The first synthesis of podocarflavone A and its analogs and evaluation of their antimycobacterial potential against Mycobacterium tuberculosis with the support of virtual screening

Article abstract of DOI:10.1080/14786419.2021.1893317

The first synthetic route developed for Podocarflavone A reported from Podocarpus macrophyllus and its analogs in 7 steps. Computational analysis for binding with the pantothenate kinase (3AVO) of Mycobacterium tuberculosis showed their docking score (ds) in the range of ?8.9 to ?9.3 Kcal/mol. MD simulations delineated the stability of the protein-ligand complexes in the TIP3P model. MMGBSA and MMPBSA values of 8d were ?42.46 Kcal/mol and ?14.58 Kcal/mol, respectively. Further in-vitro antitubercular screening of compounds 8a, 8d, and 8e against M. tuberculosis H37Ra using XRMA protocol exhibited promising antimycobacterial activity with IC₅₀ values 21.82μg/mL, 15.55 μg/mL, and 16.56 μg/mL, respectively. Compounds 8a, 8d, and 8e showed antibacterial activity with IC₅₀ values 41.56 μg/mL, 24.72 μg/mL, and 72.45 μg/mL respectively against the Staphylococcus aureus. 8a and 8d showed inhibition with IC₅₀ values 39.6 μg/mL and 27.64 μg/mL, respectively, against Bacillus subtilis. The present study could help in the further development of lead molecules against tuberculosis.

Full text of DOI:10.1080/14786419.2021.1893317

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
The first synthesis of podocarflavone A and its
analogs and evaluation of their antimycobacterial
potential against Mycobacterium tuberculosis with
the support of virtual screening
Ninad V. Puranik, Sagar Swami, Ashwini V. Misar, Ritu Mamgain, Swapnaja S.
Gulawani, Dhiman, Sarkar & Pratibha Srivastava
To cite this article: Ninad V. Puranik, Sagar Swami, Ashwini V. Misar, Ritu Mamgain,
Swapnaja S. Gulawani, Dhiman, Sarkar & Pratibha Srivastava (2021): The first synthesis of
podocarflavone A and its analogs and evaluation of their antimycobacterial potential against
Mycobacteriumꢀtuberculosis with the support of virtual screening, Natural Product Research, DOI:
10.1080/14786419.2021.1893317
To link to this article: https://doi.org/10.1080/14786419.2021.1893317
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1893317
The first synthesis of podocarflavone A and its analogs
and evaluation of their antimycobacterial potential
against Mycobacterium tuberculosis with the support of
virtual screening
b,c
a
a
Ninad V. Puranika,d , Sagar Swami , Ashwini V. Misar , Ritu Mamgain ,
ꢀ
ꢀ
Swapnaja S. Gulawania, Dhiman, Sarkarb,c and Pratibha Srivastavaa,d
b
aBioprospecting Group, Agharkar Research Institute, Pune, Maharashtra, India; Academy of Scientific
c
and Innovative Research (AcSIR), Ghaziabad, India; Combi-Chem Bio-Resource Center, Organic
d
Chemistry Division, CSIR-National Chemical Laboratory, Pune, Maharashtra, India; Savitribai Phule
Pune University, Pune, Maharashtra, India
ABSTRACT
ARTICLE HISTORY
Received 30 December 2020
Accepted 17 February 2021
The first synthetic route developed for Podocarflavone A reported
from Podocarpus macrophyllus and its analogs in
7 steps.
Computational analysis for binding with the pantothenate kinase
(3AVO) of Mycobacterium tuberculosis showed their docking score
(ds) in the range of ꢁ8.9 to ꢁ9.3 Kcal/mol. MD simulations
delineated the stability of the protein-ligand complexes in the
TIP3P model. MMGBSA and MMPBSA values of 8d were
ꢁ42.46 Kcal/mol and ꢁ14.58 Kcal/mol, respectively. Further in-vitro
antitubercular screening of compounds 8a, 8d, and 8e against M.
tuberculosis H37Ra using XRMA protocol exhibited promising anti-
mycobacterial activity with IC50 values 21.82 mg/mL, 15.55 mg/mL,
and 16.56 mg/mL, respectively. Compounds 8a, 8d, and 8e
showed antibacterial activity with IC50 values 41.56 mg/mL,
24.72 mg/mL, and 72.45 mg/mL respectively against the
Staphylococcus aureus. 8a and 8d showed inhibition with IC50 val-
ues 39.6 mg/mL and 27.64 mg/mL, respectively, against Bacillus sub-
tilis. The present study could help in the further development of
lead molecules against tuberculosis.
KEYWORDS
Podocarflavone A synthesis;
antimycobacterial activity;
docking; MD simulations;
MMPBSA; and MMGBSA
CONTACT Pratibha Srivastava
Both Ninad V. Puranik and Sagar Swami have equal contribution in this piece of work.
psrivastava@aripune.org; Dhiman Sarkar
d.sarkar@ncl.res.in
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2021.1893317.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
N. V. PURANIK ET AL.
1. Introduction
Natural products are a dynamic source for innumerable novel pharmaceutical agents
(Ahmad et al. 2016, 2017; Newman and Cragg 2016). Their isolation from natural
resources is essential for their identification, characterization, and evaluation of bio-
logical potential. Every time, their isolation from the plants causes overexploitation of
medicinal plants. With natural product synthesis, potential natural products can be
available throughout the year (Maier 2015). The flavonoid class of compounds is
prevalent among natural products (Crozier et al. 2009).
Podocarflavone A is an 8-aryl flavone reported from the leaves and twigs of plant
Podocarpus macrophyllus maki and tested for different activities (Qiao et al. 2014). This
plant belongs to the Podocarpaceae family and is distributed over tropical and sub-
tropical regions of eastern Asia and Australia. The structures of some of the com-
pounds isolated from this plant are presented in the following figure (Figure 1). Thus,
considering the promising biological activities, we contemplated developing a syn-
thetic protocol of podocarflavone A.
Tuberculosis (TB) is an acute infectious disease worldwide, and the causative agent
is Mycobacterium tuberculosis (Mtb) (WHO 2020a). In 2019, 10 million people got
infected, and 1.4 million people died from TB, though it is curable and preventable.
An estimated 58 million people have been saved from TB. A complete cure of TB is
still beyond reach, and the emergence of new pandemics such as COVID-19 can create
hurdles to accomplish it. The DOTS (Directly Observed Treatment, Short Course) ther-
apy announced by the World Health Organization (WHO) has shown an immense
effect in achieving more than 90% treatment rates. However, the lengthy period
(6–9 months) of the DOTS therapy and reoccurring gene mutations in pathogenic
strains became the prime cause of drug resistance (WHO 2020b). Hence, the burst-out
cases of multi-drug resistance (MDR) and extensive drug resistance (XDR) have
Figure 1. Structures of compounds isolated from Podocarpus macrophyllus.

NATURAL PRODUCT RESEARCH
3
emerged in recent years. The continuous search for potential new molecules with
reduced toxicity can eradicate TB from the world.
In the present study, we have reported the total synthesis of natural product podo-
carflavone A and its analogs and evaluated them for in-vitro antitubercular screening
Singh et al. 2011. The essential targets of Mtb are enzymes of the cell wall. Therefore,
docking studies of these molecules’ with cell wall enzyme pantothenate kinase (PDB
3AVO) carried out to understand their interactions (Chetnani et al. 2011). MMGBSA,
MMPBSA approach determined the thermodynamic stability of the receptor-ligand com-
plex, and MD simulations studies of 50ns demonstrated the stability of the complex in
the TIP3P model.
2. Results and discussion
Retrosynthetic analysis of podocarflavone A was planned, which confirmed 8-bromo
flavone (6) is a critical scaffold to synthesize the target molecule (8a). It could be pos-
sible either by bromination of suitably substituted flavone (5) (Route 1) or by the cyc-
lization of suitably substituted bromo chalcone (5) as shown in Route 2.
2.1. Retrosynthetic analysis
Retrosynthesis Route 1 involves the demethylation (Figure 2) of its methoxy analog
(7a), which can be synthesized from the Suzuki cross-coupling reaction (Hooshmand
et al. 2019) of 4-hydroxy phenylboronic acid with 8-bromo-5, 7-dimethoxy-2-(4-
methoxyphenyl)-4H-1-benzopyran-4-one (6). The 8-bromo flavone (6) intermediate can
be prepared from bromination of 5,7-dimethoxy-2-(4-methoxyphenyl)-4H-1-benzo-
pyran-4-one (5), which further can be obtained from cyclization of substituted chal-
cone i.e., 1-(4,6-dimethoxy-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop2-en-1-one (4)
(Arai et al. 2017). The chalcone can be synthesized using the aldol condensation of 1-
(4,6-dimethoxy phenyl-2-hydroxy)- ethanone (3) with anisaldehyde using sodium
hydroxide as
a base (Gurung et al. 2009). Intermediate 4, 6-dimethoxy-2-
Figure 2. Retrosynthetic route (Route1 and Route 2) considered for the synthesis of natural prod-
uct Podocarflavone A.

4
N. V. PURANIK ET AL.
Scheme 1. Synthesis of podocarflavone A and its analogues; Reagents: (a) AlCl3, acetyl chloride,
nitrobenzene: DCM (1:1), 0oC-90oC, 2 h; (b) DMS, K2CO3, acetone, reflux, 8 h; (c) Br2, acetic acid, 0oC
to RT, 1 h; (d) NaOH, ethanol, RT, 72 h; (e) I2, DMSO,100oC, 1 h; (f) substituted phenylboronic acid,
Pd(PPh3)4, Cs2CO3, DMF, 110oC, 6-12 h; (g) BBr3, DCM, RT, 12 h.
hydroxyacetophenone (3) can be prepared (Sheng et al. 2017) from acylated phloro-
glucinol (2), which can be synthesized by acylation of phloroglucinol using the
reported method (Zhou et al. 2017).
Route 1 could not succeed due to the formation of 6, 8-dibromo flavone instead of
monosubstituted 8-bromo flavone even on applying various parameters. This result
incited us to consider retrosynthetic route 2 for the preparation of the targeted nat-
ural molecule.
Scheme 1 represents a detailed protocol for the synthesis of podocarflavone A and
its analogs via Route 2. In this route, bromination, chalcone preparation, cyclization,
and Suzuki coupling are the key steps. The acylation of phloroglucinol using AlCl3,
acetyl chloride in dichloromethane, and nitrobenzene delivered acetylated phlorogluci-
nol (2). The yield was 75% (Zhou et al. 2017). Selective O-methylation (Sheng et al.
2017) of 2 using dimethyl sulfate, K2CO3, in acetone produced 4,6-dimethoxy-2-
hydroxyacetophenone (3) with a 90% yield. Bromination of intermediate 3 in the
presence of Br2/acetic acid at 0 ꢂC elicited into 3-bromo-4,6-dimethoxy-2-hydroxyaceto-
phenone (4) with an 80% yield (Cechinel-Filho et al. 1996). This step provided 4 hav-
ing bromo substituent at the desired position. Synthesis of 1-(3-bromo-4, 6-dimethoxy-
2-hydroxyphenyl)-3-(4-methoxyphenyl)-2-propen-1-one (5) was done by base-catalyzed
aldol condensation (Bandgar et al. 2012) of 4 with anisaldehyde, and the chalcone (5)

NATURAL PRODUCT RESEARCH
5
obtained with 95% yield. The cyclization of chalcone (5) in the presence of iodine and
dimethylsulfoxide on heating resulted into 8-bromo-5,7-dimethoxy-2-(4-methoxy-
phenyl)-4H-benzopyran-4-one (6) (Gurung et al. 2009). The Suzuki cross-coupling reac-
tion (Pan et al. 2017; Hooshmand et al. 2019) was performed for the C-C bond
formation at the 8th position of the intermediate 6 with 4-hydroxyphenyl boronic
acid produced methylated natural product Podocarflavone A (7a). The desired natural
molecule Podocarflavone A obtained by deprotection of methyl group using BBr3/
DCM/12h (Ravishankar et al. 2016). Different 4-substituted phenylboronic acids were
used to prepare the analogs of Podocarflavone A in the Suzuki coupling step
(Scheme-1). Podocarflavone A and analogs were characterized by spectroscopic ana-
lysis. The spectral data were cross-checked with the natural product reported for con-
firmation, and it was found in agreement with the isolated natural product.
Podocarflavone A (8a) was obtained after synthesis as the white powdered solid. The
LC-MS of 8a displayed a single peak and confirmed the mass of m/z 363.0. The FTIR
spectra also showed two significant peaks at 3310 cmꢁ1 and 1651 cmꢁ1 for hydroxyl
and carbonyl groups respectively. The 1H NMR (400 MHz, DMSO-d6) spectrum dis-
played a singlet d 6.37 (s, 1H, 6-H), and another singlet for proton 3-H of ring A
merged with a doublet of 3’,5’ H of the 4-hydroxyphenyl ring (C) and came as multi-
plet 6.78-6.80 (m, 3H, 3-H, 3’,5’ H). The three dublets 6.84 (d, J ¼ 8.4 Hz, 3”, 5” H), 7.25
(d, J ¼ 8.8 Hz, 2”, 6” H), 7.62 (d, J ¼ 8.8 Hz, 2’, 6’ H), belong to aromatic protons of ring
C and ring D respectively. Elegantly, we also got separate peaks for four hydroxyl
groups, which are present at 7th position of B ring at 9.49 s, OH, 7-OH), 4th position
of ring D 10.33 (s, OH, 4”) , 4th position of ring C at 10.66 (s, OH, 4’), and 5th position
of ring B at 13.11 (s, OH, 5-OH) respectively. Table S1 showed the comparative ana-
1
lysis of H NMR, 13CNMR, and IR spectra. The 13C NMR displayed signals for 21 car-
bons, which contains the signals of a carbonyl carbon (d 182.7) and 20 sp2 carbons.
The spectral analysis confirmed the total synthesis of Podocarflavone A and was
found in agreement with the isolated Podocarflaone A (Qiao et al. 2014).
2.2. Antitubercular activity
Amongst the tested compounds, compounds 8a, 8 b, 8c, 8d, and 8e exhibited promis-
ing antimycobacterial activity with IC50 values 21.82 mg/mL, 4.84ug/ml with 24.84ug/ml
, 22.54ug/ml, 15.55 mg/mL, and 16.56 mg/mL, respectively (Table S2). The compounds
8d and 8e were found more potent than the natural Podocarflavone A to inhibit Mtb.
The results showed that the conversion of the hydroxyl group with the methoxy
group restrict their inhibitory property. Replacement of the hydroxyl group at 4th pos-
ition in ring D of 8a with chloro or bromo group enhances its inhibitory potential
against Mtb. We observed the same kind of results when we checked with dihydroru-
gosaflavonoids derivatives (Puranik et al. 2018).
2.3. Antibacterial activity
During the screening, three compounds 8a, 8d, and 8e, showed potential against
Gram þ ve bacteria while there was no inhibitory activity towards Gram-ve bacteria.

6
N. V. PURANIK ET AL.
Compound 8a showed IC50 at 41.56 mg/mL 39.6 mg/mL against S. aureus and B. subtillis,
respectively. 8d showed IC50 at 24.72 mg/mL and 27.64 mg/mL against S. aureus and B.
subtillis, respectively. 8e showed IC50 at 72.45 mg/mL against S. aureus. Data are pre-
sented in Table S3.
2.4. Molecular modelling
Pantothenate kinase (3AVO) is an essential enzyme for Mtb and catalyzes the ATP-
based phosphorylation of pantothenate. It is a crucial step in the biosynthetic path-
way of coenzyme A from pantothenic acid. All the compounds were docked at the
binding pocket to identify ligand-receptor interactions. Podocaflavone A and its ana-
logs displayed strong binding properties to PanK with docking scores ꢁ7.2 to
ꢁ9.3 Kcal/mole. The docking image of 8a, 8c, 8d, and 8e are displayed in Figure
S1. The interacting residues and docking scores are presented in Table S2. Mostly
all compounds showed hydrogen-bonded interactions with Arg 238. Essential resi-
dues are Asp 129, Lys147, Tyr 235, Arg 238, Asn 277 of the active binding pocket.
All the compounds are surrounded by these amino acid residues and displayed
interactions (Figure S1). Based on their binding profiles, these compounds are
screened for anti-TB activity. Only compounds 8a, 8c, 8d, and 8e showed inhibitory
activity. Therefore, the MD simulations of active compounds (8a, 8c, 8d, and 8e)
were performed.
2.5. Molecular dynamics simulations
The docked complexes of the compounds 8a, 8c, 8d, and 8e were subjected to ana-
lyze their stability in the TIP3P water model for 50 ns. The MD simulations on mini-
mized and equilibrated complexes of 8a, 8c, 8d, and 8e were subjected to
MD simulations for 50 ns Wang et al. 2006. RMSD versus time graph of the trajectories
displayed in Figure S2. The complexes’ trajectories showed stability in the MD simula-
tions, and RMSD values are determined (Table S4).
2.6. MMGBSA and MMPBSA analysis
The thermodynamic stability of proteins was determined by MMGBSA and MMPBSA
analysis using Amber 20 Software Miller et al. 2012. The free energy values were
calculated with the help of the mmpbsa.py script of Amber 20 by using a number
of frames generated during MD simulations. It gave information about the thermo-
dynamic stability of the complex. The MMPBSA and MMGBSA values of complexes
are presented in Table S4.
3. Experimental section
All the procedures for the intermediates from 2-8 are prepared and characterized by
NMR. Their methodology is given in supplementary file 1 (Supplementary file 1). It

NATURAL PRODUCT RESEARCH
1
7
also contains Figure S1 and S2, Tables S1–S4, the Mass (LC-MS), H NMR, and 13C NMR
spectra of 7a-7e and 8a-8e.
4. Conclusion
The flavonoids are widely distributed in fruits and vegetables and are essential for
human health. Therefore, we have reported the first synthetic route involving bromi-
nation followed by chalcone preparation, cyclization, and Suzuki coupling as the key
steps to synthesize natural product Podocarflavone A, which is reported from
Podocarpus macrophyllus. The natural product 8a was successfully prepared in 7 steps.
During the synthesis, their analogs are prepared following the same route. The halo-
genated Podocarflavone A showed better results in comparison to the natural product
8a. The computational studies and in-vitro antimycobacterial screening results confirm
inhibitory activity against Mtb. These molecules can serve to discover the lead mole-
cules as antitubercular agents.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors are thankful to the Director, ARI, and the Director, NCL, Pune, India, for providing
infrastructure and financial support. S. Swami (File no: 31/011(0983)2017/EMR-01) is grateful to
the CSIR, New Delhi, India, for Senior Research Fellowship support. Dr. R. Mamgain is thankful to
the Department of Science and Technology, New Delhi, India, for WOS-A project SR/WOS-A/CS-
107/2018 and Ms. S.S. Gulawani for the support of UGC fellowship.
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