Recruitment of hexahydroquinoline as anticancer scaffold targeting inhibition of wild and mutants EGFR (EGFR^WT, EGFR^T790M, and EGFR^L858R)

Abstract

Hexahydroquinoline (HHQ) scaffold was constructed and recruited for development of new series of anticancer agents. Thirty-two new compounds were synthesised where x-ray crystallography was performed to confirm enantiomerism. Thirteen compounds showed moderate to good activity against NCI 60 cancer cell lines, with GI % mean up to 74% for 10c. Expending erlotinib as a reference drug, target compounds were verified for their inhibiting activities against EGFR^WT, EGFR^T790M, and EGFR^L858R where compound 10d was the best inhibitor with IC₅₀ = 0.097, 0.280, and 0.051 µM, respectively, compared to erlotinib (IC₅₀ = 0.082 µM, 0.342 µM, and 0.055 µM, respectively). Safety profile was validated using normal human lung (IMR-90) cells. 10c and 10d disrupted cell cycle at pre-G1 and G2/M phases in lung cancer, HOP-92, and cell line. Molecular docking study was achieved to understand the potential binding interactions and affinities in the active sites of three versions of EGFRs.

Graphical abstract

HIGHLIGHTS

• New 32 hexahydroquinoline (HHQ) analogues 6a–i, 8a–m, 10a–d, and 12a–f having the same features of EGFR inhibitors were synthesised in racemic mixtures.

• The antiproliferative activities were assessed towards 60 cancer cell lines which were efficiently inhibited by compound 10c.

• Compound 10d remarkably inhibited EGFR^WT, EGFR^T790M, and EGFR^L858R.

• Cell cycle analysis and Annexin V-based flow cytometry in the HOP-92 lung cancer cells were performed.

• The safety profile of compounds 10c and 10d was validated using normal human lung (IMR-90) cells.

• Molecular docking studies revealed that the S-isomers exhibited higher affinity than R-isomers to active sites.

Keywords:

• Hexahydroquinoline
• wild and mutant EGFR inhibition
• anticancer
• X-ray crystallography
• molecular docking

Introduction

Despite massive attempts to develop novel anticancer agents, cancer remains one of the worst diseases in the world¹. The development of resistance to several highly successful anticancer agents is a significant hurdle in the treatment of cancer². As a result, developing new anticancer drugs has gained great momentum in the sector to address the issue of resistance^3,4. According to the GLOBOCAN 2020 cancer incidence and mortality estimates, there were 19.3 million new cancer and 10 million cancer deaths⁵. One of the current strategies to manage cancer overgrowth is inhibiting a trans-membrane glycoprotein called Epidermal growth factor receptor (EGFR) due to its essential role in intracellular signalling, morphogenesis, and differentiation^6–8.

Contrary to normal cells with tightly controlled EGFR pathways, tumour cells exhibit dysregulated EGFR signalling due to receptor overexpression and/or mutation⁹. This causes angiogenesis to rise and proliferation under unfavourable conditions, leading to the development of many cancers, such as NSC lung cancer¹⁰, breast cancer¹¹, prostate cancer¹², and colon cancer¹³. EGFR is a valid therapeutic target as a result¹⁴. There are three generations of EGFR tyrosine kinase inhibitors (TKIs), each developed to overcome a mutation to the previous one¹⁵. According to studies, one of the most frequent resistance mechanisms to first-generation EGFR-TKIs was the T790M “gatekeeper” mutation in EGFR, contributing to 3% of EGFR mutations¹⁶. A further known route of resistance to first-generation EGFR-TKIs is the L858R mutation, therefore, it was necessary to develop new molecules to overcome these mutations^17,18. The mechanism by which EGFR-TKIs produce their action is by competing with ATP for the EGFR’s ATP-binding site^19–21 (Figure 1(A)). The reference drug erlotinib (first-generation EGFR-TKI) works by occupying the essential pockets of the ATP-binding site²² (Figure 1(B)). HHQ is a well-known scaffold for developing new physiologically active compounds due to its synthetic flexibility²³. HHQ scaffold shares common pharmacophoric features with ATP, as the HHQ nucleus itself can occupy the same pocket of adenine base of ATP and different substituents on the HHQ ring can play the same role of other parts of the ATP, making it easy to develop EGFR inhibitors that compete with ATP at its active site²⁴. HHQ derivatives have a wide range of biological activities, such as anti-inflammatory²⁵, antifungal²⁶, and also promising anticancer activity²⁷; therefore, many projects started to investigate them as anticancer agents. HHQ derivatives were found to exhibit their anticancer activity via different mechanisms, such as inhibition of topoisomerase²⁸, cell cycle arrest in the G2 phase²⁹, and inhibition of tyrosine kinases (EGFR)³⁰.

Figure 1. (A) ATP binding site at the EGFR viewing essential pharmacophore features. (B) The chemical structure of erlotinib (first-generation EGFR-TKI) could only reside in three pockets of the ATP binding sites.

Rational and design

EGFR has an extracellular catalytic domain to which ATP can bind, leading to the activation of EGFR^31,32. Small molecules can compete with ATP preventing its binding to the catalytic domain^33,34. The approved EGFR inhibitors are divided into; the first-generation agents, erlotinib, and gefitinib³⁵; the second-generation agents, afatinib³⁶, and dacomitinib³⁷; and most recently, osimertinib³⁸, a third-generation EGFR-TKI. Besides, many published compounds displayed promising EGFR inhibitory activity, such as compound I and compound II, which reported anticancer activity against breast cancer³⁹. Moreover, compound II revealed anticancer activity against breast carcinoma⁴⁰. Compound III showed anticancer activity against non-small cell lung cancer²⁹ (Figure 2). By reviewing the approved EGFR inhibitors, we have explored that they have shared the following pharmacophoric features; central heterocyclic ring, hydrophobic head, and hydrophobic tail that are similar to those of ATP but lacking a bioisostere of the sugar moiety. Accordingly, we have designed a series of HHQ derivatives satisfying the reported pharmacophoric features: central heterocyclic ring, HHQ ring (occupies the adenine binding region and blue coloured moiety); ester and primary amino groups that interact in the similar manner of ribose moiety (form H-bond with the ATP binding site and green coloured moiety); hydrophobic head (resides in a hydrophobic region I and red coloured moiety); finally a hydrophobic tail, phenyl ring (occupies hydrophobic region II and pink coloured moiety)⁴¹ (Figure 2).

Figure 2. The rational design of new EGFR-TKIs integrating the structural features deduced from the chemical structures of approved EGFR-TKIs (erlotinib, afatinib, and osimertinib) and compounds (I–III) with reported antitumor activity.

Results and discussion

Chemistry

The synthetic pathway to prepare the racemic mixture of 32 target compounds is outlined in Schemes 1 and 2. Intermediates, 3a–c, were synthesised via condensation reaction of aniline derivatives 1a–c and dimedone 2 in acidic media using dichloromethane (DCM) as a solvent⁴² (Scheme 1). These intermediates, 3a–c, were reacted with ethyl cyanoacetate 4 and appropriate aryl aldehydes; o-substituted benzaldehydes 5a–c, p-substituted benzaldehydes 7a–e, disubstituted benzaldehydes 9a,b, or pyridine carboxaldehydes (ortho or meta) 11a,b, in absolute ethanol as a solvent and piperidine as a catalyst via cyclocondensation mechanism^43,44 to yield targeted compounds, 6a–i, 8a–m, 10a–d, and 12a–f as a racemic mixture (Scheme 2). This reaction was accomplished in two steps; the first step was Knoevenagel condensation between aryl aldehyde and ethyl cyanoacetate leading to the formation of unsaturated nitrile⁴⁵. The second step was achieved via Michael’s addition of intermediates 3a–c to unsaturated nitrile and closure of the HHQ ring. The structures of synthesised target compounds were confirmed by IR, ¹H NMR, ¹³C NMR, mass spectroscopy, elemental analysis, and x-ray crystallography to confirm enantiomerism. The following illustrations demonstrate that the target compounds were successfully synthesised: for ¹H NMR spectra, the appearance of both sharp singlets at the range of 4.84–5.44 ppm corresponds to the proton at position 4 in the HHQ ring, a broad peak at the range of 5.97–6.82 ppm, related to NH₂ at position 2 in the HHQ ring. The disappearance of the NH peak of the intermediates 3a–c at about 6.50 ppm^46,47 and the aldehydic proton at the 9.00–10.00 ppm range was recognised⁴⁸. Regarding the ¹³C NMR spectra, the appearance of a peak in the range of 33.19–36.85 ppm is associated with the carbon atom at position 4 in the HHQ ring. In addition, the disappearance of the peaks at 100–120 ppm for the nitrile of ethyl cyanoacetate^46–48 and 190–220 ppm for the carbonyl of the aldehyde⁴⁹ was reported and supported by IR spectra, where the peaks at 2300–2250 cm⁻¹ (nitrile of ethyl cyanoacetate)⁵⁰ and 3200–3100 cm⁻¹ (NH of intermediates 3a–c) were vanished⁵¹. The molecular weights of target compounds and the mass spectroscopy results were compatible. Three elements; C, H, and N, underwent elemental analysis to ascertain their percentages, which were within ± 0.4% of theoretical values. The Supplementary file provides the spectra of the synthesised compounds.

Scheme 1. Chemical synthesis of intermediates 3a–c. Reagents and conditions: (i) gl. acetic acid (few drops), heat under reflux in DCM for 8 h.

Scheme 2. Synthesis of HHQ analogues 6a–i, 8a–m, 10a–d, and 12a–f. Reagents and conditions: (i) piperidine (few drops), heat under reflux in abs. ethanol for 24 h.

Crystal structure description for compound 6f

We have made several attempts to prepare high-quality crystals for target compounds via demanding different solvents, such as methanol, acetone, and chloroform. A good-sized crystals of compound 6f was successfully used as a representative compound to approve the enantiomerism of our target compounds⁵². A prism crystal of compound 6f (accession number 2163585) was obtained by crystallisation from methanol at room temperature. The carbon atom at position 4 in the HHQ ring of compound 6f is chiral; therefore, we may obtain either a pure enantiomer or a racemic mixture as a final product. Therefore, there is no method better than X-ray crystallography to confirm the enantiomerism of our final product⁵³. X-ray crystallography revealed a two-component disorder for compound 6f where the phenyl fragment at C8 was found to adopt two orientations that differ by a 180° rotation around the C4–C11 link; the cyclohexenone ring adopts an envelope configuration, the 1,4-dihydropyridine ring adopts a flattened boat configuration and the phenyl ring at N1 almost perpendicular to the C2/C3/C5/C10 plane^54,55. The ORTEP diagram of the R-enantiomer for compound 6f is presented below (Figure 3(A)). Using the visualiser of discovery studio software, we have found that both S-enantiomer and R-enantiomer are existing. The S-enantiomer has an HHQ ring in the plane; the ester and amino groups are directed towards the right-hand side, the two dimethyl groups are directed towards the left-hand side, and the phenyl group at position 4 in the HHQ ring is behind the plane. The R-enantiomer has the phenyl group at position 4 in the HHQ ring in front of the plane (Figure 3(B)).

Figure 3. (A) ORTEP diagram of the S-enantiomer as a representative for the racemic mixture of compound 6f obtained from single crystal x-ray data drawn at 50% thermal ellipsoid probability. Red colour: oxygen atom, brick red colour: bromine atom, blue colour: nitrogen atom, and yellow colour: chlorine atom. (B) Chemical structure of S-enantiomer of compound 6f.

Biological evaluation

In vitro preliminary anticancer activity at a single dose against 60 NCI cell lines

Thirty-two newly synthesised HHQ derivatives were subjected to preliminary anticancer screening at the USA National Cancer Institute (NCI)⁵⁶. HHQ derivatives were examined in vitro at one dose anticancer activity against total NCI 60 cancer cell line panels that include nine different types of cancers; leukaemia, NSC lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer^56–59. Nineteen derivatives failed to show cytotoxic activity against the tested cell lines reporting a GI % mean of less than 10%. These compounds are; 6b, 6g, 6h, 8a, 8d, 8f–m, and 12a–f (Tables S1 and S2) and (Figures S100, S105, S106, S108, S111, S113–120, and S129–134). The remaining thirteen derivatives 6a, 6c–f, 6i, 8b–c, 8e, and 10a–d revealed modest to good cytotoxic activity. The data were specified as a mean graph of the treated cells’ percent growth (GI% mean) and represented as a heat map, where cytotoxicity increased from the left (blue) towards the right (red), as shown in Figure 4. Leukaemia and colon cancer were the most sensitive to our target compounds; on the other hand, renal and ovarian cancers were the least sensitive.

Figure 4. Heat map data representing GI % mean of the active 13 compounds across the NCI-60 human cancer cell line panels, including their structures. *Prostate cancer.

Table 1. Effects of five doses for in vitro anticancer activity results (cytotoxic activities expressed as GI₅₀ (µM) for compound 10c against NCI sixty cancer cell lines.

Subpanel cell lines	GI50	Subpanel cell lines	GI50	Subpanel cell lines	GI50	Subpanel cell lines	GI50
Leukaemia		COLO-205	2.06	MDA-MB-435	3.21	RXF 393	1.38
CCRF-CEM	3.10	HCC-2998	2.77	SK-MEL-2	1.70	SN 12 C	3.08
HL-60(TB)	5.95	HCT-116	7.89	SK-MEL-28	6.67	TK-10	5.63
K-562	7.23	HCT-15	1.52	SK-MEL-5	1.24	UO-31	1.15
MOLT-4	9.56	HT-29	1.98	UACC-257	2.88	Prostate cancer
RPMI-8226	7.07	KM-12	2.36	UACC-62	1.26	PC-3	4.14
SR	1.47	SW-620	2.90	Ovarian cancer		DU-145	6.71
NSC Lung cancer		CNS cancer		IGROV1	7.97	Breast cancer
A549	1.88	SF-268	5.32	OVCAR-3	2.62	MCF7	1.77
EKVX	2.62	SF-295	2.03	OVCAR-4	2.83	MDA-MB-231	3.34
HOP-62	4.88	SF-539	3.78	OVCAR-5	7.26	HS 578 T	7.68
HOP-92	3.89	SNB-19	2.92	OVCAR-8	3.72	BT-549	4.58
NCI-H226	2.34	SNB-75	5.33	SK-OV-3	3.35	T-47D	1.04
NCI-H23	2.51	U251	2.46	Renal cancer		MDA-MB-468	6.83
NCI-H322-M	7.71	Melanoma		786-0	4.24	LC50 values towards 60 cell lines were more than 100 µM
NCI-H460	1.39	LOX IMVI	3.24	A498	4.54
NCI-H522	2.22	MALME-3M	2.93	ACHN	1.83
Colon cancer		M14	3.48	CAKI-1	6.82

Table 2. Inhibitory activities of the most potent 13 compounds with standard drug erlotinib against EGFR^WT, EGFR^T790M, and EGFR^L858R (the results are reported as the means of IC₅₀ values ± standard deviation for three independent replicates).

Code	IC50 ± SD (µM)			Code	IC50 ± SD (µM)
	Wild type	Mutant types			Wild type	Mutant types
	EGFR^WT	EGFR^T790M	EGFR^L858R		EGFR^WT	EGFR^T790M	EGFR^L858R
6a	0.358 ± 0.16	0.61 ± 0.005	0.49 ± 0.004	8c	0.194 ± 0.09	0.820 ± 0.006	0.812 ± 0.005
6c	0.277 ± 0.12	0.53 ± 0.004	0.50 ± 0.004	8e	0.300 ± 0.12	0.693 ± 0.005	0.351 ± 0.002
6d	0.099 ± 0.02	0.41 ± 0.002	0.25 ± 0.005	10a	0.145 ± 0.03	0.532 ± 0.004	0.490 ± 0.003
6e	0.176 ± 0.07	0.48 ± 0.003	0.37 ± 0.004	10b	0.205 ± 0.09	0.954 ± 0.008	0.878 ± 0.006
6f	0.313 ± 0.12	0.59 ± 0.004	0.48 ± 0.003	10c	0.148 ± 0.04	0.586 ± 0.003	0.623 ± 0.005
6i	0.157 ± 0.03	0.45 ± 0.003	0.23 ± 0.001	10d	0.097 ± 0.005	0.280 ± 0.001	0.051 ± 0.001
8b	0.186 ± 0.1	0.65 ± 0.004	0.77 ± 0.006	Erlotinib	0.082 ± 0.005	0.342 ± 0.001	0.055 ± 0.001

Relying on the GI % mean values (Tables S1 and S2), the 2,3- and 2,4-dichlorophenyl derivatives, 10a–d, were the most active compounds where 2,4-dichlorophenyl analogues, 10c and 10d, displayed higher activity than 2,3-dichlorophenyl derivatives, 10a and 10b. Compound 10c (GI % mean = 74) was the most active analogue and almost revealed cytotoxic activity against 58 cell lines out of the 60 screened cell lines, with activity ranging from moderate to lethal. Compound 10c exposed lethal anticancer activity against NSC lung cancer; HOP-92, melanoma; SK-MEL-2, SK-MEL-5. It reported strong anticancer activity towards leukaemia, colon, breast, renal, and prostate cancer, while it revealed moderate activity against ovarian cancer. Compound 10d (GI % mean = 48) disclosed strong anticancer activity against leukaemia, NSC lung cancer; EKVX, NCI-H226, colon cancer; HCT-116, HCT-15, prostate cancer; PC-3 and breast cancer; MCF7, T-47D, MDA-MB-468. Compound 10a (GI % mean = 35) revealed lethal anticancer activity against NSCL cancer; HOP-92, strong anticancer activity against leukaemia; MOLT-4, colon cancer; HCT-116, CNS cancer; SF-539, prostate cancer; PC-3. Compound 10b (GI % mean = 36) displayed strong anticancer activity against leukaemia; MOLT-4 colon cancer; HCT-116, HCT-15, prostate cancer; PC-3, and breast cancer; MDA-MB-468.

Para substituted phenyl derivatives, 8b,c and 8e, arose in the next place in the antitumor activity. Halogenated derivatives (8b and 8e) displayed higher cytotoxicity than non-halogenated analogues (8i, 8j, and 8 l). Compound 8b (GI % mean = 14) reported mild cytotoxicity towards NSC lung cancer; NCI-H522, colon cancer; HCT-116, HCT-15, HT29, and melanoma; UACC-62. Analogue 8e (GI % mean = 32) showed remarkable anticancer activity against leukaemia; CCRF-CEM, HL-60 (TB), K-562, MOLT-4, RPMI-8226, and NSC lung cancer; A549. Compound 8c (GI % mean = 63) was the most active analogue in this series and revealed a significant broad spectrum against the nine types of cancer as it disclosed lethal activity towards colon cancer; COLO 205 with strong anticancer activity against leukaemia; CCRF-CEM, HL-60 (TB), K-562, MOLT-4, RPMI-8226, SR, NSC lung cancer; A549, EKVX, NCI-H460, NCI-H522, colon cancer; HCT-116, HCT-15, HT29, KM12, SW-620, CNS cancer; SF-295, SF-539, SNB-19, melanoma; M14, MDA-MB-435, SK-MEL-2, SK-MEL-5, UACC-257, UACC-62, ovarian cancer; OVCAR-4, NCI/ADR-RES, renal cancer; ACHN, UO-31, prostate cancer; PC-3, and breast cancer; MCF7, T-47D, MDA-MB-468.

Moreover, ortho-substituted phenyl derivatives 6a, 6c–f, and 6i showed mild antitumor activity. Halogenated derivatives (6a and 6c–f), except for the methoxy analogue 6i (GI % mean = 36), were more cytotoxic than non-halogenated analogues (6 g–i). Compound 6a (GI % mean = 33) displayed a lethal effect against leukaemia HL-60 (TB), while it revealed strong anticancer activity towards leukaemia; CCRF-CEM, MOLT-4, RPMI-8226, SR, and prostate cancer; PC-3. Compound 6c (GI % mean = 34) disclosed strong anticancer activity against leukaemia HL-60 (TB), NSLC; NCI-H522 colon cancer; COLO 205, HCT-116, HCT-15, HT29, melanoma; SK-MEL-5, prostate cancer; PC-3, and breast cancer; T-47D, MDA-MB-468. Compound 6d (GI % mean = 12) reported mild cytotoxicity against most tested cancer cell lines. Compound 6e (GI % mean = 22) displayed a lethal effect on leukaemia HL-60 (TB), while it revealed strong anticancer activity against leukaemia; CCRF-CEM, MOLT-4, RPMI-8226, colon cancer; HCT-116, and prostate cancer; PC-3. Compound 6f (GI % mean = 24) exposed strong anticancer activity against leukaemia; HL-60 (TB), melanoma; SK-MEL-5, and prostate cancer; PC-3, while it displayed moderate activity against colon cancer; HCT-116. Compound 6i (GI % mean = 36) revealed strong anticancer activity against leukaemia; HL-60 (TB), K-562, MOLT-4, RPMI-8226, SR, colon cancer; HCT-116, prostate cancer; PC-3, and breast cancer; MCF7.

Structure–activity relationship (SAR)

We have constructed our structure–activity relationship (SAR) study based on NCI single-dose biological evaluation outcomes. Generally, we will discuss the influence of diverse substituents of two important parts on target compounds: the substituted phenyls at position N1 and the aryl groups at position-4 of the HHQ ring.

1. Regarding substituted phenyls at N1, generally the electron-withdrawing group (R = Cl) enhanced cytotoxic activity compared to the electron-donating group (R = CH₃). Relying on GI % mean results, we can confirm that:

   1. The presence of chlorine atom at the para position of phenyl ring at C-4: Analogues with unsubstituted phenyl (8c, 10c) were better in activity than substituted analogues with electron-withdrawing (Cl) (8e, 10d) or electron-donating (CH₃) groups (8d). Analogues substituted with electron-withdrawing group reported higher cytotoxic activity compared to the analogues having electron-donating groups (H > Cl > CH₃).

   2. The existence of chlorine atom at the ortho or meta positions of phenyl ring at C-4: Analogues with unsubstituted phenyl (6a, 10a) were almost equipotent to those with electron-withdrawing substituent (Cl) (6c, 10b) and more potent than analogues substituted with electron-donating groups (CH₃) (6b) (H ∼ Cl > CH₃) (6a∼6c ≫ 6b), (10a∼10b).

2. Concerning the aryl groups at position-4:

   1. Upon incorporation of ortho-substituted aryl at position 4 (target compounds 6a–i), an electron-withdrawing group, especially halogens at the ortho position of the C4 aryl enhanced anticancer activity whereas analogue bearing halogen of medium size, such as (Cl) was more potent than those having halogen of large size like (Br) (R¹; Cl > Br) (6a > 6d) in comparison with electron-donating groups (R¹ = OCH₃) (6c > 6f) (6i is an exception).

   2. Upon integration of para-substituted aryl at position 4 (analogues 8a–m), the electron-withdrawing groups at para position potentiate anticancer activity (R¹; Cl ⋙ F) in comparison with an electron-donating group (R¹ = OCH₃) (8c > 8a).

   3. Regarding disubstituted aryl at position 4 (compounds 10a–d): Disubstitution with EWG at ortho and para positions increases cytotoxic activity than ortho and meta positions (10c,d ≫ 10a,b). In contrast, pyridine analogues (12a–f), substituted with a heteroaromatic ring at the para position, do not affect biological activity. The SAR is summarised in Figure 5.

Figure 5. Summary of structure-activity relationship (SAR) of target compounds as in vitro anticancer agents against NCI 60 human cancer cell lines relying on the GI % mean values.

In vitro anticancer screening at five doses of full NCI 60 cancer cell panels

The second stage was accomplished via screening of the most active compound (10c) referred to the results of the one-dose analysis of the NCI 10c (NSC 838215) against the 60 cancer cell lines at five different concentrations (0.01, 0.1, 1, 10, and 100 µM). Cell viability was assessed using the published experimental techniques using the sulforhodamine-B (SRB) protein assay spectrophotometrically versus untreated with a test compound⁶⁰. At the end of the 48 h incubation period, the five-dose assay findings were presented for each cell line tested regarding the response parameters, GI₅₀ (needed molar concentration to inhibit 50% of cancer cell line growth)⁶¹. Compound 10c displayed magnificent anticancer activity against nearly all the tested cell lines, with GI₅₀ ranging from 1.04 to 9.56 µM. The best cytotoxic effect was observed against the T-47D breast cancer cell line (GI₅₀ = 1.04 µM), while the least cytotoxic effect was observed against the MOLT-4 leukaemia cell line (GI₅₀ = 9.56 µM). The most sensitive cancer cell lines were those having GI₅₀ > 2 µM. SR was the most sensitive leukaemia cell line with GI₅₀ = 1.47 µM. A549 and NCI-H460 were the most sensitive NSC lung cancer cell lines with GI₅₀ = 1.88 and 1.39 µM, respectively. HCT-15 and HT-29 were the most sensitive colon cancer cell lines with GI₅₀ = 1.52 and 1.98 µM, respectively. Regarding melanoma, SK-MEL-2, SK-MEL-5, and UACC-62 were the most sensitive cell lines with GI₅₀ = 1.70, 1.24, and 1.26 µM, respectively. Renal cancer was also one of the most affected cancers, specifically ACHN, RXF-393, and UO-31 cell lines with GI₅₀ =1.83, 1.38, and 1.15 µM, respectively. It revealed excellent cytotoxic activity against breast cancer, MCF7 with GI₅₀ =1.77 µM. Table 1 summarises the calculated GI₅₀ values for each of the sixty cancer cell lines for compound 10c across the nine cancer types.

Enzyme inhibition assay of EGFR^WT, EGFR^T790M, and EGFR^L858R

In the T790M mutation, methionine replaces threonine at amino acid position 790 of exon 20 of the EGFR gene⁶². This mutation alters the crystal structure of the adenosine triphosphate (ATP) binding pocket and prevents binding to the ATP binding site; hence structurally blocking first- and second-generation EGFR-TKIs and results in EGFR-TKIs resistance⁶³. The “gatekeeper” hypothesis, which holds that there is a steric clash between the larger methionine moiety (compared to threonine) on the gatekeeper side chain of EGFR^T790M and the aniline moiety of first-generation EGFR TKIs, is one of the more biochemical mechanisms of EGFR^T790M-associated resistance⁶⁴. Concerning the L858R mutation, arginine replaces leucine at the 858th amino acid of the 21st exon of the EGFR⁶⁵. It’s the most common EGFR mutation, accounting for 35% of all mutations⁶⁶. The first-generation EGFR-TKIs include erlotinib and gefitinib⁶⁷. The second-generation EGFR-TKIs (such as afatinib⁶⁸, pelitinib⁶⁹, neratinib⁷⁰, and dacomitinib which is an irreversible inhibitor with pan-HER TKI action⁷¹ created to combat resistance to first-generation EGFR-TKIs, although acquired resistance to these drugs always appears. Studies demonstrated that the EGFR gene’s T790M mutation was a typical resistance mechanism to second-generation EGFR-TKIs⁷². As a result, third-generation, EGFR-TKIs were created to overcome resistance to first- and second-generation EGFR-TKIs, and they allowed the irreversible binding to Cys797 residue in the ATP-binding site (e.g. osimertinib⁷³, rociletinib⁷⁴, and olmutinib⁷⁵.

The target compounds were designed as inhibitors of EGFR. The most potent 13 compounds against the NCI cell lines panel 6a, 6c–f, 6i, 8b,c, 8e, and 10a–d were screened for their enzymatic inhibitory activity against wild-type (EGFR^WT), L858R mutant (EGFR^L858R), and T790M mutant (EGFR^T790M) receptors using erlotinib as a reference drug. Concerning EGFR^WT results, the tested compounds showed antitumor activity ranging from 0.097 to 0.358 µM, where compound 10d reported the highest cytotoxic activity (IC₅₀ = 0.097 µM) which was very close to erlotinib (IC₅₀ = 0.082 µM). In comparison, compound 6a displayed the lowest cytotoxic activity (IC₅₀ = 0.358 µM) (Table 2). Regarding EGFR^T790M results, IC₅₀ values were ranged from 0.280 to 0.954 µM, with compound 10d recording the best cytotoxic activity (IC₅₀ = 0.280 µM), which was higher than erlotinib (IC₅₀ = 0.342 µM). On the other hand, compound 10b disclosed the lowest cytotoxic activity (IC₅₀ = 0.954 µM) (Table 2). For EGFR^L858R outcomes, IC₅₀ values were in the range of 0.051–0.878 µM. Compound 10d revealed the highest cytotoxic activity (IC₅₀ = 0.051 µM) with a privilege over erlotinib (IC₅₀ = 0.055 µM). In contrast, compound 10b showed the lowest cytotoxic activity (IC₅₀ = 0.878 µM) (Table 2). The graph of the IC₅₀ values clearly illustrated these findings (Figure 6). Accordingly, compound 10d is the most active compound with remarkable enzymatic inhibitory activities against the wild and mutant EGFRs.

Figure 6. Inhibitory activity outcomes (IC₅₀ µM) for the most potent 13 compounds 6a, 6c–f, 6i, 8b,c, 8e and 10a–d against EGFR^WT, EGFR^T790M, and EGFR^L858R.

Cytotoxic effects in vitro on IMR-90 cells (normal lung cells)

The human normal lung fibroblast (IMR-90) cell line^76,77 was used to evaluate the target compounds 10c and 10d as cytotoxic agents to assess their safety profile through their selective cytotoxicity towards cancer cells compared to normal cells using erlotinib as a reference anticancer drug. In contrast to erlotinib (IC₅₀ = 39.55 ± 1.15 μM), compounds 10c and 10d displayed less cytotoxicity towards the human normal cells, IMR-90, with IC₅₀ values of 62.17 ± 3.14 and 55.46 ± 2.57 μM, respectively. Therefore, compared to the standard anticancer treatment, erlotinib, compounds 10c and 10d are far safer and have fewer possible negative effects on normal cells.

Annexin V-FITC apoptosis assay

The most important mechanism chemotherapeutics eradicate cancer cells is apoptosis induction^78,79. Cellular alterations brought on by apoptosis include the translocation of phosphatidylserine (PS) from the inside to the outside via the plasma membrane^80,81. PS can be detected outside the plasma membrane using a sensitive probe called Annexin V, which can bind to PS^82,83. We used a FITC/AV/PI dual-staining assay with the BD FACSCalibur to perform cytometric analysis to separate the apoptosis from the necrosis mode of lung cancer HOP-92 cells (the most sensitive NSC lung cancer cell line to target compounds) death caused by the two most active compounds, 10c and 10d (BD Bio-sciences, San Jose, CA). HOP-92 cells were also overexpressed in NSC lung cancer⁸⁴. Therefore, they were selected to be subjected to apoptosis assay. HOP-92 cells were stained with AV/PI for 24 h at a mixed molar concentration of 10 µM with compounds 10c and 10d (Figure 7(A,B)). We have established that the late apoptosis ratio (upper-right quadrant of the cytogram) increased to 15.44 and 12.15% (Figure 7(A,B)) after it was 1.80% (DMSO) (Figure 7(C)). The early apoptosis ratio (lower-right quadrant of the cytogram) increased to 8.74 and 7.68% (Figure 7(A,B)) after it was 0.70% in the control sample (DMSO) (Figure 7(C)) for compounds 10c and 10d, respectively. These findings supported that the cytotoxic effects of compounds 10c and 10d were due to the apoptotic mechanism rather than the necrotic pathway. The results of compounds 10c and 10d compared to the control sample are presented as a bar chart (Figure 7(D)).

Figure 7. Apoptosis assay in flow cytometry, the effect of 10c (A), compounds 10d (B), control (C), and bar chart presentation of control, compounds 10c and 10d (D) on the percentage of Annexin V-FITC-positive staining in NSC lung cancer HOP-92 cells. The four quadrants were identified as LL: viable; LR: early apoptotic; UR: late apoptotic; UL: necrotic.

Cellular mechanistic analysis

Antitumor drugs can cause cell cycle arrest and death via activating signalling pathways^85,86. Cell growth in various cell cycle phases is measured by flow cytometry^87,88. The most potent compounds, 10c and 10d, were picked for additional investigation to discover how they could affect cell cycle progression in the NSC lung cancer cells, HOP-92 (Figure 8(A,D)). We have treated HOP-92 cells with 10 µM of compounds 10c and 10d to incubate for 24 h. We have established a remarkable increase in the percentage of cells at the pre-G1 phase 25.82% Figure 8(A) and 21.55% Figure 8(B) after being 3.05% in the control sample Figure 8(C), as well as an increase in cells at G2/M to be 34.07% Figure 8(A) and 49.21% Figure 8(B) after it was 12.98% in the control sample Figure 8(C) for compounds 10c and 10d, respectively. On the other hand, a drop in the percentage of cells in the G0/G1 to 45.75% Figure 8(A) and 25.37% Figure 8(B) instead of 55.38% (control) Figure 8(C) and a significant reduction in the proportion of cells in the S phase to become 20.18% Figure 8(A) and 25.42% Figure 8(B) rather than 31.46% (control) Figure 8(C) were detected for compounds 10c and 10d, respectively. According to these observations, Compounds 10c and 10d induce apoptosis in NSC lung cancer, HOP-92, cells via cell cycle arrest at the pre-G1 phase as well as the G2/M phase (Figure 8(D)).

Figure 8. Cell distribution in pre-G1, G0/G1, S, and G2/M phases for NSC lung cancer, HOP-92, cells after treatment with 10c (A), compounds 10d (B), and control (C). Bar chart presentation of cell distribution for control, compounds 10c and 10d (D).

In silico pharmacokinetic prediction

Regarding Molsoft software⁸⁹, the results revealed that drug-likeness scores are 0.90, 0.92, and 0.63 for erlotinib and compounds 10c and 10d, respectively. On the other hand, by looking at the SwissADME web tool radar chart⁹⁰, the compounds 10c and 10d gave better flexibility than erlotinib with a slight deviation in lipophilicity and insolubility. From these, we can conclude that compounds 10c and 10d are suggested to be drugs (Figure 9).

Figure 9. Drug-likeness model score and radar charts results of erlotinib, compounds 10c and 10d.

Molecular docking analysis

A molecular docking study was achieved to establish a deep insight into the binding mode of the R- and S-enantiomers for the most potent antitumor compounds, 10c and 10d, within the ATP-binding pocket of the crystal structures for each enzyme: EGFR^WT (PDB code: 1M17)⁹¹, EGFR^T790M (PDB code: 2JIV)⁹² and EGFR^L858R (PDB code: 4LQM) by using “molecular operating environment (MOE) version 2019.0102”⁹³. Visualisation of interactions between ligands and binding sites was accomplished via discovery studio visualiser (BIOVIA-2021.DS2021Client)^94,95. The active site of the EGFR enzyme contains essential amino acids; Lys721, Met742, Met766, Gly767, Met769, Leu694, and Asp831 which are chiefly targeted by EGFR inhibitors⁹⁶. Another essential amino acid is Cys797, the third-generation EGFR-TKIs conquer the T790M and L858R mutations resistance through covalent binding with Cys797⁹⁷. The docking results revealed that our target compounds demonstrated different types of bonds with these amino acids and illustrated the results of the biological evaluation. Looking at the literature, we find that erlotinib binds with the key amino acids in the binding site through H-bonding with Met769 and hydrophobic interactions with other amino acids, including the key amino acid Lys721 and Leu694^98,99. Considering the docking studies, compounds 10c and 10d showed a binding affinity for the active site of EGFR comparable to that found for erlotinib.

First, the original co-crystallised ligands, erlotinib, HKI, and PD-168393, were re-docked into the active sites of EGFR^WT, EGFR^T790M, and EGFR^L858R to validate the docking techniques, and the results were reported in (Table S3). Then our reference erlotinib and analogues 10c and 10d were docked into the active sites of EGFR^WT, EGFR^T790M, and EGFR^L858R enzymes. The docking results of erlotinib are provided in (Table S4). Fallouts of docking of the R- and S-enantiomers for analogues 10c and 10d, into the ATP-binding pocket of EGFR illustrated that S-enantiomer showed higher binding affinity than the R-enantiomer. This finding was also found for many other nuclei, such as tetrahydroisoquinoline¹⁰⁰, thienopyrimidine¹⁰¹, pyrrolo[2,3-d]pyrimidine¹⁰², and dihydropyrimidine¹⁰³. The alignment of S-enantiomers for compounds 10c and 10d with the co-crystallised ligands is displayed in (Figures S135–140). The R-enantiomer’s docking results for compounds 10c and 10d are detailed in (Tables S5 and S6) and (Figures S141–S146). Interactions of S-enantiomers for target compounds 10c and 10d are detailed in Figures 10–15 and Table 3.

Figure 10. The 2D (left) and 3D (right) poses for docking interactions of the S-isomer of compound 10c within the active site of EGFR (PDB code: 1M17).

Figure 11. The 2D (left) and 3D (right) poses for docking interactions of the S-isomer of compound 10d within the active site of EGFR (PDB code: 1M17).

Figure 12. The 2D (left) and 3D (right) poses for docking interactions of the S-isomer of compound 10c within the active site of EGFR (PDB code: 2JIV).

Figure 13. The 2D (left) and 3D (right) poses for docking interactions of the S-isomer of compound 10d within the active site of EGFR (PDB code: 2JIV).

Figure 14. The 2D (left) and 3D (right) poses for docking interactions of the S-isomer of compound 10c within the active site of EGFR (PDB code: 4LQM).

Figure 15. The 2D (left) and 3D (right) poses for docking interactions of the S-isomer of compound 10d within the active site of EGFR (PDB code: 4LQM).

Table 3. Fallouts of docking for S-isomers of compounds 10c and 10d within variant EGFRs.

Compound	PDB code (EGFR type)	Score (Kcal/mol)	Amino acids bind with
			Hydrophobic head	HHQ core	Hydrophobic tail
10c	1M17 (wild type)	−7.67	Val702, Ala719, Leu764, Asp831	Phe699, Lys721*, Met769, Leu820	Cys773
10d		−7.73	Val702, Ala719, Met742 Leu764, Asp831	Phe699, Lys721*, Met769, Leu820	Cys773
10c	2JIV (T790M)	−6.65	Val726, Ala743, Lys745, Leu788, Asp855	Met790, Leu844, Cys797	Leu718, Val726
10d		−7.58	Ala743, Met790, Gln791	Ile744*, Lys745, Leu788, Cys797, Leu844	Arg841
10c	4LQM (L858R)	−6.90	Val726, Lys745, Leu788, Thr854*	Phe723, Ala743, Gln791, Met793, Leu844	Cys797
10d		−7.11	Val726, Ala743, Lys745, Met766, Leu788, Met790	Phe723, Lys745*, Met793, Leu844	Cys797

All amino acids make hydrophobic interactions except for those mentioned above (*) make hydrogen bonds.

For EGFR^WT, the S-isomer of compound 10c interacts through hydrogen bonding between the oxygen atom of C = O of the ester group and Lys721. It forms hydrophobic interactions with other amino acids in the active site, which are important for activity, such as Met769. Halogen bonding interactions are also reported between 2,4-dichlorine atoms and Asp831 and Leu764, respectively (Figure 10). Compound 10d discloses hydrogen bonding interaction between the oxygen atom of C = O of the cyclohexanone ring and Lys721, in addition to forming many hydrophobic interactions with other amino acids in the active site, which are significant for activity such as Met769 and Met742. Halogen bonding interaction is also observed between the ortho chlorine atom and Asp831 (Figure 11).

Regarding EGFR^T790M, docking results of compound 10c exposed many hydrophobic attraction forces, but none of them with the amino acids that are important for activity, while compound 10d formed a hydrogen bond with Ile744 and extra substantial hydrophobic interactions. Van der Waals attraction force is detected between one methyl group on the HHQ ring of compound 10c and Cys797 (Figure 12), while for compound 10d, the Van der Waals attraction force is observed between the two methyl groups on the HHQ ring and Cys797. Moreover, analogue 10d demonstrates halogen bonding interaction between ortho chlorine atom on C4 phenyl and Gln791, giving a privilege for 10d over 10c (Figure 13).

Docking of compound 10c into EGFR^L858R revealed that a hydrogen bond is reported between the ortho chlorine atom and Thr854. Van der Waals interaction between N-phenyl and Cys797 is detected. Compound 10d forms a halogen bonding interaction between the para chlorine atom and Leu788. Van der Waals interaction between N-phenyl and Cys797 is identified (Figure 14). Besides, compound 10d discloses additional hydrophobic interactions between the two methyl groups at the HHQ ring and Phe723 and Val725 (Figure 15). This docking study revealed that the number and type of interactions for compound 10d are more effective than that for 10c, which explains its stronger inhibitory activity towards wild and mutant EGFRs (EGFR^T790M and EGFR^L858R).

As a conclusion for docking results, it is obvious that the halogenated inhibitors very efficiently displace the charged ATP ligand, mainly through halogen bonding interactions, stressing the potential role of halogen bonds in the design of new drugs and inhibitors.

Conclusion

In this work, a series of thirty-two novel HHQ derivatives (in a racemic mixture) complying the pharmacophoric features for EGFR inhibitors were designed and synthesised. X-ray crystallography was performed to confirm configuration of compound 6f. Compound 10c showed very promising anticancer activities against 60 cancer cell line subpanels of the NCI. On the other hand, compound 10d exhibited the most promising enzymatic inhibitory activity with IC₅₀ values of 0.097 µM, 0.280 µM, and 0.051 µM, respectively, against the three variants of the enzyme, EGFR^WT, EGFR^T790M, and EGFR^L858R. Furthermore, compounds 10c and 10d exhibited a total apoptosis percentage of 24.18 and 19.83%, respectively, more than that of the control (2.5%) by approximately 10- and 8-fold, respectively, towards HOP-92 lung cancer cells. Moreover, compounds 10c and 10d prompted cell cycle arrest in the pre-G1 phase in values of 25.82 and 21.55% and in the G2/M phase in values of 34.07 and 49.21%, respectively. It is worth mentioning that the biological data greatly matched the corresponding docking scores of the synthesised compounds. On the other hand, in silico studies were carried out and found that compound 10c is more promising compound to be a drug than 10d and has a privilege over erlotinib. Remarkably, compounds 10c and 10d could be considered promising lead compounds as EGFR inhibitors for further optimisation.

Experimental

Chemistry

Open-glass capillaries were used for melting point determination in a Stuart SMP30 apparatus and were uncorrected. The Sigma-Aldrich Company, and Merck company are the suppliers from which all organic reagents and solvents were purchased and were used without further purification. The progress of the reactions and the purity chick of the products was made using the developing system: ethyl acetate: n-hexane (5:2) as eluent and was visualised by exposure to UV lamp at λ 254 nm. ¹H NMR and ¹³C NMR spectra were carried out using the Bruker instrument, having a frequency of 400 MHz for ¹H NMR and 100 MHz for the ¹³C NMR spectrophotometer. Chemical shifts were recorded in ppm on the δ scale using CDCl₃ or DMSO-d₆ as solvents. Coupling constant (J) values were calculated in Hertz (Hz). Using a Thermo Scientific, ISQ Single Quadruple MS, the electron impact (EI) mass spectra were recorded. Microanalysis was performed for three elements, C, H, and N, to determine their percentages on PerkinElmer 2400 and was within ± 0.4% of theoretical values. All compounds were obtained as a racemic mixture (±) which was confirmed through x-ray crystallography and the optical rotations (α) of all synthesised compounds using a Polax-2L Polarimeter (ATAGO Co., Ltd., Saitama, Japan) where they failed to demonstrate any rotation. Several unsuccessful attempts for resolution of racemic mixtures of final products, such as crystallisation and HPLC chiral separation have been made.

General procedure for the synthesis of intermediates (3a–c)

A mixture of aniline derivatives (1a–c) (1.1 mmol) and dimedone (2) (1.0 mmol) was heated under reflux in DCM (20 ml) for 8 h using glacial acetic acid as catalyst (3 drops). After completing the reaction (checked by TLC), the solid powder was filtered off and washed with dist. water to afford yellow powders, 3a–c. Intermediate 3a reported m.p. = 180–182 °C (reported 181–183 °C⁴⁶) intermediate 3b, m.p. = 200–202 °C (reported 202–204 °C⁴⁷), and intermediate 3c, m.p. = 212–214 °C (reported 209–210 °C¹⁰⁴).

General procedure for synthesis of the substituted 2-amino-1-phenyl-1,4,6,7,8-hexahydroquinolin-3-carboxylates (6a–i), (8a–m), (10a–d), and (12a–f)

A 50 ml round bottom flask, fitted with a reflux condenser, was charged with a mixture of 3a–c (0.9 mmol), ethyl cyanoacetate 4 (1.2 mmol), appropriate aryl aldehyde; o-substituted benzaldehydes 5a–c, p-substituted benzaldehydes 7a–e, disubstituted benzaldehydes 9a,b, or o- or m-pyridine carboxaldehydes 11a,b (1.0 mmol) and a catalytic amount of piperidine (5 drops) in absolute ethanol (30 ml). The mixture was heated under reflux for 24 h. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered off, and recrystallised from methanol to yield racemic mixture of products (6a–i), (8a–m), (10a–d), and (12a–f).

(±)-Ethyl 2-amino-4–(2-chlorophenyl)-7,7-dimethyl-5-oxo-1-phenyl -1,4,5,6,7,8- hexahydroquinoline-3-carboxylate (6a)

Off-white powder, yield: 34%; m.p. 168–170 °C. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.70 (s, 3H, CH₃-dimedone), 0.86 (s, 3H, CH₃-dimedone), 1.08 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.64 (d, 1H, COCH₂, J = 16.0 Hz), 1.92 (d, 1H, COCH₂, J = 16.0 Hz), 2.16 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.91–3.93 (m, 2H, OCH₂CH₃), 5.22 (s, 1H, HHQ, 4-H), 6.82 (br. s, 2H, NH₂), 7.11 (t, 1H, Ar-H, J = 8.0 Hz), 7.24–7.27 (m, 2H, Ar-H), 7.46–7.50 (m, 3H, Ar-H), 7.61–7.67 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.38, 26.85, 28.35, 29.55, 32.23, 35.29, 42.09, 50.06, 59.24, 79.43, 113.09, 123.84, 125.80, 126.91, 129.39, 129.85, 130.04, 130.33, 133.16, 133.43, 136.45, 143.97, 149.81, 152.11, 170.19, 195.79; (+)ESI-MS (m/z): [M + H]⁺: 451.20, Anal Calcd. for C₂₆H₂₇ClN₂O₃: C; 69.25, H; 6.04, N; 6.21. Found: C; 69.01, H; 6.21, N; 6.05.

(±)-Ethyl 2-amino-4–(2-chlorophenyl)-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6b)

Off-White crystals, yield: 67%; m.p. 240–242 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.84 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.18 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.81 (d, 1H, COCH₂, J = 16.0 Hz), 2.02 (d, 1H, COCH₂, J = 16.0 Hz), 2.09 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.49 (s, 3H, CH₃), 3.96–4.10 (m, 2H, OCH₂CH₃), 5.39 (s, 1H, HHQ, 4-H), 6.31 (br. s, 2H, NH₂), 7.06 (t, 1H, Ar-H, J = 8.0 Hz), 7.16–7.29 (m, 4H, Ar-H), 7.39 (d, 2H, Ar-H, J = 8.0 Hz), 7.56 (d, 1H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.40, 21.31, 26.85, 29.59, 32.23, 35.22, 42.04, 50.06, 59.22, 79.31, 113.02, 125.81, 126.88, 129.83, 129.94, 131.02, 133.12, 133.42, 133.60, 140.31, 144.08, 150.14, 152.31, 170.21, 195.84; (+)ESI-MS (m/z): [M + H]⁺: 465.20, Anal Calcd. for C₂₇H₂₉ClN₂O₃: C; 69.74, H; 6.29, N; 6.02. Found: C; 69.98, H; 6.52, N; 6.30.

(±)-Ethyl 2-amino-4–(2-chlorophenyl)-1–(4-chlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6c)

Off-white powder, yield: 87%; m.p. 179–181 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.84 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.17 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.78 (d, 1H, COCH₂, J = 16.0 Hz), 2.01 (d, 1H, COCH₂, J = 16.0 Hz), 2.10 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.98–4.10 (m, 2H, OCH₂CH₃), 5.37 (s, 1H, HHQ, 4-H), 6.25 (br. s, 2H, NH₂), 7.06 (t, 1H, Ar-H, J = 8.0 Hz), 7.18 (t, 1H, Ar-H, J = 8.0 Hz), 7.25–7.28 (m, 1H, Ar-H), 7.33 (d, 2H, Ar-H, J = 8.0 Hz), 7.54–7.61 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.37, 26.85, 29.62, 32.29, 35.38, 42.15, 49.99, 59.35, 79.63, 113.21, 125.84, 127.04, 129.92, 130.74, 131.69, 133.29, 133.41, 134.91, 136.24, 143.63, 149.41, 151.80, 170.13, 195.79; (+)ESI-MS (m/z): [M + H]⁺: 484.81, Anal Calcd. for C₂₆H₂₆Cl₂N₂O₃: C; 64.33, H; 5.40, N; 5.77. Found: C; 64.51, H; 5.61, N; 6.00.

(±)-Ethyl 2-amino-4–(2-bromophenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6d)

Yellow powder, yield: 53%; m.p. 220–222 °C. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.70 (s, 3H, CH₃-dimedone), 0.85 (s, 3H, CH₃-dimedone), 1.07 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.64 (d, 1H, COCH₂, J = 16.0 Hz), 1.91 (d, 1H, COCH₂, J = 16.0 Hz), 2.15 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.17 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.93–4.01 (m, 2H, OCH₂CH₃), 5.19 (s, 1H, HHQ, 4-H), 6.80 (br. s, 2H, NH₂), 7.01 (t, 1H, Ar-H, J = 8.0 Hz), 7.30 (t, 1H, Ar-H, J = 8.0 Hz), 7.43 (d, 1H, Ar-H, J = 8.0 Hz), 7.49 (d, 3H, Ar-H, J = 8.0 Hz), 7.64 (d, 3H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.57, 27.01, 29.49, 32.27, 36.73, 42.13, 50.07, 59.24, 80.07, 113.74, 123.46, 126.56, 126.96, 127.12, 130.11, 130.31, 132.80, 133.17, 136.40, 146.03, 149.62, 152.03, 170.24, 195.85; (+)ESI-MS (m/z): [M + H]⁺: 495.30, Anal Calcd. for C₂₆H₂₇BrN₂O₃: C; 63.03, H; 5.49, N; 5.65. Found: C; 63.22, H; 5.34, N; 5.48.

(±)-Ethyl 2-amino-4–(2-bromophenyl)-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6e)

White powder, yield: 56%; m.p. 236–238 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.83 (s, 3H, CH₃-dimedone), 0.94 (s, 3H, CH₃-dimedone), 1.17 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.82 (d, 1H, COCH₂, J = 16.0 Hz), 2.01 (d, 1H, COCH₂, J = 16.0 Hz), 2.09 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.17 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.49 (s, 3H, CH₃), 4.00–4.13 (m, 2H, OCH₂CH₃), 5.39 (s, 1H, HHQ, 4-H), 6.32 (br. s, 2H, NH₂), 6.96 (t, 1H, Ar-H, J = 8.0 Hz), 7.20–7.23 (m, 3H, Ar-H), 7.39 (d, 2H, Ar-H, J = 8.0 Hz), 7.47 (d, 1H, Ar-H, J = 8.0 Hz), 7.53 (d, 1H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.58, 21.32, 26.99, 29.50, 32.23, 36.66, 42.08, 50.07, 59.18, 79.97, 113.67, 123.45, 126.56, 127.06, 127.69, 129.89, 132.72, 133.11, 133.54, 140.36, 146.17, 149.97, 152.22, 170.22, 195.87; (+)ESI-MS (m/z): [M + H]⁺: 509.10, Anal Calcd. for C₂₇H₂₉BrN₂O₃: C; 63.66, H; 5.74, N; 5.50. Found: C; 63.44, H; 5.53, N; 5.71.

(±)-Ethyl 2-amino-4–(2-bromophenyl)-1–(4-chlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6f)

Off-white crystals, yield: 92%; m.p. 210–212 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.69 (s, 3H, CH₃-dimedone), 0.88 (s, 3H, CH₃-dimedone), 1.14 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.65 (d, 1H, COCH₂, J = 16.0 Hz), 2.00 (d, 1H, COCH₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.24 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.94–4.02 (m, 2H, OCH₂CH₃), 4.91 (s, 1H, HHQ, 4-H), 6.80 (br. s, 2H, NH₂), 7.18–7.20 (m, 1H, Ar-H), 7.25–7.34 (m, 3H, Ar-H), 7.42 (d, 2H, Ar-H, J = 8.0 Hz), 7.59–7.67 (m, 2H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.52, 27.02, 28.33, 29.52, 32.29, 36.81, 42.21, 50.02, 59.33, 80.33, 113.89, 125.06, 126.55, 127.22, 129.47, 131.66, 132.91, 133.22, 134.89, 136.29, 145.69, 149.20, 151.70, 170.15, 195.80; (+)ESI-MS (m/z): [M + H]⁺: 529.00, Anal Calcd. for C₂₆H₂₆BrClN₂O₃: C; 58.94, H; 4.95, N; 5.29. Found: C; 59.10, H; 5.21, N; 5.12.

(±)-Ethyl 2-amino-4–(2-methoxyphenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6g)

Yellow powder, yield: 33%; m.p. 243–245 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.80 (s, 3H, CH₃-dimedone), 0.93 (s, 3H, CH₃-dimedone), 1.20 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.75 (d, 1H, COCH₂, J = 16.0 Hz), 2.02 (d, 1H, COCH₂, J = 16.0 Hz), 2.08 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.13 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.95 (s, 3H, OCH₃), 4.03–4.06 (m, 2H, OCH₂CH₃), 5.28 (s, 1H, HHQ, 4-H), 6.21 (br. s, 2H, NH₂), 6.83–6.93 (m, 3H, Ar-H), 7.09–7.13 (m, 3H, Ar-H), 7.39–7.47 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.36, 26.68, 29.72, 32.36, 33.21, 41.20, 42.24, 50.16, 55.91, 59.11, 79.94, 111.55, 113.79, 119.91, 120.27, 126.89, 129.82, 131.37, 132.07, 134.73, 136.91, 149.31, 151.96, 170.51, 195.83; (+)ESI-MS (m/z): [M + H]⁺: 447.50, Anal Calcd. for C₂₇H₃₀N₂O₄: C; 72.62, H; 6.77, N; 6.27. Found: C; 72.48, H; 7.00, N; 6.08.

(±)-Ethyl 2-amino-4–(2-methoxyphenyl)-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6h)

Yellow powder, yield: 63%; m.p. 181–183 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.78 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.80 (d, 1H, COCH₂, J = 16.0 Hz), 2.07 (d, 1H, COCH₂, J = 16.0 Hz), 2.11 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.48 (s, 3H, CH₃), 3.88 (s, 3H, OCH₃), 4.02–4.07 (m, 2H, OCH₂CH₃), 5.11 (s, 1H, HHQ, 4-H), 6.31 (br. s, 2H, NH₂), 7.20–7.23 (m, 3H, Ar-H), 7.39 (d, 2H, Ar-H, J = 8.0 Hz), 7.81 (d, 1H, Ar-H, J = 8.0 Hz), 8.36–8.38 (m, 1H, Ar-H), 8.65 (s, 1H, Ar-H),¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.44, 21.32, 27.00, 29.39, 32.46, 33.30, 41.83, 49.95, 59.45, 79.40, 113.76, 123.44, 129.64, 131.36, 133.00, 138.56, 140.69, 144.14, 144.98, 146.85, 150.34, 152.11, 169.37, 195.74; (+)ESI-MS (m/z): [M + Na]⁺: 483.20, Anal Calcd. for C₂₈H₃₂N₂O₄: C; 73.02, H; 7.00, N; 6.08. Found: C; 73.22, H; 7.25, N; 6.24.

(±)-Ethyl 2-amino-1–(4-chlorophenyl)-4–(2-methoxyphenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6i)

Yellow powder, yield: 46%; m.p. 228–230 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.79 (s, 3H, CH₃-dimedone), 0.97 (s, 3H, CH₃-dimedone), 1.22 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.74 (d, 1H, COCH₂, J = 16.0 Hz), 2.05 (d, 1H, COCH₂, J = 16.0 Hz), 2.12 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.17 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.78 (s, 3H, OCH₃), 4.05–4.09 (m, 2H, OCH₂CH₃), 5.09 (s, 1H, HHQ, 4-H), 6.17 (br. s, 2H, NH₂), 6.80 (d, 2H, Ar-H, J = 8.0 Hz), 7.30 (t, 4H, Ar-H, J = 8.0 Hz), 7.57 (d, 2H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.44, 26.73, 29.71, 32.46, 33.29, 41.90, 50.12, 55.16, 59.38, 81.68, 113.22, 116.03, 128.64, 130.75, 131.43, 135.09, 136.15, 140.03, 148.38, 151.32, 157.56, 170.03, 195.93; (+)ESI-MS (m/z): [M + H]⁺: 481.52, Anal Calcd. for C₂₇H₂₉ClN₂O₄: C; 67.42, H; 6.08, N; 5.82. Found: C; 67.20, H; 6.30, N; 6.09.

(±)-Ethyl 2-amino-4–(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-phenyl -1,4,5,6,7,8-hexahydroquinoline -3- carboxylate (8a)

Faint orange powder, yield: 94%; m.p. 215–217 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.78 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.21 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.74 (d, 1H, COCH₂, J = 16.0 Hz), 2.06 (d, 1H, COCH₂, J = 8.0 Hz), 2.11 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.20 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.06 (q, 2H, OCH₂CH₃, J = 8.0 Hz), 5.13 (s, 1H, HHQ, 4-H), 6.24 (br. s, 2H, NH₂), 7.20–7.23 (m, 2H, Ar-H), 7.33–7.37 (m, 4H, Ar-H), 7.60 (s, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.42, 23.82, 25.44, 26.61, 26.77, 29.65, 32.39, 33.34, 33.68, 41.84, 50.14, 59.28, 81.26, 114.34, 114.55, 128.47, 129.19, 130.04, 130.51, 136.58, 143.71, 149.07, 151.67, 169.93, 170.05, 195.87; (+)ESI-MS (m/z): [M + H]⁺: 435.20, Anal Calcd. for C₂₆H₂₇FN₂O₃: C; 71.87, H; 6.26, N; 6.45. Found: C; 71.56, H; 6.02, N; 6.12.

(±)-Ethyl 2-amino-1–(4-chlorophenyl)-4–(4-fluoroyphenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8b)

Shiny yellow powder, Yield: 72%; m.p. 256–258 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.78 (s, 3H, CH₃-dimedone), 0.97 (s, 3H, CH₃-dimedone), 1.20 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.74 (d, 1H, COCH_2, J = 16.0 Hz), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.14 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.22 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.04–4.11 (m, 2H, OCH₂CH₃), 5.12 (s, 1H, HHQ, 4-H), 6.21 (br. s, 2H, NH₂), 6.94 (t, 2H, Ar-H, J = 8.0 Hz), 7.29 (t, 2H, Ar-H, J = 8.0 Hz), 7.33–7.37 (m, 2H, Ar-H), 7.59 (d, 2H, Ar-H, J = 8.0 Hz),¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.40, 26.63, 29.69, 32.45, 33.64, 41.93, 50.07, 59.41, 81.34, 114.41, 114.62, 115.71, 129.10, 129.17, 130.82, 131.38, 134.92, 136.29, 143.43, 143.46, 148.56, 151.39, 159.90, 162.32, 169.89, 195.84; (+)ESI-MS (m/z): [M + Na] ⁺: 491.30, Anal Calcd for C₂₆H₂₆ClFN₂O₃: C; 66.59, H; 5.59, N; 5.97, Found: C; 66.71, H; 5.91, N; 6.10.

(±)-Ethyl 2-amino-4–(4-chlorophenyl)-7,7-dimethyl-5-oxo-1-phenyl -1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8c)

Yellow powder, yield: 76%; m.p. 215–217 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.77 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.20 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.74 (s, 1H, COCH₂), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.13 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.02–4.10 (m, 2H, OCH₂CH₃), 5.13 (s, 1H, HHQ, 4-H), 6.24 (br. s, 2H, NH₂), 7.22 (d, 2H, Ar-H, J = 8.0 Hz), 7.31–7.37 (m, 4H, Ar-H), 7.60 (d, 3H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.43, 26.84, 29.22, 29.66, 32.58, 33.16, 33.88, 41.77, 50.11, 59.35, 63.18, 80.62, 115.14, 127.51, 128.12, 129.20, 130.00, 133.96, 134.93, 136.34, 146.40, 149.23, 151.79, 166.50, 169.88, 195.85; (+)ESI-MS (m/z): [M + H]⁺: 451.00, Anal Calcd. for C₂₆H₂₇ClN₂O₃: C; 69.25, H; 6.03, N; 6.21. Found: C; 69.51, H; 6.03, N; 6.02.

(±)-Ethyl 2-amino-4–(4-chlorophenyl)-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8d)

Yellow powder, yield: 87%; m.p. 205–207 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.77 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.21 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH₂, J = 16.0 Hz), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.13 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.49 (s, 3H, CH₃), 4.04–4.08 (m, 2H, OCH₂CH₃), 5.12 (s, 1H, HHQ, 4-H), 6.28 (br. s, 2H, NH₂), 7.18–7.25 (m, 5H, Ar-H), 7.34–7.40 (m, 3H, Ar-H,), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.44, 21.31, 26.64, 29.68, 32.38, 33.90, 41.84, 50.12, 59.30, 80.51, 115.02, 127.89, 128.94, 129.22, 129.62, 131.11, 133.51, 140.41, 146.50, 149.55, 151.98, 169.90, 195.87; (+)ESI-MS (m/z): [M + H]⁺: 465.30, Anal Calcd. for C₂₇H₂₉ClN₂O₃: C; 69.74, H; 6.29, N; 6.02. Found: C; 70.00, H; 6.44, N; 6.31.

(±)-Ethyl 2-amino-1,4-bis (4-chlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8e)

Off-white powder, yield: 66%; m.p. 220–222 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.78 (s, 3H, CH₃-dimedone), 0.97 (s, 3H, CH₃-dimedone), 1.22 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.74 (d, 1H, COCH₂, J = 16.0 Hz), 2.07 (d, 1H, COCH₂, J = 16.0 Hz), 2.14 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.22 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.00–4.10 (m, 2H, OCH₂CH₃), 5.11 (s, 1H, HHQ, 4-H), 6.21 (br. s, 2H, NH₂), 7.22 (d, 2H, Ar-H, J = 8.0 Hz), 7.27–7.29 (m, 2H, Ar-H), 7.33 (d, 2H, Ar-H, J = 8.0 Hz), 7.59 (d, 2H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.41, 26.65, 29.70, 32.45, 33.85, 41.94, 50.05, 58.48, 59.46, 80.99, 115.44, 127.97, 129.62, 131.27, 131.94, 134.84, 136.34, 146.16, 148.74, 151.46, 169.83, 195.81; (+)ESI-MS (m/z): [M + Na]⁺: 507.50, Anal Calcd. for C₂₆H₂₆Cl₂N₂O₃: C; 64.33, H; 5.40, N; 5.77. Found: C; 64.57, H; 5.10, N; 6.04.

(±)-Ethyl2-amino-4–(4-methoxyphenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8f)

Shiny yellow powder, Yield: 43%; m.p. 169–171 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.54 (s, 3H, CH₃-dimedone), 0.70 (s, 3H, CH₃-dimedone), 0.98 (t, 3H, OCH₂CH₃, J = 8.0 Hz,), 1.50 (d, 1H, COCH₂, J = 16.0 Hz), 1.82 (d, 1H, COCH₂, J = 16.0 Hz), 1.88 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 1.97 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.53 (s, 3H, OCH₃), 3.78–3.87 (m, 2H, OCH₂CH₃), 4.86 (s, 1H, HHQ, 4-H), 5.97 (br. s, 2H, NH₂), 6.56 (d, 2H, Ar-H, J = 8.0 Hz); 7.10 (d, 4H, Ar-H, J = 8.0 Hz), 7.32–7.37 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.48, 26.70, 29.68, 32.39, 33.34, 35.12, 41.83, 50.18, 55.15, 59.23, 81.27, 113.20, 115.72, 128.68, 130.01, 129.82, 131.32, 136.54, 140.32, 148.94, 151.70, 157.52, 170.06, 195.52; (+)ESI-MS (m/z): [M + H] ⁺: 447.20, Anal Calcd for C₂₇H₃₀N₂O₄: C; 72.62, H; 6.77, N; 6.27; Found: C; 72.88, H; 6.95, N; 6.15.

(±)-Ethyl 2-amino-4–(4-methoxyphenyl)-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8g)

Yellow powder, yield: 63%; m.p. 181–183 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.53 (s, 3H, CH₃-dimedone), 0.70 (s, 3H, CH₃-dimedone), 0.97 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.52 (d, 1H, COCH₂, J = 16.0 Hz), 1.81 (d, 1H, COCH₂, J = 16.0 Hz), 1.87 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 1.95 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.22 (s, 3H, CH₃), 3.53 (s, 3H, OCH₃), 3.76–3.85 (m, 2H, OCH₂CH₃), 4.84 (s, 1H, HHQ, 4-H), 5.98 (br. s, 2H, NH₂), 6.55 (d, 2H, Ar-H, J = 8.0 Hz), 6.95 (d, 2H, Ar-H, J = 8.0 Hz), 7.08 (d, 2H, Ar-H, J = 8.0 Hz), 7.12 (d, 2H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.48, 21.30, 26.71, 29.69, 32.38, 33.34, 41.81, 50.19, 55.27, 59.19, 81.15, 113.17, 115.61, 128.68, 129.67, 131.08, 133.73, 140.23, 140.42, 149.20, 151.88, 157.49, 170.07, 195.65; (+)ESI-MS (m/z): [M + Na]⁺: 483.20, Anal Calcd. for C₂₈H₃₂N₂O₄: C; 73.02, H; 7.00, N; 6.08. Found: C; 73.22, H; 7.25, N; 6.24.

(±)-Ethyl 2-amino-1–(4-chlorophenyl)-4–(4-methoxyphenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8h)

Shiny yellow powder, Yield: 85%; m.p. 255–257 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.79 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.22 (t, 3H OCH₂CH₃, J = 8.0 Hz), 1.74 (d, 1H, COCH₂, J = 16.0 Hz), 2.05 (d, 1H, COCH_2, J = 16.0 Hz), 2.13 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.77 (s, 3H, OCH₃), 4.00–4.09 (m, 2H, OCH₂CH₃), 5.08 (s, 1H, HHQ, 4-H), 6.17 (br. s, 2H, NH₂), 6.80 (d, 2H, Ar-H, J = 8.0 Hz), 7.30 (t, 4H, Ar-H, J = 8.0 Hz), 7.57 (d, 2H, Ar-H, J = 8.0 Hz),¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.44, 26.74, 29.70, 32.45, 33.29, 41.91, 50.13, 55.37, 59.61, 81.71, 113.24, 116.07, 128.64, 130.74, 131.43, 135.12, 136.15, 140.04, 148.34, 151.32, 157.58, 170.04, 195.89; (+)ESI-MS (m/z): [M + H] ⁺: 481.20, Anal Calcd for C₂₇H₂₉ClN₂O₄: C; 67.42, H; 6.08, N; 5.82, Found: C; 67.56, H; 6.30, N; 5.90.

(±)-Ethyl 2-amino-4–(4-nitrophenyl)-7,7-dimethyl-5-oxo-1-phenyl -1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8i)

Yellow powder, yield: 33%; m.p. 164–166 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.76 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH₂, J = 16.0 Hz), 2.09 (d, 1H, COCH₂, J = 16.0 Hz), 2.13 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.23 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.03–4.10 (m, 2H, OCH₂CH₃), 5.24 (s, 1H, HHQ, 4-H), 6.34 (br. s, 2H, NH₂), 7.35 (d, 2H, Ar-H, J = 8.0 Hz), 7.58–7.63 (m, 5H, Ar-H), 8.14 (d, 2H, Ar-H, J = 8.0 Hz),¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.55, 27.02, 29.46, 32.25, 36.78, 42.15, 50.10, 59.21, 80.13, 113.77, 123.47, 126.53, 127.09, 130.07, 130.31, 130.44, 132.81, 133.16, 136.46, 146.04, 149.56, 152.02, 170.21, 195.76; (+)ESI-MS (m/z): [M + Na]⁺: 484.30, Anal Calcd. for C₂₆H₂₇N₃O₅: C; 67.66, H; 5.90, N; 9.10. Found: C; 67.49, H; 6.12, N; 9.24.

(±)-Ethyl 2-amino-7,7-dimethyl-1–(4-methylphenyl)-4–(4-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8j)

Brown powder, yield: 65%; m.p. 270–272 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.77 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.20 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH₂, J = 16.0 Hz), 2.05 (d, 1H, COCH_2, J = 16.0 Hz), 2.12 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.49 (s, 3H, CH₃), 4.04–4.08 (m, 2H, OCH₂CH₃), 5.12 (s, 1H, HHQ, 4-H), 6.25 (br. s, 2H, NH₂), 7.18–7.22 (m, 4H, Ar-H), 7.34–7.39 (m, 4H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.42, 21.33 26.61, 29.64, 32.41, 35.00, 41.90, 50.02, 59.44, 79.71, 114.13, 123.30, 128.72, 129.57, 131.31, 133.20, 140.68, 146.68, 146.04, 150.11, 152.13, 155.54, 169.61, 195.72; (+)ESI-MS (m/z): [M + H₂O]⁺: 491.30, Anal Calcd. for C₂₇H₂₉N₃O₅: C; 68.19, H; 6.15, N; 8.84. Found: C; 68.48, H; 6.33, N; 9.09.

(±)-Ethyl2-amino-1–(4-chlorophenyl)-7,7-dimethyl-4–(4-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8k)

Faint brown powder, yield: 65%; m.p. 198–200 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.77 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.26 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.73 (d, 1H, COCH₂, J = 16.0 Hz), 2.05 (d, 1H, COCH₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.22 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.04–4.09 (m, 2H, OCH₂CH₃), 5.11 (s, 1H, HHQ, 4-H), 6.20 (br. s, 2H, NH₂), 7.21 (d, 2H, Ar-H, J = 8.0 Hz), 7.27 (d, 2H, Ar-H, J = 8.0 Hz), 7.32 (d, 2H, Ar-H, J = 8.0 Hz), 7.57 (d, 2H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.40, 26.66, 29.68, 32.44, 33.85, 41.94, 50.07, 59.45, 81.00, 115.46, 128.13, 129.16, 130.84, 131.37, 134.87, 136.33, 146.16, 148.71, 151.47, 169.81, 195.76; (+)ESI-MS (m/z): [M + Na]⁺: 518.60, Anal Calcd. for C₂₆H₂₆ClN₃O₅: C; 62.97, H; 5.28, N; 8.47. Found: C; 63.10, H; 5.56, N; 8.29.

(±)-Ethyl 2-amino-7,7-dimethyl-4-[4-(morpholin-4-yl) phenyl]- 5-oxo-1-phenyl -1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8l)

Orange powder, yield: 44%; m.p. 193–195 °C. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.71 (s, 3H, CH₃-dimedone), 0.88 (s, 3H, CH₃-dimedone), 1.15 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.64 (d, 1H, COCH₂, J = 16.0 Hz), 1.98 (d, 1H, COCH₂, J = 16.0 Hz), 2.10 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.05 (s, 4H, morpholine), 3.71 (s, 4H, morpholine), 3.97 (m, 2H, OCH₂CH₃), 4.86 (s, 1H, HHQ, 4-H), 6.65 (br. s, 2H, NH₂), 6.84 (d, 2H, Ar-H, J = 8.0 Hz), 7.17 (d, 2H, Ar-H, J = 8.0 Hz), 7.40 (d, 2H, Ar-H, J = 8.0 Hz), 7.59–7.65 (m, 3H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.44, 26.82, 29.68, 32.45, 33.17, 41.92, 46.98, 49.67, 50.17, 59.35, 62.15, 66.43, 67.04, 81.80, 113.54, 115.41, 116.13, 128.39, 130.71, 131.42, 133.74, 135.22, 136.13, 148.27, 149.19, 151.30, 154.23, 170.06, 195.84; (+)ESI-MS (m/z): [M + Na]⁺: 524.20, Anal Calcd. for C₃₀H₃₅N₃O₄: C; 71.83, H; 7.03, N; 8.38. Found: C; 72.05, H; 7.29, N; 8.60.

(±)-Ethyl 2-amino-1–(4-chlorophenyl)-7,7-dimethyl-4-[4-(morpholin-4-yl) phenyl]-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8m)

Orange powder, yield: 71%; m.p. 175–177 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.80 (s, 3H, CH₃-dimedone), 0.97 (s, 3H, CH₃-dimedone), 1.23 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.75 (d, 1H, COCH₂, J = 16.0 Hz), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.14 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.22 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.13–3.18 (m, 4H, morpholine), 3.86–3.92 (m, 4H, morpholine), 4.02–4.14 (m, 2H, OCH₂CH₃), 5.08 (s, 1H, HHQ, 4-H), 6.17 (br. s, 2H, NH₂), 6.92 (t, 2H, Ar-H, J = 8.0 Hz), 7.29–7.34 (m, 4H, Ar-H), 7.58 (d, 1H, Ar-H, J = 8.0 Hz), 7.98 (d, 1H, Ar-H, J = 8.0 Hz),¹³C NMR (100 MHz, CDCl₃) δ ppm):14.46, 26.81, 29,69, 32.46, 33.26, 41.91, 46.94, 50.07, 59.39, 62.18, 66.43, 66.80, 81.65, 113.53, 115.96, 121.75, 128.50, 130.75, 131.41, 133.76, 135.10, 136.17, 148.51, 151.32, 154.10, 154.28, 170.03, 195.99; (+)ESI-MS (m/z): [M + H]⁺: 536.10, Anal Calcd. for C₃₀H₃₄ClN₃O₄: C; 67.22, H; 6.39, N; 7.84. Found: C; 67.50, H; 6.61, N; 7.52.

(±)-Ethyl 2-amino-4–(2,3-dichlorophenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (10a)

Yellow powder, yield: 58%; m.p. 235–237 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.84 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.17 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.80 (d, 1H, COCH_2, J = 16.0 Hz), 2.01 (d, 2H, COCH_2, J = 16.0 Hz), 2.11 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.96–4.05 (m, 2H, OCH₂CH₃), 5.44 (s, 1H, HHQ, 4-H), 6.31 (br. s, 2H, NH₂), 7.12 (t, 1H, Ar-H, J = 8.0 Hz), 7.25 (d, 2H, Ar-H, J = 8.0 Hz), 7.50 (d, 1H, Ar-H, J = 8.0 Hz), 7.61 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm); 14.39, 27.02, 29.45, 32.26, 36.57, 42.09, 50.05, 59.30, 78.92, 112.58, 126.04, 127.85, 130.17, 130.31, 131.71, 131.85, 133.06, 136.27, 146.25, 150.23, 152.28, 170.05, 195.86; ESI-MS (m/z): [M]⁺: 484.13, Anal Calcd. for C₂₆H₂₆Cl₂N₂O₃: C; 64.33, H; 5.40, N; 5.77. Found: C; 64.12, H; 5.46, N; 5.69.

(±)-Ethyl 2-amino-1–(4-chorophenyl)-4–(2,3-dichlorophenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (10b)

Yellow powder, yield: 63%; m.p. 250–252 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.84 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.15 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.78 (d, 1H, COCH_2, J = 16.0 Hz), 2.00 (d, 1H, COCH₂, J = 16.0 Hz), 2.10 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.17 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.97–4.06 (m, 2H, OCH₂CH₃), 5.41 (s, 1H, HHQ, 4-H), 6.28 (br. s, 2H, NH₂), 7.11 (t, 1H, Ar-H, J = 8.0 Hz), 7.26–7.33 (m, 4H, Ar-H), 7.48 (d, 1H, Ar-H, J = 8.0 Hz), 7.58 (d, 1H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.36, 27.01, 29.48, 32.29, 36.59, 42.14, 49.99, 59.37, 79.19, 112.78, 126.07, 127.94, 130.79, 131.66, 131.76, 131.81, 133.09, 134.75, 136.36, 146.00, 149.75, 151.97, 169.94, 195.74.; ESI-MS (m/z): [M]⁺: 518.69, Anal Calcd. for C₂₆H₂₅Cl₃N₂O₃: C; 60.07, H; 4.85, N; 5.39. Found: C; 59.90, H; 4.79, N; 5.49.

(±)-Ethyl 2-amino-4–(2,4-dichlorophenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (10c)

Yellow powder, yield: 46%; m.p. 226–228 °C. IR (KBr disc): ῡ (cm⁻¹): 3472 (NH₂ str.), 3059 (aromatic C-H str.), 2948 (aliphatic C-H str.), 1662 (C = O str.), 1495 (aliphatic C = C str.), 1266 (aliphatic C-N str.), 1209 (aliphatic C-O str.), ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.83 (s, 3H, CH₃-dimedone), 0.94 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH_2, J = 8.0 Hz), 2.01 (d, 1H, COCH₂, J = 16.0 Hz), 2.09 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.00–4.08 (m, 2H, OCH₂CH₃), 5.34 (s, 1H, HHQ, 4-H), 6.31 (br. s, 2H, NH₂), 7.14–7.17 (m, 1H, Ar-H), 7.36 (d, 3H, Ar-H, J = 8.0 Hz), 7.48–7.51 (m, 1H, Ar-H), 7.56–7.61 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm); 14.42, 26.84, 29.56, 32.24, 35.22, 42.07, 50.02, 59.31, 78.87, 112.57, 126.09, 129.47, 130.16, 130.27, 130.50, 131.68, 134.05, 134.09, 136.25, 142.65, 150.10, 152.19, 169.99, 195.82.; ESI-MS (m/z): [M]⁺: 484.67, Anal Calcd. for C₂₆H₂₆Cl₂N₂O₃: C; 64.33, H; 5.40, N; 5.77. Found: C; 64.21, H; 5.41, N; 5.84.

(±)-Ethyl 2-amino-1–(4-chorophenyl)-4–(2,4-dichlorophenyl)-7,7-dimethyl-5-oxo-1-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (10d)

Yellow powder, yield: 50%; m.p. 217–219 °C. IR (KBr disc): ῡ (cm⁻¹): 3408 (NH₂ str), 3061 (aromatic C-H str), 2960 (aliphatic C-H str), 1640 (C = O str), 1497 (aliphatic C = C str), 1210 (aliphatic C-N str), 1175 (aliphatic C-O str), ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.85 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.18 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH₂, J = 16.0 Hz), 2.00 (d, 1H, COCH₂, J = 16.0 Hz), 2.10 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.18 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 3.98–4.04 (m, 2H, OCH₂CH₃), 5.32 (s, 1H, HHQ, 4-H), 6.27 (br. s, 2H, NH₂), 7.16 (d, 1H, Ar-H, J = 8.0 Hz), 7.31 (s, 3H, Ar-H), 7.48 (d, 1H, Ar-H, J = 8.0 Hz), 7.59 (d, 2H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.38, 26.86, 29.59, 32.29, 35.34, 42.15, 49.97, 59.42, 79.13, 112.74, 126.10, 129.55, 130.79, 131,64, 131.82, 134.03, 134.23, 134.76, 136.38, 142.28, 149.59, 151.86, 169.93, 195.73ESI-MS (m/z): [M]⁺: 518.18, Anal Calcd. for C₂₆H₂₅Cl₃N₂O₃: C; 60.07, H; 4.85, N; 5.39. Found: C; 60.22, H; 4.93, N; 5.35.

(±)-Ethyl 2-amino-7,7-dimethyl-5-oxo-1-phenyl-4-(pyridin-2-yl)-1,4,5,6,7,8- hexahydroquinoline-3-carboxylate (12a)

Brown powder, yield: 93%; m.p. 158–160 °C. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.78 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH₂, J = 16.0 Hz), 2.07 (d, 1H, COCH₂, J = 16.0 Hz), 2.12 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.16 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.06 (m, 2H, OCH₂CH_3, J = 8.0 Hz), 5.12 (s, 1H, HHQ, 4-H), 6.27 (br. s, 2H, NH₂), 7.16–7.19 (m, 1H, Ar-H), 7.35 (d, 2H, Ar-H, J = 8.0 Hz), 7.60 (d, 3H, Ar-H, J = 8.0 Hz), 7.75 (d, 1H, Ar-H, J = 8.0 Hz), 8.36 (d, 1H, Ar-H, J = 8.0 Hz), 8.66 (s, 1H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.41, 26.78, 29.56, 32.43, 32.58, 32.80, 41.87, 50.04, 59.36, 80.22, 114.53, 122.80, 130.03, 130.19, 130.59, 135.70, 136.18, 143.21, 146.94, 149.75, 151.85, 169.65, 195.66; (+)ESI-MS (m/z): [M + H]⁺: 418.20, Anal Calcd. for C₂₅H₂₇N₃O₃: C; 71.92, H; 6.52, N; 10.06. Found: C; 72.10, H; 6.73, N; 10.21.

(±)-Ethyl 2-amino-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1-phenyl-4-(pyrdin-2-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (12b)

Brown powder, yield: 49%; m.p. 196–198 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.81 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.22 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.09 (d, 1H, COCH₂), 2.16 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.47 (s, 3H, CH₃), 4.08 (q, 2H, OCH₂CH₃), 5.25 (s, 1H, HHQ, 4-H), 6.36 (br. s, 2H, NH₂), 7.29 (s, 1H, Ar-H), 7.37 (d, 3H, Ar-H, J = 8.0 Hz), 7.56 (d, 3H, Ar-H, J = 8.0 Hz), 8.54 (s, 1H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.53, 21.33 26.80, 29.48, 32.54, 36.82, 41.74, 50.18, 59.13, 61.29, 77.28 (masked by solvent peaks), 121.02, 122.22, 124.94, 125.49, 129.88, 134.26, 136.58, 138.74, 152.78, 159.87, 169.78, 196.07; (+)ESI-MS (m/z): [M + Na]⁺: 454.10, Anal Calcd.for C₂₆H₂₉N₃O₃: C; 72.37, H; 6.77, N; 9.74. Found: C; 72.55, H; 7.04, N; 10.03.

(±)-Ethyl 2-amino-1–(4-chlorophenyl)-7,7-dimethyl-5-oxo-4-(pyridine-2-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (12c)

Brown powder, Yield: 60%; m.p. 256–258 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.78 (s, 3H, CH₃-dimedone), 0.96 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz,), 2.08 (d, 1H, COCH₂, J = 16.0 Hz), 2.11 (d, 1H, COCH_2, J = 16.0 Hz J = 16.0 Hz), 2.15 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.03–4.07 (m, 2H, OCH₂CH_3, J = 8.0 Hz), 5.09 (s, 1H, HHQ, 4-H), 6.29 (br. s, 2H, NH₂), 7.16 (m, 1H, Ar-H), 7.29 (d, 2H, Ar-H, J = 8.0 Hz), 7.55 (d, 2H, Ar-H, J = 8.0 Hz), 7.73 (d, 1H, Ar-H, J = 8.0 Hz), 8.33 (m, 1H, Ar-H, J = 8.0 Hz), 8.60 (s, 1H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.49, 27.03, 28.32, 29.30, 32.58, 32.82, 36.85, 41.78, 43.48, 50.08, 50.34, 59.30, 99.09, 121.38, 124.95, 129.41, 130.59, 131.72, 135.59, 135.99, 136.96, 152.48, 159.73, 169.53, 196.06; (+)ESI-MS (m/z): [M + H]⁺: 452.20, Anal Calcd for C₂₅H₂₆ClN₃O₃: C; 66.44, H; 5.80, N; 9.30, Found: C; 66.22, H; 6.09, N; 9.52.

(±)-Ethyl 2-amino-7,7-dimethyl-5-oxo-4-(pyridin-3-yl)-1,4,5,6,7,8- hexahydroquinoline-3-carboxylate (12d)

Yellow powder, Yield: 57%; m.p. 215–217 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.77 (s, 3H, CH₃-dimedone), 0.95 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.78 (d, 1H, COCH₂, J = 16.0 Hz), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.11 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.21 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.04–4.07 (m, 2H, OCH₂CH₃), 5.12 (s, 1H, HHQ, 4-H), 6.30 (br. s, 2H, NH₂), 7.21–7.24 (m, 1H, Ar-H), 7.36 (d, 2H, Ar-H, J = 8.0 Hz), 7.58–7.63 (m, 2H, Ar-H), 7.81–7.84 (m, 2H, Ar-H), 8.36–8.37 (m, 1H, Ar-H), 8.65–8.66 (d, 1H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.41, 26.84, 29.48, 32.43, 32.99, 41.84, 49.98, 59.39, 79.90, 114.23, 123.07, 130.26, 130.63, 136.02, 136.82, 143.89, 145.80, 148.45, 149.79, 151.90, 169.50, 195.68; (+)ESI-MS (m/z): [M + H]⁺: 418.50; Anal Calcd for C₂₅H₂₇N₃O₃, C; 71.92, H; 6.52, N; 10.06, Found: C; 72.00, H; 6.71, N; 10.22.

(±)-Ethyl 2-amino-7,7-dimethyl-1–(4-methylphenyl)-5-oxo-1-phenyl-4-(pyrdin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (12e)

Yellow powder, yield: 84%; m.p. 261–263 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.79 (s, 3H, CH₃-dimedone), 0.93 (s, 3H, CH₃-dimedone), 1.19 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.78 (d, 1H, COCH₂, J = 16.0 Hz), 2.02 (d, 1H, COCH₂, J = 16.0 Hz), 2.07 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.16 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.49 (s, 3H, CH₃), 3.99–4.03 (m, 2H, OCH₂CH₃), 5.27 (s, 1H, HHQ, 4-H), 6.23 (br. s, 2H, NH₂), 6.82–6.89 (m, 2H, Ar-H), 7.08–7.12 (m, 1H, Ar-H), 7.25–7.28 (m, 2H, Ar-H), 7.37–7.45 (m, 3H, Ar-H), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.36, 21.31, 26.46, 29.73, 32.34, 32.55, 33.19, 42.20, 50.17, 55,90, 59.06, 79.82, 111.34, 113.71, 119.89, 120.21, 126.83, 129.85, 130.96, 132.20, 140.01, 149.60, 152.16, 157.89, 170.52, 195.81; (+)ESI-MS (m/z): [M + H]⁺: 432.50, Anal Calcd. for C₂₆H₂₉N₃O₃: C; 72.37, H; 6.77, N; 9.74. Found: C; 72.51, H; 7.01, N; 10.02.

(±)-Ethyl 2-amino-1–(4-chlorophenyl)-7,7-dimethyl-5-oxo-4-(pyridin-3-yl)-1,4,5,6,7,8- hexahydroquinoline-3-carboxylate (12f)

Shiny white powder, Yield: 87%; m.p. 261–263 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.79 (s, 3H, CH₃-dimedone), 0.98 (s, 3H, CH₃-dimedone), 1.21 (t, 3H, OCH₂CH₃, J = 8.0 Hz), 1.77 (d, 1H, COCH₂, J = 16.0 Hz), 2.06 (d, 1H, COCH₂, J = 16.0 Hz), 2.13 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 2.22 (d, 1H, CH₂C(CH₃)₂, J = 16.0 Hz), 4.03–4.09 (m, 2H, OCH₂CH₃), 5.10 (s, 1H, HHQ, 4-H), 6.26 (br. s, 2H, NH₂), 7.18–7.21 (m, 1H, Ar-H); 7.32 (d, 2H, Ar-H, J = 8.0 Hz); 7.59 (d, 2H, Ar-H, J = 8.0 Hz); 7.75 (d, 1H, Ar-H, J = 8.0 Hz); 8.36 (d, 1H, Ar-H, J = 8.0 Hz), 8.62 (d, 1H, Ar-H, J = 8.0 Hz), ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.41, 27.07, 29.33, 32.54, 33.43, 41.92, 49.87, 59.61, 79.65, 113.97, 123.69, 131.41, 134.22, 136.66, 139.51, 143.32, 145.27, 145.93, 149.68, 151.66, 169.18, 195.65; (+)ESI-MS (m/z): [M + H]⁺: 452.50, Anal Calcd for C₂₅H₂₆ClN₃O₃, C; 66.44, H; 5.80, N; 9.30, Found: C; 66.62, H; 6.03, N; 9.52.

X-ray crystallography

A colourless prism crystal of compound 6f (accession number 2163585) was picked up as a representative sample to study the enantiomerism of our target compounds^52,53. The detailed procedures of the x-ray crystallography technique are discussed in the Supplementary file.

Biology

The comprehensive procedures of biological assays of the target HHQ analogues (6a–i, 8a–m, 10a–d, and 12a–f are presented in the Supplementary file, including; preliminary in vitro anticancer screening¹⁰⁵, EGFR kinase inhibitory assay¹⁰⁶, IMR-90 normal lung cell line for safety^76,77, Annexin V-FITC apoptosis assay¹⁰⁷, and cellular mechanistic analysis¹⁰⁸.

In silico studies

In silico studies of the representative target HHQ analogues 10c and 10d were presented in the Supplementary file. These studies include molecular docking analysis and predicting targeted compounds’ physicochemical properties and pharmacokinetics using Molsoft software and the SwissADME web tool^{91,92,109,110}. The procedures of these studies are presented in detail in the Supplementary file.

CreDIT authorship statement

Conception and the study’s design; T.F.E.-M, M.H.E.-H and H.O.T., chemical synthesis of the compounds; M.G.A.-A. and H.O.T. Biological evaluation studies on the compounds; H.O.T., O.A., A.B.M. in silico studies and molecular docking; M.G.A.-A. x-ray crystallography; K.Y, M.S. Drafting of the article; T.F.E.-M, M.H.E.-H, H.O.T., M.G.A.-A. Review and comments on the manuscript; T.F.E.-M, M.H.E.-H, O.A., and H.O.T.

Acknowledgements

The authors would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (23UQU4280008DSR003).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by Deanship of Scientific Research at Umm Al-Qura University for [Grant Code: (23UQU4280008DSR003)].