First synthesis of rugosaflavonoid and its derivatives and their activity against breast cancer

Article abstract of DOI:10.1039/c7ra04971d

Rugosaflavonoid, is a secondary metabolite isolated from the plant Rosa rugosa was synthesized in five simple steps from commercially available 3,5-dihydroxy benzoic acid involving domino aldol-Michael-oxidation reaction. This is the first report of the synthesis of rugosaflavonoid (6a). A series of its derivatives were also synthesized, characterized and evaluated for the cytotoxicity against the breast cancer MCF-7 and normal NIH3T3 cell lines. The synthetic derivatives of rugosaflavonoid showed comparable activity in both the cell lines and compounds 6d, 6e and 6f, which were found to be cytotoxic towards MCF-7 cell lines but nontoxic to NIH3T3 cell lines at 5 μM concentration. In an attempt to explore the mode of action of the best active compounds, docking on the ATP binding site of EGFR (1M17) was performed considering that EGFR over-expressed in most of the tumors. The docking score (Gscore) of 6f and standard quercetin was found to be -8.608 and -8.310 respectively.

Full text of DOI:10.1039/c7ra04971d

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First synthesis of rugosaflavonoid and its
derivatives and their activity against breast cancer†
Cite this: RSC Adv., 2017, 7, 33052
*
Ninad V. Puranik
and Pratibha Srivastava
Rugosaflavonoid, is a secondary metabolite isolated from the plant Rosa rugosa was synthesized in five
simple steps from commercially available 3,5-dihydroxy benzoic acid involving domino aldol-Michael-
oxidation reaction. This is the first report of the synthesis of rugosaflavonoid (6a). A series of its
derivatives were also synthesized, characterized and evaluated for the cytotoxicity against the breast
cancer MCF-7 and normal NIH3T3 cell lines. The synthetic derivatives of rugosaflavonoid showed
comparable activity in both the cell lines and compounds 6d, 6e and 6f, which were found to be
cytotoxic towards MCF-7 cell lines but nontoxic to NIH3T3 cell lines at 5 mM concentration. In
an attempt to explore the mode of action of the best active compounds, docking on the ATP binding
site of EGFR (1M17) was performed considering that EGFR over-expressed in most of the tumors.
The docking score (Gscore) of 6f and standard quercetin was found to be ꢀ8.608 and ꢀ8.310
respectively.
Received 3rd May 2017
Accepted 20th June 2017
DOI: 10.1039/c7ra04971d
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1. Introduction
taking place in milliseconds. Medicinal plants are one of the
best equipment for the biosynthesis of various drug based
molecules. Chromone is a valid scaffold7 in the eld of medic-
inal chemistry, due to the wide range of its biological activities,
and their structure–activity relationships have generated curi-
osity among medicinal chemists, and this has culminated into
the breakthrough of the clinical anticancer agent avopiridol,
as well as several lead molecules in other disease areas.8 Rosa
rugosa Thunb. belongs to the family of Rosaceae is a common
ornamental ower distributed in the temperate regions of
eastern Asia and widely cultivated in Yunnan Province.9 The
petals and buds of R. rugosa are frequently used as food,
incense, and Chinese medicinal materials for the cure of
stomachache, diarrhea, and gynecological ailments.10 The
literature survey has shown the presence of tannins, terpe-
noids,11–13 and avonoids14,15 in this genus. Anti-inammatory,
cytotoxic and anti-HIV activities have observed with selected
chemical ingredients isolated from R. rugosa.16 Hu et al.17 have
recently isolated and characterized cytotoxic oxepinochrome-
none and avonoids from R. rugosa. Rugosaavonoid is a new
avonoid, isolated from Rosa rugosa which showed cytotoxicity
against NB4, SHSY5Y, and MCF-7 cells. Till today the synthesis
of rugosaavonoid (Scheme 1) is not reported in the literature.
Therefore, we have focused to synthesize recently isolated
naturally occurring rugosaavonoid (6a) and its derivatives by
simple and convenient method. All the synthesized derivatives
were evaluated for their cytotoxicity against the breast cancer
cell lines MCF-7 and the normal cell lines NIH3T3.
Cancer still continues to be one of the major threats to human
society because it is widely spreading day by day and there is no
complete cure for it. The three most commonly diagnosed types
of cancer among women in 2010 were cancers of the breast,
lung, and colorectum, accounting for 52% of cancer cases in
this group. Breast cancer (BC) represents the most common
cancer among women; there were 232 672 estimated new cases
and 40 000 estimated deaths in the United States in 2014.1–3 The
toxicity allied with conventional cancer chemotherapy arises
primarily from the lack of specicity for tumor cells. It leads to
a low therapeutic index, which results in undesirable damage to
healthy organs and consequently puts restrictions on the dose
of the drug that can be administered. The majority of the
currently available anticancer drugs are designed to have
specic toxicity toward tumor cells.4,5 Several trials are being
considered to handle this predicament and thus improve the
effectiveness and tumor cell specicity of anticancer drugs.
Among these approaches, many studies have focused on natural
compounds that inhibit precisely the growth of cancer cells
more selectively than normal cells. Thus, phytoconstituents
have become the dignied category of anticancer drugs. Over
75% of non-biological anticancer drugs approved between 1981
and 2007 were either natural products or were developed based
on them.6 Therefore, the search for new anticancer agents
Bioprospecting Group, Agharkar Research Institute, G. G. Agarkar Road, Pune, 411004,
Maharashtra, India. E-mail: ninadv_puranik@yahoo.co.in; psrivastava@aripune.org
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra04971d
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Scheme 1 Synthesis of rugosaflavonoid and its derivatives using 3,5-dihydroxybenzoic acid as starting material.
methyl 2-acetyl-3,5-dimethoxybenzoate (3) using BBr3 in
dichloromethane was attempted as per the procedure reported,20
however, unfortunately only monomethylated product was ob-
tained. Thus, AlCl3 was used21 instead of BBr3 to get the desired
demethylated product in good yield. Attempt to cyclize interme-
diate methyl 2-acetyl-3,5-dihydroxybenzoate (4) to rugosaavonoid
(6a) by the literature method22 was unsuccessful, because the
reaction resulted into the formation of methyl 7-hydroxy-2-(4-
methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromene-5-carboxylate (5)
rather than rugosaavonoid (6a), which was accentuated by
2. Results and discussion
The synthesis of rugosaavonoid (6a) and its derivatives was
accomplished in 5 simple steps by selecting 3,5-dihydroxybenzoic
acid (1) as a building block (Scheme 1). In the rst step 3,5-
dihydroxybenzoic acid was methylated using dimethyl sulfate in
presence of the base,18 which produced methoxy ester (2) with
92% yield. The resultant ester was further acylated using acetyl
chloride in carbon disulde into methyl 2-acetyl-3,5-
dimethoxybenzoate (3) with 52% yield.19 Demethylation of
Table 1 Docking and cytotoxicity results of compound 6(a–j) showing cell viability analysis results after MTT assay against MCF-7 and NIH3T3
cell lines. Results are presented in % cell viability. Four different concentrations (5, 10, 15 and 20 mM) of each compound were used for
experimentsa
Conc. used
MCF7 (% cell viability)
NIH3T3 (% cell viability)
Residue involved in binding
5 mM 10 mM 15 mM 20 mM 5 mM 10 mM 15 mM 20 mM with 1M17
Docking
score
S. no.
6a
43
58
70
58
47
52
60
41
49
68
40
32
87
90
73
74
67
83
71
66
84
54
62
82
Met 769, Leu 768, Asp 831, Gly 772, Leu
694, Glu 738
Met 769, Leu 768, Leu, 694, Gly 772, Asp
831, Thr 830
Met 769, Leu 768, Gln 767, Asp 831, Thr
830, Leu 694
ꢀ5.040
ꢀ6.159
ꢀ6.661
6b
6c
6d
6e
6f
64
50
52
62
49
45
45
41
39
36
31
31
99
84
96
97
79
85
93
33
70
91
30
62
Met 769, Leu 768, Asp 831, Glu 831
Met 769, Leu 768, Asp 831, Glu 738
Met 769, Leu 768, Leu 694, Asp 831, Lys
721, Glu 738
ꢀ6.549
ꢀ6.483
ꢀ8.310
6g
6h
56
65
50
46
48
43
44
40
83
89
71
74
64
60
51
43
Met 769, Leu 768, Gln 767, Asp 831, Thr
830, Leu 694
Met 769, Leu 768, Leu, 694, Gly 772, Asp
831, Thr 830
ꢀ4.557
ꢀ4.743
6i
6j
71
54
53
49
75
48
49
67
41
47
50
92
55
89
88
55
76
82
62
72
85
65
68
Met 769, Leu 768, Asp 831, Glu 831
Met 769, Leu 768, Asp 831, Glu 738
Met 769, Lys 721, Glu 738, Asp 831
ꢀ4.743
ꢀ4.965
ꢀ8.608
Std I quercetin 90
a
All the samples run in triplicate and average of three results are presented here.
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Scheme 2 Synthesis of rugosaflavonoid derivatives using orcinol as starting material.
spectral data. This reaction was attempted 3–4 times to get the 127.11 (C-60), 128.59 (C-20), 134.55 (C-5), 157.7 (C-9), 158.55 (C-40)
required compound (6a), but it couldn't be obtained. Eventually, 162.39 (C-2), 162.54 (C-7), 169.19 (C-11), 175.68 (C-4). The IR
the nal step was carried out using the intermediate 5 with I2 and exhibited the peak at 3446 (OH), and 1735 (C]O), 1624 (C]O).
DMSO (Scheme 1), which provided the desired product (6a) Several derivatives of the rugosaavonoid using different aromatic
during 1 hour only. Rugosaavonoid was obtained as a yellow aldehydes were also synthesized. Hu et al. had reported17 13.6 mg
solid. A molecular formula of C18H14O6 was conrmed by LC-MS of rugosaavonoid (6a) from 8 kg of plant material aer several
1
m/z 326.30 [M+] and HRMS m/z 327.0853 [M + 1]+. The H NMR steps of purication. But in the current experiment, 250 mg
and 13C NMR data of 6a were obtained and compared (Table 2) rugosaavonoid (6a) was obtained from 1 g of methyl 2-acetyl-3,5-
with the reported data17 and were found in agreement with dihydroxybenzoate (4) via the intermediate 5. The derivatives were
natural product rugosaavonoid. It revealed that the compound also synthesized by replacing the ester group with the methyl
1
6a has 18 carbon and 14 protons. The H NMR of 6a showed group in rugosaavonoid moiety. These derivatives were synthe-
proton signals for methoxycarbonyl group d (3.81, s, 3H), sized using orcinol (10) as a starting material (Scheme 2), which
a methoxy group (3.86, s, 3H), and other proton peaks at d 6.79 (s, was acylated followed by the previously stated procedure of
1H, 3-H), 6.82 (d, 1H, 8-H, J ¼ 1.6 Hz), 7.10 (d, 1H, 6-H, J ¼ 2 Hz), cyclization to yield the compounds (6g–j). The rugosaavonoid
7.12 (d, 2H, 30, 50-H, J ¼ 7.2 Hz), 8.04 (d, 2H, 20, 60-H, J ¼ 7.2 Hz), and its derivatives displayed comparative results in the MTT
and a phenolic hydroxylic proton at 11.14 (s, 1H, OH). The 13C cytotoxicity assay. The details are presented in the Table 1. The
NMR of compound 6 displayed carbon signals at d 52.86 (C-11, synthetic rugosaavonoid (6a) showed 50% cytotoxicity to MCF-7
OCH3), 55.97 (C-40, OCH3), 104.25 (C-8), 105.58 (C-3), 113.55 (C- cells at 5 mM concentration, but its cytotoxicity reduced aer
10), 113.94 (C-6), 114.81 (C-30), 114.98 (C-50), 123.49 (C-10), enhancing concentration up to 20 mM with 68% cell viability of
Table 2 Comparative data of synthesized and isolated rugosaflavonoid
Paramater
Rugosaavonoid synthesized
Rugosaavonoid isolated
Mp
HRMS
226–228 ꢁ
C
Not reported
m/z 327.0863 [M + 1]+ (calcd for C18H15O6,
327.0863)
HRESIMS m/z 349.0682 [M + Na]+ (calcd for
C18H14NaO6, 349.0688)
IR (cmꢀ1
1H NMR
)
3446, 1735, 1624, 1600, 1543, 1436, 1435, 1253,
1180, 1029, 894
3416, 1702, 1657, 1610, 1565, 1456, 1432, 1287,
1182, 1028, 893
(Solvent DMSd6) d 3.81, (s, 3H, OCH3), 3.86 (s,
3H, OCH3), 6.79 (s, 1H, 3-H), 6.82 (d, 1H, 8-H, J ¼
1.6 Hz), 7.10 (d, 1H, 6-H, J ¼ 2 Hz), 7.12 (d, 2H,
30, 50-H, J ¼ 7.2 Hz), 8.04 (d, 2H, 20, 60-H, J ¼ 7.2
Hz), 11.14 (s, 1H, OH)
(Solvent pyridine-d5, 500 MHz) d 3.80 (s, 3H,
OCH3), 3.95 (s, 3H, OCH3), 6.68 (s, 1H, 3-H), 6.74
(d, 1H, 8-H, J ¼ 1.8 Hz), 6.89 (d, 1H, 6-H, J ¼ 1.8
Hz), 7.00 (d, 2H, 30,50-H, J ¼ 8.8 Hz), 7.76 (d, 2H,
20,60-H, J ¼ 8.8 Hz)
13C NMR
52.86 (OCH3), 55.97 (C-40, OCH3) 104.25 (C-8),
105.58 (C-3), 113.55 (C-10), 113.94 (C-6), 114.81
(C-30), 114.98 (C-50), 123.49 (C-10), 127.11 (C-60),
128.59 (C-20), 134.55 (C-5), 157.7 (C-9), 158.55 (C-
40), 162.39 (C-2), 162.54 (C-7), 169.19 (C-11),
175.68 (C-4)
52.4 (OCH3), 55.6 (C-40, OCH3), 103.8 (C-8),
105.2(C-3), 113.1 (C-6), 115.6 (C-30), 115.6 (C-50),
122.9 (C-10), 131.0 (C-20), 131.0 (C-60), 136.8 (C-5),
158.8 (C-9), 163.2 (C-2), 165.0 (C-7), 168.3 (C-11),
181.5 (C-4)
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Fig. 1 (a) Image of MCF-7 before treatment with 6f; (b) image of MCF-7 after treatment with 6f; (c) image of NIH3T3 before treatment with 6f; (d)
image of NIH3T3 after treatment with 6f.
MCF-7 cells. It was found to be non-toxic to NIH3T3 normal cell Gly 772, Met 769, and Leu 694 indicated hydrophobic inter-
line with 87% cell viability at the lower concentration of 5 mM. action with tyrosine kinase inhibitor, erlotinib. Therefore,
However, the toxicity increased with the higher concentration. interaction studies of rugosaavonoid compounds were
The derivative 6b showed dose dependent cytotoxicity towards carried out with EGFR (1M17) and compared with the molec-
MCF-7 and NIH3T3 cell lines. Compounds 6c and 6i showed ular docking of quercetin with 1M17. Interestingly, almost all
marginal cytotoxicity towards MCF-7 and NIH3T3 cell lines at the synthesized compounds showed non bonded interactions
lower concentrations, whereas they displayed high cytotoxicity at (Fig. 2) with the same residues such as Leu 768, Gly 772, Met
20 mM concentration. When the methoxy substituent at 40 posi- 769 and Asp 831 as observed in the crystal structure of 1M17
tion of rugosaavonoid was replaced with halogen, the with erlotinib. The protein–ligand interaction prole of 6f
compounds 6d, 6e and 6f expressed 50% cytotoxicity of MCF-7 revealed that Lys 721, Glu 738, Met 769 and Asp 831, amino
cells at the lower concentration. The synthesized derivatives of acids involved in the hydrogen bond and p–p interactions in
rugosaavonoid showed dose-dependent cytotoxicity on MCF-7 addition to hydrophobic interaction. Molecular docking score
cell lines and most of them were non-toxic to NIH3T3 cells. The of quercetin and 6f with 1M17 were found to be ꢀ8.310 and
dimethoxy derivatives 6c and 6j showed inhibition of growth of ꢀ8.608 respectively. This result is in agreement with the data
MCF-7, but they were toxic to normal cells. The images of MCF-7 published by Singh and Bast.26 Overall, docking analysis of
and NIH3T3 cells with 6f before the treatment and aer the standard quercetin and rugosaavonoid derivatives with 1M17
treatment are shown in the Fig. 1.
indicated that these derivatives had equal binding affinity
The tyrosine kinase epidermal growth factor receptor which was also well noticed from experimental cytotoxicity
(EGFR) is a transmembrane receptor central to numerous results (Table 1).
cellular process comprising cell migration, adhesion,
apoptosis and cell proliferation. The EGFR is over-expressed in
almost 90% of tumors.23,24 Protein–ligand interaction of 1M17
with EGFR-specic inhibitor25 and anticancer agent, erlotinib,
demonstrated computationally that Met 769 formed hydrogen
bond with tyrosine kinase inhibitor, whereas Leu 820, Leu 768,
3. Experimental section
3.1 Materials and method
All the chemicals used during the reactions were procured from
Spectrochem, India. 1H NMR and 13C NMR spectra were
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Fig. 2 Docking studies of rugosaflavonoid derivatives with EGFR (1M17) using discovery studio client version 4.0 (a) image of 1M17 with quercetin
without active site pocket; (b) image of 1M17 with quercetin with active site pocket (c) image of 1M17 with 6f without active site pocket; (d) image
of 1M17 with 6f with active site pocket.
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recorded at the room temperature on Varian 400 MHz spec- to obtain a crude product. The crude product was puried by
trometer and 100 MHz respectively. Chemical shi values were the column chromatography (hexane–ethyl acetate, 80 : 20) to
reported with reference to TMS as an internal standard. The acquire clean methyl 2-acetyl-3,5-dihydroxybenzoate with 68%
samples were prepared by dissolving the synthesized yield.
compounds in DMSO-d6, chemical shis were expressed in
3.2.4 Synthesis of methyl 7-hydroxy-2-(4-methoxyphenyl)-4-
d (ppm) and coupling constants (J) in hertz. The splitting oxo-3,4-dihydro-2H-chromene-5-carboxylate (5). Methyl 2-
pattern abbreviations are as follows: s, singlet; d, doublet; t, acetyl-3,5-dihydroxybenzoate (4.7 mmol) in DMSO was mixed
triplet; q, quartet; m, unresolved multiplet; dd, doublet of with anisaldehyde (4.7 mmol), I2 (0.23 mmol) and pyrrolidine
doublet. The column chromatography was performed on Merck (2.3 mmol) as per the procedure mentioned in the literature22
silica gel 60 (230–400 mesh). Analytical thin layer chromatog- and heated at reux temperature for 8 h. The progress of the
raphy was carried out on the precoated Merck silica gel 60F254 reaction was observed by the TLC. Aer completion, reaction
and iodine was used for development of compounds. IR was was allowed to cool to the room temperature and quenched with
recorded on FTIR IR Affinity ꢀ1 Shimadzu spectrophotometer. water. The aqueous layer was extracted with ethyl acetate. The
CHNS analysis was recorded on Elementar Vario El-III. Dul- organic layer was separated, dried over sodium sulfate and
becco's Modied Eagle medium (DMEM), Fetal Bovine Serum concentrated to get a crude product, which was puried by the
(FBS) and phosphate buffer saline were procured from Invi- column chromatography (hexane–ethyl acetate, 50 : 50) and
trogen. Trypsin and antibiotic solutions were procured from methyl
7-hydroxy-2-(4-methoxyphenyl)-4-oxo-3,4-dihydro-4H-
Sigma-Aldrich.
chromene-5-carboxylate was achieved in 45% yield.
3.2.5 Synthesis of methyl 7-hydroxy-2-(4-methoxyphenyl)-4-
oxo-4H-chromene-5-carboxylate (6a). I2 (0.15 mmol) was
appended to methyl 7-hydroxy-2-(4-methoxyphenyl)-4-oxo-3,4-
3.2 General procedure
3.2.1 Synthesis of methyl 3,5-dimethoxybenzoate (2). The dihydro-2H-1-benzopyran-5-carboxylate (3 mmol) in DMSO (10
intermediate 2 was prepared as per reported method by Mc ml) and reuxed for 1 h. The progress of the reaction was
Nulty and Mcleod.18 In brief, 3,5-dihydroxy benzoic acid (6 observed by the TLC. Aer completion, reaction mixture was
mmol) was taken in dry acetone. The mixture was stirred at cooled to the room temperature and quenched with water. The
40 ꢁC for 15 min, to this K2CO3 (2.5 mmol) added and the aqueous layer was extracted with ethyl acetate. The organic layer
mixture again stirred at 60 ꢁC for 10 min. Then dimethyl sulfate was separated, dried over sodium sulfate and concentrated and
(2.2 mmol) was added dropwise over a period of 30 min and the a crude product obtained, which was puried by the column
temperature was increased slowly to 80 ꢁC. The reaction mixture chromatography (hexane–ethyl acetate, 40 : 60) and obtained
was allowed to reux for 6 h. The progress of the reaction was methyl 7-hydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromene-5-
checked by TLC. Aer completion, reaction was allowed to cool carboxylate in 60% yield.
to the room temperature and ltered through the Celite bed.
Methyl 7-hydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromene-5-
The ltered mixture was concentrated to get crude product. The carboxylate (6a). Mp: 226–228 ꢁC; IR (KBr, cmꢀ1), 3446 (OH),
crude product was slowly poured on crushed ice with constant 1735 (C]O), 1624 (C]O); 1H NMR (400 MHz, DMSO-d6) d 3.81
stirring to obtain solid. The solid obtained was ltered and (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.79 (s, 1H, 3-H), 6.82 (d, 1H,
dried to get methyl 3,5-dimethoxybenzoate in 92% yield.
8-H, J ¼ 1.6 Hz), 7.10 (d, 1H, 6-H, J ¼ 2 Hz), 7.12 (d, 2H, 30, 50-H, J
3.2.2 Synthesis of methyl 2-acetyl-3,5-dimethoxybenzoate ¼ 7.2 Hz), 8.04 (d, 2H, 20, 60-H, J ¼ 7.2 Hz), 11.14 (s, 1H, OH); 13C
(3). The methyl 3,5-dimethoxybenzoate (5 mmol) was mixed NMR, (100 MHz, DMSO-d6) d 52.86 (OCH3) 55.97 (C-40, OCH3),
with acetyl chloride (25 mmol) and carbon disulde (2 ml) 104.25 (C-8), 105.58 (C-3), 113.55 (C-10), 113.94 (C-6), 114.81
under dry N2 in ice bath.19 To this AlCl3 (15 mmol) was added (C-30), 114.98 (C-50), 123.49 (C-10), 127.11 (C-60), 128.59 (C-20),
under vigorous stirring. The reaction was allowed to stir for 134.55 (C-5), 157.7 (C-9), 158.55 (C-40), 162.39 (C-2), 162.54 (C-7),
15 min. The progress of the reaction was monitored by TLC. 169.19 (C-11), 175.68 (C-4); LCMS (ESI) m/z calculated for
Aer completion, the reaction was quenched with ice and
C
18H14O6: 326.3 and found 327.0; HRMS m/z 327.0863 [M + 1]+
extracted with ethyl acetate. The organic layer was separated, elemental analysis calculated for C18H14O6: C, 66.25, H, 4.32;
dried over sodium sulfate and concentrated to get a crude found: C, 66.31, H, 4.28.
product, which was puried by the column chromatography
(hexane–ethyl acetate, 70 : 30) to obtain methyl 2-acetyl-3,5- carboxylate (6b). Mp: 240–242 ꢁC; IR (KBr, cmꢀ1), 3516 (OH),
dimethoxybenzoate in 52% yield.
1737 (C]O), 1627 (C]O); 1H NMR (400 MHz, DMSO-d6) d 2.39
Methyl
7-hydroxy-2-(4-methylphenyl)-4-oxo-4H-chromene-5-
3.2.3 Synthesis of methyl 2-acetyl-3,5-dihydroxybenzoate (s, 3H, CH3), 3.81 (s, 3H, OCH3), 6.83 (s, 1H, 3-H), 6.84 (s, 1H, 8-
(4). Methyl 2-acetyl-3,5-dimethoxybenzoate (4 mmol) was H), 7.10 (s, 1H, 6-H), 7.38 (d, 2H, J ¼ 8.0 Hz, 30, 50-H), 7.95 (d, 2H,
appended in chlorobenzene (15 ml), to this AlCl3 (10 mmol) was J ¼ 8.0 Hz, 20, 60-H), 11.18 (s, 1H, OH); 13C NMR, (100 MHz,
added slowly at room temperature20 and the reaction mixture DMSO-d6) d 21.51 (CH3), 52.88 (OCH3), 104.27 (C-8), 106.46 (C-
was heated to reux for 1 h. The progress of the reaction was 3), 113.43 (C-10), 113.68 (C-6), 126.68 (C-20, 60), 128.55 (C-10),
scrutinized by the TLC. Aer completion, the reaction was 130.14 (C-30, 50), 130.25 (C-40), 134.58 (C-5), 142.42 (C-9), 157.78
cooled to room temperature and hydrolysed using 1 N HCl. The (C-2), 162.49 (C-7) 169.14 (C-11), 175.76 (C-4); LCMS (ESI) m/z
reaction mixture was extracted with ethyl acetate. The organic calculated for C18H14O5: 310.3 and found 311.0. Elemental
layer was separated, dried over sodium sulfate and concentrated
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analysis calculated for C18H14O5: C, 69.66, H, 4.54; found: C, (s, 1H, OH); 13C NMR, (100 MHz, DMSO-d6) d 22.92 (CH3), 55.96
69.61, H, 4.49.
(C-40, OCH3), 101.38 (C-8), 106.82 (C-3), 114.95 (C-30, 50), 117.19
Methyl 2-(3,4-dimethoxyphenyl)-7-hydroxy-4-oxo-4H-chromene- (C-6), 123.73 (C-10), 128.27 (C-20, 60), 128.56 (C-10), 141.90 (C-5),
5-carboxylate (6c). Mp: 233–236 C; IR (KBr, cmꢀ1), 3444 (OH), 159.32 (C-9), 160.71 (C-40), 161.57 (C-2), 162.26 (C-7) 178.91 (C-
ꢁ
1737 (C]O), 1627 (C]O); 1H NMR (400 MHz, DMSO-d6) d 3.78 4); LCMS (ESI) m/z calculated for C17H14O4: 282.29 and found
(s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.79 (s, 283.0. Elemental analysis calculated for C17H14O4: C, 72.32, H,
1H, 3-H), 6.84 (s, 1H, 8-H), 7.13 (m, 2H, 50-H, 6-H), 7.52 (s, 1H, 20- 4.99; found: C, 72.36, H, 4.93.
H), 7.63 (d, 1H, 60-H, J ¼ 8 Hz), 11.08 (s, 1H, OH); 13C NMR, (100
7-Hydroxy-2-(4-methylphenyl)-5-methyl-4-oxo-4H-chromene
MHz, DMSO-d6) d 52.87 (OCH3), 56.17 (OCH3), 56.32 (OCH3), (6h). Mp: 252–255 C; IR (KBr, cmꢀ1), 3565 (OH), 1710 (C]O);
104.34 (C-8), 105.89 (C-3), 109.83 (C-60), 112.14 (C-50), 113.39 (C- 1H NMR (400 MHz, DMSO-d6) d 2.38 (s, 3H, CH3), 2.69 (s, 3H,
10), 113.53 (C-6), 120.29 (C-10), 123.59 (C-20), 134.51 (C-5), 149.47 CH3), 6.65 (s, 1H, 3-H), 6.72 (s, 1H, 8-H), 6.82 (s, 1H, 6-H), 7.36
(C-30), 152.34 (C-40), 157.74 (C-9), 162.36 (C-2), 162.48 (C-7), (d, 2H, 30, 50-H, J ¼ 8.4 Hz), 7.92 (d, 2H, 20, 60-H, J ¼ 7.6 Hz), 10.62
169.19 (C-11), 175.74 (C-4); LCMS (ESI) m/z calculated for (s, 1H, OH); 13C NMR, (100 MHz, DMSO-d6) d 21.54 (CH3), 22.92
ꢁ
C
19H16O7: 356.32 and found 357.0; elemental analysis calcu- (CH3), 101.40 (C-8), 107.65 (C-3), 115.16 (C-10), 117.27 (C-6),
lated for C19H16O7: C, 64.04, H, 4.52; found: C, 64.16, H, 4.59.
126.41 (C-30, C-50), 128.76 (C-10), 130.12 (C-20, C-60), 141.96 (C-
Methyl
2-(4-chlorophenyl)-7-hydroxy-4-oxo-4H-chromene-5- 40, C-5), 159.37 (C-9), 160.78 (C-2), 161.67 (C-7), 178.94 (C-4);
carboxylate (6d). Mp: 262–268 ꢁC; IR (KBr, cmꢀ1), 3645 (OH), LCMS (ESI) m/z calculated for C17H14O3: 266.29 and found
1714 (C]O), 1697 (C]O); 1H NMR (400 MHz, DMSO-d6) d 3.78 267.0. Elemental analysis calculated for C17H14O3: C, 76.67, H,
(s, 3H, OCH3), 6.80 (s, 1H, 3-H), 6.89 (s, 1H, 8-H), 7.08 (s, 1H, 6- 5.29; found: C, 76.62, H, 5.26.
H), 7.60 (d, 2H, 20, 60-H, J ¼ 8.4 Hz), 8.07 (d, 2H, 30, 50-H, J ¼ 8.8
2-(2-Fluorophenyl)-7-hydroxy-5-methyl-4-oxo-4H-chromene (6i).
Hz), 11.19 (s, 1H, OH); 13C NMR, (100 MHz, DMSO-d6) d 52.91 Mp: 252–255 ꢁC; IR (KBr, cmꢀ1), 3564 (OH), 1714 (C]O); 1H
(OCH3), 104.32 (C-8), 107.48 (C-3), 113.36 (C-10), 113.86 (C-6), NMR (400 MHz, DMSO-d6) d 2.69 (s, 3H, CH3), 6.55 (s, 1H, 3-H),
128.60 (C-20, 60), 129.62 (C-30, 50), 130.28 (C-10), 134.60 (C-5), 6.67 (s, 1H, 6-H), 6.78 (s, 1H, 8-H), 7.41–7.48 (m, 2H, 50, 60-H),
136.99 (C-9), 157.79 (C-40), 161.24 (C-2), 162.63 (C-7), 169.06 7.62–7.63 (m, 1H, 40-H), 7.98–8.0 (m, 1H, 30-H), 10.71 (s, 1H,
(C-11), 175.75 (C-4); LCMS (ESI) m/z calculated for C17H11ClO5: OH). 13C NMR, (100 MHz, DMSO-d6) d 22.90 (CH3), 101.34 (C-8),
330.71 and found 331.0; elemental analysis calculated for 112.91 (C-60), 113.01 (C-50), 114.96 (C-40), 117.24 (C-6), 117.47 (C-
C
17H11ClO5: C, 61.73, H, 3.34; found: C, 61.67, H, 3.28.
Methyl
3), 125.71 (C-10), 129.78 (C-30), 133.73 (C-20), 133.82 (C-10),
2-(4-bromophenyl)-7-hydroxy-4-oxo-4H-chromene-5- 142.13 (C-5), 156.58 (C-9), 159.54 (C-2), 161.92 (C-7), 178.58 (C-
carboxylate (6e). Mp: 275–277 ꢁC; IR (KBr, cmꢀ1), 3564 (OH), 4); LCMS (ESI) m/z calculated for C16H11FO3: 270.25 and
1737 (C]O), 1627 (C]O); 1H NMR (400 MHz, DMSO-d6) d 3.78 found 271.0. Elemental analysis calculated for C16H11FO3: C,
(s, 3H, OCH3), 6.81 (s, 1H, 8-H), 6.90 (s, 1H, 6-H), 7.07 (s, 1H, 3- 71.10, H, 4.09; found: C, 71.16, H, 4.17.
H), 7.75 (d, 2H, 20, 60-H, J ¼ 8 Hz), 8.0 (d, 2H, 30, 50-H, J ¼ 8 Hz),
2-(3,4-Dimethoxylphenyl)-7-hydroxy-5-methyl-4-oxo-4H-chro-
11.18 (s, 1H, OH); 13C NMR, (100 MHz, DMSO-d6) d 53.03 mene (6j). Mp: 231–234 ꢁC; IR (KBr, cmꢀ1), 3564 (OH), 1704 (C]
(OCH3) 104.38 (C-8), 107.41 (C-3), 113.35 (C-10), 113.93 (C-6), O); 1HMR (400 MHz, DMSO-d6) d 2.69 (s, 3H, CH3), 3.86 (s, 3H,
126.01 (C-20, C-60), 128.78 (C-30, 50), 130.59 (C-10), 132.62 (C-5), OCH3), 3.90 (s, 3H, OCH3), 6.76 (s, 1H, 3-H), 6.79 (s, 1H, 8-H),
134.62 (C-9), 157.84 (C-40), 161.52 (C-2), 162.70 (C-7), 169.19 6.91 (d, 1H, 6-H), 7.13 (d, 1H, 60-H, J ¼ 7.2 Hz), 7.17 (d, 1H, 20-H,
(C-11), 175.90 (C-4); LCMS (ESI) m/z calculated for C17H11BrO5: 7.2 Hz), 7.77 (d, 1H, 50-H, J ¼ 7.2 Hz), 10.60 (s, 1H, OH). 13C
375.17 and found 376.9, 378.9. Elemental analysis calculated for NMR, (100 MHz, DMSO-d6) d 22.90 (CH3) 58.90 (OCH3), 59.30
C
17H11BrO5: C, 54.42, H, 2.95; found: C, 54.48, H, 2.95.
Methyl
(OCH3), 101.33 (C-8), 104.20 (C-3), 110.30 (C-6), 113.35 (C-10),
2-(2-uorophenyl)-7-hydroxy-4-oxo-4H-chromene-5- 114.27 (C-10), 116.57 (C-60), 120.50 (C-20), 121.86 (C-50), 141.86
carboxylate (6f). Mp: 222–225 ꢁC; IR (KBr, cmꢀ1), 3645 (OH), (C-5), 149.83 (C-30), 153.23 (C-40), 159.30 (C-9), 160.80 (C-2),
1732 (C]O), 1697 (C]O); 1H NMR (400 MHz, DMSO-d6) d 3.81 162.55 (C-7), 178.90 (C-4); LCMS (ESI) m/z calculated for
(s, 3H, OCH3), 6.65 (s, 1H, 3-H), 6.86 (s, 1H, 8-H), 7.06 (s, 1H, 6-
C18H16O5: 312.31 and found 313.0. Elemental analysis calcu-
H), 7.41–7.5 (m, 2H, 50-H, 60-H), 7.63–7.70 (m, 1H, 40-H), 8.02– lated for C18H16O5: C, 69.21, H, 5.15; found: C, 69.17, H, 5.11.
8.06 (m, 1H, 30-H), 11.22 (s, 1H, OH); 13C NMR, (100 MHz,
DMSO-d6) d 52.81 (OCH3), 104.15 (C-8), 110.07 (C-6). 110.71 (C-
3.3 Biology
10), 116.12 (C-50), 116.30 (C-3), 125.21 (C-30), 128.93 (C-10),
128.96 (C-60), 131.36 (C-9), 136.50 (C-5), 159.11 (C-20), 161.07 (C-
40), 163.43 (C-2), 164.45 (C-7), 169.39 (C-11), 185.83 (C-4); LCMS normal NIH3T3 cell lines were obtained from National Center
3.3.1 Cell lines and culture. Breast cancer MCF-7 and
(ESI) m/z calculated for C17H11FO5: 314.26 and found 315.0. for Cell Science, Pune, India. All cell lines were cultured in
ꢁ
Elemental analysis calculated for C17H11FO5: C, 64.96, H, 3.52; a humidied atmosphere of 5% CO2 in DMEM at 37 C, sup-
found: C, 64.91, H, 3.56.
plemented with 10% FBS, penicillin (100 mg mlꢀ1) and strep-
tomycin (100 mg mlꢀ1).
7-Hydroxy-2-(4-methoxyphenyl)-5-methyl-4-oxo-4H-chromene
(6g). Mp: 245–249 C; IR (KBr, cmꢀ1), 3566 (OH), 1704 (C]O);
3.3.2 MTT assay for cytotoxicity against MCF-7 and NIH3T3
ꢁ
1H NMR (400 MHz, DMSO-d6) d 2.68 (s, 3H, CH3), 3.83 (s, 3H, cells. MCF-7 and NIH3T3 cells were grown in DMEM media
OCH3), 6.63 (s, 1H, 3-H), 6.67 (s, 1H, 8-H), 6.81 (s, 1H, 6-H), 7.08 containing 10% FBS. The effect of rugosaavonoid derivatives
(d, 2H, 20, 60-H, J ¼ 8.4 Hz), 7.97 (d, 2H, 30, 50-H, J ¼ 8.8 Hz), 10.59 on the growth of MCF-7 cells (breast cancer cell lines) and
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NIH3T3 cells was examined using the MTT assay. Cells were
subcultured in 96-well plates at a density of 2.5 ꢂ 103 cells per
well without test samples for 24 h in a nal volume of 150 ml.
Aer 24 h, cells were treated with four different concentrations
test specimens (5, 10, 15 and 20 mM in plain DMEM) in tripli-
cates and kept for 24 h in the CO2 incubator. Next day, treat-
ment media was removed and 20 ml of MTT (1.5 mg mlꢀ1 in PBS)
was added to the fresh medium. Aer three hours' of incubation
at 37 ꢁC in CO2 incubator, 180 ml DMSO was added to the each
well and plates were agitated for 1 min. Spectrophotometric
absorbance at 570 nm was measured. The percentage of
viability was calculated as per the following formula: (viable
cells)% ¼ (OD of drug-treated sample/OD of untreated sample)
ꢂ 100. Quercetin was used as the standard.
Conflict of interest
There is no conict of interest among the authors.
Acknowledgements
Authors are really thankful to Hu et al. for the isolation and
characterization of rugosaavonoid because of his team work
we are able to develop synthetic route for naturally occurring
compound (6a). We acknowledge the unconditional support of
Mr Vinod Devaraji to understand Maestro 11.2 soware to
perform the molecular docking studies. Authors are also
thankful to Agharkar Research Institute, Pune, India, for
providing nancial support and infrastructure.
3.4 Molecular docking studies
Notes and references
The molecular modelling studies were carried out on Windows
7 64-bit operating system using Maestro 11.2 soware. The
GLIDE docking application of Maestro 11.2 soware was used to
calculate GScore, which is based on an empirical scoring
function and deploy a combination of several parameters.
GScore (docking score) was calculated in kcal molꢀ1 and it
included ligand–protein interaction energies, hydrophobic
interactions, hydrogen bonds, internal energy, p–p stacking
interactions, root mean square deviation (RMSD) and des-
olvation. The X-ray crystallographic structure of erlotinib coc-
rystallized with EGFR was obtained from the protein data bank
(PDB ID: 1M17). The ATP binding site of EGFR was prepared for
docking studies in which erlotinib was removed from the active
site, hydrogen atoms were added to the structure with their
standard geometry. Active sites were observed from the
sequence analysis and soware run and it was used in pre-
dicting interactions at the active site between the selected
compounds 6f and quercetin with EGFR. The 2D structures of
the docked compounds were generated, transformed to 3D,
protonated, and energy was minimized by using Ligprep
application. Grid generation and ligand docking was performed
to obtain ligand interaction diagram with 1M17 EGFR and
docking score. Receptor–ligand interaction images were ob-
tained from discovery studio client version.
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