Synthesis, Structure, Chemical Stability, and In Vitro Cytotoxic Properties of Novel Quinoline-3-Carbaldehyde Hydrazones Bearing a 1,2,4-Triazole or Benzotriazole Moiety

A small library of novel quinoline-3-carbaldehyde hydrazones (Series 1), acylhydrazones (Series 2), and arylsulfonylhydrazones (Series 3) bearing either a 1,2,4-triazole or benzotriazole ring at position 2 was prepared, characterized by elemental analyses and IR, NMR, and MS spectra, and then subjected to in vitro cytotoxicity studies on three human tumor cell lines: DAN-G, LCLC-103H, and SISO. In general, compounds 4, 6, and 8 substituted with a 1,2,4-triazole ring proved to be inactive, whereas the benzotriazole-containing quinolines 5, 7, and 9 elicited pronounced cancer cell growth inhibitory effects with IC50 values in the range of 1.23–7.39 µM. The most potent 2-(1H-benzotriazol-1-yl)-3-[2-(pyridin-2-yl)hydrazonomethyl]quinoline (5e) showed a cytostatic effect on the cancer cell lines, whereas N′-[(2-(1H-benzotriazol-1-yl)quinolin-3-yl)methylene]-benzohydrazide (7a) and N′-[(2-1H-benzotriazol-1-yl)quinolin-3-yl)methylene]-naphthalene-2-sulfonohydrazide (9h) exhibited selective activity against the pancreas cancer DAN-G and cervical cancer SISO cell lines. Based on the determined IC50 values, the compound 5e seems to be leading compound for further development as anticancer agent.

In this context, worth noting are anticancer quinoline-based hydrazone derivatives that have been described in review articles [27,28]. Recently, quinoline-3-carbaldehyde hydrazones of type I (Figure 1) have come into the focus of our research program aimed at the discovery of novel anticancer agents. As described by Bingul et al. [29], compound I reduced the viability of SH-SY5Y neuroblastoma cancer cells and induced G 1 cell cycle arrest by upregulating the cell-cycle-related p27 Kip1 protein [29].
With the above information in mind, we decided to prepare a small library of new quinoline hydrazones (series 1) and N-acylhydrazones (series 2) bearing either 1,2,4-triazole or benzotriazole at position 2 of the quinoline ring system (Figure 1) to identify compounds with potential antitumor activity. Since N-sulfonylhydrazones have recently been studied as antiproliferative agents [30], the arylsulfonylhydrazone group was also incorporated into the target compounds ( Figure 1, series 3). In this context, worth noting are anticancer quinoline-based hydrazone derivatives that have been described in review articles [27,28]. Recently, quinoline-3-carbaldehyde hydrazones of type I ( Figure 1) have come into the focus of our research program aimed at the discovery of novel anticancer agents. As described by Bingul et al. [29], compound I reduced the viability of SH-SY5Y neuroblastoma cancer cells and induced G1 cell cycle arrest by upregulating the cell-cycle-related p27 Kip1 protein [29].
With the above information in mind, we decided to prepare a small library of new quinoline hydrazones (series 1) and N-acylhydrazones (series 2) bearing either 1,2,4-triazole or benzotriazole at position 2 of the quinoline ring system (Figure 1) to identify compounds with potential antitumor activity. Since N-sulfonylhydrazones have recently been studied as antiproliferative agents [30], the arylsulfonylhydrazone group was also incorporated into the target compounds ( Figure 1, series 3).
Due to annular tautomerism of the benzotriazole ring system, the N-heteroarylation process may take place at either the N1 or N2 nitrogen atom depending on the reaction conditions and stereochemical properties of a product [34,35]. In our case, both proton and carbon NMR spectra of the product run in  are consistent with the structure 3 (Section 3).
Due to annular tautomerism of the benzotriazole ring system, the N-heteroarylation process may take place at either the N1 or N2 nitrogen atom depending on the reaction conditions and stereochemical properties of a product [34,35]. In our case, both proton and carbon NMR spectra of the product run in  are consistent with the structure 3 (Section 3).
Structures of the benzotriazol- 1-yl (3) and benzotriazol-2-yl (3A) isomers were subjected to quantum-chemical calculations by use of the density-functional B3LYP/6-31+G* method and the SM8 (H 2 O) solvation model (Scheme 1) [36]. The computations indicated that the tautomer 3 should be more stable than 3A by 2.3 kcal/mol. Although the low energy difference suggests that both tautomers may exist in equilibrium, the N1-tautomer 3 with a higher dipole moment (µ = 6.2 Debye) than those found for the N2-tautomer 3A (µ = 2.3 Debye) should predominate over 3A in polar solvents. These results are consistent with the previous studies which indicated that in solution 1H-benzotriazole is the predominant species [37]. solvents. These results are consistent with the previous studies which indicated that in solution 1H-benzotriazole is the predominant species [37].
Compounds 2 and 3 were then subjected to reactions with hydrazine derivatives in ethanol at ambient temperature to afford the desired hydrazones 4a-e and 5a-e, respectively (Series 1, Scheme 2). The structures of the compounds 4a-e and 5a-e were confirmed by elemental analyses as well as IR, NMR, and MS spectroscopic data (see Section 3).
Regarding anticancer activity of N-acylhydrazones, we turned our attention to the results obtained by Lima et al. [38]. It was found that the -C(O)-NH-N=C-acylhydrazone scaffold of N-aroylhydrazones designed as combretastatin A4 (CA-4) analogues is bioisterically equivalent to the ethylene -CH=CH-linker. Thus, N-acylhydrazones comparable to combretastatin A4 are capable of binding to the colchicine domain on β-tubulin and may prove useful in the development of new chemotherapeutic agents with better pharmacokinetic properties than the prototype  As shown in Scheme 3, treatment of the aldehydes 2 and 3 with the appropriate aryl-and alkylhydrazides in dichloromethane under reflux in the presence of acetic acid gave rise to the formation of the corresponding N′-acylhydrazones 6a-h and 7a-h (Series 2).
Compounds 2 and 3 were then subjected to reactions with hydrazine derivatives in ethanol at ambient temperature to afford the desired hydrazones 4a-e and 5a-e, respectively (Series 1, Scheme 2). The structures of the compounds 4a-e and 5a-e were confirmed by elemental analyses as well as IR, NMR, and MS spectroscopic data (see Section 3).
Regarding anticancer activity of N-acylhydrazones, we turned our attention to the results obtained by Lima et al. [38]. It was found that the -C(O)-NH-N=C-acylhydrazone scaffold of N-aroylhydrazones designed as combretastatin A4 (CA-4) analogues is bioisterically equivalent to the ethylene -CH=CH-linker. Thus, N-acylhydrazones comparable to combretastatin A4 are capable of binding to the colchicine domain on β-tubulin and may prove useful in the development of new chemotherapeutic agents with better pharmacokinetic properties than the prototype  As shown in Scheme 3, treatment of the aldehydes 2 and 3 with the appropriate aryl-and alkylhydrazides in dichloromethane under reflux in the presence of acetic acid gave rise to the formation of the corresponding N′-acylhydrazones 6a-h and 7a-h (Series 2). The structures of the compounds 4a-e and 5a-e were confirmed by elemental analyses as well as IR, NMR, and MS spectroscopic data (see Section 3).
Regarding anticancer activity of N-acylhydrazones, we turned our attention to the results obtained by Lima et al. [38]. It was found that the -C(O)-NH-N=C-acylhydrazone scaffold of N-aroylhydrazones designed as combretastatin A4 (CA-4) analogues is bioisterically equivalent to the ethylene -CH=CHlinker. Thus, N-acylhydrazones comparable to combretastatin A4 are capable of binding to the colchicine domain on β-tubulin and may prove useful in the development of new chemotherapeutic agents with better pharmacokinetic properties than the prototype  As shown in Scheme 3, treatment of the aldehydes 2 and 3 with the appropriate aryl-and alkylhydrazides in dichloromethane under reflux in the presence of acetic acid gave rise to the formation of the corresponding N -acylhydrazones 6a-h and 7a-h (Series 2). It is well-known that N-acyl-and N-aroylhydrazones may exist as geometric isomers E/Z with respect to the C=N double bond and cis/trans amide conformers due to rotation of the amide HN-C(O) single bond ( Figure 2) [39,40]. Literature reports for N-acyl-and N-aroylhydrazones derived from aryl-and heteroaryl aldehydes indicate that these compounds may exist both in  solution [39][40][41][42] and solid phase [43][44][45][46] in the form of E-geometrical isomers. Other studies revealed the presence of the mixtures of two forms: cis and trans amide conformers of N-acyl- [39,40,[45][46][47][48][49][50][51] and N-aroylhydrazones [52,53]   Analysis of 1 H NMR spectra of the obtained N′-aroylhydrazones 6a-g and 7a-g run in  confirmed the existence of single isomers as no duplicate signals were observed. The only exception was the N′-cyclopentanecarbohydrazides 6h and 7h that in carbon and proton NMR spectra exhibited two set of resonance signals. Following the findings of Ferreira and co-workers [50], we assumed that the observed doubled signals refer to the presence of both the cis/E and trans/E amide conformers. For example, in the 13 C NMR spectrum of 6h, the signals at 172.5 ppm and 177.8 ppm referred to the carbon atoms of the amide C=O group of the cis and trans conformers, while the 1 H NMR spectrum of 6h revealed the presence of two separate singlets at 11.67 ppm and 11.42 ppm attributable to the protons of the amide C(O)-NH group. Based on the relative intensities of these signals, we concluded that in DMSO-d6 solution the N′-acylhydrazone 6h exists as a 1.3:1 mixture of equilibrating cis/E and trans/E isomers ( Figure 3).   It is well-known that N-acyland N-aroylhydrazones may exist as geometric isomers E/Z with respect to the C=N double bond and cis/trans amide conformers due to rotation of the amide HN-C(O) single bond ( Figure 2) [39,40]. Literature reports for N-acyland N-aroylhydrazones derived from aryl-and heteroaryl aldehydes indicate that these compounds may exist both in DMSO-d 6 solution [ [39][40][41][42] and solid phase [43][44][45][46] in the form of E-geometrical isomers. Other studies revealed the presence of the mixtures of two forms: cis and trans amide conformers of N-acyl- [39,40,[45][46][47][48][49][50][51] and N-aroylhydrazones [52,53] in solution. It is well-known that N-acyl-and N-aroylhydrazones may exist as geometric isomers E/Z with respect to the C=N double bond and cis/trans amide conformers due to rotation of the amide HN-C(O) single bond ( Figure 2) [39,40]. Literature reports for N-acyl-and N-aroylhydrazones derived from aryl-and heteroaryl aldehydes indicate that these compounds may exist both in  solution [39][40][41][42] and solid phase [43][44][45][46] in the form of E-geometrical isomers. Other studies revealed the presence of the mixtures of two forms: cis and trans amide conformers of N-acyl- [39,40,[45][46][47][48][49][50][51] and N-aroylhydrazones [52,53]   Analysis of 1 H NMR spectra of the obtained N′-aroylhydrazones 6a-g and 7a-g run in  confirmed the existence of single isomers as no duplicate signals were observed. The only exception was the N′-cyclopentanecarbohydrazides 6h and 7h that in carbon and proton NMR spectra exhibited two set of resonance signals. Following the findings of Ferreira and co-workers [50], we assumed that the observed doubled signals refer to the presence of both the cis/E and trans/E amide conformers. For example, in the 13 C NMR spectrum of 6h, the signals at 172.5 ppm and 177.8 ppm referred to the carbon atoms of the amide C=O group of the cis and trans conformers, while the 1 H NMR spectrum of 6h revealed the presence of two separate singlets at 11.67 ppm and 11.42 ppm attributable to the protons of the amide C(O)-NH group. Based on the relative intensities of these signals, we concluded that in DMSO-d6 solution the N′-acylhydrazone 6h exists as a 1.3:1 mixture of equilibrating cis/E and trans/E isomers ( Figure 3).   Analysis of 1 H NMR spectra of the obtained N -aroylhydrazones 6a-g and 7a-g run in  confirmed the existence of single isomers as no duplicate signals were observed. The only exception was the N -cyclopentanecarbohydrazides 6h and 7h that in carbon and proton NMR spectra exhibited two set of resonance signals. Following the findings of Ferreira and co-workers [50], we assumed that the observed doubled signals refer to the presence of both the cis/E and trans/E amide conformers. For example, in the 13 C NMR spectrum of 6h, the signals at 172.5 ppm and 177.8 ppm referred to the carbon atoms of the amide C=O group of the cis and trans conformers, while the 1 H NMR spectrum of 6h revealed the presence of two separate singlets at 11.67 ppm and 11.42 ppm attributable to the protons of the amide C(O)-NH group. Based on the relative intensities of these signals, we concluded that in  solution the N -acylhydrazone 6h exists as a 1.3:1 mixture of equilibrating cis/E and trans/E isomers ( Figure 3).
trans, E tr ans, Z cis, E cis, Z Analysis of 1 H NMR spectra of the obtained N′-aroylhydrazones 6a-g and 7a-g run in  confirmed the existence of single isomers as no duplicate signals were observed. The only exception was the N′-cyclopentanecarbohydrazides 6h and 7h that in carbon and proton NMR spectra exhibited two set of resonance signals. Following the findings of Ferreira and co-workers [50], we assumed that the observed doubled signals refer to the presence of both the cis/E and trans/E amide conformers. For example, in the 13 C NMR spectrum of 6h, the signals at 172.5 ppm and 177.8 ppm referred to the carbon atoms of the amide C=O group of the cis and trans conformers, while the 1 H NMR spectrum of 6h revealed the presence of two separate singlets at 11.67 ppm and 11.42 ppm attributable to the protons of the amide C(O)-NH group. Based on the relative intensities of these signals, we concluded that in DMSO-d6 solution the N′-acylhydrazone 6h exists as a 1.3:1 mixture of equilibrating cis/E and trans/E isomers ( Figure 3).   Next, we synthesized the quinoline-3-carbaldehyde N -sulfonylhydrazone derivatives 8a-h and 9a-h (Series 3, Scheme 4). The reactions of aldehydes 2 and 3 with arylsulfonylhydrazides proceeded smoothly in THF solution under reflux in the presence of a catalytic amount of acetic acid. The identity of the newly prepared compounds was confirmed by elemental analyses as well as the IR and NMR spectroscopic data presented in the experimental section (see Section 3).
Molecules 2018, 23, x 5 of 24 Next, we synthesized the quinoline-3-carbaldehyde N′-sulfonylhydrazone derivatives 8a-h and 9a-h (Series 3, Scheme 4). The reactions of aldehydes 2 and 3 with arylsulfonylhydrazides proceeded smoothly in THF solution under reflux in the presence of a catalytic amount of acetic acid. The identity of the newly prepared compounds was confirmed by elemental analyses as well as the IR and NMR spectroscopic data presented in the experimental section (see Section 3).

UV-Vis Studies of Hydrazones 4-9 in Aqueous Buffer
The chemical stability of the hydrazones 4-9 in phosphate-buffered saline (PBS, pH 7.4) was investigated by means of UV-Vis spectroscopy. In general, all the compounds tested proved to be stable in the PBS solution as exemplified by the hydrazides 5a and 5d and the benzenesulfonohydrazide 9c, since no new spectra with the formation of isosbestic points were observed ( Figure 4). The compound 5d ( Figure 4A) showed no noticeable time-dependent changes, whereas a decrease in the intensity of the initial spectrum of the derivative 9c ( Figure 4B) is likely due to its slow precipitation out of the PBS solution.
On the other hand, the time-dependent changes in the UV-Vis spectra of the hydrazones showed that precipitation of the derivatives 4e, 5a, 8f, and 9f is rather fast as exemplified by Figure 4C). Therefore, those poorly soluble species were excluded from a panel of compounds subjected to biological studies.

UV-Vis Studies of Hydrazones 4-9 in Aqueous Buffer
The chemical stability of the hydrazones 4-9 in phosphate-buffered saline (PBS, pH 7.4) was investigated by means of UV-Vis spectroscopy. In general, all the compounds tested proved to be stable in the PBS solution as exemplified by the hydrazides 5a and 5d and the benzenesulfonohydrazide 9c, since no new spectra with the formation of isosbestic points were observed ( Figure 4). The compound 5d ( Figure 4A) showed no noticeable time-dependent changes, whereas a decrease in the intensity of the initial spectrum of the derivative 9c ( Figure 4B) is likely due to its slow precipitation out of the PBS solution.

In Vitro Antitumor Activity
The in vitro antitumor potential of the newly synthesized quinoline-3-carbaldehyde hydrazone derivatives 4-9 was evaluated on three human cancer cell lines: the pancreatic cell line DAN-G, the large cell lung cancer cell line LCLC-103H, and the cervical cancer cell line SISO using a crystal violet microtiter plate assay as previously described [54]. This assay measures the antiproliferative activity of compounds on actively dividing cells. Primary screening of the compounds 4-9 was performed to On the other hand, the time-dependent changes in the UV-Vis spectra of the hydrazones showed that precipitation of the derivatives 4e, 5a, 8f, and 9f is rather fast as exemplified by 2-(1H-benzotriazol-1-yl)-3-(hydrazonomethyl)quinoline (5a, Figure 4C). Therefore, those poorly soluble species were excluded from a panel of compounds subjected to biological studies.

In Vitro Antitumor Activity
The in vitro antitumor potential of the newly synthesized quinoline-3-carbaldehyde hydrazone derivatives 4-9 was evaluated on three human cancer cell lines: the pancreatic cell line DAN-G, the large cell lung cancer cell line LCLC-103H, and the cervical cancer cell line SISO using a crystal violet microtiter plate assay as previously described [54]. This assay measures the antiproliferative activity of compounds on actively dividing cells. Primary screening of the compounds 4-9 was performed to indicate whether a substance possesses enough activity to inhibit cell growth by 50% at the concentration of 10 µM, which is a concentration attainable in cancer cells ( Table 1).
As revealed by the data in Table 1, the hydrazone derivatives 4, 6, and 8 bearing a triazole moiety were in general inactive with the exception of the N-sulfonylhydrazones 8b and 8g, which at a concentration of 10 µM exhibited weak to moderate cytostatic effects against all investigated cancer cell lines (percent of growth in the range of 31. .6%). On the other hand, replacement of the triazole ring with a benzotriazole moiety results in enhancement of activity as indicated by a comparison of the growth inhibitory activities of triazole-containing compounds with their corresponding benzotriazole ring counterparts (6a, 6d, and 6f-g versus 7a, 7d, and 7f-g and 8c-e and 8g-h versus 9c-e and 9g-h). This observation may arise from the higher lipophilicity of the benzotriazole analogues, which may facilitate the penetration through the tumor cell membrane and improve the targeting efficiency. Furthermore, the combined presence of a large conjugated system as well as a three-nitrogen-containing structure make the benzotriazole nucleus more susceptible to binding with enzymes or receptors in biological systems via hydrogen bonds and π-π stacking interactions [55,56].
Thus, for secondary screening aimed at determining cytotoxic potency, we selected the benzotriazole-containing compounds 5d-e, 7a, 7d, and 7f-g and 9c-e and 9g-h, which demonstrated pronounced growth inhibitory effects against at least two cancer cell lines. The results of the secondary screening are presented in Table 2 as the average IC 50 values calculated from dose-response data.
In general, the investigated compounds exhibited moderate to high growth cell inhibitory effects (IC 50 in the range of 1. .39 µM). The most potent was found to be the 2-(pyridin-2-yl)hydrazone 5e with IC 50 values ranging from 1.23 to 1.49 µM (Table 2). A reduction in cytotoxic potency by 2-to 6-fold was observed for other derivatives with acylhydrazone (compounds of type 7) or sulfonylhydrazone (compounds of type 9) moieties. However, replacing the hydrazone function with either an acylhydrazone or a sulfonylhydrazone scaffold still leads to active compounds. Hence, among the N -acylhydrazones 7 and the N -sulfonylhydrazones 9 the highest cytotoxic activity was found for compounds 7d and 9d containing a 4-chlorophenyl group ( Table 2, It should be noted that the majority of the compounds tested showed no great selectivity toward any one specific cancer cell line with the exception of the N -(benzoyl)hydrazone 7a and the N -(naphtylsulfonyl)hydrazone 9h, which were selective against the pancreatic cell line DAN-G and the cervical cancer cell line SISO (IC 50 values of 4. .59 µM) over the lung carcinoma cell line LCLC-103H (IC 50 >20 µM).  As revealed by the data in Table 1, the hydrazone derivatives 4, 6, and 8 bearing a triazole moiety were in general inactive with the exception of the N-sulfonylhydrazones 8b and 8g, which at a concentration of 10 µM exhibited weak to moderate cytostatic effects against all investigated  interactions [55,56]. Thus, for secondary screening aimed at determining cytotoxic potency, we selected the benzotriazole-containing compounds 5d-e, 7a, 7d, and 7f-g and 9c-e and 9g-h, which demonstrated pronounced growth inhibitory effects against at least two cancer cell lines. The results of the secondary screening are presented in Table 2 as the average IC50 values calculated from dose-response data. In general, the investigated compounds exhibited moderate to high growth cell inhibitory effects (IC50 in the range of 1. .39 µM). The most potent was found to be the 2-(pyridin-2-yl)hydrazone 5e with IC50 values ranging from 1.23 to 1.49 µM (Table 2). A reduction in cytotoxic potency by 2-to 6-fold was observed for other derivatives with acylhydrazone (compounds of type 7) or sulfonylhydrazone (compounds of type 9) moieties. However, replacing the hydrazone function with either an acylhydrazone or a sulfonylhydrazone scaffold still leads to active compounds. Hence, among the N′-acylhydrazones 7 and the N′-sulfonylhydrazones 9 the highest cytotoxic activity was found for compounds 7d and 9d containing a 4-chlorophenyl group (

General Information
Melting points were measured on a Boetius apparatus and are uncorrected. IR spectra were taken in KBr pellets on a Nicolet 380 FTIR 1600 spectrometer. Elemental analyses were performed on a Vario El Cube CHNS analyzer and the results are within ±0.4%. NMR spectra were recorded on a Varian Gemini 200, a Varian Unity 500, or a Bruker Avance III HD apparatus. 1 H and 13 C chemical shifts were measured relative to the residual solvent signal at 7.26 ppm and 77.0 (CDCl 3 ) or 2.50 ppm and 39.5 ppm ( . Coupling constants are shown in hertz (Hz). The mass spectra were recorded on a Shimadzu LCMS-2010 EV spectrometer equipped with an electrospray source. ESI-MS spectra were registered in a positive-or negative-ion mode. Preparative thin layer chromatography was performed on silica gel 60 PF 254 containing gypsum (Merck KGaA, Darmstadt, FRG) with the aid of Chromatotron ® using the reported solvent systems. 2-Chloroquinoline-3-carbaldehyde (1) was obtained according to the published method [57]. UV-Vis spectra were recorded with an Analytik Jena Spekol 1200 (Analytik Jena AG, Jena, Germany) in a 1.0 cm cuvette maintained at 37 • C by a thermostatically controlled cuvette holder.
Starting from 2-(1H-1,2,4-triazol-1-yl)quinoline-3-carbaldehyde (2) (0.224 g, 1 mmol) and benzenesulfonohydrazide (1 mmol), the title compound 8a was obtained after washing with hot methanol. Yield 35%; m.p. [193][194][195][196]   where OD T is the mean absorbance of the treated cells, OD c is the mean absorbance of the controls, and OD c,0 is the mean absorbance at the time the drug was added. The IC 50 values were estimated by a linear least-squares regression of the T/C corr values versus the logarithm of the substance concentration; only concentrations that yielded T/C corr values between 10% and 90% were used in the calculation. The reported IC 50 values are the averages of three independent experiments.

Conclusions
In this study, we have investigated the anticancer properties of three series of quinoline-3-carbaldehyde hydrazone derivatives possessing either 1,2,4-triazole or 1,2,3-benzotriazole rings. Analysis of the structure-activity relationships of cytotoxic activities on the human cancer cell lines of 1,2,4-triazole-containing quinolines 4, 6, and 8 and 1,2,3-benzotriazole-containing quinolines 5, 7, and 9 revealed that the less lipophilic 1,2,4-triazole derivatives are generally inactive, while the more lipophilic 1,2,3-benzotriazole analogues exhibit moderate to high cytotoxic effects. It is too early to speculate on the mechanism of action of these compounds. Nonetheless, the most active  Funding: This research received no external funding.