Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages

Article abstract of DOI:10.1002/cmdc.201700759

The enzyme Zmp1 is a zinc-containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8-hydroxyquinoline-2-hydroxamate scaffold. Among the synthesized compounds, N-(benzyloxy)-8-hydroxyquinoline-2-carboxamide (1 c) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte-derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation.

Full text of DOI:10.1002/cmdc.201700759

Accepted Article
Title: Development of potent inhibitors of the Mycobacterium
tuberculosis virulence factor Zmp1 and evaluation of their effect
on mycobacterial survival inside macrophages
Authors: Marco Paolino, Margherita Brindisi, Alessandra Vallone,
Stefania Butini, Giusepp Campiani, Chiara Nannicini,
Germano Giuliani, Maurizio Anzini, Stefania Lamponi,
Gianluca Giorgi, Diego Sbardella, Davide M Ferraris, Stefano
Marini, Massimo Coletta, Ivana Palucci, Mariachiara Minerva,
Giovanni Delogu, Ilaria Pepponi, Delia Goletti, Andrea
Cappelli, Sandra Gemma, and Simone Brogi
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To be cited as: ChemMedChem 10.1002/cmdc.201700759
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10.1002/cmdc.201700759
ChemMedChem
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Development of potent inhibitors of the Mycobacterium
tuberculosis virulence factor Zmp1 and evaluation of their effect
on mycobacterial survival inside macrophages
Marco Paolino,[†,a] Margherita Brindisi,[†,a] Alessandra Vallone,[a] Stefania Butini,[a] Giuseppe Campiani,[a]
Chiara Nannicini,[a] Germano Giuliani,[a] Maurizio Anzini,[a] Stefania Lamponi,[a] Gianluca Giorgi,[a] Diego
Sbardella,[b] Davide M. Ferraris,[c] Stefano Marini,[b] Massimo Coletta,[b] Ivana Palucci,[d] Mariachiara
Minerva,[d] Giovanni Delogu,[d] Ilaria Pepponi,[e] Delia Goletti,[e] Andrea Cappelli,[a]* Sandra Gemma,[a]*
Simone Brogi[a]
[a]
Dr M. Paolino, Dr. M. Brindisi, Dr. A. Vallone, Prof. S. Butini, Prof. G. Campiani, Dr. C. Nannicini, Dr. G. Giuliani, Prof. M. Anzini, Dr. S. Lamponi, Prof. G.
Giorgi, Prof. A. Cappelli, Prof. S. Gemma, Dr. S. Brogi
European Research Centre for Drug Discovery and Development –NatSynDrugs- and Department of Biotechnology, Chemistry, and Pharmacy
University of Siena
via Aldo Moro 1, 53100 Siena, Italy
E-mail: gemma@unisi.it; andrea.cappelli@unisi.it
†These authors contributed equally
[b]
[c]
[d]
[e]
Prof. D. Sbardella, Prof. S. Marini, Prof. M. Coletta
Department of Clinical Sciences and Translational Medicine
University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy
Dr. D. M. Ferraris
Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences
University of Piemonte Orientale “Amedeo Avogadro”, Largo Donegani 2, I-28100 Novara, Italy
Dr. I. Palucci, Dr M. Minerva, Prof. G. Delogu
Institute of Microbiology
Università Cattolica del Sacro Cuore – Fondazione Policlinico Universitario Gemelli, Rome, Italy, Largo A. Gemelli, 8 – 00168 Rome, Italy
Dr I. Pepponi, Dr. D. Goletti
Translational Research Unit
National Institute for Infectious Diseases (INMI) “L. Spallanzani”, Via Portuense, 292, 00149 Roma, Italy
Supporting information for this article is given via a link at the end of the document
Abstract: The enzyme Zmp1 is a zinc-peptidase having a critical role
in Mycobacterium tuberculosis pathogenicity. Here we describe the
identification of a small set of Zmp1 inhibitors based on a novel 8-
hydroxyquinoline-2-hydroxamate scaffold. Among the synthesized
compounds, 1c (N-(benzyloxy)-8-hydroxyquinoline-2-carboxamide)
was the most potent Zmp1 inhibitor known to date, and its binding
mode was analysed both through kinetic studies and molecular
modelling, identifying critical interactions of 1c with the zinc-ion and
residues in the active site. Effect of 1c on intracellular Mycobacterium
survival was assayed in J774 murine macrophages infected with M.
tuberculosis H37Rv or M. bovis BCG and human monocyte-derived
macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and
genotoxicity were also assessed. Overall, inhibitor 1c displayed
interesting in vitro antitubercular properties worth of further
investigation.
TB therapy, i.e. delamanid and bedaquiline,[3, 4] safer and more
effective drugs with innovative mechanisms of action are urgently
required to increase our armamentarium against MDR-TB.
M. tuberculosis evolved several virulence factors that can
interfere with the host immune response to infection.[5] Among
them, it has been demonstrated that the virulence factor Zmp1, a
Zn2+-metalloprotease essential for mycobacterial survival in
macrophages in vitro, interferes with the phagosome maturation
by inhibiting the inflammasome.[6, 7] This allows the bacterium to
survive in the phagosome and prevents fusion of the phagosome
with the lysosome. Although some findings were later debated,[8]
Zmp1 plays a key role in M. tuberculosis pathogenicity and
therefore is worth of further investigation as a putative drug
target.[9-11]
Zinc-peptidases are classically inhibited by molecules
incorporating a zinc binding group (ZBG) able to coordinate the
active site metal ion, and responsible for high-affinity and
inhibitory potency. The vast majority of zinc-peptidase inhibitors
reported to date contains a hydroxamic acid as the ZBG, although
Introduction
a
number of heterocyclic-based ZBGs have also been
identified.[12] Among them, the 8-hydroxyquinoline system
attracted our attention since it is a privileged structure and has
Tuberculosis (TB) is a disease caused by the facultative
intracellular bacterium Mycobacterium tuberculosis (M.
tuberculosis). According to the World Health Organization (WHO)
data, TB affected 10.4 million people in 2015, and 1.2 million
people were co-infected with the human immunodeficiency virus
(HIV).[1] One of the main public health challenges regarding TB is
the diffusion of multi-drug resistant (MDR)- and extensively drug
resistant (XDR)-TB infections.[2] Despite the recent new tools in
been used as metal-interacting group for
pharmacological applications.[13] In order to identify potent and
drug-like Zmp1 inhibitors, we designed novel ZBG by
hybridizing the 8-hydroxyquinoline ring with a hydroxamate
moiety (Figure 1, compounds 1a-e). For evaluating structure-
a
range of
a
1
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Zmp1 inhibitory activity and kinetic analysis
activity relationships, we also prepared the isosteric analogues
2a,b, lacking the hydroxamate oxygen, and compounds 5a,b
Novel compounds and 3,4 were tested for their in vitro
inhibitory activity against Zmp1 by using a fluorimetric assay. The
initial approach was only focused on selecting molecules
characterized by the lowest IC50 (Table 1). Gratifyingly, our design
approach led to the discovery of potent, nanomolar inhibitors of
Zmp1. To the best of our knowledge, the benzyl-derivative 1c
(IC50 = 11 nM) is the most potent Zmp1 inhibitor reported to date,
compared to a previously reported rhodamine-based inhibitor (Ki
= 94 nM).[14] Moreover, we assayed the inhibitory activity of 1c on
other Zn2+ enzymes, such as the matrix metalloproteinases
(MMPs), namely MMP-1 and MMP-2, which belong to the
collagenase and gelatinase subgroups, respectively, and are
among the most represented Zn2+ enzymes in human tissues.[15]
The IC50 of 1c on MMP-1 turned out to be >10 µM and that on
MMP-2 ≥ 1 µM (data not shown). This result emphasizes the
specificity of 1c in inhibiting Zmp1.
Figure 1. Structures of 8-hydroxyquinoline-derivatives 1a-e, 2a,b, 3, 4 and 5a,b
investigated as Zmp1 inhibitors.
lacking the free 8-hydroxy group. The activity of the novel
chemical entities was also compared to to simpler 8-
hydroxyquinoline-containing compounds such as acid 3 and
clioquinol (5-chloro-7-iodo-quinolin-8-ol, 4), a compound that is
able of interact with Zn ion but lacking the hydrophobic pendant
side chain. Derivatives 5a,b were designed to investigate the role
of the hydroxyl group at C8. We identified the potent Zmp1
inhibitor 1c (Figure 1), we characterized its kinetics of inhibition
and mode of binding (through extensive computational studies)
and evaluated the growth of M. tuberculosis H37Rv inside both
human and murine macrophages in vitro.
Scheme 1. Synthesis of compounds 1a-e, 2a,b, and 5a,b.
Table 1. Zmp1 Inhibitory Activity (IC50, µM)
Results and discussion
Chemistry
Compound
IC50 ( M)
µ
Compounds 1a-e, 2a,b and 5a,b were synthesized as described
in Scheme 1 through a straightforward synthetic approach.
Coupling of carboxylic acid 3 with commercially available O-
alkylhydroxylamines 6a-e or alkylamines 7a,b under classical
coupling conditions afforded target compounds 1a-e and 2a,b in
good overall yield. The structure of compounds 1b-d was
confirmed by X-ray crystallographic studies (Figures S1-3 of the
Supporting Information file). The 8-hydroxyquinoline-2-carboxylic
acid 3 was used as the starting material also for the synthesis of
compounds 5a. Accordingly, acid-catalyzed esterification of the
carboxylic acid followed by O-methylation afforded intermediate 8.
This latter compound was treated with LiOH to release the free
carboxylic acid 9a, which was coupled with 6c as described above
to afford 5a. Finally, the same coupling procedure was applied to
quinoline-2-carboxylic acid 9b to furnish 5b.
1a
0.115 ± 0.015
0.123 ± 0.016
0.011 ± 0.002
0.217 ± 0.034
0.189 ± 0.022
>10
1b
1c
1d
1e
2a
2b
>10
3
>10
5a
>10
5b
>10
Clioquinol (4)
>50
2
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For the most potent inhibitor 1c, a detailed kinetic analysis
was performed in order to clarify the mechanism of Zmp1
inhibition and calculate the actual Ki. Enzymatic kinetic data have
been obtained, at different concentrations of inhibitor 1c, as a
function of substrate concentration and analyzed according to the
following equation:
cleavage. The presence in Zmp1 of two binding sites for the
inhibitor 1c with two differing functional sites is the simplest
mechanism to account for the observed behavior in Figures 2A
and 2B. Obviously, several other more complex mechanisms
might describe this feature, but their use would not be justified by
the available structural information; therefore, since the simplest
mechanism in Figure 2C seems sufficient to satisfactorily
describe the observed data, we have used it throughout our
analysis. Therefore, based on Scheme reported in Figure 2C, the
dependence of obsKm on inhibitor 1c concentration [I] can be
[
E0
]
obs Km
1
1
=
+
obs
obs
k
[
S
]
k
cat
cat
(eq. 1)
where [E0] is the enzyme concentration, ν is the observed rate
(expressed as moles/s), [S] is the substrate concentration, obsKm
is the observed Michaelis-Menten equilibrium constant (reflecting
the enzyme-substrate affinity) and obskcat is the observed velocity
of the rate-limiting step. At each inhibitor 1c concentration a
described by Eq. (2a)
2
KI1 KI 2
+ KI 2
+ KI 2
[
I
]
+
[I
]
obs Km =0Km
2
KI1 KI 2
[
I
]+
[ ]
I
(Eq. 2a)
and the dependence of obskcat on inhibitor 1c concentration [I] can
dependence of Zmp1 enzymatic activity on
a fluorogenic
substrate S has allowed to obtain a value of obsKm and obskcat and
be described by Eq. (2b)
2
Figure 2 shows the dependence of obsKm (Panel A) and of
k
cat
obs
KI1 KI 2
+ KI 2
+ KI 2
[I]+ [I]
obs
k
=0kcat
cat
2
(Panel B) on the inhibitor 1c concentration. Interestingly, we
observed a bimodal behavior, characterized first by a marked
increase of obsKm, as expected for an inhibitory effect, followed by
a decrease at higher inhibitor 1c concentrations (Figure 2A); in
the case of obskcat we only observed a decrease (Figure 2B), which
parallels the first obsKm increase. This behavior underlies the
existence of two different binding sites for the molecule; the higher
affinity one displays a decrease of substrate affinity (accompanied
by a decrease of the rate of substrate cleavage), while the binding
of 1c to the second lower affinity site brings about a (partial)
recovery of substrate affinity without affecting the rate of substrate
KI1 KI 2
[I]+ [I]
(Eq. 2b)
obs
where obsKm and
k
are the parameters obtained, at a given
cat
inhibitor 1c concentration, from the analysis of the substrate
0
concentration dependence according to Eq. 1, whereas Km and
0kcat are the catalytic parameters obtained in the absence of the
inhibitor 1c. From data reported in Figure 2C it emerged that
inhibitor 1c binds with high affinity (KI1 = 11 ± 4 nM) to a first site,
inducing a dramatic 200-fold decrease of the substrate affinity (as
from a = 190, see Figure 2C).
Figure 2. (A,B) Dependence on inhibitor 1c concentration for the values of obsKm (A) and obskcat (B); error bars are within the size of the symbols. Continuous lines
correspond to the global non-linear least-squares fitting of data according to Eqs 2a and 2b, employing parameters reported in (C). Arrows indicate the values in
the absence of Inhibitor; (C) Thermodynamic equilibria for the enzyme, substrate and inhibitor interactions and parameters obtained according to the reported
scheme, where E, S and I refer to the enzyme, substrate and inhibitor 1c, respectively; Km and kcat refer to the Michaelis-Menten equilibrium constant and to the
velocity of the rate-limiting step, respectively, in the absence of inhibitor; KI1 and KI2 refer to the equilibrium dissociation constants of inhibitor 1c for the two binding
sites on the enzyme; α is the interaction parameter for the affinity (reflecting the effect of the inhibitor 1c on the substrate affinity and/or the effect of substrate on
the inhibitor affinity, with: α < 1 representing a positive interaction, which increases the substrate and/or inhibitor affinity; α > 1 representing a negative interaction,
which decreases the substrate and/or inhibitor affinity; α = 1 no interaction), and β is the interaction parameter for the rate-limiting step (reflecting the effect of the
inhibitor binding on kcat, with: β < 1 representing a negative interaction, which decreases the rate of substrate proteolytic cleavage; β > 1 representing a positive
interaction, which increases the rate of substrate enzymatic processing; β = 1 no interaction). Parameters γ and d have the same meaning, referring to the second
binding site of the inhibitor 1c.
3
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This behavior is similar to that of a competitive inhibitor, but
we also observed an effect on kcat, which undergoes a 40%
reduction (as indicated by b= 0.4, see Figure 2C and Figure 2B);
therefore, the inhibitor action should be defined as a mixed
inhibition. In any case, the value of KI1 allows to assess that, to
our knowledge, this is by far the most powerful inhibitor of
Zmp1,[14] even more than phosphoramidon, being already active
at a concentration of 10 nM.[9] A second binding site for inhibitor
1c is observed upon further increase of inhibitor concentration
(micromolar range), which is characterized by KI2 = 1.8 ± 0.6 µM.
Binding to this second site displays an activation effect on the
enzymatic activity of Zmp1, which can be attributed only to an
increase of substrate affinity; this second binding site, which is
very likely to be located far from the active site of Zmp1, acts as
an allosteric site, possibly inducing a conformational change of
the active site, which increases the substrate affinity (though not
facilitating the substrate cleavage). However, the concentration
range within which this second binding site becomes operative is
100-fold higher than for the first site, making it much less relevant
from the functional viewpoint; however, the detection of this
second activating site is important, since it envisages the
possibility that an exceedingly high local concentration of the
inhibitor 1c might induce a paradoxical effect by activating the
enzymatic activity of Zmp1, partially annihilating the strong
inhibitory effect after the binding to the first site at the catalytic
center (see Figures 2A and 2B).
dramatic drop of inhibitory potency observed for the isosteric
compound 2a, bearing a methylene in place of the hydroxamate
oxygen (2a vs 1c, Table 1), prompted us to further investigate its
binding mode (Figure 3B). Compound 2a, lacking the oxygen
atom, fails to interact with R628, so cannot correctly
accommodate its carbonyl group close to the zinc ion. The lack of
appropriate metal-coordination results in a large portion of the 8-
hydroxyquinoline moiety solvent exposed towards the lumen of
the big cavity of Zmp1. Remarkably, these different binding
modes found for 1c and 2a by means of QPLD protocol, were in
agreement with the estimated ligand binding energy ( Gbind, -
D
77.53 kcal/mol and of -40.01 kcal/mol for 1c and 2a respectively).
The QPLD protocol was also applied to compound 4 and results
are consistent with its lack of inhibitory activity at the highest
concentration tested (Figure S4 of the Supporting Information file).
Evaluation of the activity of Zmp1 inhibitor 1c
against M. tuberculosis in macrophages
As a preliminary evaluation, the anti-mycobacterial activity of the
most potent inhibitor of Zmp1 (compound 1c) was tested during
intracellular infection of M. tuberculosis in J774 murine
macrophages (Figure 4A). We chose a low multiplicity of infection
(MOI, 1:10) since at higher MOI Mtb cause toxicity early after
infection. Moreover, the drug was added 24 h post infection to
allow the activation of the Mtb evasion strategies inside the
macrophages.
Structure-activity relationships and molecular
modeling
The IC50 values of the inhibitors reported in Table 1 clearly
indicate that an intact 8-hydroxy-2-quinolyl-hydroxamate
fragment, as in compounds 1a-e, is necessary to achieve potent
Zmp1 inhibition, while the O-alkyl substituent finely tunes the
inhibition potency. All modifications of 1c affecting moieties of the
molecule potentially involved in the interaction with the zinc ion,
as in compounds 2a,b and 5a,b, resulted in a dramatic drop of
potency (Table 1). The lack of activity of compounds 3 and 4
(clioquinol) highlights the importance of the side chain attached to
the hydroxamate moiety for a correct interaction with the enzyme
binding site. In order to assess the binding mode of 1c and to
explain the SARs observed for the class of compounds here
described we performed a computational analysis by applying a
docking procedure.[16, 17] Given the characteristics of the cavity
and the presence of a charged metal ion in the binding site, we
used the QM-Polarized Ligand Docking (QPLD)[18-20] protocol as
detailed in materials and methods. The resulting docking pose is
reported in Figure 3A for the most potent inhibitor 1c.
The carbonyl group of 1c is lodged at an appropriate distance
(2.2 Å) to form a metal-coordination bond with Zn2+, while the
remaining heteroatoms form productive interactions with
neighbouring residues in the active site pocket explaining the
observed SAR. In particular, the 8-OH group of 1c establishes an
H-bond with E560, the carbonyl group, and the endocyclic
nitrogen forms two H-bond interactions with H622; the
hydroxamate NH interacts with N452, and finally, the
hydroxamate oxygen is involved in H-bond with R628.
Hydrophobic contacts, such as π-π stacking of the benzyl ring
with H493, W604 and F48 (S1’ site) and of the hydroxyquinoline
with H622 further stabilize the observed binding pose. The
Figure 3. QPLD pose of 1c (A) and 2a (B) into Zmp1 binding site (PDB ID:
3ZUK). The pictures were generated by means of PyMOL (The PyMOL
Molecular Graphics System, version 1.6-alpha, Schrödinger, LLC, New York,
2013).
4
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Compound 1c, tested at 6.5 µg/mL concentration, caused a
reduction in treated vs control of -0.63 LogCFU/106 cells.
Conversely, no effect on the viability of the avirulent strain M.
bovis BCG in J774 macrophages was observed for compound 1c
at a concentration of 6.5 µg/mL (Figure 4B), despite the fact that
a MOI of 5:1 was used. Previous studies showed that deletion of
the zmp1 gene affected intracellular survival of both M.
tuberculosis and M. bovis BCG, though the impact on the latter
was much less dramatic, probably because BCG is strongly
attenuated and cannot effectively inhibit phagosome-lysosome
fusion.[7] Hence, the restricted activity of 1c for the virulent M.
tuberculosis strain, but not for BCG, may underscore the different
role played by Zmp1 in virulent vs attenuated mycobacterial
strains. Notably, no anti-mycobacterial activity was observed for
compound 1c against extracellular M. tuberculosis (MIC > 20
µg/mL) as assessed by determination of the minimal inhibitory
concentration in axenic culture (data not shown): the result is
consistent with the previously reported lack of effects on M.
tuberculosis viability in axenic culture following Zmp1 deletion.[7]
Given these promising preliminary results, evaluation on M.
tuberculosis-infected human monocyte-derived macrophages
(hMDM) was subsequently performed (Figure 4C). Compound 1c
showed antibacterial activity against virulent M. tuberculosis in
human primary macrophages (1c added at day 1 p.i.) in a dose-
dependent manner. In fact, 1c caused -0.56, -0.42 and -0.34
reduction in logCFU/106 cells in treated vs control hMMO, at
doses of 20, 6.5 and 2.5 µg/mL, respectively. At the highest dose
tested, the effect of 1c was comparable to that of the second-line
antitubercular drug capreomycin (added at the concentration of 4
µg/mL), an important second-line drug currently used for the
treatment of MDR-TB. A similar effect was also observed when
1c was added immediately after infection (at 4 hpi, see Figure S5
of the Supporting Information file). Cytotoxicity of 1c on hMDM
was subsequently assessed and resulted in a 10% cell mortality,
comparable to that the untreated control, even at the highest
concentration of 20 µg/mL (Figure 5). Notably, at the same molar
concentration compound 4, a Zn-complexing agents with no
inhibitory activity on Zmp1, resulted in 57% toxicity.
In Vitro Toxicity and Genotoxicity Assays
The drug-like properties of 1c were further investigated by
determining cytotoxicity in vitro on mouse fibroblasts NIH3T3. A
TC50 value of 54 µM was indicative of an optimal selectivity index
(SI 5000). Investigation of the mutagenic potential of 1c was
»
assayed in the Salmonella typhimurium strains TA98 and TA100
using the Ames test (Figure S6 of the Supporting Information),[17]
indicating that 1c was not mutagenic over the concentration range
tested.
Figure 4. J774 murine macrophages were infected with (A) M. tuberculosis H37Rv (MOI of 1:10) and (B) M. bovis BCG (MOI 5:1). Intracellular bacteria were
determined by CFU counting at day 7 post-infection. Results of one representative experiment of at least three assays are shown. Statistical analysis was performed
with 2-way ANOVA with Bonferroni’s posttest; (C) Human monocyte-derived macrophages (hMDM) were infected with M. tuberculosis H37Rv (MOI of 1:1) and
incubated with the test drug for 3 days post-infection. Intracellular bacteria were evaluated by CFU counts. The result shown derive from hMDM obtained from three
different donors, each tested in triplicate. Statistical significance was determined by One-way ANOVA, Dunnett’s multiple comparison test.
5
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resulting mixture was stirred at 25 °C for 2 h, afterward the appropriate
hydroxylamine 6a-e or amine 7a,b (0.79 mmol) was added and stirring was
continued overnight. The mixture was partitioned between water and
dichlorometane and the organic layer was washed with water, dried over
sodium sulfate, and concentrated in vacuo.
N-Methoxy-8-hydroxyquinoline-2-carboxamide (1a): Brown solid (yield
35%, Mp 201-202 °C). 1H NMR (400 MHz, CDCl3) δ 3.97 (s, 3H), 7.24 (d,
J = 7.8 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H), 7.73 (br
s, 1H), 8.28-8.35 (m, 2H), 10.30 (br s, 1H). MS (ESI) m/z 241 [M + Na]+.
HPLC purity: 96.9% (Rt = 4.031 min). HRMS (ESI) m/z calcd for
C11H11N2O3: 219.0770; found, 219.0756 (M + 1)
N-(Allyloxy)-8-hydroxyquinoline-2-carboxamide
(1b):
Flash
chromatography: dichloromethane-methanol (95:5) (62% yield). An
analytical sample was obtained by recrystallization from diethyl ether-n-
hexane by slow evaporation as a pale-yellow crystalline solid (Mp 121-
122 °C). 1H NMR (400 MHz, CDCl3) δ 4.57 (d, J = 6.5 Hz, 2H), 5.26-5.37
(m, 2H), 5.99-6.09 (m, 1H), 7.24 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 8.2 Hz,
1H), 7.50-7.58 (m, 1H), 8.03 (br s, 1H), 8.23-8.37 (m, 2H), 10.58 (br s, 1H).
MS (ESI) m/z 245 [M + H]+. HPLC purity: 96.6% (Rt = 4.950 min). HRMS
(ESI) m/z calcd for C13H13N2O3: 245.0926; found, 245.0912 (M + 1).
Figure 5. Cytotoxicity was tested with the CytoTox 96® Non-Radioactive
Cytotoxicity Assay kit (Promega) according to manufacturer’s instructions.
Conclusions
N-(Benzyloxy)-8-hydroxyquinoline-2-carboxamide
(1c):
Flash-
In conclusion, Zmp1 has been claimed as a putative and novel
target for development of potential anti-TB drugs.[6, 7] However, its
role in mycobacterium survival is still under debate.[8] We
described herein the development of a series of potent inhibitors
chromatography: dichloromethane-methanol (98:2) (yield 77%). An
analytical sample was obtained by recrystallization from diethyl ether-n-
hexane as pale-yellow crystals melting at 143-144 °C. 1H NMR (400 MHz,
CDCl3) δ 5.09 (s, 2H), 7.21 (d, J = 7.6 Hz, 1H), 7.35-7.46 (m, 6H), 7.53 (t,
J = 7.8 Hz, 1H), 7.78 (br s, 1H), 8.30-8.32 (m, 2H), 10.35 (br s, 1H). 13C
NMR (75 MHz, CDCl3) δ 163.2, 153.4, 146.0, 139.0, 138.6, 132.1, 131.7,
130.3, 129.9, 129.1, 128.7, 128.0, 126.5, 121.8, 119.7, 112.0, 79.8; MS
(ESI) m/z 295 [M + H]+, 317 [M + Na]+. HPLC purity: 99.9% (Rt = 6.196
min). HRMS (ESI) m/z calcd for C17H15N2O3: 295.1083; found, 295.1067
(M + 1).
of Zmp1. The quinoline derivative 1c was used as
a
pharmacological tool in an attempt to clarify the biological role of
Zmp1. 1c was able to impair survival of M. tuberculosis in human
primary macrophages in
a dose-dependent manner when
administered at both 24 and 4 h p.i. Further work is needed to
confirm that Zmp1 is the intracellular target of 1c, but these
preliminary data indicate that the role of Zmp1 in protecting M.
tuberculosis from the host immune system is worth of further
investigation.
N-(4-Methylbenzyloxy)-8-hydroxyquinoline-2-carboxamide
(1d):
Flash-chromatography: dichloromethane-methanol (98:2) (yield 63%). An
analytical sample was obtained by recrystallization from diethyl ether-n-
hexane by slow evaporation as colorless crystals melting at 146-147 °C.
1H NMR (400 MHz, CDCl3) δ 2.22 (s, 3H), 4.98 (s, 2H), 6.98 (d, J = 7.7 Hz,
2H), 7.15-7.22 (m, 3H), 7.33 (t, J = 8.3 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H),
8.22-8.28 (m, 2H), 8.49 (br s, 1H), 11.15 (br s, 1H). MS (ESI) m/z 309 [M
+ H]+. HPLC purity: 99.5% (Rt = 6.717 min). HRMS (ESI) m/z calcd for
C18H17N2O3: 309.1239; found, 309.1223 (M + 1).
Experimental Section
Chemistry
General: Reagents were purchased from Aldrich and were used as
received. Reaction progress was monitored by TLC using Merck silica gel
60 F254 (0.040–0.063 mm) with detection by UV. Merck silica gel 60
(0.040–0.063 mm) was used for column chromatography. Melting points
were determined in Pyrex capillary tubes using an Electrothermal 8103 or
Gallenkamp apparatus and are uncorrected. 1H NMR and 13C NMR
N-(3,4-Dichlorobenzyloxy)-8-hydroxyquinoline-2-carboxamide (1e):
Flash-chromatography: dichloromethane-methanol (98:2) (yield 66%).
Compound 1e was obtained as a pale-yellow oil, which crystallized on
standing (179-180 °C). 1H NMR (400 MHz, CDCl3) δ 4.97 (s, 2H), 7.15-
7.20 (m, 3H), 7.30-7.36 (m, 2H), 7.45 (s, 1H), 7.50 (t, J = 7.9 Hz, 1H), 8.22-
8.29 (m, 2H), 11.40 (br s, 1H). MS (ESI) m/z 363 [M + H]+. HPLC purity:
96.5% (Rt = 7.472 min). HRMS (ESI) m/z calcd for C17H13N2O3Cl2:
363.0303; found, 363.0287 (M + 1).
spectra were recorded
on Varian 300 MHz or Bruker 400 MHz
spectrometers by using the residual signal of the deuterated solvent as
internal standard. Splitting patterns are described as singlet (s), doublet
(d), triplet (t), quartet (q), and broad (br); the value of chemical shifts (d)
are given in ppm and coupling constants (J) in Hertz (Hz). ESI-MS spectra
were performed by an Agilent 1100 Series LC/MSD spectrometer. RP-
HPLC were performed on a Shimadzu Prominence apparatus equipped
with LC-20AD pump, SIL-20AC HT autosampler, CTO-20AC column oven
and SPD-M20A diode array detector. The purity of all compounds tested
was ≥ 96% as determined by RP-HPLC (RP-18 Chromolith column, 4.6
mm diameter; gradient elution: solvent A: 1% TFA in ACN, solvent B: 0.5%
TFA in H2O, from 20 to 70% solvent A in 6 min, UV detection: 254 nm).
N-Phenethyl-8-hydroxyquinoline-2-carboxamide
(2a):
Flash-
chromatography: chloroform-ethyl acetate (8:2). Compound 2a was
obtained as a pale-yellow oil, which crystallized on standing (yield 89%,
Mp 171-172 °C). 1H NMR (400 MHz, CDCl3) δ 2.99 (t, J = 6.8 Hz, 2H), 3.80
(q, J = 6.7 Hz, 2H), 7.19-7.32 (m, 4H), 7.35-7.43 (m, 3H), 7.49 (s, 1H), 7.53
(t, J = 7.9 Hz, 1H), 7.91 (br s, 1H), 8.29-8.34 (m, 2H); MS (ESI) m/z 315
[M + Na]+. HPLC purity: 100% (Rt = 6.740 min). HRMS (ESI) m/z calcd for
C18H17N2O2: 293.1290; found, 293.1284 (M + 1).
General Procedure for the synthesis of 1a-e, 2a,b and 5a,b: 1-
N-(3-Phenylpropyl)-8-hydroxyquinoline-2-carboxamide (2b): Flash-
chromatography: chloroform-ethyl acetate (9:1). Compound 2b was
obtained as a pale-yellow oil, which crystallized on standing (yield 73%,
Mp 106.8-107.2 °C). 1H NMR (400 MHz, CDCl3) δ 1.99-2.11 (m, 2H), 2.75
(t, J = 7.5 Hz, 2H), 3.59 (q, J = 6.8 Hz, 2H), 7.13-7.31 (m, 6H), 7.40 (dd, J
Hydroxybenzotriazole
(0.58
mmol)
and
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (0.57 mmol) were added to a mixture of
the appropriate 2-carboxyquinoline 3 or 9a,b[21] (0.53 mmol) and N-
methylmorpholine (0.57 mmol) in N,N-dimethylformamide (5.0 mL). The
6
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10.1002/cmdc.201700759
ChemMedChem
FULL PAPER
= 8.2, 0.8 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.70 (s, 1H), 7.87 (br s, 1H),
8.29-8.35 (m, 2H). MS (ESI) m/z 307 [M + H]+. HPLC purity: 100% (Rt =
7.237 min). HRMS (ESI) m/z calcd for C19H19N2O2: 307.1447; found,
307.1438 (M + 1).
empirical method. Ligands were docked using Glide Extra Precision (XP).
QM charges were calculated by using Jaguar[32] from the electrostatic
potential energy surface of the ligand, which is generated from a single
point calculation using DFT for the QM region at 6-31G*/LACVP* basis set,
B3LYP density function. The generated poses were re-docked employing
XP method applying the calculated QM charges. Resulting post-docking
minimized poses were scored and ranked by Glide XP. The binding
energies were estimated using Prime MM-GBSA for the complexes 1c-
Zmp1 and 2a-Zmp1 as described previously.[33-35]
8-Methoxy-2-quinolylcarboxylic acid (9a): To a solution of 8 (130 mg,
0.6 mmol), prepared from 5 following a described procedure,[22] in MeOH
(3 mL) and water (2 mL), lithium hydroxide was added (36 mg, 1.5 mmol).
The reaction mixture was stirred at room temperature for 1 h. The solution
was filtered on celite to afford the title compound (120 mg, 100%) without
further purification as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 3.11 (br s, 1H), 3.96 (s, 3H), 7.22-7.24 (d, J = 8.0 Hz, 1H), 7.53-7.60 (m,
2H), 8.04-8.06 (d, J = 8.0 Hz, 1H), 8.42-8.44 (d, J = 8.0 Hz, 1H); MS (ESI)
m/z 202 [M -H]+.
Biological evaluation
Kinetic analysis of Zmp1 inhibition: Synthesized compounds underwent
a preliminary screening in order to identify the lead compound. Briefly, 10
µM inhibitor was preincubated with 0.5 nM Zmp1 in the reaction buffer (100
mM Tris-HCl, 0.1% borate, 150 mM NaCl, and 10 mM CaCl2, pH 8.0) for
15 min at 37 °C. Thereafter a fluorogenic substrate for the enzyme was
added to the reaction mixture. Notably, the MMP2/MMP7 fluorogenic
substrate displays the aminoacid sequence PLGL flanked by the
fluorophore/quencher system methoxycoumarin/dinitrophenyl-alanine-
arginine (full sequence: MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; MCA
stands for (7-methoxycoumarin-4-yl)-acetyl; Dpa stands for N-3-(2,4-
dinitrophenyl)-L-2,3-diaminopropionyl. MCA is the fluorophore, Dpa the
quencher (MMP2/MMP7 product number 03-32-5032, Calbiochem).
MMP2/MMP7 was shown to be efficiently cleaved by Zmp1 in vitro.[9, 10]
The compounds (indicated in Figure 1B) with significant inhibitory
properties were further assayed at different concentrations (ranging from
100 nM to 50 µM) in order to obtain the IC50. In addition, for compound 1c,
which displayed the highest inhibitory properties (see Figure 1B), kinetic
analysis to determine Ki was performed, as indicated below: several 1c
concentrations, ranging from 0.5 to 250 nM, were pre-incubated with 0.5
8-Methoxy-2-quinolylhydroxamic acid benzyl ester (5a): White solid
(32% yield, Mp 143-144 °C). 1H NMR (400 MHz, CDCl3): δ 8.27 (d, J = 8.5
Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.62 – 7.50 (m, 1H), 7.42 (d, J = 8.3 Hz,
1H), 7.39 – 7.25 (m, 4H), 7.08 (d, J = 7.8 Hz, 1H), 5.05 (s, 2H), 4.04 (s,
3H); MS (ESI) m/z 279 [M + H]+, 301 [M + Na]+, 579 [2M + Na]+. HRMS
(ESI) m/z calcd for C18H17N2O3: 309.1239; found, 309.1228 (M + 1).
2-Quinolylhydroxamic acid benzyl ester (5b): White solid (25% yield,
Mp 157-158 °C). 1H NMR (400 MHz, CDCl3) d 5.10 (s, 2H), 7.34-7.41 (m,
3H), 7.48-7.50 (d, J = 8.0 Hz, 2H), 7.58-7.61 (t, J = 3.04 Hz, 1H), 7.70-7.74
(t, J = 16 Hz, 1H), 7.83-7.85 (d, J = 8.0 Hz, 1H), 7.98-8.00 (d, J = 8.0 Hz,
1H), 8.08-8.31 (m, 2H), 10.35 (br s, 1H); 13C NMR (75 MHz, CDCl3) d 162.2,
149.0, 148.5, 137.9, 135.4, 132.5, 132.1, 131.7, 131.2, 130.3, 129.9, 129.1,
128.7, 128.0, 126.5, 119.0, 79.6. HPLC purity: 97.8% (Rt = 6.529 min).
HRMS (ESI) m/z calcd for C17H15N2O2: 279.1134; found, 279.1127 (M +
1).
nM Zmp1. Afterwards, the fluorogenic substrate was added at
a
concentration ranging from 1 µM to 50 µM. The analysis of the inhibitory
properties of compound 1c on MMP-2 and MMP-1 was performed
according to the following experimental conditions: 1c at various
concentrations, ranging from 50 nM to 50 µM, was preincubated for 15 min
at 37 °C with either 10 nM MMP-2 or 10 nM MMP-1 in the reaction buffer
(50 mM Tris-HCl, 10 mM CaCl2, 100 mM NaCl, pH 7.5). Thereafter, an
omni-MMP fluorogenic substrates was added at a final concentration of 20
µM (Calbiochem). In all cases, the substrate hydrolysis was recorded in an
Eclypse fluorometer (Varian) (excitation 320 nm, emission 395 nm) and
the reaction was followed until linearity was observed.
X-Ray Crystallography: Single crystals of compounds 1b-d (VC1602,
VC1222, VC1620) were submitted to X-ray data collection on an Oxford-
Diffraction Xcalibur Sapphire
3
diffractometer with
a
graphite
monochromated Mo-Kα radiation (λ = 0.71073 Å) at 293 K. The structures
were solved by direct methods implemented in SHELXS-97 program.[23]
The refinements were carried out by full-matrix anisotropic least-squares
on F2 for all reflections for non-H atoms by means of the SHELXL-97
program.[24] Crystallographic data (excluding structure factors) for the
structure in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no. CCDC
1062834 (1b), 1062832 (1c), and 1062835 (1d). Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; (fax: + 44 (0) 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk).
In vitro biological assay of 1c: J774 murine macrophages were cultured
in 48-well plates as previously indicated,[36] and infected with M.
tuberculosis H37Rv or with M. bovis BCG at a multiplicity of infection (MOI)
of 1:10 and 5:1, respectively. Following a 2 h infection, the infecting
solution were removed and cells were washed three times with PBS,
remove extracellular bacteria, and then incubated with maintaining
medium (2% fetal bovine serum). 24 h post-infection, compound 1c was
added to the infected cells at different concentrations. As a control, cells
infected with both M. tuberculosis H37Rv and with M. bovis BCG received
0 µg/mL of compound 1c. At day 7 post-infection, cells were washed three
times with PBS, lysed in 0.1% Triton X-100 and intracellular bacteria were
determined by colony forming units (CFUs) counting by serially diluting
lysates in PBS containing 0.05% Tween80 and plating on 7H11 OADC
0.05% Tween80 agar plates.[37] Colony counting was performed in
triplicate. A similar protocol was applied to human monocyte-derived
macrophages (hMDM) with a few changes. Briefly, peripheral blood
mononuclear cells (PBMCs) were isolated from buffy coats of 3 male
donors by using Ficoll (Cedarlane) and were processed to collect hMDMs
by positive selection from the PBMCs suspension by using CD14
MicroBeads, following the manufacturer’s instructions (Miltenyi Biotec).
hMDMs were seeded at 1.2·106 cells/ml in 48-well plates in X-VIVOTM 15
medium (Lonza) with human serum type AB 2% (Lonza), and incubated
for 6-7 days at 37°C in a 5% humidified atmosphere until hMDM
differentiation. No antibiotics were added to the medium. Adhered
macrophages were infected with Mtb H37Rv strain at a MOI of 1:1. As
Computational details
Protein and ligands preparation: All ligands were built and treated by
means of Maestro[25] software as described previously.[26-28] The
compounds were filtered for pan assay interference compounds (PAINS).
None of them contained sub-structural features that would label them as
“frequent hitters” in high throughput screens.[29] The three-dimensional
structure of Zmp1 (PDB ID 3ZUK) was taken from PDB, imported into
Maestro and prepared as described previously.[30, 31]
QM-Polarized Ligand Docking (QPLD), ligand binding energies:
Docking studies were performed employing the QPLD workflow
implemented in Maestro.[18, 20] QLPD utilizes Glide and Qsite software to
carry out the calculation for performing docking studies using ab initio
methodology to calculate ligand charges within the protein environment,
which is essential for charged active sites, such as metalloproteinase.
QPLD improves the partial charges on the ligand atoms in Glide docking
run by replacing them with charges generated from a quantum-mechanical
(QM) calculation on the ligand in the field of the receptor. Energy grids
were prepared using default settings within a cubic box centered on the
Zn2+ atom. Initial ligands charges were generated adopting a semi-
7
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10.1002/cmdc.201700759
ChemMedChem
FULL PAPER
described for J774 cells, hMDMs were infected and intracellular
mycobacterial CFUs were determined at 4 h and 72 h post-infection.[36]
Compound 1c was added 24h post infection at different concentrations (20,
6.5, 2.5 µg/mL) while we added a second line anti-TB drug capreomycin 4
µg/mL as positive control. Each condition was tested in triplicate.
Martin, R. L. Summers, S. Lamponi, L. Savini, I. Fiorini, M. Valoti, E.
Novellino, G. Campiani, S. Butini, J. Med. Chem. 2012, 55, 10387-10404.
[18] A. E. Cho, V. Guallar, B. J. Berne, R. Friesner, J. Comput. Chem. 2005,
26, 915-931.
[19] A. E. Cho, D. Rinaldo, J. Comput. Chem. 2009, 30, 2609-2616.
[20] Schrödinger, Suite 2011 QM-Polarized Ligand Docking protocol; Glide
version 2015.2017, Schrödinger, LLC, New York, NY, 2011; Jaguar
version 2017.2018, Schrödinger, LLC, New York, NY, 2011; QSite
version 2015.2017, Schrödinger, LLC, New York, NY, 2011.
[21] R. H. Dodd, M. Lehyaric, Synthesis 1993, 295-297.
[22] A. N. Pearce, E. W. Chia, M. V. Berridge, G. R. Clark, J. L. Harper, L.
Larsen, E. W. Maas, M. J. Page, N. B. Perry, V. L. Webb, B. R. Copp, J.
Nat. Prod. 2007, 70, 936-940.
Toxicity and Ames test: The toxicity evaluation against NIH3T3
fibroblasts and Ames test were performed as previously described
38, 39]
following literature procedures.[17,
Cytotoxicity on human
macrophages was tested with the CytoTox 96® Non-Radioactive
Cytotoxicity Assay kit (Promega) according to manufacturer’s instructions.
[23] G. M. Sheldrick, SHELXS-97, Rel. 97-2, A program for automatic solution
of crystal structures, G¨ottingen University, 1997.
Acknowledgements
[24] G. M. Sheldrick, SHELXL-97, Rel. 97-2, A program for crystal structure
refinement, G¨ottingen University, 1997.
Authors thank Prof. Menico Rizzi for plasmids of Zmp1. This work
was supported by the Italian Ministery of University and Research
(MiUR PRIN 200993WWF9 to M.C. and M. A.) and by the British
Society for Antimicrobial Chemotherapy (BSAC RESEARCH
GRANT 2016 grant number GA2016_087R to S. Brogi)
[25] Maestro, 9.2 ed. (Ed.: 9.2), Schrödinger, LLC, New York, NY, 2011, 2011.
[26] M. Brindisi, S. Maramai, S. Gemma, S. Brogi, A. Grillo, L. D. Mannelli, E.
Gabellieri, S. Lamponi, S. Saponara, B. Gorelli, D. Tedesco, T. Bonfiglio,
C. Landry, K. M. Jung, A. Armirotti, L. Luongo, A. Ligresti, F. Piscitelli, C.
Bertucci, M. P. Dehouck, G. Campiani, S. Malone, C. Ghelardini, A.
Pittaluga, D. Piomelli, V. Di Marzo, S. Butini, J. Med. Chem. 2016, 59,
2612-2632.
Keywords: 8-hydroxyquinoline-2-hydroxamate • metallo-
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8
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Entry for the Table of Contents
Starting from the 8-hydroxyquinoline privileged structure, we designed a series of inhibitors of the metallo-protease enzyme Zmp1.
This protease is a virulence factor that allows Mycobaterium tuberculosis to survive inside macrophages. The potent inhibitor 1c is
able to impair growth of M. tuberculosis inside macrophages and is not active against axenic bacteria.
9
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