Spirochromone-chalcone conjugates as antitubercular agents: Synthesis, bio evaluation and molecular modeling studies

Article abstract of DOI:10.1039/c5ra21737g

A new series of spirochromone annulated chalcone conjugates were synthesized and evaluated for their antitubercular activity against Mycobacterium tuberculosis H37Rv strain. These compounds were subjected to molecular modeling studies using docking and chemoinformatics based approaches. The docking simulations were performed against a range of known receptors for chalcone derived compounds to reveal MTB phosphotyrosine phosphatase B [MtbPtpB] protein as the most probable target based on the high binding affinity scores. Five compounds exhibit significant inhibition, showing minimum inhibitory concentration values i.e. MIC values ranging from 3.13-12.5 μg mL^-1. Further analysis of the synthesized compounds with known and in-house developed chemoinformatics tools unequivocally established their potential as anti-tubercular compounds. QSAR modeling revealed a quantitative relationship between biological activities and frontier molecular orbital energies of synthesized compounds. The predictive model can be employed further for virtual screening of new compounds in this series.

Full text of DOI:10.1039/c5ra21737g

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Muthukrishnan, M. Mujahid, P. Yogeeswari, S. Dharmarajan, M. Basavanag, E. Díaz-Cervantes, L. Bahena,
J. Robles, R. G. Gonnade, K. M and R. Vyas, RSC Adv., 2015, DOI: 10.1039/C5RA21737G.
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Spirochromone-chalcone conjugates as antitubercular agents:
synthesis, bio evaluation and molecular modeling studies
Received 00th January 20xx,
Accepted 00th January 20xx
M. Mujahid,a P. Yogeeswari,b D. Sriram,b U. M. V. Basavanag,c Erik Díaz-Cervantes,c Luis Córdoba-Bahena,c Juvencio
Robles,c R. G. Gonnade,a M. Karthikeyan,a Renu Vyas,a M. Muthukrishnan*a
DOI: 10.1039/x0xx00000x
A new series of spirochromone annulated chalcone conjugates were synthesized and evaluated for their antitubercular
activity against Mycobacterium tuberculosis H37Rv strain. These compounds were subjected to molecular modeling
studies using docking and chemoinformatics based approaches. The docking simulations were performed against a range
of known receptors for chalcone derived compounds to reveal MTB phosphotyrosine phosphatase B [MtbPtpB] protein as
the most probable target based on the high binding affinity scores. Five compounds exhibit significant inhibition, showing
minimum inhibitory concentration values i.e. MIC values ranging from 3.13-12.5 µg/ml. Further analysis of the synthesized
compounds with known and in-house developed chemoinformatics tools unequivocally established their potential as anti-
tubercular compounds. QSAR modeling revealed a quantitative relationship between biological activities and frontier
molecular orbital energies of synthesized compounds. The predictive model can be employed further for virtual screening
www.rsc.org/
of
new
compounds
in
this
series.
It is a well known fact that biologically active natural products
are recognized as evolutionarily selected and biologically
prevalidated starting point for any successful drug discovery
program. Therefore, the synthesis of natural products inspired
compound collections and their biological evaluation is a
highly promising strategy for the identification of unique
biologically relevant compound classes, especially in the
studies aimed at finding a new class of anti TB agents.2
Introduction
Tuberculosis is a devastating bacterial disease caused by
Mycobacterium tuberculosis (Mtb). It poses a major public
health risk due to the long duration of treatment (6-9 months),
coinfection with HIV and the emergence of multidrug resistant
strains etc. According to a WHO report, in 2013 alone, nearly 9
million people contracted tuberculosis and 1.5 million died
from complications of the disease.1 Despite enormous efforts
to develop antimycobacterial drug molecules, tuberculosis still Flavonoids are the most explored class of natural products as
constitutes the leading killer disease. Therefore, the discovery
and development of new types of anti TB agents acting on
novel drug targets is urgently needed.
they are widely distributed in various plants and foods such as
fruits & vegetables.3 Chalcones are an important sub class of
flavonoids that consist of open chain flavonoids in which the
two aromatic rings are fused by
a three-carbon α,β-
unsaturated carbonyl system.4 They have been reported to
possess plethora of medicinal applications which include anti-
inflammatory, antileukemic, antiulcerogenic, antimalarial,
antileishmaniosis, and anticancer activities.5 Interestingly,
many natural and natural product inspired chalcone analogues
exhibit significant antimycobacterial activity (Figure 1).6
Recently, several research groups successfully demonstrated
their mode of action by identifying their molecular targets.7
Figure 1.Natural/NP inspired chalcones with potent activity against Mtb
a.CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, Maharashtra,
India- 411008. E-mail: m.muthukrishnan@ncl.res.in; Fax: +91-20-25902629;
Ph:+91-20-25902284
B
HO
O
R
b.Medicinal Chemistry & Antimycobacterial Research Laboratory, Pharmacy Group,
Birla Institute of Technology & Science – Pilani, Hyderabad Campus, Jawahar
Nagar, Hyderabad 500 078, India
O
O
B = cyclohexyl, N-Boc piperidinyl
c. Departamento de Química
y Departamento de Farmacia, Universidad de
Guanajuato, Noria Alta S/N, Col. Noria Alta, C.P. 36050, Guanajuato, Gto., México
Figure 2. Design of spirochromone annulated chalcone
conjugates
†Electronic Supplementary Information (ESI) available: CCDC 1045946 (4f)
.
Characterization of new compounds and crystallographic data or other electronic
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format See DOI: 10.1039/b000000x
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Journal Name
A good safety profile, possibility of oral administration,8 and
heating with acetic anhydride in the presence of anhydrous
zinc chloride provided 4,6-diacetyl resorcinol
in 75% yield.11
The diacetyl compound on Kabbe condensation with
2
easy synthesis are the major factors contributing to the
growing interest in exploring the pharmacological activities of
chalcones. In this context, and in view of our long standing
interest in the chemistry of the privileged chromone motif,9
we recently reported various spirochromone derivatives
possessing 1,2,3-triazole and amino alcohol moieties that can
serve as lead molecules for developing antitubercular agents.10
These results encouraged us to further explore the
spirochromone motif as an active pharmacophore for further
diversification to exploit its anti TB potential. Herein, we
describe the synthesis of novel spirochromone annulated
chalcones, their in silico studies and in vitro screening results
against Mycobacterium tuberculosis H37Rv (Figure 2). To the
best of our knowledge, no reports on the synthesis, in silico
study and antimycobacterial activities of these spirochromone
annulated chalcone have been reported.
2
cyclohexanone or N-Boc piperidone in the presence of
pyrrolidine as a base in refluxing toluene using Dean-Stark
apparatus provided spirochromone derivatives 3a and 3b in
62% and 65% yields. Lastly, spirochromone derivatives 3a,b on
treatment with various aryl aldehydes using Claisen–Schmidt
condensation reaction produced spirochromone annulated
chalcones 4a-g
&
5a-h in moderate to good yields. The
(15 compounds) was
structure of all the new products
4
& 5
confirmed by 1H NMR, 13C NMR and mass spectral analysis. For
an example, in the 1H NMR spectra (compound 4f as a
representative example), a signal corresponding to the C-3
protons of chromanone skeleton was observed as a singlet at δ
2.73 ppm and the corresponding 13C resonance signal was
observed at δ 47.8 ppm and the spirocarbon was discernible at
δ 81.4 ppm. Further, the appearance of a sharp singlet (1H)
observed at δ 13.5 ppm in the PMR, suggested the presence of
one chelated hydroxyl group. In addition, the appearance of
two sets of doublets at δ 7.61 & 7.87, 1H each with coupling
constant 15.4 Hz were ascribable to the olefinic protons of the
chalcone moiety. Conclusive evidence for its structure was
derived from a single crystal X-ray analysis (Figure 3).
Results and discussion
Synthesis
A
three step synthetic strategy was followed for the
preparation of spirochromone annulated chalcones as outlined
in Scheme 1. As shown, the readily available resorcinol on
1
Figure 3. ORTEP diagram of the compound 4f
Anti-mycobacterial evaluation
All the new spirochromone annulated chalcone conjugates
were screened for their in vitro antimycobacterial activity
against M. tuberculosis H37Rv (ATCC27294) using an agar
dilution method. The minimum inhibitory concentration (MIC;
µg/mL) was determined for each compound. The MIC is
defined as the minimum concentration of compound required
to completely inhibit the bacterial growth. Rifampicin and
ethambutol were used as reference compounds. The MIC
values of the synthesized compounds along with the standard
drugs for comparison are reported in Table 1
.
Among the 15 spirochromone annulated chalcone conjugates
tested, five compounds 4a, 4b, 5b, 5c, and 5d were shown to
be active with MIC values in the range of 3.13-12.5 µg/mL. The
compound 5c is shown to be highly active among all the
compounds tested with a MIC value of 3.13 µg/mL. It has a
core spirochromone moiety hitherto unreported.The
Scheme 1. Reagents and conditions: (i) Ac2O, ZnCl2, 140°C,30 min,
75%; (ii) Cyclohexanone/N-Boc-piperidone, pyrrolidine, toluene, reflux,
12 h; (iii) aromatic aldehyde, 50 % aq. KOH, MeOH
preliminary SAR of the spirochromone annulated conjugates
2 | J. Name., 2012, 00, 1-3
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reveals that compounds possessing piperidinyl group at 2nd
position of the chromone ring favors better activity than the
cycloalkyl group. In addition, the activity is being influenced by
functional groups present in the chalcone moiety as well.
Halogen substitution at aromatic ring, especially fluoro
substitution enhances the activity (5b, 5c). It is also observed
that the presence of methoxy substitution at aromatic ring
doesn’t seem to influence the activity.
Table 1: In vitro antimycobacterial activity of the spirochromone annulated
chalcone conjugates
MIC
MIC
Computational studies
Entry
Compound
Entry
Compound
(µg/mL)
(µg/mL)
F
Boc
Boc
N
N
HO
O
HO
O
Methodology
1
12.5
12.5
25
10
3.13
12.5
25
F
O
5c
5d
O
O
O
O
O
4a
4b
4c
Preparation of ligands
HO
HO
The 2D structures of all compounds i.e. native ligand, compounds
4a, 4b, 5b-d, rifampicin and ethambutol were drawn and analyzed
by Marvin view. The compounds were converted to 3D structure
(.pdb) using Marvin suite 15.12 The 3D coordinates (.pdb) of each
molecule were loaded on AutoDock tools 1.5.6 (ADT) for energy
minimization. Gasteiger charges were added and the rotatable
bonds were set by the ADT and all torsions were allowed to rotate.
2
3
4
5
6
7
8
11
12
13
14
15
16
17
Cl
F
O
O
O
O
O
Boc
N
HO
HO
O
O
MeO
MeO
Cl
O
O
O
5e
H3CO
Boc
Boc
N
N
HO
O
O
O
HO
O
>25
>25
>25
>25
25
4d
O
5f
O
O
H3CO
MeO
HO
O
HO
25
OCH3
OMe
Preparation of macromolecule
O
4e
4f
O
O
5g
O
OMe
HO
O
The protein targets retrieved from RCSB Protein Data Bank are
proteins associated with metabolic functioning and proliferation of
Mycobacterium tuberculosis. Enzymes MTB phosphotyrosinephosp
hatase A [MtbPtpA] (PDB code 1U2P) and MTB phosphotyrosine
phosphatase B [MtbPtpB] (PDB code 2OZ5) proteins, Enoyl acyl
carrier protein (PDB code 2NSD), Dihydrofolate reductase (PDB
code 1DG5) served as the docking receptors. All the bound ligands
and water molecules were removed from the receptors. Polar
hydrogen atoms were added and Kollman charges were assigned to
the proteins using AutoDock tools (ADT).
Boc
N
HO
O
H3CO
>25
0.2
1.56
MeO
O
O
O
O
O
5h
O
O
HO
Rifampicin
O
4g
O
O
Boc
N
HO
O
>25
Ethambutol
5a
O
F
Boc
N
6.25
HO
O
O
O
9
5b
Table2: Molecular docking analysis of four protein targets with selected compounds. The binding energies were calculated using MOE and Autodock Vina algorithms. The energy is
measured in kcal/mole
Compound 4a
Compound 4b
Compound 5b
Compound 5c
Compound 5d
PDB
Autodock
Autodock
Autodock
Vina
Autodock
Autodock
MOE
MOE
-9.65
MOE
-9.98
MOE
MOE
target
Vina
Vina
Vina
Vina
1U2P
2OZ5
-10.00
-7.10
-6.80
-6.90
-10.15
-7.60
-11.25
-8.00
-12.41
-8.16
-12.37
-8.30
-13.90
-8.90
-13.96
-9.00
-11.36
-8.30
2NSD
1DG5
-12.20
-12.79
-8.10
-8.40
-11.84
-12.47
-7.80
-8.28
-12.95
-12.41
-8.30
-8.00
-12.37
-11.80
-7.10
-8.00
-12.51
-10.40
-6.80
-7.90
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Molecular docking
Table 3: Molecular docking studies of selected compounds on 20Z5
The molecular docking was performed and analyzed using two
state of art docking algorithms AutoDock Vina13 and MOE.14
Lamarckian genetic algorithm method implemented in the program
suite was employed to identify appropriate binding modes and
conformation of the ligand molecules. The grid map was centered
at the active site of the protein by Autogrid. The Lamarckian
genetic algorithm, the pseudo-Solis and Wets methods were
applied for minimization using default parameters. Binding energy,
intermolecular amino acids and their distance were recorded in
each ligand bound conformations. In the MOE docking simulations,
the ligands were allowed flexibility, London dG scoring function was
employed and top 30 conformations were retained.
E
n
t
Compound ID
Inter
mole
cular
dista
nce
Amino
acids
Ligan Recept
Binding
Energy
(kcal/m
ol)
d
or
involved
in
Ato
m
Atom
r
y
intermol
ecular
(Ǻ)
interactio
ns
Tyr125
1
Native Ligand
1.40
1.01
3.48
3.07
2.62
H2572
O2560
C2512
C2528
O2558
OH1069
NH543
NE1475
OG522
CG1104
-9.2
Arg59
Arg166
Ser57
Glu129
2
4a
2.96
2.96
2.89
2.44
Ser57
Ser57
His94
Arg166
O2536
O2536
O2534
O2541
OG522
OG522
ND847
NH1481
-8.2
Molecular docking analysis
With an objective to explore the potential putative targets of the
synthesized compounds among the validated Mycobacterium
receptors, we performed a docking analysis with known biological
targets reported so far for the chalcone scaffold derived anti-
tubercular compounds. In general, the validated Mtb targets for
this class of compounds are phosphotyrosine phosphatases(MtbPtp
A and B; PDB ID: 1U2P, 2OZ5), dihyrdofolate reductase (DHFR), PDB
ID: 1DG5) and enol acyl carrier reductases (PDB ID: 2NSD).15
Accordingly, automated docking was used to assess the binding
modes and conformation of the compounds synthesized in this
study. Among the 15 spirochromone-chalcone structures,
compounds 4a, 4b, 5b, 5c and 5d were considered for docking
simulations since they exhibited higher anti-tubercular activity, as
ascertained from their MIC data (Table 1). The four spirochromone
chalcones were cross docked against all potential protein targets to
reveal better binding affinities with 2OZ5, a MTB phosphotyrosine
phosphatase B [MtbPtpB] protein (Table 3). PtpB and PtpA are
protein tyrosine phosphatases, a class of regulatory enzymes that
are secreted by Mtb into the cytosol of infected macrophages.
MtbPtpB catalyzes the dephosphorylation of serine/threonine or
tyrosine and also has phosphoinositide phosphatase activity
whereas PtpA specifically dephosphorylates the tyrosine residues.
PtpA inhibits macrophages microbial activity by entering into host
macrophages, dephosphorylates the cytoplasmic protein VPS33B
and inhibits the phagosome maturation.16 MtbPtpB blocks the
signal regulated kinase and p-38 mediated by IL-6 thereby
promoting mycobacterial survival in the host.
3
4b
2.31
2.76
His94
H2537
O2541
O843
-8.3
Tyr125
O1069
4
5
6
5b
5c
3.53
Arg166
O2512
NH1481
-8.9
-9.0
-8.3
2.52
2.52
2.23
Ser57
Ser57
Glu60
O18
O18
H43
OG585
OE621
OG585
5d
2.78
2.55
Ser57
O2543
O2527
OG522
Lys164
NZ1454
Rifampicin
1.69
3.53
1.40
2.48
2.76
2.94
Glu60
H2620
H2627
O2519
O2522
O2520
O2521
Oe557
7
Phe80
O738
-7
Tyr125
Tyr125
Arg166
Arg166
OH1069
OH10c69
NH1478
NH1478
8
Ethambutol
3.07
2.41
Arg166
Phe161
O2512
O2512
N1422
-7.8
NH1481
PtpA specifically PtpA and PtpB proteins share
a structural
homology and the active site of the phosphatase enzymes lies in
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the P-loop region. However, there are certain differences in the P-
loop region and some sequential and positional variations occur in
the variable loop. These subtle distinctions in the active sites may
account for the high selectivity of the five compounds to MtbPtpB
(PDB ID: 2OZ5) than MtbPtpA (PDB ID: 1U2P). Moreover the active
site volume computed (CASTp server) showed a greater volume of
the binding pocket of PtpB (1509.9 A3) relative to PtpA (108.7 A3)
enzyme. The present compounds possess volumes ranging from
300-400 A3 which enables a better fit in the binding pocket of B
compared to A. Compound 5c performed better in the docking run
when compared with other synthesized compounds with binding
scores ranging from -7.6 to -8.0 kcal/mol against the resolved
crystal structure of 2OZ5. In another docking run, the synthesized
molecule 5c chosen for computational studies showed comparable
performance with the native ligand of receptor (entry 1, Table 3)
and slightly better performance than the known MTB drug
molecules rifampicin and ethambutol in terms of binding
efficiencies (entries 5 and 6, Table 3). It is to be noted here that
the compound 5c also possesses the best inhibitory activity in the
Mtb assay. Further, the top docked conformation of this compound
depicted a greater alignment with the native ligand pose (Figure
6B). Hence it is likely to act as good binder for the phosphatase
receptor in mycobacterium.
a) Native ligand
b) 4a
c) 4b
d) 5c
e) 5d
f) 5b
g) Ethambutol
h) Rifampicin
Figure 4: 2D protein ligand interaction maps of compounds 4a, 4b, 5b-5d, native molecule and known tuberculosis drugs ethambutol and rifampicin.
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The key intermolecular interactions between selected compounds Arginine residue was mainly involved in π-cation interaction in the
and known drugs against the phosphatase B enzyme are depicted in pocket region which helped to stabilize the protein ligand complex
Figure 4. Tyr125, Arg56, Arg166, Ser57 and Glu129 amino acids in formation. Arg166 and Tyr125 were found to interact with the
the active site interacted with native ligand with many residues oxygen atom of the pyranone ring system in compounds 4a and 4b.
depicting a high ligand exposure in the interaction map.17 Tyr125 Compound 5b showed a slightly
acts as hydrogen donor whereas Arg66 acts as hydrogen acceptor
A)
and forms a bond with the O group of the ligand. Carbonyl and
hydroxyl group based interactions were common in most of the
predicted binding poses of compounds in the active site pocket.
Specifically, Ser57 of compound 4a, Glu60 of compound 5c,
Phe80and Arg166 of rifampicin and Arg166 and Phe161 of
ethambutol made contacts with the hydroxyl group. Thr125 residue
in the receptor pocket interacted with the two carbonyl groups
present in the rifampicin structure (entry f, Figure 4). Apart from
hydrogen bonding, other stablizing intermolecular interactions such
as π-cation interaction were also observed.
Figure a:
B)
C)
Figure b:
Figure 6: Superposed docking poses for A) Pocket region of 2OZ5 with loops
(green) flanked by alpha helices(red) and beta sheets(yellow). B) Structure
alignment of docked conformation of Compound 5c (magenta)with native ligand
conformation (yellow) in the crystal structure C) Compound 5c and native ligand
in the binding pocket of receptor. The receptor surface shown is coloured
according to the electrostatic potential.
Figure 5: Stereoview depicting the binding mode of compounds 5b and 5c that
showed high in vitro anti-mycobacterial activity. Figure a depicts compound 5c in
magenta, the pocket residues are shown in cyan. Figure b depicts compound 5b
in green along with the neighbouring residues (cyan).
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Table 4: Chemoinformatics analysis of the 15 spirochromone annulated chalcone conjugates
Properties
Compounds
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
5g
5h
Lipinski Rule of Fivea
Molecular weight
380.
41
380.
41
396.
87
392.
45
422.
47
392.
45
406.
43
463.
53
481.
51
481.
52
497.
97
493.
55
493.
55
523.
58
523.
58
HB accept
HB donor
LogP
4
1
4
1
4
1
5
1
6
1
5
1
6
1
5
1
5
1
5
1
5
1
6
1
6
1
7
1
7
1
5.44
2
5.44
2
5.88
1
5.24
7
5.23
8
5.28
4
4.99
6
4.91
1
5.06
4
5.06
2
5.50
1
4.90
4
4.86
7
4.85
8
4.85
8
Chemical properties
Weiner path b
Ring count a
4228
4
4228 2152 2464 2912
2422 2664 3910 4284
4228 4228 4637 4693
5384 5332
4
4
4
4
4
5
4
4
4
4
4
4
4
4
PDL/PLL a
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
ADME properties
BBB (-3.0 – 1.2) c
0.79
4
0.83
1.62
0.2
0.08
0.22
0.05
7
0.04
0.07
0.05
8
0.06
1
0.03
0.08
3
0.19
2
0.07
4
Caco2 (nms) (<25, poor,
>500, best) c
31.6
7
30.7
4
24.0
3
37.3
8
41.8
1
21.9
4
24.7
9
33.2
7
31.8
1
31.3
3
24.3
1
33.4
3
36.7
1
39.3
8
37.0
2
HIA (50-100%) c
98.0
1
96.0
1
96.3
6
96.2
5
96.6
6
96.2
5
96.7
3
97.1
4
97.1
3
97.1
3
96.7
5
97.6
7
97.6
7
89.6
6
98.0
3
Rotatable bonds (0 - 15)a
TPSA (7.0 – 200.0) b
3
3
3
4
5
4
3
4
4
4
4
5
5
6
6
93.1
3
93.1
4
63.5
9
72.8
3
82.0
5
72.8
3
82.0
5
93.1
3
93.1
3
93.1
3
93.1
3
102.
37
102.
37
111.
6
111.
6
Toxicity properties d
DSSTox Carcinogenic
potency Mutagenecity
Neg.
(C:0.
129)
Neg.
(C:0.
102)
Neg.
(C:0.
115)
Neg.
(C:0.
153)
Neg.
(C:0.
153)
Neg.
(C:0.
196
Neg.
(C:0.
129
Neg.
(C:0.
129
Neg.
(C:0.
169
Neg.
(C:0.
153)
Neg.
(C:0.
129
Neg.
(C:0.
129
Neg.
(C:0.
129
Neg.
(C:0.
169
Neg.
(C:0.
169
DSSTox Carcinogenic
potency Mouse
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
Neg.
(C:
0.08
0)
0.08
0)
0.01
1)
0.01
1)
0.01
1)
0.01
1)
0.08
5)
0.08
5)
0.08
5)
0.01
1)
0.08
5)
0.08
5)
0.08
5)
0.08
5)
0.08
5)
a Computed using Screening Assistant (SA2) program. PDL (Progressive drug like), PLL(Progressive lead like). b Calculated using MOE(CCG) Chemoinformatics suite. c
PreADMET software. d LAZAR wherein Neg = Negative and C = Confidence value.
lower docking energy than 5c (entry 4, Table 3). Interestingly, both placed nearer to the important catalytic residue Cys 160 in the
possess similar structures, the only difference being the placement binding pocket whereas the aromatic ring of 5b lies in a different
of the fluorine atom in the ring. Perhaps the position of the fluorine plane (Figure 5b).
atom in the former probably hinders effective binding with the
Compounds 4a, 5c, 5d, rifampicin and ethambutol interacted with
pocket residue forcing the molecule to adopt a conformation that
Arg166 and Ser57 residues in the pocket region of phosphotyrosine
allows only a single π-cation interaction with arginine 166 (entry f,
phosphatase B protein. After post filtering process, the top docked
Figure 4). It is known from the x-ray crystal structure that the
protein conformation for compound 5c was chosen. Figure 6(B)
pocket region of 2OZ5 is present in a P loop region flanked by alpha
highlights the top docked pose of compound 5c aligned to the
helix chains α7, α8 and α3A displayed in Figure 6 (A). This
native ligand pose. Structure alignment of conformations was
comparable lid of α7– α8 hairpin in located on one side of the
performed using Pymol v1.7.6. Similarity in alignment between the
entrance to the to the active site and helix α3A on the hydrophobic
compounds suggests that the top docked conformation of 5c is
surface of pocket region and protects the catalytic Cys160 amino
closer to the native bioactive conformation. Figure 6(C) shows the
acid present within the P-loop PtpB resists oxidative inactivation
orientations of the compounds 5c and native ligand in the binding
better than the PtpA enzyme that does not possess this comparable
pocket of the receptor.
lid.18 As observed in Figure 5a, the more potent compound 5c is
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descriptors using PM3 and AM1 Hamiltonian for studying the
electronic properties with an objective of mapping them with the
variation in activity. The molecules were geometry optimized and
subjected to energy minimization by employing the MMFF94x force
field. The resultant 3D QSAR model yielded a R2 value of 0.87507,
RMSE 2.72 and a cross validated R2(Q2) value of 0.687. The decrease
in r squared value in the latter case can be attributed to the
presence of few outliers such as compound 5b which showed better
fit with 2D rather than 3D descriptors (Figure S2).
Chemoinformatics Analysis
A druggability check was performed for all the 15 synthesized
compounds (Table 4). Lipinski rule of 5 predictions were performed
using the Screening Assistant 2 tool.19 Most compounds displayed
no violation of the standard Rule of 5 indicating that they possess
good drug like properties.20 The notion that these compounds could
be further developed as anti-tubercular compounds was further
affirmed by ADME properties prediction using PreADMET
software.21 The compounds clearly lie in the acceptable range of
BBB model suggesting that the compounds can penetrate the Blood
Brain Barrier. It is well established fact that for a compound to be
accepted in an oral dosage form, Caco2 cell permeability values
should be above 25nms22 and the Human Intestinal Absorption(HIA)
quantities should lie in the 50-100% range.23 The selected
compound 5c, prioritized in this study fulfilled the above criteria.
TPSA (Topological polar surface area) results indicated satisfactory
values for all the 15 compounds. LAZAR (Lazy structure activity
relationships) software detects mutagenic and/or carcinogenic
properties based on the similarities in functional group with
mutagenic and/or carcinogenic compounds present in the Lazar
database.24 Based on this software, all the compounds were
predicted to be non-carcinogenic and non-mutagenic wherein the
confidence value greater than 0.025 suggested the model to give
highly reliable predictions. Thus most synthesized compounds
predicted favorable ADME predictions. To further corroborate our
studies, an in-house developed integrated suite of programs–
ChemScreener was employed. The program rapidly annotates huge
chemical libraries with more than thousand pharmacophoric (P),
toxicophoric (T) and chemophoric (C) attributes to prioritize
compounds in a virtual screening run.25 The significance of the
presence of pharmacophore and toxicophore features in a molecule
is commonly known in the medicinal chemistry literature, the term
'chemophore' refers to groups that are too reactive or inert. A
judicious selection of these features in a molecule will impact its
further development as a drug. The ChemScreener results for the
compounds 5b, 5c and 5d yielded a PTC score of 56, 33 and 25
indicating that they possess an optimum combination of maximum
pharmacophoric features and low toxicophoric and chemophoric
features. Based on the comprehensive studies performed it is
reasonable to state that the active compounds can be further
developed as lead molecules against tuberculosis.
For a comprehensive list of all the descriptors and their computed
values, please refer to the Supplementary Table S1. In the 3D model
it was observed that the main properties correlating with the
biological activity were the molecular orbital energies such as
E(LUMO), E(HOMO). Frontier orbitals of valence electrons of
compounds are known to play a major role in governing affinity
towards biomolecules, specifically low values of LUMO indicate
good electron acceptor tendency of the molecule and facilitate
better interactions with the residue atoms in the active site of the
receptor which eventually determine the biological activity. The
molecules with lower ELUMO values were found to possess better
biological activities. (See entries 1, 9-11, Table S1). All these
molecules possess a halogen atom (X= F, Cl) on the benzene ring
moiety. It is to be noted here that the docking studies also revealed
the halogen substituted benzene ring to be involved in an arene
cation type of interaction with the receptor residue atoms. The
presence of a methoxy group in the benzene system on the other
hand increased the E(LUMO) energies without affecting the
E(HOMO) energies (entries 4,5,6) however when the piperidine
group was present on the spiro skeleton, the methoxy group effect
on the energies was not so prominent. It thus appears that the
concurrent presence of halogen on the benzene ring moiety and
piperidine on the spiro conjugated framework of the molecular
system is essential for eliciting the bioactivity.
A Genetic
Programming based Symbolic Regression (GP-SR)27,28 modelling was
performed to verify the correlation of electronic energy descriptors
with bioactivity. The GP model produced a Correlation Coefficient
(CC) value of 0.9757, 0.0684(RMSE) and 1.7353 (MAPE). The GP
expression obtained is shown in the equation (1), parity plot of
experimental versus GP predicted values is given in the
Supplementary Information (Figure S3).
ꢀ = ꢁꢂ.ꢁꢃꢄꢁ.ꢅꢆꢃ∗ꢇ – 473.985 – (11.894*E) – (44.639*C) –
ꢈ.ꢉꢆꢅꢄꢊ
(128.475*D) – (459.094*B) – (13.89*D*E) – (53.352*C*B)
QSAR Modeling
Equation 1 (Where A= PM3_HOMO, B= PM3_LUMO, C=AM1_HOMO, D=
AM1_LUMO,E=MNDO_HOMO and X= Bioactivity (MIC, µg/mL)
We applied the QSAR approach to model the bioactivity of the
chalcones in our quest to gather some clues regarding the
molecular characteristics essential for biological activity. Initially, a
simple 2D regression model was built using PLS approach for the
series of chalcones using physico-chemical 2D structural descriptors
implemented in MOE package26 which yielded a coefficient of
determination 0.9449 and a root mean square error (RMSE) value
of 1.70 (Figure S1). Next, we computed the 3D QM/QC based
Thus the electronic energy descriptors based QSAR model
developed can be employed in near future for virtual screening of
not yet synthesized chalcone skeleton based molecules. To provide
an in-depth analysis of molecular orbitals and derive more accurate
energies, ab initio method was employed using DFT level of theory,
8 | J. Name., 2012, 00, 1-3
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ARTICLE
O
O
O
F
HO
N
O
O
5c
LUMO
HOMO
-6.92448 eV
-2.724675877 eV
Figure 7: Iso density surface maps of frontier orbitals of molecule 5c with best inhibitory activity as determined in the in vitro Mtb assay.
B3LYP functional and 6-31G basis set. The HOMO LUMO energies in Chemoinformatics analysis using a host of known and internally
Gaussian 0929 were -6.92448 eV and -2.724675877 eV respectively. developed tools indicated that the compounds prioritized in this
The iso density surface plots of HOMO and LUMO for the study possess good ADME profile, low toxicity and high
compound 5c possessing the best inhibitory activity are depicted in pharmacophoric attributes essential for further development as
the Figure 7. The HOMOs lie over the tricyclic spiro-chromone ring anti-tubercular agents. Further, QSAR modeling established the
system while the LUMOs are located over the fluoro benzene ring correlation between frontier molecular orbital energies and
moiety in the molecule.
biological activities of the compounds. The GP based expression can
be used for screening new molecules for their therapeutic action.
Conclusion
In summary,
a series of spirochromone annulated chalcone
Experimental Section
conjugates were synthesized for the first time using an efficient
synthetic procedure beginning from resorcinol. All these new
compounds were characterized by 1H NMR, 13C NMR and mass
spectral analysis. The in vitro antimycobacterial evaluation of all the
synthesized compounds showed that five compounds possess
moderate to good antimycobacterial activity. Noticeably,
compound 5c is most potent compound in vitro with a MIC value of
3.13 µg/mL, against MTB. Molecular docking studies were utilized
to explore the putative targets among the known chalcone specific
protein receptors. In theory the compounds showed greater affinity
towards the mycobacterium phosphatase Ptp B enzyme, these
studies will provide guidelines for future experimental efforts.
General Methods
Solvents were purified and dried by standard procedures prior to
use. 1H NMR and 13C NMR spectra were recorded on a Bruker AC-
200 NMR spectrometer. Spectra were obtained in CDCl3.
Monitoring of reactions was carried out using TLC plates Merck
Silica gel 60 F254 and visualization with UV light (254 and 365 nm),
I2 and anisaldehyde in ethanol as development reagents. HRMS
(ESI) were recorded on
a ORBITRAP mass analyser (Thermo
Scientific, QExactive). Mass spectra were recorded at an ionization
energy 70 eV on API Q Star Pulsar spectrometer using electrospray
ionization. Ten fold serial dilutions of each test compound/drug
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were incorporated into Middlebrook 7H11 agar medium with OADC Synthesis of spirochromone annulated chalcone conjugates (4-5):
General procedure:
To a well-stirred mixture of (3a or 3b) (0.7 mmol) and substituted
Growth Supplement. Inoculum of M. tuberculosis H37Rv were
prepared from fresh Middlebrook 7H11 agar slants with OADC
Growth Supplement adjusted to 1mg/mL (wet weight) in Tween 80
aldehydes (0.77 mmol) in methanol (3 mL) at room temperature
was slowly added 50% KOH solution (5 mL) dropwise. The resulting
reaction mixture was stirred for 12 h at 60 oC. After completion of
the reaction (by TLC), solvent was removed under reduced pressure
and the residue was dissolved in ethyl acetate. The organic layer
was washed with 5% HCl followed by brine, dried and concentrated.
The residue was purified by column chromatography (silica gel, pet
ether/ ethyl acetate, 75:25) to yield pure product 4-5. Spectroscopic
data of compounds 4-5 are given below.
(0.05%) saline diluted to 100-2 to give
a concentration of
approximately 1007 cfu/mL. A 5 μL amount of bacterial suspension
was spotted into 7H11 agar tubes containing 10-fold serial dilutions
of drugs per mL. The tubes were incubated at 37 °C, and final
readings were recorded after 28 days. The minimum inhibitory
concentration (MIC) is defined as the minimum concentration of
compound required to give complete inhibition of bacterial growth.
1,1'-(4,6-dihydroxy-1,3-phenylene)bis(ethan-1-one) (2)
(E)-6-(3-(4-fluorophenyl)acryloyl)-7-hydroxyspiro[chromane-2,1'-
To a mixture of resorcinol (5 g, 45.5 mmol) and acetic anhydride
(9.3 g, 91 mmol) was added zinc chloride (10 g, 73.4 mmol) and the
resulting mixture was heated at 140 °C for 30 minutes. The hot
mixture was allowed to attain ambient temperature followed by
addition of 50% dilute HCl (140 mL) and the solution was allowed to
stand for 1 h. The orange crude product was obtained by filtration
with suction, washed with distilled water till the colour of the
filtrate is nearly colourless. The crude product was crystallized using
ethanol to yield 4,6-diacetylresorcinol 2 (6.6 g, 75%); m.p. 178-79
oC; 1H NMR (200 MHz, CDCl3): δH = 2.64 (s, 6H), 6.42 (s, 1H), 8.21 (s,
1H), 12.93 (s, 2H); 13C NMR (50 MHz, CDCl3): δ 202.4 (CO, 2
carbons), 168.8 (C, 2 carbons), 136.2 (CH, 2 carbons), 113.5 (C),
104.9 (CH), 26.0 (CH3, 2 carbons).
cyclohexan]-4-one (4a)
o
1
Yellow solid; m.p. 156-57 C, H NMR (200 MHz, CDCl3): δH = 1.46-
1.77 (m, 8H), 1.97-2.05 (m, 2H), 2.73 (s, 2H), 6.50 (s, 1H), 6.95-7.19
(m, 2H), 7.51 (dd, J = 15.4, 9.3 Hz, 1H), 7.65-7.74 (m, 2H), 7.86 (dd,
15.4, 3.1Hz, 1H), 13.5 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 192.3
(CO), 190.6 (CO), 169.9 (C), 165.2 (C), 161.8 (C), 144.4 (CH), 130.9
(CH, 2 carbons), 130.7 (CH), 130.6 (C), 119.2 (CH), 116.4 (CH, 2
carbons), 115.2 (C), 113.9 (C), 105.4 (CH), 81.4 (C), 47.8 (CH2), 35.0
(CH2, 2 carbons), 24.9 (CH2), 21.3 (CH2, 2 carbons); HRMS(ESI): m/z
Calcd for C23H21FO4 [M+H]+ 381.1497, found 381.1495.
(E)-6-(3-(2-fluorophenyl)acryloyl)-7-hydroxyspiro[chromane-2,1'-
cyclohexan]-4-one (4b)
o
1
6-acetyl-7-hydroxyspiro[chromane-2,1'-cyclohexan]-4-one (3a)
Yellow solid; m.p. 188-89 C; H NMR (200 MHz, CDCl3): δH = 1.46-
1.77 (m, 8H), 1.97-2.03 (m, 2H), 2.73 (s, 2H), 6.50 (s, 1H), 6.95-7.24
(m, 2H), 7.38-7.49 (m, 1H), 7.69-7.82 (m, 2H), 8.03 (d, J=15.4 Hz,
1H), 8.55 (s, 1H), 13.49 (s, 1H); 13C NMR (50 MHz, CDCl3): δC = 192.6
(CO), 190.6 (CO), 170.0 (C), 165.3 (C), 159.3 (C), 138.2 (CH), 132.4
(CH), 130.8 (CH), 129.5(CH), 124.6 (CH), 122.0 (CH), 121.9 (C), 116.1
(CH), 115.3 (C), 114.0 (C), 105.4 (CH), 81.5 (C), 47.8 (CH2), 35.1 (CH2,
2 carbons), 25.0 (CH2), 21.4 (CH2, 2 carbons); HRMS(ESI): m/z Calcd
for C23H21FO4 [M+H]+ 381.1497, found 381.1496.
To a solution of 2 (5 g, 24.7 mmol) in ethanol (20 mL) was added
cyclohexanone (6 mL, 49 mmol) followed by addition of pyrollidine
(5.7 mL, 49 mmol) and the resulting mixture was refluxed using
Dean-Stark apparatus for 12 h. After completion of the reaction,
solvent was removed under reduced pressure and the residue was
dissolved in ethyl acetate. The organic layer was washed with 1N
HCl followed by dilute NaHCO3, brine and concentrated. The crude
product was purified using column chromatography (silica gel, pet
ether/ ethyl acetate, 95:5) to yield 3a (4.2 g, 62%) as a colourless
solid. 1H NMR (200 MHz, CDCl3): δH = 1.44-1.75 (m, 8H), 1.94-2.03
(m, 2H), 2.63 (s, 3H), 2.70 (s, 2H), 6.48 (s, 1H), 8.36 (s, 1H), 12.82 (s,
1H); 13C NMR (50 MHz, CDCl3): δ 203.3 (CO), 190.5 (CO), 168.6 (C),
165.1 (C), 131.8 (CH), 115.0 (C), 113.9 (C), 105.1 (CH), 81.3 (C), 47.7
(CH2), 34.9 (CH2, 2 carbons), 26.4 (CH3), 24.9 (CH2), 21.3 (CH2, 2
carbons); MS: m/z 297 [M+Na]+.
(E)-6-(3-(2-chlorophenyl)acryloyl)-7-hydroxyspiro[chromane-2,1'-
cyclohexan]-4-one (4c)
o
1
Yellow solid; m.p. 162-63 C; H NMR (200 MHz, CDCl3): δH = 1.52-
1.70 (m, 8H), 1.97-2.03 (m, 2H), 2.73 (s, 2H), 6.52 (s, 1H), 7.34-7.39
(m, 2H), 7.43-7.48 (m, 1H), 7.62 (d, J = 15.5 Hz, 1H), 7.82-7.86 (m,
1H), 8.30 (d, J = 15.5 Hz, 1H), 8.55 (s, 1H), 13.45 (s, 1H); 13C NMR (50
MHz, CDCl3): δC 192.2 (CO), 190.6 (CO), 170.0 (C), 165.3 (C), 141.4
(CH), 135.8 (C), 132.7 (C), 131.6 (CH), 130.7 (CH), 130.3 (CH), 128.0
(CH), 127.1 (CH), 122.0 (CH), 115.2 (C), 113.9 (C), 105.4 (CH), 81.4
(C), 47.8 (CH2), 35.0 (CH2, 2 carbons), 24.9 (CH2), 21.3 (CH2, 2
carbons); HRMS(ESI): m/z Calcd for C23H21ClO4 [M+H]+ 397.1201,
found 397.1203.
tert-butyl 6-acetyl-7-hydroxy-4-oxospiro[chromane-2,4'-piperidin
e]-1'-carboxylate (3b)
The same procedure as in the preparation of 3a was used with the
N-Boc piperidone (5.9 g, 21.5 mmol) affording spirocompound 3b as
a colorless solid (5.2 g, 65 %); m.p. 181-82 oC. 1H NMR (200 MHz,
CDCl3): δH = 1.46 (s, 9H), 1.56-1.71 (m, 2H), 1.97-2.04 (m, 2H), 2.64
(s, 3H), 2.72 (s, 2H), 3.16-3.27 (m, 2H), 3.88-3.92 (m, 2H), 6.48 (s,
1H), 8.38 (s, 1H), 12.85 (s, 1H); 13C NMR (50 MHz, CDCl3): δ 203.6
(CO), 189.4 (CO), 168.8 (C), 164.4 (C), 154.5 (C), 131.9 (CH), 115.3
(C), 113.7 (C), 105.2 (CH), 79.8 (C), 79.1 (C), 47.6 (CH2), 34.2 (CH2),
28.3 (CH3, 3 carbons), 26.4 (CH3).
(E)-7-hydroxy-6-(3-(4-methoxyphenyl)acryloyl)spiro[chromane-
2,1'-cyclohexan]-4-one (4d)
o
1
Yellow solid; m.p. 164-65 C; H NMR (200 MHz, CDCl3): δH = 1.47-
1.67 (m, 8H), 1.96-2.03 (m, 2H), 2.72 (s, 2H), 3.87 (s, 3H), 6.50 (s,
1H), 6.94 (dd, J = 8.8, 2.8 Hz, 2H), 7.50 (d, J = 15.4 Hz, 1H), 7.63 (d, J
= 7.8 Hz, 2H), 7.87 (dd, J = 15.2, 8.0 Hz, 1H), 8.55 (s, 1H), 13.73 (s,
10 | J. Name., 2012, 00, 1-3
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1H); 13C NMR (50 MHz, CDCl3): δC 192.5 (CO), 190.7 (CO), 170.0 (C), carbons), 128.9 (CH, 2 carbons), 119.3 (CH), 115.7 (C), 113.7 (C),
165.0 (C), 162.1 (C), 145.7 (CH), 130.8 (CH, 2 carbons), 130.7 (CH), 105.5 (CH), 79.9 (C), 79.2 (C), 47.7 (CH2, 2 carbons), 47.6 (CH2), 34.2
127.1 (C), 116.8 (CH), 115.3 (C), 114.5 (CH, 2 carbons), 113.8 (C), (CH2, 2 carbons), 28.3 (CH3, 3 carbons); HRMS(ESI): m/z Calcd for
105.3 (CH), 81.3 (C), 55.4 (CH3), 47.8 (CH2), 35.0 (CH2, 2 carbons), C27H29NO6 [M+Na]+ 486.1887, found 486.1897.
24.9 (CH2), 21.3 (CH2, 2 carbons); HRMS(ESI): m/z Calcd for C23H24O5
tert-butyl (E)-6-(3-(4-fluorophenyl)acryloyl)-7-hydroxy-4-oxospiro
[M+H]+ 393.1697, found 393.1697.
[chromane-2,4'-piperidine]-1'-carboxylate (5b)
(E)-6-(3-(2,4-dimethoxyphenyl)acryloyl)-7-hydroxyspiro[chroma
Yellow solid; m.p. 177-78 oC; 1H NMR (400 MHz, CDCl3): δH = 1.47 (s,
ne-2,1'-cyclohexan]-4-one (4e)
9H), 1.63-1.69 (m, 2H), 2.02 (bd, J = 13.7 Hz, 2H), 2.74 (s, 2H), 3.21-
1
Yellow solid; m.p. 215-16 oC; H NMR (200 MHz, CDCl3): δH = 1.51-
3.24 (m, 2H), 4.09-4.14 (m, 2H), 6.53 (s, 1H), 6.96 (d, J = 8.1 Hz, 2H),
1.77 (m, 8H), 1.97-2.03 (m, 2H), 2.71 (s, 2H), 3.88 (s, 3H), 3.93 (s,
7.54 (d, J = 15.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 15.1 Hz,
1H), 8.57 (s, 1H), 13.77 (s, 1H); 13C NMR (100 MHz, CDCl3): δC 192.6
3H), 6.48-6.49 (m, 2H), 6.54 (dd, J = 8.5, 2.4 Hz, 1H), 7.63 (d, J = 1.6
Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 8.17 (d, J = 15.6 Hz, 1H), 8.56 (s, 1H),
13.88 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 193.1 (CO), 190.7 (CO),
(CO), 189.7 (CO), 170.3 (C), 164.3 (C), 161.7 (C), 154.6 (CO), 146.2
(CH), 130.9 (CH, 2 carbons), 130.6 (C), 126.9 (CH), 116.6 (CH), 115.8
170.1 (C), 164.9 (C), 163.6 (C), 160.7 (C), 141.4 (CH), 131.3 (CH),
(C), 115.0 (C), 113.6 (CH, 2 carbons), 105.4 (CH), 79.9 (C), 79.1 (C),
130.5 (CH), 117.3 (CH), 116.7 (C), 115.6 (C), 113.7 (C), 105.6 (CH),
47.7 (CH2, 3 carbons), 34.3 (CH2, 2 carbons), 28.4 (CH3, 3 carbons);
HRMS(ESI): m/z Calcd for C27H28FNO6 [M+Na]+ 504.1793, found
105.2 (CH), 98.4 (CH), 81.2 (C), 55.6 (CH3), 55.5 (CH3), 47.9 (CH2),
35.1 (CH2, 2 carbons), 25.0 (CH2), 21.4 (CH2, 2 carbons); HRMS(ESI):
504.1798.
m/z Calcd for C25H26O6 [M+H]+ 423.1802, found 423.1801.
tert-butyl (E)-6-(3-(2-fluorophenyl)acryloyl)-7-hydroxy-4-oxospiro
(E)-7-hydroxy-6-(3-(3-methoxyphenyl)acryloyl)spiro[chromane-
[chromane-2,4'-piperidine]-1'-carboxylate (5c)
2,1'-cyclohexan]-4-one (4f)
1
Yellow solid; m.p. 174-75 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.63-1.69 (m, 2H), 2.01-2.05 (m, 2H), 2.75 (s, 2H), 3.21-3.24 (m,
2H), 3.89-3.90 (m, 2H), 6.54 (s, 1H), 7.14-7.25 (m, 2H), 7.42-7.47 (m,
1H), 7.71-7.78 (m, 2H), 8.06 (d, J = 15.4 Hz, 1H), 8.57 (s, 1H), 13.50
(s, 1H); 13C NMR (50 MHz, CDCl3): δC 192.6 (CO), 189.5 (CO), 170.1
(C), 164.5 (C), 160.5 (C), 154.6 (CO), 138.5 (CH), 130.9 (C), 129.6
(CH), 124.6 (CH), 122.4 (C), 121.8 (CH), 116.4 (CH), 116.2 (CH) 115.6
(C), 113.7 (C), 105.5 (CH), 79.9 (C), 79.2 (C), 47.7 (CH2, 3 carbons),
34.3 (CH2, 2 carbons), 28.3 (CH3, 3 carbons); HRMS(ESI): m/z Calcd
for C27H28FNO6 [M+Na]+ 504.1793, found 504.1792.
Yellow solid; m.p. 162-63 oC; H NMR (200 MHz, CDCl3): δH = 1.48-
1.70 (m, 8H), 1.97-2.04 (m, 2H), 2.73 (s, 2H), 3.90 (s, 3H), 6.52 (s,
1H), 6.98-7.04 (m, 1H), 7.19-7.20 (m, 1H), 7.28-7.42 (m, 2H), 7.61 (d,
J = 15.4 Hz, 1H), 7.87 (d, J = 15.4 Hz, 1H), 8.57 (s, 1H), 13.50 (s, 1H);
13C NMR (50 MHz, CDCl3): δC 192.6 (CO), 190.6 (CO), 170.0 (C), 165.2
(C), 160.0 (C), 145.8 (CH), 130.7 (CH), 130.0 (CH), 121.5 (CH), 121.4
(C), 119.8 (CH), 117.1 (C), 117.0 (CH), 113.9 (C), 113.6 (CH), 105.4
(CH), 81.4 (C), 55.4 (CH3), 47.8 (CH2), 35.0 (CH2, 2 carbons), 25.0
(CH2), 21.3 (CH2, 2 carbons); HRMS(ESI): m/z Calcd for C25H24O5
[M+H]+ 393.1697, found 393.1703.
tert-butyl (E)-6-(3-(2-chlorophenyl)acryloyl)-7-hydroxy-4-oxospiro
[chromane-2,4'-piperidine]-1'-carboxylate (5d)
(E)-6-(3-(benzo[d][1,3]dioxol-5-yl)acryloyl)-7-hydroxyspiro[chrom
ane-2,1'-cyclohexan]-4-one (4g)
1
Yellow solid; m.p. 183-84 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.58-1.73 (m, 2H), 2.00-2.05 (m, 2H), 2.75 (s, 2H), 3.17-3.29 (m,
2H), 3.88-3.93 (m, 2H), 6.55 (s, 1H), 7.36-7.50 (m, 3H), 7.61 (d, J =
15.5 Hz, 1H), 7.82-7.87 (m, 1H), 8.33 (d, J = 15.5 Hz, 1H), 8.57 (s,
1H), 13.49 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 192.3 (CO), 189.5
(CO), 170.1 (C), 164.5 (C), 159.3 (C), 154.6 (CO), 141.7 (CH), 135.8
(C), 132.6 (C), 131.8 (CH), 130.9 (CH), 130.4 (CH), 128.0 (CH), 127.2
(CH), 121.9 (CH), 113.7 (C), 105.5 (CH), 79.9 (C), 79.2 (C), 47.7 (CH2,
3 carbons), 34.3 (CH2, 2 carbons), 28.3 (CH3, 3 carbons); HRMS(ESI):
m/z Calcd for C27H28ClNO6 [M+Na]+ 520.1497, found 520.1500.
Yellow solid; m.p. 162-63 oC; H NMR (200 MHz, CDCl3): δH = 1.52-
1.77 (m, 8H), 1.97-2.03 (m, 2H), 2.73 (s, 2H), 6.06 (m, 2H), 6.51 (s,
1H), 6.85 (d, J = 7.9 Hz, 1H), 7.17 (dd, J = 8.2, 1.2 Hz, 1H), 7.23-7.24
(m, 1H), 7.46 (d, J = 15.5 Hz, 1H), 7.82 (d, J = 15.5 Hz, 1H), 8.54 (s,
1H), 13.66 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 192.4 (CO), 190.6
(CO), 170.0 (C), 165.1 (C), 150.4 (C), 148.5 (C), 145.6 (CH), 130.5
(CH), 128.8 (C), 125.8 (CH), 117.4 (CH), 115.3 (C), 113.8 (C), 108.7
(CH), 107.0 (CH), 105.3 (CH), 101.7 (CH2), 81.3 (C), 47.8 (CH2), 35.0
(CH2, 2 carbons), 24.9 (CH2), 21.3 (CH2, 2 carbons); HRMS(ESI): m/z
Calcd for C24H22O6 [M+H]+ 407.1489, found 407.1489.
tert-butyl (E)-7-hydroxy-6-(3-(3-methoxyphenyl)acryloyl)-4-oxo
spiro[chromane-2,4'-piperidine]-1'-carboxylate (5e)
tert-butyl 6-cinnamoyl-7-hydroxy-4-oxospiro[chromane-2,4'-pip
eridine]-1'-carboxylate (5a)
Yellow solid; m.p. 162-63 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.61-1.67 (m, 2H), 2.00-2.07 (m, 2H), 2.73 (s, 2H), 3.16-3.29 (m,
2H), 3.84-3.89 (m, 5H), 6.52 (s, 1H), 6.96-7.02 (m, 1H), 7.17-7.20 (m,
1H), 7.31-7.41 (m, 2H), 7.59 (d, J = 15.2 Hz, 1H), 7.87 (d, J = 15.2 Hz,
1H), 8.56 (s, 1H), 13.56 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 192.6
(CO), 189.5 (CO), 170.2 (C), 164.5 (C), 160.3 (C), 154.6 (CO), 146.2
(CH), 135.6 (CH), 130.8 (CH), 130.0 (CH), 121.5 (CH), 119.6 (C), 117.1
Yellow solid; m.p. 165-66 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.61-1.69 (m, 2H), 1.99-2.04 (m, 2H), 2.75 (s, 2H), 3.22-3.24 (m,
2H), 3.87-3.96 (m, 2H), 6.54 (s, 1H), 7.46-7.47 (m, 3H), 7.66-7.72 (m,
3H), 7.94 (d, J = 15.5 Hz, 1H), 8.59 (s, 1H), 13.58 (s, 1H); 13C NMR (50
MHz, CDCl3): δC 192.6 (CO), 189.6 (CO), 170.2 (C), 164.4 (C),154.6
(CO), 146.1 (CH), 134.3 (C), 131.2 (CH), 130.8 (CH), 129.0 (CH, 2
This journal is © The Royal Society of Chemistry 20xx
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(CH), 115.7 (C), 113.7 (CH), 113.7 (C), 105.5 (CH), 79.9 (C), 79.2 (C), DST, New Delhi for the award of a women scientist fellowship
55.4 (CH3), 47.7 (CH2, 3 carbons), 34.3 (CH2, 2 carbons), 28.3 (CH3, 3 (LS-201/2011). All the authors thank the anonymous referee
carbons); HRMS(ESI): m/z Calcd for C28H31NO7 [M+Na]+ 516.1993, for providing suggestions to improve the manuscript.
found 516.2006.
tert-butyl (E)-7-hydroxy-6-(3-(4-methoxyphenyl)acryloyl)-4-oxo
spiro[chromane-2,4'-piperidine]-1'-carboxylate (5f)
Notes and references
1
2
(a) Global Tuberculosis Report 2014, World Health
Organization, Geneva, 2014; (b) A. Koul, E. Arnoult, N.
Lounis, J. Guillemont and K. Andries Nature 2011, 469, 483.
(a) D. J. Newman and G. M. Cragg, in Natural Products in
Medicinal Chemistry, ed. S. Hanessian, Wiley-VCH Verlag
GmbH & Co. KgaA, 1st edn., 2014, part.1, pp 1-41; (b) S.
Basu, B. Ellinger, S. Rizzo, C. Deraeve, M. Schurmann, H.
Preut, H. D. Arndt and H. Waldmann, Proc. Nat. Acad. Sci.,
2011, 108, 6805; (c) S. Wetzel, R. S. Bon, K. Kumar and H.
Waldmann, Angew. Chem. Int. Ed., 2011, 50, 10800; (d) M.
Manger, M. Scheck, H. Prinz, J. P. V. Kries, T. Langer, K.
Saxena, H. Schwalbe, A. Furstner, J. Rademann and H.
Waldmann, Chem Bio Chem., 2005, 6, 1749.
J. K. Lin and M. S. Weng, in The Science of Flavonoids, ed. E.
Grotewold, Springer, New York, 2006, Ch. 8, pp 239-268.
(a) D. N. Dhar, The Chemistry of Chalcones and Related
Compounds, John Wiley & Sons, New York, 1981.
L. D. Chiaradia, A. Mascarello, M. Purificacao, J. Vernal, M. N.
S. Cordeiro, M. E. Zenteno, A. Villarino, R. J. Nunes, R. A.
Yunes and H. Terenzi, Bioorg. Med. Chem. Lett., 2008, 18,
6227.
(a) Y. M. Lin, PCT, WO 01/21164A2, 2001; (b) Y. M. Lin, Y.
Zhou, M. T. Flavin, L. M. Zhou, W. Nie and F. C. Chen, Bioorg.
Med. Chem., 2002, 10, 2795; (c) Z. Nowakowska, Eur. J. Med.
Chem., 2007, 42, 125; (d) R. H. Hans, E. M. Guantai, C.
Lategan, P. J. Smith, B. Wan, S. G. Franzblau, J. Gut, R. J.
Rosenthal and K. Chibale, Bioorg. Med. Chem. Lett., 2010, 20,
942.
(a) L. D. Chiaradia, P. G. A. Martins, M. N. S. Cordeiro, R. V. C.
Guido, G. Ecco, A. D. Andricopulo, R. A. Yunes, J. Vernal, R. J.
Nunes and H. Terenzi, J. Med. Chem., 2012, 55, 390; (b) A.
Mascarello, M. Mori, L. D. Chiaradia-Delatorre, A. C. O.
Menegatti, M. F. Delle, F. Ferrari, R. A. Yunes, R. J. Nunes, H.
Terenzi, B. Botta and M. Botta, PLoS One, 2013, 8, e77081;
(c) A. Mascarello, L. D. Chiaradia, J. Vernal, A. Villarino, R. V.
C. Guido, P. Perizzolo, V. Poirier, D. Wong, P. G. A. Martins, R.
J. Nunes, R. A. Yunes, A. D. Andricopulo, Y. Av-Gay and H.
Terenzi, Bioorg. Med. Chem., 2010, 18, 3783.
B. W. Vanhoecke, F. Delporte, E. Van Braeckel, A. Heyerick,
H. T. Depypere, M. Nuytinck, D. De Keukeleire, and M. E.
Bracke, In Vivo. 2005, 19, 103.
(a) M. Muthukrishnan, U. M. V. Basavanag and V. G. Puranik,
Tetrahedron Lett. 2009, 50, 2643; (b) M. Muthukrishnan and
O. V. Singh, Synth. Commun. 2008, 38, 3875; (c) M.
Muthukrishnan, P. S. Patil, S. V. More and R. A. Joshi,
Yellow solid; m.p. 176-77 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.60-1.73 (m, 2H), 2.00-2.06 (m, 2H), 2.74 (s, 2H), 3.18-3.29 (m,
2H), 3.89-3.90 (m, 5H), 6.53 (s, 1H), 6.95 (d, J = 8.7 Hz, 2H), 7.50 (d, J
= 15.7 Hz, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 15.7 Hz, 1H), 8.58
(s, 1H), 13.76 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 192.5 (CO), 189.7
(CO), 170.2 (C), 164.2 (C), 162.2 (C), 154.6 (CO), 146.0 (CH), 130.8
(CH, 2 carbons), 130.6 (CH), 127.0 (C), 116.7 (CH), 115.7 (C), 114.4
(CH, 2 carbons), 113.5 (C), 105.3 (CH), 79.9 (C), 79.0 (C), 55.4 (CH3),
47.6 (CH2, 3 carbons), 34.3 (CH2, 2 carbons), 28.4 (CH3, 3 carbons);
HRMS(ESI): m/z Calcd for C28H31NO7 [M+H]+ 494.2173, found
494.2175.
3
4
5
tert-butyl (E)-6-(3-(2,4-dimethoxyphenyl)acryloyl)-7-hydroxy-4-
oxospiro[chromane-2,4'-piperidine]-1'-carboxylate (5g)
Yellow solid; m.p. 160-61 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.66-1.72 (m, 2H), 1.99-2.05 (m, 2H), 2.73 (s, 2H), 3.16-3.31 (m,
2H), 3.88-3.89 (m, 5H), 3.93 (s, 3H), 6.48-6.49 (m, 1H), 6.51 (s, 1H),
6.54-6.60 (m, 1H), 7.62-7.63 (m, 1H), 7.67 (d, J = 8.1 Hz, 1H), 8.18 (d,
J = 15.4 Hz, 1H), 8.57 (s, 1H), 13.93 (s, 1H); 13C NMR (50 MHz,
CDCl3): δC 193.0 (CO), 189.7 (CO), 170.3 (C), 164.1 (C), 163.7 (C),
160.7 (C), 154.6 (CO), 141.7 (CH), 131.3 (CH), 130.6 (CH), 116.9 (CH),
116.5 (C), 115.9 (C), 113.5 (C), 105.5 (CH), 105.3 (CH), 98.3 (CH),
79.8 (C), 78.9 (C), 55.6 (CH3), 55.5 (CH3), 47.7 (CH2, 3 carbons), 34.2
(CH2, 2 carbons), 28.3 (CH3, 3 carbons); HRMS(ESI): m/z Calcd for
C29H33NO8 [M+Na]+ 546.2098, found 546.2126.
6
7
tert-butyl (E)-6-(3-(2,5-dimethoxyphenyl)acryloyl)-7-hydroxy-4-
oxospiro[chromane-2,4'-piperidine]-1'-carboxylate (5h)
Yellow solid; m.p. 150-51 oC; 1H NMR (200 MHz, CDCl3): δH = 1.47 (s,
9H), 1.70-1.72 (m, 2H), 2.00-2.07 (m, 2H), 2.74 (s, 2H), 3.16-3.29 (m,
2H), 3.84-3.86 (m, 5H), 3.91 (s, 3H), 6.53 (s, 1H), 6.88-7.03 (m, 2H),
7.19 (d, J = 3.0 Hz, 1H), 7.70 (d, J = 15.4 Hz, 1H), 8.21 (d, J = 15.4 Hz,
1H), 8.59 (s, 1H), 13.72 (s, 1H); 13C NMR (50 MHz, CDCl3): δC 193.0
(CO), 189.5 (CO), 170.1 (C), 164.3 (C), 154.5 (CO), 153.6 (C), 153.4
(C), 141.4 (CH), 130.8 (CH), 123.7 (C), 120.0 (CH), 118.2 (CH), 115.8
(C), 113.7 (CH), 113.6 (C), 112.4 (CH), 105.3 (CH), 79.8 (C), 79.0 (C),
56.0 (CH3), 55.9 (CH3), 47.7 (CH2), 47.6 (CH2, 2 carbons), 34.2 (CH2, 2
carbons), 28.3 (CH3, 3 carbons); HRMS(ESI): m/z Calcd for C29H33NO8
[M+Na]+ 546.2098, found 546.2099.
8
9
Mendeleev Commun. 2005
, 100; (d) O. V. Singh, M.
Muthukrishnan and R. Gopan, Synth. Commun. 2005, 35,
2723; (e) O. V. Singh, M. Muthukrishnan and M.
Sundaravadivelu, Indian J. Chem. 2005, 44B, 2575; (f) R.
Vyas, M. Karthikeyan, G. Nainaru and M. Muthukrishnan J
Comb. H. Tr. Scrn., 2015, 18, 624.
Acknowledgments
10 (a) M. Muthukrishnan, M. Mujahid, P. Yogeeswari and D.
Sriram, Tetrahedron Lett. 2011, 52, 2387; (b) M. Mujahid, R.
G. Gonnade, P. Yogeeswari and D. Sriram, M.
Muthukrishnan, Bioorg. Med. Chem. Lett., 2013, 23, 1416.
11 A. A. A. Emara and A. A. A. Abou-Hussen, Spectrochim Acta
A. 2006, 64, 1010.
12 ChemAxon Marvin Suite 15 (Accessed on: 17th June
2015:https://www.chemaxon.com/download/marvin-suite/)
13 O. Trott, A. J. Olson, Journal of Comput. Chem. 2010, 31, 455.
Financial support from the CSIR Network projects (CSC0130,
BSC0121 and CSC0108) is gratefully acknowledged. J. Robles
acknowledges financial support from CONACYT (Research
grant 168474). U.M.V.-B. (378051/252139). E. D.-C.
acknowledges support from CONACYT graduate scholarship
(368973/250842). L. C.-B. acknowledges support from
CONACYT graduate scholarship (327057/265238). RV thanks
12 | J. Name., 2012, 00, 1-3
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14 Chemical Computing Group Inc., Molecular Operating
Environment Software, Montreal, 2010.
15 F. Macaev, V. Boldescu, S. Pogrebnoi and G. Duca, Medicinal
Chemistry, 2014, 4, 487.
16 C. Grundner, H. L. Ng and T. Alber, Structure, 2005, 13, 1625.
17 C. Grundner, D. Perrin, R. H. Van Huijsduijnen, D. Swinnen, J.
Gonzalez, C. L. Gee and T. Alber, Structure, 2007, 15, 499.
18 K. A. Rawls, P. T. Lang, J. Takeuchi, S. Imamura, T. D.
Baguley, C. Grundner, T. Alber and J. A. Ellman. Bioorganic &
Medicinal Chemistry Letters 2009, 19, 6851.
19 V. Le Guilloux, A. Arrault, L. Colliandre, S. Bourg, P. Vayer and
L. Morin-Allory, J. cheminformatics, 2012, 4, 20.
20 C. A. Lipinski, Drug Discovery Today: Technologies, 2004, 1,
337.
21 S.K. Lee, S. H. Park, I. H. Lee and No, K. T. PreAD-MET Ver. v2.
0, BMDRC: 2007, Seoul. Korea.
22 M. Tripathi, S. I. Khan, A. Thakur, P. Ponnan and D. S. Rawat,
New J. Chem., 2015, 39, 3474.
23 S. Ren and E. J. Lien, Caco-2 cell permeability vs human
gastro-intestinal absorption: QSPR analysis. In Progress in
Drug Research (pp. 1-23), 2000, Birkhäuser Basel.
24 A. Maunz, M. Gutlein, M. Rautenberg, D. Vorgrimmler, D.
Gebele and C. Helma Fron. Pharmacol. 2013, 4, 38.
25 M. Karthikeyan, D. Pandit and R. Vyas, J Comb. H. Tr. Scrn.,
2015, 18, 544.
26 Molecular Operating Environment (MOE), 2013.08; Chemical
Computing Group Inc., 1010 Sherbooke St. West, Suite #910,
Montreal, QC, Canada, H3A 2R7, 2015
.
27 R. Vyas, P. Goel, M Karthikeyan, S.S. Tambe and B.D.
Kulkarni, Lett. Drug Des. Discov. 2014, 11, 1112.
28 R. Vyas, P. Goel and S. Tambe. Applications of genetic
programming in chemistry and chemical engineering, in: A.H.
Gandomi, A.H. Alavi, C. Ruan (Eds). Handbook of Genetic
Programming
applications,
Springer,
2015,
http://dx.doi.org/10.1007/978-3-319-20883-1-5, Chapter
(in press).
29 M. J. Frisch et al., Gaussian 09, Revision A.01, Gaussian Inc,
5
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Graphical Abstract
Spirochromone-chalcone conjugates as antitubercular agents: synthesis, bio evaluation and
molecular modeling studies
M. Mujahid, P. Yogeeswari, D. Sriram, U. M. V. Basavanag, Erik Díaz-Cervantes, Luis
Córdoba-Bahena, Juvencio Robles, R. G. Gonnade, M. Karthikeyan, Renu Vyas and M.
Muthukrishnan
We report new spiro chromone scaffold derived molecules possessing invitro anti-tubercular
activities. QSAR based molecular modeling studies correlated the bioactivities with the
frontier molecular orbital energies.