Design and synthesis of Quinazolinone, Benzothiazole derivatives bearing guanidinopropanoic acid moiety and their Schiff bases as cytotoxic and antimicrobial agents

2.1 Materials and methods

All the starting materials and solvents were purchased from commercially available sources. The melting points were determined on THERMONIC MODEL-C-LMP-1, Campbell melting point apparatus (uncorrected). ¹H NMR were recorded on Brucker NRC-IISC 400/DRX-500/AV-III 500(S)/(L) AV-700 NMR spectrometer, ESI-MS were recorded with a Mariner System 5304 mass spectrometer and Elementary analysis was performed on a CHN-O-Rapid instrument within ±0.4% of the theoretical values at IIT Madras, Chennai. All the reactions were monitored using thin layer chromatography (TLC) using glass plate coated with silica gel G. TLC plates were developed in iodine and UV and chloroform: methanol (1:1) was taken as mobile phase.

2.2 Synthesis

2.2.1

2.2.1 Series I

Compounds 1–6, (6-substituted Benzo[d]thiazol-2-amine derivatives) and compounds 7–12, (1-(6-substituted Benzo[d]thiazol-2-yl)thiourea were prepared from the corresponding substituted anilines and from the compounds 1–6 respectively by using earlier reported methods (Pankaj et al., 2010) (Scheme 1).

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Scheme 1

Synthesis of BTGP (13–18) from aryl amines (13–18).

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2.2.1.1

2.2.1.1 Synthesis of 3-(3-(6-substituted Benzo[d]thiazol-2-yl)guanidino) propanoic acid derivatives (BTGP) (Scott et al., 2001) from 1-(6-substituted Benzo[d]thiazol-2-yl)thioureas (compounds 13–18, Scheme 1)

To the stirring mixture of compounds 7–12 containing Silica gel G (0.02 mol, 0.01g), Cupric sulphate pentahydrate (0.01 g), and Triethylamine (2 mL) in Tetrahydrofuran (25 mL), 1.78 g (0.02 mol) of β-alanine were added and stirring continued for 5–6 h, then kept at room temperature for 24 h. The crude product formed was recrystallised from methanol.

2.3 STEP III: 6-Chloro-N-(2,2-dimethyl-1,2,3,8a-dihydroquinazolin-4(4aH)-ylidene)benzo[d] thiazol-2-amine (Compound-41)

Black solid, Yield, 79%, m.p.,121 °C, (IR) υ_max(KBr/cm⁻¹), 3169 (N–H str), 1633 (C⚌N), 1519, 1399 (C–CH₃), 1333 (C–N), 810 (C–Cl), 1298 (C–S), ¹H NMR (δ-ppm): 7.87 (m, 2H, C_6–7-H of Dihydroquinazolinone ring), 7.74–7.75 (m, 2H, C_5,7-H of Benzothiazole ring), 7.55–7.57 (m, 1H, C₄-H of Benzothiazole ring), 6.58–6.63 (m, 2H, C_6–7-H of Dihydroquinazolinone ring), 2.48–2.49 (s, 6H, dimethyl). ESI-MS: 346.88 [M⁺].

Anal. Calcd for C₁₇H₁₇ClN₄S (344.86); C, 59.21; H, 4.97; N, 16.25. Found: C, 59.65; H, 4.63; N, 16.35.

3.1 In vitro cytotoxicity

3.2 Antimicrobial evaluation

4.1 Chemistry

Series 1 compounds 3-(3-(6-substitutedbenzo[d]thiazol-2-yl) guanidino) propanoic derivatives (13–18) were synthesized according to earlier reported method in the literature, shown in scheme 1. Compounds 13–18 prepared in 3 steps, the first step involved reaction of various substituted aromatic amines, ammoniumthiocyanate and bromine in glacial acetic acid under cold condition at 0–10 °C afforded 6-substituted benzo[d]thiazol-2-amine derivatives (1–6) which further treated with saturated ammoniumthiocynate and Conc. Hydrochloric acid (second step) to yield the corresponding thioureas (7–12). When the compounds 7–12, in third step treated with β-alanine dissolved in tetrahydro furan containing silica gel G, CuSO₄.5H₂O and triethyl amine (accelerates desulphuration) afforded the guanidinopropanoic acid derivatives 13–18 (Scheme 1) as a final compound of series one.

Series 2 Compounds 3-(3-(2-substituted-4-oxoquinazolin-3(4H)-yl) guanidino) propanoic acid derivatives (34–38) were prepared in 4 steps as shown in scheme 2. Initially, intermediate compounds 2-substituted quinazolin-4(3H)-one derivative (19–23) were prepared by refluxing with substituted aldehyde in ethanol for 3 h at 70 °C, when the formed non isolable intermediate Schiff bases were refluxed with CuCl₂ in ethanol for 3 h at 70 °C produced the compounds 19–23. In compounds 19–23, amino group was introduced at N-3 of Quinazolinone by treating with hydrazine hydrate, ethylene glycol in acidic medium, refluxing the reaction mixture for 3 h to afford 3-amino-2-substituted quinazolin-4(3H)-one derivative (24–28). Compounds 19–23 do not form hydrazone when they are treated with hydrazine hydrate since the Quinazolinones 19–23 are cyclic amides (–NH–C⚌O), (Scheme 2). The method adopted for the prearation of Quinazolinone thioureas 29–33 and Quinazolinone guanidino propanoic acid derivatives 34–38 (scheme 2) were similar to the preparation 7–12 and 13–18 respectively.

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Scheme 2

Synthesis of QGP derivatives (34–38) from anthranilamide.

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Series 3 compounds Schiff bases (41–54) were prepared from substituted benzo[d]thiazol-2-amines (1–7) and 2-methyl-2-substituted-2,3-dihydroquinazolin-4(1H)-ones (39–40), shown in scheme 3. Intermediate compounds 39–40 were synthesised by treating anthranilamide with acetone and acetophenone (refluxed for 90 minutes) in presence of p-toluenesulfonic acid monohydrate as cyclising agent, Dihyhydroquinazolinones (39–40) formed (via non isolable imine formation and cyclization). Finally, Schiff base derivatives (41–54) were prepared by reacting the 2-substituted-2,3-dihydroquinazolin-4(1H)-ones (39–40) and substituted benzo[d]thiazol-2-amine(1–7) (Scheme 1, first step compounds) then refluxed for 10–16 h, the amino group of compounds 1–7 reacted with keto group of compounds 39–40 in ethanol to afford the Schiff base derivatives (41–54) (scheme 3).

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Scheme 3

Synthesis of Schiff base derivatives (41–54) from Benzothiazole-2-amines (1–7) and 2,3-dihydroquinazolin-4(1H)-ones (39–40).

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Infrared (IR) spectroscopy of the compounds 7–12 and 29–33, showed characteristic bands at 1051–1196 by the thiocarbonyl moiety C⚌S. Carboxylic acid group of the compounds 13–18, 34–38 showed characteristic broad bands at the range of about 3379–3216 cm⁻¹ by the -OH stretching of the carboxylic acid, bands at about 1692–1679 cm⁻¹ by the carboxylic C⚌O stretching. C⚌N absorption bands appeared in the region of at 1493–1430 cm⁻¹.

Compounds 41–54 showed strong absorption bands in the region of 1390–1210 cm⁻¹ by C⚌N, while the absorption bands for –NH₂ and C⚌O were absent in compounds 41–54. The ¹H NMR spectrum of the compounds 13–18 and 34–38 exhibited three broad singlets, one at about 9.575–11.187 ppm by the carboxylic acid proton, second at 7.585–7.788 by imine proton and third at 2.985–3.376 by –NH proton. Adjacent two methylene protons of the compounds 13–18 shown two triplets at 3.137–3.315 (J = 7.4 Hz) and 2.560–2.481 (J = 7.4 Hz). The compounds 34–38 were also shown two similar triplets at 2.495–2.485 (J = 7 Hz) and 1.156–1.120 (J = 7). Presence of two triplets with the same coupling constant in the compounds 13–18 and 34–38 confirmed presence of two adjacent methylene protons. In addition, methyl proton of compound 34 exhibited singlet at 1.452, phenolic OH proton and methoxy proton of compound 36 exhibited singlets at 6.387 and 3.946 respectively. Methoxy protons of compound 38 exhibited singlet at 3.489–3.498. Compound 41–54 were shown singlet at 7.859–7.888 ppm, which were assigned to third position –NH of dihydro Quinazolinone. Sulphanilamide group of compounds 17, 46 and 52 exhibited broad singlet at 2.121- 2.864 while carboxylic acid protons attached to Benzothiazole ring of compounds 15,45 and 53 exhibited one broad singlet at 10.955–11.718. The phenolic -OH group of compounds 45 and 54 were also shown broad singlet at about 9.486–11.243 ppm.

In mass spectrum, thiourea intermediates showed base peak due to cleavage of [C₇H₈N₂]^+• at m/z = 138 and another intense peak at about m/z 108 due to deamination from base peak with relative intensity of 60%. The obtained molecular ion m/z values of the final guanidinopropanoic acid derivatives were consistent with the molecular weight of compounds and the base peak was formed after the successive removal of propanoic acid moiety with one intense peak due to desulphuration from base peak with relative intensity of 62%.

4.2 In vitro cytotoxicity

The synthesized BTGP derivatives 13–18, QGP 34–38 and Schiff base derivatives 43–45, 48–53 (20 compounds) were evaluated for antiproliferative activities against human cancer cell line HeLa. The results were summarized in Table 1. All the compounds showed antiproliferative activities against the HeLa cell line with the half maximal inhibitory concentration (IC₅₀) values of 2–550 μM (except Schiff base derivatives IC₅₀ values of 743–875 μm) and compound 18 showed the most potent activity with a mean IC₅₀ of 2 μM which was close to the value of the positive control, doxorubicin. Structure–activity relationship (SAR) analysis indicated that Benzothiazole bearing guanidinopropanoic acid 13–18 showed stronger activities than the Quinazolinone bearing guanidinopropanoic acid 34–38, with all the IC₅₀ values below 100 μM. In the SAR further study of 13–18, the –OH, –COOH, and –SO₂NH₂ substituted at sixth position of Benzothiazole ring played an important role for the activity. Compounds 18 with –OH substitution at sixth showed the most potent activity against the HeLa cell lines, with the mean IC₅₀ of 2 μM. From these results it is evident that hydrogen bonding substituent is necessary for the potent activity at the sixth position of Benzothiazole ring, further size of these groups also play important role in the activity, activity order is OH > COOH > SO₂NH₂. From the results it is clear that smaller hydrogen bonding group (donor Hydrogen containing) at sixth position of Benzothiazole ring necessary for the potent activity.

4.3 In vitro antibacterial activity

Twenty-three compounds (7–17, 34–38, 44–46, 49–52 and 54) were evaluated for in vitro antimicrobial activity against 11 pathogenic bacteria and 10 pathogenic fungus. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial growth around the disks in millimetre. The minimum inhibitory concentration (MIC) measurement was determined for all the compounds using the twofold serial dilution method. The MIC (μg/mL) values were recorded. Most of the tested exhibited differential activity against tested bacteria and fungi.

From the results of antibacterial activity it is evident that 6 compounds (11, 14, 34, 38, 44 and 46), 11 compounds (7, 10, 11, 12, 13, 14, 15, 11, 35, 36, 38 and 49), 4 compounds (10, 12, 17 and 49), 2 compounds (15 and 17), 3 compounds (34, 38, 44), 5 compounds (8, 16, 34, 36, 38) and 3 compounds (34, 38 and 49) were more active against Pseudomonas aeruginosa, Salmonella paratyphyi, Vibrio cholera, Bacillus lentus, Rhodosporium rubrum, Staphylococcus albus and Staphylococcus aureus, respectively than the reference drug ciprofloxacin and compounds 11, 14, 34, 38 and 49 showed the broad spectrum of activity (Table 2).

The results of antifungal activity indicated that, Compounds 34, 51, 45 and 37 were more potent against Monascus purpureus, Aspergillus fumigates, Aspergillus parasiticus and Microsporum gypseum than the reference drug Clotrimazole (Table 3).

Collectively, compounds 11, 14, 38 and 49 are considered to be the most active antimicrobial members identified in this study with a broad spectrum of antibacterial activity against both Gram positive and Gram negative bacteria. Particularly, compounds 14 was superior over the ciprofloxacin against Pseudomonas aeruginosa (14, MIC 3.125 and Cp MIC 19.5 μg/mL) and compound 49 highly active against Salmonella paratyphyi (49, MIC 3.125 and Cp MIC 43.4 μg/mL). Compound 34 was more active against Monascus purpureus (34, MIC 3.125 and Clotrimazole MIC 30 μg/mL).