Synthesis and antiproliferative activity studies of new functionalized pyridine linked thiazole derivatives

2.1 Chemistry

Melting points were determined on Gallenkamp electric device and are uncorrected. The infrared spectra (potassium bromide discs) were registered on Thermo Scientific Nicolet iS10 FTIR spectrometer. The ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were acquired on JEOL’s 500 MHz NMR spectrometer. The mass spectra were determined on Quadrupole GC-MS (DSQII) mass spectrometer at 70 eV. Elemental analyses of carbon, hydrogen and nitrogen were estimated on Perkin Elmer 2400 analyzer.

2.2 MTT cytotoxicity assay

The technical method of MTT assay depends on the reduction of yellow 3-(4,5-methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into purple formazan product, mainly by mitochondrial reductase activity inside the living cells. The cells used in cytotoxicity assay were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells suspended in the medium (2 × 10⁴ cells/mL) were plated in 96-well culture plates and incubated at 37 °C in a 5% CO₂ incubator for 12 h. The tested sample (2 μL) was added to the cells (2 × 10⁴) in 96-well plates and cultured at 37 °C for 3 days. The cultured cells were mixed with 20 μL of MTT solution and incubated for 4 h at 37 °C. The supernatant was carefully removed from each well and 100 μL of DMSO were added to each well to dissolve the formazan crystals, which were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a microplate reader using a test wavelength of 570 nm. The results were expressed as the IC₅₀ values, which inducing 50% inhibition of cell growth of the treated cells when compared to the growth of control cells.

3.1 Chemistry

Initially, the nucleophilic substitution of chlorine form 2-chloro-N-(pyrid-2-yl)acetamide (1) by the interaction with 4-hydroxybenzaldehyde was successfully carried out in the presence of DMSO and potassium carbonate at room temperature to furnish 2-(4-formylphenoxy)-N-(pyrid-2-yl)acetamide (2) (Ma et al., 2011) (Scheme 1). The key compound, 2-(4-formylphenoxy)-N-(pyrid-2-yl)acetamide (2), was utilized as a precursor for the construction of pyridine-thiazole hybrids through building of thiazole ring systems at the formylphenoxy part. Thus, the synthetic plan of the present part starts by condensation of the formyl group in the key 2 with thiosemicarbazide. The condensation proceeded by heating in ethanol in the presence of glacial acetic acid to afford the corresponding thiosemicarbazone derivative 3 with 71% yield. The target pyridine-thiazole compounds 4a and 4b were obtained by the reaction of equimolar quantities of thiosemicarbazone derivative 3 with phenacyl bromide and/or 4-chlorophenacyl bromide, respectively. The IR, ¹H NMR, ¹³C NMR and mass spectral analyses were utilized to elucidate the structures of these hybrids 4a and 4b.

Close

Scheme 1

Synthesis of pyridine-thiazole hybrids 4a and 4b.

Export to PPT

The cyclocondensation of thiosemicarbazone derivative 3 with ethyl bromoacetate was performed in boiling ethanol in the presence of sodium acetate to produce 2-(4-((2-(4-oxothiazolin-2-yl)hydrazono)methyl)phenoxy)-N-(pyrid-2-yl)acetamide (5), which undergoes Knoevenagel condensation with three different benzaldehyde derivatives (namely; 4-tolualdehyde, 4-anisaldehyde and/or 4-chlorobenzaldehyde). Such condensation was proceeded by refluxing the reaction mixture in ethanol and piperidine to afford the conforming 5-arylidene-thiazolin-4-one derivatives 6a-c (Scheme 2). The structures of pyridine-thiazole hybrids 5 and 6 were established based on their correct spectral analyses. For example, the IR spectrum of pyridine-thiazolin-4-one derivative 5 displayed absorption at 1708 cm⁻¹ for the carbonyl group of the new constructed thiazole moiety. The lower absorption of this carbonyl group (1691–1698 cm⁻¹) in compounds 6a-c is attributed to the conjugation with exocyclic double bond. The ¹H NMR spectrum of 5 showed the protons of methylene (thiazolin-4-one moiety) as singlet at 4.11 ppm. The ¹H NMR spectra of compounds 6a-c clearly indicated the lack of this signal and instead displayed a singlet signal of the olefinic proton in the region from 7.76 to 7.78 ppm. The reaction of 3 with diethyl bromomalonate was performed in boiling ethanol and sodium acetate to yield ethyl 4-oxo-2-(2-(4-(2-oxo-2-(pyrid-2-ylamino)ethoxy)-benzylidene)hydrazinyl)thiazolin-5-carboxylate (7). The plausible reaction mechanism for the formation of 7 can be initiated by replacement the bromine atom of diethyl bromomalonate by sulfur atom of thiosemicarbazide to form an open chain-sulfide. This subsequently undergoes in situ elimination of ethanol molecule to give final thiazole ring system 7 in the light of modified Hantzsch-Thiazole synthesis.

Close

Scheme 2

Synthesis of pyridine-thiazole hybrids 5, 6 and 7.

Export to PPT

The synthesis of 5-acetylthiazole derivative 8 was achieved via treatment of thiosemicarbazone 3 with 3-chloropentane-2,4-dione in refluxing ethanol and triethylamine (Scheme 3). The structure of pyridine-thiazole hybrid 8 was established because of its agreement spectral data. The ¹H NMR spectrum of 8 showed two singlet signals for the protons of methyl groups (thiazole-COCH₃ and thiazole-CH₃) at 2.58 and 2.76 ppm, respectively. The details of NMR signals were described in the experimental section. The acetyl group of pyridine-thiazole hybrid 8 was successfully converted into to the conjugated thiazole-enaminone derivative 9 by heating with dimethylformamide-dimethylacetal (DMF-DMA) in xylene. The enaminone part of pyridine-thiazole hybrid 8 was utilized as a precursor for the synthesis of pyrazole ring system through treatment with hydrazine hydrate in refluxing ethanol to furnish the corresponding thiazolyl-pyrazole compound 10. The spectroscopic tools (IR, ¹H NMR and ¹³C NMR) were employed to confirm the structures of thiazole-enaminone 9 and its corresponding thiazolyl-pyrazole 10. The IR spectrum of thiazole-enaminone 9 showed the conjugated carbonyl absorption band at 1687 cm⁻¹ as well as the absorption bands at 3272, 3185 cm⁻¹ for the NH groups. Moreover, ¹H NMR spectrum of thiazole-enaminone 9 showed the characteristic signals for the conjugated enaminone part as two singlet signals at 3.12 and 3.28 ppm (two methyl groups) and singlet for one proton (C⚌CH-N) at 8.12 ppm. These signals were completely disappeared from the ¹H NMR spectrum of compound 10 and instead showed the characteristic doublet signals of the pyrazole ring system at 6.64 ppm (pyrazole-H4) and 7.78 ppm (pyrazole-H5), respectively, with coupling constant = 4.00 Hz.

Close

Scheme 3

Synthesis of pyridine-thiazole hybrids 8, 9 and 10.

Export to PPT

3.2 In vitro anticancer activity

The cytotoxicity of ten pyridine linked thiazole derivatives and their precursors was screened against four cancer cell lines; liver carcinoma (HepG2), laryngeal carcinoma (Hep-2), prostate cancer (PC3), breast cancer (MCF-7) and normal fibroblast cells (WI38) by the MTT assay (Mohamed et al., 2017; Abd El-Meguid et al., 2019) at the National Cancer Institute (Egypt). The results of cytotoxicity are listed (Table 1) in terms of IC₅₀ values and compared with the known anticancer reference drug 5-fluorouracil (5-Fu). Some of the synthesized pyridine-thiazole compounds 7 and 10 exhibited good cytotoxicity with pronounced activity to inhibit the growth of MCF-7 and HepG2 cancer cells. Structure activity relationship (SAR) results of the tested pyridine-thiazole hybrids gives an insight concerning the structural features related to anticancer activity. Firstly, cyclization of the thiosemicarbazone 3 with ethyl bromomalonate to produce compound 7 in which the thiazolin-4-one ring system is substituted at the fifth position with ethyl carboxylate group enhances the cytotoxic effectiveness toward MCF-7 and HepG2 cancer cells with IC₅₀ values 5.36 and 6.78 μM, respectively. Its activity is higher than the anticancer reference drug 5-fluorouracil that exhibit potency with IC₅₀ values 6.14 and 7.20 μM for MCF-7 and HepG2 cancer cells, respectively. The pyridine-thiazole hybrid 10 that substituted with pyrazole moiety at the fifth position of thiazole ring system exhibited the next remarkable activity against the same cancer cells with IC₅₀ = 5.84 and 8.76 μM, respectively. For the pyridine-4-arylidenethiazolin-4-one derivatives 6a-c, it was observed that compound 6c (Ar = 4-ClC₆H₄) revealed strong cytotoxicity with IC₅₀ = 10.63 μM toward against breast cancer cell line. This is higher than that of compound 6b (Ar = 4-MeOC₆H₄) which exhibited moderate inhibition with IC₅₀ = 12.68 μM, although the latter compound 6b (Ar = 4-MeC₆H₄) exhibited sensible inhibition toward MCF-7 cell line with IC₅₀ = 15.42 μM. These results find support from the order of pharmacological activity that follow the arrangement of substituents (Abbas and Abd El-Karim, 2019; Al-Anazi et al., 2019): 4-Cl > 4-OCH₃ > 4-CH₃. The pyridine-thiazole hybrids 4, 5 and 8 showed acceptable cytotoxic activities towards MCF-7 and HepG2 cell lines to announce IC₅₀ values in the range 18.51–27.16 μM. The introduction of enaminone moiety in compound 9 caused dramatic decrease in the cytotoxic activity towards MCF-7 and HepG2 cell lines (IC₅₀ = 27.63 and 30.36 μM) when compared with the activity of all the other synthesized hybrids. Further examination of the synthesized pyridine-thiazole hybrids was performed to explore their cytotoxicity on the normal cell line (WI38). The results indicated that most of these compounds affected lesser extent of cytotoxicity, their IC₅₀ ranged from 22.82 to 77.14 μM. Fortunately, the promising selectivity of compounds 7 and 10 as cytotoxic agents for MCF-7 and HepG2 cell lines, they showed weak cytotoxic effects on normal cell lines (WI38).

3.3 Molecular docking

The molecular docking (Al-Dawood et al., 2016) was employed to discuss the binding mode of synthesized pyridine-thiazole derivatives through Rho-associated protein kinase (ROCK-1) via utilizing MOE v10.2015.10 program. The Rho-associated kinase (ROCK-1) is critical for cancer cell migration and invasion, suggesting it may be useful therapeutic target (Patel, et al., 2012). The protein structure of PDB ID: 3TWJ was selected from protein data bank (PDB). One of the famous anti-cancer chemotherapy drugs for colon and breast cancer is Fluorouracil, which was utilized as reference ligand to undertake the molecular docking studies toward ROCK-1 protein. Fluorouracil is a pyrimidine analog that is a nucleoside metabolic inhibitor for the treatment of breast cancer (Choi and Kim, 2009). The interactions of the newly designed hybrids with active sites are summarized in Table 2.

2-(4-Formylphenoxy)-N-(pyrid-2-yl) acetamide derivative 2 displayed small binding over two different types of interactions. Four intermolecular hydrogen bonds between the nitrogen atom of amidic group with Ser 108 (2.81 Å), N-atom of pyridine ring with Phe 110 (3.41 Å), and O-atom of amide function with Glu 379 (2.90 Å). Two π-π interactions between both of O atom of Aldehydic group with Arg 84 and Benzene ring with Glu 88 through intermolecular distance (3.02 Å), (4.27 Å), respectively. This aldehyde precursor 2 exhibited binding energy score, S = −5.8000 kcal/mol (Fig. S1). Thiosemicarbazone derivative 3 displayed three intermolecular hydrogen bonds. The first intermolecular hydrogen bond was displayed between NH of Thiosemicarbazone moiety with Asp 374 (2.92 Å) (Fig. S2). Where, the second and third hydrogen bonds are formed between NH₂ of thiosemicarbazone moiety with both of Asp 374 and Arg 84 through intermolecular distances (3.21 Å) and (3.10 Å), respectively. All of these interactions presented good binding energy score, S = −6.7686 kcal/mol.

The pyridine-thiazole compound 4a delivered binding through formation of two intermolecular hydrogen bonds in addition to one π-π interaction (Fig. S3). One hydrogen bond was formed between the sulfur atom of the thiazole ring and Ser 108 (3.20 Å), second intermolecular hydrogen bond was displayed between oxygen atom of the amide moiety and Ala 86 (3.00 Å), and π-π interaction was exhibited between the pyridine ring with Ala 86 (3.65 Å). It is noticeable that derivative 4a was revealed a better interaction energy score (S = −6.8745 kcal/mol). The second derivative 4b presented four intermolecular hydrogen bonds. The first and the second interactions were existed between the S-atom of the thiazole ring and both of Ser 108 (3.09 Å), and Arg 147(3.15 Å) of amino acids of 3TWJ. Meanwhile, the third hydrogen bond was raised between the O-atom of amidc group with Ala 86 via intermolecular distance (3.04 Å), and the fourth was arisen between Glu 379 and the nitrogen atom of the hydrazo moiety (3.08 Å) (Fig. S4). Derivative 4b was shown a score of binding with the amino acids of 3TWJ (S = −7.1059 kcal/mol).

Meanwhile, compound 5 revealed two dissimilar types of interactions (Fig. 2). The first was an intermolecular hydrogen bond between the nitrogen atom of the amide function with Asp 347 (2.94 Å), and π-π interactions were exhibited between the pyridine ring with Arg 84 (3.40 Å). Compound 5 was exhibited reasonable binding score with the amino acids of 3TWJ (S = −6.6614 kcal/mol). The pyridine-thiazole derivative 6a displayed three π-π interactions in addition to one intermolecular hydrogen bond. The first π-π interaction was demonstrated between pyridine ring with Arg 84 (4.09 Å). The second and the third hydrogen bonds were arisen between the benzene ring with Glu 379, and thiazolin-4-one ring with Thr 380 through two intermolecular distances (4.19 Å) and (4.18 Å), respectively. Meanwhile, the intermolecular hydrogen bond was exhibited H-donor on the amidic nitrogen with between Asp 347 (3.58 Å). All of these intermolecular forces were displayed binding energy score, S = −7.4179 kcal/mol (Fig. 3). In addition, pyridine-thiazole derivative 6b demonstrated two types of interactions. The first was exhibited intermolecular hydrogen bond between the S- atom of thiazolin-4-one moiety with Glu 111 (3.92 Å), the second π-π interaction was arisen between Pyridine ring with Phe 381 via intermolecular forces (3.96 Å). All of these bindings were revealed better binding energy score, S = −8.0093 kcal/mol (Fig. 4). Moreover, the pyridine-thiazole derivative 6c displayed four intermolecular hydrogen bonds. The first was displayed between the hydrogen donor on the amidic nitrogen with Ser 108 (3.06 Å). Where, the rest of hydrogen bonds were displayed between both of the two nitrogen atoms of hydrazo moiety, and S atom of thiazolin-4-one ring with Phe 381through intermolecular distance (3.27 Å), (4.34 Å), and (3.16 Å), respectively. Derivative 6c was showed given binding energy score, S = −7.7215 kcal/mol (Fig. 5).

Close

Fig. 2

The interaction of pyridine-thiazole hybrid 5 with Rho-associated protein kinase 1.

Export to PPT

Close

Fig. 3

The interaction of pyridine-thiazole hybrid 6a with Rho-associated protein kinase 1.

Export to PPT

Close

Fig. 4

The interaction of pyridine hybrid 6b with Rho-associated protein kinase 1.

Export to PPT

Close

Fig. 5

The interaction of pyridine-thiazole hybrid 6c with Rho-associated protein kinase 1.

Export to PPT

The pyridine-thiazole derivative 7 presented three intermolecular hydrogen bonds. The first and the second intermolecular hydrogen bonds were demonstrated between the hydrogen donor of both of hydrazo N1 and S-atom of thiazolidinone ring with Asp 374 through intermolecular distance (2.96 Å) and (3.59 Å), respectively. While, the third hydrogen bond was established between the oxygen atom of the thiazolin-4-one ring with Glu 379 (3.27 Å). All of these bindings were displayed binding energy score, S = −7.2041 kcal/mol (Fig. 6).

Close

Fig. 6

The interaction of pyridine-thiazole hybrid 7 with Rho-associated protein kinase 1.

Export to PPT

Furthermore, pyridine-thiazole compound 8 exhibited one intermolecular hydrogen bond in addition to two π-π interactions. The hydrogen bond was demonstrated between the hydrogen donor on the nitrogen atom of the amidic moiety with the Asp 374 (3.04 Å), and the two π-π interactions were risen between both of the thiazole ring with Thr 380 and the benzene ring with Glu 379 through intermolecular distances (4.44 Å) and (3.96 Å), respectively. All of these interactions were revealed binding energy score, S = −7.1284 kcal/mol (Fig. S5). Meanwhile, enaminone compound 9 revealed two intermolecular hydrogen bonds and one π-π interaction. The two hydrogen bonds were demonstrated between both of the nitrogen atom of the hydrazo group and the S-atom of the thiazole ring with Glu 379 through intermolecular distances (3.27 Å) and (4.16 Å), respectively. However, the π-π interaction was displayed between the pyridine ring with the Gln 391 through intermolecular distance (3.52 Å). Somewhere, these bindings were disclosed binding energy score, S = −7.0980 kcal/mol (Fig. S6).

Moreover, derivative 10 presented two intermolecular hydrogen bonds. The first one was shown between the hydrogen donor of NH of pyrazole ring with Ala 86 through intermolecular distance (3.18 Å). Where, the second hydrogen donor was exhibited between S-atom of the thiazole ring with Glu 111 I intermolecular distance (3.90 Å). All of these bindings were disclosed binding energy score, S = −7.5528 kcal/mol (Fig. 7).

Close

Fig. 7

The interaction of pyridine-thiazole hybrid 10 with Rho-associated protein kinase 1.

Export to PPT

The mechanism of antitumor action. Key components of the Rho/Rho-associated coiled-coil containing protein kinase (ROCK) signalling pathway. Various extracellular stimuli (growth factors and hormones) bind to cell membrane receptors, which subsequently act upon guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) to regulate activation of Rho GTPase proteins. Once in its GTP-bound active state, Rho GTPase binds to ROCK (ROCK1/2) to stimulate key downstream effectors (Chin et al., 2015).