Design, Synthesis, and Antifungal Activity of Isochroman-Fused Coumarins against Rhizoctonia solani

We dedicate this article to the fond memory of our beloved Late Prof. Ashok K. Prasad

Abstract

This study presents the synthesis and antifungal assessment of novel isochroman-fused coumarin derivatives against Rhizoctonia solani. The compounds were synthesized using an efficient and environmentally benign approach and demonstrated strong in silico binding affinity to CYP51. In vitro assays further confirmed their antifungal activity, with one compound exhibiting the highest potency, achieving an ED₅₀ value of 3.59 μM. These findings underscore the potential of isochroman-fused coumarins as eco-friendly rice sheath blight control agents.

Rice is a crucial dietary staple for 67% of the global population, making its protection from disease critical for ensuring food security.[1] [2] Among the various threats to rice cultivation, fungal diseases are particularly devastating, leading to substantial yield losses.[3–5] Rhizoctonia solani, a major fungal pathogen of sheath blight disease, ranks as the second most critical fungal disease affecting rice crops worldwide.[6–8] According to UN data from 2009-2010, Rhizoctonia solani not only impacts rice but also other essential crops such as wheat, maize, potatoes, and soybeans, further amplifying its agricultural significance.[9,10]

While numerous fungicides are available to mitigate fungal diseases, the prolonged use of existing fungicides has raised concerns regarding environmental pollution, food safety, and the emergence of resistant fungal strains.[11] [12] This highlights the urgent need for new antifungal agents with improved efficacy and safety profiles.

Isochroman is a privileged heterocyclic scaffold with broad applications in drug discovery due to its diverse pharmacological activities.[13] Numerous isochroman-based compounds have shown therapeutic potential in central nervous system disorders,[14] antibacterial,[15] antihypertensive,[16] anticancer,[17] antioxidant,[18] and anti-inflammatory conditions.[19] Additionally, natural products containing isochroman moieties contribute significantly to biological efficacy.[20] For instance, fusarentin (A) exhibits insecticidal and fungicidal properties, underscoring the antifungal relevance of the isochroman core (Figure [1]).[21] Isochroman-based secondary metabolites, such as B and C extracted from Penicillium expansum, have demonstrated potent antifungal activity against Lasiodiplodia theobromae (Figure [1]).[22] Similarly, isochroman analogue D has shown activity against Cryptococcus neoformans, with a minimum inhibitory concentration (MIC) of 35 μg/mL (Figure [1]).[23] Synthetic isochroman derivatives (E–I) have also displayed significant antifungal activity against pathogens such as Cryptococcus neoformans and Saccharomyces cerevisiae (Figure [1]), reinforcing the therapeutic importance of this scaffold in antifungal drug development.[24] [25] [26]

Coumarins represent another class of biologically active benzopyrones with over 1,300 derivatives exhibiting antibacterial, antifungal, antiviral, anti-inflammatory, and anticancer activities.[27] [28] The integration of heterocyclic moieties into coumarin structures such as azetidine, thiazolidine, thiazole, and oxadiazole has enhanced their antimicrobial potency and selectivity.[29,30] Given their pharmacological versatility, coumarin hybrids have emerged as promising therapeutic candidates for combating fungal infections.[31]

To combat these issues, herein we introduce novel isochroman-fused coumarins as potential antifungal agents. These hybrid molecules combine the structural features of both isochroman and coumarin, both of which are known for their diverse pharmacological activities, including antifungal properties. To the best of the knowledge, this is the first exploration of the synthesis and antifungal potential of these fused compounds. The compounds were synthesized through an efficient, one-pot, two-step process and evaluated for their effectiveness against Rhizoctonia solani, with the goal of creating potent, selective, and eco-friendly next-generation fungicides.

Chemistry

This study focused on the synthesis of isochroman-fused coumarin derivatives through a one-pot, two-step palladium-catalyzed reaction, emphasizing an environmentally benign approach. Initially, substituted 4-hydroxycoumarins 1a–h were synthesized via an environmentally benign acetylation of phenols followed by reaction with diethyl carbonate, following established procedures described in the literature.[32] This approach aligns with recent advancements in green chemistry, emphasizing solvent-free or low-toxicity solvent systems and recyclable catalysts, as reported in various studies on coumarin synthesis.[33] For instance, the Pechmann condensation and Knoevenagel condensation methods have been widely employed for synthesizing coumarins under optimized conditions using eco-friendly solvents and catalysts, ensuring high yields and reduced environmental impact.[34]

The synthesis of isochroman-fused coumarin derivatives was systematically optimized by evaluating key reaction parameters such as solvent, base, catalyst, temperature, and reaction time. Initially, the reaction between 4-hydroxycoumarin (1a) and 2-bromobenzyl bromide (2a) was carried out using Pd(OAc)₂ as a catalyst, K₂CO₃ as a base, and DMF as the solvent at 80 °C for 5 h, yielding only 36% of the desired product (4a, Table [1], entry 1). Alternative solvents such as diethyl carbonate (DEC), PEG-400, γ-valerolactone, and acetonitrile were tested but resulted in negligible or no product formation (Table [1], entries 2–5). However, propylene carbonate emerged as the most effective solvent, increasing the yield to 50% (Table [1], entry 6). Its environmentally benign nature and ability to stabilize intermediates likely contributed to this improvement. Subsequently, the effect of various bases was explored. Among the bases tested (K₂CO₃, Na₂CO₃, Cs₂CO₃, NaH), K₂CO₃ was found to be the most effective, facilitating optimal product formation (Table [1], entries 7–9).

Table 1 Optimization of the Reaction Conditions for the Synthesis of 6H,11H-Isochromeno[4,3-c]chromen-11-one (4a) from 4-Hydroxy Coumarin (1a)^a and 2- Bromobenzyl Bromide (2a)

Entry	Solvent	Catalyst ( 10 mol%)	Base (3 equiv)	Time (h)	Temp (°C)	Yield (%)b
1	DMF	Pd(OAc)2	K2CO3	5	80	36
2	DEC	Pd(OAc)2	K2CO3	5	80	22
3	PEG-400	Pd(OAc)2	K2CO3	5	80	N.R.
4	γ-valerolactone	Pd(OAc)2	K2CO3	5	80	N.R.
5	acetonitrile	Pd(OAc)2	K2CO3	5	80	N.R.
6	propylene carbonate	Pd(OAc)2	K2CO3	5	80	50
7	propylene carbonate	Pd(OAc)2	Na2CO3	5	80	N.R.
8	propylene carbonate	Pd(OAc)2	Cs2CO3	5	80	N.R.
9	propylene carbonate	Pd(OAc)2	NaH	5	80	N.R.
10	propylene carbonate	PdCl2	K2CO3	5	80	54
11	propylene carbonate	Pd(PPh3)2Cl2	K2CO3	5	80	N.R.
12	propylene carbonate	PdCl2	K2CO3	5	100	58
13	propylene carbonate	PdCl2	K2CO3	5	110	65
14	propylene carbonate	PdCl2	K2CO3	4	120	68
15	propylene carbonate	PdCl2	K2CO3	4	130	61
16	acetone	PdCl2	K2CO3	4	120	12
17	toluene	PdCl2	K2CO3	4	120	trace
18	benzene	PdCl2	K2CO3	4	120	N.R.
19	propylene carbonate	PdCl2	(C2H5)3N	4	120	N.R.
20	propylene carbonate	PdCl2	DIPEA	4	120	N.R.
21	propylene carbonate	PdCl2	DIPA	4	120	N.R.
22	propylene carbonate	PdCl2	DBU	4	120	15
23	propylene carbonate	Pd2(dba)3	K2CO3	4	120	32
24	propylene carbonate	Pd(PPh3)4	K2CO3	4	120	trace
25	propylene carbonate	Ni(COD)2	K2CO3	4	120	43
26	propylene carbonate	NiCl2	K2CO3	4	120	N.R.
27	propylene carbonate	PdCl2	K2CO3	4	120	tracec
28	propylene carbonate	PdCl2	K2CO3	4	120	60d

^a Reaction conditions: 1a (1.0 equiv), 2-bromobenzyl bromide 2a (1.0 equiv), base (3 equiv) in solvent (5 mL).

^b Isolated yields, N.R. = no reaction.

^c 2-Chlorobenzyl bromide instead of 2-bromobenzyl bromide.

^d 2-Iodobenzyl bromide instead of 2-bromobenzyl bromide.

The palladium catalyst was another critical parameter. Replacing Pd(OAc)₂ with PdCl₂ significantly improved the yield to 54% (Table [1], entry 10). PdCl₂ is known for its higher activity in oxidative addition reactions compared to Pd(OAc)₂, which could explain the improved yield. In contrast, Pd(PPh₃)₂Cl₂ resulted in no reaction (Table [1], entry 11), possibly due to the steric bulk of the phosphine ligands hindering substrate coordination or oxidative addition.

Temperature optimization revealed that increasing the reaction temperature enhanced the yield. Raising the temperature from 80 °C to 120 °C progressively increased the yield (Table [1], entries 12–14), with the highest yield of 68% achieved at 120 °C. This suggests that higher temperatures facilitate the reaction kinetics, overcoming activation barriers. However, further increasing the temperature to 130 °C resulted in a decreased yield (61%, Table [1], entry 15), likely due to side reactions, substrate decomposition, or catalyst deactivation. This observation highlights the importance of identifying the optimal temperature window for maximizing product formation. The reaction time was optimized at 4 h under the best conditions. Prolonged reaction times did not significantly improve yields but increased the likelihood of side reactions. A shift from propylene carbonate to solvents like acetone, toluene, and benzene (Table [1], entries 16–18) drastically reduced the product yield. Screening alternative organic bases such as triethylamine, DIPEA, DIPA, and DBU (Table [1], entries 19–22) under otherwise optimal conditions resulted in no reaction or low yields, reaffirming the superiority of K₂CO₃. When the other catalysts were explored Pd₂(dba)₃ and Pd(PPh₃)₄ (Table [1], entries 23 and 24), moderate and trace product formation was observed, respectively. Notably, switching to nickel-based catalysts; Ni(COD)₂ and NiCl₂, (Table [1], entries 25 and 26) provided modest to no yields, further confirming the essential role of PdCl₂ in facilitating the cyclization process. Additionally, the impact of varying the halogen substituent on the benzyl bromide was examined; using 2-chlorobenzyl bromide resulted in only trace product (Table [1], entry 27), while 2-iodobenzyl bromide afforded a 60% yield (Table [1], entry 28).

The optimized conditions – PdCl₂ (10 mol%), K₂CO₃ (3 equiv), propylene carbonate as solvent, and heating at 120 °C for 4 h – resulted in a 68% yield of the isochroman-fused coumarin derivative. This yield, while moderate, was achieved under environmentally benign conditions, utilizing a green solvent and a relatively mild base. The use of PdCl₂ as a catalyst aligns with established palladium-catalyzed transformations, and the choice of propylene carbonate significantly contributes to the overall sustainability of the process.

Plausible Mechanism

Based on the literature report,[35] [36] a possible mechanism is outlined in Scheme [2]. The formation of isochroman-fused coumarins involves a sequential nucleophilic substitution and a palladium-catalyzed cyclization. Initially, 4-hydroxycoumarin (1a) is deprotonated by K₂CO₃, yielding the nucleophilic enolate 1a′. This enolate then reacts with 2-bromobenzyl bromide (2a) via an S_N2 mechanism, generating the O-alkylated intermediate 3a. The catalytic cycle commences with the reduction of Pd(II) to Pd(0). Oxidative addition of Pd(0) to the C–Br bond of 3a forms the organopalladium complex 5. Subsequently, concerted metalation–deprotonation (CMD) activates the sp² carbon, leading to intramolecular cyclization and the formation of the palladacycle 6. Finally, reductive elimination releases the desired product 4a and regenerates the Pd(0) catalyst. This environmentally benign synthesis, utilizing propylene carbonate, efficiently produces the fused coumarin under mild conditions.

With the optimal reaction parameters established, a diverse library of isochroman-fused coumarins 4a–i was efficiently synthesized. Utilizing substituted 4-hydroxycoumarins 1a–h and 2-bromobenzyl bromide derivatives 2a,b, the one-pot, two-step protocol, employing PdCl₂ (10 mol%), K₂CO₃ (3 equiv), and propylene carbonate at 120 °C for 4 h, yielded the desired products in 50–68% yield (Scheme [1]). The observed yield variations correlated with electronic and steric effects of the substituents on the coumarin and benzyl moieties. Structural confirmation of all synthesized compounds was achieved through ¹H NMR, ¹³C NMR, and HRMS analysis, with single-crystal X-ray diffraction providing definitive structural elucidation for compound 4b (Figure [2]). The successful application of this optimized protocol underscores its robustness and versatility in generating novel isochroman-fused coumarins 4a–i for subsequent antifungal evaluation against Rhizoctonia solani.

In Silico Studies

Molecular docking, a critical computational tool in drug discovery, was employed to predict the binding interactions of the synthesized isochroman-fused coumarin derivatives with sterol 14α-demethylase (CYP51, PDB ID: 3GW9). CYP51, a key enzyme in ergosterol biosynthesis, is essential for fungal cell membrane integrity, making it a strategic target for antifungal drug development.[37] AutoDock Vina was utilized to analyze the binding affinity and interaction profiles of the synthesized compounds, aiming to understand their potential as CYP51 inhibitors.[37]

The docking results revealed that all synthesized compounds exhibited superior binding affinities compared to the reference antifungal agent, hexaconazole (–7.4 kcal/mol). Notably, compounds 4b, 4f, and 4i demonstrated the most favorable binding energies, with values of –8.2 kcal/mol, –8.1 kcal/mol, and –8.1 kcal/mol, respectively (Table [2]). These more negative binding energies suggest stronger binding interactions and a higher potential for effective CYP51 inhibition. Detailed analysis of the binding interactions between the synthesized compounds and the active site residues of CYP51 was performed, providing insights into the molecular basis of their binding (Figure S20).

The observed enhanced binding affinities indicate that the synthesized isochroman-fused coumarins may exhibit stronger target engagement and potentially superior antifungal activity compared to hexaconazole. The docking poses of compounds 4b, 4f, and 4i, presented in Figure [3], illustrate their specific interactions within the CYP51 active site, highlighting key residues involved in ligand binding. These visual representations provide valuable insights into the spatial orientation and interactions of the compounds within the enzyme’s binding pocket.

Bioefficacy Evaluation: In Vitro Fungicidal Activity

The antifungal efficacy of the synthesized isochroman-fused coumarin derivatives 4a–i was evaluated against the plant pathogen Rhizoctonia solani through in vitro studies. The half-maximal effective concentration (ED₅₀), representing the compound concentration required to inhibit 50% fungal growth, was determined from at least four independent experiments.

The compounds were tested at five concentrations (10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.62 μM) using potato dextrose agar (PDA) culture media. The results demonstrated significant antifungal activity, with all synthesized compounds effectively suppressing Rhizoctonia solani growth. Notably, compound 4e exhibited the highest antifungal potency, with an ED₅₀ value of 3.59 μM.

The antifungal activity of each compound, along with statistical parameters including fiducial limits, chi-square values, and regression equations, is summarized in Table [3]. Comparative performance of the synthesized compounds and the reference antifungal agent, hexaconazole, is illustrated graphically in Figures [4] and 5.

These in vitro findings highlight the potential of isochroman-fused coumarin derivatives as promising antifungal agents for managing Rhizoctonia solan induced infections in crops. The observed potency of these compounds suggests their potential as eco-friendly alternatives to conventional fungicides. However, further in vivo studies, including field trials, are essential to validate their efficacy under real-world conditions and assess their potential for commercial application.

In conclusion, this study has demonstrated the successful synthesis and evaluation of isochroman-fused coumarin derivatives as effective antifungal agents against Rhizoctonia solani.[38] [39] The efficient, green synthetic methodology provided a series of compounds that exhibited potent in vitro antifungal activity. In silico docking studies confirmed strong binding to CYP51, validating the target engagement. These findings highlight the potential of these novel compounds as promising alternatives to conventional fungicides, offering an environmentally responsible approach to controlling rice sheath blight and protecting crop yields. Further in vivo evaluations are warranted to assess their field efficacy and explore their potential for agricultural applications. Additionally, investigations into structure–activity relationships and mechanism of action will further optimize these compounds for improved antifungal performance.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

We appreciate the assistance provided by University Science Instrumentation Centre (USIC) and Department of Chemistry, University of Delhi for recording NMR, HRMS and IR data.

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• 38 General Procedure for the Synthesis of Isochroman-Fused Coumarins 4a–i 4-Hydroxy coumarins 1a–h (1 equiv) and 2-bromobenzyl bromide 2a,b (1 equiv) were dissolved in propylene carbonate (5 mL) in the presence of potassium carbonate base (3 equiv) and 10 mol% PdCl₂ as a catalyst in a 50 mL round-bottomed flask. The reaction mixture stirred for 4 h at 120 °C. Chloroform was then used to extract the resultant reaction mixture (3 × 30 mL) after the indication of completion of the reaction by TLC. The combined extracts were washed with brine solution, then dried using anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure under a vacuum resulting in the isolation of the crude product. The crude product thus obtained was purified by silica gel chromatography using ethyl acetate in petroleum ether as a gradient solvent system. The separated pure products were characterized by ¹H, ¹³C NMR spectroscopy, mass spectroscopy, and IR spectroscopy.
• 39 6H,11H-Isochromeno[4,3-c]chromen-11-one (4a) It was obtained as a white solid in 68% yield using 5% ethyl acetate in petroleum ether as an eluent, mp 105–107 °C; R _ƒ = 0.4 (8% ethyl acetate in petroleum ether). IR (KBr): 1702, 1612, 1490, 1332, 1286, 1091, 913, 750 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 8.56 (1 H, d, J = 7.9 Hz), 7.86–7.88 (1 H, m), 7.57 (1 H, t, J = 8.4 Hz), 7.42 (1 H, t, J = 7.6 Hz), 7.32 (3 H, dt, J = 15.2, 7.7 Hz), 7.13 (1 H, d, J = 7.4 Hz), 5.41 (2 H, s). ¹³C NMR (100.6 MHz, CDCl₃): δ = 161.3, 160.3, 153.0, 132.6, 129.2, 128.4, 127.5, 126.8, 125.0, 124.2, 124.1, 123.2, 116.7, 115.3, 102.8, 69.9. HRMS (ESI): m/z calcd for C₁₆H₁₁O₃ [M + H]⁺: 251.0703; found: 251.0705.

Corresponding Author

Publication History

Received: 10 March 2025

Accepted after revision: 19 May 2025

Accepted Manuscript online:
20 May 2025

Article published online:
02 July 2025

© 2025. Thieme. All rights reserved

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• References and Notes

• 1 John DA, Babu GR. Front. Sustain. Food Syst. 2021; 5: 644559
  PubMedSearch in Google Scholar
• 2 Shiferaw B, Smale M, Braun HJ, Duveiller E, Reynolds M, Muricho G. Food Secur. 2013; 5: 291
  Search in Google Scholar
• 3 Iqbal J, Zia-ul-Qamar, Yousaf U, Asgher A, Dilshad R, Qamar FM, Bibi S, Rehman SU, Haroon M. In Sustainable Agriculture in the Era of the OMICs Revolution . Springer International Publishing; Cham: 2023: 241
  Search in Google Scholar
• 4 Ayaz M, Li CH, Ali Q, Zhao W, Chi YK, Shafiq M, Ali F, Yu XY, Yu Q, Zhao JT, Yu JW. Molecules 2023; 28: 6735
  PubMedSearch in Google Scholar
• 5 Strange RN, Scott PR. Annu. Rev. Phytopathol. 2005; 43: 83
  PubMedSearch in Google Scholar
• 6 Singh A, Rohilla R, Singh US, Savary S, Willocquet L, Duveiller E. Can. J. Plant Pathol. 2002; 24: 65
  Search in Google Scholar
• 7 Senapati M, Tiwari A, Sharma N, Chandra P, Bashyal BM, Ellur RK, Bhowmick PK, Bollinedi H, Vinod KK, Singh AK, Krishnan SG. Front. Plant Sci. 2022; 13: 881116
  PubMedSearch in Google Scholar
• 8 Singh A, Rohila R, Willocquet SS. L, Singh US. Indian Phytopathol. 2003; 56: 434
  Search in Google Scholar
• 9 Aydın MH. Türkiye Tarımsal Araştırmalar Dergisi 2022; 9: 118
  Search in Google Scholar
• 10 Almeida F, Rodrigues ML, Coelho C. Front. Microbiol. 2019; 10: 214
  PubMedSearch in Google Scholar
• 11 Wu TL, Zhang BQ, Luo XF, Li AP, Zhang SY, An JX, Zhang ZJ, Liu YQ. Ind. Crops Prod. 2023; 191: 115975
  Search in Google Scholar
• 12 Upadhyay RK, Saini KK, Deswal N, Singh T, Tripathi KP, Kaushik P, Shakil NA, Bharti AC, Kumar R. RSC Adv. 2022; 12: 24412
  PubMedSearch in Google Scholar
• 13 Shen HC. Tetrahedron 2009; 65: 3931
  CrossrefSearch in Google Scholar
• 14 Xie S, Li X, Yu H, Zhang P, Wang J, Wang C, Xu S, Wu Z, Liu J, Zhu Z, Xu J. Bioorg. Med. Chem. 2019; 27: 2764
  PubMedSearch in Google Scholar
• 15 Patel GM, Deota PT. Heterocycl. Commun. 2014; 20: 299
  Search in Google Scholar
• 16 Xie S, Li X, Yu H, Zhang P, Wang J, Wang C, Xu S, Liu J, Zhu Z, Wu Z, Xu J. Bioorg. Med. Chem. 2019; 27: 2764
  PubMedSearch in Google Scholar
• 17 Manivel P, Sharma A, Maiyalagan T, Rajeswari MR, Khan FN. Phosphorus, Sulfur Silicon Relat. Elem. 2010; 185: 387
  Search in Google Scholar
• 18 Mateos R, Madrona A, Pereira-Caro G, Domínguez V, Cert RM, Parrado J, Sarria B, Bravo L, Espartero JL. Food Chem. 2015; 173: 313
  PubMedSearch in Google Scholar
• 19 Wang J, Han G, Wei Y, Jiang G. Chin. J. Chem. 1998; 15: 112
  Search in Google Scholar
• 20 Tamanna Tamanna, Kumar M, Joshi K, Chauhan P. Adv. Synth. Catal. 2020; 362: 1907
  CrossrefSearch in Google Scholar
• 21 Dillon MP, Simpson TJ, Sweeney JB. Tetrahedron Lett. 1992; 33: 7569
  Search in Google Scholar
• 22 He G, Matsuura H, Takushi T, Kawano S, Yoshihara T. J. Nat. Prod. 2004;  67: 1084
  PubMedSearch in Google Scholar
• 23 Inagaki T, Kaneda K, Suzuki Y, Hirai H, Nomura E, Sakakibara T, Yamauchi Y, Huang LH, Norcia M, Wondrack JA, Sutcliffe JA, Kojima SA, Kojima N. J. Antibiot. 1998; 51: 112
  Search in Google Scholar
• 24 Saeed A, Mumtaz A. J. Saudi Chem. Soc. 2017;  21: 186
  Search in Google Scholar
• 25 Rusman Y, Held BW, Blanchette RA, Wittlin S, Salomon CE. J. Nat. Prod. 2015;  78: 1456
  PubMedSearch in Google Scholar
• 26 McMullin DR, Nsiama TK, Miller JD. J. Nat. Prod. 2014;  77: 206
  PubMedSearch in Google Scholar
• 27 Stefanachi A, Francesco L, Leonardo P, Marco C, Angelo C. Molecules 2018; 23: 1
  Search in Google Scholar
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• 38 General Procedure for the Synthesis of Isochroman-Fused Coumarins 4a–i 4-Hydroxy coumarins 1a–h (1 equiv) and 2-bromobenzyl bromide 2a,b (1 equiv) were dissolved in propylene carbonate (5 mL) in the presence of potassium carbonate base (3 equiv) and 10 mol% PdCl₂ as a catalyst in a 50 mL round-bottomed flask. The reaction mixture stirred for 4 h at 120 °C. Chloroform was then used to extract the resultant reaction mixture (3 × 30 mL) after the indication of completion of the reaction by TLC. The combined extracts were washed with brine solution, then dried using anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure under a vacuum resulting in the isolation of the crude product. The crude product thus obtained was purified by silica gel chromatography using ethyl acetate in petroleum ether as a gradient solvent system. The separated pure products were characterized by ¹H, ¹³C NMR spectroscopy, mass spectroscopy, and IR spectroscopy.
• 39 6H,11H-Isochromeno[4,3-c]chromen-11-one (4a) It was obtained as a white solid in 68% yield using 5% ethyl acetate in petroleum ether as an eluent, mp 105–107 °C; R _ƒ = 0.4 (8% ethyl acetate in petroleum ether). IR (KBr): 1702, 1612, 1490, 1332, 1286, 1091, 913, 750 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 8.56 (1 H, d, J = 7.9 Hz), 7.86–7.88 (1 H, m), 7.57 (1 H, t, J = 8.4 Hz), 7.42 (1 H, t, J = 7.6 Hz), 7.32 (3 H, dt, J = 15.2, 7.7 Hz), 7.13 (1 H, d, J = 7.4 Hz), 5.41 (2 H, s). ¹³C NMR (100.6 MHz, CDCl₃): δ = 161.3, 160.3, 153.0, 132.6, 129.2, 128.4, 127.5, 126.8, 125.0, 124.2, 124.1, 123.2, 116.7, 115.3, 102.8, 69.9. HRMS (ESI): m/z calcd for C₁₆H₁₁O₃ [M + H]⁺: 251.0703; found: 251.0705.

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