Ninad V. Puranik and
Pratibha Srivastava*
Bioprospecting Group, Agharkar Research Institute, G. G. Agarkar Road, Pune, 411004, Maharashtra, India. E-mail: ninadv_puranik@yahoo.co.in; psrivastava@aripune.org
First published on 29th June 2017
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.
Scheme 1 Synthesis of rugosaflavonoid and its derivatives using 3,5-dihydroxybenzoic acid as starting material. |
S. no. | Conc. used | Residue involved in binding with 1M17 | Docking score | |||||||
---|---|---|---|---|---|---|---|---|---|---|
MCF7 (% cell viability) | NIH3T3 (% cell viability) | |||||||||
5 μM | 10 μM | 15 μM | 20 μM | 5 μM | 10 μM | 15 μM | 20 μM | |||
a All the samples run in triplicate and average of three results are presented here. | ||||||||||
6a | 43 | 58 | 60 | 68 | 87 | 74 | 71 | 54 | Met 769, Leu 768, Asp 831, Gly 772, Leu 694, Glu 738 | −5.040 |
6b | 58 | 47 | 41 | 40 | 90 | 67 | 66 | 62 | Met 769, Leu 768, Leu, 694, Gly 772, Asp 831, Thr 830 | −6.159 |
6c | 70 | 52 | 49 | 32 | 73 | 83 | 84 | 82 | Met 769, Leu 768, Gln 767, Asp 831, Thr 830, Leu 694 | −6.661 |
6d | 64 | 62 | 45 | 36 | 99 | 97 | 93 | 91 | Met 769, Leu 768, Asp 831, Glu 831 | −6.549 |
6e | 50 | 49 | 41 | 31 | 84 | 79 | 33 | 30 | Met 769, Leu 768, Asp 831, Glu 738 | −6.483 |
6f | 52 | 45 | 39 | 31 | 96 | 85 | 70 | 62 | Met 769, Leu 768, Leu 694, Asp 831, Lys 721, Glu 738 | −8.310 |
6g | 56 | 50 | 48 | 44 | 83 | 71 | 64 | 51 | Met 769, Leu 768, Gln 767, Asp 831, Thr 830, Leu 694 | −4.557 |
6h | 65 | 46 | 43 | 40 | 89 | 74 | 60 | 43 | Met 769, Leu 768, Leu, 694, Gly 772, Asp 831, Thr 830 | −4.743 |
6i | 71 | 53 | 48 | 41 | 92 | 88 | 82 | 85 | Met 769, Leu 768, Asp 831, Glu 831 | −4.743 |
6j | 54 | 49 | 49 | 47 | 55 | 55 | 62 | 65 | Met 769, Leu 768, Asp 831, Glu 738 | −4.965 |
Std I quercetin | 90 | 75 | 67 | 50 | 89 | 76 | 72 | 68 | Met 769, Lys 721, Glu 738, Asp 831 | −8.608 |
Paramater | Rugosaflavonoid synthesized | Rugosaflavonoid isolated |
---|---|---|
Mp | 226–228 °C | Not reported |
HRMS | m/z 327.0863 [M + 1]+ (calcd for C18H15O6, 327.0863) | HRESIMS m/z 349.0682 [M + Na]+ (calcd for C18H14NaO6, 349.0688) |
IR (cm−1) | 3446, 1735, 1624, 1600, 1543, 1436, 1435, 1253, 1180, 1029, 894 | 3416, 1702, 1657, 1610, 1565, 1456, 1432, 1287, 1182, 1028, 893 |
1H NMR | (Solvent DMSd6) δ 3.81, (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.79 (s, 1H, 3-H), 6.82 (d, 1H, 8-H, J = 1.6 Hz), 7.10 (d, 1H, 6-H, J = 2 Hz), 7.12 (d, 2H, 3′, 5′-H, J = 7.2 Hz), 8.04 (d, 2H, 2′, 6′-H, J = 7.2 Hz), 11.14 (s, 1H, OH) | (Solvent pyridine-d5, 500 MHz) δ 3.80 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.68 (s, 1H, 3-H), 6.74 (d, 1H, 8-H, J = 1.8 Hz), 6.89 (d, 1H, 6-H, J = 1.8 Hz), 7.00 (d, 2H, 3′,5′-H, J = 8.8 Hz), 7.76 (d, 2H, 2′,6′-H, J = 8.8 Hz) |
13C NMR | 52.86 (OCH3), 55.97 (C-4′, OCH3) 104.25 (C-8), 105.58 (C-3), 113.55 (C-10), 113.94 (C-6), 114.81 (C-3′), 114.98 (C-5′), 123.49 (C-1′), 127.11 (C-6′), 128.59 (C-2′), 134.55 (C-5), 157.7 (C-9), 158.55 (C-4′), 162.39 (C-2), 162.54 (C-7), 169.19 (C-11), 175.68 (C-4) | 52.4 (OCH3), 55.6 (C-4′, OCH3), 103.8 (C-8), 105.2(C-3), 113.1 (C-6), 115.6 (C-3′), 115.6 (C-5′), 122.9 (C-1′), 131.0 (C-2′), 131.0 (C-6′), 136.8 (C-5), 158.8 (C-9), 163.2 (C-2), 165.0 (C-7), 168.3 (C-11), 181.5 (C-4) |
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. |
The tyrosine kinase epidermal growth factor receptor (EGFR) is a transmembrane receptor central to numerous 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-specific inhibitor25 and anticancer agent, erlotinib, demonstrated computationally that Met 769 formed hydrogen bond with tyrosine kinase inhibitor, whereas Leu 820, Leu 768, Gly 772, Met 769, and Leu 694 indicated hydrophobic interaction with tyrosine kinase inhibitor, erlotinib. Therefore, interaction studies of rugosaflavonoid compounds were carried out with EGFR (1M17) and compared with the molecular docking of quercetin with 1M17. Interestingly, almost all the synthesized compounds showed non bonded interactions (Fig. 2) with the same residues such as Leu 768, Gly 772, Met 769 and Asp 831 as observed in the crystal structure of 1M17 with erlotinib. The protein–ligand interaction profile of 6f revealed that Lys 721, Glu 738, Met 769 and Asp 831, amino acids involved in the hydrogen bond and π–π interactions in addition to hydrophobic interaction. Molecular docking score of quercetin and 6f with 1M17 were found to be −8.310 and −8.608 respectively. This result is in agreement with the data published by Singh and Bast.26 Overall, docking analysis of standard quercetin and rugosaflavonoid derivatives with 1M17 indicated that these derivatives had equal binding affinity which was also well noticed from experimental cytotoxicity results (Table 1).
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. |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra04971d |
This journal is © The Royal Society of Chemistry 2017 |