Syntheses of 2-methoxyestradiol and eugenol template based diarylpropenes as non-steroidal anticancer agents

Abstract

Syntheses of 2-methoxyestradiol (1) and eugenol (6) template based conformationally flexible and rigid diarylpropenes, 14(a–l) and 20(a–e), as nonsteroidal anticancer agents have been performed. The synthesized compounds were evaluated for their anticancer activity in in vitro using a panel of human cancer cell lines viz. MCF-7, A549, DU 145, KB and MDA-MB-231by SRB assay. Compounds 14i, 14k and 15a showed significant anticancer activity at IC₅₀ between 10.27 μM to 27.91 μM in different cancer cell lines. The most active molecule, 14k, inhibited proliferation of cells by inducing apoptosis and arresting the cell cycle at the G2/M phase. In vitro toxicity of these compounds (14i, 14k and 15a) in healthy hepatic monocyte (THP-1) cells showed high selectivity of compounds towards cancerous vs. healthy cells.

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Introduction

2-Methoxyestradiol (2ME2, 2), a metabolite of 17β-estradiol (1), possess potent antiproliferative, antiangiogenic and proapoptotic activities and is now an investigational drug for cancer chemotherapy under the trade name Panzem® (Fig. 1).^1–8 In humans, 2ME2 is formed via transformation of estradiol (1) to 2-hydroxyestradiol (2OHE2) which is finally converted into 2ME2 with the help of hepatic cytochrome P450 and catechol-O-transferase enzymes.^9,10 The antiproliferative activity of 2ME2 is independent of estrogen receptors (ERs) since it has very weak interactions with ERs, rather it acts through its binding with the colchicine binding site of β-tubulin protein.^11–15

Fig. 1 Estradiol derivatives, cytotoxic phytomolecules and designed Prototype I and II.

Structurally, the presence of a phenyl ring bearing a hydroxy and methoxy group in the tetracyclic steroidal framework is a prime requirement for biological activity of 2ME2. Similarly, some natural products such as combretastatin CA4 (3), isoeugenol (4) and dehydrozingerone (5) which also possess a phenyl ring bearing a hydroxy and methoxy group similar to 2ME2 (2) are reported to have diverse biological activities including anticancer activity.^16,17

Furthermore, eugenol (6), another structurally similar natural product, present in ocimum sanctum L (sweet basil) and clove (syzygium aromaticum L) displays diverse biological activities such as antimicrobial, antioxidant, and anticancer activities etc. (Fig. 1).^18,19 In two independent studies, the weak anticancer activity of 6 was potentiated by use of gemcitabine (7) and 2ME2 respectively in combination therapy.^20,21 These observations concluded that eugenol (6) has synergetic effect on cell proliferation inhibition. In literature, a substituted diarylpropene derivative isolated from Dalbergia paviflora (leguminosae) has been reported for significant cell proliferation inhibitory activity.²² Following this lead, Ito et al. synthesized different non-target specific diarylpropene analogues as cancer chemo-preventive agents and evaluated them in a single cancer cell line using curcumin as positive control.²³

In view of the fact that main clinical demerit of 2ME2 is limited bioavailability due to its transformation into glucuronoids and 17β-hydroxydehydrogenase (17β-HSD) mediated conversion of 2ME2 into 2-methoxyestrone (2ME1), there is a need to develop non-steroidal mimics of 2ME2 which can have similar mode of action and devoid of many side effects associated with 2ME2.^24,25

Realizing the biological potential of diarylpropene pharmacophore and its suitability with the objective of present study, different target oriented (i.e. estrogen receptor(ER) and/or tubulin protein), 2-methoxyestradiol (2ME2) and eugenol (6) template based conformationally flexible and rigid diarylpropenes of type I and II have been synthesized as non-steroidal cancer chemotherapeutic agents and evaluated for their cytotoxic activity in different cancer cell lines (specially in ER + cell line) for cancer chemotherapy (Fig. 1).

The proposed prototypes will have a phenyl ring bearing a hydroxy and methoxy group similar to 2ME2 (2), CA4 (3) and eugenol (6), and a cytostatic portion which could possibly encompass the region homologous to the rings C and D complex of 2ME2.

Results and discussion

Chemistry

The synthesis of prototype-I was started through base catalyzed Aldol condensation of substituted acetophenone (9) and benzaldehyde (10) at room temperature which gave diaryl propenones (11) in 82–92% yields (Scheme 1). NMR spectroscopic analyses of diarylpropenone derivatives (11) revealed trans stereochemistry across the C=C bond. Diarylpropenones (11) were subjected to catalytic hydrogenation using 10% palladium adsorbed on charcoal (Pd/C) in ethylacetate–methanol mixture (8 : 2) at normal pressure and temperature for about 3 h which yielded diarylpropanones (12) in 66–93% yields. Subsequently, reduction of 12 using sodium borohydride at room temperature yielded diarylpropanols (13) as racemic mixture in quantitative yields. The racemic mixture of compound 13 on dehydration in presence of HCl in ethanol at reflux for 1 h gave desired diarylpropenes (14) in 48–85% yields.

Scheme 1 Reagent and conditions (a) KOH in ethanol, 35–40 °C; (b) 10% Pd/C, ethylacetate–methanol (1 : 1), 35–40 °C; (c) sodium borohydride, dry ethanol, 35–40 °C; (d) HCl, methanol, reflux. ^a In case of hydroxy derivatives of 10, R = THP which was deprotected during workup and gave hydroxy derivative of 11.

Proton NMR spectroscopic analyses of diarylpropene derivatives (14) showed coupling constant (J value) in the range of 15.6 to 15.9 Hertz for olefinic proton which revealed trans stereochemistry across the C=C bond. For SAR study, compound 15 was proposed to be synthesized from compound 14k. Synthesis of 15 was done through catalytic hydrogenation of 14k using 10% palladium adsorbed on charcoal (Pd/C) in ethylacetate–methanol mixture (8 : 2) at normal pressure and temperature for about 1 h, yielded 15 in 78–90% yield.

The synthesis of target compounds of prototype (II) was made using same methodology used for synthesis of prototype-I (Scheme 2). Cyclic ketones (16) such as 6-methoxytetralone, 5-methoxyindanone, 4,5-dimethoxyindanone and substituted benzaldehyde (10) were used as starting materials to generate chalcones (17) which on reduction of C=C double bond using catalytic hydrogenation gave compound 18 as racemic mixture. Compound 18 on sodium borohydride reduction yielded 19 as diasteromeric mixture. Compound 19 on dehydration under acidic reaction conditions yielded compound 20. Generally, yields at each step of the synthesis were good and comparable to the corresponding open chain analogues (Scheme 1).

Scheme 2 Reagent and conditions (a) (i) KOH in ethanol, 35–40 °C (ii) HCl; (b) 10% Pd/C, ethylacetate–methaol (1 : 1), 35–40 °C; (c) sodium borohydride, dry ethanol, 35–40 °C; (d) HCl, methanol, reflux. ^a In case of hydroxy derivatives of 10, R = THP which was deprotected during workup and gave hydroxy derivative of 17.

The synthesized compounds were characterized by the use of different spectroscopic techniques viz. NMR, IR, mass spectrometry.

HPLC analysis

The purity of compounds 14 (a–l), 15 (a–c) and 20 (a–e) was determined using high-low chromatographic approach on reverse phase solid matrix C₁₈ coupled with photo diode array (PDA) detector with gradient mobile phase composition of acetonitrile–water (80 : 20, v/v) with and without acid additives at a flow rate of 1.0 mL min⁻¹. Peak area normalization method of high-low chromatographic analysis was adopted to determine the chromatographic purity and the concentration of every identified HPLC peak of compounds in the absence of primary standards. The purity of the compounds ranged 98–99%.

Biology

The compounds 14 (a–l), 15(a–c) and 20 (a–e) were investigated for their anticancer activity in in vitro model in a panel of human cancer cell lines viz. A549 (lung carcinoma), DU 145 (prostate carcinoma), KB (oral carcinoma contaminated with HeLa cells), MCF-7 (ER+ breast adenocarcinoma) and MDA-MB-231 (ER− breast adenocarcinoma) using SRB assay. Further, Docking experiments were performed for the most active compound 14k using β-tubulin protein and estrogen receptor α to predict the possible mode of action.

In vitro anticancer activity

The anticancer activity of compounds, 14 (a–l), 15 (a–c) and 20 (a–e) is described by half maximal inhibitory concentration (IC₅₀) value in Table 1. Most of the compounds have shown anticancer activity at IC₅₀ value between 10.27 μM to 81.97 μM in different cancer cell lines under study. In these experiments, tamoxifen (TAM), 2-methoxyestradiol (2ME2) and eugenol (6) were used as positive control (Table 1). Out of twenty compounds, compounds 14i, 14k and 15a showed significant anticancer activity at IC₅₀ between 10.27 μM to 27.91 μM in different cancer cell lines comparable to tamoxifen (TAM), 2-methoxyestradiol (2ME2) and eugenol (6) (Table 1). Compound 14k was found to be most active compound among two series.

Table 1 In vitro anticancer activity of compounds 14 (a–l), 15 (a–c) and 20 (a–e) using SRB assay (IC₅₀ in μM)^b,c and in vitro cytotoxicity in THP-1 Cells using resazurin assay (CC₅₀ in μM)

		IC50 (μM) (mean ± SE)					CC50 (μM)
S no	Compound	MCF-7	A549	DU145	KB	MDA-MB-231	THP-1
a Although the compounds had been tested thrice in this cell line, value of one assay was >50 (a) or >100 (b), therefore it could not be included in calculation of mean and SE.b Values are represented as mean IC50 value ± SE of three independent experiments, (−) = not done.c Cell lines used: A549 (lung carcinoma), DU 145 (prostate carcinoma), KB (oral carcinoma contaminated with HeLa cells), MCF-7 (ER+ breast adenocarcinoma) and MDA-MB-231 (ER− breast adenocarcinoma).
1	14a	66.32 ± 8.55	37.00 ± 3.72	44.60 ± 3.59	20.10 ± 4.47	60.84 ± 4.58	—
2	14b	78.72 ± 6.54	55.90 ± 6.64	72.22 ± 2.70	58.11 ± 7.31	>100	—
3	14c	54.59 ± 2.03	48.35 ± 4.99	60.01 ± 2.98	42.58 ± 4.58	66.44 ± 1.66	—
4	14d	54.57 ± 1.03	50.69 ± 5.77	58.34 ± 3.59	41.05 ± 4.47	65.79 ± 0.17	—
5	14e	>50	>50	>50	>50	—	—
6	14f	>100	>100	>100	>100	>100	—
7	14g	68.74^a	52.40^a	>100	53.83 ± 11.30	>100	—
8	14h	45.28 ± 26.78	23.06^a	31.49^a	38.01 ± 27.49	>100	—
9	14i	16.03 ± 1.54	16.45 ± 0.45	26.53 ± 7.19	19.12 ± 10.47	—	150
10	14j	34.44 ± 2.13	32.67 ± 1.48	>50	38.752^a	—	—
11	14k	14.27 ± 1.11	10.27 ± 0.86	17.99 ± 12.96	—	—	300
12	14l	56.45 ± 1.77	47.66 ± 5.52	64.29 ± 2.33	39.14 ± 4.48	65.23 ± 1.16	—
13	20a	48.08 ± 0.85	46.85 ± 6.95	59.81 ± 1.91	31.94 ± 2.83	56.96 ± 1.43	—
14	20b	>100	>100	>100	46.23^a	>100	—
15	20c	71.80 ± 4.95	66.39 ± 6.77	81.97 ± 4.23	51.53 ± 6.05	>100	—
16	20d	58.82^a	>100	>100	53.84 ± 19.11	>100	—
17	20e	79.70^a	67.16^a	>100	56.66 ± 18.69	>100	—
18	15a	13.42 ± 4.03	11.73 ± 1.34	21.56 ± 5.25	14.97 ± 9.61	—	300
19	15b	>100^a	>100^a	>100^a	>100^a	>100^a	300
20	15c	57.80 ± 9.96	14.45^a	33.69^a	<6.25^a	—	100
21	Eugenol	40.69^a	33.50 ± 1.14	33.19 ± 2.74	38.58^a	—	—
22	TAM	8.74 ± 1.17	10.37 ± 0.84	12.14 ± 0.62	11.40 ± 2.02	8.41 ± 1.15	—
23	2ME2	>30^a	7.51^a	—	<1.73^a	>30^a	—

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In vitro toxicity in healthy hepatomonocyte (THP-1) cells

Compound 14i, 14k and 15 (a–c) were further evaluated for their inherent toxicity in healthy hepatic monocyte (THP-1) cells in vitro to know their selectivity towards cancerous cell vs. healthy cells. The toxicity results showed that compounds 14k, 15a and 15b did not show any toxicity to the healthy cells at 300 μM concentration whereas, compound 14i and 15c was devoid of any toxicity to the healthy THP-1 cells at 150 μM and 100 μM concentrations respectively. The toxicity assessment results and anticancer activities of (IC₅₀ value) of these compounds (14i, 14k and 15a) showed many fold selectivity towards cancerous cell vs. healthy cells.

Cell division cycle study

Being a potent molecule of the both series, compound 14k was further studied for detailed biological characterization. For this purpose, estrogen receptor (ER) positive human breast cancer cell line (MCF-7) was selected for subsequent biological assays. The effect of 14k on cell division cycle was studied. After 24 h incubation with the compound at 15 μM concentration (near IC₅₀ value), 14k showed significant accumulation of G2/M population compared to the vehicle treated controls (Fig. 2a–c). This was associated with significant decrease in cells at S and G0/G1 phase.

Fig. 2 Effect of 14k on cell division cycle (a, b) MCF-7 cells were treated with 14k at IC₅₀ concentrations for 24 h. After staining with PI, cells were subjected to flow cytometry. (c) Histogram showing average population cells in various phases (G1, G2, S) of cell cycle (mean ± S.E. of three independent assays, each performed in duplicate). #P < 0.05, *P < 0.001 compared with vehicle treated controls.

We further investigated ability of 14k to trigger apoptosis in MCF-7 cells. Cells were exposed to the compound at IC₅₀ concentration for 24 h, stained with DAPI and were observed under fluorescent microscope for fragmented nuclei, a hallmark of apoptosis. Compound 14k caused marked fragmentation of MCF-7 cellular nuclei in comparison to the vehicle control group (Fig. 3).

Fig. 3 14k induced apoptosis in MCF-7 cells, MCF-7 cells were treated with the compound at IC₅₀ concentrations for 24 h at 37 °C. Cells were stained with DAPI and images acquired on a fluorescent microscope (Nikon, Japan) using 20× objective.

Structure–activity relationship (SAR) study

Further, the structure–activity (SAR) relationship of synthesized compounds revealed that position of double bond in these molecules had great impact on their biological activity. Compound 14h and 14k are positional isomers with respect to the position of double bond (C=C). Both compounds bear a methoxyphenyl group and a trimethoxyphenyl group linked together by –HC=CH–CH₂ linker. Compound 14k showed better activity (at IC₅₀ 10.27 μM to 17.99 μM) than 14h (at IC₅₀ 23.06 μM to 45.28 μM) in all cancer cell lines. The observed difference in their activity might be due to position of the double bond. Based on their observation, Amato et al. have shown that ring A of 2ME2 is homologous to the ring C of colchicines (8) and ring B of the combretastin-A4 (CA4, 3) and the remaining nonplanar part is homologous to the rings C and D complex.²⁶ Similarly, it is possible that structural arrangement of compound 14k allows it to interact with β-tubulin for its cytotoxic activity in general and has ER mediated selectivity for cytotoxic activity in ER positive cells (MCF-7 cells) in particular. This was further supported by docking experiment (discussed below). Removal of double bond from 14k (compound 15a) did not affect anticancer activity of 14k, however, in case of 14i, removal of C=C double bond (compound 15b) abolished the biological activity. The anticancer activity of 14k and 15a in ER positive cancer cells (MCF-7 cells) can be assumed due to better structural complementarities of these derivatives with ligand binding pocket of ER.

Docking study

To verify our assumptions, docking experiments were performed (Table 2) for compound 14k on β-tubulin and estrogen receptor-α (Fig. 4b and 5b). Docking of 14k at colchicine binding site showed good binding within colchicine binding site of β-tubulin with binding energy of −5.34 kcal mol⁻¹ and inhibitory constant (K_i value) of 121.87 μM. It had a hydrogen bond (H-bond) interactions via oxygen atom of trimethoxyphenyl group with bond distance of 3.06 Å with Cys β241amino acid of β-tubulin similar to colchicine (5) and 2ME2 (2).^27,28 Further, docking of 14k on ER-α [PDB:1A52] showed one H-bond interaction between trimethoxyphenyl group of 14k with Leu327 of ligand binding pocket (LBP) having bond distance of 2.85 Å in a region encompassed by rings C and D (Fig. 5a). The binding energy and inhibitory constant for 14k were found to be −5.9 kcal mol⁻¹ and 47.33 μM respectively (Fig. 5b). Interestingly, a virtual hydroxyl derivative of 14k (14k′) showed one H-bond interaction between trimethoxyphenyl group and Ile326 in a region encompassed by rings C and D of 17β-estradiol (1) with bond distance of 3.27 Å (Fig. 5c). Additionally, there is one more H-bond interaction between hydroxyl group of ring A of 14k′ and Glu353 with bond distance of 2.87 Å. The binding energy and inhibition constant for 14k′ were found to be −5.26 kcal mol⁻¹ and 138.5 μM respectively.

Table 2 Docking interactions of 2ME2, 14k on β-tubulin and 14k, 14k′ and 17-β-estradiol, on estrogen receptor-α (ERα)

Name/Pubchem ID	Structure	Binding energy (kcal mol^−1)	Ki value (inhibitory constant)	Hydrogen bond	Number of rotatable bonds
(A) β-tubulin receptor
2-methoxyestradiol (2ME-2) (CID_66414)		−6.42	19.67 μM	Cys241: HG, Lys352: HZ3, Asn349: O	3
14k		−5.34	121.87 μM	Cys241: HG	7
(B) Estrogen receptor
17-β-Estradiol		−9.83	62.57 nM	His524: HD1, Arg394: HH21, Glu353: OE1	3
14k		−5.9	47.33 μM	Leu327: HN	7
14k′		−5.26	138.5 μM	Glu353: OE2, Ile326:HN	7

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Fig. 4 (a and b) Docking poses of 2-methoxyestradiol (2ME2, 2) & 14k within colchicine binding site of β-tubulin.

Fig. 5 (a) Docking pose of compound 17β-estradiol (1) of within LBD of Estrogen Receptor-α, (b) Docking pose of compound 14K within LBD Estrogen Receptor-α, (c) Docking pose of compound 14K′ within LBD of within LBD of Estrogen Receptor-α.

Conclusion

In conclusions, 2-methoxyestradiol (1) and eugenol (6) template based conformationally flexible and rigid diarylpropenes of type I and II, established significant anticancer activity. Compounds 14i, 14k and 15a showed potential cytotoxicites against various human cancer cell lines and were found to be non-toxic against healthy hepatic monocyte (THP-1) cells. Anti-cancer activity of the most potent molecule (14k) was found to be mediated through induction of apoptosis by arresting cells at G2/M phase of division cycle. In the both series, conformationally flexible diarylpropenes were more active than structurally rigid analogues possibly due to molecular flexibility. Further structural modifications of these diarylpropenes may lead to a good lead in future for development of ER selective anticancer drug molecules.

Experimental section

General

The reagents and the solvents used in this study were of analytical grade and used without further purification. All the reactions were monitored on Merck aluminium thin layer chromatography (TLC, UV_{254 nm}) plates. Column chromatography was carried out on silica gel (60–120 mesh). The melting points were determined on Buchi melting point M560 apparatus in open capillaries and are uncorrected. Commercial reagents were used without purification.¹H and ¹³C NMR spectra were recorded on a Bruker WM-300 (300 MHz) using CDCl₃ and DMSO-d₆ as the solvent. Chemical shift are reported in parts per million shift (δ-value) based on the middle peak of the solvent (CDCl₃–DMSO-d₆) (δ 75.00 ppm for ¹³C NMR) as the internal standard. Signal patterns are indicated as s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet; brm, broad multiplet. Coupling constants (J) are given in Hertz. Infrared (IR) spectra were recorded on a Perkin-Elmer AX-1 spectrophotometer in KBr disc and reported in wave number (cm⁻¹). ESI mass spectra were recorded on Shimadzu LC-MS and LC-MS-MS APC3000 (Applied Biosystems) after dissolving the compounds in acetonitrile and methanol.

General procedure for the synthesis of compounds (11a–m)

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (11k). In a round bottomed flask compound 9 (R₁ = R₃ = H, R₂ = OCH₃, 2.00 g, 13.00 mmol) was dissolved into methanol (20 mL) and methanolic KOH (10%, 20 mL) was added to this solution drop wise on ice bath. After 20 minutes, compound 10 (R′=R′′=OCH₃, R=CH₃, 2.62 g, 13.00 mmol) dissolved in methanol (15 mL) was added to reaction mixture. The reaction mixture was allowed to stir at ice bath for 30 minutes and afterwards at room temperature for 12 h. The progress of reaction was monitored using TLC. After completion of reaction, solvent was evaporated followed by drop wise addition of dilute HCl was made and pH of the content was adjusted to acidic. The content was extracted with ethyl acetate. The organic layer was then separated and dried over anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude material was purified by crystallization by methanol which yielded pure compound 11k as yellow solid. Yield: (3.58 g), 82%; m.p. 134–135 °C; R_f: 0.34 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3448, 1656, 1601, 1504, 1460, 1220, 1118, 1029, 998; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.83–3.91 (m, 12H, 4 × OCH₃), 6.85 (s, 2H, ArH), 6.97 (d, J = 9.0 Hz, 2H, ArH), 7.40 (d, J = 15.6 Hz, 1H, CH), 7.70 (d, J = 15.3 Hz, 1H, CH), 8.02 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 55.89 (OCH₃), 56.63 (2 × OCH₃), 61.39 (OCH₃), 106.02 (2 × C), 114.24 (2 × C), 121.66, 130.99, 131.20 (2 × C), 131.54, 140.71, 144.53, 153.88, 163.81 (2 × C), 189.09 (C=O); ESIMS (C₁₉H₂₀O₅): m/z = 329 [M + H]⁺ .

3-(4-Hydroxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (11a). Yield: (2.52 g), 77%; m.p. Oil °C; R_f: 0.22 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3369, 2933, 1651, 1576, 1509, 1340, 1156, 1124, 1028, 793; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.93–3.95 (m, 12H, 4 × OCH₃), 6.95 (d, J = 8.1 Hz, 1H, ArH), 7.11 (d, J = 1.5 Hz, 1H, ArH), 7.22–7.42 (m, 4H, ArH), 7.75 (d, J = 15.6 Hz, 1H, CH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 56.45 (OCH₃), 56.87 (2 × OCH₃), 61.35 (OCH₃), 106.66 (2 × C), 110.93, 115.37, 119.99, 123.39, 127.89, 134.23, 145.57, 147.27, 148.77, 153.54 (3 × C), 189.88 (C=O); ESIMS (C₁₉H₂₀O₆): m/z = 345 [M + H]⁺, 367 [M + Na]⁺.

3-(4-Hydroxy-3-methoxyphenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (11b). Yield: (2.84 g), 82%; m.p. 128–129 °C; R_f: 0.20 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3333, 1644, 1588, 1559, 1510, 1024, 801, 722; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.92–3.95 (m, 9H, 3 × OCH₃), 6.04 (bs, 1H, OH), 6.90–6.96 (m, 2H, ArH), 7.11 (s, 1H, ArH), 7.20–7.29 (m, 1H, ArH), 7.36–7.41 (d, J = 15.3 Hz, 1H, CH), 7.57–7.65 (m, 2H, ArH), 7.69 (d, J = 11.7 Hz, 1H, CH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 56.42 (OCH₃), 56.47 (2 × OCH₃), 110.38, 110.67, 111.31, 115.32, 119.79, 123.27, 123.43, 128.06, 131.98, 144.78, 147.25, 148.59, 149.65, 153.55, 189.60 (C=O); ESIMS (C₁₈H₁₈O₅): m/z = 315 [M + H]⁺, 337 [M + Na]⁺, 353 [M + K]⁺.

3-(4-Hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (11c). Yield: (3.10 g), 82%; m.p. 157–158 °C; R_f: 0.30 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3317, 1651, 1591, 1426, 1278, 1220, 1172, 1025, 981, 820; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.87 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 6.03 (s, 1H, OH), 6.91–6.98 (m, 3H, ArH), 7.12 (s, 1H, ArH), 7.19–7.25 (m, 1H, ArH), 7.38 (d, J = 15.3 Hz, 1H, CH), 7.68 (d, J = 14.4 Hz, 1H, CH), 8.02 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 55.90 (OCH₃), 56.42(OCH₃), 110.46, 114.20 (2 × C), 115.28, 119.93, 123.57, 128.05, 131.14 (2 × C), 131.72, 144.79, 147.21, 148.54, 163.69, 189.27(C=O); ESIMS (C₁₇H₁₆O₄): m/z = 285 [M + H]⁺, 307 [M + Na]⁺.

1-(Benzo[d][1,3]dioxol-6-yl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (11d). Yield: (3.34 g), 92%; m.p. 153–154 °C; R_f: 0.50 (10% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3265, 1641, 1596, 1563, 1501, 1450, 1258, 1034, 843, 801; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.95 (s, 3H, OCH₃), 6.05 (s, 3H, OH and OCH₂), 6.88 (d, J = 8.1 Hz, 1H, ArH), 6.94 (d, J = 8.1 Hz, 1H, ArH), 7.11 (s, 1H, ArH), 7.19 (d, J = 8.1 Hz, 1H, ArH), 7.32 (d, J = 15.6 Hz, 1H, CH), 7.51 (s, 1H, ArH), 7.63 (d, J = 8.1 Hz, 1H, ArH), 7.73 (d, J = 15.6 Hz, 1H, CH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 56.43 (OCH₃), 102.23 (CH₂), 108.28, 108.84, 110.44, 115.29, 119.76, 123.66, 124.90, 127.97, 133.62, 145.04, 147.24, 148.63 (2 × C), 151.63, 188.77 (C=O); ESIMS (C₁₇H₁₄O₅): m/z = 299 [M + H]⁺, 321 [M + Na]⁺.

1-(Benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (11e). Yield: (2.68 g), 78%; m.p. 147–148 °C; R_f: 0.44 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2905, 2840, 1648, 1580, 1502, 1441, 1249, 1031, 929, 805; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.83 (s, 3H, OCH₃), 6.03 (s, 2H, CH₂), 6.85–6.92 (m, 3H, ArH), 7.75 (d, J = 15.6 Hz, 1H, CH), 7.50–7.63 (m, 4H, ArH), 7.35 (d, J = 15.6 Hz, 1H, CH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 55.79 (OCH₃), 102.20 (CH₂), 108.27, 108.83, 114.81 (2 × C), 119.85, 124.85, 128.16, 130.51 (2 × C), 133.68, 144.47, 148.64, 151.91, 162.00, 188.69 (C=O); ESIMS (C₁₇H₁₄O₄): m/z = 305 [M + Na]⁺.

1,3-Bis(4-methoxyphenyl)prop-2-en-1-one (11f). Yield: (2.89 g), 81%; m.p. 98–99 °C; R_f: 0.40 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2954, 2841, 1655, 1594, 1506, 1330, 1253, 1216, 1168, 1018, 817; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.84 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 6.91–6.98 (m, 4H, ArH), 7.41 (d, J = 15.3 Hz, 1H, CH), 7.59 (d, J = 8.7 Hz, 2H, ArH), 7.77 (d, J = 15.6 Hz, 1H, CH), 8.02 (d, J = 9.0 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 55.79 (OCH₃), 55.86 (OCH₃), 114.19 (2 × C), 114.80 (2 × C), 120.04, 128.26, 130.48 (2 × C), 131.09 (2 × C), 131.80, 144.20, 161.93, 163.68, 189.19 (C=O); ESIMS (C₁₇H₁₆O₃): m/z = 269 [M + H]⁺.

1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (11g). Yield: (2.98 g), 90%; m.p. 86–87 °C; R_f: 0.15 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3057, 3005, 1650, 1571, 1507, 1248, 1158, 1026, 823, 796, 766; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.85–3.96 (m, 9H, 3 × OCH₃), 6.91–6.95 (m, 3H, ArH), 7.43 (d, J = 15.6 Hz, 1H, CH), 7.58–7.69 (m, 4H, ArH), 7.75–7.81 (d, J = 15.6 Hz, 1H, CH); ¹³C MR (CDCl₃, 75 MHz, δ ppm): 55.80 (OCH₃), 56.45 (2 × OCH₃), 110.40, 111.26, 114.80 (2 × C), 119.80, 123.22, 128.23, 130.51 (2 × C), 132.02, 144.22, 149.63, 153.51, 161.94, 189.05 (C=O); ESIMS (C₁₈H₁₈O₄): m/z = 299 [M + H]⁺, 321 [M − H]⁺.

1-(3,4,5-Trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (11h). Yield: (2.81 g), 90%; m.p. 99–100 °C; R_f: 0.24 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2939, 2838, 1655, 1573, 1457, 1028, 993, 821, 707; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 3.77 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.81 (s, 6H, 2 × OCH₃), 6.99 (d, J = 9.0 Hz, 2H, ArH), 7.43 (s, 2H, ArH), 7.74–7.77(m, 4H, ArH and CH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 55.31 (OCH₃), 56.18 (2 × OCH₃), 60.22 (OCH₃), 106.58 (2 × C), 114.74 (2 × C), 119.84, 128.21, 130.82 (2 × C), 134.19, 142.98, 144.04, 153.83 (2 × C), 162.19, 188.19 (C=O); ESIMS (C₁₉H₂₀O₅): m/z = 329 [M + H]⁺.

3-(3,4,5-Trimethoxyphenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (11i). Yield: (3.58 g), 90%; m.p. 126–127 °C; R_f: 0.20 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2998, 2947, 2833, 1657, 1583, 1511, 1465, 1416, 1268, 1124, 1023, 818, 766; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.81–3.85 (m, 15H, 5 × OCH₃), 6.87–6.94 (m, 3H, ArH), 7.43 (d, J = 15.6 Hz, 1H, CH), 7.62–7.74 (m, 3H, CH and ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 56.48 (2 × OCH₃), 56.64 (2 × OCH₃), 61.39 (OCH₃), 106.04 (2 × C), 110.34, 111.25, 121.46, 123.41, 130.97, 131.77, 140.72, 144.54, 149.70, 153.67, 153.87 (2 × C), 188.97 (C=O); ESIMS (C₂₀H₂₂O₆): m/z = 381[M + Na]⁺, 397[M + K]⁺.

3-(4-Hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (11j). Yield: (2.69 g), 90%; m.p. 155–156 °C; R_f: 0.20 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3404, 2937, 1586, 1509, 1236, 1125, 1002, 830; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.85–3.95 (m, 9H, 3 × OCH₃), 6.89 (d, J = 8.7 Hz, 2H, ArH), 7.27 (s, 2H, ArH), 7.36 (d, J = 15.3 Hz, 1H, CH), 7.51–7.56 (m, 2H, ArH), 7.75 (d, J = 15.6 Hz, 1H, CH); ¹³C MR (CDCl₃, 75 MHz, δ ppm): 55.75 (2 × OCH₃), 60.22 (OCH₃), 105.36, 115.60 (2 × C), 117.76, 125.50, 129.89 (2 × C), 133.34, 144.54, 152.45 (2 × C), 159.70, 188.54 (C=O); ESIMS (C₁₈H₁₈O₅): m/z = 313 [M − H]⁺, 315 [M + H]⁺.

3-(4-Hydroxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (11l). Yield: (2.68 g), 79%; m.p. 179–180 °C; R_f: 0.30 (30% ethyl acetate–hexane); ¹H NMR (DMSO-d₆, 300 MHz, δ ppm): 3.38 (s, 3H, OCH₃), 6.82 (d, J = 8.4 Hz, 2H, ArH), 7.05 (d, J = 8.4 Hz, 2H, ArH), 7.60–7.74 (m, 4H, CH and ArH), 8.11 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, δ ppm): 55.56 (OCH₃), 113.98 (2 × C), 115.84 (2 × C), 118.45, 125.96, 130.75 (2 × C), 130.82, 130.92 (2 × C), 143.68, 160.00, 163.03, 187.30 (C=O); ESIMS (C₁₆H₁₄O₃): m/z = 255 [M + H]⁺, 277 [M + Na]⁺.

3-(3,4,5-Trimethoxyphenyl)-1-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (11m). Yield: (2.65 g), 64%; m.p. 183–186 °C; R_f: 0.20 (30% ethyl acetate–hexane); ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.89–3.96 (m, 12H, 4 × OCH₃), 6.24 (s, 1H, OH), 6.85 (s, 2H, ArH), 6.98 (d, J = 7.5 Hz, 1H, ArH), 7.41 (d, J = 15.6 Hz, 1H, CH), 7.62–7.65 (m, 2H, ArH), 7.71 (d, J = 15.6 Hz, 1H, CH); (CDCl₃, 75 MHz, δ ppm): 56.34 (OCH₃), 56.66 (2 × OCH₃), 61.37 OCH₃, 106.13 (2 × C), 110.96, 114.17, 121.43, 124.04, 130.98, 131.47, 140.82, 144.52, 147.38, 150.83, 153.90 (2 × C), 188.90 (C=O); ESIMS (C₁₉H₂₀O₆): m/z = 345 [M + H]⁺, 367 [M + Na]⁺.

General procedure for the synthesis of compounds (12a–m)

3-(3,4,5-Trimethoxyphenyl)-1-(4-methoxyphenyl)propan-1-one (12k). Compound 11k (1.00 g, 3.04 mmol) was taken in a round bottomed flask and dissolved in ethyl acetate–methanol mixture (8 : 2). The mixture was flushed with nitrogen gas and palladium charcoal (Pd/C, 5% wt) (0.10 g) was added and hydrogen gas was passed to it for 3 h. The progress of reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated and crude material was worked up using ethyl acetate and water. The organic layer was the separated, dried anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude was purified by column chromatography on silica gel (100–200 mesh size) using ethyl acetate–hexane (12 : 88) as eluents which yielded pure compound 12k as white solid. Yield: (0.88 g), 88%; m.p. 94–95 °C; R_f: 0.52 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2938, 2837, 1683, 1251, 1178, 1124, 1012, 845; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.99 (t, J = 7.5 Hz, 2H, CH₂), 3.23 (t, J = 7.5 Hz, 2H, CH₂), 3.81–3.85 (m, 12H, 4 × OCH₃), 6.45 (s, 2H, ArH), 6.92 (d, J = 8.7 Hz, 2H, ArH), 7.93 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 31.21 (CH₂), 40.59 (CH₂), 55.85 (OCH₃), 56.50 (2 × OCH₃), 61.22 (OCH₃), 105.87, 114.14 (2 × C), 130.42, 130.69 (2 × C), 137.66 (2 × C), 153.61 (3 × C), 163.90, 198.21 (C=O); ESIMS (C₁₉H₂₂O₅): m/z = 353 [M + H]⁺, 369 [M + K]⁺.

3-(4-Hydroxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)propan-1-one (12a). Yield: (0.93 g), 92%; m.p. 122–123 °C; R_f: 0.54 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3411, 2934, 2840, 1667, 1265, 1236, 1127, 1029, 998, 851; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.98 (t, J = 7.3 Hz, 2H, CH₂), 3.22 (t, J = 7.3 Hz, 2H, CH₂), 3.86–3.90 (m, 12H, 4 × OCH₃), 5.55 (s, 1H, OH), 6.73, (bs, 2H, ArH), 6.70 (d, J = 7.8 Hz, 1H, ArH), 7.22 (s, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 30.56 (CH₂), 41.11 (CH₂), 56.30 (OCH₃), 56.71 (2 × OCH₃), 61.33 (OCH₃), 106.00 (2 × C), 111.65, 114.82, 121.28, 132.61, 133.61, 143.02, 144.42, 146.88, 153.47 (2 × C), 198.65 (C=O); ESIMS (C₁₉H₂₂O₆): m/z = 345 [M − H]⁺, 369 [M + Na]⁺.

3-(4-Hydroxy-3-methoxyphenyl)-1-(3,4-dimethoxyphenyl)propan-1-one (12b). Yield: (0.83 g), 82%; m.p. 141–142 °C; R_f: 0.25 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3355, 1648, 1019, 792; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.88–2.93 (m, 2H, CH₂), 3.12–3.17 (m, 2H, CH₂), 3.79 (s, 3H, OCH₃), 3.84 (bs, 6H, 2 × OCH₃), 5.46 (s, 1H, OH), 6.65–6.67 (m, 2H, ArH), 6.75–6.81 (m, 2H, ArH), 7.45–7.51 (m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.18 (CH₂), 36.76 (CH₂), 49.71 (OCH₃), 55.63 (OCH₃), 56.49 (OCH₃), 104.83, 107.83, 114.28 (2 × C), 129.72, 130.26 (2 × C), 132.11, 149.41, 149.88, 155.99, 158.51, 207.04 (C=O); ESIMS (C₁₈H₂₀O₅): m/z = 339 [M + Na]⁺, 354 [M + K]⁺.

3-(4-Hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)propan-1-one (12c). Yield: (0.86 g), 85%; m.p. 112–113 °C; R_f: 0.50 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3504, 1671, 978, 822, 780; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.91 (bs, 2H, CH₂), 3.24 (t, J = 7.6 Hz, 2H, CH₂), 3.81 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 6.72 (bs, 2H, ArH), 6.89 (s, 1H, ArH), 7.00 (d, J = 9.0 Hz, 2H, ArH), 7.33 (s, 1H, OH), 7.98 (d, J = 9.0 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 30.20 (CH₂), 40.36 (CH₂), 55.40 (OCH₃), 55.74 (OCH₃), 112.52, 114.06 (2 × C), 115.17, 121.11, 130.54 (2 × C), 130.63, 133.38, 145.20, 147.72, 163.86, 197.63(C=O); ESIMS (C₁₇H₁₈O₄): m/z = 287 [M + H]⁺, 309 [M + Na]⁺.

1-(Benzo[d][1,3]dioxol-6-yl)-3-(4-hydroxy-3-methoxyphenyl)propan-1-one (12d). Yield: (0.66 g), 66%; m.p. 80–81 °C; R_f: 0.31 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3495, 2930, 1677, 1251, 1033, 982, 928, 866, 823; ¹H NMR (CDCl₃, 1d DMSO-d₆, 300 MHz, δ ppm): 2.94–2.99 (m, 2H, CH₂), 3.15–3.20 (m, 2H, CH₂), 3.86 (s, 3H, OCH₃), 5.54 (s, 1H, OH), 6.02 (s, 2H, OCH₂O), 6.65–6.77 (m, 2H, ArH), 6.81–6.86 (m, 2H, ArH), 7.42 (s, 1H, ArH), 7.54 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 1d DMSO-d₆, 75 MHz, δ ppm): 30.54 (CH₂), 40.94 (CH₂), 56.30 (OCH₃), 102.22 (CH₂), 108.26, 111.59, 114.78, 121.26, 124.65, 132.24, 133.64, 144.36, 146.85, 148.59 (2 × C), 152.11, 197.91 (C=O); ESIMS (C₁₇H₁₆O₅): m/z = 299 [M − H]⁺, 323 [M + Na]⁺.

1-(Benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)propan-1-one (12e). Yield: (0.92 g), 92%; m.p. 51–52 °C; R_f: 0.64 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2911, 1666, 1242, 1034, 814, 785; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 2.91 (t, J = 7.5 Hz, 2H, CH₂), 3.22 (t, J = 7.5 Hz, 2H, CH₂), 3.73 (s, 3H, OCH₃), 6.08 (s, 2H, CH₂), 6.82 (d, J = 8.4 Hz, 2H, ArH), 6.91 (d, J = 8.1 Hz, 1H, ArH), 7.18 (d, J = 8.4 Hz, 2H, ArH), 7.42 (d, J = 1.5 Hz, 1H, ArH), 7.63 (d, J = 8.1 Hz, 1H, ArH); ¹³C NMR (acetone-D₆, 75 MHz, δ ppm): 29.59 (CH₂), 40.26 (CH₂), 54.94 (OCH₃), 102.45 (CH₂), 107.72, 108.11, 114.09 (2 × C), 124.57, 129.72 (2 × C), 130.40, 133.88, 148.64, 152.05, 158.52, 197.10; ESIMS (C₁₇H₁₆O₄): m/z = 307 [M + Na]⁺.

1,3-Bis(4-methoxyphenyl)propan-1-one (12f). Yield: (0.86 g), 86%; R_f: 0.42 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2934, 1680, 1032, 823, 742, 691; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 2.98 (t, J = 7.5 Hz, 2H, CH₂), 3.34 (t, J = 7.5 Hz, 2H, CH₂), 3.77 (s, 6H, 2 × OCH₃), 6.86 (d, J = 8.7 Hz, 2H, ArH), 7.23 (d, J = 8.4 Hz, 2H, ArH), 7.53 (d, J = 8.1 Hz, 2H, ArH), 8.03 (d, J = 7.5 Hz, 2H, ArH); ESIMS (C₁₇H₁₈O₃): m/z = 261 [M + H]⁺, 283 [M + Na]⁺.

1-(3,4,5-Trimethoxyphenyl)-3-(4-methoxyphenyl)propan-1-one (12h). Yield: (0.80 g), 80%; m.p. 91–92 °C; R_f: 0.53 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3003, 2935, 1675, 1246, 1122, 1008, 816, 763; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.99 (t, J = 7.3 Hz, 2H, CH₂), 3.22 (t, J = 7.5 Hz, 2H, CH₂), 3.77 (s, 3H, OCH₃), 3.89 (bs, 9H, 3 × OCH₃), 6.84 (d, J = 8.4 Hz, 2H, ArH), 7.15–7.19 (m, 4H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 29.93 (CH₂), 40.97 (CH₂), 55.66 (OCH₃), 56.71 (2 × OCH₃), 61.31 (OCH₃), 106.02 (2 × C), 114.39 (2 × C), 129.78 (2 × C), 132.62, 133.73, 143.03, 153.48 (2 × C), 158.47, 198.53 (C=O); ESIMS (C₁₉H₂₂O₅): m/z = 353 [M + Na]⁺, 369 [M + K]⁺.

1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)propan-1-one (12g). Yield: (0.81 g), 80%; m.p. 64–65 °C; R_f: 0.31 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3447, 3004, 2931, 1668, 1263, 1156, 1026, 889, 822, 767; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.99 (t, J = 7.6 Hz, 2H, CH₂), 3.21 (t, J = 7.6 Hz, 2H, CH₂), 3.77 (s, 3H, OCH₃), 3.91–3.93 (m, 6H, 2 × OCH₃), 6.82–6.87 (m, 3H, ArH), 7.16 (d, J = 8.1 Hz, 2H, ArH), 7.52–7.58 (m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 30.02 (CH₂), 40.65 (CH₂), 55.66 (OCH₃), 56.38 (OCH₃), 56.43(OCH₃), 110.44, 110.63, 114.34 (2 × C), 123.04, 129.75 (2 × C), 130.60, 133.80, 149.45, 153.65, 158.39, 198.45 (C=O); ESIMS (C₁₈H₂₀O₄): m/z = 323 [M + Na]⁺, 339 [M + K]⁺.

3-(3,4,5-Trimethoxyphenyl)-1-(3,4-dimethoxyphenyl)propan-1-one (12i). Yield: (0.93 g), 93%; m.p. 95–96 °C; R_f: 0.28 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2941, 2839, 1678, 1009, 872, 776; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 2.90–2.95 (m, 2H, OCH₂), 3.26–3.31 (m, 2H, CH₂), 3.67 (s, 3H, OCH₃), 3.78 (s, 6H, 2 × OCH₃), 3.85–3.87 (m, 6H, 2 × OCH₃), 6.59 (s, 2H, ArH), 7.01 (d, J = 8.4 Hz, 1H, ArH), 7.52 (d, J = 2.1 Hz, 1H, ArH), 7.66 (dd, J = 2.1 and 2.1 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 31.00 (CH₂), 39.99 (CH₂), 55.00 (OCH₃), 55.87(2 × OCH₃), 55.69 (OCH₃), 59.96 (OCH₃), 106.30 (2 × C), 110.89, 111.00, 122.90, 130.61, 137.02, 137.77, 149.65, 153.78 (2 × C), 154.00, 197.64 (C=O); ESIMS (C₂₀H₂₄O₆): m/z = 283 [M + Na]⁺, 399 [M + K]⁺.

3-(4-Hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)propan-1-one (12j). Yield: (0.68 g), 68%; m.p. 141–142 °C; R_f: 0.29 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3439, 2990, 1664, 1230, 1124, 999, 826, 775; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 2.89 (t, J = 7.2 Hz, 2H, CH₂), 3.24–3.29 (m, 2H, CH₂), 3.78 (s, 3H, OCH₃), 3.87 (s, 6H, 2 × OCH₃), 6.73 (d, J = 9.0 Hz, 2H, ArH), 7.09 (d, J = 7.8 Hz, 2H, ArH), 7.29 (s, 2H, ArH), 8.15 (bs, 1H, OH); ¹³C NMR (acetone-D₆, 75 MHz, δ ppm): 29.97 (CH₂), 40.70 (CH₂), 56.30 (2 × OCH₃), 60.38 (OCH₃), 106.29 (2 × C), 115.74 (2 × C), 129.98 (2 × C), 132.86, 133.14, 153.95(3 × C), 156.26, 198.30 (C=O); ESIMS (C₁₈H₂₀O₅): m/z = 315 [M + H]⁺, 339 [M + 2H + Na]⁺.

3-(4-Hydroxyphenyl)-1-(4-methoxyphenyl)propan-1-one (12l). Yield: (0.85 g), 85%; m.p. 82–83 °C; R_f: 0.40 (30% ethyl acetate–hexane); ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.97 (t, J = 7. 5 Hz, 2H, CH₂), 3.21 (t, J = 7.6 Hz, 2H, CH₂), 3.85 (s, 3H, OCH₃), 6.06 (bs, 1H, OH), 6.78 (d, J = 8.4 Hz, 2H, ArH), 6.92 (d, J = 9.0 Hz, 2H, ArH), 7.08 (d, J = 8.1 Hz, 2H, ArH), 7.94 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 29.56 (CH₂), 40.37 (CH₂), 55.44 (OCH₃), 113.73 (2 × C), 115.36 (2 × C), 129.44 (2 × C), 129.76, 130.42 (2 × C), 133.04, 154.14, 163.51, 198.86 (C=O); ESIMS (C₁₆H₁₆O₃): m/z = 257 [M + H]⁺, 279 [M + Na]⁺.

3-(3,4,5-Trimethoxyphenyl)-1-(4-hydroxy-3-methoxyphenyl) propan-1-one (12m). Yield: (0.80 g) 80%; R_f: 0.0.37 (30% ethyl acetate–hexane); ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.99 (t, J = 7.5 Hz, 2H, CH₂), 3.21 (t, J = 7.5 Hz, 2H, CH₂), 3.79–3.83 (m, 12H, 4 × OCH₃), 6.46 (s, 2H, ArH), 6.97 (d, J = 8.1 Hz, 1H, ArH), 7.60–7.63 (m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 31.20 (CH₂), 40.63 (CH₂), 55.85 (OCH₃), 56.23 (2 × OCH₃), 61.22 (OCH₃), 105.87, 114.14 (2 × C), 130.42, 130.69, 137.66 (2 × C), 153.61 (3 × C), 156.8, 163.90, 198.21 (C=O); ESIMS (C₁₉H₂₂O₆): m/z = 346 [M + H]⁺.

General procedure for the synthesis of compounds (13a–m)

1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propan-1-ol (13k). In a round bottomed flask, compound 12k (0.86 g, 2.60 mmol) was dissolved in methanol and kept at 0 °C for 15–20 minutes. To this mixture sodium borohydride (NaBH_4, 0.29 g, 7.80 mmol), was added and reaction was transferred to room temperature after half an hour. The progress of reaction was monitored by TLC (30% ethyl acetate–hexane). After completion of reaction, solvent was taken off and the reaction mixture was treated with NH₄Cl and worked up using ethyl acetate and water. The organic layer was the separated, dried anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude solid material was purified by washing it with hexane which yielded pure compound 13k as white solid in 98% (3.58 g), yield which was further processed for hydrolysis as such. The experimental procedure used for synthesis of compound 13a–m is same as described for compound 13k. The molar ratio of reactants was same for synthesis of all derivatives.

General procedure for the synthesis of compounds (14a–m)

5-(4-Methoxycinnamyl)-1,2,3-trimethoxybenzene (14k). In a round bottomed flask, compound 13k (0.83 g, 2.50 mmol) was dissolved in ethanol and conc. HCl (1 mL) was added to it. The reaction mixture was transferred for reflux. The progress of reaction was monitored by TLC (30% ethyl acetate–hexane). After completion of reaction, solvent was taken off and the reaction mixture was worked up using ethyl acetate and water. The organic layer was the separated, dried over anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude solid material was purified by column chromatography on silica gel (100–200 mesh size) using hexane as eluents which yielded pure compound 14k as white solid. Yield: (0.44 g), 54%; m.p. 61–62 °C; R_f: 0.45 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2936, 2835, 1593, 1507, 1459, 1330, 1239, 1120, 1017, 832; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.46 (d, J = 6.6 Hz, 2H, CH₂), 3.80–3.84 (m, 12H, 4 × OCH₃), 6.16–6.24 (m, 1H, CH), 6.39–6.45 (m, 3H, CH and ArH), 6.84 (d, J = 8.7 Hz, 2H, ArH), 7.30 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 40.09 (CH₂), 55.69 (OCH₃), 56.51 (2 × OCH₃), 61.25 (OCH₃), 106.00 (2 × C), 114.38 (2 × C), 127.19, 127.67 (2 × C), 130.65, 130.95, 136.00, 153.65 (3 × C), 159.35; ESIMS (C₁₉H₂₂O₄): m/z = 337 [M + Na]⁺, 353 [M + K]⁺.

4-(3,4,5-Trimethoxycinnamyl)-2-methoxyphenol (14a). Yield: (0.38 g), 48%; m.p. Oil; R_f: 0.37 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3424, 2937, 2837, 1582, 1509, 1458, 1236, 1124, 787; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.46 (d, J = 6.0 Hz, 2H, CH₂), 3.80–3.89 (m, 12H, 4 × OCH₃), 5.52 (s, 1H, OH), 6.21–6.28 (m, 1H, CH), 6.35 (d, J = 15.9 Hz, 1H, CH), 6.58 (s, 2H, ArH), 6.72–6.75 (m, 2H, ArH), 6.86 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 39.35 (CH₂), 56.33 (OCH₃), 56.49 (2 × OCH₃), 61.30 (OCH₃), 103.70 (2 × C), 111.70, 114.76, 121.76, 129.60, 131.03, 132.31, 133.66, 137.99, 144.51, 146.95, 153.72 (2 × C); ESIMS (C₁₉H₂₄O₆): m/z = 331 [M + H]⁺, 329 [M − H]⁺.

4-(3,4-Dimethoxycinnamyl)-2-methoxyphenol (14b). Yield: (0.59 g), 63%; m.p. 103–134 °C; R_f: 0.4 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3488, 1513, 1462, 1264, 1139, 1025, 814, 766; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.46 (d, J = 6.3 Hz, 2H, CH₂), 3.87 (bs, 9H, 3 × OCH₃), 5.52 (bs, 1H, OH), 6.17–6.24 (m, 1H, CH), 6.37 (d, J = 15.9 Hz, 1H, CH), 6.73–6.91 (m, 6H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 39.43 (CH₂), 56.22 (OCH₃), 56.34 (2 × OCH₃), 109.03, 111.57, 111.64, 114.71, 119.53, 121.71, 128.16, 130.78, 131.07, 132.61, 144.42, 146.91, 148.86, 149.44; ESIMS (C₁₈H₂₀O₄): m/z = 323 [M + Na]⁺.

4-(4-Methoxycinnamyl)-2-methoxyphenol (14c). Yield: (0.42 g), 50%; m.p. 92–93 °C; R_f: 0.57 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3435, 1606, 1512, 1459, 1435, 1265, 1237, 1028, 834, 786; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.41 (d, J = 6.6 Hz, 2H, CH₂), 3.76 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 6.18–6.28 (m, 1H, CH), 6.40 (d, J = 15.6 Hz, 1H, CH), 6.66–6.86 (m, 5H, ArH), 7.32 (d, J = 8.7 Hz, 2H, ArH), 7.38 (s, 1H, OH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 39.01 (CH₂), 55.02 (OCH₃), 55.75 (OCH₃), 112.52, 114.26 (3 × C), 115.27, 121.29, 127.54 (2 × C), 127.94, 130.21, 130.80, 145.33, 147.82, 159.44; ESIMS (C₁₇H₁₈O₃): m/z = 293 [M + Na]⁺, 309 [M + K]⁺.

4-(3-(Benzo[d][1,3]dioxol-6-yl)allyl)-2-methoxyphenol (14d). Yield: (0.66 g), 85%; m.p. 78–79 °C; R_f: 0.6 (10% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3455, 1607, 1500, 1444, 1253, 1035, 880, 794; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.45 (d, J = 6.3 Hz, 2H, CH₂), 3.87 (s, 3H, OCH₃), 5.53 (s, 1H, OH), 5.93 (bs, 2H, CH₂), 6.11–6.21 (m, 1H, ArH), 6.32–6.37 (d, J = 15.6 Hz, 1H, ArH), 6.72–6.80 (m, 4H, CH and ArH), 6.85–6.91(m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 39.32 (CH₂), 56.31 (OCH₃), 101.37 (CH₂), 105.95, 108.64, 111.57, 114.71, 120.95, 121.65, 128.30, 130.73, 132.45, 132.54, 144.40, 146.90, 147.20, 148.37; ESIMS (C₁₇H₁₆O₄): m/z = 285 [M + H]⁺, 307 [M + Na]⁺, 323 [M + K]⁺.

5-(3-(4-Methoxyphenyl)prop-1-enyl)benzo[d][1,3]dioxole (14e). Yield: (0.42 g), 54%; m.p. oil; R_f: 0.80 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2924, 1609, 1508, 1443, 1246, 1038, 934, 816; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.46 (d, J = 6.6 Hz, 2H, CH₂), 3.79 (s, 3H, OCH₃), 6.49 (s, 2H, CH₂), 6.11–6.21 (m, 1H, CH), 6.34 (d, J = 15.6 Hz, 1H, CH), 6.69–6.90 (m, 5H, ArH), 7.14 (d, J = 8.4 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 38.72 (CH₂), 55.69 (OCH₃), 101.34 (CH₂), 105.97, 108.61, 114.34 (2 × C), 120.90, 128.39, 129.95 (2 × C), 130.71, 132.52, 132.72, 147.17, 148.36, 158.49; ESIMS (C₁₇H₁₆O₃): m/z = 307 [M + K]⁺.

1,3-Bis(4-methoxyphenyl)prop-1-ene (14f). Yield: (0.37 g), 48%; m.p. 63–64 °C; R_f: 0.85 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2953, 2902, 1609, 1510, 1243, 1032, 823; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 3.44 (d, J = 6.6 Hz, 2H, CH₂), 3.76(bs, 6H, 2 × OCH₃), 6.18–6.28 (m, 1H, CH), 6.40 (d, J = 15.9 Hz, 1H, CH), 6.85 (d, J = 8.4 Hz, 4H, ArH), 7.15 (d, J = 8.1 Hz, 2H, ArH), 7.32 (d, J = 8.7 Hz, 2H, ArH); ¹³C NMR (acetone-D₆, 75 MHz, δ ppm): 38.48 (CH₂), 54.97 (OCH₃), 55.02 (OCH₃), 114.17 (2 × C), 114.20 (2 × C), 127.54 (2 × C), 127.81, 129.82 (2 × C), 130.36, 130.76, 132.82, 158.66, 159.48; ESIMS (C₁₇H₁₈O₂): m/z = 253 [M − H]⁺, 255[M + H]⁺, 277 [M + Na]⁺.

1,2-Dimethoxy-4-(3-(4-methoxyphenyl)prop-1-enyl)benzene (14g). Yield: (0.47 g), 60%; m.p. 48–49 °C; R_f: 0.61 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2930, 2839, 1605, 1509, 1268, 1240, 1126, 1019, 966, 818; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.48 (d, J = 6.3 Hz, 2H, CH₂), 3.80–3.88 (m, 9H, 3 × OCH₃), 6.15–6.25 (m, 1H, CH), 6.37 (d, J = 15.9 Hz, 1H, CH), 6.78–6.91 (m, 5H, ArH), 7.17 (d, J = 8.4 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 38.82 (CH₂), 55.69 (OCH₃), 56.20 (OCH₃), 56.34 (OCH₃), 109.05, 111.60, 114.32 (2 × C), 119.52, 128.23, 130.02 (2 × C), 130.75, 131.13, 132.79, 148.84, 149.43, 158.49; ESIMS (C₁₈H₂₀O₃): m/z = 283 [M − H]⁺, 285 [M + H]⁺, 307 [M + Na]⁺.

1,2,3-Trimethoxy-5-(3-(4-methoxyphenyl)prop-1-enyl)benzene (14h). Yield: (0.38 g), 48%; m.p. 69–71 °C; R_f: 0.78 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2936, 2835, 1583, 1508, 1459, 1419, 1243, 1123, 1027, 819; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.51 (d, J = 6.0 Hz, 2H, CH₂), 3.82–3.96 (m, 12H, 4 × OCH₃), 6.23–6.32 (m, 1H, CH), 6.52 (d, J = 15.6 Hz, 1H, CH),6.61 (s, 2H, ArH), 6.84–6.91 (m, 2H, ArH), 7.12–7.20 (m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 38.77 (CH₂), 55.69 (OCH₃), 56.50 (2 × OCH₃), 61.32 (OCH₃), 103.59 (2 × C), 114.35 (2 × C), 129.69, 130.06 (2 × C), 131.00, 132.49, 133.70, 153.64 (3 × C), 158.54; ESIMS (C₁₉H₂₂O₄): m/z = 337 [M + Na]⁺.

1,2-Dimethoxy-4-(3-(3,4,5-trimethoxyphenyl)prop-1-enyl)benzene (14i). Yield: (0.40 g), 51%; m.p. 67–68 °C; R_f: 0.71 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2934, 2835, 1588, 1510, 1458, 1236, 1127, 1024, 823; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.47 (d, J = 6.6 Hz, 2H, CH₂), 3.82–3.88 (m, 15H, 5 × OCH₃), 6.16–6.24 (m, 1H, CH), 6.37–6.45 (m, 3H, CH and ArH), 6.72 (d, J = 8.4 Hz, 1H, ArH), 6.80 (d, J = 9.3 Hz, 1H, ArH), 6.90 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 40.09 (CH₂), 56.25 (OCH₃), 56.35 (OCH₃), 56.53 (2 × OCH₃), 61.25 (OCH₃), 106.10 (2 × C), 109.21, 111.67, 119.58, 127.50, 130.98, 131.18, 136.48, 148.99, 149.49, 153.68 (3 × C); ESIMS (C₂₀H₂₄O₅): m/z = 367 [M + Na]⁺, 369 [M + 2 + Na]⁺.

4-(3,4,5-Trimethoxycinnamyl)phenol (14j). Yield: (0.41 g), 52%; m.p. 124–125 °C; R_f: 0.46 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3355, 3001, 2935, 2836, 1584, 1511, 1454, 1418, 1237, 1125, 1003, 830, 781; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.44 (dd, J = 0.9 and 0.5 Hz, 2H, CH₂), 3.80–3.85 (m, 9H, 3 × OCH₃), 6.18–6.37 (m, 2H, CH), 6.57 (s, 2H, ArH), 6.78 (d, J = 8.4 Hz, 2H, ArH), 7.10 (d, J = 8.4 Hz, 2H, ArH); ESIMS (C₁₈H₂₀O₄): m/z = 301 [M + H]⁺, 323 [M + Na]⁺.

4-(4-Methoxycinnamyl)phenol (14l). Yield: (0.39 g), 50%; m.p. 73–74 °C; R_f: 0.43 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3370, 1605, 1511, 1242, 1026, 831; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.39 (d, J = 6.6 Hz, 2H, CH₂), 3.73 (s, 3H, OCH₃), 4.95 (bs, 1H, OH), 6.08–6.17 (m, 1H, CH), 6.31 (d, J = 15.9 Hz, 1H, CH), 6.70–6.79 (m, 4H, ArH), 6.95 (d, J = 8.4 Hz, 2H, ArH), 7.19–7.24 (m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 38.84 (CH₂), 55.74 (OCH₃), 114.38 (2 × C), 115.71 (2 × C), 127.64 (2 × C), 127.94, 130.20 (2 × C), 130.54, 130.84, 133.00, 154.31, 159.19; ESIMS (C₁₆H₁₆O₂): m/z = 293 [M + Na]⁺, 309 [M + K]⁺.

3-(3,4,5-Trimethoxyphenyl)-1-(4-hydroxy-3-methoxyphenyl) prop-1-ene (14m). Yield: (0.45 g), 57%; oil; R_f: 0.30 (30% ethyl acetate–hexane); ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.39 (d, J = 6.3 Hz, 2H, CH₂), 3.75–3.86 (m, 12H, 4 × OCH₃), 5.54 (s, 1H, OH), 6.04–6.14 (m, 1H, CH), 6.29–6.34 (m, 1H, CH), 6.38 (s, 2H, ArH), 6.76–6.82 (m, 3H, ArH); ESIMS (C₁₉H₂₂O₅): m/z = 331 [M + H]⁺, 353 [M + Na]⁺.

General procedure for the synthesis of (15a–c)

1,2,3-Trimethoxy-5-(3-(4-methoxyphenyl)propyl)benzene (15a). Compound 14k (0.80 g, 2.54 mmol) was taken in a round bottomed flask and dissolved in ethyl acetate–methanol mixture (8 : 2). The mixture was flushed with nitrogen gas and palladium charcoal (Pd/C, 5% wt) (0.08 g) was added and hydrogen gas was passed to it for 1 h. The progress of reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated and crude material was worked up using ethyl acetate and water. The organic layer was the separated, dried anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude was purified by column chromatography on silica gel (100–200 mesh size) using ethyl acetate–hexane (12 : 88) as eluents which yielded pure compound 15 as white solid. Yield: (0.68 g), 85%; m.p. 56–57 °C; R_f: 0.80 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2933, 2837, 1588, 1509, 1457, 1242, 1125, 1006, 829; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 1.90–2.00 (m, 2H, CH₂), 2.59–2.66 (m, 4H, 2 × CH₂), 3.82–3.87 (m, 12H, 4 × OCH₃), 6.42 (s, 2H, ArH), 6.87 (d, J = 8.7 Hz, 2H, ArH), 7.14 (d, J = 8.4 Hz, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 33.58 (CH₂), 34.96 (CH₂), 36.19 (CH₂), 55.66 (OCH₃), 56.47 (2 × OCH₃), 61.52 (OCH₃), 105.75 (2 × C), 114.16 (2 × C), 129.72 (2 × C), 134.65, 136.50, 138.54, 153.48 (2 × C), 158.19; ESIMS (C₁₉H₂₄O₄): m/z = 316 [M]⁺, 339 [M + Na]⁺.

1,2-Dimethoxy-4-(3-(3,4,5-trimethoxyphenyl)propyl)benzene (15b). Yield: (0.72 g), 90%; m.p. 52–53 °C; R_f: 0.30 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2939, 2837, 1589, 1514, 1463, 1239, 1130, 1029, 1003, 823, 766; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 1.90–2.01 (m, 2H, CH₂), 2.59–2.66 (m, 4H, 2 × CH₂), 3.85–3.89 (m, 12H, 4 × OCH₃), 6.42 (s, 2H, ArH), 6.74–6.77 (m, 2H, ArH), 6.81–6.84 (d, J = 7.8 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 33.48, 33.46, 36.21, 56.26 (OCH₃), 56.36 (2 × OCH₃), 61.22 (OCH₃), 105.86 (2 × C), 111.77, 112.35, 120.67 (2 × C), 135.26, 136.61, 138.45, 147.66, 149.30, 153.50; ESIMS (C₂₀H₂₆O₅): m/z = 347[M + H]⁺, 369 [M + Na]⁺.

2-Methoxy-4-(3-(3,4,5-trimethoxyphenyl)propyl)phenol (15c)

Yield: (0.63 g), 78%; m.p. 83–84 °C; R_f: 0.45 (30% ethyl acetate–hexane); ¹H NMR (CDCl₃, 300 MHz, δ ppm): 1.79–1.89 (m, 2H, CH₂), 2.48–2.53 (t, J = 2.7 Hz, 4H, 2 × CH₂), 3.73–3.82 (m, 12H, 4 × OCH₃), 5.51 (s, 1H, CH), 6.32 (s, 2H, ArH), 6.57–6.60 (dd, J = 1.8 and 1.8 Hz, 1H, ArH), 6.71–6.75(m, 2H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 33.35, 35.20, 36.13, 56.42, 56.48 (2 × OCH₃), 61.23, 105.83 (2 × C), 111.04, 115.09, 120.13, 135.97, 136.56, 138.51, 145.16, 145.91, 153.48 (2 × C); ESIMS (C₁₉H₂₄O₅): m/z = 333 [M + H]⁺.

General procedure for the synthesis of compounds (17a–e)

2-(4-Hydroxy-3-methoxybenzylidene)-3,4-dihydro-6-methoxynaphthalen-1(2H)-one (17a). In a round bottomed flask compound 16 (R₁ = R₂ = H, n = 2) (2.00 g, 11.36 mmol) was dissolved into methanol (20 mL) and methanolic KOH (10%, 20 mL) was added to this solution drop wise on ice bath. After 20 minutes, compound 10 (R′ = OCH₃, R = H, 2.63 g, 11.36 mmol) dissolved in methanol (15 mL) was added to reaction mixture. The reaction mixture was allowed to stir at ice bath for 30 minutes and afterwards at room temperature for 12 h. The progress of reaction was monitored using TLC. After completion of reaction, solvent was evaporated followed by drop wise addition of dilute HCl and pH of the content was adjusted to acidic. The content was extracted with ethyl acetate. The organic layer was then separated and dried over anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude material was purified by crystallization by methanol which yielded pure compound 17a as yellow solid. Yield: (2.53 g), 72%; m.p. 129–130 °C; R_f: 0.18 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3182, 1637, 1597, 1556, 1522, 1255, 1127, 1093, 1034, 867, 826, 765; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.88–2.93(m, 2H, CH₂), 3.12 (bs, 2H, CH₂), 3.86(s, 3H, OCH₃), 3.90(s, 3H, OCH₃), 5.93(s, 1H, OH), 6.70(s, 1H, ArH), 6.86 (dd, J = 2.4 &2.1 Hz, 1H, ArH), 6.96–7.03 (m, 3H, ArH), 7.78 (s, 1H, ArH), 8.09 (d, J = 8.7 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 27.71 (CH₂), 29.61 (CH₂), 55.83 (OCH₃), 56.83(OCH₃), 112.66, 113.19, 113.65, 114.85, 124.08, 127.58, 128.75, 131.09, 134.14, 136.77, 145.95, 146.70, 146.82, 163.90, 187.15(C=O); ESIMS (C₁₉H1₈O₄): m/z = 309[M − 1]⁺, 333 [M + Na]⁺.

2-(4-Hydroxy-3-methoxybenzylidene)-2,3-dihydro-5-methoxyinden-1-one (17b). Yield: (3.02 g), 83%; m.p. 199–201 °C; R_f: 0.09 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3490, 1681, 1624, 1589, 1521, 1267, 1092, 1028, 837, 811; ¹H NMR (DMSO-d₆, 300 MHz, δ ppm): 3.75–3.79 (m, 6H, 2 × OCH₃), 3.94 (bs, 2H, CH₂), 6.79–6.82 (d, J = 8.1 Hz, ArH), 6.90–6.93 (d, J = 8.4 HZ, 1H, ArH), 7.09–7.14 (m, 2H, ArH), 7.21–7.47 (m, 4H, CH and ArH), 7.59–7.62 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, δ ppm): 32.80, 56.52, 56.60, 110.92, 115.23, 149.53, 148.67, 133.24, 133.12, 133.24, 148.67, 149.53, 153.60, 165.53, 192.53 (C=O); ESIMS (C₁₈H₁₆O₄): m/z = 297 [M + H]⁺, 323 [M + Na]⁺.

2-(4-Hydroxy-3-methoxybenzylidene)-2,3-dihydro-5,6-dimethoxyinden-1-one (17c). Yield: (2.92 g), 86%; m.p. 140–141 °C; R_f: 0.11 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3424, 1672, 1578, 1505, 1311, 817, 714; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.79–3.85 (m, 11H, 3 × OCH₃ and CH₂), 6.77–6.87 (m, 2H, ArH), 7.01–7.07 (m, 2H, ArH), 7.16 (s, 1H, CH), 7.36 (s, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.41 (CH₂), 55.28 (OCH₃), 56.42 (OCH₃), 58.58 (OCH₃), 105.26, 107.65, 113.96, 116.03, 125.12, 127.86, 131.50, 132.91, 133.21, 144.94, 147.86, 148.57, 149.85, 155.48, 193.45 (C=O); ESIMS (C₁₉H₁₈O₅): m/z = 327 [M + H]⁺, 349 [M + Na]⁺.

2-(4-Methoxybenzylidene)-2,3-dihydro-5-methoxyinden-1-one (17d). Yield: (2.55 g), 74%; m.p. 145–146 °C; R_f: 0.15 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 1684, 1597, 1504, 1256, 1125, 1022, 808; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.84–3.92 (m, 8H, CH and 2 × OCH₃), 6.91–6.97 (m, 4H, ArH), 7.55–7.60 (m, CH and ArH), 7.82 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.92 (CH₂), 55.77 (OCH₃), 56.05 (OCH₃), 110.13, 114.81 (2 × C), 115.49, 126.42, 128.71, 132.03, 132.72 (2 × C), 132.91, 133.34, 152.76, 161.03, 165.44, 193.27 (C=O); ESIMS (C₁₈H₁₆O₃): m/z = 303 [M + Na]⁺, 319 [M + K]⁺.

2-(4-Methoxybenzylidene)-2,3-dihydro-5,6-dimethoxyinden-1-one (17e). Yield: (2.90 g), 90%; m.p. 191–192 °C; R_f: 0.46 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 1684, 1597, 1504, 1256, 1125, 1022, 808; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.83–4.20 (m, 11H, CH₂ and 3 × OCH₃), 6.92–6.94 (m, 3H, ArH), 7.29 (s, 1H, CH), 7.52–7.58 (m, 3H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.55 (CH₂), 55.76 (OCH₃), 56.53 (OCH₃), 56.64 (OCH₃), 105.49, 107.61, 114.81 (2 × C), 128.75, 131.68, 132.60 (2 × C), 132.67, 133.56, 145.02, 150.00, 155.62, 161.02, 193.57 (C=O); ESIMS (C₁₉H₁₈O₄): m/z = 333 [M + Na]⁺.

General procedure for the synthesis of compounds (18a–e)

2-(4-Hydroxy-3-methoxybenzyl)-3,4-dihydro-6-methoxynaphthalen-1(2H)-one (18a). Compound 17a (1.00 g, 3.23 mmol) was taken in a round bottomed flask and dissolved in ethyl acetate–methanol mixture (8 : 2). The mixture was flushed with nitrogen gas and palladium charcoal (Pd/C, 5% wt) (0.10 g) was added and hydrogen gas was passed to it for 3 h. The progress of reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated and crude material was worked up using ethyl acetate and water. The organic layer was the separated, dried anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude was purified by column chromatography on silica gel (100–200 mesh size) using ethyl acetate–hexane (12 : 88) as eluents which yielded pure compound 18a as white solid. Yield: (0.72 g), 72%; m.p. 114–115 °C; R_f: 0.40 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3353, 2932, 1663, 850; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 1.67 (s, 2H, CH₂), 2.56–2.68 (m, 2H, CH₂), 2.85–2.89 (m, 2H, CH₂), 3.35–3.39 (m, 1H, CH), 3.84–3.86 (m, 6H, 2 × OCH₃), 5.53 (s, 1H, OH), 6.66–6.74 (m, 3H, ArH), 6.80–6.84 (m, 2H, ArH), 8.03 (d, J = 9.0 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 28.00 (CH₂), 29.29 (CH₂), 35.87(CH₂), 49.73 (CH), 55.81 (OCH₃), 56.32 (OCH₃), 112.11, 112.85, 113.59, 114.54, 122.35, 126.55, 130.38, 132.41, 144.36, 146.87, 146.95, 163.90, 198.75 (C=O); ESIMS (C₁₉H₂₀O₄): m/z = 335 [M + Na]⁺.

2-(4-Hydroxy-3-methoxybenzyl)-2,3-dihydro-5-methoxyinden-1-one (18b). Yield: (0.59 g), 59%; m.p. 120–121 °C; R_f: 0.37 (40% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3376, 1681, 1593, 1517, 1251, 849, 814; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.63 (dd, J = 9.6 and 9.9 Hz, 1H, CH of CH₂), 2.80 (dd, J = 3.0 and 3.0 Hz, 1H, CH of CH₂), 2.89–2.98 (m, 1H, CH), 3.10 (dd, J = 7.5 and 7.5 Hz, 1H, CH of CH₂), 3.22–3.28 (dd, J = 3.9 and 4.2 Hz, 1H, CH of CH₂), 3.84 (s, 6H, OCH₃), 5.55 (s, 1H, OH), 6.68–6.73 (m, 2H, ArH), 6.81–6.83 (m, 2H, ArH), 6.88 (d, J = 8.4 Hz, 1H, ArH), 7.69 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 31.29 (CH₂), 32.44 (CH₂), 49.67, 56.01 (OCH₃), 56.29 (OCH₃), 110.09, 111.84, 114.60, 115.82, 122.03, 126.01, 130.30, 131.91, 144.52, 146.92, 157.16, 165.84, 206.63 (C=O); ESIMS (C₁₈H₁₈O₄): m/z = 299 [M + H]⁺, 321 [M + Na]⁺.

2-(4-Hydroxy-3-methoxybenzyl)-2,3-dihydro-5,6-dimethoxyinden-1-one (18c). Yield: (0.70 g), 70%; m.p. 137–138 °C; R_f: 0.27 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3387, 2988, 2939, 1676, 1216, 1035, 972, 846, 803, 665: ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.57–2.65 (m, 1H, CH of CH₂), 2.76 (d, J = 16.8 Hz, 1H, CH), 2.93 (bs, 1H, CH), 3.04–3.09 (m, 1H, CH), 3.25 (d, J = 13.8, Hz, 1H, CH), 3.83–3.92 (m, 9H, 3 × OCH₃), 5.06 (s, 1H, OH), 6.68–6.73 (m, 2H, ArH), 6.84 (bs, 2H, ArH), 7.25 (s, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.17 (CH₂), 37.39 (CH₂), 49.79, 55.29 (OCH₃), 56.49 (OCH₃), 56.59 (OCH₃), 104.79, 107.86, 111.86, 114.62, 122.00, 129.71, 131.95, 144.53, 146.92, 149.51, 149.91, 156.04, 207.12 (C=O); ESIMS (C₁₉H₂₀O₅): m/z = 329 [M + H]⁺.

2-(4-Methoxybenzyl)-2,3-dihydro-5-methoxyinden-1-one (18d). Yield: (0.87 g), 87%; m.p. 85–86 °C; R_f: 0.57 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2916, 2841, 1689, 1251, 1023, 840, 689; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.58–2.66 (m, 1H, CH of CH₂)2.78 (dd, J = 3.6 and 3.6 Hz, 1H, CH of CH₂), 2.89–2.98 (m, 1H, CH), 3.09 (dd, J = 7.5 and 7.8 Hz, 1H, CH of CH₂), 3.28 (dd, J = 4.2 and 4.2 Hz, 1H, CH of CH₂), 3.78 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 6.80–6.90 (m, 4H, ArH), 7.14 (d, J = 8.4 Hz, 2H, ArH), 7.69 (d, J = 8.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.50(CH₂), 36.68 (CH₂), 49.61 (CH), 55.63 (OCH₃), 55.99 (OCH₃), 110.11, 114.31 (2 × C), 115.75, 126.04, 130.27 (3 × C), 132.10, 157.04, 158.54, 165.81, 206.46 (C=O); ESIMS (C₁₈H₁₈O₃): m/z = 283 [M + H]⁺, 305 [M + Na]⁺.

2-(4-Methoxybenzyl)-2,3-dihydro-5,6-dimethoxyinden-1-one (18e). Yield: (0.73 g), 73%; m.p. 122–123 °C; R_f: 0.13 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2947, 2926, 1691, 1602, 1506, 1464, 1259, 810, 778; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.58–2.65 (m, 1H, CH of CH₂), 2.71–2.78 (dd, J = 3.0 and 3.0 Hz, 1H, CH of CH₂), 2.89–2.97 (m, 1H, CH), 3.01–3.09 (m, 1H, CH of CH₂), 3.24–3.30 (dd, J = 4.2 and 4.2 Hz, 1H, CH of CH₂), 3.77 (s, 3H, OCH₃), 3.82–3.925 (m, 6H, 2 × OCH₃), 6.81 (d, J = 8.7 Hz, 3H, ArH), 7.12–7.18 (m, 3H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 32.18 (CH₂), 36.76 (CH₂), 49.71 (CH), 55.63 (OCH₃), 56.49 (OCH₃), 56.57 (OCH₃), 104.83, 107.83, 114.28 (2 × C), 129.72, 130.26 (2 × C), 132.11, 149.41, 149.88, 155.99, 158.51, 207.04 (C=O); ESIMS (C₁₉H₂₀O₄): m/z = 313 [M + H]⁺, 335 [M + Na]⁺.

General procedure for the synthesis of compounds (19a–e)

In a round bottomed flask, compound 18a (0.60 g, 1.92 mmol) was dissolved in methanol and kept at 0 °C for 15–20 minutes. To this mixture sodium borohydride (NaBH_4, 0.22 g, 5.76 mmol), was added and reaction was transferred to room temperature after half an hour. The progress of reaction was monitored by TLC (30% ethyl acetate–hexane). After completion of reaction, solvent was taken off and the reaction mixture was treated with NH₄Cl and worked up using ethyl acetate and water. The organic layer was the separated, dried anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude solid material was purified by washing it with hexane which yielded pure compound 19a as white solid in 98% (0.54 g) yield which was further processed for hydrolysis as such. The experimental procedure used for synthesis of compound 19b–m is same as described above. The molar ratio of reactants was same for synthesis of all derivatives.

General procedure for the synthesis of compounds (20a–e)

4-((1,2-Dihydro-7-methoxynaphthalen-3-yl)methyl)-2-methoxyphenol (20a). In a round bottomed flask, compound 19a (0.97 g, 2.93 mmol) was dissolved in ethanol and conc. HCl (1 mL) was added to it. The reaction mixture was transferred for reflux. The progress of reaction was monitored by TLC (30% ethyl acetate–hexane). After completion of reaction, solvent was taken off and the reaction mixture was worked up using ethyl acetate and water. The organic layer was the separated, dried over anhydrous sodium sulphate (Na₂SO₄) and concentrated. The crude solid material was purified by column chromatography on silica gel (100–200 mesh size) using hexane as eluents which yielded pure compound 20a as white solid. Yield: (0.75 g), 82%; m.p. 72–73 °C; R_f: 0.50 (20% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3386, 2935, 1607, 1511, 1432, 1266, 1240, 1152, 1033, 802, 738; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 2.17 (t, J = 7.9 Hz, 2H, CH₂), 2.76 (t, J = 7.9 Hz, 2H, CH₂), 3.42 (s, 2H, CH₂), 3.79 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 5.53 (s, 1H, OH), 6.21 (s, 1H, CH), 6.67–6.73 (m, 4H, ArH), 6.861 (d, J = 8.4 Hz, 1H, ArH), 6.94 (d, J = 8.1 Hz, 1H, ArH); ¹³C NMR (CDCl₃, 75 MHz, δ ppm): 27.07 (CH₂), 29.15 (CH₂), 43.83 (CH₂), 55.69 (OCH₃), 56.32 (OCH₃), 111.51, 111.80, 113.98, 114.54, 122.17, 123.44, 126.89, 128.36, 131.94, 136.55, 138.93, 144.41, 146.87, 158.70; ESIMS (C₁₉H₂₀O₃): m/z = 319 [M + Na]⁺, 335 [M + K]⁺.

2-Methoxy-4-((6-methoxy-1H-inden-2-yl)methyl)phenol (20b). Yield: (0.60 g), 66%; m.p. 108–109 °C; R_f: 0.58 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3434, 2932, 1604, 1515, 1473, 1275, 1237, 1034, 869, 821; ¹H NMR (acetone-d₆, 300 MHz, δ ppm): 3.22 (bs, 2H, CH₂), 3.68 (s, 2H, CH₂), 3.74 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 6.41 (s, 1H, CH), 6.69–6.78 (m, 4H, ArH), 6.85 (bs, 1H, ArH), 6.96 (bs, 1H, ArH), 7.12 (d, J = 8.1 Hz, 1H, ArH), 7.36 (bs, 1H, OH); ¹³C NMR (acetone-d₆, 75 MHz, δ ppm): 37.43 (CH₂), 40.85 (CH₂), 55.16 (OCH₃), 55.74 (OCH₃), 110.60, 111.92, 112.73, 115.22, 120.55, 121.60, 126.79, 132.07, 138.85, 145.36, 145.66, 147.80, 148.24, 157.87; ESIMS (C₁₈H₁₈O₃): m/z = 283 [M + H]⁺, 305 [M + Na]⁺.

2-methoxy-4-((5,6-dimethoxy-1H-inden-2-yl)methyl)phenol (20c). Yield: (0.65 g), 71%; m.p. 116–117 °C; R_f: 0.38 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3412. 2963, 2924, 1607, 1513, 1487, 1461, 1298, 1269, 855; ¹H NMR (acetone-d₆, 300 MHz, δ ppm): 3.18 (s, 2H, CH₂), 3.68 (s, 2H, CH₂), 3.75–3.89 (m, 9H, 3 × OCH₃), 6.39 (s, 1H, CH), 6.67–6.77 (m, 2H, ArH), 6.84–6.89 (m, 2H, ArH), 7.15 (s, 1H, ArH); ¹³C NMR (acetone-d₆, 75 MHz, δ ppm): 37.43 (CH₂), 40.73 (CH₂), 55.77 (OCH₃), 55.95 (OCH₃), 56.22 (OCH₃), 105.56, 109.72, 112.75, 115.21, 121.58, 127.17, 132.19, 136.27, 138.86, 145.37, 147.58, 147.81, 149.15, 149.30; ESIMS (C₁₉H₂₀O₄): m/z = 313 [M + H]⁺, 335 [M + Na]⁺.

2-(4-methoxybenzyl)-6-methoxy-1H-indene (20d). Yield: (0.45 g), 50%; m.p. 83–84 °C; R_f: 0.6 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 3006, 2927, 2837, 1606, 1511, 1464, 1232, 1243, 1179, 1106, 1030, 859, 809, 745; ¹H NMR (acetone-D₆, 300 MHz, δ ppm): 3.21 (s, 2H, CH₂), 3.70–3.83 (m, 8H, CH₂ and 2 × OCH₃), 6.39 (s, 1H, ArH), 6.75 (d, J = 8.1 Hz, 1H, ArH), 6.84–6.96 (m, 3H, ArH), 7.11–7.18 (m, 3H, CH and ArH); ¹³C NMR (acetone-D₆, 75 MHz, δ ppm): 36.89 (CH₂), 40.83 (CH₂), 54.96 (OCH₃), 55.16 (OCH₃), 110.60, 111.95, 114.16 (2 × C), 120.56, 120.89, 126.89, 130.08 (2 × C), 132.70, 145.63, 148.12, 157.92, 158.68; ESIMS (C₁₈H₁₈O₂): m/z = 267 [M + H]⁺, 265 [M − H]⁺.

2-(4-methoxybenzyl)-5,6-dimethoxy-1H-indene (20e). Yield: (0.57 g), 62%; m.p. 94.0–95 °C; R_f: 0.6 (30% ethyl acetate–hexane); IR (KBr, ν_max cm⁻¹): 2941, 2834, 1609, 1507, 1460, 1320, 1242, 1101, 1033, 853, 823; ¹H NMR (CDCl₃, 300 MHz, δ ppm): 3.20 (s, 2H, CH₂), 3.79–3.87 (m, 11H, CH₂ and 3 × OCH₃), 6.39 (s, 1H, CH), 6.83–6.85 (m, 3H, ArH), 6.95 (bs, 1H, ArH), 7.13 (d, J = 8.4 Hz, 2H, ArH); ¹³C NMR (acetone-D₆, 75 MHz, δ ppm): 36.89 (CH₂), 40.70 (CH₂), 54.96 (OCH₃), 55.91 (OCH₃), 56.17 (OCH₃), 105.46, 109.60, 114.13 (2 × C), 127.27, 130.07 (2 × C), 132.81, 136.20, 138.76, 147.55, 149.03, 149.27, 158.64; ESIMS (C₁₉H₂₀O₃): m/z = 297 [M + H]⁺, 319 [M + Na]⁺.

HPLC analysis

Liquid chromatography based generic methods are widely applicable for the pharmaceutical industry to identify, quantify and determine the purity of small organic compounds. The purity of compounds was determined using high-low chromatographic approach. LC-PDA-MS (Shimadzu, Japan) consisting of an analytical column (Phenomenex, C₁₈ 250 × 4.6 mm, 5 mm), pumps (LC-20AD), injector and PDA (SPD-M20A), was used for analysis. Gradient elution covering a wide range of solvent polarity viz. methanol–water, acetonitrile–water with and without acid additives, was tried on diverse stationary phases. Finally, a better separation was achieved with a mobile phase composition of acetonitrile–water (80 : 20, v/v). A flow rate of 1.0 mL min⁻¹ and column temperature of 27 °C was maintained throughout the run.

In vitro anticancer activity

Assessment of cell growth inhibition. In vitro anticancer activity of test compounds was studied by Sulphorhodamine B (SRB) dye based plate assay. In brief, 10⁴ cells per well were added in 96-well culture plates and incubated at 37 °C in 5% CO₂ concentration. After overnight incubation of cells, serial dilutions of test compound were added to the wells. Untreated cells served as control. After 48 h, cells were fixed with ice-cold Tri-chloroacetic acid (50% w/v, 100 μl per well), stained with SRB (0.4% w/v in 1% acetic acid, 50 μl per well), washed and air-dried. Bound dye was solubilised with 10 mM Tris base (150 μl per well) and absorbance was read at 540 nm on a plate reader. The cytotoxic effect of compound was calculated as % inhibition in cell growth as per formula: [1-(Absorbance of drug treated cells/Absorbance of untreated cells) × 100]. Determination of 50% inhibitory concentration (IC₅₀) was based on dose-response curves.

Cell cycle analysis by flowcytometry. Cell cycle distribution was measured in concentration and time dependent manner by flow cytometric analysis of PI-stained cellular DNA, as described earlier.¹³ Briefly, MCF-7 cells (4 × 10⁵ per well) were seeded in 6-well culture plate and grown overnight (37 °C, 5% CO₂). Compound treated cells were harvested by trypsinization and fixed (30 min, 4 °C) with ice-cold 70% ethanol at indicated time points. The pellets were washed with PBS and re-suspended in a solution containing PI (20 mg ml⁻¹), Triton X100 (0.1%) and RNase (1 mg ml⁻¹) in PBS. After incubation (45 min, in the dark, 37 °C), cells were analysed on a FACS Calibur flow cytometer (BD Biosciences). Distribution of cells in different phases of cell cycle was calculated using “Cell Quest” software.

Detection of fragmented nuclei by DAPI staining. To observe the nuclear morphology, cells (10⁴ per well) were grown overnight in 96-well plates and treated with 14k at IC₅₀ concentration. After 24 h incubation with test compound, cells were fixed with 4% formaldehyde (50 μl per well), washed with PBS and permeabilized with 0.1% Triton X100 (50 μl per well). After washing with PBS, cells were stained with DAPI (2 μg mL⁻¹, 50 μl per well) for 15 min in dark. Images were acquired with 20× objective using a fluorescent microscope (Nikon, Japan).

In vitro cytotoxicity in THP-1 cells. The compounds were tested for its cytotoxicity against THP-1 cell line in vitro using resazurin assay (Roy et al., 2011). Briefly, 10⁴–10⁶ cells per well were seeded in 96-well plate in 200 μl RPMI supplemented with heat inactivated fetal bovine serum (10% v/v) and incubated at 37 °C with 5% CO₂ for overnight. Cells were exposed to different dilutions of test compounds and standard (Rifampin) in triplicate ranging from 400 to 6.25 μM concentration with two fold dilutions. After 48 h 20 μl of resazurin (0.02% w/v) was added in each well and further incubated for 4–5 h. The colour change from purple to pink was assessed visually and fluorescence was measured at 530 ± 25 nm and 590 ± 25 nm for excitation and emission respectively in Synergy Biotek plate reader. CC₅₀ values (50% cytotoxic concentrations) were calculated by plotting fluorescence values using Microsoft excel template.

Ligand and protein preparation for docking experiments

Estrogen receptor (PDB: 1A52; 17-β-estradiol bound) and β-tubulin (PDB: 1SA0; colchicine bound) were retrieved from RCSB Protein Data Bank. Ligand Explorer viewer and PDBsum (http://www.ebi.ac.uk/pdbsum/) were used to inspect the binding site residues and their interactions with bound ligands. This showed that 17β-estradiol has interactions via hydrogen bond with His524, Arg394 and Glu353 in bound state with chain A. Surrounding residues at this active site included Leu346, Leu387, Leu391, Phe404, Met388, Ala350, Ile424 and Leu525. Whereas, colchicine bound active site of β-tubulin protein showed colchicine interaction via hydrogen bond with Cys241 in bound state with chain B. Surrounding residues at this active site also included Leu255, Asn258, Ala316, Ile378, Leu242, Val238, Ala250 and Lys254.

The test compounds were drawn using chemsketch (http://www.acdlabs.com) and the SMILES format of these structures were converted into 3D coordinate file in .pdb format using online SMILE translation tool (http://cactus.nci.nih.gov/translate/). Energy minimization of these compounds was performed using ligand preparation tool of Discovery studio 2.5 and PRODRG server (http://davapc1.bioch.dundee.ac.wy.prodrg). Estrogen receptor chain A and β-tubulin chain B were used for docking studies after removing heteroatoms and water molecules. Proteins were energy minimized using Swiss-pdb viewer 4.01 (http://www.expasy.org/spdbv) by applying GROMOS96 force field to compute energy and to execute energy minimization. AutoDock 4.2 software (http://www.scripps.edu) was used to execute docking experiments following Lamarckian Genetic algorithm (hybrid of genetic and local search algorithm) and using default docking parameters. Best docked conformation of each ligand based on docking parameters and binding site residues interactions were selected among top 10 docked conformations of each ligand generated during docking. LigPlot Plus version v.1.4.4 (Roman Laskowski, 2009) was used to generate schematic diagram of protein-ligand interactions for a given ligand in a PDB file. Colchicine and 2-methoxyestradiol (known tubulin inhibitors) were also docked on β-tubulin and results obtained were compared with test compounds, diarylpropene derivatives on β-tubulin. Similarly, 17β-estradiol (known estrogen receptor alpha inhibitor) was docked on estrogen receptor to compare the results obtained with test compounds, diarylpropene derivatives on estrogen receptor alpha.

Acknowledgements

The authors thank the Director, CSIR-CIMAP and Council of Scientific Industrial Research (CSIR) for financial support. The work was carried out under project ChemBio (BSC-203) to AG and GAP 0115 (sponsored by DST, India) to JS.

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