In silico exploration of acetic acid driven multicomponent synthesis: design, characterization, and antioxidant evaluation of spiroacridines and spiroquinolines

Abstract

In this study, a highly efficient green synthetic protocol was developed for the synthesis of spiroacridines and spiroquinolines via a multicomponent reaction of 3,4-methylenedioxyaniline, isatin and cyclic 1,3-diones in glacial acetic acid. This metal-free multicomponent approach offers mild reaction conditions, shorter reaction time and easy product purification without column chromatography. Frontier molecular orbitals (FMOs), various quantum chemical descriptors (QCDs) and molecular electrostatic potential (MEP) surfaces were computed using density functional theory (DFT) with a B3LYP/6-311G+(d,p) basis set. All newly synthesized compounds were screened for their antioxidant properties. The compounds 4a and 4v are the most potent ABTS and DPPH radical scavengers to the standard ascorbic acid. In silico molecular docking studies were conducted to determine the binding affinities of the most potent 4a and 4v with targeted antioxidant protein, LD-carboxypeptidase, which show a strong binding affinity of −10.5 kcal mol⁻¹ and −10.7 kcal mol⁻¹, respectively. The most potent antioxidants 4a and 4v were evaluated for in silico drug-likeness and ADME prediction.

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Sustainability spotlight

We synthesized a novel spiroacridines and spiroquinolines using one pot multicomponent reactions of 3,4-methylenedioxyaniline, isatin and cyclic 1,3-diones in presence of glacial acetic acid. This reactions protocol covers broad sustainable approaches such as metal-free, mild reaction conditions, shorter reaction time and easy product purification without using column chromatography. The claimed derivatives provides a promising antioxidants property and drug likeness properties.

1. Introduction

Reactive oxygen species (ROS) play an integral role in the development of many major illnesses, including cancer, diabetes, arteriosclerosis, heart disease, and cataracts.¹ The detrimental effects of free radicals that may cause biological damage are referred to as oxidative stress. Free radicals have a pathogenic role in the development of a wide range of chronic degenerative illnesses in humans, including cancer and autoimmune, inflammatory, and cardiovascular neurodegenerative disorders.^2,3 Free radicals are chemical entities that possess an unpaired electron. By grasping an electron from a neighbouring biomolecule, these free radicals initiate a chain reaction in the human body, causing them to behave abnormally in living systems. Antioxidants are compounds that donate electrons to free radicals in order to transform them into non-harmful substances. In a nutshell, antioxidants protect the body from oxidative stress by suppressing the oxidation process.⁴ As a result, the necessity to produce antioxidant drugs has grown in recent years.

In present times, the use of sustainable synthetic protocols has received much more attention for the construction of novel heterocycles.⁵ For the construction of various heterocycles of biological interest, multicomponent reactions are most commonly used to form multiple C–C and C–N bonds without the isolation of an intermediate. MCRs are more vulnerable than classical multistep synthesis owing to their higher reaction efficiency and straightforward operation.^6,7 New spiroacridines and spiroquinolines were made using this multicomponent synthetic approach. It has encouraged the synthetic community to seek out more environmentally friendly syntheses and reactions in order to speed up preparation while using less energy and effort.^1,8

Nitrogen-containing heterocycles are significant in medicinal chemistry and drug design, as evidenced by their preponderance in marketed drugs.⁹ Spiroindolines have grabbed the interest of researchers due to their privileged structural moiety and occurrence in a wide range of bioactive natural products and synthesised drugs.¹⁰ Acridines and quinolines are interesting moieties in heterocyclic chemistry that attract the most attention to chemical modifications and are promising chemical entities both synthetically and medicinally due to their diverse pharmacological applications such as anticancer,^11,12 SRC kinase inhibitory,^13,14 antioxidant,^15,16 and antiallergic activity.¹⁷Fig. 1 depicts several acridine,¹⁸ quinoline,^19,20 spiroindoline²¹ and 1,3-benzodioxole²² conjugates with promising biological applications. The antioxidant activity of the spiroacridine and spiroquinoline moieties has received little attention. We were inspired to create acridine and quinoline-containing spiroacridines and spiroquinolines as a result of this.

Fig. 1 Acridine, quinoline, 1,3-benzodioxole and spiroindoline-based biologically active compounds.

In continuation to our efforts towards the construction of novel spiro molecules,^16,23–26 herein, we explored a metal-free multicomponent synthesis^1,27–29 of spiroacridines and spiroquinolines via a one-pot reaction of 3,4-methylenedioxyaniline, isatin and cyclic 1,3-diones in the presence of glacial acetic acid, which is vital for sustainability (Scheme 1). To acquire theoretical insights, the frontier molecular orbital (FMO), the molecular electrostatic potential (MEP), and several quantum chemical descriptors (QCDs) were computed using the DFT using the B3LYP/6-311G+(d,p) basis set. All the newly synthesised spiro molecules were screened for their antioxidant properties.

Scheme 1 Multicomponent synthesis of 4a from 3,4-methylenedioxyaniline 1, isatins 2a–h, and cyclic 1,3-diones 3a–c.

2. Results and discussion

2.1. Chemistry

In this protocol, we selected 3,4-methylenedioxyaniline 1 (1 mmol), isatin 2a (1 mmol), and dimedone 3a (1 mmol) as a model reaction in order to achieve our target spiro heterocyclic moiety 7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione 4a under catalyst-free conditions via multicomponent synthesis. In order to check the effect of temperature and solvents, we optimized our model reaction by using a variety of solvents at different temperatures. In the initial phase, we initiated our model reaction employing environmentally friendly solvents, water and ethanol, under room temperature conditions (Table 1, entries 1 and 2). However, no discernible transformation of isatin 2a into our target compound 4a was observed. In pursuit of our desired molecule 4a, we executed our model reaction by elevating the temperature from room temperature to reflux. Subsequently, our model reaction was conducted utilizing ethanol at its reflux temperature (Table 1, entry 3). During this experiment, we noted the production occurring within the reaction medium at its reflux temperature. Thin-layer chromatography analysis indicated a full transformation of 2a into 4a, accompanied by the emergence of a novel fluorescence spot on the TLC plate. After that, we performed our model reaction using different organic solvents such as methanol, ethyl L-lactate, acetic acid, DMF and acetonitrile (Table 1, entries 4–8). The best outcomes were achieved when glacial acetic acid was used (Table 1, entry 6). In the presence of glacial acetic acid, we observed rapid transformation of 2a into 4a within just 5 minutes under reflux conditions. Therefore, we selected it as the best optimum condition for the synthesis of our target moiety 7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione 4a. It is noteworthy that acetic acid is the sole solvent containing acidic hydrogen among all solvents, playing a dual role as both the catalyst and solvent. This dual functionality enhances reaction efficiency and reduces overall reaction time.

Table 1 Optimization of reaction for the preparation of compound 4a^a

Entry	Solvent^b	Temperature	Time	Conversion relative to isatin^c
a Reaction conditions: 1 mmol 3,4-methylenedioxyaniline 1, 1 mmol isatin 2a and 1 mmol dimedone 3a, reflux. b 3 mL solvent. c Observed from TLC analysis.
1	Water	rt	6 h	NR
2	Ethanol	rt	6 h	NR
3	Ethanol	Reflux	45 min	100%
4	Methanol	Reflux	45 min	100%
5	Ethyl L-lactate	Reflux	90 min	100%
6	Acetic acid	Reflux	5 min	100%
7	DMF	Reflux	4 h	Incomplete
8	Acetonitrile	Reflux	60 min	100%

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Using this best optimum condition, we systematically explored the substrate scope for synthesizing spiroacridines 4a–p and spiroquinolines 4q–x. This exploration involved utilizing 3,4-methylenedioxyaniline 1 (1 mmol), various substituted isatins 2a–h (1 mmol), and diverse cyclic 1,3-diones 3a–c (1 mmol) in the presence of glacial acetic acid at reflux temperature. In the presence of various substituted isatins 2a–h and various cyclic 1,3-diones, all reactions proceeded smoothly and furnished final moieties 4a–x with good isolated yield ranging from 68–78% within the stipulated time range. The results are summarized in Table 2. Fortunately, we discovered the solidification of the final product following the reaction transformation in each set of studies. The primary investigation for the confirmation of the desired product is carried out by the thin layer chromatography method using 70% ethyl acetate and 30% n-hexane as the mobile phase. The structures of all newly synthesized compounds are characterized by ¹H NMR, ¹³C NMR and HRMS spectral analysis.

Table 2 Substrate scope of the synthesis of spiroacridines 4a–p and spiroquinolines 4q–x^a

a Reaction conditions: 1 mmol 3,4-methylenedioxyaniline 1, 1 mmol isatins 2a–h and 1 mmol cyclic 1,3-diones 3a–c, 3 mL glacial acetic acid, reflux.

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2.2. Density functional theory calculations

2.2.1. Frontier molecular orbital (FMO) analysis. In this study, we analysed frontier molecular orbitals (FMOs) to gain insights into stability, reactive properties, molecular interactions and physicochemical properties of newly synthesised spiroacridines 4a–p and spiroquinolines 4q–x by using density functional theory (DFT) calculations. The lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) are the two primary molecular orbitals associated with reactions with other molecular structures, respectively. The DFT/B3LYP method with a 6-311G+(d,p) basis set was used to calculate the energies of HOMO and LUMO and their orbital energy gap E_gap of spiro compounds 4a–x. The HOMO–LUMO graphical depiction of molecules 4a, 4i and 4q is shown in Fig. 2. Positive and negative areas are represented by red and green colours, respectively. Compounds 4a, 4i and 4q contain a dimedone, cyclohexane 1,3-dione and cyclopentane 1,3-dione ring moiety, respectively. A lower orbital energy gap denotes more reactivity and less stability. As can be seen from Fig. 2, 4a with a dimedone ring moiety has higher reactivity and less stability than 4i and 4q, whereas 4q with a cyclopentane 1,3-dione ring moiety has the highest stability and less reactivity.

Fig. 2 The HOMO–LUMO energy diagram of molecules 4a, 4i and 4j calculated at DFT/B3LYP/6-311G+(d,p) in the gaseous phase.

HOMO and LUMO energies are also used to illustrate various quantum chemical descriptors (QCDs) such as ionisation potential (IP), electron affinity (EA), chemical hardness (η) and softness (σ), electronegativity (χ), chemical potential (μ), electrophilicity index (ω), nucleophilicity index (ε_γ) and maximum charge transfer index (ΔN_max). These QCDs were computed using the B3LYP/6-311G+(d,p) basis set, according to Koopman's theorem.³⁰ These QCDs play an essential role in emphasising the chemical behaviour and reactivity of the target molecule. The computed QCDs of spiro molecules 4a–x are listed in Table 3.

Table 3 Calculated quantum chemical descriptors of spiro molecules 4a–x^a

Molecule	E HOMO (eV)	E LUMO (eV)	E g (eV)	IP (eV)	EA (eV)	η (eV)	σ (eV)	χ (eV)	μ (eV)	ω (eV)	ε γ (eV)	N (eV)
a DFT/B3LYP/6-311G+(d,p) was used in a gaseous state.
4a	−5.530	−1.363	4.167	5.530	1.363	2.083	0.240	3.447	−3.447	2.851	−7.181	1.654
4b	−5.645	−1.487	4.158	5.645	1.487	2.079	0.240	3.566	−3.566	3.058	−7.413	1.715
4c	−5.649	−1.484	4.165	5.649	1.484	2.082	0.240	3.567	−3.567	3.054	−7.427	1.713
4d	−5.506	−1.340	4.166	5.506	1.340	2.083	0.240	3.423	−3.423	2.812	−7.129	1.643
4e	−5.641	−1.473	4.168	5.641	1.473	2.084	0.240	3.557	−3.557	3.036	−7.413	1.707
4f	−5.429	−1.285	4.144	5.429	1.285	2.072	0.241	3.357	−3.357	2.719	−6.956	1.620
4g	−5.831	−2.547	3.284	5.831	2.547	1.642	0.305	4.189	−4.189	5.343	−6.878	2.551
4h	−5.627	−1.460	4.167	5.627	1.460	2.084	0.240	3.544	−3.544	3.013	−7.384	1.701
4i	−5.533	−1.360	4.173	5.533	1.360	2.086	0.240	3.446	−3.446	2.847	−7.191	1.652
4j	−5.651	−1.480	4.171	5.651	1.480	2.085	0.240	3.565	−3.565	3.047	−7.435	1.710
4k	−5.656	−1.485	4.170	5.656	1.485	2.085	0.240	3.571	−3.571	3.057	−7.446	1.712
4l	−5.509	−1.336	4.173	5.509	1.336	2.086	0.240	3.422	−3.422	2.807	−7.140	1.640
4m	−5.646	−1.472	4.174	5.646	1.472	2.087	0.240	3.559	−3.559	3.035	−7.428	1.706
4n	−5.462	−1.309	4.152	5.462	1.309	2.076	0.241	3.385	−3.385	2.760	−7.029	1.631
4o	−5.842	−2.543	3.299	5.842	2.543	1.649	0.303	4.192	−4.192	5.328	−6.915	2.542
4p	−5.634	−1.460	4.173	5.634	1.460	2.087	0.240	3.547	−3.547	3.014	−7.401	1.700
4q	−5.621	−1.204	4.416	5.621	1.204	2.208	0.226	3.413	−3.413	2.637	−7.536	1.545
4r	−5.736	−1.322	4.413	5.736	1.322	2.207	0.227	3.529	−3.529	2.822	−7.787	1.599
4s	−5.701	−1.306	4.394	5.701	1.306	2.197	0.228	3.504	−3.504	2.793	−7.698	1.595
4t	−5.596	−1.181	4.415	5.596	1.181	2.208	0.226	3.388	−3.388	2.600	−7.480	1.535
4u	−5.732	−1.153	4.579	5.732	1.153	2.289	0.218	3.442	−3.442	2.588	−7.881	1.504
4v	−5.568	−1.153	4.415	5.568	1.153	2.207	0.227	3.360	−3.360	2.558	−7.418	1.522
4w	−5.920	−2.614	3.306	5.920	2.614	1.653	0.302	4.267	−4.267	5.508	−7.053	2.582
4x	−5.720	−1.306	4.415	5.720	1.306	2.207	0.227	3.513	−3.513	2.795	−7.755	1.591

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2.2.2. Molecular electrostatic potential analysis. A mapping of electrostatic potential onto a surface with a constant electron density is called the molecular electrostatic potential, or MEP. The molecular electrostatic potential (MEP) surface is often used to forecast the locations or sections of a molecule that would initially attract an approaching electrophile or nucleophile. The MEP surface of all spiro molecules 4a–x was calculated using the DFT/B3LYP/6-311G+(d,p) basis set in the gaseous phase. The MEP surface plots of 4a, 4i and 4q are shown in Fig. 3. Different MEP surface values are represented by the different colours in the plots. Here, the dark blue colour indicates the strongest attraction and is most susceptible to nucleophilic attack whereas the dark red colour indicates the strongest repulsion and is most susceptible to electrophilic attack. The majority of the negative areas lie above the O atom of oxygen of both carbonyl groups while the positive area lies above the N atom of nitrogen. According to this settlement, the O atom in this area participates in intermolecular interactions as an acceptor and the N atom in this area participates in intramolecular interactions as a donor in molecules 4a–x.

Fig. 3 Molecular electrostatic surface diagram of molecules 4a, 4i and 4j calculated at DFT/B3LYP/6-311G+(d,p) in the gaseous phase.

2.3. In vitro antioxident activity

To identify antioxidant properties of all newly synthesized spiroacridines 4a–p and spiroquinolines 4q–x, in vitro 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was tested. As a standard, antioxidant ascorbic acid was used. For ABTS and DPPH radical scavenging assay, it exhibits 41.84 ± 0.25 μM and 90.10 ± 0.70 μM IC₅₀ values, respectively. Table 4 displays the antioxidant activities of 4a–x. All spiro compounds show good radical scavenging activity. Among them 4a (ABTS, IC₅₀ = 22.52 ± 0.82 μM; DPPH, IC₅₀ = 19.07 ± 0.23 μM) and 4v (ABTS, IC₅₀ = 21.27 ± 0.58 μM; DPPH, IC₅₀ = 59.15 ± 0.29 μM) are the most potent radical scavengers against both ABTS & DPPH radicals which display excellent scavenging activity to standard ascorbic acid (Fig. 4). Meanwhile, all spiro compounds 4a–x are good ABTS radical scavengers. Spiroacridines 4b (ABTS, IC₅₀ = 36.27 ± 0.74 μM), 4h (ABTS, IC₅₀ = 34.18 ± 0.88 μM), 4i (ABTS, IC₅₀ = 23.75 ± 0.83 μM), 4n (ABTS, IC₅₀ = 24.58 ± 0.76 μM), and 4p (ABTS, IC₅₀ = 22.80 ± 0.74 μM) and spiroquinolines 4r (ABTS, IC₅₀ = 35.85 ± 0.34 μM), 4u (ABTS, IC₅₀ = 23.19 ± 0.23 μM), and 4x (ABTS, IC₅₀ = 27.30 ± 0.26 μM) show excellent ABTS radical scavenging activity.

Table 4 In vitro ABTS and DPPH radical scavenging activities of spiroacridines 4a–x and spiroquinolines 4q–x

Compound name	ABTS radical scavenging activity	DPPH radical scavenging activity
	IC50 ± SEM^a (μM)	IC50 ± SEM^a (μM)
a SEM (standard error mean). b Standard for ABTS and DPPH radical scavenging activity. Bold values show the lowest IC50s.
4a	22.52 ± 0.82	19.07 ± 0.23
4b	36.27 ± 0.74	156.64 ± 0.49
4c	66.48 ± 0.19	768.70 ± 0.27
4d	66.92 ± 0.53	686.08 ± 0.72
4e	56.39 ± 0.42	695.75 ± 0.64
4f	76.40 ± 0.31	253.01 ± 0.52
4g	48.12 ± 0.36	318.36 ± 0.37
4h	34.18 ± 0.88	179.63 ± 0.48
4i	23.75 ± 0.83	245.18 ± 0.23
4j	57.70 ± 0.19	897.43 ± 0.18
4k	46.31 ± 0.26	427.61 ± 0.28
4l	48.81 ± 0.38	364.29 ± 0.45
4m	48.97 ± 0.85	942.74 ± 0.34
4n	24.58 ± 0.76	262.80 ± 0.72
4o	42.89 ± 0.38	503.96 ± 0.16
4p	22.80 ± 0.74	901.44 ± 0.83
4q	42.75 ± 0.59	526.25 ± 0.44
4r	35.58 ± 0.34	1088.76 ± 0.58
4s	47.96 ± 0.19	924.27 ± 0.32
4t	46.96 ± 0.42	119.50 ± 0.34
4u	23.19 ± 0.23	904.15 ± 0.46
4v	21.27 ± 0.58	59.15 ± 0.29
4w	112.09 ± 0.66	726.31 ± 0.41
4x	27.30 ± 0.26	231.09 ± 0.17
Ascorbic acid^b	41.84 ± 0.25	90.10 ± 0.70

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Fig. 4 Potent ABTS and DPPH radical scavengers.

2.4. In silico docking study

Following the observation of outstanding antioxidant activity exhibited by the potent spiroacridine 4a and spiroquinoline 4v, a subsequent docking study was conducted. Gram-positive Listeria monocytogenes is a widespread intracellular pathogen which is responsible for numerous outbreaks of foodborne illness.³¹ So, for the docking study, we have chosen the Listeria monocytogenes targeted antioxidant protein in the LD-carboxypeptidase domain. According to the docking analysis, 4a and 4v exhibit proficient binding to the receptor's active sites, specifically site-1 and site-3, with respective binding affinities of −10.5 kcal mol⁻¹ and −10.7 kcal mol⁻¹, which are subsequently better than −5.4 kcal mol⁻¹, exhibited by the standard drug (ascorbic acid) employed in the antioxidant activity investigation (Fig. 5). Among the receptor's two active sites, 4a interacts with the active site-1 via conventional H-bonds, π-cation type of interaction, carbon–hydrogen bonds, π–π T-shapes, and unfavorable donor–donor type of interaction. The H-atom attached to the indole ring of spiroacridine 4a shows unfavorable donor–donor type interaction with TYR A:174 (1.94 Å). The carbonyl oxygen of the indole ring and an oxygen atom of 1,3-benzodioxole show conventional H-bonding with ARG A:19 (2.33 Å) and GLY A:85 (2.92 Å), respectively. An aryl ring of 1,3-benzodioxol of 4a interacts with ARG A:19 (3.65 Å) and HIS A:296 (5.28 Å) through π-cation and π–π T-shaped types of interactions, respectively. Furthermore, the methylene group of 1,3-bonzodioxol of 4a interacts with GLY A:85 (3.01 Å) through a carbon–hydrogen bond.

Fig. 5 Molecular docking study of 4a and 4v: (A) 4a was docked on Listeria monocytogenes to identify the potential binding sites in the LD-carboxypeptidase domain; (B) interaction diagram of 4a; (C) 4v docked on Listeria monocytogenes to identify the potential binding sites in the LD-carboxypeptidase domain; (D) interaction diagram of 4v.

4v interacts with the receptor at active site-3 through conventional H-bonding, π–π stacked type of bond, carbon–hydrogen bonds, π–π T-shapes, and π-cation, alkyl, π-sigma, and π-alkyl types of bonds. Carbonyl oxygen atoms of the quinoline ring and indole ring of 4v interact with TRP A:173 (2.88 Å) and HIS A:296 (4.67 Å) through conventional H-bonds and π–π stacked type of bond, respectively. An aryl ring of indole of 4a interacts with ILE A:83 (5.49 Å), TYR A:174 (4.88 Å) and TRP A:173 (5.50 Å) through π-alkyl, π–π stacked and π–π T-shaped interactions, respectively. The methoxy group attached to the aryl ring of 4a interacts with ILE A:83 (5.30 Å) and TYR A:174 (4.88 Å) through alkyl and π-sigma types of bonds, respectively. Furthermore, an aryl ring of 1,3-benzodioxol interacts with HIS A:296 (4.67 Å) and ARG A:19 (4.95 Å) through π–π T-shaped and π-cation types of bonds. These interactions make the binding of 4a and 4v strong enough in the binding sites of the Listeria monocytogenes targeted antioxidant protein of the LD-carboxypeptidase domain. So, 4a and 4v inhibit radical growth and show a good minimum inhibitory concentration value.

2.5. In silico ADME prediction

Prediction of absorption, distribution, metabolism, and excretion (ADME) properties before experimental trials is an essential aspect of the drug discovery and development process. The most potent antioxidants 4a and 4v were evaluated for in silico drug-likeness and ADME prediction. Several filters such as the Lipinski filter, Ghose filter, Veber filter, Egan filter and Muegge filter help predict drug-likeness based on their physicochemical properties. It was observed that both 4a and 4v obeyed all necessary rules and had no violation of such rules, proving them to be important drug candidate. The calculated physicochemical properties of potent compounds are summarized in Table S1 (mentioned in the ESI†).

The calculated bioavailability score was found to be 0.55 for 4a and 4v. BOILED-Egg delineation was used to predict high gastrointestinal absorption (HIA) and blood–brain barrier (BBB) permeability of 4a and 4v. BOILED-Egg delineation of these 4a and 4v is illustrated in Fig. 6 “The white region is the physicochemical space of molecules with the highest probability of being absorbed by the gastrointestinal tract, and the yellow region (yolk) is the physicochemical space of molecules with the highest probability to permeate to the brain”.³²4v shows high gastrointestinal absorbance, and it has no blood–brain permeability, while 4a shows blood–brain permeability. Blue dots for 4a and 4v denoted that they are P-gp substrates. From the SVM model, 4a was found to be a CYP1A2 inhibitor except for 4v, whereas both 4a and 4v were found to be inhibitors of CYP2C19, CYP2C9, CYP2D6 and CYP3A4 which can perform metabolic biotransformation. Furthermore, there were no PAINS and Brenk filter alerts of toxicity found for 4a and 4v.

Fig. 6 BOILED Egg model of compounds 4a and 4v.

3. Experimental

3.1. General

All chemicals used in this synthesis are commercially available and used without further purification. Melting points were obtained by the open capillary tube method and are uncorrected. High-resolution mass spectra (HRMS) were acquired using an Agilent technologies model G6564 QTOF. Nuclear magnetic resonance spectra (¹H NMR & ¹³C NMR) were obtained using a Bruker 500 MHz WB FT-NMR spectrometer having proton noise decoupling mode with a standard 5 mm probe using DMSO-d₆ solution. Abbreviations used for the ¹H NMR signal are as follows: s = singlet, d = doublet, t = triplet, q = quartet, qu = quintet, dd = double doublet, td = triple doublet, and m = multiplet. The chemical shifts are reported in parts per million and coupling constants (J) are provided in Hertz.

3.2. General procedure for the synthesis of spiroacridines 4a–p and spiroquinolines 4q–x

In a 50 mL round bottom flask, a mixture of 3,4-methylenedioxy aniline 1 (1 mmol), substituted isatins 2a–h (1 mmol) and cyclic 1,3-diones 3a–c (1 mmol) in 3 mL glacial acetic acid was refluxed for 5–8 min. On completion of the reaction (TLC), 5 mL of distilled water was added to the reaction mixture and stirred at room temperature for complete precipitation of the product. The furnished solid mass was filtered off, dried and then thoroughly washed again with 5 mL 5% ethyl acetate solution in n-hexane to afford the desired products 4a–x.

3.2.1. 7′,7′-Dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4a). White solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.25 (s, 1H, NH), 9.51 (s, 1H, NH), 7.08–7.05 (m, 1H, ArH), 6.81–6.76 (m, 3H, ArH), 6.52 (s, 1H, ArH), 5.97 (s, 1H, ArH), 5.90 (s, 1H, OCH̲_aH_bO), 5.86 (s, 1H, OCH̲_bH_aO), 2.50–2.36 (m, 2H, CH₂), 2.08 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.92 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.04 (s, 3H, CH₃), 0.99 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 190.7, 179.7, 150.9, 145.6, 142.3, 140.0, 138.9, 128.7, 126.0, 121.7, 120.8, 114.6, 107.8, 104.2, 102.5, 100.1, 95.8, 50.2, 49.0, 39.5, 31.1, 27.4, 25.7; HRMS (ESI-MS) m/z calcd for C₂₃H₂₁N₂O₄ [M + H]⁺: 389.1501, found: 389.1518.

3.2.2. 5-Chloro-7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4b). Off-white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.40 (s, 1H, NH), 9.60 (s, 1H, NH), 7.13 (dd, J = 8.2 Hz, 2.2 Hz, 1H, ArH), 6.83 (d, J = 8.2 Hz, 1H, ArH), 6.75 (d, J = 2.1 Hz, 1H, ArH), 6.55 (s, 1H, ArH), 5.97 (s, 1H, ArH), 5.93 (s, 1H, OCH̲_aH_bO), 5.88 (s, 1H, OCH̲_bH_aO), 2.46 (d, J = 4.5 Hz, 2H, CH₂), 2.07 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.98 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.04 (s, 3H, CH₃), 1.01 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 190.9, 179.3, 151.3, 145.9, 142.5, 140.6, 139.1, 128.7, 126.0, 124.2, 121.6, 113.7, 109.3, 104.0, 101.9, 100.2, 96.0, 50.5, 48.9, 39.4, 31.2, 27.0, 26.1; HRMS (ESI-MS) m/z calcd for C₂₃H₁₉ClN₂O₄ [M]⁺: 422.1033, found: 422.1038.

3.2.3. 5-Bromo-7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4c). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.42 (s, 1H, NH), 9.60 (s, 1H, NH), 8.32 (s, 1H, ArH), 7.26 (dd, J = 8.5 Hz, 2 Hz, 1H, ArH), 6.86 (d, J = 2.0 Hz, 1H, ArH), 6.79 (d, J = 8.5 Hz, 1H, ArH), 6.55 (s, 1H, ArH), 5.98 (s, 1H, ArH), 5.93 (s, 1H, OCH̲_aH_bO), 5.88 (s, 1H, OCH̲_bH_aO), 2.46 (s, 2H, CH₂), 2.07 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.99 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.04 (s, 3H, CH₃), 1.01 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 190.9, 179.2, 151.3, 145.9, 142.5, 141.0, 139.5, 128.8, 128.6, 124.3, 111.9, 109.9, 104.0, 101.9, 100.2, 96.0, 78.0, 48.9, 39.4, 31.1, 27.9, 27.0, 26.1; HRMS (ESI-MS) m/z calcd for C₂₃H₂₀BrN₂O₄ [M + H]⁺: 467.0606, found: 467.0602.

3.2.4. 5,7′,7′-Trimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4d). White solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.14 (s, 1H, NH), 9.49 (s, 1H, NH), 6.86 (d, J = 8 Hz, 1H, ArH), 6.69 (d, J = 7.5 Hz, 1H, ArH), 6.57 (s, 1H, ArH), 6.51 (s, 1H, ArH), 5.95 (s, 1H, ArH), 5.90 (s, 1H, OCH̲_aH_bO), 5.86 (s, 1H, OCH̲_bH_aO), 2.11 (s, 2H, CH₂), 1.96–1.90 (m, 2H, CH₂), 1.29 (s, 3H, ArCH₃), 1.04 (s, 3H, CH₃), 1.00 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 190.9, 179.3, 151.4, 146.0, 142.6, 141.1, 139.6, 128.9, 128.7, 124.4, 113.7, 112.0, 110.0, 104.1, 102.0, 100.3, 96.1, 49.0, 39.5, 31.2, 28.0, 27.6, 27.1, 26.2; HRMS (ESI-MS) m/z calcd for C₂₄H₂₃N₂O₄ [M + H]⁺: 403.1658, found: 403.1654.

3.2.5. 7-Chloro-7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4e). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.68 (s, 1H, NH), 9.61 (s, 1H, NH), 7.13 (d, J = 8 Hz, 1H, ArH), 6.82 (t, J = 8 Hz, 1H, ArH), 6.74 (d, J = 7 Hz, 1H, ArH), 6.54 (s, 1H, ArH), 5.97 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_aH_bO), 5.87 (s, 1H, OCH̲_bH_aO), 2.45 (m, 2H, CH₂), 2.09 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.93 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.04 (s, 3H, CH₃), 0.98 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 192.3, 181.3, 152.5, 147.2, 143.9, 141.5, 140.5, 130.2, 127.6, 123.3, 122.0, 116.2, 109.4, 105.8, 104.1, 101.7, 97.4, 51.8, 50.6, 41.1, 32.6, 29.0, 27.3; HRMS (ESI-MS) m/z calcd for C₂₃H₁₉ClN₂O₄ [M]⁺: 422.1033, found: 422.1038.

3.2.6. 5-Methoxy-7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4f). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.08 (s, 1H, NH), 9.50 (s, 1H, NH), 6.72 (d, J = 8.5 Hz, 1H, ArH), 6.64 (d, J = 8.5 Hz, 1H, ArH), 6.52 (s, 1H, ArH), 6.33 (s, 1H, ArH), 5.98 (s, 1H, ArH), 5.91 (s, 1H, OCH̲_aH_bO), 5.87 (s, 1H, OCH̲_bH_aO), 3.58 (s, 3H, OCH₃), 2.08 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.94 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.91 (s, 2H, CH₂), 1.04 (s, 3H, CH₃), 1.00 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 192.0, 180.5, 152.5, 147.1, 143.6, 141.8, 140.3, 129.8, 127.1, 125.4, 122.8, 114.8, 110.5, 105.2, 103.1, 101.4, 97.2, 51.7, 50.1, 40.6, 32.3, 28.2, 27.3, 21.1; HRMS (ESI-MS) m/z calcd for C₂₄H₂₀N₂O₄Na [M + Na] + [−H₂O]: 423.1321, found: 423.1113.

3.2.7. 7′,7′-Dimethyl-5-nitro-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4g). Greenish yellow solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.04 (s, 1H, NH), 9.75 (s, 1H, NH), 8.10 (dd, J = 8.5 Hz, 2.5 Hz, 1H, ArH), 7.57 (d, J = 2 Hz, 1H, ArH), 7.03 (d, J = 9 Hz, 1H, ArH), 6.58 (s, 1H, ArH), 6.00 (s, 1H, ArH), 5.93 (s, 1H, OCH̲_aH_bO), 5.89 (s, 1H, OCH̲_bH_aO), 2.07 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.99 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.90 (s, 2H, CH₂), 1.04 (s, 3H, CH₃), 1.03 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 191.4, 179.7, 151.8, 146.4, 143.0, 141.6, 140.0, 129.3, 129.2, 124.9, 114.2, 112.4, 110.4, 104.5, 102.5, 100.7, 96.5, 49.4, 39.9, 31.6, 28.4, 27.5, 26.6; HRMS (ESI-MS) m/z calcd for C₂₃H₁₉N₃O₆ [M]⁺: 433.1274, found: 433.1240.

3.2.8. 5-Fluoro-7′,7′-dimethyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4h). White solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.27 (s, 1H, NH), 9.57 (s, 1H, NH), 6.92–6.88 (m, 1H, ArH), 6.81–6.78 (m, 1H, ArH), 6.60 (dd, J = 8 Hz, 2.5 Hz, 1H, ArH), 6.53 (s, 1H, ArH), 5.97 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_aH_bO), 5.87 (s, 1H, OCH̲_bH_aO), 2.44 (m, 2H, CH₂), 2.07 (d, J = 16 Hz, 1H, COCH̲_aH_b), 1.96 (d, J = 16 Hz, 1H, COCH̲_bH_a), 1.04 (s, 3H, CH₃), 1.00 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm):192.5, 181.1, 159.3, 157.5, 154.4, 147.2, 143.7, 141.9 (d, J = 25 Hz), 137.7, 130.0, 115.2, 113.5 (d, J = 90 Hz), 110.8 (d, J = 95 Hz), 109.7 (d, J = 30 Hz), 105.4, 104.8, 101.5, 97.3, 62.3, 52.2, 36.8, 27.6, 25.8, 21.3; HRMS (ESI-MS) m/z calcd for C₂₃H₂₀FN₂O₄ [M + H]⁺: 407.1407, found: 407.1393.

3.2.9. 7′,8′-Dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4i). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.24 (s, 1H, NH), 9.54 (s, 1H, NH), 7.08–7.04 (m, 1H, ArH), 6.80–6.77 (m, 3H, ArH), 6.52 (s, ArH), 5.95 (s, 1H, ArH), 5.90 (s, 1H, OCH̲_aH_bO), 5.85 (s, 1H, OCH̲_bH_aO), 2.57 (t, J = 6 Hz, 2H, CH₂), 2.05–2.18 (m, 2H, CH₂), 1.83–1.92 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 192.6, 181.4, 154.4, 147.2, 143.8, 141.5, 140.6, 130.1, 127.6, 123.5, 121.9, 116.2, 109.4, 105.5, 101.7, 97.5, 97.2, 51.9, 37.1, 27.8, 21.5; HRMS (ESI-MS) m/z calcd for C₂₁H₁₆N₂O₄ [M]⁺: 360.1110, found: 360.1110.

3.2.10. 5-Chloro-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4j). Light pink, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.40 (s, 1H, NH), 9.64 (s, 1H, NH), 7.13 (dd, J = 8 Hz, 2 Hz, 1H, ArH), 6.83 (d, J = 8.5 Hz, 1H, ArH), 6.79 (d, J = 2.5 Hz, 1H, ArH), 6.53 (s, 1H, ArH), 5.96 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_aH_bO), 5.87 (s, 1H, OCH̲_bH_aO), 3.77 (qu, J = 12 Hz, 6 Hz, 2H, CH₂), 2.58 (dd, J = 11 Hz, 5 Hz, 2H, CH₂), 2.16–2.12 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 179.5, 153.2, 145.9, 142.4, 140.7, 139.0, 128.5, 125.9, 124.2, 121.8, 113.6, 109.3, 103.9, 103.3, 100.2, 95.9, 60.9, 50.5, 35.4, 26.1, 24.3, 19.9; HRMS (ESI-MS) m/z calcd for C₂₁H₁₅ClN₂O₄ [M]⁺: 394.0720, found: 394.0734.

3.2.11. 5-Bromo-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4k). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.41 (s, 1H, NH), 9.63 (s, 1H, NH), 7.26 (dd, J = 8.5 Hz, 2 Hz, 1H, ArH), 6.90 (d, J = 2 Hz, 1H, ArH), 6.78 (d, J = 8 Hz, 1H, ArH), 6.55 (s, 1H, ArH), 5.97 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_aH_bO), 5.88 (s, 1H, OCH̲_bH_aO), 3.78 (qu, J = 6 Hz, 2.5 Hz, 1H, 2H, CH₂), 2.59 (dd, J = 12 Hz, 5.5 Hz, 2H, CH₂), 2.17–2.12 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 191.2, 179.3, 153.2, 145.9, 142.4, 141.2, 139.4, 128.8, 128.5, 124.5, 113.7, 112.0, 109.8, 104.0, 103.3, 100.2, 95.9, 50.5, 35.4, 26.1, 24.3, 19.9; HRMS (ESI-MS) m/z calcd for C₂₁H₁₆BrN₂O₄ [M + H]⁺: 439.0293, found: 439.0289.

3.2.12. 5-Methyl-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4l). Off white solid, mp greater than 300 °C; ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 191.1, 179.3, 153.2, 145.8, 142.4, 141.1, 139.4, 128.8, 128.5, 124.4, 113.6, 111.9, 109.8, 103.9, 103.3, 100.1, 95.9, 60.9, 50.5, 35.3, 26.1, 24.3, 19.8; HRMS (ESI-MS) m/z calcd for C₂₂H₁₉N₂O₄ [M + H]⁺: 375.1345, found: 375.1343.

3.2.13. 7-Chloro-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4m). Brownish black solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.68 (s, 1H, NH), 9.65 (s, 1H, NH), 7.14 (dd, J = 8 Hz, 1 Hz, 1H, ArH), 6.82 (t, J = 7.5 Hz, 1H, ArH), 6.76 (d, J = 7 Hz, 1H, ArH), 6.55 (s, 1H, ArH), 5.96 (s, 1H, ArH), 5.93 (s, 1H, OCH̲_aH_bO), 5.88 (s, 1H, OCH̲_bH_aO), 2.59 (t, J = 6.5 Hz, 2H, CH₂), 2.21–2.07 (m, 2H, CH₂), 1.94–1.84 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 179.6, 153.3, 145.9, 142.5, 140.8, 139.0, 128.6, 126.0, 124.3, 121.8, 113.7, 109.3, 104.0, 103.3, 100.2, 96.0, 61.0, 50.6, 35.4, 26.2, 24.3; HRMS (ESI-MS) m/z calcd for C₂₁H₁₅ClN₂O₄ [M + H]⁺: 395.0799, found: 395.0796.

3.2.14. 5-Methoxy-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4n). White solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.07 (s, 1H, NH), 9.52 (s, 1H, NH), 6.71 (d, J = 8.5 Hz, 1H, ArH), 6.64 (dd, J = 8 Hz, 2.5 Hz, 1H, ArH), 6.51 (s, 1H, ArH), 6.36 (d, J = 2.5 Hz, 1H, ArH), 5.96 (s, 1H, ArH), 5.90 (s, 1H, OCH̲_aH_bO), 5.86 (s, 1H, OCH̲_bH_aO), 3.59 (s, 1H, OCH₃), 2.57 (t, J = 5.5 Hz, 2H, CH₂), 2.15–2.07 (m, 2H, CH₂), 1.91–1.88 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 192.8, 180.9, 154.8, 147.4, 144.0, 142.8, 141.0, 130.4, 130.1, 126.1, 115.2, 113.6, 111.4, 110.1, 105.6, 104.9, 101.8, 97.5, 62.5, 52.1, 37.0, 25.9, 21.5; HRMS (ESI-MS) m/z calcd for C₂₂H₁₈N₂O₅ [M]⁺: 390.1216, found: 390.1179.

3.2.15. 5-Nitro-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4o). Light brown solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 11.04 (s, 1H, NH), 9.77 (s, 1H, NH), 8.09 (dd, J = 8.5 Hz, 2 Hz, 1H, ArH), 7.61 (d, J = 2 Hz, 1H, ArH), 7.02 (d, J = 8.5 Hz, 1H, ArH), 6.58 (s, 1H, ArH), 5.99 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_aH_bO), 5.88 (s, 1H, OCH̲_bH_aO), 2.67–2.57 (m, 2H, CH₂), 2.18–2.09 (m, 2H, CH₂), 1.90–1.88 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 193.0, 181.7, 155.2, 148.5, 147.7, 144.2, 142.6, 141.0, 130.2, 125.4, 118.6, 114.5, 109.6, 105.5, 104.6, 101.9, 97.7, 51.9, 36.8, 27.7, 21.4; HRMS (ESI-MS) m/z calcd for C₂₁H₁₆N₃O₆ [M + H]⁺: 406.1039, found: 406.1037.

3.2.16. 5-Fluoro-7′,8′-dihydro-5′H-spiro[indoline-3,10′-[1,3]dioxolo[4,5-b]acridine]-2,9′(6′H)-dione (4p). White solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.28 (s, 1H, NH), 9.60 (s, 1H, NH), 6.92–6.87 (m, 1H, ArH), 6.78 (dd, J = 8.5 Hz, 4 Hz, 1H, ArH), 6.64 (dd, J = 8 Hz, 2.5 Hz, 1H, ArH), 6.53 (s, 1H, ArH), 5.96 (s, 1H, ArH), 5.91 (s, 1H, OCH̲_aH_bO), 5.87 (s, 1H, OCH̲_bH_aO), 3.77 (qu, J = 12 Hz, 6 Hz, 2H, CH₂), 2.57 (t, J = 6 Hz, 2H, CH₂), 2.15–2.08 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 192.6, 181.3, 159.5, 157.7, 154.6, 147.4, 143.9, 142.1 (d, J = 30 Hz), 137.9, 130.1, 115.4, 113.7 (d, J = 95 Hz), 111.0 (d, J = 100 Hz), 109.9 (d, J = 35 Hz), 105.6, 105.0, 101.7, 97.5, 52.4, 37.0, 26.0, 21.5; HRMS (ESI-MS) m/z calcd for C₂₁H₁₆FN₂O₄ [M + H]⁺: 379.1094, found: 379.1090.

3.2.17. 6,7-Dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4q). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.38 (s, 1H, NH), 10.15 (s, 1H, NH), 7.15 (t, J = 7.5 Hz, 1H, ArH), 6.87–6.81 (m, 3H, ArH), 6.58 (s, 1H, ArH), 5.94 (s, 1H, ArH), 5.90 (s, 2H, OCH₂O), 2.73 (d, J = 5.5 Hz, 2H, CH₂), 2.22 (d, J = 5.5 Hz, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 198.0, 178.5, 165.3, 146.8, 143.3, 139.9, 139.0, 130.8, 127.3, 125.2, 123.5, 114.1, 110.3, 107.5, 105.5, 101.0, 97.1, 50.6, 32.3, 25.0; HRMS (ESI-MS) m/z calcd for C₂₀H₁₅N₂O₄ [M + H]⁺: 347.1032, found: 347.1043.

3.2.18. 5′-Chloro-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4r). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.53 (s, 1H, NH), 10.22 (s, 1H, NH), 7.20 (dd, J = 8.5 Hz, 2 Hz, 1H, ArH), 6.88 (d, J = 8 Hz, 1H, ArH), 6.84 (d, J = 2.5 Hz, 1H, ArH), 6.60 (s, 1H, ArH), 5.96 (s, 1H, OCH̲_aH_bO), 5.94 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_bH_aO), 2.73 (dd, J = 10.5 Hz, 4.5 Hz, 2H, CH₂), 2.23 (t, J = 5 Hz, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 199.0, 179.5, 166.3, 147.8, 144.3, 140.9, 131.8, 128.3, 126.2, 124.5, 115.1, 111.3, 108.5, 106.5, 102.0, 98.1, 51.6, 33.3, 26.0; HRMS (ESI-MS) m/z calcd for C₂₀H₁₄ClN₂O₄ [M + H]⁺: 381.0642, found: 381.0641.

3.2.19. 5′-Bromo-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4s). Light brown solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.57 (s, 1H, NH), 10.54 (s, 1H, NH), 7.33 (dd, J = 8 Hz, 2 Hz, 1H, ArH), 6.94 (d, J = 2 Hz, 1H, ArH), 6.84 (d, J = 8.5 Hz, 1H, ArH), 6.60 (s, 1H, ArH), 5.95 (s, 1H, OCH̲_aH_bO), 5.94 (s, 1H, ArH), 5.92 (s, 1H, OCH̲_bH_aO), 2.72 (t, J = 8.5 Hz, 2H, CH₂), 2.23 (t, J = 5 Hz, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 197.5, 177.8, 164.8, 146.2, 142.7, 139.7, 138.8, 130.2, 129.5, 125.6, 113.5, 112.4, 110.3, 106.9, 104.9, 100.4, 96.5, 49.9, 31.7, 23.1; HRMS (ESI-MS) m/z calcd for C₂₀H₁₄BrN₂O₄ [M + H]⁺: 425.0136, found: 425.0133.

3.2.20. 5′-Methyl-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4t). Light brown, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.29 (s, 1H, NH), 10.15 (s, 1H, NH), 6.93 (d, J = 7.5 Hz, 1H, ArH), 6.74 (d, J = 7.5 Hz, 1H, ArH), 6.62 (s, 1H, ArH), 6.57 (s, 1H, ArH), 5.93 (s, 1H, OCH̲_aH_bO), 5.90 (s, 1H, OCH̲_bH_aO), 5.88 (s, 1H, ArH), 2.71 (dd, J = 10 Hz, 4.5 Hz, 2H, CH₂), 2.22–2.20 (m, 2H, CH₂), 2.14 (s, 3H, CH₃); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 199.0, 179.8, 166.1, 147.5, 144.1, 139.3, 138.5, 131.7, 131.2, 128.6, 125.0, 116.1, 109.5, 109.2, 106.6, 101.9, 97.4, 51.4, 33.4, 24.6, 21.1; HRMS (ESI-MS) m/z calcd for C₂₁H₁₇N₂O₄ [M + H]⁺: 361.1188, found: 361.1189.

3.2.21. 7′-Chloro-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4u). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.85 (s, 1H, NH), 10.26 (s, 1H, NH), 7.22 (d, J = 8.5 Hz, 1H, ArH), 6.88 (t, J = 7.5 Hz, 1H, ArH), 6.79 (d, J = 7.5 Hz, 1H, ArH), 6.60 (s, 1H, ArH), 5.95 (s, 1H, OCH̲_aH_bO), 5.94 (s, 1H, ArH), 5.91 (s, 1H, OCH̲_bH_aO), 2.73 (t, J = 5 Hz, 2H, CH₂), 2.43–2.21 (m, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm):199.0, 179.6, 166.2, 147.8, 144.3, 139.7, 131.7, 128.4, 123.7, 123.2, 115.2, 114.0, 108.7, 106.5, 102.0, 98.1, 79.6, 52.2, 33.3, 24.7; HRMS (ESI-MS) m/z calcd for C₂₀H₁₄ClN₂O₄ [M + H]⁺: 381.0642, found: 381.0677.

3.2.22. 5′-Methoxy-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4v). Light brown solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.22 (s, 1H, NH), 10.16 (s, 1H, NH), 6.79 (d, J = 8.5 Hz, 1H, ArH), 6.74–6.71 (m, 1H, ArH), 6.58 (s, 1H, ArH), 6.39 (d, J = 2.5 Hz, 1H, ArH), 5.94 (s, 1H, OCH̲_aH_bO), 5.91 (s, 1H, OCH̲_bH_aO), 5.90 (s, 1H, ArH), 3.61 (s, 3H, OCH₃), 2.74–2.70 (m, 2H, CH₂), 2.22 (t, J = 5 Hz, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 199.0, 179.7, 166.2, 155.6, 147.5, 144.1, 139.5, 135.2, 131.7, 115.9, 112.8, 111.4, 110.1, 109.0, 106.5, 101.9, 98.0, 55.8, 51.8, 33.4, 24.6; HRMS (ESI-MS) m/z calcd for C₂₁H₂₀N₃O₅ [M + NH₄]⁺: 394.1403, found: 394.1426.

3.2.23. 5′-Nitro-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4w). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.56 (s, 1H, NH), 10.23 (s, 1H, NH), 7.32 (dd, J = 8.5 Hz, 2 Hz, 1H, ArH), 6.94 (d, J = 1.5 Hz, 1H, ArH), 6.83 (d, J = 8 Hz, 1H, ArH), 6.59 (s, 1H, ArH), 5.95 (s, 1H, OCH̲_aH_bO), 5.94 (s, 1H, ArH), 5.91 (s, 1H, OCH̲_bH_aO), 2.71 (t, J = 7.5 Hz, 2H, CH₂), 2.3 (t, J = 5 Hz, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 198.3, 179.0, 165.5, 154.9, 146.8, 143.4, 138.8, 134.5, 131.0, 115.2, 112.1, 110.7, 109.4, 108.3, 105.8, 101.2, 97.3, 55.1, 32.7, 23.9; HRMS (ESI-MS) m/z calcd for C₂₀H₁₄N₃O₆ [M + H]⁺: 392.0883, found: 392.0882.

3.2.24. 5′-Fluoro-6,7-dihydrospiro[cyclopenta[b][1,3]dioxolo[4,5-g]quinoline-9,3′-indoline]-2′,8(5H)-dione (4x). Off white solid, mp greater than 300 °C; ¹H-NMR (500 MHz, DMSO-d₆) (δ, ppm): 10.43 (s, 1H, NH), 10.22 (s, 1H, NH), 7.00–6.96 (m, 1H, ArH), 6.85 (dd, J = 8.5 Hz, 4.5 Hz, 1H, ArH), 6.69 (dd, J = 7.5 Hz, 2 Hz, 1H, ArH), 6.59 (s, 1H, ArH), 5.95 (s, 1H, ArH), 5.92 (s, 2H, OCH₂O), 2.72 (d, J = 4 Hz, 2H, CH₂), 2.22 (d, J = 3 Hz, 2H, CH₂); ¹³C-NMR (125 MHz, DMSO-d₆) (δ, ppm): 197.4, 178.2, 164.7, 146.1, 142.6, 138.1, 136.5, 130.2, 130.6, 113.6, 113.0 (d, J = 95 Hz), 110.5 (d, J = 95 Hz), 108.9 (d, J = 30 Hz), 107.0, 104.9, 100.4, 96.5, 78.0, 50.3, 31.7, 23.1; HRMS (ESI-MS) m/z calcd for C₂₀H₁₄FN₂O₄ [M + H]⁺: 365.0938, found: 365.0937.

3.3. Computational details

The Gaussian 16 software package³³ was utilized for computational investigations and all DFT calculations were performed using the B3LYP method with a 6-311G+(d,p) basis set in the ground state. The chemical reactivity and stability of the molecule were investigated using Becke's three parameter (B3LYP) diffusion functionals at the 6-311G+(d,p) basis set. The frontier molecular orbital (FMO) and molecule electrostatic potential (MEP) surface analyses were accomplished by the DFT/B3LYP method using a 6-311G+(d,p) basis set. The orbital energy gap (E_gap) and other quantum chemical descriptors (QCDs) were calculated from frontier molecular orbitals (FMOs) with the same conceptual method and by using well-known equations. Gauss view 6.1.1 (ref. 34) was used for geometry optimization and data visualization.

3.4. ABTS radical scavenging assay

The ABTS free radical cation scavenging activity of compounds was determined by a standard procedure. First, a 7 mM concentrated solution of ABTS was prepared and then a 2.45 mM concentrated solution of potassium persulfate was added to the ABTS solution. After adding potassium persulfate to the ABTS solution, the solution was kept in a dark place for 12–16 h at room temperature. The sample solutions of the test compound were prepared in absolute alcohol ranging from 0.01 mg mL⁻¹ to 1 mg mL⁻¹. Then the test sample was added to the ABTS solution and incubated for 30 min at 37 °C. The absorbance was measured at a wavelength of 734 nm, and the procedure was repeated for ascorbic acid as a reference standard. The % inhibition of radical scavenging activity was determined using the given formula.

3.5. DPPH radical scavenging assay

The free radical scavenging activity of DPPH was tested using a standard procedure. 0.3 mM DPPH concentrated solution in ethanol was incubated for 30 min at 37 °C with various test samples. The absorption of the sample was taken at 517 nm. The same procedure was followed for ascorbic acid as for the standard. The % inhibition of radical scavenging activity was determined using the given formula.

3.6. In silico molecular docking

3.6.1. Molecular docking. The AlphaFold structure of the Listeria monocytogenes targeted antioxidant protein of the LD-carboxypeptidase domain (ID: AF-A0A823DAK7-F1-model_v4) was imported from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/entry/A0A823DAK7, 11/01/2024, 01:26 PM) (ebi.ac.uk). Autodock Vina software³⁵ was used for molecular docking simulations of 4a and 4v. Autodock Tools version 1.5.7 is used for pre-processing. In order to analyze the bound conformations and interactions, Discovery Studio Visualizer was used.

3.6.2. Ligand preparation for docking study. ChemBio3D 22.0.0 was used to draw the 3D structure of spiroacridine 4a and spiroquinoline 4v. The energy was minimized to a minimum RMS gradient of 0.000 in each interaction. All structures were saved in SDFile (*.sdf and *.sd) file format which was then transformed into a .pdb file using Open Babel for input to the Autodock tool. The ligand structure was then saved in the (.pdbqt) file format.

3.6.3. Preparation of the protein structure. The AlphaFold crystal structure of the Listeria monocytogenes targeted antioxidant protein of the LD-carboxypeptidase domain (ID: AF-A0A823DAK7-F1-model_v4) with the per-residue model confidence score (pLDDT) between 0 and 100 was imported (as .pdb file) from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/entry/A0A823DAK7, 11/01/2024, 01:26 PM) (ebi.ac.uk). A circle with a volume of 20 × 20 × 20 ų at centre 9.637000, 4.581000, and −0.419000 (site-1) and −0.363000, 8.831000, and −4.919 (site-3) were set as the grid. As the AlphaFold structure of LD-carboxypeptidase was free from crystal water, polar hydrogens were added with the help of Discovery Studio Visualizer. Finally, with the help of ADT, Kolmann and Gasteiger charges were added to each atom of protein and merged with the non-polar hydrogen atoms to form the protein structure. Then, the structure was saved in PDBQT file format.

3.7. In silico ADMET prediction

In silico ADMET prediction of the most potent antioxidants 4a and 4q were calculated using web tool SwissADME (http://www.swissadme.ch/).³⁶

4. Conclusion

In conclusion, we have developed a green synthetic approach for the synthesis of spiroacridines and spiroquinolines via a multicomponent reaction of 3,4-methylenedioxyaniline, isatin and cyclic 1,3-diones in glacial acetic acid. The key features of this protocol are the metal-free multicomponent approach, mild reaction conditions, faster reaction time, moderate to good yield and easy product purification without column chromatography. FMOs, various QCDs and MEP of all spiro molecules were obtained using DFT calculations using the B3LYP method with a 6-311G+(d,p) basis set. Furthermore, the antioxidant properties of all newly synthesized compounds were evaluated. According to in silico molecular docking studies of the most potent 4a and 4v with the targeted antioxidant protein, LD-carboxypeptidase, they show a strong binding affinity of −10.5 kcal mol⁻¹ and −10.7 kcal mol⁻¹, respectively. In addition, 4a and 4v are the most potent radical scavengers as they possess good drug-likeness as well as in silico ADME prediction.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

SGP, PJP, DBU and HMP are grateful to the Department of Chemistry, Sardar Patel University, for providing computational & lab facilities. SGP is grateful to the UGC, New Delhi, for a UGC-JRF (NTA Ref. No. 201610157514; dated: 01.04.2021). MPP and DBU are thankful to the Knowledge Consortium of Gujarat for the SHODH fellowship (Student Reference No. 2021016434 & 2021016413, respectively).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4su00024b

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