An efficient and metal-free synthesis of 5,7-dimethylcyclopentenon[2,3- c ]coumarin

Abstract

An improved and alternative method for the metal-free synthesis of 5,7-dimethylcyclopentenon[2,3-c]coumarin as a possible useful aflatoxin template in the production of molecular imprinting polymers with affinity toward natural aflatoxin molecules is reported. A synthesis of 5,7-dimethylcyclopentenon[2,3-c]coumarin from 3,5-dimethoxyphenol via a Pechmann condensation involved five steps and represents an alternative commercially viable approach over existing synthetic protocols in the literature. Operational simplicity, inexpensive key raw materials, short reaction times, and excellent yields are remarkable features of this approach. The aflatoxin template was provided in 55%–67% overall yield from 3,5-dimethoxyphenol with >99% purity, evaluated by high-performance liquid chromatography, ¹H NMR, ¹³C NMR, high-resolution mass spectrometry, and liquid chromatography–mass spectrometry.

Introduction

Aflatoxins belong to a large family of polyketide natural products known as mycotoxins due to their toxicity that originate from the secondary metabolites of a variety of ubiquitous fungal species. This particular group of toxins is produced by the molds Aspergillus flavus ¹ and Aspergillus Parasiticus ² that contaminate a variety of agricultural commodities and, when consumed by animals, can also transfer into edible animal by-products such as meat, milk, and milk by-products. The problems caused by aflatoxins are significant due to their high toxicity and carcinogenicity, aflatoxin B1 (AFB1) being classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC), and is tightly regulated in both food and feedstuffs. Although biological, physical and chemical mitigation procedures and improved sampling techniques, analytical methods, and surveillance programs are available to monitor and control aflatoxin contamination, the “hot spot” nature of their occurrence is making contamination unavoidable. ³ In this context, there is a need to develop suitable devices for the removal of aflatoxins from contaminated agricultural and animal products, and more particularly from milk.

Molecularly imprinted polymers (MIPs) are the polymeric materials that exhibit molecular recognition for a target molecule. The MIP polymerization reaction is performed in the presence of the target molecule or a suitable molecular mimic or surrogate that is used as template. To create an “imprint,” the template is then removed by washing with an appropriate solvent, leaving a cavity in the polymer in the shape of the template. When the MIP encounters the molecule of interest, the molecule binds in the cavity through non-covalent interactions and regio- and stereo-chemical compatibility. The target molecule can then be removed by washing with a solvent. ⁴ In the case of aflatoxins, there are calls for alternatives to the use of the targeted molecules for MIP preparation because, despite their commercial availability, they suffer from high prices with limited quantities available. Moreover, they could place workers at risk of serious health hazards from handling large quantities of aflatoxins during large-scale MIP preparation and the final MIP could itself represent a hazard due to the risk of template bleeding (desorption) when using the MIP as a mitigation or diagnostic device in food and/or liquids. In this respect, 5,7-dimethylcyclopentenon[2,3-c]coumarin (aflatoxin template (AFT)) has been selected, synthesized, and used as a template as an AFB1 epigone in the production of the corresponding MIP, with a structural similarity of 82% with the AFB1 molecule (based on the maximum common substructure (MCS) shared among compound pairs and a Tanimoto coefficient of 0.8261) and between 70% and 90% homology with other members of the aflatoxin group, such as the AFB1 metabolization product commonly found in milk, aflatoxin M1 (AFM1, maximum common subgraph (MCS) Tanimoto coefficient of 0.7917), using cheminformatics measurements.^5,6

In the literature, Trost et al. ⁷ reported a synthesis of 5,7-dimethylcyclopentenon[2,3-c]coumarin by a Lewis acid catalyzed cyclization of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid. Asao et al. also reported an improved process for the production of 5,7-dimethylcyclo-pentenon[2,3-c]coumarin. ⁸ Zhou and Corey ⁹ reported the total synthesis of aflatoxin B2 using an asymmetric [3 + 2] cycloaddition step, which contains the similar basic skeleton of the AFT. Bhute et al. reported a multi-step protocol for the synthesis of 5,7-dimethylcyclo-pentenon[2,3-c]coumarin from dimethoxyphenol via cyclization of an ester using polyphosphoric acid (PPA), although a major disadvantage of this method is the very low yield, that is, the overall yield of the AFT from 3,5-dimethoxyphenol is 13%–14%. ¹⁰ Moreover, the reported methods required harsh reaction conditions, expensive reagents that were not readily available, the use of large quantities of organic solvents and/or toxic metal catalysts, and tedious work-up procedures leading to unsatisfactory yields. Considering these issues, there is room for a more efficient synthetic alternative for the production of 5,7-dimethylcyclo-pentenon[2,3-c]coumarin. Recently, we reported a synthesis of AFTs and MIPs together with their applications to the detection of mycotoxins. ¹¹ In this report, we describe our efforts to meet these challenges for the synthesis of 5,7-dimethylcyclopentenon[2,3-c]coumarin via cyclization of a mono carboxylic acid using PPA so providing a high yield, a commercially viable, simple, and scalable process, which precludes the use of hazardous reagents or toxic metal catalysts.

Results and discussion

4-(Chloromethyl)-5,7-dimethoxy-2H-chromen-2-one (1) was prepared in >95% yield by a Pechmann condensation of 3,5-dimethoxyphenol with ethyl-4-chloroacetoacetate in the presence of a mixture of acetic acid and sulfuric acid. Diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate (2) was obtained in >86% yield by the alkylation of diethylmalonate with 4-(chloromethyl)-5,7-dimethoxy-2H-chromen-2-one in the presence of a mixture of KI, 18-Crown-6, and t-BuOK in acetonitrile. Subsequent hydrolysis of diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate with NaOH in ethanol gave 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonic acid (3) in >97% yield. 3-(5,7-Dimethoxy-2-oxo-2H-chromen-4-yl) propanoic acid (4) was obtained in >91% yield upon partial decarboxylation of 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonic acid in p-xylene under reflux for 2 h. The final product, 5,7-dimethylcyclopentenon[2,3-c]coumarin (the aflatoxin B1 template, AFT) was obtained in the range of 15%–75% yield by the cyclization of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid in the presence of a metal catalyst acting as a Lewis acid, ² whereas the final product was obtained in >92% yield by the cyclization of the 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid in PPA. The overall yields are summarized in Table 1.

Table 1.

The effect of different Lewis acid catalysts and polyphosphoric acid (PPA) on the cyclization of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid to give 5,7-dimethylcyclopentenon[2,3-c]coumarin.

Acid catalyst	Conditions a	Yield b (%)
AlCl3	Equiv. of AlCl3; (COCl)2; CH2Cl2; room temperature	15
AlCl3	Equiv. of AlCl3; (COCl)2; CH2Cl2; room temperature	32
Sc(OTf)3	Eq. of Sc(OTf)3; (COCl)2; CH2Cl2; room temperature	45
Sc(OTf)3 + LiClO4	1.0 equiv. of Sc(OTf)3; 5.0 equiv. of LiClO4; CH3NO2; 60 °C	75
Polyphosphoric acid (PPA) c	10 weight equiv. of PPA, 80 °C	92

a

Milliequivalents of acid catalyst with respect to the milliequivalents of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid.

b

Isolated yield of 5,7-dimethylcyclopentenon[2,3-c]coumarin.

c

10 g of PPA were used for 1 g of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid.

Overall, AFT was provided in about a 65% yield with >99% purity by high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC-MS) from 3,5-dimethoxyphenol in the new synthetic approach using metal-free cyclization methodology (Scheme 1).

Scheme 1.

Synthesis of 5,7-dimethylcyclopentenon [2,3-c]coumarin (AFT). Reagents and conditions: (a) ethyl-4-chloroacetoacetate, glacial acetic acid, H₂SO₄, 55–60 °C, 12 h; (b) diethyl malonate, acetonitrile, KI, 18-Crown-6, t-BuOK, room temperature, 12 h; (c) aqueous NaOH, ethanol, 60–62 °C, 2 h; (d) p-xylene, reflux temperature, 4 h; (e) dry nitromethane, Sc(OTf)₃, LiClO₄, 60 °C, 1 h; (f) polyphosphoric acid (PPA), 75–80 °C, 4 h.

Conclusion

In conclusion, we have developed an efficient, scalable, non-infringing, commercially viable, and alternative approach for the total synthesis of pure 5,7-dimethylcyclopentenon[2,3-c]coumarin from 3,5-dimethoxyphenol using a new synthetic protocol via the key intermediates: 4-(chloromethyl)-5,7-dimethoxy-2H-chromen-2-one, diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate, 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl) malonic acid, and 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid, using a Pechmann condensation, alkylation, hydrolysis, partial decarboxylation, and improved cyclization reaction conditions. The key and final step involved in this synthesis was the cyclization of 5,7-dimethoxycoumarin-4-propanoic acid with PPA instead of an expensive Lewis acid metal catalyst that gave 5,7-dimethylcyclopentenon[2,3-c]coumarin with improved yields. Pure 5,7-dimethylcyclo-pentenon[2,3-c]coumarin was obtained with an overall yield of >65% from 3,5-dimethoxyphenol through this a five-step synthetic methodology.

Experimental

All starting materials, reagents, and solvents are commercially available and were used without further purification. Melting points were determined with an X-4 apparatus and are uncorrected. The NMR spectra were recorded on a Bruker Ascend 400 spectrometer using tetramethylsilane (TMS) as an internal standard. The reactions were monitored by thin-layer chromatography (TLC; HG/T2354-92, GF254), and compounds were visualized on TLC with UV light. The purity of products was determined by direct infusion into an electrospray ionization ion mobility quadrupole time-of-flight mass spectrometer (Vion^®, Waters Corp., Milford, MA, USA) operated in positive ion mode and coupled to an Acquity^® ultra performance liquid chromatography (UPLC) system (Waters Corp.). Molecular structural similarity was measured using ChemMine Tools (www.chemmine.ucr.edu) using two different algorithms calculation, that could be briefly described as: (1) atom pairs structural descriptor (AP Tanimoto coefficient) and/or (2) MCS Tanimoto coefficient shared among common pairs, both including subsequently the size of the compound and size of shared.^5,6

Synthesis of 4-(chloromethyl)-5,7-dimethoxy-2H-chromen-2-one (1)

A reactor was charged with 3,5-dimethoxyphenol (25 g, 0.162 mol) and glacial acetic acid (50 mL) under argon stream at room temperature (RT), and the mixture was stirred at 40–45 °C to give a clear solution. The reaction mixture was cooled to 8 °C, and then a solution of ethyl-4-chloroacetoacetate (26.6 g, 0.162 mol) in glacial acetic acid (12.5 mL), and sulfuric acid (12.5 mL) was added dropwise for 15 min at 8–10 °C. The reaction mixture was stirred for 1 h at RT. Temperature of the reaction mixture was slowly raised to 60 °C, and stirred for 12 h at 55–60 °C. The reaction mixture was cooled to 40 °C and then water (150 mL) was added dropwise for 15 min at 40–45 °C. The mixture was stirred for 2 h at RT to precipitate the product. The product was filtered and washed with water (2 × 25 mL). The crude wet product was then mixed with methanol (50 mL), the residual slurry was stirred for 30 min at 0–5 °C. The product was filtered and washed with cold methanol (2 × 25 mL), and the product was dried in a vacuum oven for 12 h at 35–40 °C to give 4-(chloromethyl)-5,7-dimethoxy-2H-chromen-2-one (39.2 g, 95.2% yield) as a white amorphous solid. ¹H NMR (400 MHz, CDCl₃): δ 3.86 (s, 3H), δ 3.90 (s, 3H), δ 4.90 (s, 2H), δ 6.33 (d, 1H, J = 2.4 hz), δ 6.46 (s, 1H), δ 6.48 (d, 1H, J = 2.4 hz). m.p. = 155–156 °C (lit. ¹² 155–157 °C).

Synthesis of diethyl 2-[(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl] malonate (2)

To a solution of diethylmalonate (3.65 g, 0.0228 mol) in anhydrous acetonitrile (60 mL) at RT was added a mixture of 4-(chloromethyl)-5,7-dimethoxy-2H-chromen-2-one (5 g, 0.019 mol), potassium iodide (0.4 g, 0.0024 mol), and 18-Crown-6 (0.63 g, 0.0024 mol), under argon atmosphere. The heterogeneous reaction mixture was stirred for 30 min at RT. Potassium-t-butoxide (2.17 g, 0.0193 mol) was added lot-wise for 5 min at 25–30 °C, and the reaction mixture was stirred for 12 h at RT. Acetonitrile and other volatiles were removed by distillation under reduced pressure at 35–40 °C to give the crude product. The crude product was dissolved in a mixture of ethyl acetate (100 mL) and water (20 mL), the residual organic layer was then separated and concentrated to dryness under reduced pressure at 35–40 °C to give diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl) malonate (6.21 g, 86.5% yield) as a pale yellow colored solid. ¹H NMR (400 MHz, CDCl₃): δ 1.26 (t, 6H, J = 7.2 hz), δ 3.47 (d, 2H, J = 7.2 hz), δ 3.73 (t, 1H, J = 6.8 hz), δ 3.85 (s, 3H), δ 3.86 (s, 3H), δ 4.16-4.28 (m, 4H), δ 6.02 (s, 1H), δ 6.32 (d, 1H, J = 2.4 hz), δ 6.47 (d, 1H, J = 2.4 hz). ¹³C NMR (100 MHz, CDCl₃): δ 170.8, 162.9, 160.8, 157.8, 156.7, 154.8, 112.2, 104.8, 98.5, 93.5, 61.8, 56.4, 55.9, 50.8, 35.3, 14.9. High-resolution mass spectrometry (HRMS) calcd for C₁₉H₂₂O₈: 378.1315; found: 378.1402. m.p. = 238–240 °C.

Synthesis of 2-[(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl]malonic acid (3)

A reactor was charged with a mixture of diethyl 2-[(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl] malonate (5.0 g, 0.013 mol) in ethanol (20 mL, four volumes) at RT. A solution of sodium hydroxide (1.058 g, 0.026 mol) in water (20 mL) was added in one lot at RT. The reaction mixture was then slowly heated to 60 °C and stirred for 2 h at 60–62 °C. The reaction mixture was cooled to RT and then stirred for 3 h at RT. Ethanol and other volatiles were removed by distillation under reduced pressure at 35–40 °C to give the crude product. Water (50 mL) was added to the crude product and pH of the reaction mixture was adjusted to 1.0 with concentrated hydrochloric acid. The product was then extracted with ethyl acetate (2 × 50 mL), and the two pooled organic extract was washed with water (50 mL). Solvent was removed under reduced pressure after drying the ethyl acetate extracts over anhydrous sodium sulfate (0.5 g) to give 2-[(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl]malonic acid (4.068 g, 97.2% yield) as a pale yellow colored solid. ¹H NMR (400 MHz, CDCl₃): δ 3.48 (d, 2H, J = 7.2 hz), δ 3.78 (t, 1H, J = 7.0 hz), δ 3.85 (s, 3H), δ 3.86 (s, 3H), δ 6.02 (s, 1H), δ 6.32 (d, 1H, J = 2.4 hz), δ 6.47 (d, 1H, J = 2.4 hz), δ 11.58 (bs, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 178.6, 162.7, 160.6, 157.5, 156.3, 154.4, 111.8, 104.6, 98.3, 93.4, 56.6, 55.4, 54.7, 34.8. HRMS calcd for C₁₅H₁₄O₈: 322.0689; found: 322.0712. m.p. = 294–296 °C.

Synthesis of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid (4)

A heterogeneous reaction mixture of 2-[(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl]malonic acid (4 g, 0.0124 mol) in anhydrous p-xylene (40 mL, 10 volumes) was slowly heated to the reflux temperature of p-xylene under argon atmosphere, and then stirred for 4 h at the same temperature. The reaction mixture was cooled to RT, followed by isolation of the product by filtration. The product was washed with n-heptane (2 × 10 mL) and dried in a vacuum oven for 12 h at 40–45 °C to give 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid (3.15 g, 91.5% yield) as a pale yellow colored solid. ¹H NMR (400 MHz, CDCl₃): δ 2.68 (t, 2H, J = 7.6 hz), δ 3.24 (t, 2H, J = 7.6 hz), δ 3.86 (s, 3H), δ 3.89 (s, 3H), δ 6.03 (s, 1H), δ 6.33 (d, 1H, J = 2.0 hz), δ 6.47 (d, 1H, J = 2.0 hz), δ 11.52 (bs, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 177.8, 162.5, 160.4, 157.3, 156.4, 154.2, 111.6, 104.3, 98.4, 93.2, 56.2, 55.1, 34.6, 33.7. HRMS calcd for C₁₄H₁₄O₆: 278.0790; found: 278.0815. m.p. = 267–269 °C.

Literature method: typical procedure for the synthesis of 5,7-dimethylcyclopentenon[2,3-c]coumarin (AFT) using a Lewis acid

A reactor was charged with a mixture of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid (2 g, 0.0072 mol), scandium trifluoromethanesulfonate (2 g, 0.0072 mol), and lithium perchlorate in anhydrous nitromethane (200 mL, 100 volumes) under argon atmosphere at RT. The reaction mixture was slowly heated to 60 °C and stirred for 1 h at 60 °C, then cooled to RT. The mixture was diluted with methylene chloride (250 mL), dried over anhydrous Na₂SO₄, and concentrated to dryness under reduced pressure at 25–30 °C to give the crude product. Pure 5,7-dimethylcyclopentenon[2,3-c]coumarin (1.40 g, 75% yield, HPLC purity: 99.5% (HPLC conditions: Column: Acquity UPLC^® BEH C18 1.7 μm 2.1 mm × 50 mm from Waters, Solvent: 1:1 (v/v) ratio of methanol and water amended with 0.1% formic acid, flow rate: 0.5 mL/min, RT: 2.05 min)) was isolated as a white solid using flash chromatography eluting with 5% methanol:methylene chloride. ¹H NMR (400 MHz, DMSO-d₆): δ 2.52 (dd, 2H, J = 5.4 hz and J = 5.1 hz), δ 3.35 (dd, 2H, J = 5.4 hz and J = 5.1 hz), δ 3.88 (s, 3H), δ 3.92 (s, 3H), δ 6.51 (d, 1H, J = 1.7 hz), δ 6.62 (d, 1H, J = 1.7 hz). ¹³C NMR (100 MHz, CDCl₃): δ 201.4, 176.6, 166.5, 160.3, 158.8, 155.6, 118.1, 103.9, 96.2, 93.8, 56.1, 55.9, 35.5, 28.8. LC-MS m/z: 261 (MH⁺). m.p. = 230–232 °C (lit. ⁷ 230 °C).

Improved method: synthesis of 5,7-dimethylcyclopentenon[2,3-c]coumarin (AFT) using PPA

A heterogeneous reaction mixture of 3-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)propanoic acid (2 g, 0.0072 mol) in PPA (20 g, 10 volumes) was slowly heated to 75 °C and stirred for 4 h at 75–80 °C under argon atmosphere. The reaction mixture was cooled to 10 °C and then diluted with ice-cold water (100 mL) under stirring for 15 min. The product was then filtered, and washed with water (2 × 10 mL) to give the crude product. The crude product was recrystallized from methanol (10 mL), and then dried in a vacuum oven for 12 h at 35–40 °C to afford 5,7-dimethylcyclopentenon[2,3-c]coumarin (1.72 g, 92% yield, HPLC purity: 99.7% (HPLC conditions: Column: Acquity UPLC^® BEH C18 1.7 μm 2.1 mm × 50 mm from Waters, Solvent: 1:1 (v/v) ratio of methanol and water amended with 0.1% formic acid, flow rate: 0.5 mL/min, RT: 2.05 min)) as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 2.52 (dd, 2H, J = 5.4 hz and J = 5.1 hz), δ 3.35 (dd, 2H, J = 5.4 hz and J = 5.1 hz), δ 3.88 (s, 3H), δ 3.92 (s, 3H), δ 6.51 (d, 1H, J = 1.7 hz), δ 6.62 (d, 1H, J = 1.7 hz). ¹³C NMR (100 MHz, CDCl₃): δ 201.4, 176.6, 166.5, 160.3, 158.8, 155.6, 118.1, 103.9, 96.2, 93.8, 56.1, 55.9, 35.5, 28.8. LC-MS m/z: 261 (MH⁺). m.p. = 230–232 °C (lit. ⁷ 230 °C).