Synthesis and Biological Importance of 2-(thio)ureabenzothiazoles

Abstract

The (thio)urea and benzothiazole (BT) derivatives have been shown to have a broad spectrum of biological activities. These groups, when bonded, result in the 2-(thio)ureabenzothizoles (TBT and UBT), which could favor the physicochemical and biological properties. UBTs and TBTs are compounds of great importance in medicinal chemistry. For instance, Frentizole is a UBT derivative used for the treatment of rheumatoid arthritis and systemic lupus erythematosus. The UBTs Bentaluron and Bethabenthiazuron are commercial fungicides used as wood preservatives and herbicides in winter corn crops. On these bases, we prepared this bibliography review, which covers chemical aspects of UBTs and TBTs as potential therapeutic agents as well as their studies on the mechanisms of a variety of pharmacological activities. This work covers synthetic methodologies from 1935 to nowadays, highlighting the most recent approaches to afford UBTs and TBTs with a variety of substituents as illustrated in 42 schemes and 13 figures and concluded with 187 references. In addition, this interesting review is designed on chemical reactions of 2-aminobenzothiazoles (2ABTs) with (thio)phosgenes, iso(thio)cyanates, 1,1′-(thio)carbonyldiimidazoles [(T)CDI]s, (thio)carbamoyl chlorides, and carbon disulfide. This topic will provide information of utility for medicinal chemists dedicated to the design and synthesis of this class of compounds to be tested with respect to their biological activities and be proposed as new pharmacophores.

1. Introduction

Studies on urea derivatives as biological modulators of intracellular targets have showed the importance of the urea group that, when incorporated in small molecules, display a broad range of biological activities highlighting the importance of this group in drug development and medicinal chemistry. Many drugs containing the urea group such as Cabozatenib, Sorafenib, and Regorafenib used as anticancer drugs have been developed [1,2]. The urea NH moiety is regarded as a hydrogen bond donor, while its oxygen atom, an excellent acceptor, gives the capability for interacting with a variety of protein targets in several ways to this group. Moreover, the urea moiety favors aqueous solubility due to the strong intermolecular hydrogen bonding formation with different solvents. These interactions play an important role in biological structures and functions, regarding protein and nucleic acid folding, molecular recognition, allostery, signal transduction, and enzymatic catalysis [3].

In general, (un)symmetrical (thio)ureas have been shown to have a broad spectrum of biological and pharmacological activities. These compounds are very interesting due to their use as anti-HIV, antiviral, antimalarial, cytotoxic, anti-inflammatory, antifungal, antimicrobial, antidiabetic, anti-tuberculosis, anti-HCV, and CNS drugs [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. Among them, several aryl thiourea derivatives have been used in medicine, industry, and agriculture [28,29,30,31,32]. Moreover, thiourea moiety has been incorporated in many tyrosine kinase inhibitors due to its ability in the formation of hydrogen bonds in the ATP binding cavity of enzymes [33]. For instance, the thiourea derivative YH345A has shown strong protein farnesyl transferase inhibition activity [4]. On the other hand, some heterocyclic thioureas have shown powerful DNA topoisomerase inhibitory activity [10], technological applications such as fluorescence properties [34], corrosion determination of some metals [35], catalysts in chemical reactions [36,37,38,39], and for the extraction of toxic metals [40].

From the drugs used in today’s medicine, many of them possess a heterocyclic nucleus as benzothiazole (BT). Nowadays, BT is a very important phamacophore in medicinal chemistry due to their diversity of pharmacological properties. Developments in biological evaluation of this class of heterocyclic molecules produced changes and promoted the design of new molecules based on their mechanisms of action. BT, one of the family of benzazoles, is an aromatic heterobicyclic compound with a benzene nucleus fused with a thiazole ring. The BT nucleus has been found in marine or terrestrial natural compounds with diverse biological activities. Since 1950, medicinal chemists have been interested in the synthesis of BT derivative compounds, since the pharmacological profile of Riluzole (6-trifluoromethoxy-2-aminobenzothiazole) was found to be a clinically anticonvulsant drug [41,42]. Since then, BT has been an important scaffold with a wide array of interesting biological activities and therapeutic functions. For instance, BT derivatives have been used in the treatment of various diseases such as neurodegenerative disorders, local brain ischemia, central muscle relaxants, and cancer. In the 2000s, few reviews describing the synthetic strategies and biological activities of the BT nucleus were found reported in the literature [43,44,45,46,47]. In the past decade, thirty reviews were found about synthetic methodologies and medicinal activities associated with the BT core as a highly important scaffold for drug development, which has increased rapidly. These reviews were focused on research highlighting anticancer and antimicrobial activities, as well as anticonvulsant, anti-inflammatory, antifungal, antioxidant, antitubercular, antimalarial, antileishmanial, anti-Alzheimer, antitubercular, antidiabetic, and miscellaneous activities [48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73]. Among them, some BT derivatives presented to be useful for the treatment of various diseases including neurodegenerative disorders, local brain ischemia, Huntington’s disease, and cancer. A mini-review about BT derivatives, such as antimicrobial and antiviral [74] and four as anticancer agents, were found [75,76,77,78]. It is worth mentioning that at least ten reviews about this topic were found in the literature from last year until now [79,80,81,82,83,84,85,86,87,88,89]. On the other hand, a review from 2015 to 2020 about the pharmacological activities of BT-related patents was recently reported [90]. Moreover, in 2020, BT derivatives were found to act as multifunctional effectiveness such as antioxidant, sunscreen (filter), antifungal, and anti-proliferative agents [91]. In 2021, we published a literature review on research progress about the condensation of o-amino-thiophenoles with carboxylic acids, acid chlorides, amides, nitriles, esters, thioesters, and ortho-esters, including carbon dioxide (CO₂) as starting materials to access substituted BTs [92].

Nowadays, researchers continuously work on the design and synthesis of BT molecules to obtain more effective derivatives that can be used as drugs [93,94,95,96,97,98,99,100,101,102,103,104]. For instance, scientists are fighting to find drugs against viruses such as influenza and coronavirus. In this sense, two article reviews about the synthesis and structure–activity relationship, as well as various methods to evaluate the antiviral activity against specific viruses of BT derivatives, recently appeared in the literature [105,106].

Since UBTs synthesized by Kauffmann in 1935 were found to be local anesthetics, potent hypoglycemics, and antibacterial agents, these kinds of compounds have had the interest of medicinal chemists [107,108,109,110]. For instance, Frentizole [6-methoxy-phenylureabenzothiazole], Figure 1, a non-toxic UBT drug (IC₅₀ = 200 μM), was approved by the FDA for the treatment of rheumatoid arthritis and systemic lupus erythematosus and has been tested for the immunosuppressive and super-immunosuppressive dose levels on the resistance of the mice to viral infections, Figure 1 [111,112]. It was also found that the mean survival time of specific pathogen-free male mice pre-treated with Frentizole or Azathioprine at 100, 50 or 25 mg/kg and infected with herpes simplex and influenza virus was reduced. On the other hand, it is well known that the combination of UBT and TBT derivatives leads to inhibitors of DNA topoisomerase or HIV reverse transcriptase (e.g., III, Figure 1) [113,114,115]. In addition, the UBTs Bentaluron and Bethabenthiazuron (Tribunil or Ormet) are commercial fungicides used, respectively, as wood preservative and herbicide in winter corn crops [116,117].

Due to the current relevance of BT derivatives, we focused our attention on (thio)ureabenzothiazole derivatives to prepare a literature review from 1935 to now about the methods of the synthesis of this kind of compound to be analyzed with respect to their biological activity studies. Despite a large number of urease inhibitors being reported and marketed, there is still need of more potent inhibitors with fewer side effects and more efficacy.

In general, the official nomenclature of (thio)ureas is on the base of numbering the N1, C2=O(S) and N3 of the urea group. Some authors consider this group to be N, C=O, N’, so in this case, the BT group in (T)BTs is considered to be N-benzothiazol-2-yl or N1-benzothiazol-2-yl group. The alkyl substituents on N′ or N3 are represented as N′-alkyl or N3-alyl. In this work, we consider whether (thio)ureabenzothiazoles can be shortened to (T)UBT. Any substitution on the BT ring and (thio)urea groups was placed on the left of the final name, respectively. For instance, in 6-methyl-methylUBT, the first methyl group is on the 6^th position of the BT ring, and the second methyl is on the N3 atom on the urea group. Any di- or tri-substitution on the (thio)urea group can be differentiated by using N or N1 and N′ or N3-substitutions.

In general, in this review, 2-aminobenzothiazoles 2ABTs or substituted-2ABTs are starting materials used for the synthesis of TBT or UBT. The introduction of the carbamide group into 2ABTs to obtain UBTs or TBTs were phosgene, isocyanates or isothiocyanates, 1,1′-carbonyldiimidazole (CDI) or 1,1′-thiocarbonyldiimidazole (TCDI), carbamoyl chlorides, and carbon disulfide, as well as some new methodologies, which are commented on. We hope this review will provide information about UBTs and TBTs and their biological activities.

2. Results of the Literature Review

In 1935, for the first time, Kaufmann synthesized several substituted UBTs 2–3, bis-6-chloro UBT 4, substituted phenyl-UBTs 5a–e, and substituted phenyl-TBTs 5f–h from the reaction of the corresponding substituted 2ABTs with urea, cyanic acid, phosgene, benzene-isocyanate, and –thiocyanate in chloroform as solvent, Scheme 1 [107].

The same Kauffmann′s methods were used to prepare phenyl-UBTs 6, substituted TBTs 7 and bis-substituted UBTs 8 in order to examine their antibacterial, acaricidal, and insecticidal activities, toxicity in mice, and relationship between chemical structures and biological actions, Scheme 2 [108].

Later, the respective 2ABT or 2-N-alkyl-ABT were treated with an alky- or aryl-iso(thio)cyanate or carbamoyl chloride in an aprotic solvent to yield around 81% of the urea compounds 9, Figure 2 [110]. Refluxing toluene for 4 h was used in the case of substituted-aryl-UBTs and refluxing tetrahydrofuran (THF) for 6 h in the case of substituted-aryl-UBTs. All compounds were tested for immunosuppression in the sheep erythrocyte assay in mice and for antiviral activity against Coxsackie A21 (Coe) virus infection in mice. The most potent immunosuppressant was substituted ureanaphtothiazoles 9a. One of them was 250 times more active as azathioprine in the sheep erythrocyte test in mice. R² must be an aryl group to be active. For R¹ = H, the best R² aryl groups were the halogen substituted pClPh, mClPh, and oFPh. In the immunosuppressive structure–activity relationship (SAR), the best activity was observed for R groups in the 4^th position on the BT ring, for example, 4Cl is even active when R² is cyclohexyl. When R¹ = Me, the potency decreases to the 50 mg/kg range. In antiviral SAR, the antiviral activity showed to be dependent on the nature of R²: if, for example, in R² = 1-naphthyl, adamantyl, and aryl groups, the compounds are quite active.

In 1973, 4,6-disubstituted-β-bromo-propyl-UBTs 10a–g and 4,6-disubstituted-ethenoyl UBTs 11a–g were prepared from the reaction of 4,6-disubstituted-2ABTs with β-bromo-propionyl-isocyanate to be cyclized to produce l-(4,6-disubstituted-benzothiazol-2-yl) dihydrouracils 12, Scheme 3 [118]. A mixture of ether/THF was used for 10a,f,g or THF for 10b–e. The reaction mixture refluxed for 2 h. All compounds were shown to have antibacterial, antifungal, and antiprotozoal in vitro effects. The minimal inhibition concentration of 6-thiocyanate-β-bromo-propionyl-UBT 10f was 50 μg/mL for all tested organisms, which represent a high efficiency.

For instance, Frentizole, 6-methoxy-phenylUBT depicted in Figure 1, was synthetized, and crystals were obtained from ethanol/water to be studied with respect to its structure in the solid state [119].

The reported phenyl-UBT 13 was obtained from the reaction of 2ABT with an excess of phenyl-isocyanate in boiling benzene and recrystallized from EtOH, EtOH-H₂O, or 1,4-dioxane, Scheme 4 [120]. This compound was tested for cytokinin-like activity. The results showed compound 13 inhibited chlorophyll synthesis, perhaps by interfering with the chlorophyll biosynthetic pathway, but not by acting as cytokinin-like. This inhibition was not overcome by the addition of zeatin riboside (ZR).

In 2014, it was reported the first Pd catalyzed C–N coupling of BT-2-ol with phenyl urea via a two-step process involving in situ C–OH bond activation using phosphonium salt as an efficient catalyst system to give out rapid coupling for the synthesis of phenyl-UBT 13 in excellent yield (90%), Scheme 4 [121].

A series of 6-substitutedUBTs 14 inhibitors of p56^lck were prepared to elucidate their SAR, selectivity, and cell activity in the T-cell proliferation assay, Scheme 5 [122]. The urea derivatives 14 were prepared from the corresponding 6-substituted 2ABT when treated with an alkyl- or aryl-isocyanate in dichloromethane and pyridine to form UBTs 14. Alternatively, the 6-substituted-2ABTs treated with phenyl chloroformate in aqueous THF in the presence of potassium bicarbonate in the form of a phenyl carbamate, which is treated with an amine in THF form the ureas 14. Compound 14r was found to be a potent and selective Lck inhibitor with good cellular activity (IC₅₀ = 0.004 μM) against T-cell proliferation.

A series of 6-(2,6-dichlorobenzoamidyl)-alkyl-UBTs 15 was prepared from the reaction of N-6-(2,6-dichlorobenzoamidyl)-2ABT with the corresponding alkyl-isocyanate in pyridine as a dissolvent at 60 °C for 16 h, Scheme 6 [123]. The cytotoxicity text results of all compounds against tumorigenic cell lines were shown. The UBT derivatives 15a–e had the same potency as the amide or urethane derivatives. Selective cytotoxicity against a tumorigenic cell line, WI-38 VA-13 subline 2RA (VA-13), was observed, but not against the normal parental cell line, WI-38. EC₅₀ (ng/mL): 15a = 32, 15b = 30, 15c = 28, 15d = 290, and 15e = 150.

Several substituted-hepta-O-acetyl-β-D-lactosyl-TBTs 16 were prepared in 64–90% by the condensation of hepta-O-acetyl-β-D-lactosyl-isothiocyanate with substituted-2ABTs, Scheme 7 [124]. The structures of these new lactosyl-TBTs have been established on the basis of IR, NMR, and mass spectral studies.

A series of 45 different 6-substituted-aryl-UBTs 17, 19, and 20 and 6-substituted-aryl-TBTs 18 were synthesized from the reaction of the corresponding 6-substituted-2ABT with CDI or TCDI in stirring CH₃CN at room temperature, then reacted with amines in DMF on heating 100 °C, Figure 3 [125]. Using an ELISA-based screening assay, the Frentizole SAR was identified as a novel UBT inhibitor of the interaction of amyloid beta peptide (Aβ) and Aβ-binding alcohol dehydrogenase (ABAD) [Aβ-ABAD], recently implicated in the pathogenesis of Alzheimer’s disease (AD), with a 30-fold improvement in potency. In summary, all synthetized compounds were identified as micro molar inhibitors of the Aβ-ABAD interaction. The compounds 19h and 19l showed the most potent inhibition with IC₅₀ values of 6.46 and 6.56 μM, respectively.

The phenyl-TBT 21 has been isolated from thermal transformation of 3-phenylamino-5-phenylimino-1,2,4-dithiazole via intramolecular rearrangement through intermediate A, Scheme 8 [126]. Analogous thermal or acidic catalyzed rearrangements have been described [127].

A series of aryl-UBTs 22 has been synthesized in 40–60% yield from the reaction of the substituted 2ABT with the corresponding aryl-isocyanate in presence of N,N′-dimethylformamide (DMF), triethylamine (TEA), and dimethylaminopyridine (DMAP) stirring for 8h at room temperature, Scheme 9 [128]. The resultant compounds were evaluated for their antiproliferative profiles in human SK-Hep-1 (liver), MDA-MB-231 (breast), and NUGC-3 (gastric) cell lines. Compounds 22e, 22g, and 22h had the potential to moderate inhibitory activities. More of these compounds have been investigated for their ability to inhibit Raf-1 activity. However, they exhibited moderate to poor inhibition.

The 1,1-bistrifluoromethyl-1-alkyl-UBTs 23a–c was prepared by addition of 2ABT to the corresponding bis-fluoroalkyl-isocyanates, Figure 4 [129]. These UBTs were evaluated for their in vitro antiproliferative activities against the human cancer cell lines. The most sensitive cell lines relative to the tested compounds were: 23c SNB-75 (CNS cancer, log GI₅₀ = 5.84) and 24b UO-31 (renal cancer, log GI₅₀ = 5.66), and SR (leukemia, log GI₅₀ = 5.44) human cancer cells.

A series of 6-trifluoromethoxy-TBTs 24a–f from 6-trifluoromethoxy-2ABT (Riluzole), a neuroprotective drug in many animal models of brain disease have been synthesized, Figure 5 [130]. The biological activity of synthetized TBTs was preliminarily tested by means of an in vitro protocol of ischemia/reperfusion injury. The results demonstrated that 24a–d significantly attenuated neuronal injury. Selected for the testing of their antioxidant properties, compounds 24a–d were shown to be endowed with a direct reactive oxygen species (ROS) scavenging activity. Compounds 24a and 24b were also evaluated for their activity on voltage-dependent Na⁺ and K⁺ currents in neurons from rat piriform cortex. At 50 μM, compound 24b inhibited the transient Na⁺ current to a much smaller extent than Riluzole, whereas 24d was almost completely ineffective.

Some 4/6-substituted-phenyl-TBTs 25 were synthesized by the reaction of substituted-2ABT with the corresponding substituted phenyl-isothiocyanate in absolute ethanol, Scheme 10 [131]. Compounds 25 were condensed with malonic acid in acetyl chloride to obtain 1-(4/6-substituted-BT-2-yl)-3-phenyl-2-thiobarbituric acid derivatives 26. All the synthesized compounds were characterized by elemental analysis, infrared (IR), hydrogen nuclear magnetic resonance ¹H NMR, and Mass spectral studies. Compounds 25 and 26 were screened for their entomological and antibacterial activities. All the synthesized compounds were screened for antibacterial activity at a concentration of 200 μg/mL and 100 μg/mL in DMF using streptomycin and ceftazidime as standard against Gram positive and negative bacteria. Among the synthesized compounds, 26a, 26f, and 26g compounds were found to possess broad-spectrum activities. However, the activities were much less than those of standard antibacterial agents used. These compounds also showed potent antiulcer, anti-inflammatory, and antitumor activities as well as antifeedant activity and acaricidal activity against Spodoptera litura and Tetranychus urticae, respectively. From the results, these compounds would be better used in drug development to combat bacterial infections and would be better used as antifeedant and acaricidal activities in the future as well.

Firooznia et al., synthesized three 4,7-disubstituted-piperidine UBTs 27a–c from the reaction of the 4,7-disubstituted-2ABT with para nitro phenyl chloroformate, followed by the displacement of p-nitro-phenol with an appropriately substituted amine, Scheme 11 [132]. The synthesized compounds were evaluated for their selectivity against the A_2A and A₁ receptors. Antagonists of the A_2B receptor were used as potential therapeutic agents in models of diabetes, asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. Compound 27c displayed excellent A_2B potency, as well as good A_2A and A₁ selectivity, IC₅₀ (A_2B cAMP) = 20 nm, Ki (A₁ binding) = 690 nm, and Ki (A_2A binding) = 530 nm. A SAR study revealed that compounds with urea derivatives, enhance A_2B potency.

The 6-substituted-benzyl UBTs 28a–g were synthetized from the reaction of 6-substituted-2ABT with the in situ generated substituted benzyl-isocyanates obtained from the benzylamine and triphosgene, Scheme 12 [133]. To improve yields, n-butyl-lithium was used to activate 2ABT (24–70%). In the case of 28g (R = CN), the 6-tetrazole p-methoxybenzyl-UBT 29g was obtained in 91% using sodium azide NaN₃ in microwave radiation. All compounds were evaluated for their inhibition of glycogen synthease kinase-3 (GSK-3) activity. Several compounds were identified to reduce in vitro GSK-3β activity beneath 50% at a concentration of 10 μM. The SAR of the library justified the synthesis of the UBT 29g (IC₅₀ = 140 nM), which displayed more than twofold enhanced activity compared with the reference compound 1-(4-methoxybenzyl)-3-(5-nitrothiazol-2-yl)urea (AR-A014418: IC₅₀ = 330 nM).

A series of sixteen differentially substituted-(phenyl)-methyl-phosphonate-TBTs 30a–p were synthetized from substituted 2ABTs and O,O′-di-alkyl-isothiocyanate-(phenyl)methyl-phosphonates in heating dry CH₃CN at 90 °C in 30 min under microwave irradiation with a power input of 120 W, Scheme 13 [134]. The products were obtained in good to excellent yields (40–81%), shorter reaction times, milder reaction conditions, and simple purification procedures. The compounds possessed broad-spectrum insecticidal and antiviral activities against Tobacco Mosaic Virus (TMV) in vivo. Two compounds, 30a (R¹ = 6F, R = ⁿPr) and 31l (R¹ = OMe; R = ⁿBu), had remarkably high in vitro insecticidal activities against Plutella xylostella compared with the control insecticide Avermectin. Furthermore, all were associated with moderate to good anti-TMV activities.

Caputo et al. synthesized two sets of 6-substituted Aryl-UBTs 31a–e and 32a–e by reacting substituted 2ABTs with aryl-isocyanates in dry dichloromethane at room temperature (31a–e, 60–85% and 32a–e, 81–93%) and evaluated in an in vitro primary anticancer assay against a panel of 60 human tumor cell lines, Scheme 14 [135]. Compounds 32a and 32c showed good anticancer activities, more marked for compound 32c. All compounds in a preliminary in vitro assay as inhibitors of the ubiquitin-activating enzyme (E1) lacked significant activity. The UBT scaffold 32c showed considerable growth inhibitory activities against different human tumor cell lines such as leukemia (log GI₅₀ value −5.93), non-small cell lung (log GI₅₀ value −6.0), colon cancer (log GI₅₀ value −5.89), CNS cancer (log GI₅₀ value −5.73), melanoma (log GI₅₀ value −5.89), ovarian cancer (log GI₅₀ value −5.74), renal cancer (log GI₅₀ value −5.90), prostate cancer (log GI₅₀ value −5.72), and breast cancer (log GI₅₀ value −6.0) as compared with the reference drug 5-fluorouracil NSC 19893.

The appropriate 4/6-arylsubstituted-2ABT and the respective aryl-isothiocyanate was reacted in a mixture of DMAP (5 mol %) in DMF to furnish 6/4-arylsubstituted-TBT derivatives 33a–l in 83–91% yield, Figure 6 [115]. All compounds were evaluated for cytotoxic activity against two human monocytic cell lines (U 937, THP-1) and a mouse melanoma cell line (B16-F10). Based on their IC₅₀ values, almost all compounds had significant antiproliferative activity on U 937 and B16-F10 cells, with 33b, 33e, 33f, 33k, 33c, and 33h being the most actives. Compound 33e demonstrated to have the best antiproliferative activity against the U-937 cell line. The IC₅₀ values of compound 33e were higher (16.23 ± 0.81 μM), (47.73 ± 2.39 μM), and (34.58 ± 1.73 μM) as compared to standard compound etoposide IC₅₀ values (17.94 ± 0.89 μM), (18.69 ± 0.94 μM), and (2.16 ± 0.11 μM)) against U-937, B16-F10 and THP-1 cell lines, respectively.

Azam and coworkers synthetized aryl-UBT derivatives 34a–s from the reaction of 2ABT in acetonitrile as dissolvent with the dropwise addition of the appropriate aryl-isocyanate stirred at room temperature until the completion of the reaction (1–4 h), Scheme 15 [136]. All compounds were tested as anti-Parkinsonian agents with an improved pharmacological profile in haloperidol-induced catalepsy and oxidative stress in mice. All compounds were active in alleviating haloperidol-induced catalepsyin mice. Furfuryl 34i and 2-methoxy 34c emerged as potent agents. With exception of 2-chloro, 5-trifluoromethyl-substituted analog 34q, and halogen substituted derivatives 34n–p exhibited moderate antiparkinsonian activity. Molecular docking studies of these compounds with adenosine A2A receptor exhibited very good binding interactions and warrants further studies to confirm their binding with human A2A receptor for the design and development of better therapy for Parkinson′s disease PD.

The 6-methoxy-m-methoxyUBTs 35a, 6-methoxy-m-methoxyTBTs 35b, and their hydrolyzed compounds 36a,b were synthesized via amide coupling starting from the reaction of 6-methoxy-2ABT with m-methoxy-benzo-isocyanate or m-methoxy-benzo-isothiocyanate in pyridine at 100 °C for 20 h, respectively, Scheme 16 [137]. The ether cleavage of the methoxy groups was carried out with boron tribromide (BBr₃) in methylene chloride (CH₂Cl₂) at −278 °C to room temperature to yield compounds 36a and 36b. The three-unit bridge resulted in the inactive urea 36a against human 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1), but the more lipophilic thiourea 36b showed a moderate inhibitory activity (70b: 62% inhibition at 1 µM).

Patents from 2007 to 2012 reported the reaction of 6-bromo-5-iodo-2ABT or 7-bromo,5-iodo-2ABT with ethyl-isocyanate in 1,4-dioxane at 80°C for 12 h under N₂ to afford the corresponding 1-(5.6- or 1-(5,7-dihalogen)ethyl-UBTs, Figure 7 [138,139,140,141,142]. In addition, one hundred and thirty-eight 1-(4-fluoro-5-bromo-7-substituted-ethylUBTs were obtained from the reaction of the corresponding dihalogenide-2ABT with methyl(chloro)thioformate, phenyl-chloroformate, or p-nitrophenyl chloroformate in the presence of a base as pyridine or TEA in a solvent as dichloromethane, chloroform, carbon tetrachloride or mixtures, Figure 7. The resultant 1-(5,6- or 1-(5,7-dihalogenide-BT-2-yl)-3-ethylurea in DMF/H₂O was treated with pyridine-3-boronic acid and potassium phosphate (K₃PO₄) to generate the corresponding 6-fluoro-5-substituted-ethylUBT 37, 5,7-disubstituted-ethylUBTs compounds 38–41, and 4-fluoro-5,7-disubstituted-ethylUBT as 42 in approximately 65% yield.

Ethyl-UBTs 37–42 were tested against Gram-positive and Gram-negative pathogens. The minimum inhibitory concentration (MIC₉₀) values for 37 were found to be: S. aureus = 0.06 μg/mL, S. pneumoniae = 0.015 μg/mL, S. epidermidis = 0.03 μg/mL, and E. faecalis = 0.25 μg/mL.

Compound 38 exhibited important inhibitory activity against S. aureus GyrB (IC₅₀ 0.014 μg/mL) and strong antibacterial activity against VR E. faecalis, VR E. faecium, FQR S. pneumoniae, S. aureus, and M. catarrhalis with a MIC value of less than 25 μg/mL. The solubility and antimicrobial profiles of 39 were improved at physiological pH from 6.25 μg/mL to 50 μg/mL by bonding a carboxylic acid on pyridine group (39), while maintaining antibacterial activity against target pathogens (S. aureus GyrB IC₅₀ 0.001 μg/mL, E. faecalis MIC 0.12 μg/mL). In addition, benzothiazole ethyl ureas 40 and 41 were synthesized by changing the carboxylate in compound 38 by a cyclic amine [78,79]. Both compounds inhibited the turnover of ATP by both the DNA gyrase and topoisomerase IV enzymes, with mean IC_50s ranging from 0.0033 μg/mL to 0.046 μg/mL. These compounds were also tested against six major Gram-positive pathogens, exhibiting potent inhibition with MIC₉₀ values from 0.015 μg/mL for S. pneumoniae to 0.25 μg/mL for S. aureus, compared with the MIC₉₀ values of 2 μg/mL and 4 μg/mL, respectively, for linezolid and 16 μg/mL and 16 μg/mL, respectively, for levofloxacin. The tested compounds were also potent against S. aureus drug-resistant strains, including VRE, MRSA, VRSA, linezolid-non-susceptible S. aureus, daptomycin-non-susceptible, methicillin-resistant S. epidermidis, penicillin resistant S. pneumoniae, and FQ-S. pneumoniae strains. Moreover, no cross-resistance was observed caused by the mechanisms responsible for conferring resistance to these antibiotics. Both compounds also showed a promising pharmacokinetic profile in rats and mice [78].

Two additional patents described the synthesis of 4-fluoro-5,7-disubstitutedbenzothiazole ureas as compound 42 [75]. All compounds were tested for their DNA supercoiling assays for GyrB inhibition and a DNA relaxation assay for ParE inhibition. Under both assay conditions, few compounds showed activity in the sub-micromolar range (IC₅₀ values 30–550 nM) and were found to be active in antibacterial assays against Gram-positive pathogens.

The same procedure was used to synthetize a series of 6-substituted-ethylUBTs 43, 44 and 6-substituted-R¹ethylUBTs 45 as effective inhibitors against wild-type (wt) and T₃₁₅I mutant Bcr-Abl Kinases, Scheme 17 [143]. Through a structure-based drug design, Nocodazole was modified, varying the C2 and C6 groups. It was found that the introduction of polar groups at the terminal ethyl group enhanced physicochemical properties and potency in cellular inhibition. Several compounds inhibited the kinase activity of both wild-type Bcr-Abl and the T₃₁₅I mutant with IC₅₀ values (43a, IC₅₀(wt) = 0.06 nM and IC₅₀(T₃₁₅I) = 0.11 nM) and exhibited good antiproliferative effects on Ba/F₃ cell lines transformed with either wild-type or T₃₁₅I mutant Bcr-Abl.

By using ethyl isocyanate in 1,4-dioxane at 80 °C overnight, the 5-OBn and 7-bromo-2ABT was transformed to the 5,7-disubstituted-ethylUBT 46 in 75% yield, subsequent pyridine-2-yl group was coupled to the C-7 to afford compound 47 in 70% yield, Scheme 18 [144]. Compound 47 was Bn-deprotected (97%), converted to the triflate (68%), and a Miyaura borylation reaction yielded 88% of the key borinic acid intermediate 48 for further derivatization through a Suzuki cross-coupling of the corresponding 5-bromo pyrimidine with 49, followed by saponification to yield 5,7-disubstituted-ethylUBTs 49a–d, 50a–h, and 51a–d, Scheme 18. The synthetized compounds were tested as bacterial DNA gyrase and topoisomerase IV inhibitors. Antibacterial properties were demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, and Haemophilus influenzae. Compounds 50a and 50b had IC₅₀ values for S. aureus topoisomerase IV (0.012 and 0.008 μg/mL, respectively) comparable to their S. aureus DNA gyrase ATPase IC₅₀ values. These compounds also showed specificity for bacterial topoisomerases, with no inhibition of human topoisomerase II. The compounds 49a–h bearing a α-substituent to the carboxylic acid group, resulting in excellent solubility and favorable pharmacokinetic properties.

Compounds 50a and 50b in Scheme 18 were evaluated for their biochemical, antibacterial, and pharmacokinetic properties [145]. Both compounds inhibited the ATPase activity of GyrB and ParE with IC₅₀ of < 0.1 μg/mL. Prevention of DNA supercoiling by DNA gyrase was observed. These compounds inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC₉₀s against clinical isolates ranged from 0.015 μg/mL for Streptococcus pneumoniae to 0.25 μg/mL for Staphylococcus aureus. No cross-resistance with common drug-resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound 50a or 50b and other antibiotics, as the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were < 2.3 × 10⁻¹⁰ with compound 50a and < 5.8 × 10⁻¹¹ with compound 50b at concentrations to 8 folds the MICs. These values indicate a multi-targeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound 50b showed an approximately 3-fold improvement over compound 50a in terms of both the clearance and the area under the concentration–time curve. The measured oral bioavailability of compound 50b was 47.7%.

Palmer et al. used the same procedure to obtain a series of 5-substituted alcohol-containing ethyl UBTs 52a–s, 53a–g, and 54a–k, which were identified to have superior antibacterial activity and drug-like properties, Figure 8 [146]. The 5-substituted alcohol-containing ethyl-UBTs 52a–s were prepared to improve the pharmacokinetic profile. The SAR study of these series revealed that these compounds had potent antibacterial activity against a primary panel of pathogenic bacteria compared to the carboxylic acid 50c, Scheme 18. Compounds 53a–c displayed potent antibacterial activity against S. aureus 29213 (MIC 0.03–0.06 μg/mL), S. pyogenes (MIC 0.06–0.12 μg/mL), and H. influenza 49247a (MIC 0.25–1 μg/mL). Compounds 52a–c also displayed significant inhibitory activity against purified S. aureus T173 GyrB (IC₅₀ 0.25 μg/mL all cases) and S. pyogenes ParE (IC₅₀ to 0.5–1 μg/mL). Compounds 52a, d–m were investigated for the scope and limitations of the alcohol-containing substituents. In general, compounds showed potency in the nanomolar range against DNA gyrase ATPase in a malachite green assay. The introduction of bulky groups at the tertiary, pseudo benzylic position of the pyrimidine ring resulted in a remarkable loss of activity, particularly against the ParE mutant strain of S. pyogenes (52d diethyl, IC₅₀ 8 μg/mL; 52f cyclohexyl ring system, IC₅₀ > 16 μg/mL). However, increasing on-target ATPase inhibition was observed when a heteroatom was introduced into the alicyclic 6-membered ring (52g–j), IC_50s S. aureus GryB 0.25–8; S. pyogenes ParE 1–8 μg/mL), suggesting a conformational role, possibly a flattening of the ring. Reduced antibacterial activity against S. aureus 29213 (MIC > 16 μg/mL), S. pyogenes 51339 (MIC 8 μg/mL; IC₅₀ > 16 μg/mL), and H. influenza 49247a (MIC 16 μg/mL) was observed when the ring was contracted to an azetidine (52k) and leaving the free NH group more exposed. However, strong potency (MIC 0.06–0.5 μg/mL) across the entire tested bacterial stains (with IC_50s values of 0.5 and 2 μg/mL against S. aureus GryB and S. pyogenes ParE, respectively) was observed in the case of physically constrained oxetane analogue 53m.

The secondary alcohols 52n–q, s and the diol-containing molecule 52r were explored to enhance the solubility. The extension of R¹ to a longer chain alkyl and small cycloalkyl groups 52n, 52o, 52q exhibited no effect on the potency of the series toward the enzyme. However, the large t-butyl moiety 52p showed a substantial drop in activity against Haemophilus influenza. Moreover, a remarkable drop in activity against S. aureus in the presence of serum was observed. The morpholine group in 52s and diol in 52r, designed to enhance hydrophilicity, was well tolerated. Further, the extension of the diol-containing series resulted in compounds 53b–f. In this series, the introduction of small groups did not hamper antibacterial activity significantly. However, diol 53d showed a relative intolerance of the large rings. Compounds 54a–f enhanced hydrophilicity and reduced protein binding resulted in potent Gram-positive antibacterial activity. Further, introduction of alcohol-containing moieties at the C5 position of the BT core and modifications at the C7 position yielded compounds 54a–k. These compounds with enhanced hydrophilicity and reduced protein binding resulted in potent Gram-positive antibacterial activity. Compounds 54f–h containing 5-membered rings substituted with ether or alcohol containing moieties, enhanced the unbound fraction from 9% to 19%. However, this enhancement was partially compensated for by a moderate increase in MIC. The substitution of azetidine moieties in compounds 54i–k enhanced this property further.

In a typical reaction, ortho-, meta-, or para-tolyl-isocyanates were treated with 2ABT in the presence of 1,4-dioxane at room temperature to synthetize three tolyl-UBTs 55a–c to evaluate their inhibitory effects on α-chymotrypsin enzyme, Figure 9 [147]. It was found the para-substituted N-(1,3-benzothiazol-2-yl)-N′-(4-methylphenyl) urea 55c derivative substantially inhibited α-chymotrypsin activity (IC₅₀ = 20.6 ± 0.06 μM). Due to the presences of the bulky 1,3-benzothiazol-2-yl group at one nitrogen of the urea bridge, the activity is enhanced by the reduction of the steric hindrance in the order ortho > metha > para.

The 6-bromo-2ABT was reacted successively with CDI and substituted ethylamine to yield 6-bromo ethyl-UBTs 56 in 74–90%, Scheme 19 [148]. Intermediates 56 were reacted successively with bis(pinacolato)diboron and intermediate 57, catalyzed by PdCl₂(dppf), to yield a series of 6-(2,3-disubstituted pyridine-5-yl)-ethyl-substituted-UBTs 58a–l in 33–51%. The antiproliferative activities of the synthesized compounds in vitro were evaluated against HCT116, MCF-7 U87MG, and A549 cell lines. The compounds with potent antiproliferative activity were tested for their acute oral toxicity and inhibitory activity against PI3Ks and mTORC1. The results indicate that the compounds with R¹ = 2-dialkylaminoethyl moiety 58b, 58f, 58k, and 58l retain the antiproliferative activity and inhibitory activity against PI3K and mTOR. In addition, their acute oral toxicity reduced dramatically. Moreover, compound 58f can effectively inhibit tumor growth in a mouse S180 homograft model. These findings suggest that derivatives 58f, 58k, and 58l can serve as potent PI3K inhibitors and anticancer agents with low toxicity.

Using the same procedure, compounds 59m–q were synthetized in a 40–45% yield to compare the activities with different substituents at the 3-position of pyridine ring, Figure 10. In the case of pyridine ring 59m, the activity dramatically dropped.

The 6-sulfonamide-2ABT was reacted with one equivalent of carbon disulfide and one or two equivalents of dimethyl sulfate in the presence of one equivalent or two equivalents of sodium hydroxide to afford compounds 60 in 54% and 62 in 67%, Scheme 20. Furthermore, compound 60 was reacted with ethylenediamine or piperazine in dioxane at room temperature to give the bis-TBTs 61a or 61b, in 21 and 20.7%, respectively. Under the same reaction conditions, compound 62 produced the bis-Methyl-iso-TBTs 63a or 63b in 14 and 17%, respectively [149]. All compounds were investigated as inhibitors of the four isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), the cytosolic CA I and II, and the tumor-associated isozymes CA IX and XII. Docking studies showed favorable interactions between the scaffolds of these new inhibitors and the active sites of the investigated CA isoforms. Compounds 61a,b and 63a,b acted as highly potent inhibitors of the tumor-associated hCA IX and hCA XII with K_Is in the nanomolar range (2.1–2.6 and 4.3–4.8 nM), compared to the standard drugs AAZ (25 and 5.8 nM) and EZA (34 and 22 nM). The cytosolic isozyme hCA II was also inhibited with K_Is ranging from 3.5 to 4.7 nM, compared with the standard drugs (12 and 8 nM). On the other hand, from molecular docking studies, compounds 61a,b and 63a,b were found as potent inhibitors against the slow cytosolic isoform hCA I with K_Is in the range of 12.0–37.4 nM, compared to the standard drugs (250 and 25 nM).

A series of seventeen different picoline-amide based UBT derivatives 64a, 64b, 65, and 66a–n, were synthesized as shown in Scheme 21 [150]. The treatment of 2ABT with the appropriate aliphatic isocyanate in dioxane afforded compounds 64a and 64b in approximately 87% yield, while the thiourea derivative 65 was obtained in only a 10% yield from the reaction of 2ABT with ethyl-iso-thiocyanate in pyridine. On the other hand, the target compounds 66a–n were prepared via a two-step one-pot reaction. The 2ABT was first converted into its isocyanate intermediate by the treatment of CDI in DMF and followed by the reaction with suitable aliphatic amine. All compounds were tested for their inhibitory activities against the wild-type ABL at 10 μM and showed strong enzymatic inhibition, 93.3–100%. Their IC₅₀ values were found to be 18.2–285 nM. Compounds 64a, 65, 66a, 66f, 66g, 66i, 66l, and 66n were tested over the mutant type ABLT315I and displayed promising potency with IC₅₀ values of 39.9–511 nM.

The 3,3,3-trifluoroethyl-isocyanate was used for the synthesis of a series of substituted tri-fluoroethyl-UBTs 67a–d in 70–95% yield, Scheme 22 [151]. These compounds were tested at a single high dose (10⁻⁵ M). The moderate anticancer activity against some types of cancer on the individual human cell lines for leukemia, non-small cell lung cancer and renal cancer were shown. All compounds showed anticancer activity on individual cell lines. Activity was notable on individual cell lines against leukemia, non-small cell lung cancer, and renal cancer. The COMPARE algorithm analysis revealed that possible mechanisms of action of these ureas include alkylating agent 67c, DNA antimetabolites 67b–d, RNA/DNA antimetabolites 67d, topoisomerase II inhibitor 67b, and antimitotic agent 67d.

In 2016, a three-component reaction of o-amino-thiophenol disulfide, copper cyanide, and aryl cyanates were used for the synthesis of N,N-dimethyl-UBT 68 and phenyl-UBTs 69, Scheme 23 [152]. This transformation is based on an oxidative copper-mediated S-cyanation as a key step, then a cyclization sequence enabling a rapid and efficient synthesis of 2ABT, which reacts with the corresponding electrophile to produce thiourea 68 in 37% or urea 69 in 55%.

A series of substituted p-benzene-sulfonamide-TBTs 70a–c were synthesized from substituted 2ABTs and p-isothiocyanate-benzene-sulfonamide as starting materials, Scheme 24 [153]. The structures of the compounds were established from elemental analyses, IR, ¹H-NMR, ¹³C-NMR, and mass spectral data analysis. All compounds were evaluated for their in vitro anticancer activity against various cancer cell lines. Compound 70a exhibited good activity higher than or comparable to the reference drugs, 2′7′dichlorofluorescein (DCF) and Doxorubicin, except the breast cancer line. As a trial to suggest the mechanism of action of the active compounds, the molecular docking on the active site of the mitogen kinase enzyme (MK-2) was performed, and the results showed compound 70a may represent a good candidate for further biological investigations as anticancer agents.

A series of 6-substituted-alkyl(aryl)-UBTs and 6-substituted-alkyl(aryl)-TBTs 71a–v were synthesized from 6-substituted-2ABTs and alkyl/cycloalkyl/aryl-isocyanates or isothiocyanates in refluxing toluene, Scheme 25 [154]. The reaction rates and yields depended on substituents introduced on the BT ring and the isocyanates and isothiocyanates. The reaction rate was greater with an electron-donating group on the BT ring such as ethyl C₂H₅ or methyl CH₃. The rate was very low with an electron-withdrawing group, such as nitrogen dioxide NO₂. Isocyanates were more reactive than isothiocyanates due to the greater electronegativity of the oxygen atom compared to that of the sulfur atom. Compounds were tested as myorelaxants and inhibitors of insulin secretion. Compounds 71u and 71v showed high myorelaxant activity. The 6-substituted-alkyl-UBTs 71f, 71g, and 71t–v containing strong electron-withdrawing groups as NO₂, CN at the 6-position, and an alkyl group linked to the urea or the thiourea function were found to be the most potent compounds. Some compounds were tested on rat pancreatic islets provoked a marked inhibition of insulin secretion, among which 71a exhibited a clear tissue selectivity for pancreatic β-cells.

A series of UBTs 72a–m tethered with a pyridyl-amide moiety by ether linkage at the 6-position of BT was synthesized as potent anticancer sorafenib analogs, Scheme 26 [155]. The synthesis was achieved by treating the 6-substituted 2ABT with the corresponding aryl isocyanate in either anhydrous DMF or acetonitrile under argon atmosphere for compounds 72a–j. Compounds 72k–m were prepared via a two-step one-pot reaction. The 6-substituted 2ABT was converted into the isocyanate by treatment with CDI in DMF, followed by the reaction with the proper aromatic amine. Twelve of these derivatives were analyzed for its antiproliferative activity over a panel of 60 human cancer cell lines at a single-dose concentration of 10 μM. Compounds 72a–d were more potent than the sorafenib used in the treatment of renal cell carcinoma, and their GI₅₀ values were determined. Compound 72b showed good inhibitory activities against ACHN (renal cancer cells lines) and A-498 (human kidney carcinoma cell line) with GI₅₀ values of 0.542 µM and 1.02 µM, respectively. This compound also showed efficacy against UO-31 and RXF 393 cell lines. Compounds 72a and 72d exhibited excellent antiproliferative activities with low IG₅₀ values of 1.85 and 2.10 µM against RCC and ACHN cell lines, respectively. The SAR study revealed that sorafenib analogues possess anti proliferative activity due to the presence of both urea spacer and phenyl di-substitution. Compound 72b demonstrated the highest CLogP value was the most lipophilic and potent derivative in the low µM range.

To the in situ generated 2-isocyanateBT from substituted 2ABT and CDI in DMF at room temperature, the appropriate aniline was added, and the reaction mixture was heated at 100 °C for 3 h to generate the corresponding UBTs 73a and 73b, Scheme 27 [156]. The in vitro anticancer evaluation of 73b showed substantial broad-spectrum antiproliferative activity against 60 human cancer cell lines. It showed GI₅₀ values of 51.4 and 19 nM against leukemia K-562 and colon carcinoma KM12 cell lines, respectively. The kinase screening of compound 73b revealed its nanomolar-level inhibitory activity of certain oncogenic kinases implicated in both tumorigenesis and angiogenesis. Compound 73b displays IC₅₀ values of 0.82, 3.81, and 53 nM toward Tie2, TrkA, and ABL-1(wild-type and T315I mutant) kinases, respectively. Moreover, 73b is orally bioavailable with a favorable in vivo pharmacokinetic profile. Compound 73b may serve as a promising candidate for the further development of potent anticancer chemotherapeutics.

Molecular modeling study was performed to evaluate the recognition of a TBT 74, and its Cu(II), Co(II), and Ni(II) metal complexes were synthesized, Scheme 28 [157]. They were characterized by micro analysis, IR, ¹H-NMR, EPR, UV-Visible spectral analyses, molar conductance, and thermal analysis studies. These studies revealed that the metal complexes have distorted octahedral geometry. The recognition of target compounds at the 3MNG binding pocket was evaluated by molecular modeling. The copper complex selectively binds to the crucial amino acid residues in the active site of 3MNG. The in vitro antioxidant activity of the ligand and its metal complexes was assayed by radical scavenging activity (DPPH, H₂O₂ and NO) and ferric-reducing antioxidant power (FRAP) methods. The ligand showed moderate antioxidant activity, whereas the metal complexes exhibited better antioxidant activity than that of the ligand, with the copper complex being the most potent antioxidant.

Selected 6-subtituted-2ABTs were activated with one equivalent of CDI to obtain their corresponding intermediates B in excellent yields (90–95%), which were treated with a substituted aromatic amine to produce two series of 6-substituted-aryl-UBTs 75a–zt in moderate-to-excellent yields (36–99%) along with novel insights into the structure and activity relationships for the inhibition of amyloid-beta binding alcohol dehydrogenase (ABAD), Scheme 29 [158]. Compounds 75zg and 75zi showed potent ABAD inhibition, whereas compound 75zi exhibited comparable cytotoxicity with the Frentizole standard; however, this was a one-fold higher cytotoxicity than the parent Riluzole standard. The calculated and experimental physical–chemical properties of the most potent compounds showed promising features for blood–brain barrier penetration, BBB.

On the other hand, compounds 75a–zb in Scheme 29 were evaluated for their inhibitory activity on CK1 and their potential to cross the BBB was predicted using the central nervous system–multiparameter optimization (CNS-MPO) model and eventually parallel artificial membrane permeability assay (PAMPA) [159]. Several compounds were found to be sub-micromolar CK1 inhibitors, identified using compound 75q as being the best hit (IC₅₀ = 0.16 mM/1.92 mM for CK1_δ resp. CK1_ε). However, compounds 75e and 75s were shown to be low micromolar inhibitors of both CK1 and ABAD, and hence they present a potential novel class of dual-acting anti-AD therapeutics. The results of PAMPA for 75e and 75s suggest that the compounds should be able to penetrate into the brain.

A series of 6-substituted aryl-(thio)urea-BTs and GBTs 76a–o was synthetized, Scheme 30 [160]. The synthesis started with the activation of the corresponding 6-substituted 2ABT using CDI in refluxing DMF or TCDI in refluxing acetonitrile (MeCN), Scheme 30. In the next step, the reactive imidazolyl intermediate C was treated with the corresponding aniline in DMF at 60 °C to obtain the 6-substituted thio(urea) products 76. Guanidine analogues were prepared by treating the corresponding thiourea with mercury oxide in methanolic ammonia solution. Compound 76l was identified as the most promising hit compound with good inhibitory activity (IC₅₀ = 3.06 ± 0.40 µM) and an acceptable cytotoxicity profile comparable to the Frentizole. The acceptable physical–chemical properties of the guanidine compound 76l suggest its capability to permeate through the BBB, making compound 76l a novel lead structure for further development and biological assessment.

The 5-substituted-TBTs 77a–d were prepared in excellent yields (60–68%) by the reaction of 5-substituted 2ABTs with carbon disulfide (CS₂) and dimethyl sulfate followed by ammonolysis of the intermediate D, Scheme 31 [161]. The cytotoxic activity was screened for antitumor activity against human breast cancer cells (MCF-7), human cervix epithelial carcinoma (HeLa), a human colon cancer cell line (HT-29), a human leukemia cell line (K-562), and a mouse neuroblastoma cell line (Neuro-2a) using cisplatin as a reference by MTT assay. The results provide experimental evidence that the compounds induce apoptosis in cancer cell lines. According to flow cytometry results, the treatment of HT-29 cells with 77d produced a large population of apoptotic cell (79.45%), which was 1.2-fold higher than that produced by cisplatin (65.28%) at the same concentration.

Fluoroquinolone-TBT derivatives 78a–n were synthetized from a mixture of 6-substituted 2ABT, fluoroquinolone, and CS₂ in basic medium (NaHCO₃/DMF) and refluxed for 10 h, Scheme 32 [162]. All compounds were tested against bacterial strains. Some compounds exhibited excellent antibacterial activity against Staphylococcus auerus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa bacterial strains. Among all compounds, the 6-nitro substituted UBT, along with norfloxacin 78b and gatifloxacin 78l, showed MIC₅₀ μg/mL when tested against S. auerus. Moreover, compounds 78d, 78f, and 78l showed superior MIC (15, 10, and 15 μg/mL, respectively) against B. subtilis. The results reveal that the compounds have significant antibacterial potential and are suitable candidates for further exploration.

Fifteen differentially substituted-TBTs 79a–o were synthetized from the reaction of commercially available 2ABT and the corresponding alkyl- or aryl-isothiocyanate in acetonitrile, Scheme 33 [163]. Substituted-TBTs 79a–g were cyclized to 1,3-thiazolidin-4-ones 80a–g. Molecular structure of compounds 79d, 79m, 79o, and 80c was determined by X-ray crystallography. All compounds were evaluated on their cytotoxicity against human leukemia/lymphoma- and solid tumor-derived cell lines and of their antiviral activity against HIV-1 and representatives of ssRNA and dsDNA viruses. Compound 80e showed activity against the HIV-1 wild type and against variants carrying clinically relevant mutations. A colorimetric enzyme immunoassay clarified its mode of action as a non-nucleoside inhibitor of the reverse transcriptase.

A mixture of 6-methoxy-2ABT, BMZ derivatives, CS₂, basic medium (NaHCO₃) and DMF was refluxed for 10 h. to afford a series of N-(6-methoxybenzo[d]thia-zol-2-yl)-2-substituted phenyl-1H-benz[d]imidazole-1-carbothioamides 81a–h, Scheme 34 [164]. All compounds were characterized and screened for their in vitro antimicrobial activity against selected bacterial and fungal strains. These compounds were also evaluated for their antimalarial activity against P. falciparum. Antimicrobial activity screening results showed compounds 81b against P. aeruginosa, 81c against S. aureus and E. coli, 81d against E. coli and P. aeruginosa, and 81g against E. coli emerged as prospective antibacterial leads with excellent activity (MIC 12.5–62.5 μg/mL). Only one fungal strain, C. albicans, was susceptible towards synthesized compounds. On the other hand, compounds 81c and 81h exhibited antimalarial activity with IC₅₀ values of 0.18 and 0.11 μg/mL as compared to standard drugs chloroquine (IC₅₀ 0.020 μg/mL) and quinine (IC₅₀ 0.268 μg/mL).

Compound AHS-211 was modified to prove the role of the urea linker to preserve the bioactive conformation and led to the development of 6-ydroxy-arylUBTs 84a–c as promising selective Dyrk1A inhibitors, Scheme 35 [165]. The compounds 84a–c were synthetized through the reaction of the 6-methoxy-2ABT with phenyl-isocyanates in DMF at room temperature to generate the 6-methoxy-arylUBTs 82a–c, Scheme 35, or alternatively through the reaction of 6-methoxy-2ABT with phenyl-chloroformate in the presence of pyridine as a base and dioxane as solvent to give the carbamate as intermediate, which was further in situ reacted with amine derivatives in refluxing dioxane to give the 6-methoxy-ethylsubstitutedUBTs 83a and 83b, Scheme 35. UBT 83b was hydrolyzed with aqueous KOH solution in THF at room temperature for 14 h, then the carboxylic acid of 82c was reacted with the corresponding amine and HBTU at room temperature overnight to generate 6-methoxy-arylUBTs 85a,b. Compounds 82a and 85a,b were ether dealkylated in the presence of Phosphorous tribromide (PBr₃₎ in CH₂Cl₂ as a dissolvent at room temperature for 20 h to generate compounds 84a–c. Although the extension with the 4-benzyl amide in compound 84b enhanced the potency toward Dyrk1A (IC₅₀ = 0.063 μM) by more than 2-fold compared with 84a and AHS-211, it did not show any improvement in selectivity over Dyrk1B. On the other hand, compound 83c was almost similar to AHS-211 in potency against Dyrk1A, yet it showed more than a 15-fold selectivity for Dyrk1A over Dyrk1B. Importantly, both 84b and 84c displayed superior selectivity for Dyrk1A over Dyrk2 when compared with AHS-211. Additionally, 84c displayed good selectivity for Dyrk1A over Clk1, one of the most common off targets of many reported Dyrk1A inhibitors, with a selectivity factor > 7 (IC₅₀ of 84c against Clk1 = 730 nM, [ATP] = 15 μM). The design concept of attaching a urea linker to the BT core, followed by a scaffold expansion, led to a new class of potent and selective Dyrk1A inhibitors. The best compound, 84c, did not inhibit the homologous Dyrk2 isoform and even showed a remarkable 15-fold selectivity over the most closely related isoform Dyrk1B. In addition, the activity toward the atypical kinase haspin that was strongly inhibited by most of the previously reported Dyrk1A inhibitors was abolished with 84c. Together with the favorable balance of lipophilic/hydrophilic functions, further testing of 84c as a potential agent for the treatment of Dyrk1A-related neuropathological disorders could be envisaged.

From the 6-aminoethyl-UBT obtained from the reaction of 6-nitro-2ABT with ethyl isocyanate in refluxing toluene 24 h in the presence of TEA, followed by reduction of the nitro group to amine, Scheme 36 [166]. A series of ethyl-urea derivatives of 6-amino-2ABT, named 86a–I, were synthetized and evaluated for E. coli DNA gyrase inhibition using an in vitro DNA gyrase supercoiling assay. The most potent DNA gyrase inhibitors were 6-substituted UBTs 86c–e, 86g, and 86h with IC₅₀ values in the low micromolar range. The most promising inhibitors identified were evaluated against selected Gram-positive and Gram-negative bacterial strains. Compound 86d showed a MIC of 50μ M against an E. coli efflux pump-defectives train, which suggests that the efflux decreases the on-target concentrations of these compounds.

Three series of 6-substituted-aryl UBTs 87a–l, 88a–g, and 89a–d were designed and synthetized, Figure 11 [167]. The synthesis proceeded according to the two-step reaction process; 6-methoxy-2ABT reacted with CDI to give the mono-substituted intermediate. This intermediate was subsequently reacted with the respective substituted aniline to give a final 6-substituted-aryl UBTs 87a–l. To obtain compounds 87f, 87g, and 88i, the N-Boc protective group was cleaved under acidic conditions. Compound 90a was prepared from the 2(N-Me-amino)-BT with the corresponding 3-chloro, 4-methoxy-phenylisocyanate in THF at room temperature. All compounds were evaluated for in vitro 17β-HSD10 inhibitory ability. Compounds 87d and 88c showed the most promising 17β-HSD10 inhibitory activity in enzymatic assays, although the orthogonal screens indicated that 88c could be inhibiting 17β-HSD10 in an unfavorable manner. Key structure–activity relationships were established and validated with a urea linker, and a 4-phenolic moiety with a 3-halogen substitution confirmed to be essential for compound 17β-HSD10 inhibitory ability. Furthermore, a bulky 6-substitution (e.g., t-butyl) on BT appeared to be the most promising, potentially occupying the chemical space more effectively within the binding site. Positively, the most promising compounds were also shown to have an inhibitory effect at a cellular level with limited cytotoxicity, and all hit compounds displayed a more favorable kinetic mechanism of action (reversible mixed inhibition) to other previously published work. These findings provided significant structural activity insight into the 17β-HSD10 inhibitor compound design and were the most promising observations to date. With further hit optimizing and neuronal cellular evaluation to determine if these compounds are protective against Aβ-mediated cytotoxicity, this could potentially lead to a novel class of therapeutics for AD.

The 2,3-cyclization reaction of unsymmetrical 6-substituted-TBTs F with 2-bromoacetophenone in the presence of TEA afforded N-(6-substitued-1,3-benzothiazol-2-yl)-4-phenyl-1,3-thiazol-2(3H)-imine compounds 90a–d, Scheme 37 [168]. Also, 2-Me-carbamodithioate-BTs E were reacted with methyl anthranilate to give the in situ thiourea intermediate G, which was cyclized to afford 3-(benzo[d]thiazol-2-yl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one derivatives 90a–d. Compounds 90a–d were evaluated for anti-tumor activity against MCF-7, MDA-MD-231, HT-29, HeLa, Neuro-90a, K-562, and L-929 cell lines, and the results from the MTT-assay revealed the best cytotoxicity for 90b compound (IC₅₀ = 5.94 ± 1.98 μM).

The 2-dithiomethylcarboimidateBT 92, derived from 2ABT, was reacted with l-glycine, l-alanine, l-phenyl-glycine, l-phenyl-alanine, l-valine, and l-leucine to afford a series the isolable sodium salts of the SMe-iso-thiourea carboxylates 93a–f, whose hydrolysis of the SMe group and methylation of the isolable UBT-carboxylates with methyl iodide in stirring DMF as solvent affords the UBTs methyl esters 94a–f, Scheme 38 [169]. The structures of synthesized compounds were established by ¹H and ¹³C NMR, and the structures of methyl esters SMe-iso-thiourea-BTs 93a–f were derived from (l)-glycine, (l)-alanine, (l)-phenyl-glycine, and (l)-leucine by X-ray diffraction analysis.

A library of ten 6-substituted-p-methoxybenzylUBTs 95a–j was designed and synthetized, Scheme 39 [170]. Molecular modeling studies with the proposed covalent inhibitors were performed using the Maestro Schrödinger Drug Discovery Suite. The biological activities of the compounds were also tested.

The in situ generated ammonium hydrochloride salt of p-methoxy-benzyl-amine was reacted with CDI for 7 min to yield the intermediate N-(4-methoxybenzyl) carbamoyl-imidazole H in a quantitative yield, which was coupled with the corresponding 6-substituted 2ABT to form 6-substituted-p-methoxybenzylUBTs 95a–j and to improve the literature yields of 95a and 95h, which were increased from 70 and 26% to 99% and quantitative yield, respectively.

Compounds 95a–c, 95b,c, 95f,g, and 95j, were evaluated for GSK-3β inhibitory activity using a luminescent assay. The potent GSK-3β inhibitor AR-A014418 (IC₅₀ = 0.072 ± 0.043) was used as reference standard.

The inhibitory ability of its BT-equivalent, 95a (28g, Scheme 12), is known in the literature.

The HMK inhibitor 95c was more potent than the corresponding acetyl-derivative, 95b, confirming the structure–activity influence of the halomethyl-ketone moiety to increase inhibitory activity. Furthermore, the biological activities of the vinyl and ethyl ketones 95f and 95g were low, whereas the acrylamide 95j displayed insignificant inhibition.

A set of 60 substituted-aryl-UBTs 96a–zzc and the 6Cl-(3Cl,4OHphenyl)-TBT 97 was prepared from the corresponding substituted-2ABT when treated with CDI or TCDI, followed by the addition of the corresponding substituted aniline, Figure 12 [171]. The compounds were evaluated for their inhibitory ability and mechanism of action against the human 17β-HSD10. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution on the phenyl ring moiety. Among these, compounds 96zx, 96zzc exhibited IC₅₀ values of 1–2 µM and showed an uncompetitive mechanism of action with respect to aceto-acetyl-CoA. These uncompetitive UBTs inhibitors of 17β-HSD10 were considered promising compounds as potential drugs for neurodegenerative diseases.

Several Frentizole derivatives 98a–k were selected by molecular docking as potential nicotinate mononucleotide adenylyl-transferase (NadD) inhibitors for antimicrobial activity evaluation, Figure 13 [172]. Compounds 98f and 98h–k showed antimicrobial activity against Gram-positive bacteria-Staphylococcus Aureus, Methicillin-resistant Staphylococcus aureus (except for 98f and 98j), Staphylococcus epidermidis (except for 98j), and Vancomycin-resistant Enterococcus (only 98k). However, even the best obtained MICs and MBCs were substantially higher than values corresponding to standard benzalkonium bromide (BAC₁₄). Therefore, none of the tested derivatives can be considered to be a novel promising antimicrobial agent.

Chiral UBT 99a and chiral (T)UBTs 99b,c were synthesized from the reaction of 2ABT with sodium hydride NaH in DMF, followed by addition of the respective chiral-(1-isocyanatoethyl)benzene, and stirred for 12 h at room temperature, Scheme 40 [173]. All compounds were in vitro evaluated for their antimicrobial activity against B. cereus, S. aureus, E. coli, and P. aeruginosa. The results indicated all compounds were only active in Gram-positive bacteria. The UBT 99a and TBTs 99b and 99c with either R- or S-configurations or had no antibacterial properties.

As depicted in Scheme 41, forty nine combined substituted-aryl-UBTs 100a–p that were synthetized from substituted-2ABTs and substituted-phenyl-isocyanates in ketone by an easy and cheap synthetic route, Scheme 41 [174]. Synthetized compounds were studied on their antibacterial activity against Gram-positive and Gram-negative strains. Compounds 100f and 100j showed the highest antimicrobial activity against Staphylococcus aureus; they were more active than triclocarban (TCC), with MIC values of 8 µg/mL versus 16 µg/mL of TCC. Moreover, compound 100c was much more active than TCC against Enterococcus faecalis, a Gram-positive bacterium that is strongly responsible for nosocomial infections. Finally, compound 100p, even though less active than the others, exerted an interesting bactericidal action.

An iodine-catalyzed reaction of 6-substituted-2ABTs and isocyanides for the synthesis of 6-substituted-substituted UBTs 101a–i (48–60%) via a metal-free isocyanide insertion reaction was reported, Scheme 42 [175]. Introducing a simple method for the synthesis of desired UBT skeletons and the use of more acceptable iodine molecules instead of expensive transition metal catalysts are the most important advantages of this strategy.

It is important to mention that several substituted-substituted TBTs had been used since 1968 as intermediates to synthetize 2-guanidinebenzothiazoles (GBTs), for example, substituted-substituted-TBTs 102 were obtained as isolable intermediates from the reaction of substituted-2ABTs with alkyl- or aryl-thiocyanates followed by the amination-desulfurization reaction with PbO, HgCl₂, HgO, CuSO₄.5H₂O, CH₃I to afford GBTs 103, Scheme 43 [176,177,178,179,180,181,182,183,184].

Also, interesting 6-substituted-sugar TBTs 104a–c were obtained in 56–77% yields from the reaction of the respective 6-substituted-2ABT and the corresponding per-O-acetylated isothiocyanate lactose derivative in dry pyridine, which were transformed to the respective GBT derivative, Figure 14 [185].

3. Conclusions

From the literature review, benzothiazole, was found to be a bioactive and structurally simple benzofuzed heterocyclic compound that plays an important role in medicinal chemistry. It was observed that, depending on the functional group present on the BT molecule, the group plays an important role in the physicochemical properties. On the quest to discover better medicinal agents, researchers should understand the relative contributions of each functional group on the BT ring. It can become a part of the development and discovery of new drugs with potential biological activity. During the last decade, efforts have been taken to synthesize medicinally important BT derivatives, and, among them, researchers have discovered many (T)UBT derivatives showing promising biological activities.

In the present review, efforts were taken to summarize the different methodologies used for the synthesis of (T)UBT derivatives along with their biological activity. In general, 2ABTs were reacted with (alkyl-)aryl-iso(thio)cyanates, 1′-(thio)carbonyldiimidazole (T)CDI, carbamoyl chlorides, and carbon disulfide for the synthesis of (T)UBTs. It is hoped that this review will benefit budding researchers in the field of benzothiazole urea-based drug designing.

This topic is interesting, because it will provide information of utility for medicinal chemists dedicated to the design and synthesis of this class of compounds to be tested with respect to their biological activities and be proposed as new pharmacophores.