Molybdate sulfuric acid (MSA): an efficient reusable catalyst for the synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones under solvent-free conditions and evaluation of their in vitro bioassay

Abstract

An efficient green synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones has been achieved under solvent-free conditions by the reaction of 1H-benzo[d]imidazol-2-amine, various aldehydes and 1,3-dicarbonyl compounds in the presence of molybdate sulfuric acid (MSA) as a catalyst. Higher product yields were isolated easily using this reusable MSA catalyst, and environmentally benign reaction conditions in shorter reaction time are the merits of this reaction. All the newly synthesized compounds were tested for their anti-oxidant and anticancer activities. Most of them showed good to excellent bio-activity in both the experiments.

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Recently, the second highest cause of death worldwide is a class of cancer disease caused by uncontrolled cell growth.¹ Even though more than 50 years have passed since the discovery of the toxic action of nitrogen mustards on cancer cells still there is a need to develop effective cytotoxic agents. In addition, reactive oxygen species (ROS) are oxygen centred free radicals, which are generated in the human body and would cause damage to lipids, proteins and DNA and thus may lead to various diseases such as cancer, drug-associated toxicity, and inflammation. Furthermore, radical reactions play significant role in the development of life limiting chronic diseases such as cancer, ageing, diabetes, arteriosclerosis and others.² Herein, either natural or synthetic anti-oxidants are molecules that have the capability of interacting with free radicals and stopping their chain reactions before essential vital molecules are damaged.³ Thus, they are recently considered as the drug candidates to counter these multifarious diseases causing metabolic free-radical reactions by protecting cells and organisms.

Nitrogen-containing heterocycles play a major role in the pharmaceutical and agrochemical industries because of their often potent physiological properties, which have resulted in numerous applications such as antibacterial,⁴ antifungal,⁵ antiviral,⁶ antioxidant,⁷ anti-inflammatory agents⁸ and anticancer activities.⁹ Among a large variety of N-containing heterocyclic compounds, tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones (A) have received considerable attention because of their pharmacological properties and clinical applications.¹⁰ Moreover, they were found to possess multiple biological activities, such as antimicrobial,¹¹ antifungal,¹² anticancer,¹³ anti-inflammatory,¹⁴ anticonvulsant,¹⁵ antihypertensive,¹⁶ antihistaminic,¹⁷ analgesic¹⁸ and anti-HIV¹⁹ activities.

Therefore, the synthesis of the tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones has attracted much attention in organic synthesis. To date, only few methods have been reported for the synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones.²⁰ However, these procedures also are limited in scope because of relatively long reaction time and the use of an organic solvent, expensive ionic liquids and catalysts. Therefore, the search continues for a better green catalyst for the synthesis of heterocycles containing tetrahydrobenzo-[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones ring fragment in terms of operational simplicity, cost effectiveness, environmentally benign practical procedure, economic viability and greater selectivity.

In this context, multi-component, one-pot syntheses under solvent-free conditions have materialized as an efficient and powerful strategy in modern synthetic organic chemistry because synthesis of complex organic molecules from simple and readily available substrates can be achieved in a very fast and efficient manner without isolation of any intermediates.²¹ The multi-component reactions (MCRs) contribute to these requirements of an environmentally friendly process by reducing the synthesis steps, energy consumption and waste production. Therefore, the development of new MCRs and improvement of the known MCRs are popular areas of research in current synthetic organic chemistry.

In addition, sulfonic acid-containing catalysis has developed considerable interest in various disciplines of science, including organic synthesis, because of the prime advantage that, in most of the cases, these catalysts can be recovered without measurable changes in their catalytic activity and selectivity and straightforward work-up, availability, eco-friendly reaction conditions, reusability and ability to promote a wide range of reactions;²² therefore, they can be used in continuous flow reactions. In this study, molybdate sulfuric acid (MSA) as a proficient proton source was found to be synthetically useful in modern organic reactions.²³ It has numerous advantages over conventional acid catalysts, such as ease of handling, stability, low cost, and easy recyclability because of its insolubility in most organic solvents. Thus, it has been selected as a solid heterogeneous alternative to sulfuric acid.

Previously reported studies revealed that there are no reports on the synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones by MSA catalysis under solvent-free conditions. Therefore, the health risks stated above and the need for the development of more effective anti-oxidant and anticancer drug molecules encouraged us to design and synthesise new chemical compounds with high efficiency, low toxicity and broad spectrum of bio-activity.

In continuation of our efforts to develop a better synthetic procedure in terms of operational simplicity, economic viability, greater selectivity and as well as the interest in applications of heterogeneous-catalyzed organic reactions²⁴ in green chemical synthetic approaches, herein, we report a one-pot synthesis by coupling of 1H-benzo[d]imidazol-2-amine (1), various aldehydes (2a–z & a′) and 1,3-dicarbonyl compounds (3a, 3b) by using MSA as a reusable catalyst at 80 °C under green condition (Scheme 1) to synthesise the tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones (4a–z & 4a′). Bioassay studies of the title compounds showed them to have potential activity.

Scheme 1 MSA catalyzed synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones.

Chemistry

The reaction of 1H-benzo[d]imidazol-2-amine (1, 1 mmol), 2-pyridinecarboxaldehyde (2a, 1 mmol) and dimidine (3a, 1 mmol) under neat conditions at 80 °C was run initially in the absence of catalyst to standardize the experimental conditions (Scheme 1). Desired product, 4a, is not formed even after 6 h stirring. After 10 h of stirring only a trace amount of product was formed (Table 1, entry 1). The same reaction when performed in the presence of 5 mol% of the PS/PTSA 4a is formed in 40% yield within 4 h (Table 1, entry 2). The product 4a formed was confirmed by spectroscopic analysis.

Encouraged by this result, to optimize the product yield, the same reaction was carried out with various sulfonic acid containing catalysts such as glucose sulfonic acid (GSA), phosphosulfonic acid (PSA), PEG-SO₃H and MSA. The results are summarized in Table 1. Among all the screened catalysts MSA was found to be superior with respect to reaction time and product yield (Table 1, entry 6). Later, the quantity of the catalyst required for obtaining the maximum product yield was studied by using 1 mol%, 2 mol%, 5 mol% and 10 mol% of MSA and the obtained yields were 65, 78, 92 and 92%, respectively (Table 1, entries 6–9). This showed that 5 mol% of MSA was sufficient and further increases in the quantity of catalyst did not show significant improvement in the reaction rate and product yields. It shows that the rate of the reaction and product yield is dependent on the nature and quantity of the catalyst (Table 1, entry 9).

Table 1 Influence of the catalyst for the synthesis of 3,3-dimethyl-12-(pyridin-2-yl)-3,4,5,12-tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-one (4a)^a

Entry	Catalyst (mol%)	Time (min)	Yield^b (%)
a Reaction of 1H-benzo[d]imidazol-2-amine (1, 1 mmol), Pyridinecarboxaldehyde (2a, 1 mmol) and dimidine (3, 1 mmol) under neat condition at 80 °C.b Isolated yield.c Catalyst was reused four times.
1	Neat	600	Trace
2	PT/PTSA (30 mg)	240	40
3	GSA (5)	170	65
4	PSA (5)	130	66
5	PEG-SO3H (30 mg)	110	75
6^c	MSA (5)	20	92, 90, 89, 86
7	MSA (1)	60	65
8	MSA (2)	40	78
9	MSA (10)	20	92

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The influence of various organic solvents at different reaction temperatures on the model reaction with 5 mol% of MSA and without catalyst was investigated. In toluene, THF, CH₂Cl₂, CH₃CN, and DMF rate of the reaction was slow and in lower product yields were obtained (Table 2, entries 1–6). The same reaction in ethanol improved both the reaction rate as well as product yield (Table 2, entry 7). But highest product yield was observed in solvent-free conditions at 80 °C (Table 2, entry 8) and thus could be attributed to a uniform distribution of the eutectic mixture of reactants in it.

Table 2 Optimization of reaction conditions of solvent and temperature for the synthesis of 3,3-dimethyl-12-(pyridin-2-yl)-3,4,5,12-tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-one (4a)^a

Entry	Solvent	Temp (°C)	Yield^b (%)
			Catalyst-free	MSA (5 mol%)
a Reaction conditions: 1H-benzo[d]imidazol-2-amine (1, 1 mmol), pyridinecarboxaldehyde (2a, 1 mmol) and dimidine (3, 1 mmol) at 20 min.b Isolated yield.
1	Toluene	110	32.4	48.3
2	Chlorobenzene	120	35.2	55.7
3	CH2Cl2	75	39.2	58.4
4	CCl4	75	30.4	45.1
5	THF	65	43.2	54.2
6	Acetonitrile	65	38.8	55.4
7	Ethanol	70	65.4	80.4
8	Solvent-free	80	25.3	92.0

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In order to investigate the catalytic activity and the possibility of the catalyst recyclability and reusability, MSA was recovered from the reaction mixture by simple filtration in ethyl acetate. The separated catalyst was dried in a vacuum oven at 100 °C and was reused for subsequent experiments under similar reaction conditions (Table 1, entry 6). The results showed that the catalyst could be effectively reused for at least four consecutive cycles without appreciable loss in its catalytic activity. The recyclability data demonstrated the high stability of the catalyst under the reaction conditions (Fig. 2).

Fig. 1 Effect of solvent on product yields on model reaction.

Fig. 2 Reusability of the MSA catalyst.

The effect of temperature on the reaction in various solvents and without solvent was also tested (Table 2, entries 1–7). Irrespective of the solvent, even at high boiling solvent the yield was poor (Fig. 1). But under solvent-free conditions a higher (80%) product yield was observed at 80 °C (Table 2, entry 8). Solvation of the substrates with solvents which reduced their effective interaction might be the primary cause for the drop in the product yield.

To compare the advantage of the use of MSA as a catalyst over others, the reaction was performed with a few representative examples. The results showed that MSA is a superior catalyst for the model reaction. All other reported procedures required either expensive and/or high catalyst loading (Table 3).

Table 3 Comparison of various reported catalysts with MSA

Entry	Catalyst/solvent/temperature (°C)	Catalyst load (mol%)	Time (min)	Yield (%)	Reference
1	[bimm^+][BF4]^−/90	3 mL	420	90	20f
2	Iodine/acetonitrile/80	10	15	88	20d
3	Sulfamic acid/acetonitrile/reflux	5	20	90	20e
4	Silica gel/MW ethanol/120	—	6	92	20b
5	MSA/solvent-free/80	5	20	92	Present work

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Having optimized the reaction conditions for the synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-one (4a–z & 4a′) using MSA as the catalyst under solvent-free conditions, were applied for the synthesis of various aldehydes. As shown in Table 4. MCRs produced excellent product yields for a wide range of aromatic aldehydes bearing both electron-donating and electron-withdrawing substituents. It is noteworthy that no reports exist for the synthesis of bioactive tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones from aliphatic and heterocyclic aldehydes. In this procedure the pure products were isolated by simple filtration without chromatography or a cumbersome work-up procedure. After the reaction, the catalyst can be separated from the product and reused without a significant decrease in its catalytic activity. The structures of the new compounds were determined by ¹H NMR, ¹³C NMR and HRMS. The data of 4z and 4a′ compounds were agreed with reported compounds.

Table 4 Synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones^a

Entry	Aldehyde (R)	Compound	Yield^b (%)	Time (min)	MP (°C)
a Reaction of 1H-benzo[d]imidazol-2-amine (1, 1 mmol), various aldehydes (2a–z & a′ 1 mmol), 1,3-dicarbonyl compounds (3, 1 mmol) catalysed by MSA under solvent-free at 80 °C.b Isolated yield.
1		4a	92	20	340–342
2		4b	94	26	282–284
3		4c	90	30	270–272
4		4d	91	26	301–303
5		4e	92	28	296–298
6		4f	89	30	310–312
7		4g	92	25	240–242
8		4h	91	24	271–273
9		4i	89	30	288–290
10		4j	88	35	308–310
11		4k	89	32	221–223
12		4l	88	30	278–280
13		4m	92	35	305–307
14		4n	90	31	298–300
15		4o	91	29	311–313
16		4p	93	25	309–311
17		4q	92	30	353–355
18		4r	93	25	378–380
19		4s	85	37	312–314
20		4t	91	32	375–377
21		4u	88	38	304–306
22		4v	89	40	378–380
23		4w	85	35	36–367
24		4x	90	30	310–312
25		4y	89	35	258–260
26		4z	91	20	355–357
27		4a′	92	14	325–327

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Pharmacological screening

In recent years, an increased interest has been observed for using antioxidants for medical purposes. Thus, drugs have been considered for preventing and/or treatment of diseases that have anti-oxidant and free radical scavenging properties. An effective anti-oxidant protects our body from free radicals that cause oxidative stress or “cellular rust”, which can lead to a host of severe medical conditions. Because anti-oxidants help to prevent cancer and other cardiovascular diseases, herein, we screened the anti-oxidant activity and the cytotoxic activity of all the synthesized title compounds.

Anti-oxidant activity

Free radicals generated in many bioorganic redox processes may damage various organs of the body by inducing oxidative processes, and they have also been implicated in a number of life-limiting chronic diseases and aging. The high reactivity of free radicals within the body can be neutralized by electrons or hydrogen radicals that are donated by anti-oxidants and thus free radical scavengers (Scheme 2). To explore the free radical scavenging ability of the newly synthesized compounds, we carried out different types of in vitro assay experiments such as, 1,1-diphenyl-2-picrylhydrazyl (DPPH),²⁵ hydroxyl (H₂O₂)²⁶ and reducing power (RP)²⁷ radical scavenging activity methods to develop potent anti-oxidants. Herein, we measured the in vitro inhibitory concentration of 50% (IC₅₀) (eqn (1)) with reference to Vitamin-C as a standard anti-oxidant. Most of the titled compounds exhibited good in vitro anti-oxidant activity in these representative methods (Table 5).where A_cont. is the absorbance of the control (containing all reagents except the test compound, blank sample) and A_test is the absorbance of the test compound.

Scheme 2 Radical scavenging activity of the title compounds (4a–z and 4a′) due to the labile nature of hydrogen by the tautomerisation of unpaired electrons on nitrogen atom.

Table 5 Anti-oxidant activity of test compounds (4a–z & 4a′)

Compound	Inhibition%
	DPPH	H2O2	RP
4a	80.9	82.4	86.2
4b	87.9	88.1	83.1
4c	88.7	89.2	84.9
4d	82.8	84.8	84.5
4e	78.2	84.1	83.9
4f	87.1	83.3	87.9
4g	65.4	61.7	68.5
4h	65.1	60.8	66.8
4i	81.4	81.7	82.0
4j	77.2	71.4	78.8
4k	77.9	75.2	79.2
4l	78.6	81.6	83.7
4m	78.1	82.8	83.8
4n	86.4	81.3	82.1
4o	86.1	80.8	83.2
4p	81.2	79.6	83.5
4q	75.3	73.6	76.9
4r	78.3	77.1	78.8
4s	80.7	80.8	81.8
4t	87.2	81.8	86.9
4u	79.8	78.6	80.2
4v	77.6	74.3	79.2
4w	79.7	76.7	79.5
4x	64.9	60.4	65.8
4y	64.3	60.1	64.1
4z	78.3	79.8	80.2
4a′	84.9	83.8	85.4
Vitamin-C	89.8	89.2	93.5

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DPPH scavenging activity

One of the widely accepted and often used tools for estimating free radical scavenging activity of anti-oxidants is nitrogen centred stable DPPH free radical, which is purple in colour, because it is a rapid, simple and inexpensive method. During of free radical scavenging activity, it accepts an electron or a hydrogen radical from testing anti-oxidant and forms a non-radical DPPH (Scheme 2) resulting in its de-colorization which is in stoichiometry. DPPH has been used to measure anti-oxidant properties by measuring the change in the absorbance produced in this reaction.

Most of the title compounds showed good to excellent DPPH radical scavenging activity (Table 5). These compounds have good radical and/or hydrogen donating capacity (Scheme 2). Among them, 4c, 4b, 4t, 4f, 4n, 4o, 4d, 4i, 4p, and 4a showed the highest scavenging activity of 88.7%, 87.9%, 87.2%, 87.1%, 86.4%, 86.1%, 82.8%, 81.4%, 81.2% and 80.9%, respectively, when compared with other compounds. The remaining compounds also exhibited good DPPH radical scavenging activity when compared with the ascorbic acid (89.8%), which was used as the positive control (Fig. 3).

Fig. 3 Anti-oxidant activity of the title compounds – all the radical scavenging activities of the title compounds represented with the standard Vitamin C. All data are expressed as mean ± S.D. (n = 3).

Hydroxyl radical (H₂O₂) scavenging activity

Hydroxyl radical is one of the most ROS that attacks almost every molecule in the body. It was measured based on the competition between hydroxyl radicals generated by compounds and deoxyribose. Most of the newly synthesized title compounds showed good to excellent hydroxy radical scavenging activity (Table 5). When compared with others, compounds 4c, 4b, 4d, 4e, 4f, 4m, 4a, 4t, 4i and 4l showed excellent OH radical scavenging activity of 89.2%, 88.1%, 84.8%, 84.1%, 83.3%, 82.8%, 82.4%, 81.8%, 81.7% and 81.6%, respectively. All the remaining compounds exhibited good hydroxyl radical scavenging activity values, which indicate that the title compounds can be considered as potential anti-oxidants when compared to ascorbic acid (89.2%), which was used as the positive control (Fig. 3).

Reducing power (RP) scavenging activity

The potential anti-oxidant activity of a compound may be indicated by its significant reducing power capability. The reducing power of the title compounds (Table 5) increased with increasing availability of either non-bonding electrons or labile hydrogen. The results showed (Fig. 3) that hydroxy substituted compounds, 4f, 4t, 4a, 4c, 4d, 4e, 4m, 4l, 4p and 4o exhibited more scavenging activity of 87.9%, 86.9%, 86.2%, 84.9%, 84.5%, 83.9%, 83.8%, 83.2%, 83.5% and 83.2%, respectively. However, the aliphatic moiety as side group compounds 4g, 4h, 4x, and 4y showed the least scavenging activity of 68.5%, 66.8%, 65.8% and 64.1%, respectively, when compared with the positive control ascorbic acid (93.5%).

Anticancer activity

Now-a-days, the anticancer activity of N-containing heterocyclic/acyclic compounds against various anticancer cell lines were reported frequently. As the titled compounds showed excellent anti-oxidant activity, our attention was turned to estimate their anticancer activity, because of they are auxiliary drugs co-administered with various formulations.

All the newly synthesized compounds were assessed in vitro for cytotoxic activity against HeLa (human cervical cancer) and SK-BR-3 (human breast adenocarcinoma) cell lines. The number of live cells were measured after 24 h of treatment (MTT assay) and their cytotoxic activity²⁸ was determined by calculating the half maximal inhibitory concentrations (IC₅₀) values, which are presented in Table 6.

Table 6 Cytotoxic activity^a of test compounds (4a–z & 4a′) against HeLa and SK-BR-3 cell lines^b

Compound	(IC50 in μg mL^−1)^c
	HeLa	SK-BR-3
a With different concentrations of test compounds are treated on exponentially growing cells for 24 h and cell growth inhibition was analyzed by MTT assay;b decrease in mean percentage of cell number of five independent experiments was used to calculate the linear regression equation;c 50% concentration decrease of cell number in the absence of an inhibitor as compared with that of the control cultures. The values, in mean ± SE (SE = standard error) are five individual observations.
4a	16.00 ± 0.35	14.80 ± 0.58
4b	35.95 ± 0.45	26.44 ± 0.69
4c	27.41 ± 0.46	26.66 ± 0.58
4d	30.91 ± 0.61	30.56 ± 0.66
4e	26.49 ± 0.75	25.63 ± 0.53
4f	14.41 ± 0.52	13.02 ± 0.52
4g	17.23 ± 0.73	13.01 ± 0.39
4h	13.54 ± 0.60	11.79 ± 0.60
4i	26.01 ± 0.62	25.01 ± 0.70
4j	36.39 ± 0.48	37.93 ± 0.61
4k	39.00 ± 0.75	37.81 ± 1.02
4l	47.68 ± 1.01	46.22 ± 0.80
4m	47.75 ± 0.98	43.70 ± 0.62
4n	65.50 ± 0.47	65.95 ± 0.73
4o	39.93 ± 0.80	38.04 ± 0.68
4p	36.99 ± 0.84	32.86 ± 0.58
4q	41.48 ± 1.03	36.50 ± 0.60
4r	79.66 ± 1.60	73.67 ± 0.51
4s	49.07 ± 1.29	42.53 ± 0.58
4t	89.65 ± 1.52	86.69 ± 0.57
4u	41.13 ± 1.07	35.81 ± 0.66
4v	57.93 ± 0.91	55.84 ± 0.70
4w	66.21 ± 1.66	54.65 ± 0.50
4x	21.31 ± 0.88	17.32 ± 0.40
4y	23.34 ± 0.82	17.20 ± 0.79
4z	29.59 ± 0.98	19.89 ± 0.82
4a′	27.92 ± 1.28	18.56 ± 0. 91
Etoposide	9.75 ± 0.55	5.32 ± 0.42
Camptothecin	1.86 ± 0.33	1.48 ± 0.11

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The results showed that compounds 4h, 4f, 4a, 4g, 4x, and 4y possessed higher anti-proliferative activity against the two cell lines compared with the standard anticancer drugs, Etoposide and Camptothecin. Among them, compounds 4h and 4f have the highest activity (13.54 ± 0.60 and 14.41 ± 0.52 μg mL⁻¹ for HeLa and 11.79 ± 0.60 and 13.01 ± 0.39 μg mL⁻¹ for SK-BR-3 cells). Comparatively higher level of activity was observed for the remaining compounds; however, the compounds 4n, 4r, 4t, 4v and 4w showed less activity on both cell lines.

Structure activity relationship (SAR)

The potent nature of title N-containing heterocyclic compounds were expected to be more active because of the presence of heteroatoms, which contained non-bonding electron pairs or labile hydrogen on heteroatoms (Scheme 2, A → A1/A2), and they can interact easily with ROS/cancerous cell lines.

The anti-oxidant bioassay results showed that the compounds that are possessed with good electron donor/labile hydrogen on the basic core structure (A) could affect the ability of title compounds to interact with the target by peripheral and thereby influence the scavenging activity on ROS. Moreover, compounds, 4g, 4h, 4x, and 4y, which possess an aliphatic moiety on core structure, A, showed the least scavenging activity. However, when it comes to the anti-proliferative activity, these compounds show the highest inhibitory activity. From these observations, it may be concluded that the aliphatic side groups also exhibit good activity in both the types (anti-oxidant and anticancer) of bioassay. The other compounds also exhibited good anticancer activity.

We have demonstrated a green and efficient method for the synthesis of a series of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones (4a–z & 4a′) in excellent yields via a one-pot multi-component reaction using a catalytic amount of MSA under solvent-free conditions. The important features of this protocol are environmental acceptability, economic viability, less reaction time, high yields, purification of products by non-chromatographic method, cleaner reaction profiles, atom efficiency and recyclability of MSA, which qualify it as the best method for the synthesis of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones (4a–z & 4a′). The title compounds were screened for their in vitro anti-oxidant activity and most of the compounds were found to be effective against ROS. The majority of them also have excellent in vitro anticancer activity on two human cancer cell lines, HeLa and SK-BR-3, compared with those of standard drugs.

Experimental

Preparation of MSA

A suspension of anhydrous sodium molybdate (20 mmol, 4.118 g) was added to dry n-hexane (25 mL) in a 100 mL round bottom flask, which was equipped with an overhead stirrer and kept in an ice bath. To this solution, chlorosulfonic acid (0.266 mL, 40 mmol) was added drop wise for 30 min and stirred for 1.5 h (Scheme 3). The reaction mixture was gradually poured into 25 mL of chilled distilled water with stirring. MSA was separated by filtration and it was washed 5–6 times with cold distilled water until its filtrate tests negative for chloride ions. It was dried at 120 °C for 5 h, and obtained in 91% yield as a bluish powder.

Scheme 3 Synthesis of MSA.

Molybdate sulfuric acid, which showed good thermal stability, decomposed at 354 °C. The overlaid FT-IR spectra of sodium molybdate and molybdate sulfuric acid (MSA) are shown in Fig. S1.† As shown in the spectrum of MSA, the characteristic bands of both anhydrous sodium molybdate and OSO₃ group shifted evidently to higher wavenumbers. The well-defined bands at 3600–3000 cm⁻¹ are related to OH stretching, the band at 1635 cm⁻¹ is related to the H–O–H bending mode of the lattice water, and the bands at 1300–1100 cm⁻¹ might be the asymmetric and symmetric stretching modes of S=O. A strong band at 827 cm⁻¹ in the FT-IR spectrum of sodium molybdatewas assigned to the stretching mode of Mo–O. This band was shifted to 1100 cm⁻¹ and appeared as an overlapped band with S=O stretching bands in the spectrum of MSA. Broadening of the absorbance band positioned at 3600–3000 cm⁻¹ was due to the rapid exchanges of acidic hydrogen via H-bonding, and it reveals the formation of MSA.

The commercial Na₂MoO₄ have shown the main diffraction peaks at 16.88, 27.78, 32.68, 48.98, 52.18 and 57.18 of 2 a.u, referred to the 111, 220, 311, 422, 511 and 440 diffraction planes. The XRD pattern of the prepared MSA shows a series of new peaks with large shift from the original peaks of Na₂MoO₄ indicating that random distribution of planes on MSA (Fig. S2†). These can be attributed to the formation of MSA as a new phase system, whereas broadening of all peaks showed less crystallization structure and more amorphous shape of MSA.

Synthesis of 3,3-dimethyl-12-(pyridin-2-yl)-3,4,5,12-tetrahydrobenzo [4,5]imidazo[2,1-b]quinazolin-1(2H)-one (4a)

A mixture of 1H-benzo[d]imidazol-2-amine (1, 1 mmol), pyridinecarboxaldehyde (2a, 1 mmol) and dimidine (3a, 1 mmol) and MSA (5 mol%) was stirred at 80 °C under solvent-free conditions for 20 min (Table 4, entry 1). The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was washed with ethyl acetate and filtered to recover the catalyst. The filtrate was evaporated, and the crude product was recrystallized using ethanol to obtain pure 4a in excellent yield (92%). The spent MSA catalyst from different experiments was combined, and then washed with ether and dried overnight in a vacuum oven and reused. Compounds 4b–z & 4a′ were also synthesized by adopting this procedure.

Biological assays

DPPH free radical reduction method

The nitrogen centered stable free radical DPPH gives a strong absorption maximum at λ = 517 nm, which is suitable for spectrophotometric studies. The test compounds in solutions (100 μM) were added to dioxane/ethanol solution (100 μM) of DPPH. The tubes were kept for 20 min at ambient temperature and the absorbance was measured at λ = 517 nm. The % scavenging of the DPPH radical was expressed using eqn (1).

Determination of hydrogen peroxide (H₂O₂) scavenging activity

Hydrogen peroxide scavenging activity of compounds was determined using a 40 mM solution of H₂O₂ that was prepared in phosphate buffered saline (50 mM, PBS, pH 7.4). The absorbance value of H₂O₂ mixture was determined at 230 nm using a spectrophotometer. To the solution of the compound (100 μM) in 4 mL distilled water, 0.6 mL of hydrogen peroxide-PBS. Moreover, the absorbance of the analyte mixture was determined at λ = 230 nm after 10 min against a blank solution (parent compound with PBS) without H₂O₂. In a similar way, ascorbic acid was measured in place of the test compound and absorbance was measured after 10 minutes against a blank solution.

Reducing power

The principle involved here was that increase in the absorbance of the reaction mixtures indicates an increase in the anti-oxidant activity. In order to form coloured complexes, the test compounds (at 25, 50, 75, and 100 mg mL⁻¹ concentrations in methanol) were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide [(K₃Fe(CN)₆)] (1% w/v). The resulting mixture was incubated for 20 min at 50 °C, and then 2.5 mL of trichloroacetic acid (TCA, 10% w/v) was added. After centrifuging the mixture for 10 min at 3000 rpm, 2.5 mL of distilled water and 0.5 mL of FeCl₃ (0.1%, w/v) were added to the upper layer of the solution (2.5 mL) and the UV absorbance was determined at 700 nm against a blank sample using a spectrophotometer. For each compound measured the mean values from three independent tests and was found less than 2% standard deviations.

Anticancer activity

Cell culture

Cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HeLa cells were cultured at 37 °C in T-75 tissue culture flasks (Nunc, Denmark) in a 5% CO₂ humidified incubator using appropriate media supplemented with DMEM containing 10% heat-inactivated FBS. Similarly, SK-BR-3 cells were cultured in RPMI media. In 96 well microtiter plate each well was seeded the cells with 100 μL medium in identical conditions at a final density of 2. The cells were treated with test compounds in different concentrations (0.1–100 μg mL⁻¹) in DMSO (carrier solvent) and incubated overnight with three replicates for each in a final volume of 200 μL. Then 10 μL of MTT (5 mg mL⁻¹) was added to each well after 24 h and the plate was incubated for 4 h at 37 °C in the dark again. The formazan crystals after removing the media along with MTT were solubilized in DMSO (100 μL per well). Finally, MTT reduction was measured by reading the absorbance at 570 nm using GENios® microplate reader (Tecan Austria GmbH, Austria). Effects of the tilte compounds on the cell viability were measured by untreated cells added with DMSO as a control. Linear regression analysis was performed on the collected data and the regression lines were plotted for the best straight-line fit. By using the corresponding regression equation, the IC₅₀ concentrations were calculated.

Conclusion

We have reported a green and efficient method for the synthesis of a series of tetrahydrobenzo[4,5]imidazo[2,1-b]quinazolin-1(2H)-ones (4a–z & 4a′) in excellent yields via a one-pot multi-component reaction by using a catalytic amount of MSA under solvent-free conditions. The environmental acceptability, economic viability, lowered reaction time, high product yields, easy purification of products, cleaner reaction profile, higher atom efficiency and reusability of MSA qualify this method as the best one for the synthesis of these compounds. The majority of these compounds have good anti-oxidant and excellent in vitro anticancer activity on two human cancer cell lines, HeLa and SK-BR-3, compared with those of standard drugs.

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Footnote

† Electronic supplementary information (ESI) available: Analytical and spectral data and NMR spectra were provided as supplementary data for all compounds. See DOI: 10.1039/c4ra13440k

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