Skeletal rearrangement in Biginelli reaction for the synthesis of chromenoquinoline fused spirohydantoins and their in silico cytotoxicity study

Abstract

Although Biginelli reaction is known to generate dihydropyrimidinones having wide arrays of phramacological properties, herein we report an unprecedented skeletal rearrangement in Biginelli reaction to synthesize pharmacologically versatile imidazolidine-2,4-diones, popularly known as hydantoins. Here a mixture of isatin, 4-hydroxycoumarin and 1,3-dimethylurea reacts in the presence of a catalytic amount of p-TsOH to generate spirohydantoins fused with chromenoquinoline moiety. Skeletal rearrangement of the 5-membered keto-lactam ring of isatin to a structurally constrained 5-membered ring in the form of spirohydantoins is the most interesting feature of this protocol. Nonetheless, the reaction introduces a novel molecular architecture bearing medicinally important spirohydantoin and chromenoquinoline scaffolds. In-silico molecular docking simulation of the synthesized compounds against human kinesin Eg5 (PDB ID: 2IEH) along with 17 other tumor-associated targets demonstrated strong binding efficiency, indicating that these compounds merit further biological evaluation to validate their anticancer potential.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-45715-7.

Introduction

Hydantoins emerged in 1860s and became a constant in the clinical lexicon since 1916 when 5-ethyl-5-phenylhydantoin was introduced as a less toxic alternative to antihypnotic drug phenobarbital. Discovery of 5,5-diphenylhydantoin, popularly known as phenytoin, expanded its scope of clinical applications that include treatment of epilepsy, migraine, dizziness, hiccups, myocardial infarction, burns, and wounds¹. Later, the spirohydantoins (Fig. 1) became a subject of intense scrutiny in medicinal chemistry due to their broad therapeutic profiles that include antitumor^2–4, antidiabetic^5–7, antipsychotic⁸, antiepileptic^9,10 antibacterial^11,12, and antidepressant and anxiolytic^8,13 activities. In recent years, chromenoquinolines are also subject of extensive pharmacological studies due to their remarkable bioactivity along with high fluorescence quantum yield^14–16. As a pharmaceutical, the substituted chromenoquinolines exhibit remarkable properties, such as anti-inflammatory activities, and serotonin- and oestrogen- receptor functions^17–19.

Fig. 1.

Some well-known drugs with hydantoin scaffold.

Biginelli reaction is one of the oldest multicomponent reactions that gives 3,4-dihydropyrimidin-2(1 H)-ones (DHPMs) from the reaction of a mixture of aromatic aldehyde, ethyl acetoacetate and urea, mostly under acid catalysis^20–22. The DHPMs show a wide range of biological activities such as calcium channel blockers, anti-inflammatory, antifungal, anticancer, anti-HIV, anti-tubercular, antibacterial, antifilarial, antihyperglycemic, antihypertensive, anticonvulsant, analgesic, anti-TRPA1 or anti-SARS, antioxidant, among others^23–30. Widely known DHPMs such as monastrol, enastron and piperastrol amplify the importance of DHPMs as promising targets in cancer chemotherapy through inhibition of Kinesin-5, a protein that is involved in the regulation and function of mitosis^31,32.

Skeletal rearrangement reactions, that involve simultaneous cleavage and formation of bonds in the reaction process to form new molecular architecture, have attracted profound interest among synthetic chemist^33,34. Among the cyclic molecules, synthesis of spiroisatins is one the most studied area in organic synthesis due to its extremely high reactivity of the C-3 keto-group³⁵. Given the rich pharmacological portfolio of molecules bearing isatin^36–44, coumarin^45–49, and dihydropyrimidinone^50–52 scaffolds, we recently reported an L-prolineH₂SO₄ ionic liquid catalysed Biginelli reaction of isatin with 4-hydroxycoumarin and urea (thiourea) for synthesis of dihydropyrimidinones⁵³. Surprisingly, the same reactants underwent an unexpected skeletal rearrangement of the isatin nucleus upon using 1,3-dimethylurea to generate a chromenoquinoline based hydantoin derivative (Scheme 1), albeit in smaller yield (20%). Literature studies revealed that only a handful of methods are available for synthesis of spirohydantoins, the most common one being the Bucherer–Bergs reaction of ketones with potassium cyanide and ammonium carbonate^54–56. Basarab and Briones reported a two-step method where magnesium triflate catalyzed a tert-amino effect reaction (T-reaction) involving ethyl 3-(2-(dialkylamino)-phenyl)-2-nitroacrylates leading to tetrahydroquinoline nitroester derivatives which were finally transformed to the corresponding spirohydantoin derivatives⁵⁷. Here we present a one-step multicomponent method for synthesis of spirohydantoins fused with highly exquisite chromenoquinoline molecular scaffold that is formed due to skeletal rearrangement in the Biginelli reaction.

Scheme 1.

Dihydropyrimidinone vs. spirohydantoin in the Biginelli reaction.

Results and discussion

Given the fact that Biginelli reaction is rarely studied with substituted urea, we conducted a model reaction of isatin, 4-hydroxycoumarin, and 1,3-dimethyl urea in the presence of L-proline.H₂SO₄ ionic liquid in acetonitrile (Table 1, entry 1) to get an off-white precipitate. Of course, the reaction was very slow and hardly gave 20% conversion in 24 h. The product was isolated simply by filtration followed by washing with ice-cold ethanol. Finally, the product was purified by recrystallization from ethanol. The IR spectra of the product were in sharp contrast to corresponding dihydropyrimidinones with two sharp C = O stretching bands at 1763 and 1703 cm^-1 which were attributed to the characteristic carbonyl group at C-2 and C-4 of hydantoin ring⁵⁸. The structure of the product was then confirmed by various NMR experiments and HRMS data. In ¹H NMR, two methyl groups of the hydantoin moiety appeared as singlets at δ 3.08 and δ 2.50 ppm, each integrating for three protons. The quaternary carbon of the compound showed a peak at δ 65.0 ppm and the carbonyl group (-C = O) of the chromene ring appeared at δ 159.5 ppm in ¹³C NMR. While the two carbonyl groups (-C = O) of the hydantoin ring appeared at δ 175.0 ppm and δ 155.9 ppm, the peaks at δ 89.6, 115.1, 116.8, 117.7, 117.9, 120.1, 125.7, 126.3, 127.2, 130.8, 136.2, 136.3, 145.6, and 152.2 ppm indicated the sp² carbons of the aromatic region of the chromene and quinoline rings.

Table 1.

Optimization of reaction conditions^{a, b}.

Entry	Catalyst (mol %)	Solvent	T/oC	t/h	%Yield
1	L-proline·H2SO4 (20)	MeCN	80	24	20
2	L-proline (20)	MeCN	80	24	–
3	H2SO4 (20)	MeCN	80	24	10
4	I2 (20)	MeCN	80	24	–
5	MeCO2H (20)	MeCN	80	24	40
6	CF3CO2H (20)	MeCN	80	24	45
7	PTA (20)	MeCN	80	24	48
8	BF3.Et2O (20)	MeCN	80	24	38
9	p-TsOH (20)	MeCN	80	24	60
10	p-TsOH (30)	MeCN	80	24	83
11	p -TsOH (30)	MeCN	80	12	82
12	p-TsOH (30)	MeCN	80	8	65
13	p-TsOH (30)	1,4-Dioxane	100	12	30
14	p-TsOH (30)	Water	100	12	–
15	p-TsOH (30)	DMF	130	12	–
16	p-TsOH (30)	MeCN	50	24	–

^aReactions are carried out at 1 mmol scale.

^bPure yields are reported.

Since we obtained a very intriguing molecule, a host of readily available acid catalysts (entries 2–9) were screened to identify the optimum catalyst to effect the transformation. Most of them facilitated the formation of the desired product, albeit in low to moderate yields. But the use of 30 mol% of p-TsOH turned out to be most efficient, affording the product in 83% yield in 24 h of heating under reflux in acetonitrile. Encouraged by the result, the reaction time was reduced further to 12 h. To our pleasure, complete consumption of the starting materials was observed to give 82% isolated yield. Upon reducing the reaction time further to 8 h, significant drop in yield was noted (entry 12). The model reaction was screened in other solvents with higher boiling points in order to study the effect of solvent and temperature. Upon screening the reaction in 1,4-dioxane and water at their boiling points, inferior yields were observed (entries 13, 14). The similar observation was made when the reaction mixture was heated to 130 °C using DMF as a solvent. Likewise, the reaction did not work at 50 °C also in acetonitrile medium even for an extended duration probably due to poor solubility of the starting material in acetonitrile at that temperature. Based on the optimization studies, it was concluded that the reaction works at optimum with a catalytic amount of p-TsOH (30 mol%) upon heating under reflux in acetonitrile.

Given the fact that synthesis of spiroheterocycles is often challenging due to their inherent structural strains resulting molecular stability, we extended the substrate scope of the reaction to achieve structurally complex spirohydantoins bearing biologically relevant chromenoquinoline scaffold. Under the optimized conditions, all the reactions gave excellent yields (Chart 1). The reactions of 1,3-dimethylurea with a variety of substituted 4-hydroxycoumarins and isatin derivatives have also shown excellent conversion.

Chart 1.

Synthesis of chromenoquinoline fused spirohydantoins^{a, b}.

In order to confirm the structure of the compounds beyond any doubt, repeated attempts were made to obtain single crystal of all the synthesized compounds. Unfortunately, precipitation occurred in each attempt. But we could confirm the structure from HMBC experiment. HMBC analysis of compound 1j (Fig. 2) showed correlation of an N-methyl proton (δ 3.08 ppm) with two carbonyl carbons at δ 174.0 ppm and δ 155.7 ppm confirming the presence of an imide group of the hydantoin moiety. The second methyl group of -NCH₃ at δ 2.51 ppm also exhibited correlation with the carbonyl carbon at δ 155.7 ppm confirming the presence of hydantoin ring. The second methyl group of -NCH₃ at δ 2.51 ppm also showed a correlation with the spiro carbon at δ 64.42 ppm to establish a 3-bond coupling between the -NCH₃ protons of hydantoin ring with spiro-fused framework. Finally, the¹H-¹³ C correlations observed between aromatic -CH proton of the quinoline ring (singlet, δ 7.88 ppm) and the spiro carbon (δ 64.42 ppm) established the connectivity between the quinoline ring and the spiro centre.

Fig. 2.

HMBC correlation.

Inspired by the findings with 1,3-dimethylurea, we screened the reaction of isatin and 4-hydroxycoumarin with 1,3-dimethylthiourea under the optimized conditions for an extended time period (Scheme 2) to observe that the reactions do not give the desired products even after heating under reflux till 24 h.

Scheme 2.

Reaction with 1,3-dimethylthiourea.

Given the structural similarity of 4-hydroxy-6-methyl-2-pyrone with 4-hydroxycoumarin, we investigated its reaction with isatin and 1,3-dimethylurea under the optimized conditions (Scheme 3). To our pleasure, the reaction was found complete within 12 h to give the desired product in 60% yield. Unfortunately, when the reaction was extended to a few more isatin derivatives, complex inseparable mixture of products was obtained.

Scheme 3.

Reaction with 4-hydroxy-6-methyl-2-pyrone.

To understand the plausible mechanism, we conducted a series of reactions (Scheme 4). When an equimolar mixture of 1,3-dimethyl urea and 4-hydroxycoumarin was reacted, the reaction did not proceed, and the starting materials were recovered. The reaction of an equimolar mixture of isatin and 4-hydroxycoumarin gave a polar product upon stirring with reflux for 6 h, albeit the starting materials were not consumed completely. The polar product was confirmed to be the Knoevenagel product, (I), from ¹H NMR and HRMS data. When the Knoevenagel product (I) was put into a reaction with 1,3- dimethyl urea, the reaction gave only a trace amount of the product after stirring at reflux for 24 h. Of course, the reaction of 1,3-dimethyl urea and isatin gave an intermediate (II) in just 2 h with complete consumption of the starting materials as indicated by thin-layer chromatography. Since the intermediate was difficult to isolate because of its high polarity, we added a solution of 4-hydroxycoumarin in acetonitrile directly into the reaction mixture after complete consumption of the starting materials. The reaction was complete within 10 h to give a white precipitate that was filtered and recrystallized to get 79% isolated yield of the desired product, 1a. Interestingly, the reaction of an equimolar reaction of isatin and 1,3-dimethylthiourea in the presence of p-TsOH under similar reaction conditions did not give any product. The observation may be ascribed to better nucleophilicity of the sulfur atom in thiourea than that of the amine under acidic condition leading to possible formation of a highly reversible intermediate (III), rather than the formation of the intermediate IV [Scheme 4 (v)] which is critical for the formation of the desired product.

Scheme 4.

Mechanistic study through controlled reactions.

Based on the above observations, a plausible mechanism was proposed (Fig. 3). The acid catalyst, p-TsOH activated the C-3 carbonyl group of isatin upon heating under reflux to react with 1,3-dimethylurea to give the intermediate (II). The intermediate (II) undergoes a 1,2-addition reaction with 4-hydroxycoumarin to form the intermediate (V) which undergoes ring opening of the oxindole ring to form the hydantoin ring (VI) by reacting with amine residue of 1,3-methylurea. In the final step, protonation of the chromene carbonyl group activates it towards intramolecular nucleophilic attack by the amine of the aniline residue, which subsequently undergoes dehydration through a series of intermediates (VII – IX) followed by deprotonation to afford the desired product.

Fig. 3.

Plausible mechanism.

Molecular Docking analysis

Recognizing the hydantoin scaffolds are linked to cytotoxic and antiproliferative effects^59–61, we explored the potential interaction of the synthesized molecules through molecular docking study against Eg5 or kinesin spindle protein (KSP). KSP is a molecular motor protein associated with the transport of vesicles, microtubules, organelles by converting chemical energy from hydrolysis of ATP to mechanical energy that plays an essential role in the formation of mitotic spindles in human body⁶². Thus, inhibition of KSP leads to mitotic arrest by mitotic checkpoint activation and apoptosis of cells⁶³. As cancer cells predominantly depends on mitosis, molecules that inhibits KSP are of great interests.

The Eg5 protein with the PDB ID: 2IEH was chosen as the target protein from Protein Data Bank (www.rcsb.org), where a keto derivative of well-known antitumor drug monastrol, Mon-97, is co-crystallized in the active site⁶³. Autodock 4.2 was used for docking simulation⁶⁴. The 3D structure of all the synthesized compounds was optimized using DFT based B3LYP method. 6–31 + G(d, p) basis set was used for C, H, N and O atoms and lanl2dz for Cl and Br atoms⁶⁵. The protein was cleaned by removing all non-standard residues and chain A was selected for docking analysis using ChimeraX⁶⁶. The addition of polar hydrogens and Kollman charges were carried out using Autodock 4.2 itself.

To validate the docking protocol, the co-crystallized Mon-97 was redocked with the protein. The grid box was adjusted to encompass the whole protein with the X, Y and Z co-ordinates 24.938, 2.508 and 103.398 Å, respectively, with the number of grid points set to 126 × 126 × 122 along the X, Y and Z direction and a grid spacing of 0.492 Å. Docking was performed by 10 number of genetic algorithm (GA) runs. The ligand was observed to occupy the same binding pocket similar to that of Mon-97 co-crystal. The root mean square deviation (RMSD) of the binding pose of the ligand shows only 0.106 Å (good when RMSD ≤ 2Å) deviation from the original co-crystallized pose indicating a very good alignment and validating the reliability of the docking parameters used. After the validation, the synthesized molecules (1a-1p) were subjected to docking with the optimized grid settings.

The analysis reveals that the compounds 1a, 1b, 1c and 1e showed very good docking scores of 9.41, -9.28, -9.35 and − 9.05 kCalmol^-1 with inhibition constants of 127.59 nM, 156.34 nM, 139.96 nM, 232.18 nM, respectively (Table 2). The binding pocket is similar to the original Mon-97 ligand. Interestingly, the nitro-substituted compounds exhibited a preference for an alternative binding site, potentially the allosteric site of the protein. Other compounds also demonstrated good binding affinities.

Table 2.

Binding scores and inhibition constants of the compounds with 2IEH.

Compd.	Binding energy (kCal mol−1)	Inhibition constant
1a	− 9.41	127.59 nM
1b	− 9.28	156.34 nM
1c	− 9.35	139.96 nM
1d	− 8.82	340.84 nM
1e	− 9.05	232.18 nM
1f	− 8.56	535.75 nM
1 g	− 8.95	275.57 nM
1 h	− 8.10	1.16 µM
1i	− 8.06	1.24 µM
1j	− 8.17	1.03 µM
1k	− 7.98	1.41 µM
1 L	− 8.12	1.11 µM
1 m	− 8.78	364.34 nM
1n	− 8.47	619.43 nM
1o	− 7.43	3.57 µM
1p	− 8.15	1.07 µM
Mon-97	− 9.72	75.59 µM

Compound 1a with the maximum docking score exhibits hydrophobic interactions like alkyl, π-alkyl, π-cation, π-anion, π-π, π-sigma, van-der wall contacts with residues Arg221, Ala218, Glu116, Glu118, Arg119, Ala133, Pro137, Leu160, Leu214, Phe239, Trp127, Gly217, Ile136, Gly134, Trp127, Glu215, Tyr211 within the binding pocket (Fig. 4a and b). The present docking results strongly support the hypothesis that the synthesized molecules may act as effective KSP inhibitors. Subsequently, 17 additional cancer-related target proteins from the PDB- namely 2 × 2R⁶⁷, 2 × 7D⁶⁸, 2 × 7E⁶⁸, 3KEN⁶⁹, 4A50⁷⁰, 1A26⁷¹, 1BIX⁷², 1TE6⁷³, 1NOW⁷⁴, 2VCJ⁷⁵, 2RCW⁷⁶, 3CRY⁷⁷, 3HQU⁷⁸, 2RJR⁷⁹, 4B7P⁸⁰, 5P21⁸¹, and 6V34⁸², of which first 5 were KSPs, were selected and docked with the synthesized compounds. All targets demonstrated satisfactory binding profiles (See SI), suggesting these molecules may possess broader binding capabilities warranting further in-vitro evaluation.

Fig. 4.

(a) 3D representation of compound 1a (atoms are specified) in the binding pocket of 2IEH; (b) 2D representation of compound 1a (carbon- grey, nitrogen-blue, oxygen-red) in the binding pocket of 2IEH.

Conclusion

In summary, we have unveiled a novel Biginelli reaction pathway that, for the first time, enables the construction of an imidazole core rather than formation of a pyrimidine ring. The reaction unravelled formation of pharmacologically important hydantoin ring fused with another highly potent molecular architecture in the form of chromenoquinolines, all in one step. Preliminary mechanistic studies revealed that the reaction proceeds via an unprecedented skeletal rearrangement of the isatin nucleus, giving rise to a structurally unique and biologically important chromenoquinoline fused spirohydantoin framework. It may also be highlighted that; this transformation establishes the first synthetic route to chromenoquinoline derivatives directly from an isatin scaffold. Simple reaction conditions and no chromatographic separation are added highlights of the transformation. The method not only expand the synthetic versatility of the Biginelli reaction but also open new avenues for the development of complex and strained biologically relevant heterocyclic molecular architectures. Molecular docking simulation predominantly against kinesin spindle protein 2IEH and other 17 cancer-related target proteins revealed that all designed compounds exhibited strong binding energies and low inhibition constants, interacting effectively with key active site residues. These results highlight their promising inhibitory potential and provide valuable molecular insights that lay groundwork for future in-vitro and in-vivo validation studies.

Experimental

All reagents and solvents were commercial grade and were used directly without further purification. The melting points were recorded from Buchi M-500 melting point apparatus. IR spectra were recorded on a Bruker Alpha ІІ FT-IR spectrometer. ¹H and¹³ C NMR spectra were recorded on a Bruker Advance ІІ (400 MHz) spectrometer using TMS as internal reference. Chemical shifts for¹ H NMR spectra are reported (in parts per million) relative to internal tetramethylsilane (Me₄Si = 0.0 ppm) with DMSO-d₆ as solvent. ¹³C NMR spectra were recorded at 100 MHz. Chemical shifts for¹³ C NMR spectra are reported (in parts per million) relative to internal tetramethylsilane (Me₄Si = 0.0 ppm) with DMSO-d₆ as solvent. ¹H NMR data are reported in the order of chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet, bm = broad multiplet), coupling constant in hertz (Hz) and number of protons. TLC plates were visualized by UV. The HRMS was recorded on a WATERS, Xevo G2-XS QT mass spectrometer. Elemental analysis was recorded on a Perkin–Elmer-2400 CHN/S analyser.

General procedures for preparation of Spirohydantoins

To an equimolar mixture of 4-hydroxycoumarin (162 mg, 1 mmol), isatin (147 mg, 1 mmol), and 1,3-dimethylurea (88 mg, 1 mmol) in a round bottom flask charged with a magnetic stirring bar was added 2 mL of acetonitrile and 30 mol% of p-TsOH (51 mg, 0.3 mmol) and the suspension was stirred at 80 °C for 12 h. The resulting mixture was cooled to room temperature and the solid product was filtered off. The solid precipitate was washed with ethanol (5 ml) thrice to remove any unreacted starting materials. The solid crude product was then dissolved in ethanol and kept overnight to get the pure recrystallized product.

Spectroscopic data of the compounds

1’,3’-Dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1a): White solid. Yield: 82%; m.p. > 300 °C; IR (KBr, cm^− 1): ν 3409, 3283, 3004, 1763, 1703, 1618, 1539, 1492, 1107, 751 cm^− 1. ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H, NH), 8.46 (d, J = 7.6 Hz, 1H, Ar-H), 7.80 (m, 1H, Ar-H), 7.53 (m, 4 H, Ar-H), 7.22 (m, 2 H, Ar-H), 3.08 (s, 3 H, CH₃), 2.50 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 175.0, 159.5, 155.9, 152.2, 145.6, 136.3, 136.2, 130.8, 127.2, 126.3, 125.7, 120.1, 117.9, 117.7, 116.8, 115.1, 89.6, 65.0, 25.5, 25.4 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₅N₃O₄ 362.1135; found 362.1136.

2-Chloro-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1b): White solid. Yield 85%; m.p > 300 °C; IR (KBr, cm^− 1): ν 3446, 3279, 2926, 1758, 1701, 1623, 1567, 1538, 1486, 1338, 1040, 826 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.63 (s, 1H, NH), 8.56 (s, 1H, Ar-H), 7.63 (bm, 4 H, Ar-H), 7.20–7.16 (m, 2 H, Ar-H), 3.02 (s, 3 H, CH₃), 2.44 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.9, 159.5, 155.7, 151.6, 145.6, 136.1, 133.3, 130.7, 128.9, 127.1, 125.6, 123.3, 119.7, 117.7, 117.6, 114.5, 89.5, 64.8, 25.4, 25.3 ppm. Elemental analysis for C₂₀H₁₄ClN₃O₄: Calc. C 60.69, H 3.57, N 10.62. Found C 60.72, H 3.58, N 10.59.

1’,2,3’-Trimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1c): White solid. Yield 80%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3416, 3085, 1732, 1707, 1663, 1612, 1585, 1348 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.45 (s, 1H, NH), 8.16 (s, 1H, Ar-H), 7.54–7.38 (m, 3 H, Ar-H), 7.30 (d, J = 8.4 Hz, 1H, Ar-H), 7.09 (m, 2 H, Ar-H), 2.97 (s, 3 H, CH₃), 2.41 (s, 3 H, CH₃), 2.38 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 175.1, 160.0, 155.8, 151.0, 146.4, 136.3, 134.5, 134.2, 130.6, 127.1, 125.4, 123.2, 117.5, 117.4, 112.6, 88.9, 64.9, 25.4, 25.2, 21.0 ppm. Elemental analysis for C₂₁H₁₇N₃O₄: Calc. C 67.19, H 4.56, N 11.19. Found C 67.21, H 4.59, N 11.14.

2-Bromo-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1d): White solid. Yield 89%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3412, 3010, 1734, 1709, 1664, 1610, 1581, 1345 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.63 (s, 1H, NH), 8.73 (s, 1H, Ar-H), 7.91–7.52 (m, 4 H, Ar-H), 7.22 (bm, 2 H, Ar-H), 3.07 (s, 3 H, CH₃), 2.50 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.8, 159.4, 155.7, 152.0, 145.5, 136.1, 136.0, 130.6, 127.1, 126.2, 125.6, 120.0, 117.7, 117.6, 116.7, 115.0, 89.5, 64.8, 25.4, 25.3 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₄BrN₃O₄ 440.0240, 442.0220; found 440.0244, 442.0243.

9-chloro-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1e): White solid. Yield 88%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3430, 3397, 2923 1731, 16, 1660, 1610, 1574, 1346 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.64 (s, 1H, NH), 8.33 (d, J = 7.6 Hz, 1H, Ar-H), 7.70 (t, J = 7.6 Hz, 1H, Ar-H), 7.52–7.41 (m, 4 H, Ar-H), 7.16 (s, 1H, Ar-H), 2.97 (s, 3 H, CH₃), 2.43 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.6, 159.7, 155.8, 152.8, 146.4, 135.4, 133.8, 130.9, 128.9, 126.6, 124.8, 123.8, 119.6, 117.7, 112.8, 95.5, 88.9, 64.7, 25.5, 25.4 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₄ClN₃O₄ 396.0746, found 396.0746.

2,9-Dichloro-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1f): White solid. Yield 90%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3452, 3272, 3124, 2948, 1762, 1707, 1623, 1568, 1532, 1484, 1341, 1019, 878 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.70 (s, 1H, NH), 8.54 (s, 1H, Ar-H), 7.80 (d, J = 8.8 Hz, 1H, Ar-H), 7.57–7.51 (m, 3 H, Ar-H), 7.23 (s, 1H, Ar-H), 3.03 (s, 3 H, CH₃), 2.49 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.4, 159.4, 155.7, 151.6, 145.5, 135.3, 133.4, 131.0, 129.1, 129.0, 126.6, 123.4, 119.8, 119.6, 119.6, 114.4, 89.5, 64.6, 25.5, 25.4 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₃Cl₂N₃O₄ 430.0356, found 430.0355.

9-Chloro-1’,2,3’-trimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1g): White solid. Yield 86%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3453, 3279, 2922, 1761, 1708, 1625, 1579, 1532, 1486, 1338, 1026, 874 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.61 (s, 1H, NH), 8.19 (s, 1H, Ar-H), 7.58–7.53 (m, Ar-H), 7.37 (d, J = 8.4 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 3.03 (s, 3 H, CH₃), 2.48 (s, 6 H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.6, 159.8, 155.7, 151.0, 146.3, 135.4, 134.6, 134.2, 130.9, 128.8, 126.6, 123.3, 119.6, 119.5, 117.5, 112.5, 88.9, 64.7, 25.4, 25.3, 21.1 ppm. Elemental analysis for C₂₁H₁₆ClN₃O₄: Calc. C 61.55, H 3.94, N 10.25. Found C 61.58, H 3.98, N 10.21. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₁H₁₆ClN₃O₄ 410.0902 found 410.0900.

2-Bromo-9-chloro-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1h): White solid. Yield 88%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3450, 2926, 1762, 1708, 1663, 1624, 1565, 1346 cm^− 1.¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H, NH), 8.59 (s, 1H, Ar-H), 7.84 (dd, J = 8.8, 2.0 Hz, 1H, Ar-H), 7.73 (dd, J = 8.8, 2.0 Hz, 1H, Ar-H), 7.58 (d, J = 8.8 Hz, 1H, Ar-H), 7.52 (d, J = 8.4 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H), 3.08 (s, 3 H, CH₃), 2.54 (s, 3 H, CH₃) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ 174.5, 159.4, 155.8, 151.6, 145.6, 135.6, 133.9, 133.5, 129.3, 129.1, 123.3, 119.9, 119.9, 119.8, 117.0, 114.3, 89.6, 64.6, 25.5, 25.4 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₄BrClN₃O₄ 473.9851, 475.9830; found 473.9854, 475.9820.

1’,3’-Dimethyl-9-nitrospiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1i): White solid. Yield 84%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3408, 3010, 1734, 1707, 1660, 1610, 1574, 1484 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 11.10 (s, 1H, NH), 8.48 (d, J = 8.0 Hz, 1H, Ar-H), 8.39 (dd, J = 9.2, 2.4 Hz, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 7.85–7.80 (m, 2 H, Ar-H), 7.63–7.55 (m, 2 H, Ar-H), 3.12 (s, 3 H, CH₃), 2.59 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.3, 159.6, 155.8, 153.0, 146.7, 144.0, 141.7, 134.1, 126.3, 125.0, 124.0, 123.3, 118.9, 118.3, 117.8, 112.7, 90.6, 64.5, 25.6, 25.5 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₄N₄O₆: 407.0986; found 407.0982.

2-Chloro-1’,3’-dimethyl-9-nitrospiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1j): White solid. Yield 86%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3408, 3010, 1770, 1710, 1630, 1570, 1544, 1512, 1332 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 11.06 (s, 1H, NH), 8.57 (s, 1H, Ar-H), 8.36–8.31 (m, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 7.83 (dd, J = 9.2, 2.4 Hz, 1H, Ar-H), 7.71 (d, J = 9.2 Hz, 1H, Ar-H), 7.56 (d, J = 8.8 Hz, 1H, Ar-H), 3.07 (s, 3 H, CH₃), 2.53 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 174.1, 159.6, 155.7, 151.7, 145.8, 144.1, 141.5, 133.7, 129.1, 126.4, 123.5, 123.3, 119.8, 118.8, 118.2, 114.2, 91.2, 64.4, 25.6, 25.5 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₄N₄O₆: 441.0596; found 441.0595.

1’,2,3’-Trimethyl-9-nitrospiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1k): White solid. Yield 83%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3408, 3010, 1771, 1708, 1632, 1570, 1544, 1512, 1332 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz) δ 10.91 (s, 1H, NH), 8.27 (d, J = 7.9 Hz, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 7.80 (s, 1H, Ar-H), 7.66 (d, J = 8.4 Hz, 1H, Ar-H), 7.52 (d, J = 7.4 Hz, 1H, Ar-H), 7.33 (d, J = 7.8 Hz, 1H, Ar-H), 2.98 (s, 3 H, CH₃), 2.42 (s, 6 H, 2CH₃). ppm¹³ C NMR (DMSO-d₆, 100 MHz, ) δ 174.4, 159.7, 155.8, 143.9, 141.7, 138.4, 135.0, 134.4, 126.3, 123.4, 123.3, 120.0, 118.8, 118.3, 117.6, 112.3, 90.5, 64.5, 25.6, 25.5, 21.1 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₁H₁₆N₄O₄: 421.1143; found 421.1141.

2-Bromo-1’,3’-dimethyl-9-nitrospiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1l): White solid. Yield 85%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3408, 3010, 1768, 1705, 1621, 1576, 1547, 1512, 1335 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz) δ 11.00 (s, 1H, NH), 8.63 (s, 1H, Ar-H), 8.29 (d, J = 8.0 Hz, 1H, Ar-H), 7.99–7.83 (m, 2 H, Ar-H), 7.64 (d, J = 8.8 Hz, 1H, Ar-H), 7.43 (d, J = 8.8 Hz, 1H, Ar-H), 3.01 (s, 3 H, CH₃), 2.44 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz, ) δ 174.1, 159.2, 155.7, 152.1, 145.7, 144.1, 141.5, 136.5, 126.4, 123.3, 120.1, 118.8, 118.2, 116.9, 114.6, 91.2, 64.4, 25.6, 25.5 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₃BrN₄O₆ 485.0091, 487.0071; Found 485.0085, 487.0080.

174.1, 159.2, 155.7, 152.1, 145.7, 144.1, 141.5, 136.5, 126.4, 123.3, 120.1, 118.8, 118.2, 116.9, 114.6, 91.2, 64.4, 25.6, 25.5.

9-Bromo-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12 H)-trione, (1m): White solid. Yield 86%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3408, 3010, 1767, 1708, 1629, 1570, 1544, 1512, 1332 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz): δ 10.66 (s, 1H, NH), 8.62 (s, 1H, Ar-H), 7.82 (d, J = 8.4 Hz, 1H, Ar-H), 7.56–7.51 (m, 3 H, Ar-H), 7.29–7.19 (m, 2 H, Ar-H), 3.08 (s, 3 H, CH₃), 2.51 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz): δ 175.0, 159.5, 155.8, 151.6, 145.6, 136.1, 133.3, 130.8, 129.0, 127.1, 125.7, 123.3, 119.8, 117.7, 117.6, 114.5, 89.5, 64.8, 25.4, 25.3 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₂₀H₁₄BrN₃O₄: 440.0240, 442.0220; Found 440.0225, 442.0224.

9-Bromo-2-chloro-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1n): White solid. Yield 88%; m.p.>300 °C; IR (KBr, cm^− 1): ν 3396, 3274, 1764, 1704, 1620, 1567, 1529, 1347 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz, ) δ 10.57 (s, 1H, NH), 8.37 (s, 1H, Ar-H), 7.64–7.50 (m, 2 H, Ar-H), 7.36–7.29 (m, 2 H, Ar-H), 7.16 (s, 1H, Ar-H), 2.87 (s, 3 H, CH₃), 2.32 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz) δ 174.5, 159.4, 158.1, 155.7, 151.6, 145.5, 135.6, 133.9, 133.4, 129.4, 129.0, 123.3, 119.9, 116.9, 114.4, 114.3, 89.6, 64.5, 25.5, 25.4 ppm. Elemental analysis for C₂₀H₁₃BrClN₃O₄: Calc. C 50.61, H 2.76, N 8.85. Found C 50.62, H 2.78, N 8.81.

9-Bromo-1’,2,3’-trimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1o): White solid. Yield 83%; m.p > 300 °C; IR (KBr, cm^− 1): ν 3452, 3278, 1765, 1705, 1625, 1581, 1531, 1484, 1326 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz, ) δ 10.62 (s, 1H, NH), 8.19 (s, 1H, Ar-H), 7.66 (d, J = 8.4 Hz, 1H, Ar-H), 7.57 (d, J = 8.4 Hz, 1H, Ar-H), 7.49 (d, J = 8.8 Hz, 1H, Ar-H), 7.38 (d, J = 8.0 Hz, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 3.03 (s, 3 H, CH₃), 2.47 (s, 6 H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz, ) δ 174.7, 159.8, 155.8, 151.0, 146.3, 135.8, 134.6, 134.2, 133.7, 129.4, 123.3, 120.0, 119.8, 117.5, 116.7, 112.5, 89.1, 64.6, 25.5, 25.4, 21.1 ppm. Elemental analysis for C₂₁H₁₆BrN₃O₄: Calc. C 55.52, H 3.55, N 9.25. Found C 55.58, H 3.59, N 9.22.

2,9-Dibromo-1’,3’-dimethylspiro[chromeno[4,3-b]quinoline-7,4’-imidazolidine]-2’,5’,6(12H)-trione, (1p): White solid. Yield 86%; m.p > 300 °C; IR (KBr, cm^− 1): ν 3442, 1753, 1700, 1632, 1568, 1535, 1352 cm^− 1. ¹H NMR (DMSO-d₆, 400 MHz, ) δ 10.75 (s, 1H, NH), 8.71 (s, 1H, Ar-H), 7.96 (dd, J = 8.8, 1.9 Hz, 1H, Ar-H), 7.73 (dd, J = 8.7, 2.0 Hz, 1H, Ar-H), 7.51 (dd, J = 8.8, 2.1 Hz, 2 H, Ar-H), 7.37 (s, 1H, Ar-H), 3.08 (s, 3 H, CH₃), 2.54 (s, 3 H, CH₃) ppm. ¹³C NMR (DMSO-d₆, 100 MHz, ) δ 174.5, 159.4, 155.7, 151.6, 145.5, 135.6, 133.9, 133.4, 129.4, 129.0, 123.3, 119.9, 119.8, 116.9, 114.4, 89.7, 64.5, 25.5, 25.4 ppm. Elemental analysis for C₂₀H₁₃BrN₃O₄: Calc. C 46.27, H 2.52, N 8.09. Found C 46.29, H 2.58, N 8.05.

1,3,3’-trimethylspiro[imidazolidine-4,10’-pyrano[4,3-b]quinoline]-1’,2,5(5’H)-trione, (2): White solid. Yield 60%; m.p. >300 °C; IR (KBr, cm^− 1): ν 3444, 3252, 1757, 1700, 1661, 1662, 1586, 1536, 1489, 1453 cm^− 1. ¹H NMR (400 MHz, DMSO-d₆): δ 10.56 (s, 1H, NH), 7.33 (t, J = 7.2 Hz, 1H, Ar-H), 7.10–6.95 (m, 3 H, Ar-H), 2.92 (s, 3 H, CH₃), 2.34 (s, 3 H, CH₃), 2.17 (s, 3 H, CH₃).¹³ C NMR (100 MHz, DMSO-d₆): δ 175.0, 162.7, 161.3, 155.7, 150.0, 136.4, 130.6, 127.4, 124.8, 117.9, 116.9, 97.7, 85.7, 64.6, 25.3, 25.1, 20.0 ppm. HRMS (ESI-TOF) m/z: [M + H]⁺ calc. for C₁₇H₁₅N₃O₄ 326.1135; found 326.1130.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Mezhubeinuo, HB, and APS carried out the experimental studies involving 4-hydroxycoumarin and 1,3-dimethylurea, and 4-hydroxy-6-methyl-2-pyrone and 1,3-dimethylthiourea. HB carried out the *in-silico* cytotoxicity studies. GB conceptualized, supervised, and prepared the manuscript in consultation with HB.

Data availability

The data underlying this study are available in the published article and its Supplementary Information.

Declarations

Competing interests

The authors declare no competing interests.