Introduction

Thiazoles have attracted a great deal of attention because of their diverse biological activities. Thiamine (vitamin \(\hbox {B}_{1}\), 1) is one of the B-complex vitamins that contains a thiazole ring, which is vitally important to keep a living organism operating properly. Commercial drugs, such as sulfathiazole 2 (antimicrobial drug), ritonavir 3 (antiretroviral drug), and abafungin 4 (antifungal drug) contain a thiazole moiety (Fig. 1).

Synthesized compounds containing a thiazole moiety have been used for the treatment of allergies [1], hypertension [2], inflammation [3], human immunodeficiency virus (HIV) infections [4], and schizophrenia [5]. Thiazoles also exhibit other biological activities, such as antibacterial [6, 7], antitumor [8], analgesic [9], anticonvulsant [10], antimelanogenesis [11], antipsychotic [12], and antioxidant [13]. Also, thiazoles can act as cyclin-dependent kinase (CDK) inhibitors [14] and \(\upbeta \)-glucuronidase inhibitors [15].

Analogs such as 2-substituted-6-fluorobenzo[d]thiazoles [16], (thiazol-2-yl)hydrazine derivatives [17]. 3-Allyl-2-(substituted imino)-4-phenyl-3H-thiazoles, and 2,2\(^\prime \)-(1,3-phenylene)bis(3-substituted-2-imino-4-phenyl-3H-thiazole) derivatives [18] showed anti-candida, antibacterial, and acetylcholinesterase activities. Moreover, thiazoles have been used as semiconducting materials [19], thiazole-iridium complexes as phosphorescent organic light-emitting diodes [20], and naphthol-substituted thiazoles as ratiometric fluorescent chemosensors [21].

Fig. 1
figure 1

Vitamin \(\hbox {B}_{1}\) and drugs containing a thiazole moiety

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Fig. 2
figure 2

Thiazoles accessed via one-pot synthesis

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Recently, our research group reported the one-pot synthesis of new thiazolyl-pyrazoline derivatives 5–6 [22], bis-thiazoles 7–8 [23], and thiazolylpyridazinones 9 [24] exhibiting antibacterial activity, and new 1,4-dihydropyridines 10 bearing a thiazole moiety exhibiting high antioxidant activity [25] (Fig. 2). In continuation of our previous investigation [26], we report herein the one-pot MCR synthesis of new hydrazinyl-thiazole analogs and their biological activities.

Results and discussion

Chemistry

New 2,4-disubstituted hydrazinyl-thiazoles 14a–g were synthesized via the one-pot reaction of different aldehydes/ketones 11a–g, thiosemicarbazide 12, and 4-methoxy phenacyl bromide 13 in the presence of AcOH in EtOH under reflux conditions (Scheme 1). A literature survey indicated that hydrazone-thiazoles are generally synthesized in a two-step procedure. To the best of our knowledge, this is the first one-pot synthesis report for 14 analogs [27–30].

At the onset of our research, we explored the one-pot, multi-component preparation of 14a in EtOH at room temperature in the absence of a catalyst (entry 1) which after 6h afforded the desired product in 56 % yield. In the presence of AcOH, the yield of 14a increased from 56 to 60 % (entry 2). Then, the scope of the reaction was investigated under reflux conditions. As shown in Table 1, 14a was obtained after 2h in EtOH or DMF in high yield (entries 4 & 8). Since DMF is a toxic solvent, EtOH was chosen as it is an eco-friendly and green solvent.

Table 1 Optimization of 14a
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Scheme 1
scheme 1

Synthesis of new thiazoles 14a–g

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Our results show that the products are obtained in high yields and are easily purified using non-chromatographic methods. The advantages of this method are simple set-up and product isolation, reproducibility, using cheap and eco-friendly solvent.

In most cases, upon addition of 13 to a solution of 12 in EtOH after 2 h under reflux condition with aldehydes/ketones 11a–g in the presence of few drop of AcOH, the desired product precipitated. This is a good sign for the start of the reaction. Hydrazinyl-thiazoles 14a, 14b, and 14c were obtained as pure solids without the need for further purification.

The structures of hydrazinyl-thiazoles 14a–g were determined by IR, \(^{1}\hbox {H}\) NMR, and \(^{13}\hbox {C}\) NMR spectra. In the IR spectra, the disappearance of the carbonyl and thiosemicarbazone N–H stretching vibration bands indicates the participation of thioamide moiety in the cyclization reaction formation of desired products. In \(^{1}\hbox {H}\) NMR spectra, the presence of a singlet at 7.09–6.06 ppm confirms the thiazole ring closure. Also, the \(\hbox {OCH}_{3}\) signal appeared as a singlet at 3.87–3.67 ppm. Other aromatic protons appeared in the expected region around 8.94–6.68 ppm. The N–H signal appeared as a broad singlet at 14.50–11.45 ppm. In addition, the N–H signal can be endo- or exocyclic. However, according to literature [31], the signal at 14.50–11.45 ppm is related to an exocyclic N-H. The \(^{13}\hbox {C}\) NMR spectra of 14a–g provided the expected number and types of carbons. The C–H of the thiazole ring appeared at 100.4–98.7 ppm. The signal at 55.9–55.3 is attributed to \(\hbox {OCH}_{3}\) and other aromatic carbons appeared at 112.6–171.5 ppm.

In order to apply this procedure to carbonyl compounds with steric hindrance, we investigated the 3MCRs of camphor and 2-adamantanone 15 with thiosemicarbazide 12, and 4-methoxy phenacyl bromide 13. The reaction of camphor was unsuccessful and produced several by-products. However, 15 led to mixture of products 16A–16D in moderate yield (Scheme 2).

Scheme 2
scheme 2

Synthesis of thiazoles 16A–D from adamantanone and proposed structures

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Fig. 3
figure 3

DPPH radical scavenging activity of hydrazinyl-thiazoles 14a–g

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The \(^{1}\hbox {H}\) NMR spectra did not show a single product; rather, it showed a mixture of two sets of stereoisomer 16A/B and C/D. The protons of the adamantane moiety appeared at 2.20–1.25 ppm, \(\hbox {OCH}_{3}\) of isomers 16C/D appeared at expected region at 3.96–3.72 ppm, and the proton of the thiazole moiety as well as other aromatic protons appeared at 7.80–6.71 ppm. Several signals at 3.25–3.01 and 2.74–2.58 ppm corresponded to CH–N=N isomers C and D.

Biology

Antioxidant assay

Using DPPH (diphenylpicrylhydrazyl) is one of the simplest methods to evaluate antioxidant activity. DPPH is a stable, free radical of violet color. When a compound donates a radical hydrogen atom to a DPPH molecule, it reduces DPPH and so the absorbance of DPPH decreases. In brief, the higher the antioxidant activity, the lighter the violet hue. The antioxidant activity of hydrazinyl-thiazoles 14a–g was evaluated by the DPPH radical scavenging activity method. In this method, a decrease in absorption band at 517 nm indicates that the test compound possesses antioxidant activity. The radical scavenging activity of 14a–g was screened at a concentration of 4000–62.5 \(\upmu \hbox {g}/\hbox {mL}\) and monitored at 517 nm. Also, \(\hbox {IC}_{50}\) values (the concentration of 14a–g to scavenge 50 % of DPPH radical concentration) were calculated from line equation which can be obtained from Fig. 3. Ascorbic acid was used as standard. All hydrazinyl-thiazoles 14a–g showed dose-dependent antioxidant activity (Fig. 3).

Scheme 3
scheme 3

Proposed mechanism of radical scavenging activity

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The \(\hbox {IC}_{50}\) values of 14a–g are in the range 2.90–0.23 \(\upmu \hbox {M}\) (Table 2). It is worth noting that hydrazinyl-thiazole 14c has the lowest \(\hbox {IC}_{50}\) value \((0.23\,\upmu \hbox {M})\) while compound 14a has the highest \(\hbox {IC}_{50}\) value \((2.90\,\upmu \hbox {M})\) compared to the \(\hbox {IC}_{50}\) value of ascorbic acid (antioxidant compound) \((1.30\,\upmu \hbox {M})\). The higher antioxidant activity is reflected in a lower \(\hbox {IC}_{50}\). Compounds 14a–g were determined to exhibit potent radical scavenging activity in a DPPH assay with \(\hbox {IC}_{50}\) values in the following order14c, 14g, 14b, 14e, ascorbic acid, 14f, 14d, and 14a respectively.

According to the literature [32] and our recent report [25], the high antioxidant activity of 14a–g is attributed to the presence of the thiazole ring. The N–H at the \(\hbox {C}_{2}\) position of the thiazole ring can readily donate a hydrogen radical to DPPH radical and form a radical of the test compound. Therefore, the radical is highly resonance-stabilized through the thiazole ring and =C–N–NH–C moiety (Scheme 3).

Table 2 \(\hbox {IC}_{50}\) values for DPPH radical scavenging activity of hydrazinyl-thiazoles 14a–g
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In addition to the presence of the thiazole moiety, the high antioxidant activity of 14g can be due to the presence of benzylic hydrogens. These benzylic hydrogens are easily converted to the stable benzyl radical by DPPH. Furthermore, the antioxidant activity of 14b and 14c can be due to the presence of aliphatic hydrogens which can form tertiary, secondary, and primary carbon radicals. As it is mentioned in Table 2, these compounds exhibit better activity than ascorbic acid. Therefore, the presence of benzylic and aliphatic radicals as well thiazole and =C–N–NH–C moieties possibly play an effective role in antioxidant activity (Scheme 3).

Antibacterial assay

Products 14a–g were screened for their in vitro antibacterial activity against Gram-positive and Gram-negative bacteria strains including Staphylococcus aureus (S. aureus), Micrococcus luteus (M. luteus), Escherichia coli (E. coli), Pseudomonas aeruginosa (Ps. aeruginosa), Bacillus subtilis (B. subtilis), Aeromonas hydrophila (A. hydrophila) using a well-diffusion method. DMSO was used as negative control and showed no activity against the above bacterial strains. Penicillin G and Cefixime were used as positive controls (Table 3).

Table 3 Antibacterial activity of hydrazinyl-thiazoles 14a–g \((4000\,\upmu \hbox {g}/\hbox {mL})\) as zone of inhibition in millimeter
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Hydrazinyl-thiazoles 14a–g were evaluated for their antibacterial activity at a concentration of \(4000\,\upmu \hbox {g}/\hbox {mL}\) in DMSO. The experiments were performed in triplicate. The results are presented as \(\hbox {mean}\pm \hbox {standard}\) deviation in millimeter. According to the results, only 14d was active against all six bacterial strains, and 14f had the highest antibacterial activity against S. aureus and M. luteus. Compound 14d was more active against E. coli and also showed significant inhibition activity against B. subtilis. In addition, 14e showed significant inhibition activity against Ps. aeruginosa. Only 14b showed good antibacterial activity against A. hydrophila.

Some reports showed moderate antibacterial activity of hydrazinyl-thiazoles. For example, Bharti and co-workers [33] studied the antibacterial activity of several hydrazinyl-thiazole derivatives, finding that their thiazoles showed no antibacterial activity against E. coli and only a few were active against Ps. aeruginosa. However, in our study, 14c–e had moderate antibacterial activity against E. coli, while 14e good antibacterial activity against Ps. aeruginosa. Moreover, Lee et al. [34] studied the antibacterial activity of hydrazinyl-thiazoles on several bacterial strains. According to their report, none of their synthesized compounds had antibacterial activity against Ps. aeruginosa, and only a few of them had good activity against S. aureus, B. subtilis, and E. coli. Also, the hydrazinyl-thiazoles showed no antibacterial activity against A. hydrophila.

Fig. 4
figure 4

In vitro cytotoxic activity of 14a on human melanoma cancer cell lines A375. Results are presented as the percentage of cell \(\hbox {viability}\pm \hbox {SD}\) using the MTT protocol

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Anticancer assay

Melanoma is a cancer that develops in melanocytes (the pigment cells present in the skin). It is known that melanoma can spread to other parts of the body causing serious illness and death. Melanoma can be more serious than two other forms of skin cancers, e.g., basal-cell cancer (BCC) and squamous-cell cancer (SCC). The primary treatment for melanoma is surgery. There are a few drugs used in chemotherapy of melanoma cancer; however, none of them contain hydrazinyl-thiazole moiety, e.g., dacarbazine and temozolomide contain imidazole and carboxamide moieties, respectively. An in vitro cytotoxicity study of 14a was carried out on human melanoma cancer cell lines A375 and Hu-02 (normal human skin cell lines) obtained from the National Centre for Cell Science, Tehran, Iran. Cell viability of 14a was evaluated using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay protocol at concentrations of 0.5, 0.25, 0.125, 0.075, 0.037 mg/mL in DMSO. DMSO was used as negative control. The MTT method is based on the reduction of soluble MTT by mitochondrial reductase of the viable cells to insoluble purple crystals of formazan ((E, Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan). The time of exposure was 24 h and 48 h (Fig. 4). Results are reported as the percent of cell viability \(\pm \) standard deviation. Statistical analysis demonstrated that 14a had cytotoxic activity on Hu-02 (69.4 %) and A375 (58.8 %) cell lines at 24 h time of exposure and on Hu-02 (64.3 %) and A375 (51.2 %) at 48 h time of exposure at a concentration of 0.5 mg/mL \((\hbox {P}\le 0.05)\).

The \(\hbox {LC}_{50}\) values of 14a are shown in Table 4. The \(\hbox {LC}_{50}\) values are between 1.19 and 0.55 mg/mL. As it is shown, the \(\hbox {LC}_{50}\) values on melanoma cancer cells (A375) are lower than on skin cells (Hu-02). However, increasing the time of exposure from 24 h to 48 h did not have a significant effect on the \(\hbox {LC}_{50}\) values.

Table 4 The \(\hbox {LC}_{50}\) values of cytotoxic effects of 14a on melanoma cancer cell lines (A375) \((\hbox {mean}\pm \hbox {SD})\)
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Conclusions

Several new hydrazinyl-thiazoles derivatives 14a–g were synthesized via one-pot reaction of aldehydes/ketones 11a–g, thiosemicarbazide 12, and 4-methoxy phenacyl bromide 13 through a reliable procedure for high yields and high purity. Hydrazinyl-thiazoles 14a, 14b, and 14c were obtained as pure solids without further purification. All products showed high antioxidant activity in comparison to ascorbic acid. Compounds 14b and 14e had the highest antioxidant activity; their \(\hbox {IC}_{50}\) was less than that of ascorbic acid. Compounds 14a–g showed low to moderate antibacterial activity. Compound 14d possessed the highest antibacterial activity against B. subtilis. Compound 14a had cytotoxic activity at a concentration of 0.5 mg/mL upon 24 h and 48 h time of exposure on melanoma cancer cell lines.

Experimental section

Materials and instruments

Starting materials, reagents, biological cultures, and solvents were obtained from commercial suppliers Fluka, Merck, Sd-fine, Quelab and were used without further purification. All reactions were monitored by silica gel-coated TLC plates (\(60\hbox { F}_{254}\) Merck). IR spectra were recorded on a Shimadzu IR-470 spectrophotometer in anhydrous KBr. \(^{1}\hbox {H}\) NMR and \(^{13}\hbox {C}\) NMR spectra were recorded on a 400 MHz and 500 MHz Bruker spectrometers using DMSO-\(\hbox {d}_{6}\) and \(\hbox {CDCl}_{3}\) as solvents. Chemical shifts are expressed relative to TMS as singlet (s), doublet (d), triplet (t), multiple (m), doublet of doublet (dd), doublet of triplet (dt), triplet of triplet (tt), doublet of doublet of doublet (ddd), and quintet (quin). Coupling constants are expressed in Hertz (Hz). Elemental analyses were carried out on a Carlo–Erba EA1110 CNNO-S analyzer. Melting points were determined with a Mettler Fp5 apparatus, and were uncorrected. Absorbance in the antioxidant assay was recorded on an Unico 2100 spectrophotometer.

General procedure for the synthesis of hydrazinyl-thiazoles 14a–g

To a solution of thiosemicarbazide 12 (1 mmol) in EtOH, aldehydes/ketones 11a–g (1 mmol) and a few drops of AcOH were added and this mixture was refluxed and stirred. After a few minutes (1–15 min), 4-methoxy phenacyl bromide 13 (1 mmol) was added and the reaction was refluxed and stirred until completion (1.5–2 h). Thin layer chromatography (TLC) was used to monitor the progress of the reaction (EtOAc:n-hexane 3:6). After completion of the reaction, the reaction mixture was cooled down to room temperature. The resulting solid was filtered and washed with or recrystallized in EtOH.

2-(2-(6-Methoxy-3,4-dihydronaphthalen-1(2H)-ylidene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (14a):

Cream solid (0.3 g, 80 %); mp 233–236\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 3210 (stretch N–H), 3100 (stretch C–H aromatic), 2920, 2830 (stretch C–H aliphatic), 1610 (stretch C=N), 1570, 1500 (stretch C=C), 1250, 1240, 1045 (stretch C–O), 825, 760, 700 (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f }\)(83 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (400 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 12.46 (s, 1H, NH), 8.02 (d, \(J = 8.8\hbox {Hz}\), 1H), 7.67 (d, \(J = 8.0\hbox { Hz}\), 2H), 6.99 (d, \(J = 8.8\hbox { Hz}\), 2H), 6.82 (dd, \(J = 8.8\), 2.8 Hz, 1H), 6.68 (d, \(J = 2.4\hbox { Hz}\), 1H), 6.06 (s, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 2.90 (t, \(J = 6.4\hbox { Hz}\), 2H), 2.79 (t, \(J = 6.0\hbox { Hz}\), 2H), 2.02 (quin, \(J = 6.3\hbox { Hz}\), 2H) ppm; \(^{13}\hbox {C}\) NMR (100 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 169.3, 161.2, 160.7, 154.0, 142.5, 127.1, 126.9, 123.7, 121.5, 114.7, 113.5, 112.6, 99.1, 55.4, 55.3, 29.7, 27.2, 21.6 ppm; Anal. calcd. for \(\hbox {C}_{21}\hbox {H}_{21}\hbox {N}_{3}\hbox {O}_{2}\hbox {S}\): C, 66.49, H, 5.56, N, 11.10. Found: C, 66.51; H, 5.59; N, 11.06 %.

4-(4-Methoxyphenyl)-2-(2-(4-phenylcyclohexylidene)hydrazinyl)thiazole (14b)

Orange solid (0.3 g, 85 %); mp 186–189\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 3210 (stretch N–H), 3020 (stretch C–H aromatic), 2920 (stretch C–H aliphatic), 1610 (stretch C=N), 1560, 1540, 1510 (stretch C=C), 1250, 1040 (stretch C–O), 820, 740, 690 (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (80 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (400 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 12.45 (s, 1H, NH), 7.67 (d, \(J = 8.8\hbox { Hz}\), 2H), 7.33 (t, \(J = 7.2\hbox { Hz}\), 2H), 7.26–7.22 (m, 3H), 7.01 (d, \(J = 8.8\hbox { Hz}\), 2H), 6.56 (s, 1H), 3.86 (s, 3H), 3.32 (d, \(J = 14.8\hbox { Hz}\), 1H), 2.88 (tt, \(J= 12\), 3.3 Hz, 1H), 2.72 (d, \(J = 14.4\hbox { Hz}\), 1H), 2.47 (dt, \(J = 14\), 4.9 Hz, 1H), 2.32 (dt, \(J = 14.1\), 5.3 Hz, 1H), 2.27–2.17 (m, 2H), 1.79 (ddd, \(J= 25.5\), 13.1, 4.3 Hz, 2H) ppm; \(^{13}\hbox {C}\) NMR (125 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 170.1, 164.2, 161.5, 145.1, 140.8, 129.0, 127.5, 127.1, 127.0, 120.3, 115.4, 98.8, 55.8, 43.5, 35.2, 34.6, 33.3, 29.4 ppm; Anal. calcd. for \(\hbox {C}_{22}\hbox {H}_{23}\hbox {N}_{3}\hbox {OS}\): C, 70.03; H, 6.11; N, 11.09. Found: C, 69.98; H, 6.15; N, 11.12 %.

2-(2-(Heptan-4-ylidene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (14c)

Orange solid (0.26 g, 83 %); mp 127–131\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 3210 (stretch N–H), 3100 (stretch C–H aromatic), 2950, 2850 (stretch C–H aliphatic), 1610 (stretch C=N), 1560, 1540, 1505 (stretch C=C), 1250, 1020 (stretch C–O), 830 (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (82 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (400 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 12.39 (s, 1H, NH), 7.66 (d, \(J = 8.2\hbox { Hz}\), 2H), 6.99 (d, \(J = 7.8\hbox { Hz}\), 2H), 6.53 (s, 1H), 3.85 (s, 3H), 2.49 (t, \(J = 9\hbox { Hz}\), J = 8 Hz, 2H), 2.34 (t, \(J = 7.6\hbox { Hz}\), 2H), 1.70–1.61 (m, 4H), 1.11 (t, \(J = 7.2\hbox { Hz}\), 3H), 0.98 (t, \(J = 7.4\hbox { Hz}\), 3H) ppm; \(^{13}\hbox {C}\) NMR (125 MHz, \(\hbox {DMSO}-d_{6}\)) \({\updelta }\) 170.2, 166.1, 161.5, 140.7, 127.5, 120.4, 115.3, 98.7, 55.8, 38.9, 33.8, 19.8, 19.7, 14.6, 14.1 ppm; Anal. calcd. for \(\hbox {C}_{17}\hbox {H}_{23}\hbox {N}_{3}\hbox {OS}\): C, 64.35; H, 7.28; N, 13.21. Found: C, 64.30; H, 7.31; N, 13.17 %.

2-(2-((4-Chlorophenyl)(phenyl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (14d)

Orange crystal (0.32 g, 78 %); mp 239–242\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 3120 (stretch C–H aromatic), 1610 (stretch C=N), 1580, 1560, 1540 (stretch C=C), 1250, 1030 (stretch C–O), 830, 740, 690 (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (73 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (400 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 12.07 (s, 1H, NH), 7.69 (d, \(J = 8.8\hbox { Hz}\), 2H), 7.66 (d, \(J = 8.4\hbox { Hz}\), 2H), 7.60 (dd, \(J = 7.8\), 1.3 Hz, 2H), 7.49 (tt, \(J = 6.6\), 1.8 Hz, 1H), 7.42 (tt, \(J = 8.8\), 1.2, 2H), 7.35 (d, \(J = 8.8\hbox { Hz}\), 2H), 6.85 (d, \(J = 8.8\hbox { Hz}\), 2H), 6.63 (s, 1H) 3.85 (s, 3H) ppm; \(^{13}\hbox {C}\) NMR (125 MHz, \(\hbox {CDCl}_{3}/\hbox {DMSO}-d_{6}\)) \({\updelta }\) 170.0, 161.3, 137.2, 135.7, 131.3, 130.7, 130.6, 130.4, 129.7, 129.6, 128.9, 128.5, 128.3, 127.6, 115.1, 114.6, 100.4, 55.7 ppm; Anal. calcd. for \(\hbox {C}_{23}\hbox {H}_{18}\hbox {C}\hbox {lN}_{3}\hbox {OS}\): C, 65.81; H, 4.29; N, 9.98. Found: C, 65.85; H, 4.33; N, 10.03 %.

4-(4-Methoxyphenyl)-2-(phenyl(pyridin-2-yl)methylene)hydrazinyl)thiazole (14e)

Red solid (0.27 g, 71 %); mp 215–220\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 1605 (stretch C=N), 1550, 1510, 1480 (stretch C=C), 1240, 1050 (stretch C–O), 830, 760, 730, 700 (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (74 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (400 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 14.51 (s, 1H, NH), 8.93 (d, \(J = 3.6\hbox { Hz}\), 1H), 7.83–7.77 (m, 3H), 7.64–7.60 (m, 2H), 7.52–7.39 (m, 5H), 6.97 (d, \(J = 8.8\hbox { Hz}\), 2H), 6.78 (s, 1H), 3.87 (s, 3H) ppm; \(^{13}\hbox {C}\) NMR (125 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 171.5, 160.6, 150.2, 148.6, 138.7, 136.0, 129.9, 129.6, 129.3, 128.7, 128.6, 127.6, 127.3, 126.0, 124.7, 114.6, 100.3, 55.3 ppm; Anal. calcd. for \(\hbox {C}_{22}\hbox {H}_{18}\hbox {N}_{4}\hbox {OS}\): C, 68.41; H, 4.72; N, 14.53. Found: C, 68.36; H, 4.67; N, 14.48 %.

2-Methoxy-4-((2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)methyl)-6-nitrophenol (14f)

Red solid (0.3 g, 75 %); mp 207–209\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 3250 (stretch N–H), 3100 (stretch C–H aromatic), 2980, 2830 (stretch C–H aliphatic), 1610 (stretch C=N), 1570, 1480 (stretch C=C), 1540, 1340 (stretch \(\hbox {NO}_{2}\)), 1250, 1050 (stretch C–O), 840, 760, 740, 690 (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (60 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (500 MHz, \(\hbox {DMSO}-d_{6}\)) \({\updelta }\,\sim 11.45\) (s, 1H, NH), 7.95 (s, 1H), 7.74 (d, \(J = 8.76\hbox { Hz}\), 2H), 7.67 (dd, \(J = 1.52\hbox { Hz}\), 1H), 7.49 (dd, \(J = 1.48\hbox { Hz}\), 1H), 7.09 (s, 1H), 6.92 (d, \(J = 8.7\hbox { Hz}\), 2H), 3.90 (s, 3H), 3.74 (s, 3H) ppm; \(^{13}\hbox {C}\) NMR (125 MHz, \(\hbox {DMSO}-d_{6}\)) \({\updelta }\) 168.8, 159.6, 151.1, 150.6, 144.2, 140.2, 138.1, 128.3, 127.6, 126.2, 115.1, 114.8, 113.1, 102.3, 57.4, 55.9 ppm; Anal. calcd. for \(\hbox {C}_{18}\hbox {H}_{16}\hbox {N}_{4}\hbox {O}_{5}\hbox {S}\): C, 54.03; H, 4.06; N, 13.97. Found: C, 53.97; H, 4.02; N, 14.04 %.

4-(4-Methoxyphenyl)-2-(2-(2,4,6-trimethylbenzylidene)hydrozinyl)thiazole (14g)

Cream solid (0.26 g, 75 %); mp 229–233\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 3400 (stretch N–H), 3100 (stretch C–H aromatic), 2950, 2820 (stretch C–H aliphatic), 1610 (stretch C=N), 1510 (stretch C=C), 1250, 1020 (stretch C–O), 850, 750, 740, (OOP. C–H) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (75 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (500 MHz, \(\hbox {DMSO}-d_{6}\)) \({\updelta }\sim 13.00\) (s, 1H, NH), 8.65 (s, 1H), 7.52 (d, \(J = 8.6\hbox { Hz}\), 2H), 6.81 (d, \(J = 6.9\hbox { Hz}\), 2H), 6.75 (s, 2H), 6.56 (s, 1H), 3.67 (s, 3H), 2.31 (s, 6H), 2.13 (s, 3H) ppm; \(^{13}\hbox {C}\) NMR (125 MHz, \(\hbox {DMS0}-d_{6}\)) \({\updelta }\,\hbox {DMSO}-d_{6}\)) \({\updelta }\sim 170\), 161.3, 151.2, 141.1, 140.9, 139.1, 130.4, 127.9, 126.8, 120.6, 115.0, 100.2, 55.7, 22.2, 21.5 ppm. Anal. calcd. for \(\hbox {C}_{20}\hbox {H}_{21}\hbox {N}_{3}\hbox {OS}\): C, 68.37; H, 6.05; N, 11.94. Found: C, 68.32; H, 5.98; N, 11.98 %.

2-(Adamantan-2-ylidenehydraono)-4-(4-methoxyphenyl)-2,3-dihydrothiaole (16)

Orange solid (0.17g, 50 %); mp 140–148\(\,^{\circ }\hbox {C}\) (from EtOH); IR (KBr) \(\upnu _\mathrm{max}\) 2917, 2849 (stretch C-H aliphatic), 1605 (stretch C=N), 1549, 1503 (stretch C=C) \(\hbox {cm}^{-1}\); \(\hbox {R}_{f}\) (35 %, hexane: EtOAc 6:3); \(^{1}\hbox {H}\) NMR (500 MHz, \(\hbox {CDCl}_{3}\)) \({\updelta }\) 7.80–6.71 (m, Ar, NH, H-thiazole), 3.96–3.72 (OCH\(_{3})\), 3.01–2.55 (CH–C=N of C and D), 2.20–1.25 (CH and \(\hbox {CH}_{2}\) adamantan) ppm.

Biological methods

DPPH radical scavenging assay

The DPPH radical scavenging activity of 14a–d was evaluated according to the literature [25]. A 2,2-diphenyl-2-picrylhydrazyl (DPPH) solution was prepared by dissolving an appropriate amount of DPPH in MeOH to give a concentration of \(6.25 \times 10^{-5}\hbox { M}\). Compounds 14a–g and DPPH with different concentrations (4000, 2000, 1000, 500, 250, 125, \(62.5\,\upmu \hbox {g}/\hbox {mL}\)) in MeOH were prepared. Then, 0.1 mL of each hydrazinyl-thiazole solution was added to 3.9 mL of DPPH solution and was shaken vigorously. Samples were kept in darkness for 30 min and then their absorbance was measured at 517 nm. MeOH was used as blank. Radical scavenging activity was calculated as follows:

where \(A_\mathrm{control}\) is the absorbance of the negative control (containing all reagents except test compounds), \(A_\mathrm{sample}\) the absorbance of the test compounds. \(\hbox {IC}_{50}\) values of the test compounds were determined by plotting the radical scavenging activity percentage against concentration of the test compound.

Antibacterial assay

The antibacterial activity of hydrazinyl-thiazoles was evaluated biologically using a well-diffusion method. First, nutrient agar and nutrient broth cultures were prepared according to manufacturer’s instructions and were incubated at \(37\,^{\circ }\hbox {C}\). After incubation for the appropriate time, a suspension of \(30\,\upmu \hbox {L}\) of each bacterium was added to the nutrient agar plates. Cups (5 mm in diameter) were cut in the agar using sterilized glass tube. Each well received \(30\,\upmu \hbox {L}\) of the test compounds at concentration of \(4000\,\upmu \hbox {g}/\hbox {ml}\) in DMSO. Then, plates were incubated at \(37\,^{\circ }\hbox {C}\) for 24 h, after this time the zone of inhibition was measured. The values are expressed in millimeters (mm). The experiments were performed in triplicate. The results are reported as \(\hbox {mean}\pm \hbox {SD}\) of zone of inhibition in millimeter. Antibacterial activity of each hydrazinyl-thiazole was compared with Penicillin G and Cefixime as standards. DMSO was used as negative control.

In vitro anticancer assay

The anticancer activity of the hydrazinyl-thiazole 14a was determined against melanoma cancerous cell lines Hu-02 and A375 using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The melanoma cancerous cell lines were provided by the Iranian Biological Resource Center, Tehran. The MTT method is based on the reductive cleavage of the tetrazolium ring of MTT into insoluble purple formazan. \(100\,\upmu \hbox {L}\) of the cell suspension were added to each well of 96-well microplate and then incubated for 12–24 h at \(37\,^{\circ }\hbox {C}\) in a \(\hbox {CO}_{2}\) incubator. Compound 14a was diluted in concentrations of 0.037–0.5 mg/mL in DMSO by two-fold serial dilution. DMSO was used as negative control. Different concentrations of a test compound were added to each well and then incubated for 24 and 48 h at \(37\,^{\circ }\hbox {C}\) in a \(\hbox {CO}_{2}\) incubator. Then, \(10\,\upmu \hbox {L}\) of MTT (5 mg/mL) stock solution in PBS were added to each well and incubated for 4 h, the upper solution was removed, and \(100\,\upmu \hbox {L}\) of DMSO were added to the media. The plates were shaken slowly until the insoluble crystals of the produced formazan dissolved. The plates were kept for 2–4 h in the dark. After that, the plates were read at 360 nm on an ELISA reader. The experiments were performed in triplicate.

The percentage of cell viability was calculated using the following formula:

The \(\hbox {LC}_{50}\) values (lethal concentration of the test compound required to kill 50 % of sample population) were determined by plotting cell viability percentage against concentration of the sample.