Novel Polyhydroquinoline-Hydrazide-Linked Schiff’s Base Derivatives: Multistep Synthesis, Antimicrobial, and Calcium-Channel-Blocking Activities

Polyhydroquinoline (PHQ) are the unsymmetrical Hantzsch derivatives of 1,4-dihydropyridines with several biological applications. In this work, twenty-five (3–27) new Schiff’s base derivatives of polyhydroquinoline hydrazide were synthesized in excellent to good yields by a multi-component reaction. The structures of the synthesized products (1–27) were deduced with the help of spectroscopic techniques, such as 1H-, 13C -NMR, and HR-ESI-MS. The synthesized products (1–27) were tested for their antibacterial and in vitro calcium -channel-blocking (CCB) potentials using the agar-well diffusion method, and isolated rat aortic ring preparations, respectively. Among the series, sixteen compounds were found to inhibit the growth of Escherichia coli and Enterococcus faecalis. Among them, compound 17 was observed to be the most potent one at a dose 2 µg/mL, with an 18 mm zone of inhibition against both bacteria when it was compared with the standard drug amoxicillin. Eight compounds showed CCB activity of variable potency; in particular, compound 27 was more potent, with an EC50 value of 0.7 (0.3–1.1) µg/mL, indicating their CCB effect.


Chemistry
We have successfully synthesized twenty-five new biologically/pharmacologically active polyhydroquinoline Schiff's base derivatives through multi-step reactions. In the first step, 3-ethoxy-4-hydroxybenzaldehyde was reacted with ethyl chloroacetate in the presence of potassium carbonate in dimethylformamide (DMF) solvent to produce esterified aldehyde. In the second step, the esterified aldehyde was reacted with dimedone, ethyl acetoacetate, and ammonium acetate in ethanol [43] to produce polyhydroquinoline (1). In the third step, a mixture of polyhydroquinoline and hydrazine hydrate was dissolved in ethanol to produce polyhydroquinoline hydrazide (2). Finally, polyhydroquinoline hydrazide (2) was further reacted with various substituted aliphatic/aromatic aldehydes in the presence of acid using ethanol solvent in order to produce the desired Schiff's base derivatives of polyhydroquinoline 3-27 (Scheme 1, Table 1). ified aldehyde. In the second step, the esterified aldehyde was reacted with dimedone, ethyl acetoacetate, and ammonium acetate in ethanol [43] to produce polyhydroquinoline (1). In the third step, a mixture of polyhydroquinoline and hydrazine hydrate was dissolved in ethanol to produce polyhydroquinoline hydrazide (2). Finally, polyhydroquinoline hydrazide (2) was further reacted with various substituted aliphatic/aromatic aldehydes in the presence of acid using ethanol solvent in order to produce the desired Schiff's base derivatives of polyhydroquinoline 3-27 (Scheme 1, Table 1). Scheme 1. Synthesis of Schiff's base derivatives based on polyhydroquinoline nucleus. ified aldehyde. In the second step, the esterified aldehyde was reacted w ethyl acetoacetate, and ammonium acetate in ethanol [43] to produce polyh (1). In the third step, a mixture of polyhydroquinoline and hydrazine hy solved in ethanol to produce polyhydroquinoline hydrazide (2). Finally, p oline hydrazide (2) was further reacted with various substituted aliphatic/ hydes in the presence of acid using ethanol solvent in order to produce the d base derivatives of polyhydroquinoline 3-27 (Scheme 1, Table 1).
Scheme 1. Synthesis of Schiff's base derivatives based on polyhydroquinoline nucl ified aldehyde. In the second step, the esterified aldehyde was reacted with dimedone, ethyl acetoacetate, and ammonium acetate in ethanol [43] to produce polyhydroquinoline (1). In the third step, a mixture of polyhydroquinoline and hydrazine hydrate was dissolved in ethanol to produce polyhydroquinoline hydrazide (2). Finally, polyhydroquinoline hydrazide (2) was further reacted with various substituted aliphatic/aromatic aldehydes in the presence of acid using ethanol solvent in order to produce the desired Schiff's base derivatives of polyhydroquinoline 3-27 (Scheme 1, Table 1).
Scheme 1. Synthesis of Schiff's base derivatives based on polyhydroquinoline nucleus. presence of potassium carbonate in dimethylformamide (DMF) solvent to p ified aldehyde. In the second step, the esterified aldehyde was reacted wit ethyl acetoacetate, and ammonium acetate in ethanol [43] to produce polyhy (1). In the third step, a mixture of polyhydroquinoline and hydrazine hydr solved in ethanol to produce polyhydroquinoline hydrazide (2). Finally, pol oline hydrazide (2) was further reacted with various substituted aliphatic/ar hydes in the presence of acid using ethanol solvent in order to produce the de base derivatives of polyhydroquinoline 3-27 (Scheme 1, Table 1).
Scheme 1. Synthesis of Schiff's base derivatives based on polyhydroquinoline nucleu presence of potassium carbonate in dimethylformamide (DMF) solvent to produce esterified aldehyde. In the second step, the esterified aldehyde was reacted with dimedone, ethyl acetoacetate, and ammonium acetate in ethanol [43] to produce polyhydroquinoline (1). In the third step, a mixture of polyhydroquinoline and hydrazine hydrate was dissolved in ethanol to produce polyhydroquinoline hydrazide (2). Finally, polyhydroquinoline hydrazide (2) was further reacted with various substituted aliphatic/aromatic aldehydes in the presence of acid using ethanol solvent in order to produce the desired Schiff's base derivatives of polyhydroquinoline 3-27 (Scheme 1, Table 1).
Scheme 1. Synthesis of Schiff's base derivatives based on polyhydroquinoline nucleus.

Antibacterial Bioassay
The antibacterial activities of the newly synthesized compounds (1-27) against Escherichia coli (gram-negative bacterium) and Enterococcus faecalis (gram-positive bacterium) were evaluated by measuring the zone of inhibition (ZOI) using the agar-well diffusion method [44,45]. The tested compounds were compared with the zone of inhibition that was produced by the amoxycillin. The results were recorded and interpreted accordingly were found the most potent at a dose 2 µg/mL, with 18 mm ZOI against E. coli and E. faecalis when compared with the standard amoxicillin. Two compounds (8 and 16) displayed a good inhibition of 16 mm at a higher concentration (2 µg/mL) against E. coli and a promising inhibition of 18 mm in the case of E. faecalis. Similarly, compounds 2, 26, 13, 3, and 12 showed moderate to good inhibition, while compounds 9, 18, 5, and 27 attributed non-significance inhibition (8-10 mm) ( Table 2). Furthermore, compounds 4, 6, 7, 10, 11, 14, 15, 19, 20, 23, and 27 did not show any inhibition from the lower to the higher concentrations. Escherichia coli (gram-negative bacterium); Enterococcus faecalis (gram-positive bacterium). Above 18 mm (significant activity), 16-18 mm (good activity), 13-15 mm (low activity), 9-12 mm (non-significant), below 9 mm (no activity).

In Vitro Calcium-Channel-Blocking Study in Isolated Aorta from SD Rats
An aortic ring was incubated in normal Kreb's solutions and the cumulative addition of the tested compounds (1-27) determined a vasorelaxant response against high K + -induced contraction. The results have suggested that compound 27 was the most potent compound, with 100% vasorelaxation in the isolated aortic ring preparation, with an EC 50 value of 0.7 (0.3-1.1) µg/mL (Figure 1 Figure S1).
In the isolated rat aortic ring preparation, the synthesized compounds were investigated against high K + (80 mM)-induced contraction. The high K + depolarizes the smooth muscle cell membrane and opens the voltage-dependent calcium channels (VDCCs), resulting in an influx of extracellular calcium (Ca ++ ) and an activation of the contractile machinery [46]. Some of the compounds (27, 26, 14, 7, 23, 17, 19, and 6) displayed a significant vasorelaxant response against high K + -induced contraction. It is well known that the contraction of the smooth muscles, such as rat aortic ring preparation, is dependent upon an increase in the cytoplasmic concentration of calcium (Ca ++ ) ions for activating the contractile element [47]. The increase in intracellular Ca ++ occurs either via influx through VDCCs or via its release from intracellular stores in the sarcoplasmic reticulum [48]. Some In the isolated rat aortic ring preparation, the synthesized compounds were investigated against high K + (80 mM)-induced contraction. The high K + depolarizes the smooth muscle cell membrane and opens the voltage-dependent calcium channels (VDCCs), resulting in an influx of extracellular calcium (Ca ++ ) and an activation of the contractile machinery [46]. Some of the compounds (27, 26, 14, 7, 23, 17, 19, and 6) displayed a significant vasorelaxant response against high K + -induced contraction. It is well known that the contraction of the smooth muscles, such as rat aortic ring preparation, is dependent upon an increase in the cytoplasmic concentration of calcium (Ca ++ ) ions for activating the contractile element [47]. The increase in intracellular Ca ++ occurs either via influx through VDCCs or via its release from intracellular stores in the sarcoplasmic reticulum [48]. Some of these synthesized compounds caused the inhibition of high K + -induced contraction in the isolated rat aortic ring preparations, indicating their blocking effect on VDCCs. The findings from our current investigation provide the mechanistic pharmaco-

Conclusions
Novel polyhydroquinoline derivatives (1-27) were synthesized in good to excellent yields using a standard procedure. All the synthesized derivatives were confirmed with the help of various spectroscopic techniques, such as HR-ESI-MS, 1 H-NMR, and 13 C-NMR, and, finally, were screened for their antimicrobial, and in vitro calcium-channel-blocking (CCB) potential using the agar-well diffusion method and isolated rat aortic ring preparations, respectively. Among the series, sixteen compounds were found to inhibit the growth of Escherichia coli and Enterococcus faecalis. Compound 17 was found to be the most potent at a dose of 2 µg/mL, with an 18 mm zone of inhibition against E. coli and E. faecalis when compared with the standard drug amoxicillin. Eight compounds showed CCB activity of variable potency; compound 27 was more potent, with an EC 50 value of 0.7 (0.3-1.1) µg/mL, indicating a CCB effect. It can be concluded that due to the active potential of the synthesized derivatives, the medicinal chemists need to investigate these compounds in more detail in the field of medicinal chemistry.

General
All of the chemicals used were analytical grade and were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification. Thin-layer chromatography (TLC) was performed on Merck Silica gel 60 F 254 plates using the solvent system ethyl acetate/n-hexane. The melting points were recorded on a Stuart apparatus. Modern high-resolution electrospray ionization spectroscopy (HR-ESIMS) (Agilent 6530 LC Q-TOF, manufactured in USA/EU, made in Singapore) was used to confirm the masses of the synthesized compounds. 1 H-and 13 C-NMR spectra were recorded on a nuclear magnetic resonance (NMR) spectrometer (BRUKER, Zürich, Switzerland) spectrometer at 600 MHz and 150 MHz, respectively, using MeOD and CDCl 3 solvents. The abbreviations that have been used in the work are as follows: s: singlet, d: doublet, t: triplet, m: multiplet and J: coupling constant to explain NMR signals in Hertz (Hz) and chemical shifts (δ). The values were expressed in parts per million (ppm). The structures of all compounds were confirmed with the help of HRESIMS and 1D ( 1 H-and 13 C) NMR spectroscopy.

Animals
The Sprague-Dawley rats (200-250 g) and mice (20-25 g) of either sex used in the study were housed in the animal house of the COMSATS University Islamabad Abbottabad campus in a controlled environment. The animals were given tap water ad libitum and a standard diet.

Synthesis of Ethyl-2-(2-ethoxy-4-formylphenoxy) Acetate
In a 100 mL round bottomed (RB) flask, 3-ethoxy-4-hydroxy benzaldehyde (3 g, 0.018 moles) was dissolved into 30 mL DMF solvent and potassium carbonate (K 2 CO 3 ) was added to it. The reaction was continuously stirred for 30 min at 120 • C. After 30 min, ethyl chloroacetate (1.9 mL) was added to it and it was refluxed for 8-10 h. The product formation was checked with thin-layer chromatography (TLC) in a solvent system n-hexane and ethyl acetate (7:3). Upon completion, the reaction mixture was cooled to room temperature and poured into ice-cold distilled water. The precipitates that were formed were filtered, washed with an excess of water, dried under air, weighed, and recrystallized with ethanol to obtain the pure esterified product.
White amorphous powder was as follows:

Synthesis of Polyhydroquinoline (1)
Ethyl-2-(2-ethoxy-4-formylphenoxy)acetate ( Figure S2) (3.27 g) and dimedone (1.8 g) were dissolved into 100 mL RB flask, dissolved in 30 mL absolute ethanol, and stirred for 30 min. Then, ethyl acetoacetate (1.65 mL) and ammonium acetate (3.96 g) were added to the reaction mixture and refluxed for 6-7 h. The progress of the reaction was monitored by TLC using system n-hexane and ethyl acetate (7:3), respectively. After the reaction was complete, as indicated by TLC, the reaction mixture was poured into cold water. The resulting precipitate was filtered, washed with an excess of water and hot n-hexane, dried, and collected for further reaction. The obtained product was further confirmed by different spectroscopic techniques, such as NMR ( 1 H-, 13 C) and HR-ESI-MS.

Synthesis of Polyhydroquinoline Hydrazide (2)
Polyhydroquinoline 1 (5.01 g) and hydrazine hydrate (1.5 mL) were taken into a 100 mL RB flask in 15 mL ethanol solvent and refluxed for 4-5 h to produce the desired product (hydrazide) in good yield. The product formation was checked by TLC using a solvent system (n-hexane: ethyl acetate, 3:7). After the completion of the reaction, it was cooled to room temperature and poured into a beaker containing ice-cold distilled water. Precipitates were formed, which were filtered and washed with an excess of water to remove un-reacted hydrazine. The product was dried at room temperature and recrystallized from ethanol to obtain the desired compound in pure form. The formation of the product was further confirmed by mass and NMR. 4.6. General Procedure for the Synthesis of Schiff's Base Derivatives of Polyhydroquinoline  Twenty-five polyhydroquinoline-based Schiff's base derivatives were synthesized from the desired hydrazide 2. Various substituted aromatic/aliphatic aldehydes were reacted with hydrazide of polyhydroquinoline in 100 mL RB flask containing 15 mL ethanol solvent, a catalytic amount of glacial acetic acid was added, and it was stirred for 20-30 min ( Table 1). The polyhydroquinoline hydrazide (120 mg) was then added to the reaction mixture and refluxed for 2-3 h. The progress of the reaction was monitored by TLC using a solvent system of hexane and ethyl acetate (6:4). After the completion of the reaction, the mixture was cooled to room temperature and poured into ice-cold distilled water. Precipitates were formed, filtered, washed with hot n-hexane to remove un-reacted aldehydes, and dried under air to obtain pure compounds 3-27. In some cases, no precipitates were formed, so the reaction mixture was extracted with ethyl acetate to obtain pure products. The desired products were collected, weighed, and confirmed by different spectroscopic techniques, such as mass and NMR.    Figure S1: The effect of synthesized compounds against high K+ (80 mM) induced contraction (n = 3-5), values represented as mean ± SEM using two-way ANOVA; Figure S2: 1H-NMR and 13C-NMR spectra of ethyl-2-(2-ethoxy-4formylphenoxy)acetate; Figure S3: 1 H-NMR and 13 C-NMR spectra of compound 1; Figure S4: 1 H-NMR and 13 C-NMR spectra of compound 2; Figure S5: Mass, 1 H-NMR, and 13 C-NMR spectra of compound 3; Figure S6: Mass, 1 H-NMR, and 13 C-NMR spectra of compound 4; Figure S7: Mass, 1 Hand 13 C-NMR spectra of compound 5; Figure S8: Mass, 1 H-and 13 C-NMR spectra of compound 6; Figure S9: Mass, 1 H-and 13 C-NMR spectra of compound 7; Figure S10: Mass, 1 H-and 13 C-NMR spectra of compound 8; Figure S11: Mass, 1 H-and 13 C-NMR spectra of compound 9; Figure S12: Mass, 1 H-and 13 C-NMR spectra of compound 10; Figure S13: Mass, 1 H-and 13 C-NMR spectra of compound 11; Figure S14: Mass, 1 H-and 13 C-NMR spectra of compound 12; Figure S15: Mass, 1 Hand 13 C-NMR spectra of compound 13; Figure S16: Mass, 1 H-and 13 C-NMR spectra of compound 14; Figure S17: Mass, 1 H-and 13 C-NMR spectra of compound 15; Figure S18: Mass, 1 H-and 13 C-NMR spectra of compound 16; Figure S19: Mass, 1 H-and 13 C-NMR spectra of compound 17; Figure S20: Mass, 1 H-and 13 C-NMR spectra of compound 18; Figure S21: Mass, 1 H-and 13 C-NMR spectra of compound 19; Figure S22: Mass, 1 H-NMR and 13 C-NMR spectra of compound 20; Figure S23: Mass and 1 H-NMR spectra of compound 21; Figure S24: Mass, 1 H-and 13 C-NMR spectra of compound 22; Figure S25: Mass, 1 H-and 13 C-NMR spectra of compound 23; Figure S26: Mass, 1 H-and 13 C-NMR spectra of compound 24; Figure S27: Mass, 1 H-and 13 C-NMR spectra of compound 25; Figure S28: Mass, 1 H-and 13 C-NMR spectra of compound 26; Figure S29. HR-ESI-MS, 1 H-and 13

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Data Availability Statement:
The spectroscopic data presented in this study are available in the supporting information.