Tetrahydroquinolinone derivatives as potent P-glycoprotein inhibitors: design, synthesis, biological evaluation and molecular docking analysis

Tetrahydroquinolinones bearing a phenyl ring with electron-withdrawing substitution showed remarkable P-glycoprotein inhibitory activity and significantly increased rhodamine accumulation in MES-SA/DX5 cells.

Abstract

P-glycoprotein (P-gp) is a transmembrane efflux pump that has been associated with ineffective cancer chemotherapy and multidrug resistance (MDR). Chemical inhibitors of P-gp could have potential cancer therapeutic applications by preventing or reversing MDR. To exploit this, we designed twenty-five tetrahydroquinolinone analogs bearing pyridyl methyl carboxylate at C₃ and different substituents at C₄ as MDR reversal agents. The inhibitory effects of the synthesized compounds against P-gp were assessed by flow cytometric determination of rhodamine 123 accumulation in P-gp over-expressing MES-SA/DX5 cells. Fluorescence imaging of intracellular rhodamine 123 accumulation in MES-SA/DX5 cells was also performed. Furthermore, the effect of active derivatives on the reduction of doxorubicin's IC₅₀ in MES-SA/DX5 cells was evaluated using MTT assay. Molecular docking was used to confirm the binding mode of some of the synthesized compounds. Five compounds in group A, bearing a 2-pyridyl methyl ester substituent at the C₃ position, significantly increased rhodamine accumulation at 25 μM comparable to verapamil, a well-established P-gp inhibitor, while only 2 compounds in group B bearing 3-pyridyl methyl ester at the same position had this effect. This study shows that tetrahydroquinolinones containing methyl pyridine esters could represent an attractive scaffold for the discovery of P-gp inhibitors as MDR reversal agents in cancer cells.

Introduction

Cancer is a main cause of death worldwide and the annual number of new cancer cases is estimated to rise from 14 million in 2012 to 22 million within the next 2 decades.1 Various chemotherapeutic agents are available for the treatment of cancer.2,3 However, a major impediment to successful chemotherapy is multidrug resistance (MDR), which may occur due to the over-expression of ATP-binding cassette (ABC) membrane transporter family members including P-glycoprotein (P-gp), the breast cancer-resistance protein (BCRP) and the multidrug resistance-associated protein 1 (MRP1) in cancer cells.4–6 These transporters considerably reduce the intracellular amount of anticancer drugs by an ATP-dependent efflux mechanism.7

P-gp, a 170 kDa glycoprotein, is the first ABC efflux pump recognized to be responsible for the failure of chemotherapeutic agents in tumor cells.7 Several structurally diverse P-gp substrates have been reported so far, including drugs such as anticancer agents, antihistamines, immunosuppressive drugs, antivirals, antibiotics, antihypertensives, analgesics, HIV-protease inhibitors and steroids.8–11

Clinical studies have revealed a negative correlation between patients' response to chemotherapeutic agents in sarcomas, ovarian cancer, breast cancer, hematological malignancies such as leukemia and the level of P-gp expression in their tumors.12–15 Therefore; P-gp inhibitors that efficiently reverse P-gp-mediated MDR could significantly improve current cancer therapies.

1,4-Dihydropyridines (1,4-DHPs), known as calcium channel blocker (CCB) agents, are primarily used for the treatment of cardiovascular diseases16 but have also been shown to possess anticancer, anticonvulsant, anti-inflammatory, neuroprotectant and antitubercular properties.17,18 In addition to these biological effects, DHPs can effectively reverse MDR, mainly by a P-gp inhibitory mechanism.19–24 For example, DHPs such as nifedipine (compound 1) (Fig. 1), niguldipine, nimodipine and nicardapine have been shown to inhibit MDR.25,26 In this context, we have recently investigated DHPs containing alkyl pyridine carboxylate at the C₃ position (compounds 2 and 3) (Fig. 1) as potent MDR reversal agents.19,20,27 On the other hand, quinidine, a natural compound with two fused rings, was reported as an effective multidrug resistance inhibitor of the human leukemic cell line K562/ADM (compound 4) (Fig. 1).28

Fig. 1. Structure of some previously reported MDR reversal agents structurally related to the designed compounds of this study.

In order to investigate the effects of fusing a DHP ring with a cyclohexanone ring on MDR reversal activity, we designed novel tetrahydroquinolinone analogs based on the structure of quinidine, nifedipine and other MDR reversal agents. To define a good structure–activity relationship (SAR) and find new and more effective MDR reversal compounds, we designed tetrahydroquinolinone derivatives bearing different substituents including aliphatic, heteroaromatic and aromatic moieties either with electron-donating or with electron-withdrawing groups at the C₄ position while having 2-pyridyl methyl carboxylate (group A compounds) or 3-pyridyl methyl carboxylate (group B compounds) substituents at the C₃ position. To this end, twenty-five tetrahydroquinolinone derivatives were synthesized and all the target compounds were evaluated for their P-gp inhibitory activity on the MES-SA/DX5 cell line using flow cytometry technique, MTT assay and fluorescence imaging. Finally, in order to obtain insight about the binding mode of the derivatives in the active site of P-gp, molecular docking analysis was performed.

Results and discussion

Design

The tetrahydroquinolinone compounds were designed based on previously reported MDR reversal agents (compounds 1–4) (Fig. 1).19–21,25–28 Therefore, we considered several structural features in our design (Fig. 2).

Fig. 2. Structural design of novel MDR reversal agents based on previously reported MDR reversal scaffolds.

-The DHP ring was chosen because the MDR inhibitory activity of DHP pharmacophore has been reported in previous studies.19–21

-With the aim of studying the effect of fusing the DHP core with a cyclohexanone ring on the MDR reversal activity, we designed the tetrahydroquinoline scaffold based on the structure of quinidine.

-Methyl pyridine carboxylates at the C₃ position were introduced to enhance the MDR reversal activity. Furthermore, to explore the importance of nitrogen position in the pyridine ring on reversal of multidrug resistance, pyridin-2-ylmethyl carboxylate and pyridin-3-ylmethyl carboxylate substituents were presented in group A and B compounds, respectively.

-To define better structure–activity relationships (SARs), different substituents (aliphatic, heteroaromatic and aromatic moieties either with electron-donating or with electron-withdrawing groups) were inserted at the C₄ position.

Synthesis

Twenty-five tetrahydroquinolinone analogs were successfully synthesized by the reactions explained in Experimental section, and the structures were confirmed by IR, ¹H NMR, ¹³C NMR, MS and elemental analysis. The chemical structures and physical properties of the synthesized compounds are mentioned in Table 1. Acetoacetates (2a and 2b) were obtained via the reaction of pyridin-2-ylmethanol (1a) or pyridin-3-ylmethanol (1b) with 2,2,6-trimethyl-4H-1,3-dioxin-4-one. The final products were synthesized by the reaction of the obtained acetoacetate (2a or 2b) with different aldehydes and 1,3-cyclohexandione in the presence of excess amounts of ammonium acetate (Scheme 1).

Table 1. Structure, physical properties and drug likeness score of synthesized compounds.

Compound	R	M.P. (°C)	Yield (%)	R f	Log P a	HBA b	HBD c	TPSA d	nRB e	MW f
A1	Phenyl	171	41	0.40	2.719	5	1	67.76	5	353.00
A2	3-Nitrophenyl	169	12	0.47	1.881	7	1	113.58	6	398.99
A3	4-Nitrophenyl	128	13	0.39	1.881	7	1	113.58	6	398.99
A4	2-Nitrophenyl	178	18	0.45	1.881	7	1	113.58	6	398.99
A5	4-Cyanophenyl	158	32	0.45	1.727	6	1	91.55	5	379.00
A6	4-Chlorophenyl	128	46	0.31	2.241	5	1	67.76	5	387.97
A7	4-Bromophenyl	149	34	0.32	2.417	5	1	67.76	5	431.92
A8	4-Methoxyphenyl	128	36	0.40	1.904	6	1	76.99	6	380.99
A9	2-Furyl	148	23	0.47	1.401	6	1	76.99	5	344.99
A10	2-Thienyl	150	26	0.38	1.766	5	1	93.06	5	360.97
A11	Propyl	134	21	0.41	2.600	5	1	67.76	6	317.00
A12	4-Hydroxyphenyl	235	26	0.28	1.583	6	2	87.99	5	370.00
A13	3-Hydroxyphenyl	245	12	0.26	1.583	6	2	87.99	5	370.00
A14	3,4,5-Trimethoxyphenyl	174	68	0.38	2.173	8	1	95.45	8	436.98
B1	Phenyl	211	45	0.39	2.350	5	1	67.76	5	353.00
B2	3-Hydroxyphenyl	214	24	0.28	1.214	6	2	87.99	5	370.00
B3	4-Nitrophenyl	194	16	0.41	1.512	7	1	110.90	6	398.99
B4	2-Nitrophenyl	158	13	0.40	1.512	7	1	110.90	6	398.99
B5	4-Cyanophenyl	208	25	0.47	1.358	6	1	91.55	5	379.00
B6	4-Chlorophenyl	217	47	0.40	1.872	5	1	67.76	5	387.97
B7	4-Bromophenyl	233	43	0.37	2.048	5	1	67.76	5	431.92
B8	4-Methoxyphenyl	212	62	0.41	1.535	6	1	76.99	6	380.99
B9	2-Furyl	159	24	0.47	1.032	6	1	76.99	5	344.99
B10	2-Thienyl	172	60	0.36	1.397	5	1	93.06	5	360.97
B11	Propyl	105	17	0.42	2.231	5	1	67.76	6	317.00

^aLogarithm of partition coefficient between n-octanol and water (log P).

^bNumber of hydrogen bond acceptors (HBA).

^cNumber of hydrogen bond donors (HBD).

^dTopological polar surface area (TPSA).

^eNumber of rotatable bonds (nRB).

^fMolecular weight (MW).

Scheme 1. Procedure for the synthesis of tetrahydroquinolinone analogs (A1–A14 and B1–B11).

Drug-likeness properties

As demonstrated in Table 1, the synthesized analogs have log P, TPSA and MW in the range of 1.03–2.71, 67.76–113.58 and 317–436 g mol^–1, respectively. In addition, all the compounds exhibit fewer than 7 hydrogen bond acceptors (HBAs), fewer than 2 hydrogen bond donors (HBDs) and fewer than 8 rotatable bonds. Therefore, all the synthesized tetrahydroquinolinone derivatives could serve as suitable drug-like candidates with no violation of Lipinski's rule of five and the Veber rule.33,34

Flow cytometric analysis of rhodamine 123 accumulation

In order to evaluate the inhibitory effect of our compounds on the P-gp pump, the intracellular accumulation of Rh123, a substrate of P-gp, was measured by determination of its fluorescence intensity using a flow cytometric assay in MES-SA/DX5 cells. This experiment was also performed on MES-SA cells for comparison.

A representative histogram of the effect of different doses of compound A3 on the intracellular accumulation of Rh123 in resistant MES-SA/DX5 cells is shown in Fig. 3. A dose-dependent response can be observed as the histogram shifts to the right. All the active compounds except A4 and B4 showed a dose-dependent effect. The results are expressed as the ratio of the geometric mean (Gmean) of the fluorescence intensity of MES-SA/DX5 cells treated with the tested compounds to the Gmean of the fluorescence intensity of untreated MES-SA/DX5 cells (Fig. 4). Compounds bearing phenyl rings with ortho- or para-substituted electron-withdrawing groups at the C₄ position of the tetrahydroquinolinone core, such as A3, A4, A5, A7, B3, B4 and B7 possessed the highest Gmean of fluorescence intensity compared to control (cells without drug) (Fig. 4). A4, A7 and B4 with Gmean-to-Gmean of control ratios of 7.36, 6.16 and 6.22, respectively, at 25 μM, were more active than verapamil with the ratio of 5.54 at the same concentration (Fig. 4). The inhibitory effect of the 2-pyridyl ester derivatives, A3 and A5, with the respective 4-nitrophenyl and 4-cyanophenyl moieties at C₄ at 25 μM was almost near that of verapamil at the same concentration. In general, 2-pyridyl methyl ester derivatives (A series) demonstrated superior inhibitory activity over their 3-pyridyl-methyl ester (B series) counterparts, e.g. Rh123 accumulation was higher in cells treated with A7 compared to B7 and in cells incubated with A5 compared to B5 at all three tested concentrations (5, 25, and 100 μM). All compounds containing phenyl, 3-nitrophenyl, heteroaromatic and aliphatic moieties, hydroxyphenyl and trimethoxyphenyl at C₄ (A1, A2, A9–A14, B1, B2, and B9–B11) demonstrated the lowest Rh123 accumulation in both groups A and B.

Fig. 3. Dose-dependent effect of compound A3 on the retention of rhodamine 123 in MES-SA/DX5 cells. Cells were resuspended in RPMI 1640 and treated with A3 as a P-gp inhibitor at 5, 25 and 100 μM. After incubation for 20 min, 5 μM Rh123 was added for 20 min at 37 °C and then the cells were centrifuged, washed twice with ice-cold PBS and resuspended in PBS. Then 20 000 cells were counted using a flow cytometer with excitation and emission wavelengths of 488 nm and 530 nm, respectively. Histograms were drawn using WINMDI version 2.9.

Fig. 4. Flow cytometric detection of rhodamine 123 efflux from MES-SA/DX5 cells. Cells were suspended in RPMI 1640 and treated with verapamil (positive control) or synthesized P-gp inhibitors. After incubation for 20 min, 5 μM Rh123 was added and incubated for another 20 min at 37 °C and then the cells were centrifuged and washed twice with ice-cold PBS and resuspended in PBS. The fluorescence caused by the presence of Rh123 in cells was measured using a flow cytometer with excitation and emission wavelengths of 488 nm and 530 nm, respectively. The effects of the synthesized compounds bearing pyridin-2-ylmethyl carboxylate (A) and pyridin-3-ylmethyl carboxylate (B) are shown; VRP: verapamil. *The difference between the Gmean of fluorescence intensities in the presence of the test compound is significantly different from that of untreated control cells (P < 0.05). The Gmean of the fluorescence intensity of untreated control cells was 218 ± 8.1 (SEM).

As anticipated, none of the tested compounds showed a significant increase in Rh123 accumulation (Gmean of fluorescence intensity) compared to control in non-resistant MES-SA cells (Fig. 5).

Fig. 5. Flow cytometric detection of rhodamine 123 efflux from MES-SA cells. Cells were suspended in RPMI 1640 and treated with verapamil (positive control) or synthesized P-gp inhibitors. After incubation for 20 min, 5 μM Rh123 was added and incubated for another 20 min at 37 °C and then the cells were centrifuged and washed twice with ice-cold PBS and resuspended in PBS. The fluorescence caused by the presence of Rh123 in cells was measured using a flow cytometer with excitation and emission wavelengths of 488 nm and 530 nm, respectively. The effects of the synthesized compounds bearing pyridin-2-ylmethyl carboxylate (A) and pyridin-3-ylmethyl carboxylate (B) are shown; VRP: verapamil. *The difference between the Gmean of fluorescence intensities in the presence of the test compound is significantly different from that of untreated control cells (P < 0.05). The Gmean of fluorescence intensity of untreated control cells was 1780 ± 6.4 (SEM).

The structures of the most potent derivatives are illustrated in Fig. 6. According to the data presented in Fig. 4 a structure–activity relationship (SAR) can be deduced as follows:

Fig. 6. Structures of the most active derivatives.

-The position of nitrogen in the alkyl pyridine ester substituent at C₃ is substantial for P-gp inhibitory activity. Thus, considering the log P values in Table 1, it can be stated that the more lipophilic 2-pyridyl methyl carboxylate substituents (group A) were better P-gp modulators than 3-pyridyl methyl carboxylate substituents (group B).

-Flow cytometry analysis revealed that most of the tetrahydroquinolinone derivatives, except those with hydroxyphenyl, propyl and heteroaromatic moieties at the C₄ position, remarkably increased Rh123 accumulation compared to control and had shown a concentration-dependent modulatory activity (except A4 and B4). Therefore, the alternation of functions at the C₄ position also has a significant impact on MDR reversal activity.

-Introducing a nitrophenyl moiety at the C₄ position, as in A3 and A4, as a hydrogen bond acceptor function led to a noticeable increase in activity as compared to A1 in all tested concentrations.

-Compounds bearing a nitro moiety at the para (A3) or ortho (A4) position of the 4-phenyl residue showed greater P-gp inhibitory activities compared to A2, which has a nitro moiety at the meta position.

-As concluded from the flow cytometry results, compounds A6, B6 (bearing 4-chlorophenyl at C₄), A7 and B7 (bearing 4-bromophenyl at C₄) had considerable effects on the P-gp inhibitory activity due to their increased lipophilicity. Compound A7 induced a 2.2-, 4.3- and 3.1-fold increase in Rh123 accumulation as compared to A1 at 5, 25 and 100 μM, respectively.

-Generally, it can be deduced that placing electron-withdrawing groups on the ortho and para positions of the 4-phenyl ring would improve MDR reversal activity.

-Introducing a 4-methoxy functional group at the phenyl ring, as in A8, led to more than twofold increase in the activity as compared to A1 in all tested concentrations, but increasing the number of methoxy groups on the phenyl ring caused A14 to be an inactive compound.

-Flow cytometry results showed that compounds A9 and B9 were not effective P-gp inhibitors. The activity was slightly better in A10 (log P = 1.766) and B10 (log P = 1.397) than A9 (log P = 1.401) and B9 (log P = 1.032) at 25 and 100 μM, respectively, due to the improved lipophilicity.

-Introduction of a propyl group at the C₄ position of the central core in A11 and B11 did not lead to active analogs, perhaps because of their lower lipophilicity. Therefore, increasing the length of the alkyl group and introducing hydrogen bond acceptor groups such as NO₂ or OCH₃ may be alternatives to refine the activity for these compounds. Group A and B6 compounds could not be studied for their ability to reduce doxorubicin's IC₅₀ at 25 μM because they showed direct cytotoxicity at this concentration.

MDR reversal assay

In order to confirm the findings of the Rh123 accumulation assay, the ability of active compounds to reduce the IC₅₀ of doxorubicin against MES-SA/DX5 cells was evaluated as an indication of their MDR reversal activity. As illustrated in Fig. 7, compounds A3, A6, A7 and B7 induced the greatest increase in cytotoxicity of doxorubicin at 5 μM. Evidently, the position of the nitrogen atom in the pyridine ring is important for the MDR reversal effect. Generally, 2-pyridyl ester derivatives (group A) induced a significant decrease in IC₅₀ of doxorubicin at 5 μM, whereas for the 3-pyridyl methyl ester counterpart (group B), except for B7, this occurred at a higher concentration. In these experiments, the IC₅₀ value of the direct cytotoxic effect of doxorubicin in the absence of any test compound was 4.4 μM ± 0.28 (SEM).

Fig. 7. Determination of doxorubicin resistance reversal in MES-SA/DX5 cells. Reduction of doxorubicin's IC₅₀ by synthesized compounds was measured in MES-SA/DX5 cells. Cells were seeded in 96-well microplates and incubated for 24 h, and then compounds at two concentrations were added to wells and after 1.5 hours of incubation, cells were treated with doxorubicin. The plates were further incubated for 48 h, MTT assay was performed and IC₅₀ of doxorubicin was calculated in the absence or presence of synthesized compounds and percent reduction of IC₅₀ values were calculated. *The difference between the IC₅₀ value of doxorubicin in the presence of the test compound is significantly different from the IC₅₀ value of doxorubicin in the absence of the test compound (P < 0.05).

Enhancement of intracellular rhodamine 123 accumulation

Fluorescence imaging was done for some active compounds (A3, A4, A5, A7, B3 and B4) to confirm our findings quantitatively. As illustrated in Fig. 8, an increase in fluorescence in MES-SA/DX5 cells was observed in the presence of synthesized compounds in a dose-dependent manner.

Fig. 8. Fluorescence microscopy of intracellular rhodamine 123 accumulation in MES-SA/DX5 cells. Cells were seeded into 6-well plates and incubated for 48 h. Afterwards, the cells were treated with compounds (A3, A4, A5, A7, B3 and B4) at 25 μM for 20 min followed by Rh123 at the final concentration of 5 μM for another 20 min. Cells were then washed three times with ice-cold PBS, resuspended in cold PBS and visualized using a Nikon eclipse Ti-U fluorescence microscope using blue filter (510–560 nm). The control represents cells that were not exposed to any synthesized compound.

Molecular docking studies

Docking results revealed that compounds A3, A7 and B3 are well located within the active site. Binding models of these compounds and nifedipine are depicted in Fig. 9. Generally, in all three active compounds (A3, A7 and B3) a key hydrogen bond interaction could be seen between NH of the tetrahydroquinolinone ring and the carbonyl side chain of Gln441 (distance: A3, 2.07 Å; A7, 2.06 Å; B3, 1.91 Å), whereas this binding was not established by the inactive compound A12. The nitro moieties in compounds A3 and B3 were accommodated into the receptor via two H-bond interactions with Arg543 (distance: A3, 2.12 Å; B3, 2.28 Å) and Ser474 (distance: A3, 2.42 Å; B3, 2.35 Å). It appears that the binding pose of A3 and A7 (containing 2-pyridyl methyl carboxylate) is slightly different from that of compound B3 (containing 3-pyridyl methyl carboxylate) (Fig. 10). Consequently, A3 and A7 established a hydrogen bond interaction between carbonyl of 2-pyridyl methyl carboxylate moiety and the hydroxyl group of Ser474 (distance: A3, 2.12 Å; A7, 2.17 Å), but in the case of B3, a H-bond interaction was established between the nitrogen atom of the pyridine ring and the backbone NH of Gln475 (distance: 2.32 Å). The orientation of A12, an inactive compound, is completely different from that of A3, A7 and B3 (Fig. 10). Compound A12 was accommodated into the receptor via two hydrogen bonds; one hydrogen bond was observed between the hydroxyl group of A12 and the oxygen side chain of Thr911 (distance: 2.09 Å) and the other was between the carbonyl group of the cyclohexene ring and the NH side chain of Arg543 (distance; 2.08 Å). Comparative binding modes of A3, A7, B3 and the inactive compound A12 are illustrated in Fig. 10. Binding interaction and docking results are listed in Table 2.

Fig. 9. Docking result of compounds A3 (a), A7 (b), B3 (c) and A12 (d) in the active site of P-gp. Ligands are displayed as yellow sticks, while the core amino acid residues are displayed as cyan sticks.

Fig. 10. Comparative binding modes of compounds A3 (yellow), A7 (green), B3 (cyan) and A12 (orange) in the active site of P-gp. The pyridyl methyl carboxylate moiety in B3 was differently oriented in the active site compared to compounds A3 and B3, while the orientation of A12 was totally different from those of A3, A7 and B3.

Table 2. Docking results of A3, A7, A12, B3 and nifedipine into the P-gp binding site.

Compound	ΔG (kcal mol–1)	K i (nM)	H-bond interaction		Distance (Å)
			Atom of ligand	Amino acid
A3	–11.27	3.35	NH[combining low line]	Gln441	2.07
			Nitrogen (NO2)	Arg543	2.12
			Nitrogen (NO2)	Ser474	2.42
			C O (carboxylate)	Ser474	2.12
A7	–10.58	17.47	NH[combining low line]	Gln441	2.06
			C O (carboxylate)	Ser474	2.17
			Nitrogen (pyridine ring)	Asn903	1.93
B3	–10.07	44.37	NH[combining low line]	Gln441	1.91
			Nitrogen (NO2)	Arg543	2.28
			Nitrogen (NO2)	Ser474	2.35
			Nitrogen (pyridine ring)	Gln475	2.32
A12	–9.41	49.59	OH[combining low line]	Thr911	2.09
			C O (cyclohexenone ring)	Arg543	2.08
Nifedipine	–8.95	276.70	Nitrogen (NO2)	Arg547	1.97
			C O (carboxylate)	Arg543	2.16

Conclusion

In conclusion, a novel series of tetrahydroquinolinone derivatives containing pyridyl alkyl carboxylate at C₃ as inhibitors of P-gp were designed. After synthesis of the designed compounds, their biological activities were evaluated. It appears that the tetrahydroquinolinone scaffold is a suitable structure to find effective modulators of P-gp. As biological evaluation results revealed, the P-gp inhibitory potential is dependent on the substitutions at the C₃ and C₄ positions of tetrahydroquinolinone core. Consequently, derivatives bearing 2-pyridyl methyl carboxylate have superior activity over their counterparts with 2-pyridyl methyl carboxylate, and compounds containing a phenyl moiety with ortho or para electron-withdrawing substitution (such as 4-nitrophenyl, 2-nitrophenyl, 4-cyanophenyl and 4-bromophenyl moieties) at the C₄ position of the central core showed the highest P-gp inhibitory activity. The binding modes of the potent compounds A3, A7 and B3 inside the P-gp active site showed that these compounds were well accommodated in the active site and key H-bond interactions were observed between ligands and Gln441, Ser474, Arg543, Gln475 and Asn903 of the active site.

Experimental section

Chemicals

Pyridine-2-yl-methanol, pyridine-3-yl-methanol, 1,3-cyclohexandione and aldehydes were purchased from Merck, Darmstadt, Germany. Rhodamine 123, thiazolyl blue tetrazolium bromide (MTT) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one were from Sigma-Aldrich, Saint Louis, MO, USA. RPMI 1640, Dulbecco's phosphate-buffered saline and penicillin G/streptomycin were products of Biosera, Ringmer, UK. Fetal bovine serum (FBS) was from Invitrogen, San Diego, CA, USA. Doxorubicin and cisplatin were obtained from Ebewe Pharma, Unterach, Austria.

Apparatuses

Infrared spectra were obtained on a Perkin-Elmer spectrometer (KBr disk) (Perkin-Elmer, Waltham, MA). Proton nuclear magnetic resonance and carbon-13 nuclear magnetic resonance spectra were determined on a Bruker 300 spectrometer. Chemical shifts (δ) are reported as ppm and tetramethylsilane was used as internal standard. Mass spectra were recorded with an Agilent spectrometer (Agilent Technologies 9575c inert MSD, USA). Elemental analysis was done at the Microanalytical Department, Central Laboratories for Research, Shiraz University of Medical Sciences, and results were within 0.4% of the calculated value. Melting points were obtained using a hot stage apparatus (Electrothermal, Essex, UK).

Cell lines

Human uterine sarcoma cells (MES-SA) and their MDR subline MES-SA/DX5 with P-gp over-expression were obtained from Sigma-Aldrich. Cells were cultured in RPMI 1640 medium containing FBS (10%) penicillin (100 units per ml) and streptomycin (100 μg ml^–1) and were kept at 37 °C in a humidified incubator with 5% CO₂. Both cell lines were grown in monolayer cultures. MES-SA/DX5 cells were cultured in medium containing 100 nM doxorubicin.

Synthesis

Synthesis of aryl acetoacetate (2a and 2b)

A solution of pyridin-2-ylmethanol or pyridin-3-ylmethanol (20 mmol, 2.18 g) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (20 mmol, 2.84 g) was refluxed in 10 ml xylene at 150 °C for 1–3 h. The completion of the reaction was checked using thin layer chromatography (TLC). After cooling to room temperature, xylene was removed from the reaction mixture. The final pure product was afforded as a pale yellow oil by purifying the reaction mixture using silica gel column chromatography with petroleum ether–ethyl acetate as the mobile phase.27

Pyridine-2-yl-methyl-3-oxobutanoate (2a)

Yield: 89%. Yellow oil. IR (KBr): υ 1747 (C O, ester), 1720 (C O, ketone), 2973 cm^–1 (C–H aromatics).

Pyridine-3-yl-methyl-3-oxobutanoate (b

Yield: 92%. Yellow oil. IR (KBr): υ 1741 (C O, ester), 1713 (C O, ketone), 2963 cm^–1(C–H aromatics).

General procedure for the synthesis of tetrahydroquinolin-5(1H)-one derivative (A1–A14, B1–B11)

A solution of aryl acetoacetate (1 mmol, 193.2 mg), 1,3-cyclohexandione (1 mmol, 112.1 mg), ammonium acetate (5 mmol, 385.4 mg) and the corresponding aldehyde (1 mmol) was protected from light and refluxed in ethanol (7 ml) for 3–8 h. Formation of the product and completion of the reaction were confirmed by TLC. The reaction mixture was purified using TLC with chloroform–ethanol as the mobile phase (90–10%). The product was recrystallized to give the pure compounds.

Pyridin-2-ylmethyl 2-methyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A1)

Recrystallized from ethanol. Pale yellow crystals. M.p.: 171 °C. ¹H NMR (CDCl₃, 300 MHz) δ_H (ppm): 1.83–1.90 (m, 2H, cyclohexenone), 2.19–2.32 (m, 7H, cyclohexenone and DHP–CH[combining low line]_{3[combining low line]}), 5.03 and 5.20 (AB system, 2H, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 5.12 (s, 1H, DHP–C₄–H[combining low line]), 6.73 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.01–7.25 (m, 7H, phenyl, pyridine–H-5 and NH), 7.41 (td, 1H, J = 7.8, 0.75 Hz, pyridine–H-4), 8.42 (d, 1H, J = 4.5 Hz, pyridine–H-6). MS (EI), m/z (%): 375 (M⁺, 5), 297 (34), 282 (100), 238 (5), 209 (4), 188 (48), 160 (14), 133 (4), 93 (21), 65 (5). IR (KBr): υ 3276 (NH), 3081 (CH-aromatic), 2931 (CH-aliphatic), 1682, 1633 cm^–1 (CO). Found: C, 73.65; H, 5.94; N, 7.41%. Anal. calcd for C₂₃H₂₂N₂O₃: C, 73.78; H, 5.92; N, 7.48%.

Pyridin-2-ylmethyl 2-methyl-4-(3-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A2)

Recrystallized from ethyl acetate. Yellow crystals. M.p.: 169 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.79–1.94 (m, 2H, cyclohexenone), 2.20–2.25 (m, 2H, cyclohexenone), 2.36 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.50–2.52 (m, 2H, cyclohexenone, overlapped with DMSO), 5.02 and 5.16 (AB system, 2H, J_AB = 13.5 Hz, COOCH[combining low line]_{2[combining low line]}, overlapped with DHP–C₄–H[combining low line]), 5.06 (s, 1H, DHP–C₄–H[combining low line]), 7.00 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.26–7.30 (m, 1H, pyridine–H-5), 7.49 (t, 1H, J = 7.8 Hz phenyl–H-5), 7.51–7.61 (m, 1H, phenyl–H-6), 7.66 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 7.92–7.99 (m, 2H, phenyl–H-2,4), 8.47 (dd, 1H, J = 4.8, 0.9 Hz, pyridine–H-6), 9.42 (brs,1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 18.84, 21.16, 26.46, 36.60, 37.00, 66.16, 102.26, 111.03, 121.40, 121.68, 122.48, 123.28, 130.03, 134.79, 137.13, 147.57, 147.91, 149.49, 150.10, 152.23, 156.38, 166.54, 195.30. MS (EI), m/z (%): 420 (M + 1⁺, 2), 402 (9), 327 (100), 297 (35), 281 (12), 188 (51), 160 (17), 133 (5), 93 (47), 65 (7). IR (KBr): υ 3293 (NH), 3069 (CH-aromatic), 2942 (CH-aliphatic), 1705, 1643 (CO), 1521, 1344 cm^–1 (NO₂). Found: C, 65.69; H, 5.10; N, 9.09%. Anal. calcd for C₂₃H₂₁N₃O₅: C, 65.86; H, 5.05; N, 10.02%.

Pyridin-2-ylmethyl 2-methyl-4-(4-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A3)

Recrystallized from ethyl acetate. Yellow crystals. M.p.: 128 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.71–1.94 (m, 2H, cyclohexenone), 2.18–2.24 (m, 2H, cyclohexanone), 2.35 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.48–2.51 (m, 2H, cyclohexenone, overlapped with DMSO), 5.02–5.16 (m, 3H, COOCH[combining low line]_{2[combining low line]} and DHP–C₄–H[combining low line]), 7.01 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.26–7.30 (m, 1H, pyridine–H-5), 7.40 (d, 2H, J = 8.7 Hz, phenyl–H-2,6), 7.65 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.04 (d, 2H, J = 8.7 Hz, phenyl–H-3,5), 8.49 (dd, 1H, J = 4.8, 0.6 Hz, pyridine–H-6), 9.41 (brs, 1H, NH). MS (EI), m/z (%): 419 (M⁺, 2), 402 (4), 327 (100), 297 (19), 280 (9), 188 (32), 160 (11), 93 (40), 65 (5). IR (KBr): υ 3281 (NH), 3026 (CH-aromatic), 2946 (CH-aliphatic), 1693, 1649 (CO), 1505, 1341 cm^–1 (NO₂). Found: C, 65.65; H, 5.07; N, 9.97%. Anal. calcd for C₂₃H₂₁N₃O₅: C, 65.86; H, 5.05; N, 10.02%.

Pyridin-2-ylmethyl 2-methyl-4-(2-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A4)

Recrystallized from ethyl acetate. Yellow crystals. M.p.: 178 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.73–1.95 (m, 2H, cyclohexenone), 2.10–2.25 (m, 2H, cyclohexenone), 2.37 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.49–2.53 (m, 2H, cyclohexenone, overlapped with DMSO), 5.05 and 5.10 (2H, AB system, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 5.74 (s, 1H, DHP–C₄–H[combining low line]), 6.77 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.26–7.30 (m, 1H, pyridine–H-5), 7.35–7.41 (m, 1H, phenyl–H-4), 7.49–7.52 (m, 1H, phenyl–H-6), 7.61–7.71 (m, 3H, pyridine–H-4 and phenyl–H-3,5), 8.48 (d,1H, J = 4.2 Hz, pyridine–H-6), 9.36 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 18.86, 21.10, 26.55, 32.53, 36.96, 65.71, 102.70, 111.57, 120.9, 123.00, 124.06, 127.38, 131.34, 133.30, 137.02, 142.53, 147.28, 148.48, 149.27, 151.89, 156.73, 166.66, 194.86. MS (EI), m/z (%): 419 (M⁺, 2), 402 (12), 327 (100), 297 (25), 280 (10), 188 (30), 160 (10), 133 (7), 93 (36), 65 (7). IR (KBr): υ 3260 (NH), 3050 (CH-aromatic), 2939 (CH-aliphatic), 1697, 1641 (CO), 1530, 1335 cm^–1 (NO₂). Found: C, 65.99; H, 5.03; N, 9.90%. Anal. calcd for C₂₃H₂₁N₃O₅: C, 65.86; H, 5.05; N, 10.02%.

Pyridin-2-yl methyl 4-(4-cyanophenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A5)

Recrystallized from ethyl acetate. Yellow crystals. M.p.: 158 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.71–1.92 (m, 2H, cyclohexenone), 2.15–2.24 (m, 2H, cyclohexenone), 2.34 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.48–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 5.02 and 5.12 (m, 3H, COOCH[combining low line]_{2[combining low line]} and DHP–C₄–H[combining low line]), 6.97 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.27 (m,1H, pyridine–H-5), 7.33 (d, 2H, J = 8.4 Hz, phenyl–H-3,5), 7.64 (d, 2H, J = 8.4 Hz, phenyl–H-2,6), 7.69 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.50 (d, 1H, J = 4.8 Hz, pyridine–H-6), 9.39 (brs, 1H, NH). MS (EI), m/z (%): 399 (M⁺, 3), 307 (100), 297 (27), 263 (5), 234 (4), 205 (5), 188 (40), 160 (12), 133 (4), 93 (40), 65 (5). IR (KBr): υ 3282 (NH), 3023 (CH-aromatic), 2892 (CH-aliphatic), 2223 (CN), 1691, 1649 cm^–1 (CO). Found: C, 72.24; H, 5.31; N, 10.50%. Anal. calcd for C₂₄H₂₁N₃O₃: 72.16; H, 5.30; N, 10.52%.

Pyridin-2-yl methyl 4-(4-chlorophenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A6)

Recrystallized from ethanol. Yellow crystals. M.p.: 128 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.71–1.93 (m, 2H, cyclohexenone), 2.19–2.30 (m, 2H, cyclohexenone), 2.33 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.46–2.50 (m, 2H, cyclohexanone, overlapped with DMSO), 4.96 (s, 1H, DHP–C₄–H[combining low line]), 5.04 and 5.15 (AB system, 2H, J_AB = 13.5 Hz, COOCH[combining low line]_{2[combining low line]}), 6.96 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.16 (d, 2H, J = 8.4 Hz, phenyl–H-2,6), 7.23 (d, 2H, J = 8.4 Hz, phenyl–H-3,5), 7.26–7.31 (m, 1H, pyridine–H-5), 7.67 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.51 (d, 1H, J = 4.8, pyridine–H-6), 9.31 (brs, 1H, NH). MS (EI), m/z (%): 409 (M + 1⁺, 1), 318 (42), 317 (32), 316 (100), 297 (35), 272 (5), 188 (39), 160 (12), 133 (4), 93 (32), 65 (5). IR (KBr): υ 3260 (NH), 3026 (CH-aromatic), 2889 (CH-aliphatic), 1689, 1614 cm^–1 (CO). Found: C, 67.72; H, 5.17; N, 6.81%. Anal. calcd for C₂₃H₂₁ClN₂O₃: C, 67.56; H, 5.18; N, 6.85%.

Pyridin-2-yl methyl 4-(4-bromophenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A7)

Recrystallized from ethanol. Yellow crystals. M.p.: 149 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.68–1.93 (m, 2H, cyclohexenone), 2.15–2.23 (m, 2H, cyclohexenone), 2.33 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.43–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 4.94 (s, 1H, DHP–C₄–H[combining low line]), 5.04 and 5.15 (AB system, 2H, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 6.94 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.11 (d, 2H, J = 8.4 Hz, phenyl–H-2,6), 7.27–7.31 (m, 1H, pyridine–H-5), 7.36 (d, 2H, J = 8.4 Hz, phenyl–H-3,5), 7.67 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.50 (dd, 1H, J = 4.8, 0.6 Hz, pyridine–H-6), 9.31 (brs, 1H, NH). MS (EI), m/z (%): 453 (M⁺, 1), 362 (98), 361 (20), 360 (100), 316 (5), 297 (36), 281 (4), 237 (4), 188 (57), 160 (21), 133 (6), 93 (61), 65 (11). IR (KBr): υ 3281 (NH), 3027 (CH-aromatic), 2935 (CH-aliphatic), 1689, 1614 cm^–1 (CO). Found: C, 70.09; H, 4.68; N, 6.21%. Anal. calcd for C₂₃H₂₁BrN₂O₃: C, 60.94; H, 4.67; N, 6.18%.

Pyridin-2-ylmethyl 4-(4-methoxyphenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A8)

Recrystallized from ethanol. Yellow crystals. M.p.: 128 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.68–1.93 (m, 2H, cyclohexenone), 2.18–2.22 (m, 2H, cyclohexenone), 2.32 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.42–2.50 (m, 2H, cyclohexenone overlapped with DMSO), 3.68 (s, 3H, phenyl–OCH[combining low line]_{3[combining low line]}), 4.92 (s, 1H, DHP–C₄–H[combining low line]), 5.05 and 5.15 (AB system, 2H J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 6.73 (d, 2H, J = 8.4 Hz, phenyl–H-3,5), 6.95 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.06 (d, 2H, J = 8.7 Hz, phenyl–H-2,6), 7.26–7.30 (m, 1H, pyridine–H-5), 7.66 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.51 (d, 1H, J = 4.2 Hz, pyridine–H-6), 9.21 (brs, 1H, NH). MS (EI), m/z (%): 404 (M⁺, 4), 312 (100), 297 (19), 268 (7), 239 (5), 224 (4), 188 (28), 160 (10), 133 (4), 93 (14), 65 (5). IR (KBr): υ 3283 (NH), 3075 (CH-aromatic), 2949 (CH-aliphatic), 1704, 1645 (CO), 1029, 1250 cm^–1 (COC). Found: C, 71.39; H, 5.97; N, 6.91%. Anal. calcd for C₂₄H₂₄N₂O₄: 71.27; H, 5.98; N, 6.93%.

Pyridin-2-ylmethyl 4-(furan-2-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A9)

Recrystallized from ethyl acetate. Pale brown crystals. M.p.: 148 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.76–1.97 (m, 2H, cyclohexenone), 2.23–2.27 (m, 2H, cyclohexenone), 2.30 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.42–2.50 (m, 2H, cyclohexenone overlapped with DMSO), 5.10–5.25 (m, 3H, COOCH[combining low line]_{2[combining low line]} and DHP–C₄–H[combining low line]), 5.84 (d, 1H, J = 3.00 Hz, furyl–H-3), 6.23–6.25 (m, 1H, furyl–H-4), 7.22 (d, 1H, J = 7.8 Hz, pyridine–H-3), 7.29–7.33 (m, 1H, pyridine–H-5), 7.39 (m, 1H, furyl–H-5), 7.77 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.52 (dd, 1H, J = 5.1, 0.9 Hz, pyridine–H-6), 9.36 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 18.76, 21.26, 26.53, 29.90, 37.09, 66.02, 100.30, 104.77, 108.48, 110.66, 121.42, 123.22, 137.32, 141.44, 147.36, 149.45, 152.55, 156.86, 158.82, 166.78, 195.01. MS (EI), m/z (%): 364 (M⁺, 2), 297 (4), 272 (100), 244 (12), 228 (18), 188 (11), 160 (7), 143 (7), 93 (23), 65 (8). IR (KBr): υ 3282 (NH), 3072 (CH-aromatic), 2939 (CH-aliphatic), 1699, 1647 cm^–1 (CO). Found: C, 69.01; H, 5.51; N, 7.68%. Anal. calcd for C₂₁H₂0N₂O₄: 69.22; H, 5.53; N, 7.69%.

Pyridin-2-yl methyl 2-methyl-5-oxo-4-(thiophen-2-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A10)

Recrystallized from ethyl acetate. Orange crystals. M.p.: 150 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.83–2.04 (m, 2H, cyclohexenone), 2.30–2.34 (m, 2H, cyclohexenone), 2.34 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.53–2.56 (m, 2H, cyclohexenone overlapped with DMSO), 5.20 and 5.27 (AB system, 2H, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 5.32 (s, 1H, DHP–C₄–H[combining low line]), 5.73 (d, 1H, J = 3.30 Hz, thienyl–H-3), 6.87–6.90 (m, 1H, thienyl–H-4), 7.17 (d, J = 7.8 Hz, pyridine–H-3), 7.24 (dd, 1H, J = 5.1, 1.2 Hz, thienyl–H-5), 7.33–7.37 (m, 1H, pyridine–H-5), 7.78 (td, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.57 (d, 1H, J = 4.5 Hz, pyridine–H-6), 9.48 (brs, 1H, NH). MS (EI), m/z (%): 381 (M + 1⁺, 2), 297 (5), 288 (100), 270 (5), 244 (16), 215 (5), 188 (19), 160 (9), 133 (4), 93 (34), 65 (7). IR (KBr): υ 3282 (NH), 3084 (CH-aromatic), 2953 (CH-aliphatic), 1683, 1634 cm^–1 (CO). Found: C, 66.51; H, 5.28; N, 7.33%. Anal. calcd for C₂₁H₂₀N₂O₃S: C, 66.29; H, 5.30; N, 7.36%.

Pyridin-2-ylmethyl 2-methyl-5-oxo-4-propyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A11)

Recrystallized from ethyl acetate. Pale yellow crystals. M.p.: 134 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 0.74 (t, 3H, J: 6.9 Hz, CH₂–CH₂–C[combining low line]H[combining low line]_{3[combining low line]}), 1.01–1.23 (m, 4H, C[combining low line]H[combining low line]_{2[combining low line]}–[combining low line]CH[combining low line]_{2[combining low line]}–CH₃), 1.74–1.94 (m, 2H, cyclohexenone), 2.12–2.29 (m, 5H, cyclohexenone and DHP–CH[combining low line]_{3[combining low line]}), 2.38–2.42 (m, 2H, cyclohexenone), 3.88 (1H, t, J = 5.4 Hz, DHP–C₄–H[combining low line]), 5.14 and 5.21 (2H, AB system, J_AB = 13.5 Hz, COOCH[combining low line]_{2[combining low line]}), 7.30–7.36 (2H, m, pyridine–H-3,5), 7.82 (1H, td, J = 7.8, 1.8 Hz, pyridine–H-4), 8.53 (1H, d, J = 4.2 Hz, pyridine–H-6), 9.07 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 14.77, 17.93, 18.71, 21.42, 26.54, 29.46, 37.30, 39.23, 65.96, 102.71, 110.86, 121.75, 123.28, 137.34, 146.93, 149.54, 152.57, 156.90, 167.29, 195.56. MS (EI), m/z (%): 340 (M⁺, 4), 297 (100), 248 (16), 206 (5), 188 (72), 160 (17), 133 (4), 105 (9), 93 (4), 65 (4). IR (KBr): υ 3285 (NH), 3075 (CH-aromatic), 2947 (CH-aliphatic), 1698 cm^–1 (CO). Found: C, 70.37; H, 7.14; N, 8.26%. Anal. calcd for C₂₀H₂₄N₂O₃: C, 70.56; H, 7.11; N, 8.23%.

Pyridin-2-ylmethyl 4-(4-hydroxyphenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A12)

Recrystallized from ethyl acetate. Pale yellow crystals. M.p.: 235 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.75–1.98 (m, 2H, cyclohexenone), 2.23–2.28(m, 2H, cyclohexenone), 2.36 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.48–2.56 (m, 2H, cyclohexenone, overlapped with DMSO), 4.93 (s, 1H, DHP–C₄–H[combining low line]), 5.10 and 5.20 (AB system, 2H, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 6.62 (d, 2H, J = 8.4 Hz, phenyl–H-3,5), 6.98–7.03 (m, 3H, pyridine–H-3 and phenyl–H-2,6), 7.32–7.36 (m, 1H, pyridine–H-5), 7.73 (td, 1H, J = 7.5, 1.5 Hz, pyridine–H-4), 8.56 (d,1H, J = 4.5 Hz, pyridine–H-6), 9.17 (brs, 1H, NH), 9.23 (brs, 1H, NH). MS (EI), m/z (%): 390 (M⁺, 2), 298 (100), 254 (5), 225 (5), 188 (18), 188 (30), 160 (7), 93 (16), 65 (11), 39 (4). IR (KBr): υ 3286 (NH), 3082 (CH-aromatic), 2951 (CH-aliphatic), 1683, 1632 cm^–1 (CO). Found: C, 70.97; H, 5.69; N, 7.14%. Anal. calcd for C₂₃H₂₂N₂O₄: C, 70.75; H, 5.68; N, 7.17%.

Pyridin-2-ylmethyl 4-(3-hydroxyphenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A13)

Recrystallized from ethyl acetate. Pale yellow crystals. M.p.: 245 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.73–1.93 (m, 2H, cyclohexenone), 2.19–2.22 (m, 2H, cyclohexenone), 2.32 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.43–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 4.93 (s, 1H, DHP–C₄–H[combining low line]), 5.06 and 5.16 (AB system, 2H, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 6.49 (d, 1H, J = 7.5 Hz, phenyl–H-4), 6.60–6.62 (m, 2H, phenyl–H-2,6), 6.93–6.99 (m,2H, pyridine–H-3 and phenyl–H-5), 7.25–7.30 (m,1H, pyridine–H-5), 7.66 (t, 1H, J = 7.5 Hz, pyridine–H-4), 8.49–8.51 (m, 1H, pyridine–H-6), 9.14 (brs, 1H, NH), 9.24 (brs, 1H, OH). MS (EI), m/z (%): 390 (M⁺, 6), 298 (100), 254 (6), 225 (4), 206 (4), 188 (30), 160 (30), 133 (3), 93 (14), 65 (5). Found: C, 70.61; H, 5.69; N, 7.15%. Anal. calcd for C₂₃H₂₂N₂O₄: C, 70.75; H, 5.68; N, 7.17%

Pyridin-2-ylmethyl 2-methyl-5-oxo-4-(3,4,5-trimethoxyphenyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (A14)

Recrystallized from ethyl acetate. Pale yellow crystals. M.p.: 174 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.77–194 (m, 2H, cyclohexenone), 2.21–2.25 (m, 2H, cyclohexenone), 2.32 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.46–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 3.59 (s, 3H, phenyl–4-OCH[combining low line]_{3[combining low line]}), 3.61 (s, 6H, phenyl–3,5-OCH[combining low line]_{3[combining low line]}), 4.95 (s, 1H, DHP–C₄–H[combining low line]), 5.10 and 5.18 (AB system, 2H, J_AB = 13.8 Hz, COOCH[combining low line]_{2[combining low line]}), 6.42 (s, 2H, phenyl–H-2,6), 7.07 (d, 1H, J = 8.1 Hz, pyridine–H-3), 7.28–7.32 (m, 1H, pyridine–H-5), 7.67–7.72 (m, 1H, pyridine–H-4), 8.51–8.53 (d, 1H, J = 4.5 Hz, pyridine–H-6), 9.12 (brs, 1H, NH). MS (EI), m/z (%): 464 (M⁺, 2), 433 (4), 372 (100), 328 (11), 297 (36), 188 (35), 160 (13), 133 (4), 93 (14), 65 (5). IR (KBr): υ 3276(NH), 3073 (CH-aromatic), 2935 (CH-aliphatic), 1702, 1647 (CO), 1070, 1230 cm^–1 (COC). Found: C, 67.42; H, 6.05; N, 6.00%. Anal. calcd for C₂₆H₂₈N₂O₆: C, 67.23; H, 6.08; N, 6.03%.

Pyridin-3-ylmethyl 2-methyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B1)

Recrystallized from ethyl acetate. Pale yellow crystals. M.p.: 211 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.70–1.91 (m, 2H, cyclohexenone), 2.17–2.22 (m, 2H, cyclohexenone), 2.30 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.46–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 4.91 (s, 1H, DHP–C₄–H[combining low line]), 5.02 and 5.11 (AB system, 2H, J_AB = 12.9 Hz, COOCH[combining low line]_{2[combining low line]}), 7.05–7.18 (m, 5H, phenyl–H-2,3,4,5,6), 7.30–7.34 (m, 1H, pyridine–H-5), 7.52 (d, 1H, J = 7.5 Hz, pyridine–H-4), 8.44 (s, 1H, pyridine–H-2), 8.49 (d, 1H, J = 4.2 Hz, pyridine–H-6), 9.24 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 18.76, 21.20, 26.48, 36.02, 37.15, 62.97, 103.13, 111.77, 123.89, 126.21, 127.87, 127.94, 128.21 128.32, 132.76, 136.04, 146.55, 148.05, 149.42, 149.49, 151.58, 167.02, 195.19. MS (EI), m/z (%): 374 (M⁺, 4), 297 (100), 282 (30), 283 (6), 216 (14), 180 (4), 161 (7), 133 (4), 92 (9), 65 (6). IR (KBr): υ 3202 (NH), 3060 (CH-aromatic), 2951 (CH-aliphatic), 1667, 1634 cm^–1 (CO). Found: C, 73.53; H, 5.94; N, 7.51%. Anal. calcd for C₂₃H₂₂N₂O₃: C, 73.78; H, 5.92; N, 7.48%.

Pyridin-3-yl methyl 4-(3-hydroxyhenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B2)

Recrystallized from ethyl acetate and ethanol. Pale yellow crystals. M.p.: 214 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.66–1.93 (m, 2H, cyclohexenone), 2.18–2.20 (m, 2H, cyclohexenone) 2.30 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.44–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 4.87 (s, 1H, DHP–C₄–H[combining low line]), 5.04 and 5.12 (AB system, 2H, J_AB = 12.9 Hz, COOCH[combining low line]_{2[combining low line]}), 6.46–6.49 (m, 1H, phenyl–H-4), 6.54–6.57 (m, 2H, phenyl–H-2,6), 6.93 (t, 1H, J = 7.5 Hz, phenyl–H-5), 7.29–7.34 (m, 1H, pyridine–H-5), 7.53 (dt, 1H, J = 7.8, 1.5 Hz, pyridine–H-4), 8.46 (d, 1H, J = 1.5 Hz, pyridine–H-2), 8.49 (dd, 1H, J = 4.8, 1.5 Hz, pyridine–H-6), 9.13 (brs, 1H, NH), 9.21 (brs, 1H, OH). MS (EI), m/z (%): 390 (M⁺, 11), 312 (14), 297 (100), 254 (4), 216 (4), 161 (7), 133 (4), 93 (18). IR (KBr): υ 3276 (NH), 3072 (CH-aromatic), 2938 (CH-aliphatic), 1699, 1645 cm^–1 (CO). Found: C, 70.57; H, 5.64; N, 7.19%. Anal. calcd for C₂₃H₂₂N₂O₄: C, 70.75; H, 5.68; N, 7.17%.

Pyridin-3-ylmethyl 2-methyl-4-(4-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B3)

Recrystallized from ethyl acetate. Yellow crystals. M.p.: 194 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.70–1.93 (m, 2H, cyclohexenone), 2.16–2.23 (m, 2H, cyclohexenone), 2.33 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.46–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 4.98–5.13 (m, 3H, COOCH[combining low line]_{2[combining low line]} and DHP–C₄–H[combining low line]), 7.29–7.33 (m, 1H, pyridine–H-5), 7.35 (d, 2H, J = 9.0 Hz, phenyl–H-2,6), 7.55 (d, 2H, J = 7.8 Hz, pyridine–H-4), 8.02 (d, 2H, J = 9.0 Hz, phenyl–H-3,5), 8.40 (s, 1H, pyridine–H-2), 8.48 (d, 1H, J = 4.5 Hz, pyridine–H-6), 9.40 (brs, 1H, NH). MS (EI), m/z (%): 419 (M⁺, 9), 402 (14), 327 (38), 297 (100), 205 (9), 161 (11), 133 (5), 92 (16), 65 (7). IR (KBr): υ 3209 (NH), 3076 (CH-aromatic), 2948 (CH-aliphatic), 1667, 1645 (CO), 1496, 1324 cm^–1 (NO₂). Found: C, 65.70; H, 5.06; N, 10.06%. Anal. calcd for C₂₃H₂₁N₃O₅: C, 65.86; H, 5.05; N, 10.02%.

Pyridin-3-yl methyl 2-methyl-4-(2-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B4)

Recrystallized from ethyl acetate. Yellow crystals. M.p.: 158 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.62–1.87 (m, 2H, cyclohexenone), 2.10–2.17 (m, 2H, cyclohexenone), 2.30 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.42–2.45 (m, 2H, cyclohexenone), 4.94–5.03 (2H, m, COOCH[combining low line]_{2[combining low line]}), 5.63 (s, 1H, DHP–C₄–H[combining low line]), 7.22–7.41 (m, 4H, pyridine–H-4 and phenyl–H-4,5,6), 7.54 (t, 1H, J = 7.2 Hz, pyridine–H-5), 7.62 (d, 1H, J = 7.8 Hz, phenyl–H-3), 8.28 (s, 1H, pyridine–H-2), 8.43 (d, 1H, J = 3.6 Hz, pyridine–H-6), 9.33 (brs, 1H, NH). MS (EI), m/z (%): 419 (M⁺, 6), 402 (9), 327 (26), 297 (100), 161 (18), 133 (7), 92 (12), 65 (4). IR (KBr): υ 3207 (NH), 3077 (CH-aromatic), 2940 (CH-aliphatic), 1698, 1635 (CO), 1528, 1380 cm^–1 (NO₂). Found: C, 65.59; H, 5.08; N, 9.97%. Anal. calcd for C₂₃H₂₁N₃O₅: C, 65.86; H, 5.05; N, 10.00%.

Pyridin-3-yl methyl 4-(4-cyanophenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B5)

Recrystallized from ethanol. Yellow crystals. M.p.: 208 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.70–1.92 (m, 2H, cyclohexenone), 2.16–2.22 (m, 2H, cyclohexenone), 2.32 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.45–2.50 (m, 2H, cyclohexenone, overlapped with DMSO) 4.95 (s, 1H, DHP–C₄–H[combining low line]), 5.1 and 5.00 (AB system, 2H, J_AB = 12.9 Hz, COOCH[combining low line]_{2[combining low line]}), 7.28 (d, 2H, J = 8.4 Hz, phenyl–H-3,5), 7.31–7.35 (m, 1H, pyridine–H-5), 7.53 (dt, 1H, J = 6.00, 1.8 Hz, pyridine–H-4), 7.61 (d, 2H, J = 8.4 Hz, phenyl–H-2,6), 8.40 (d, 1H, J = 1.5 Hz, pyridine–H-2), 8.50 (dd, 1H, J = 4.5, 1.5 Hz, pyridine–H-6), 9.36 (brs, 1H, NH). MS (EI), m/z (%): 399 (M⁺, 7), 307 (55), 297 (100), 263 (9), 234 (4), 205 (11), 161 (11), 133 (5), 92 (16), 65 (7). IR (KBr): υ 3208 (NH), 3069 (CH-aromatic), 2954 (CH-aliphatic), 2228 (CN), 1668, 1643 cm^–1 (CO). Found: C, 72.01; H, 5.30; N, 10.51%. Anal. calcd for C₂₄H₂₁N₃O₃: 72.16; H, 5.30; N, 10.52%.

Pyridin-3-yl methyl 4-(4-chlorophenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B6)

Recrystallized from ethanol. Pale yellow crystals. M.p.: 217 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.70–1.92 (m, 2H, cyclohexenone), 2.14–2.22 (m, 2H, cyclohexenone), 2.31 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.41–2.47 (m, 2H, cyclohexenone, overlapped with DMSO), 4.89 (s, 1H, DHP–C₄–H[combining low line]), 5.02 and 5.10 (AB system, 2H, J_AB = 12.6 Hz, COOCH[combining low line]_{2[combining low line]}), 7.10 (d, 2H, J = 8.7 Hz, phenyl–H-2,6), 7.20 (d, 2H, J = 8.7 Hz, phenyl–H-3,5), 7.31–7.35 (m, 1H, pyridine–H-5), 7.53 (dt, 1H, J = 8.1, 1.8 Hz, pyridine–H-4), 8.44 (d, 1H, J = 1.5 Hz, pyridine–H-2), 8.50 (dd, 1H, J = 4.5, 1.5 Hz, pyridine–H-6), 9.29 (brs, 1H, NH). MS (EI), m/z (%): 408 (M⁺, 4), 318 (12), 317 (7), 316 (34), 297 (100), 272 (7), 205 (4), 161 (7), 133 (4), 92 (12), 65 (4). IR (KBr): υ 3208 (NH), 3079 (CH-aromatic), 2954 (CH-aliphatic), 1664, 1649 cm^–1 (CO). Found: C, 67.40; H, 5.19; N, 6.87%. Anal. calcd for C₂₃H₂₁ClN₂O₃: C, 67.56; H, 5.18; N, 6.85%.

Pyridin-3-yl methyl 4-(4-bromophenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B7)

Recrystallized from ethanol. Pale yellow crystals. M.p.: 233 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.72–1.91 (m, 2H, cyclohexenone), 2.14–2.21 (m, 2H, cyclohexenone), 2.30 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.44–2.50 (m, 2H, cyclohexenone overlapped with DMSO), 4.87 (s, 1H, DHP–C₄–H[combining low line]), 5.02 and 5.10 (AB system, 2H, J_AB = 12.6 Hz, COOCH[combining low line]_{2[combining low line]}), 7.05 (d, 2H, J = 8.4 Hz, phenyl–H-2,6), 7.29–7.35 (m, 3H, pyridine–H-5 and phenyl–H-3,5), 7.53 (dt, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.45 (d, 1H, J = 1.8 Hz, pyridine–H-2), 8.50 (dd, 1H, J = 4.8, 1.5 Hz, pyridine–H-6), 9.28 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_H (ppm): 18.78, 21.16, 26.45, 35.88, 37.08, 63.02, 102.62, 111.37, 119.26, 123.87, 130.17, 131.17, 132.69, 136.10, 146.88, 147.39, 149.48, 149.56, 151.73, 166.82, 195.19. MS (EI), m/z (%): 452 (M⁺, 3), 362 (18), 361 (3), 360 (20), 297 (100), 237 (3), 205 (5), 180, 161 (7), 133 (4), 92 (11), 65 (5). IR (KBr): υ 3207 (NH), 3079 (CH-aromatic), 2952 (CH-aliphatic), 1667, 1634 cm^–1 (CO). Found: C, 70.21; H, 4.68; N, 6.16%. Anal. calcd for C₂₃H₂₁BrN₂O₃: C, 60.94; H, 4.67; N, 6.18%.

Pyridin-3-yl methyl 4-(4-methoxyphenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B8)

Recrystallized from ethyl acetate and ethanol. Yellow crystals. M.p.: 212 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.68–1.90 (m, 2H, cyclohexenone), 2.12–2.22 (m, 2H, cyclohexenone), 2.32 (3H, s, DHP–CH[combining low line]_{3[combining low line]}), 2.44–2.50 (m, 2H, cyclohexenone, overlapped with DMSO), 3.67 (s, 3H, phenyl–OCH₃), 4.84 (s, 1H, DHP–C₄–H[combining low line]), 5.02 and 5.10 (AB system, 2H, J_AB = 12.6 Hz, COOCH[combining low line]_{2[combining low line]}), 6.70 (d, 2H, J = 8.7 Hz, phenyl–H-3,5), 7.00 (d, 2H, J = 8.7 Hz, phenyl–H-2,6), 7.30–7.35 (m, 1H, pyridine–H-5), 7.54 (d, 1H, J = 8.1 Hz, pyridine–H-4), 8.46 (d, 1H, J = 1.5 Hz, pyridine–H-2), 8.49 (dd, 1H, J = 4.5, 1.5 Hz, pyridine–H-6), 9.28 (brs, 1H, NH). MS (EI), m/z (%): 404 (M⁺, 7), 312 (98), 297 (100), 268 (14), 205 (5), 161 (10), 133 (4), 92 (18), 65 (9). IR (KBr): υ 3205 (NH), 3077 (CH-aromatic), 2956 (CH-aliphatic), 1668, 1634 (CO), 1029, 1250 cm^–1 (COC). Found: C, 71.51; H, 5.95; N, 6.90%. Anal. calcd for C₂₄H₂₄N₂O₄: 71.27; H, 5.98; N, 6.93%.

Pyridin-3-yl methyl 4-(furan-2-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B9)

Recrystallized from ethyl acetate. Light brown crystals. M.p.: 159 °C. ¹H NMR (CDCl₃, 300 MHz) δ_H (ppm): 1.94–2.06 (m, 2H, cyclohexenone), 2.27–2.47 (m, 7H, cyclohexanone, and DHP–CH[combining low line]_{3[combining low line]}), 5.08 and 5.26 (AB system, 2H, J_AB = 12.9 Hz, COOCH[combining low line]_{2[combining low line]}), 5.30 (s, 1H, DHP–C₄–H[combining low line]), 5.81 (d, 1H, J = 3 Hz, furyl–H-3), 6.20 (t, 1H, J = 1.8 Hz, furyl–H-4), 6.84 (s, 1H, DHP–NH[combining low line]), 7.18 (1H, d, J = 0.6 Hz, furyl–H-5), 7.24–7.26 (m, 1H, pyridine–H-5), 7.60 (d, 1H, J = 7.5 Hz, pyridine–H-4), 8.54 (m, 2H, pyridine–H-2,6). MS (EI), m/z (%): 364 (M⁺, 3), 308 (6), 272 (100), 244 (7), 228 (24), 161 (6), 133 (2), 92 (10), 65 (5). IR (KBr): υ 3277 (NH), 3077 (CH-aromatic), 2943 (CH-aliphatic), 1674, 1631 cm^–1 (CO). Found: C, 68.95; H, 5.51; N, 7.70%. Anal. calcd for C₂₁H₂0N₂O₄: 69.22; H, 5.53; N, 7.69%.

Pyridin-3-yl methyl 2-methyl-5-oxo-4-(thiophen-2-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B10)

Recrystallized from ethyl acetate. White crystals. M.p.: 172 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 1.83–2.01 (m, 2H, cyclohexenone), 2.23–2.33 (m, 2H, cyclohexenone), 2.35 (s, 3H, DHP–CH[combining low line]_{3[combining low line]}), 2.52–2.55 (m, 2H, cyclohexenone, overlapped with DMSO), 5.15–5.26 (3H, m, COOCH[combining low line]_{2[combining low line]} and DHP–C₄–H[combining low line]), 5.26 (s, 1H, DHP–C₄–H[combining low line]), 6.67 (d, 1H, J = 3.3 Hz, thienyl–H-3), 6.85–6.88 (m, 1H, thienyl–H-4), 7.23 (dd, 1H, J = 5.1, 1.2 Hz, thienyl–H-5), 7.39–7.43 (m, 1H, pyridine–H-5), 7.69 (dt, 1H, J = 7.8, 1.5 Hz, pyridine–H-4), 8.56–8.57 (m, 2H, pyridine–H-2,6), 9.45 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 18.75, 21.27, 26.42, 30.88, 37.09, 63.12, 102.73, 111.22, 123.11, 123.95, 126.91, 132.79, 136.06, 146.97, 149.47, 149.50, 151.79, 151.91, 152.03, 166.82, 195.09. MS (EI), m/z (%): 380 (M⁺, 7), 297 (29), 288 (100), 270 (3), 244 (36), 215 (4), 188 (5), 161 (9), 133 (4), 92 (12), 65 (7). IR (KBr): υ 3200 (NH), 3066 (CH-aromatic), 2947 (CH-aliphatic), 1667, 1635 cm^–1 (CO). Found: C, 66.09; H, 5.30; N, 7.35%. Anal. calcd for C₂₁H₂₀N₂O₃S: C, 66.29; H, 5.30; N, 7.36%.

Pyridin-3-yl methyl 2-methyl-5-oxo-4-propyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (B11)

Recrystallized from ethyl acetate. Pale yellow crystals. M.p.: 105 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ_H (ppm): 0.71 (t, 3H, J = 6.9 Hz, CH₂–CH₂–C[combining low line]H[combining low line]_{3[combining low line]}), 1.00–1.14 (m, 4H, C[combining low line]H[combining low line]_{2[combining low line]}–[combining low line]CH₂–CH₃), 1.77–1.92 (2H, m, cyclohexenone), 2.11–2.22 (m, 5H, cyclohexenone and DHP–CH[combining low line]_{3[combining low line]}), 2.37–2.43 (m, 2H, cyclohexenone), 3.83 (t, 1H, J = 5.4 Hz, DHP–C₄–H[combining low line]), 5.10 and 5.20 (AB system, 2H, J_AB = 12.9 Hz, COOCH[combining low line]_{2[combining low line]}), 7.40–7.44 (m, 1H, pyridine–H-5), 7.78 (dt, 1H, J = 7.8, 1.8 Hz, pyridine–H-4), 8.53 (dd, 1H, J = 4.8, 1.5 Hz, pyridine–H-6), 8.59 (d, 1H, J = 1.5 Hz, pyridine–H-2), 9.02 (brs, 1H, NH). ¹³C NMR (DMSO-d₆, 75 MHz) δ_C (ppm): 14.70, 17.94, 18.69, 21.40, 26.52, 29.39, 37.27, 39.27, 62.93, 102.65, 110.87, 124.02, 132.97, 136.30, 146.90, 149.54, 149.68, 152.51, 167.38, 195.54. MS (EI), m/z (%): 341 (M + 1⁺, 3), 297 (100), 205 (14), 161 (19), 133 (9), 105 (11), 92 (18), 65 (17). IR (KBr): υ 3289 (NH), 3029 (CH-aromatic), 2937 (CH-aliphatic), 1698, 1649 cm^–1 (CO). Found: C, 70.39; H, 7.12; N, 8.26%. Anal. calcd for C₂₀H₂₄N₂O₃) require C, 70.56; H, 7.11; N, 8.23%.

Drug-likeness calculation

We have calculated the drug-likeness properties of all compounds using DruLiTo 1 software. The compounds were input to the software interface in SDF format to compute the properties.

Biological evaluation

Flow cytometric analysis of rhodamine 123 accumulation

MES-SA and MES-SA/DX5 cells were suspended in serum-free RPMI at 5 × 10⁵ cells per ml. Cells were treated with the synthesized compounds or verapamil as a positive control and incubated for 20 min at 37 °C. For MES-SA/DX-5 cells, doxorubicin was removed from the medium one day before the experiment. Then 5 μM rhodamine 123 (Rh123) was added to each tube. After incubation for another 20 min at 37 °C, cells were centrifuged and washed twice with ice-cold PBS. Cells were resuspended in 500 μL cold PBS and kept on ice before analysis using a FACSCalibur flow cytometer (Becton Dickinson, USA). The fluorescence emission caused by the presence of Rh123 in cells was detected at 488 nm excitation and 530 nm emission. The geometric mean (Gmean) of fluorescence intensity values was determined using WINMDI 2.9 (TSRI, USA).

MDR reversal assay

MES-SA/DX5 cells were seeded into 96-well plates at a density of 5 × 10⁴ and incubated for 24 h at 37 °C. Afterwards, the synthesized compounds were added to each well (in triplicate) at two different concentrations, with negligible cytotoxic activity (cell viability >85%). Cells were incubated for 90 min, and then three different concentrations of doxorubicin were added. After incubation for another 48 h at 37 °C, cell viability was estimated by MTT reduction assay: 80 μl of medium was removed and 80 μl MTT solution was added at a final concentration of 0.5 mg ml^–1. Plates were incubated for 4 h at 37 °C to allow the formazan crystals to be formed and then 200 μl DMSO was added to each well to dissolve the crystals. Absorbance was determined at 570 nm with background correction at 655 nm using a Bio-Rad microplate reader (Model 680). The percentage of inhibition of viability compared to control was assessed for each concentration of compound and IC₅₀ values were computed using CurveExpert software version 1.34 for Windows.

Fluorescence microscopy of cellular rhodamine 123 uptake

MES-SA/DX5 cells were seeded at a density of 2 × 10⁵ cells per ml in 6-well plates and incubated for two days at 37 °C. Drugs were added to each well and incubated for 20 min and then cells were treated with Rh123 at a final concentration of 5 μM and incubated for another 20 min. After incubation, cells were washed three times with ice-cold PBS, resuspended in cold PBS and visualized with a Nikon eclipse Ti-U fluorescence microscope using blue filter (510–560 nm).

Molecular docking analysis

In order to obtain a clear idea about the binding modes of the studied tetrahydroquinolinones in the DHP binding site of P-gp, docking was performed for compounds A3, A7, A12 and B3 by using AutoDock 4.2 software. Since the crystal structure of P-gp is not available, we applied the 3D structure of human P-gp, which has been previously provided by our research group, by performing homology modeling and MD simulation on the X-ray structure of Apo murine P-gp (PDB entry: ; 3G5U).29,30

The structures were sketched and minimized using molecular mechanics (MM⁺) and then semi-empirical methods. All the aforementioned procedures were carried out using HYPERCHEM 7.0 software. AutoDock Tools 4.2 was used to prepare ligands and protein for the docking process. The grid box dimensions were set to 60 × 60 × 60 with 0.375 Å grid spacing and the grids' center was placed at residue Arg905 (x = 93.12, y = 68.17, z = 124.91), involved in the substrate binding site.31,32 The Lamarckian genetic search algorithm was applied, and the number of GA runs was set to 80. The other parameters were left at program default values.

Conflicts of interest

The authors declare no competing interests.