Design and synthesis of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives via the Diels–Alder reaction: molecular docking, antibacterial activity, ADMET analysis and photophysical properties

Abstract

A series of fused tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives was successfully synthesized via the Diels–Alder reaction. Molecular docking studies were conducted to understand the interaction modes between the synthesized hybrid compounds and the receptor bacterial strains of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Notably, the in silico results demonstrated that compound 19l (−8.7 kcal mol⁻¹ with E. coli and −8.4 kcal mol⁻¹ with S. aureus) and 19p (−8.7 kcal mol⁻¹ with E. coli and −9.1 kcal mol⁻¹ with S. aureus) exhibited good binding values. Additionally, the in vitro antibacterial studies showed that compounds 19l and 19p demonstrated excellent antibacterial activities, with a zone of inhibition (ZI) of 17 mm and a minimum inhibitory concentration (MIC) of 12.5 μg mL⁻¹ against both E. coli and S. aureus, which were comparable to the performance of the standard antibiotic ciprofloxacin. Further, the bioavailability was assessed through virtual ADMET parameters, which suggested that most of the compounds possessed favorable pharmacokinetic profiles. To further enrich the study, photophysical properties of all the synthesized molecules were also examined using UV-visible and fluorescent spectroscopies.

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1. Introduction

Bacterial infections have become a major problem globally.^1,2 The misuse and overuse of antibiotics have led to the emergence of antimicrobial resistance (AMR), resulting in rapid rise of antibiotic-resistant strains of bacteria known as.^3,4 Antibiotic-resistant infections result in severe illnesses, prolonged hospital stays, higher medical costs, and treatment failures. Over the past two decades, microbial infections have significantly increased.⁵ Each year, there is a death range of approximately 70 000 worldwide due to antimicrobial resistance, and it is estimated that it may increase to 10 million cases per year by 2050.^6–8 Despite the discovery and development of numerous new anti-microbial drugs, there is still an increase in bacterial infections, and their treatment is still a major challenge for public health.^9,10 Therefore, the discovery of novel, cost-effective antimicrobial agents is necessary to mitigate the proliferation of these pathogens.

Among the promising scaffolds for antibacterial drug development, the 2H-chromene core has garnered significant attention owing to its diverse biological activities.^11–13 Studies have demonstrated that the 2H-chromene derivatives exhibit potent antibacterial properties, along with other pharmacological effects such as anticancer, anti-inflammatory, anti-tubercular, and anti-HIV activities.^14–23 This versatile scaffold provides a robust platform for designing novel therapeutic agents with improved efficacy and selectivity against bacterial pathogens.

Similarly, maleimides are the nitrogen-containing heterocyclic motifs which have shown substantial promise in biomedical research. For instance, N-disubstituted maleimides represent a valuable structural motif in drug discovery, contributing to the development of novel therapeutic agents targeting bacterial infections.^24–27 By combining the structural features of 2H-chromene and maleimide, there is an opportunity to enhance antibacterial efficacy through hybrid molecules that leverage the biological potency of both scaffolds (Fig. 1).^24–27

Fig. 1 Structures of some selected bioactive molecules of 2H-chromenes and maleimide.

Previous studies have explored the synthesis of chromene-based maleimide hybrids using various chemical transformations. In 2006, Park et al. first reported the synthesis of novel benzopyran-based scaffolds through Diels–Alder reactions.²⁸ Subsequent work by the same research group in 2010 demonstrated the biological potential of these hybrids in treating prostate cancer, diabetes, and obesity.^29,30 However, their synthetic methods relied on expensive and carcinogenic reagents, such as trifluoromethanesulfonic anhydride and tin catalysts, limiting their scalability and environmental compatibility.

To address these limitations and develop a more cost-effective and eco-friendly approach, we present an efficient synthesis of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives. Our strategy involves phosphorus tribromide and palladium catalyst, which are more economical and environmentally benign alternatives. This method also explored diverse substituents on the chromene framework and the para position of the styrene ring, enhancing the scope of structural modifications.

As part of our ongoing efforts to develop tricyclic benzopyran derivatives as antibacterial agents, we recently synthesized a series of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives and evaluated their potent antibacterial activities. Molecular docking studies provided insights into their binding affinities and possible mechanisms of action. Furthermore, in silico ADMET analyses were conducted to assess their pharmacokinetic properties, and in vitro antibacterial assays were performed against two pathogenic bacterial strains to evaluate their efficacy. Spectroscopic studies, including UV-visible and fluorescence analyses, were also carried out to investigate the impact of various substituents on the photophysical properties of the synthesized compounds. This comprehensive investigation aims to advance the development of potent, cost-effective antibacterial agents to combat the rising challenge of antibiotic resistance.

2. Result and discussion

2.1. Chemistry

2.1.1 Synthesis. Given our significant research focus on synthesizing chromene-fused six-membered carbacycles using efficient and environmentally friendly methods, our initial studies included the synthesis of the parent pharmacophore, i.e., 2,2-dialkyl-4-styryl-2H-chromene 17a. Our experiment began with the aldol condensation of o-hydroxy acetophenone 11(a–b) and substituted ketones 12(a–d), followed by intramolecular cyclization in the presence of pyrrolidine, leading to the formation of 2,2-disubstituted chromones 13(a–e). Treatment of PBr₃ with these chromones 13(a–e) in neat condition afforded 4-bromo-2,2-disubstituted-2H-chromene 15(a–e).^31–37 Having 4-bromo-2,2-dialkyl-2H-chromene 15(a–e), we intended to couple 15(a–e) with substituted styrene 16(a–c) to synthesize 2,2-dialkyl-4-styryl-2H-chromene 17(a–n) according to the known literature method (Scheme 1).^14,15

Scheme 1 Synthesis of 2,2-disubstituted chromones 13(a–e), 4-bromo-2,2-dialkylsubstituted-2H-chromenes 15(a–e) and 2,2-disubstituted-4-styryl-2H-chromene 17(a–n).

After successfully synthesizing the parent pharmacophore 17a, we synthesized the target hybrid molecule 19a using commercially available N-methyl maleimide 18a. To synthesize the fused carbacycle 19a through a Diels–Alder reaction, we screened the reaction conditions by altering the solvent, time and temperature.

According to the previously reported literature,^14,15 the reaction was first carried out in different solvents, including DCM, THF, 1,4-dioxane, DMF and benzene at their respective boiling points for about 12–24 hours, which resulted in poor to moderate yield (entries 1–6). However, the reaction did not proceed in H₂O (entry 7). We observed an increase in the product yield when the reaction temperature was raised from 80 °C to 100 °C using toluene as the solvent while maintaining the same reaction time. However, the starting materials were not fully consumed at this stage. Upon further increasing the temperature to 120 °C, we achieved excellent yield within 4 hours. This improvement may be due to the energy barrier of the reactants being overcome at higher temperatures, thereby facilitating the reaction (entries 8–10). The results are illustrated in Table 1. After the successful completion of the reaction, product 19a was characterized using ¹H NMR, ¹³C NMR and HRMS analyses.

Table 1 Exploring the reaction conditions for the Diels–Alder reaction to synthesize 19a

Entry^a	Maleimide equiv.	Solvent	Temp. (°C)	Time (h)	Yield (%)
a Reaction conditions: 17a (1 equiv.), 18 (3 equiv.), toluene (2–3 mL); isolated yield; n.r. = no reaction.
1	1	DCM	35	24	12
2	2	DCM	35	24	22
3	2	THF	60	24	28
4	2	1,4-Dioxane	100	24	32
5	2	DMF	150	24	37
6	2	Benzene	80	12	51
7	2	H2O	100	24	NR
8	2	Toluene	80	12	59
9	2	Toluene	100	12	76
10	3	Toluene	120	04	91

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2.1.2 Characterization. The structure of product 19a was elucidated using the spectroscopic technique ¹H, ¹³C NMR and HRMS. In the proton NMR spectrum, the characteristic H₁₃ proton appeared at 6.59–6.57 ppm (m, 1H). Four stereogenic H₃, H₁₄, H₂₁ and H₂₅ protons attributed to the peaks at 2.65 ppm (s, 1H), 3.73–3.70 ppm (m, 1H), 3.38–3.34 ppm (m, 1H) and 3.52–3.49 ppm (m, 1H), respectively, which clearly proved the incorporation of the maleimide ring in the 4-styryl-2H-chromene core. The two-methyl protons H₁₁ and H₁₂ appeared as two sharp singlets at 1.30 ppm and 1.82 ppm, respectively. Furthermore, the N-methyl proton displayed one sharp singlet at 2.65 ppm, confirming that 19a was formed. The remaining aromatic protons appeared at their respective regions, signifying the conformation of the structure. In the ¹³C NMR, the two characteristic carbonyl carbons appeared at δ 175.7 (C₂₂) ppm and δ 175.3 (C₂₄) ppm, respectively. The signal at δ 29.1 ppm corresponded to the N-methyl carbon. Two methyl carbons of the chromene core appeared at δ 24.8 ppm and δ 26.0 ppm. The signals at the region δ 75.1 ppm represent the quaternary C₂ carbon of the chromene framework and all other carbon signals resonate at appropriate positions. In the HRMS spectrum, the experimental molecular ion peak appeared at 374.1780 [M + H]⁺, which is equivalent to the calculated ion peak at 374.1756, confirming the formation of 19a (Fig. 2).

Fig. 2 ¹H and ¹³C NMR spectral data analysis of compound 19a.

After optimizing the reaction conditions and spectroscopic characterization, we next explored the scope and generality of the reaction on various substrates, as shown in Scheme 2.

Scheme 2 Synthesis of a library of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q).

2,2-Disubstituted-4-styryl-2H-chromene 17(a–n) initially reacted with N-methyl maleimide 18a, resulting in tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives in good to excellent yield. From Scheme 2, it is observed that the yield of the cyclized product decreases with an increase in the alkyl chain length at the R₂ position of the chromene moiety. Compounds having 2,2-dimethyl substituents at the R₂ position of 19(a–c) displayed excellent yield (91–94%). Further, by modifying the methyl group to ethyl group 19(d–f), there is a slight decrease in the yield of the cyclized product (88–91%). Similarly, having 2,2-dipropyl substituents in 19(g–i), the yield decreases significantly (83–89%). However, compounds 19(j–n) show remarkable yields (84–90%) when a methyl group is introduced at the R₁ position of the chromene framework.

Furthermore, to increase the substrate scope and check the yield of the cyclized product, 2,2-disubstituted-4-styryl-2H-chromene 17(o–q) was further reacted with N-phenyl maleimide 18b to give the desired derivatives in good yield (73–79%). It has been concluded that N-methyl maleimide derivatives give higher yield as compared to N-phenyl maleimide, as shown in Scheme 2. The structures of cycloadducts 19(a–q) were confirmed by ¹H, ¹³C, and HRMS spectroscopic techniques.

2.1.3 X-ray crystallographic analysis. The single X-ray crystallographic data is represented in Fig. 3. (CCDC-2321501), which represents the four new stereogenic protons, are in the same face.³⁸

Fig. 3 Single crystal X-ray representation of compound 19i.

2.1.4 Plausible mechanism. The plausible mechanism of 2,4,4-trimethyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19a is shown in Scheme 3. The Diels–Alder reaction between 2,2-dimethyl-4-styryl-2H-chromene 17a with N-methyl maleimide 18a yields a six-membered ring with four stereocenters. In the Diels–Alder reaction, the endo approach predominates over the exo approach due to secondary interactions; hence, we presumed the stereochemistry of four stereogenic hydrogens are in the same face and the plane of ring is influenced by the endo approach of the dienophile with diene in a transition state.

Scheme 3 Proposed mechanism of 19a.

2.1.5 Transformations of 11-(4-methoxyphenyl)-2-methyl-4,4-dipropyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19i. To demonstrate the synthetic utility of our designed molecule from previously described methods,³⁹ we proposed a derivatization reaction, as described in Scheme 4. Initially, the cyclo-adduct11-(4-methoxyphenyl)-2-methyl-4,4-dipropyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19i reacted with DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) (1.5 equiv.) in the presence of toluene at 100 °C for 30 min to afford the aromatized product 11-(4-methoxyphenyl)-2-methyl-4,4-dipropylchromeno[3,4-e]isoindole-1,3(2H,4H)-dione 21 in excellent yield. After the synthesis and confirmation of the structure from ¹H NMR,¹³C NMR and HRMS spectroscopic techniques, we further progressed to study the in silico and in vitro antimicrobial activity and ADMET study of all the synthesized cyclized products 19(a–q).

Scheme 4 Synthesis of 11-(4-methoxyphenyl)-2-methyl-4,4-dipropylchromeno[3,4-e]isoindole-1,3(2H,4H)-dione 21.

3. Biological studies

3.1. Molecular docking study

Molecular docking studies of the synthesized hybrid tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19(a–q) derivatives were performed to identify the binding interaction with the DNA gyrase inhibitor. The Gram-negative bacterial strain E. coli and Gram-positive bacterial strain S. aureus are two human pathogenic bacterial strains of the DNA gyrase inhibitor used for molecular modelling. The crystal structure of E. coli (PDB ID: 3G7E) and S. aureus (PDBID: 3G7B) were retrieved, and the heteroatoms and water molecules were removed during the docking procedure. Autodock tool 4.2.0 was used to determine the binding affinity scores, and the Discovery Studio v 2017 program was employed in visualizing the intermolecular binding interaction of the docked complex. In this study, seventeen newly designed hybrid molecules were successfully docked, and the binding affinity values ranged from −5.6 kcal mol⁻¹ to −8.7 kcal mol⁻¹ in the case of E. coli and −6.9 kcal mol⁻¹ to −9.1 kcal mol⁻¹ in the case of S. aureus. The current investigation revealed that compound 19l possessing diethyl at the R₂ position of the chromene ring, methyl at the R₁ position, hydrogen at the R₃ position and methyl at the R₄ position showed very good yield −8.7 kcal mol⁻¹ with E. coli DNA gyrase and −8.4 kcal mol⁻¹ with S. aureus DNA gyrase. Furthermore, compound 19p possesses dimethyl at the R₂ position of the chromene ring, methyl at the R₃ position and phenyl group at the R₄ position, demonstrating robust binding interactions with a binding energy value of −8.7 kcal mol⁻¹ for E. coli (PDB ID: 3G7E) and −9.1 kcal mol⁻¹ for S. aureus (PDBID: 3G7B). The synthesized compounds interacted with bacterial DNA gyrase through various interactions such as van der Waals, conventional hydrogen bond, carbon–hydrogen bond, alkyl, π–alkyl, π–anion, π–cation, and π–sigma. The docking scores and their binding interactions are displayed in Table 2.

Table 2 Docking result scores and binding interactions of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives

Sl no.	E. coli (PDB ID: 3G7E)		S. aureus (PDBID: 3G7B)
	Score (kcal mol^−1)	Binding interactions	Score (kcal mol^−1)	Binding interactions
19a	−7.1	Arg62, Ala39, Pro65, Lys89	−8.1	Ile79, Ile63, Ala38
19b	−6.2	Asn32, Phe90, Ile80, Ala76, Pro65, Ile64	−7.8	Asn31, Ile63, Ala38
19c	−7.9	Pro65, Asp35, Phe90, His102, Ala39, Lys89, Ile64, Ala76, Ile80	−8.2	Arg61, Ser32, Ala38, Asn31, Ile79, Pro64, Ile63
19d	−7.9	Arg62, Pro65, Lys89, Ala39, Ile64	−8.3	Asn31, Pro64
19e	−7.4	Arg62, Leu38, Lys89, Val97	−8.3	Asn31, Glu35, Arg61, Ile63, Ala38
19f	−6.5	Phe90, Arg62, Leu38, Pro65, Ala39, Lys89	−7.0	Asn31, Glu35, Pro64, Ile79, Ile63, Ala75
19g	−6.7	Arg62, Leu38, His41, Ala39, Lys89	−7.9	Glu35, Arg61, Ile79, Ile63, Pro64
19h	−5.6	Phe90, Glu36, Arg62, Ile80, Pro65, Ile64, Ala39, Lys89	−7.9	Arg61, Ile63, Ile79, Pro64, Ala38
19i	−8.2	Asp35, Pro65, Ile64, Ile80, Leu38, Ala39, Lys89	−6.9	Asn31, Pro64, Ile63
19j	−6.9	Leu38, Glu36, Lys89, Ala39, Asp35, Val97	−9.1	Glu35, Arg61, Asn31, Ile63, Ile79, Ile129, Pro64
19k	−6.9	Ala39, Arg62, Glu36, Pro65, Ala39	−8.2	Arg61, Ile63, Ile79, Ala38
19l	−8.7	Thr151, Ile64, Pro65, Ala33, Glu36, Arg62, Phe90, Lys89, Ala39, Ile80	−8.4	Asn31, Arg61, Ile79, Pro64, Ile63, Ala38
19m	−7.1	Arg62, Glu36, Asp35, Lys89, His102, Ala39	−8.4	Asn31, Arg61, Glu35, Gly62, Pro64, Ile63
19n	−8.3	Arg62, Ala86, Asp59, Lys89, Val106, Val29, Ala33, Ile64, Pro65, Ile80	−8.4	Asn31, Ile79, Ile63
19o	−6.4	Arg62, Lys89, Asn32, Pro65, Ile64, Phe90	−8.6	Arg61, Ile63, Asn31, Ile79, Pro64
19p	−8.7	Arg62, Val97, Lys89, Ala39, Ile64, Pro65	−9.1	Asn31, Glu35, Arg61, Ile63, Ile79, Pro64
19q	−7.5	Arg62, Glu36, Asp35, Ala39, Lys89, Val97	−8.7	Glu35, Arg61, Ile79, Ile63, Pro64

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From Table 2, it is noted that compound 19l has very good binding interactions with the active pocket site of E. coli and S. aureus DNA gyrase. The docked complex with DNA gyrase E. coli displays a series of van der Waals interactions with specific amino acid residues Asp92, Arg122, Asp91, Ala76, Asp59, Gly63, Gly61, Gly88, Ala86, Gly87, His102, Asp35, Leu38, carbon hydrogen bonds with Pro65, Thr151, Ile64, Ala33, π–cation and π–anion interactions with Arg62, Glu36, alkyl interaction with Ala39, Phe90, Lys89, Pro65, Ile64, and π–alkyl interaction with Ile80, Ala39, Lys89 for compound 19l. Similarly, compound 19l held into the active cavities with the DNA gyrase enzymes of S. aureus (PDBID: 3G7B) via van der Waals interactions with Ala75, Asp34, Glu35, Ser32, Gly62, Ser83, conventional hydrogen bond with Asn31, π–cation interaction with Arg61, π–sigma interaction with ILE79, alkyl interaction with Ile63, Pro64 and π-alkyl interaction with Ala38, Ile63 and Pro64. Compound 19p also displayed a remarkable result for both the docked complexes: van der Waals interactions with Gly99, Ser98, Gly100, His102, Asp35, Gly88, Ile80, Ala76, Phe90, Glu36, Asp91, conventional hydrogen bond with Arg62, alkyl interaction with Val97, Lys89, π–alkyl interaction with Ala39, Pro65, Ile64 in the case of E. coli DNA gyrase. Alternatively, S. aureus shows van der Waals interactions with Arg98, Gly62, Ser32, Asp58, Thr127, Ile129, Ile28, Asp34, conventional hydrogen bond with Asn31, π–cation and π–anion interactions with Arg61, Glu35, π–sigma with Ile63, alkyl and π–alkyl interaction with Ile79 and Pro64 for compound 19p. From the docking score, it is observed that the compounds 19l and 19p display promising results in terms of antibacterial activities. The 2D and 3D diagrams of the most potent compounds, 19l and 19p, against E. coli DNA gyrase and S. aureus DNA gyrase, are represented in Fig. 4 and 5, respectively.

Fig. 4 2D interactions of the most potent compounds (A) compound 19l and (B) Compound 19p with Gram-negative bacterial DNA gyrase (PDBID: 3 G7E). (C) Compound 19l and (D) compound 19p with Gram-positive bacterial DNA gyrase (PDBID: 3G7B).

Fig. 5 Most potent molecular docking studies of maleimide derivatives. The images depict the protein and ball-stick models. (A) Compound 19l and (B) compound 19p with Gram-negative bacterial DNA gyrase (PDBID: 3G7E). (C) Compound 19l and (D) Compound 19p with Gram-positive bacterial DNA gyrase (PDBID: 3G7B).

3.2. Antibacterial evaluation

A series of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q) were investigated for in vitro antibacterial activities against two human bacterial strains. All the synthesized derivatives were evaluated against Gram-negative E. coli and Gram-positive S. aureus via the agar well diffusion method using the standard drug ciprofloxacin. The ZI (zone of inhibition) and MIC (minimum inhibitory concentration) values of those molecules against these bacteria are also determined and the molecules are found to be active against both the bacterial strains, as represented in Table 3.

Table 3 Antibacterial activities of the newly synthesized tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q). (Abbreviations: ZI, zone of inhibition; MIC, minimum inhibitory concentration. *Standard drug: ciprofloxacin)

Sl. no.	E. coli		S. aureus
	ZI (mm)	MIC (μg mL^−1)	ZI (mm)	MIC (μg mL^−1)
19a	15	25	16	25
19b	13	50	15	25
19c	16	25	16	25
19d	16	25	16	25
19e	15	25	16	25
19f	14	50	14	50
19g	14	50	15	25
19h	12	50	15	25
19i	16	50	14	50
19j	14	50	17	12.5
19k	14	50	16	25
19l	17	12.5	17	12.5
19m	15	25	16	25
19n	16	25	16	25
19o	14	50	17	12.5
19p	17	12.5	17	12.5
19q	15	25	16	25
^StdCiprofloxacin	—	6.25	—	6.25

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Among all the tested compounds, compound 4,4-diethyl-2,8-dimethyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19l and 4,4-dimethyl-2-phenyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19p were found to be the most potent compounds with a Z1 of 17 mm against both E. coli and S. aureus bacterial strains. Furthermore, we performed a cell viability assay to determine the minimum inhibitory concentration (MIC) of the compounds. The MIC results revealed that both E. coli and S. aureus displayed potent activity with an MIC value of 12.5 μg mL⁻¹ for both compounds 19l and 19p. However, from the antibacterial study, we found that compound 2-methyl-4,4-dipropyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19h possessed notably poor antimicrobial activity with a ZI value of 12 mm and 15 mm for E. coli and S. aureus, respectively. The respective ZI values and MIC values of all the tested compounds are illustrated graphically in Fig. 6 and 7, respectively.

Fig. 6 Graphical representation of the in vitro antimicrobial (ZI) assay of the synthesized tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives.

Fig. 7 Graphical representation of the in vitro antimicrobial (MIC) assay of the synthesized tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives compared with the standard drug ciprofloxacin.

3.3. Structure activity relationship (SAR) studies

From the in silico molecular docking study, we found that compounds, 4,4-diethyl-2,8-dimethyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19l and 4,4-dimethyl-2-phenyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19p, have good binding scores of −8.7 kcal mol⁻¹ and −8.7 kcal mol⁻¹ for E. coli and −8.4 kcal mol⁻¹ and −9.1 kcal mol⁻¹ for S. aureus DNA gyrase, respectively. It also demonstrated effective antibacterial activity with a MIC value of 12.5 μg mL⁻¹ against both E. coli and S. aureus bacterial strains for both molecules 19l and 19p. Furthermore, it is observed that compound 2-methyl-4,4-dipropyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione 19h tivity with a MIC value of 50 μg mL⁻¹ for E. coli and 25 μg mL⁻¹ for S. aureus bacterial strains. Also, compound 19h has a poor binding affinity score (−5.6 kcal mol⁻¹ for E. coli and −7.9 kcal mol⁻¹ for S. aureus), as compared to all the synthesized molecules.

The structure–activity relationship studies reveal that the compound is more favorable towards antibacterial activity, particularly when the chromene scaffold is associated with a diethyl group at the R₂ position with a methyl group at the R₁ position, hydrogen at the R₃ position with N-methyl maleimide (19l) as well as with dimethyl at the R₂ position, methyl at the R₃ position with N-phenyl maleimide (19p). However, very poor results were observed in the case of compound 19h, which is associated with the dipropyl group at the R₂ position and methyl at the R₃ position with N-methyl maleimide. The pictorial representation of the SAR study is illustrated in Fig. 8.

Fig. 8 Pictorial representation of the SAR of the tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3 aH)-dione derivatives 19(a–q).

3.4. Estimation of physicochemical, pharmacokinetic and ADMET properties

The in silico pharmacokinetic study using ADMET was carried out for drug development cascades. The pharmacokinetic and physicochemical properties of seventeen synthesized molecules 19(a–q) were evaluated using the SwissADME tool, comparing them to the standard drug ciprofloxacin.^40,41 Subsequently, the Lipinski's Rule of Five (RO5) was used for the physicochemical properties.⁴² It states that molecular weight (MW) should be ≤500 g mol⁻¹, octanol–water partition coefficient (cLog P) should be ≤5, number of hydrogen bond acceptors (HBA) should be ≤10, number of hydrogen bond donors (HBD) should be ≤5 and topological polar surface area (tPSA) should be ≤140 Å for a potential compound to be an orally active drug molecule.^43–45

Additionally, the topological polar surface area (TPSA), which is inversely related to the percentage absorption (%ABS = 109–0.345 × TPSA), was proposed as a potential alternative to counting hydrogen bonding groups for estimating the absorption percentages. The analysis of the results indicates that the synthesized derivatives exhibit favorable physicochemical properties, with hydrogen bond acceptors (HBA) ranging from 3 to 4, TPSA values between 36.13 and 44.06 Å, and an acceptable number of hydrogen bond donors.

Again, the toxicity of the potent molecules 19l and 19p was determined using ProTox II online software. ProTox II tool determines the toxicity levels and the 50% lethal dose (LD₅₀).^20,46–48 From Table 4, compound 19l showed a Class 5 toxicity level with an estimated LD₅₀ of 2200 mg kg⁻¹, whereas compound 19p showed a Class 5 toxicity level with an estimated LD₅₀ of 3900 mg kg⁻¹ (Fig. 9). Consequently, the blood–brain-barrier (BBB), human-intestinal-absorption (HIA), and Caco-2-permeability were evaluated. From the result, it was concluded that most of the compounds are less toxic and suitable as oral bioactive drug molecules.

Table 4 Calculation of Lipinski's ‘rule of 5′ for the tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q)

Sl. no	Compound code	RO5					% ABS^f	Toxicity		Pharmacokinetics
		MW^a	HBA^b	HBD^c	mlogP^d	tPSA^e		Class	LD50^g (mg kg^−1)	BBB^h	HIA^i	Caco-2^j
a Molecular weight in g mol^−1.b Number of H-bond acceptors.c Number of H-bond donors.d Partition coefficient (lipophilicity).e Topological polar surface area in Å.f % of Absorbance.g 50% lethal dose.h Blood brain barrier permeant.i Human-intestinal-absorption.j Caco-2-permeability.
1	19a	373	3	0	3.69	36.13	96.53	5	2200	0.93	1	0.63
2	19b	387	3	0	4.13	36.13	96.53	5	2200	0.93	1	0.63
3	19c	403	4	0	3.65	43.67	93.93	5	2200	0.87	1	0.60
4	19d	401	3	0	4.35	36.52	96.40	5	2200	0.97	1	0.63
5	19e	415	3	0	4.79	36.52	96.40	5	2200	0.97	1	0.63
6	19f	431	4	0	4.31	44.06	93.79	5	2200	0.93	1	0.60
7	19g	429	3	0	5.37	36.52	96.40	5	2200	0.98	1	0.63
8	19h	443	3	0	5.81	36.52	96.40	5	2200	0.98	1	0.63
9	19i	459	4	0	5.32	44.06	93.79	5	2200	0.97	1	0.60
10	19j	387	3	0	4.13	36.13	96.53	5	2200	0.93	1	0.63
11	19k	417	4	0	4.09	43.67	93.93	5	2200	0.87	1	0.60
12	19l	415	3	0	4.79	36.52	96.40	5	2200	0.97	1	0.63
13	19m	429	3	0	5.24	36.52	96.40	5	2200	0.97	1	0.63
14	19n	445	4	0	4.75	44.06	93.79	5	2200	0.93	1	0.60
15	19o	435	3	0	4.98	35.48	96.75	5	3900	0.95	1	0.59
16	19p	449	3	0	5.42	35.48	96.75	5	3900	0.94	1	0.58
17	19q	463	3	0	5.64	35.87	96.62	5	3900	0.98	1	0.61
18	Ciprofloxacin	331	5	2	1.28	74.57	83.27	4	1000	0.72	0.97	0.64

---

Fig. 9 Bioavailability radar plot of the standard antibiotic ciprofloxacin compared with synthesized compounds 19l and 19p.

A radar chart was plotted to describe the six physicochemical parameters: lipophilicity (LIPO), size (SIZE), polarity (POLAR), solubility (INSOLU), saturation (INSATU), and flexibility (FLEX). As illustrated in Fig. 9, the physicochemical properties of compounds 19l and 19p generally fall within the acceptable range (pink region) (Fig. 10).^2,49

Fig. 10 Theoretical toxicity results of ciprofloxacin and synthesized compounds 19l and 19p calculated using ProTox 3.0.

After the ADMET analysis, from the literature survey, we found that both 2H-chromene and maleimide show fluorescent behavior.^50–52 Hence, we studied the UV and fluorescence activity of all the synthesized tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q).

4. Photophysical properties

4.1. UV-visible studies

The UV-visible spectra of seventeen synthesized tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dionederivatives 19(a–q) were recorded in a Shimadzu single monochromator UV-2600 spectrophotometer at 15 μm concentration. Initially, the cyclic derivative 19a was dissolved in DMSO at 15 μM concentration, and the spectra showed two peaks at 262 nm (n to π*) and 313 nm (π to π*) transitions. Notably, compounds 19b and 19c showed peaks at 262 nm and 313 nm, 263 nm and 312 nm, respectively. The graph also revealed an isosbestic point at 291 nm, as illustrated in Fig. 11. Additionally, the spectra for compounds 19d, 19e, and 19f showed absorption maxima at 262 nm for the n to π* transition and 312 nm for the π to π* transition, with the same isosbestic point at 291 nm. Similarly, compounds 19g, 19h and 19i displayed λ_max at 262 nm, 262 nm and 263 nm for the n to π* transition and 313 nm, 312 nm and 313 nm for the π to π* transition, respectively. It was observed that increasing the chain length at the R₂ position in the chromene moiety resulted in a shift from hypochromic to hyperchromic in the intensity of the UV-visible spectra.

Fig. 11 UV-visible spectra of compounds 19(a–q) in DMSO medium.

On the other hand, a blue shift to red shift (from 312 to 321 nm) was observed in compound 19j when a methyl group was introduced at the R₁ position of the chromene framework, accompanied by a gradual decrease in optical density (OD). Similarly, a slight change in absorption maxima was noted in compound 19k compared to 19j. In a similar fashion, the absorption maxima and optical density of compounds 19l, 19m, and 19n were compared, showing peaks at 265 nm, 265 nm, and 266 nm for the n to π* transition, and 320 nm, 321 nm, and 320 nm for the π to π* transition, respectively. An isosbestic point appeared at 297 nm for compounds 19l, 19m, and 19n. Furthermore, the UV-visible spectra of three different synthesized molecules (19o, 19p, and 19q), where a phenyl group was introduced at the R₄ position, showed a slight decrease in intensity compared to compounds 19a, 19b and 19c. The overall UV-visible spectra are shown in Fig. 11.

4.2. Fluorescence studies

To examine the fluorescence properties of the series of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q), fluorescence experiments were conducted using fluorescence spectrometry Hitachi-7000 keeping the fluorescence slit width at 2.5 nm for each. A specific concentration (15 μm) was maintained for all synthesized compounds during the assay. At the beginning of our experiment, compound 19a showed a wavelength of 352 nm with an optical density of 6151. Compound 19b, which has a methyl group introduced at the R₃ position of the 4-styrylaromatic ring, showed a recorded wavelength of 352 nm with a slight increase in OD to 8421. Similarly, compounds 19c, 19d, 19e, 19f, 19g, 19h, and 19i showed the same λ_max (352 nm) value with a gradual increase in OD values ranging from 8053 to 9784. It is noted that the increase in the chain length at the R₂ position in the chromene analogue led to a corresponding increase in the optical density value (6151–9784).

However, a blue shift to red shift (from 352 to 363 nm) was observed for compound 19j, which exhibited notably strong fluorescence intensity amongst all the synthesized derivatives. Also, the compounds 19k, 19l and 19n have the same wave number at 361 nm with OD values of 6615, 6516 and 5127, respectively. Consequently, compounds 19o, 19p, and 19q displayed similar λ_max at 353 nm, comparable to compounds 19a, 19b, and 19c. Finally, it was observed that all the synthesized derivatives demonstrated good fluorescent activity, as illustrated in Fig. 12.

Fig. 12 Fluorescence spectra of compounds 19(a–q) in DMSO medium.

5. Conclusion

In this study, a class of fused tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives were synthesized with good to excellent yields without the need for column chromatography. The reaction was carried out under mild conditions, yielding compounds with good binding affinities, potent antibacterial activity, and favorable photophysical properties, making this method highly efficient and practical. The in vitro results revealed that compounds 19l and 19p showed the most potent antibacterial activity against the tested pathogenic strains. Also, these results have been explained by the binding energy parameters determined from molecular docking studies, which showed that the selected compounds can bind with bacterial DNA gyrase more efficiently, similar to the antibiotic ciprofloxacin. Interestingly, the ADMET parameter prediction indicated that the majority of the synthesized conjugates have an acceptable pharmacokinetic profile with a nontoxic characteristic. Moreover, the photophysical properties are suitable for in vivo high-resolution microscope imaging. We propose that in the future, this strategy can be applied to medicinal chemistry for the synthesis of natural products with pharmacological activity and drug-containing structure of tetrahydrochromeno isoindole dione derivatives.

6. Experimental section

The commercially available reactants and reagents required for this reaction were purchased from the standard supplier (Sigma-Aldrich, TCI) and were used without further purification. All the reactions were carried out in anhydrous conditions. The progress of each reaction was monitored by thin-layer chromatography (TLC) on silica gel 60 (F254) coated with aluminum plates. Visualization of spots on the TLC plate was accomplished with UV light (254 nm). ¹H NMR spectra were recorded on a 400 MHz (100 MHz for ¹³C NMR) JEOL NMR spectrometer using CDCl₃ as solvent. High-resolution mass spectra (HRMS) were recorded using a Bruker micro TOF-QII mass spectrometer at IISER-Berhampur. The chemical shift was calibrated to tetramethyl silane (TMS) as an internal reference. Chemical shifts (δ) are given in ppm and coupling constants (J) in Hertz (Hz). The following abbreviations are used to indicate the signal multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Melting points were determined using a SMP-10 digital apparatus and were uncorrected. The UV-visible and fluorescent spectra were taken in a Shimadzu single monochromator UV-2600 spectrophotometer and HITACHI F-7000 fluorescence spectrometer machine at Sambalpur University.

7. Experimental procedure

7.1. General experimental procedure for the preparation of substituted chroman-4-one 13(a–e)

In a 100 mL clean and dry round bottom (RB) flask, substituted o-hydroxyacetophenone (1.0 equiv.) 11(a–b), substituted ketone (3 equiv.) 12(a–c) and pyrrolidine (0.5 equiv.) were taken. The mixture was stirred under a reflux medium for about 6–8 h. The reaction progress was monitored by TLC and was found to be completed after 6–8 h. After cooling to room temperature, the reaction mixture was quenched using water and extracted with ethyl acetate. The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude product was purified by column chromatography using ethyl acetate/hexane to afford the pure compound 13(a–e). All compounds were characterized by NMR.

7.2. General experimental procedure for the synthesis of 4-bromo-2H-chromene derivatives 15(a–e)

Substituted chroman-4-one, (1.0 equiv.) 13(a–e) and phosphorus tribromide (PBr₃) 14 (3 equiv.) were taken in a clean and dry round bottom flask and stirred under reflux condition for 30–40 min. Set up by a trap using cotton and anhydrous calcium chloride. After completion, the reaction mixture was quenched using ice water and extracted with ethyl acetate. The organic layer was washed with brine, separated, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by column chromatography using ethyl acetate/hexane to furnish the pure compound 15(a–e). All compounds were characterized by NMR data.

7.3. General experimental procedure for the synthesis of 4-styryl-2H-chromene derivatives 17(a–n)

Substituted 4-bromo-2H-chromene, (1.0 equiv.) 15(a–e), substituted styrene (2.0 equiv.) 16(a–c), palladium(II) complex (0.1 equiv.), K₂CO₃ (3.0 equiv.) and 1.5 mL of dry DMF were taken in the same vial and subjected to microwave irradiation at 120 °C, 100 W for about 20 min. The reaction was monitored by TLC and was found to be completed after 20 min. After completion, the reaction mixture was quenched using water and extracted with ethyl acetate. The organic layer was washed with brine, separated, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flask column chromatography using ethyl acetate/hexane to afford the pure compound 17(a–n). It gives two isomer which is not separated.

7.4. General experimental procedure for the synthesis of tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione derivatives 19(a–q)

Substituted 4-styryl-2H-chromene, (1.0 equiv.) 17(a–n), maleimide (3.0 equiv.) 18(a–b), and 3 mL of dry toluene were taken in a sealed tube and stirred under reflux condition at 120 °C for 4 h. The reaction was monitored by TLC and was found to be completed after 4 h. Then, the reaction was cooled at room temperature, washed with hexane and filtered to afford a pure white solid compound 19(a–q). The compound was characterized by NMR and HRMS spectral data.

7.5. General experimental procedure for the synthesis of 11-(4-methoxyphenyl)-2-methyl-4,4-dipropylchromeno[3,4-e]isoindole-1,3(2H,4H)-dione (21)

11-(4-Methoxyphenyl)-2-methyl-4,4-dipropyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3 aH)-dione(1.0 equiv.) 19i, DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) (1.5 equiv.) and 2 mL of dry toluene were taken in a clean and dry sealed tube and allowed to stir at 100 °C for 30 min. The progress of the reaction was monitored by TLC and after completion of the reaction, the pure compound 21 was obtained using column chromatography using ethyl acetate/hexane. After synthesis, the compound was characterized using¹H NMR, ¹³C NMR and HRMS spectroscopic techniques.

7.5.1 2,4,4-Trimethyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19a). White solid (91% yield), M.P. = 258–260, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.44 (dd, J₁₂ = 1.6 Hz, J₁₃ = 8.0 Hz, 1H), 7.36–7.32 (m, 2H), 7.28–7.25 (m, 3H), 7.12–7.08 (m, 1H), 6.88–6.81 (m, 2H), 6.59–6.57 (m, 1H), 3.73–3.70 (m, 1H), 3.52–3.49 (m, 1H), 3.38–3.34 (m, 1H), 2.65 (s, 4H), 1.82 (s, 3H), 1.30 (s, 3H).¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.3, 152.7, 138.7, 133.2, 129.5, 128.8 (2C), 128.3 (2C), 127.3, 122.5, 121.1, 120.9 (2C), 119.0, 75.1, 48.1, 45.7, 42.8, 42.6, 29.1, 26.0, 24.8. HRMS (ESI) calculated for C₂₄H₂₄NO₃ [M + H]⁺ 374.1756, found 374.1780.

7.5.2 2,4,4-Trimethyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19b). White solid (94% yield), M.P. = 253–255, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.43 (dd, J₁₂ = 1.2 Hz, J₁₃ = 7.6 Hz, 1H), 7.19 (s, 1H), 7.14 (s, 3H), 7.11–7.07 (m, 1H), 6.87–6.80 (m, 2H), 6.56–6.54 (m, 1H), 3.70–3.67 (m, 1H), 3.51–3.47 (m, 1H), 3.35–3.31 (m, 1H), 2.65 (s, 4H), 2.31 (s, 3H), 1.81 (s, 3H), 1.30 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.4, 152.6, 136.8, 135.6, 133.0, 129.4, 129.1 (2C), 128.7 (2C), 122.5, 121.2, 121.1, 120.9, 118.9, 75.1, 48.2, 45.6, 42.6, 42.5, 29.1, 26.0, 24.7, 21.1. HRMS (ESI) calculated for C₂₅H₂₆NO₃ [M + H]⁺ 388.1913, found 388.1903.

7.5.3 11-(4-methoxyphenyl)-2,4,4-trimethyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19c). White solid (92% yield), M.P. = 267–269, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.43 (dd, J₁₂ = 1.2 Hz, J₁₃ = 7.6 Hz, 1H), 7.19–7.17 (m, 2H),7.17–7.16 (m, 1H), 7.11–7.07 (m, 1H), 6.88–6.80 (m, 3H), 6.53–6.51 (m, 1H), 3.76 (s, 3H), 3.69–3.66 (m, 1H), 3.50–3.47 (m, 1H), 3.32–3.29 (m, 1H), 2.65 (s, 4H), 2.09 (s, 3H), 1.81 (s, 3H), 1.30 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.8, 175.4, 158.7, 152.6, 132.9, 130.6, 129.8 (2C), 129.4, 122.5, 121.4, 121.0, 120.9, 118.9, 113.7 (2C), 75.1, 55.2, 48.1, 45.6, 42.5, 42.1, 29.1, 25.9, 24.7. HRMS (ESI) calculated for C₂₅H₂₆NO₄ [M + H]⁺ 404.1862, found 404.1877.

7.5.4 4,4-Diethyl-2-methyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19d). White solid (88% yield), M.P. = 249–251,¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.50 (dd, J₁₂ = 1.2 Hz, J₁₃ = 8.0 Hz, 1H), 7.43–7.40 (m, 2H), 7.35–7.33 (m, 3H), 7.19–7.15 (m, 1H), 6.95 (dd, J₁₂ = 0.8 Hz, J₁₃ = 8.4 Hz, 1H), 6.91–6.87 (m, 1H), 6.64–6.62 (m, 1H), 3.80–3.77 (m, 1H), 3.56–3.53 (m, 1H), 3.45–3.41 (m, 1H), 2.73 (s, 4H), 2.43–2.28 (m, 2H), 1.81–1.75 (m, 1H), 1.52–1.47 (m, 1H), 1.08 (t, J = 7.6 Hz, 3H), 0.84 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.6, 175.4, 152.1, 138.6, 133.4, 129.5, 128.8 (2C), 128.3 (2C), 127.2, 122.4, 121.4, 120.9, 120.8, 118.9, 79.3, 48.4, 43.2, 42.8, 42.3, 27.3, 25.5, 24.8, 8.3, 7.5. HRMS (ESI) calculated for C₂₆H₂₈NO₃ [M + H]⁺ 402.2069, found 402.2081.

7.5.5 4,4-Diethyl-2-methyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19e). White solid (91% yield), M.P. = 245–247, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.49 (dd, J₁₂ = 1.2 Hz, J₁₃ = 7.6 Hz, 1H), 7.22 (s, 4H), 7.19–7.15 (m, 1H), 6.95 (dd, J₁₂ = 1.2 Hz, J₁₃ = 8.4 Hz, 1H), 6.90–6.86 (m, 1H), 6.62–6.60 (m, 1H), 3.77–3.74 (m, 1H), 3.55–3.51 (m, 1H), 3.42–3.38 (m, 1H), 2.73 (s, 3H), 2.72–2.71 (m, 1H), 2.38 (s, 3H), 2.36–2.27 (m, 2H), 1.80–1.75 (m, 1H), 1.54–1.46 (m, 1H), 1.08 (t, J = 7.6 Hz, 3H), 0.83 (t, J = 7.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.5, 152.1, 136.8, 135.5, 134.2, 133.3, 129.4, 129.1 (2C), 128.6 (2C), 122.4, 121.2, 120.9, 118.9, 79.3, 48.5, 43.2, 42.5, 42.4, 27.3, 25.5, 24.8, 21.1, 8.3, 7.5. HRMS (ESI) calculated for C₂₇H₃₀NO₃ [M + H]⁺ 416.2226, found 416.2214.

7.5.6 4,4-Diethyl-11-(4-methoxyphenyl)-2-methyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19f). White solid (89% yield), M.P. = 254–256, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.49 (dd, J₁₂ = 0.8 Hz, J₁₃ = 8.0 Hz, 1H), 7.25–7.23 (m, 2H), 7.18–7.14 (m, 1H), 6.95–6.93 (m, 3H), 6.90–6.86 (m, 1H), 6.59–6.57 (m, 1H), 3.83 (s, 3H), 3.76–3.73 (m, 1H), 3.54–3.50 (m, 1H), 3.39–3.35 (m, 1H), 2.73 (s, 4H), 2.42–2.27 (m, 2H), 1.82–1.73 (m, 1H), 1.54–1.45 (m, 1H), 1.08 (t, J = 7.6 Hz, 3H), 0.83 (t, J = 7.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.8, 175.4, 158.7, 152.1, 133.2, 130.6, 129.8 (2C), 129.4, 122.3, 121.4, 121.3, 120.9, 118.9, 113.7 (2C), 79.3, 55.2, 48.4, 43.2, 42.3, 42.2, 27.3, 25.5, 24.8, 8.2, 7.5. HRMS (ESI) calculated for C₂₇H₃₀NO₄ [M + H]⁺ 432.2175, found 432.2183.

7.5.7 2-Methyl-11-phenyl-4,4-dipropyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19g). White solid (83% yield), M.P. = 260–262, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.43 (dd, J₁₂ = 1.6 Hz, J₁₃ = 8.0 Hz, 1H), 7.36–7.32 (m, 2H), 7.28–7.25 (m, 3H), 7.11–7.07 (m, 1H), 6.87–6.79 (m, 2H), 6.56–6.54 (m, 1H), 3.71–3.68 (m, 1H), 3.44–3.41 (m, 1H), 3.36–3.32 (m, 1H), 2.66 (s, 3H), 2.64 (s, 1H), 2.24–2.15 (m, 2H), 1.62–1.52 (m, 2H), 1.37–1.22 (m, 4H), 1.00 (t, J = 7.2 Hz, 3H), 0.68 (t, J = 7.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.6, 175.3, 152.1, 138.6, 133.5, 129.4, 128.8 (2C), 128.3 (2C), 127.2, 122.3, 121.3, 120.9, 120.8, 118.9, 78.8, 48.4, 43.7, 42.8, 42.4, 37.9, 36.1, 24.8, 17.1, 16.6, 14.6, 14.2. HRMS (ESI) calculated for C₂₈H₃₂NO₃ [M + H]⁺ 430.2382, found 430.2392.

7.5.8 2-Methyl-4,4-dipropyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19h). White solid (89% yield), M.P. = 257–259, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.42 (dd, J₁₂ = 0.8 Hz, J₁₃ = 7.6 Hz, 1H), 7.14 (s, 4H), 7.11–7.07 (m, 1H), 6.86–6.79 (m, 2H), 6.54–6.52 (m, 1H), 3.68–3.65 (m, 1H), 3.43–3.40 (m, 1H), 3.33–3.29 (m, 1H), 2.66 (s, 3H), 2.63 (s, 1H), 2.31 (s, 3H), 2.22–2.16 (m, 2H), 1.63–1.53 (m, 2H), 1.35–1.25 (m, 4H), 1.00 (t, J = 7.2 Hz, 3H), 0.68 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.4, 152.0, 136.8, 135.6, 133.4, 129.4, 129.1 (2C), 128.6 (2C), 122.3, 121.3, 121.1, 120.8, 118.9, 78.8, 48.5, 43.6, 42.5, 42.4, 37.9, 36.1, 24.8, 21.1, 17.2, 16.6, 14.6, 14.2. HRMS (ESI) calculated for C₂₉H₃₄NO₃ [M + H]⁺ 444.2539, found 444.2552.

7.5.9 11-(4-methoxyphenyl)-2-methyl-4,4-dipropyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19i). White solid (85% yield), M.P. = 261–263, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.49 (dd, J₁₂ = 1.2 Hz, J₁₃ = 7.6 Hz, 1H), 7.25–7.23 (m, 2H), 7.18–7.13 (m, 1H), 6.95–6.85 (m, 4H), 6.58–6.56 (m, 1H), 3.83 (s, 3H), 3.74–3.71 (m, 1H), 3.48–3.46 (m, 1H), 3.38–3.34 (m, 1H), 2.73 (s, 3H), 2.70 (s, 1H), 2.29–2.24 (m, 2H), 1.70–1.61 (m, 3H), 1.39–1.33 (m, 3H), 1.07 (t, J = 7.2 Hz, 3H), 0.76 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.4, 158.7, 152.1, 133.3, 130.6, 129.8 (2C), 129.4, 122.3, 121.3, 121.2, 120.8, 118.9, 113.7 (2C), 78.8, 55.2, 48.4, 43.6, 42.4, 42.2, 37.9, 36.1, 24.8, 17.1, 16.6, 14.6, 14.2. HRMS (ESI) calculated for C₂₉H₃₄NO₄ [M + H]⁺ 460.2488, found 460.2479.

7.5.10 2,4,4,8-Tetramethyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole 1,3(2H,3aH)-dione (19j). White solid (88% yield), M.P. = 246–248, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.36–7.32 (m, 2H), 7.28–7.24 (m, 4H), 6.91 (dd, J₁₂ = 1.2 Hz, J₁₃ = 8.0 Hz, 1H), 6.78–6.75 (m, 1H), 6.56–6.54 (m, 1H), 3.72–3.69 (m, 1H), 3.51–3.48 (m, 1H), 3.37–3.34 (m, 1H), 2.65 (s, 3H), 2.64 (s, 1H), 2.20 (s, 3H), 1.80 (s, 3H), 1.28 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.3, 150.5, 138.7, 133.3, 130.3, 130.2, 128.8 (2C), 128.3 (2C), 127.2, 122.7, 120.6, 120.5, 118.7, 74.9, 48.1, 45.6, 42.8, 42.6, 29.1, 26.0, 24.8, 20.8. HRMS (ESI) calculated for C₂₅H₂₆NO₃ [M + H]⁺ 388.1913, found 388.1903.

7.5.11 11-(4-Methoxyphenyl)-2,4,4,8-tetramethyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19k). White solid (90% yield), M.P. = 247–249, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.22 (d, J = 1.6 Hz, 1H), 7.19–7.16 (m, 3H), 6.90 (dd, J₁₂ = 1.6 Hz, J₁₃ = 8.0 Hz, 1H), 6.88–6.85 (m, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.50–6.49 (m, 1H), 3.76 (s, 3H), 3.69–3.64 (m, 1H), 3.48–3.45 (m, 1H), 3.31–3.28 (m, 1H), 2.65 (s, 3H), 2.62 (s, 1H), 2.19 (s, 3H), 1.79 (s, 3H), 1.28 (s, 3H).¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.9, 175.4, 158.7, 150.5, 133.1, 130.7, 130.3, 130.1, 129.8 (2C), 122.7, 121.1, 120.5, 118.7, 113.7 (2C), 74.9, 55.2, 48.2, 45.5, 42.6, 42.1, 29.0, 25.9, 24.8, 20.7. HRMS (ESI) calculated for C₂₆H₂₈NO₄ [M + H]⁺ 418.2018, found 418.2027.

7.5.12 4,4-Diethyl-2,8-dimethyl-11-phenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19l). White solid (84% yield), M.P. = 250–252, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.44–7.40 (m, 2H), 7.36–7.32 (m, 3H), 7.31–7.30 (m, 1H), 6.98 (dd, J₁₂ = 1.6 Hz, J₁₃ = 8.0 Hz, 1H), 6.87–6.85 (m, 1H), 6.62–6.60 (m, 1H), 3.80–3.77 (m, 1H), 3.55–3.52 (m, 1H), 3.45–3.41 (m, 1H), 2.74 (s, 3H), 2.69 (s, 1H), 2.42–2.29 (m, 2H), 2.27 (s, 3H), 1.82–1.74 (m, 1H), 1.52–1.45 (m, 1H), 1.08 (t, J = 7.6 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.7, 175.4, 149.9, 138.7, 133.5, 130.3, 130.0, 128.8 (2C), 128.3 (2C), 127.2, 122.6, 121.0, 120.6, 118.7, 79.1, 48.4, 43.2, 42.8, 42.4, 27.2, 25.5, 24.9, 20.8, 8.3, 7.5. HRMS (ESI) calculated for C₂₇H₃₀NO₃ [M + H]⁺ 416.2226, found 416.2238.

7.5.13 4,4-Diethyl-2,8-dimethyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19m). White solid (89% yield), M.P. = 251–253, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.29 (d, J = 1.6 Hz, 1H), 7.23 (s, 4H), 6.97 (dd, J₁₂ = 1.6 Hz, J₁₃ = 8.4 Hz, 1H), 6.86–6.84 (m, 1H), 6.59–6.57 (m, 1H), 3.76–3.73 (m, 1H), 3.53–3.50 (m, 1H), 3.41–3.37 (m, 1H), 2.73 (s, 3H), 2.68 (s, 1H), 2.38 (s, 3H), 2.35–2.29 (m, 2H), 2.27 (s, 3H), 1.79–1.74 (m, 1H), 1.50–1.44 (m, 1H), 1.07 (t, J = 8.0 Hz, 3H), 0.82 (t, J = 7.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.8, 175.5, 149.9, 136.8, 135.6, 133.4, 130.3, 130.0, 129.1 (2C), 128.6 (2C), 122.6, 121.1, 120.9, 118.7, 79.1, 48.5, 43.2, 42.5, 42.4, 27.2, 25.5, 24.8, 21.1, 20.8, 8.3, 7.5. HRMS (ESI) calculated for C₂₈H₃₂NO₃ [M + H]⁺ 430.2382, found 430.2387.

7.5.14 4,4-Diethyl-11-(4-methoxyphenyl)-2,8-dimethyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19n). White solid (86% yield), M.P. = 258–260, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.29 (d, J = 1.6 Hz, 1H), 7.27–7.24 (m, 1H), 6.99–6.94 (m, 3H), 6.86–6.84 (m, 1H), 6.56–6.55 (m, 1H), 3.84 (s, 3H), 3.75–3.72 (m, 1H), 3.52–3.50 (m, 1H), 3.39–3.35 (m, 1H), 2.74 (s, 3H), 2.68 (s, 1H), 2.41–2.29 (m, 2H), 2.27 (s, 3H), 2.17 (s, 3H), 1.79–1.74 (m, 1H), 1.50–1.44 (m, 1H), 1.07 (t, J = 7.6 Hz, 3H), 0.82 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 175.8, 175.5, 158.6, 149.9, 133.4, 130.7, 130.3, 130.0, 129.8 (2C), 122.6, 121.1, 121.0, 118.7, 113.7 (2C), 79.1, 55.2, 48.4, 43.1, 42.4, 42.1, 27.2, 25.5, 24.9, 20.8, 8.3, 7.5. HRMS (ESI) calculated for C₂₈H₃₂NO₄ [M + H]⁺ 446.2331, found 446.2325.

7.5.15 4,4-Dimethyl-2,11-diphenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19o). White solid (76% yield), M.P. = 263–265, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.56 (dd, J₁₂ = 1.2 Hz, J₁₃ = 7.6 Hz, 1H), 7.41–7.36 (m, 4H), 7.35–7.27 (m, 2H), 7.25–7.18 (m, 3H), 6.95–6.89 (m, 2H), 6.84–6.82 (m, 2H), 6.81–6.76 (m, 1H), 3.91–3.89 (m, 1H), 3.75–3.72 (m, 1H), 3.63–3.59 (m, 1H), 2.87–2.84 (m, 1H), 1.92 (s, 3H), 1.44 (s, 3H).¹³C NMR (100 MHz, CDCl₃): δ (ppm) 174.7, 174.5, 152.5, 138.5, 133.8, 131.5, 129.6, 128.9 (2C), 128.7 (2C), 128.4 (2C), 128.2, 127.3, 126.2, 122.5, 121.1, 120.9, 120.6, 118.9, 75.2, 48.4, 45.8, 42.8, 42.3, 28.9, 25.7. HRMS (ESI) calculated for C₂₉H₂₆NO₃ [M + H]⁺ 436.1913, found 436.1926.

7.5.16 4,4-Dimethyl-2-phenyl-11-(p-tolyl)-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19p). White solid (79% yield), M.P. = 260–262, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.49 (dd, J₁₂ = 2.0 Hz, J₁₃ = 7.6 Hz, 1H), 7.22–7.20 (m, 3H), 7.18–7.08 (m, 5H), 6.87–6.81 (m, 2H), 6.76–6.74 (m, 2H), 6.69–6.67 (m, 1H), 3.82–3.79 (m, 1H), 3.66–3.63 (m, 1H), 3.52–3.48 (m, 1H), 2.79–2.78 (m, 1H), 2.28 (s, 3H), 1.84 (s, 3H), 1.36 (s, 3H).¹³C NMR (100 MHz, CDCl₃): δ (ppm) 174.8, 174.6, 152.5, 136.9, 135.4, 132.7, 131.6, 129.5, 129.1 (2C), 128.8 (2C), 128.7 (2C), 128.1, 126.2 (2C), 122.5, 121.2, 121.1, 120.6, 118.9, 75.3, 48.5, 45.8, 42.5, 42.3, 28.9, 25.6, 21.1. HRMS (ESI) calculated for C₃₀H₂₈NO₃ [M + H]⁺ 450.2069, found 450.2048.

7.5.17 4,4-Diethyl-2,11-diphenyl-3b,4,11,11a-tetrahydrochromeno[3,4-e]isoindole-1,3(2H,3aH)-dione (19q). White solid (73% yield), M.P. = 264–266, ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.55 (dd, J₁₂ = 1.2 Hz, J₁₃ = 7.6 Hz, 1H), 7.41–7.40 (m, 4H), 7.34–7.30 (m, 1H), 7.26–7.15 (m, 4H), 6.96–6.94 (m, 1H), 6.91–6.83 (m, 3H), 6.78–6.76 (m, 1H), 3.91–3.88 (m, 1H), 3.70–3.67 (m, 1H), 3.62–3.58 (m, 1H), 2.84–2.83 (m, 1H), 2.49–2.44 (m, 1H), 2.34–2.29 (m, 1H), 1.87–1.81 (m, 1H), 1.58–1.52 (m, 1H), 1.10 (t, J = 7.6 Hz, 3H), 0.86 (t, J = 7.6 Hz, 3H).¹³C NMR (100 MHz, CDCl₃): δ (ppm) 174.6, 174.5, 151.9, 138.5, 133.2, 131.6, 129.5, 128.8 (2C), 128.7 (2C), 128.4 (2C), 128.1, 127.2, 126.1 (2C), 122.3, 121.1, 120.9 (2C), 118.9, 79.3, 48.8, 43.3, 42.8, 42.1, 27.2, 25.3, 8.2, 7.5. HRMS (ESI) calculated for C₃₁H₃₀NO₃ [M + H]⁺ 464.2226, found 464.2223.

7.5.18 11-(4-Methoxyphenyl)-2-methyl-4,4-dipropylchromeno[3,4-e]isoindole-1,3(2H,4H)-dione (21). Light green liquid (78% yield), ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.87 (s, 1H), 7.64 (dd, J₁₂ = 1.6 Hz, J₁₃ = 8.0 Hz, 1H), 7.54–7.48 (m, 2H), 7.30–7.25 (m, 1H), 7.02–6.91 (m, 4H), 3.88 (s, 3H), 3.12 (s, 3H), 2.72–2.64 (m, 2H), 2.05–1.97 (m, 2H), 1.53–1.43 (m, 2H), 1.23–1.16 (m, 2H), 0.85 (t, J = 7.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 167.5, 167.2, 159.9, 154.4, 139.7, 136.5, 136.4, 131.4, 130.6 (2C), 128.9, 128.8, 128.7, 127.5, 123.7, 121.2, 119.1117.6, 113.4 (2C), 84.5, 55.3, 41.2 (2C), 24.0, 17.5 (2C), 14.4 (2C). HRMS (ESI) calculated for C₂₉H₃₀NO₄ [M + H]⁺ 456.2175, found 456.2165.

8. Method of crystal growth

Compound 19i was first dissolved in a minimal amount of ethyl acetate, followed by the addition of drops of hexane. This solution was then placed in a sample vial and stored in a dark area for 4 to 5 weeks to enable gradual evaporation. After 5 weeks, white needle-like crystals of compound 19i were formed.

9. Molecular docking studies of compound 19(a–q) against bacterial DNA gyrase of E. coli and S. aureus

All the synthesized derivatives 19(a–q) were carefully drawn with proper stereochemistry and the 2D structures were converted to optimized 3D structures. Molecular docking studies were conducted using Autodock Tools version 4.2.0. The host proteins, including the bacterial DNA gyrase inhibitors for Gram-negative bacterial strain E. coli (PDB ID: 3 G7E) and Gram-positive bacterial strain S. aureus (PDB ID: 3 G7B), were downloaded and retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/). Before docking the complex, water molecules and other hetero-atoms were removed from these host protein structures using PyMOL (https://www.pymol.org/).^53,54 Also, polar hydrogen bonds were added, and a grid box was set to 60 × 60 × 60 for the xyz axes (covering ligand binding pocket). 0.375 Å is the grid space for 100 genetic algorithm runs.^2,55 The two programs, ‘autodock’ and ‘autogrid’, were used to determine the binding affinity scores for the host–guest interaction. The 2D structures of ligand–receptor interactions were visualized using PyMOL and BIOVIA Discovery Studio v 2017.

10. Antibacterial evaluation of compound 19(a–q) against bacterial DNA gyrase of E. coli and S. aureus

The in vitro antibacterial activity of all the synthesized molecules was tested using the agar-well diffusion method against E. coli (MTCC614) and S. aureus (MTCC7443) bacterial strains. Ciprofloxacin served as the standard antibiotic. Initially, the compounds were dissolved in DMSO and tested on Mueller–Hinton agar plates to assess the zones of inhibition (ZI). For the minimum inhibitory concentration (MIC) assay, the compounds were prepared at various concentrations (6.25, 12.5, 25, 50 and 100 μg mL⁻¹ and 200 μg mL⁻¹) and tested in a 96-well plate format to evaluate their bacterial inhibitory properties.

11. Physicochemical, pharmacokinetic and ADME studies

The ADME and pharmacokinetic properties of all the synthesized compounds were studied on the free online server Swiss ADME (https://www.swissadme.ch/). Initially, chemical structures were drawn on Marvin to generate SMILE and inserted directly on the webpage to initiate the prediction process.^56–58 The toxicity evaluation of the synthesized compounds 19(a–q) was determined using ProTox II free software.

12. Photophysical study

The photophysical (UV-visible and fluorescence) studies were performed for all the synthesized compounds using the Shimadzu single monochromator UV-2600 spectrophotometer and HITACHI F-7000 fluorescence spectrometer. Initially, the compounds were dissolved in DMSO at a uniform concentration (15 μm), and the spectra were recorded.⁵⁹

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Sonali Priyadarshini Parida: conceptualization, investigation, methodology, formal analysis, data correction, writing-review & editing; Seetaram Mohapatra: supervision, resources, funding acquisition, editing; Suhasini Mohapatra: writing original draft, conceptualization, writing-review & editing; Tankadhar Behera: UV-visible & fluorescent study; Sabita Nayak: conceptualization, validation, supervision, funding acquisition, editing; software, resources; Chita Ranjan Sahoo: software, antibacterial studies.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The author, Seetaram Mohapatra, expresses gratitude to the Department of Science and Technology (DST), Odisha (ST-SCST-MISC-0011-2023, Bhubaneswar, 25-05-2023). He also acknowledges the Centre of Excellence in Environment and Public Health, Department of Higher Education, Government of Odisha (Grant no 26913/HED/HE-PTC-WB-02-17 OHEPEE). Author Sabita Nayak extends thanks to Mukhyamantri Research Innovation (MRI) for Extramural Research Grant-2024(24 EM/CH/35). The author, Sonali Priyadarshini Parida, also acknowledges DST, Odisha program (2692/ST, Bhubaneswar, 16-7-2020) for providing the financial support as a project assistant. Author Suhasini Mohapatra acknowledges the financial support from SERB-SURE, New Delhi (Ref. SUR/2022/000257). Additionally, the author is thankful to DST-FIST, New Delhi (SR/FST/CSI-243/2012) for the NMR facility.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 2321501. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5ra02212f

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