Green Acid Mediated Synthesis of Imidazo[1,2‐a]Pyrimidine–Thiazole/1,3,4‐Thiadiazole Hybrids: Design, Characterization and In‐Vitro Biological Evaluation

ABSTRACT

This work reports the design and development of two series of imine‐linked imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole derivatives using a molecular hybridization approach as anti‐tubercular agents. A total of thirty‐seven new hybrid compounds (B1‐B37) were synthesized and characterized. One final molecule, B20, was structurally confirmed by single‐crystal x‐ray diffraction analysis. All the final compounds (B1‐B37) were tested for activity against the Mycobacterium tuberculosis H37Rv strain. Compounds B5, B26, and B30 exhibited significant inhibitory effects, with MICs of 12.5, 12.5, and 6.25 µg/mL, respectively. All novel final compounds also displayed effective antibacterial action against four bacterial strains S. aureus, E. coli, B. subtilis, and P. aeruginosa. Toxicity tests on mouse fibroblast (L929) cell lines revealed that the highly active anti‐TB compounds (B5, B26, and B30) were nontoxic. Furthermore, the outcomes of the in silico ADME evaluation, DFT analysis, molecular docking studies (targeting CYP121 and InhA), and dynamic simulation demonstrated the potent compounds' great potential as prospective scaffolds for the development of novel antitubercular drugs.

A new set of imidazo[1,2‐a]pyrimidine‐thiazole/thiadiazole hybrids were developed through the molecular hybridization concept. The thiazole and thiadiazole derivatives were synthesized using water as a solvent, and the final compounds were synthesized using citric acid as an eco‐friendly catalyst. One of the compounds exhibited potent anti‐TB activity with an MIC of 6.25 µg/mL.

1. Introduction

The cause of tuberculosis (TB) is the bacterium M. tuberculosis, which is disseminated through the air by coughing or bacterial expulsion from infected people [1]. In 2024, WHO report estimates that 10.8 million individuals globally were affected by TB. About 1.25 million people died from the infection, including 0.161 million people with HIV and 1.09 million people without HIV [2]. Mycobacteria multiply rapidly in weeks, moving through the lymph and blood to infect various organs and cells of the body [3]. If exposed to a patient with drug‐resistant TB in an overcrowded hospital region with poor ventilation, up to 50% of the patients could become infected within a day [4]. H37Rv and H37Ra are frequently used strains in TB research, with H37Rv being virulent and H37Ra being avirulent. These strains are commonly employed as standard strains for testing new anti‐TB drugs [5]. The cell wall of M. tuberculosis is rich in complex lipids, particularly mycolic acids, which make up more than half of its envelope. Their hydrophobic nature prevents the entry of many substances, including drugs [6]. In the H37Rv bacterial strain, the genome InhA is a trans‐enoyl‐acyl carrier protein (ACP) reductase crucial for synthesizing mycolic acid. InhA uses NADH to reduce 2‐trans‐enoyl‐ACP, resulting in NAD+ and a reduced enoyl thioester‐ACP, which is essential for mycolic acid production [7, 8]. The TB epidemic has worsened in recent years due to a lack of new treatment options; most present medicines are several decades old, and the growth of multidrug‐resistant (MDR‐TB) and extensively drug‐resistant (XDR‐TB) strains. MDR‐TB is susceptible to the two important first‐line TB medicine, isoniazid and rifampicin. It has less effective cure rates, often between 50%–70%. XDR‐TB is resistant to isoniazid, rifampicin, fluoroquinolones, and second‐line injectable TB drugs. Detection takes several weeks, resulting in a low rate of success (30%–50%), specifically in HIV patients [9]. The current standard drug therapy for MDR‐TB and XDR‐TB is prolonged and ineffective in many cases. To enhance existing treatments, newly developed anti‐TB medications should have a few crucial features. New anti‐TB medications aim to decrease treatment duration and need less frequent dosage, while also demonstrating high bactericidal action against both active and latent bacteria. They must be effective against MDR and XDR strains, particularly from new chemical classes with novel mechanisms of action. Furthermore, they should be safer, well‐tolerated, appropriate for pregnant women, children, and HIV‐1 patients, and the cost should be low.

In our previous work, we reported the molecular hybridization of the imidazo[1,2‐a]pyrimidine scaffold with thiazole through a hydrazinyl linker. In contrast, in the present study, we introduced an imine (C═N) linkage between the imidazo[1,2‐a]pyrimidine and thiazole/thiadiazole moieties. Furthermore, in our previous work, different functional groups were primarily incorporated onto the imidazo[1,2‐a]pyrimidine ring. In the present study, however, we systematically introduced diverse functionalities into both the imidazo[1,2‐a]pyrimidine and thiazole/thiadiazole moieties. This structural modification and expanded functional‐group diversification were designed to enhance the molecular properties and, consequently, improve the biological potency of the synthesized compounds against the bacterial strain [10].

Pyrimidine accounts for approximately 16% of the top six‐membered nitrogen rings in US FDA‐approved medications. Thiazole is commonly found in five‐membered rings, particularly in β‐lactam antibiotics, which account for 67% of all thiazole‐based medicines [11]. The mesoionic property of the 1,3,4‐thiadiazole ring enables these heterocycles to pass through cell membranes and interact with biological targets, resulting in high oral absorption and bioavailability [12]. Currently, three pyrimidine‐based drugs TBA‐7371, GSK 286, and SPR720 are in clinical trials. GSK 286 (Phase I) targets mycobacterial cholesterol metabolism (I). TBA‐7371 inhibits DprE1 and kills TB bacteria (MBC 0.78–1.56 µM) in animal lungs (II). SPR720 (Phase II) hits DNA gyrase (GyrB) (III). US FDA states that SPR720 is a Qualified Infectious Disease Product (QIDP) for treating lung infections caused by nontuberculous mycobacteria (NTM) and M. tuberculosis (M. tb) (Figure 1) [13, 14].

FIGURE 1.

Pyrimidine‐derived anti‐TB molecules that are currently under clinical testing and pyrimidine, Thiazole/thiadiazole derived anti‐TB molecules.

Recent research results reveal that compounds with imidazole[1,2‐a]pyrimidine – thiazole/1,3,4‐thiadiazol‐2‐amine frameworks have potent antitubercular activity. These structural classes have demonstrated promising effects in suppressing M. tuberculosis in current investigations. Deohate reported pyrimidine‐thidiazole compounds and tested their anti‐TB efficacy. The compounds containing 3‐chloro and 4‐chloro phenyl substituents had the maximum potency, with MIC values of 6.25 µM (IV) [15]. Reddyrajula and Dalimba reported molecules of imidazol[1,2‐a]pyrimidine‐triazole hybrids. The molecules containing methyl, methoxy, nitro, and cyano groups showed high activity, with a MIC value of 1.56 µg/mL (V) [16]. Panchani synthesized benzo[4,5]imidazo[1,2‐a]pyrimidine derivatives and tested their anti‐TB efficacy. Compounds having aromatic rings containing electronegative groups such as 4‐chloro, 4‐bromo, and 4‐fluoro showed high activity against both latent and active M. tuberculosis H37Rv strain [17]. Shaikh et al. reported the design of thiazole‐ and 1,3,4‐thiadiazole‐linked target molecules and evaluated their in vitro antitubercular activity against the H37Ra strain. Among them, ethyl 2‐(2‐((5‐amino‐1,3,4‐thiadiazol‐2‐yl)thio)acetamido)‐5‐methylthiazole‐4‐carboxylate exhibited substantial activity, with an MIC value of 7.12 µg/mL (VI) [18]. Patel et al. reported molecules of 1,3,4‐thiadiazole derivatives, one of which, N‐(5‐(4‐nitrophenyl)‐1,3,4‐thiadiazol‐2‐yl)benzanide, showed significant inhibitory activity against both the H37Rv and MDR‐TB strains, with a MIC of 9.87 µM (VII) [19]. Recent studies have reported antitubercular compounds containing pyrimidine, thiazole, and thiadiazole scaffolds, with their activities summarized in Table 1.

TABLE 1.

Summary of Recent Antitubercular Compounds.

Years	References	Chemical scaffold	Representative compound	M. tuberculosis strain	MIC (µg/mL)/(µM)
2023	Pflégr et al. [20].	Pyrimidine‐1,3,4‐oxadiazole	4l	M. tuberculosis H37Rv	2µM
2023	Hemeda et al. [21]	Pyrimidine‐tethered benzothiazole	9c	M. tuberculosis ATCC 25177	0.24 µg/mL
2024	Shehta and Alsaiari n.d [22].	Phthalimide‐pyrimidine	6f	M. tuberculosis H37Ra,25177	0.48 µg/mL
2024	Raghu et al. [23]	Pyrimidine‐linked thiazolidinedione	5a	M. tuberculosis H37Rv	0.22 µM
2024	Shaikh et al. [24].	Thiadiazole‐thiazole	3	M. tuberculosis H37Ra.	3.90 µg/mL
2025	Talamadla et al. [25]	2‐(1H‐Pyrazol‐1‐yl) Pyrimidine	F‐2, F‐5 and F‐9	M. tuberculosis H37Rv strain.	6.25 µg/mL
2025	Panigrahi and Sahu [26]	1,3,4‐Thiadiazole	5b, and 6b	M. tuberculosis H37RV, strain	4 µg/mL
2026	Kumar et al. [27].	Thiazole‐acetamide	5c	M. tuberculosis H37Ra	16 µg/mL

We synthesized a novel group of hybrid compounds (B1‐B37) by combining imidazo[1,2‐a]pyrimidine cores with thiazole/1,3,4‐thiadiazol‐2‐amine molecules in a single molecular framework. This method was stimulated by literature findings that highlighted the significant antitubercular properties of imidazo[1,2‐a]pyrimidine and thiazole/thiadiazol‐based derivatives. These two distinctive pharmacophores are successfully combined via an imine linkage to generate a unified hybrid scaffold. Our synthetic strategy is founded on the principles of green chemistry. Notably, the essential thiazole and thiadiazole intermediates are effectively synthesized using water as an environmentally friendly solvent. Furthermore, the final hybrid molecules were synthesized through a Schiff base reaction that utilizes citric acid, a natural, biodegradable organic acid, as a green catalyst, making the entire process sustainable and environmentally conscientious. Furthermore, inserting various substituents at critical functional positions, represented by R¹, R², and R³ (Scheme 2), allows for structural optimization, which not only increases compound diversification but also enhances the compounds' biological function.

SCHEME 2.

Synthesis of imine‐linked imidazole[1,2‐a]pyrimidine—thiazole/1,3,4‐thiadiazol derivatives: Reagents and conditions: (v) Citric acid, molecular sieves, absolute ethanol, 24‐30 h, reflux.

2. Results and Discussion

2.1. Chemistry

The synthetic approach for imine‐mediated imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazol hybrids (B1‐B37) is demonstrated in Schemes 1 and 2. First step entails, commercially available 2‐aminopyrimidine (1) was reacted with appropriate substituted phenacyl bromides 2(a‐f), resulting in the production of substituted imidazo[1,2‐a]pyrimidine derivatives 3(a‐f) by a cyclization process. In the second step, compounds 3(a‐f) underwent a Vilsmeier‐Haack formylation reaction with POCl₃ and DMF, resulting in the formation of imidazo[1,2‐a]pyrimidine‐carbaldehyde derivatives 4(a‐f) [10]. Substituted phenacyl bromides 5(a‐e) were reacted with thiourea (6) in water as a solvent, yielding the desired 2‐aryl‐thiazol‐4‐amine derivatives 7(a‐e). The intermediate 2‐amino‐1,3,4‐thiadiazole 10(a‐c) was produced by reacting a substituted phenyl carboxylic acid 8(a‐c) with thiosemicarbazide (9) in the presence of POCl₃ and then treating with water. The final compounds (B1‐B37) were produced via a Schiff base reaction, which involves reacting appropriate imidazole[1,2‐a]pyrimidine with 2‐aryl‐thiazol‐4‐amine 10(a‐e) or 5‐aryl‐1,3,4‐thiadiazol‐2‐amine 7(a‐c) in the presence of an acid and absolute ethanol. The intermediates and final compounds' (B1‐B37) structures were confirmed by ¹H NMR, ¹³C NMR, and mass spectrometry (Figures S1–S147). The ¹H NMR spectrum of molecule (3) showed a singlet peak at ∼ δ 8.6 ppm, indicating the successful formation of the imidazo[1,2‐a] ring (‐─H) by cyclization. Compound 4(a‐f) ¹H NMR spectrum showed a singlet peak at ∼ δ 10.09 ppm, indicating the formation of the aldehyde (─CHO) functionality. The synthesis of compounds 7(a‐e) was verified by a broad singlet peak at ∼ δ 7.0 ppm, attributable to two protons of the NH₂ group, and a singlet for a proton at ∼ δ 6.8 ppm, showing the successful formation of the thiadiazole ring (─CH). The formation of compounds 10(a‐c) was confirmed by a broad singlet at ∼ δ 7.4 ppm in the ¹H NMR spectrum, confirming the presence of NH₂ in the 1,3,4‐thiadiazol ring. The ¹H NMR spectra of final compounds B1‐B25 show a singlet peak at ∼ δ 9.4 ppm, suggesting the presence of the imine (HC═N) linkage, and a singlet peak with one proton at ∼ δ 7.3 ppm, confirming the existence of the thiazole ring (─CH). In addition, all further aromatic proton signals are consistent with the expected structure of the molecules. For the targeted compounds B26‐B27, a singlet at ∼ δ 9.2 ppm in the ¹H NMR spectra confirms the successful formation of the imine (HC═N) linkage between the imidazo[1,2‐a]pyrimidine and 1,3,4‐thiadiazol moieties. Furthermore, all additional aromatic proton signals correlate with the expected molecular structures. The ¹³C NMR spectra of the final compounds B1‐B37 show a particular peak around ∼ δ 138 ppm, referred to as the imine carbon (C─N), and another signal at ∼ δ 172 ppm, corresponding to the carbon connected with two nitrogen atoms and one sulfur atom. This confirms the presence of a thiazole/1,3,4‐thiadiazole ring in the structure. Single crystals of the substance B20 were grown by dissolving it in DCM/PET and allowing the solvent to gently evaporate at room temperature. After 8‐10 days, crystals measuring 0.4 × 0.20 × 0.15 mm³ were produced. The compound structure of B20 was clearly confirmed by single‐crystal x‐ray diffraction (SC‐XRD) research, as shown in Figure 2 and Table 2.

SCHEME 1.

Synthesis of intermediates: Reagents and conditions: (i) Acetone, reflux, 12 h (ii) POCl₃, DMF, 100°C, 2 h (iii) H₂O, RT, 6 h (iv) POCl₃, H₂O, reflux, 4 h.

FIGURE 2.

ORTEP figure displaying the compound's B20 X‐ray crystal structure.

TABLE 2.

Crystal Data and Structure Refinement Parameters for B20.

Identification code	B20	ρcalcg/cm3	1.444
Empirical formula	C22H14ClN5S	μ/mm−1	2.940
Formula weight	415.89	F (000)	856.0
Temperature/K	298.74(10)	Crystal size/mm3	0.38 × 0.32 × 0.28
Crystal system	monoclinic	Radiation	Cu Kα (λ = 1.54184)
Space group	P21/n	2Θ range for data collection/°	7.808 to 148.414
a/Å	12.19895(19)	Index ranges	−14 ≤ h ≤ 14, ‐9 ≤ k ≤ 6, ‐26 ≤ l ≤ 26
b/Å	7.49731(10)	Reflections collected	18598
c/Å	21.2612(3)	Independent reflections	3744 [Rint = 0.0462, Rsigma = 0.0315]
α/°	90	Data/restraints/parameters	3744/0/262
β/°	100.3215(14)	Goodness‐of‐fit on F2	1.037
γ/°	90	Final R indexes [I≥2σ (I)]	R1 = 0.0411, wR2 = 0.1157
Volume/Å3	1913.07(5)	Final R indexes [all data]	R1 = 0.0460, wR2 = 0.1218
Z	4	Largest diff. peak/hole / e Å−3	0.24/‐0.26
CCDC Deposition Number: 2518115

2.2. Biological Studies

2.2.1. Anti‐Tubercular Activity Studies

We developed two novel verities of imine‐bonded imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole hybrids (B1‐B37) and assessed their antitubercular efficacy against the M. tuberculosis H37Rv strain (ATCC 27294). Figures 3 and 4 indicate the MIC values (in µg/mL) of compounds B1‐B37, together with the standard antitubercular medications for comparison. The studied molecules had MIC values ranging from 6.25 to 50 µg/mL (Table 2). Compound diversification was carried out by inserting different substituents (─CH₃, ─OCH₃, ─Cl, ─Br) on the phenyl rings attached to imidazo[1,2‐a]pyrimidine (R¹) and thiazole/thiadiazole (R²/R³) rings. It was observed that these structural variations resulted only a moderate change in the MIC values of the derivatives. Three compounds, B5, B26, and B30 showed significant activity with MIC in the rage 6.25 ‐ 12.5 µg/mL. Among these, compound B30 had the highest activity, with a MIC of 6.25 µg/mL. It is evident from Table 3 that a set of 25 compounds displayed promising activity with MIC in the range of 6.25–25 µg/mL whereas remaining 9 compounds showed moderate activity with MIC of 50 µg/mL.

FIGURE 3.

Antitubercular activity of compounds B1–B25 against M. tuberculosis H37Rv strain.

FIGURE 4.

Antitubercular activity of compounds (B26‐B37) against M. tuberculosis H37Rv (PZA: Pyrazinamide, and STM: Streptomycin).

TABLE 3.

The target compounds' MIC values (B1‐B37).

Compound code	R1	R2(B1‐B25)/ R3(B26‐B37)	Mol Wt	MIC µg/mL	MIC µM
B1	CH3	CH3	409.1361	25	61.10
B2	CH3	OCH3	425.1310	25	58.80
B3	CH3	F	413.1110	25	60.51
B4	CH3	Cl	429.0815	25	58.26
B5	CH3	H	395.1205	12.5	31.63
B6	OCH3	CH3	425.1310	25	58.80
B7	OCH3	OCH3	441.1259	25	56.67
B8	OCH3	F	429.1060	50	116.52
B9	OCH3	Cl	445.0764	50	112.34
B10	OCH3	H	411.1154	25	60.81
B11	F	CH3	413.1110	25	60.51
B12	F	OCH3	429.1060	25	58.26
B13	F	F	417.0860	50	119.87
B14	F	Cl	433.0564	50	115.45
B15	F	H	399.0954	50	125.28
B16	Cl	CH3	429.0815	25	58.26
B17	Cl	OCH3	445.0764	25	56.17
B18	Cl	F	433.0564	25	57.72
B19	Cl	Cl	449.0269	25	55.67
B20	Cl	H	415.0658	25	60.23
B21	H	CH3	395.1205	25	63.27
B22	H	OCH3	411.1154	25	60.81
B23	H	F	399.0954	25	62.64
B24	H	Cl	415.0658	50	120.46
B25	H	Ph	381.1048	50	131.19
B26	OCH3	CH3	426.1263	12.5	29.33
B27	CH3	CH3	410.1314	25	60.95
B28	Cl	CH3	430.0767	50	116.25
B29	Br	CH3	474.0262	50	105.47
B30	OCH3	F	430.1012	6.25	14.01
B31	CH3	F	414.4624	25	58.12
B32	Cl	F	434.0517	25	55.55
B33	Br	F	478.0012	25	50.61
B34	OCH3	H	412.1106	25	60.66
B35	CH3	H	396.1157	25	63.11
B36	Cl	H	416.0611	25	60.08
B37	Br	H	460.0106	25	54.34

While evaluating the activity profile of first set of compounds (B1‐B25), which include differently substituted thiazole hybrids, compound B5 with a ─CH₃ group at R¹ showed the most promising activity with a MIC value of 12.5 µg/mL. Replacing the ─CH₃ group at R¹ with other substituents (OCH₃, F, Cl, and H) did not enhance the inhibition activity. In fact, the activity is reduced in some of the derivatives as compared with their analogs with a ─CH₃ group at R¹. Among different substituents at R² (CH₃, OCH₃, F, Cl, and H), compounds with no substituents (i.e., R² = H) showed better or similar activity (except B25) as compared to other molecules of the individual series with a fixed substituent at R¹. So, among the thiazole derivatives (B1‐B25), a ─CH₃ group at R¹ with no substituent on the phenyl group attached to the thiazole ring (i.e., R² = H) are the favorable structural features to enhance the inhibition activity. Among the thiadiazole derivatives (B26‐B37), compounds with a—OCH₃ substituent at R¹ (B26 and B30) showed better activity. The favorable substituents at R³ are a ─CH₃ group (B26) or a ─F group (B30).

According to the SAR analysis, B5 contains a p‐methyl group that increases lipophilicity and may enhance hydrophobic interactions, while its phenyl‐thiazole moiety provides an aromatic surface for favorable interactions. In B26, the electron‐donating methoxy group provides an additional hydrogen‐bond acceptor, while the 1,3,4‐thiadiazole ring offers additional N and S atoms and modifies the electronic properties. Whereas, B30 and B26 structurally differ at the R³ substituent group. The methoxy group in B26 is replaced with a fluorine atom in B30. Fluorine can alter lipophilicity and molecular polarity without substantially increasing molecular size. This change improved the activity two‐fold, with B30 showing an MIC of 6.25 µg/mL compared with 12.5 µg/mL for B26, suggesting that fluorine substitution may provide a favorable electronic, steric, and lipophilic balance.

2.2.2. Antibacterial Activity Studies

The antibacterial activity of all target compounds (B1–B37) against four bacterial strains S. aureus, B. subtilis, P. aeruginosa, and E. coli was evaluated at a concentration of 100 µg/mL. The diffusion method was used to evaluate the antibacterial activity study, and the zones of inhibition were estimated used to millimeters (mm). Ciprofloxacin was the standard reference drug. Table 4 summarizes the outcomes, including the inhibition zone results for the control and all final molecules. A most of the studied compounds demonstrated significant antibacterial activity. Specifically, in the S. aureus strain, compounds B22, B26, and B36 showed good effectiveness, exhibiting zones of inhibition of 13 ± 0.18, 13 ± 0.12, and 18 ± 0.27 mm, respectively. The inhibition zone values of the remaining compounds ranged from 8 to 12 mm, indicating moderate activity. For B. subtilis strain, compounds B20 and B22 showed good inhibition with zone of inhibition value 18 ± 0.18 mm and 13 ± 0.22 mm, respectively. Remaining compounds showed inhibition zone values ranging from 8 to 12 mm. Compound B30 showed a significant activity in the study against E. coli, with a zone of inhibition of 21 ± 0.15 mm. The next set of most active compounds, B16, B22, and B34, displayed inhibition zones of about 13 mm. The inhibition zone values of the remaining compounds ranged from 7 to 12 mm, indicating moderate activity. A majority of the compounds showed zones of inhibition of 8‐9 mm against the P. aeruginosa strain, indicating that the nature of the substituents had an insignificant impact.

TABLE 4.

Antibacterial activity of compounds B1–B37 (zone of inhibition, mm).

Compounds code	Zone of inhibition in mm
	Gram‐positive bacteria		Gram‐negative bacteria
	S. aureus (ATCC 23235)	B. subtilis (ATCC 6051)	E. coli (ATCC 25922)	P. aeruginosa (ATCC 27853)
B1	8 ± 0.29	8 ± 0.31	7 ± 0.22	8 ± 0.12
B2	8 ± 0.32	9 ± 0.07	8 ± 0.18	9 ± 0.08
B3	10 ± 0.14	10 ± 0.11	8 ± 0.22	8 ± 0.22
B4	11 ± 0.18	11 ± 0.17	8 ± 0.08	9 ± 0.21
B5	9 ± 0.09	8 ± 0.19	12 ± 0.09	8 ± 0.07
B6	8 ± 0.22	9 ± 0.22	8 ± 0.14	8 ± 0.08
B7	11 ± 0.31	8 ± 0.06	8 ± 0.22	8 ± 0.10
B8	10 ± 0.11	8 ± 0.13	8 ± 0.28	8 ± 0.11
B9	11 ± 0.15	11 ± 0.15	8 ± 0.31	9 ± 0.15
B10	12 ± 0.17	12 ± 0.17	10 ± 0.17	8 ± 0.17
B11	8 ± 0.19	8 ± 0.22	8 ± 0.19	9 ± 0.14
B12	9 ± 0.21	10 ± 0.29	11 ± 0.06	8 ± 0.12
B13	10 ± 0.11	8 ± 0.33	8 ± 0.28	8 ± 0.21
B14	8 ± 0.13	8 ± 0.35	8 ± 0.30	9 ± 0.20
B15	8 ± 0.14	9 ± 0.22	8 ± 0.11	8 ± 0.18
B16	8 ± 0.18	8 ± 0.28	13 ± 0.21	8 ± 0.11
B17	9 ± 0.21	10 ± 0.22	11 ± 0.31	9 ± 0.22
B18	11 ± 0.26	10 ± 0.11	8 ± 0.18	9 ± 0.23
B19	9 ± 0.28	11 ± 0.16	9 ± 0.27	8 ± 0.05
B20	11 ± 0.24	18 ± 0.18	11 ± 0.25	8 ± 0.09
B21	12 ± 0.14	12 ± 0.11	8 ± 0.32	8 ± 0.11
B22	13 ± 0.18	13 ± 0.22	13 ± 0.22	8 ± 0.12
B23	12 ± 0.17	12 ± 0.29	8 ± 0.33	8 ± 0.15
B24	12 ± 0.10	11 ± 0.28	9 ± 0.16	8 ± 0.18
B25	11 ± 0.09	12 ± 0.22	12 ± 0.10	8 ± 0.23
B26	13 ± 0.12	11 ± 0.25	9 ± 0.08	9 ± 0.22
B27	10 ± 0.18	11 ± 0.18	8 ± 0.22	8 ± 0.20
B28	8 ± 0.19	12 ± 0.19	9 ± 0.13	9 ± 0.17
B29	8 ± 0.21	11 ± 0.14	8 ± 0.19	8 ± 0.11
B30	8 ± 0.22	10 ± 0.17	21 ± 0.15	9 ± 0.26
B31	9 ± 0.28	12 ± 0.19	9 ± 0.22	8 ± 0.22
B32	8 ± 0.32	11 ± 0.11	8 ± 0.27	8 ± 0.24
B33	10 ± 0.30	8 ± 0.09	12 ± 0.28	8 ± 0.13
B34	11 ± 0.29	8 ± 0.13	13 ± 0.21	8 ± 0.14
B35	12 ± 0.28	8 ± 0.15	8 ± 0.21	9 ± 0.11
B36	18 ± 0.27	8 ± 0.19	9 ± 0.19	8 ± 0.18
B37	9 ± 0.25	9 ± 0.23	11 ± 0.13	8 ± 0.11
Ciprofloxacin	36 ± 0.25	30 ± 0.22	35 ± 0.27	30 ± 0.21

2.3. Cytotoxicity

For the compounds which exhibited effective antitubercular activity (MIC ≤ 12.5 µg/mL), we evaluated in vitro cytotoxicity against mouse fibroblast (L929, CVCL 0462) cells using the MTT assay (Tables S1–S3). Figure 5 shows the proportion of cell growth inhibition for active compounds at various concentrations (3.125, 6.25, 12.5, 25, 50, 75, and 100 µg/mL). The results showed that all investigated substances had negligible toxicity, with IC₅₀ values greater than 400 µM (Table 5).

FIGURE 5.

The graphical visualization of the cell viability of the most active molecules against mouse fibroblast (L929) cells.

TABLE 5.

MIC values, IC₅₀, and SI values of active compounds.

Compound Code	MIC (µM) *	IC50 (µM) a	SI b
B5	31.63	427.05	13.50
B26	29.33	448.05	15.27
B30	14.01	547.17	39.05

^a Concentration of the substance that causes 50% inhibition of the VERO cell lines.

^b SI = IC₅₀ against Vero cells/* MIC against M. tuberculosis H37Rv

2.4. Computational Studies

2.4.1. In Silico ADME Studies

A drug development scientist's principal task is to identify and eliminate compounds that are sensitive to failure in clinical trials. According to statistics, out of six chemical entities that enter human clinical trials, only one successfully becomes approved for market as a medicine. According to regulatory agency data, around 37% of failures are due to poor pharmacokinetic qualities (such as ADME), 20% are discontinued owing to high toxicity, 28% fail due to insufficient efficacy, and approximately 5% are dropped for market reasons [28]. The pharmacokinetic and physicochemical features of novel molecules imidazol[1,2‐a]pyrimidine‐thiazole/1,3,4‐thidiazole derivatives (B1‐B37) were examined utilizing the QikProp module through ADME analysis. Table 6 shows the pharmacokinetic parameters for the final compounds (B1‐B37). These factors consists of molecular weight (Mol. Wt), polar surface area (PSA), number of hydrogen bond donors and acceptors (HB and HA), partition coefficient (QP log Po/w), human oral absorption (% HOA), blood‐brain partition coefficient (QP log BB), interaction with human serum albumin (QP log khsa), aqueous solubility (QP log S), and permeability through Caco‐2 cell membranes. Lipinski's Rule of Five (RO5) is commonly employed to measure the oral bioavailability of pharmacological compounds. This rule states that a compound has good oral bioavailability if it has a molecular weight (MW) of less than 500 Da, a partition coefficient (CLogP) of less than five, no more than five HBD, and no more than ten HBA. The RO5 examination of the newly synthesized compounds (B1‐B37) revealed that all satisfied these criteria, indicating good oral bioavailability (Table S4). PSA determines hydrogen bonding and polarity, which aids in predicting absorption and bioavailability. Compounds B1‐B37 had high oral bioavailability, with PSA values ranging from 46.45 to 72.24 Ų. The target compounds showed adequate water solubility (‐6.308 to ‐7.689), which is crucial for assessing intestinal absorption of drug molecules (QPlogS ≤ 0.5). Their Blood/Brain partition coefficient (QPlogBB) values, which range from ‐0.669 to 0.178, are within the acceptable range, showing the capacity to penetrate the blood‐brain barrier. In addition, the human serum albumin binding coefficient (QPlogKhsa) was between 0.434 and 0.958, which falls within acceptable bounds. According to the results, all targeted compounds exhibited 100% oral absorption in humans.

TABLE 6.

ADME and physico‐chemical properties of the target molecules (B1‐B37).

Compound Code	MW (≤500Da a	HBD (≤5) b	HBA (≤10) c	QPlog P(o/w) (≤5) d	QPlogS (≤0.5) e	nRB (0–15) f	PSA (≤140Å) g	QPPCaco (<25 nm/s is low; >500 nm/s is high h	QPlogKhsa (− 1.5 to 1) i	QPlogBB (− 3.0 to 1.2) j	%OA (>80 % is high:<25 % is low k
B1	409.1361	0	5	5.734	−7.382	3	46.45	2823.30	0.958	−0.126	100
B2	425.1310	0	5.75	5,625	−7.633	4	54.67	1981.5	0.867	−0.413	100
B3	413.1110	0	5	5.646	−7.143	3	46.45	2823.98	0.83	0	100
B4	429.0815	0	5	5.911	−7.535	3	46.46	2823.08	0.911	0.053	100
B5	395.1205	0	5	5.407	−6.77	3	46.46	2823.30	0.783	−0.11	100
B6	425.1310	0	5.75	5.471	−6.928	4	54.70	2816.34	0.76	−0.187	100
B7	441.1259	0	6.5	5.217	−6.497	5	62.9	2825.25	0.566	−0.246	100
B8	429.1060	0	5.75	5.382	−6.69	4	54.71	2813.93	0.632	−0.061	100
B9	445.0764	0	5.75	5.648	−7.081	4	54.72	2813.87	0.714	−0.008	100
B10	411.1154	0	5.75	5.143	−6.315	4	54.72	2812.39	0.586	−0.171	100
B11	413.1110	0	5	5.645	−7.138	3	46.47	2826.83	0.829	0.001	100
B12	429.1060	0	5.75	5.382	−6.682	4	54.68	2828.39	0.632	−0.058	100
B13	417.0860	0	5	5.558	−6.906	3	46.49	2825.73	0.702	0.125	100
B14	433.0564	0	5	5.825	−7.299	3	46.5	2815.09	0.784	0.178	100
B15	399.0954	0	5	5.319	−6.532	3	46.50	2815.43	0.656	0.015	100
B16	429.0815	0	5	5.911	−7.529	3	46.50	2823.28	0.911	0.054	100
B17	445.0764	0	5.75	5.469	−7.076	4	54.68	2826.50	0.713	−0.005	100
B18	433.0564	0	5	5.824	−7.298	3	46.50	2814.85	0.784	0.178	100
B19	449.0269	0	5	6.089	−7.689	3	46.50	2815.0	0.865	0.231	100
B20	415.0658	0	5	5.585	−6.923	3	46.51	2815.08	0.737	0.068	100
B21	395.1205	0	5	5.406	−6.765	3	46.47	2827.21	0.783	−0.109	100
B22	411.1154	0	5.75	5.143	−6.308	4	54.69	2827.59	0.585	−0.168	100
B23	399.0954	0	5	5.319	−6.532	3	46.49	2816.36	0.656	0.016	100
B24	415.0658	0	5	5.585	−6.924	3	46.49	2815.31	0.737	0.068	100
B25	381.1048	0	5	5.081	−6.765	3	46.49	2815.80	0.609	−0.094	100
B26	426.1263	0	6.25	4.864	−6.757	4	72.24	1261.21	0.61	−0.651	100
B27	410.1314	0	5.5	5.124	−7.208	3	64.04	1256.40	0.807	−0.595	100
B28	430.0767	0	5.5	5.455	−8.067	3	64	888.43	0.867	−0.669	100
B29	474.0262	0	5.5	5.381	−7.481	3	64	1260.85	0.786	−0.402	100
B30	430.1012	0	6.25	4.772	−6.505	4	72.22	1264.68	0.481	−0.515	100
B31	414.1063	0	5.5	5.036	−6.972	3	64.06	1255.12	0.68	−0.462	100
B32	434.0517	0	5.5	5.21	−7.112	3	63.99	1261.54	0.631	−0.277	100
B33	478.0012	0	5.5	5.289	−7.232	3	63.99	1260.85	0.656	−0.268	100
B34	412.1106	0	6.25	4.534	−6.134	4	72.22	1263.69	0.434	−0.623	100
B35	396.1157	0	5.5	4.797	−6.598	3	64.05	1255.76	0.633	−0.569	100
B36	416.0611	0	5.5	4.971	−6.738	3	63.98	1261.24	0.584	−0.384	100
B37	460.0106	0	5.5	5.05	−6.859	3	63	1261.03	0.61	−0.374	100

^a Molecular Weight.

^b Number of hydrogen bond donors.

^c Number of hydrogen bond acceptors.

^d Logarithm of partition coefficient between n‐octanol and water.

^e Aqua solubility parameter.

^f Number of rotatable bonds.

^g Polar Surface Area.

^h Caco‐2 Cell permeability.

ⁱ Human serum albumin binding co‐efficient.

^j Blood/Brain partition co‐efficient.

^k Percentage of oral absorption.

2.5. Molecular Docking Studies

Molecular docking assessment are often used for studying how ligands interact with a protein's binding site, providing binding energy estimates and aiding in the understanding of fundamental molecular mechanisms. All of the synthesized compounds (B1‐B37) were screened against two target enzymes to examine the binding approach of imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole hybrids: cytochrome P450 monooxygenase (CYP121, PDB ID: 4KTF) with the co‐crystallized ligand 1TM, and enoyl‐acyl carrier protein reductase (InhA, PDB ID: 1P44) with the co‐crystallized ligand GEQ. To ensure the viability of the active site, the standard ligands were re‐docked into the appropriate protein targets. The results demonstrated that the ligands preserved the same interaction patterns as those found in the crystal structures.

2.5.1. Docking Studies With Inh A

InhA is an essential enzyme in the type II fatty acid production pathway. The production of mycolic acid by Mycobacterium requires Tyr158, a crucial amino acid residue that interacts with long‐chain fatty acyl substrates [29]. The most potent compounds showed pi– stacking and H‐bonding interactions with the Tyr158 and Phe149 residues. The binding affinities of most active derivatives, B5, B26, and B30, are ‐7.9, ‐8.0, and ‐8.8 Kcal/mol, respectively (Table 7). The docking poses of these three molecules are depicted in Figure 6. Compound B5 displayed pi–pi interactions with the Tyr 158 residue. The imidazo[1,2‐a]pyrimidine core is connected to a methyl‐substituted phenyl ring and thiazole ring, which facilitates these interactions (Figure 6a,b). Compound B26 exhibited pi–pi stacking interactions with the Tyr158 residue through the 1,3,4‐thiadiazole ring and methoxy‐substituted phenyl ring on the imidazo[1,2‐a]pyrimidine scaffold. Also, it showed a pi‐cationic interaction with the Lys 165 residue through methyl‐substituted phenyl ring attached to the 1,3,4‐thidiazole ring (Figure 6c,d). Compound B30 has a H‐boning interaction with the target. This interaction happens between the Tyr 158 residue and the 1,3,4‐thiadiazole ring. In addition, it exhibits two pi–pi stacking interactions with the Tyr158 residue through its 1,3,4‐thiadiazole ring and methoxy‐substituted phenyl ring connected to the imidazo[1,2‐a]pyrimidine scaffold. It also showed pi‐cationic infarctions with Lys 165 residues through the fluro‐substituted phenyl ring connected to the 1,3,4‐thiazole ring (Figure 6e,f).

TABLE 7.

Binding energy and interactions of target compounds (B5, B26, and B30) with receptor 1P44 and 4KTF.

	Receptor 1P44		Receptor 4KTF
Compound code	Docking score (Kcal/mol)	Pi–Pi stacking	Docking score (Kcal/mol)	Pi–Pi stacking
B5	−7.9	Tyr 158	−7.8	Phe 168, Phe230
B26	−8.0	Tyr 158, Phe149 Lys 165 (pi‐cation)	−8.1	Trp 182, Phe 168
B30	−8.8	Tyr158 Lys 165 (pi‐cation) Tyr 158 (H‐bonding)	−8.6	Trp 182, Phe168

FIGURE 6.

The 2D (left) and 3D (right) docking poses of compound B5 (a and b), B26 (c and d), and B30 (e and f) with receptor 1P44.

2.5.2. Docking Studies With CYP121

Another new anti‐TB target is the enzyme CYP121, which is responsible for generating a unique carbon‐carbon bond. It connects two tyrosine residues within cyclodityrosine, resulting in the formation of mycocyclosin [30]. The proposed compounds demonstrated pi–pi stacking and hydrogen bond interactions with the amino acid residues in the active site of CYP121. Most of these compounds engaged in pi–pi stacking with the Trp182 and Phe168 residues. As shown in Table 7, the best anti‐TB compounds B5, B26, and B30 had binding affinities of ‐7.8, ‐8.1, and ‐8.6 Kcal/mol, respectively. Compound B5 showed two pi–pi stacking interactions with the Phe 230 and Phe 168 residues. The phenyl ring linked to the thiazole moiety and methyl‐substituted phenyl ring connected to the imidazo[1,2‐a]pyrimidine scaffold drove the major of these interactions (Figure 7a,b). Compound B26 displayed two pi–pi stacking contacts with the Phe 168 and Trp 182 residues. The 1,3,4‐thiadiazole and methyl‐substituted phenyl ring connected to the thiadiazole moiety are responsible for these interactions (Figure 7c,d). Compound B30 displayed two pi–pi stacking contacts with the Trp182 and Phe168 residues. Trp182 interacts with the F‐substituted phenyl ring, whereas Phe168 interacts with the 1,3,4‐thiadiazole ring (Figure 7e,f). Therefore, incorporating a thiazole or 1,3,4‐thiadiazole ring into the imidazo[1,2‐a]pyrimidine framework increased the likelihood of pi–pi stacking and H‐bonding interactions with critical residues of the InhA, such as Tyr158 and Phe148. In CYP121, interactions with the Trp182 and Phe 168 residues most likely contribute to hybrid molecules enhancing the activity against the M. tuberculosis H37Rv strain.

FIGURE 7.

The 2D (left) and 3D (right) docking poses of compound B5 (a and b), B26 (c and d), and B30 (e and f) with receptor 4KTF.

2.6. DFT Studies

Understanding the energy properties of molecules is extremely important while researching drugs. Potential relationships between a drug and its target include covalent bonds, ionic forces, ion‐dipole and dipole‐dipole interactions, hydrogen bonding, charge transfer, and hydrophobic effects. DFT is frequently used in computer‐aided drug design, and it has been demonstrated to be reliable in predicting covalent, ionic, and hydrogen bonding interactions [31]. Molecular orbitals are essential to the transfer of electrons in chemical reactions. The LUMO is the orbital with the lowest energy level that can take on electrons, and the HOMO is the orbital that can hold the highest energy electrons. Various chemical characteristics are frequently ascertained using the HOMO and LUMO energy values. Descriptors of global reactivity include parameters like global softness (S), hardness (η), electrophilicity index (ω), electronegativity (χ), global nucleophilicity (N), and chemical potential (μ). Molecules having a small HOMO‐LUMO energy gap (ΔE) are considered soft, as they have reduced kinetic stability, can readily modify their electronic structure, and are more reactive [32]. Molecules with a large HOMO‐LUMO energy gap (ΔE) are classified as hard due to their higher kinetic stability, lesser reactivity, and reduced susceptibility to electronic configuration changes. Target molecules have HOMO and LUMO frontier orbitals that are evenly spread across the molecule. In compound B5, the HOMO electron density concentrates on the thiazole ring and its linked phenyl ring, whereas the LUMO electron density is dispersed over the imidazo[1,2‐a]pyrimidine moiety and the imine linkage (Figure 8a). In compounds B26 and B30, the HOMO electron density concentrates mainly on the thiadiazole ring and the methoxy‐substituted phenyl ring, whereas the LUMO electron density is concentrated over the imidazo[1,2‐a]pyrimidine unit and the imine linkage (Figures 9a and 10a). The energy band gaps for molecules B5, B26, and B30 were calculated to be 2.7159, 2.6845, and 2.6825 eV, respectively (Table 8). Electron affinity (EA) denotes a molecule's ability to receive electrons, whereas ionization potential (IP) shows its ability to donate electrons. The studied molecule has greater IP values relative to EA indicate that they are more likely to transfer electrons than to take them. This was additionally reinforced by the fact that all final molecules demonstrated electron‐donating (nucleophilic) activity, with electronegativity values lower than IP values. A strong nucleophile has a low electrophilicity index (ω < 3) and a high nucleophilicity value (N), while a strong electrophile has a high index (ω > 3). The molecular electrostatic potential (MEP) map depicts how electrostatic potential (EP) is spread over various atomic locations while accounting for partial charge distribution, electronegativity of important atoms, and their mutual interactions. Figures 8–10b,c show the MEP profiles of compounds B5, B26, and B30. The EP values are represented by a color scale, with red highlighting places of strong negative potential, blue indicating areas of greatest positive potential, and green corresponding to zones of neutral potential [33]. The red areas in these compounds are primarily found around the nitrogen atoms of the imidazo[1,2‐a]pyrimidine and thiazole/1,3,4 thiadiazole ring, indicating that they are nucleophilic. The EP characteristics suggest that these regions either participate in pi–pi stacking and hydrogen bonding interactions or preferentially connect with areas of opposite potential within the binding pocket.

FIGURE 8.

Frontier Molecular Orbitals (a), Electron density surface (b) and Electrostatic potential (c) of compound B5.

FIGURE 9.

Frontier Molecular Orbitals (a), Electron density surface (b), Electrostatic potential (c) of compound B26.

FIGURE 10.

Frontier Molecular Orbitals (a), Electron density surface (b), Electrostatic potential (c) of compound B30.

TABLE 8.

The global reactivity parameters of B5, B26, and B30 using DFT studies.

Comp a	HOMO (eV) b	LUMO (eV) c	ΔE (eV) d	IP (eV) e	EA (eV) f	η (eV) g	σ (eV) h	μ (eV) i	ω (eV) j	χ (eV) k
B5	−5.0081	−2.2922	2.7159	5.0081	2.2922	1.3579	0.3681	−1.3579	0.6789	3.6501
B26	−5.0984	−2.4139	2.6845	5.0984	2.4139	1.3422	0.3725	−1.3422	0.6711	3.7561
B30	−5.2288	−2.5463	2.6825	5.2288	2.5463	1.3412	0.3728	−1.3412	0.6706	3.8875

^a Compound Code.

^b HOMO = Highest occupied molecular orbital.

^c LUMO = Lowest unoccupied molecular orbital.

^d Bandgap (∆E) = E_HOMO—E_LUMO .

^e Ionization potential (IP) =—E_HOMO .

^f Electron affinity (EA) =—E_LUMO .

^g Chemical hardness (η) = (IP‐EA)/2.

^h Chemical softness (σ) = 1/2 η.

ⁱ Chemical potential (μ) =—η.

^j Electrophilicity index (ω) = η/2.

^k Electronegativity (χ) = (IP + EA)/2.

2.7. Molecular Dynamics Studies

Using GROMACS software, a 100 ns molecular dynamics (MD) simulation was used to examine the stability and conformational flexibility of the B30–1P44 ligand–protein complex. RMSD analysis was performed to assess the structural deviations and overall stability of the protein and ligand–protein complex throughout the MD simulation. The RMSD profiles of 1P44 and the 1P44–B30 complex remained relatively stable over the 100 ns simulation, with only minor fluctuations observed. Following the initial equilibration phase, the protein backbone RMSD remained predominantly within the range of 1.8–2.1 Å, with only transient deviations during the trajectory. The ligand RMSD also remained comparatively stable, indicating that B30 retained its position within the binding pocket and maintained a stable bound conformation throughout the simulation. Hydrogen‐bond studies revealed that the complex maintained 0‐2 protein‐ligand hydrogen bonds, with one bond occurring more frequently. The H‐bond distances ranged between 2.4–3.5 Å, indicating stable and geometrically favored interactions. The Rg analysis indicated that the 1P44‐B30 complex remained compact and stable, with Rg values ranging from 18.1–18.7 Å (mean ≈18.43 Å). Minor fluctuations suggest typical structural changes, not major protein unfolding. The MM‐PBSA analysis revealed predominantly negative binding free energies throughout the 100 ns simulation, indicating a favorable and persistent interaction between B30 and 1P44. The binding‐energy distribution was focused at ‐25 to ‐27 Kcal/mol, indicating the complex's strong energetic stability (Figure 11). RMSF analysis revealed that the majority of protein residues had low to moderate flexibility, with higher fluctuations restricted to certain flexible areas, with a maximum fluctuation of around 3.5 Å. The ligand showed modest flexibility at its outer atoms but maintained an overall stable structure within the binding pocket (Figure 12).

FIGURE 11.

(a) Root mean square deviation (RMSD), (b) Hydrogen bond analysis, (c) Radius of gyration (Rg), (d) MM‐PBSA binding free energy profile analysis of the 1P44‐B30 complex during a 100 ns simulation.

FIGURE 12.

Root mean square fluctuation (RMSF) analysis of the 1P44‐B30 complex illustrating residue‐wise flexibility of the protein and atom‐wise mobility of the bound ligand during the 100 ns simulation.

3. Conclusions

Two distinct classes of 37 novel imidazo[1,2‐a]pyrimidine‐thiazole/thiadiazole hybrid molecules (B1–B37) were created using a molecular hybridization technique. The structures of all intermediate and final compounds were characterized by using ¹H NMR, ¹³C NMR, and mass spectrometry. In the antitubercular study, all of the tested compounds showed promising effectiveness against the Mycobacterium TB H37Rv strain (ATCC 27294). Compounds B5, B26, and B30 possess the highest potency with MIC values of 12.5, 12.5, and 6.25 µg/mL, respectively. Cytotoxicity investigations demonstrated that these active anti‐TB compounds are safe for normal cell lines (SI > 10), confirming their potential as therapeutic agents. Additionally, most of the compounds that were targeted showed substantial antibacterial activity against all four tested bacterial strains. Furthermore, the imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole derivatives' significant binding affinity to the active sites of the target enzymes InhA and CYP121 supported their observed in vitro activity. In silico ADME study revealed that the majority of the drug molecules have good oral bioavailability. DFT tests indicated other advantageous electronic features, such as low electron affinity, a narrow HOMO‐LUMO energy gap, reduced chemical hardness, high chemical potential, and increased chemical softness. A 100 ns MD simulation confirmed the stability and compactness of the 1P44–B30 complex. Favorable binding energy and stable interactions supported the strong binding of B30 within the active site. These characteristics indicate improved responsiveness and stronger binding connections with the receptor. Thus, imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole compounds (B1–B37) have significant promise as lead scaffolds for developing novel anti‐TB compounds.

4. Experimental Section

4.1. Materials and Methods

All the chemicals utilized in this work were purchased from reputable vendors, such as Alfa Aesar, TCI, and Sigma Aldrich. Thin‐layer chromatography (TLC) on alumina plates coated with silica gel initial was used to monitor the reactions' development (Merck 60 F254). The generated spots were seen in a UV chamber using a (8:3) combination of petroleum ether and ethyl acetate as the mobile phase. A digital melting point device was used to measure the melting points of the produced compounds without any adjustments. On a Bruker FT‐NMR spectrometer, which operates at 400 MHz for ¹H‐NMR and 100 MHz for ¹³C‐NMR, spectroscopic investigations were done. The internal reference standard used was tetramethylsilane (TMS), and the solvents employed were CDCl₃ and DMSO‐d₆. The coupling constants are given in Hertz, and the chemical shift values are presented in parts per million (δ). Standard abbreviations, such as “s” for singlet, “d” for doublet, “t” for triplet, “q” for quartet, “m” for multiplet, and “dd” for doublet of doublets, were used to indicate the splitting patterns in the ¹H‐NMR spectra. Bruker TopSpin 4.1.4 software was utilized for the processing and analysis of spectral data. Additionally, all intermediates and final products' high‐resolution mass spectra (HR‐MS/Matric Assisted Laser Deposition Ionization (MALDI)) were acquired using a Waters Xevo QTOF MS/Bruker system that was outfitted with electrospray ionization (ESI).

4.2. Synthesis and characterization

4.2.1. General Procedure for the Synthesis of Substituted thiazol‐2‐amines 7(a‐e)

In a 100 mL round‐bottom flask, appropriate phenacyl bromide (5a‐5e) (5 g, 1.0 equivalent) and thiourea (1.5 equivalent) were mixed with water (20 mL) and stirred the reaction mixture for 4 h at room temperature. The progress of the reaction was monitored using TLC. After completion, the reaction mixture was put into ice‐cold water, and then treated with a 25% ammonia solution to basify the reaction mixture. A white‐colored solid persisted after complete basification of the reaction. The desired product was collected by filtration, cleaned thoroughly with cold water, and dried.

4.2.1.1. 4‐(p‐Tolyl)thiazol‐2‐amine (7a)

White solid, Yield: 75 %; m.p.: 225°C–226°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.68 (2H, d, J = 8.1 Hz), 7.17 (2H, d, J = 8.0 Hz), 7.00 (2H, s), 6.91 (1H, s), 2.30 ppm (3H, s). ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.53, 150.37, 136.80, 132.76, 132.76, 129.47, 125.94, 101.00, 21.24 ppm. HR‐MS (m/z) = 191.0582 [M + H]⁺.

4.2.1.2. 4‐(4‐Methoxyphenyl)thiazol‐2‐amine (7b)

White solid, Yield:85 %; m.p.: 210‐211°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.72 (2H, d, J = 8.8 Hz), 6.99 (2H, s), 6.92 (2H, d, J = 8.9 Hz), 6.81 (1H, s), 3.76 ppm (3H, s). ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.53, 158.97, 150.14, 128.32, 127.28, 114.26, 99.78, 55.53 ppm. HR‐MS (m/z) = 207.0518 [M + H]⁺.

4.2.1.3. 4‐(4‐Fluorophenyl)thiazol‐2‐amine (7c)

White solid, Yield: 82 %; m.p.: 104°C–105°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.86‐7.80 (2H, m), 7.20‐7.16 (2H, m), 7.06 (2H, s), 6.97 ppm (1H, s). ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.76, 163.06, 160.63, 149.25, 132.03, 132.00, 127.95, 127.87, 115.81, 115.60, 101.66, 101.65 ppm. HR‐MS (m/z) = 195.0347 [M + H]⁺.

4.2.1.4. 4‐(4‐Chlorophenyl)thiazol‐2‐amine (7d)

White solid, Yield:80 %; m.p.: 170°C–171°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.82 (2H, d, J = 7.6 Hz), 7.42 (2H, d, J = 7.7 Hz), 7.12 (2H, s), 7.06 ppm (1H, s). ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.83, 148.94, 134.16, 132.01, 128.92, 127.68, 102.77 ppm. HR‐MS (m/z) = 211.0025 [M + H]⁺.

4.2.1.5. 4‐Phenylthiazol‐2‐amine (7e)

White solid, Yield: 90 %; m.p.: 150°C–151°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.80 (2H, d, J = 7.4 Hz), 7.37 (2H, t, J = 7.5 Hz), 7.26 (1H, t, J = 7.3 Hz), 7.05 (2H, s), 7.00 ppm (1H, s).¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.64, 150.31, 135.40, 128.90, 127.62, 126.00, 101.94 ppm. HR‐MS (m/z) = 177.0425 [M + H]⁺.

4.2.2. General Procedure for the Synthesis of Substituted thiadiazole‐2‐amines 10(a‐c)

The substituted phenyl carboxylic acid derivatives (8a‐8c) (5.0 g, 1.0 equivalent), thiosemicarbazide (1.0 equivalent), and POCl₃ (1.5 equivalent) were heated at 75°C and stirred continuously for 30 min. After that, the reaction mixture was allowed to cool to ambient temperature and then water (15 mL) was added to it. The resultant mixture was then refluxed for 4 h. After cooling, the solution was carefully adjusted to pH 8 by adding 50% NaOH dropwise while stirring. To acquire the final target compounds, the resultant solid was filtered and then refined by recrystallization from ethanol.

4.2.2.1. 5‐(4‐Tolyl)‐1,3,4‐thiadiazol‐2‐amine (10a)

White solid, Yield: 88 %; m.p.: 215°C–216°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.64 (2H, d, J = 8.0 Hz), 7.40 (2H, s), 7.28 (2H, d, J = 7.9 Hz), 2.34 ppm (3H, s). ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.67, 156.97, 139.75, 130.11, 128.77, 126.69, 21.34 ppm. HR‐MS (m/z) = 192.0526 [M + H] ⁺.

4.2.2.2. 5‐(4‐Flourophenyl)‐1,3,4‐thiadiazol‐2‐amine (10b)

White solid, Yield: 80 %; m.p.: 234°C–235°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.76 (2H, d, J = 7.68 Hz), 7.47(2H, m), 7.43 ppm (2H, m) ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.78, 163.06, 160.64, 149.26, 132.02, 131.99, 127.95, 127.87, 115.81, 115.59, 101.65 ppm. HR‐MS (m/z) = 196.0272 [M + H]⁺.

4.2.2.3. 5‐Phenyl‐1,3,4‐thiadiazol‐2‐amine (10c)

White solid, Yield: %; m.p.: 225°C–226°C; ¹H NMR (400 MHz, DMSO‐d₆): δ = 7.83(2H, s,), 7.21 (2, t, J = 8 Hz), 7.08 (2H, s), 6.98 ppm (1H, s). ¹³C NMR (100 MHz, DMSO‐d₆): δ = 168.98, 156.84, 131.46, 130.03, 129.59, 126.76 ppm. HR‐MS (m/z) = 178.0385 [M + H]⁺.

4.2.3. General Procedure for Synthesis of (E)‐1‐(2‐(imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4)thiazol/1,3,4‐thiadiazole‐2‐yl)methanimine Derivatives (B1‐B37)

In a 50 mL round‐bottom flask, an equimolar quantity of imidazo[1,2‐a]pyrimidine‐3‐carbaldehyde (1.0 equivalent) was reacted with a substituted thiazol/1,3,4‐thiadiazol‐2‐amine in ethanol (15 mL) under reflux conditions. Citric acid (1.5 equivalent) was used as a catalyst. Molecular sieves were added to the reaction mixture to use in the abolition of water produced during the reaction. The mixture was refluxed for 24‐30 h. After completion, the hot mixture was vacuum‐filtered to collect the crude solid, which was then refined by recrystallization with hot ethanol. The final products were obtained at a yield of 60%–85%.

4.2.3.1. (E)‐1‐(2‐(p‐Tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(p‐tolyl)thiazol‐2‐yl)methanimine (B1)

Yellow solid, Yield:71%; m.p.: 250°C–251°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, dd, J = 1.7, 6.7 Hz), 9.40 (1H, s), 8.79 (1H, dd, J = 1.8, 4.0 Hz), 7.83 (4H, dd, J = 8.3, 9.9 Hz), 7.39 (2H, d, J = 7.8 Hz), 7.30 (1H, s), 7.24 (2H, d, J = 7.9 Hz), 7.18‐7.16 (1H, m), 2.47 (3H, s), 2.39 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.57, 158.04, 153.94, 153.25, 151.55, 150.89, 140.30, 138.17, 137.70, 131.70, 129.89, 129.78, 129.58, 129.43, 126.16, 116.28, 110.60, 110.50, 21.48, 21.30 ppm. HR‐MS (m/z): = 410.1370[M + H]⁺.

4.2.3.2. (E)‐N‐(4‐(4‐Methoxyphenyl)thiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl) Methanimine (B2)

Yellow solid, Yield:76%; m.p.: 219°C–220°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, d, J = 6.3 Hz), 9.39 (1H, s), 8.78 (1H, s), 7.90 (2H, d, J = 8.36 Hz), 7.82 (2H, d, J = 7.68), 7.30(1H, s), 7.23 (2H, m), 7.18 (1H, t, J = 4.52 Hz), 7.12 (2H, d, J = 8.4 Hz), 3.92 (3H, s), 2.39 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 161.28, 153.22, 151.51, 138.18, 137.67, 131.70, 131.39, 129.44, 126.16, 116.02, 114.57, 110.51, 110.44, 55.47, 21.30 ppm. HR‐MS (m/z): = 426.1318 [M + H]⁺.

4.2.3.3. (E)‐N‐(4‐(4‐Fluorophenyl)thiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl) Methanimine (B3)

Yellow solid, Yield: 69 %; m.p.: 281°C–282°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.34 (1H, d, J = 6.7 Hz), 9.42 (1H, s), 8.83 (1H, dd, J = 4.0 Hz), 7.84 (4H, m), 7.75 (2H, d, J = 8.28 Hz), 7.36 (1H, s), 7.26‐7.22 (3H, m), 2.42 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.36, 156.68, 153.84, 152.42, 150.98, 150.74, 138.22, 136.71, 131.84, 130.62, 129.44, 126.16, 115.34, 116.15, 110.86, 110.82, 21.34 ppm. MALDI (m/z) = 414.118 [M + H]⁺.

4.2.3.4. (E)‐N‐(4‐(4‐Chlorophenyl)thiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl) Methanimine (B4)

Yellow solid, Yield: 72 %; m.p.: 322°C–323°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.31 (1H, d, J = 5.8 Hz), 9.42 (1H, s), 8.84 (1H, s), 7.84 (4H, dd, J = 7.9, 15.6 Hz), 7.59 (2H, d, J = 8.0 Hz), 7.42 (3H, t, J = 8.1 Hz), 7.22 (1H, t, J = 5.0 Hz), 2.50 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.91, 161.34, 158.31, 153.38, 152.60, 151.78, 150.99, 140.42, 137.69, 134.06, 132.87, 131.39, 129.88, 129.81, 128.92, 127.51, 124.82, 114.59, 111.53, 111.47, 110.69, 110.60, 21.49 ppm. MALDI (m/z) = 430.301 [M + H]⁺.

4.2.3.5. (E)‐N‐(4‐Phenylthiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl)methanimine (B5)

Green solid, Yield: 65 %; m.p.: 188°C–189°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, dd, J = 1.7, 6.7 Hz), 9.40 (1H, s), 8.79 (1H, s), 7.92 (4H, dd, J = 8.3, 9.9 Hz), 7.45 (6H, m), 7.19 (1H, t, 4.32 Hz), 2.47 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.70, 158.12, 153.83, 153.30, 151.64, 140.34, 137.72, 134.38, 129.88, 129.80, 128.76, 128.30, 126.27, 116.27, 111.27, 110.64, 21.49 ppm. HR‐MS (m/z) = 396.1211 [M + H]⁺.

4.2.3.6. (E)‐1‐(2‐(4‐Methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(p‐tolyl)thiazol‐2‐yl) Methanimine (B6)

Yellow solid, Yield:85%; m.p.: 278°C–279°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.34 (1H, d, J = 5.3 Hz), 9.41 (1H, s), 8.82 (1H, d, J = 2.0 Hz), 7.88 (4H, t, J = 8.5 Hz), 7.42 (2H, d, J = 7.8 Hz), 7.22 (1H, s), 7.22 (1H, q, J = 3.6 Hz), 6.99 (2H, d, J = 8.6 Hz), 3.88 (3H, s), 2.50 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.55, 159.74, 158.00, 153.66, 153.24, 151.45, 150.88, 140.28, 137.70, 129.88, 129.78, 129.58, 127.57, 127.35, 116.28, 114.10, 110.60, 109.55, 55.36, 21.49 ppm. HR‐MS (m/z): = 426.1316 [M + H]⁺.

4.2.3.7. (E)‐1‐(2‐(4‐Methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(4‐methoxyphenyl) thiazol‐2‐yl)methanimine (B7)

Yellow solid, Yield:80 %; m.p.: 290°C–291°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, dd, J = 2.0, 6.8 Hz), 9.39 (1H, s), 8.79 (1H, dd, J = 2.0, 4.2 Hz), 7.90‐7.84 (4H, m), 7.18 (1H, s), 7.18‐7.15 (1H, m), 7.12 (2H, d, J = 8.8 Hz), 6.97 (2H, d, J = 8.8 Hz), 3.92 (3H, s), 3.86 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.62, 161.27, 159.74, 157.79, 153.63, 153.22, 151.40, 150. 91, 137.67, 131.39, 127.56, 124.92, 116.03, 114.57, 114.10, 110.52, 109.50, 55.48, 55.36 ppm. HR‐MS (m/z): = 442.1266 [M + H]⁺.

4.2.3.8. (E)‐N‐(4‐(4‐Fluorophenyl)thiazol‐2‐yl)‐1‐(2‐(4‐methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)methanimine (B8)

Yellow solid, Yield: 68 %; m.p.: 318°C–319°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.17 (1H, dd, J = 1.7, 6.7 Hz), 9.31 (1H, s), 8.92 (1H, dd, J = 1.6, 4.2 Hz), 7.99 (2H, d, J = 8.4 Hz), 7.90 (3H, d, J = 10.2 Hz), 7.73 (2H, d, J = 8.4 Hz), 7.54 (1H, q, J = 3.7 Hz), 7.03 (2H, d, J = 8.6 Hz), 3.80 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.91, 161.32, 158.02, 153.32, 152.78, 151.62, 137.67, 131.40, 128.04, 127.96, 124.85, 115.78, 115.57, 114.58, 110.78, 55.49 ppm. MALDI (m/z) = 430.112 [M + H]+.

4.2.3.9. (E)‐N‐(4‐(4‐Chlorophenyl)thiazol‐2‐yl)‐1‐(2‐(4‐methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)methanimine (B9)

Yellow solid, Yield: 68 %; m.p.: 278°C–279°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.21 (1H, d, J = 4.8 Hz), 9.31 (1H, s), 8.72 (1H, s), 7.82 (2H, d, J = 7.5 Hz), 7.72 (2H, d, J = 8.0 Hz), 7.49 (2H, d, J = 7.8 Hz), 7.29 (1H, s), 7.11 (1H, s), 7.04 (2H, d, J = 8.0 Hz), 3.85 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 150.63, 137.70, 131.12, 129.32, 127.57, 114.16, 109.99, 55.36 ppm. MALDI (m/z) = 446.189 [M + H]⁺.

4.2.3.10. (E)‐1‐(2‐(4‐Methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐phenylthiazol‐2‐yl) methanimine (B10)

Yellow solid, Yield: 72 %; m.p.: 322°C–323°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, dd, J = 2.1, 6.8 Hz), 9.40 (1H, s), 8.79 (1H, dd, J = 2.1, 4.2 Hz), 7.92 (4H, q, J = 6.3 Hz), 7.45 (2H, t, J = 7.5 Hz), 7.36 (2H, s), 7.18 (1H, q, J = 3.7 Hz), 7.11 (2H, d, J = 8.8 Hz), 3.92 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.76, 161.30, 157.90, 153.80, 153.27, 151.57, 150.93, 137.68, 134.40, 131.39, 128.76, 128.30, 126.26, 124.87, 116.01, 114.58, 111.22, 110.56, 55.48 ppm. HR‐MS (m/z) = 412.1164 [M + H]⁺.

4.2.3.11. (E)‐1‐(2‐(4‐Fluorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(p‐tolyl)thiazol‐2‐yl) methanimine (B11)

Yellow solid, Yield: 68 %; m.p.: 288°C–289°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.32 (1H, dd, J = 1.8, 6.7 Hz), 9.41 (1H, s), 8.81 (1H, dd, J = 1.9, 4.0 Hz), 7.94 (2H, d, J = 6.7 Hz), 7.81 (2H, d, J = 8.0 Hz), 7.58 (2H, m), 7.31 (1H, s), 7.24 (2H, d J = 8 Hz), 7.20 (1H, m), 2.39 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.34, 156.72, 153.97, 153.46, 150.93, 150.76, 138.29, 137.72, 131.86, 131.78, 129.47, 126.14, 116.34, 116.12, 110.84, 110.81, 21.31 ppm. MALDI (m/z) = 414.475 [M + H]⁺.

4.2.3.12. (E)‐1‐(2‐(4‐Fluorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(4‐methoxyphenyl)thiazol‐2‐yl)methanimine (B12)

Yellow solid, Yield: 65 %; m.p.: 316°C–317°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, q, J = 3.0 Hz), 9.39 (1H, s), 8.81 (1H, t, J = 2.1 Hz), 7.94 (2H, q, J = 4.7 Hz), 7.85 (3H, d, J = 8.6 Hz), 7.28 (1H, s), 7.25 (1H, s), 7.21 (1H, q, J = 3.8 Hz), 6.98 (2H, d, 8.6), 3.85 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.32, 159.80, 153.70, 153.45, 150.83, 137.71, 131.86, 131.78, 127.55, 127.25, 116.34, 116.12, 114.14, 110.80, 109.90, 55.36 ppm. MALDI (m/z) = 430.415 [M + H]⁺.

4.2.3.13. (E)‐1‐(2‐(4‐Fluorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(4‐fluorophenyl)thiazol‐2‐yl)methanimine (B13)

Yellow solid, Yield: 76 %; m.p.: 288°C–289°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.31 (1H, dd, J = 2.0, 6.8 Hz), 9.41 (1H, s), 8.82 (1H, dd, J = 2.1, 4.2 Hz), 7.94‐7.86 (4H, m), 7.59 (3H, t, J = 7.5 Hz), 7.30 (1H, s), 7.32 (1H, m), 7.13 ppm (1H, t, J = 7.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 156.83, 153.50, 151.01, 137.72, 134.31, 131.87, 131.78, 128.80, 128.38, 126.25, 116.36, 116.14, 111.60, 110.83 ppm. MALDI (m/z) = 418.094 [M + H]⁺.

4.2.3.14. (E)‐N‐(4‐(4‐Chlorophenyl)thiazol‐2‐yl)‐1‐(2‐(4‐fluorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)methanimine (B14)

Yellow solid, Yield: 88 %; m.p.: 226°C–227°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.29 (1H, dd, J = 3.4, 3.4 Hz), 9.37 (1H, s), 8.82 (1H, d, J = 2.1 Hz), 7.94 (2H, m), 7.85 (2H, d, J = 8.4 Hz), 7.41 (3H, d, J = 8.4 Hz), 7.30 (2H, d, J = 8.5 Hz), 7.23 ppm (1H, q, J = 3.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 172.68, 153.58, 151.17, 150.86, 137.70, 134.16, 132.80, 131.87, 131.80, 128.96, 127.50, 116.37, 116.15, 111.83, 110.87 ppm. MALDI (m/z) = 434.062 [M + H]⁺.

4.2.3.15. (E)‐1‐(2‐(4‐Fluorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐phenylthiazol‐2‐yl) methanimine (B15)

Yellow solid, Yield: 72 %; m.p.: 332°C–333°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.31 (1H, dd, J = 2.1, 6.8 Hz), 9.41 (1H, s), 8.82 (1H, dd, J = 2.1, 4.2 Hz), 7.94‐7.86 (4H, m), 7.59 (3H, m), 7.30 (1H, s), 7.22 (1H, q, J = 3.7 Hz), 7.14 ppm (2H, t, J = 8.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 172.60, 165.30, 164.07, 162.81, 156.93, 153.54, 152.85, 151.05, 150.83, 137.68, 131.87, 131.78, 130.61, 128.59, 128.56, 128.04, 127.96, 116.36, 116.14, 115.82, 115.61, 111.13, 110.85 ppm. MALDI (m/z) = 400.391 [M + H]⁺.

4.2.3.16. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(p‐tolyl)thiazol‐2‐yl) methanimine (B16)

Yellow solid, Yield: 87 %; m.p.: 300°C–301°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, d, J = 5.1 Hz), 9.40 (1H, s), 8.81 (1H, d, J = 2.1 Hz), 7.89 (2H, d, J = 8.3 Hz), 7.81 (2H, d, J = 7.9 Hz), 7.57 (2H, d, J = 8.4 Hz), 7.33 (1H, s), 7.24 (3H, m), 2.40 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.24, 156.41, 154.02, 153.53, 150.74, 138.30, 137.73, 131.59, 131.12, 129.48, 129.34, 126.15, 116.51, 110.93, 110.85, 21.31 ppm. MALDI (m/z) = 430.417 [M + H]⁺.

4.2.3.17. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(4‐methoxyphenyl)thiazol‐2‐yl)methanimine (B17)

Yellow solid, Yield: 82 %; m.p.: 330°C–331°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.24 (1H, d, J = 6.7 Hz), 9.33 (1H, s), 8.74 (1H, d, J = 2.2 Hz), 7.82 (5H, dd, J = 8.5, 13.8 Hz), 7.50 (2H, d, J = 8.3 Hz), 7.15‐7.12 (1H, m), 6.91 (2H, d, J = 8.5 Hz), 3.79 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.22, 159.81, 153.52, 150.63, 137.74, 131.13, 129.34, 127.56, 116.53, 114.15, 110.86, 110.00, 55.37 ppm. MALDI (m/z) = 446.082 [M + H]⁺.

4.2.3.18. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(4‐fluorophenyl)thiazol‐2‐yl)methanimine (B18)

Yellow solid, Yield: 80 %; m.p.: 228°C–229°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.31 (1H, dd, J = 1.8, 6.8 Hz), 9.41 (1H, s), 8.85 (1H, dd, J = 1.9, 4.1 Hz), 7.91‐7.88 (4H, m), 7.60 (2H, d, J = 8.4 Hz), 7.44 (2H, d, J = 8.5 Hz), 7.40 (1H, s), 7.25 ppm (1H, q, J = 3.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 172.51, 156.61, 153.62, 152.90, 150.87, 137.72, 136.44, 131.12, 129.36, 128.05, 127.97, 116.48, 115.84, 115.62, 111.24, 110.92 ppm. MALDI (m/z) = 434.061 [M + H]⁺.

4.2.3.19. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(4‐chlorophenyl)thiazol‐2‐yl)methanimine (B19)

Yellow solid, Yield: 90 %; m.p.: 230°C–231°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.29 (1H, dd, J = 2.0, 6.8 Hz), 9.38 (1H, s), 8.83 (1H, dd, J = 2.0, 4.2 Hz), 7.88 (4H, q, J = 6.8 Hz), 7.57 (2H, d, J = 8.5 Hz), 7.41 (2H, d, J = 8.6 Hz), 7.37 (1H, s), 7.23 ppm (1H, q, J = 3.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 172.58, 156.70, 153.64, 152.70, 150.98, 150.85, 137.70, 136.46, 134.17, 132.76, 131.12, 130.84, 129.35, 128.96, 127.50, 116.46, 111.92, 110.92 ppm. MALDI (m/z) = 450.379 [M + H]⁺.

4.2.3.20. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐phenylthiazol‐2‐yl) methanimine (B20)

Yellow solid, Yield: 78 %; m.p.: 300°C–301°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.30 (1H, d, J = 5,4 Hz), 9.41 (1H, s), 8.82 (1H, s) 7.91(4H, m), 7.57 (2H, d, J = 7 Hz), 7.44 (4H, m), 7.21 ppm (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.70, 158.14, 153.83, 153.30, 151.63, 150.92, 140.32, 137.70, 134.40, 129.88, 129.80, 129.56, 128.75, 128.30, 126.27, 116.27, 111.27, 110.62 ppm. MALDI (m/z) = 416.071 [M + H]⁺.

4.2.3.21. (E)‐1‐(2‐Phenylimidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐(p‐tolyl)thiazol‐2‐yl)methanimine (B21)

Yellow solid, Yield: 88 %; m.p.: 296°C–297°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.35 (1H, dd, J = 2.1, 6.8 Hz), 9.43 (1H, s), 8.83 (1H, dd, J = 2.1, 4.2 Hz), 7.96 (2H, d, J = 6.64 Hz), 7.83 (2H, d, J = 8.0 Hz), 7.62 (3H, d, J = 7.7 Hz), 7.34 (1H, s), 7.27 (2H, d, J = 7.9 Hz), 7.23 (1H, q, J = 3.7 Hz), 2.41 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.46, 157.86, 153.96, 153.35, 151.43, 150.85, 138.21, 137.74, 132.42, 131.65, 130.00, 129.45, 129.03, 126.15, 116.46, 110.72, 110.65, 21.31 ppm. MALDI (m/z) = 396.1211 [M + H]+.

4.2.3.22. (E)‐N‐(4‐(4‐Methoxyphenyl)thiazol‐2‐yl)‐1‐(2‐phenylimidazo[1,2‐a]pyrimidin‐3‐yl) methanimine (B22)

Green solid, Yield: 88 %; m.p.: 330°C–331°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.32 (1H, dd, J = 2.1, 6.8 Hz), 9.41 (1H, s), 8.81 (1H, s), 7.94 (2H, d, J = 6.68 Hz), 7.85 (2H, d, J = 8.1 Hz), 7.59 (3H, d, J = 7.7 Hz), 7.21 (2H, m), 6.9 (2H, d, J = 8.5 Hz), 3.85 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 172.90, 161.58, 161., 153.30, 152.80, 151.65, 151.60, 137.64, 131.38, 129.88, 129.78, 128.05, 127.97, 116.00, 115.76, 114.86, 110.76, 110.56, 55.47 ppm. HR‐MS (m/z) = 412.1162 [M + H]⁺.

4.2.3.23. (E)‐N‐(4‐(4‐Fluorophenyl)thiazol‐2‐yl)‐1‐(2‐phenylimidazo[1,2‐a]pyrimidin‐3‐yl) methanimine (B23)

Yellow solid, Yield: 88 %; m.p.: 338°C–339°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.31 (1H, dd, J = 2.1, 6.8 Hz), 9.40 (1H, s), 8.82 (1H, dd, J = 2.1, 4.2 Hz), 7.94‐7.90 (4H, m), 7.46 (2H, t, J = 4.4 Hz), 7.42 (1H, s), 7.39 (1H, d), 7.37 (2H, q, J = 3.7 Hz), 7.28 ppm(1H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 153.42, 152.85, 151.56, 137.71, 132.38, 130.05, 129.99, 129.04, 128.75, 127.97, 116.44, 115.79, 111.57, 110.95, 110.77 ppm. MALDI (m/z) = 400.112 [M + H]⁺.

4.2.3.24. (E)‐N‐(4‐(4‐Chlorophenyl)thiazol‐2‐yl)‐1‐(2‐phenylimidazo[1,2‐a]pyrimidin‐3‐yl) methanimine (B24)

Yellow solid, Yield: 88 %; m.p.: 318°C–319°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.29 (1H, dd, J = 2.1, 6.8 Hz), 9.37 (1H, s), 8.82 (1H, d, J = 2.0 Hz), 7.94‐7.90 (2H, m), 7.85 (2H, d, J = 8.36 Hz), 7.41 (3H, m), 7.30 (3H, d, J = 8.5 Hz), 7.23 ppm (1H, q, J = 8.7 Hz. ¹³C NMR (100 MHz, CDCl₃): δ = 167.60. 151.74, 149.19, 148.81, 146.03, 132.98, 129.54, 127.54, 126.60, 124.04, 123.63, 121.51, 120.02, 111.75, 106.95, 106.13 ppm. MALDI (m/z) = 416.312 [M + H]⁺.

4.2.3.25. (E)‐1‐(2‐Phenylimidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(4‐phenylthiazol‐2‐yl)methanimine (B25)

Yellow solid, Yield: 88 %; m.p.: 324°C–325°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.31 (1H, dd, J = 3.3 Hz), 9.41 (1H, s), 8.82 (1H, dd, J = 2.1 Hz), 7.94 (5H, m), 7.59 (3H, m), 7.30 (1H, s), 7.22 (1H, q, J = 3.6 Hz), 7.13 ppm (2H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 172.57, 157.93, 153.84, 153.36, 151.44, 150.86, 138.22, 137.74, 132.43, 131.65, 130.00, 129.46, 129.04, 126.16, 116.47, 110.73, 110.65 ppm. HR‐MS (m/z) = 382.1125 [M + H]⁺.

4.2.3.26. (E)‐1‐(2‐(4‐Methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐(p‐tolyl)‐1,3,4‐thiadiazol ‐2‐yl)methanimine (B26)

Yellow solid, Yield: 80 %; m.p.: 326°C–327°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.28 (1H, dd, J = 1.8, 6.8 Hz), 9.26 (1H, s), 8.84 (1H, dd, J = 1.9, 4.1 Hz), 7.89 (4H, t, J = 8.1 Hz), 7.31 (2H, d, J = 8.0 Hz), 7.24 (1H, q, J = 3.7 Hz), 7.13 (2H, d, J = 8.7 Hz), 3.93 (3H, s), 2.44 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 173.85, 166.68, 161.54, 159.13, 154.31, 153.87, 151.36, 141.61, 137.81, 131.40, 129.85, 127.87, 127.49, 124.46, 116.03, 114.69, 110.92, 55.50, 21.54 ppm. HR‐MS (m/z) = 427.1271 [M + H]⁺.

4.2.3.27. (E)‐N‐(5‐(p‐Tolyl)‐1,3,4‐thiadiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl) methanimine (B27)

Yellow solid, Yield: 82 %; m.p.: 188°C–189°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.28 (1H, dd, J = 1.6, 6.7 Hz), 9.24 (1H, s), 8.83 (1H, dd, J = 1.8, 4.4 Hz), 7.86 (4H, d, J = 6.2 Hz), 7.39 (2H, d, J = 8.2 Hz), 7.30 (2H, d, J = 8.4 Hz), 7.24 (1H, m), 2.44 (3H, s), 2.42 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 154.43, 153.88, 141.63, 140.71, 137.83, 129.92, 129.89, 129.85, 129.77, 127.52, 111.01, 21.53, 21.48 ppm. HR‐MS (m/z) = 411.1319 [M + H]⁺.

4.2.3.28. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐(p‐tolyl)‐1,3,4‐thiadiazol‐2‐yl)methanimine (B28)

Yellow solid, Yield: 70 %; m.p.: 344°C–345°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.28 (1H, dd, J = 1.9, 6.8 Hz), 9.25 (1H, s), 8.88 (1H, dd, J = 2.0, 4.1 Hz), 7.88 (4H, d, J = 6.9 Hz), 7.59 (2H, d, J = 8.4 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.27 (1H, d, J = 4.3 Hz), 2.44 ppm(3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 173.47, 167.06, 157.74, 154.15, 153.69, 151.22, 141.75, 137.85, 136.77, 131.10, 130.49, 129.88, 129.47, 127.76, 127.52, 116.46, 111.27, 21.55 ppm. MALDI (m/z) = 431.082 [M + H]⁺.

4.2.3.29. (E)‐1‐(2‐(4‐Bromophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐(p‐tolyl)‐1,3,4‐thiadiazol‐2‐yl)methanimine (B29)

Yellow solid, Yield: 73 %; m.p.: 325°C–326°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.29 (1H, d, J = 6.8 Hz), 9.25 (1H, s), 8.88 (1H, t, J = 2.0 Hz), 7.88 (2H, d, J = 7.7 Hz), 7.81 (2H, d, J = 7.2 Hz), 7.75 (2H, d, J = 8.2 Hz), 7.32 (2H, d, J = 7.8 Hz), 7.26 (1H, s), 2.44 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 173.46, 167.05, 157.76, 154.17, 153.69, 151.22, 141.76, 137.85, 132.42, 131.31, 130.93, 129.88, 127.75, 127.52, 125.14, 116.44, 111.28, 21.55 ppm. MALDI (m/z) = 475.148 [M + H]⁺.

4.2.3.30. (E)‐N‐(5‐(4‐Florophenyl)‐1,3,4‐thiadiazol‐2‐yl)‐1‐(2‐(4‐methoxyphenyl)imidazo[1,2‐a] pyrimidin‐3‐yl)methanimine (B30)

Yellow solid, Yield: 71 %; m.p.: 321°C–322°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.24 (1H, dd, J = 1.7, 6.7 Hz), 9.25 (1H, s), 8.82 (1H, dd, J = 1.8, 4.0 Hz), 7.97 (2H, m), 7.89 (2H, d, J = 8.6 Hz),7.23‐7.15 (3H, m), 7.10 (2H, d, J = 8.6 Hz), 3.90 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 161.62, 154.44, 153.95, 137.77, 131.41, 129.58, 129.58, 129.49, 124.44, 116.50, 116.28, 114.44, 110.94, 55.49 ppm. MALDI (m/z) = 431.353 [M + H]⁺.

4.2.3.31. (E)‐N‐(5‐(4‐Florophenyl)‐1,3,4‐thiadiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl)methanimine (B31)

Yellow solid, Yield: 74 %; m.p.: 339°C–340°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.26 (1H, dd, J = 1.9, 6.8 Hz), 9.26 (1H, s), 8.84 (1H, dd, J = 2.0, 4.1 Hz), 7.98 (2H, dd, J = 5.3 Hz), 7.81 (2H, d, J = 7.9 Hz), 7.40 (2H, d, J = 7.8 Hz), 7.24 (3H, m), 2.47 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 173.44, 167.07, 154.15, 152.24, 141.78, 137.80, 136.74, 131.14, 130.56, 129.92, 128.22, 127.76, 126.54, 116.48, 111.64, 21.46 ppm. MALDI (m/z) = 415.146 [M + H]⁺.

4.2.3.32. (E)‐N‐(5‐(4‐Florophenyl)‐1,3,4‐thiadiazol‐2‐yl)‐1‐(2‐(4‐chlorophenyl)imidazo[1,2‐a] pyrimidin‐3‐yl)methanimine (B32)

Yellow solid, Yield: 68 %; m.p.: 340°C–341°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.25 (1H, dd, J = 1.8, 6.7 Hz), 9.25 (1H, s), 8.86 (1H, dd, J = 2.0, 4.1 Hz), 7.97 (2H, m), 7.85 (2H, d, J = 8.4 Hz), 7.57 (2H, d, J = 8.4 Hz), 7.27 (1H, s), 7.21 ppm (2H, t, J = 8.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 173.82, 157.91, 154.21, 153.79, 137.84, 136.81, 131.20, 131.11, 130.49, 129.48, 129.21, 127.60, 116.47, 111.30 ppm. MALDI (m/z) = 435.062 [M + H]⁺.

4.2.3.33. (E)‐1‐(2‐(4‐Bromophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐(4‐florophenyl)‐1,3,4‐thiadiazol‐2‐yl)methanimine (B33)

Yellow solid, Yield: 70 %; m.p.: 312°C–313°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.25 (1H, d, J = 6.6 Hz), 9.24 (1H, s), 8.86 (1H, d, J = 2.1 Hz), 7.97 (2H, m), 7.78 (2H, d, J = 8.4 Hz), 7.72 (2H, d, J = 8.3 Hz), 7.27 (1H, s), 7.21 ppm (2H, t, J = 8.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 154.23, 153.79, 137.85, 132.43, 131.32, 131.21, 130.91, 130.49, 129.21, 127.61, 125.18, 111.31 ppm. MALDI (m/z) = 479.206 [M + H]⁺.

4.2.3.34. (E)‐1‐(2‐(4‐Methoxyphenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)methanimine (B34)

Yellow solid, Yield: 78 %; m.p.: 334°C–345°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.26 (1H, t, J = 3.3 Hz), 9.27 (1H, s), 8.82 (1H, d, J = 2.1 Hz), 7.97 (2H, t, J = 3.4 Hz), 7.88 (2H, d, J = 8.6 Hz), 7.49 (3H, d, J = 2.0 Hz), 7.23 (1H, q, J = 3.6 Hz), 7.10 (2H, d, J = 8.6 Hz), 3.91 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 174.22, 165.63, 165.29, 161.59, 159.41, 154.46, 153.98, 151.45, 137.80, 131.41, 129.58, 129.50, 126.94, 124.41, 116.51, 116.29, 116.03, 114.71, 110.97, 55.50 ppm. HR‐MS (m/z) = 413.118 [M + H]⁺.

4.2.3.35. (E)‐N‐(5‐Phenyl‐1,3,4‐thiadiazol‐2‐yl)‐1‐(2‐(p‐tolyl)imidazo[1,2‐a]pyrimidin‐3‐yl) methanimine (B35)

Yellow solid, Yield: 80 %; m.p.: 336°C–337°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.26 (1H, dd, J = 1.9, 6.8 Hz), 9.26 (1H, s), 8.84 (1H, dd, J = 2.0, 4.1 Hz), 7.98 (2H, q, J = 8,76 Hz), 7.81 (2H, d, J = 8.4 Hz), 7.40 (2H, d, J = 8.0 Hz), 7.24 (4H, m), 2.47 ppm (3H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 160.56, 149.82, 149.23, 136.03, 133.05, 125.15, 125.11, 124.85, 124.76, 124.38, 111.76, 111.53, 106.30, 22.83 ppm. HR‐MS (m/z) = 397.122 [M + H]⁺.

4.2.3.36. (E)‐1‐(2‐(4‐Chlorophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)methanimine (B36)

Yellow solid, Yield: 76 %; m.p.: 327°C–328°C; ¹H NMR (400 MHz, CDCl₃): δ =: 10.31 (1H, d, J = 6.5 Hz), 9.41 (1H, s), 8.82 (1H, s), 7.86 (4H, m), 7.75 (2H, d, J = 7.8 Hz), 7.42 (3H, m), 7.22 ppm (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 154.26, 153.82, 137.82, 132.44, 131.31, 129.63, 129.54, 116.55, 116.44, 116.32, 111.32 ppm. MALDI (m/s) = 417.278 [M + H]⁺.

4.2.3.37. (E)‐1‐(2‐(4‐Bromophenyl)imidazo[1,2‐a]pyrimidin‐3‐yl)‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)methanimine (B37)

Yellow solid, Yield: 78 %; m.p.: 334°C–335°C; ¹H NMR (400 MHz, CDCl₃): δ = 10.28 (1H, d, J = 6.5 Hz), 9.28 (1H, s), 8.88 (1H, s), 7.99 (2H, s), 7.81 (2H, d, J = 7.8 Hz), 7.55 (2H, d, J = 8.0 Hz), 7.53 (3H, s), 7.30 ppm (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 154.28, 153.84, 137.83, 132.44, 131.32, 129.63, 129.55, 116.56, 116.34, 111.34 ppm. MALDI (m/z) = 462.094 [M + H]⁺.

4.3. Biological Studies

4.3.1. In Vitro Antitubercular Activity

The antitubercular potential of the target molecules (B1‐B37) was evaluated against M. tuberculosis employing the microplate Alamar Blue assay method (MABA). Resazurin is used in this approach because it is heat‐stable, nontoxic, and permeable to the cells. To stop test medium evaporation during incubation, a sterile 96‐well microplate with U‐shaped wells was filled with 200 µL of sterile deionized water in each well. Prepared target molecules and reference medication were dissolved in DMSO and diluted in two‐fold dilutions to generate concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.6, 0.8, 0.4, and 0.2 µg/mL. For all wells of Middlebrook 7H9 broth, add 0.2% glycerol and 10% OADC. Then, 100 µL of M. tuberculosis H37Rv (ATCC 27294) inoculum was added [34]. The test substances were then diluted tenfold. The plates were parafilm‐sealed and incubated for seven days at 37°C. Following the initial incubation, each well received a freshly produced mixture of Alamar Blue reagent and 10% Tween 80 in a 1:1 ratio, and the plates were incubated at 37°C for an additional 24 h. A noticeable color change occurred in the wells, with blue indicating inhibition of bacterial growth and pink representing active bacterial growth. The minimum inhibitory concentration (MIC) was established as the smallest concentration of a substance that totally inhibits bacterial development. Streptomycin (STM) and pyrazinamide (PZA) were used as standard reference medications for comparison. This method is similar to that recommended by the National Committee for Clinical Laboratory Standards for the determination of MIC in triplicate.

4.3.2. In Vitro Bacterial Studies

All target molecules (B1–B37) were tested for antibacterial activity through the zone of inhibition method against four bacterial types: two Gram‐negative strains, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853), and two Gram‐positive strains, Staphylococcus aureus (ATCC 23235) and Bacillus subtilis (ATCC 6051). Sterilized nutrient agar media (100 mL) was poured into two 250‐mL conical flasks and allowed to cool to room temperature. Bacterial subcultures grown for 18–24 h were then added to the media and properly mixed to ensure an even distribution of the organisms. The inoculated agar was then put into equal numbers of sterile petri plates, each carrying approximately 45‐50 mL of medium, and allowed to harden. Following solidification, a sterile cork borer was used to create 6 mm diameter wells in the agar. To make solutions at the necessary concentration (100 µg/mL), the test substance was dissolved in DMSO, and 1 mL of each solution was carefully poured into the wells. The Petri plates were then incubated inverted at 37°C for 24‐48 h. Following incubation, zones of growth inhibition formed around the wells, and their diameters (in mm) were measured and compared to the standard medicines [35, 36].

4.3.3. Cytotoxicity Studies

To assess the drug‐testing capability of the newly synthesized hybrid derivatives, L929 cell lines were used. These cell lines produced from mouse fibroblasts were obtained from the National Centre for Cell Sciences (NCCS) in Pune, India. Cells were plated in 96‐well flat‐bottom microplates and incubated overnight at 37°C, 95% humidity, and 5% CO₂. Samples were added at different amounts (100, 50, 25, 12.5, 6.25, and 3.12 µg/mL), and cells were incubated for 24 h. After incubation, the wells were rinsed twice with PBS. Then, 20 µL of MTT solution was put into each well, and the plate was subjected to incubation at 37°C. After 4 h, 100 µL of DMSO was applied to dissolve the formazan crystals. The absorbance at 570 nm was recorded with a microplate reader [37].

4.4. Computational Studies

4.4.1. In Silico ADME Studies

The main causes for drug discovery failure are toxicity, ineffectiveness, and poor pharmacokinetic properties. Absorption, distribution, metabolism, and excretion (ADME), is a phrase used to characterize pharmacokinetic behavior. These characteristics are mostly dictated by a drug's basic chemical makeup, including its MW, structure, ionization behavior, water solubility, and lipophilicity. Designing medications that are both safe and effective requires an understanding of a compound's ADME profile. The pharmacokinetic characteristics of the target compounds were predicted in this work using Schrödinger's QikProp program.

4.4.2. In Silico Molecular Docking Studies

A molecular docking analysis was carried out to examine the potential binding interactions of imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole hybrid derivatives (B1‐B37) with the target enzymes M. tuberculosis InhA (PDB ID: 1P44) and CYP121 (PDB ID: 4KTF). The InhA/CYP121 enzymes were chosen for molecular docking investigations due to recent discoveries demonstrating a high binding affinity for specific Imidazo[1,2‐a]pyrimidine and thiazol/1,3,4‐thiadiazole derivatives. Initially, the ligands were sketched using the 2D Sketcher and prepared at neutral pH using the LigPrep module (Schrödinger LLC, New York, USA, version 2022). During ligand preparation, the structures were energy‐minimized using the OPLS‐2005 force field, and the appropriate stereochemistry was assigned based on the generated three‐dimensional (3D) coordinates. The structure of protein retrieved from the Protein Data Bank (https://www.rcsb.org/) had been generated using the Protein Preparation Wizard, which includes methods such as deleting heteroatoms and water molecules [38]. The receptor grid was generated using Glide, with the co‐crystallized ligand defining the active‐site region and a van der Waals scaling factor of 0.9. In XP method, ligand atoms are observed as flexible, allowing them to change position and conformations to generate the optimum interactions with the protein's active site [39]. The docking grid was centered at X = 14.07, Y = 14.18, and Z = 8.69 Å. The redocked pose was then superimposed onto the experimentally determined crystallographic pose. The resulting RMSD value of 1.456 Å indicated good agreement between the redocked and native ligand conformations, thereby validating the reliability of the applied docking protocol. The docking data were examined using docking scores together with other molecular interactions, such as hydrogen bonding and π‐π stacking, produced between the imidazo[1,2‐a]pyrimidine‐thiazole/1,3,4‐thiadiazole hybrids and the amino acid residues situated in the active regions of the target proteins.

4.4.3. DFT Studies

Comprehending pharmaceutical products compounds' stability, reactivity, and interaction patterns requires assessing their energy properties. Drug design has a strong emphasis on evaluating drug‐target interactions, which may include covalent bonds, dipole‐dipole, ion‐dipole interactions, and hydrogen bonding, or hydrophobic effects. The reliability of density functional theory (DFT) in forecasting the strength of these interactions is essential for assessing its efficacy in computer‐aided medication development. All of the proposed molecules were studied utilizing Schrödinger's Jaguar platform to get HOMO, LUMO, and MESP values. This study was carried out utilizing the B3LYP functional (Lee‐Yang‐Parr correlation) with the 6‐31G++ (d, p) basis set, as well as hybrid DFT, which integrates the Becke three‐parameter exchange and the Lee‐Yang‐Parr correlation potential [40]. DFT can figure out important electronic properties that affect drug behavior, such as HOMO‐LUMO energies, band gap (∆E), electron affinity (EA), dipole moment, chemical softness (σ), IP, electronegativity (χ), electrophilicity index (ω), chemical potential (μ), and nucleophilicity (N). These characteristics are crucial in defining a molecule's reactivity, stability, and possible biological interactions [41].

4.4.4. Molecular Dynamic Stimulation

To assess the structural stability and dynamic behavior of the 1P44‐B30 protein–ligand complex, a 100 ns MD simulation was carried out using GROMACS 2026.2. Discovery Studio was used to create the protein, and the CHARMM27 force field was used with ligand parameters from Swiss‐Param [42]. A TIP3P water‐filled dodecahedral box was used to solvate the complex, neutralize it with Na⁺/Cl⁻ ions, and then minimize its energy [43]. After NVT and NPT equilibration at 300 K and 1 bar, respectively, a 100 ns production run with a 2‐fs time step was conducted [44]. PME was used for long‐range electrostatics, and LINCS was employed for bond restrictions. After periodic boundary correction, centering, and fitting, the trajectory was examined for RMSD, RMSF, radius of gyration (Rg), SASA, and hydrogen‐bond interactions to evaluate the complex's stability, flexibility, compactness, solvent accessibility, and binding behavior.

Author Contributions

Dinesha P: conceptualization, investigation, writing – original draft, methodology, validation, formal analysis, data curation. Shivakumar Naik: methodology. Udayakumar D: conceptualization, funding acquisition, writing – original draft, writing – review and editing, validation, project administration, supervision. Sampat Parayya Vibhutimath: methodology. Rajkumar Reddy Rajula: methodology, validation. Revanasiddappa B.c: methodology, supervision. Nanditha Bhat: methodology, validation. Naveena S. Veeranagaiah: methodology, validation.

Funding

There is no specific funding to this work.

Conflicts of Interest

The authors declare no conflicts of interest

Supporting information

Supporting File 1: cbdv71857‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The structural characterization data (¹H NMR, ¹³C NMR, and HR‐MS Spectra of the compounds) are provided in the Supporting Information.