Design, synthesis, in vitro and in silico studies of 2, 3-diaryl benzofuran derivatives as antitubercular agents

Article abstract of DOI:10.1016/j.bioorg.2020.103784

As a part of our drug discovery program for anti-tubercular agents, a total of seventeen 2, 3-diaryl benzofuran hybrids were designed, synthesized and screened for their anti-tubercular potential against Mycobacterium tuberculosis H37Ra avirulent strain. Out of seventeen, four derivatives showed significant activity against M. tuberculosis H37Ra avirulent strain (ATCC 25177) with MIC value ranging from 12.5 to 50 μg/mL but out of four, one derivative (9E) was significantly active (MIC 12.5 μg/mL), which was further supported by the molecular docking energy (?8.4 kcal/mol) with respect to the first line anti-tubercular drug, isoniazid (?6.2 kcal/mol) on the target Polyketide Synthase-13. All the derivatives were also evaluated for their cytotoxicity against the normal lung cell line L-132 by the MTT assay and no toxicity was observed up to 27.4 μg/mL concentration. This report on the antitubercular potential of benzofuran derivatives may be of great help in anti-tubercular drug development.

Full text of DOI:10.1016/j.bioorg.2020.103784

Journal Pre-proofs
Design, Synthesis, In vitro and In silico Studies of 2, 3-diaryl Benzofuran De-
rivatives as Antitubercular agents
Balakishan Bhukya, Aparna Shukla, Vinita Chaturvedi, Priyanka Trivedi,
Shailesh Kumar, Feroz Khan, Arvind S. Negi, Santosh Kumar Srivastava
PII:
S0045-2068(20)30076-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.103784
YBIOO 103784
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
10 January 2020
12 March 2020
20 March 2020
Please cite this article as: B. Bhukya, A. Shukla, V. Chaturvedi, P. Trivedi, S. Kumar, F. Khan, A.S. Negi, S.
Kumar Srivastava, Design, Synthesis, In vitro and In silico Studies of 2, 3-diaryl Benzofuran Derivatives as
Antitubercular agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103784
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Design, Synthesis, In vitro and In silico Studies of 2, 3-diaryl Benzofuran Derivatives
as Antitubercular agents
Balakishan Bhukyaa, Aparna Shuklab, Vinita Chaturvedic, Priyanka Trivedic, Shailesh
Kumard, Feroz Khan, Arvind S. Negia* and Santosh Kumar Srivastavaa*
aMedicinal Chemistry Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow,
India-226015, bMetabolic
&
Structural Biology Department, CSIR-Central Institute of Medicinal and
c
Aromatic Plants, Lucknow-226015, Biochemistry Division, Central Drug Research Institute, Lucknow, India-
226001, dDepartment of Applied Chemistry, Babasaheb Bhimrao Ambedkar University, Lucknow-India 2260
*Address correspondence Medicinal Chemistry Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow,
India-226015; Tel: +91-522-2718581; Fax: +91-522-2342666; E-mail: skscimap@gmail.com
CIMAP/PUB/2020/MAR/14
CIMAP publication No.:
Abstract: As a part of our drug discovery program for anti-tubercular agents, a total of
seventeen 2, 3-diaryl benzofuran hybrids were designed, synthesized and screened for their
anti-tubercular potential against Mycobacterium tuberculosis H37Ra avirulent strain. Out of
seventeen, four derivatives showed significant activity against M.tuberculosis H37Ra Virulent
strain (ATCC 25177) with MIC value ranging from 12.5-50 µg/mL But out of four, one
derivative (9E) was significantly active (MIC 12.5ug/mL), which was further supported by the
molecular docking score (-8.4) with respect to the first line anti-tubercular drug, isoniazid (-
6.2) on the target Polyketide Synthase-13. All the derivatives were also evaluated for their
cytotoxicity against the normal lung cell line L-132 by the MTT assay and no toxicity was
observed up to 27.4 µg/mL concentration. This report on the antitubercular potential of
benzofuran derivatives may be of great help in anti-tubercular drug development.
Keywords: Benzofuran, semi-synthetic derivatives, anti-tubercular activity, in-silico studies.
INTRODUCTION
Tuberculosis (TB) is a contagious air-borne bacterial disease caused by the infection of
Mycobacterium tuberculosis (MTB), which mainly affects the lungs. According to global
tuberculosis, report-2018 Tuberculosis (TB) is one of the top 10 causes of death worldwide in
2017. It is also the leading killer of people with HIV and a major cause of death related to
antimicrobial resistance. TB is treated with drugs as given in Figure 1 with a complex
treatment regimen, consisting of multiple antibiotics that target diverse cellular processes. In
2017 ten million new TB cases were reported worldwide mainly affecting men 5.8 million,
women 3.2 million and children’s 1 million. In 2017 1.6 million people were died from
Tubercular disease, including 0.3 million among people with HIV. Over a period of time, the
pathogen has become resistant to the two most powerful drugs like rifampicin and
fluoroquinolones. Globally in 2017, 558000 people developed TB cases that were resistant to
rifampicin (RR-TB) and 160000 cases of Multidrug resistance as well as rifampicin resistance
(MDR-TB/ RR-TB). Although, there are several antitubercular drug molecules in pipeline as
shown in Figure 2, but to tackle this situation, still there is an urgent need to explore new
anti-TB molecules [1, 2]. Natural products play a major role in the discovery of new drug
leads as they are having novel structures, excellent biological activities and absolute
selectivity. Almost 50% of the FDA approved drugs are either the natural products or natural
product derivatives [3, 4, 5].

HO
O
OH
O
O
O
OH OH
F
O
S
F
F
NH
N
F
H
N
N
O
O
O
N
NH2
N
N
O
N
O
OH
O
N
O
No2
N
NH
N
TBI-223 phase-1
Macozinone phase-1
Rifampicin
O
Isoniazid
O
R
R
O
N
O
O
F
OH
HN
F
N
O
OH
H
N
NH
N
Cl
O
N
F
Cl
N
N
H
F
HO
N
HN
Ethambutol
OPC-167832 phase-2
Telacebec phase-2
Ciprofloxacin
O
F
O
F
F
F
F
F
O
OH
N
O
O
N
HN
N
O
NH2
NH2
N
O
N
O2N
N
Pretomanid phase-3
Bedaquiline phase-3
Pyrazinamide
Cycloserine
Figure-2: New anti-tubercular drugs in pipeline
Figure-1: Anti-tubercular drugs already in the market
It has been reported that naturally isolated flavonoids possess 37.78% more antitubercular
activity than any other class of natural compounds. In this regard benzofuran derivatives
have shown versatile biological activities (not less than 47 types of activities [6] and some of
the benzofuran derivatives are already in the market (Rifampicin) while few are in the
pipelines. Recently it has been reported that benzofuran [7] pharmacophore is of significant
importance in the development of antitubercular drugs [8]. Hence, as a part of our drug
discovery program for anti-tubercular agents [9-14], design and synthesis of three 2,3-di aryl
benzofuran prototypes (Figure 3) was carried out and all the derivatives were in-vitro
evaluated for their antitubercular potential against Mtb H37Ra strain. Further, in-silico and
cytotoxicity studies were also carried out for the most active derivatives.
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
F
O
HO
O
O
HO
10B
O
O
9
O
8
O
Prototype-2
Prototype-1
Prototype-3
Figure (3): Designed prototypes
GENERAL EXPERIMENT PROCEDURE

Melting points were determined on E-Z Melt automated melting point apparatus (Stanford
Research System, USA) in open glass capillaries and were uncorrected. Reagents and other
chemicals were procured from Merck India Limited, Avra Synthesis, India and used without
purification. Reactions were monitored on Merck pre-coated silica gel TLC-GF254 aluminum
sheets and detection was done under UV cabinet (254nm and 365nm) and Compounds
1
were purified over silica gel (60-120 and 100-200 mesh) and characterized by H and 13C
NMR, ESI-MS, and ESI-HRMS. The NMR spectra were obtained on Bruker Avance-500
MHz Spectrometers. Chemical shifts are given in δ ppm values with tetramethylsilane (TMS)
1
as reference. H–1H coupling constant (J) values are given in Hz. ESI mass spectra were
recorded on Shimadzu LC-MS and High-Resolution Mass (HRMS) on Agilent 6545 Q-TOF
after dissolving the compounds in methanol.
Synthesis of 2, 3-di aryl benzofuran prototypes:
The synthesis of 2, 3-di aryl benzofuran prototypes such as prototype-1, prototype-2 and
prototype-3 were synthesized as shown in scheme-1, scheme-2 and scheme-3.
Scheme-1
The scheme-1 was started with 3’, 5’- dihydroxybenzoic acid-1 as a starting material. Then
compound-1 methylated with concentrated sulphuric acid in methanol at 80C temperature to
get 2. Then 2 was iodinated with N-iodo succinimide performed in methanol to get 3.
I
HO
OH
i
ii
HO
OH
HO
OH
HO
O
O
O
O
O
1
3
2
iii
O
O
O
I
I
O
O
O
O
O
HO
OH
v
O
6
iv
O
O
4
vi
5
O
O
O
8A R= H
O
O
8B R=Mehtyl
8C R= Ethyl
8D R= Prophyl
8E R=Isopropyl
8F R= n-Butyl
8G R=Isopentyl
O
O
O
O
O
O
vii
O
RO
O
O
O
O
7
8
Scheme-1-Reagent and condition: (i) Conc-H2SO4, methanol, temp 80oC, 4h, 98%; (ii) NIS,
methanol, rt, 16h, 98%: (iii) K2CO3, methyl iodide, DMF, rt, 15h 98% : (iv) (Ph3P)2PdCl2, CuI,
aryl iodide, triethylamine, 60 ˚C, 5h, 98%: (v) iodine, CH2Cl2, rt, 23h, 99%: (vi) Trimethoxy
phenyl boronic acid, NaHCO3, (Ph3P)2PdCl2, DMF/H2O (4:1), 80oC, 7h, 96%: (vii) KOH,
MeoH/ H2O (9:1), 80oC, 6h, 98%.

Then methylation has carried out in the presence of potassium carbonate and methyl iodide
in DMF to get compound-4. By using compound-4 and 4-methoxy phenylacetylene exploiting
the reaction of Sonogashira cross-coupling reaction afforded compound -5. But Sonogashira
carbon-carbon bond formation reaction, aryl halides which contain electron-donating groups
have given higher yields and electron-withdrawing groups were provided lower yields. The
iodo cyclization of compound-5 was done at rt by using molecular iodine as a reagent to get
6. With the compound-6 and 3’, 4’, 5’- Trimethoxy phenylboronic acid in the presence of
Suzuki-cross coupling reaction to getting 7, which was demethylated in presence of 10%
KOH in methanol and water (9:1) to get final pharmacophore 8.
Synthesis of Methyl 3, 5-dihydroxybenzoate (2): - The reaction was started with 3, 5-
dihydroxybenzoic acid (1g, 0.00649 mol), in methanol (10 mL), and concentrated sulfuric
acid (96% pure in water, 0.5 ml, 0.0093 mmol) slowly added to the above solution. The
reaction mixture was heated to reflux for 3h. The reaction mixture was cooled to room
temperature and then solvent removed under reduced pressure. The residue was extracted
with ethyl acetate (50mL) and washed with water (10mL). The organic layer was dried over
anhydrous sodium sulfate, filtered and evaporated. The crude residue so obtained was
purified by column chromatography (hexane-ethyl acetate 14%), which afforded compound 2
as a white solid in 94% yield.
Synthesis of Methyl 3,5-dihydroxy-4-iodobenzoate (3):- To a stirred solution of methyl 3,
5-dihydroxybenzoate (250 mg, 1.488 mmol) in methanol (1 mL) N-iodosuccinamide (351.5
mg, 1.57 mmol) was added at 0 0C. After stirring at room temperature for 16 h, the reaction
mixture was poured into Ice-cold water (5mL) and then this solution was transferred into a
separatory funnel and slowly saturated aqueous solution of sodium thiosulfate (5 mL) was
added to it, which after 5 minutes resulted in the formation of a white solid compound. The
mixture was extracted thrice with EtOAc (5-7 mL). The combined organic layer was dried
over Na2SO4 and solvent removed on rotary evaporator, which afforded the crude product-3.
Synthesis of Methyl 4-iodo-3, 5-dimethoxybenzoate (4): - To a stirred solution of methyl
3, 5-dihydroxy-4-iodobenzoate (230 mg, 0.78 mmol) in DMF (1.5 mL) K2CO3 (386.5 mg, 3.9
mmol) was added at rt. After 20 min of stirring at room temperature, methyl iodide (0.15 mL,
2.35 mmol) was added and the resulting solution was further stirred for 15 h at RT. After
completion of the reaction, the mixture was extracted thrice with EtOAc. The combined
organic layer was dried with Na2SO4 and concentrated on rotary evaporator. The crude so
obtained was purified by column chromatography, which afforded a half white compound-4
(230mg, 81%).
General procedure for synthesis of Methyl 3, 5-dimethoxy-4-(2-(4-methoxy phenyl)
ethynyl) benzoate (5, 5A and 5B):- To a stirred solution of methyl 4-iodo-3, 5-
dimethoxybenzoate (150.0 mg, 0.510 mmol) in triethyl amine (6 mL), PdCl2 (PPh3)2 (45.0
mg, 0.064 mmol, 3 mol-%), CuI (12.0 mg, 0.063 mmol, 3 mol-%) and p-methoy-phenyl
acetylene (79.0 mg, 0.598 mmol) for compound 5; 1-Ethylnyl 4-Fluorobenzene (71.0 mg,
0.591 mmol) for compound 5A and methyl 4-ethynylbenzoate (95.0 mg, 0.593 mmol) for
compound 5B were added and after being heated at 600C for 5h, the reaction mixture was
cooled to rt and then extracted thrice with (50 mL) EtOAc. The combined EtOAc was dried
over Na2SO4 and concentrated on rotary evaporator. The crude residue so obtained was
purified by column chromatography (4% EtOAc/hexane), which afforded a solid compound-5

(140mg, 92%); a yellowish solid compound-5A (130 mg, 85%) and a solid compound-5B
(100 mg, 65%).
General procedure for synthesis of methyl 3-iodo-4-methoxy-2-(4-methoxyphenyl)
benzofuran-6-carboxylate (6, 6A and 6B):- To a solution of alkyne-5, 5A and 5B (725 mg)
in CH2Cl2 (60 mL) I2 (1140 mg) was gradually added in CH2Cl2 (15 mL) and the mixture was
stirred at rt for 22 h. The reaction mixture was then diluted with (30 mL) of DCM and stirring
was continued further for 10 min at rt. The excess amount of I2 was removed by washing
with a saturated aqueous solution of Na2S2O3. The organic layer was dried over Na2SO4 and
solvent removed under vacuum. The crude product so obtained was purified by column
chromatography, which afforded compound-6 (720mg, 96%), 6A (700mg, 93%) and 6B
(65%).
Methyl4-methoxy-3-(3,4,5-trimethoxy) 2(4methoxyphenyl) benzofuran-6-carboxylate
(8B) :- To a solution of iodobenzofuran-6 (500 mg, 1.14 mmol) in DMF/water (4:1, 20.5 mL),
3,4,5-trimethoxy phenyl boronic acid (350 mg, 1.3 mmol), sodium bicarbonate (133.4 mg,
1.6 mL) and PdCl2(PPh3)2 (40 mg, 0.057 mmol) were added. The solution was, then stirred
for 10 min at room temperature and then heated at 80 0C for 10h. After cooling the reaction
mixture, it was extracted thrice with EtOAc (50 mL). The organic layer was dried over
Na2SO4 and concentrated on rotary evaporator. The crude so obtained was purified by
column chromatography (4% EtOAc/hexane), which afforded solid compound-8B (630 mg,
89%).
4-methoxy-3-(3,4,5-trimethoxyphenyl)-(4methoxyphenyl) benzofuran-6-carboxylic acid
(8A):- To a solution of final compound-8A (300 mg, 0.63 mmol) in methanol/water (9:1, 27.3
mL) potassium hydroxide (100 mg, 1.785 mmol) was added and the solution was heated for
0
6h at 80 C. The methanol was removed on rotary evaporator and the crude mixture was
extracted thrice with EtOAc. The combined organic layer was dried over Na2SO4 and solvent
removed on rotary evaporator. The residue so obtained was purified by column
chromatography (4% EtOAc/hexane), which afforded a solid compound 8A (285 mg, 97%).
Common method for the preparation of the substituted esters (8C-8G): - To a solution
of acid compound 8A (20 mg) in methanol (7 mL) was added 3drops of concentrated
sulphuric acid and heated for 1h at 80 0C. The methanol was removed on rotary evaporator
and the crude residue was extracted thrice with EtOAc. The organic layer was dried over
Na2SO4 and solvent removed on rotary evaporator. The crude was purified by column
chromatography (4% EtOAc/hexanes), which afford the desired derivatives (8C-8G; 21- 23
mg, 90-98%).
Scheme-2
In scheme-2 compound 6A and 3’, 4’, 5’-trimethoxy phenylboronic acid were coupled by
Suzuki cross-coupling reaction to afford 7A. Further 7A was demethylated with 10% KOH in
methanol and water (9:1) to afford final pharmacophore 9A.
Common method for the preparation of the substituted esters (9B-9I): - To a solution of
acid compound-9A (20 mg) in methanol (7 mL) 3drops of concentrated sulphuric acid was
added. The solution was heated for 1h at 80 0C. The methanol was removed under vacuum.
The residue obtained was extracted thrice with (50 mL) EtOAc. The organic layer was dried
over Na2SO4 and the solvent reduced under vacuum. The crude so obtained was purified by

column chromatography (4% EtOAc/hexane), which afforded the desired products 9B-9I (21-
23mg; 90-98%).
O
O
F
O
O
O
I
(i)
F
F
O
HO
OH
O
O
O
B
O
O
7A
(ii)
O
O
6A
O
O
5A
O
O
O
O
O
9A R= H
9B R=Mehtyl
9C R= Ethyl
O
O
O
O
O
9D R= Prophyl
9E R=Isopropyl
9F R= n-Butyl
9G R=Isobutyl
9H R=Isopentyl
9I R=2-Butanol
O
F
HO
O
(iii)
F
O
RO
9A
O
O
9B-9I
Scheme-2-Reagent and condition: (i) Trimethoxy phenylboronic acid, NaHCO3, (Ph3P)2
˚
PdCl2, DMF/H2O (4:1), 80 C, 7h, 96%: (ii)KOH, methanol/ water (9:1), 80 ˚C, 6h, 98%. (iii)
Conc-H2So4, methanol, temp 80oC, 4h, 98%.
Scheme-3
In scheme-3 the 3’, 4’, 5’- trimethoxy phenylboronic acid was hooked up to compound-6B by
Suzuki cross-coupling reaction to afford the desired compound-10A.
HO
OH
B
O
O
O
O
O
O
O
O
O
I
O
O
O
O
O
O
O
O
O
i
O
O
O
10A
6B
O
5B
O
O
Scheme-3-Reagent and condition: (i) Trimethoxy phenylboronic acid, NaHCO3, (Ph3P)2
PdCl2, DMF/H2O (4:1), 80 ˚C, 7h, 40%.
In-vitro anti-tubercular assay using M. tuberculosis (avirulent strain):
Determination of MIC:
The Minimum Inhibitory Concentration (MICs) of 2,3-diaryl benzofuran derivatives and
antitubercular drugs INH were determined against Mtb H37Ra by Agar dilution assay [15].

Briefly, 2,3-diaryl benzofuran derivatives were dissolved in dimethyl sulphoxide (DMSO) to
make stock (5 mg/mL). Serial dilutions from stocks were also made in DMSO. To 1.9 mL MB
7H11 agar medium (in tubes, temp. 45-50ºC, with OADC supplement), 0.1 mL of a
compound or DMSO (negative control) was added. The contents were mixed and allowed to
solidify as slants. Three weeks old culture of Mtb H37Ra was harvested from L-J medium
and its suspension (1 mg/mL equivalent to 108 bacilli approximately) was made in normal
saline (containing 0.05% Tween-80). 10 µl of 1:10 dilution of this suspension (~105 bacilli)
was inoculated into each tube and incubated at 37 ºC for 4 weeks. The lowest concentration
of a compound up to which there was no visible growth of bacilli was its minimal inhibitory
concentration (MIC).
In silico studies
Identification of anti-Mtb targets, target preparation and molecular docking
The probable anti-Mtb targets were searched through STITCH database v5.0
(http://stitch.embl.de) and from reported literatures. Stitch database develop score-based
interaction network between standard known compound and protein targets. The 3D
crystallographic structures of identified targets viz., polyketide synthase 13 (PDB: 5V3X),
catalase peroxidase (PDB: 1SJ2), dihydrofolate reductase (PDB: 4M2X) and enoyl-ACP
reductase (PDB: 4TRO) were retrieved through protein databank (http://www.pdb.org). The
protein and ligand structures were prepared by using Autodock Vina (MGL, La Jolla, USA).
Initially Autodock Vina prepared extended PDBQT file for receptor and ligands. The method
included addition of polar hydrogens, atomic partial charges and AD4 atom type conversion,
providing information about torsional degree of freedom. Afterward the receptor was
implanted in 3D grid system with prob atoms at each grid point. The electrostatic and
desolvation potentials of each atom type (C, O, N & H) with every grid point was calculated.
Further, ligand conformation energy was calculated through grids. A docking protocol
validation was performed by redocking study of crystallised ligand TAM1. The RMSD value
for redocked and co-crystallised ligand was calculated by using Molsoft-chemist v3.8-6a
2018 software. A low RMSD value of 1.47 standardised the docking protocol. The receptor-
ligand interaction results were analyzed through Discovery Studio visualizer.
Computational assessment for oral bioavailability and potential toxicity
The two major reasons for the new drug candidate’s failure are the poor bioavailability and
high toxicity. Hence, the oral bioavailability of the studied compounds were assessed
through Lipinski’s rule of five (RO5). While the toxicity parameters of the studied derivatives
were assessed though the standard computational assessment tool TOPKAT (DS v3.5).
TOPKAT utilizes its patented optimal predictive space (OPS) to calculate different toxicity
parameters for query set compounds (Enslein, 1988). The oral bioavailability, blood-brain-
barrier (BBB) penetration capacity and gastrointestinal (GI) absorption were assessed
though DS ADME plot. This included the calculated parameters such as, plasma protein
binding, CYP2D6 metabolism, hepatotoxicity, mouse and rat male/female carcinogenicity,
developmental toxicity (DT), skin and ocular irritancy etc.
RESULTS & DISCUSSION
As part of our drug discovery program, the starting material 3’, 5’- dihydroxybenzoic acid was
converted to pharmacophore 8 as given in scheme 1. Later on, using this pharmacophore,

seven derivatives (8A to 8G) were prepared. Similarly, using 3’, 4’, 5’-Trimethoxy
phenylboronic acid (6A) as starting material pharmacophore 9 was prepared as given in
scheme 2 and using this Pharmacophore-9 nine derivatives (9A to 9I) were prepared.
Compound-10A was prepared by hooking up 3’, 4’, 5’-trimethoxy phenylboronic acid to
compound 6B by Suzuki cross-coupling reaction as given in scheme 3. It was also observed
that in Suzuki cross-coupling carbon-carbon bond formation reaction, the aryl groups
containing electron-donating groups resulted in higher yields while the electron-withdrawing
groups gave lower yields. All the compounds were characterized with the use of 1H, 13C
NMR and Mass spectroscopic data. Further, all the derivatives were evaluated for their
antitubercular activity against the H37Ra avirulent strain (ATCC 25177) using isoniazid as
the standard drug and the results are presented in (Table 1).
Table 1: MIC of 2, 3-diaryl benzofuran hybrids derivatives
MIC
S.No
1.
Code
8A
8B
8C
8D
8E
8F
(ug/mL)
Not Active
Not Active
50 ug/mL
Not Active
Not Active
Not Active
Not Active
50 ug/mL
Not Active
Not Active
Not Active
12.5 ug/mL
Not Active
50 ug/mL
Not Active
Not Active
Not Active
(0.025) ug/mL
++++++
2.
3.
4.
5.
6.
7.
8G
9A
9B
9C
9D
9E
9F
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
9G
9H
9I
10A
Std Drug INH
No drug control

From the Table-1, it is evident that several derivatives were active in the range of 12.5 to
50µg/mL. Among all, derivative 9E was the most active (MIC 12.5 µg/mL) followed by the
derivatives 8C, 9A and 9G (MIC 50 µg/mL) while the starting material 7 (8B) was inactive.
Further, Structure Activity Relationship (SAR) for these derivatives against the H37Ra
avirulent strain can be derived. From the Table-1, it is evident that with the free acid group
(8A) was inactive, but on increasing the ester chain length from methyl to ethyl, propyl, iso-
propyl, n-butyl and iso-pentyl (8B to 8G) the activity dramatically increased in ethyl ester
(8C) which may be due to increase in ester chain length, but after that increase in chain
length resulted in loss of activity. The reason may be that the electron-donating aliphatic
chains should be of medium size, which could fit well at the receptor site. Similarly, when we
compare between the structures of 8A and 9A, there is only difference of –OCH3 and –F
groups at the C-4 (p) position of phenyl ring, but there was significant difference in their
activity. The derivative 8A was inactive while 9A was active (MIC 50 µg/mL). Further, when
other methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, iso-pentyl and 2-butanyl (9B to 9I)
ester derivatives of 9A were evaluated for their antitubercular potential, it was found that
isopropyl ester derivative (9E) was most active (MIC 12.5 µg/mL) followed by iso-butyl ester
(MIC 50 µg/mL) derivative (9G), which is the same to that of starting material 9A (MIC 50
µg/mL). This significant increase in 9E activity may be further correlated to the increased
electron-donating power of branched aliphatic chains of medium size, which could fit well at
the receptor site. The last (4-methoxycarbonyl) phenyl benzofuran derivative (10A) was
found inactive. From the above it may be concluded that (4-fluoro) phenyl group with
medium size branched aliphatic chains played an important role in enhancing the
antitubercular activity.
O
O
O
O
F
O
O
O
MIC 12.5 (ug/mL)
9E
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
O
F
Prototype-1
O
HO
O
O
O
O
O
O
O
O
8C
O
MIC 50 (ug/mL)
9A
MIC 50 (ug/mL)
O
O
O
F
HO
O
O
O
O
Prototype-2
F
O
O
O
MIC 50 (ug/mL)
9G
Figure 4: Best molecules of this series

Molecular docking and mode of action study of derivatives on identified Mtb targets:
Mycolic acid is one of the most essential components for Mtb virulence and drug resistance.
Till now more than 20 enzymes have been discovered which are involved in Mtb mycolic
acid synthesis. These are long chained fatty acids found abundantly in Mtb cell walls which
provide high resistance to the bacteria against several antibiotics. Polyketide synthase 13
(PK13) is one of the essential enzymes involved in advance phase of mycolic acid synthesis
in Mtb. PK13 exhibit five catalytic domains viz., one acyl transferase domain, two acyl carrier
domains, one β-keto acyl-synthase domain and the C-terminal thioester (TE) domain that
collectively contribute to the mycolic acid synthesis process. TM16 is a recently discovered
benzofuran class based highly potent PK13 inhibitor. TM16 binds at TE domain of PK13 and
block the availability of its catalytic centre for substrate binding. Here the synthesised active
derivatives viz., 8C, 9A, 9E and 9G belong to the benzofuran class therefore we performed a
molecular docking study against PK13. Based on STITCH database three additional putative
targets namely catalase-peroxidase, dihydrofolate reductase and enoyl-ACP reductase were
prioritized for ligand-target binding studies (Figure 6).
The results of docking binding affinity of studied derivatives are compiled in (Table 2 and
Table 4).
Table 2: Molecular docking results of derivatives 8C, 9A, 9E, 9G against Mtb target PK13
Name
Docking
binding
energy
PK13 (PDB: 5V3X) binding pocket residues
within 4Å radius
Key amino
acid & H-
bond length
(Å)
(kcal/mol)
ISONIAZID
(positive
control)
SER-1533, LEU-1534, VAL-1562, ASN-1640, ILE-
1643, ASP-1644, TYR-1663, ALA-1667, TYR-1674, (2.90)
HIS-1699
TYR-1663
-6.2
TM16 (PK13
TYR-1582, GLN-1633, SER-1636, TYR-1637, ASN- ASP-1644
co-crystallized -9.6
ligand)
1640, ARG-1642, ILE-1643, ASP-1644, TYR-1663,
HIS-1664, ASP-1666, ALA-1667, PHE-1670, GLU-
1671, TYR-1674
(2.55)
ALA-1477, TRP-1532, SER-1533, TYR-1582, ASN-
1640, ILE-1643, ASP-1644, TYR-1663, HIS-1664,
ASP-1666, ALA-1667, PHE-1670, GLU-1671, HIS-
1699, ILE-1700
SER-1533
(3.07)
8C
9A
9E
9G
-8.3
-8.6
-8.7
-8.8
ALA-1477, TRP-1532, SER-1533, ASN-1640, ILE-
1643, ASP-1644, TYR-1663, HIS-1664, PHE-1670,
GLU-1671, HIS-1699, ILE-1700
SER-1533
(3.06)
ALA-1477, TRP-1579, TYR-1582, VAL-1611, GLN-
1633, TYR-1637, ASN-1640, HIS-1664, ASP-1666,
ALA-1667, PHE-1670, GLU-1671, TYR-1674, ILE-
1700
GLN-1633
(2.92)
ALA-1477, TRP-1579, TYR-1582, GLN-1633, TYR-
1637, ASN-1640, HIS-1664, ASP-1666, ALA-1667,
PHE-1670, GLU-1671, TYR-1674, ILE-1700
GLN-1633
(3.03)
Analysis of docking results against target PK13 indicate, the studied derivatives viz., 8C, 9A,
9E and 9G showed good binding affinity in comparison to control drug isoniazid. Derivatives

9E and 9G showed better binding affinity of -8.7 kcal/mol and -8.8 kcal/mol than derivative
8C and 9A with
(b)
(a)
(d)
(c)
(f)
(e)
Figure 5: A 2D representation of binding mode of positive control Mtb drug Isoniazid and
PK13 co-crystallized benzofuran based inhibitor named ‘TM16’ and synthesized novel

derivatives 8C, 9A, 9E and 9G. (a) propose binding pose of Isoniazid on PK13 binding site
with docking binding energy -6.2 kcal/mol, (b) re-docking study of co-crystallized ligand
TM16 on PK13 TE domain (benzofuran based inhibitor), (c) binding pose of derivative 8C on
PK13 binding site with docking binding energy -8.3 kcal/mol, (d) binding pose of derivative
9A on PK13 binding site with docking binding energy -8.6 kcal/mol, (e) binding pose of
derivative 9E on PK13 binding site with docking binding energy -8.7 kcal/mol and (f) binding
pose of derivative 9G on PK13 binding site with docking binding energy -8.8 kcal/mol.
Figure 6: Mtb target prediction for positive control isoniazid through web-based tool STITCH
v5.0 (http://stitch.embl.de). inhA, nat, folA and katG stands for enoyl-ACP reductase,
arylamine V-acetyltransferase, dihydrofolate reductase and catalase peroxidase
respectively.
Table 3: TOPKAT predicted toxicity and carcinogenic potency of positive control INZ
(ISONIAZID) and synthesized derivatives, 8C, 9A, 9E and 9G
Hep
atot
oxic
ity
Ames
mutagenicit
y
Nam
e
Mouse Female Mouse Male
Rat Female
Rat Male
DTP
Multi
Carcinogen
Non-
Carcinogen
Non-
Carcinogen
Non-
Carcinogen
Non-
Carcinogen
INH
8C
9A
9E
Multi-Carcinogen Multi-Carcinogen Non-Carcinogen
Mutagen
True Toxic
True Toxic
True Toxic
True Toxic
True Toxic
Single-
Carcinogen
Single-
Carcinogen
Single-
Carcinogen
Single-
Non-
Mutagen
Non-
Mutagen
Non-
Mutagen
Non-
Mutagen
Multi-Carcinogen
Non-Carcinogen
Non-Carcinogen
Non-Carcinogen
Non-Carcinogen
Single-
Carcinogen
Carcinogen
Single-
Carcinogen
9G
Non-Carcinogen
DTP; developmental toxicity

Table 4 : Compliance of docking binding energy results of derivatives 8C, 9A, 9E and 9G on
putative Mtb targets catalase peroxidase, dihydrofolate reductase and enoyl-ACP reductase.
Name
Catalase-
peroxidase reductase
Dihydrofolate Enoyl
ACP
(PDB:
1SJ2)
(PDB: 4M2X) reductase
(PDB:
4TRO)
ISONIAZID -6.0
(positive
-5.6
-5.9
control)
8C
9A
9E
9G
-7.8
-8.5
-9.7
-8.9
-8.5
-8.5
-8.5
-9.0
-8.0
-8.6
-8.8
-8.9
binding energy of -8.3 kcal/mol and -8.6 kcal/mol respectively. However, the redocking study
of TM16 the known PK13 inhibitor displayed highest binding affinity of -9.6 kcal/mol (Table
2). Further, the ligand-receptor interaction analysis revealed all derivatives bind well at
inhibitor binding site of PK13. The interaction results displayed in (Table 2) indicated that the
derivatives 8C and 9A form hydrogen bond with amino acid residue SER-1533 and
trimethoxy phenyl fragment of the derivatives.
However they also indicated an unfavourable acceptor-acceptor hindrance with PK13
binding site amino acid residue HIS-1699 and trimethoxy phenyl moiety of 8C and 9A which
is highlighted with red colour in Figure 5. On the other hand derivatives 9E and 9G are
making hydrogen bond with carbonyl oxygen present at benzofuran moiety and amino acid
residue GLN-1633. While, no unfavourable interaction was observed in the case of 9E and
9G derivatives. All derivatives also showed number of non-covalent interactions such as π-π
T-shaped, π-π stacked, π-σ, π-alkyl and halogen interaction with aromatic rings of
compounds (Figure 5). Here, figure 5 illustrate aromatic rings of benzofuran moiety of
derivatives 8C and 9A make π-π T-shaped interaction with aromatic ring of amino acid
residue PHE1670. Whereas derivatives 9E and 9G are making π-π stacked based
interaction with amino acid residue PHE-1670. PK13 amino acid residue PHE1670 is located
near to the acyl binding pocket and play critical role in TM16 binding. Derivatives 9E and 9G
make hydrogen bond (green dotted line) of length 2.9 Å and 3.0 Å with amino acid residue
GLN-1633. They also showed halogen-based interaction with fluoride group and binding site
amino acid residue ALA-1667. Also, the results of docking binding affinity of derivatives 8C,
9A, 9E and 9G against other predicted targets viz., KatG, FolA and inhA indicated that the
derivatives possess good binding affinity in comparison to FDA approved drug isoniazid
(Table-4). Analysis of ligand-target interactions showed the derivatives make number of
interactions with hydrophobic binding sites of targets KatG, FolA & inhA.
Pharmacokinetic and toxicity assessment results
The toxicity predictions results related to male/female mouse and rat, ames mutagenicity,
indicate active derivative 9E is non-carcinogenic and non-mutagenic in nature. However,
other derivatives viz., 8C, 9G and 9A may show hepatotoxicity and developmental toxicity
when prolong used. All studied derivatives showed less oral lethality in rat (LD50) compared
to drug isoniazid. A good plasma protein binding capacity of studied derivatives display high

biological half-life that influence the biological efficacy of derivatives. All studied derivatives
were predicted to be CYP2D6 non-inhibitor which indicate the compounds are safe for drug-
drug based interactions. Derivatives 9E and 9G were predicted to be safe for skin and ocular
irritancy and sensitization parameters. All derivatives are aerobically degradable means they
are non-persistent in nature (Table 3 and 4). Results of Lipinski’s rule of five showed all the
studied derivatives violate single rule of RO5 by crossing the cut-off limit for LogP value i.e.,
5. Compliance of predicted toxicity parameters and RO5 are given in table (5).
Table 5: Parameter calculation for Lipinki’s rule of five (RO5) violations
ALogP98
PSA_2D (Å2)
Molecular
weight
No. of H- No. of
Acceptor Donor
No. of
violation
Name
-0.423
5.584
5.231
6.183
6.388
67.912
83.435
86.39
74.505
74.505
137.139
492.517
452.428
494.508
508.535
2
7
6
6
6
2
0
1
0
0
0
1
1
1
2
INH
8C
9A
9E
9G
Conclusion:
In summary, 2, 3-diaryl benzofuran derivatives were precisely designed and synthesized by
the concise method. All the seventeen novel benzofuran synthesized derivatives were
screed for their antitubercular activity against the H37Ra avirulent strain (ATCC 25177)
using isoniazid as the standard drug. Among the seventeen derivatives, four showed
antitubercular activity of which one (9E) was significantly active (MIC 12.5 ug/mL). From this
study it may be concluded that (4-fluoro) phenyl group with medium size branched aliphatic
chains played an important role in enhancing the antitubercular activity in benzofuran.
Further, molecular docking results revealed that 9E binds well at inhibitor binding site of Mtb
drug target polyketide synthase 13 with binding energy of -8.7 kcal/mol. Amino acid residues
GLN-1633 and PHE-1670 playing major role in hydrogen bonding and non-covalent based
interactions with derivative 9E. The computational oral bioavailability and toxicity
assessment indicated that the derivative 9E may show good pharmacokinetic properties and
less toxicity. However, further studies are needed for QSAR guided lead optimization. These
results may be of great help in anti-tubercular drug development from a very common,
inexpensive synthon.
CONFLICT OF INTEREST
The authors confirm that they have no conflict of interest to declare.
ACKNOWLEDGEMENTS
The authors are thankful to the Director CIMAP and Director CDRI for their keen interest
and encouragement in carrying out the present work. One of us (SKS) gratefully
acknowledges ICMR for the award of Emeritus Medical Scientist.

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Supplementary file
NMR Spectroscopy and Mass spectrometry data
Spectroscopic data:
Methyl 4-iodo-3, 5-dimethoxy benzoate (4): 1H NMR (500MHz, CDCl3): δ 7.09 (s, 2H, H-2
& 6), 3.87 (s, 3H, OCH3, H-8), 3.5 (s, 6H, 2OCH3, H-9 & 10 ).13C NMR (CDCl3, 125MHz)
ppm 131.48 (C, C-1), 104.36 (CH, C-2&6), 84.60 (C, C-4), 159.18 (C, C-3&5), 165.85 (CO,
C-7), 52.51 (CH3, C-8), 56.73 (OCH3, C-9&10).
Methyl 3, 5-dimethoxy-4-(2-(4-methoxy phenyl) ethynyl) benzoate (5): 1H NMR
(500MHz, CDCl3): δ 7.35 (s, 2H, H-2 & 6), 3.81 (s, 3H, OCH3, H-8), 3.79 (s, 9H, 3OCH3, H-
9, 10 & 7’’) 7.36 (d, 2H,H-2’’ & 6’’), 7.84 (d, 2H, H-3’’ & 5’’). 13C NMR (CDCl3, 125MHz) ppm
131.79 (C, C-1), 104.68 (CH, C-2 & 6), 159.40 (C, C-3 & 5), 84.60 (C, C-4), 166.50 (CO, C-
7). 52.26 (CH3, C-8), 55.17 (OCH3, C-9 & 10), 93.00 (C, C-1’ & 2’ ), 115.00 (C, C-1’’), 133.09
(CH, C-2’’ & 6’’), 113.78 (C, C-3’’ & 5’’ ), 162.60 (C, C-4’’ ), 56.17 (OCH3, C-7’’).
1
Methyl 3-iodo-4-methoxy-2-(4-methoxyphenyl) benzofuran-6-carboxylate (6): H NMR
(500MHz, CDCl3): δ 7.37 (s, 1H, H-5), 7.83 (s, 1H, H-7), 8.09 (d, 2H, H-2’ & 6’), 7.02 (d, 2H,
H-3’ & 5’), 3.86 (s, 3H, OCH3, H-7’), 3.94 (s, 3H, OCH3, H-1’’), 4.00 (s, 3H, OCH3, H-1’’’’). 13C
NMR (CDCl3, 125 MHz): ppm 155.38 (C, C-2), 55.82 (C, C-3), 153.36 (C, C-4), 106.36 (CH,
C-5), 126.96 (C, C-6), 105.05 (CH, C-7), 160.57 (C, C-8), 122.25 (C, C-9), 124.16 (C, C-1’),
129.63 (CH, C-2’ & 6’), 113.88 (CH, C-3’ & 5’), 162.70 (C, C-4’), 53.24 (CH3, C-7’), 55.41
(CH3, C-1’’), 167.03 (CO, C-1’’’), 52.35 (CH3, C-1’’’’).
Methyl 4-methoxy-3-(3,4,5-trimethoxy)-2-(4methoxyphenyl) benzofuran-6-carboxylate
(7 and 8B) :
8''
7''
O
4''
1''
O
5''
6''
3''
O
1'''
5
9''
5'
O
4
2''
6'
1'
3
7'
9
1'''''
O
1
4'
2
O
O
8
6
3'
2'
1''''
O
7
8B
0
Solid with mp 149.9 C. 1H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5), 7.83 (s, 1H, H-7),
8.08 ( d, 2H, H-2’ & 6’ J= 9.0 Hz), 6.97 (d, 2H, H-3’ and 5’ J= 8.5 Hz), 6.56 (s, 1H, H-2’’ &
6’’) 6.58 (s, 1H, H-6’’) 3.84 (s, 9H, OCH3, H-7’,7’’ & 9’’), 3.94 (OCH3, 3H , H-8’’), 3.94 (s, 3H,
13
OCH3, H-1’’’), 3.99 (s, 3H, OCH3, H-1’’’’’). C NMR (CDCl3, 125 MHz): ppm 154.41 (C, C-2),
60.85 (C, C-3), 153.36 (C, C-4), 106.36 (CH, C-5), 126.97 (C, C-6), 105.05 (CH, C-7),
155.37 (C, C-8), 122.24 (C, C-9), 124.15 (C, C-1’), 129.62 (CH, C-2’ & 6’), 113.88 (CH, C-3’
& 5’), 160.58 (C, C-4’), 53.23 (CH3, C-7’), 138.09 (C,C-1’’)’.105.19 (C, C-2’’& 6’’), 153.53 (C,

C-3’’ &5’’), 139.30 (C, C-4’’), 55.81 (OCH3, C-7’’ & 9’’), 56.07 (OCH3. C-8’’), 55.40 (CH3, C-1’’’),
167.01 (CO, C-1’’’’), 52.34 (OCH3, C-1’’’’’) ESI-MS (MeOH): For C27H26O8, 517 [M+K]+.
4-methoxy-3-(3,4,5-trimethoxyphenyl)-2-(4methoxyphenyl)
benzofuran-6-carboxylic
acid (8A): Solid with mp 242.5 0C . H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5), 8.15 (s,
1H, H-7), 8.00 (d, 2H, H-2’ & 6’ J= 6.5 Hz), 7.04 ( d, 2H, H-3’ & 5’ J= 6.5 Hz ), 6.57 (s, 1H, H-
2’’ ) 6.59 (s, 1H, H-6’’) , 3.83 (s, 9H, OCH3, H-7’ , 7’’ & 9’’), 3.94 (OCH3, 3H , H-8’’), 3.75 (s,
3H, OCH3, H-1’’’), ppm: 13C NMR (CDCl3, 125 MHz): δ 152.53 (C, C-2), 119.97 (C, C-3),
151.07 (C, C-4), 103.77 (CH, C-5), 127.44 (C, C-6), 103.32 (CH, C-7), 152.20 (C, C-8),
112.11 (C, C-9), 121.79 (C, C-1’), 129.68 (CH, C-2’ & 6’), 112.58 (CH, C-3’ & 5’), 151.07 (C,
C-4’), 58.20 (CH3, C-7’), 129.91 (C,C-1’’), 103.77 (C, C-2’’& 6’’), 152.20 (C, C-3’’ & 5’’ ),
129.91 (C, C-4’’), 53.46 (OCH3, C-7’’ & 9’’), 53.94 (OCH3, C-8’’), 53.46 (CH3, C-1’’’), 158.42
(CO, C-1’’’’) ppm. ESI-MS (MeOH): for C26H24O8, 465 [M+H] +.
1
Ethyl 4-methoxy-3-(3, 4, 5-trimethoxyphenyl)-2-(4-methoxyphenyl) benzofuran-6-
carboxylate (8C) : white amorphous. In 1H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5), 7.78
(s, 1H, H-7), 7.82 ( d, 2H, H-2’ & 6’ J= 8.5 Hz), 7.01 (d, 2H, H-3’ and 5’ J= 9.0 Hz), 6.57 (s,
1H, H-2’’ ) 6.59 (s, 1H, H-6’’) , 3.85 (s, 9H, OCH3, H-7’ , 7’’ & 9’’), 3.87 (s, 3H , OCH3, H-8’’),
4.00 (s, 3H, OCH3, H-1’’’), 4.40 (m, CH2, 2H, H-1’’’’’), 1.42 (t, 3H, CH3, H-2’’’’’ J= 6.5 Hz). 13C
NMR (CDCl3, 125 MHz): ppm 154.41 (C, C-2), 61.26 (C, C-3), 153.35 (C, C-4), 106.30 (CH,
C-5), 126.73 (C, C-6), 105.06 (CH, C-7), 154.97 (C, C-8), 122.82 (C, C-9), 124.20 (C, C-1’),
129.61 (CH, C-2’ & 6’), 113.88 (CH, C-3’ & 5’), 160.32 (C, C-4’), 53.23 (CH3, C-7’), 139.30
(C,C-1’’)’.105.22 (C, C-2’’& 6’’), 153.35 (C, C-3’’ &5’’), 139.30 (C, C-4’’), 55.80 (OCH3, C-7’’ &
9’’), 56.08 (OCH3. C-8’’), 55.40 (CH3, C-1’’’), 167.01 (CO, C-1’’’’), 61.09 (CH2, C-1’’’’’), 14.38
(CH3, C-2’’’’’). ESI-MS for C28H28O8 (MeOH): 493 [M+H] +
Propyl
4-methoxy-3-(3,4,5-trimethoxyphenyl)-2-(4-methoxyphenyl)benzofuran-6-
carboxylate (8D) : White amorphous. In 1H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5), 7.78
(s, 1H, H-7), 7.79 (d, 2H, H-2’ & 6’ J= 8.5 Hz), 6.99 (d, 2H, H-3’ & 5’ J= 7.0 Hz ), 6.96 (s, 1H,
H-2’’ ) 6.98 (s, 1H, H-6’’), 3.85 (s, 9H, OCH3, H-7’,7’’ & 9’’), 4.00 ( s, 3H , OCH3, H-8’’), 3.85
(s, 3H, OCH3, H-1’’’), 4.31 (t, 2H, CH2, H-1’’’’’ J= 6.5 Hz) 1.82 (m, 2H, CH2, H-2’’’’’) 1.4 (t, 3H,
CH3, H-3’’’’’ J= 7.0 Hz). 13C NMR (CDCl3, 125 MHz): ppm 154.41 (C, C-2), 66.67 (C, C-3),
153.83 (C, C-4), 106.49 (CH, C-5), 126.61 (C, C-6), 105.08 (CH, C-7), 154.97 (C, C-8),
122.81 (C, C-9), 124.20 (C, C-1’), 129.60 (CH, C-2’ & 6’), 113.88 (CH, C-3’ & 5’), 160.33 (C,
C-4’), 52.35 (CH3, C-7’), 139.30 (C,C-1’’)’.105.24 (C, C-2’’& 6’’), 153.83 (C, C-3’’ &5’’), 139.30
(C, C-4’’), 55.78 (OCH3, C-7’’ & 9’’), 56.07 (OCH3. C-8’’), 55.39 (CH3, C-1’’’), 167.06 (CO, C-
1’’’’), 66.67 (CH2, C-1’’’’’) 22.20 (CH2, C-2’’’’’) 10.58 (CH3, C-3’’’’’). ESMS for C29H30O8 (MeOH):
507 [M+H] +
Isopropyl 4-methoxy-3-(3, 4, 5-trimethoxyphenyl)-2-(4-methoxyphenyl) benzofuran-6-
1
carboxylate (8E): Viscous compound. In H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5),
8.08 (s, 1H, H-7), 7.84 (d, 2H, H-2’ & 6’ J= 6.5 Hz), 7.01 (d, 2H, H-3’& 5’ J= 9.0 Hz), 6.57
(s, 1H, H-2’’ ) 6.59 (s, 1H, H-6’’), 3.86 (s, 9H, OCH3, H-7’ , 7’’ & 9’’), 3.87 (s, 3H , OCH3, H-
8’’), 3.87 (s, 3H, OCH3, H-1’’’), 5.27 (m, 1H, CH, H-1’’’’’), 1.40 (d, 6H, 2XCH3, H-2’’’’’ & 3’’’’’). 13C
NMR (CDCl3, 125 MHz): ppm 154.41 (C, C-2), 115.90 (C, C-3), 153.32 (C, C-4), 106.24
(CH, C-5), 126.59 (C, C-6), 105.05 (CH, C-7), 155.24 (C, C-8), 122.32 (C, C-9), 124.07 (C,
C-1’), 129.59 (CH, C-2’ & 6’), 113.88 (CH, C-3’ & 5’), 160.54 (C, C-4’), 55.40 (CH3, C-7’),
132.23 (C,C-1’’)’.105.19 (C, C-2’’& 6’’), 153.53 (C, C-3’’ &5’’), 139.31 (C, C-4’’), 55.81 (OCH3,

C-7’’ & 9’’), 56.07 (OCH3. C-8’’), 55.40 (CH3, C-1’’’), 166.46 (CO, C-1’’’’), 68.70 (CH, C-1’’’’’),
22.71 (CH3, C-2’’’’’ & 3’’’’’). ESMS for C29H30O8 (MeOH): 507 [M+H] +
n-Butyl
4-methoxy-3-(3,4,5-trimethoxyphenyl)-2-(4-methoxyphenyl)
benzofuran-6-
1
carboxylate (8F): Viscous compound. In H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5),
8.09 (s, 1H, H-7), 7.78 (d, 2H, H-2’ & 6’ J= 9.0 Hz), 7.35 (d, 2H, H-3’& 5’ J= 9.5 Hz), 6.82 (s,
1H, H-2’’ ) 6.96 (s, 1H, H-6’’), 3.84 (s, 9H, OCH3, H-7’ , 7’’ & 9’’), 3.99 (OCH3, 3H , H-8’’),
3.89 (s, 3H, OCH3, H-1’’’), 4.34 (t, 2H, CH2, H-1’’’’’ J= 6.5 Hz) 1.77 (m, 2H, CH2, H-2’’’’’) 1.50
(m, 2H, CH2, H-3’’’’’), 0.87 (t, 3H, CH3, H-4’’’’’ J= 7.0 Hz). 13C NMR (CDCl3, 125 MHz): ppm
154.40 (C, C-2), 115.11 (C, C-3), 153.53 (C, C-4), 106.47 (CH, C-5), 126.60 (C, C-6), 104.34
(CH, C-7), 154.96 (C, C-8), 122.80 (C, C-9), 124.17 (C, C-1’), 129.76 (CH, C-2’ & 6’), 113.87
(CH, C-3’ & 5’), 160.32 (C, C-4’), 52.34 (CH3, C-7’), 132.07 (C,C-1’’)’.106.24 (C, C-2’’& 6’’),
153.35 (C, C-3’’ &5’’), 139.29 (C, C-4’’), 55.78 (OCH3, C-7’’ & 9’’), 56.07 (OCH3. C-8’’), 55.39
(CH3, C-1’’’), 167.02 (CO, C-1’’’’), 66.11 (CH2, C-1’’’’’) 31.94 (CH2, C-2’’’’’) 19.35 (CH2, C-3’’’’’),
13.82 (CH3, C-4’’’’’) ppm. ESMS for C30H32O8 (MeOH): 539 [M+H+NH4] +
Isopentyl 4-methoxy3-(3, 4, 5-trimethoxyphenyl)-2-(4-methoxyphenyl) benzofuran-6-
1
carboxylate (8G): Viscous compound. In H NMR (500MHz, CDCl3): δ 7.37 (s, 1H, H-5),
8.10 (s, 1H, H-7), 7.79 (d, 2H, H-2’ & 6’ J= 8.5 Hz),, 7.00 (d, 2H, H-3’& 5’ J= 8.5 Hz), 6.97 (s,
1H, H-2’’ ) 6.99 (s, 1H, H-6’’), 3.86 (s, 9H, OCH3, H-7’ , 7’’ & 9’’), 3.88 (s, 3H , OCH3, H-8’’),
4.00 (s, 3H, OCH3, H-1’’’), 4.38 (t, 2H, CH2, H-1’’’’’ J= 6.0 Hz) 1.70 (m, 2H, CH2, H-2’’’’’), 1.82
(m, 1H, CH, H-3’’’’’), 1.29 (s, 6H, CH3, H-4’’’’’ & 5’’’’’). 13C NMR (CDCl3, 125 MHz): ppm 154.96
(C, C-2), 114.36 (C, C-3), 153.35 (C, C-4), 106.48 (CH, C-5), 126.61 (C, C-6), 104.34 (CH,
C-7), 155.31 (C, C-8), 122.80 (C, C-9), 124.19 (C, C-1’), 129.60 (CH, C-2’ & 6’), 113.88 (CH,
C-3’ & 5’), 160.32 (C, C-4’), 53.23 (CH3, C-7’), 130.27 (C, C-1’’), 106.24 (C, C-2’’& 6’’),
153.35 (C, C-3’’ &5’’), 139.31 (C, C-4’’), 55.79 (OCH3, C-7’’ & 9’’), 56.07 (OCH3. C-8’’), 55.40
(CH3, C-1’’’), 167.05 (CO, C-1’’’’), 63.79 (CH2, C-1’’’’’), 37.50 (CH2, C-2’’’’’), 25.39 (CH, C-3’’’’’),
22.71 (2CH3, C-4’’’’’ & 5’’’’’) ppm. ESMS for C31H34O8 (MeOH): 552 [M+H] +
2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl) benzofuran-6-carboxylicacid
(9A): Half white solid with mp 253.0 0C. In 1H NMR (500MHz, CDCl3): δ 7.71 (bs, 1H, H-7),
7.44 (bs, 1H, H-5), 7.44 ( d, 2H, H-2’ & 6’ J= 8.0 Hz), 7.45 (d, 2H, H-3’& 5’ J= 8.0 Hz), 6.88
(s, 2H, H-2’’ & 6’’) , 3.83 (s, 9H, OCH3, H-7’’ , 8’’ , 9’’ ), 3.81 (s, 3H, OCH3, H-1’’’). ppm 13C
NMR (CDCl3, 125 MHz): δ 139.28 (C, C-2), 119.32 (C, C-3), 151.83 (C, C-4), 104.23 (CH, C-
5), 128.56 (C, C-6), 104.42 (CH, C-7), 161.08 (C, C-8), 123.50 (C, C-9), 125.52 (C, C-1’),
128.56 (CH, C-2’ & 6’), 116.30 (CH, C-3’ & 5’), 163.04 (C, C-4’), 132.24 (C, C-1’’), 104.42
(C, C-2’’& 6’’), 160.68 (C, C-3’’&5’’), 133.05 (C, C-4’’), 56.15 (OCH3, C-7’’, 8’’, 9’’&1’’’), 167.00
(CO, C-1’’’’). ESI-MS (MeOH): for C25H21FO7, 453 [M+H]+.
Methyl
2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl)
benzofuran-6-
carboxylate( 9B) : Viscous compound . In 1H NMR (500MHz, CDCl3): δ 7.38 (bs, 1H, H-5),
7.84 (bs, 1H, H-7), 8.13 (d, 2H, H-2’ & 6’ J= 9.0 Hz), 7.19 (d, 2H, H-3’& 5’ J= 9.0 Hz), 7.16 (s,
2H, H-2’’ & 6’’), 3.93 (s, 12H, OCH3, H- 7’’ , 8’’ , 9’’ & 1’’’), 4.00 (s, 3H , OCH3, H-1’’’’’). 13C
NMR (CDCl3, 125 MHz): ppm 154.55 (C, C-2), 115.69 (C, C-3), 153.59 (C, C-4), 106.39
(CH, C-5), 127.57 (C, C-6), 105.15 (CH, C-7), 154.36 (C, C-8), 123.92 (C, C-9), 125.94 (C,
C-1’), 128.55 (CH, C-2’ & 6’), 115.51 (CH, C-3’ & 5’), 162.31 (C, C-4’), 130.10 (C,C-1’’),
105.15 (C, C-2’’& 6’’), 153.59 (C, C-3’’ &5’’), 132.33 (C, C-4’’), 55.81 (OCH3, C-7’’ & 9’’), 56.35
(OCH3. C-8’’), 54.61 (CH3, C-1’’’), 166.85 (CO, C-1’’’’), 52.34 (OCH3, C-1’’’’’). ESMS for
C26H23O7 (MeOH): 467 [M+H] +

Ethyl 2-(4-fluorophenyl)-4-methoxy-3-(3, 4, 5-trimethoxyphenyl) benzofuran-6-
carboxylate (9C): M.P: 103.5 0C .In 1H NMR (500MHz, CDCl3): δ 7.58 (bs, 1H, H-5), 7.57
(bs, 1H, H-7), 7.56 (d, 2H, H-2’ & 6’ J= 8.5 Hz), 7.02 (d, 2H, H-3’& 5’ J= 8.5 Hz), 7.16 (s, 2H,
H-2’’ & 6’’) , 3.96 (s, 12H, OCH3, H- 7’’ , 8’’ , 9’’ & 1’’’), 4.34 (m, 2H , CH2, H-1’’’’’), 1.40 (t, 3H ,
CH3, H-2’’’’’ J= 6.5 Hz), ppm: 13C NMR (CDCl3, 125 MHz): ppm 154.55 (C, C-2), 115.69 (C,
C-3), 153.59 (C, C-4), 106.39 (CH, C-5), 127.57 (C, C-6), 105.15 (CH, C-7), 154.36 (C, C-8),
123.92 (C, C-9), 125.94 (C, C-1’), 128.55 (CH, C-2’ & 6’), 115.51 (CH, C-3’ & 5’), 162.31 (C,
C-4’), 130.10 (C,C-1’’), 105.15 (C, C-2’’& 6’’), 153.59 (C, C-3’’ &5’’), 132.33 (C, C-4’’), 55.81
(OCH3, C-7’’ & 9’’), 56.35 (OCH3. C-8’’), 54.61 (CH3, C-1’’’), 166.09 (CO, C-1’’’’), 61.41 (CH2, C-
1’’’’’), 14.10 (CH3, C-2’’’’’) ppm. ESI-MS (MeOH): for C27H25 FO7 , 481 [M+H] +
n-Propyl 2-(4-fluorophenyl)-4-methoxy-3-(3, 4, 5-trimethoxyphenyl) benzofuran-6-
1
carboxylate (9D): Yellowish viscous compound. In H NMR (500MHz, CDCl3): δ 7.24 (s,
1H, H-5), 7.87 (s, 1H, H-7), 7.56 (d, 2H, H-2’ & 6’ J= 8.5 Hz), 7.14 (d, 2H, H-3’& 5’ J= 7.5 Hz),
7.03 (s, 1H, H-2’’ ) 7.04 (s, 1H, H-6’’), 3.95 (s, 9H, OCH3, H-7’’ , 9’’ & 1’’’), 4.00 (s, 3H , OCH3,
H-8’’), 4.30 (t, 2H, CH2, H-1’’’’’ J= 6.5 Hz) 1.79 (m, 2H, CH2, H-2’’’’’), 0.87 (t, 3H, CH3, H-3’’’’’J=
7.0 Hz). 13C NMR (CDCl3, 125 MHz): ppm 151.95 (C, C-2), 119.59 (C, C-3), 152.81 (C, C-4),
106.00(CH, C-5), 126.92 (C, C-6), 104.41 (CH, C-7), 156.43 (C, C-8), 114.07 (C, C-9),
126.99 (C, C-1’), 126.99 (CH, C-2’ & 6’), 116.13 (CH, C-3’ & 5’), 163.58 (C, C-4’), 131.42
(C,C-1’’)’.105.15 (C, C-2’’& 6’’), 156.43 (C, C-3’’ &5’’), 139.29 (C, C-4’’), 56.03 (OCH3, C-7’’ , 8’’
, 9’’& 1’’’), 166.15 (CO, C-1’’’’), 67.04 (CH2, C-1’’’’’), 22.70 (CH2, C-2’’’’’), 10.50 (CH3, C-3’’’’’).
ESMS for C28H27FO7 (MeOH): 495 [M+H] +
Isopropyl
2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl)
benzofuran-6-
1
carboxylate (9E): viscous compound. In H NMR (500MHz, CDCl3): δ 7.23 (s, 1H, H-5),
7.58 (s, 1H, H-7), 7.56 (d, 2H, H-2’ & 6’ J= 9.0 Hz), 7.02 (d, 2H, H-3’& 5’ J= 9.0 Hz), 6.59 (s,
1H, H-2’’ ) 6.61 (s, 1H, H-6’’) , 3.96 (s, 12H, OCH3, H-7’’, 8’’, 9’’ & 1’’’), 5.27 (m, 1H, CH, H-
1’’’’’), 1.39 (s, CH3, 6H, H-2’’’’’ & 3’’’’’). 13C NMR (CDCl3, 125 MHz): ppm 150.31 (C, C-2),
115.43 (C, C-3), 150.31 (C, C-4), 106.24 (CH, C-5), 126.59 (C, C-6), 105.05 (CH, C-7),
155.24 (C, C-8), 122.32 (C, C-9), 124.07 (C, C-1’), 129.59 (CH, C-2’ & 6’), 113.88 (CH, C-3’
& 5’), 160.54 (C, C-4’), 55.40 (CH3, C-7’), 132.23 (C,C-1’’)’.105.19 (C, C-2’’& 6’’), 153.53 (C,
C-3’’ &5’’), 139.31 (C, C-4’’), 55.81 (OCH3, C-7’’ & 9’’), 56.07 (OCH3. C-8’’), 55.40 (CH3, C-1’’’),
166.46 (CO, C-1’’’’), 68.70 (CH, C-1’’’’’), 22.71 (CH3, C-2’’’’’ & 3’’’’’). ESMS for C28H27 FO7
(MeOH): 495 [M+H] +
n-Butyl-2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl)
benzofuran-
6-
1
carboxylate (9F): Viscous compound. In H NMR (500MHz, CDCl3): δ 7.24 (s, 1H, H-5),
7.58 (s, 1H, H-7), 7.56 (d, 2H, H-2’ & 6’ J= 9.0 Hz), 7.02 (d, 2H, H-3’& 5’ J= 9.0 Hz), 6.94 (s,
1H, H-2’’ ) 7.01 (s, 1H, H-6’’), 3.95 (s, 9H, OCH3, H-7’’ , 9’’ & 1’’’), 4.00 (s, 3H , OCH3, H-8’’),
4.34 (t, 2H, CH2, H-1’’’’’ J= 6.5 Hz), 1.77 (m, 2H, CH2, H-2’’’’’), 1.46 (m, 2H, CH2, H-3’’’’’), 0.87
(t, 3H, CH3, H-4’’’’’ J= 7.0 Hz). 13C NMR (CDCl3, 125 MHz): ppm 142.77 (C, C-2), 119.60 (C,
C-3), 151.96 (C, C-4), 106.53 (CH, C-5), 126.92 (C, C-6), 104.41 (CH, C-7), 156.43 (C, C-8),
114.07 (C, C-9), 126.99 (C, C-1’), 129.68 (CH, C-2’ & 6’), 115.60 (CH, C-3’ & 5’), 163.58 (C,
C-4’), 130.62 (C,C-1’’)’.105.16 (C, C-2’’& 6’’), 151.96 (C, C-3’’ &5’’), 139.28 (C, C-4’’), 56.32
(OCH3, C-7’’ , 8’’ , 9’’& 1’’’), 166.17 (CO, C-1’’’’), 65.35 (CH2, C-1’’’’’) 33.83 (CH2, C-2’’’’’) 22.70
(CH2, C-3’’’’’), 14.12 (CH3, C-4’’’’’). ESMS for C29H29FO7 (MeOH): 509 [M+H] +
tert-Butyl-2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl)benzofuran-6-
0
1
carboxylate (9G): Yellowish solid with mp 159.5 C . In H NMR (500MHz, CDCl3): δ 7.19

(s, 1H, H-5), 7.54 (bs, 1H, H-7), 7.52 (d, 2H, H-2’ & 6’ J= 8.5 Hz), 6.98 (d, 2H, H-3’& 5’ J= 8.5
Hz), 6.97 (s, 1H, H-2’’ ) 6.98 (s, 1H, H-6’’), 3.91 (s, 12H, OCH3, H-7’’, 8’’, 9’’ & 1’’’), 1.20 (s,
9H, 3xCH3, H-2’’’’’ , 3’’’’’ & 4’’’’’ ). 13C NMR (CDCl3, 125 MHz): ppm 152.76 (C, C-2), 119.59 (C,
C-3), 152.76 (C, C-4), 104.49 (CH, C-5), 123.24 (C, C-6), 99.12 (CH, C-7), 156.34 (C, C-8),
114.01 (C, C-9), 123.77 (C, C-1’), 133.62 (CH, C-2’ & 6’), 115.54 (CH, C-3’ & 5’), 163.53 (C,
C-4’), 56.24 (CH3, C-7’), 131.64 (C,C-1’’)’.105.14 (C, C-2’’& 6’’), 153.50 (C, C-3’’ &5’’), 139.22
(C, C-4’’), 56.24 (OCH3, C-7’’, 8’’, 9’’& 1’’’), 165.77 (CO, C-1’’’’), 80.97 (C, C-1’’’’’), 29.65 (CH3,
C-2’’’’’ , 3’’’’’ & 4’’’’’). ESMS for C29H29 FO7 (MeOH): 509 [M+H] +
iso-Pentanyl,
2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl)benzofuran-6-
1
carboxylate (9H): Viscous compound. In H NMR (500MHz, CDCl3): δ 7.14 (s, 1H, H-5),
7.70 (s, 1H, H-7), 7.52 (d, 2H, H-2’ & 6’ J= 8.5 Hz), 7.02 (d, 2H, H-3’& 5’ J= 9.0 Hz), 7.01 (s,
1H, H-2’’ ) 6.59 (s, 1H, H-6’’), 3.95 (s, 9H, OCH3, H-7’’ , 9’’ & 1’’’), 4.00 (s, 3H , OCH3, H-8’’),
4.38 (t, 2H, CH2, H-1’’’’’J= 6.0 Hz) 1.68 (m, 2H, CH2, H-2’’’’’), 1.76 (m, 1H, CH, H-3’’’’’), 0.87 (s,
6H, 2XCH3, H-4’’’’’ & H-5’’’’’). 13C NMR (125MHz, CDCl3): ppm 143.17 (C, C-2), 115.36 (C, C-
3), 160.78 (C, C-4), 104.40 (CH, C-5), 122.91 (C, C-6), 104.40 (CH, C-7), 160.78 (C, C-8),
115.18 (C, C-9), 122.09 (C, C-1’), 130.93 (CH, C-2’ & 6’), 115.36 (CH, C-3’ & 5’), 166.11 (C,
C-4’), 130.93 (C,C-1’’)’.105.16 (C, C-2’’& 6’’), 160.78 (C, C-3’’ &5’’), 139.34 (C, C-4’’), 55.95
(OCH3, C-7’’ , 8’’ , 9’’& 1’’’), 166.11 (CO, C-1’’’’), 64.06 (CH2, C-1’’’’’) 37.08 (CH2, C-2’’’’’) 24.51
(CH, C-3’’’’’), 22.37 (CH3, C-4’’’’’ and 5’’’’’). ESMS for C30H31 FO7 (MeOH): 423 [M+H] +
sec-Butyl-2-(4-fluorophenyl)-4-methoxy-3-(3,4,5-trimethoxyphenyl)benzofuran-6-
1
carboxylate (9I): Viscous compound. In H NMR (500MHz, CDCl3): δ 7.19 (s, 1H, H-5),
7.54 (bs, 1H, H-7), 7.84 (d, 2H, H-2’ & 6’ J= 8.5 Hz), 7.00 (d, 2H, H-3’& 5’ J= 7.0 Hz), 6.93 (s,
1H, H-2’’ ) 6.94 (s, 1H, H-6’’), 3.91 (s, 12H, OCH3, H-7’’, 8’’, 9’’ & 1’’’), 1.43 (d, 3H, CH3, H-1’’’’’
J= 7.0 Hz), 4.24 (m, CH, 1H, H-2’’’’’ ), 1.92 (m, CH2,H-3’’’’’ ), 0.82 (t, 3H, CH3, H-4’’’’’ J= 6.5 Hz
). 13C NMR (CDCl3, 125 MHz): ppm 153.09 (C, C-2), 119.48 (C, C-3), 153.09 (C, C-4),
106.03 (CH, C-5), 130.60 (C, C-6), 99.20 (CH, C-7), 156.34 (C, C-8), 113.62 (C, C-9),
123.54 (C, C-1’), 131.10 (CH, C-2’ & 6’), 115.29 (CH, C-3’ & 5’), 163.57 (C, C-4’), 133.55 (C,
C-1’’)’ 105.12 (C, C-2’’& 6’’), 153.09 (C, C-3’’ &5’’), 139.11 (C, C-4’’), 56.24 (OCH3, C-7’’, 9’’&
1’’’), 62.03 (OCH3, C-8’’), 166.24 (CO, C-1’’’’), 22.57 (CH3, C-1’’’’’), 65.32 (CH, C-2’’’’’), 29.58
(CH2 , C-3’’’’’), 8.46 (CH3, C-4’’’’’). ESMS for C29H29 FO7 (MeOH): 509 [M+H] +
Methyl-2-(4-methoxycarbonyl)
phenyl)-4-methoxy-3-(3,4,5-trimethoxy
phenyl)
0
1
benzofuran-6-carboxylate (10A) : Melting point: 150.4-160 C. In H NMR (500MHz,
CDCl3): δ 7.65 (s, 1H, H-5), 8.02 (s, 1H, H-7), ), 8.01 (d, 2H, H-2’ & 6’ J= 8.0 Hz), 7.64 (d, 2H,
H-3’& 5’ J= 8.0 Hz), 7.24 (s, 1H, H-2’’ ) 6.25 (s, 1H, H-6’’), 3.92 (s, 6H, OCH3, H-7’’ & 9’’),
3.94 (s, 3H , OCH3, H-8’’), 3.96 (s, 6H, OCH3, H-1’’’’’ & 8’). C NMR (CDCl3, 125 MHz): ppm
13
142.85 (C, C-2), 115.90 (C, C-3), 151.89 (C, C-4), 108.93 (CH, C-5), 124.06 (C, C-6), 105.69
(CH, C-7), 161.17 (C, C-8), 123.51 (C, C-9), 124.06 (C, C-1’), 129.42 (CH, C-2’ & 6’), 114.08
(CH, C-3’ & 5’), 131.36 (C, C-4’), 52.25 (CH3, C-7’), 135.18 (C, C-1’’), 104.56 (C, C-2’’& 6’’),
151.89 (C, C-3’’ &5’’), 139.30 (C, C-4’’), 52.50 (OCH3, C-7’’ , 8’ & 9’’), 56.37 (OCH3. C-8’’),
52.25 (OCH3, C-1’’’), 166.53 (CO, C-7’), 166.67 (CO, C-1’’’), 52.34 (OCH3, C-1’’’’’). ESMS for
C28H26O9 (MeOH): 507 [M+H] +

O
O
O
O
O
HO
O
8A
O
O
O
O
O
O
HO
O
8A
O

O
O
O
O
O
O
O
O
8B
O
O
O
O
O
O
O
O
8B

O
O
O
O
O
O
O
8C
O
O
O
O
O
O
O
O
8C
O

O
O
O
O
F
HO
O
O
9A
O
O
O
O
F
HO
O
O
9A

O
O
O
O
F
O
O
O 9B
O
O
O
O
F
O
O
O 9B

O
O
O
O
F
O
O
9E
O
O
O
O
O
F
O
O
9E
O

O
O
O
O
F
O
O
O
9G
O
O
O
O
F
O
O
O
9G

O
O
O
O
O
O
O
O
10A
O
O
O
O
O
O
O
O
O
10A
O