Design, synthesis, in vitro antiproliferative effect and in situ molecular docking studies of a series of new benzoquinoline derivatives

2.1 Chemistry

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Scheme 1

Synthesis of benzoquinoline derivatives: 1. R = 4-CH₃C₆H₄ (CMMP 1), 2. R = 4-NO₂C₆H₄ (CNMP 2), 3. R = 4-(CH₃)₂NC₆H₄ (CPMP 3), 4. R = 2-Theinyl (CRMP 4), 5. R = 1-Naphthyl (CTMP 5).

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Scheme 2

Reaction mechanism for the synthesis of benzoquinoline derivatives.

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Fig. 1

Structures of 2-amino-4-subistituted-5,6-dihydrobenzo[h]quinoline-3 carbonitrile derivatives.

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2.2 Anticancer activity

2.3 Molecular docking

2.3.1

2.3.1 Breast cancer

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Fig. 2

3D docking, site view & 2D of the CMMP (1) (2-Amino-4-(p-tolyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile) and 3EQM protein cancer of breast cancer protein.

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Table 2 Docking score and energy of the compounds and 3EQM protein cancer of breast cancer protein.

mol	S	rmsd_refine	E_conf	E_place	E_score1	E_refine	E_score2
CMMP (1)	−7.24	0.78	−28.29	−77.10	−10.40	−27.16	−7.24
	−7.03	0.98	−28.90	−83.32	−9.94	−28.83	−7.03
	−6.88	1.01	–32.85	−73.07	−10.73	−34.00	−6.88
	−6.87	2.32	−38.88	−97.65	−10.36	−35.17	−6.87
	−6.82	1.29	−36.64	−87.97	−9.93	−29.06	−6.82
CNMP (2)	−7.55	0.73	−0.55	−79.68	−10.61	−36.75	−7.55
	−7.54	0.86	9.13	−79.41	−11.17	−27.95	−7.54
	−7.53	1.33	3.78	−81.73	−10.84	−36.80	−7.53
	−7.46	1.32	−1.90	−101.67	−10.74	−36.30	−7.46
	−7.41	1.16	1.33	−96.69	−10.72	−37.65	−7.41
CPMP (3)	−6.61	0.74	−15.18	−70.33	−9.50	−26.14	−6.61
	−6.48	0.95	−20.08	−86.56	−9.12	−30.38	−6.48
	−6.47	1.02	−20.15	−77.36	−8.94	−31.02	−6.47
	−6.41	4.98	−20.99	−69.87	−10.30	−29.63	−6.41
	−6.37	2.33	−18.56	−69.25	−9.11	−26.41	−6.37
CRMP (4)	−6.86	1.05	−18.54	−81.17	−9.65	−30.58	−6.86
	−6.80	0.90	−16.94	−77.32	−9.67	−28.40	−6.80
	−6.76	0.90	−21.62	−71.69	−9.81	−25.78	−6.76
	−6.73	1.30	–22.96	−82.51	−10.06	–32.29	−6.73
	−6.70	1.18	−1.72	−83.66	−9.39	−35.69	−6.70
CTMP (5)	−7.41	1.40	−19.25	−68.66	−10.38	–32.46	−7.41
	−7.34	0.70	–22.89	−90.73	−10.57	−28.94	−7.34
	−7.27	3.05	−25.14	−74.92	−9.98	−39.27	−7.27
	−7.17	1.11	−16.44	−73.64	−9.89	–33.62	−7.17
	−7.15	1.71	–23.67	−99.32	−11.01	−34.25	−7.15

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Table 3 Interaction table between the compounds and 3EQM protein cancer of breast cancer protein.

	Ligand	Receptor	Interaction	Distance E	(kcal/mol)
CMMP (1)	N 41	CA MET 303	H-acceptor	3.59	−0.9
	6-ring	N ALA 438	pi-H	4.23	−1.1
CNMP (2)	O 37	NE1 TRP 141	H-acceptor	3.39	−1.4
	O 37	NH2 ARG 145	H-acceptor	3.14	−1.3
	O 38	NH2 ARG 115	H-acceptor	3.08	−1.1
	O 38	NE ARG 435	H-acceptor	2.97	−2.9
	O 38	NH2 ARG 435	H-acceptor	3.17	−1.9
CPMP (3)	N 36	SD MET 311	H-donor	4	−0.6
CRMP (4)	N 24	SD MET 311	H-donor	3.54	−1.7
	6-ring	CA GLY 439	pi-H	4.33	−0.7
CTMP (5)	N 23	O ARG 435	H-donor	2.98	−1.7

2.3.2

2.3.2 Colon cancer

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Fig. 3

3D docking, site view & 2D of the CMMP (1) (2-Amino-4-(p-tolyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile) and 3IG7 protein cancer of colon-cancer protein.

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2.3.3

2.3.3 Hepato cancer

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Fig. 4

3D docking, site view & 2D of the CMMP (1) (2-Amino-4-(p-tolyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile) and 4FM9 protein cancer of hepato-cancer protein.

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3.1 Antiproliferative effect

Three different human tumor cell lines that include colon carcinoma HT29, hepatocellular carcinoma HePG2, and Caucasian breast adenocarcinoma MCF7 were taken into consideration and used to investigate an in vitro antiproliferative effect (cytotoxic activity) of the all synthesised derivatives (Fig. 1) viz. 2-amino-4-(p-tolyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (CMMP, 1), 2-Amino-4-(4-nitrophenyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (CNMP, 2), 2-Amino-4-(dimethylamino)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile(CPMP,3),2-Amino-4-(thiophen-3-yl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile(CRMP,4) and 2-Amino-4-(naphthalen-1-yl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile(CTMP,5) through MTT assay(https://www.dojindo.eu.com/Protocol/Dojindo-Cell-Proliferation-Protocol.pdf, 2019). Table 1 demonstrates the LC₅₀ (μM) values (concentration of the test benozquinoline derivatives that were responsible to lead the death of 50% of the cells in 24 h (lethal dose)). The obtained data showed that all the different cancer cell lines which were taken into account displayed dissimilar degree of sensitivity activities against all of the above mentioned benzoquinoline based derivatives that were evaluated in this study. In the colon cancer cell line (HT29), compounds 2 and 5 (viz. LC₅₀ 21.23 and 30.12 μM, respectively) revealed distinctive sensitivity in comparison to the referenced anticancer drug, doxorubicin (LC₅₀ 40.0 μM). Meanwhile, the compound 2 superseded the doxorubicin (LC₅₀ 40.0 μM) activity, however compounds 1, 3 and 4 (LC₅₀ 42.64, 85.84 and 74.7 μM, respectively) displayed moderate cytotoxic effect towards the HT29 with compound 2 being the most potent among them. However, the MCF 7 (human breast cancer cell line) showed a significant sensitivity towards four of the investigated structure analogues and their LC₅₀ ranged 8.24–54.45 μM, in comparison to doxorubicin (LC₅₀ 4.0 μM). Between all the examined compounds, the highest activity was shown by compounds 2 and 5 (LC₅₀ 8.24 and 10.12 μM, respectively). While the hepatocellular carcinoma HepG2 was found to be the slightest sensitive as the growth of this cell line was impacted by the three structure analogues of benzoquinoline only. Moreover, compounds 2 and 5 were found to show a potent growth inhibition potential as evidenced from their small LC₅₀ values (26.15 and 25.20 μM, respectively), that represented around 40–60% of the activity of doxorubicin (LC₅₀ 4.0 μM). This implied that the compounds 1, 3 and 4 had a good cytotoxic effect against the all cell lines used in this study. The compound 2 was found to be the most active member in the current research work with distinct efficacy towards both the MCF 7 (human breast cancer) (about 50% of the activity of doxorubicin; LC₅₀ 8.24 versus 4.0 μM, respectively) and HT29 (the colon carcinoma) (almost two-fold as active as doxorubicin; LC₅₀ 21.23 versus 40 μM, respectively). Existence of two cyano functional groups on the double bonded carbons (olefinic) especially nitro group (being the most active substituent) appeared to structurally influence the cytotoxic activity of the coded compound 2 among others (Larsson et al., 2020).

3.2 Molecular docking

Molecular docking is one of the proficient ways to calculate as well as surmise the kind of interaction and binding sites with the interacting molecules. MOE modeling program was employed in order to visualise and calculate the binding sites and their docking scores of the coded compounds viz. CMMP (1), CNMP (2), CPMP (3), CRMP (4) and CTMP (5) with each of the three different cancer protein enzymes coded as 3EQM for human breast, 3IG7 for colon and 4FM9 for hepatocellular carcinoma cell lines, respectively. Among the all energies calculated, the least binding energy revealed the highest activity which was observed by the ranking poses generated by scoring functions which are given in Tables 2, S4 and S6, respectively. The best score was obtained by the 2 in all the three cancer proteins and the scores were −7.55, −6.90 and −6.90, respectively. List of hydrogen bonds between all the synthesized compounds figure with coenzymes of the chosen proteins are respectively presented in Tables 3, S5 and S7. The best fitted poses adopted by the compounds docked into enzymes viz. 3EQM, 3IG7 and 4FM9 are shown in Figs. 2, 3 and 4, respectively (Meng et al., 2011; Pantsar and Poso, 2018).

Molecular Operating Environment (MOE) is one of the molecular docking routines that is employed in order to recognize an accurate docking studies between the compounds and the target proteins. The docking pose and types of interaction were aligned and agreed with the experimental LD₅₀ of these compounds against the three cancer proteins (Figs. 2-4; S1-S6). For the 3EQM protein “human breast cancer”, the compound 2 showed the high docking score through hydrogen bonding between two oxygen atoms of the nitro group towards Trp 141, Arg 145, Arg115 and Arg 435 amino acid residues. The hydrogen bond varied from 2.97 to 3.39 A° and the energy stabilization by −1.1 to −2.9 K cal. While in the case of 3IG7 protein “colon cancer”, the compound 2 showed the high docking score by the hydrogen accepting ability of one of the oxygen atoms of nitro group towards Lys 33 amino acid residue with hydrogen bond “3.26 A°” and energy stabilization by −3.3 K cal., nitrogen atom of the same nitro group demonstrated hydrogen bond “3.53 A^o and energy stabilization by −2.1 K cal. with Leu 83 amino acid residue as well as hydrogen –π-stacking with 6-membered ring of Gly 11 “3.99 A° and −0.9 K cal.”. On the other hand, it was found that the 2 exhibited the high docking score according to its interaction with 4FM9 protein “hepatocellular carcinoma cell lines” through one of the oxygens of nitro group with Arg 727 residue as a hydrogen bond viz. 3.01 A° and −2.80 K cal., respectively (Ferreira et al., 2015; Singh et al., 2019). These hydrogen bonding, ionic and hydrophobic bonding interactions with the key amino acid residues of the identified binding pockets based on the above output, stabilized the structure of the target receptor. All the docked pose with the least binding energy has the highest affinity, thereby, is considered the best docked conformation. Moreover, analysis of these docked ligands with these proteins brought into focus a very vital interaction operating at the molecular level, this is due to the fact that the docking program (MOE) was able to reproduce experimentally observed binding modes, as in this findings, it identifies the particular target-ligand conformation (Pantsar and Poso, 2018; Boittier et al., 2020).

4.1 Instrumentation

Gallenkamp device was used to determine the melting point of all benzoquinoline derivatives. Bruker DPX-400 FT NMR spectrometer was used to determine the proton and carbon 13 spectra. Tetramethylsilane (TMS) and DMSO‑d₆ were used as an internal standard and solvent, respectively. The chemical shifts were measured as δ in ppm and splitting patterns were labelled as q for quartet, m for multiplet, d for doublet and s for singlet, respectively. Shimadzu FT-IR 8400S spectrophotometer was used to measure the infrared spectra by following the KBr pellet method. 2400 Perkin Elmer Series 2 analyzer was used to carry out the elemental analysis and the observed values were found to vary with the practical values within the range of ± 0.4%. All synthetic reactions were observed through TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the UV-lamp at λ 254 was used to detect the spots of the final products on the TLC plate. The three kinds of distinct cancer cell lines namely breast (MCF-7), colon (HT29) and hepatocellular carcinoma were directly procured from the Sigma-Aldrich Co. (St Louis, MO, USA).

4.2 General method for the synthesis of arylidene derivatives

A combination of the suitable carbonyl compounds i.e. aldehydes (10 mmol), malononitrile i.e. propanedinitrile (10 mmol) and piperidine (azinane) (catalytic amount of it) in ethyl alcohol (25 mL) were agitated for about sixty minutes at room temperature (25 °C). Afterwards, the reaction mixture was then poured onto water (200 mL) and left undisturbed overnight. Subsequently, the solid product as precipitate was obtained through filtration process, washed carefully with plenty of H₂O then completely dried. Later, it was recrystallized with the suitable organic solvents to obtain in pure form (Qandalee et al., 2013).

4.3 General procedure for synthesis of 2-Amino-4-subistituted-5,6-dihydrobenzo[h]-quinoline-3-carbonitrile.

A combination of arylidene derivative (Hussaini, 2016), 1-tetralone (1.46 g, 10 mmol), 1 (10 mmol) and excess ammonium acetate (6.2 g, 80 mmol) in an ethanol (30 mL) (absolute) was heated for about 4–6 h under reflux. The completion of reaction was closely examined by the TLC plates run in different binary solvent systems. After comprehensive conversion (with the TLC indication), the separated solid material during heating was cautiously obtained through filtration process and recrystallized from a mixture of ethyl alcohol and a few drops of DMF as needles shaped fine crystals (Asiri et al., 2011).

2-amino-4-(p-tolyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (1) [CMMP (R = 4-CH₃C₆H₄)] 1: Recrystallized from DMF/ethanol as needles (3.2 g, 82%), m.p. 150–152 °C. ν _max (cm⁻¹, KBr): 2205 (CN); 3332, 3482 (NH₂). ¹HNMR [400 MHz, DMSO‑d₆, δ (ppm)]: δ 2.36 (s, 3H, CH₃); 2.49 (m, 2H, H-5), 2.70 (m, 2H, H-6), 7.28–8.20 (m, 10H, Ar H + NH₂). ¹³CNMR [150 MHz, DMSO‑d₆, δ (ppm)]: 21.35 (CH₃), 23.72 (C-5), 27.94 (C-6); 114.87 (CN); 86.41, 125.55, 126.84, 127.42, 127.41, 128.20, 129.82, 129.83, 130.10, 132.46, 134.25, 139.22, 150.23, 159.82, 160.23 (ArC). Anal% Calcd for C₂₁H₁₇N₃: C, 81.00; H, 5.50; N, 13.49. Found: C, 80.98; H, 5.53; N, 13.51.

2-Amino-4-(4-nitrophenyl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (2) [CNMP (R = 4-NO₂C₆H₄)] 2: Recrystallized from ethanol as needles (3.2 g, 79%), m.p. 190–192 °C. ν _max (cm⁻¹, KBr): 2228 (CN), 3238 (NH₂).¹HNMR [400 MHz, DMSO‑d₆, δ (ppm)]: 2.51 (m, 2H, H-5); 2.73 (m, 2H, H-6); 6.81–8.05 (m, 10H, Ar H + NH₂), 7.98 (s, 1H, NH). ¹³CNMR [150 MHz, DMSO‑d₆, δ (ppm)]: 23.69 (C-5), 27.79 (C-6), 112.53 (CN), 115.47, 116.73, 121.76, 125.20, 125.30, 126.40, 128.50, 130.60, 139.23, 147.37, 147.59, 159.85, 161.95, 162.73 (ArC). Anal.% Calcd for C₂₀H₁₄N₄O₂: C, 70.17; H, 4.12; N, 16.37. Found: C, 70.21; H, 4.01; N, 16.51.

2-Amino-4-(dimethylamino)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (3) [CPMP (R = 4-(CH₃)₂NC₆H₄)] 3: Recrystallized from ethanol as needles (3.6 g, 82%), m.p. 178–180 °C. ν _max (cm⁻¹, KBr): 2209 (CN); 3362, 3478 (NH₂). ¹HNMR [400 MHz, DMSO‑d₆, δ (ppm)]: 3.10 (s, 6H, CH₃); 2.54 (m, 2H, H-5), 2.71 (m, 2H, H-6), 6.85–8.17 (m,10H, Ar H + NH₂). ¹³CNMR [150 MHz, DMSO‑d₆, δ (ppm)]: 41.38 (CH₃), 24.14 (C-5), 27.85 (C-6), 113.55 (CN), 85.8, 112.41, 112.52, 122.80, 125.42, 126.93, 127.52, 128.46, 128.7, 130.78, 139.52, 150.22, 155.3, 159.91, 160.35 (ArC). Anal.% Calcd for C₁₆H₁₆N₄: C, 72.70; H, 6.10; N, 21.20. Found: C, 70.68; H, 6.13; N, 21.22.

2-Amino-4-(thiophen-3-yl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (4) [CRMP (R = 2-Theinyl)] 4: Recrystallized from ethanol as needles (3.1 g, 77%), m.p. 175–176 °C. ν _max (cm⁻¹, KBr): 2212 (CN); 3355, 3432 (NH₂). ¹HNMR [400 MHz, DMSO‑d₆, δ (ppm)]: 2.57 (m, 2H, H-5),2.75 (m, 2H, H-6), 7.22–8.17 (m, 9H, Ar H + NH₂). ¹³CNMR [150 MHz, DMSO‑d₆, δ (ppm)]: 24.02 (C-5), 27.44 (C-6), 113.8 (CN), 85.22, 126.84, 126.82, 127.91, 128.54, 128.44, 128.62, 128.90, 130.75, 134.22, 139.63, 139.80, 146.92, 159.78, 160.23 (ArC). Anal.% Calcd for C₁₈H₁₃N₃S: C, 71.26; H, 4.32; N, 13.85. Found: C, 71.23; H, 4.35; N, 13.82.

2-Amino-4-(naphthalen-1-yl)-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (5) [CTMP (R = 1-Naphthyl)] 5: Recrystallized from ethanol as needles (3.4 g, 80%), m.p. 220–222 °C. ν _max (cm⁻¹, KBr): 2220 (CN); 3333, 3480 (NH₂). ¹HNMR [400 MHz, DMSO‑d₆, δ (ppm)]: 2.56 (m, 2H, H-5), 2.74 (m, 2H, H-6); 7.30–8.10 (m,13H, Ar H + NH₂). ¹³CNMR [150 MHz, DMSO‑d₆, δ (ppm)]: 24.05, 27.82 (CH₂), 113.70 (CN), 85.54, 124.22, 125.54, 125.71, 126.62, 126.64, 127.50, 128.38, 128.41, 128.62, 128.92, 130.40, 132.79, 134.25, 134.98, 138.89, 139.3, 150.35, 159.62, 161.29 (ArC). Anal.% Calcd for C₂₄H₁₇N₃: C, 82.97; H, 4.93; N, 12.10. Found: C, 82.95; H, 4.96; N, 12.13.

4.4 In vitro cytotoxicity investigation via MTT assay

All the synthesized derivatives of benzoquinoline were tested for their in vitro cytotoxic effect by following an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method (Cell Counting Kit-8 (WST-8 based), Dojindo Molecular Technologies, Inc. Cat.# CK04-01) (Denizot and Lang, 1986). Three distinct human cancer cell lines, viz., colon carcinoma HT29, Caucasian breast adenocarcinoma MCF7 and hepatocellular carcinoma HepG2 as well as a normal non transformed human foreskin fibroblast Hs27 cell line were used for abovementioned cytotoxicity investigation. The in vitro cytotoxicity investigation through MTT assay was carried out with slight modification. Briefly, all experiments were carefully carried out in a laminar flow cabinet biosafety class II (Baker, SG403INT, Stanford, ME, USA) level particularly within a sterile region. Cells were cultured in batches for a specific period of 10 days and then carefully seeded at 10 × 10³ cells/well (concentration) in a freshly prepared growth medium. This process was carried in a 96-well microtiter plastic plate for twenty four hours under 5% CO₂ with the help of a water jacketed CO₂ incubator (Sheldon, TC2323, Cornelius, OR, USA) at 37 °C. Media was cautiously extracted, freshly prepared medium without serum was then transferred, and cells (negative control) were subjected to incubation only. Leter, they were treated with various strengths of all the synthesized quinoline derivatives investigated at 100–50-25–12.5–6.25–3.125–1.56–0.78 μg/mL. All the synthesized quinoline derivatives were dissolved in DMSO which was taken as a vehicle. The final concentration of the DMSO solution was not increased more than 0.2% for any cell preparations. DMEM was used for MCF 7 cell line and RPMI 1640 medium was used for HT29 and HepG2 cell lines suspensions and then 1% antibiotic–antimycotic mixture (10,000 μg/mL streptomycin sulphate, 10,000 IU/mL penicillin potassium and 25 μg/mL amphotericin B), and 1% L-glutamine were also added in 96-well flat bottom microplate at 37 °C under 5% CO₂. After incubation process for 24 h, aspiration of the medium was carried out and then 40 μL of MTT reagent (2.5 μg/mL) was vigilantly transferred to every well and then plate was further kept for incubation for another four hours at 37 °C under 5% CO₂. Afterwards, 10% sodium dodecyl sulphate (SDS) (200 μL) in deionized water was separately/individually transferred to every well to terminate the reaction by dissolving the crystals formed and then plate was again put for incubation all night at 37 °C. At 595 nm, the absorbance was measured using microplate multiwall reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA). 620 nm was used as a reference wavelength in this experiment. The values are displayed in Table 1 as LC₅₀ (μM) (toxic amount that was responsible to kill 50% cells in the period of 24 h) for all the synthesized quinoline based derivatives investigated in this study (Sylvester, 2011; Larsson et al., 2020).

4.5 Molecular docking methodology

All the scores obtained in the molecular docking process were calculated and characterized through the special software package known as Molecular Operating Environment (MOE). Initially, the 3D structures of all the synthetic compounds tested were built with the help of Chem3D ultra 12.0 software [Molecular Modeling and Analysis; Cambridge Soft Corporation] and subsequently all the compounds were evaluated for energy minimization through MOPAC and finally kept as MDL MolFile (*.mol). Protein Data Bank (https://www.rcsb.org/pdb/ welcome.do) was used to recover and generate the target crystal structures of breast protein cancer (PDB code = 1HK7 and PDB code = 3EQM), colon protein cancer (PDB code = 3IG7) and hepatocellular carcinoma (PDB code = 4FM9) (Mashat et al., 2019). For the optimization process, all bound cofactors and waters ligands were disconnected from the protein structure and the hydrogen atoms were finally attached to it. The active sites were sequestered and taken as dummies atoms. MMFF94x force field was used to apportion all the parameters and charges (Abdellattif et al., 2018). Upon generation of the alpha-site spheres through the use of MOE’s site finder module, the molecules structural model was docked on the surface of the interior of the cancer protein through the MOE’s DOCK module. For executing all the calculations, intel(R) core(TM)i7, 3.8 GHz based machine running MS Windows 10 as the operating system was used. London dG scoring function was used to execute the Dock scoring in MOE software and two unrelated refinement methods were further used to upgrade. Auto rotatable bonds were subsequently permitted for the top five binding poses that were targeted for the analysis to attain the best possible score (Althagafi et al., 2019). The database browser was then employed in order to compare the docking poses to the ligand in the co-crystallized structure as well as to acquire RMSD of the docking pose. Later, the binding free energy and hydrogen bonds between the synthesized molecules and amino acid residues in the receptors were calculated in order to rank the binding affinity of the molecules to the protein molecules taken into account. Hydrogen bond length below 3.5 A° was used to ascertain the intensity of the hydrogen bonding between the synthesized quinoline derivatives and amino acids in the receptors. Moreover, RMSD of the synthesized quinoline derivatives’ position was further matched to the docking pose employed in ranking. Both the types of interactions as well as the RMSD of the ligand (native) within the receptor’s structure were taken as standard docked model (Abdel-Rhman et al., 2019).

4.6 Statistical analysis

A statistical significance was confirmed among the samples in question and the cells with vehicle (negative control) using independent t-test by SPSS 20 program.