Identification of two novel thiophene analogues as inducers of autophagy mediated cell death in breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2021.116112

Natural compounds isolated from different medicinal plants remain one of the major resources of anticancer drugs due to their enormous chemical diversity. Studies suggested therapeutic potential for various tanshinones, key bioactive lipophilic compounds

Full text of DOI:10.1016/j.bmc.2021.116112

Bioorg. Med. Chem. 37 (2021) 116112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of two novel thiophene analogues as inducers of autophagy
mediated cell death in breast cancer cells
,
,
,
Chandrima Gaina 1, Aparna Sarkarb 1, Shrea Burala 1, Moumita Rakshitb, Jeet Banerjeeb,
Ankita Deyb, Nabendu Biswasc, Gandhi K. Karb *, Abhik Sahaa
,
,*
a School of Biotechnology, Presidency University, Second Campus, Plot No. DG/02/02, Premises No. 14-0358, Action Area-ID, New Town, Kolkata 700156, West
Bengal, India
b Department of Chemistry, Presidency University, 86/1 College Street, Kolkata 700073, West Bengal, India
c Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata 700073, West Bengal, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Natural compounds isolated from different medicinal plants remain one of the major resources of anticancer
drugs due to their enormous chemical diversity. Studies suggested therapeutic potential for various tanshinones,
key bioactive lipophilic compounds from the root extracts of Salvia miltiorrhiza Bunge, against multiple cancers
including breast carcinoma. We designed, synthesized and evaluated anti-cancer properties of a series of
condensed and doubly condensed furophenanthraquinones of tanshinone derivatives on two breast cancer lines -
MCF7 and MDA-MB-231. We identified two thiophene analogues - compounds 48 and 52 with greater anti-
proliferative efficiency (~4 fold) as compared to the natural tanshinones. Mechanistically, we showed that
both compounds induced autophagy mediated cell death and partial but significant restoration of cell death in
the presence of autophagy inhibitor further supported this notion. Both compounds transcriptionally activated
several autophagy genes responsible for autophagosome formation along with two death regulators – GADD34
Breast cancer
Tanshinones
Thiophene analogues
Autophagy
Abbreviations: Tan-I, tanshinone-I; Tan-IIA, tanshinone-IIA; NMR, nuclear magnetic resonance; FT-IR, Fourier-transform infrared spectroscopy; TFA, CF3CO2H,
trifluoroacetic acid; TFAA, (CF3CO)2O, trifluoroacetic anhydride; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium(0); Et3N, triethylamine; DMF, dimethylforma-
mide; hrs, hours; NaBH4, sodium borohydride; EtOH, ethanol; r.t., room temperature; MsCl, methanesulfonyl chloride; LiCl, lithium chloride; KCN, potassium cy-
anide; Aq., aqueous; KOH, potassium hydroxide; Na2HPO4-H2O, sodium phosphate dibasic; MeOH, methanol; PBr3, phosphorus tribromide; CHCl3, chloroform; atm.,
atmosphere; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; HRMS, high-resolution mass spectrometry; ESI, electrospray ionization; LC/MS, liquid chromatog-
raphy–mass spectrometry; MTT, (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide); ER, estrogen receptor; PR, progesterone receptor; Her2, human
epidermal growth factor receptor 2; DMSO, dimethyl sulfoxide; μM, micromolar; NADPH, nicotinamide adenine dinucleotide phosphate; nm, nanometer; IC50, the
concentration that gives half-maximal response; EtBr, ethidium bromide; AO, acridine orange; DNA, deoxyribonucleic acid; PI, propidium iodide; PARP1, poly [ADP-
ribose] polymerase 1; Ex, excitation; Em, emission; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra
large; BCL2L1, BCL2 like 1; Bim, Bcl-2 interacting mediator of cell death; BCL2L11, BCL2 like 11; Beclin 1, Coiled-Coil, Moesin-Like BCL2-Interacting Protein; Bax,
BCL2-associated X, apoptosis regulator; Bak, BCL2-antagonist/killer 1; LC3, microtubule associated protein 1 light chain 3 alpha/beta; p62/SQSTM1, sequestosome
1; ATG, autophagy related gene; PCR, polymerase chain reaction; RNA, ribonucleic acid; ATG16L1, autophagy related 16 like 1; ATG16L2, autophagy related 16 like
2; GABARAP, GABA (Gamma aminobutyric acid) type A receptor-associated protein; GABARAPL1, GABA type A receptor-associated protein like 1; GABARAPL2,
GABA type A receptor-associated protein like 2; PIK3CG, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma; UVRAG, UV radiation resistance
associated gene; DDIT3, DNA damage inducible transcript 3; CHOP, C/EBP (CCAAT/enhancer-binding protein)-homologous protein; GADD34, growth arrest and
DNA-damage-inducible protein 34; PPP1R15A, protein phosphatase 1 regulatory subunit 15A; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase;
EIF2AK3, eukaryotic translation initiation factor 2 alpha kinase 3; ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; ERN1, endoplasmic
reticulum to nucleus signaling 1; eIF2α, eukaryotic translation initiation factor 2 alpha; ATF4, activating transcription factor 4; cDNA, complementary DNA; ACTB,
actin beta; B2M, beta-2-microglobulin; HPRT1, hypoxanthine phosphoribosyltransferase 1; RPLP0, ribosomal protein lateral stalk subunit P0; CQ, choroquine; GFP,
green fluorescence protein; mCherry, mFruits family of monomeric red fluorescent proteins (mRFPs); DCM, dichloromethane; Et2O, diethyl ether; P.E, petroleum
ether; EtOAc, ethyl acetate; TLC, thin-layer chromatography; H2SO4, sulfuric acid; KBr, potassium bromide; NaHCO3, sodium bicarbonate; Na2SO4, sodium sulfate;
M.P, melting point; QTOF, Quad Time Of Flight; HCl, hydrochloric acid; FITC, fluorescein isothiocyanate.
* Corresponding authors.
E-mail addresses: gandhi.chem@presiuniv.ac.in (G.K. Kar), abhik.dbs@presiuniv.ac.in (A. Saha).
1
Equal Contribution.
https://doi.org/10.1016/j.bmc.2021.116112
Received 14 January 2021; Received in revised form 2 March 2021; Accepted 5 March 2021
Available online 17 March 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
and CHOP for inducing cell death. Altogether, our studies provide strong evidence to support compounds 48 and
52 as promising leads for further development as anticancer agents through modulating autophagy mechanism.
1. Introduction
potency and the drug-like properties of tanshinone molecules.
In an aim to identify more potent tanshinone analogues, we designed
Salvia miltiorrhiza Bunge (Danshen) has been widely used in tradi-
tional Chinese medicine to treat a number of ailments particularly car-
diovascular diseases for centuries.1,2 Subsequently, modern
pharmacological studies identified several bioactive molecules - the
hydrophilic phenolic acids and the lipophilic tanshinones from dry roots
of Danshen exhibiting pro-cardiovascular effects such as expanding the
coronary artery and increasing blood flow in the coronary artery, along
with anti-oxidant, anti-inflammation, liver protection, immune regula-
tion as well as anticancer properties.2–4
and synthesized eight compounds ꢀ 15, 23, 25, 27, 36, 43, 48 and 52
and tested their anti-proliferative activities in two breast cancer lines
MCF7 and MDA-MD-231. Recently, thiophene, a sulphur containing
heterocyclic scaffold, has emerged as an attractive option for synthesis
of library of small molecules exhibiting diverse biological activities
including – anti-oxidant, anti-inflammatory, anti-microbial as well as
anti-cancer properties.21,22 Herein, we identified two novel thiophene
analogues – compounds 48 and 52 exhibiting increased anti-tumour
potency as compared to the natural tanshinones both Tan-I (1) and
Tan-IIA (2). Our results evidently demonstrated that both compounds 48
and 52 induced autophagy mediated cell death in two breast cancer cell
lines.
The lipophilic components, a large number of condensed furo-
phenanthraquinones and their di-, tetra- and hexahydro derivatives
(tanshinones) including tanshinone-I (Tan-I, 1), tanshinone-IIA (Tan-
IIA, 2), tanshinone-IIB (3), tanshindiol-B (4), tanshindiol-C (5), cryp-
totanshinone (6), nortanshinone (7), dihydrotanshinone-I (8),
isotanshinone-II (9) and dihydroisotanshinone-II (10) among others
with the core nucleus, phenathro[1,2-b]furan-10,11-dione and phena-
thro[4,3-b]furan-4,5-dione, have been isolated from dried roots of
different Salvia species5,6 (Fig. 1).
2. Results and discussion
2.1. Synthesis and characterization
Owing to the increasing biological importance of condensed furo-
phenanthraquinones within tanshinone molecules, both synthetic
chemists and biologists expressed increased attention in synthesis of a
variety of tanshinone derivatives and subsequent exploration of their
biological activities particularly as anti-cancer agents.5,11,20 Likewise, in
order to synthesize novel furophenanthroquinones and their analogues,
we developed a novel route for synthesis of phenathro[1,2-b]furan-
10,11-dione and phenathro[4,3-b]furan-4,5-dione starting from suitable
1/2-(2-furyl)-3,4-dihydronaphthalen-2-carbaldehyde derivatives as re-
ported earlier.23,24 The method has great potential in synthesis of both
“S” and “U” shaped tetracyclic furophenanthroquinones as well as tri-
cyclic furonaphthoquinones with various substituents in ABCD (or BCD
rings in case of furonaphthoquinone) rings of the core nucleus.23,24
Herein, we synthesised phenanthro[2,1-b]furan-10,11-dione (15), as
nuclear analogue of tanshinones following similar strategy. To this end,
we used (2-Bromo-naphthalen-1-yl)-acetic acid methyl ester (11) and 3-
furanboronic acid as starting material (Scheme 1).
Structurally, all the isolated furophenanthraquinones contains a
tetracyclic framework either in “S” shaped form (phenanthro[1,2-b]
furan-10,11-dione derivatives and its di-, tetra- or hexa-hydro de-
rivatives) or in “U” shaped form (phenanthro[4,3-b]furan-4,5-dione
derivatives or its dihydro derivatives) (Figs. 2 and S1).
Among these, particularly Tan-IIA (2) and to a lesser extent Tan-I (1)
have been extensively investigated and reported to exert diverse bio-
logical properties. A growing body of evidence indicated potent anti-
cancer properties of tanshinone derivatives against multiple cancers
such as cervical cancer, colorectal cancer, gastric cancer, oesophageal
cancer, hepatocellular carcinoma, lung cancer, oral cancer, ovarian
cancer, osteosarcoma, prostate cancer, and breast carcinoma both in
vitro and in vivo.7–18 Despite the potential, further clinical development
of tanshinone molecules as anti-cancer agents has been decelerated
largely due to its weak potency and exceptionally low aqueous solubil-
ity.19,20 As a result, a number of research groups are currently being
engaged on structural optimization to enhance both the anti-cancer
The compound 11 was subjected to Pd (0) catalyzed Suzuki Coupling
Fig. 1. Various tetracyclic furophenanthraquinone derivatives isolated from dry root extracts of different Salvia species.
2

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
Reaction with 3-furanboronic acid to furnish [2-(furan-3-yl)-naph-
thalen-1-yl] acetic acid methyl ester (12) as a colourless solid in 70%
yield. In IR spectra, the compound showed a strong absorption at 1750
cmꢀ 1 characteristics of the ester carbonyl group. 1H NMR of the com-
pound 12 was also in agreement with the assigned structure. Alkaline
hydrolysis of the ester 12 afforded the corresponding acid 13 in 74%
yield. The corresponding FT-IR spectroscopic analysis also demon-
strated a strong absorption at 1690 cmꢀ 1 for COOH group. The car-
boxylic acid 13 was then cyclised in the presence of TFA/TFAA to
furnish the phenol 14 in 77% yield. 1H NMR spectra confirmed the
assigned structure of the phenol 14. At the final step of the synthesis, the
target quinone molecule 15 was achieved as a dark red solid in 56%
yield, by Fremy’s salt oxidation of phenol 14. The quinone 15 was
characterized by usual spectroscopic data (1H NMR, 13C NMR, FT-IR and
Mass spectroscopy).
As depicted in Scheme 4, synthesis of a regioisomeric ‘U’-shaped
novel thienophenanthraquinone, 10-methoxyphenanthro[3,4-b]thio-
phene-4,5-dione (27) commenced from 3,4-dihydro-1(3-thienyl)-7-
methoxynaphthalene-2-carbaldehyde25 (26) as starting material by
following the similar protocol as reported earlier5 with good to very
good yields.
Next, modifications were introduced in both rings A and D of the core
nucleus present in the naturally occurring furophenanthraquinone,
isotanshinone-II molecule. It is well known that bioisosteric relationship
exists between benzene and thiophene ring where ‘CH = CH’ is
considered as equivalent to ‘S’. So the synthesis of bisthiophene
condensed naphthoquinones, viz. 9-methylnaphtho[1,2-b:7,8-b′]bis-
thiophene-4,5-dione (36) and 9-methylnaphtho[2,1-b:7,8-b′]bisthio-
phene-4,5-dione (43) were achieved where ring A of phenanthro[4,3-b]
furan-4,5-dione (the core nucleus of isotanshinone II) was replaced by a
2-methylthiophene moiety while ring D was modified with a thiophene
moiety (Fig. 4).
Another novel ‘U’ shaped furophenanthraquinone i.e. phenanthro
[3,4-b]furan-4,5-dione (the core nucleus of isotanshinone II) was syn-
thesized starting from 1-bromo-2-naphthaldehydes (17) and 3-furanbor-
onic acid (Scheme 2). The two key steps involve in this methodology
were i) Suzuki coupling of an aryl halide and furanboronic acid to form
aryl-furyl bond and ii) development of o-quinone functionality by
oxidation of a phenolic moiety (Fig. 3).
Several reports suggested that thiophene analogues represent a rich
source of potential scaffolds in search of novel bioactive compounds as
anti-micriobial, anti-inflammatory as well as anti-cancer agents.26,27 It
would be worthwhile to explore the possibility in generating new
compounds by fusing differently substituted moieties which may offer
enhanced pharmacological activities. Therefore, to explore many more
systematic modifications on thiophene moiety warrants further inves-
tigation. As described in Fig. 4, the thiophene analogue compound 36
may be considered as Bioisostere of thienophenanthraquinone and
compound 43 is the nuclear analogue of ‘U’-shaped thieno-
phrenanthraquinone. The target molecules 36 and 43 were synthesised
from a common precursor 4-bromo-6,7-dihydro-2-methylbenzo[b]thio-
phene-5-carbaldehyde (29) (which was obtained from 6,7-dihydro-2-
methylbenzo[b]thiophen-4(5H)-one28 (28) via Vilsmeier-Haack reac-
tion) and two different aryl boronic acids, 2-thiopheneboronic acid and
3- thiopheneboronic acid, respectively instead of 2/3-furanboronic acids
following Scheme 5 and Scheme 6.
The compound 18 was synthesized by the Suzuki coupling of 1-
bromo-2- naphthaldehyde (17) with 3-furanboronic acid using Pd
(PPh3)4 as the catalyst, Et3N as the base and DMF as the solvent at 100 ◦C
under Argon atmosphere (Scheme 2). The aldehyde 17 was efficiently
prepared from 1-tetralone using modified Vilsmeier-Haack reaction,
followed by aromatisation, as reported earlier.23 After confirming the
structure by spectroscopic analysis, the furyl naphthaldehyde (18) was
then reduced with NaBH4 to the alcohol 19, which on treatment with
mesyl chloride, lithium chloride in the presence of s-collidine, furnished
the corresponding chloromethyl derivative, which without further pu-
rification was directly subjected to reaction with KCN in DMF to obtain
the corresponding cyanomethyl derivative 20. The nitrile 20 on hy-
drolysis (KOH, EtOH-H2O) afforded the carboxylic acid 21 as a light
brown solid and was then cyclised to the corresponding furophenan-
threnol 22 in excellent yield on treatment with a mixture of TFA and
TFAA at 0 ◦C. The phenol 22 was then oxidized by Fremy’s salt to the
target o-quinone 23 as a brick red solid in 77% yield.
The target compound 36 was synthesized starting from 4-bromo-6,7-
dihydro-2-methylbenzo[b]thiophene-5-carbaldehyde (29) and 2-thio-
phene boronic acid (Scheme 5). Bromoaldehyde 29 was obtained from
the ketone 28 in 73% yield on reaction with PBr3/DMF in CHCl3. Suzuki
coupling of compound 29 with 2-thiopheneboronic acid followed by
aromatization (DDQ, benzene, reflux) of the resulting product 30 fur-
nished compound 31 in very good yields. Following the protocol of
Next, we intended to modify the D ring of the isotanshinone-II core
nucleus by replacing the furan ring with a thiophene ring. To this end,
we utilized thiophene boronic acid instead of furan boronic acid for the
aryl-thienyl bond formation by Suzuki reaction. The synthesis of phe-
nanthro[4,3-b]thiophene-4,5-dione5 (25) as the thiophene analogue of
the ABCD ring system of isotanshinone-II was achieved using the same
reaction sequence, which was previously employed in the synthesis of
“U” shaped furophananthraquinones starting from 3,4-Dihydro 1-(2-
thienyl)naphthalene-2-carbaldehyde25 (24) and 2-thiophene boronic
acid (Scheme 3).5 All the compounds were subsequently characterized
by standard spectroscopic evaluation (vide experimental).
–
–
–
functional group transformation CHO → CH2OH → CH2Cl →
–
CH2CN, synthesis of the nitrile derivative 33, a colourless solid was
achieved in very good to excellent yields in all steps. Alkaline hydrolysis
(KOH, EtOH-H2O, reflux) of the nitrile derivative produced the car-
boxylic acid 34 in 50% yield. The carboxylic acid (34) was then cyclised
(TFAA-TFA, 0 ◦C) to 9-methylnaphtho[1,2-b:7,8-b′]bisthiophen-4-ol
(35) in excellent yield. Finally oxidation of compound 35 with Fremy’s
salt produced the target compound 9-methylnaphtho[1,2-b:7,8-b′]bis-
thiophene-4,5-dione (36) as a deep violet solid in 77% yield. Following a
Fig. 2. Core nucleus of “S”, “U” shaped furophenanthraquinone and furonaphthoquinone.
3

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
similar sequence of reactions, synthesis of the regioisomeric compound
9-methylnaphtho[2,1-b:7,8-b′]bisthiophene-4,5-dione (43) was ach-
ieved in eight steps, starting from the bromoalfdehyde 29 and 3-thiophe-
neboronic acid (Scheme 6).
presence of the major functional groups on different analogues. The
calculated molecular weights of the individual compounds were estab-
lished by the presence of [M+1]+ or [M+Na]+ molecular ion peaks in
HRMS data. 1D NMR analysis confirmed the expected chemical shifts of
the different H and C present in each compound.
Anticancer activities of various naphthoquinone derivatives
condensed with heterocyclic moiety such as furan, dihydrofuran, thio-
phene, pyran among others have been widely reported and many
distinct mechanisms of action have been attributed to them.29,30
Importantly, cyclic ether-fused tricyclic naphthoquinones are major
pharmacophores due to their significant antibacterial as well as anti-
cancer properties.30 However, tricyclic naphthoquinones have not yet
been reported from natural sources. Nevertheless, a number of reports
indicated the synthesis of this class of theino/furonaphthoquinones.31–34
Notably, a number of naphthoquinones including naphtho[2,1-b]thio-
phene-4,5-diones, naphtho[1,2-b]thiophene-4,5-diones and naphtho
[2,3-b]thiophene-4,9-diones demonstrated potential anticancer activ-
ities.35,36 We have developed a synthetic route for the furonaph-
thoquinones as described earlier.24
General screening for in vitro anti-proliferative activities of
synthesized compounds: The growth inhibitory effects of all the eight
synthesized derivatives of natural tanshinones – compounds 1 (Tan-I)
and 2 (Tan-IIA) were initially assessed against two human breast cancer
cell lines i.e. MCF-7 and MDA-MB-231 using MTT assays to select the
most promising ones for further screenings and mechanistic evaluation.
While they have many phenotypic and genotypic variations, both MCF7
and MDA-MB-231 breast cancer cells are invasive in nature. However, as
compared to hormone dependent MCF7 cells (estrogen and progester-
one receptor positive; ER+PR+), MDA-MB-231 cells (triple negative,
ERꢀ PRꢀ Her2ꢀ ) represent
phenotype.37,38
a more aggressive form breast cancer
Cells plated in 96 well plate were either left untreated (DMSO con-
trol) or treated with various compounds with increasing concentrations
Next, we projected to synthesis two more thienonaphthoquinone
derivatives as a extension of our methodology starting from commer-
cially available methyl(2-bromophenyl)-acetate (44) and thio-
pheneboronic acids and subsequently achieved compounds 48 and 52
via a novel and considerably short synthetic route (Scheme 7 and
Scheme 8). As depicted in Scheme 7, the naphthoquinone 48, as a
thiophene analogue, simulates BCD rings of isotanshinone-II core nu-
cleus. Pd(0)-catalysed Suzuki coupling of the ester 44 with 2-thiophene-
boronic acid produced methyl 2-[2-(2-thienyl)phenyl]acetate (45) as a
pale yellow oil in good yield. In IR spectrum, it showed strong absorp-
(0–40 μM). 24 h post-treatment cells were subjected to MTT assay as
described in the Experimental Section to determine the cell viability.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
mediated cell-viability is a colorimetric assay, where the NADPH-
dependent oxidoreductase enzymes released from viable cells convert
the yellow tetrazolium dye to insoluble purple formazan crystals. The
absorbance measured at 540 nm of the purple crystals solubilized in
DMSO indicate the percentage of viable cells. Subsequently IC50 values
were calculated for each compound in both cell lines and are summa-
rized in Table 1. Overall, the results confirmed that furophenan-
thraquinone could serve as a potential scaffold for new anticancer agents
with a cytotoxic activity in the micromolar range. MTT assay revealed
that two naphthoquinone derivatives 48 (IC50 = 1.94 in MCF7 and 2.59
tion at 1738 cmꢀ 1 for the CO2CH3 functionality. 1H NMR, 13C NMR
–
and HRMS data also justified the assigned structure (vide experimental).
Subsequent hydrolysis of 45 to the carboxylic acid 46, followed by
cyclisation to naphtho[1,2-b]thiophen-4-ol (47) was achieved in excel-
lent yield. The carboxylic acid 46 exhibited a strong absorption band
μM in MDA-MB-231) and 52 (IC50 = 1.85 in MCF7 and 2.95 μM in MDA-
1
around 1693 cmꢀ 1 for the presence of CO2H group. H NMR, 13C NMR
MB-231), bearing thiophene moieties, induced most significant growth
inhibition compared to the rest of the compounds, in both breast cancer
lines (Table 1). In both cell lines, these derivatives were found to be
around 4-fold more potent than the natural tanshinones – compounds 1
–
and HRMS analysis also justified the assigned structure of the compound
47.
Oxidation of the intermediate phenol 47 with Fremy’s salt furnished
the target molecule 48 in very good yield (scheme 8). In IR spectrum, the
compound 48 exhibited a broad absorption band around 1659 cmꢀ 1 for
the presence of carbonyl groups. 1H NMR (400 MHz, CDCl3) spectral
data of the compound 48 is in conformity with the assigned structure.
13C NMR [(100 MHz, CDCl3): δ = 124.72, 126.08, 127.80, 129.10,
129.73, 130.28, 132.41, 135.85, 136.55, 151.27, 173.86, 180.47 ppm]
as well as HRMS data [(ESI+): m/z [M+H]+ calcd for C12H7O2S:
(IC50 = 7.92 in MCF7 and 7.96 μM in MDA-MB-231) and 2 (IC50 = 7.79
in MCF7 and 7.79 μM in MDA-MB-231). While compound 27, another
thiophene analogue, retained anti-proliferative activity parallel to those
of the parent tanshinones (1, 2) in MCF7 and MDA-MB-231, with IC50 of
8.02 and 6.00 μM, respectively, the other five compounds 15, 23, 25, 36
and 43 were remarkably less potent (~2–3-fold) compared to the nat-
ural tanshinones with IC50 range from 12.79 to 23.43 µM in both cell
lines.
215.0089; found: 214.9340 and [M+Na] calcd for
C12H6O2SNa:
236.9986; found: 236.9225] also supported the structure.
Compounds 48 and 52 exhibit anti-proliferative activities by
inducing cell apoptosis: The two most potent thiophene analogues
compounds 48 and 52 along with compound 27 and two naturally
occurring tanshinone molecules (1 and 2) were selected for further an-
alyses. In order to corroborate compounds 48 and 52 mediated
enhanced anti-proliferative activities, alterations of cell morphology
were observed in a phase contrast microscope in both MCF7 and MDA-
MB-231 lines at different time points (0, 6, 12 and 24 h) at 2.5 µM
concentration (Fig. S53A-B). Both compounds 48 and 52 treatment
caused a drastic difference in cell death percentage in comparison to that
Following a similar reaction sequence starting from same ester 44
and 3-thiopheneboronic acid, the synthesis of naphtho[2,1-b]thiophene-
4,5-dione (52) was accomplished successfully (Scheme 8).
In sum, we successfully synthesized a total of eight novel phe-
nanthtraquinone derivatives with thiophene and furan moieties as tan-
shinone analogues including compounds 15, 23, 25, 27, 36, 43, 48 and
52. The structures of synthesized analogues were confirmed using
relevant spectroscopic techniques – FT-IR, 1H NMR, 13C NMR and
HRMS. Fourier-transform infrared (FT-IR) analysis confirmed the
Scheme 1. Synthesis of phenanthro[2,1-b]furan-10,11-dione (15).
Reagents and conditions: (i) 3-Furanboronic acid, TEA, Pd(PPh3)4, DMF, 110 ◦C, 7 h, 70%. (ii) 20% aqueous KOH solution, methanol, reflux, 9 h, 74%. (iii) TFAA,
TFA, r.t, 12 h, 75%. (iv) Fremy’s salt, 1/6 M aqueous solution of Na2HPO4, MeOH, 0 ◦C to r.t, 12 h, 57%.
4

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
Scheme 2. Synthesis of Phenanthro[3,4-b]furan-4,5-dione (23).
Reagents and conditions: i) 3-furanboronic acid, Pd(PPh3)4 (1 mol%), Et3N, DMF, 100 ◦C, 10 hrs under Aratm., 61%; ii) NaBH4, EtOH, r.t., 2 hrs, 97%; iii) MsCl, s-
collidine, LiCl, DMF, 0 ◦C-r.t., overnight; iv) KCN, DMF, 7 hrs, 76%; v) Aq. KOH, EtOH, reflux, 18 hrs, 82%; vi) TFAA-TFA, 0 ◦C-r.t., overnight, 83%; vii) Fremy’s salt,
Na2HPO4-H2O, MeOH, overnight, 77%.
of natural tanshinones Tan-I (1) and Tan-IIA (2) (Fig. S53A-B). In
agreement with the MTT assays, compounds 48 and 52 treatment
resulted in significant reduction in cell number and size of both MCF7
and MDA-MB-231 cells in a time dependent manner (Fig. S53A and
S53B, respectively). In contrast, compound 27 showed similar activity
as that of the parent compounds 1 and 2 (Fig. S53A-B).
To determine whether the cytotoxic effect of compounds 48 and 52
on breast cancer cells was induced by apoptosis, cells were either left
untreated (DMSO control) or treated with 2.5 μM compounds 1, 2, 27,
48 and 52 for 24 h, stained with 1:1 solution of ethidium bromide (EtBr)
and acridine orange (AO) and viewed in a fluorescence microscope.
While AO stains live cells as green, EtBr stains the DNA as red in case of
apoptosis and necrosis. Therefore, apoptotic cell are observed as distinct
red nucleus in live green cells, while non-viable or necrotic cells are fully
stained red. The representative microscopy images of each panel
demonstrated a significant increase of cell death in response to com-
pounds 48 and 52 treatment compared DMSO control and other com-
pounds including 1, 2 and 27 (Fig. S53C). The percentage of apoptotic
and necrotic cells were calculated with respect to live cells and plotted as
bar diagrams (Fig. S53D). As expected, the results demonstrated that
compounds 48 and 52 induced cell death ~2-fold more than that of Tan-
IIA (Fig. S53E). Compound 27 showed similar activity as that of Tan-
IIA, while at this concentration Tan-I did not show any cell death
(Fig. S53D).
Fig. 3. Synthetic strategy for “U” shaped furophenanthraquinone nucleus.
Scheme 3. Synthesis of Phenanthro[4,3-b]thiophene-4,5-dione (25).
To further validate this data, the cell apoptosis assay was performed
by Annexin V/PI staining of drug treated cells using a similar experi-
mental setup. The percentage of overall apoptotic cells was determined
from the sum of early apoptosis (Annexin V+/PIꢀ ) and late apoptosis
(Annexin V+/PI+) in both MCF7 and MDA-MB-231 cell lines (Fig. 5A
and 5B, respectively). In agreement with the previous results, com-
pounds 48 and 52 exhibited significant stronger effects (>2–20 folds) on
apoptotic induction as compared to the natural tanshinones – com-
pounds 1 and 2 along with compound 27 in both cell lines (Fig. 5A and
5B). Although compounds 48 and 52 treatment showed comparable
total cell death (74.7% and 77.1% in MCF7; 66.3% and 66.4% in MDA-
MB-231), compound 52 induced more apoptosis than that of compound
48 (37.0% vs. 54.9% in MCF7; 22.8% vs. 39.6% in MDA-MB-231) (Fig.
5A and 5B, bar diagrams). Interestingly, the results indicated that
among the two natural tanshinones, compound 1 (Tan-I) distinctively
induced apoptosis in MCF7 line (15.21% vs. 1.62%), in contrast com-
pound 2 (Tan-IIA) mediated apoptosis was specific to triple negative
breast cancer line (9.57% vs. 3.21%) (Fig. 5A and 5B, bar diagrams).
Importantly, in addition to cell apoptosis compounds 48 and 52 also
demonstrated a significant increase in other types of cell death including
necrosis (Fig. 5A and 5B). To re-validate compounds 48 and 52
mediated enhanced apoptotic induction, western blot analyses was
performed against cleaved PARP1, a hallmark of apoptosis (Fig. 5A). As
Scheme
4. Synthesis
of
10-methoxyphenanthro[3,4-b]thiophene-4,5-
dione (27).
Fig. 4. Bis(heteroannulated)naphthoquinones 36 and 43.
5

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Bioorganic & Medicinal Chemistry 37 (2021) 116112
Scheme 5. Synthesis of 9-methylnaphtho[1,2-b:7,8-b′]bisthiophene-4,5-dione (36).
Reagents and Conditions: i) PBr3, DMF, CHCl3, 0 ◦C-r. t., 16 hrs, 73%; ii) 2-thiopheneboronic acid, Pd(PPh3)4, Et3N, DMF, 110 ◦C, N2 atm., 10 hrs, 61%; iii) DDQ,
benzene, reflux, 24 hrs, 86%; iv) NaBH4, EtOH, r.t., 2 hrs, 91%; v) MsCl, LiCl, s-collidine, DMF, 0 ◦C overnight; vi) KCN, DMF, 11 hrs, 84%; vii) KOH, EtOH-H2O
reflux, 20 hrs, 50%; viii) TFAA-TFA, 0 ◦C overnight, 80%; ix) Fremy’s salt, 1/6M Na2HPO4 buffer solution, MeOH, 0–5 ◦C, overnight, 77%.
Scheme 6. Synthesis of 9-methylnaphtho[2,1-b:7,8-b′]bisthiophene-4,5-dione (43).
Reagents and Conditions: i) 3-thiopheneboronic acid, Pd(PPh3)4, Et3N, DMF, 110 ◦C, N2 atm., 10 hrs, 67%; ii) DDQ, benzene, reflux, 24 hrs, 89%; iii) NaBH4, EtOH,
r.t., 2 hrs, 94%; iv) MsCl, LiCl, s-collidine, DMF, 0 ◦C overnight; v) KCN, DMF, 11 hrs, 89%; vi) KOH, EtOH-H2O reflux, 20 hrs, 58%; vii) TFAA-TFA, 0 ◦C overnight,
93%; viii) Fremy’s salt, 1/6M Na2HPO4 buffer solution, MeOH, 0–5 ◦C, overnight, 83%.
similar to Annexin/PI staining, compound 52 and to a lesser extent (~2-
fold) compound 48 treatments led to a marked increase of PARP
cleavage in both cell lines (Fig. 5C and 5D).
death mechanisms often arise concurrently that includes ‘programmed
cell death type I’ or apoptosis and ‘programmed cell death type II’ or
autophagy. While apoptosis is the primary cell death mechanism,
autophagic pathway in general plays a cytoprotective role through
which cancer cells avoid various deleterious effects caused by
Compounds 48 and 52 induce both apoptosis and autophagy: In
response to several anticancer therapies, two essential forms of cell
Scheme 7. Synthesis of naphtho[1,2-b]thiophene-4,5-dione.
Reagents and conditions: (i) 2-thiopheneboronic acid, Et3N, DMF, Pd(PPh3)4, 110 ◦C, N2 atm., 6 hrs, 65%; (ii) KOH, EtOH, H2O, reflux, 12 hrs, 78%; (iii) TFAA-
TFA, 0 ◦C, overnight, 80%; (iv) Fremy’s salt, 1/6 M aq. Na2HPO4, MeOH, r.t., overnight, 72%.
Scheme 8. Synthesis of naphtho[2,1-b]thiophene-4,5-dione.
Reagents and conditions: (i) 3-thiopheneboronic acid, Et3N, DMF, Pd(PPh3)4, 110 ◦C, N2, 5 hrs, 72%; (ii) KOH, EtOH, H2O, reflux, 10 hrs, 83%; (iii) TFAA-TFA,
0 ◦C, overnight, 75%; (iv) Fremy’s salt, MeOH, 1/6 M aq. Na2HPO4, r. t., overnight, 86%.
6

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Bioorganic & Medicinal Chemistry 37 (2021) 116112
Table 1
IC50 values of tanshinone derivatives against breast cancer lines.#
Compounds
IUPAC name
IC50
(μ
M) (MCF7)
IC50 (μM) (MDA-MB231)
1
1,6-dimethylnaphtho[1,2-g][1]benzofuran-10,11-dione
1,6,6-trimethyl-8,9-dihydro-7H-naphtho[1,2-g][1]benzofuran-10,11-dione
Phenanthro[2,1-b]furan-10,11-dione
7.927 ± 0.37
7.7935 ± 1.27
14.411 ± 2.65
15.7625 ± 3.062
12.7865 ± 0.81
8.02 ± 0.91
7.962 ± 1.28
7.695 ± 0.176
20.875 ± 1.68
17.45 ± 3.12
15.61 ± 3.01
6.00 ± 0.28
2
15
23
25
27
36
43
48
52
Phenanthro[3,4-b]furan-4,5-dione
Phenanthro[4,3-b]thiophene-4,5-dione
10-methoxyphenanthro[3,4-b]thiophene-4,5-dione
9-methylnaphtho[1,2-b:7,8-b’]bisthiophene-4,5-dione
9-methylnaphtho[2,1-b:7,8-b’]bisthiophene-4,5-dione
naphtho[1,2-b]thiophene-4,5-dione
17.5225 ± 0.53
14.566 ± 2.85
1.945 ± 0.06
1.8565 ± 0.10
23.4285 ± 7.84
20.565 ± 2.59
2.595 ± 1.03
2.955 ± 1.18
naphtho[2,1-b]thiophene-4,5-dione
#
Cells were treated with indicated compounds for 24 h and the cell viability was determined by MTT assay. Each experiment was independently repeated three
times.
chemotherapeutic agents.39–41 Accordingly, in many human cancers
viewed under confocal microscope (Fig. 6B). Live cell confocal micro-
scopy revealed drastic amplification of LC3 puncta formation (lipidated
LC3-II attached with the autophagosmal membrane) coincided with
lysosomes in response to all three tanshinone derivatives compounds 27,
48 and 52 treatment as compared to DMSO control and two parent
natural tanshinones, compounds 1 and 2 (Fig. 6B), indicating that
tanshinone analogues might deregulate autophagy pathway.
including breast cancer autophagy induction potentially contributes to
attain drug resistance through counteracting apoptosis triggered during
chemotherapy.41–43 Research suggests that autophagy induction ap-
pears to play a dual role in both luminal and triple negative breast
cancers.44 For example, tetrandrine, a bis-benzylisoquinoline alkaloid,
promotes autophagy mediated cell death in MCF7 line.45 Additionally, a
growing body of evidence suggested that natural tanshinones and their
derivatives induce cell death in multiple cancer cell lines through
manipulating autophagy pathway.46–49 Mechanistically, both apoptosis
and autophagy cascades share regulatory mechanisms essentially
controlled by Bcl-2 family members, where anti-apoptotic proteins Bcl-2
(BCL2) or Bcl-xL (BCL2L1) block apoptosis and autophagy by seques-
tering primarily two other family members BH3-only proteins - pro-
apoptotic Bim (BCL2L11) and pro-autophagic Beclin 1 (BECN1).50–53
Collectively, Bim and Beclin 1 appear to play central roles in regulating
the balance between autophagy and apoptosis, which in turn decides
cancer cell death or cell survival in response to chemotherapeutic
agents.50
In response to various stresses autophagy is activated independent or
dependent of transcriptional regulation.57,59,60 In order to determine the
underlying mechanisms of compounds 48 and 52 induced autophagy
pathway, real time PCR analyses were carried out using total RNA
extracted from MCF7 cells either left untreated (DMSO control) or
treated with 2.5 μM tanshinones and three derivatives for 24 h (Fig. 6C).
The real time PCR profiled a total of thirty four key regulatory genes that
are critically involved in regulating the autophagy pathway as described
in Table S1. The primer sequences for this study are listed in Table S2.
The relative expression (ΔCt value) of each transcript from average
value of three housekeeping genes including ACTB, B2M and GAPDH
was represented as heatmap (log2) using 2ꢀ ΔΔCt method after normali-
zation of the raw data in respect to DMSO treated MCF7 cells (Fig. 6C).
The results demonstrated that as similar to western blot analyses,
treatment of all three tanshinone derivatives compounds 27, 48 and 52
along with Tan-IIA (2) transcriptionally activated pro-apoptotic Bcl-2
family member BCL2L11 gene expression (Fig. 6C). Among other pro-
apoptotic Bcl-2 family members, although to a lesser extent, treat-
ments of compounds 48 and 52 led to a specific transcriptional activa-
tion of BAD gene (Fig. 7C). In general, compound 48 as compared
compound 52 appeared to be more potent transcriptional activator. A
number of autophagy related genes including ATG16L1, ATG16L2,
GABARAP, GABARAPL1, GABARAPL2, PIK3CG, UVRAG involved in
autophagosome biogenesis were significantly up-regulated in com-
pounds 48 and 52 treated cells (Fig. 6C). In addition, compound 48 and
52 treatment transcriptionally activated two unfolded protein response
(UPR) genes DDIT3 (CHOP) and PPP1R15A (GADD34) (Fig. 6C). Taken
together, the above results suggested that compounds 48 and 52 might
induce cell death by promoting autophagy flux through transcriptional
activation of genes involved in autophagosome formation under stress
conditions.
The previous results indicated that compounds 48 and 52 caused cell
death partly by apoptotic induction. We next wondered that whether
compounds 48 and 52 induced cell apoptosis through mitochondrial
regulation of Bcl-2 family member proteins (Fig. 6A). While there was
no change of pro-apoptotic Bcl-2 proteins including Bax and Bak along
with anti-apoptotic Bcl-2 proteins such as Bcl-2 and Bcl-xL, the relative
level of pro-apoptotic Bim expression was dramatically increased in
response to all three tanshinone derivatives compounds 27, 48 and 52 in
MCF7 cells (Fig. 6A). Furthermore, compounds 48 and 52, but not 27
induced the pro-autophagic Bcl-2 family member protein Beclin 1 (Fig.
6A). Upon various cellular insults, Beclin 1 initiates autophagosme
formation54 and thus it does not indicate true completion of autophagy
pathway (known as autophagy flux), where the sequestered cytoplasi-
mic materials are degraded after fusion between autophagosomes and
lysosomes.55 When autophagy is triggered, cleavage of LC3-I to LC3-II
occurs with the assistance of ATG3 and ATG7. Accumulation of lipi-
dated LC3-II leads to formation of a complex of ubiquitin-like system
consisting of ATG5, ATG12 and ATG16, which further helps in the
autophagosome formation. p62 (SQSTM1) is an adaptor protein, which
binds to the autophagosomal membrane along with LC3-II and delivers
the ubiquitinated cytoplasmic cargo for degradation within the autoly-
sosome (a fusion between autophagosome and lysosomal structure).55,56
Moreover, p62 is itself degraded and represents a good indicator for
autophagy flux.56–58 Western blot analyses clearly demonstrated that
compounds 27, 48 and 52 treatment in MCF7 cells significantly
enhanced ratio of LC3-II:LC3-I (densitometric analyses of LC3-II cleav-
age over LC3-I) as well as p62 degradation (Fig. 6A). To further sub-
stantiate this data, MCF7 cells transiently transfected with GFP-tagged-
LC3 expressing plasmid were either left untreated (DMSO control) or
Compounds 48 and 52 transcriptionally activate UPR regulators
in MCF7 cells: Owing to aberrant cell proliferation as well as in
response to various cell stresses such as growth factor deprivation and
chemotherapy, cancer cells often encounter endoplasmic reticulum
stress as a consequence of accumulation of mis-folded and un-folded
proteins.61,62 This initiates an Unfolded Protein Response (UPR) trig-
gered by three tarnsmembrane receptors - PERK (EIF2AK3), ATF6 and
IRE1 (ERN1) signaling cascades.61,63 In order to alleviate the UPR
response, autophagy is subsequently activated in which the mis-/un-
folded protein aggregates are degraded and allows cancer cells to grow
in such a hostile conditions by providing free amino acids as energy
resource.61,64 However, in case of prolonged stress conditions, UPR
treated with 2.5
μM tanshinones and their three analogues. 24 h post-
treatment the cells were stained with lyso-tracker and hoechest and
7

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Bioorganic & Medicinal Chemistry 37 (2021) 116112
Fig. 5. Cell death induced by tanshinones (1 and 2) and their derivatives - compounds 27, 48 and 52: ~0.5 × 105 (A) MCF7 and (B) MDA-MB-231 cells plated into
each well of six-well plates were either left untreated (DMSO control) or treated with 2.5 μM natural tanshinones (compounds 1 and 2) and their three derivatives
(compounds 27, 48 and 52) for 24 h were subjected to apoptosis assay using annexin V/propidium iodide (PI) staining. FITC labeled annexin V binding (Ex = 488
nm; Em = 350 nm) and PI staining were detected using BD Accuri C6 Flow Cytometry Analyzer. Error bars represent standard deviations of duplicate assays of two
independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. ~5 × 106 (C)**** MCF7 and (D) MDA-MB-231 cells treated with drugs using a
similar experimental set up, were subjected to western blot analyses with the indicated antibodies. GAPDH blot was used as loading control and protein bands were
quantified by Odyssey imager software and indicated as bar diagrams at the bottom of corresponding lanes.
triggers apoptotic cell death by activating a number of death regula-
biogenesis and to maintain autophagy flux.71 As described in Fig. 6C,
compounds 48 and 52 mediated specific transcriptional activation of
CHOP (DDIT3) and GADD34 (PPP1R15A) as well as to comprehend
possible mechanism of dual apoptosis and autophagy induction, led us
to further explore the overall transcriptional changes in UPR and related
pathway in response to these compounds. To this end, total RNA was
isolated from MCF7 cells with or without the treatment with compounds
48 and 52 for 24, cDNA was prepared and subjected to PCR microarray
analyses (Fig. 7A), which profiles a total of 84 key regulatory genes that
are critically involved in regulating the UPR signalling cascade as
described in Table S3. As described in the Experimental Section, in
response to compounds 48 and 52 treatment, the relative expression
(ΔCt) of each transcript from average value of five housekeeping genes
(ACTB, B2M, GAPDH, HPRT1 and RPLP0) in the array platform was
represented as heatmap (log2) using 2ꢀ ΔΔCt method after normalization
tors.63,65 PERK-mediated eIF2
α phosphorylation leads stabilization of
the transcription factor ATF4, which in turn triggers transcriptional
activation of several genes involved in both autophagy and
apoptosis.66,67 Among these, CHOP (CEBP (CCAAT/enhancer-binding
protein) homologous protein/growth arrest and DNA-damaged-
inducible protein 153 or GADD153) transcription factor plays a dual
role in both inducing apoptosis and limiting autophagy through the
transcriptional control of specific downstream target genes in response
to cellular stress signals.68,69 In contrast, PPP1R15A (Growth arrest and
DNA damage-inducible protein 34 or GADD34), which is also activated
by PERK-eIF2α-ATF4 pathway, negatively regulates this cascade via a
feedback mechanism by inducing eIF2
α
dephosphorylation.70 More-
over, GADD34 plays an essential role in autophagy during stress con-
ditions and initiates translation of several genes required for lysosomal
8

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Bioorganic & Medicinal Chemistry 37 (2021) 116112
Fig. 6. Compounds 48 and 52 induce both apoptosis and autophagy: (A) ~5 × 106 MCF7 cells either left untreated (DMSO control) or treated with 2.5
μM natural
tanshinones (compounds 1 and 2) and their three derivatives (compounds 27, 48 and 52) for 24 h were subjected to western blot analyses with the indicated
antibodies. Protein band intensities in were quantified by Odyssey imager software and indicated either as bar diagrams or LC3-II/I ratio at the bottom of each
corresponding lane. Representative gel pictures are shown of two independent experiments. GAPDH blot was performed as loading control. (B) ~1 × 106 MCF7 cells
grown at 70% confluency on 35 mm dish embedded with 12 mm glass viewing areatransiently transfected with GFP-LC3 expressing construct were either left
untreated (DMSO control) or treated with 2.5 µM drugs for 24 h as stated above and subjected for live cell confocal microscopy. Nuclei and lysosomes were
counterstained with Hoechst 33,342 and LysoTracker Red DND-99, respectively. Each panel of confocal images is representative pictures of two independent ex-
periments. Scale bars, 5 μm. The bar diagrams below represent the mean value of LC3 puncta counting from at least 10 cells of 3 different fields. Error bars represent
standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001, respectively. ns = non significant. (C) Using
the similar experimental set up as described in (A), total RNA was isolated and subjected to cDNA preparation followed by quantitative real-time PCR (qPCR)
analyses for the selected cell genes. The relative changes in transcripts (log2) using the 2ꢀ ΔΔCt method are represented as heatmap in comparison to DMSO control
using GAPDH, B2M and RPLPO as housekeeping genes. Two independent experiments were carried out in similar settings and results represent as an average value
for each transcript.
of the raw data in respect to DMSO treated cells (Fig. 7A). The results
demonstrated that along with DDIT3 and PPP1R15A, several UPR reg-
ulators particularly the IRE1 signalling cascade were transcriptionally
activated in response to the drug treatment (Fig. 7A). Western blot
analyses further validated the expressions of ATF4, CHOP and GADD34
in drug treated cells as compared to the DMSO control (Fig. 7B).
Overall, the results revealed that compounds 48 and 52 stimulate cell
death by manipulating autophagy-UPR network particularly inducing
expression of several death regulators – GADD34 and CHOP.
in a dose dependent fashion (Fig. 8A and 8C). In order to establish the
notion of autophagy mediated cell death, MCF7 cells treated with 2.5 µM
either compound 48 or 52 were further treated with an autophagy in-
hibitor, chloroquine (CQ) (Fig. 8B and 8D). The anti-malarial drug CQ
blocks autophagy flux by escalating the lysosomal acidic pH and blocks
the fusion between autophagosome and lysosome, which in turn leads to
inhibition of p62 degradation and accumulation of lipidated LC3-II
forms.58,72 In fact, the results demonstrated that CQ co-administration
resulted in significant reduction (~2-fold) of cell apoptosis (decreased
cleaved PARP1 expression) induced by either compounds 48 or 52 alone
(Fig. 8B and 8D). Effect of CQ, was further demonstrated by accumu-
lation of autophagy markers both p62 and LC3 cleavage in western blot
analyses (Fig. 8B and 8D). Additionally, live cell confocal microscopy
analyses of MCF7 cells transiently co-transfected with plasmids
Compounds 48 and 52 promote autophagy mediated cell death:
Hitherto, the results pointed towards a model that compounds 48 and 52
might regulate cell death through enhancing autophagy flux. Indeed,
both compounds 48 and 52 induced apoptosis (PARP cleavage) and
autophagy flux (LC3-II conversion and p62 degradation) simultaneously
9

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Bioorganic & Medicinal Chemistry 37 (2021) 116112
Fig. 7. Compounds 48 and 52 transcriptionally
activate UPR regulators: ~10 × 106 MCF7 cells
either left untreated (DMSO control) or treated
with 2.5 μM compounds 48 or 52 for 24 h, were
harvested for either total RNA extraction fol-
lowed by cDNA generation according to manu-
facturer’s protocol or protein extraction for
western blot analyses. (A) Amplified cDNAs were
then subjected to PCR-microarray analyses for
“Unfolded Protein Response (UPR)” pathway
from SABiosciences in accordance with manu-
facturer’s instructions. The relative changes in
transcripts (log2) using the 2ꢀ ΔΔCt method were
represented as a heat map in comparison to
control cells taking average of five housekeeping
genes ACTB, B2M, GAPDH, HPRT1 and RPLP0.
Data analysis (algorithm provided by the manu-
facturer) also incorporated the quality check to
include or exclude the real-time PCR data points
for individual genes. Two independent experi-
ments were carried out in similar settings and
results represent as an average value for each
transcript. (B) Western blot analyses were carried
out using the indicated antibodies. Protein band
intensities (pixels) in each lane were quantified
by Odyssey imager software and presented as
either by bar diagrams at the bottom of each
picture.
expressing GFP-tagged LC3 and mCherry-tagged p62 in response to in-
dividual treatment of tanshinone analogues and CQ clearly revealed that
both compounds 48 and 52 significantly induced distinct puncta pattern
of GFP-LC3 molecule co-localized with reduced mCherry-p62 expression
as compared CQ treatment alone (Fig. 8E).
appreciation of natural products in drug discovery together with po-
tential anticancer properties of natural tanshinones, the markedly
improved anti-proliferative activities of tanshinone analogues - com-
pounds 48 and 52 merit further meticulous investigations for successful
development as anticancer agents. In addition to elucidating the un-
derlying molecular mechanism of autophagy mediated cell death,
thorough in vivo studies need to be conducted for further validation as
well as to establish the optimal dose for potential therapeutic applica-
tion. Moreover, toxicological studies should be performed to envisage
both short-term and long-term possible side effects. Furthermore,
studies to assess bioavailability, pharmacokinetics, and pharmacody-
namics need be carried out in order to ultimately develop the two
thiophene analogues as anticancer drugs against various subtypes of
breast cancers.
In order to further establish whether these thiophene analogues of
natural tanshinones could induce autophagy mediated cell death, we
performed cell viability assays in both MCF7 and MDA-MB-231 cells
individually treated with 2.5 µM compounds 48 and 52 with increasing
doses (25, 50 and 100 µM) of CQ for 24 h (Fig. 8F and 8G). Although CQ
treatment alone caused cell death due to its well-known cytotoxity at
higher doses (100 µM), it significantly inhibited cell-killing activities
exerted by compounds 48 and 52 in a dose dependent fashion (Fig. 8F
and 8G). In agreement to this, previously Tan-IIA demonstrated auto-
phagy mediated cell death in human osteosarcoma MG-63 cells at higher
doses and the effect could be somewhat alleviated in the presence of
CQ.7 Taken together, the results above evidently demonstrated that the
two novel thiophene analogues - compounds 48 and 52 are potent
autophagy inducers and thereby promoting cell death.
4. Experimental section
General Procedures: All reactions were carried out using oven-
dried or flame dried clean glass wares. All reagents were purchased
from commercial suppliers in the highest purity grade available and
used without further purification. The solvents, chloroform (CHCl3),
dichloromethane (DCM), diethyl ether (Et2O), ethanol (EtOH), dime-
thylformamide (DMF), Benzene, ethanol (EtOH), methanol (MeOH),
Petroleum ether (P.E) and ethyl acetate (EtOAc) were purchased from E.
Merck (India) Ltd. or SRL (India) Ltd. and distilled prior to use. P.E refers
to the fraction boiling in the range 60–80 ◦C. All common solvents and
reagents (CHCl3, DMF, Et3N, benzene, s-collidine, etc.) were dried as
per literature procedure. Furanboronic acids, thiophene boronic acids,
1-tetralone, 4-methyl-1-tetralone, 6-methoxy-1-tetralone, cyclohexa-
none, cyclopentanone, cyclooctanone, 4-tert-butylcyclohexanone, tet-
rakis(triphenylphosphine)palladium(0), 2,3-dichloro-5,6-dicyanobenz
oquinone (DDQ), CDCl3 and DMSO‑d6 were purchased from Sigma
Aldrich (now Merck). Trifluoroacetic anhydride (TFAA), trifluoroacetic
acid (TFA), phosphorous tribromide, Pd/C, sodium borohydride
(NaBH4), Mesyl chloride and s-collidine were purchased from
3. Conclusions
In sum, we successfully synthesized a total of eight novel phe-
nanthtraquinones derivatives with thiophene and furan moieties as
tanshinone analogues through utilizing diversity-oriented synthesis73, a
synthetic approach that engages efficient incorporation of multiple
molecular scaffolds in the library for the discovery of novel biologically
active small molecules. Some modifications were made in the ring A or
both ring A and D of the core nucleus present in natural occurring
furophenanthraquinone: Isotanshinone-II. In addition, we also prepared
two novel thienonaphthoquinone derivatives 48 and 52 by simulating
BCD ring of Isotanshinone-II core nucleus via a novel synthetic route.
Overall, our study supports the notion of development of two thiphene
analogues - compounds 48 and 52 towards the treatment of breast
cancer through modulating autophagy pathway. Given the growing
10

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
Fig. 8. Compounds 48 and 52 promote autophagy mediated cell death: ~5 × 106 MCF7 cells were either left untreated (DMSO control) or treated with increasing
doses (0, 1.25, 2.5 and 5 μM) of compounds (A) 48 or (C) 52. Using a similar experimental setting, MCF7 cells were either individually treated with 2.5 μM
compounds (B) 48 or (D) 52 or in combination with 50 μM Chloroquine (CQ). (A-D) 24 h post-treatment cells were harvested and subjected to western blots were
performed with the indicated antibodies. GAPDH blot was performed as loading control. The relative intensities of LC3 protein bands were quantified using the
software provided by Odyssey CLx Imaging System and shown as LC3-II/I ratio at the bottom of LC3 blot Representative gel pictures are shown of at least two
independent experiments. (E) ~1 × 106 MCF7 cells transiently transfected with constructs expressing GFP-LC3 and mCherry-p62 for 24 h were left untreated (DMSO
control) or treated with 2.5 μM compounds 48 or 52 or 50 μM CQ for another 24 h and subjected to live cell confocal microscopy after staining the nucleus with
Hochest 33342. Each panel in (E) corresponds to single experiment of two independent experiments. Scale bars, 5
plated into each well of six-well plates were either left untreated (DMSO control) or treated with 2.5
μ
m. (F-G) ~0.5 × 105 MCF7 and MDA-MB-231 cells
μ
M compounds (F) 48 or (G) 52 in the presence and absence of
increasing concentrations (25, 50 and 100 μ
M) of CQ for 24 h at 37 ◦C in a humidified CO2 chamber and subjected cell viability assays using MTT as described in the
Experimental Section. Error bars represent standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001,
respectively. ns = non significant.
Spectrochem, India. Fremy’s salt (potassium nitrosodisulfonate) was
prepared in the laboratory as per literature procedure. Anhydrous so-
dium sulfate (Na2SO4) was used for drying solutions.
JMSAX505HA. High-resolution-mass spectra (HRMS) were recorded on
either a TRACE GC ULTRA POLARIS Q or a Waters Qtof Micro YA263
mass spectrometer. All compounds tested in biological assays are >95%
pure.
Purification by column chromatography was done using silica gel
(100–200 mesh, 230–400 mesh) (E. Merck, India or SRL) using P.E
60–80 ◦C and EtOAc as eluents, unless otherwise mentioned. Pre-coated
silica gel 60 GF254 TLC sheets on aluminium plate (E. Merck, Germany)
were used for thin-layer chromatography (TLC). Melting points were
recorded in open capillaries using conc. H2SO4 bath or electrical melting
apparatus and are uncorrected.
(2-Furan-3-yl-naphthalen-1-yl)-acetic acid methyl ester (12):
0.16 g (1.42 mmol) 3-furanboronic acid, 1 ml (7.2 mmol) triethylamine
and 0.016 g (1–2 mol%) Pd(PPh3)4 were added to a solution of 0.300 g
(1.08 mmol) compound 11 in 3 ml DMF. Now, the reaction mixture was
allowed to heat at 110 ◦C with constant stirring under argon atmosphere
for about 7 hr. After cooling to room temperature, it was poured into ice
cold water and extracted with ether. The ether layer was washed with
10 ml ice cold 5% NaHCO3 solution followed by 10 ml ice cold 5% brine
solution. It was collected and dried over anhydrous Na2SO4 and solvent
was removed. The collected compound was purified by column chro-
matography over silica gel (230–400 mesh) (EtOAc: P.E = 10:90). The
compound solidified in presence of ethanol (yield 0.200 g, 70%). M.p:
75–77 ◦C. IR: 1750 cmꢀ 1. 1H NMR (400 MHz, CDCl3): δ 7.90–7.92 (m,
1H), 7.72–7.80 (m, 2H), 7.56–7.57 (m, 1H), 7.37–7.50 (m, 4H), 6.54 (d,
Infrared spectra were recorded on KBr pellets using a Shimadzu
Fourier Transform
(FT-IR) 8300 spectrophotometer to confirm the presence of the main
functional groups present in each of the synthesized tanshinone analogs.
1H NMR (500 MHz or 400 MHz or 300 MHz) and 13C NMR (125 MHz or
100 MHz or 75 MHz) spectra were recorded on a Bruker AVANCE III
500 MHz or 400 MHz or 300 MHz NMR spectrometer using tetrame-
thylsilane as the internal standard. The ESIMS was recorded on a JEOL
11

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
1H, J = 2.4 Hz), 4.11 (s, 2H), 3.64 (s, 3H). MS (QTOF ES+): found,
267.0729 [M+H]+; calcd for C17H15O3, 267.1021.
755, 3428. 1H NMR (400 MHz, CDCl3): δ 4.663 (s, 2H), 6.371 (d, 1H, J =
1.6 Hz), 7.24–7.44 (m, 4H), 7.436 (d, 1H, J = 1.8 Hz), 7.55–7.65 (m,
2H), 7.713 (d, 1H, J = 8.0 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ =
62.3, 108.8, 124.8, 125.7, 127.2, 127.4, 127.5, 128.4, 138.5, 142.9,
129.3, 132.0, 132.1, 133.7, 134.7 ppm.
3-(2-Furan-3-yl-naphthalen-1-yl)-acetic acid (13): 1 ml of 20%
aqueous KOH solution was added to a solution of 0.20 g (0.75 mmol) of
the compound 12 in 3 ml ethanol and was allowed to reflux for 9 hr. The
reaction mixture was quenched with some crushed ice water, acidified
with cold concentrated HCl and filtered. The collected residue was dis-
solved in 5–10 ml saturated solution of NaHCO3 and further acidified
with cold conc. HCl. The white solid appeared was filtered, the residue
was washed with ice cold water and collected (0.14 g, 74%) M.P: 160 ◦C.
IR: 1690 cmꢀ 1. MS (QTOF ES+): found, 253.0873 [M+H]+; calcd for
2-Cyanomethyl-1-(3-furyl)naphthalene (20): A mixture of 19
(0.224 g, 1.0 mmol), s-collidine (0.2 ml, 1.3 mmol), mesyl chloride (0.1
ml, 1.3 mmol) and lithium chloride (0.07 mg, 1.5 mmol) in DMF (8 ml)
were stirred overnight at 0–5 ◦C under anhydrous condition. The reac-
tion mixture was then poured into crushed ice and extracted with ether
(3 × 20 ml). The organic layer was washed with ice-cold aq. 5% HCl,
thoroughly with cold water and dried (anhyd. Na2SO4). The solvent was
removed to furnish brown oil (0.174 g, 72%). This crude product (0.243
g, 1.0 mmol) was dissolved in dry DMF (2 ml), and KCN (0.1 g, 1.5
mmol) was added to it and stirred for 6 hrs at rt and then for 1 h at 50 ◦C.
The reaction mixture was poured into ice water (5 ml) and extracted
with ether (3 × 20 ml). The organic layer was washed thoroughly with
ice-cold water, dried and the solvent removed. The crude product was
purified by column chromatography to furnished 20 as a light yellow
C
16H13O3, 253.0865.
Phenanthro[2,1-b]furan-11-ol (14): 0.07 g (0.28 mmol) of com-
pound 13, 0.89 ml trifluoroacetic anhydride and 0.18 ml trifluoroacetic
acid was allowed to react at room temperature for overnight. It was then
poured into ice cold saturated solution of NaHCO3, stirred well,
extracted with DCM. The organic layer was collected, washed with cold
NaHCO3 solution, finally dried over anhydrous Na2SO4 and solvent
removed. The compound (a faint brown solid) was purified by column
chromatography (silica gel/230–400 mesh and benzene as elluent)
solid. Yield 0.177 g, 76%. M.p 116–118 ◦C; IR (νmax): 758, 2240 cmꢀ 1
;
(yield 0.05 g, 75%), m.p: 98 ◦C. IR: 1240, 1590, 3430 cmꢀ 1
.
1H NMR
1H NMR (400 MHz, CDCl3): δ 3.747 (s, 2H), 6.494 (s, 1H), 7.453 (dt, 1H,
J = 17.6 Hz & 20.8 Hz), 7.50 (d, 1H, J = 8.0 Hz), 7.51 (s, 1H), 7.59 (d,
1H, J = 8.8 Hz), 7.675 (s, 1H), 7.725 (d, 1H, J = 8.4 Hz), 7.868 (d, 1H, J
= 8.0 Hz), 7.898 (d, 1H, J = 8.8 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ
= 22.7, 112.6, 125.8, 126.3, 126.4, 126.8, 128.0, 129.0, 141.2, 143.8,
118.4, 120.7, 130.0, 132.9, 133.3 ppm.
(400 MHz, CDCl3): δ 8.57 (d, 1H, J = 8.4 Hz), 8.09 (s, 1H), 7.61 (ddd,
1H, J = 6.8 Hz), 8.0 (d, 1H, J = 8.8 Hz), 7.92 (d, 1H, J = 8 Hz), 7.75–7.80
(m, 2H), 7.55–7.62 (m, 2H), 7.32 (d, 1H, J = 2 Hz). MS (QTOF ES+):
found, 235.0864 [M+H]+; calcd for C16H11O2, 235.0759.
Phenanthro[2,1-b]furan-10,11-dione (15): To an aqueous solu-
tion of 14 ml 1/6 M of Na2HPO4, 0.170 g (0.63 mmol) of Fremy’s salt
was added and stirred at 0 ◦C. Now, to this stirred solution, a solution of
the compound 14 (0.05 g, 0.21 mmol) in MeOH (7 ml) was added drop
wise. The reaction mixture was allowed to stir at 0–5 ◦C for 4 hr and then
left in the freeze for overnight. The dark red solid was filtered, washed
with 5 ml ice cold water and collected. The fluorescent orange com-
pound was crystallized with benzene (yield 0.03 g, 57%), m.p greater
than 200 ◦C. IR: 770, 1050, 1430, 1600, 1650 cmꢀ 1. 1H NMR (400 MHz,
DMSO‑d6): δ 9.43 (d, 1H, J = 8.8 Hz), 8.12 (d, 1H, J = 8 Hz), 7.80–7.84
(m, 2H), 7.63–7.73 (m, 2H), 7.52–7.56 (m, 1H), 6.98 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ ¼ 170.9, 167.1, 152.6, 137.2, 136.9, 134.3, 132.4,
132.2, 130.9, 129.8, 128.8, 127.4, 126.0, 124.4, 123.5, 110.2 ppm. MS
(QTOF ES+): found, 249.0634 [M+H]+; calcd for C16H9O3, 249.0552.
1-(3-furyl)-2-naphthaldehyde (18): A mixture of the bromoalde-
hyde 17 (0.235 g, 1.0 mmol), 3-furanboronic acid (0.155 g, 1.2 mmol.),
Et3N (0.5 ml, 3.5 mmol) and DMF (2 ml) was stirred under argon for 30
min. Pd(PPh3)4 (2 mol%) was added to it. The mixture was then heated
with stirring under argon at 100 ◦C until (10 hr) the reaction was
completed. The reaction mixture was then cooled to r.t, poured into ice-
cold water and extracted with ether (3 × 20 ml). The organic layer was
washed successively with aq. NaHCO3, brine and dried. Removal of the
solvent afforded the crude product which was purified by column
chromatography, followed by recrystalisation from Pet Ether to furnish
2-Carboxymethyl-1-(3-furyl)naphthalene (21): A solution of 20
(0.23 g, 1 mmol) in 30% KOH (4 ml in 10 ml EtOH) was refluxed for 18
hrs. Excess EtOH was distilled off, the residual solution was diluted with
ice-cold water (5 ml) and extracted with ether (2 × 15 ml). The aq. part
was acidified with aq. 5% HCl under ice cold condition, and the oil that
separated was extracted with DCM (3 × 20 ml). The combined organic
extract washed with cold water, dried and the solvent removed. The
residue was dissolved in aq. NaHCO3, acidified with aq. 5% HCl in ice-
cold condition, and the separated solid was filtered. It was crystallised
from PE-EtOAc to furnish 21 as a light brown solid. Yield 0.206 g, 82%.
Mp: 154–156 ◦C. IR (νmax): 1707, 3105 cmꢀ 1
;
1H NMR (400 MHz,
CDCl3): δ 3.679 (s, 2H), 6.408 (s, 1H), 7.30–7.46 (m, 4H), 7.545 (d, 1H,
J = 1.2 Hz), 7.759 (d, 1H, J = 8.0 Hz), 7.767 (d, 2H, J = 8.4 Hz) ppm; 13
C
NMR (100 MHz, CDCl3): δ = 39.3, 113.1, 125.8, 126.3, 126.4, 127.5,
127.9, 128.2, 141.4, 143.1, 121.4, 129.2, 130.1, 132.8, 177.3 ppm. HR-
MS: calcd for C16H12O3 252.0786 [M+], found 252.0791.
Phenanthro[3,4-b]furan-4-ol (22): TFA (1 ml) was added slowly
to a mixture of 0.075 g 58 (0.075 g, 0.30 mmol) and TFAA (3 ml) at 0 ◦C
and the solution stirred overnight under anhydrous condition. The
mixture was poured into ice-cold saturated aq. NaHCO3 and extracted
thoroughly with DCM (3 × 20 ml). The organic layer was washed suc-
cessively with aq. NaHCO3 and H2O and dried. Removal of solvent
afforded the crude product which was purified by column chromatog-
raphy to furnish 22 as a yellow solid. Yield 58 mg, 83%; M.p
150–152 ◦C; IR (νmax): 3439 cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ 7.315
(s, 1H), 7.58 (dt, 1H, J = 1 7.6 Hz & 2 0.8 Hz), 7.698 (dt, 1H, J = 1 8.0 Hz
and 2 1.6 Hz), 7.758 (s, 2H), 7.841 (d, 1H, J = 2.0 Hz), 7.909 (d, 1H, J =
2.0 Hz), 7.946 (dd, 1H, J = 1 8.0 Hz & 2 0.8 Hz), 8.891 (d, 1H, J = 8.4
Hz) ppm; 13C NMR (100 MHz, CDCl3): δ = 109.1, 109.4, 124.4, 125.2,
126.1, 126.4, 127.1, 128.7, 144.8, 119.4, 123.9, 130.73, 130.76, 131.7,
141.0, 144.0 ppm.
an orange solid. Yield 0.135 g, 61%. M.p 86–88 ◦C. IR (
ν
max, cmꢀ 1):
1671; 1H NMR (400 MHz, CDCl3): δ 6.649 (s, 1H), 7.519 (t, 1H, J = 8.0
Hz), 7.59 (s, 1H), 7.627 (t, 1H, J = 8.0 Hz), 7.686 (d, 1H, J = 1.6 Hz),
7.904 (d, 2H, J = 8.8 Hz), 7.97 (d, 1H, J = 8.4 Hz), 8.032 (d, 1H, J = 8.8
Hz), 10.148 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ = 113.9, 122.2,
127.0, 127.2, 128.3, 128.6, 128.9, 142.7, 143.5, 192.6, 118.6, 132.2,
132.8, 136.0, 137.2 ppm. HR-MS (ESI): calcd for C15H10O2: 222.0681
[M+], found 222.0688.
1-(3-Furyl)-2-hydroxymethylnaphthalene (19): To the stirred
solution of the aldehyde 18 (0.222 g, 1 mmol) in EtOH (10 ml), taken in
a 50 ml r.b flask, NaBH4 0.04 g (1.0 mmol) was added pinch by pinch.
The stirring was continued at r.t. for further 2 hrs. Excess EtOH was
removed by distillation and the reaction mixture was diluted with 5 ml
ice water and extracted with DCM (3 × 15 ml). The organic layer was
washed thoroughly with water and dried. Removal of the solvent
afforded the crude product which purified by column chromatography
to furnish 19 as a colourless oil. Yield 0.217 g, 97%. IR (νmax, cmꢀ 1):
Phenanthro[3,4-b]furan-4,5-dione (23): A solution of 57 (0.025
g, 0.1 mmol) in MeOH (4 ml) was added drop wise. Stirring was
continued overnight at rt. The reaction mixture was then extracted with
DCM, dried and the solvent removed. The crude product was purified by
crystallisation from MeOH. The purified product appeared as a brick red
solid. Yield 19 mg, 77%. Mp: 170–172 ◦C. IR (νmax): 1665, 1693 cmꢀ 1
;
1H NMR (500 MHz, CDCl3): δ 7.464 (br s,1H), 7.68 (d, 1H, J = 9.5 Hz),
7.671 (d, 1H, J = 9.5 Hz), 7.86–7.93 (m,3H), 8.184 (d, 1H, J = 8.5 Hz),
8.52–8.57 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ = 112.3, 125.2,
12

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
125.3, 128.3, 129.2, 129.5, 130.2, 150.6, 128.6, 128.8, 129.9, 135.2,
137.7, 166.9, 181.3 ppm; HRMS: calcd for C16H8O3: 248.0473 [M+],
found 248.0472.
the previous synthesis, asoff white solid; Yield: 270 mg (1 mmol, 84%);
mp:106–108 ◦C; IR(KBr) νmax: 2358 cmꢀ 1; 1H NMR (500 MHz, CDCl3): δ
= 2.53 (s, 3H), 3.72 (s, 2H), 6.76 (s, 1H), 7.07 (d, J = 3.0 Hz,1H), 7.19 (t,
J = 4.5 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 5.0 Hz, 1H), 7.79
(d, J = 8.5 Hz, 1H) ppm; 13C NMR (125 MHz, CDCl3): δ = 16.3, 22.1,
118.4, 121.5, 122.9, 123.8, 125.8, 126.8, 127.6, 128.2, 128.3, 138.2,
139.5, 142.0, 142.8 ppm; HRMS (ESI+): m/z [M+Na]+ calcd for
4-Bromo-6,7-dihydro-2-methylbenzo[b]thiophene-5-carbalde-
hyde (29): 2.3 ml PBr3 (24.4 mmol) was added drop wise to an ice cold
mixture of 2.5 ml DMF (27.2 mmol) in 10 ml chloroform at 0–5 ◦C and
the stirring was continued for 30 min protecting from moisture. A so-
lution of 2-methyl-6,7-dihydrobenzo[b]thiophen-4(5H)-one (28) (1.28
gm, 7.7 mmol) in 5 ml chloroform was added drop wise to it. The re-
action mixture was gradually allowed to attain r.t. and stirring was
continued for another 16 hrs when the reaction was found to be
completed. It was poured into ice cold saturated sodium acetate solu-
tion, stirred well for about 5 min and extracted with chloroform (3 × 20
ml). Combined chloroform layer was washed successively with cold
brine solution, NaHCO3 solution and finally with brine solution and
dried (anhyd. Na2SO4). After removal of the solvent, resulting crude
product was purified by column chromatography [silica gel. (100–200
mesh) / P.E (60–80 ◦C)-EtOAC: 1:50] to obtain the title compound 29 as
C15H11NaNS2: 292.0231; found: 292.0236.
2-[2-Methyl-4-(2-thienyl)benzo[b]thiophen-5-yl]acetic
acid
(34): 260 mg (0.97 mmol) of the nitrile derivative 33 on hydrolysis with
4 ml 30% KOH in EtOH (reflux for 20 hrs) afforded the compound 34 as
a white solid after usual work up and purification. Yield: 140 mg (0.49
mmol, 50%); mp: 170–172 ◦C; IR (KBr) νmax: 1699 cmꢀ 1; HRMS (ESI+):
m/z [M+Na]+ calcd for C15H12O2NaS2: 311.0176; found: 311.1094.
9-Methylnaptho[1,2-b:7,8-b′]bisthiophen-4-ol (35): Cyclization
of the carboxylic acid 34 (120 mg, 0.42 mmol) with a mixture of 4.2 ml
trifluoroacetic anhydride and 1.5 ml trifluoroacetic acid, afforded the
compound 35 as a dark coloured semi solid mass, after usual work up
and purification. Yield: 90 mg (0.33 mmol, 80%); 1H NMR (500 MHz,
CDCl3): δ = 2.78 (s, 3H), 5.47 (br s, 1H), 7.15 (s, 1H), 7.62 (d, J = 5.0 Hz,
1H), 7.65 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 5.5 Hz, 1H), 7.82 (d, J = 8.5
Hz, 1H) 8.03 (s, 1H) ppm; 13C NMR (125 MHz, CDCl3): δ = 16.6, 107.0,
120.3, 120.5, 120.8, 122.2, 123.0, 125.2, 130.3, 130.7, 136.3, 137.0,
141.2, 148.5, 211.40 ppm; HRMS (ESI+): m/z [M+H]+ calcd for
C15H11OS2: 271.0173; found: 271.3785.
light yellow solid. Yield 1.45 gm (73%), mp: 70–72 ◦C; IR (KBr) νmax
:
1634 cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ = 2.38 (s, 3H), 2.69–2.64 (m,
2H), 2.79–2.75 (m, 2H) 6.90 (s, 1H), 9.97 (s, 1H) ppm; 13C NMR (100
MHz, CDCl3): δ = 15.2, 22.5, 23.9, 124.7, 129.1, 135.3, 135.6, 137.1,
142.0, 192.4 ppm; HRMS (ESI+): m/z [M+H]+ calcd. for C10H10O81Br:
258.9615; found: 258.9158.
6,7-Dihydro-2-methyl-4-(2-thienyl)benzo[b]thiophene-5-car-
baldehyde (30): 700 mg (2.69 mmol) the bromoaldehyde 29 was
subjected to Suzuki coupling reaction (reaction time 10 hrs) with 2-thi-
opheneboronic acid (410 mg, 3.2 mmol) under the identical reaction
condition, as used previously, afforded the compound 30 as orange
yellow solid after usual work up and purification. Yield: 430 mg (1.65
9-Methylnaphtho[1,2-b:7,8-b′]bisthiophen-4,5-dione (36): The
phenolic compound 35 (80 mg, 0.3 mmol) on oxidation with 450 mg of
Fremy’s salt in 1/6 (M) Na2HPO4 solution (30 ml) and MeOH (12 ml) for
overnight at 0–4 ◦C, afforded the o-quinone derivatives 36 as a deep
violet solid, after usual work up and purification. Yield: 65 mg (0.23
mmol, 61%), mp: 54–56 ◦C; IR (KBr) νmax: 1645 cmꢀ 1
;
1H NMR (400
mmol, 77%); mp: 227–228 ◦C; IR (KBr) νmax: 1686 & 1657 cmꢀ 1
;
1H
MHz), (CDCl3): δ = 2.37 (s, 3H), 2.78–2.83 (m, 2H), 2.83–2.91 (m, 2H),
6.52 (s, 1H), 7.0 (d, 1H, J = 3.2 Hz), 7.09 (d, ill split, J ~5 Hz, 1H), 7.46
(dd, J = 4.8 Hz & 1.6 Hz, 1H), 9.75 (s,1H) ppm; 13C NMR (75 MHz,
CDCl3): δ = 15.1, 21.8, 22.6, 124.1, 126.9, 127.5, 130.2, 131.8, 135.8,
135.9, 136.7, 141.3, 143.5, 192.2 ppm; HRMS (ESI+): m/z
[M+H]+calcd for C14H13OS2: 261.0330; found: 261.1271 & [M+Na]+
calcd for C14H12NaOS2: 283.0227; found: 283.1413.
NMR (400 MHz, CDCl3 + DMSO‑d6): δ = 2.67 (s, 3H), 7.53 (d, J = 5.6
Hz, 1H), 7.69 (d, J = 5.6 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.91 (d, J =
8.4 Hz, 1H), 8.14 (s, 1H) ppm; 13C NMR (125 MHz, CDCl3): δ = 21.6,
125.8, 128.2, 129.1, 131.2, 131.9, 133.0, 139.7, 142.1, 151.1, 152.6,
178.9, 185.0 (2C short, sample was poorly soluble even in DMSO‑d6;
possibly 2 quaternary carbons didn’t appeared with this number of
scanning) HRMS (ESI+): m/z [M+H]+ calcd for C15H9O2S2: 284.9966;
found: 284.8027 & [M+Na]+ calcd for C15H8O2NaS2: 306.9863; found:
306.7510.
2-Methyl-4-(2-thienyl)benzo[b]thiophene-5-caraldehyde (31):
420 mg (1.61 mmol) of compound 30 and 440 mg (1.94 mmol) of DDQ
in 20 ml dry benzene was refluxed (24 hrs) protecting from moisture and
after usual work up,furnished the compound 31 as pale yellow solid.
Yield: 360 mg (1.40 mmol, 86%), mp: 104–106 ◦C; IR (KBr) νmax: 1676
6,7-Dihydro-2-Methyl-4-(3-thienyl)-benzo[b]thiophene-5-car-
baldehyde (37): 705 mg (2.71 mmol) the bromoaldehyde 29 was
subjected to Suzuki coupling reaction with 3-thiopheneboronic acid
(410 mg, 3.2 mmol) under the identical reaction condition, afforded the
compound 37 after usual work up and purification as pale yellow solid;
yield: 475 mg (1.82 mmol, 67%); mp: 55–56 ◦C; IR (KBr)νmax: 1649
cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ = 2.31 (s, 3H), 2.71–2.75 (m, 2H),
2.81–2.85 (m, 2H), 6.31 (s, 1H), 7.02 (dd, J = 1.2 & 4.8 Hz,1H), 7.24
(dd, ill split, J = 1.2 & 2.8 Hz, 1H), 7.35 (dd, J = 3.2 & 4.8 Hz, 1H), 9.59
(s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ = 15.2, 21.5, 22.8, 124.0,
125.9, 126.4, 129.2, 130.6, 135.7, 136.0, 136.9, 141.5, 146.1, 192.6
ppm; HRMS (ESI+): m/z [M+H]+ calcd for C14H13OS2: 261.0330;
found: 261.0750 & [M+Na]+ calcd for C14H12OS2Na: 283.0227; found:
283.689.
cmꢀ 1
;
1H NMR (400 MHz, CDCl3): δ = 2.47 & 2.50 (both s, total 3H),
6.95 (br s, 1H), 7.09 and 7.1 (both d, ill split, J = 4.8 Hz, total 1H), 7.13
& 7.14 (both d, J = 3.2 Hz, total 1H), 7.46 & 7.48 (both br s, ill split,
total 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 9.97 (s, 1H)
ppm; 13C NMR (75 MHz, CDCl3): δ = 16.2, 121.4, 121.5, 122.3, 127.2,
127.3, 130.0, 131.4, 133.1, 135.8, 140.9, 142.7, 144.8, 191.9 ppm;
HRMS (ESI+): m/z [M+H]+ calcd for C14H11OS2: 259.0173; found:
259.0110 & [M+Na]+ calcd for C14H10NaOS2: 281.0071; found:
280.9917.
[2-Methyl-4-(2-thienyl)benzo[b]thiophene-5-yl]methanol
(32): 350 mg (1.35 mmol) of the aldehyde 31 on reduction with NaBH4
(1.35 mmol) in ethanol for 2 hrs at r.t. and after usual work up, afforded
the compound 32 as a yellowish oil. Yield: 320 mg (1.23 mmol, 91%); 1H
NMR (300 MHz, CDCl3): δ = 2.13 (br s, 1H), 2.49 & 2.54 (both s, total
3H), 4.59 & 4.68 (both s, total 2H), & 6.78 & 6.88 (both s, total 1H), 7.03
(d, J = 2.4 Hz, 1H), 7.11 & 7.12 (both d, J = 3.6 Hz, total 1H), 7.37 (d, J
= 8.4 Hz, 1H), 7.39–7.41 (m, 1H), 7.71 (d, J = 8.1 Hz, 1H) ppm; NMR
(400 MHz, CDCl3): δ = 16.2, 63.0, 121.5, 122.2, 123.8, 126.0, 127.1,
127.2, 127.8, 135.9, 138.7, 138.8, 141.3, 141.5 ppm; HRMS (ESI+): m/z
[M]+ calcd for C14H12OS2: 260.0330; found: 260.1869.
2-Methyl-4-(3-thienyl)benzo[b]thiophene-5-carbaldehyde
(38): 420 mg (1.61 mmol) of compound 37 and 440 mg (1.94 mmol) of
DDQ in 20 ml dry benzene was refluxed for 24 hrs protecting from
moisture. After usual work up, the compound 38 was obtained as pale
yellow solid.yield: 370 mg (1.43 mmol, 89%); mp: 102–103 ◦C; IR (KBr)
νmax: 1673 cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ = 2.56 (s, 3H), 6.91 (s,
1H), 7.23 (dd, J = 0.8 & 4.8 Hz, 1H), 7.36 (dd, J = 2.8 & 4.8 Hz, 1H),
7.51 (dd, J = 2.8 & 4.8 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4
Hz, 1H), 9.99 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ = 16.3, 121.6,
121.7, 121.8, 125.9, 126.1, 130.0, 130.9, 136.1, 136.2, 140.5, 142.5,
145.2, 192.4 ppm; HRMS (ESI+): m/z [M+H]+ calcd for C14H11OS2:
259.0173; found: 259.0254.
2-[2-Methyl-4-(2-thienyl)benzo[b]thiophene-5-yl]acetonitrile
(33): The compound 32 (310 mg, 1.19 mmol) was converted to the
nitrile derivative 33 under the identical reaction condition, as used for
13

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
[2-Methyl-4-(3-thienyl)benzo[b]thiophen-5-yl]methanol (39):
MHz, CDCl3): δ 3.57 (s, 3H), 3.67 (s, 2H), 6.96–7.01(m, 2H), 7.21–7.26
(m, 4H), 7.34 (dd, J = 2 Hz and 8.0 Hz, 1H) ppm;13C NMR (100 MHz,
CDCl3): δ = 39.1, 52.0, 125.7, 127.0, 127.2, 127.2, 128.2, 130.7, 131.2,
132.7, 134.7, 141.9, 172.2 ppm; HRMS (ESI+): m/z [M+Na]+ calcd for
C13H12O2SNa: 255.0456; found: 254.8899.
360 mg (1.39 mmol) of the aldehyde 38 on NaBH4 (1.39 mmol)
reduction in EtOH (2 hrs), afforded the compound 39 as colourless
viscous liquid; yield: 340 mg (1.31 mmol, 94%); 1H NMR (400 MHz,
CDCl3): δ = 2.34 (s, 3H), 2.58 (br s, 1H), 4.33 (s, 2H), 6.59 (br s, 1H),
6.93 (dd, J = 1.2 & 4.8 Hz, 1H), 7.07 (dd, J = 1.2 & 3.2 Hz, 1H), 7.16 (d,
J = 8.0 Hz, 1H), 7.21 (dd, J = 2.8 & 4.8 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 16.3, 63.0, 121.5, 121.7, 123.9,
124.2, 125.4, 129.5, 130.2, 134.6, 138.6, 139.0, 140.5, 141.1 ppm;
HRMS (ESI+): m/z [M+Na]+ calcd for C14H12OS2Na: 283.0227; found:
283.2804.
2-[2-(2-Thienyl)phenyl]acetic acid (46): To a solution of the
compound 45 (310 mg, 1.34 mmol) in ethanol (3 ml), 4 ml 30% aq. KOH
solution was added. It was refluxed for 12 hrs. Excess ethanol was
removed by distillation and the residue was diluted with ice-water (5
ml). It was extracted with ether (2 × 15 ml).The aqueous part was
acidified with 1:1 HCl under ice-cold condition. The precipitated acid
was filtered and washed thoroughly with cold water and dried. The
crude acid 46 thus obtained was re-dissolved in saturated aq. NaHCO3
solution, filtered to remove any suspended impurities. The filtrate was
cooled in ice bath was acidified 1:1 aq. HCl. The acid 46 precipitated as
white solid was filtered and dried to furnish the title compound 46 as off
white solid. Yield: 240 mg, 78%. M.P: 185–186 ◦C. IR (KBr) νmax: 1693
cmꢀ 1; HRMS (ESI+): m/z [M+Na]+ calcd for C12H10O2SNa: 241.0299;
found: 241.1128.
2-[2-Methyl-4-(3-thienyl)benzo[b]thiophen-5-yl]acetonitrile
(40): The alcohol 39 (310 mg, 1.19 mmol) was converted to the cyanide
40 (under the identical reaction condition), as a pale yellow low melting
solid; yield: 285 mg (1.06 mmol, 89%); IR (KBr) νmax: 2242 cmꢀ 1
;
1H
NMR (400 MHz, CDCl3): δ = 2.45 (s, 3H), 4.54 (s, 2H), 6.67 (s, 1H), 7.10
(dd, J = 1.4 & 5.2 Hz, 1H), 7.27 (dd, J = 1.4 & 2.4 Hz, 1H), 7.34 (d, J =
8.4 Hz, 1H), 7.38 (dd, J = 2.4 & 5.2 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 16.2, 121.5, 123.9, 124.1, 125.4,
129.4, 130.4, 134.6, 138.5, 139.1, 140.6, 141.2 (3C Short, possibly three
quaternary carbons didn’t appeared with this number of scanning) ppm;
HRMS (ESI+): m/z [M+Na]+ calcd for C15H11NS2Na: 292.0231; found:
292.0465.
Naphtho[1,2-b]thiophene-4-ol (47): To an ice cold magnetically
stirred mixture of 230 mg (1 mmol) of the acid 46 and 9 ml of tri-
fluoroacetic anhydride, 2.5 ml trifluoroacetic acid was injected slowly at
0–4 ◦C and stirred further for 1 hr protecting from moisture and then left
in the refrigerator for overnight. The reaction mixture was decomposed
with crushed ice and extracted with dichloromethane thoroughly. The
organic layer was washed with cold aq. NaHCO3 solution, cold brine
solution and dried (anhyd. Na2SO4). Removal of the solvent and puri-
fication of the crude product by plate chromatography (Silica gel GF254;
benzene), the phenol 47 was obtained as semi solid mass. Yield: 160 mg
(0.8 mmol, 80%); 1H NMR (400 MHz, CDCl3): δ = 5.48 (br s, 1H), 7.01
(s, 1H), 7.43–7.46 (m, 2H), 7.51 (d, J = 5.4 Hz, 1H), 7.63 (d, J = 5.4 Hz,
1H), 7.76 (d, J = 7.4 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 105.4, 122.6, 123.2, 123.7, 124.2, 124.5, 125.9,
126.5, 127.0, 127.4, 134.4, 148.7 ppm; HRMS (ESI+): m/z [M+H]+
calcd for C12H9OS: 201.0296; found: 201.0602.
2-[2-Methyl-4-(3-thienyl)benzo[b]thiophen-5-yl]acetic
acid
(41): 260 mg (0.97 mmol) of the nitrile derivative 40 on hydrolysis with
4 ml 30% KOH in EtOH under reflux for 20 hrs, afforded the compound
41 as a white solid after usual work up and purification. Yield: 160 mg
(0.56 mmol, 58%); IR (KBr) νmax: 1697 cmꢀ 1; HRMS (ESI+): m/z
[M+H]+ calcd for C15H13O2S2: 289.0357; found: 289.0103 & m/z
[M+Na]+ calcd for C15H12O2S2Na: 311.0176; found: 310.9653.
9-Methylnaphtho[2,1-b:7,8-b′]bisthiophen-4-ol (42): Cycliza-
tion of the carboxylic acid 41 (120 mg, 0.42 mmol) with a mixture of 4
ml trifluoroacetic anhydride and 1.5 ml trifluoroacetic acid under the
identical reaction condition, afforded the compound 42 as a reddish
semi solid mass, after usual work up and purification. yield: 105 mg
(0.39 mmol, 93%); 1H NMR (500 MHz, CDCl3): δ = 2.76 (s, 3H), 5.66 (br
s, 1H), 7.18 (s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 5.5 Hz, 1H),
7.82 (d, J = 8.5 Hz, 1H), 8.00 (s, 1H), 8.4 (d, J = 5.0 Hz, 1H) ppm; 13C
NMR (125 MHz, CDCl3): δ = 29.8, 95.9, 105.5, 107.0, 108.3, 120.6,
122.4, 125.3, 126.3, 129.4, 131.5, 193.9 (three Carbon short, possibly
three quaternary carbons didn’t appeared with this number of scanning)
ppm; HRMS (ESI+): m/z [M+H]+ calcd for C15H11OS2: 271.0173;
found: 270.9474.
Naphtho[1,2-b]thiophene-4,5-dione (48): To a vigorously stirred
solution of 900 mg Fremy’s salt in 20 ml aq. 1/6M disodium hydrogen
phosphate at 0–5 ◦C, a solution of the phenol 47 (130 mg, 0.65 mmol) in
MeOH (8 ml) was added drop wise. The colour of the solution quickly
changes from violet to reddish brown with formation of dark red to deep
red ppt. Stirring was continued at 0–5 ◦C for an additional hour and the
mixture was left in the refrigerator for overnight. The reaction mixture
was then extracted with dichloromethane, washed with cold water,
dried (anhyd. Na2SO4) and solvent removed. Crude o-quinone derivative
thus obtained on further purification by preparative TLC (Silica gel
GF254 mesh; benzene-methanol, 5:1) followed by recrystallisation from
MeOH afforded the title compound 48 as deep orange solid. Yields: 100
mg (0.47 mmol), 72%; mp: 207–208 ◦C. IR (KBr) νmax: br peak 1659
9-Methylnaphtho[2,1-b:7,8-b′]bisthiophene-4,5-dione
(43):
Oxidation of the phenolic compound 42 (80 mg, 0.3 mmol) with 450 mg
(1.68 mmol) of Fremy’s salt, under the identical reaction condition,
afforded the compound 43 as a deep red solid, after usual work up and
purification. yield: 70 mg (0.25 mmol, 83%); mp: 230–232 ◦C; IR (KBr)
νmax: broad strong peak at 1652 cmꢀ 1; 1H NMR (500 MHz, CDCl3): δ =
2.61 (s, 3H), 7.47 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 5.2 Hz,
1H), 7.84 (d, J = 5.2 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H) ppm; HRMS
(ESI+): m/z [M+H]+ calcd for C15H9O2S2: 284.9966; found: 285.1825 &
[M+Na]+ calcd for C15H8O2S2Na: 306.9863; found: 307.1755.
cmꢀ 1
.
1H NMR (400 MHz, CDCl3): δ = 7.19 (d, J = 5.2 Hz, 1H), 7.34
(ddd, J = 0.8, 7.6 & 7.6 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.46 (d, J =
5.2 Hz, 1H), 7.53 (ddd, J = 1.6, 7.6 & 7.6 Hz, 1H), 8.00 (d, J = 0.8 and
7.2 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ = 124.7, 126.0, 127.8,
129.1, 129.7, 130.2, 132.4, 135.8, 136.5, 151.2, 173.8, 180.4 ppm;
HRMS (ESI+): m/z [M+H]+ calcd for C12H7O2S: 215.0089; found:
214.9340 and [M+Na]+ calcd for C12H6O2SNa: 236.9986; found:
236.9225.
Methyl 2-[2-(2-thienyl)phenyl]acetate (45): A stirred mixture of
500 mg (2.18 mmol) of bromo ester 44, 2-thiopheneboronic acid (2.62
mmol), triethylamine (5.45 mmol) in 4 ml of DMF was degasified for 25
min by bubbling N2 through it. Now to it, Pd(PPh3)4 catalyst (2 mol%)
was added quickly and degasified for another 15 min. The mixture was
then heated at about 110 ◦C for 6 hrs under N2 atmosphere. When the
reaction was completed (checked by TLC), the mixture was poured into
ice water (15–20 ml) and extracted thoroughly with ether (3 × 20 ml).
Organic layer was washed with ice water, 5% aq. NaHCO3 solution and
finally with ice water. Removal of solvent afforded the crude product
which on purification by column chromatography [silica gel 100–200
mesh / P.E (60–80 ◦C)-EtOAc, 15:1] furnished the compound 45 as a
yellow oil. Yield: 330 mg, 65%. IR (KBr) νmax: 1738 cmꢀ 1; 1H NMR (400
Methyl 2-[2-(3-thienyl)phenyl]acetate (49): 500 mg (2.18 mmol)
of the bromoester 44 was subjected to Suzuki coupling reaction with 3-
thiopheneboronic acid (2.62 mmol) under the identical reaction con-
dition, as used for the preparation of the compound 45, and 365 mg
(1.57 mmol, 72%) of the compound 49 (yellowish oil) was obtained
after usual work up and purification. IR (KBr) νmax: 1727 cmꢀ 1. 1H NMR
(400 MHz, CDCl3): δ = 3.55 (s, 3H), 3.57 (s, 2H), 7.03 (dd, J = 1.6 and
4.8 Hz, 1H), 7.17 (dd, J = 1.6 and 3.2 Hz, 1H), 7.21–7.27 (m, 5H) ppm;
13C NMR (100 MHz, CDCl3): δ = 38.9, 52.0, 123.2, 125.4, 127.2, 127.6,
129.0, 130.3, 130.5, 132.1, 137.2, 141.2, 172.4 ppm. HRMS (ESI+): m/z
14

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
[M+Na]+ calcd for C13H12O2SNa: 255.0456; found: 254.8899.
2-[2-(3-Thienyl)phenyl]acetic acid (50): 340 mg (1.47 mmol) of
the ester 49 on hydrolysis with 4 ml ethanolic 30% KOH, under the
identical reaction condition, as used for the preparation of the com-
pound 46 afforded the compound 50 as a white solid, after usual work
up and purification. Light brown solid. Yield: 280 mg (1.22 mmol, 83%);
mp: 188–190 ◦C. IR (KBr) νmax: 1699 cmꢀ 1; HRMS (ESI+): m/z [M+Na]+
calcd for C12H10O2SNa: 241.0299; found: 241.1128.
Briefly, ~5 × 103 MCF7 or MDA-MB-231 cells seeded in a 96 well plate
were treated with drugs in increasing concentrations (0–40 μM). 24 h
post-treatment MTT was added at a concentration of 0.5 mg/ml and
after 3 h the media was discarded and 200 L of DMSO was added in
μ
each well to dissolve the insoluble purple formazan crystals. The
absorbance was measured at 540 nm using a microplate reader (Synergy
H1; Bio Tek) and subsequently IC50 of respective compounds were
calculated from plotted graphs. Experiments were performed in dupli-
cate and were independently repeated two times.
Naphtho[2,1-b]thiophene-4-ol (51): Cyclization of the carboxylic
acid 50 (260 mg, 1.13 mmol) with the mixture of 7 ml trifluoroacetic
anhydride and 3 ml trifluoroacetic acid, under the identical reaction
condition, as used for the preparation of the compound 47 afforded after
usual work up the compound 46 and purification of it the phenol 51 was
obtained as a brown low melting solid. Yield: 170 mg (0.85 mmol, 52%).
1H NMR (400 MHz, CDCl3): δ = 6.89 (br s, 1H), 7.17 (s, 1H), 7.41(d, J =
5.2 Hz,1H), 7.43–7.48 (m, 2H), 7.64 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 7.6
Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ =
105.4, 108.4, 121.2, 123.4, 124.2, 124.3, 125.8, 125.9, 127.5, 129.4,
148.6, 162.6 ppm; HRMS (ESI+): m/z [M+H]+ calcd for C12H9OS:
201.0296; found: 201.0256.
Cell morphology analysis: ~5 × 104 MCF7 or MDA-MB-231 cells
plated in 6 well plates were either left untreated (DMSO control) or
treated with 2.5 μM drugs. The morphological changes and character-
istics for cell death were captured using Fluorescence cell imager (BIO-
RAD, Hercules, CA, USA) at different time intervals (0, 6, 12 and 24 h).
Apoptosis assay by EtBr/AO staining: ~5 × 104 MCF7 cells plated
in 6-well plates with a coverslip inside in each well were either left
untreated (DMSO control) or treated with 2.5
μM two natural tan-
shinones (compounds 1 and 2) along with compounds 27, 48 and 52. 24
h post-treatment the media was discarded, washed with PBS and sub-
sequently stained with 1:1 solution of ethidium bromide (EtBr) and
acridine orange (AO) (0.4 mg/ml each). The cells were then immedi-
ately washed with 1 × PBS and mounted on a slide using Fluoromount
(Invitrogen, Thermo Fisher Scientific Inc.). Apoptosis was assessed by
identifying apoptotic, viable and nonviable cells using a Carl Zeiss
Fluorescence microscope with an Apotome.2 attachment (Carl Zeiss AG,
Germany) with a blue excitation (480 nm) and a barrier filter (515–530
nm). Typically, AO stains viable cells as green, while EtBr stains the
nucleus as red (bright orange) in response to apoptotic induction. The
apoptosis and cell viability assessments were performed based on 200
cells per sample and represented as bar diagrams.
Naphtho[2,1-b]thiophene-4,5-dione (52): The phenolic com-
pound 51 (130 mg, 0.65 mmol) on oxidation with 900 mg of Fremy’s salt
under the identical reaction condition, as used for the preparation of the
compound 47, afforded the o-quinone 52 as a deep yellow solid, after
usual work up and purification. Yield: 120 mg (0.56 mmol, 86%). Mp:
210–212 ◦C. IR (KBr) νmax: 1634 and 1651 cmꢀ 1
.
1H NMR (400 MHz,
CDCl3): δ = 7.34–7.38 (m,1H), 7.45 (d, J = 4.8 Hz 1H), 7.56–7.60 (m,
2H), 7.79 (d, J = 5.2 Hz, 1H), 8.03 (dd, J = 0.8 and 7.6 Hz, 1H) ppm; 13
C
NMR (100 MHz, CDCl3): δ = 124.5, 125.1, 129.7, 130.0, 130.8, 133.0,
135.8, 138.7, 146.7, 172.5, 180.9 ppm (one quarternary carbon was not
observed possibly due to insufficient number of scanning); HRMS
(ESI+): m/z [M+H]+ calcd for C12H7O2S: 215.0089; found: 215.1627
and [M+Na]+ calcd for C12H6O2SNa: 236.9986; found: 237.1626.
Drugs, antibodies and plasmids: Tanshinone I (T5330), Tan-
shinone IIA (T4952) and Chloroquine diphosphate salt (C6628) were
purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).
Apoptosis assay by annexin V/PI staining: Cell apoptosis induced
by tanshinones and the three derivatives – compounds 27, 48 and 52
was determined by Annexin V-FITC Apoptosis Staining/Detection Kit
(ab14085) according to manufacturer’s protocol (Abcam Cambridge,
UK). Briefly, ~5 × 105 MCF7 or MDA-MB-231 cells were either left
untreated (DMSO control) or treated with different tanshinone ana-
logues. 24 h post-treatment cells were collected by centrifugation and
re-suspended in 500 µL of 1X binding buffer. Next, cells were stained
with 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) (Sigma-
Aldrich Corp. St. Louis, MO, USA) for 5 min at room temperature in the
dark. FITC labelled Annexin V binding (Ex = 488 nm; Em = 350 nm) and
PI staining were detected using FITC signal detector by the phycoery-
thrin emission signal detector in BD Accuri C6 Flow Cytometry Analyzer
(BD Biosciences, San Jose, CA, USA). Experiments were performed in
duplicate and presented the data as an average with SE+/ꢀ .
Mouse monoclonal antibodies directed against p62/SQSTM1
(Clone# 3/P62 lck ligand), Beclin1 (Clone# 20/Beclin) and cleaved
PARP (Clone# Asp214) were purchased from BD Biosciences (Franklin
Lakes, NJ, USA). Rabbit monoclonal antibody directed against Bcl-2
(D55G8), Bcl-xL (54H6), Bax (D2E11), Bim (C34C5), Bak1 (D4E4) and
mouse monoclonal antibody against CHOP (L63F7) were purchased
from Cell Signaling Technology Inc. (Danvers, MA). Mouse monoclonal
antibodies against GAPDH (0411), p53 (DO-1), MAPLC3B (G-9), ATF4
(C-20) and GADD34 (D8) were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). DyLight 800/680 conjugated (for western
blots) and Alexa Fluor 488/555 tagged (for confocal micoscopy) sec-
ondary antibodies anti-mouse, anti-rabbit, anti-sheep IgG (H + L) were
purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
pBabePuro-GFP-LC3 construct expressing GFP-tagged LC3B was a
kind gift from Jayanta Debnath (Addgene plasmid# 22405) and
mCherry-tagged p62 plasmid expressing plasmid was generously pro-
vided by Edward M. Campbell (Loyola University Chicago, IL, USA)
Cell lines and Cell culture: Human breast cancer MCF7 (ER+ PR+
Her2ꢀ ) cells were obtained from Gaurisankar Sa (Bose Institute, Kol-
kata) and MDA-MB-231 (ERꢀ PRꢀ Her2ꢀ ) were obtained from Ranjana
Pal (Presidency University, Kolkata). MCF7 and MDA-MB-231 cell lines
were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
(Gibco, Invitrogen, Thermo Fisher Scientific Inc.) and Roswell Park
Memorial Institute medium (RPMI) (Gibco, Invitrogen, Thermo Fisher
Scientific Inc.), respectively at 37 ◦C in a humidified environment that
contains 5% CO2. The culture medium was supplemented with 10% FBS
(Fetal Bovine Serum; Gibco/Invitrogen, Inc., USA) and 1% penicillin-
streptomycin solution (Sigma-Aldrich Corp., St. Louis, MO).
Western blot analysis: ~10 × 106 MCF7 or MDA-MB-231 cells were
either left untreated (DMSO control) or treated with 2.5 μM tanshinone
analogues. 24 h post-treatment, cells were harvested, washed with ice
cold 1X PBS (Gibco/Invitrogen, Inc., USA), and subsequently lysed in
0.5 ml ice cold RIPA buffer (Sigma-Aldrich Corp., St. Louis, MO) with
protease inhibitor (Cell Signalling Technology Inc., Danvers, MA, USA).
Protein samples were estimated by Bradford reagent (BIO-RAD, Hercu-
les, CA, USA). Samples were boiled in laemmeli buffer (BIO-RAD, Her-
cules, CA), fractionated by SDS-PAGE and transferred to a 0.45 µm
nitrocellulose membrane (BIO-RAD, Hercules, CA). The membranes
were then probed with specific antibodies incubated overnight at 4 ◦C
followed by incubation with appropriate infrared-tagged/DyLight sec-
ondary antibodies for 1 h at 37 ◦C and viewed on an Odyssey CLx Im-
aging System (LiCor Inc., Lincoln, NE, USA). Image analysis and
quantification measurements were performed using the Odyssey
Infrared Imaging System application software (LiCor Inc., Lincoln, NE,
USA).
RNA extraction and real-time PCR analysis: Total RNA was iso-
lated from ~10 × 106 MCF7 or MDA-MB-231 cells either left untreated
(DMSO control) or treated with 2.5 µM tanshinone analogues for 24 h
using TRIzol reagent according to the manufacturer’s instructions
Cytotoxicity assay by MTT: Cell viability was measured using MTT
(Sigma-Aldrich Corp., St. Louis, MO) as per manufacturer’s instructions.
15

C. Gain et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116112
(Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA), fol-
lowed by cDNA preparation using iScript cDNA synthesis kit (BIO-RAD,
Hercules, CA, USA) as per manufacturer’s protocol. RNA and cDNA
quality and quantity were checked using Synergy H1 Multimode
Microplate Reader (BioTek Instruments, Inc., VT, USA). qPCR analysis
was performed using iTaq Universal SYBR Green Supermix (BIO-RAD,
Hercules, CA, USA) in CFX Connect™ Real-Time PCR detection System
(BIO-RAD, Hercules, CA, USA) with the following thermal profile – 1
cycle: 95 ◦C for 10 min; 40 cycles: 95 ◦C for 15 sec followed by 60 ◦C for
1 min; and finally the dissociation curve at – 95 ◦C for 1 min, 55 ◦C 30
min, and 95 ◦C for 30 sec. Unless and otherwise stated, each sample was
performed in duplicate and calculation was made using a 2ꢀ ΔΔCT
method to quantify relative expression compared with housekeeping
gene control. The real time PCR primers used in this study were designed
from Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-bl
ast/) and listed in Table S2. Real-time PCR primers were obtained
from Integrated DNA Technologies, Inc. (Coralville, IA, USA).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We sincerely thank to Jayanta Debnath (University of California, San
Francisco, USA), Edward M. Campbell (Loyola University Chicago, IL,
USA), Gaurisankar Sa (Bose Institute, Kolkata, India) and Ranjana Pal
(Presidency University, Kolkata, India) for providing reagents, plasmids
and cell lines. We also thank Pralay Majumder (Presidency University,
Kolkata, India) for technical support in using Microscopy Facility at
Dept. Life Sciences, Presidency University, Kolkata, India. A.S. is a
Wellcome Trust/DBT India Alliance Intermediate Fellow. C.G. is the
recipient of UGC-NET Senior Research Fellowship, India.
Transfection: For confocal microscopy, ~10 × 105 MCF7 cells were
transiently transfected with plasmids expressing GFP-tagged LC3 or
mCherry-tagged p62 using Lipofectamine 3000 according to manufac-
turer’s protocol (Invitrogen, Thermo Fisher Scientific Inc., Waltham,
MA, USA). 24 h post-transfection cells were either left untreated or
treated with natural tanshinones or different synthetic compounds for
another 24 h and subjected to live-cell confocal microscopy analyses.
Confocal microscopy: For live cell imaging, ~1 × 106 MCF7 cells
grown at 70% confluency on 35 mm dish embedded with 12 mm glass
viewing area (Nunc, Thermo Fisher Scientific Inc., Waltham, MA, USA)
were transfected with GFP-LC3 or mCherry-p62 expressing constructs as
described above. 24 h post-transfection, cells were either left untreated
(DMSO control) or treated with 2.5 µM drugs for another 24 h. Hoechst
3342 solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) and
LysoTracker Red DND-99 (Invitrogen, Thermo Fisher Scientific Inc.,
Waltham, MA, USA) were used to stain the nucleus and the acidic ly-
sosomes, respectively. The images were obtained by a Leica DMi8
Confocal Laser Scanning Microscope and analyzed in the Leica Appli-
cation Suite X (LAS X) software (Leica Microsystems GmbH, Wetzlar,
Germany).
Financial Disclosure
This work was supported by grants from Wellcome Trust/DBT India
Alliance Intermediate Fellowship research grant (IA/I/14/2/501537),
Department of Biotechnology (DBT), Govt. of India (BT/PR8123/MED/
30/990/2013) and Department of Science and Technology (DST), Govt.
of India (CRG/2018/001044) to AS and Department of Science and
Technology (DST), Govt. of India (SR/S1/OC-30/2011) to GKK. The
funders had no role in study design, data collection and analysis, deci-
sion to publish, or preparation of the manuscript.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116112.
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