New chromane-based derivatives as inhibitors of mycobacterium tuberculosis salicylate synthase (mbti): Preliminary biological evaluation and molecular modeling studies

Article abstract of DOI:10.3390/molecules23071506

Tuberculosis is the leading cause of death from a single infectious agent worldwide; therefore, the need for new antitubercular drugs is desperate. The recently validated target salicylate synthase MbtI is the first enzyme involved in the biosynthesis of

Full text of DOI:10.3390/molecules23071506

molecules
Article
New Chromane-Based Derivatives as Inhibitors of
Mycobacterium tuberculosis Salicylate Synthase
(MbtI): Preliminary Biological Evaluation and
Molecular Modeling Studies
Elena Pini 1,† ID , Giulio Poli 2,†, Tiziano Tuccinardi 2 ID , Laurent Roberto Chiarelli 3
,
ID
Matteo Mori 1 ID , Arianna Gelain 1, Luca Costantino 4, Stefania Villa 1, Fiorella Meneghetti 1,
and Daniela Barlocco 1
*
ID
1
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via L. Mangiagalli 25,
20133 Milano, Italy; elena.pini@unimi.it (E.P.); matteo.mori@unimi.it (M.M.); arianna.gelain@unimi.it (A.G.);
stefania.villa@unimi.it (S.V.); daniela.barlocco@unimi.it (D.B.)
Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy; giulio.poli@unipi.it (G.P.);
tiziano.tuccinardi@unipi.it (T.T.)
Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, Università degli Studi di Pavia,
via Ferrata 9, 27100 Pavia, Italy; laurent.chiarelli@unipv.it
Dipartimento Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, via Campi 103,
41121 Modena, Italy; luca.costantino@unimore.it
2
3
4
*
†
Correspondence: fiorella.meneghetti@unimi.it; Tel.: +39-02-5031-9306
These authors contributed equally.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 May 2018; Accepted: 19 June 2018; Published: 21 June 2018
Abstract: Tuberculosis is the leading cause of death from a single infectious agent worldwide; therefore,
the need for new antitubercular drugs is desperate. The recently validated target salicylate synthase
MbtI is the first enzyme involved in the biosynthesis of mycobactins, compounds able to chelate iron,
an essential cofactor for the survival of Mycobacterium tuberculosis in the host. Here, we report on the
synthesis and biological evaluation of chromane-based compounds as new potential inhibitors of MbtI.
Our approach successfully allowed the identification of a novel lead compound (
promising activity against this enzyme (IC50 = 55 M). Molecular modeling studies were performed in
order to evaluate the binding mode of and rationalize the preliminary structure-activity relationships,
1), endowed with a
µ
1
thus providing crucial information to carry out further optimization studies.
Keywords: antimycobacterial agent; siderophore; mycobactin; iron; consensus docking; chorismate;
MD simulation
1. Introduction
Tuberculosis (TB), one of the deadliest infectious diseases in the world with about 1.7 million
deaths reported in 2016, is caused by Mycobacterium tuberculosis (Mtb) [1,2]. Antitubercular therapy has
existed since the late 1940s [ ], but the long duration of the prescribed treatment (6–12 months) often
3
causes low patient compliance. Another problem is that Mtb can survive for extended periods of time
in its non-replicative or persistent state, becoming resistant to conventional forms of chemotherapy.
Patient non-compliance, coupled with the ability of Mtb to enter the persistent state, has contributed
to the alarming emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB)
mycobacterial strains [
rifampicin (MDR-TB), and resistance against second-line injectable drugs like fluoroquinolones is a
growing concern (XDR-TB) [ ]. Drug-resistant TB poses a serious threat to existing control programs
4,5]. Mtb has developed resistance to the first-line drugs isoniazid and
6
Molecules 2018, 23, 1506; doi:10.3390/molecules23071506
www.mdpi.com/journal/molecules

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since the therapy becomes less effective, more complex and more expensive, compared to that of
drug-susceptible forms. There are a number of novel anti-TB compounds in clinical trials, and two new
drugs have been recently approved: delamanid and bedaquiline [
on the effectiveness, safety, and tolerability of bedaquiline is urgently required, because its severe side
effects (mainly QT prolongation) could be potentially life-threatening [ ]. Considering these issues,
11] and the development of new scaffolds to sustain the TB drug
7]. In particular, more information
8
the identification of novel targets [
pipeline are imperative [12].
9–
Mtb survival in the host is highly dependent on the availability of iron; this cofactor is made
available through two different strategies: the synthesis of siderophores (mycobactins), compounds
able to chelate iron, and the uptake/metabolism of host heme. Water-soluble carboxymycobactins
and lipophilic mycobactins, compounds that vary according to the nature of their lipophilic moiety,
are able to solubilize iron and compete with the iron-binding proteins (e.g., transferrin, lactoferrin,
ferritin) of the host [9]. MbtI is the first enzyme involved in the mycobactin biosynthetic pathway;
it catalyzes the interconversion of chorismate to salicylic acid, via the intermediate isochorismate [
(Scheme 1).
9]
Scheme 1. Reactions catalyzed by MbtI, the salicylate synthase of Mtb.
This enzyme is a valuable target to develop new drugs able to counteract Mtb infection, since it
has been structurally and biochemically characterized [13], and it is essential for the survival of the
mycobacterium under iron-deficient conditions [14–16]. Moreover, it is absent in the host, making
MbtI inhibitors potentially safe for humans. Several strategies have been followed for the discovery of
MbtI inhibitors [17]. In particular, substrate (chorismate and isochorismate) analogues, transition state
analogues and compounds originated from HTS have been identified [9,17,18].
Inspired by the work of Aldrich and co-workers [19], which focused on the synthesis of analogues
of the first transition state (TS-1) of the isochorismate partial reaction (see Scheme 1), we observed that
the bicyclic chromane and 4-oxochromane motifs, if bearing a carboxylic moiety at C5 position, could be
suitable scaffolds to mimic the transition states formed in the reaction catalyzed by MbtI. In particular,
the chromanone nucleus caught our attention as a privileged bicyclic scaffold in medicinal chemistry
and core structural element of compounds endowed with antitubercular activity [20
the chroman-4-one framework has been reported to be an integral part of several antimycobacterial
agents [21 23]. Encouraged by these data, we addressed our efforts to the research of new MbtI
inhibitors bearing this scaffold. In this work, we assayed against MbtI compounds , chromane
and chromanone derivatives available in our in-house library. The best inhibitor demonstrated a
,21]. Moreover,
–
1–5
1

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promising activity, which was investigated by means of computational tools. This encouraging result
could pave the way for further improvements on this promising scaffold.
2. Results and Discussion
2.1. Synthesis
Compounds
synthesis started from the condensation of methyl 3,5-dihydroxybenzoate and crotonoyl chloride or acryloyl
chloride in the presence of boron trifluoride etherate as base, which led to chromanones and , respectively.
1–5 were obtained through the synthetic route outlined in detail in Scheme 2. Their
1
2
Successively, the hydroxyl group was quantitatively converted into the corresponding p-toluenesulfonyl
derivative 1a, using sodium hydroxide and p-toluenesulfonyl chloride [24]. The elimination of the O-tosyl
group was performed using Ni Raney, according to the method described by Kenner and Murray [25],
affording the intermediate
3. The isolation of carboxylchromane 4, though in a low yield, was performed by
hydrogenation in the presence of Pd/BaSO4 [26], since the more frequently used reduction with Pd/C [27
]
and the Clemmensen reaction [28] were too drastic. Unsuccessfully, we also tried other methods involving
the use of zinc acetate, zinc trifluoroacetate, tin/HCl, the Wolff-Kishner reduction with hydrazine and
a zinc-aluminum chloride hexahydrate-THF-water system, a promising chemoselective green reagent
and catalyst in organic synthesis [29]. The hydroxyl chromane derivative
reductive condition used for , starting from . Finally, the methyl ester (1b) of
a common procedure (Fischer-Speier esterification).
5
was obtained in the same
4
1
1 was obtained according to
Scheme 2. Synthetic route for compounds
CH2=CHCOCl, BF3Et2O, PhNO2, N2, 24 h, reflux; b. HCl, overnight, 0 C; c. water, steam-distillation;
1
–
5
. Reagents and conditio◦ns: (i) a. CH3CH=CHCOCl or
yield: 40% for 1; yield: 35% for 2; (ii) NaOH, TsOCl, THF, overnight, rt; yield: 82%; (iii) Ni Raney,
NaHCO3, water; overnight, rt; quantitative yield; (iv) Pd/BaSO4, H2, ethanol, 6 h, 5 atm., rt; yield: 70%
for 4; 75% for 5; (v) CH3OH, conc. H2SO4, 24 h, reflux; quantitative yield.

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2.2. Biological Evaluation and Preliminary Structure-Activity Relationships (SAR)
The chromane and chromanone derivatives reported in Table 1 were submitted to biological
evaluation to assess their MbtI inhibitory activity. The results were compared with that of one of the
most potent MbtI inhibitor reported to date, (1-carboxyprop-1-enyloxy)-2-hydroxybenzoic acid (I) [9],
synthesized following the procedure reported in the literature [18] and used as a positive control.
Table 1. In vitro activity of compounds 1–5 and I.
Code
I
Structure
MbtI Residual Activity at 100 µM
MbtI IC50 (µM)
11.6 ± 2.4
11.0 ± 3.9
1
1b
2
23.3 ± 3.4
55.8 ± 4.2
>75
ND
HO
O
O
O
28.1 ± 2.9
61.3 ± 5.3
HO
3
>75
ND
4
>75
ND
5
50.1 ± 7.1
ND
ND: not determined.
Among the tested compounds,
of MbtI catalytic activity at a concentration of 100
5-fold less potent than the reference . The absence of the methyl group in
did not influence the inhibitory effect, as demonstrated by the similar IC50 values of 1 and 2 (Figure 1).
1
revealed a remarkable potency, showing more than 75% inhibition
M and an IC50 of 55.8 4.2 M; therefore, is less than
showed that this substituent
µ
±
µ
1
I
2

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Figure 1. Effects of
1 ( ) and 2 (ꢀ) against MbtI enzyme activity compared with I (H). IC50 values were
determined by assaying the enzyme at subsaturating concentrations of chorismic acid (50 µM).
Since the 7-hydroxyl function was demonstrated to be a key structural element to enhance the
antibacterial activity of the 4-chromanone nucleus [30], we tested the chromanone and chromane
both lacking the 7-hydroxyl group. They displayed very weak activities, with less than 25% inhibition
of MbtI catalytic activity at 100 M, showing that this group plays a key role for MbtI inhibition
3
4,
µ
too. Previously, it was found that small and polar functionalities at the 4-position of the chromane
scaffold played important roles against Mtb [24]. Our finding showed that the presence of the carbonyl
group in the chromanone scaffold has not a prominent role in retaining MbtI inhibitory potency.
As reported in Table 1, compound 5 showed a halved inhibitory activity compared to 1. In order to
assess if the carboxylic group was essential for the activity of the best candidate 1, the corresponding
ester derivative 1b was synthesized and tested. This compound showed a very low inhibitory activity
on MbtI, thus confirming the importance of the interaction between this function and the target
enzyme [9].
2.3. Binding Mode Evaluation of 1–5 within the MbtI Catalytic Site
To suggest a binding mode for this class of derivatives, the interaction of 1 with MbtI was analyzed
by means of docking and molecular dynamics (MD) simulations. As a first step, a consensus docking
method was applied as it is known to predict the ligand-binding pose better than single docking
programs [31]. By using this kind of approach, a ligand is docked into the target protein by means
of different docking procedures. Then, among the different best-ranked poses (originated by the
different docking methods) the pose in common with the largest number of docking procedures is
considered as the best docking pose. The
was then subjected to 100 ns of MD simulation with explicit water molecules, as described in the
Experimental Section. Figure 2 shows the main interactions between and MbtI: the carboxylic group
of the compound chelates the magnesium ion and it is involved in an H-bond with the nitrogen
backbone of G241, while the aromatic ring shows a cation- interaction with K438, that is also present
in the crystallographic conformation of inside the binding pocket [18]. The hydroxyl group of forms
a stable H-bond with the oxygen backbone of G270 that cannot be formed by , which however shows
interaction between the close residue H334 and its alkene moiety (see Figure S1). Regarding the
1-MbtI complex obtained by means of this docking strategy
1
π
I
1
I
a π-π

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interactions of the 2-methyl-2H-pyran-4(3H)-one scaffold of
1, the carbonyl oxygen shows an H-bond
with the charged nitrogen of K205, the methyl substituent shows lipophilic interactions with the side
chains of L402 and L404, whereas the oxygen of the bicyclic ring does not seem to establish important
interactions. The chromanone
1
is thus able to form two different H-bonds, which are not observed
is able to better fit into the catalytic pocket of MbtI, forming
in the MbtI- complex. Nevertheless,
I
I
additional van der Waals interactions with the residues surrounding its aromatic ring. Moreover,
thanks to the presence of an additional carboxylic group, the ligand can establish a further H-bond
interaction with Y385 (Figure S1).
Figure 2. Minimized average structure of 1 docked at MbtI binding site.
In order to better evaluate the binding mode of this series of compounds in relation to the derived
SAR, the same consensus docking and MD simulation protocols applied to
1
were also used to predict
are
the putative binding disposition of 1b within the MtbI catalytic site. As shown in Figure 3, 2–5
–5
predicted to assume a similar binding mode, with their carboxylic moiety chelating the magnesium ion
and H-bonding G421, and with their bicyclic scaffold predominantly sandwiched between K438 from
one side and T361 from the other side. All compounds form a cation-π interaction with K438 and show
hydrophobic contacts with the methyl group of T361, as well as with L404. This result suggests that all
these derivatives are able to bind to the enzyme due to the presence of the fundamental carboxylic
group. However,
they lack the hydroxyl group (Figure 3B,C), displayed only a minimal activity. Interestingly, although
is predicted to form an H-bond with K205, it shows a comparable activity to , suggesting that the
3 and 4, which cannot form the H-bond with G270 in their predicted binding mode as
3
4
carbonyl group contributes only marginally to the activity. This supports the SAR data highlighting that
the 7-hydroxyl group represents another key structural element, together with the carboxylic group,
to enhance the inhibitory activity of the ligands. Indeed,
forms an H-bond with G270 through the 7-hydroxyl group, maintains considerable inhibitory activity
compared to . Finally, as expected on the basis of the predicted binding mode of , the 2-methyl
group of the chromanone scaffold does not contribute to the affinity of the enzyme, since , which
showed a binding mode very similar to , retained total inhibitory activity. On the contrary, 1b was
5, which does not interact with K205 but
1
1
2
1
not predicted to assume the binding mode shown by its analogues. This compound is not able to
chelate the magnesium ion and thus it shows a completely reversed binding disposition, where the
key H-bonds formed by 1 are lost (see Figure S2).

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Figure 3. Minimized average structure of compounds
2 (A), 3 (B), 4 (C) and 5 (D) docked into MbtI
binding site.
3. Materials and Methods
3.1. General Chemistry
Commercial chemicals and solvents were of reagent grade, purchased from suppliers (Sigma-Aldrich,
St. Louis, MO, USA) and used as received. Anhydrous solvents were used without further drying. Silica
gel plates on glass support (0.25 nm, Sigma-Aldrich) were used to follow the course of the reactions and
visualized under UV lamp operating at wavelengths of 254 or 365 nm; when necessary, the spots were
evidenced using a KMnO4 solution. Silica gel 60 (Sigma-Aldrich pore size 60 Å, 63–200 µm) was used
for the purification of intermediates and final compounds. All tested compounds were characterized by
1
the means of FT-IR, NMR, and MS to check their purity. H and 13C NMR and bidimensional analyses
were acquired at ambient temperature with a Varian-Oxford 300 MHz instrument, operating at 300 MHz
for 1H and 75 MHz for 13C. Chemical shifts are expressed in ppm (
δ) from tetramethylsilane resonance
in the indicated solvent (TMS:
δ = 0.0 ppm), while J-couplings are given in Hertz. The APT sequence
was used to distinguish methine and methyl carbon signals from those of methylene and quaternary
carbons. FT-IR spectra were collected using the Spectrum One Perkin Elmer (Waltham, MA, USA) FT-IR

Molecules 2018, 23, 1506
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Spectrometer in the spectral region between 4000 and 600 or 450 cm−1 for solid or liquid compounds,
respectively, and analyzed by transmittance technique with 32 scans and 4 or 8 cm−1 resolution. Solid
samples were mixed in a mortar with KBr (1:100) and pressed by a hydraulic press (10 tons) into small
tablets. For liquid samples one drop was placed between two plates of sodium chloride. MS analyses
were carried out with a Thermo Finnigan (Waltham, MA, USA) LCQ Advantage system, equipped with a
quaternary pump, a Diode Array Detector (working wavelength: 254 nm) and a MS spectrometer, with an
Electrospray ionization source and an Ion Trap mass analyzer (ionization: ESI positive or ESI negative;
capillary temperature: 250 ◦C; source voltage: 5.50 kV; source current: 4.00
5.50 V and 7.50 V respectively; intermultipole lens voltage: 16.00 V; trap DC offset voltage:
The purity of the compounds was assessed by means of high-resolution mass spectrometry using a Waters
SYNAPT G2 Q-TOF mass spectrometer.
µ
A; multipole 1 and 2 offset:
−
−
−
−10.00 V).
3.1.1. Synthesis of 7-Hydroxy-2-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-carboxylic acid (
7-Hydroxy-4-oxo-3,4-dihydro-2H-benzopyran-5-carboxylic acid (2)
1
) and of
) or
A mixture of methyl 3,5-dihydroxybenzoate (2.0 g, 11.8 mmol), crotonoyl chloride (for
1
acryloyl chloride (for ) (19.0 mmol), boron trifluoride etherate (4.5 mL, 35.7 mmol) and nitrobenzene
2
◦
(12 mL) was heated at 85 C for 24 h, under nitrogen atmosphere. Then, the black mixture was poured
into 5 M HCl (60 mL) and nitrobenzene was removed by steam distillation. The hot tarry aqueous
distillation residue was filtered, and 5 M HCl (24 mL) was added to the clear red filtrate before cooling
the mixture in an ice-bath. The solution was then extracted with diethyl ether (3
combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo giving
the crude products.
×
25 mL), and the
Compound
ethyl acetate/light petroleum ether gave pink crystals. Yield 40%, m.p. 251–252 C. 1H-NMR (300 MHz
DMSO-d6): 12.81 (broad s. exch. D2O, 1H, COOH), 10.79 (broad s. exch. D2O, 1H, OH), 6.32 (s,
2H, H6, H8), 4.59–4.57 (m, 1H, H2), 2.58–2.56 (m, 2H, H3, H30 ), 1.38 (d, 3H, J = 6.2 Hz, CH3) ppm.
13C-NMR (75 MHz, DMSO-d6):
189.75, 170.61, 164.29, 163.81, 138.41, 110.53, 109.32, 103.60, 74.85,
44.28, 21.10 ppm. FT-IR (KBr):
1 was obtained as a partially crystalline orange-brown solid. Crystallization from
◦
,
δ
δ
ν
3320, 2994, 2913, 1709, 1665, 1600, 1581, 1420, 1294, 1229, 1164 cm−1
.
MS (ESI): m/z calcd for C11H10O5 222.19 found m/z 221.28 [M − H]−.
HRMS (ESI-QTOF) calcd. for C11H10O5: [M − H]− 221.0450; found: 221.0451.
Compound 2 was purified by column chromatography (ethyl acetate/methanol 8:2) to afford pink
crystals. Yield 35%, m.p. (dec) 220–230 C. 1H-NMR (300 MHz, DMSO-d6):
1H, COOH), 9.90 (broad s. exch. D2O, 1H, OH), 7.07 (d, 1H, J = 2.6 Hz, H6), 6.62 (d, 1H, J = 2.6 Hz
H8), 3.17 (t, 2H, J = 6.6 Hz, H2), 2.69 (t, 2H, J = 6.6 Hz, H3) ppm. 13C-NMR (75 MHz, DMSO-d6):
δ 13.00 (broad s. exch. D2O,
◦
,
δ
168.65, 168.02, 156.83, 153.61, 131.37, 115.54, 113.47, 107.62, 28.79, 21.01 ppm. FT-IR (KBr):
ν 3319, 3081,
1746, 1688, 1625, 1588, 1405, 1287, 1264, 1168, 1150, 1129 cm−1. MS (ESI): m/z calcd for C10H8O5 208.04,
found m/z 207.74 [M − H]−, m/z 415.34 [2M + Na]+.
HRMS (ESI-QTOF) calcd. for C10H8O5: [M − H]− 207.0293; found: 207.0296.
3.1.2. Synthesis of 2-Methyl-4-oxo-7-(toluene-p-sulfonyloxy)-3,4-dihydro-2H-benzopyran-5-carboxylic
acid (1a)
Compound
1 (0.90 g, 4.1 mmol) was dissolved in THF (11 mL) and a 15% NaOH solution (6 mL)
was added. To the resulting red solution cooled at 0 ◦C, p-tosyl chloride (0,77 g, 4.0 mmol) in THF
(6 mL) was added dropwise, and the reaction mixture was stirred overnight at room temperature.
A 10% HCl solution (10 mL) was then added until pH 2–3 and the resulting mixture was extracted
with ethyl acetate (2
×
25 mL). The combined organic layers were washed with water (2
×
10 mL)
and dried over anhydrous Na2SO4, filtered and concentrated in vacuo. Crystallization from ethyl
◦
1
acetate/light petroleum ether gave colorless needles of 1a. Yield 83%, m.p. 161–162 C. H-NMR
(300 MHz, DMSO-d6): 10.55 (broad s. exch. D2O, 1H, COOH), 7.81 (d, 2H, J = 8.0 Hz, H3Ts, H5Ts),
7.49 (d, 2H, J = 8.0 Hz, H2Ts, H6Ts), 6.80 (d, 1H, J = 2.2 Hz, H6), 6.63 (d, 1H, J = 2.2 Hz, H8) , 4.75–4.64
δ

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(m, 1H, H2), 2.83–2.63 (m, 2H, H3, H30 ), 2.42 (s, 3H, CH3Ts), 1.38 (d, 3H, J = 6.2 Hz, CH3) ppm. FT-IR
(KBr):
ν
3375, 3328, 3078, 2984, 2919, 1713, 1699, 1598, 1383, 1297, 1193, 1180 cm−1. MS (ESI): m/z calcd
for C18H16SO7 376.30, found m/z 377.32 [M + H]+.
3.1.3. Synthesis of 2-Methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-carboxylic acid (3)
To a stirred solution of 1a (1.25 g, 3.3 mmol) in 0.2 M NaHCO3 (75 mL), a suspension of Raney
Nickel (1:5 w/w) catalyst was added. The mixture was stirred over a period of 6 h at room temperature.
Then, the catalyst was filtered through a celite pad; the filtrate was acidified with 4 M HCl (15 mL)
until pH 2–3 and extracted with diethyl ether (3
over anhydrous Na2SO4, filtered and evaporated in vacuo, affording the pure
m.p. 167–168 ◦C. 1H-NMR (300 MHz, DMSO-d6):
12.85 (broad s. exch D2O, 1H, COOH), 7.54 (t,
1H, J = 8.4, Hz, H7), 7.07 (d, 1H, J = 8.4 Hz, H6), 6.93 (d, 1H, J = 7.3 Hz, H8), 4.70–4.63 (m, 1H, H2),
2.71–2.57 (m, 2H, H3, H30 ), 1.41 (d, 3H, J = 6.2 Hz, CH3) ppm. 13C-NMR (75 MHz, DMSO-d6):
191.58,
3338, 2979,
×
25 mL). The combined organic layers were dried
3. Quantitative yield,
δ
δ
170.79, 161.76, 136.29, 136.26, 120.07, 119.41, 117.62, 74.83, 44.51, 21.05 ppm. FT-IR (KBr):
ν
2918, 1703, 1683, 1596, 1484, 1298 cm−1. MS (ESI): m/z calcd for C11H10O4 206.19 found m/z 207.87
[M + H]+.
HRMS (ESI-QTOF) calcd. for C11H10O4: [M − H]− 205.0501; found: 205.0502.
3.1.4. Synthesis of 2-Methyl-3,4-dihydro-2H-benzopyran-5-carboxylic acid (4)
Pd/BaSO4 (264 mg, 0.8 mmol) was stirred in anhydrous ethanol (5 mL), then
3 (90 mg, 0.4 mmol),
previously dissolved in anhydrous ethanol (5 mL), was added. The resulting mixture was reduced
with hydrogen in autoclave at 5 atm for 6 h at room temperature. After completion of the reaction,
the catalyst was filtered off on a celite pad, the filter cake was rinsed with ethanol, and the filtrate was
evaporated under reduced pressure. The residue was dissolved in ethyl acetate (5 mL), diluted with
water (2 mL), acidified with 10% HCl (2 mL) until pH = 2, and extracted with ethyl acetate (3
The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo to
×
5 mL).
give a solid residue, which was then purified by chromatographic column (ethyl acetate/methanol
1
9:1), to obtain
4
as an oil. Yield 70%. H-NMR (300 MHz, DMSO-d6):
δ
12.64 (broad s. exch D2O, 1H,
COOH), 7.35 (dd, 1H, J = 7.6, 1.3 Hz, H6), 7.14 (t, 1H, J = 7.6, Hz, H7), 6.92 (d, 1H, J = 7.6 Hz, H8),
4.14–4.06 (m, 1H, H2), 3.03 (ddd, 1H, J = 18.0, 5.8, 3.2 Hz, H3), 2.95 (ddd, 1H, J = 18.0, 11.5, 6.2 Hz, H30 ),
2.05–1.96 (m, 1H, H4), 1.61–1.47 (m, 1H, H4’), 1.32 (d, 3H, J = 6.2 Hz, CH3) ppm. 13C-NMR (75 MHz,
DMSO-d6):
(KBr):
δ 168.93, 155.60, 131.93, 126.98, 123.45, 122.44, 120.60, 71.76, 28.91, 23.84, 21.44 ppm. FT-IR
ν
3429.8, 2968.8, 2928.8, 2645.1, 1687.9, 1594.4, 1457.8, 1278.7, 1139.9, 1109.1, 801.2 cm−1. MS (ESI):
m/z calcd for C11H12O3 192.20, found m/z 191.20 [M − H]−.
HRMS (ESI-QTOF) calcd. for C11H12O3: [M − H]− 191.0708; found: 191.0709.
3.1.5. Synthesis of 7-Hydroxy-2-methylchroman-5-carboxylic acid (5)
Pd/BaSO4 (0.55 g, 1.62 mmol) was stirred in anhydrous ethanol (15 mL), then 1 (0.2 g, 0.90 mmol),
dissolved in 10 mL of anhydrous ethanol, was charged. The resulting mixture was stirred under
hydrogen flow in autoclave at 5 atm for 6 h at room temperature. After completion of the reaction,
the catalyst was filtered through a celite pad, the filter cake was rinsed with ethanol, and the filtrate
was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (10 mL) and then
washed with 2 M HCl (3
evaporated in vacuo to give
DMSO-d6) 12.64 (broad s, exch. D2O, 1H, COOH), 9.40 (broad s, exch. D2O 1H, OH), 6.82 (d,
×
5 mL). The organic layer was dried over anhydrous Na2SO4, filtered and
5
◦
as a yellow solid. Yield 75%, m.p. 168–170 C. 1H-NMR (300 MHz,
δ
J = 2.6 Hz, 1H, H6), 6.32 (d, J = 2.6 Hz, 1H, H8), 4.21–3.87 (m, 1H, H2), 2.96 (dd, J = 5.6, 3.1 Hz, 1H, H3),
2.90 (dd, J = 5.6, 3.1 Hz, 1H, H30 ), 2.01–1.86 (m, 1H, H4), 1.63–1.37 (m, 1H, H40 ), 1.27 (d, J = 6.2 Hz,
3H, CH3) ppm.13C-NMR (75 MHz, DMSO-d6):
δ
168.87, 156.42, 156.06, 132.18, 114.15, 110.27, 107.09,
71.79, 29.21, 23.30, 21.43 ppm. FT-IR(KBr):
ν 3370, 2972, 2945, 2618, 1697, 1622, 1589, 1491, 1410,1308,
1132 cm−1. MS (ESI): m/z calcd for C11H12O4 208.07, found m/z 207.21 [M − H]−.

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HRMS (ESI-QTOF) calcd. for C11H12O4: [M − H]− 207.0657; found: 207.0657.
3.1.6. Synthesis of 7-Hydroxy-4-oxo-3,4-dihydro-2H-benzopyran-5-methyl ester (1b)
To a cold stirred solution of
L) was added dropwise at 0 C. The solution was heated at reflux overnight. Afterwards methanol
1
(90 mg, 0.405 mmol) in anhydrous MeOH, a solution of 97% H2SO4
◦
(43
µ
was evaporated in vacuo and the residue was dissolved in a NaHCO3 saturated solution (5 mL) and
then extracted with Et2O (3 6 mL). The combined organic layers were washed with brine, dried over
anhydrous sodium sulfate, and then concentrated in vacuo to obtain a light pink powder. Yield 80%,
m.p. 166–168 ◦C. 1H-NMR (300 MHz, DMSO-d6):
10.89 (broad s. exch. D2O, 1H, OH), 6.37 (s, 2H,
×
δ
H6, H8), 4.63–4.55 (m, 1H, H2), 3.73 (s, 3H, COOCH3), 2.62–2.56 (m, 2H, H3,), 1.37 (d, 3H, J = 6.2 Hz,
CH3) ppm. 13C-NMR (75 MHz, DMSO-d6):
δ
189.85, 164.15, 163.65, 136.34, 110.70, 109.51, 103.95, 94.88,
3258, 2958, 2924, 1736, 1652, 1609, 1580, 1501, 1428, 1300,
74.70, 52.68, 43.94, 20.80 ppm. FT-IR (KBr):
ν
1235, 1159 cm−1. MS (ESI): m/z calcd for C12H12O5 236.07 found m/z 235.33 [M − H]−.
HRMS (ESI-QTOF) calcd. for C12H12O5 [M − H]− 235.0606; found: 235.0606.
3.2. MbtI Inhibitory Activity
MbtI was produced in recombinant form as previously reported [17]. Enzymatic activity was
determined by measuring the production of salicylic acid, by a fluorimetric assay (Ex. = 305 nm,
Em. = 420 nm), slightly modified from Vasan et al. [32]. Assays were performed at 37 ◦C in a final
volume of 400
pH 7.5, 5 mM MgCl2, 1–2
chorismic acid. Inhibition assays were performed at subsaturating concentrations of chorismic acid
(50 M), in the presence of the compound at 100 M (stock dissolved 20 mM in DMSO). Blank control
µL, using a Perkin-Elmer LS3 fluorimeter. The reaction mixture contained: 50 mM Hepes
µM MbtI, and the reactions were started by the addition of the substrate,
µ
µ
was performed by adding DMSO. The solubility of the compounds was evaluated in the solution in
which the biochemical assays were performed (up to 100 µM concentration).
For IC50 determinations, the enzyme activities were measured in the presence of compound and
values were estimated according to the following equation:
ꢀ
ꢁ
[I]
A[1] = A[0]x 1 −
,
[I] + IC50
where A[1] is the enzyme activity at inhibitor concentration [I] and A[0] is the enzyme activity without
inhibitor.
3.3. Docking Calculations
The crystal structure of MbtI, in complex with the inhibitor
I (PDB code: 3VEH) was taken
from the Protein Data Bank [33]. The complex was placed in a rectangular parallelepiped water box.
Magnesium ion was inserted, analyzing its disposition and interactions into the structure 2FN1 (PDB
code) [34]. After adding hydrogen atoms, the protein complexed with its inhibitor was minimized
using Amber14 software [35] and ff14SB force field at 300 K. The complex was placed in a rectangular
parallelepiped water box, an explicit solvent model for water (TIP3P) was used and the complex was
solvated with a 10 Å water cap. Sodium ions were added as counter ions to neutralize the system.
Two steps of minimization were then carried out; in the first stage, we kept the protein fixed with a
position restraint of 500 kcal/mol
In the second stage, we minimized the entire system through 5000 steps of steepest descent followed
by conjugate gradient (CG) until a convergence of 0.05 kcal/Å mol. The ligand was built by means of
·
Å2 and we solely minimized the positions of the water molecules.
·
Maestro [36] and was then minimized in a water environment (using the generalized Born/surface
area model) by means of Macromodel [37]. The minimization was performed using the CG, the MMFFs
force field, and a distance-dependent dielectric constant of 1.0 until the ligands reached a convergence
value of 0.05 kcal
·
Å−1 mol−1. Five different docking procedures were applied and for each docking
·

Molecules 2018, 23, 1506
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calculation only the best-scored pose was taken into account [38
–40]. The docking calculations were
carried out by using GOLD 5.1 with the ASP, CSCORE CHEMPLP and GSCORE scoring functions [41
]
and PLANTS [42] employing the procedures previously described [43]. Each ligand was docked
into the binding site of MbtI by using the different docking procedures, then the root-mean-square
deviation (RMSD) of each of these docking poses against the remaining docking results was evaluated
using the rms analysis software of the GOLD suite. The most populated cluster of solutions was then
considered and subjected to molecular dynamic (MD) simulations.
3.4. MD Simulations
All simulations were performed using AMBER, version 14 [35]. MD simulations were carried out
using the ff14SB force field at 300 K in a rectangular parallelepiped water box. The TIP3P explicit solvent
model for water was used. Sodium ions were added as counter ions to neutralize the system. Prior to
MD simulations, two steps of minimization were carried out using the same procedure described above.
Particle mesh Ewald (PME) electrostatics and periodic boundary conditions were used in the simulation.
The time step of the simulations was 2.0 fs with a cutoff of 10 Å for the non-bonded interactions,
while SHAKE was employed to keep all bonds involving hydrogen atoms rigid. Constant-volume
periodic boundary MD was carried out for 0.5 ns, during which the temperature was raised from 0
to 300 K. Then 99.5 ns of constant pressure periodic boundary MD was carried out at 300 K using
the Langevin thermostat to maintain constant the temperature of our system. All the
α carbons of
the protein were blocked with a harmonic force constant of 10 kcal/mol
·
Å2. General Amber force
field (GAFF) parameters were assigned to the ligands, while partial charges were calculated using the
AM1-BCC method as implemented in the Antechamber suite of AMBER 14.
4. Conclusions
A small set of compounds incorporating the chromane motif was evaluated for the inhibition of
MbtI enzymatic activity. The approach successfully led to the disclosure of the hit
1
provided with
an encouraging activity against MbtI (IC50 value of 55
µM), which is less than 5-fold lower than that
showed by the reference inhibitor I.
On the basis of the acquired data, evidencing the importance of the carbonyl group and the
7-hydroxyl function as key structural elements to gain MbtI inhibition, we identified a novel structural
chemotype, represented by compound
reported. These preliminary observations could provide the basis for the design of new inhibitors.
Molecular modeling studies allowed to identify a reliable binding mode of within the MbtI
1, for which anti-MbtI properties had never been previously
1
catalytic site and to rationalize the preliminary SAR data based on the predicted-ligand protein
interactions, thus paving the way for lead optimization studies focused on the development of more
potent analogues. It should be highlighted that
1 retains some of the interactions evidenced by I in the
crystallographic conformation inside the binding pocket, but it also allows to evidence a new possible
role for the oxygen backbone of G270, thus identifying a new anchoring point for the design of MbtI
inhibitors. Indeed, all compounds endowed with a considerable activity showed an interaction with
G270 through their 7-hydroxyl group in their predicted binding mode. Finally, the importance of
the carboxylic group for the inhibitory activity of the ligands was demonstrated by the inactivity of
the methyl ester 1b, for which a completely different binding mode, lacking the key ligand–protein
interactions, was predicted.
Overall, these new findings could enhance the possible perspectives in antitubercular drug discovery.
Supplementary Materials: The supplementary materials are available online. The IR, MS and NMR spectra of
compounds are provided. Figure S1: minimized average structure of compound 1; Figure S2: predicted binding
mode of compound 1b into MbtI catalytic site.
Author Contributions: G.P. and T.T. carried out the computational studies; E.P., M.M., A.G., and S.V. designed
and synthesized the compounds, L.R.C. and L.C. carried out the biological experiments, F.M. and D.B. supervised
the whole study and wrote the paper.

Molecules 2018, 23, 1506
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Acknowledgments: This work was funded by University of Milan (Linea B).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of compounds 1, 1b, 3, 4 and 5 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).