Total Synthesis of Psoralidin, an Anticancer Natural Product

Abstract

A base catalyzed condensation of phenyl acetate with acid chloride followed by intramolecular cyclization and microwave assisted cross metathesis reaction lead to the first total synthesis of psoralidin, a natural product with a broad range of biological activities, in a highly convergent and regio-selective manner.

Introduction

Psoralidin (1) was first isolated from seeds of Psoralea corylifolia Linn in 1948 by Chakravarti et al.¹ and later its structure was corrected by Khastgir et al. in 1961.² It was found to be an active ingredient of many Indian and Chinese traditional herbal medicines.³ Psoralidin (1) exhibits a variety of biological activities like antioxidant,⁴ antibacterial,⁵ antidepressant activities,^6,7 and it shows inhibitory activities against protein tyrosine phosphatase 1B, which plays a major role in the negative regulation of insulin signaling.⁸ In addition, psoralidin inhibits antigen-IgE-induced degranulation in RBL-2H3 cells⁹ and acts as a potent antidepressant by strongly inhibiting forskolin-induced corticotrophin releasing factor (CRF) gene transcription.^6,7 Psoralidin also has great potential as an anticancer agent, showing cytotoxic effects on gastric (SNU-1 and SNU-16), colon (HT-29) and breast (MCF-7) cancer cell lines.^10,11 Recently, we reported that psoralidin (1) inhibits protein kinase Akt phosphorylation and thus inhibits the growth of androgen independent prostate cancer cells both in vitro and in vivo.¹²

Psoralidin (1) is a member of the coumestane family of natural products having an isopentenyl group at the C-2 position of coumesterol (4). In 1961 Nashipuri & Pyne¹³ reported the synthesis of dihydropsoralidin (2) and confirmed the structure of the natural product. Jain et al.¹⁴ reported the synthesis of some prenylated coumestanes as analogues of psoralidin, and most recently Fürstner et al.¹⁵ synthesized and corrected the proposed structure of phaseol (3), a structural isomer of 1. However, so far, there is no report on the synthesis of psoralidin (1). In our ongoing work on the investigation of chemical constituents of rasagenthi lehyam, a complementary and alternative medicine for prostate cancer used as Ayurveda and Siddha medicine in different parts of India, we isolated 1 as the active principle of this mixture of 38 different botanicals, metals and fats.¹² However, the isolated natural product was found to be insoluble in water and therefore less effective for in vivo studies. To overcome this problem, we plan to generate a synthetic library of psoralidin analogues which can be utilized to study structure-activity-relationships (SAR), which in turn will also pave the way to synthesize an active analogue or derivative with increased water solubility. Herein, we report an efficient and general synthesis of 1 which can be easily adopted for the synthesis of various analogues.

Many routes to prepare coumestanes can be found in the literature, but most of them are complicated and require multistep syntheses.^16–24 Retro-synthetically, psoralidin (1) can be reduced to 5 through opening of the lactone and furan rings, and 5 in turn can be obtained from two relatively simple starting materials, namely 2,4-dimethoxy-phenylacetic acid methylester (6) and 2,4-dimethoxy-5-prenyl benzoylchloride (7) (Scheme 1). This strategy was expected to be flexible enough for the preparation of various analogues, considering the facile preparation of starting materials. Al-Maharik & Botting²⁴ used a similar strategy for their synthesis of ¹³C-labelled coumesterol (4).

Scheme 1.

Retrosynthetic Analysis

Results and Discussion

We started our synthesis from 1,5-dibromo-2,4-dimethoxybenzene (9),²⁵ which was prepared from resorcinol (8) through bromination with Br₂/CHCl₃ followed by O-methylation of the hydroxyl groups using K₂CO₃/MeI. Br/Mg exchange with i-PrMgCl at −10 °C followed by quenching with prenylbromide provided 10 exclusively in 65% yield. When carrying out the same reaction with n-BuLi, diprenylated and mono-debrominated side products were found that were not easy to separate. Acid 11 was obtained by replacing the bromine atom of 10 using n-BuLi and CO₂ gas that was bubbled through the reaction mixture. The other starting molecule, compound 6, was prepared by the condensation of 2,4-dimethoxy benzaldehyde and hippuric acid following a literature procedure.²⁶ Treatment of acid 11 with oxalyl chloride in presence of catalytic amounts of DMF produced acid chloride 7, which without further purification was reacted with the anion obtained by treatment of 6 with LDA, to produce the desired compound 5 in overall 82% yield. The structure of 5 was confirmed by spectroscopic analysis. At this point it was expected that demethylation followed by intramolecular cyclization would yield 1 in a one pot reaction. The demethylation of the four methoxy groups was carried out with BBr₃ at 0 °C, but the attempted in situ cyclization ended up with isopsoralidin (12) instead of 1.² The structure of 12 was confirmed by comparison with reported data (Scheme 2).^13,27 The formation of 12 can be explained by the generation of a stable tertiary carbocation produced from the prenyl group in acidic conditions, which is attacked by the ortho hydroxy group to yield the undesired dihydropyran ring. A similar reaction of a prenyl group was reported by Molyneux & Jurd.²⁷

Scheme 2.

To avoid this undesired reaction, we modified our strategy, and planned to introduce the prenyl group at the concluding step of the reaction sequence. Thus, we started with acid 13,²⁸ which was prepared from 9 by treatment with n-BuLi/CO₂ (Scheme 3). The reaction was optimized using less than one equivalent of n-BuLi, which provided a single product 13 in good (81%) yields. Reaction of the corresponding acid chloride 14 with 6 under similar conditions as above provided 15, which in turn was demethylated and cyclized to 16 in 78% yield. The structures of compounds 13 – 16 were confirmed by ¹H and ¹³C NMR spectroscopy as well as HRMS. Protection of the two phenolic OH-groups of 16 as MOM-ethers using diisopropylethylamine (i-Pr₂NEt) and methoxy methyl chloride (MOMCl) gave compound 17 in 65% yield. It was anticipated that Br/Mg or Br/Li exchange of 17 followed by electrophilic addition of prenyl bromide would complete the synthesis. However, all attempts to convert 17 with i-PrMgBr/prenyl bromide or n-BuLi/prenyl bromide under various reaction conditions ended up with complex product mixtures. A Pd(0)-catalyzed Ullmann type cross coupling reaction also failed to give any product.

Scheme 3.

In the search for a specific functional group which can (i) survive the acidic conditions necessary for the cyclization reaction and (ii) can be easily transformed to a prenyl group, we considered the olefin cross-metathesis reaction. Recently Hastings et al.²⁹ used olefin cross-metathesis reaction in the presence of Grubbs 2^nd generation catalyst to convert an allyl side chain to prenylated compound in very good yields. We hoped that a possible primary or secondary carbocation produced from an allyl group under acidic conditions would not be stable enough to cause an undesired cyclization reaction, like in case of the tertiary carbocation from the prenyl residue described above. We prepared the allylated compound 18 from 9 using i-PrMgBr and allyl bromide. A similar reaction sequence using n-BuLi/CO₂ and 6/LDA as described above provided compounds 19 and 21, respectively. Demethylation and cyclization produced the expected allylated compound 22 in 85% yields along with very small amounts of the dibrominated side product 23 (1%, Scheme 4). Compounds 18, 19, 21, 22 and 23 were characterized by ¹H and ¹³C NMR spectroscopy as well as HRMS for structure verification.

Scheme 4.

Our attempt to perform the alkene cross-metathesis reaction of 22 in the presence of a Grubbs 2^nd generation catalyst and 2-methyl-2-butene initially failed, when using identical reaction conditions as described by Hastings et al.²⁹ Even stirring the reaction at room temperature for 72 h or refluxing the mixture for 48 h with repeated addition of Grubbs catalyst and 2-methyl-2-butene failed to give any isolable product. To check the feasibility of the reaction, we performed the same reaction on 18 which produced prenylated compound 10 in very good yields. Since compound 22 might require a higher temperature for the cross-metathesis reaction, which was limited in this case by both the chosen solvent and the reagent 2-methyl-2-butene, we decided to carry out the reaction in a sealed tube in a microwave oven to allow higher reaction temperature and to avoid loss of starting material.³⁰ Thus, the final step of the total synthesis was achieved by heating a mixture of 22, Grubbs 2^nd generation catalyst and 2-methyl-2-butene in CH₂Cl₂ in a sealed tube microwave reactor at 100 °C for 15 minutes. The reaction produced 1 in 73% yield along with compound 24 as minor side product. All spectroscopic data of the synthetic compound matched those of the natural product (Table 1, see supporting information), except for the melting point, which was significantly higher for the synthetic compound than reported for the natural product. Since there is no ambiguity about the structure of the synthetic compound, we assume that melting point of the natural product was lowered by minor impurities.

Conclusions

In summary, we achieved the first total synthesis of psoralidin (1) in a convergent and highly regioselective manner. The synthetic scheme described here is straight-forward and efficient, the longest linear sequence is only five steps starting from commercially available resorcinol. The process is readily adaptable and flexible as substitutions can be introduced in both the phenyl acetate and the allyl benzoyl chloride starter molecules to furnish numerous analogues for SAR studies. Further structural diversification could be achieved on the level of the concluding Grubbs-reaction. Biological assays to determine and compare the efficacy of 16, 22, 23 with that of 1, as well as syntheses of various regio-isomers are in progress.

Experimental Section³¹

Psoralidin (1)

Grubbs 2^nd generation catalyst (3 mg, 0.0035 mmol) was added to a degassed solution of 22 (20 mg, 0.06 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 semi preparative HPLC (symmetry C₁₈, 7 μm, solvent gradient 9:1 CH₃CN:H₂O) to get compound 1 (16 mg, 73%) as white solid along with minor amount of side product 24 (1 mg, 5%). R_f 0.4 (50% ethyl acetate in hexane); Mp > 400 °C (lit¹ 315 °C); ν_max (film) cm⁻¹ 1719, 1629, 1597, 1419, 1370, 1260, 1092; ¹H (DMSO-d₆, 500 MHz) δ 7.68 (d, 1H, J = 8.0 Hz), 7.62 (s, 1H), 7.17 (d, 1H, J = 2.0 Hz), 6.94 (dd, 1H, J = 8.0, 2.0 Hz), 6.93 (s, 1H), 5.35 (t, 1H, J = 7.5 Hz), 3.32 (d, 2H, J = 7.5 Hz), 1.74 (s, 3H), 1.70 (s, 3H); ¹³C (DMSO-d₆, 125 MHz) δ 159.6 (C), 159.0 (C), 157.8 (C), 157.0 (C), 156.0 (C), 152.9 (C), 132.6 (C), 126.5 (C), 121.8 (CH), 121.0 (CH), 120.6 (CH), 114.7 (C), 114.0 (CH), 103.8 (C), 102.4 (CH), 102.0 (C), 98.8 (CH), 27.6 (CH₂), 25.7 (CH₃), 17.7 (CH₃); HRMS (EI+) m/z 336.1008 ([M]⁺, C₂₀H₁₆O₅, requires 336.0998).

(E)-2-(but-2-enyl)-3,9-dihydroxy-6H-benzofuro[3,2-c]chromen-6-one (24)

R_f 0.35 (50% ethyl acetate in hexane); Mp > 400 °C; ν_max (film) cm⁻¹ 1718, 1637, 1629, 1267, 1213, 1093; ¹H (DMSO- d₆, 500 MHz) δ 7.68 (d, 1H, J = 8.5 Hz), 7.64 (s, 1H), 7.17 (d, 1H, J = 2.0 Hz), 6.94 (dd, 1H, J = 8.5, 2.0 Hz), 6.90 (s, 1H), 5.70-5.50 (m, 2H), 1.67 (d, 3H, J = 6.0 Hz); ¹³C (DMSO-d₆, 125 MHz) δ 159.6 (C), 157.8 (C), 157.0 (C), 155.9 (C), 153.1 (C), 128.7 (CH), 126.3 (CH), 126.2 (CH), 125.3 (C), 121.3 (C), 120.5 (CH), 118.6 (C), 114.7 (CH), 113.9 (CH), 102.4 (C), 98.7 (CH), 31.9 (CH₂), 17.8 (CH₃); HRMS (EI+) m/z 322.0847 ([M]⁺, C₁₉H₁₄O₅, requires 322.0841).

Methyl 3-[2,4-Dimethoxy-5-(3-methyl-but-2-enyl)-phenyl]-2-(2,4-dimethoxy-phenyl)-3-oxo-propionate (5)

To a stirred solution of compound 11 (340 mg, 1.36 mmol) in CH₂Cl₂ (8 mL) under N₂ atmosphere, DMF (one drop) and oxalyl chloride (0.18 mL, 2.04 mmol) were added. The mixture was stirred for 3 h at room temperature and then the solvent was removed under vacuum to yield acid chloride 7, which was directly used for the next reaction without further purification.

A solution of compound 6 (430 mg, 2.1 mmol) in THF (5 mL) was added at − 78 °C to a solution of LDA, prepared from n-BuLi (2.5 M in THF, 1.1 mL, 2.72 mmol) and i-Pr₂NH (0.38 mL, 2.72 mmol) in THF (6 mL) at 0 °C under N₂ atmosphere. The this way produced yellowish anion was stirred at − 78 °C for 45 min and then a solution of crude acid chloride 7 in THF (10 mL) was added dropwise. The reaction mixture was stirred at the same temperature for another 45 min and then at room temperature for 4 h. The reaction was quenched by the addition of 10% HCl (10 mL), 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 column chromatography to yield 5 (495 mg, 82%) as yellowish oily compound. R_f 0.3 (50% ethyl acetate in hexane); ν_max (CHCl₃) cm⁻¹ 2359, 2341, 1738, 1666, 1604, 1507, 1463, 1270, 1211, 1029; ¹H (CDCl₃, 500 MHz) δ 7.72 (s, 1H), 7.01 (d, 1H, J = 8.0 Hz), 6.41 (s, 1H), 6.40 (d, 1H, J = 8.0 Hz), 6.30 (s, 1H), 5.91 (s, 1H), 5.22 (t, 1H, J = 7.5 Hz), 3.82 (s, 3H), 3.76 (s, 3H), 3.75 (s, 3H), 3.72 (s, 3H), 3.70 (s, 3H), 3.18 (d, 2H, J = 7.5 Hz), 1.68 (s, 3H), 1.64 (s, 3H); ¹³C (CDCl₃, 125 MHz) δ 193.0 (C), 171.1 (C), 162.6 (C), 160.3 (C), 159.6 (C), 157.9 (C), 132.7 (CH), 132.3 (CH), 130.3 (CH), 122.6 (C), 122.0 (C), 118.2 (C), 115.9 (C), 104.1 (CH), 98.5 (CH), 94.2 (CH), 57.3 (CH₃), 55.6 (CH₃), 55.5 (CH₃), 55.4 (CH₃), 55.3 (CH₃), 52.2 (CH), 27.6 (CH₂), 25.9 (CH₃), 17.8 (CH₃); HRMS (EI+) m/z 442.1993 ([M]⁺, C₂₅H₃₀O₇, requires 442.1992).

1-Bromo-2,4-dimethoxy-5-(3-methyl-but-2-enyl)-benzene (10)

Compound 9 (3.10 g, 10.47 mmol) was added to a solution of i-PrMgCl (1 M in THF, 13 mL, 13 mmol) at − 10 °C under N₂ atmosphere. After 45 min of stirring, prenyl bromide (1.3 mL, 11.5 mmol) was added dropwise and the mixture was allowed to stir at the same temperature for 30 min and then at room temperature for 2h. 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 × 75 mL). The organic layer was washed with water (2 × 30 mL), brine (20 mL), dried (Na₂SO₄), filtered and concentrated. The resulting crude compound was purified by column chromatography to yield 10 (1.94 g, 65%) as colorless liquid along with 550 mg of unreacted starting material 9. R_f 0.5 (5% ethyl acetate in hexane); ν_max (CHCl₃) cm⁻¹ 1599, 1500, 1463, 1295, 1208, 1029; ¹H (CDCl₃, 500 MHz) δ 7.21 (s, 1H), 6.44 (s, 1H), 5.21 (t, 1H, J = 7.0 Hz), 3.87 (s, 3H), 3.82 (s, 3H), 3.19 (d, 2H, J = 7.0 Hz), 1.72 (s, 3H), 1.67 (s, 3H); ¹³C (CDCl₃, 125 MHz) δ 157.5 (C), 154.8 (C), 133.1 (C), 133.0 (CH), 124.0 (C), 122.2 (CH), 101.6 (C), 96.8 (CH), 56.6 (CH₃), 55.9 (CH₃), 27.6 (CH₂), 26.0 (CH₃), 17.9 (CH₃); HRMS (EI+) m/z 284.0414 ([M]⁺, C₁₃H₁₇O₂Br, requires 284.0412).

2,4-Dimethoxy-5-(3-methyl-but-2-enyl)-benzoic acid (11)

n-BuLi (2.5 M in THF, 2.1 mL, 5.34 mmol) was added dropwise to a stirred solution of 10 (1.27 g, 4.45 mmol) in THF (15 mL) at −78 °C under N₂ atmosphere. After 30 min CO₂ gas was passed through the solution for 45 min and then it 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 × 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 11 (930 mg, 84%) as white solid. R_f 0.1 (50% ethyl acetate in hexane); Mp: 98–99 °C; ν_max (film): cm⁻¹ 1719, 1616, 1442, 1278,1168, 1020; ¹H (CDCl₃, 500 MHz): δ 7.77 (s, 1H), 6.41 (s, 1H), 5.16 (t, 1H, J = 7.0 Hz), 3.97 (s, 3H), 3.84 (s, 3H), 3.14 (d, 2H, J = 7.0 Hz), 1.64 (s, 3H), 1.60 (s, 3H); ¹³C (CDCl₃, 125 MHz): δ 166.2 (C), 162.6 (C), 158.5 (C), 133.6 (CH), 132.9 (C), 123.9 (CH), 121.6 (C), 108.9 (C), 94.5 (CH), 56.7 (CH₃), 55.7 (CH₂), 27.6 (CH₃), 25.8 (CH₃), 17.7 (CH₃); HRMS (EI+): m/z 250.1206 ([M]⁺, C₁₄H₁₈O₄, requires 250.1205).

Methyl 3-(5-bromo-2,4-dimethoxy-phenyl)-2-(2,4-dimethoxy-phenyl)-3-oxo-propionate (15)

This compound was prepared as yellowish oil (320 mg, 74%) from compound 13 and 6, following an analogous reaction sequence as described above for the synthesis of compound 5 (for the exact procedure, see supporting information). R_f 0.3 (50% ethyl acetate in hexane); Mp 136–137 °C; ν_max (film): cm⁻¹ 1730, 1668, 1613, 1590, 1508, 1465, 1334, 1271, 1213, 1155, 1021; ¹H (CDCl₃, 500 MHz) δ 8.10 (s, 1H), 7.00 (d, 1H, J = 9.0 Hz), 6.43 (s, 1H), 6.42 (d, 1H, J = 9.0 Hz), 6.34 (s, 1H), 5.88 (s, 1H), 3.91 (s, 3H), 3.80 (s, 3H), 3.76 (s, 6H), 3.72 (s, 3H); ¹³C (CDCl₃, 125 MHz) δ 192.1 (C), 170.8 (C), 160.6 (C), 160.5 (C), 160.1 (C), 157.9 (C), 136.0 (CH), 130.4 (CH), 120.2 (C), 115.3 (C), 104.3 (CH), 103.2 (C), 98.7 (CH), 95.8 (CH), 57.3 (CH₃), 56.6 (CH₃), 55.7 (CH₃), 55.6 (CH₃), 55.4 (CH₃), 52.4 (CH); HRMS (EI+) m/z 452.0477 ([M]⁺, C₂₀H₂₁O₇Br, requires 452.0471).

2-Bromo-3,9-dihydroxy-benzo[4,5]furo[3,2-c]chromen-6-one (16)

BBr₃ (1 M in CH₂Cl₂, 1.72 mL, 1.72 mmol) was added at 0 °C to a solution of 15 (130 mg, 0.29 mmol) in CH₂Cl₂ (6 mL) under N₂ atmosphere. The mixture was stirred at same temperature for 10 h and water (10 mL) was added. CH₂Cl₂ was removed in vacuum and the mixture was heated to reflux for 1 h. The water layer was extracted with ethyl acetate (2 × 30 mL), the organic layer was washed with water (2 × 15 mL), brine (10 mL), dried (Na₂SO₄) and concentrated. Crude product was purified by column chromatography to get 16 (78 mg, 78%) as white solid. R_f 0.3 (50% ethyl acetate in hexane); Mp 362–364 °C; ν_max (film) cm⁻¹ 1719, 1634, 1599, 1418, 1368, 1272, 1085; ¹H (DMSO- d₆, 500 MHz) δ 8.08 (s, 1H), 7.68 (d, 1H, J = 8.5 Hz), 7.15 (d, 1H, J = 2.0 Hz), 7.03 (s, 1H), 6.94 (dd, 1H, J = 8.5, 2.0 Hz); ¹³C (DMSO-d₆, 125 MHz) δ 158.2 (C), 157.5 (C), 157.3 (2C, C), 156.1 (C), 153.4 (C), 125.0 (CH), 120.8 (CH), 114.5 (C), 114.2 (CH), 107.0 (C), 105.4 (C), 103.9 (CH), 102.8 (C), 98.7 (CH); HRMS (EI+) m/z 345.9475 ([M]⁺, C₁₅H₇O₅Br, requires 345.9477).

2-Bromo-3,9-bis-methoxymethoxy-benzo[4,5]furo[3,2-c]chromen-6-one (17)

i -Pr₂NEt (0.05 mL, 0.29 mmol) was added to a solution of 16 (40 mg, 0.11 mmol) in CH₂Cl₂ (6 mL) at room temperature. The mixture was stirred for 15 min and then MOMCl (0.02 mL, 0.25 mmol) was added. The reaction was stirred for 10 h and quenched by the addition of water (5 mL). The aqueous layer was extracted with ethyl acetate (2 × 20 mL), the organic layer was washed with water (2 × 10 mL), brine (10 mL), dried (Na₂SO₄) and concentrated. The crude product was purified by column chromatography to get 17 (35 mg, 65%) as white solid. R_f 0.8 (30% ethyl acetate in hexane); Mp 212–214 °C; ν_max (film) cm⁻¹ 1758, 1629, 1490, 1353, 1257, 1163, 1072, 957; ¹H (CDCl₃, 500 MHz) δ 8.14 (s, 1H), 7.94 (d, 1H, J = 8.5 Hz), 7.35 (s, 1H), 7.29 (s, 1H), 7.14 (d, 1H, J = 8.5 Hz), 5.33 (s, 2H), 5.25 (s, 2H), 3.53 (s, 3H), 3.51 (s, 3H); ¹³C (CDCl₃, 125 MHz) δ 159.0 (C), 158.0 (C), 157.2 (C), 156.5 (C), 156.3 (C), 153.9 (C), 125.6 (CH), 122.0 (CH), 117.6 (C), 115.5 (CH), 109.3 (C), 108.2 (C), 104.7 (CH), 102.7 (C), 99.9 (CH), 95.5 (CH₂), 95.2 (CH₂), 56.9 (CH₃), 56.4 (CH₃); HRMS (EI+) m/z 433.9992 ([M]⁺, C₁₉H₁₅O₇Br, requires 434.0001).

Supplementary Material

supporting. Supporting Information Available.

General experimental methods, experimental details and characterization data for compounds 9, 12, 13, 15, 18, 19, 21–23 and copies of ¹H and ¹³C NMR spectra of all new compounds. This material is available free of charge via the internet at http://pubs.acs.org.

Figure 1.

Chemical structures of psoralidin and natural products