Synthesis of Psoralidin derivatives and their anticancer activity: First synthesis of Lespeflorin I

Abstract

Synthetic scheme for the preparation of a number of different derivatives of anticancer natural product Psoralidin is described. A convergent synthetic approach is followed using simple starting materials like substituted phenyl acetic esters and benzoic acids. The developed synthetic route leads us to complete the first synthesis of an analogous natural product Lespeflorin I₁, a mild melanin synthesis inhibitor. Preliminary bioactivity studies of the synthesized compounds are carried out against two commonly used prostate cancer cell lines. Results show that the bioactivity of the compounds can be manipulated by the simple modification of the functional groups.

Graphical Abstract

Introduction

Psoralidin (1)¹ is a member of an emerging class of natural product called prenylated coumestane which contains one or more isopentenyl group in the nucleus of 6H-benzofuro[3,2-c]chromen-6-one (Fig. 1). The natural product, in the crude mixture of its producer Psoralea corylifolia Linn, has been used as a traditional medicine in India and China from ancient time.² The compound itself also show a wide range of bioactive properties like antioxidant,³ antidepressant,⁴ antibacterial,⁵ anti-diabetic, ⁶ antiviral,⁷ anti-inflammatory,⁸ and anti-osteoporosis.⁹ It has also been found to be active against different types of cancer cells like gastric, breast, colon, bone, and stomach.¹⁰ Research from different groups, including us, have shown that Psoralidin targets multiple signaling cascades and inhibits the proliferation of castration resistant prostate cancer cells by down regulation of MAPkinase signaling with little or almost zero toxicity.^11a–e In the same prostate cancer cells the compound also induces reactive oxygen species (ROS) mediated apoptosis.^11d The IC₅₀ values of Psoralidin (1) against two different prostate cancer cell lines namely, PC-3 and DU-145 are 60 μM and 45 μM, respectively. Our group is working on the anticancer properties of Psoralidin for a long time.^11a–d But the relatively high IC₅₀ value of the compound coupled with inherent low solubility brings a great deal of difficulty in further biological studies. It is to be mentioned that chemotherapeutic agents such as Docetaxel (IC₅₀ ~5nM),^11f,g Cabazitaxel (IC₅₀ ~ 10nM)^11h and Mitoxantrone (IC₅₀ ~ 5 μM)¹¹ⁱ have been reported to be active against prostate cancer cells (PC-3). Therefore, it is assumed that only a synthetic modification might help to improve the activity of the compound leading to further practical application.

Fig. 1.

Psoralidin and structurally similar bioactive natural products.

There are a number of different methodologies reported in the literature for the synthesis of coumestanes.¹² But only a few of those processes are adopted for the prenylated coumestanes,^12i–l mainly because of the sensitivity of the compounds towards strong reagents and conditions. Thus the synthesis of analogs of Psoralidin is always a difficult task. Earlier, we have reported a convergent synthetic pathway for the total synthesis of Psoralidin.^12l In this article, we have shown that a number of different derivatives of Psoralidin (1) can be prepared by the modification of our earlier reported^12l pathway. In this context, we have also synthesized Lespeflorin I₁ (2), a 8-hydroxy analog of Psoralidin. The compound was isolated from the roots of Lespedeza floribunda Bunge and reported to act as a mild melanin synthesis inhibitor in normal human epidermal melanocytes.¹³ The activities of all the synthesized compounds were tested against two different prostate cancer cell lines to get a preliminary idea about the structure-activity relationship of the parent natural product.

Results and discussion

We started with the attempt to synthesize derivatives like 14 & 15, where the prenyl group of Psoralidin (1) was either absent or replaced with -Me (Scheme 1). The target was to evaluate the role of the existing prenyl group in the bioactivity pattern of parent molecule. In this point it is to be mentioned that the prenyl group is the most sensitive functional group of the molecule which makes the synthesis process more difficult.^12l The synthetic scheme started with 1-bromo-2,4-dimethoxybenzene (7) and 1-bromo-5-methyl-2,4-dimethoxybenzene (8) for the synthesis of 14 & 15, respectively. Compound 8 was prepared from 1,5-dibromo-2,4-dimethoxy benzene by selective replacement of one of the bromine atom with methyl. Metallation of the bromine atom of 7 and 8 with n-butyl lithium followed by treatment with carbon dioxide produced acids 9 and 10, respectively. Addition of LDA generated anion of methyl 2-(2,4-dimethoxyphenyl)acetate (11) to acid chlorides of 9 and 10, prepared by the treatment of oxallyl chloride, produced compounds 12 and 13. It is to be noted that compound 11 was prepared by the perkin condensation of 2,4-dimethoxybenzaldehyde and hippuric acid following a reported procedure.¹⁴ Finally, BBr₃ mediated one pot demethylation and cyclization sequence gave the desired Psoralidin derivatives 14 and 15. Structural assignments of the new compounds were carried out using regular spectroscopic techniques like IR, ¹H, ¹³C NMR and HRMS.

Scheme 1.

Synthesis of derivatives with substitution at C-2.

In the subsequent experiment, we decided to evaluate the function of the two hydroxyl groups and also the importance of their relative positions in the parent natural product. To this end we targeted four derivatives 29–32, where compound 29 and 30 had one of the hydroxyl groups missing from the C-3 and C-9 position of the parent Psoralidin, respectively. On the other hand, compound 31 and 32 carried all the same functional groups with altered position at C-8, C-9 and C-2, C-3, respectively. The starting materials for the synthesis were phenyl acetates 11, 16, 17 and benzoic acid derivatives 18–20 (Scheme 2). The acetates 16 and 17 were prepared from corresponding hydroxyphenylacetic acids by methylation. The compound 18 was prepared from 4-allyl-2-bromo-1-methoxybenzene. Compound 19 and 20 were prepared in two steps from 1,5-dibromo-2,4-dimethoxybenzene and 1,4-dibromo-2,5-diemthoxybenzene, respectively, by sequential replacement of two bromine atoms with allyl and CO₂ (See supporting information). The acids 18–20 were converted to corresponding acid chlorides and condensed with the LDA generated anions of phenyl acetates to produce compounds 21–24. Next, the BBr₃-mediated demethylative cyclization yielded allylated coumestane derivatives 25–28. Finally, a Grubb’s metathesis reaction with 2-methylbut-2-ene provided the desired Psoralidin derivatives 29–32. The structures of all the compounds were confirmed by ¹H and ¹³C NMR spectroscopy, as well as HRMS.

Scheme 2.

Synthesis of Psoralidin analogs 29–32.

Next, it was attempted to introduce extra hydroxyl group in the parent nucleus of Psoralidin (1). To this end, we conceived the idea that hydroxyl group might increase the activity as well as solubility of the molecule. So, we targeted to synthesize three derivatives 2, 46, and 47, placing one excess hydroxyl group each in the C-8, C-4, and C-10 position of the natural product, respectively (Scheme 5). As discussed earlier, compound 2 is a natural product namely, Lespeflorine I₁, which shows a mild melanin synthesis inhibitory property in normal human epidermal melanocytes.¹³ Following a similar synthetic strategy as described earlier, we needed the phenylacetate derivatives 11, 33, 34 and benzoic acids 19, 35 (Scheme 3). Synthesis of compound 33 was reported in the literature starting from 2,4,5-trimethoxybenzoic acid in six linear steps.¹⁵ To avoid the lengthy synthetic procedure and to follow a common synthetic route for all the similar compounds, we used an analogous procedure described for the synthesis of compound 11.¹⁴ We started with methoxy substituted benzaldehyde and used three convergent steps i.e., a perkin type condensation with hippuric acid, refluxing with NaOH/H₂O₂, and esterification, to produce all the three compounds 11, 33, and 34 in good overall yield (see supporting information). Compound 35 was prepared from 1,5-dibromo-2,3,4-trimethoxy benzene by sequential replacement of two bromine atoms with allyl and CO₂ (see supporting information). LDA mediated condensation of 33 & 11 with 19 & 35 produced compounds 36 & 37, respectively. Similar reaction between 34 and 19 produced 38. Next, BBr₃ mediated one pot demethylative cyclization produced allylated derivatives 39–41 (Scheme 3). The structures of compounds 36–41 were confirmed by IR, ¹H and ¹³C NMR spectroscopy, as well as HRMS.

Scheme 5.

Synthesis of Lespeflorine I₁ (2) and analogs

Scheme 3.

Synthesis of allylated coumestanes 39–41

Now, it was expected that a Grubb’s catalyzed cross metathesis reaction with 2-methyl-2-butene would be sufficient to provide the targeted Psoralidin derivatives. But, the 2^nd generation Grubb’s catalyzed cross metathesis reaction of both 39 and 40 with 2-methyl-2-butene ended up with producing a complex mixture of products (Scheme 4). In fact, it was observed that addition of the catalyst in the solution of 39 and 40 instantaneously destroyed the compounds at room temperature. In this context it should be mentioned that a similar reaction could easily be carried out during the total synthesis of Psoralidin.^14l Possibly the presence of a catechol type motif in 39 and 40 made the compounds more vulnerable to oxidation. To stop the plausible transition metal catalyzed oxidation of the free catechol moiety, we decided to protect the hydroxyl groups with some easily removable functional groups. So, triacetates 42 and 43 were prepared from 39 and 40, respectively, by the treatment with acetic anhydride in the presence of triethylamine. Now the cross metathesis reaction with 2-methyl-2-butene, catalyzed by 2^nd generation Grubb’s catalyst at room temperature, produced 44 and 45 in very good yield. The experiment confirmed the role of the catechol moiety in the earlier reaction. But, we faced another hurdle during the deprotection of acetyl group. All of the attempted acetyl deprotection strategies ended up with the formation of a complex mixture of products. Even very mild basic condition like NaHCO₃/MeOH¹⁶ turned out to be harsh enough to destroy the compounds. We were unable to use any acidic reagents because of the reported sensitivity of the compounds towards very mild acidic condition.^12l An attempted reaction at neutral environment using neutral alumina¹⁷ in the conventional or microwave heating condition also failed to produce any isolable product.

Scheme 4.

Synthesis of triacetates and their deprotection

To cope with this type of extremely sensitive compounds, we needed a very labile protecting group which could be opened up in neutral condition without using any acidic or basic reagent. A possible use of benzyl as a protecting group, and hence a H₂/Pd-C mediated reductive deprotection, was not considered because of the possibility of double bond reduction as well as palladium metal catalyzed oxidation. In this context, a cyclic carbonate protecting group was found to be a best fit to solve the problem. The cyclic carbonates can protect two ortho-hydroxy groups and open up easily by refluxing with water only. Thus, two ortho-hydroxyl groups of the compounds 39–41 were protected as a cyclic carbonate by refluxing with pyridine and phosgene in toluene.¹⁸ An attempt to purify the products proved to be difficult because of the inherent instability of the cyclic carbonate protecting group which eventually opened up to give the starting materials back. So the product, after the removal of toluene and pyridine, was subjected to 2^nd generation Grubb’s catalyzed cross metathesis reaction without any attempt of purification. Again in the next step, the crude product was subjected to a deprotection reaction by refluxing with water.¹⁹ Final purification of the product, thus formed, yielded compound 2, 46, and 47, as colorless solid (Scheme 5). As expected, all the physical and spectral characteristics of compound 2 were identical to the reported data of natural Lespeflorin I₁ (Table S1 in supporting information). Structures of other two compounds were also confirmed using IR, ¹H, ¹³C NMR spectroscopy as well as HRMS.

Now, the activities of the synthesized compounds were compared with the parent natural product using two prostate cancer cell lines namely, PC-3 and DU-145. The prostate cancer cell lines were chosen because of the reported activity of Psoralidin against castration resistant prostate cancer cells.^11a–e For the experiment, the cells were treated with the dimethyl sulfoxide (DMSO) solution (final concentration of 0.002%) of the synthesized compounds and the cell viability was quantified using the trypan blue exclusion assay.²⁰ The natural product Psoralidin was used as a positive control and the vehicle (DMSO) as a negative control. Experiments on compound 14–15 and 25–28 showed that none of the compounds was able to carry out a reasonable inhibition of the growth of the cancer cells. The experiment established that the prenyl group was an essential functionality to preserve the bioactivity of the molecule. Similar was the result for compounds 29 and 30 which showed that the hydroxyl groups were also indispensable for the activity of the molecule. The compounds 31 and 32 showed very faint activity compared to the parent Psoralidin (1) (Table 1) confirming the relative positions of functional groups as equally important for the biological activity of the parent compound. We got a better result with the compounds having extra hydroxyl group. The natural product Lespeflorin I₁ (2) was almost equally active to Psoralidin (1) in PC3 cell line (IC₅₀ ~68 μM). In case of DU145 cell line, the activity was even better (IC₅₀ ~4 μM) (Table 1). Among the other two compounds, 47 was less active compared to Psoralidin (1). But, compound 46 showed very good activity against both the cell lines. With the IC₅₀ value of ~14 μM and ~1 μM in PC3 and DU145 cell line, respectively, it was 5 to 45 times more active compared to the parent natural product 1. Although, these results are not sufficient to explain the exact reason of the enhancement of the activity of compound 46, it may be assumed that the hydroxyl group in the C-4 position increases the hydrogen bonding with the target protein. On the other hand, the -OH group in the C-10 position of 47 most probably increases the steric hindrance and inhibits the desired interaction between protein and C9-OH. As expected, due to the acetyl protection of the free hydroxyl groups, compound 44 and 45 were completely inactive.

Table 1.

Comparison of anti-proliferative activity of Psoralidin and synthetic derivatives in two different prostate cancer cell lines

Compound	Cytotoxicity PC3 cells (IC50, μM)a	Cytotoxicity DU145 cells (IC50, μM)a
Psoraldin (1)	60 ± 3	45 ± 5
Lespeflorin I1 (2)	68 ± 5	4 ± 1
31	125 ± 10	110 ± 10
32	95 ± 8	58 ± 5
46	14 ± 2	1 ± 0.5
47	120 ± 10	76 ± 8

^aMeasured in triplicate using trypan blue exclusion assay method.

Conclusion

In conclusion, we synthesized a few different derivatives of Psoralidin by the elimination of an existing functional group or addition of a new functional group in the original skeleton of the natural product. The activities of the synthetic compounds were tested against two different prostate cancer cell lines. Structure-activity relationship (SAR) study revealed that introduction of a hydroxyl group in the appropriate position of the molecule increased the activity in many fold. We have also reported the first synthesis of Lespeflorine I₁ (2). The compound was found to be active against prostate cancer cell lines in addition to its earlier known melanin synthesis inhibitory property. The success of this work encourages future exploration of related structures to obtain more active compound.

Experimental

General Remarks

Reactions in anhydrous condition were conducted in flame-dried glass apparatus under N₂ atmosphere. THF was freshly distilled from sodium and benzophenone and DMF, CH₂Cl₂ and i-Pr₂NH was distilled from CaH₂. Chromatographic separations were performed using silica gel 60–120 and 230–400. All commercially available reagents were used as received. Melting points are uncorrected. Infrared (IR) spectra were recorded using an FT-IR spectrometer, either as a pressed thin KBr disks or as chloroform solutions on sodium chloride disks. ¹H and ¹³C NMR spectra were recorded in Fourier transform mode at 500 (¹H) / 125 (¹³C) MHz in the indicated solvents. High resolution (HRMS) mass spectra were obtained using electron impact (EI) ionization techniques on a magnetic sector instrument at a resolution greater than 10,000. Microwave reactions were carried out either on a Biotage Initiator 2.0 instrument or Discover labmate focused^™ microwave applicator (standard configuration, temperature control, external IR temperature sensor, fixed hold time). HPLC purification was carried out using a Symmetry Prep C₁₈7μm column (19 × 150 mm) on a binary LC system.

Cell culture

Human prostate cancer cell lines PC-3 and DU-145 were grown in Dulbecco’s modified Eagle’s medium and RPMI-1640, respectively, and supplemented with 10% fetal bovine serum with antibiotics. Psoralidin and derivatives were dissolved in dimethyl sulfoxide (DMSO) and the cells were treated with DMSO at a final concentration of 0.002%. Cell viability was quantified using the trypan blue exclusion assay. After 24h treatment with Psoralidin and Psoralidin derivatives, cells were washed with PBS. A collection of supernatants and adherent cells obtained by trypsinization were incubated in 0.4% trypan blue (Corning, Manassas, VA) at 1:1 ratio and pipetted onto a hematocytometer and manually counted under a microscope at ×20 magnification. ²⁰ Psoralidin was used as a positive control and vehicle (DMSO) as a negative control.

Lespeflorin I₁ (2).¹³

This compound was prepared from 39 in three steps without purification of any intermediates. To a stirred solution of 39 (14 mg, 0.04 mmol) in toluene (20 mL), pyridine (0.3 mL) and phosgene (0.3 mL) was added. The mixture was refluxed under nitrogen atmosphere for 3 h and then allowed to cool. 10% HCl (5 mL) was added under ice cold condition and the mixture was extracted with ether (30 mL) immediately. The organic layer was washed with water (2 × 10 mL), brine (10 mL), dried (Na₂SO₄) and concentrated under vacuum. Remaining toluene was removed by repeated co-evaporation with ethyl acetate. The brown solid compound, thus produced, was dissolved in CH₂Cl₂ (20 mL) and degassed. To this mixture degassed 2-methyl-2-butene (2 mL) and Grubb’s 2^nd generation catalyst (3 mg, 0.0035 mmol) was added and allowed to stir at room temperature for 12 h under nitrogen atmosphere. After completion of the reaction, as checked by TLC, it was concentrated under vacuum and extracted with ethyl acetate (2 × 20 mL). The organic layer was washed with water (2 × 10 mL) and concentrated to get a light brown solid which was dissolved in THF (5 mL) and water (5 mL). The mixture was refluxed for 1h and then THF was removed. Extraction with ethyl acetate (2 × 20 mL) and subsequent washing with water (2 × 10 mL), brine (10 mL), drying (Na₂SO₄) and concentration produced crude compound 2. It was finally purified by reverse phase semi preparative HPLC (symmetry C₁₈, 7 μm, solvent gradient 9:1 CH₃CN:H₂O) to get the natural product Lespeflorin I₁ (2) (9.5 mg, 62% over three steps) as a brownish solid. R_f 0.5 (1:3:9 methanol : ethyl acetate : hexane); mp > 360 °C, chars at 210 °C; ν_max (film) cm⁻¹ 1718, 1629, 1490, 1267, 1161, 1002; ¹H NMR (DMSO-d₆, 500 MHz) δ ) δ 10.73 (brs, 1H, OH), 9.55 (brs, 1H, OH), 9.42 (brs, 1H, OH), 7.60 (s, 1H, C1-H), 7.23 (s, 1H, C7-H), 7.18 (s, 1H, C10-H), 6.91 (s, 1H, C4-H), 5.35 (t, 1H, J = 8.0 Hz, C2′-H), 3.32 (d, 2H, J = 8.0 Hz, C1′-H₂), 1.74 (s, 3H, CH₃), 1.70 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.2 (C), 158.7 (C), 158.0 (C), 152.7 (C)148.9 (C), 145.6 (C), 144.4 (C), 132.6 (C), 126.4 (C), 121.8 (CH), 120.8 (CH), 114.2 (C), 104.9 (CH), 104.1 (C), 102.4 (CH), 102.2 (C), 99.0 (CH), 27.6 (CH₂), 25.7 (CH₃), 17.7 (CH₃); HRMS (EI+) m/z 352.0950 ([M]⁺ C₂₀H₁₆O₆, requires 352.0946).

1-Bromo-2,4-dimethoxy-5-methylbenzene (8)

4,6-Dimethoxy-1,3-dibromobenzene²¹ (1.50 g, 5.07 mmol) was added to a solution of i-PrMgCl (1 M in THF, 7 mL, 7 mmol) at −10 °C under N₂ atmosphere. After 45 min, methyl iodide (0.5 mL, 7.7 mmol) was added drop wise, and the mixture was allowed to stir at the same temperature for 30 min and then at rt for 4 h. The reaction was quenched by the addition of 10% HCl (15 mL). THF was removed in vacuum, and the mixture was extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with water (2 × 30 mL) and brine (20 mL), dried (Na₂SO₄), filtered, and concentrated. The resulting crude compound was purified by column chromatography to get 8 (950 mg, 81%) as a colorless liquid. R_f 0.6 (1:10 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1555, 1222, 1143, 1045; ¹H NMR (CDCl₃, 500 MHz): δ 7.24 (s, 1H, C6-H), 6.42 (s, 1H, C3-H), 3.87 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 2.1 (s, 3H, CH₃); ¹³C NMR (CDCl₃,125 MHz): δ 157.8 (C), 154.6 (C), 133.8 (CH), 120.0 (C), 100.8 (C), 96.2 (CH), 56.3 (CH₃), 55.5 (CH₃), 15.1 (CH₃); HRMS (EI+) m/z 229.9946 ([M]⁺ C₉H₁₁BrO₂, requires 229.9942).

2,4-dimethoxybenzoic acid (9).²²

n-BuLi (2.5 M in THF, 3.3 mL, 8.3 mmol) was added drop wise to a stirred solution of 1-bromo-2,4-dimethoxybenzene (7) (1.5 g, 6.9 mmol) in THF (15 mL) at −78 °C under N₂ atmosphere. After 30 min, CO₂ gas was passed through the solution during 45 min of time and the mixture was allowed to warm up to room temperature. THF was removed in vacuum and the mixture was treated with saturated NaHCO₃ solution (40 mL). The water layer was washed with ethyl acetate (2 × 20 mL) and then acidified with conc. HCl. The mixture was extracted with ethyl acetate (2 × 75 mL). The organic layer was washed with water (2 × 30 mL), brine (30 mL), dried (Na₂SO₄), filtered and concentrated. Recrystallization from ethyl acetate produced 9 (1.15 g, 91%) as white solid. R_f 0.3 (1:1 ethyl acetate : hexane); mp 103–105 °C (lit.²² 107–109 °C); ¹H NMR (CDCl₃, 500 MHz): δ 8.10 (d, 1H, J = 9.0 Hz, C6-H), 6.61 (dd, 1H, J = 9.0, 2.0 Hz, C5-H), 6.50 (d, 1H, J = 2.0 Hz, C3-H), 4.01 (s, 3H, OMe), 3.86 (s, 3H, OMe); ¹³C NMR (CDCl₃, 125 MHz): δ 179.9 (C), 165.2 (C), 160.2 (C), 135.2 (CH), 110.1 (C), 106.2 (CH), 98.5 (CH), 56.4 (CH₃), 55.7 (CH₃).

Methyl 2,3-bis(2,4-dimethoxyphenyl)-3-oxopropanoate (12).^12c

To a stirred solution of acid 9 (320 mg, 1.76 mmol) in CH₂Cl₂ (10 mL) under N₂ atmosphere, DMF (one drop) and oxalyl chloride (0.23 mL, 2.6 mmol) were added. The mixture was stirred for 3 h at room temperature and then the solvent was removed under vacuum. The crude product acid chloride was directly used for the next reaction without further purification. A solution of compound 11¹⁴ (554 mg, 2.64 mmol) in THF (6 mL) was added at −78°C to a freshly prepared solution of LDA, prepared from n-BuLi (2.5 M in THF, 1.44 mL, 3.52 mmol) and i-Pr₂NH (0.49 mL, 3.52 mmol) in THF (5 mL) at 0 °C under N₂ atmosphere. The thus produced yellowish anion was stirred at −78 °C for 45 min and then a solution of crude acid chloride in THF (10 mL) was added drop wise. The reaction was stirred at the same temperature for another 45 min and then at room temperature for 4 h. 10% HCl (10 mL) was added to quench the reaction, THF was removed in vacuum and the water layer was extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with water (2 × 20 mL), brine (20 mL), dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography to get 12 (500 mg, 76%) as yellowish oily compound. R_f 0.3 (1:1 ethyl acetate : hexane); ¹H NMR (CDCl₃, 500 MHz): δ 7.89 (d, 1H, J = 9.0 Hz, C6′-H), 7.03 (d, 1H, J = 8.5 Hz, C6″-H), 6.49 (dd, 1H, J = 9.0, 2.0 Hz, C5′-H), 6.44 (s, 1H, C3′-H), 6.41 (d, 1H, J = 8.5 Hz, C5″-H), 6.36 (d, 1H, J = 2.0 Hz, C3″-H), 5.95 (s, 1H, C2-H), 3.78 (s, 3H, OMe), 3.75 (s, 3H, OMe), 3.74 (s, 3H, OMe), 3.73 (s, 3H, OMe), 3.72 (s, 3H, CO₂Me); ¹³C NMR (CDCl₃, 125 MHz): δ 193.1 (C), 171.0 (C), 164.9(C), 160.7 (C), 160.3(C), 157.9 (C), 133.5 (CH), 130.3 (CH), 119.5 (C), 115.6 (C), 105.7 (CH), 104.2 (CH), 98.5 (CH), 98.0 (CH), 57.2 (CH₃), 55.5 (CH₃), 55.5 (CH₃), 55.2 (CH₃), 55.2 (CH₃), 52.2 (CH); HRMS (EI+) m/z 374.1370 ([M]⁺ C₂₀H₂₂O₇, requires 374.1366).

Methyl 3-(2,4-dimethoxy-5-methylphenyl)-2-(2,4-dimethoxyphenyl)-3-oxopropanoate (13)

This compound was prepared from compound 8 in three steps without purifying the intermediate acid and acid chloride. n-BuLi (2.5 M in THF, 0.87 mL, 2.18 mmol) was added drop wise to a stirred solution of 8 (420 mg, 1.82 mmol) in THF (10 mL) at −78 °C under N₂ atmosphere. After 30 min CO₂ gas was passed through the solution during 45 min of time and the mixture was allowed to warm up to room temperature. THF was removed in vacuum and the mixture was treated with saturated NaHCO₃ solution (30 mL). The water layer was washed with ethyl acetate (2 × 15 mL) and then acidified with conc. HCl. The mixture was extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with water (2 × 20 mL), brine (20 mL), dried (Na₂SO₄), filtered and concentrated to get the acid 10 (320 mg). Without further purification it was dissolved in CH₂Cl₂ (10 mL) under N₂ atmosphere, DMF (one drop) and oxalyl chloride (0.23 mL, 2.6 mmol) were added. The mixture was stirred for 3 h at room temperature and then the solvent was removed in vacuum to yield the corresponding acid chloride, which was again directly used for the next reaction without further purification. A solution of compound 11¹⁴ (514 mg, 2.45 mmol) in THF (7 mL) was added at −78 °C to a freshly prepared solution of LDA, prepared from n-BuLi (2.5 M in THF, 1.3 mL, 3.26 mmol) and i-Pr₂NH (0.45 mL, 3.26 mmol) in THF (5 mL) at 0 °C under N₂ atmosphere. The thus produced yellowish anion was stirred at −78 °C for 45 min and then a solution of crude acid chloride, prepared earlier, in THF (10 mL) was added drop wise. The reaction was stirred at the same temperature for another 45 min and then at room temperature for 4 h. 10% HCl (10 mL) was added to quench the reaction, THF was removed in vacuum and the water layer was extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with water (2 × 20 mL), brine (20 mL), dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography to get 13 (480 mg, 68% in three steps) as yellowish oily compound. R_f 0.5 (1:1 ethyl acetate : hexane); ν_max (CHCl₃) cm⁻¹ 1732, 1659, 1608, 1508, 1463, 1211, 1029; ¹H NMR (CDCl₃, 500 MHz): δ 7.71 (s, 1H, C6′-H), 7.01 (d, 1H, J = 8.5 Hz, C6″-H), 6.43 (s, 1H, C3′-H), 6.41 (d, 1H, J = 8.5 Hz, C5″-H), 6.28 (s, 1H, C3″-H), 5.94 (s, 1H, C2-H), 3.82 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.71 (s, 3H, CO₂CH₃), 2.10 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 125 MHz): δ 193.1 (C), 171.1 (C), 163.0 (C), 160.3 (C), 159.6 (C), 157.9 (C), 133.1 (CH), 130.1 (CH), 118.9 (C), 118.0 (C), 115.8 (C), 104.1 (CH), 98.4 (CH), 94.1 (CH), 57.2 (CH₃), 55.5 (CH₃), 55.3 (CH₃), 55.2 (CH₃), 55.2 (CH₃), 52.1 (CH), 15.3 (CH₃); HRMS (EI+) m/z 388.1529 ([M]⁺ C₂₁H₂₄O₇, requires 388.1521).

3,9-Dihydroxy-6H-benzofuro[3,2-c]chromen-6-one (14).^14c

BBr₃ (1 M in CH₂Cl₂, 6.1 mL, 6.1 mmol) was added at 0 °C to a solution of 12 (450 mg, 1.23 mmol) in CH₂Cl₂ (15 mL) under N₂ atmosphere. The mixture was stirred at same temperature for 10 h and quenched by the addition of water (10 mL). CH₂Cl₂ was removed in vacuum and the mixture was heated to reflux for 1 h. The water layer was extracted with ethyl acetate (2 × 40 mL). The organic layer was washed with water (2 × 20 mL), brine (20 mL), dried (Na₂SO₄) and concentrated. The crude product was purified by column chromatography to get compound 14 (270 mg, 82%) as brown solid. R_f 0.3 (1:1 ethyl acetate : hexane); mp >360 °C (lit.

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350–360 °C); ¹H NMR (DMSO-d₆, 500 MHz): δ 7.80 (d, 1H, J = 8.5 Hz, C7-H), 7.66 (d, 1H, J = 8.5 Hz, C1-H), 7.13 (d, 1H, J = 2.0 Hz, C10-H), 6.92 (dd, 1H, J = 8.5, 2.0 Hz, C8-H), 6.89 (dd, 1H, J = 8.5, 2.0 Hz, C2-H), 6.88 (d, 1H, J = 2.0 Hz, C4-H); ¹³C NMR (DMSO-d₆, 125 MHz): δ 161.2 (C), 159.5 (C), 157.6 (C), 157.0 (C), 156.0 (C), 154.7 (C), 122.7 (CH), 120.7 (CH), 114.6 (C), 114.0 (CH), 113.8 (CH ), 104.2 (C), 103.1 (CH), 102.1 (C), 98.7 (CH); HRMS (EI+) m/z 268.0376 ([M]⁺ C₁₅H₈O₅, requires 268.0372).

3,9-Dihydroxy-2-methyl-6H-benzofuro[3,2-c]chromen-6-one (15)

This compound was prepared by the BBr₃ mediated demethylative cyclization of 13 following the identical procedure as described for the transformation of 12 to 14. Yield 83%; R_f 0.4 (1:1 ethyl acetate : hexane); mp >360 °C; ν_max (film) cm⁻¹ 1720, 1631, 1600, 1424, 1237, 1093; ¹H NMR (DMSO-d₆, 500 MHz) δ 7.69 (s, 1H, C1-H), 7.66 (d, 1H, J = 8.0 Hz, C7-H), 7.13 (d, 1H, J = 2.0 Hz, C10-H), 6.92 (dd, 1H, J = 8.0, 2.0 Hz, C8-H), 6.89 (s, 1H, C4-H), 2.21 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz): δ 159.5 (C), 159.4 (C), 157.8 (C), 157.0 (C), 155.9 (C), 152.9 (C), 123.0(C), 122.4 (CH), 120.6 (CH), 114.7 (C), 113.9 (CH), 103.7 (C), 102.1 (CH), 101.9 (C), 98.7 (CH), 15.6 (CH₃); HRMS (EI+) m/z 282.0523 ([M]⁺ C₁₆H₁₀O₅, requires 282.0528).

Methyl 2-(2-methoxyphenyl)acetate (16).²³

To a stirred solution of 2-hydroxyphenylacetic acid (1.04 g, 6.8 mmol) in acetone (30 mL), K₂CO₃ (2.83 g, 20.5 mmol) and MeI (1.06 mL, 17.1 mmol) was added. The mixture was stirred at room temperature for 12 h and then acetone was removed at vacuum. Water (20 mL) was added to the mixture and extracted with ethyl acetate (2 × 30 mL). The organic layer was washed with water (2 × 20 mL), brine (20 mL), dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography to get 16 (1.10 g, 89%) as colorless oil. R_f 0.5 (1:20 ethyl acetate : hexane); ¹H NMR (CDCl₃, 500 MHz) δ 7.24 (d, 1H, J = 8.0 Hz, C6′-H), 7.16 (d, 1H, J = 8.0 Hz, C3′-H), 6.92-6.85 (m, 2H, C4′-H & C5′-H), 3.80 (s, OCH₃), 3.67 (s, 3H, CO₂CH₃), 3.62 (s, 2H, C2-H₂).

Methyl 2,5-dimethoxyphenylacetate (17).²⁴

To a stirred solution of 2,5-dimethoxyphenylacetic acid (17a, 1.01 g, 5.1 mmol) in dry methanol (10 mL), SOCl₂ (0.56 mL, 7.65 mmol) was added at 0 °C. The reaction was stirred at room temperature for 4 h and then methanol was removed at vacuum. Residue was dissolved in ethyl acetate (50 mL) and washed with water (2 × 20 mL), brine (20 mL), dried (Na₂SO₄) and concentrated. Chromatographic purification produced 17 (980 mg, 91%) as colorless oil. R_f 0.4 (1:10 ethyl acetate : hexane); mp 62–64 °C; ¹H NMR (CDCl₃, 500 MHz) δ 6.80-6.74 (m, 3H, C3′-H, C4′-H & C6′-H), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.67 (s, 3H, CO₂CH₃), 3.59 (s, 2H, C2-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 171.7 (C), 153.2 (C), 151.5 (C), 123.8 (C), 116.8 (CH), 112.4 (CH), 111.2 (CH), 55.6 (CH₃), 55.2 (CH₃), 51.4 (CH₃), 35.4 (CH₂); HRMS (EI+) m/z 210.0896 ([M]⁺ C₁₁H₁₄O₄, requires 210.0892).

5-Allyl-2-methoxybenzoic acid (18)

This semi solid compound was prepared from 4-allyl-2-bromo-1-methoxybenzene, following a similar procedure as described for the transformation of 7 to 9. Yield 97%; R_f 0.4 (1:1 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1639, 1498, 1428, 1246, 1178, 1016; ¹H NMR (CDCl₃, 500 MHz): δ 7.92 (d, 1H, J = 2.5 Hz, C6-H), 7.34 (dd, 1H, J = 8.0, 2.5 Hz, C4-H), 6.95 (d, 1H, J = 8.0 Hz, C3-H), 5.98-5.80 (m, 1H, C2′-H), 5.10-4.98 (m, 2H, C3′-H₂), 4.00 (s, 3H, OCH₃), 3.31 (d, 2H, J = 7.0 Hz, C1′-H₂); ¹³C NMR (CDCl₃, 125 MHz): δ 166.4 (C), 156.6 (C), 136.4 (CH), 134.9 (CH), 133.0 (C), 132.6 (CH), 117.0 (CH₂), 115.9 (C), 111.7 (CH), 56.3 (CH₃), 38.5 (CH₂); HRMS (EI+) m/z 192.0784 ([M]⁺ C₁₁H₁₂O₃, requires 192.0786).

4-Allyl-2,5-dimethoxybenzoic acid (20)

This compound was obtained as white solid from 1-allyl-4-bromo-2,5-dimethoxybenzene (See supporting information) following an analogous reaction sequence as described for the transformation of 7 to 9. Yield 94%; R_f 0.3 (1:1 ethyl acetate : hexane); mp 103–105°C; ν_max (film) cm⁻¹ 1682, 1509, 1430, 1273, 1219, 1039; ¹H NMR (CDCl₃, 500 MHz) δ 7.51 (s, 1H, C6-H), 6.81 (s, 1H, C3-H), 5.98-5.82 (m, 1H, C2′-H), 5.10-5.00 (m, 2H, C3′-H₂), 3.97 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.35 (d, 2H, J = 7.0 Hz, C1′-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 165.7 (C), 152.2 (C), 151.7 (C), 136.6 (CH), 135.1 (CH), 116.6 (CH₂), 115.3 (C), 113.5 (C), 113.4 (CH), 57.0 (CH₃), 55.8 (CH₃), 34.3 (CH₂); HRMS (EI+) m/z 222.0899 ([M]⁺ C₁₂H₁₄O₄, requires 222.0892).

Methyl 3-(5-allyl-2-methoxyphenyl)-2-(2,4-dimethoxyphenyl)-3-oxopropanoate (21)

This compound was obtained as yellowish oil from compound 18 and 11 following an identical method as described for the preparation of 12. Yield 84%; R_f 0.4 (1:3 ethyl acetate : hexane); ν_max (CDCl₃) cm⁻¹ 1737, 1679, 1610, 1508, 1463, 1159, 1032; ¹H NMR (CDCl₃, 500 MHz) δ 7.63 (d, 1H, J = 2.5 Hz, C6′-H), 7.24 (dd, 1H, J = 8.5, 2.5 Hz, C4′-H), 7.05 (d, 1H, J = 8.0 Hz, C6″-H), 6.81 (d, 1H, J = 8.5 Hz, C3′-H), 6.42 (dd, 1H, J = 8.0, 2.5 Hz, C5″-H), 6.41 (d, 1H, J = 2.5 Hz, C3″-H), 5.94 (s, 1H, C2-H), 5.94-5.84 (m, 1H, C2‴-H), 5.08-4.95 (m, 2H, C3‴-H₂), 3.77 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 3.72 (s, 3H, CO₂CH₃), 3.30 (d, 2H, J = 6.5 Hz, C1‴-H₂). ¹³C NMR (CDCl₃, 125 MHz) δ 195.0 (C), 170.6 (C), 160.5 (C), 157.9 (C), 157.2 (C), 137.1 (CH), 134.3 (CH), 132.2 (C), 131.2 (CH), 130.4 (CH), 126.4 (C), 116.0 (C), 115.3 (CH₂), 111.7 (CH), 104.2 (CH), 98.5 (CH), 57.4 (CH₃), 55.5 (CH₃), 55.3 (CH₃), 55.2 (CH₃), 52.2 (CH), 39.0 (CH₂); HRMS (EI+) m/z 384.1568 ([M]⁺ C₂₂H₂₄O₆, requires 384.1572).

Methyl 3-(5-allyl-2,4-dimethoxyphenyl)-2-(2-methoxyphenyl)-3-oxopropanoate (22)

This compound was obtained as yellowish oil from compound 16 and 19 following an analogous reaction sequence as described for the preparation of 12. Yield 80%; R_f 0.5 (1:3 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1737, 1658, 1604, 1463, 1272, 1129, 1027; ¹H NMR (CDCl₃, 500 MHz) δ 7.75 (s, 1H, C6′-H), 7.26-7.21 (m, 1H, C6″-H), 7.15-7.11 (m, 1H, C5″-H), 6.95-6.90 (m, 2H, C4″-H & C3″-H), 6.31 (s, 1H, C3′-H), 6.02 (s, 1H, C2-H), 5.98-5.80 (m, 1H, C2‴-H), 5.05-4.90 (m, 2H, C1‴-H₂), 3.81 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.72 (s, 3H, CO₂CH₃), 3.27 (d, 2H, J = 6.5 Hz, C1‴-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 192.7 (C), 170.9 (C), 162.6 (C), 159.9 (C), 156.9 (C), 136.5 (CH), 132.8 (CH), 129.7 (CH), 128.8 (CH), 123.4 (C), 121.2 (C), 120.5 (CH), 118.3 (C), 115.6 (CH2), 110.4 (CH), 94.3 (CH), 57.9 (CH₃), 55.6 (CH₃), 55.3 (CH₃), 52.2 (CH₃), 33.4 (CH₂); HRMS (EI+) m/z 384.1570 ([M]⁺ C₂₂H₂₄O₆, requires 384.1573).

Methyl 3-(5-allyl-2,4-dimethoxyphenyl)-2-(2,5-dimethoxyphenyl)-3-oxopropanoate (23)

This compound was obtained as yellowish oil from compound 17 and 19 following an identical reaction sequence as described for the preparation of 12. Yield 86%; R_f 0.5 (1:3 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1730, 1685, 1630, 1450, 1220, 1070; ¹H NMR (CDCl₃, 500 MHz) δ 7.73 (s, 1H, C6′-H), 6.77 (s, 1H, C6″-H), 6.81-6.69 (m, 2H, C3″-H & C4″-H), 6.31 (s, 1H, C3′-H), 5.97 (s, 1H, C2-H), 5.95-5.85 (m, 1H, C2‴-H), 5.05-4.95 (m, 2H, C3‴-H₂), 3.84 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 3.72 (s, 3H, OCH₃), 3.68 (s, 3H, CO₂CH₃), 3.25 (d, 2H, J = 6.5 Hz, C1‴-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 192.5 (C), 170.6 (C), 162.6 (C), 159.9 (C), 153.3 (C), 151.2 (C), 136.4 (CH), 132.7 (CH), 124.4 (C), 121.2 (C), 118.3 (CH), 116.0 (CH₂), 115.6 (CH), 113.2 (CH), 111.4 (C), 94.4 (CH), 57.9 (CH₃), 56.1 (CH₃), 55.8 (CH₃), 55.5 (CH₃), 55.2 (CH₃), 52.2 (CH), 33.4 (CH₂); HRMS (EI+) m/z 414.1674 ([M]⁺ C₂₃H₂₆O₇, requires 414.1678).

Methyl 3-(4-allyl-2,5-dimethoxyphenyl)-2-(2,4-dimethoxyphenyl)-3-oxopropanoate (24)

This compound was obtained as yellowish oil from compound 20 and 11 following the similar procedure as described for the preparation of 12. Yield 83%; R_f 0.5 (1:3 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1736, 1672, 1611, 1508, 1400, 1210, 1039; ¹H NMR (CDCl₃, 500 MHz) δ 7.37 (s, 1H, C6′-H), 7.02 (d, 1H, J = 9.0 Hz, C6″-H), 6.71 (s, 1H, C3′-H), 6.41 (s, 1H, C3″-H), 6.38 (d, 1H, J = 9.0 Hz, C5″-H), 5.99 (s, 1H, C2-H), 5.96-5.82 (m, 1H, C2‴-H), 5.08-4.98 (m, 2H, C3‴-H₂), 3.71 (s, 3H, OCH₃), 3.69 (s, 3H, OCH₃), 3.67 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃), 3.64 (s, 3H, CO₂CH₃), 3.32 (d, 2H, J = 6.5 Hz, C1‴-H); ¹³C NMR (CDCl₃, 125 MHz) δ 193.3 (C), 170.3 (C), 160.0 (C), 157.5 (C), 153.0 (C), 150.8 (C), 135.6 (CH), 135.3 (CH), 129.7 (CH), 123.7 (C), 115.9 (CH₂), 115.1 (C), 113.3 (C), 111.5 (CH), 103.8 (CH), 98.0 (CH), 56.9 (CH₃), 55.3 (CH₃), 55.1 (CH₃), 54.9 (CH₃), 54.7 (CH₃), 51.6 (CH), 34.0 (CH₂); HRMS (EI+) m/z 414.1677 ([M]⁺ C₂₃H₂₆O₇, requires 414.1679).

2-Allyl-9-hydroxy-6H-benzofuro[3,2-c]chromen-6-one (25)

This compound was prepared by the BBr₃ mediated demethylative cyclization of 21 as described for the synthesis of 14. Yield 72%; R_f 0.3 (1:1 ethyl acetate : hexane); mp 217–219 °C; ν_max (film) cm⁻¹ 1707, 1625, 1508, 1250, 1076; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.29 (brs, 1H, OH), 7.65 (d, 1H, J = 8.0 Hz, C7-H), 7.61 (s, 1H, C1-H), 7.39-7.34 (m, 2H, C4-H & C3-H), 7.11 (d, 1H, J = 2.0 Hz, C10-H), 6.92 (dd, 1H, J = 8.0, 2.0 Hz, C8-H), 6.00-5.92 (m, 1H, C2′-H), 5.18-5.05 (m, 2H, C3′-H₂), 3.41 (d, 2H, J = 7.0 Hz, C1′-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 158.5 (C), 157.9 (C), 157.7 (C), 156.6 (C), 151.3 (C), 137.2 (CH), 137.1 (C), 132.3 (CH), 121.4 (CH), 120.5 (CH), 117.3 (CH), 117.0 (CH₂), 114.7 (C), 114.6 (C), 112.1 (CH), 105.5 (C), 98.9 (CH), 26.1 (CH₂); HRMS (EI+) m/z 292.0733 ([M]⁺ C₁₈H₁₂O₄, requires 292.0736).

2-Allyl-3-hydroxy-6H-benzofuro[3,2-c]chromen-6-one (26)

The BBr₃ mediated demethylative cyclization of 22 to synthesize this compound was similar to that described for the synthesis of 14. Yield 75%; R_f 0.3 (1:1 ethyl acetate : hexane); mp 262–264 °C; ν_max (film) cm⁻¹ 1697, 1625, 1597, 1419, 1257, 1141, 1012; ¹H NMR (Acetone-d₆, 500 MHz) δ 8.01-7.96 (m, 1H, C7-H), 7.75 (s, 1H, C1-H), 7.74-7.72 (m, 1H, C10-H), 7.50-7.46 (m, 2H, C8-H & C9-H), 7.00 (s, 1H, C4-H), 6.12-6.06 (m, 1H, C2′-H), 5.25-5.10 (m, 2H, C3′-H₂), 3.52 (d, 2H, J = 7.0 Hz, C1′-H₂); ¹³C NMR (Acetone-d₆, 125 MHz) δ 160.9 (C), 159.3 (C), 157.5 (C), 155.1 (C), 154.3 (C), 136.2 (CH), 125.1 (CH), 124.0 (C), 123.8 (C), 123.0 (C), 122.4 (CH), 120.7 (C), 115.8 (CH), 111.7 (CH₂), 104.5 (C), 102.8 (CH), 33.1 (CH₂); HRMS (EI+) m/z 292.0733 ([M]⁺ C₁₈H₁₂O₄, requires 292.0735).

2-allyl-3,8-dihydroxy-6H-benzofuro[3,2-c]chromen-6-one (27)

This white solid compound was prepared by the BBr₃ mediated demethylative cyclization of 23 following a similar protocol for the synthesis of 14. Yield 71%; R_f 0.3 (1:4:12 methanol:ethyl acetate : hexane); mp 322–324 °C; ν_max (film) cm⁻¹ 1724, 1636, 1599, 1420, 1272, 1163, 1009; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.74 (brs, 1H, OH), 9.60 (brs, 1H, OH), 7.49 (d, 1H, J = 9.0 Hz, C10-H), 7.46 (s, 1H, C1-H), 7.19 (d, 1H, J = 3.0 Hz, C7-H), 6.83 (dd, 1H, J = 9.0, 3.0 Hz, C9-H), 6.81 (s, 1H, C4-H), 6.05-5.95 (m, 1H, C2′-H), 5.20-5.05 (m, 2H, C3′-H₂), 3.30 (d, 2H, J = 6.5 Hz, C1′-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 160.8 (C), 159.3 (C), 157.5 (C), 155.1 (C), 153.4 (C), 148.4 (C), 136.1 (CH), 125.0 (C), 124.0 (CH), 121.6 (CH), 116.5 (CH₂), 114.1 (C), 112.2 (C), 105.2 (CH), 103.6 (C), 102.3 (CH), 101.7 (CH), 33.0 (CH₂); HRMS (EI+) m/z 308.0691 ([M]⁺ C₁₈H₁₂O₅, requires 308.0684).

3-Allyl-2,9-dihydroxy-6H-benzofuro[3,2-c]chromen-6-one (28)

This white solid compound was prepared by the BBr₃ mediated demethylative cyclization of 24, similar to that described for the preparation of 14. Yield 74%; R_f 0.3 (1:4:12 methanol : ethyl acetate : hexane); mp 285–290 °C; ν_max (film) cm⁻¹ 1686, 1555, 1415, 1245, 1101, 1025; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.06 (brs, 1H, OH), 7.66 (d, 1H, J = 8.5 Hz, C7-H), 7.19 (s, 1H, C1-H), 7.15 (s, 1H, C10-H), 7.14 (s, 1H, C4-H), 6.92 (d, 1H, J = 8.5 Hz, C8-H), 6.05-5.85 (m, 1H, C2′-H), 5.20-5.00 (m, 2H, C3′-H₂), 3.33 (d, 2H, J = 6.0 Hz, C1′-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 158.3 (C), 157.5 (C), 157.4 (C), 156.2 (C), 152.0 (C), 146.1 (C), 135.8 (CH), 132.0 (C), 120.9 (CH), 117.7 (CH), 116.5 (CH₂), 114.6 (C), 114.2 (CH), 110.4 (C), 104.7 (C), 104.4 (CH), 98.6 (CH), 33.9 (CH₂); HRMS (EI+) m/z 308.0681 ([M]⁺ C₁₈H₁₂O₅, requires 308.0685).

9-Hydroxy-2-(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c]chromen-6-one (29)

Grubb’s second-generation catalyst (3 mg, 0.0035 mmol) was added to a degassed solution of 25 (20 mg, 0.07 mmol) in CH₂Cl₂ (10 mL) and 2-methyl-2-butene (2 mL). The solution was heated in a sealed tube at 100 °C for 15 min by a microwave reactor (Biotage Initiator 2.0, standard configuration, temperature control, external IR temperature sensor, fixed hold time). CH₂Cl₂ was removed in vacuum, and the mixture was purified by reverse phase semipreparative HPLC (symmetry C₁₈, 7 μm, solvent gradient 9:1 CH₃CN:H₂O) to get compound 29 (17 mg, 77%) as a white solid. R_f 0.4 (1:1 ethyl acetate : hexane); mp 110–112 °C; ν_max (film) cm⁻¹ 1735, 1629, 1508, 1443, 1375, 1093; ¹H NMR (DMSO-d₆, 500 MHz) δ 7.91 (d, 1H, J = 8.0 Hz, C7-H), 7.73 (s, 1H, C1-H), 7.40-7.32 (m, 2H, C4-H & C3-H), 7.14 (s, 1H, C10-H), 6.97 (d, 1H, J = 8.0 Hz, C8-H), 5.35 (t, 1H, J = 6.0 Hz, C2′-H), 3.44 (d, 2H, J = 6.0 Hz, C1′-H₂), 1.77 (s, 3H, CH₃), 1.75 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.7 (C), 158.8 (C), 156.9 (C), 156.3 (C), 151.8 (C), 138.8 (C), 133.9 (C), 131.9 (CH), 129.6 (CH), 122.4 (CH), 122.2 (CH), 120.6 (C), 117.5 (CH), 114.4 (C), 112.8 (CH), 106.1 (C), 99.2 (CH), 33.9 (CH₂), 26.0 (CH₃), 18.1 (CH₃); HRMS (EI+) m/z 320.1053 ([M]⁺ C₂₀H₁₆O₄, requires 320.1049).

3-hydroxy-2-(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c]chromen-6-one (30)

This white solid compound was prepared by the Grubb’s metathesis reaction of 26, following the same procedure as described for the synthesis of 29. Yield 72%; R_f 0.4 (1:1 ethyl acetate : hexane); mp 238–240 °C; ν_max (film) cm⁻¹ 1719, 1635, 1418, 1260, 1141, 1007; ¹H NMR (DMSO-d₆, 500 MHz) δ 7.92 (d, 1H, J = 8.0 Hz, C7-H), 7.85 (d, 1H, J = 8.0 Hz, C10-H), 7.69 (s, 1H, C1-H), 7.52-7.44 (m, 2H, C8-H & C9-H), 6.95 (s, 1H, C4-H), 5.37 (t, 1H, J = 6.0 Hz, C2′-H), 3.33 (d, 2H, J = 6.0 Hz, C1′-H₂), 1.75 (s, 3H, CH₃), 1.71 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ 160.7 (C), 159.8 (C), 157.6 (C), 154.5 (C), 153.6 (C), 132.6 (C), 126.7 (C), 126.1 (CH), 125.2 (CH), 123.2 (C), 121.6 (CH), 121.5 (CH), 120.3 (CH), 112.0 (CH), 103.4 (C), 102.5 (CH), 101.6 (C), 27.5 (CH₂), 25.6 (CH₃), 17.7 (CH₃); HRMS (EI+) m/z 320.1051 ([M]⁺ C₂₀H₁₆O₄, requires 320.1048).

3,8-Dihydroxy-2-(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c]chromen-6-one (31)

The Grubb’s metathesis reaction for the synthesis of this compound from 27 is similar to that described for 29. Yield 79%; R_f 0.5 (1:4:12 methanol:ethyl acetate : hexane); mp >360 °C, chars at 240 °C; ν_max (film) cm⁻¹ 1718, 1630, 1426, 1268, 1163, 1005; ¹H NMR (DMSO-d₆, 500 MHz) δ 9.72 (brs, 1H, OH), 7.63 (s, 1H, C1-H), 7.61 (d, 1H, J = 9.0 Hz, C10-H), 7.24 (d, 1H, J = 2.5 Hz, C7-H), 6.92 (s, 1H, C4-H), 6.88 (dd, 1H, J = 9.0, 2.5 Hz, C9-H), 5.34 (t, 1H, J = 8.0 Hz, C2′-H), 3.31 (d, 2H, J = 8.0 Hz, C1′-H₂), 1.73 (s, 3H, CH₃), 1.70 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ 161.1 (C), 159.8 (C), 157.8 (C), 155.3 (C), 153.5 (C), 148.5 (C), 132.7 (C), 126.7 (C), 124.2 (CH), 121.7 (CH), 121.3 (CH), 114.2 (C), 112.4 (CH), 105.2 (CH), 103.6 (C), 102.5 (C), 101.7 (CH), 27.6 (CH₂), 25.7 (CH₃), 17.7 (CH₃); HRMS (EI+) m/z 336.0999 ([M]⁺ C₂₀H₁₆O₅, requires 336.0997).

2,9-Dihydroxy-3-(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c]chromen-6-one (32)

This white solid compound was produced by the Grubb’s metathesis reaction of 28, following the same procedure as described for the synthesis of 29. Yield 82%; R_f 0.5 (1:4:12 methanol : ethyl acetate : hexane); mp > 360 °C, chars at 230 °C; ν_max (film) cm⁻¹ 1685, 1629, 1560, 1438, 1383, 1251, 1097; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.20 (brs, 1H, OH), 7.72 (d, 1H, J = 8.0 Hz, C7-H), 7.25 (s, 1H, C1-H), 7.24 (s, 1H, C10-H), 7.21 (s, 1H, C4-H), 6.97 (d, 1H, J = 8.0 Hz, C8-H), 5.32 (t, 1H, J = 7.5 Hz, C2′-H), 3.33 (d, 2H, J = 7.5 Hz, C1′-H₂), 1.72 (s, 3H, CH₃), 1.70 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.0 (C), 158.1 (C), 157.9 (C), 156.7 (C), 152.6 (C), 146.7 (C), 134.1 (C), 133.4 (C), 121.7 (CH), 121.4 (CH), 117.8 (CH), 115.0 (C), 114.7 (CH), 110.6 (C), 105.0 (C), 104.8 (CH), 99.1 (CH), 28.8 (CH₂), 25.9 (CH₃), 19.5 (CH₃); HRMS (EI+) m/z 336.0995 ([M]⁺ C₂₀H₁₆O₅, requires 336.0998).

Methyl 2-(2,4,5-trimethoxyphenyl)acetate (33).^15,24

A mixture of 2,4,5-trimethoxybenzaldehyde (4 g, 20.4 mmol), hippuric acid (4.38 g, 24.5 mmol), powdered fused sodium acetate (1.76 g, 21.42 mmol), and acetic anhydride (6.16 mL, 65.28 mmol) was heated on an oil bath at 120 °C. The mixture turned almost solid and then gradually liquefied (15–20 min). The temperature was decreased to 100 °C and allowed to stir for 3 h. The reaction was cool to room temperature; ethanol (50 mL) was added and allowed to stand overnight. The product was filtered by suction while washing with ice-cold alcohol (2 × 100 mL), hot water (2 × 100 mL) and dried to get a yellowish powder. 10% sodium hydroxide (20 mL) was added to this and refluxed for 10 h. After cooling, 40% NaOH (10 mL) was added and then 30% hydrogen peroxide (10 mL) diluted with water (20 mL) was added cautiously maintaining the temperature of the solution below 15 °C using an ice-salt bath. The solution was allowed to stand at room temperature for overnight and then acidified by the addition of conc. HCl. The acidic solution thus formed was extracted with benzene (2 × 100 mL), washed with water (2 × 40 mL), brine (40 mL) and dried (Na₂SO₄). Removal of the benzene produced brown oily compound which was dissolved in dry methanol (20 mL). Conc. H₂SO₄ (5 mL) was added and the mixture was refluxed for 4 h. Methanol was distilled off and the remaining solution was extracted with ethyl acetate (2 × 40 mL). The organic layer was washed with water (2 × 20 mL), brine (20 mL) and dried (Na₂SO₄). Concentration and silica gel column chromatographic purification produced 33 (3.2 g, 65%) as white solid. R_f 0.5 (1:3 ethyl acetate :hexane); mp 45–47 °C (lit.²⁴ 44–46 °C); ¹H NMR (CDCl₃, 500 MHz) δ 6.72 (s, 1H, C6′-H), 6.51 (s, 1H, C3′-H), 3.86 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.67 (s, 3H, CO₂CH₃), 3.55 (s, 2H, C2-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 172.7 (C), 151.9 (C), 149.1 (C), 143.1 (C), 114.9 (CH), 114.4 (C), 97.9 (CH), 56.8 (CH₃), 56.6 (CH₃), 56.3 (CH₃), 52.1 (CH₃), 35.2 (CH₂); HRMS (EI+) m/z 240.0993 ([M]⁺ C₁₂H₁₆O5, requires 240.0998).

Methyl 2,3,4-trimethoxyphenylacetate (34).²⁵

This compound was obtained as colorless oil from 2,3,4-trimethoxybenzaldehyde (34a) using an identical procedure as followed for the preparation of compound 33. Yield 58%. R_f 0.5 (1:3 ethyl acetate : hexane); ¹H NMR (CDCl₃, 500 MHz) δ 6.86 (d, 1H, J = 8.5 Hz, C6′-H), 6.60 (d, 1H, J = 8.5 Hz, C5′-H), 3.86 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 3.68 (s, 3H, CO₂CH₃), 3.56 (s, 2H, C2-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 171.8 (C), 152.8 (C), 151.5 (C), 141.7 (C), 124.4 (CH), 120.2 (C), 106.7 (CH), 60.1 (CH₃), 60.0 (CH₃), 55.3 (CH₃), 51.2 (CH₃), 34.7 (CH₂); HRMS (EI+) m/z 240.0995 ([M]⁺ C₁₂H₁₆O5, requires 240.0998).

5-Allyl-2,3,4-trimethoxybenzoic acid (35)

This white solid compound was obtained from 1-Allyl-5-bromo-2,3,4-trimethoxybenzene (See supporting information) following the similar procedure as described for the transformation of 7 to 9. Yield 93%; R_f 0.3 (1:1 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1660, 1518, 1222, 1013; ¹H NMR (CDCl₃, 500 MHz) δ 7.56 (s, 1H, C6-H), 5.96-5.70 (m, 1H, C2′-H), 5.05-4.82 (m, 2H, C3′-H₂), 3.98 (s, 3H, O CH₃), 3.86 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.26 (d, 2H, J = 6.0 Hz, C1′-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 167.0 (C), 156.5 (C), 152.7 (C), 145.7 (C), 136.2 (CH), 130.0 (C), 127.7 (CH), 117.2 (C), 116.2 (CH₂), 62.3 (CH₃), 60.9 (CH₃), 60.8 (CH₃), 33.9 (CH₂); HRMS (EI+) m/z 252.0994 ([M]⁺ C₁₃H₁₆O₅, requires 252.0998).

Methyl 3-(5-allyl-2,4-dimethoxyphenyl)-3-oxo-2-(2,4,5-trimethoxyphenyl)propanoate (36)

This compound was obtained as yellowish oil from compound 33 and 19 following the similar procedure as described for the preparation of 12. Yield 81%. R_f 0.4 (1:1 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1732, 1651, 1519, 1211, 1028; ¹H NMR (CDCl₃, 500 MHz) δ 7.70 (s, 1H, C6′-H), 6.70 (s, 1H, C6″-H), 6.50 (s, 1H, C3′-H), 6.31 (s, 1H, C3″-H), 5.96 (s, 1H, C2-H), 5.95-5.84 (m, 1H, C2‴-H), 5.02-4.94 (m, 2H, C3‴-H₂), 3.85 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.72 (s, 3H, CO₂CH₃), 3.25 (d, 2H, J = 5.5 Hz, C1‴-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 192.8 (C), 170.5 (C), 162.1 (C), 159.4 (C), 151.1 (C), 148.8 (C), 142.4 (C), 136.1 (CH), 132.2 (CH), 120.7 (C), 118.0 (C), 115.1 (CH₂), 114.0 (C), 113.5 (CH), 96.9 (CH), 94.0 (CH), 56.7 (CH₃), 56.1 (CH₃), 55.9 (CH₃), 55.6 (CH₃), 55.1 (CH₃), 54.9 (CH₃), 51.7 (CH), 32.9 (CH₂); HRMS (EI+) m/z 444.1785 ([M]⁺ C₂₄H₂₈O₈, requires 444.1784).

Methyl 3-(5-allyl-2,3,4-trimethoxyphenyl)-2-(2,4-dimethoxyphenyl)-3-oxopropanoate (37)

This compound was obtained as yellowish oil from compound 35 and 11 following the identical procedure as described for the preparation of 12. Yield 76%; R_f 0.4 (1:3 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1720, 1640, 1510, 1210, 1020; ¹H NMR (CDCl₃, 500 MHz) δ 7.32 (s, 1H, C6′-H), 7.10 (d, 1H, J = 8.5 Hz, C6″-H), 6.43 (d, 1H, J = 8.5 Hz, C5″-H), 6.42 (s, 1H, C3″-H), 5.93 (s, 1H, C2-H), 5.92-5.82 (m, 1H, C2‴-H), 5.05-4.95 (m, 2H, C3‴-H₂), 3.81 (s, 6H, OCH₃), 3.76 (s, 6H, OCH₃), 3.75 (s, 3H, OCH₃), 3.72 (s, 3H CO₂CH₃), 3.29 (d, 2H, J = 6.5 Hz, C1‴-H₂); ¹³C NMR (CDCl₃, 125 MHz) δ 194.6 (C), 170.5 (C), 160.6 (C), 157.9 (C), 156.1 (C), 152.9 (C), 146.0 (C), 136.7 (CH), 130.5 (CH), 129.0 (C), 126.7 (C), 126.2 (CH), 116.1 (CH₂), 115.3 (C), 104.4 (CH), 98.8 (CH), 61.5 (CH₃), 61.0 (CH₃), 60.8 (CH₃), 56.5 (CH₃), 55.7 (CH₃), 55.5 (CH₃), 52.5 (CH), 34.1 (CH₂); HRMS (EI+) m/z 444.1780 ([M]⁺ C₂₄H₂₈O₈, requires 444.1784).

Methyl 3-(5-allyl-2,4-dimethoxyphenyl)-3-oxo-2-(2,3,4-trimethoxyphenyl)propanoate (38)

This yellowish oily compound was obtained from 34 and 19, using an identical reaction sequence as described for the synthesis of 12. Yield 83%; R_f 0.4 (1:1 ethyl acetate : hexane); ν_max (film) cm⁻¹ 1738, 1651, 1604, 1468, 1270, 1096; ¹H NMR (CDCl₃, 500 MHz) δ 7.73 (s, 1H, C6′-H), 6.85 (d, 1H, J = 8.5 Hz, C6″-H), 6.60 (d, 1H, J = 8.5 Hz, C5″-H), 6.33 (s, 1H, C3′-H), 5.92 (s, 1H, C2-H), 5.92-5.85 (m, 1H, C2‴-H), 5.05-4.95 (m, 2H, C3‴-H₂), 3.85 (s, 3H, OCH₃), 3.83 (s, 6H, OCH₃), 3.81 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.71 (s, 3H, CO₂CH₃), 3.26 (d, 2H, J = 6.0 Hz, C1‴-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 192.1 (C), 170.3 (C), 162.1 (C), 159.3 (C), 152.7 (C), 151.2 (C), 141.4 (C), 135.9 (CH), 132.0 (CH), 123.5 (C), 120.6 (CH), 120.4 (C), 117.7 (C), 115.0 (CH₂), 106.5 (CH), 94.0 (CH), 60.3 (CH₃), 60.0 (CH₃), 57.3 (CH₃), 55.2 (CH₃), 55.0 (CH₃), 54.8 (CH₃), 51.5 (CH), 32.8 (CH₂); HRMS (EI+) m/z 444.1780 ([M]⁺ C₂₄H₂₈O₈, requires 444.1784).

2-Allyl-3,8,9-trihydroxy-6H-benzofuro[3,2-c]chromen-6-one (39)

This brown solid compound was prepared by the BBr₃ mediated demethylative cyclization of 36, similar to that described for the preparation of 14. Yield 75%. R_f 0.3 (1:3:9 methanol : ethyl acetate : hexane); mp 340–342 °C; ν_max (film) cm⁻¹1693, 1631, 1501, 1414, 1255, 1150, 1010; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.64 (brs, 1H, OH), 9.46 (brs, 1H, OH), 9.38 (brs, 1H, OH), 7.49 (s, 1H, C1-H), 7.21 (s, 1H, C7-H), 7.14 (s, 1H, C10-H), 6.84 (s, 1H, C4-H), 6.02-5.91 (m, 1H, C2′-H), 5.14-5.04 (m, 2H, C3′-H₂), 3.32 (d, 2H, J = 6.5 Hz, C1′-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.0 (C), 158.5 (C), 157.8 (C), 152.7 (C), 148.8 (C), 145.6 (C), 144.4 (C), 136.2 (CH₂), 124.9 (C), 121.2 (C), 116.4 (CH), 114.2 (C), 104.9 (CH), 104.1 (C), 102.4 (CH), 102.2 (C), 99.0 (CH), 33.1 (CH₂); HRMS (EI+) m/z 324.0636 ([M]⁺ C₁₈H₁₂O₆, requires 324.0634).

2-Allyl-3,4,9-trihydroxy-6H-benzofuro[3,2-c]chromen-6-one (40)

This white solid compound was prepared by the BBr₃ mediated demethylative cyclization of 37, following the same procedure as described for the transformation of 12 to 14. Yield 81%; R_f 0.3 (1:3:9 methanol : ethyl acetate : hexane); mp 258–260 °C; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.03 (brs, 1H, OH), 9.99 (brs, 1H, OH), 9.69 (brs, 1H, OH), 7.68 (d, 1H, J = 8.5 Hz, C7-H), 7.18 (s, 1H, C1-H), 7.15 (s, 1H, C10-H), 6.93 (d, 1H, J = 8.5 Hz, C8-H), 6.10-5.90 (m, 1H, C2′-H), 5.18-5.00 (m, 2H, C3′-H₂), 3.39 (d, 1H, J = 6.5 Hz, C1′-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.8 (C), 157.6 (C), 157.0 (C), 156.1 (C), 147.8 (C), 141.9 (C), 136.4 (CH), 132.4 (C), 124.9 (C), 120.7 (CH), 116.3 (CH₂), 114.8 (C), 114.0 (CH), 111.5 (CH), 104.1 (C), 102.0 (C), 98.7 (CH), 33.6 (CH₂); HRMS (EI+) m/z 324.0636 ([M]⁺ C₁₈H₁₂O₆, requires 324.0634).

2-Allyl-3,9,10-trihydroxy-6H-benzofuro[3,2-c]chromen-6-one (41)

This white solid compound is the result of BBr₃ mediated demethylative cyclization of 38, as described for the synthesis of 14. Yield 82%; R_f 0.3 (1:3:9 methanol : ethyl acetate : hexane); mp >360 °C, chars at 232 °C; ν_max (film) cm⁻¹ 1724, 1629, 1528, 1395, 1137, 1079; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.77 (brs, 1H, OH), 9.60 (brs, 1H, OH), 9.59 (brs, 1H, OH), 7.69 (s, 1H, C1-H), 7.16 (d, 1H, J = 8.5 Hz, C7-H), 6.95 (d, 1H, J = 8.5 Hz, C8-H), 6.94 (s, 1H, C4-H), 6.10-5.98 (m, 1H, C2′-H), 5.20-5.08 (m, 2H, C3′-H₂), 3.41 (d, 2H, J = 7.0 Hz, C1′-H₂); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.6 (C), 158.8 (C), 157.9 (C), 153.0 (C), 144.9 (C), 144.8 (C), 144.7 (C), 136.2 (CH), 131.2 (C), 125.1 (C), 121.5 (CH), 116.6 (CH₂), 116.3 (C), 114.4 (CH), 109.8 (CH), 104.0 (C), 102.4 (CH), 33.1 (CH₂); HRMS (EI+) m/z 324.0633 ([M]⁺ C₁₈H₁₂O₆, requires 324.0633).

2-Allyl-6-oxo-6H-benzofuro[3,2-c]chromene-3,8,9-triyl triacetate (42)

To a stirred solution of 39 (50 mg, 0.15 mmol) in CH₂Cl₂ (10 mL), triethyl amine (0.07 mL, 0.52 mmol), acetic anhydride (0.05 mL, 0.49 mmol), and DMAP (2 mg) was added. The reaction mixture was allowed to stir at room temperature for 6 h. After completion, as confirmed by TLC, water (10 mL) was added. The mixture was extracted with chloroform (40 mL) and the organic layer was washed with water (2 × 15 mL), brine (15 mL), dried (Na₂SO₄), and concentrated. Chromatographic purification produced 42 (65 mg, 94%) as white solid. R_f 0.2 (1:3 ethyl acetate : hexane); mp 278–280 °C; ¹H NMR (CDCl₃, 500 MHz) δ 7.89 (s, 1H, C1-H), 7.84 (s, 1H, C7-H), 7.55 (s, 1H, C10-H), 7.24 (s, 1H, C4-H), 6.00-5.85 (m, 1H, C2′-H), 5.25-5.05 (m, 2H, C3′-H₂), 3.40 (d, 2H, J = 6.5 Hz, C1′-H₂), 2.35 (s, 3H, OAc), 2.34 (s, 6H, OAc); ¹³C NMR (CDCl₃, 125 MHz) δ 168.8 (C), 168.6 (C), 168.4 (C), 161.0 (C), 157.6 (C), 152.9 (C), 152.6 (C), 152.0 (C), 141.5 (C), 140.5 (C), 134.9 (C), 130.1 (C), 123.1 (CH), 121.6 (CH₂), 117.7 (C), 115.9 (CH), 112.2 (CH), 110.5 (C), 107.7 (CH), 105.5 (C), 34.4 (CH₂), 21.1 (CH₃), 20.9 (CH₃), 20.7 (CH₃); HRMS (EI+) m/z 450.0952 ([M]⁺ C₂₄H₁₈O₉, requires 450.0951).

2-Allyl-6-oxo-6H-benzofuro[3,2-c]chromene-3,4,9-triyl triacetate (43)

This compound was obtained as white solid by the acetylation of 40 using an analogous procedure used during the synthesis of 42. Yield 90%. R_f 0.6 (1:1 ethyl acetate : hexane); mp 232–234 °C; ν_max (film) cm⁻¹ 1761, 1754, 1750, 1715, 1640, 1500, 1285; ¹H NMR (CDCl₃, 500 MHz) δ 8.08 (d, 1H, J = 8.0 Hz, C7-H), 7.76 (s, 1H, C1-H), 7.47 (d, 1H, J = 1.5 Hz, C10-H), 7.20 (dd, 1H, J = 8.0, 1.5 Hz, C8-H), 5.98-5.85 (m, 1H, C2′-H), 5.25-5.10 (m, 2H, C3′-H₂), 3.41 (d, 2H, J = 6.5 Hz, C1′-H₂), 2.42 (s, 3H, OAc), 2.35 (s, 6H, OAc); ¹³C NMR (CDCl₃, 125 MHz) δ 169.4 (C), 167.6 (C), 167.3 (C), 159.9 (C), 156.6 (C), 155.5 (C), 149.8 (C), 145.2 (C), 144.4 (C), 134.5 (CH), 131.6 (C), 130.8 (C), 122.1 (CH), 121.0 (C), 119.7 (CH), 119.2 (CH), 118.0 (CH₂), 111.3 (C), 106.2 (CH), 105.5 (C), 34.5 (CH₂), 21.3 (CH₃), 20.5 (CH₃), 20.5 (CH₃); HRMS (EI+) m/z 450.0952 ([M]⁺ C₂₄H₁₈O₉, requires 450.0951).

2-(3-Methylbut-2-en-1-yl)-6-oxo-6H-benzofuro[3,2-c]chromene-3,8,9-triyl triacetate (44)

Grubb’s second-generation catalyst (4 mg, 0.004 mmol) was added to a degassed solution of 42 (30 mg, 0.07 mmol) in CH₂Cl₂ (10 mL) and 2-methyl-2-butene (3 mL). The mixture was allowed to stir at room temperature for 12 h. Once the reaction is complete, as checked by TLC, it was concentrated and purified by silica gel column chromatography to afford 44 (27 mg, 85%) as white solid. R_f 0.4 (1:3 ethyl acetate : hexane); mp 261–263 °C; ¹H NMR (CDCl₃, 500 MHz) δ 7.90 (s, 1H, C1-H), 7.82 (s, 1H, C7-H), 7.57 (s, 1H, C10-H), 7.22 (s, 1H, C4-H), 5.26 (t, 1H, J = 7.5 Hz, C2′-H), 3.33 (d, 2H, J = 7.5 Hz, C1′-H₂), 2.35 (s, 3H, OAc), 2.33 (s, 6H, OAc), 1.79 (s, 3H, CH₃), 1.73 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 168.8 (C), 168.6 (C), 168.4 (C), 161.1 (C), 157.8 (C), 152.7 (C), 152.6 (C), 152.1 (C), 141.5 (C), 140.5 (C), 134.9 (C), 131.7 (C), 122.7 (CH), 121.7 (C), 120.6 (CH), 115.9 (CH), 112.1 (CH), 110.5 (C), 107.7 (CH), 105.4 (C), 28.6 (CH₂), 26.0 (CH₃), 21.1 (CH₃), 20.9 (CH₃), 20.7 (CH₃), 18.2 (CH₃); HRMS (EI+) m/z 478.1262 ([M]⁺ C₂₆H₂₂O₉, requires 478.1264).

2-(3-Methylbut-2-en-1-yl)-6-oxo-6H-benzofuro[3,2-c]chromene-3,4,9-triyl triacetate (45)

This white solid powder like compound was prepared by a 2^nd generation Grubb’s catalyzed olefin metathesis reaction of 43 following an identical procedure as described for the synthesis of 44. Yield 84%. R_f 0.3 (1:3 ethyl acetate : hexane); mp 212–214 °C; ν_max (film) cm⁻¹ 1765, 1760, 1755, 1710, 1629, 1490, 1280; ¹H NMR (CDCl₃, 500 MHz) δ 8.06 (d, 1H, J = 8.5 Hz, C7-H), 7.71 (s, 1H, C1-H), 7.47 (d, 1H, J = 1.5 Hz, C10-H), 7.18 (dd, 1H, J = 8.5, 1.5 Hz, C8-H), 5.25 (t, 1H, J = 7.0 Hz, C2′-H), 3.33 (d, 2H, J = 7.0 Hz, C1′-H₂), 2.41 (s, 3H, OAc), 2.34 (s, 3H, OAc), 2.35 (s, 3H, OAc), 1.79 (s, 3H, CH₃), 1.71 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 169.4 (C), 167.8 (C), 167.4 (C), 160.1 (C), 156.7 (C), 155.5 (C), 149.8 (C), 145.0 (C), 144.4 (C), 135.1 (C), 132.4 (C), 131.5 (C), 122.1 (CH), 121.2 (C), 120.2 (CH), 119.6 (CH), 118.9 (CH), 111.3 (C), 106.2 (CH), 105.5 (C), 28.7 (CH₂), 25.9 (CH₃), 21.3 (CH₃), 20.5 (CH₃), 20.5 (CH₃), 18.1 (CH₃); HRMS (EI+) m/z 478.1262 ([M]⁺ C₂₆H₂₂O₉, requires 478.1264).

3,4,9-Trihydroxy-2-(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c]chromen-6-one (46)

This colorless solid compound was synthesized in three steps from 40, without purification of the intermediates, following an identical procedure as described for the synthesis of 2. Yield 60% in three steps. R_f 0.5 (1:3:9 methanol : ethyl acetate : hexane); mp 250–252 °C; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.03 (brs, 1H, OH), 9.95 (brs, 1H, OH), 9.66 (brs, 1H, OH), 7.70 (d, 1H, J = 9.0 Hz, C7-H), 7.19 (s, 1H, C1-H), 7.18 (s, 1H, C10-H), 6.94 (d, 1H, J = 9.0 Hz, C8-H), 5.34 (t, 1H, J = 6.0 Hz, C2′-H), 3.33 (d, 2H, J = 6.0 Hz, C1′-H₂), 1.73 (s, 3H, CH₃), 1.70 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ 159.8 (C), 157.6 (C), 157.0 (C), 156.0 (C), 147.8 (C), 141.7 (C), 132.4 (C), 132.3 (C), 126.3 (C), 122.0 (C), 120.6 (CH), 114.7 (C), 113.9 (CH), 110.9 (CH), 104.0 (C), 101.9 (C), 98.7 (CH), 27.9 (CH₂), 25.6 (CH₃), 17.8 (CH₃); HRMS (EI+) m/z 352.0947 ([M]⁺ C₂₀H₁₆O₆, requires 352.0946).

3,9,10-Trihydroxy-2-(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c]chromen-6-one (47)

This white solid compound was prepared in three consecutive steps, starting from 41 as described for the synthesis of 2. Yield 64% in three steps. R_f 0.5 (1:3:9 methanol : ethyl acetate : hexane); mp > 360 °C, chars at 215 °C; ν_max (film) cm⁻¹ 1718, 1629, 1508, 1383, 1264; ¹H NMR (Acetone-d₆, 500 MHz) δ 7.71 (s, 1H, C1-H), 7.28 (d, 1H, J = 7.5 Hz, C7-H), 7.03 (d, 1H, J = 7.5 Hz, C8-H), 6.96 (s, 1H, C4-H), 5.44 (t, 1H, J = 6.5 Hz, C2′-H), 3.44 (d, 2H, J = 6.5 Hz, C1′-H₂), 1.78 (s, 3H, CH₃), 1.77 (s, 3H, CH₃); ¹³C NMR (Acetone -d₆, 125 MHz) δ 161.1 (C), 159.7 (C), 158.8 (C), 154.4 (C), 145.6 (C), 145.4 (C), 133.7 (C), 132.1 (C), 127.6 (C), 122.8 (CH), 122.3 (CH), 118.4 (C), 115.0 (CH), 111.5 (CH), 105.8 (C), 103.9 (C), 103.5 (CH), 28.6 (CH₂), 26.0 (CH₃), 18.0 (CH₃); HRMS (EI+) m/z 352.0946 ([M]⁺ C₂₀H₁₆O₆, requires 352.0947).