Metal-chelating properties and antiviral activity of some 2-hydroxyphenyl amides

Article abstract of DOI:10.1016/j.poly.2017.03.032

Influenza virus is an hot topic in medicinal chemistry and great efforts are ongoing for the discover of new antivirals able to overcome problems related to resistant strains and adverse side effects of current drugs. Influenza virus endonuclease is an attractive target for antiviral drug development and in particular the strategy to chelate the metal ion(s) within the active site proved to be an efficient mode to inhibit enzymatic activity. Our previous findings revealed that 2-hydroxyamide derivatives are able to chelate Mg²⁺ ions, forming complexes with different stoichiometric ratios. Here we report on the activity of the three ligands N-(4-fluorobenzyl)-2-hydroxybenzamide, N-(4-fluorobenzyl)-2,3-dihydroxybenzamide, and N1,N3-bis(4-fluorobenzyl)-2-hydroxyisophthalamide, containing the salicylic group, and their Mg²⁺ complexes (7)–(9), evaluated by means of virus yield assay in influenza virus-infected MDCK cells and vRNP reconstitution assay in HEK293T cells. In some cases, promising anti-influenza activities with EC₅₀ values in the low micromolar range were found. As a contribute to clarify the activity in cells of the ligands, here we also present a study on the their coordinating properties towards the other essential metal ion Cu(II), carried out by potentiometric and calorimetric measurements.

Full text of DOI:10.1016/j.poly.2017.03.032

Accepted Manuscript
Metal-chelating properties and antiviral activity of some 2-hydroxyphenyl
amides
M. Carcelli, E. Fisicaro, C. Compari, L. Contardi, D. Rogolino, C. Solinas, A.
Stevaert, L. Naesens
PII:
S0277-5387(17)30220-6
DOI:
http://dx.doi.org/10.1016/j.poly.2017.03.032
POLY 12547
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 January 2017
17 March 2017
19 March 2017
Please cite this article as: M. Carcelli, E. Fisicaro, C. Compari, L. Contardi, D. Rogolino, C. Solinas, A. Stevaert,
L. Naesens, Metal-chelating properties and antiviral activity of some 2-hydroxyphenyl amides, Polyhedron (2017),
doi: http://dx.doi.org/10.1016/j.poly.2017.03.032
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Metal-chelating properties and antiviral activity of some
2-hydroxyphenyl amides
M. Carcelli,*a,b E. Fisicaro,c C. Compari,c L. Contardi,c D. Rogolino,a,b C. Solinas,d A. Stevaert,e L.
Naesense
aDipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, University of
Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy and bCIRCMSB (Consorzio Interuniversitario
di Ricerca in Chimica dei Metalli nei Sistemi Biologici), Parma Unit, University of Parma, Italy,
cDipartimento di Scienze degli Alimenti e del Farmaco, University of Parma, Parco Area delle Scienze
27/A, 43124 Parma, Italy, dDipartimento di Chimica e Farmacia, Università di Sassari, Via Vienna 2, I-
07100 Sassari, Italy, eRega Institute for Medical Research, KU Leuven – University of Leuven, B-3000
Leuven, Belgium
*Corresponding author:
email: mauro.carcelli@unipr.it
Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale
Università di Parma
Phone: +39 0521 905427
Fax: +39 0521 905557
1

Abstract
Influenza virus is an hot topic in medicinal chemistry and great efforts are ongoing for the discover of
new antivirals able to overcome problems related to resistant strains and adverse side effects of current
drugs. Influenza virus endonuclease is an attractive target for antiviral drug development and in particular
the strategy to chelate the metal ion(s) within the active site proved to be an efficient mode to inhibit
enzymatic activity. Our previous findings revealed that 2-hydroxyamide derivatives are able to chelate
Mg2+ ions, forming complexes with different stoichiometric ratios. Here we report on the activity of the
three ligands N-(4-fluorobenzyl)-2-hydroxybenzamide, N-(4-fluorobenzyl)-2,3-dihydroxybenzamide, and
N1,N3-bis(4-fluorobenzyl)-2-hydroxyisophthalamide, containing the salicylic group, and their Mg2+
complexes (7)-(9), evaluated by means of virus yield assay in influenza virus-infected MDCK cells and
vRNP reconstitution assay in HEK293T cells. In some cases, promising anti-influenza activity with EC50
values in the low micromolar range were found. As a contribute to clarify the activity in cells of the
ligands, here we also present a study on the their coordinating properties towards the other essential metal
ion Cu(II), carried out by potentiometric and calorimetric measurements.
Keywords: chelating ligands; magnesium complexes; ITC measurements; influenza virus
endonuclease; antiviral activity.
1. Introduction
Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA
genome, belonging to the Orthomyxoviridae.1 Seasonal human influenza A or B virus infections are an
important cause of morbidity and mortality;2 moreover there is a permanent risk of sudden influenza
pandemics, such as the notorious ‘Spanish flu’ in 19183 and the swine-origin H1N1 pandemic in 2009.4
Influenza vaccination is a widely used prophylactic measure, whereas two classes of anti-influenza
2

virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or the viral
neuraminidase (zanamivir and oseltamivir). The M2 inhibitors have limited clinical utility due to their
central nervous system side effects, in addition resistance is a growing concern for both M2 inhibitors
and oseltamivir.5,6,7,8,9 Therefore, there is an urgent need for new antiviral compounds, preferably based
upon novel pharmacophores and different modes of action.10
The influenza virus polymerase is widely recognized as an attractive target for antiviral drug
development: inhibition of its PA endonuclease, in particular, has deserved much attention in recent
years.11,12,13 The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and
PA.14,15 The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter).16,17,18 The
number and type of ions present in the catalytic site are not definitely clarified: it seems to contain
one,17,19 two,16,20 or even three21 manganese(II) or, more probably,22,23 magnesium(II) ions as metal
cofactors. Anyway, the two-metal-ion model is supported by several biochemical findings.24,25 Diverse
PA-binding agents with metal-chelating properties have been identified as influenza endonuclease
inhibitors (Figure 1): 2,4-dioxobutanoic acid derivatives,26,27 flutimide and its derivatives,28 tetramic
acids,29 5-hydroxypyrimidin-4-one derivatives,30 marchantins31 and green tea catechins, like
epigallocatechin-3-gallate.32,33 These inhibitors share a common pharmacophoric motif that is likely
responsible for sequestration of the metal ions in the catalytic site of PA-Nter.
In recent years, we focused our research on chemical scaffolds that are able to chelate the metal ions in
the PA-Nter active site, resulting in inhibition of influenza PA endonuclease activity.34,35,36 More
specifically, considering the functional resemblance between the active sites of HIV integrase and PA-
Nter,12 we focused here our attention on N-(4-fluorobenzyl)-2-hydroxybenzamide, N-(4-fluorobenzyl)-
2,3-dihydroxybenzamide, and N1,N3-bis(4-fluorobenzyl)-2-hydroxyisophthalamide (HL1, H2L2, and
HL3, Scheme 1), previously tested as HIV integrase inhibitors,37 and containing the salicylic group.
These molecules have different coordinating abilities (Figure 2); in particular, HL1, H2L2, and HL3
are able to chelate Mg2+ ions, forming complexes with different stoichiometric ratios (Scheme 1).34
3

Here we report on the activity of HL1-HL3 and the Mg2+ complexes (7)-(9), evaluated by means of
virus yield assay in influenza virus-infected MDCK cells and vRNP reconstitution assay in HEK293T
cells. Even if it is premature to advance hypothesis about the mechanism of action of these compounds
in cells, it seemed reasonably to connect the activity of HL1-HL3 also to their possibility to chelate the
metal ions in the PA-Nter active site. The importance to advance in the understanding of the
coordination chemistry of HL1-HL3, move us to investigate also their chemistry in solution with
copper(II) ion, another essential trace element.
2. Experimental
2.1
Chemistry. All reagents of commercial quality were used without further purification. Purity of
compounds was determined by elemental analysis and verified to be ≥95% for all synthesized
molecules. NMR spectra were recorded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The
ATR-IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by
using a diamond crystal plate in the range of 4000-400 cm-1. Elemental analyses were performed by
using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation.
Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI)
time-of-flight Micromass 4LCZ spectrometer. MS spectra were acquired in positive EI mode by means
of a DEP-probe (Direct Exposure Probe) mounting on the tip a Re-filament with a DSQII Thermo
Fisher apparatus, equipped with a single quadrupole analyzer. HL1, H2L2 and HL3 and the
corresponding magnesium complexes 7-9 were synthesized according to slightly modified literature
procedures34 (see Supporting information for the chemical characterization).
2.2
Potentiometric titrations. The Cu(II) stock solutions were prepared from CuCl2·2H2O (Merck)
and bidistilled water and were acidified by adding HCl to avoid Cu(II) hydrolysis. The concentration of
the excess H+ was obtained by titrating with KOH. Cu(II) concentration was determined by using
4

EDTA as a titrant in the presence of concentrated ammonia using Fast Sulfon Black as indicator.
Equilibrium constants for protonation and complexation were determined by means of potentiometric
titrations in methanol:water=9:1 v/v solution at ionic strength of 0.1 M KCl. The titrations were carried
out under nitrogen in the pH range 2.5-11 by using 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, and controlled by a homemade software. Temperature (25 0.1 °C) was controlled to
0.1°C by using a thermostatic circulating water bath (ISCOGTR 2000 IIx). 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 method38 allowing for 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). Appropriate aliquots of ligand solution, prepared
by weight, were titrated with standard KOH (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 nitrogen, were used throughout. The experimental procedure to reach high
accuracy in the determination of the equilibrium constants in this mixed solvent has been described in
detail elsewhere.39 The protonation constants of HL1, H2L2 and HL3 were obtained by titrating 20 ml
of samples of each ligand (3 x 10-3 M). For obtaining the complex formation constants, titrations were
performed in different ligand/metal ratios (from 1 up to 4). At least two measurements (about 60
experimental points each) were performed for each system. The software HYPERQUAD40 was used
for obtaining the speciation and the logarithm of the stability constants (log βpqr) from titration data.
βpqr = [MpLqHr]/[M]p[L]q[H]r is the cumulative formation constant for the equilibrium reaction pM +
qL + rH = MpLqHr, in which M indicates the metal, L the completely deprotonated ligand and H the
proton. Charges are omitted for simplicity.
2.3
Isothermal titration calorimetry (ITC). ITC measurements were carried out on a CSC model
5300 N-ITC III isothermal titration calorimeter (Calorimetry Sciences Corporations, USA) at 25°C.
5

KOH solution (0.065–0.145 M) in methanol:water = 9:1 at 0.1 M KCl ionic strength was injected in
steps of 5 µL into a 960 µL reaction cell by a 250 µL syringe with an interval of 400-500 s between
two successive injections, with stirring speed of 150 revolutions per minute (rpm), following the
procedure previously described.41 For obtaining the protonation heat of the ligand, the cell was filled
up with a solution of the ligand in methanol:water = 9:1, 0.1 M KCl ionic strength. Dilution heats were
subtracted by carrying out blank experiments, in which the KOH solution was injected into the same
solvent, without ligand. The molar enthalpy change for the reaction H++ OH−= H2O was obtained by
titrating a solution of HCl (in the cell) by KOH, in the burette, both prepared in methanol:water = 9:1
at ionic strength 0.1 M KCl. It results -20.6 0.4 kJ mol-1.41 The experimental peaks were integrated
and corrected for the dilution heats by the NanoAnalyze (TA Instrument) software. The data so
obtained were elaborated by the Hyp∆H software,41,42 assuming the protonation constants from
potentiometric measurements.
2.4
Cells and media. Madin-Darby canine kidney (MDCK) cells (a kind gift from Dr. M.
Matrosovich, Marburg, Germany) and human embryonic kidney 293T (HEK293T) cells (purchased
from Thermo Fisher Scientific, Waltham, MA) were cultivated in Dulbecco's modified Eagle medium
supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate.
During virus experiments, the MDCK cells were maintained in MDCK infection medium, consisting of
Ultra MDCK medium (Lonza, Basel, Switzerland) supplemented with 0.0225% sodium bicarbonate, 2
mM L-glutamine, and 2 µg/ml tosyl phenylalanyl chloromethyl keton (TPCK) - treated trypsin (Sigma-
Aldrich, St. Louis, MO). Cells were incubated in a humidified atmosphere containing 5% CO2.
2.5
vRNP reconstitution assay. The procedure to determine the inhibitory effect of the compounds
on influenza vRNPs reconstituted in HEK293T cells, is described in full detail elsewhere.43 Briefly, the
four relevant plasmids (i.e. the expression plasmids for PB1, PB2, PA and NP) were combined with the
firefly luciferase reporter plasmid, and co-transfected into HEK293T cells using Lipofectamin 2000
6

(Invitrogen, Life Technologies, Gent, Belgium). After incubation at 37°C for 24 h in the presence of
serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI)
was used to determine luciferase activity. The 50% effective concentration (EC50) was defined as the
compound concentration causing 50% reduction in the vRNP-driven luciferase signal, as compared to
cells receiving medium instead of compound. These EC50 values were calculated by non-linear
regression analysis using GraphPad Prism software, on data from 2-5 experiments. In parallel,
compound cytotoxic activity was determined in untransfected HEK293T cells which had been
incubated with serial dilutions of the compounds for 24 h, using the MTS cell viability assay (CellTiter
96 AQueous One Solution Cell Proliferation Assay, Promega). These spectrophotometric data were used
to calculate the 50% cytotoxic concentration (CC50), i.e. the concentration reducing cell viability by
50%, as compared to wells receiving medium instead of compound. Ribavirin (Virazole; ICN
Pharmaceuticals, Costa Mesa, CA) was included as the reference compound.
2.6
Virus yield assay. We previously published in full detail the virus yield assay to determine the
anti-influenza virus activity in MDCK cell cultures. 44 Briefly, one day prior to infection, MDCK cells
were seeded into 96-well plates at 25,000 cells per well. At day 0, serial dilutions of the test
compounds were added, immediately followed by infection with influenza A/PR/8/34 virus. After 24 h
incubation at 35°C, the virus amount in the supernatants was estimated by determining the viral
genome copy number in a one-step quantitative real-time reverse transcription (qRT)-PCR assay
(CellsDirect One-Step qRT-PCR kit; Invitrogen), with influenza virus M1-specific primers and probe.
The EC99 and EC90 values were calculated by interpolation from data of 2-3 experiments, and defined
as the compound concentration causing respectively a 2-log10 and 1-log10 reduction in viral RNA
(vRNA) copy number, as compared to the virus control receiving no compound. In parallel, the CC50
values after 24 h incubation with compounds were determined in uninfected MDCK cells, using the
spectrophotometric MTS cell viability assay described above, respectively. Ribavirin was included as
the reference compound.
7

3. Results and Discussion
Salicylamides (1)-(3) were obtained with good yields (Scheme 1);34 deprotection of the hydroxyl
groups was then carried out with 1M solution of boron tribromide to obtain HL1, H2L2 and HL3
(Scheme 1). The protonation constants of the salicylic OH are 9.51(1) for HL1, 9.11(1) for H2L2, and
7.28(1) for HL3; the second acidity constant for H2L2 is greater than 12, out of the working range of
the glass electrode.34 The thermodynamic study of the protonation of the ligands was completed by
performing ITC experiments and the results are shown in Table 1; in Figure 3 the comparison
between experimental and computed cumulative heats for HL1 are reported (data for H2L2 and HL3
.
.
are in Supporting information). The complexes Mg(L1)2 3H2O (7), Mg2(L2)2 2.5H2O (8) and
.
Mg(L3)2 3H2O (9) (Scheme 1) were synthesized and characterized (see Supporting information) as
previously disclosed.34 We want to evaluate the activity of HL1, H2L, and HL3 in influenza virus-
infected cells. In the cell the ligands HL1, H2L, and HL3 could interact with ions different from the
Mg(II) of the active site of PA-Nter, so their coordinating properties towards copper(II), another
essential trace element, are also investigated for comparison. As far as the potentiometric titrations of
the ligand/Cu(II) systems are concerned, we had to deal with poor solubility in the systems HL1/Cu(II)
and HL3/Cu(II). In particular, for HL1 we found the presence of a precipitate in the pH range 6 – 8 for
metal:ligand ratio 1:2 and around pH = 6 for the metal:ligand ratio 1:4. We suppose that the species
CuHL1 is poorly soluble. The precipitate is formed above pH 8 (1:4 ratio) and above pH 9 (1:2 ratio)
with HL3, probably due the formation of insoluble hydroxylated species. Only the experimental points
in which the solution appeared clear were processed by the software HYPERQUAD40. The models of
speciation in solution for the ligands and Cu(II), showing the best statistical parameters and the best fit
between experimental and computed potentiometric titration curves, are shown in Table 2. The
distribution diagrams as a function of the pH, evaluated for metal to ligand ratio 4:1, [Cu(II)] = 25
8

mM, are reported in Figure 4. As expected, the ligands have a greater affinity towards Cu(II) than
towards Mg(II) (see Supporting information for the distribution diagrams and the formation constants
with Mg(II)).34 This can give rise to some doubts regarding the stability of the Mg(II) complexes (7)-
(9) in cells, even if, obviously, Cu(II) is not uncoordinated in cells and the resulting equilibria are more
complex. While Mg(II) starts to bind the ligand at a pH value at which it is already deprotonated,
probably forming outer sphere complexes, Cu(II) is able to lower the pKa value of the ligand of a few
units, forming complex species already at acidic pH. HL1 and HL3 give rise to the same species in
solution, namely CuL, CuL2 and CuL2H-1, with similar affinity. In the case of HL1, the precipitate
formed around pH = 7 could be the neutral specie CuL2, that then re-solubilizes forming CuL2H-1.
Moreover, for HL1 we were able to find out also the species CuL2H-2, formed beyond pH = 10 and
probably due to the dissociation of two water molecules completing the coordination sphere of the
Cu(II) ion. It was not possible to search for this species in the case of HL3, because the solution is
turning opaque beyond pH = 9, probably due to the precipitation of hydroxylated compounds. H2L2
gives rise to the same species as HL1, but seems unable to form the complex CuL2. At physiological
pH the species CuL2 is the most abundant for HL3, it reaches only a few percent for HL1 and it is
completely absent for H2L2. These last two ligands at neutral pH form preferably the species ML. We
have also examined the hypothesis of the formation in solution of di-metallic species M2L2, but they
were always rejected by the software.
3.1
Anti-influenza virus activity in enzymatic and cellular assays. The hydroxybenzamide-based
ligands HL1-HL3 and their magnesium(II) complexes (7)-(9) were previously tested for their ability to
inhibit the PA endonuclease activity in an enzymatic assay with recombinant PA-Nter (Table 3):34 (8)
proved to be the most active compound, with an IC50 of 18 µM, which is 3-fold higher than that of the
reference compound DPBA, but lower than the value of the corresponding free ligand H2L2 (IC50 = 33
µM). On the contrary, HL1, HL3 and the complexes (7) and (9) did not inhibit the endonuclease
reaction at 500 µM, the highest concentration tested. These results of the enzymatic assays, moved us
9

now to evaluate these compounds in two cellular assays, a reporter-based vRNP reconstitution assay in
HEK293T cells and a virus yield assay in MDCK cells (Table 3). In the vRNP reconstitution assay,
which is well suited to determine the activity and selectivity of possible PA endonuclease inhibitors in
cell culture, the effect of the compounds on the activity of reconstituted vRNPs is measured. HL3 and
the corresponding complex (9) displayed the most potent antiviral activity, with EC50 values of 6.7 and
3.1 µM respectively (Table 3), and a selectivity index (ratio of CC50 to EC50) of 10. In the virus yield
assay on the other hand, the magnesium complexes showed the best antiviral profile, with EC90 values
of 81, 87 and 8.5 µM for (7), (8) and (9), respectively.
Comparison of the biological activities reported in Table 3 shows for some compounds a discrepancy
between anti-influenza virus activity in cells and inhibition in the PA-Nter enzymatic assay. This is an
indication that these compounds probably act also on a PA-unrelated target in virus-infected cell
cultures. Their antiviral activity could be related to inhibition of influenza virus entry, viral RNA
synthesis, or a stage beyond viral RNA synthesis (possibly virus maturation or release). In order to
clearly define the antiviral target of these different molecules, specific mechanistic experiments are
currently ongoing.
4.1
CONCLUSIONS
Salicylamides have been studied for their biological properties for a long time.45 In the present work we
show that salicylamides HL1-HL3 and their Mg(II) complexes (7)-(9) have interesting activity against
influenza virus in cells. It is worth of note that, in some cases, the best antiviral profiles are shown by the
metal complexes. Even in these cases, however, it is difficult to know if the complexes are the effective
drug or, more properly, pro-drugs. In the biological medium, in fact, there are metal ions and ligands that
can behave as competitors. The complexing properties of the salicylamides in solution suggested that their
activity can be due to the possibility to chelate the Mg(II) ions in the active site of influenza virus
10

endonuclease. However, the copper(II) complexes of HL1-HL3 are more stable than the corresponding
magnesium(II) ones: in cells free copper(II) is present at low concentrations, but it is difficult to think that
HL1-HL3 can act as uncomplexed ligands. On the other side, in cells the magnesium(II) complexes (7)-
(9) could give rise to transmetallation. In conclusion, care has to be taken in the individuation of the
chemical species responsible for the biological activity. Another, possibly correlated, notable point to be
considered is that some compounds (HL1 or HL3, for example) are more active in cells than in the
enzyme assays. Analysis of the (multitarget?) mechanism of action and full pharmocological properties of
similar salicylamides and their metal complexes are warranted in follow-up studies.
Abbreviations
CC50
50% cytotoxic concentration
Direct Exposure Probe
DEP
DPBA
2,4-dioxo-4-phenylbutanoic acid
EC50, EC90, EC9950%, 90%, 99% maximal effective concentration
ESI-MS
Electrospray mass spectral analyses
human embryonic kidney 293T cells
Isothermal Titration Calorimetry
Madin-Darby canine kidney cells
N-terminal domain of PA
HEK293T cells
ITC
MDCK cells
PA-Nter
vRNPs
viral ribonucleoproteins
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
11

ACKNOWLEDGMENTS
“Centro Interdipartimentale Misure Giuseppe Casnati” of the University of Parma are thanked for
facilities.
12

Table 1. Thermodynamic parameters for the protonation of the ligands under study. In brackets the
standard deviation on the last figure.
logKa
∆G (kJ mol-1) ∆H (kJ mol-1) ∆S (J K-1mol-1)
HL1
H2L2
HL3
9.51(1)
-54.27(2)
-51.99(2)
-41.54(2)
-10.9(1)
-16.9(5)
-8.6(2)
145(2)
117(4)
110(2)
9.11(1)
7.28(1)
13

Table 2. Logarithms of the formation constants (βpqr = [MpLqHr]/[M]p[L]q[H]r) in methanol:water = 9:1
v/v, I = 0.1 M KCl at 25 °C for the ligands under study with Cu(II). SDs are given in parentheses.
p
q
r
HL1
H2L2
HL3
1
1
1
1
1
2
2
2
0
5.66(37)
8.99(47)
0.10(38)
-12.02(38)
8.74(6)
5.42(6)
9.22(10)
0.82(8)
0
-1
-2
4.53(7)
-6.70(11)
14

Table 3. Inhibitory activity of ligands HL1-HL3 and Mg2+ complexes (7)-(9) in the enzymatic assay with
influenza virus PA-Nter endonuclease,34 or in cellular influenza virus assays based on virus yield or vRNP
reconstitution.
Enzyme assay with
PA-Ntera
Virus yield assay in influenza virus-infected
MDCK cellsb
vRNP reconstitution assay in
HEK293T cellsc
Antiviral Activity
Cytotoxicity
Activity
Cytotoxicity
Compound
IC50
>500
33
>500
>500
18
>500
5.5
-
EC99
>200
>50
>200
126
•125
13
EC90
>200
>50
>200
81
MCC
CC50
148
EC50
35
20
6.7
104
24
3.1
-
CC50
155
79
HL1
H2L2
HL3
(7)
(8)
(9)
DPBAd
Ribavirin
>200
50
>200
•200
113
50
>180
>144
>151
>194
41
74
>200
44
30
87
8.5
-
13
-
8.5
-
•200
-
>200
-
>200
9.3
aPublished results in the enzymatic PA-Nter endonuclease assay.34
bMDCK (Madin-Darby canine kidney) cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the
compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR. The EC99 and EC90 values represent
the compound concentrations (in µM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively. The cytotoxicity,
assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS
cell viability assay, in µM).
cHEK293T (human embryonic kidney 293T) cells were co-transfected with the four vRNP-reconstituting plasmids and the
luciferase reporter plasmid in the presence of the test compounds. The EC50 value represents the compound concentration (in
µM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection. The CC50 (in µM), i.e.
the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.
dDPBA, 2,4-dioxo-4-phenylbutanoic acid, used as reference.
15

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Scheme, Figure and Table captions
Scheme 1. Synthesis of the ligands HL1-HL3 and the corresponding magnesium complexes (7)-(9)a
Figure 1. Some representative influenza endonuclease inhibitors with, in bold, their chelating chemotype.
Figure 2. One metal (HL1) versus two metals binding (H2L2 and HL3) chelating modes.
Figure 3. a) Raw data from the ITC titration for the protonation of HL1. Upward peaks indicate an
exothermic reaction. Each peak corresponds to a single 5 µl injection. b) Comparison between
experimental and computed cumulative heats (output from Hyp∆H software). Experimental conditions:
HL1 9.5 µmoles, titrant KOH 0.1 M.
Figure 4. Distribution diagrams for the systems ligand:Cu=2:1, [Cu2+] = 1.25 mM. a) Ligand = HL1, in
the pH range 6-8 a precipitate is formed. b) Ligand = H2L2. c) Ligand = HL3, the solution becomes
cloudy above pH=9. The monodeprotonated ligands are indicated as L; charges are omitted for
simplicity.
Table 1. Thermodynamic parameters for the protonation of the ligands under study. In brackets the
standard deviation on the last figure.
Table 2. Logarithms of the formation constants (βpqr = [MpLqHr]/[M]p[L]q[H]r) in methanol:water = 9:1
v/v, I = 0.1 M KCl at 25 °C for the ligands under study with Cu(II). SDs are given in parentheses.
21

Table 3. Inhibitory activity of ligands HL1-HL3 and Mg2+ complexes (7)-(9) in the enzymatic assay with
influenza virus PA-Nter endonuclease,34 or in cellular influenza virus assays based on virus yield or vRNP
reconstitution.
22

23

24

25

26

27

Graphical Abstract Synopsis
2-hydroxyamide pharmachophore can chelate metal cofactor(s) within influenza virus endonuclease. 2-
Hydroxyamide ligands and their magnesium complexes have interesting antiviral activity on infected
cells. However, investigation of solution behaviour of this pharmachophore highlighted higher stability
for copper(II) complexes vs magnesium ones, rising questions about the effective form of active
species.
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29