Heterocyclic Scaffold of Interest: Pyrimido [2, 3-b] Benzothiazole as Emerging Multifunctional Bioactive Agents

The search for promising chemical molecules for therapeutic activity is a growing trend in medicinal chemistry. Amongst the heterocyclic compounds, benzothiazole derivatives play a very important role as drugs in various diseases and disorders, and are present in various marketed drugs. The pyrimidine also plays an important role in medicinal chemistry and is present in important drug substances (Fig.1).

These two compounds (benzothiazole and pyrimidine), with beneficial biological activity, play a significant part in biological research due to their presence. The chemistry of fused heterocycles is known to fascinate the field of investigation in therapeutic chemistry. The ring is formed by the fusion of benzothiazole and pyrimidine is known as pyrimido [2, 3-b]benzothiazole. (Fig.2).

The condensed benzothiazoles ¹, pyrimido[2,1-b]benzothiazoles have a considerable place in the research area, especially in synthetic as well as in pharmaceutical chemistry, because of their potent and significant pharmacological activities. Literature surveys revealed that pyrimido[2,1-b]benzothiazoles exhibit a wide spectrum of biological and pharmacological properties, such as antitumor ², antiviral ³, anti-fungal ⁴, antiproliferative ⁵, antimicrobial ⁶, anti-inflammatory ⁷, muscle relaxant, sedative, and anti-allergic activities ⁸.  Considering all the above findings, with potent activity. The review emphasizes the pyrimido[2,1-b]benzothiazole derivatives along with their biological and medicinal activities.

Anti-bacterial Activity

Baheti et al. developed novel 15-iminobenzothiazolo[2,3-b]pyrimido[5,6-e]pyrimido[2,3-b]benzohiazol-14(H)-one 13a-l and its 3,10-disubstituted derivatives, and reported antibacterial activity against  S.aureus, B.subtilis, E.coli, and S.typhi with a zone of inhibition from 9 to 13mm, as standard drug Streptomycin and Penicillin exhibited zone of inhibition 18,22,15 and 16 mm respectively ⁹.

Singh et al. developed a series of 2-substituted-9-chloro-fluropyrimido[2,1-b]benzothiazole-3-cynao-4(H)-one 14a-i and tested their antibacterial, anti-inflammatory, and antifungal properties in vitro and in vivo. The selected compounds showed activity against B.subtilis, S.aureus, Pseudomonas, and E.coli by the cup plate method, which exhibited zone of inhibition 8-22 mm against the standard of Procain Pencillin and Streptomycin for 50 µg and 100 µg concentration, with 20,22,18,21, and 19,21,20,22 mm of inhibition. It also used the minimum inhibitory concentration method for the same bacteria with relatively significant inhibitory action in the range of 50-800 µ/ml  ¹⁰.

Kumar et al. generated 4-Phenyl-2H-pyrimido[2,1-b]benzothiazole-2-ones. The antimicrobial activities of compounds 15a-f were investigated against a variety of microorganisms, including gram-positive or negative B.coagulans, B.substilis, P.aeruginosa, and S.aureus, using the disk diffusion method in the 1.3-15.6 mm zone of inhibition against the standard of Chloramphenicol (100 µg/ml) with a significance value of 12.3 mm ¹¹.

Bhoi et al. produced the 4H-pyrimido[2,1-b]benzothiazole derivative of new ethyl-2-methyl-4-(pyridine-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate derivatives 16 a-l. A one-pot, three-component microwave-aided synthesis was used to test its antibacterial, antioxidant, and antitubercular activities against Mycobacterium tuberculosis H37RV, in which the antibacterial activity in these bacteria E.aerogens, E.coli, M.luteus and B.cereus against the Streptomycin as a standard which having 24 mm zone of inhibition in which same compounds are more or less activity compared to standard within 17 to 30 mm ¹².

Badne et al. synthesised a novel synthesis and biological activity of 2-substituted derivatives of 3-cyano-4-imino-2-methylthio-8-methoxy-4H-pyrimido[2,1-b][1,3]benzothiazole 17a-f, which were assessed for antibacterial activity range of 6-12 mm for S.aureus, B.subtilis, E.coli, and S.typhi against Norflaxacin as a reference, with 14,24,20, and 16 mm of zone of inhibition at 25 µg/disc ⁶.

Sayed et al. synthesized pyrimido[2,1-b]benzothiazole 18 a-d with an active methylene group using selected N-, O-, and C- nucleophiles and tested its antimicrobial effectiveness against active antibacterial agents with more than 12 mm of zone of inhibition against Streptomycin and Ampicillin as a standard for S.aureus, B.subtilis, P.aeruginosa, and E.coli, bacteria in a range of less than 6 mm to more than 12mm ¹³.

Sahu et al. synthesised 4H-pyrimido-[2,1-b]benzothiazole derivatives of curcumin 19a-h in the microwave with good yield and showed antibacterial effectiveness against ciprofloxacin as a standard with a range of 0.6 to 1.25 µM/mL Minimum inhibitory concentration (MIC) for S.aureus, S.typhi, P.aeruginosa, E.coli, B.cereus and P. rettgeri bacteria in the range of 0.625 to 20 µM/mL ¹⁴.

Gupta et al. synthesised the synthesis of pyrimido[2,1-b]benzothiazole 20a-j, followed by ring closure, and tested it for antibacterial activity against Meropenem, Vancomycin, with a zone of inhibition of 16,15 mm as a standard for E.coli, Enterobacter, and S. aureus bacteria with a range of 7 to 15 mm ¹⁵.

Eris, Kin et al. synthesized and studied 4-imino-3-arylazo-4H-pyrimido [2,1-b] [1,3] benzothiazole-2-oles 21 a-m, for antimicrobial activity against standard Ciprofloxacin with 24 to 28 ± 1.2 mm of zone of inhibition for S.aureus, B.subtilis, K.prneumoniae and E.coli 16 to 32 mm. The dyes were obtained by coupling carbocyclic amine-based diazonium chloride with compound ¹⁶.

Ubale et al. synthesised 6-imino-7-oxo-benzothiazolo[2,3-b]pyrimido[5,6-e]pyrimido[2,3-b][1,3] benzothiazole 22a-g. Selected newly synthesized compounds were tested for antimicrobial activity against Penicillin, with 26, 28 mm of zone of inhibition for E.coli and B.subtilis, with 9 to 24 mm. Additionally, they also showed antioxidant activity ¹⁷.

Bondock et al. reported that novel polyheterocyclic ring complexes derived from 2-oxo-2H-pyrimido[2,1-b]benzothiazole-3-carbonitrile 23a-f, were tested for antibacterial activity against Streptomycin, Chloramphenicol as a standard 6.25 µg/mL Minimum inhibitory concentration (MIC) and around 38 mm of zone of inhibition for B. subtilis, S. pyogenes, E.coli, and P.aeruginosa with the range of 3.125 -100 µg/mL, 14 to 44 mm using the agar plate method ¹⁸.

Pawde et al. recently synthesized the thiazolo pyrimido pyrimido benzothiazole derivative 12,13-diimino-9-methyl-thiazolo[2,3-a]-4H-pyrimido[4,5-d]-4H-pyrimido[2,1-b]benzothiazole 24a-g, demonstrated significant antibacterial activity against Penicillin as a standard with 26,28 mm of zone of inhibition for E.coli and  B. subtilis bacteria in the range of 4 to 14 mm using the disc diffusion method, and antioxidant activity using the DPPH assay with penicillin and ascorbic acid as standards, respectively ¹⁹.

Bhoi et al. synthesized a variety of unique N'-isonicotinoyl-2-methyl-4(pyridine-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide 25 a-n analogues using a standard approach. All synthesized analogues are tested in vitro for antibacterial and anti-mycobacterial activity against the Mycobacterium tuberculosis H37Rv strain. The minimum inhibitory concentration ranges from 6.25 to 62.5 µg/mL, with the most powerful molecule at 6.25 µg/mL ²⁰.

Badne et al. synthesised 2-oxo-3-cyano-4 thiomethyl-10-methoxy-14-oxo-14H-pyrazolo (5,6-d) pyrimido (6,7-c) pyrimido [2,3-b] benzothiazole 26a-f had synthesized which were tested for their antimicrobial activities in a range of 10-19 mm of zone of inhibition for S.aureus and E.coli using the Disc diffusion technique at a temperature 37^?C against Penicillin as a standard, with 24 and 20 mm, respectively ²¹.

Venkatesh et al. reported the synthesis of novel substituted Phenyl-1,5-dihydro-2H-benzo[4,5]thiazolo[3,2-a]pyrimido[4,5-d]pyrimidine 27 a-m derivatives as a one-pot synthesis against different bacterial strains with Minimum inhibitory concentration (MIC) values of 50-500 µg/mL when compared to the standard drugs Ciprofloxacin, Bavistin for S.aureus, S. typhi, P.syringae, X.campestris, E. coli, A.flavus, F.oxysporum, C.albicans, C.keratinophilum bacteria, and also showed antifungal activity ²².

Bhosale et al. synthesized 3-amino-8-chloro-4-oxo-(2H)/Aryl/Heteryl-pyrazolo[3',4':4,5]. pyrimido[2,1-b][1,3] benzothiazoles 28 a-i were physiologically tested for gram-positive species S.aureus and B.subtilis, as well as gram-negative species E.coli and S.typhi, using the paper disc diffusion method (14,24,20,16) zone of inhibition using Norfloxacin as the standard ²³.

Sain et al. reported new and simple ways for synthesizing ethyl-8-chloro-4-(4-substituted phenyl)-2[(Nethoxyphthalimido)amino]-4H-pyrimidino[2,1-b1,3]benzothiazole-3-carboxlates 29 a–c & 6-chloro-N-[3-{2-(4-substitutedphenyl)ethenyl} [1-N ethoxyphthalimido quinoxaline-2(1H)-ylidene] -1,3-benzothiazole-2-amines.All of the compounds demonstrated good to moderate antibacterial activity against Ciprofloxacin, 16-18 mm zone of inhibition for P.mirabilis, K. pneumonia, E.coli, and P.aureoginosa, with the range of 7 to 27 mm ²⁴.

Sahu et al. prepared 4H-pyrimido[2,1-b]benzothiazole derivatives 30a-h, used Petra/osiris/molinspiration (POM) analyses with the advantage of short reaction time, no side products, and easy to work with evaluated in vitro antimicrobial activity with ciprofloxacin (for antibacterial strains) with 1.62 or 3.25 µM/mL Minimum inhibitory concentration for S. aureus, P. aeruginosa, S. typhi, E. coli, B. cereus, and P. rettegeri ²⁵.

Anticancer Agents

Waghamare et al. synthesised and in-vitro anticancer activity of 3-cyano-6,9-dimethyl 4-imino 2-methylthio-4H-pyrimido[2,1-b][1,3] benzothiazole and its 2-substituted derivatives 31a exhibited better anticancer activity against 60  cell cancer lines panels one of which in presence of two methyl and –SCH₃ groups in nucleus showed against lung cancer, renal cancer and breast cancer, like wise in presence of p-CH3 and p-OCH3 group some of it exhibited remarkable percentage growth inhibition against HOP-92 (Lung cancer), UACC-62 (Melanoma), and HOP-92 (Lung cancer), UACC-62 (Melanoma). The presence of p-Cl group exhibited excellent activity against K-562, RPMI-8226 (Leukemia), HOP- 92 (Lung cancer), UO-31 (Renal cancer) cell lines panel, also due to presence of due to presence of heteryl cyclic amines at 2-positions exhibited inhibitory effect against CAKI-1, UO-31 (Renal cancer), MCF-7 (Breast cancer) and K-562 (Leukemia), CAKI-1, UO-31 (Renal cancer), PC-3 (Prostrate cancer) ²⁶.

Labhsetwar et al. designed and synthesized fused pyrimido benzothiazoles, such as 8-Chloro-3-cyano-4-imino-2-methylthio-4H-pyrimido[2,1-b][1,3] benzothiazole and its 2- substituted derivatives 32a and to evaluate at National Cancer Institute, Maryland, USA for their in vitro anticancer activity towards 60 human cancer cell lines at a 5 concentration level in which the parent with –SCH₃, 4-nitro phenoxy substituent most potent against HL-60 (TB) cell line (Leukemia). However, the presence of acetyl acetylonyl group at 2-position against TK-10 cell line (Renal cancer), Compounds having 4-nitro aniline, pyrolidino, ethyl acetoacetynl substituent at 2 position showed antitumor activity against HCC-2998 cell line (Colon cancer), and other compounds exhibited inhibitory cytotoxicity against SF-539 cell line (CNS). All synthesised compounds displayed cytotoxicity response across cell lines, sensitive cell lines exhibit GI50 values <10-8 M and insensitive cell lines >10-4 M. For second-stage screening, some compounds demonstrate cytotoxic properties for the design of new non-toxic anticancer drugs   ²⁷.

Prasad et al. synthesised remarkable inhibitory effects of 9-chloro-3-cyano-8-fluoro-2-metylthio-4-oxo-4H-pyrimido[2,1-b][1,3]benzothiazole and its 2-sustituted derivatives 33a-d which had been prepared by the reaction of 2-amino-7-chloro-6-fluro-benzothiazole with ethyl-2-cyano-3,3-bismethylthioacrylate for their in-vitro anticancer activity against Doxorubicin standard within range of value inactive to 200 µg/ml IC₅₀ for  MCF-7 (Breast cancer), HeLa (Epithelial cervix cancer), A549 (lung cancer), B16 (Mouse melanoma) and Hepg2 (liver cancer) respectively  ²⁸.

Nagarapu et al. reported the synthesis of novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate 34a-j derivatives and their cytotoxicity effect against human breast adenocarcinoma MDA-MB-231 and MCF-7 tumour cell lines ²⁹.

Gabr et al. synthesised pyrimido[2,1-b]benzothiazole derivatives 35a-l which shows the broad spectrum of antitumour activity at a single dose a (10 uM) activity against a panel of 60 cancer cell lines at the GI50 and  TGI levels,  then some prominent compounds are further studied for five dose level screening which gives GI₅₀ = 0.44 mM, TGI =1.2 mM and LC₅₀ MG-MID = 6.6 mM) and 4 (GI₅₀ = 0.77 mM, TGI = 2.08 mM and LC₅₀ MG-MID = 11.74 mM potent value in this study for CCRF-CEM (Leukemia), NCI-H522( Non small cell lung cancer), HT29 (Colon cancer), SNB-75 (CNS cancer), LOX IMVI (Melanoma), IGROV1(Ovarian cancer), CAKI-1 (Renal cancer), PC-3 (Prostate cancer) and MDA-MB-468 (Breast cancer) ³⁰.

Chadegani et al. synthesised 2-aminobenzothiazole, benzaldehyde derivatives, β-ketoester, β-diketone or malonate derivatives in solvent-free conditions, providing the corresponding pyrimido[2,1-b]benzothiazole derivatives 36a-e 60-70% yields, optimistic time, high bond forming efficiency,solvent-free reaction conditions ³¹.

Maged et al. synthesised new derivatives of bicyclic fused rings with bridgehead nitrogen of 3-substituted imidazo/pyrimidine derivatives 37a-k, reacting 2-aminobenzothiazole, acetyl acetone, and different substituted aldehydes in a one-pot reaction, which have biological activities against colon cancer at concentrations ranging from 12.5 to 400 μg/mL ³².

El-Sherbeny et al. synthesised certain pyrimido[2,1-b]benzothiazole 38a-j for in vitro, which exhibited a broad spectrum antitumor activity with different selective cell lines using 2-amino-4-chlorobenzothiazole ³³.

Sahu et al. synthesised 4H-pyrimido[2,3-b]benzothiazole derivatives 39a-h using aromatic aldehydes, ethyl acetoacetate, and 2-aminobenzothiazole in the presence of L-proline under solvent conditions using Petra/Osiris/molinspiration(POM) analysis for antitumour activity on human cell lines MCF-7, HELA, and Hep-G2 against Doxorubicin as standard ³⁴.

Antifungal activity

Chidrawar et al. synthesised MCR reaction of 2-amino-6-nitro-benzothiazole and bis methylthio methylene malonitrile to give 3-Cyano-4-imino-2-methylthio-8-nitro-pyrimido[2,1-b][1,3]benzothiazole 40 a which showed broad-spectrum antifungal activity against Fluconazole used as a standard with 23 and 21mm zone of inhibition, Penicillium sp., and Aspergillus Niger by well diffusion method in the range of 8 to 19 mm ³⁵.

Chidrawar et al. had synthesised simple, efficient method for 2-substituted derivatives of  3-cyano-4,14-diimino-2-methylthio-10-nitropyrimido[2,3-b]pyrazolo[3,4-e]pyrimido[2,3b][1,3]-benzothiazole 41 a by one step multicomponent reaction by heating of 3-amino-4-imino-8-nitro-2H-pyrazolo[3,4-e]pyrimido[2,3-b][1,3]benzothiazole and bis (methylthio) methylene malononitrile independently with aromatic amines/phenols/heterylamines/compounds containing active methylene group respectively in the presence of dimethyl formamide and catalytic amount of anhydrous potassium carbonate. All the derivatives are screened for antifungal activity by microbial assay method against Fluconazole as a standard, 23 and 21 mm zone of inhibition for Penicillium sp. and Aspergillus niger species in the range of 7 to 23 mm ³⁶.

Inflammatory activity

Nalawade et al. synthesised new 4H-pyrimido compounds which has the ability of [1,3]benzothiazole-3-carboxylates to reduce inflammation and have been assessed. Many of the chemicals in this series exhibited noteworthy action. Ethyl-(4R)-2-amino-6-chloro-4-(3,4,5-trimethoxyphenyl)-4H-pyrimido, on the other hand, [2,1-b] [1, 3] -benzothiazole-3-carboxylate 42 a-j has been found to possess the most promising anti-inflammatory activity against diclofenac sodium as standard with 94.73 % inhibition of denaturation ³⁷.

Hilal et al. synthesised 8-fluoro-2-methyl-4H-pyrimido [2,1-b][1,3]benzothiazole-4-one 43a showed antifungal activity against griseofulvin, nystatin, and hymexazol as a standard, with 24.3 to 81 % inhibition of different bacteria like M.canis, F. tricinctum, P.ultimum, P.aphanidermatum, and P.middletonii  in the range of 44.4  to 100 % as compared to reference compounds. And 8-fluoro-2,3-trimethylene-4H-pyrimido [2,1-b][1,3]benzothiazole-4-ones via condensation of β-keto esters with 2-aminopyridine derivatives, which were characterized by elemental analysis ³⁸.

Miscellaneous activity

The reported activity of pyrimido(2,3-b)benzothiazole has created interest in the scientific community to synthesize different derivatives of the molecule by using different catalysis, as given in the following table. The application of a catalyst leads to an advantage of improving its yield and purity of that product, as reported by them.