Solvent- and Catalyst-Free One-Pot Green Bound-Type Fused Bis-Heterocycles Synthesis via Groebke-Blackburn-Bienaymé Reaction/SNAr/Ring-Chain Azido-Tautomerization Strategy.

A new, efficient, green, endogenous water-triggered, solvent- and catalyst-free ultrasound-assisted one-pot Groebke-Blackburn-Bienaymé reaction/SNAr/ring-chain azido-tautomerization strategy to synthesize bound-type fused bis-heterocycles imidazo or benzo[d]imidazo[2,1-b]thiazoles and 1,5-disubstituted tetrazole (1,5-DsT) containing quinoline moiety is described, which allows synthesis of two types of fused heterocycles in one step under mild green conditions. Antibacterial and antiamebic activities of selected newly synthesized compounds were carried out against three bacterial species: Gram-positive bacterium Staphylococcus aureus ATCC 6538 and Gram-negative bacteria Pseudomonas aeruginosa ATCC 13384 and Escherichia coli O55 and against one amebic species: Entamoeba histolytica.

Introduction

Bis-heterocycles are molecules that can be formed by two linked, bound, spacer, or fused heterocyclic scaffolds. The purpose behind the combination of two heterocycles is to improve their potential applications in different areas such as chelating agents and metal ligands, complexes or metal–organic framework precursors, as well as electrical materials, and mainly in the development of bioactive molecules and drugs.[1−3] In this context, tetrazolo[1,5-a]quinolines[4−6] and imidazo[2,1-b]thiazoles[7] are important fused heterocycles that exhibit a wide variety of biological activities. For example, imidazo[2,1-b]thiazole I demonstrated to be a potent antidiabetic,[8] benzo[d]imidazo[2,1-b]thiazole II displayed a strong antifungal activity compared to standard fluconazole,[9] and compound III showed an antibacterial activity comparable to that of ampicillin (Figure ).[10]

Figure 1

Target compounds and some bis-heterocycles with biological activity.

The common methodologies to synthesize tetrazolo[1,5-a]quinolines need harsh conditions.[11,12] Recently, the synthesis of the fused tetrazolo[1,5-a]quinoline scaffold using trimethylsilyl azide, a less toxic azide source, at room temperature (RT) in quantitative yield through a SNAr/ring-chain azido-tautomerization process was reported.[13] Among the one-pot reported methods for the synthesis of imidazo[2,1-b]thiazoles and their benzo[d]fused analogues, the common method consists of the construction of an imidazole ring, which involves the condensation of 2-aminothiazole derivatives with 1,2-difunctionalized units, such as α-haloketones or α-haloesters.[14,15] However, these methodologies have disadvantages such as low to moderate yields, use of toxic chemicals, reflux conditions, and limited substrate scope.

The Groebke–Blackburn–Bienaymé reaction (GBBR)[16−18] is one of the most important class of isocyanide-based multicomponent reactions (IMCRs)[19−22] and one of the most efficient methods for the synthesis of fused imidazo heterocycles.[23] The GBBR takes place between an aldehyde, 2-aminoazine, and an isocyanide, generally in the presence of a suitable catalyst, such as Brønsted acids, Lewis acids, ionic liquids, solid-supported acids, and miscellaneous and organic bases.[24,25] Particularly, only two reports describing the synthesis of imidazo[2,1-b]thiazoles and their benzo[d]fused analogues using different catalysts, such as Yb(OTf)3[26] and H3PO4/Al2O3,[27] under solvent-free conditions are available. However, these methods have limitations such as high temperatures, the use of some expensive catalysts, and nonheterocyclic substituent at C-3 of the imidazole ring. Interestingly, there is only one report toward the synthesis of imidazo[2,1-b]thiazoles in water under catalyst-free conditions.[28]

It is highlighted that only one previously reported GBBR methodology is available toward benzo[d]imidazo[2,1-b]thiazoles 4a–e under solvent- and catalyst-free conditions, which was carried out at 160 °C in good to excellent yields (81–95%) (Scheme a).[29] However, the scope was limited, and the authors described only four examples, with less complexity of the final compounds. Moreover, the use of high temperature and limited substrate scope is the major drawbacks of this methodology. It is noteworthy to mention that the use of heterocyclic aldehydes in the GBBR for the synthesis of bound-type bis-heterocyclic imidazo[2,1-b]thiazoles analogues has been practically unexplored. In this context, the 2-chloro-3-formyl-quinoline aldehyde has been used in the GBBR under catalytic conditions such as γ-Fe2O3@HAp-Pr-NHSO3H[30] or using stoichiometric amounts of ammonium chloride.[31] However, the great disadvantage of these synthetic protocols is the limited substrate scope toward imidazothiazoles. Also, it is highlighted that in these protocols, the expensive and noncommercial catalyst or nongreen solvent, long reaction times, and high temperature were required.

Scheme 1

Closer and Previous Works Related to the Synthesis of Imidazo[2,1-b]thiazoles and Benzo[d]imidazo[2,1-b]thiazoles

As a part of our ongoing research program, in past few years, we have reported a novel one-pot synthesis based on IMCR methodologies or strategies to synthesize bis-heterocycles containing scaffolds of interest in medicinal chemistry, coordination chemistry, and in optics.[13,32−39] In 2016, we reported the one-pot synthesis of bound-type bis-heterocycles containing the imidazo[2,1-b]thiazole 8a–k and benzo[d]imidazo[2,1-b]thiazole scaffolds 10a–l σ bound with different heterocycles, under the catalyst-free microwave (MW)-assisted GBBR (Scheme b).[40] It is important to mention that this later methodology represents the first catalyst-free one-pot synthesis of bound-type bis-heterocycles in which imidazo[2,1-b]thiazoles were coupled with various heterocyclic systems such as quinoline, chromone, and julolidine, and the scope of this methodology was confirmed by the use of aldehydes with different structural and electronic nature. To the best of our knowledge, the synthesis of bound-type bis-heterocycles containing imidazo[2,1-b]thiazole or benzo[d]imidazo[2,1-b]thiazole scaffolds and their analogues coupled with complex heterocyclic substituent at the C-3 position of the imidazole ring under green conditions via solvent- and catalyst-free GBBR assisted by ultrasound irradiation (USI) has not yet been reported. On the contrary, in all previously reported multicomponent reaction (MCR) processes, to synthesize fused tetrazolo[1,5-a]quinolines analogues, the strategy report here is the first in which the tetrazole ring was formed in situ and high complexity of the substituent at the C-3 position of the quinoline ring was resulted from the functionalization of aldehyde via the GBBR under green conditions.

The one-pot synthesis of bound-type bis-heterocycles containing imidazo[2,1-b]thiazoles and their benzo[d]fused analogues via green or eco-friendly conditions involving the strategy one-pot GBBR/post-transformations is an almost practically unexplored field. Surprisingly, only one previously reported GBBR/Pictet–Spengler strategy is available toward imidazo[2,1-b]thiazole-based polyheterocycles. However, the use of noncommercial heterogeneous acid catalyst was necessary.[41]

As far as we know, the one-pot green synthesis of fused tetrazolo[1,5-a]quinoline analogues functionalized with a heterocyclic substituent at the C-3 position of the quinoline ring has not been reported neither via one-pot nor stepwise methodology.

As a part of our research program in the development of green or eco-friendly strategies,[13,42] herein, we describe the catalyst- and solvent-free one-pot green synthesis of bis-heterocycles containing two different fused heterocycles such as imidazo or benzo[d]imidazo[2,1-b]thiazoles and 1,5-DsT with quinoline moiety via the strategy one-pot GBBR/post-transformation under mild and green conditions.

The efficient and novel one-pot GBBR/post-transformation strategy is a contribution in the design and development of the eco-friendly IMCR strategies[43] toward the synthesis of bis-heterocycles (Figure ), having the following advantages such as (a) it permits functionalization at the C-3 position in both heterocycles (imidazole and quinoline), (b) increases the complexity of the previously functionalized heterocycle, (c) one-pot synthesis of two different fused heterocycles, and (d) works under mild and green conditions. Thus, their application to synthesize unsymmetrical bound-type bis-heterocycles containing fused heterocycles as imidazo[2,1-b]thiazole and 1,5-DsT with quinoline via ultrasound-assisted one-pot GBBR/SNAr/ring-chain azido-tautomerization process under mild, catalyst- and solvent-free conditions is described here. The combination of GBBR with these processes improves their synthetic potential and increases the molecular complexity of the GBB product. The latter one is resulting from the synthesis of two fused heterocycles in the one-pot process minimizing the use of reagents such as solvents and catalysts, which are commonly needed in the GBBR (Scheme c).

Figure 2

Toward the synthesis of fused bis-heterocycles via the developed one-pot strategy based on GBBR and SNAr/ring-chain azido-tautomerization post-transformations.

The USI is responsible for the cavitation effect, growth, and implosive collapse of bubbles in liquid. The USI in MCRs has emerged as a green synthetic approach because of the milder reaction conditions, shorter reaction times, and high yields.[44] Reactions in solvent-free and in high-viscosity solvents are favorable for sonication.[45]

Results and Discussion

In order to find the optimum conditions for each of the three different processes (GBBR/SNAr/ring-chain azido-tautomerization) involved in the synthetic strategy toward the synthesis of unsymmetrical bound-type bis-heterocycles 12a, 2-chloro-3-formyl-quinoline 5, 2-aminothiazole 7a, cyclohexyl isocyanide 6a, and TMSN311 were selected as the model reaction (Table ). In concordance with our main line research, green solvents and moderate temperatures were studied to optimize the reaction conditions. Initially, the one-pot process was carried out in water at RT (Table , entry 1) and under heating (Table , entry 2). However, in both cases, the starting materials remained completely unconsumed. When the reaction was carried out in ethanol at RT (Table , entry 3) and under reflux conditions (Table , entry 4), the desired product 12a was obtained in trace amount and 27% and in 43% after heating at 80 °C for 12 h under solvent-free conditions (Table , entry 5).

Table 1

Optimization of the Reaction Conditionsa

entry	conditions	temp (°C)	time (h)	yieldb (%)
1	H2O	25	24	NR
2	H2O	80	12	NR
3	EtOH	25	24	trace
4	EtOH	80	12	27
5	neat	80	12	43
6	H2O/USI	25	5	NR
7	H2O/USI	60	2	trace
8	EtOH/USI	60	2	15
9	neat/USI	25	5	trace
10	neat/USI	60	1	91

Reaction conditions: equimolar amounts of 5, 6a, 7a, and 11.

Yield of isolated product. USI (42 kHz). NR = no reaction.

The GBBR product was not achieved when the reaction was performed in water under USI at RT (Table , entry 6). The same reaction in heating under USI in water (Table , entry 7) or ethanol (Table , entry 8) gave only trace and 15% of compound 12a. Under neat USI conditions at RT, only trace amount of product 12a was formed (Table , entry 9). To our delight, a further improvement was achieved when the reaction was carried out at 60 °C under USI and solvent-free conditions for 1 h, yielding the expected compound 12a in 91% (Table , entry 10). Finally, we found the optimal conditions as follows: when 5 (1.0 equiv) reacted with 6a (1.0 equiv), 7a (1.0 equiv), and 11 (1.0 equiv) via catalyst- and solvent-free conditions at 60 °C under USI (Table , entry 10).

Having optimized reaction conditions, we next explored the scope of the method by varying 2-aminoazines and isocyanides and keeping the aldehyde component constant. As seen in Table , imidazo[2,1-b]thiazoles 12a–h were synthesized in good to excellent yields (84–98%). The best results were obtained by using 2-aminothiazole or 2-aminothiazole-5-carbonitrile with cyclohexyl-, tert-butyl- and phenethyl isocyanide 90–98% (12a–c, 12e–g). On the other hand, the benzo[d]imidazo[2,1-b]thiazoles 13a–h were synthesized in good to excellent yields (79–94%). Excellent yields were obtained 91–94% in the case of 2-aminobenzothiazole (13a–c). Slight lower yields 79–90% were obtained after using 2-amino-6-fluorobenzothiazole (13e–h). Particularly, the use of p-methoxybenzyl isocyanide produced the lowest yields 79–88% with all of the 2-aminoazines (12d, 12h, 13d, and 13h).

Table 2

Substrate Scopea

The reaction was carried out with using equimolar quantities of 5, 6, 11, and 7 or 9.

Suitable crystal of compound 12b (Figure ) was obtained, and the structure was confirmed through X-ray diffraction analysis (CCDC 1582632; see the Supporting Information for additional details).

Figure 3

ORTEP diagram of compound 12b.

A plausible mechanism is outlined in Scheme . First, the GBBR mechanism starts with the condensation of 2-aminothiazole 7a with aldehyde 5 to form imine 14, which may follow two possible pathways to give the expected GBBR product. First, the well-documented and previously suggested[18] [4 + 1] cycloaddition with isocyanide 6 (path A), and second, it may also undergo nonconcerted path via α-isocyanide addition to form nitrilium intermediate 15 followed by 5-exodig cyclization to afford precursor 16 (path B).

Scheme 2

Plausible One-Pot GBBR/SNAr/Ring-Chain Azido-Tautomerization Mechanism

Recently, we have reported the mechanistic computational studies using DFT approach for the catalyst-free GBBR,[40] where we found that only the nonconcerted pathway is allowed under catalyst-free conditions. On the basis of these previous results, we proposed that the reaction follows a nonconcerted route (path B). It highlights the important role of the acidic proton at the C-4 position of the quinoline ring and the condensed water in stabilization of imine, allowing the α-isocyanide addition to generate nitrilium intermediate 15. This later undergoes 5-exodig cyclization to give precursor 16, followed by spontaneous 1,3-hydride shift to give the GBBR product 17. To confirm this hypothesis, we carried out DFT computational studies (see the Supporting Information for additional details). Figure shows the energy profile for the GBBR mechanism, taking water-stabilized imine intermediate 14 as a starting point. Axis “y” shows the Gibbs free energy (ΔG°) in kcal·mol–1, and axis “x” shows the reaction coordinate where we found TS14→15, which leads to the water-stabilized nitrilium intermediate 15. This reaction step has an energy barrier of 20.3 kcal·mol–1. The formation of nitrilium intermediate 15 is an endergonic process (17.4 kcal·mol–1) with respect to the imine intermediate 14. Then, following the intrinsic reaction coordinate (IRC), we found TS15→16, which leads to the intermediate 16. This reaction step presents a little energy barrier of 3.6 kcal·mol–1. The formation of intermediate 16 is an exergonic process (−3.7 kcal·mol–1) with respect to the imine intermediate 14. It is noteworthy that stabilization due to the interaction of the water molecule along the reaction coordinate is maintained in the intermediates 14 to 15 and their TS, which suggests an important role to obtain low energy barriers.

Figure 4

Energy profile of the GBBR mechanism calculated using DFT at the M06-2X/6-311+G(d,p)//M06-2X/6-311G(d) level of theory.

After 1,3-H shift, the interaction with water is broken by obtaining the final product which is favored thermodynamically by −20.9 kcal·mol–1 with respect to the imine intermediate 14. Thus, a conclusion after analyzing this energy profile is that the α-addition of isocyanide to the imine intermediate 14 is the slowest and determinant step throughout the GBBR mechanism. Thus, 5-exo-dig cyclization of nitrilium 15 to intermediate 16 is the faster and spontaneous step throughout the GBBR mechanism. Besides, as the barrier to form 16 from nitrilium 15 is not very large, it may be difficult to observe this intermediate experimentally. Finally, the reaction of TMSN3 with water (formed during imine condensation) produces hydrazoic acid (HN3) and liberation of TMS-O-TMS.[42]

Next, HN3 protonates the nitrogen of quinoline GBB product 17 to form iminium ion 18 which undergoes SNAr addition by the azide anion B to generate intermediate 19. The subsequent elimination of chloride ion in 19 takes place to generate precursor 20, in which the azide group undergoes ring-chain azido-tautomerization to furnish the tetrazolo[1,5-a]quinoline σ-bound with the imidazo[2,1-b]thiazole framework 12a.

Antiamebic Activity

Amebiasis is a human intestinal infection caused by Entamoeba histolytica characterized by bloody diarrhea, which often leads to death mainly in tropical countries.[46] In turn, protozoan parasites have also become serious health problems worldwide because of their ability to resist the current drugs. In this context, the synthesis of novel compounds and evaluation for their pharmacological properties will always worth to be investigated. Previously, we reported the synthesis of chromone-tetrazoles[32] and fluorine-containing chromone-tetrazoles[33] with in vitro activity against the parasite E. histolytica. The IC50 of these compounds was in the range of 57.1–67.3 μg/mL. In the present study, we evaluated the antiamebic effect of two representative newly synthesized compounds 12f and 13g based on previous reports where similar compounds shown good antibacterial activities.[9,10] Compound 13g has no antiamebic activity at the maximum concentration tested of 320 μg/mL; meanwhile, compound 12f exhibits an IC50 = 140 μg/mL (Supporting Information). Although the IC50 against E. histolytica is relatively high, the compound seems to be specific to the protozoa because it has no antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. This specificity could be important in order to not disturb the gut microbiota because it is essential for human functions including digestion, energy metabolism, and immune pathways of the host.[47] Further work is necessary to intensify the potency of 12f compound by changing the structure of substituent groups in order to modify its bioactivity against E. histolytica and on other pathogenic protozoa such as Trichomonas vaginalis and Giardia lamblia.

Antibacterial Activity

Additionally, we screened compounds 12f and 13g against three bacterial species: Gram-positive bacterium S. aureus ATCC 6538, Gram-negative bacteria P. aeruginosa ATCC 13384 and E. coli. Unfortunately, the tested compounds were found to be inactive against the above-mentioned bacterial species.

All synthesized compounds were characterized by spectroscopic techniques such as 1H and 13C NMR, high-resolution mass spectrometry (HRMS), and IR.

Conclusions

The sonication-assisted one-pot green IMCR/post-transformation strategy developed here under solvent- and catalyst-free is one contribution toward the synthesis of bound-type fused bis-heterocycles (a) imidazole analogues containing complex substituents at C-3 of imidazole and (b) the fused 1,5-DsT with quinoline which have a fused imidazole analogue at C-3. Additionally, this strategy has advantages as all processes take place under mild and green conditions. This is the first one-pot green GBBR strategy of bound-type fused bis-heterocycles imidazole analogues under mild, sonication-assisted, solvent- and catalyst-free conditions. USI reduced the reaction time and increased the yields. This GBBR/post-transformation strategy allowed the construction of two fused heterocycles with high complexity in one-pot under green conditions in excellent yields. The generated endogenous water plays dual role in the formation of two fused heterocycles, first stabilizing imine and nitrilium intermediate to form fused imidazole and second in the formation of fused tetrazole via in situ generating hydrazoic acid. This is the first protocol in which the energy profile analysis of the intermediates involved in the GBBR mechanism shows that the acidic proton at the C-4 position of the quinoline ring and the endogenous water played a central role in the stabilization of both imine and nitrilium intermediates and favored the nonconcerted route (path B) via the 5-exo-dig cyclization, which was not certain in our previous report.[34]

The strategy herein reported have advantages over the previously reported one-pot MCR protocols toward fused tetrazolo[1,5-a]quinoline analogues that the tetrazole ring was created in situ using mild, green, and neat conditions via endogenous water-triggered formation of hydrazoic acid by single-proton exchange with TMSN3. The design and development of one-pot IMCR strategies coupled with post-transformation are not only valuable as a powerful synthetic tool to increase the complexity of the molecules which allows the construction of polyheterocycles containing diverse privileged scaffolds but also very important in the field of green chemistry under mild, solvent- and catalyst-free conditions. In addition, we have demonstrated that compound 12f is moderately antiamebic active against E. histolytica showing an IC50 = 140 μg/mL. Though the tested compounds failed to show strong antibacterial and antiamebic activities, this study could help to synthesize bioactive imidazo[2,1-b]thiazole analogues using different substituents.

Experimental Section

General Information

All reactions were carried out in a 10 mL stoppered glass tube. Reactions were monitored by silica gel TLC plates, using a mixture of hexane and ethyl acetate as eluents. Flash column chromatography was performed using silica gel (230–400 mesh) and mixtures in different proportions of hexanes with ethyl acetate as a mobile phase. Melting points were determined on a Fisher-Johns apparatus and were uncorrected. All solvents were distilled prior use. 2-Aminothiazoles were purchased from Sigma-Aldrich and used without further purification. 1H and 13C NMR spectra were acquired on Bruker ADVANCE III spectrometers (500 MHz) at 295 K in CDCl3. Chemical shifts are reported in parts per million (δ/ppm). Internal reference for 1H NMR spectra is with respect to TMS at 0.0 ppm. Internal reference for 13C NMR spectra is with respect to CDCl3 at 77.0 ppm. Coupling constants are reported in hertz (J/Hz). Multiplicities of the signals are reported using the standard abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). IR spectra were recorded on a PerkinElmer 100 FT-IR spectrometer using neat compounds, and the wavelengths are reported in reciprocal centimeters (ν/cm–1). HRMS samples were ionized by ESI+ and recorded via the TOF method. MW-assisted reactions were performed in the closed vessel mode using a monomodal CEM Discover unit. The reaction progress was monitored by TLC, and the spots were visualized under UV light (254 or 365 nm). Flash column chromatography was performed using silica gel (230–400 mesh), and a mixture of petroleum ether with AcOEt (7:3 v/v) as a mobile phase. Melting points were determined on a Fisher-Johns apparatus and were uncorrected. All starting materials were used without further purification. The solvents were distilled and dried according to standard procedures.

General Procedure for the Synthesis of Imidazo[1,2-a]thiazole-tetrazolo[1,5-a]quinolines (12a–h) and Benzo[d]imidazo[2,1-b]thiazole-tetrazolo[1,5-a]quinolines (13a–h)

In a dry 10 mL tube, a mixture of 2-chloro-3-formylquinoline 5 (1.0 equiv), 5-substituted-2-aminothiazole or 6-substituted-2-aminobenzothiazole (1.0 equiv), an isocyanide (1.0 equiv), and the subsequent addition of trimethylsylilazide was heated under USI (60 °C, 42 kHz) for 60 min. The products were purified by silica gel column chromatography (hexane/ethyl acetate) to afford the corresponding imidazo[2,1-b]thiazoles 12a–h and their benzo[d]fused analogues 13a–h. All compounds were characterized by melting points, NMR (1H, 13C), FTIR, and HRMS.

Characterization Data

N-Cyclohexyl-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazol-5-amine (12a)

Yellow solid; yield 91%; melting range = 175–176 °C; Rf = 0.35 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3475, 1569, 1549, 1296; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.60 (d, J = 6.8 Hz, 1H), 8.56 (s, 1H), 7.95 (d, J = 6.4 Hz, 1H), 7.71 (dd, J = 6.8, 6.0 Hz, 1H), 7.62 (dd, J = 6.4, 5.6 Hz, 1H), 7.35 (d, J = 3.6 Hz, 1H), 6.91 (d, J = 7.2 Hz, 1H), 6.74 (d, J = 3.6 Hz, 1H), 2.83–2.77 (m, 1H), 1.80–1.77 (m, 2H), 1.59–1.55 (m, 2H), 1.46–1.43 (m, 1H), 1.08–1.00 (m, 3H), 0.83–0.77 (m, 2H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 146.2, 145.9, 133.5, 129.9, 129.3, 129.1, 128.4, 128.2, 128.1, 125.3, 120.7, 117.8, 116.8, 112.6, 57.8, 34.0 (2C), 25.8, 25.3 (2C); HRMS (ESI+) m/z: calcd for C20H20N7S+, 390.1495; found, 390.1533.

N-(tert-Butyl)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazol-5-amine (12b)

Pale yellow solid; yield 98%; melting range = 136–137 °C; Rf = 0.24 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3282, 1670, 1532, 1284; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.62 (d, J = 6.4 Hz, 1H), 8.55 (s, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.75 (dd, J = 6.4, 6.0 Hz, 1H), 7.64 (dd, J = 6.4, 6.0 Hz, 1H), 7.50 (d, J = 3.2 Hz, 1H), 6.77 (d, J = 4.5 Hz, 1H), 6.03 (s, 1H) 0.93 (s, 9H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 146.8, 146.2, 133.7, 131.1, 130.2, 129.9, 129.5, 129.1, 128.3, 124.8, 121.1, 118.7, 116.7, 112.2, 56.0, 29.6 (3C); HRMS (ESI+) m/z: calcd for C18H18N7S+, 364.1339; found, 364.1348.

N-Phenethyl-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazol-5-amine (12c)

White solid; yield 97%; melting range = 155–157 °C; Rf = 0.44 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3250, 1598, 1561, 1282; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.64–8.54 (m, 2H), 8.02–7.99 (m, 1H), 7.75 (t, J = 6.0 Hz, 1H), 7.65 (t, J = 6.0 Hz, 1H), 7.19 (s, 1H), 7.13 (d, J = 3.2 Hz, 1H), 6.98–6.93 (m, 5H), 6.80 (d, J = 3.2 Hz, 1H), 3.36 (t, J = 5.6 Hz, 2H), 2.73 (t, J = 5.6 Hz, 2H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 145.7, 143.7, 139.2, 134.5, 130.6, 129.4, 129.2, 129.1 (2C), 129.0, 128.9, 128.5, 128.4 (2C), 127.3, 126.7, 124.8, 119.3, 116.7, 111.8, 50.6, 36.9; HRMS (ESI+) m/z: calcd for C22H18N7S+, 412.1339; found, 412.1341.

N-(4-Methoxybenzyl)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazol-5-amine (12d)

Yellow solid; yield 84%; melting range = 129–131 °C; Rf = 0.26 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3275, 1610, 1568, 1277; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.64 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.0, 7.2 Hz, 1H), 7.68 (dd, J = 8.0, 7.2 Hz, 1H), 7.38 (d, J = 4.4 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 4.05 (s, 2H), 3.41 (s, 3H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 158.7, 146.4, 145.9, 133.0, 130.7, 130.0, 129.7 (2C), 129.5, 129.2, 129.0, 128.4, 128.3, 125.0, 120.3, 117.6, 116.6, 113.3 (2C), 112.9, 55.0, 53.0; HRMS (ESI+) m/z: calcd for C22H18N70S+, 428.1288; found, 428.1277.

5-(Cyclohexylamino)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazole-2-carbonitrile (12e)

Pale yellow solid; yield 95%; melting range = 240–241 °C; Rf = 0.59 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3475, 1569, 1549, 1296; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ: 8.68 (d, J = 6.8 Hz, 1H), 8.61 (s, 1H), 8.02 (d, J = 6.4 Hz, 1H), 7.96 (s, 1H), 7.82 (dd, J = 6.4, 5.6 Hz, 1H), 7.71 (dd, J = 6.4, 5.6 Hz, 1H), 7.04 (d, J = 6.8 Hz, 1H), 2.86–2.81 (m, 1H), 1.84–1.82 (m, 2H), 1.67–1.65 (m, 2H), 1.54–1.52 (m, 1H), 1.23–1.07 (m, 5H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 145.9, 143.8, 134.0, 130.6, 130.4, 129.6, 129.4, 129.3, 128.6, 127.5, 124.9, 119.5, 116.8, 112.1, 98.4, 58.1, 34.0 (2C), 25.6, 25.2 (2C); HRMS (ESI+) m/z: calcd for C21H19N8S+, 415.1448; found, 415.1455.

5-(tert-Butylamino)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazole-2-carbonitrile (12f)

Orange solid; yield 96%; melting range = 216–218 °C; Rf = 0.55 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3311, 1685, 1552, 1312; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.70 (d, J = 6.8 Hz, 1H), 8.58 (s, 1H), 8.09 (s, 1H), 8.05 (d, J = 6.4 Hz, 1H), 7.86 (dd, J = 6.4, 5.6 Hz, 1H), 7.73 (dd, J = 6.4, 5.6 Hz, 1H), 6.12 (s, 1H), 0.97 (s, 9H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 146.0, 145.0, 135.1, 131.9, 131.1, 131.0, 129.9, 129.4, 128.6, 128.4, 124.7, 120.0, 116.9, 112.1, 98.2, 56.4, 29.7 (3C); HRMS (ESI+) m/z: calcd for C19H17N8S+, 389.1291; found, 389.1296.

5-(Phenethylamino)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazole-2-carbonitrile (12g)

Yellow solid; yield 90%; melting range = 207–209 °C; Rf = 0.53 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3237, 1577, 1560, 1293; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.67 (d, J = 8.4 Hz, 1H), 8.56 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.19–7.11 (m, 6H), 3.37–3.34 (m, 2H), 2.82 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 145.8, 143.8, 139.3, 134.6, 130.6, 129.5, 29.3, 129.2 (2C), 129.1, 129.0, 128.6, 128.5 (2C), 127.4, 126.7, 124.8, 119.4, 116.8, 111.9, 98.2, 50.6, 37.0; HRMS (ESI+) m/z: calcd for C23H17N8S+, 437.1291; found, 437.1291.

5-((4-Methoxybenzyl)amino)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazole-2-carbonitrile (12h)

Pale yellow solid; yield 88%; melting range = 178–180 °C; Rf = 0.53 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3335, 1611, 1564, 1252; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.08 (s, 1H), 8.05 (d, J = 6.4 Hz, 1H), 7.81–7.77 (m, 3H), 7.61 (t, J = 6.0 Hz, 2H), 6.90 (d, J = 6.8 Hz, 2H), 6.54 (d, J = 6.8 Hz, 1H), 3.96 (s, 2H), 3.52 (s, 3H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 159.3, 148.4, 147.4, 143.7, 140.6, 136.3, 131.1, 130.6, 130.5, 129.6 (2C), 128.5, 128.0, 127.6, 127.1, 126.9, 126.8, 114.0 (2C), 112.0, 98.5, 55.2, 52.9; HRMS (ESI+) m/z: calcd for C23H17N80S+, 453.1241; found, 453.1253.

N-Cyclohexyl-2-(tetrazolo[1,5-a]quinolin-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-amine (13a)

Yellow solid; yield 91%; melting range = 149–150 °C; Rf = 0.18 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3255, 1586, 1565, 1253; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.70 (d, J = 6.8 Hz, 1H), 8.65 (s, 1H), 8.15 (d, J = 6.4 Hz, 1H), 8.04 (d, J = 6.0 Hz, 1H), 7.82–7.79 (m, 1H), 7.72–7.69 (m, 2H), 7.49 (dd, J = 6.8, 5.6 Hz, 1H), 7.36 (dd, J = 6.8, 5.6 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 2.87–2.82 (m, 1H), 1.95–1.92 (m, 2H), 1.69 (br s, 1H), 1.62–1.58 (m, 2H), 1.50–1.47 (m, 1H), 1.11–1.04 (m, 4H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 146.2, 144.8, 135.5, 133.3, 130.4, 130.1, 129.7, 129.5, 129.1, 128.6, 128.4, 126.5, 125.2, 125.0, 124.1, 120.4, 116.8, 114.9, 58.2, 33.5 (2C), 25.8, 25.5 (2C); HRMS (ESI+) m/z: calcd for C24H22N7S+, 440.1652; found, 440.1653.

N-(tert-butyl)-2-(tetrazolo[1,5-a]quinolin-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-amine (13b)

Pale green solid; yield 94%; melting range = 285–287 °C; Rf = 0.59 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3270, 1572, 1533, 1282; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.70 (d, J = 6.8 Hz, 1H), 8.63 (s, 1H), 8.57 (d, J = 6.4 Hz, 1H), 8.06 (d, J = 6.4 Hz, 1H), 7.84 (t, J = 6.0 Hz, 1H), 7.72 (t, J = 6.0 Hz, 1H), 7.68 (d, J = 6.4 Hz, 1H), 7.46 (d, J = 6.0 Hz, 1H), 7.35 (t, J = 6.0 Hz, 1H), 0.96 (s, 9H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 146.4, 145.5, 133.8, 133.7, 133.4, 130.6, 130.4, 130.3, 129.8, 129.4, 128.5, 125.9, 125.1, 124.9, 124.1, 120.9, 116.9, 116.1, 57.0, 29.3 (3C); HRMS (ESI+) m/z: calcd for C22H20N7S+, 414.1495; found, 414.1503.

N-phenethyl-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazol-5-amine (13c)

Yellow solid; yield 91%; melting range = 212–213 °C; Rf = 0.47 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3243, 1566, 1550, 1259; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.59 (d, J = 8.0 Hz, 1H), 8.53 (s, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.29–7.22 (m, 2H), 7.04–6.97 (m, 5H), 6.86 (br s, 1H), 3.30–3.25 (m, 2H), 2.74 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 146.0, 144.8, 139.0, 136.3, 132.9, 130.3, 130.1, 129.4, 129.1, 128.9 (2C), 128.7, 128.5, 128.4, 128.3 (2C), 126.5, 126.4, 125.1, 125.0, 124.0, 120.1, 116.7, 114.8, 51.3, 36.3; HRMS (ESI+) m/z: calcd for C26H20N7S+, 462.1495; found, 462.1507.

N-(4-Methoxybenzyl)-6-(tetrazolo[1,5-a]quinolin-4-yl)imidazo[2,1-b]thiazol-5-amine (13d)

Yellow solid; yield 81%; melting range = 181–182 °C; Rf = 0.46 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3264, 1569, 1544, 1242; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.63 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.25 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.68 (7, J = 7.6 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 6.24 (d, J = 8.4 Hz, 2H), 4.02 (br s, 2H), 3.21 (s, 3H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 158.5, 145.8, 145.2, 134.6, 133.1, 131.2, 130.4, 130.0, 129.9 (2C), 129.7, 129.2, 129.0, 128.7, 128.3, 126.7, 125.2, 124.9, 124.1, 119.9, 116.6, 114.8, 112.9 (2C), 54.8, 53.5; HRMS (ESI+) m/z: calcd for C26H20N70S+, 478.1445; found, 478.1456.

N-Cyclohexyl-7-fluoro-2-(tetrazolo[1,5-a]quinolin-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-amine (13e)

Yellow solid; yield 85%; melting range = 281–282 °C; Rf = 0.54 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3253, 1570, 1549, 1294; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.47 (s, 1H), 8.15 (dd, J = 7.2, 4.0 Hz, 1H), 8.07 (d, J = 6.8 Hz, 1H), 7.89 (d, J = 6.4 Hz, 1H), 7.77 (ddd, J = 6.8, 5.6, 1.2 Hz, 1H), 7.60 (ddd, J = 6.8, 5.6, 1.2 Hz, 1H), 7.42 (dd, J = 6.4, 2.0 Hz, 1H), 7.19 (ddd, J = 7.2, 6.8, 2.0 Hz, 1H), 3.41–3.39 (m, 1H), 2.75 (br s, 1H), 1.80–1.77 (m, 2H), 1.58–1.55 (m, 2H), 1.46–1.44 (m, 1H), 1.08–1.01 (m, 4H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 160.8, 158.9, 149.0, 147.4, 143.6, 140.6, 134.4, 131.9, 131.0 (d, J = 207.9 Hz), 130.8, 128.6, 128.4, 128.0, 127.6, 127.5, 115.2 (d, J = 7.0 Hz), 113.8 (d, J = 19.2 Hz), 111.2 (d, J = 21.6 Hz), 57.5, 33.8 (2C), 25.8, 24.8 (2C); HRMS (ESI+) m/z: calcd for C24H21N7SF+, 458.1558; found, 458.1560.

N-(tert-Butyl)-7-fluoro-2-(tetrazolo[1,5-a]quinolin-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-amine (13f)

Yellow solid; yield 90%; melting range = 284–286 °C; Rf = 0.58 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3250, 1574, 1535, 1281; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.71 (d, J = 6.8 Hz, 1H), 8.60 (s, 1H), 8.54 (dd, J = 7.2, 3.6 Hz, 1H), 8.07 (d, J = 6.4 Hz, 1H), 7.85 (ddd, J = 6.4, 5.6, 0.8 Hz, 1H), 7.74 (ddd, J = 6.4, 5.6, 0.8 Hz, 1H), 7.40 (dd, J = 6.4, 2.0 Hz, 1H), 7.19 (ddd, J = 7.2, 6.8, 2.0 Hz, 1H), 5.62 (s, 1H), 0.96 (s, 9H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 160.8, 158.9, 146.4, 145.1, 133.8, 133.7, 132.8 (d, J = 204.9 Hz), 130.6, 130.1, 129.8, 129.3, 128.5, 124.9, 121.1, 116.9, 116.7 (d, J = 6.9 Hz), 113.5 (d, J = 19.5 Hz), 111.1 (d, J = 21.6 Hz), 55.0, 29.3 (3C); HRMS (ESI+) m/z: calcd for C22H19N7SF+, 432.1401; found, 432.1405.

7-Fluoro-N-phenethyl-2-(tetrazolo[1,5-a]quinolin-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-amine (13g)

Pale yellow solid; yield 88%; melting range = 231–232 °C; Rf = 0.40 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3264, 1573, 1533, 1283; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.59 (d, J = 7.6 Hz, 1H), 8.52 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.65–7.58 (m, 2H), 7.29 (dd, J = 8.0, 2.0 Hz, 1H), 7.08–6.99 (m, 5H), 6.94 (d, J = 8.8, 2.4 Hz, 1H), 6.81 (d, J = 6.8 Hz, 1H), 3.23 (q, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3; 25 °C; TMS): δ 161.0, 158.6, 146.0, 144.4, 139.1, 136.2, 131.7 (d, J = 10.0 Hz), 130.2, 129.4, 129.1, 129.0 (2C), 128.8, 128.5, 128.4, 128.3 (2C), 126.5, 125.0, 120.0, 116.7, 115.6 (d, J = 8.8 Hz), 114.0 (d, J = 23.9 Hz), 111.1 (d, J = 27.2 Hz), 51.4, 36.4; HRMS (ESI+) m/z: calcd for C26H19N7SF+, 480.1401; found, 480.1409.

7-Fluoro-N-(4-methoxybenzyl)-2-(tetrazolo[1,5-a]quinolin-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-amine (13h)

Yellow solid; yield 79%; melting range = 248–249 °C; Rf = 0.38 (hexane–AcOEt = 7/3 v/v); FT-IR (ATR) νmax/cm–1: 3270, 1572, 1536, 1298; 1H NMR (400 MHz; CDCl3; 25 °C; TMS): δ 8.63 (d, J = 8.4 Hz, 1H), 8.30 (dd, J = 8.8, 4.4 Hz, 1H), 8.24 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.45 (dd, J = 8.0, 2.4 Hz, 1H), 7.24 (td, J = 8.8, 2.4 Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H), 6.57 (br s, 1H), 6.24 (d, J = 8.4 Hz, 2H), 3.99 (br s, 2H), 3.21 (s, 3H); 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ 158.4, 145.7, 145.2, 134.6, 133.1, 131.2, 130.3, 129.9, 129.8 (2C), 129.6, 129.1, 128.9, 128.6, 128.2, 126.6, 125.1, 124.8, 124.1, 119.9, 116.5, 114.8, 112.9 (2C), 54.7, 53.4; HRMS (ESI+) m/z: calcd for C26H19N7OSF, 496.1350; found, 496.1366.

Antiamebic Assay

The virulent strain of E. histolytica (HM-1-IMSS) was grown axenically at 37 °C in the TYI-S-33 medium supplemented with 10% heat-inactivated bovine serum. Trophozoites at the exponential phase of growth were used in all experiments. The antiamebic activity of the synthesized imidazo[2,1-b]thiazole analogues 12f and 13g dissolved in dimethyl sulfoxide (DMSO) was assessed using the standard dilution micromethod and subculture.[33] Trophozoites (2 × 104) in 100 mL of TYI-S-33 were introduced in each well of the 96-well microtiter plate (Nunc Thermo Scientific) and allow the parasites to adhere to the bottom of the well at 37 °C for 2 h; then, 100 mL of different concentrations (10–320 μg/mL) of each compound tested in TYI-S-33 was added and incubated at 37 °C for 24 h. The trophozoites were detached by chilling at 4 °C for 10 min and transferred to new culture tubes with fresh medium without antibiotic and incubated for 48 h at 37 °C. The final number of parasites was determined with a hemocytometer, and the percentages of growth inhibition were calculated by comparison with the control culture. Each test included metronidazole (U.S.P. Poulenc, Lt. Montreal) as a standard amebicidal drug and a control containing DMSO. The concentration of DMSO did not exceed 1.5% in all assays performed. Each assay was performed in triplicated and repeated two times.

Antibacterial Assay

The antibacterial activity of the synthesized compounds was assessed by employing a standard dilution method. The assay was carried out with several bacterial species, the Gram-positive bacterium S. aureus ATCC 6538, and the Gram-negative bacteria P. aeruginosa ATCC 13384 and E. coli O55. The inoculum was an overnight culture of each bacterial species in the LB broth diluted in the same media to a final concentration of 100 CFU/mL. The new synthesized compounds were dissolved in DMSO to a concentration of 20 mg/mL. Further dilutions were performed in the LB broth containing each bacteria species to reach a final concentration range of 10–320 μg/mL. The bacterial growth was detected by optical density determination at 600 nm (Spectrophotometer GeneQuant-pro Amersham). The antibiotic cefotaxime (Sigma) was used as control bactericidal tests of the reference strains.