
recrystallized from water/ethanol, giving rise to a white powder. Yield:
3
1H, Ar-H, 3JHH = 7.4 Hz); 7.51 (t, 2H, Ar-H, JHH = 7.8 Hz); 3.85 (s,
97%. Mp: 234−235 °C (dec). IR (cm−1): νCO = 1687, 1618. H
1
3H, CH3); 3.68 (s, 3H, CH3); 3.17 (m, 1H, CH, 3JHH =6 0.4 Hz); 1.39
NMR (DMSO-d6): δ 15.41 (s, br, OH); 13.43 (s, br, 1H, NH); 8.85
3
(d, 6H, CH3, JHH = 6.8 Hz). ESI/MS (+, m/z): 330 [M+].
3
(s, 1H, CH); 8.23 (s, 1H, Ar-H); 8.00 (d, 1H, Ar-H, JHH = 9 Hz);
3
Synthesis of N-(4-Fluorobenzyl)-5-hydroxy-2-isopropyl-1-
methyl-6-oxo-1,6-dihydroxypyrimidine-4-carboxylate (HL2).
An amount of 2.9 g (8.7 mmol) of 7 was dissolved in 40 mL of
methanol in a sealed tube, and to the mixture was added dropwise 2
equiv (2.2 g) of 4-fluorobenzylamine. The mixture was refluxed for 3
h. After the mixture was cooled at room temperature, an amount of 10
mL of 1 M NaOH was added. The solution was stirred for 30 min at
room temperature, then diluted with 20 mL of water and extracted
with CH2Cl2. The aqueous layer was treated with 1 N HCl. At neutral
pH a white precipitate appeared, which was filtered off and washed
with water. Yield: 80%. IR (cm−1): νNH = 3339; νCO = 1673, 1636.
1H NMR (DMSO-d6): δ 12.14 (s, br, 1H, OH); 9.21 (s, br, 1H, NH);
7.81 (d, 1H, Ar-H, JHH = 8.1 Hz); 7.27 (m, 5H, Ar-H); 4.14 (s, 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. ESI/
MS (+, m/z): 279 [MH+]. Crystallization from chloroform afforded
crystals suitable for X-ray diffraction analysis.
Synthesis of N′-Hydroxy-2-methylpropanimideamide (3).
Hydroxylamine chlorohydride (3 g, 43 mmol) was dissolved in 30 mL
of methanol, and to the mixture was added KOH (2.7 g, 48 mmol).
After the mixture was stirred for 30 min, KCl was filtered off and
washed several times with methanol. Isobutyronitrile (3 g, 43 mmol)
was added and the reaction mixture refluxed for 24 h. On elimination
of the solvent, a yellow oil was obtained. Yield: 85%. 1H NMR
(CDCl3): δ 8.08 (s, br, OH); 4.58 (s, br, 2H, NH2); 2.45 (m, 1H, CH,
3
3
7.38 (dd, 2H, Ar-H, JHH3 = 7.0 Hz); 7.17 (dd, 2H, Ar-H, JHH = 8.7
Hz); 4.49 (d, 2H, CH2, JHH = 6.3 Hz); 3.52 (s, 3H, CH3); 3.15 (m,
1H, CH, 3JHH = 6.6 Hz); 1.25 (d, 6H, CH3, 3JHH = 6.6 Hz). 13C NMR
(DMSO-d6): δ 168.4 (Hd); 160.8 (d, JC−F = 324 Hz); 157.9 (Hb);
155.7 (Ha); 145.5; 134.8; 129.2 (d, JC−C = 10 Hz); 124.9 (Hc); 115.0
(d, JC−C = 28 Hz); 41.4; 30.8; 30.3; 20.4. ESI/MS (+, m/z): 319 [M+].
Crystallization from chloroform afforded crystals suitable for X-ray
diffraction analysis.
3JHH = 6.9 Hz); 1.19 (dd, 6H, CH3, JHH = 6 Hz). ESI/MS (+, m/z):
3
102 [M+].
Synthesis of Dimethyl 2-(1-Amino-2-methyl-
propylidenaminooxy)but-2-endioate (4). An amount of 3.7 g
(36 mmol) of 3 was dissolved in 20 mL of distilled CHCl3 under
anhydrous conditions. To the mixture was added dropwise a 15 mL
solution of dimethylacetylene dicarboxylate (6.3 g, 44.6 mmol). The
reaction mixture turned yellow. It was refluxed for 24 h and then dried
under vacuum. A brown oil was obtained. Quantitative yield. 1H NMR
(CDCl3) major isomer: δ 5.87 (s, 1H, CH); 4.74 (s, br, 2H, NH2);
3.97 (s, 3H, CH3); 3.72 (s, 3H, CH3); 2.50 (overlapping signals, CH);
1.20 (overlapping signals, CH3). Minor isomer: δ 5.76 (s, 1H, CH);
5.52 (s, br, 1H, NH); 5.22 (s, br, 1H, NH); 3.84 (s, 3H, CH3); 3.74 (s,
3H, CH3).
Synthesis of MgL2 ·2H2O (8). HL1 (97 mg, 0.35 mmol) was
1
dissolved in 4 mL of methanol and 6 mL of dimethylsulfoxide, and to
the mixture was added 1.2 equiv of 0.1 M NaOH. The solution was
stirred at room temperature for 30 min, and then Mg(OH)2 (10 mg,
0.17 mmol) was added. The reaction mixture was stirred for 24 h at
room temperature. On elimination of the solvent a white powder was
isolated, which was washed with water and dried under vacuum. Yield:
1
78%. IR (cm−1): νNH = 3356; νCO= 1597, 1569, 1420. H NMR
Synthesis of Methyl 5-Hydroxy-2-isopropyl-6-oxo-1,6-dihy-
droxypyrimidine-4-carboxylate (5). An amount of 36 mmol of 4
(8.8 g) was dissolved in 115 mL of o-xylene, obtaining a brown
solution that was refluxed for 48 h. The reaction was monitored by
TLC (CH2Cl2/MeOH, 3%). On evaporation of the solvent, a dark oil
was obtained. It was dissolved in warm ethyl acetate. When the
mixture was cooled at −18 °C, a brown solid precipitated, which was
(DMSO-d6): δ 16.66 (s, br, 1H, NH); 8.78 (s, 1H, CH); 8.03 (s,
1H, Ar-H); 7.67 (br, 2H, Ar-H); 7.28 (m, br, 5H, Ar-H) 4.11 (s, 2H,
CH2). ESI/MS (+, m/z): 581 [MH+]; 603 [M + Na+].
Synthesis of MnL2 ·3H2O (9). HL1 (97 mg, 0.35 mmol) was
1
dissolved in 30 mL of methanol and 3 mL of dimethylsulfoxide, and to
the mixture was added 1.2 equiv of 0.1 M NaOH. The solution was
stirred at room temperature for 30 min, and then an aqueous solution
of Mn(CH3COO)2 (44 mg, 0.17 mmol) was added. Immediately, a
yellow precipitate was formed, which was stirred for an additional 4 h.
The solution was concentrated on vacuum and the solid filtered off
further recrystallized from ethyl acetate. Yield: 30%. IR (cm−1): νOH
=
1
3167; νCO = 1704, 1672. H NMR (DMSO-d6): δ 12.66 (s, br, 1H,
OH); 10.17 (s, br, 1H, NH); 3.81 (s, 3H, CH3); 2.77 (m, 1H, CH,
3JHH = 6.4 Hz); 1.16 (d, 6H, CH3, 3JHH = 6.7 Hz). ESI/MS (+, m/z):
and washed with water. Yield: 82%. IR (cm−1): νNH = 3446; νCO
=
212 [M+].
1584, 1560, 1407. ESI/MS (+, m/z): 634 [M + Na+]. X-ray diffraction
Synthesis of Methyl 5-Benzyloxy-2-isopropyl-6-oxo-1,6-
dihydroxypyrimidine-4-carboxylate (6). An amount of 2.3 g
(11 mmol) of 5 and an equimolar amount of benzoic anhydride were
suspended in 30 mL of dry pyridine under anhydrous conditions. After
1 h the solution was clear. It was stirred at room temperature for 48 h.
The reaction was monitored by TLC (ethyl acetate, 100%). The
quality crystals were obtained by recrystallization from DMSO as
1
MnL2 ·2DMSO.
Synthesis of MgL2 ·3H2O (10). HL2 (96 mg, 0.3 mmol) was
2
dissolved in 20 mL of methanol, and to the mixture was added 1.2
equiv of 0.1 M NaOH. After the mixture was stirred at room
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temperature for 30 min, an aqueous solution of Mg-
(CH3COO)2·4H2O (0.15 mmol) was slowly added and a white
precipitate appeared. The reaction mixture was stirred at room
temperature for 3 h, and the precipitate was filtered off and washed
with water. Yield: 77%. IR (cm−1): νNH = 3358; νCO= 1660, 1621. 1H
NMR (DMSO-d6): δ 9.57 (s, br, 1H, NH); 7.21 (s, br, 2H, Ar-H);
6.93 (s, br, 2H, Ar-H); 4.35 (s, 2H, CH2); 3.44 (s, 3H, NCH3); 3.05
(m, 1H, CH); 1.18 (t, 6H, CH3). 13C NMR (DMSO-d6): δ 166.9 (br,
Hd); 165.4 (br, Hb); 160.8 (d, JC−F = 324 Hz); 155.0 (br, Ha); 144.7;
135.8; 129.2 (d, JC−C = 10 Hz); 125.9 (br, Hc); 114.6 (d, JC−C = 28
Hz); 41.0; 30.1; 30.0; 20.8. Recrystallization from methanol/water
stored under nitrogen to avoid oxidation. Equilibrium constants for
protonation and complexation reactions were determined by means of
potentiometric measurements, carried out in methanol/water = 9:1 v/
v solution at ionic strength 0.1 M KCl and 25 ± 0.1 °C, at pH 2.5−11
under N2. Temperature was controlled to ±0.1 °C by using a
thermostated circulating water bath (ISCO GTR 2000 IIx).
Appropriate aliquots of ligand solution, prepared by weight, were
titrated with standard KOH (solvent of methanol/water = 9:1 v/v, I =
0.1 M KCl) with and without metal ions, applying constant speed
magnetic stirring. Freshly boiled methanol and double-distilled water,
kept under N2, were used throughout. The experimental procedure in
order to reach very high accuracy in the determination of the
equilibrium constants in this mixed solvent has been described in
detail elsewhere.71 The protonation constants of the ligands were
obtained by titrating 20−50 mL samples of each ligand (3 × 10−3 to 9
× 10−3 M). For the complex formation constants, the titrations were
performed with different ligand/metal ratios (1 up to 5). At least two
measurements (about 60 experimental points each) were performed
for each system. Potentiometric titrations were carried out by a fully
automated apparatus equipped with a CRISON GLP 21-22 digital
voltmeter (resolution, 0.1 mV) and a 5 mL Metrohm Dosimat 655
autoburet, both controlled by a homemade software in BASIC,
working on an IBM computer. The electrodic chain (Crison 5250 glass
electrode and 0.1 M KCl in methanol/water = 9:1 v/v calomel
electrode, Radiometer 401) was calibrated in terms of [H+] by means
of a strong acid−strong base titration by Gran’s method,72 allowing the
determination of the standard potential, E° (371.5 ± 0.4 mV), and of
the ionic product of water, Kw (pKw = 14.40 ± 0.05) in the
experimental conditions used. The software HYPERQUAD59 was used
to evaluate the protonation and complexation constants from emf data.
Biological Materials, Chemicals, and Enzymes. All com-
pounds 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.
afforded crystals of MgL2 ·2H2O suitable for X-ray diffraction analysis.
2
Synthesis of MnL2 ·3H2O (11). Compound 11 was prepared
2
using the same protocol as described for 10 using Mn-
(CH3COO)2·4H2O. The solution became immediately bright yellow
and was stirred for 3 h at room temperature. On concentration of the
solution, a yellow precipitate appeared, which was filtered off and
washed with water. Yield: 70%. IR (cm−1): νNH = 3375; νCO= 1616
(br). ESI/MS (+, m/z): 714 [M + Na+].
Synthesis of CoL2 ·2.5H2O (12). Compound 12 was prepared
2
using the same protocol as described for 10 using Co-
(CH3COO)2·4H2O. The solution turned light red, and the reaction
mixture was stirred at room temperature for 3 h. The solvent was
eliminated on vacuum, and the residue was suspended in diethyl ether,
filtered off, and washed with water. A red powder was obtained. Yield:
74%. IR (cm−1): νNH = 3375; νCO= 1621 (br). Recystallization from
methanol/water, 5/1, afforded crystals of CoL2 ·2H2O suitable for X-
2
ray diffraction analysis.
Synthesis of ZnL2 ·2H2O (13). Compound 13 was prepared
2
using the same protocol as described for 10 using Zn-
(CH3COO)2·2H2O. The solution remained colorless and was stirred
at room temperature for 3 h. On concentration of the solution, a white
precipitate appeared, which was filtered off and washed with water.
1
Yield: 70%. IR (cm−1): νNH = 3364; νCO = 1616 (br). H NMR
(DMSO-d6): δ 10.00 (s, br, 1H, NH); 7.25 (s, br, 2H, Ar-H); 7.03 (s,
br, 2H, Ar-H); 4.42 (s, 2H, CH2); 3.51 (s, 3H, NCH3); 3.11 (m, 1H,
CH); 1.19 (t, 6H, CH3)..
Preparation of Oligonucleotide Substrates. The oligonucleo-
tides 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′-processing and strand transfer 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 temperature, and run through a spin 25 minicolumn (USA
Scientific) to separate annealed double-stranded oligonucleotide from
unincorporated material.
Integrase Assays. To determine the extent of 3′-processing and
strand transfer, wild-type IN was preincubated at a final concentration
of 200 nM with the inhibitor in reaction 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. Then 20 nM of the 5′-end 32P-labeled linear
oligonucleotide substrate was added, and incubation was continued
for an additional 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).
X-ray Crystallography. Single crystal X-ray diffraction data were
collected at room temperature (293 K) using graphite monochro-
mated Mo Kα radiation (λ = 0.710 73 Å) on a APEX2 CCD
diffractometer for 9 and 12 and using Cu Kα (λ = 1.541 78 Å) on a
Siemens AED diffractometer equipped with scintillation counter for
10, HL1, and HL2. Lorentz polarization and absorption corrections
were applied.63,64 Structures were solved by direct methods using
SIR9765 and refined by full-matrix least-squares on all F2 using
SHELXL9766 implemented in the WingX package.67 Hydrogen atoms
were partly located from Fourier difference maps and partly
introduced in idealized positions riding on their carrier atoms.
Anisotropic displacement parameters were refined for all non-
hydrogen atoms. Table 1 summarizes crystal data and structure
determination results. Hydrogen bonds were analyzed with
SHELXL9766 and PARST97,68 and extensive use was made of the
Cambridge Crystallographic Data Centre packages69,70 for the analysis
of crystal packing.
Crystallographic data (excluding structure factors) for HL1, HL2, 9,
10, and 12 have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publications CCDC 829359−829363.
Copies of the data can be obtained free of charge on application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax, (+44) 1223-
336-033; e-mail, deposit@ccdc.cam.ac.uk).
Potentiometric Measurements. The Mg(II), Mn(II), and
Co(II) stock solutions were prepared from MgCl2·6H2O (Aldrich),
MnCl2·4H2O (Janssen), and anhydrous CoCl2 (Aldrich). Zn(II) stock
solution was prepared by dissolving zinc granules (Aldrich) with HCl.
Their concentrations were determined using EDTA as a titrant. For
Mn(II) and Mg(II), the sodium salt of Eriochrome black T in the
presence of triethanolamine and hydroxylamine chloride was used as
an indicator, while for Co(II) and Zn(II), titrations were performed in
the presence of sodium acetate and hexamethylenetetramine, using
xylenol orange as indicator. The Co(II) solutions were prepared and
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
calculated using the following equation:
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Article
that is highly conserved among retroviral/retrotransposon integrases
and bacterial insertion sequence transposases. Mol. Cell. Biol. 1992, 12,
2331−2338.
where C, N, and D are the fractions of the 21-mer substrate
converted to 19-mer (3′-proc product) or ST products for
DNA alone, DNA plus IN, and IN plus drug, respectively. The
IC50 values were determined by plotting the logarithm of drug
concentration versus percent inhibition to obtain the
concentration that produced 50% inhibition.
(8) Chiu, T. K.; Davies, D. R. Structure and function of HIV
Integrase. Curr. Top. Med. Chem. 2004, 4, 965−979.
(9) Kirschberg, T.; Parrish, J. Metal chelators as antiviral agents. Curr.
Opin. Drug Discovery 2007, 10, 460−472.
(10) Rouffet, M.; Cohen, S. M. Emerging trends in metalloprotein
inhibition. Dalton Trans. 2011, 40, 3445.
ASSOCIATED CONTENT
* Supporting Information
■
S
(11) Pais, G. C. G.; Burke, T. R. Novel aryl diketo-containing
inhibitors of HIV-1 integrase. Drugs Future 2002, 27, 1101−1111.
(12) Dayam, R.; Sanchez, T.; Neamati, N. Diketo acid
pharmacophore. 2. Discovery of structurally diverse inhibitors of
HIV-1 integrase. J. Med. Chem. 2005, 48, 8009−8015.
(13) Kawasuji, T.; Yoshinaga, T.; Sato, A.; Yodo, M.; Fujiwara, T.;
Kiyama, R. A platform for designing HIV integrase inhibitors. Part 1:
2-Hydroxy-3-heteroaryl acrylic acid derivatives as novel HIV integrase
inhibitor and modeling of hydrophilic and hydrophobic
pharmacophores. Bioorg. Med. Chem. 2006, 14, 8430−8445.
(14) Dayam, R.; Neamati, N. Active site binding modes of the beta-
diketoacids: a multi-active site approach in HIV-1 integrase inhibitor
design. Bioorg. Med. Chem. 2004, 12, 6371−6381.
Figures S1−S4 illustrating the crystal packing of ligands HL1
and HL2 and of complexes 9 and 10, respectively; Table S1
listing crystal data and structure refinement for compounds
HL1, HL2, 9, 10, and 12; Table S2 listing elemental analysis
data for ligands 2, 5, 6, 8−13, and complexes HL1 and HL2.
This material is available free of charge via the Internet at
http://pubs.acs.org. Crystallographic data (excluding structure
factors) for HL1, HL2, 9, 10, and 12 have been deposited with
the Cambridge Crystallographic Data Centre as Supplementary
Publications Nos. CCDC 829359−829363. Copies of the data
can be obtained free of charge on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, U.K. (fax, (+44) 1223-336-
033; e-mail, deposit@ccdc.cam.ac.uk).
(15) Pais, G. C. G.; Zhang, X.; Marchand, C.; Neamati, N.;
Cowansage, K.; Svarovskaia, E. S.; Pathak, V. K.; Tang, Y.; Nicklaus,
M.; Pommier, Y.; Burke, T. R. Jr. Structure activity of 3-aryl-1,3-diketo-
containing compounds as HIV-1 integrase inhibitors. J. Med. Chem.
2002, 45, 3184−3194.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 0521 905419. E-mail: dominga.rogolino@unipr.it.
■
(16) Zhao, X. Z.; Maddali, K.; Vu, B. C.; Marchand, C.; Hughes, S.
H.; Pommier, Y.; Burke, T. R. Jr. Examination of halogen substituent
effects on HIV-1 integrase inhibitors derived from 2,3-dihydro-6,7-
dihydroxy-1H-isoindol-1-ones and 4,5-dihydroxy-1H-isoindole-
1,3(2H)-diones. Bioorg. Med. Chem. Lett. 2009, 19, 2714−2717.
(17) Egbertson, M. S. HIV integrase inhibitors: from diketoacids to
heterocyclic templates: a history of HIV integrase medicinal chemistry
at Merck West Point and Merck Rome (IRBM). Curr. Top. Med.
Chem. 2007, 7, 1251−1272.
ACKNOWLEDGMENTS
The authors thank the “Centro Interfacolta
■
̀
Misure Giuseppe
Casnati” and the “Laboratorio di Strutturistica Mario Nardelli”
of the University of Parma, Italy, for facilities. We thank Dr.
Maria Orecchioni and Paolo Fiori for assistance with NMR
spectroscopy, Dr. Fabrizio Carta for his help on the preparation
of the ligand HL2, and Giuseppe Foroni for technical assistance.
(18) Billich, A. S-1360 Shionogi-GlaxoSmithKline. Curr. Opin. Invest.
Drugs 2003, 4, 206−209.
̀
M.S. is grateful to Universita di Sassari, Italy, for their partial
financial support. The work in N.N.’s laboratory was supported
by funds from the Campbell Foundation.
(19) Garvey, E. P.; Johns, B. A.; Gartland, M. J.; Foster, S. A.; Miller,
W. H.; Ferris, R. G.; Hazen, R. J.; Underwood, M. R.; Boros, E. E.;
Thompson, J. B.; Weatherhead, J. G.; Koble, C. S.; Allen, S. H.;
Schaller, L. T.; Sherrill, R. G.; Yoshinaga, T.; Kobayashi, M.; Wakasa-
Morimoto, C.; Miki, S.; Nakahara, K.; Noshi, T.; Sato, A.; Fujiwara, T.
The naphthyridinone GSK364735 is a novel, potent human
immunodeficiency virus type 1 integrase inhibitor and antiretroviral.
Antimicrob. Agents Chemother. 2008, 52, 901−908.
ABBREVIATIONS USED
■
HIV-1, human immunodeficiency virus type 1; IN, HIV-1
integrase; DKA, β-diketo acid; HPCA, hydroxypyrimidinecar-
boxamide; QCA, quinolonecarboxylic acid; SSTI, selective
strand transfer inhibitor
(20) Boros, E. E.; Edwards, C. E.; Foster, S. A.; Fuji, M.; Fujiwara, T.;
Garvey, E. P.; Golden, P. L.; Hazen, R. J.; Jeffrey, J. L.; Johns, B. A.;
Kawasuji, T.; Kiyama, R.; Koble, C. S.; Kurose, N.; Miller, W. H.;
Mote, A. L.; Murai, H.; Sato, A.; Thompson, J. B.; Woodward, M. C.;
Yoshinaga, T. Synthesis and antiviral activity of 7-benzyl-4-hydroxy-
1,5-naphthyridin-2(1H)-one HIV integrase inhibitors. J. Med. Chem.
2009, 52, 2754−2761.
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