INTRODUCTION:

In the 20^th century chemotherapy has revolutionized the treatment of infective diseases since the innovation of antibacterial dyes by Paul Ehrlich, covered the way to a great victory for human health and long life. The development of resistance against currently used antimicrobial drugs led to an invigorated curiosity of the researchers in infective diseases to develop new chemical entities to battle them^1-3. Patient morbidity, costs of treatment, rates of hospitalization, and use of broad-spectrum agents are remarkably increased by antimicrobial resistance^4-6.

Tuberculosis is a deadly disease usually caused by Mycobacterium tuberculosis. It has killed an estimated one billion people over the last two spans and even remains stop ten causes of death in the world. According to the 2018 report of WHO, 5, 58,000 people developed rifampicin-resistant (RR TB), multidrug- resistant tuberculosis (MDR-TB) or extensively drug- resistant (XDR TB) in the world.

Therefore, it is essential to develop rational chemotherapeutic agents to delay the emergence of resistance and, ideally, shorten the duration of therapy of this infection^7-9.

In cancer cells, however, damaged DNA is not repaired. People can inherit damaged DNA, which results in 10 percent of all cancers. Known causes of cancer such as environmental factors like ultraviolet rays, smoking and viruses like oncogenic viruses may code for growth factors and may amplify them, may control cell death suppressor gene or tumor suppressor genes. Carcinomas are the most common type of cancer; these tumors arise from the cells that cover external and internal body surfaces. The most frequent cancers of this type are lung, breast, colon and prostate cancer.

Nowadays available anticancer therapeutics was limited due to their toxicity properties and also their developed resistance against to that drug. Also, in the market available anticancer drugs are not unambiguous. The main goal of the drug discovery process was to developed new anticancer therapeutic agents with their two controlled the development of resistance against the existences of cancer. There is urgency for development of innovative and cost effective anticancer therapeutic agents.^10-12

Benzimidazole is a lead molecule for the most of the biological agent use in the pharmaceutical industry. It consists of fused benzene ring with heterocyclic aromatic imidazole. The existence of imidazole creates it a resourceful heterocycles with an extensive range of biological activities such as antiulcer (Gastric H⁺/K⁺-ATPase inhibitors), antihypertensive, anti-inflammatory, anticonvulsant, analgesic, antiprotozoal, antitrichinellosis, antidiabetic, anti-HIV, antimicrobial, antitubercular, anticancer, antihistaminic, antioxidant, antiviral, antiparasitic agents, diuretic, and DNA binding activities.^13-26

Encouraged by the upstairs findings and in the persistence of our work on dihydrobenzimidazole thiopyranooxazinone derivatives, we herein report the synthesis and in vitro evaluation of dihydrobenzimidazole thiopyranooxazinone derivatives as potent biological agents.

MATERIALS AND METHODS:

The chemicals of analytical grade required for the synthesis of coupled mercaptobenzimidazole derivatives were purchased from Sigma-Aldrich and SD fine chemicals (India). Synthesized compounds were determined for its melting points with the help of precision melting point apparatus and were uncorrected. Completion of reaction was confirmed by TLC on silica gel-G plates and the spots were visualized in UV chamber or iodine chamber. IR spectra of intermediates and derivatives compound were recorded by using on KBr pellets on a Jasco FTIR-460 plus spectrophotometer and vibrational frequencies expressed in cm^-1.¹ HNMR spectra were recorded on BRUKER 400 MHz spectrometer in deuterated DMSO using tetramethylsilane (TMS) as internal standard and chemical shifts were recorded as δ (parts per million).

General procedure:^27-29

STEP 1:

Synthesis of Mercaptobenzimidazole (DPK3A):

Procedure:

O-phenylenediamine (10.8g, 0.1moles) treated with carbon disulfide (7.67g, 0.1moles) in the presence of potassium hydroxide (5.65g, 0.1moles), 100ml of 95% ethanol, and 15ml of water used as a solvent in a round bottom flask was refluxed on a water bath for three hours. After the completion of the reaction, the reaction mixture was cooled and filtered. After that, 1-1.5g of activated charcoal was added carefully in the filtrate and refluxed on the water bath for 10 minutes; the activated charcoal was removed by filtration. The filtrate was treated with 100ml of warm water at 60-70⁰C for 10 minutes. Dilute acetic acid was poured into the reaction mixture for acidification with gentle agitation to yield shiny crystals as a product, which is further kept in a refrigerator for three hours to allow the complete crystallization process. The obtained solid product was separated through the Buchner funnel and dried at 40⁰C overnight and recrystallized from the ethanol.

Analytical and spectral data of DPK3B:

R_f value 0.67, Yield 73.33%; M.P. 300-305°C; FTIR (KBr, cm^–1):3155 (N-H), 2993 (C-H, Ar), 1512 (C=C, Ar), 1357 (C-N), 655 (C-S).

STEP 2:

Synthesis of Benzimidazole sulfonyl carbonyl acetic acid (DPK3B):

Procedure: Mercaptobenzimidazole (150mg, 0.1moles) was treated with Meldrum acid (184mg, 0.1moles) in the presence of anhydrous 1, 4- dioxane (5ml, 0.1moles) used as a solvent in a round bottom flask was refluxed on a water bath for four hours. After the completion of the reaction, the reaction mixture was cooled and filtered. After that, the filtrate was introduced into a separatory funnel and partitioned with ethyl acetate and saturated sodium bicarbonate. From the partitioned solution, the mixture to separate out aqueous layer and acidified at pH 1-2 by adding carefully concentrated hydrochloric acid. Further, the acidified solution extracted several times with methylene chloride (dichloromethane). The obtained extract was dried over the magnesium sulfate and concentrates to assume targeted product and recrystallized from the benzene or hexane.

Analytical and spectral data of DPK3B:

R_f value 0.67, Yield 57.80%; M.P. 325-330°C; FTIR (KBr, cm^–1):3155 (N-H), 2993 (C-H, Ar), 2576 (O-H, COOH), 1627 (C=O), 1512 (C=C, Ar) 1357 (C-N), 655 (C-S).

STEP 3:

Synthesis of Dihydrobenzimidazole hydroxyl thio pyranone (DPK3C):

Procedure: Benzimidazole sulfonyl carbonyl acetic acid (98mg, o.1moles) treated with polyphosphoric acid (1g, 0.1moles, 116%) or Eaton’s reagent in an Erlenmeyer flask was stirred at 120^oC for 6-8 hours. After the completion of the reaction, the reaction mixture was cooled to room temperature and then added 10ml of water with vigorous stirring. The obtained precipitate was filtered off and washes with water. The precipitate was dried in the air and recrystallized with ethanol.

Analytical and spectral data of DPK3C:

R_f value 0.75, Yield 51.54%; M.P. 283-287°C; FTIR (KBr, cm^–1): 3503 (O-H), 3155 (N-H), 2993 (C-H, Ar), 1643 (C=O), 1512 (C=C, Ar), 1357 (C-N), 655 (C-S).

STEP 4:

General procedure for synthesis of dihydrobenzimidazole thiopyranooxazinone derivatives (DPK3d1-DPK3d5):

General Procedure:

Dihydrobenzimidazole hydroxythiopyranone (1g, 0.1 moles) treated with an aromatic aldehyde (1.5g, 0.1 moles) in the presence ethanol 10ml used as a solvent in a round bottom flask was refluxed on a water bath for 6-8 hours. After that, the reaction the mixture was cooled to room temperature; the obtained precipitate was filtered off and dried it. The targeted product was recrystallized with ethanol or dimethylformamide.

Table 2: Substitution of aromatic aldehyde and amine

Compound Code	Ar-CHO	R-NH₂
DPK3d1	P-Chlorobenzaldehyde	Aniline
DPK3d2	P-Chlorobenzaldehyde	P-Nitroaniline
DPK3d3	Benzaldehyde	M-Chloroaniline
DPK3d4	Benzaldehyde	P-Nitroaniline
DPK3d5	Benzaldehyde	O-Nitroaniline

RESULT AND DISCUSSION:

From the literature survey, it was revealed that the benzimidazole has been reported to develop number of molecules have exposed various potent pharmacological activities. In this research studies, we have reported synthesized dihydrobenzimidazole thiopyranooxazinone derivatives. These newly synthesized derivatives were screened against in vitro antimicrobial, antitubercular and anticancer activities. These compounds structurally elucidated by analytical studies such as determining physical constant and spectral studies such FTIR, ¹HNMR, showed satisfactory results.

In vitro antimicrobial activity:

In vitro antimicrobial activity of synthesized dihydrobenzimidazole thiopyranooxazinone derivatives were screened by the tube dilution method against Escherichia coli (Gram-negative bacteria/ATCC 25922), Bacillus subtilis (Gram-positive bacteria/ATCC 6051), and Aspergillus niger (fungal strain/ATCC 6275). Synthesized compounds having observed MIC values are showed in Table 3. Some of the dihydrobenzimidazole thiopyranooxazinone derivatives were found to be highly efficient as antimicrobial agents. All the synthesized compounds showed average antibacterial activity as compared to standard ciprofloxacin. Compounds DPKd1 to DPK3d5 shown potent antifungal activity in the comparison of standard fluconazole.

Table 3: Antimicrobial activity, MIC values of synthesized compounds.

Sr. No.	Compound Code	MIC in µg/ml
		Antibacterial activity		Antifungal activity
		*B. subtilis*	*E. coli*	*A. niger*
1.	DPK3d1	50	25	1.6
2.	DPK3d2	50	100	3.12
3.	DPK3d3	100	100	6.25
4.	DPK3d4	50	50	3.12
5.	DPK3d5	50	100	6.25
6.	Ciprofloxacin	2	2	-
7.	Fluconazole	-	-	8
8.	Control (DMF)	-	-	-

Figure 2: Graphical representation of antimicrobial activity, MIC values of synthesized compounds against Escherichia coli, Bacillus subtilis, and Aspergillus niger.

In vitro antitubercular activity:

In vitro antitubercular activity of synthesized dihydrobenzimidazole thiopyranooxazinone derivatives were screened by the Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (H37Rv strain, ATCC 27294). Synthesized compounds having observed MIC values are showed in Table 4. Compounds DPK3d1 shown potent activity and also, rest of the compounds shown moderate activities in the comparison of standard antitubercular drugs such as Pyrazinamide, Ciprofloxacin, and Streptomycin.

Table 4: Antitubercular activity, MIC values of synthesized compounds.

Sr. No.	Compound Code	MIC in μg/ml	Sr. No.	Compound Code	MIC in μg/ml
1	DPK3d1	1.6	5	DPK3d5	6.25
2	DPK3d2	12.5	6	Pyrazinamide	3.12
3	DPK3d3	6.25	7	Ciprofloxacin	3.12
4	DPK3d4	12.5	8	Streptomycin	6.25

Figure 3: Graphical representation of antitubercular activity, MIC values of synthesized compounds against Mycobacterium tuberculosis (H37Rv strain).

In vitro anticancer activity:

In vitro anticancer activity of synthesized dihydrobenzimidazole thiopyranooxazinone derivatives were screened by the sulforhodamine B assay (SRB) against human lung cancer cell line A-549. The examined compounds having observed GI50 values are showed in Table 4. Compounds DPK3d3, DPK3d4 and DPK3d5 shown highly potent cytotoxic nature against human lung cancer cell line A-549 as compared Adriamycin.

Table 4: Anticancer activity, GI50 values of synthesized compounds against Human Lung Cancer Cell Line (A-549).

Sr. No.	Compound Code	Human Lung Cancer Cell Line A-549
		% Control Growth
		Drug Concentrations (µg/ml)
		Average Values
		10	20	40	80
1.	DPK3d1	89.9	93.1	91.3	104.3
2.	DPK3d2	93.8	92.2	89.6	99.1
3.	DPK3d3	98.2	104.8	103.1	126.1
4.	DPK3d4	95.4	99.1	99.8	116.0
5.	DPK3d5	100.6	106.2	101.1	104.9
6.	Adriamycin	20.7	20.6	19.7	34.2

Figure 3: Graphical representation of anticancer activity with GI50 values of synthesized compounds against human lung cancer cell line A-549.

Structure- activity relationship of dihydrobenzimidazole thiopyranooxazinone derivatives:

In vitro evaluation studies (antimicrobial, antitubercular and anticancer activities) of dihydrobenzimidazole thiopyranooxazinone derivatives, the following SAR may be assumed:

1. The dihydrobenzimidazole thiopyranooxazinone derivatives having antimicrobial studies conclude that, obtained compounds shown potent antifungal activity in comparison of antibacterial activity.

2. Antitubercular activities studies of the dihydrobenzimidazole thiopyranooxazinone derivatives shown that the synthesized compounds have a very good interaction with target sites and has need of supplementary in vivo studies to confirm the antitubercular activity.

3. The dihydrobenzimidazole thiopyranooxazinone derivatives having cytotoxic studies conclude that, there should be slight structural modifications to develop affinity of drug to the binding of a molecule to the target site.

4. The above results also indicated a fact that different structural requirements are essential for a compound to show different activities. The structure-activity relationship amongst the dihydrobenzimidazole thiopyranooxazinone derivatives outcomes are summarized as follows:

Figure 4: Basic nucleus of dihydrobenzimidazole thiopyranooxazinone derivatives.

Presence of basic nucleus dihydrobenzimidazole thiopyranooxazinone essential for antimicrobial, antitubercular and anticancer activities.

At position Ar:

Presence of unsubstituted benzene ring essential for cytotoxic activity.

Presence of substituted benzene ring essential for antimicrobial and antitubercular activities. Substitution of -Cl (Chloro group) in the benzene ring at para position shown potent to moderate antimicrobial and antitubercular activities.

At position R:

Presence of substituted benzene ring essential for antimicrobial, antitubercular and anticancer activities.

At para position substitution of -Cl (Chloro group) shown potent antimicrobial activity and moderate to good antitubercular, anticancer activity.

At ortho position substitution of -NO2 (Nitro group) and -Cl (Chloro group) shown potent antitubercular, anticancer activity and moderate to good antimicrobial activity.

CONCLUSION:

Dihydrobenzimidazole thiopyranooxazinone derivatives were efficiently synthesized and screened for their in vitro antimicrobial, antitubercular and anticancer activities. From the antimicrobial results confirmed that antifungal activity most potent in the comparison of antibacterial activity. Compounds DPK3d1 shown potent antitubercular agent as compared with standard drugs. The results of anticancer activity exhibited that majority of the derivatives inhibited 50% cell growth of human lung cancer cell line (A549), especially; compounds DPK3d3, DPK3d4 and DPK3d5 shown potent cytotoxic agent as compared to standard Adriamycin.

ACKNOWLEDGMENTS:

The authors would like to acknowledge Savitribai Phule Pune University, Maharashtra, India for providing a good spectral data analysis facility. We are also thankful to Dr. Kishore G. Bhat, Maratha Mandal's Central Research Laboratory Belgaum, for the antimicrobial and antitubercular screening of the synthesized compounds. Also, thankful to Dr. Nirmal Kumar, Advanced Centre for Treatment, Research and Education in Cancer, (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai for the anticancer screening of the synthesized compounds.

CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

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Received on 03.04.2020 Modified on 31.05.2020

Research J. Pharm. and Tech 2021; 14(3):1453-1458.

DOI: 10.5958/0974-360X.2021.00259.6

Compound code	Molecular Formula	M.P. (^oC)	R_fValue	% Yield	FTIR (KBr cm^-1)	NMR) (δ ppm)
DPK3d1	C₃₁H₂₃Cl₂N₃O₂S	287-289^oC	0.67	75	3464 (N-H), 3109 (C-H, Ar), 1627 (C=O), 1450 (C=C, Ar), 1033 (C-N), 964 (C-O-C), 879 (Cl), 709 (C-S)	10.5 (bs, 1H, NH), 7.80 (s, 1H, CH), 7.10-7.60 (m, 3H, Ar-H), 2.50 (s, 1H, CH).
DPK3d2	C₃₁H₂₂Cl₂N₄O₄S	294-296^oC	0.65	80	3286 (N-H), 3039 (C-H, Ar), 1666 (C=O), 1442 (C=C, Ar), 1342 (NO₂, Ar), 1141 (C-N), 1087 (C-O-C), 779 (Cl), 686 (C-S)	¹H NMR (500MHz, DMSO-d6): δ 12.50 (s, 1H, NH), 10.50 (s, 1H, NH), 7.90-8.00 (s, 1H, CH), 6.50-7.60 (m, 3H, Ar-H), 2.50 (s, 1H, CH)
DPK3d3	C₃₁H₂₄ClN₃O₂S	270-272^oC	0.54	63	3232 (N-H), 3039 (C-H, Ar), 1666 (C=O), 1442 (C=C, Ar), 1180 (C-O-C), 1149 (C-N), 748 (Cl), 655 (C-S)	--
DPK3d4	C₃₁H₂₄N₄O₄S	281-282^oC	0.73	94	3255 (N-H), 3093 (C-H, Ar), 1643 (C=O), 1442 (C=C, Ar), 1357 (NO₂, Ar), 1211(C-O-C), 1149 (C-N), 648 (C-S)	13.00 (s, 1H, NH), 10.00 (s, 1H, NH), 8.50 (s, 1H, CH), 6.50-8.20 (m, 3H, Ar-H), 2.50 (s, 1H, CH)
DPK3d5	C₃₁H₂₄N₄O₄S	283-285^oC	0.68	88	3302 (N-H), 3047 (C-H, Ar), 1635 (C=O), 1481 (C=C, Ar), 1365 (NO₂, Ar), 1149 (C-O-C), 1087 (C-N), 601 (C-S)	13.00 (s, 1H, NH), 10.00 (s, 1H, NH), 8.00 (s, 1H, CH), 7.50-7.70 (m, 3H, Ar-H), 2.50 (s, 1H, CH)