Introduction

Despite the recent significant increase in the discovery of compounds with antimicrobial activities, the use of these compounds remains rather limited due to the difficulties of application, high risks of toxicity, drug resistance, undesired side effects, pharmacokinetic deficiencies and/or inadequate antimicrobial activities. Hence, synthetic organic chemists have been interested in developing biological active compounds with minimal side effects.

Imidazole and 1,3,4-thiadiazole are well known heterocyclic compounds widely used in pharmaceutical chemistry (Kim et al. 2013; Padmavathi et al. 2011; Singh et al. 2016; El-Gohary and Shaaban 2013). The synthesis and characterization of imidazole and 1,3,4-thiadiazole derivatives having various biological activities are still being investigated today (Banu et al. 2010; Romagnoli et al. 2015; Kamal et al. 2014). These heterocyclic systems in which imidazole and 1,3,4-thiadiazole rings are fused together with a bridgehead nitrogen atom are referred to as imidazo[2,1-b][1,3,4]thiadiazoles (Alwan et al. 2015).

Imidazo[2,1-b][1,3,4]thiadiazoles and their heterocyclic derivatives are known to exhibit several biological activities, including antibacterial (Luo et al. 2013; Chandrakantha et al. 2014; Atta et al. 2011), antifungal (Lata et al. 2015), antimicrobial (Lamani et al. 2009; Alegaon and Alagawadi 2011), anti-inflammatory (Kadi et al. 2007; Gadad et al. 2008; Jadhav et al. 2008), and antituberculosis (Ramprasad et al. 2015; Alegaon et al. 2012) activities. Being a bio-isostere of Levamisole (which is used to regulate and strengthen the immune system), these compounds have attracted many researchers studying anticancer activities (Tegginamath et al. 2013; Patel et al. 2015; Noolvi et al. 2011; Terzioglu and Gursoy 2003; Noolvi et al. 2012; Kumar et al. 2014; Karki et al. 2011). In addition, several studies conducted with imidazo[2,1-b][1,3,4]thiadiazoles and their derivatives reported that these compounds was being used in various industries, such as dye, herbicide and insecticide production (Bakherad et al. 2010; Jalhan et al. 2012). Hence, imidazo[2,1-b][1,3,4]thiadiazoles and derivatives have become important members of the heterocyclic compounds in pharmaceutical chemistry.

The most commonly used method to synthesize imidazo[2,1-b][1,3,4]thiadiazole derivatives is the reaction of 2-amino-1,3,4-thiadiazole derivatives with appropriate phenacyl bromide derivatives (Tzitzikas et al. 2013). However, a new method known as Suzuki-Miyaura cross-coupling reactions has recently been used to synthesize these compounds (Copin et al. 2012; Kotha et al. 2002).

In the light of the aforementioned literature survey, the aims of this study include the following: the synthesis of imidazo[2,1-b][1,3,4]thiadiazole derivatives with potential biological activities; the characterization of these compounds with various spectroscopic methods; and the investigation of in vitro antifungal activity. The computational studies were also utilized to gain insight into the binding mode of the compounds and the effect of substituents on the activities. The synthetic method used to synthesize these compounds is shown below in Scheme 1.

Scheme 1
scheme 1

Synthesis of substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives 4-21

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Results and discussion

Chemistry

In the first part of the study, we synthesized 2-amino-1,3,4-thiadiazole derivatives 2a, b from the reactions of nitrile compounds 1a, b with thiosemicarbazide in trifluoroacetic acid (TFA) at 60 °C with high yields (75 and 88%). 2-Amino-1,3,4-thiadiazole derivatives 2a, b were obtained as specified in the literature (Er et al. 2014; Er et al. 2016).

In the infrared (IR) spectra of 2-amino-1,3,4-thiadiazole derivatives 2a, b, two peaks were observed at 3265, 3088 cm−1 which corresponding to the symmetric and asymmetric absorption bands of –NH2 group.

The structures of 2-amino-1,3,4-thiadiazole derivatives 2a, b were confirmed by 1H NMR spectroscopy, as well. In the 1H nuclear magnetic resonance (NMR) spectra, the proton signals of -NH2 group in 2a, b which bonded to the 1,3,4-thiadiazole ring at the C-2 position were recorded as a singlet in the range between 7.04–7.09 ppm corresponding to two protons. Proton peaks belonging to the -NH2 group of these compounds 2a, b disappeared as a result of the proton-deuterium exchange performed with D2O. The methylene (–CH2) protons bonding the thiophene and phenyl groups to the thiadiazole ring at the 5-position were observed as a singlet corresponding to two protons in the range between 4.02–4.36 ppm.

The structures of 2-amino-1,3,4-thiadiazole derivatives 2a, b were also confirmed by the 13C NMR spectrum. C-2 carbon signals of the 2-amino-1,3,4-thiadiazole ring in these compounds were recorded in the range between 159.07, 157.57 ppm, and C-5 carbon signals were recorded in the range between 169.75, 169.44 ppm. In the 13C NMR spectra, it was observed that the resonance values of carbons at the C-2 and C-5 positions of the 2-amino-1,3,4-thiadiazole ring were highly compatible with these types of compounds in the literature (Sancak et al. 2007). Other spectral data pertaining to the carbon skeleton of the compounds fully support the suggested structures. Experimental part, the physical properties and the spectral data related to compounds 2a, b are provided in detail in the Supplementary Material Section.

In the second part of this study, we synthesized imidazo[2,1-b][1,3,4]thiadiazole derivatives 4–21, the target compounds, by the reactions of 2-amino-1,3,4-thiadiazole derivatives 2a, b with phenacyl bromide derivatives 3 in absolute ethyl alcohol with moderate-high yields (52–69%) (Scheme 1). According to the IR spectral data, -NH2 symmetric and asymmetric absorption bands of the compounds 2a, b observed at 3265, 3088 cm−1 were disappeared for the compounds 4–21. This IR data demonstrates the most important evidence for the formation of imidazo[2,1-b][1,3,4]thiadiazole derivatives 4–21.

Also, the -NH2 group proton signals observed in the 7.04–7.09 ppm range disappeared in the 2-amino-1,3,4-thiadiazole derivatives which were the precursors of imidazo[2,1-b][1,3,4]thiadiazole derivatives 4–21 in the 1H NMR spectra. Instead, a singlet was observed that corresponds to one proton in the 8.48–8.94 ppm range representing the C5-H signals in imidazo[2,1-b][1,3,4]thiadiazole derivatives 4–21, which also provides distinct evidence for the formation of these compounds. This data is consistent with the literature (Lamani et al. 2009; Karki et al. 2011).

Signals that appeared in the 109.60–113.61 ppm range and the 143.17–145.68 ppm range in 13C NMR spectra of these compounds are important evidence of ring cyclization. These signals correspond to the C5 and C6 carbons of imidazo[2,1-b][1,3,4]thiadiazole derivatives. Other 1H NMR and 13C NMR spectra data of these compounds are given in detail in the experimental part. The 1H NMR and 13C NMR spectra of all synthesized compounds are given in the Supplementary Material Section.

In addition, the mass spectra of all synthesized compounds were observed to be as expected and supported by molecular ion peaks.

The structures of compounds 7, 8, 10, and 17 were also confirmed by X-ray diffraction analysis. The crystal structures and crystallographic data of compounds 7, 8, 10, and 17 are shown in Fig. 1 and Table 1, respectively. In addition, rotational disorder was observed on the thiophene ring of the compound 10. Bond lengths, bond angles and packing structures of compounds 7, 8, 10, and 17 are given in the Supplementary Material Section.

Fig. 1
figure 1

The crystal structures of compounds 7, 8, 10, and 17

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Table 1 Structural parameters of compounds 7, 8, 10, and 17
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Molecular docking studies

Molecular docking studies were performed to see possible binding modes of the synthesized compounds. A high resolution (1.6 Å) X-ray crystal structures of trichothecene 3-O-acetyltransferase (PDB ID: 2RKV) in complexed with Coenzyme A and T-2 mycotoxin (ZBA, native ligand) was used in calculations. Although it is well known that the docking software performs well in generating biologically active conformations of the ligands and identifying the binding modes to the target, however, the present scoring functions are not expected to discriminate between active and inactive compounds in such a success rate (Ece and Sevin 2013; Mascarenhasa and Ghoshal 2008). Docking scores of the compounds 4–21 together with that of the native ligand, ZBA are given in Table 2.

Table 2 Docking score and selected molecular properties of ZBA and the compounds 4–21
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Glide docking performed well in ranking the most active compound 10 which has the second highest docking score amongst the synthesized compounds. ZBA has the highest docking score and it fits the binding pocket by making hydrogen bonds with Tyrosine 413 and Histidine 156 (Fig. 2). Although compound 10 does not make hydrogen bonds with the amino acid side chains of the target, it has a higher solvent exposure area that can in consequence, make the compound accommodate easily in the binding pocket without much conformational changes.

Fig. 2
figure 2

2D ligand interaction diagram for ZBA (top) and compound 10 (bottom)

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Binding surfaces of the target and ZBA, compounds 10 and 17 (the native ligand, the most and the least active compounds towards Alternaria solani, respectively) were generated and colored by electrostatic potential map (Fig. 3). Red color indicates high electron density regions while blue color shows low electron density zones. The binding conformation of compound 10 and ZBA look very similar. Compound 10 has favorable electrostatic interactions with the target. Low electron density region of the ligand interacts with the high electron density region in the active site. On the other hand, unfavorable interactions can be observed for the ligand 17. Yet, its binding conformation is very different from ZBA and compound 10 (Fig. 3).

Fig. 3
figure 3

Binding surface of the target together with ZBA (left), compound 10 (middle) and compound 17 (right)

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In vitro antifungal activity and SAR (structure-activity relationship) studies

Antifungal activity values (inhibition zone, percentage inhibition) of all synthesized compounds (2a, 2b, and 4–21) against plant pathogens are shown in Tables 3 and 4. All compounds used against plant pathogens showed varying levels of antifungal activity. Thiram 80% (reference drug) was used as a positive control. It showed 69% inhibition against Alternaria solani, 64% against Fusarium oxysporum f. sp. Lycopersici, and 43% against Vertcillium dahliae. Dimethylsulfoxide (DMSO), which was used as the negative control, showed no antifungal activity. Synthesized compounds were taken in four different doses (5, 2.5, 1.25 and 0.625 mg/mL) and showed 12–46% inhibition against Alternaria solani, 9–42% against Fusarium oxysporum f. sp. Lycopersici, and 2–32% against Vertcillium dahliae. Each compound showed varying levels of antifungal activity depending on the plant pathogens. The inhibition rates of compounds in the positive control demonstrate that the compounds produced moderate to high activities. We also calculated the LD50 values of the compounds (Fig. 4). According to these values, the most affected fungus type was Alternaria solani, followed by Fusarium oxysporum f. sp. Lycopersici and Vertcillium dahliae, respectively. LD50 dose values were in the 6.8–14.4 mg/mL range for Alternaria solani, in the 9.4–20.3 mg/mL range for Fusarium oxysporum f. sp. Lycopersici, and in the 8.6–43.3 mg/mL range for Vertcillium dahliae. Considering these inhibition rates and LD50 dose values, the highest antifungal activity was related to compounds 8, 9, 10, 11, and 16 for Alternaria solani; compounds 2a, 6, 10, 11, 13, 15, 19, and 20 for Fusarium oxysporum f. sp. Lycopersici; and compounds 2b, 6, 10, 11, 12, 13, 16, 19, 20, and 21 for Vertcillium dahliae.

Table 3 Antifungal activity (inhibition zone) of all compounds against plant pathogens
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Table 4 Percentage inhibition of all new compounds against plant pathogens
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Fig. 4
figure 4

LD50 doses of all compounds against plant pathogens. LD50 values were calculated from dose-response by Probit analysis

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Furthermore, a structure-activity relationship (SAR) was also studied. Our primary aim in the SAR study was the modification of R group with thiophene and 3,4-dimethoxyphenyl moieties. Then, different substitution patterns were carefully selected as R1 to confer the effect of different electronic environments on the activities. Thus, electron donating groups such as methoxy, phenyl and napthyl, and electron withdrawing groups such as nitro, cyano, fluoro, chloro, and bromo were chosen as substituents on structure of the target compounds (Fig. 5).

Fig. 5
figure 5

SAR of imidazo[2,1-b][1,3,4]thiadiazole derivatives as antifungal agents

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In general, when R is the thiophene ring, the compounds 4–12 showed a higher activity (Tables 3 and 4, Fig. 4). Steric hindrance of 3,4-dimethoxyphenyl compared to thiophene could be attributed to this observation. As can be seen from Fig. 3, binding conformation of the least active compound 17 (towards Alternaria solani) is very different from that of the native ligand ZBA and the most active compound 10. We believe that the bulky group forces compound 17 to fit into the binding pocket in a vertical position relative to ZBA and compound 10.

In an attempt to see the substitution effect on the observed activities, we calculated highest occupied molecular orbital (HOMO) energy values and also octanol-water partition coefficients (QPlogPo/w) which shows the hydrophilic/hydrophobic property of the compounds studied. According to the QPlogPo/w values tabulated in Table 2, hydrophilic character is clearly one of the crucial factors that enhances activity. The active compounds also seem to have low HOMO energy values. Compound 10 which is found to be the highest active compound towards all three pathogens, has the lowest octanol-water partition coefficient. It also has one of the lowest HOMO energy value which results from the electron withdrawing substituent, –CN. The least active compound 17 towards Alternaria solani has high HOMO energy as can be expected from electron donating –OCH3 group.

The combination of experimental findings on SAR study and computational works revealed that the presence of electron-withdrawing groups and less bulky groups at the para position on benzene ring have significant effects on the antifungal activity.

Conclusion

In conclusion, imidazo[2,1-b][1,3,4]thiadiazole derivatives, the target compounds of this study, were synthesized with simple and practical methods. The structures of synthesized compounds were elucidated by various spectroscopic methods, including IR, 1H NMR, 13C NMR, elemental analysis, mass spectroscopy and X-ray diffraction analysis. The in vitro antifungal activity of the synthesized compounds was evaluated against plant pathogens. Most of the compounds showed considerable antifungal activity. In addition, molecular docking studies were performed for all synthesized compounds to determine the potential binding mode of inhibitors. From the SAR study and with the help of computational works, it was observed that the presence of electron-withdrawing and low steric groups at the para position on benzene ring enhanced the antifungal activity. The best active compound 10 was found to have the lowest octanol/water partition coefficient, high HOMO energy and high docking score.

The molecular docking results, along with the biological assay data, show that all the synthesized compounds have promising biological activities and might be novel potential drug candidates.

Experimental section

General methods

The 1H NMR and 13C NMR spectra of the compounds were measured in DMSO-d6 using an Agilent NMR VNMRS spectrometer at 400 MHz and 100 MHz, respectively. Chemical shift values are given in ppm (δ) with tetramethylsilane as the internal standard. The IR spectra were recorded in a Bruker Optics Alpha Fourier transform-infrared in attenuated total reflectance (ATR). The mass spectra were measured with a Thermo TSQ Quantum Access Max LC-MS/MS spectrometer. Elemental analyses were performed for a LECO 932 CHNS (Leco-932, St. Joseph, MI, USA) instrument and the results were within ± 0.4% of the theoretical values. Melting points were recorded on a Thermo Scientific IA9000 series apparatus and were uncorrected. The nitrile derivatives (1a–b) were procured from commercial suppliers (Biostar-Turkey). All the chemicals, reagents and solvents were directly purchased from Sigma-Aldrich (St. Louis, MO) and were used without further purification, unless mentioned specifically.

Crystallographic analysis

A suitable crystal was selected and placed (Bruker 2008) on a ‘Bruker APEX-II CCD’ diffractometer. The crystal was kept at 293(2) K during the data collection process. Using Olex2 (Dolomanov et al. 2009), the structure was determined by the ShelXT (Sheldrick 2015) structure solution program using Direct Methods and refined with the ShelXL (Sheldrick 2015) refinement package using Least Squares minimization. The positions of hydrogen atoms were obtained geometrically according to the overlap method. When placing hydrogen atoms geometrically, the bond length was fixed as aromatic C–H 0.93 Å, methylene C-H2 at 0.97 Å, and methyl C–H3 at 0.96 Å.

Fungal isolate

The following plant pathogens were used in the antifungal activity studies: Alternaria solani, Fusarium oxysporum f.sp. lycopersici and Vertcillium dahliae. The fungal pathogens were isolated from tomatoes in Antalya, Turkey. The pathogens were grown on a PDA (potato dextrose agar) medium at 22 ± 2 °C for about 7 days.

Computational section

Ligand and protein preparation

Before going any further in computational works, all the compounds have to be prepared for calculations. Hence, the compounds 4–21 were prepared using LigPrep module of Schrödinger suite. Optimized potential liquid simulations 3 (OPLS3) force field was used for minimizations (Harder et al. 2015). All possible states of the compounds at pH 7.0 ± 2.0 were generated.

Molecular docking studies were carried out using X-ray crystal structures of trichothecene 3-O-acetyltransferase (PDB ID: 2RKV) in complexed with Coenzyme A and T-2 mycotoxin (ZBA) having a resolution of 1.6 Å. Prior to calculations, this raw crystal structure was prepared using the Protein Preparation Wizard (PrepWizard) in Maestro of Schrödinger software package (Schrödinger 2016a). As a first step, hydrogen atoms were added and any water and heteroatoms except native ligand (ZBA) were removed in the PrepWizard. The protein structure was then refined by correcting the missing side chain atoms and assigning the bond orders. After the optimization step, in order to remove the steric clashes between the atoms, the refined structure was further minimized using OPLS3 force field.

Molecular docking

The docking calculations were performed using the Glide SP (standard precision) module of Schrödinger Suite (Friesner et al. 2004; Halgren et al. 2004; Friesner et al. 2006). A grid that represents the binding pocket was generated using the default settings. The native ligand was used as a reference to choose the center and size of the receptor grid.

Calculation of molecular properties

HOMO enery values were calculated using Wavefunction’s Spartan ‘16 parallel suite (Spartan 16, Wavefunction Inc., Irvine CA). The equilibrium geometry and orbital energies of each compound were calculated at ground state using Semi-Empirical Parametric model number 3 (PM3). QikProp module of Schrodinger was used to calculate octanol-water partition coefficients of the compounds (Schrödinger 2016b).

Synthesis

General procedure for the synthesis of imidazo[2,1-b][1,3,4]thiadiazole derivatives 4–21

In a two-necked flask, 2-amino-1,3,4-thiadiazole derivatives 2a–b (0.004 mol) were dissolved in absolute ethanol (30 mL). Phenacyl bromide derivatives 3a–i (0.04 mol) were also dissolved in absolute ethanol (20 mL) and then added drop by drop to this solution at room temperature with the assistance of a dropping funnel. The mixture was then refluxed and stirred for 12–16 h. The progress of reaction was monitored by thin layer chromatographyat appropriate time intervals. The excess solvent was removed under reduced pressure and neutralized by an aqueous sodium carbonate (Na2CO3) solution. The solution was filtered and washed with deionized water. The solid matter was recrystallized from acetone. The synthesized compounds were dried with P2O5 in a vacuum oven. The physical properties and spectral data derived from the obtained products are listed below.

6-Phenyl-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (4)

Light yellow crystals, yield 0.74 g (62%), mp 167–169 °C (from Acetone); IR (ATR, cm−1): 3110 (Ar–CH), 2979 (Aliph. CH), 1594 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.68 (s, 2H, -CH2), Thiophene-H [7.02 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 4.0 Hz, 1H)], Phenyl-H [7.25 (t, J = 12.0 Hz, 1H), 7.38 (t, J = 12.0, 2H), 7.83 (d, J = 8.0 Hz, 2H)], Imidazole-H [8.64 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.65 (–CH2), Thiophene-C [125.08 (CH), 126.80 (CH), 127.72 (CH), 145.41 (C)], Phenyl-C [127.82 (CH), 128.09 (CH), 129.10 (CH), 134.30 (C)], Imidazole-C [110.77 (CH), 145.32 (C)], Thiadiazole-C [137.83 (C), 164.43 (C)]; MS: m/z 298.18 (M+1, 100). Anal. Calcd. for C15H11N3S2: C, 60.58; H, 3.73; N, 14.13. Found: C, 60.64; H, 3.79; N, 14.16.

6-(4-Bromophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (5)

Light gray crystals, yield 0.89 g (59%), mp 176–178 °C (from Acetone); IR (ATR, cm−1): 3128 (Ar–CH), 2981 (Aliph. CH), 1596 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.68 (s, 2H, –CH2), Thiophene-H [7.02 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 4.0 Hz, 1H)], Phenyl-H [7.57 (dd, J = 4.0, 2.0 Hz, 2H), 7.78 (dd, J = 4.0, 2.0 Hz, 2H)], Imidazole-H [8.70 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.65 (–CH2), Thiophene-C [126.83 (CH), 127.06 (CH), 127.82 (CH), 145.56 (C)], Phenyl-C [120.59 (C), 128.10 (CH), 132.03 (C), 133.59 (CH)], Imidazole-C [111.24 (CH), 144.23 (C)], Thiadiazole-C [137.76 (C), 164.77 (C)]; MS: m/z 376.19 (M+, 95), 378.15 (M+2, 100). Anal. Calcd. for C15H10BrN3S2: C, 47.88; H, 2.68; N, 11.17. Found: C, 47.84; H, 2.65; N, 11.14.

6-(4-Chlorophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (6)

Gray crystals, yield 70.81 g (61%), mp 170–172 °C (from Acetone); IR (ATR, cm−1): 3124 (Ar–CH), 2974 (Aliph. CH), 1594 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.68 (s, 2H, –CH2), Thiophene-H [7.02 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 4.0 Hz, 1H)], Phenyl-H [7.43 (dd, J = 3.6, 2.0 Hz, 2H), 7.84 (dd, J = 3.6, 2.0 Hz, 2H)], Imidazole-H [8.68 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.64 (–CH2), Thiophene-C [126.73 (CH), 126.82 (CH), 127.82 (CH), 145.54 (C)], Phenyl-C [128.11 (CH), 129.13 (CH), 132.07 (C), 133.22 (C)], Imidazole-C [111.20 (CH), 144.20 (C)], Thiadiazole-C [137.76 (C), 164.73 (C)]; MS: m/z 332.30 (M+1, 100). Anal. Calcd. for C15H10ClN3S2: C, 54.29; H, 3.04; N, 12.66. Found: C, 54.34; H, 3.00; N, 12.63.

6-(4-Fluorophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (7)

Light yellowish crystals, yield 0.81 g (64%), mp 172–173 °C (from Acetone); IR (ATR, cm−1): 3139 (Ar–CH), 2941 (Aliph. CH), 1585 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.68 (s, 2H, –CH2), Thiophene-H [7.02 (q, J = 8.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H)], Phenyl-H [7.22 (t, J = 12.0 Hz, 2H), 7.86 (q, J = 12.0, 2H)], Imidazole-H [8.62 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.63 (–CH2), Thiophene-C [126.80 (CH), 127.04 (CH), 127.81 (CH), 145.36 (C)], Phenyl-C [116.08 (CH), 128.09 (CH), 130.87 (C), 163.16 (C)], Imidazole-C [110.62 (CH), 144.50 (C)], Thiadiazole-C [137.80 (C), 164.46 (C)]; MS: m/z 315.96 (M+, 100). Anal. Calcd. for C15H10FN3S2: C, 57.12; H, 3.20; N, 13.32. Found: C, 57.22; H, 3.25; N, 13.35.

6-(4-Methoxyphenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (8)

Light yellow crystals, yield 0.84 g (64%), mp 166–167 °C (from Acetone); IR (ATR, cm−1): 3127 (Ar–CH), 2923 (Aliph. CH), 1599 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.76 (s, 3H, –OCH3), 4.67 (s, 2H, –CH2), Thiophene-H [7.02 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 4.0 Hz, 1H)], Phenyl-H [6.95 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0, 2H)], Imidazole-H [8.51 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.63 (–CH2), 55.56 (-OCH3), Thiophene-C [126.78 (CH), 126.98 (CH), 127.80 (CH), 145.48 (C)], Phenyl-C [114.52 (CH), 126.42 (C), 128.04 (CH), 159.12 (C)], Imidazole-C [109.60 (CH), 145.03 (C)], Thiadiazole-C [137.89 (C), 163.92 (C)]; MS: m/z 328.19 (M+1, 100). Anal. Calcd. for C16H13N3OS2: C, 58.69; H, 4.00; N, 12.83. Found: C, 58.64; H, 4.04; N, 12.88.

6-(4-Nitrophenyl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (9)

Yellow solid, yield 0.81 g (59%), mp 216–218 °C (from Acetone); IR (ATR, cm−1): 3125 (Ar–CH), 2947 (Aliph. CH), 1597 (C=N), 1520, 1336 (NO2); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.71 (s, 2H, -CH2), Thiophene-H [7.03 (s, 1H), 7.14 (s, 1H), 7.50 (d, J = 4.0 Hz, 1H)], Phenyl-H [8.09 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0, 2H], Imidazole-H [8.94 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.67 (–CH2), Thiophene-C [126.90 (CH), 127.84 (CH), 128.20 (CH), 145.89 (C)], Phenyl-C [124.67 (CH), 125.70 (CH), 140.86 (C), 146.55 (C)], Imidazole-C [113.53 (CH), 143.17 (C)], Thiadiazole-C [137.60 (C), 165.77 (C)]; MS: m/z 343.22 (M+1, 100). Anal. Calcd. for C15H10N4O2S2: C, 52.62; H, 2.94; N, 16.36. Found: C, 52.65; H, 2.98; N, 16.31.

4-(2-(Thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl)benzonitrile (10)

Gray crystals, yield 0.62 g (52%), mp 210–212 °C (from Acetone); IR (ATR, cm−1): 3109 (Ar–CH), 2930 (Aliph. CH), 2277 (C≡N), 1589 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.70 (s, 2H, -CH2), Thiophene-H [7.03 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 4.0 Hz, 1H)], Phenyl-H [7.85 (dd, J = 4.0, 2.0 Hz, 2H), 8.01 (dd, J = 4.0, 2.0 Hz, 2H], Imidazole-H [8.87 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.66 (-CH2), 119.48 (C≡N), Thiophene-C [126.87 (CH), 127.83 (CH), 128.18 (CH), 146.17 (C)], Phenyl-C [112.95 (C), 125.54 (CH), 133.21 (CH), 138.82 (C)], Imidazole-C [109.75 (CH), 143.55 (C)], Thiadiazole-C [137.64 (C), 165.50 (C)]; MS: m/z 322.82 (M+, 100). Anal. Calcd. for C16H10N4S2: C, 59.61; H, 3.13; N, 17.38. Found: C, 59.58; H, 3.18; N, 17.35.

6-([1,1′-Biphenyl]-4-yl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (11)

Gray solid, yield 0.97 g (65%), mp 190–191 °C (from Acetone); IR (ATR, cm−1): 3093 (Ar–CH), 2961 (Aliph. CH), 1580 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.69 (s, 2H, –CH2), Thiophene-H [7.02 (s, 1H), 7.13 (s, 1H), 7.50 (s, 1H)], Phenyl-H [7.69 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.0, 2H)], Phenyl-Phenyl-H [7.35 (t, J = 8.0 Hz, 1H), 7.45–7.43 (m, 4H)], Imidazole-H [8.70 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.65 (–CH2), Thiophene-C [125.62 (CH), 126.82 (CH), 128.09 (CH), 145.45 (C)], Phenyl-C [127.82 (CH), 129.62 (CH), 133.46 (C), 140.16 (C)], Phenyl-Phenyl-C [126.90 (CH), 127.54 (CH), 129.39 (CH), 139.31 (C)], Imidazole-C [110.96 (CH), 145.06 (C)], Thiadiazole-C [137.84 (C), 164.50 (C)]; MS: m/z 373.85 (M+, 100). Anal. Calcd. for C21H15N3S2: C, 67.53; H, 4.05; N, 11.25. Found: C, 67.60; H, 4.09; N, 11.29.

6-(Naphthalen-2-yl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazole (12)

Gray solid, yield 0.76 g (55%), mp 170–172 °C (from Acetone); IR (ATR, cm−1): 3109 (Ar–CH), 2942 (Aliph. CH), 1584 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 4.70 (s, 2H, –CH2), Thiophene-H [7.03 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 4.0 Hz, 1H)], Naphthyl-H [7.50–7.47 (m, 2H), 7.93–7.87 (m, 3H), 7.99 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H)], Imidazole-H [8.77 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 31.67 (-CH2), Thiophene-C [126.84 (CH), 127.84 (CH), 128.62 (CH), 145.61 (C)], Naphtyl-C [123.18 (CH), 123.86 (CH), 126.24 (CH), 128.08 (CH), 128.13 (CH), 128.37 (CH), 131.79 (C), 132.82 (C), 133.68 (C)], Imidazole-C [111.33 (CH), 145.38 (C)], Thiadiazole-C [137.81 (C), 164.56 (C)]; MS: m/z 347.95 (M+, 100). Anal. Calcd. for C19H13N3S2: C, 65.68; H, 3.77; N, 12.09. Found: C, 65.65; H, 3.81; N, 12.14.

2-(3,4-Dimethoxybenzyl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazole (13)

White solid, yield 0.94 g (67%), mp 170–171 °C (from Acetone); IR (ATR, cm−1): 3125 (Ar–CH), 2923 (Aliph. CH), 1591 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.73 (s, 6H, 2-OCH3), 4.33 (s, 2H, -CH2), (OCH3)2Ar–H [6.93 (q, J = 12.0 Hz, 2H), 6.99 (s, 1H)], Phenyl-H [7.24 (t, J = 12.0 Hz, 1H), 7.38 (t, J = 12.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H)], Imidazole-H [8.61 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 37.01 (–CH2), 55.96 (–OCH3), (OCH3)2Ar–C [110.67 (CH), 112.48 (CH), 121.74 (CH), 129.08 (C), 148.68 (C), 149.36 (C)], Phenyl-C [125.05 (CH), 127.66 (CH), 128.57 (CH), 134.36 (C)], Imidazole-C [113.31 (CH), 145.38 (C)], Thiadiazole-C [145.25 (C), 165.75 (C)]; MS: m/z 352.26 (M+1, 100). Anal. Calcd. for C19H17N3O2S: C, 64.94; H, 4.88; N, 11.96. Found: C, 64.99; H, 4.85; N, 11.92.

6-(4-Bromophenyl)-2-(3,4-dimethoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole (14)

Yellow solid, yield 1.19 g (69%), mp 172–174 °C (from Acetone); IR (ATR, cm−1): 3120 (Ar–CH), 2924 (Aliph. CH), 1592 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.72 (s, 6H, 2-OCH3), 4.33 (s, 2H, -CH2), (OCH3)2Ar–H [6.93 (q, J = 12.0 Hz, 2H), 6.99 (s, 1H)], Phenyl-H [7.57 (d, J = 8.0 Hz, 2H), 7.78 (t, J = 8.0 Hz, 2H)], Imidazole-H [8.67 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 37.01 (–CH2), 55.96 (–OCH3), (OCH3)2Ar–C [111.14 (CH), 112.48 (CH), 120.52 (CH), 128.52 (C), 148.69 (C), 149.36 (C)], Phenyl-C [121.75 (C), 127.03 (CH), 132.02 (C), 133.65 (CH)], Imidazole-C [113.30 (CH), 145.63 (C)], Thiadiazole-C [144.08 (C), 166.08 (C)]; MS: m/z 430.39 (M+, 60), 432.35 (M+2, 90). Anal. Calcd. for C19H16BrN3O2S: C, 53.03; H, 3.75; N, 9.76. Found: C, 53.11; H, 3.78; N, 9.79.

6-(4-Chlorophenyl)-2-(3,4-dimethoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole (15)

Yellow solid, yield 0.94 g (61%), mp 163–165 °C (from Acetone); IR (ATR, cm−1): 3121 (Ar–CH), 2923 (Aliph. CH), 1592 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.72 (s, 6H, 2-OCH3), 4.33 (s, 2H, -CH2), (OCH3)2Ar-H [6.93 (q, J = 12.0 Hz, 2H), 6.99 (s, 1H)], Phenyl-H [7.43 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H)], Imidazole-H [8.66 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 37.01 (–CH2), 55.95 (-OCH3), (OCH3)2Ar–C [111.10 (CH), 112.48 (CH), 121.74 (CH), 129.11 (C), 148.70 (C), 149.36 (C)], Phenyl-C [126.71 (CH), 128.52 (CH), 132.00 (C), 133.29 (C)], Imidazole-C [113.30 (CH), 145.62 (C)], Thiadiazole-C [144.05 (C), 166.04 (C)]; MS: m/z 386.21 (M+1, 100). Anal. Calcd. for C19H16ClN3O2S: C, 59.14; H, 4.18; N, 10.89. Found: C, 59.19; H, 4.15; N, 10.88.

2-(3,4-Dimethoxybenzyl)-6-(4-fluorophenyl)imidazo[2,1-b][1,3,4]thiadiazole (16)

White solid, yield 0.77 g (52%), mp 147–149 °C (from Acetone); IR (ATR, cm−1): 3107 (Ar–CH), 2952 (Aliph. CH), 1594 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.72 (s, 6H, 2-OCH3), 4.33 (s, 2H, –CH2), (OCH3)2Ar-H [6.91 (q, J = 12.0 Hz, 2H), 6.99 (s, 1H)], Phenyl-H [7.22 (dd, J = 8.0, 4.0 Hz, 2H), 7.85 (dd, J = 8.0, 4.0 Hz, 2H)], Imidazole-H [8.60 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 36.99 (-CH2), 55.96 (-OCH3), (OCH3)2Ar-C [110.53 (CH), 112.48 (CH), 121.74 (CH), 128.56 (C), 148.68 (C), 149.36 (C)], Phenyl-C [116.07 (CH), 127.00 (CH), 130.93 (C), 160.65 (C)], Imidazole-C [113.29 (CH), 145.42 (C)], Thiadiazole-C [144.34 (C), 165.81 (C)]; MS: m/z 369.86 (M+, 100). Anal. Calcd. for C19H16FN3O2S: C, 61.77; H, 4.37; N, 11.37. Found: C, 61.65; H, 4.39; N, 11.42.

2-(3,4-Dimethoxybenzyl)-6-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (17)

Yellow crystals, yield 0.95 g (62%), mp 167–169 °C (from Acetone); IR (ATR, cm−1): 3115 (Ar–CH), 2943 (Aliph. CH), 1595 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.73 (s, 6H, 2-OCH3), 3.75 (s, 3H, –OCH3), 4.32 (s, 2H, -CH2), (OCH3)2Ar-H [6.92–6.88 (m, 2H), 6.99 (d, J = 8.0 Hz, 1H)], Phenyl-H [6.95 (dd, J = 4.0, 2.0 Hz, 2H), 7.75 (dd, J = 8.0, 4.0 Hz, 2H)], Imidazole-H [8.48 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 36.98 (-CH2), 55.56 (–OCH3), 55.96 (–OCH3), (OCH3)2Ar–C [109.50 (CH), 112.48 (CH), 121.71 (CH), 128.63 (C), 148.68 (C), 149.36 (C)], Phenyl-C [114.51 (CH), 126.38 (C), 127.03 (CH), 159.08 (C)], Imidazole-C [113.28 (CH), 145.31 (C)], Thiadiazole-C [145.08 (C), 165.25 (C)]; MS: m/z 382.23 (M+1, 100). Anal. Calcd. for C20H19N3O3S: C, 62.97; H, 5.02; N, 11.02. Found: C, 62.91; H, 5.05; N, 11.10.

2-(3,4-Dimethoxybenzyl)-6-(4-nitrophenyl)imidazo[2,1-b][1,3,4]thiadiazole (18)

Yellow solid, yield 0.92 g (58%), mp 174–176 °C (from Acetone); IR (ATR, cm−1): 3115 (Ar–CH), 2926 (Aliph. CH), 1591 (C=N), 1514, 1335 (–NO2); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.73 (s, 6H, 2-OCH3), 4.33 (s, 2H, –CH2), (OCH3)2Ar-H [6.93 (q, J = 12.0 Hz, 2H), 7.00 (s, 1H)], Phenyl-H [8.09 (d, J = 8.0 Hz, 2H), 8.25 (d, J = 8.00 Hz, 2H)], Imidazole-H [8.92 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 36.99 (–CH2), 55.96 (-OCH3), (OCH3)2Ar–C [110.53 (CH), 112.48 (CH), 121.74 (CH), 129.10 (C), 148.69 (C), 149.36 (C)], Phenyl-C [124.69 (CH), 125.71 (CH), 140.85 (C), 146.57 (C)], Imidazole-C [113.61 (CH)], 143.42 (C)], Thiadiazole-C [144.34 (C), 165.81 (C)]; MS: m/z 396.94 (M+, 100). Anal. Calcd. for C19H16N4O4S: C, 57.57; H, 4.07; N, 14.13. Found: C, 57.62; H, 4.15; N, 14.18.

4-(2-(3,4-Dimethoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl)benzonitrile (19)

White solid, yield 0.98 g (65%), mp 170–172 °C (from Acetone); IR (ATR, cm−1): 3090 (Ar–CH), 2940 (Aliph. CH), 2228 (C≡N), 1575 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.72 (s, 6H, 2-OCH3), 4.34 (s, 2H, –CH2), (OCH3)2Ar–H [6.92 (q, J = 12.0 Hz, 2H), 6.99 (s, 1H)], Phenyl-H [7.84 (d, J = 8.0 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H)], Imidazole-H [8.84 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 37.03 (-CH2), 55.97 (-OCH3), (OCH3)2Ar-C [109.68 (CH), 112.47 (CH), 121.77 (CH), 128.42 (C), 148.71 (C), 149.37 (C)], Phenyl-C [112.85 (C), 125.51 (CH), 133.18 (CH), 138.88 (C)], 119.48 (C≡N), Imidazole-C [113.32 (CH), 146.24 (C)], Thiadiazole-C [143.39 (C), 166.80 (C)]; MS: m/z 376.86 (M+, 100). Anal. Calcd. for C20H16N4O2S: C, 63.81; H, 4.28; N, 14.88. Found: C, 63.87; H, 4.33; N, 14.91.

6-([1,1′-Biphenyl]-4-yl)-2-(3,4-dimethoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole (20)

Gray solid, yield 1.01 g (59%), mp 167–169 °C (from Acetone); IR (ATR, cm−1): 3123 (Ar–CH), 2919 (Aliph. CH), 1592 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.73 (s, 6H, 2-OCH3), 4.33 (s, 2H, –CH2), (OCH3)2Ar–H [6.93 (q, J = 12.0 Hz, 2H), 6.99 (s, 1H)], Phenyl-H [7.69 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H)], Phenyl-Phenyl-H [7.25 (t, J = 12.0 Hz, 1H), 7.38 (t, J = 12.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H)], Imidazole-H [8.61 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 37.00 (-CH2), 55.96 (–OCH3), (OCH3)2Ar–C [110.67 (CH), 112.48 (CH), 121.74 (CH), 129.08 (C), 148.68 (C), 149.37 (C)], Phenyl-C [125.05 (CH), 127.66 (CH), 128.57 (C), 134.37 (C)], Imidazole-C [113.30 (CH), 145.38 (C)], Phenyl-Phenyl-C [125.59 (CH), 126.90 (CH), 127.34 (CH), 129.39 (C)], Thiadiazole-C [145.25 (C), 165.75 (C)]; MS: m/z 428.17 (M+1, 100). Anal. Calcd. for C25H21N3O2S: C, 70.24; H, 4.95; N, 9.83. Found: C, 70.21; H, 4.99; N, 9.78.

2-(3,4-Dimethoxybenzyl)-6-(naphthalen-2-yl)imidazo[2,1-b][1,3,4]thiadiazole (21)

White solid, yield 0.98 g (61%), mp 192–193 °C (from Acetone); IR (ATR, cm−1): 3059 (Ar–CH), 2984 (Aliph. CH), 1590 (C=N); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.73 (s, 6H, 2-OCH3), 4.35 (s, 2H, –CH2), (OCH3)2Ar–H [6.94 (q, J = 12.0 Hz, 2H), 7.01 (s, 1H)])], Naphthyl-H [7.51–7.44 (m, 2H), 7.93–7.87 (m, 3H), 7.98 (t, J = 12.0 Hz, 1H), 8.37 (s, 1H)], Imidazole-H [8.77 (s, 1H)]; 13C NMR (100 MHz, DMSO-d6, δ ppm): 37.03 (-CH2), 55.98 (–OCH3), (OCH3)2Ar–C [111.24 (CH), 112.50 (CH), 121.78 (CH), 128.61 (C), 148.70 (C), 149.37 (C)], Naphtyl-C [123.11 (CH), 123.85 (CH), 126.22 (CH), 126.90 (CH), 128.07 (CH), 128.35 (CH), 128.57 (CH), 131.84 (C), 132.79 (C), 133.68 (C)], Imidazole-C [113.32 (CH), 145.68 (C)], Thiadiazole-C [145.22 (C), 165.90 (C)]; MS: m/z 401.99 (M+, 100). Anal. Calcd. for C23H19N3O2S: C, 68.81; H, 4.77; N, 10.47. Found: C, 68.85; H, 4.74; N, 10.41.

Biological activity

In vitro effect of the compounds against plant pathogens

The antifungal activity of the compounds was determined by the agar well diffusion method (Garammar 1976). Different concentrations (5, 2.5, 1.25 and 0.625 mg/mL) of compounds were obtained after they were dissolved in DMSO. DMSO was used (100 µL/well) as a negative control. A commercial dose of Thiram 80% was used as a positive control. The PDA was poured into 90 mm petri plates (~20 mL/plate−1). Four (5 mm diameter) wells were opened by a sterile cork borer at a position of 30 mm from the centre on the PDA plate. Each plate contained four different concentrations (5, 2.5, 1.25, and 0.625 mg/mL) of all compounds. Each dose (100 µL/well) was added to the well and kept at room temperature for 2 h during the diffusion period. The PDA plates were incubated (in the centre of the PDA) with 5 mm plugs from seven-day-old cultures. The plates were then incubated at 22 ± 2 °C for 7 d. The experiment was designed with three replicates and repeated two times. All inhibition zones were recorded. All antifungal activity values were determined by measuring the inhibition zone diameter between the pathogen and the well. The percentage of inhibition was calculated according to the following formula; (Vyas et al. 2006)

$${\rm{\% }}\,{\rm{Inhibition}}\,{\rm{ = }}\,{\rm{Inhibition}}\,{\rm{zone}}\,{\rm{in}}\,{\rm{treatment/Control*}}\,{\rm{x}}\,{\rm{100}}$$

*Control: Mycelial growth of negative control