Synthesis and Biological Evaluation of 2-Substituted Quinoline 6-Carboxamides as Potential mGluR1 Antagonists for the Treatment of Neuropathic Pain

Abstract

A series of 2-amino and 2-methoxy quinoline-6-carboxamide derivatives have been synthesized and their metabotropic glutamate receptor type 1 (mGluR1) antagonistic activities were evaluated in a functional cell-based assay. The compound 13c showed the highest potency with IC₅₀ value of 2.16 µm against mGluR1. Finally, in vivo evaluation of 13c in the rat spinal nerve ligation (SNL) model exhibited weak analgesic effects with regard to both mechanical allodynia and cold allodynia.

Glutamate is the excitatory neurotransmitter in the central nerve system (CNS). The signaling action of glutamate is regulated through glutamate receptors, which contribute to the glutamate-mediated postsynaptic excitation of neural cells. Thus, they play an important role in neural communication, memory formation, learning, and regulation.¹⁾

There are two kinds of glutamate receptors, ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) according to their activation mechanism triggering a postsynaptic current. iGluRs rapidly form the ion channel pore upon binding to glutamate, whereas mGluRs indirectly activate ion channels on the plasma membrane via a G-protein signaling pathway. mGluRs belong to family C G protein-coupled receptors (GPCRs) characterized by a large extracellular domain that contains the glutamate binding site. There are eight different types of mGluRs, labeled mGluR1 to mGluR8, divided into three groups based on their sequence similarity, signal transduction and pharmacology.²⁾

mGluR1, one of the receptors in Group I, is predominantly localized in the postsynaptic region and widely distributed in the CNS including hippocampus, cerebellum, thalamic nuclei and spinal cord. Stimulating the mGluR1 causes phospholipase C (PLC) to hydrolyze phosphoinositide into inositol triphosphate (IP3), which in turn binds to the IP3 receptor on the endoplasmic reticulum, elevating intracellular calcium levels.^3,4)

According to the previous studies, mGluR1 is involved in various CNS related disorders such as epilepsy,^5,6) anxiety,^7–9) stroke,¹⁰⁾ psychotic disorder^11–13) and pain.^14–21) In particular, the role of glutamate and mGluR1 in pain states has been well studied. Glutamate, released from primary afferent neurons, may be responsible for persistent activation of spinal neurons and hypersensitivity to painful stimuli.^22,23) Pharmacological studies have shown that the intrathecal administration of mGluR1 agonists can induce spontaneous pain behavior which can be reduced by the effect of mGluR1 antagonists.^24,25) Furthermore, treatment of neuropathic pain model (by constriction injury of the sciatic nerve) with selective mGluR1 antibodies significantly attenuated cold hyperalgesia and mechanical allodynia.²⁶⁾ These evidences have supported a role for mGluR1 in neuropathic pain states.

To date, several mGlu1 negative allosteric modulators (NAMs) have been discovered and proved to be effective in vivo experiments.²⁷⁾ For examples, A-841720 (1), a potent and mGluR1 antagonist, achieved excellent efficacy at 100 µmol/kg (ED₅₀=22 µmol/kg, intraperitoneally (i.p.)) in the spinal nerve ligation (SNL) model of neuropathic pain.²⁸⁾ JNJ16259685 (2) exhibited an excellent mGluR1 antagonistic activity with IC₅₀ value of 1.21 nm, and its in vivo efficacy has been reported in the rat formalin.²⁹⁾ In addition, morphine-like analgesic effects were observed with compound 3 in an elecrtomyogpraph pinch model (EPM).³⁰⁾

During our efforts toward development of novel drugs for treatment of neuropathic pain, we have initiated a program to discover mGluR1 NAMs as potential therapeutic targets. Considering the structures of several mGluR1 NAMs (Fig. 1), we have designed quinoline derivatives having a set of amines on the both substituents R¹ and R², which were illustrated in Fig. 2. In particular, in comparison with mGluR1 antagonists 2, 3 and 4³¹⁾ in Fig. 1, we assumed that hydrophobic groups at R¹ and cyclic amino carbonyl groups at R² could be crucial inhibitory components. Furthermore, change of these substituents in quinolines might mediate drug-like properties to improve in vitro metabolic stability of the designed compounds.

Fig. 1. Representative mGluR1 Negative Allosteric Modulators

Fig. 2. The Structure of Quinoline Derivatives 5

In this study, we report our progress in the synthesis of 2,6-disubsituted quinolines as potential mGluR1 NAMs and their biological evaluation for treatment of neuropathic pain.

Results and Discussion

Chemistry

The synthesis of compounds 12–16 is described in Chart 1. Following the literature procedure,^32,33) we first prepared 6-bromoquinolin-2(1H)-one 8 in three steps; formation of acyl chloride, amidation with 4-bromoaniline and cyclization. Treatment of 8 with POCl₃ afforded the corresponding 2-chloroquinoline 9, which was subjected to coupling reactions with various amines or sodium methoxide to give 2-substitued 6-bromoquniolines 10. Lithiation of 10 followed by addition of CO₂ gas led to formation of carboxylic acids 11.²⁹⁾ Finally, amide coupling reactions of 11 with a set of amines in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) gave quinoline derivatives 12–16. Alternatively, we successfully obtained compound 17 using palladium-catalyzed coupling reactions to introduce a cyano group to quinolines 10 at the 6-position. Hydrolysis of 17 followed by methylation³⁴⁾ yielded a mixture of amides, which was easily separated by column chromatography to afford 12b and 12c.

Chart 1. Synthesis of 2-Substituted Quinoline-6-carboxamides 12–16

Reagents and conditions: a) (COCl)₂, 0°C to rt, 12 h then 120°C, 30 m, 89%; b) 4-bromoaniline, pyr, DCM, rt, 3 h, 80%; c) H₂SO₄, 0°C to rt, 3 h, 89%; d) POCl₃, 120°C, 6 h, 99%; e) various amines (neat) or NaOMe, MeOH (for R¹=OMe) 80–110°C, 6–40 h, 66–93%; f) nBuLi, CO₂ (g), THF, −78°C to rt, 1–3 h then 1 n HCl, 37–86%; g) HBTU, amines, TEA, DMF, 23–110°C, 24 h, 5–93%; h) CuCN, Pd(PPh₃)₄, DMF, 120°C, 48 h, 93%; i) KOH, tBuOH, 100°C, 24 h, 97%; j) MeBr, 50% aq. NaOH, Bu₄NHSO₄, PhH, 100°C, 2.5 h, 18% (12b), 71% (12c).

Biological Assay

The mGluR1 antagonistic activities of our synthesized compounds 12–16 were examined by a functional cell based assay measuring the ability of the compounds to inhibit the mobilization of calcium by a high concentration of glutamate in human mGluR1/HEK293 cells using FDSS6000 system. Investigating the effect of cyclic amines at the 2-position of quinolines, we found that 2-pyrrolidyl or 2-piperidinylquinolines 12 and 13 showed considerably good inhibitory activities with higher than 40% inhibition values at the concentration of 10 µm when small amide groups (R²=Me or Et, and R³=H) were located at the 6-position of quinolines. On the other hand, when R¹ was piperazine, 4-methyl/ethylpiperazine, or morpholine, most quinoline derivatives except 14h and 15b exhibited weak antagonistic activity (% inhibition values <40%) regardless of 6-amide substituents. Additionally, we replaced cyclic amines with methoxy substituent at R¹. However, such a modification did not significantly improve a negative modulation of mGluR1. In fact, the highest inhibitory value increased up to only 58% at the concentration of 10 µm when R² and R³ are equal to cyclopropyl and hydrogen. Based on the structure–activity relationship (SAR) information from Table 1, the important pharmacophore components are summarized in Fig. 3. Indeed, a hydrophobic and larger substituent (cyclic amine) at R¹ is responsible for high potency for mGluR1. The size of a secondary amide group at the 6-position of quinolines seems to be another essential factor to enhance mGluR1 antagonistic activity.

Table 1. In Vitro Inhibitory Activity of the Synthesized Compounds against mGluR1

Compounds	R1	R2		R3	% Inhibition (mGluR1)a)
					10 µm	1 µm
12a	Pyrrolidine	H		H	16.55	24.75
12b	Pyrrolidine	Methyl		H	65.97	33.14
12c	Pyrrolidine	Methyl		Methyl	35.85	14.68
12d	Pyrrolidine	Ethyl		H	56.22	11.35
12e	Pyrrolidine	Propyl		H	47.15	2.71
12f	Pyrrolidine	Octyl		H	44.07	18.06
12g	Pyrrolidine	Phenyl		H	15.65	14.82
12h	Pyrrolidine	Benzyl		H	16.78	20.71
12i	Pyrrolidine	Morpholine			25.84	20.91
12j	Pyrrolidine	4-(4-Fluorophenyl)piperazine			15.57	20.03
12k	Pyrrolidine	4-(2,4-Dimethylphenyl)piperazine			30.22	14.98
13a	Piperidine	Methyl		H	72.86	33.59
13b	Piperidine	Methyl		Methyl	21.90	11.34
13c	Piperidine	Ethyl		H	76.76	32.71
13d	Piperidine	Propyl		H	61.97	24.80
13e	Piperidine	Propyl		Propyl	46.75	26.29
13f	Piperidine	Isopropyl		H	5.98	9.61
13g	Piperidine	Cyclopropyl		H	56.21	9.89
13h	Piperidine	Phenyl		H	9.29	16.94
13i	Piperidine	Benzyl		H	21.07	24.03
13j	Piperidine	Morpholine			24.50	11.79
13k	Piperidine	4-Phenylpiperazine			13.84	0.99
14a	Piperazine	Ethyl		H	28.73	−4.98
14b	Piperazine	Propyl		Propyl	18.80	0.64
14c	Piperazine	H		H	16.50	2.85
14d	4-Methylpiperazine	Methyl		H	9.31	16.38
14e	4-Methylpiperazine	Isopropyl		H	23.15	17.15
14f	4-Ethylpiperazine	Methyl		Methyl	15.01	4.05
14g	4-Ethylpiperazine	Isopropyl		H	22.70	8.50
14h	4-Ethylpiperazine	Methyl		H	40.89	10.89
15a	Morpholine	H		H	30.25	5.68
15b	Morpholine	Methyl		H	48.80	19.35
15c	Morpholine	Ethyl		H	13.99	13.61
15d	Morpholine	Propyl		H	16.45	24.30
15e	Morpholine	Isopropyl		H	29.63	14.08
15f	Morpholine	Methyl		Methyl	31.45	4.07
15g	Morpholine	Propyl		Propyl	27.03	11.12
15h	Morpholine	Cyclohexyl		H	35.38	8.35
15i	Morpholine	4-(4-Fluorophenyl)piperazine			31.43	16.93
15j	Morpholine	2,4-(Dimethylphenyl)piperazine			28.68	−0.46
16a	MeO-	Methyl		H	6.85	5.04
16b	MeO-	Methyl		Methyl	3.09	16.73
16c	MeO-	Ethyl		H	53.09	27.45
16d	MeO-	Isopropyl		H	38.41	15.82
16e	MeO-	Cyclopropyl		H	58.10	27.34
16f	MeO-	Pyrrolidine			−0.51	13.12
16g	MeO-	Piperidine			23.95	16.67
16h	MeO-	4-Methylpiperazine			32.97	11.40

a) Ca²⁺ flux assay using glutamate as agonist.

Fig. 3. Structure–Activity Relationship (SAR) Study of the Synthesized Compounds

Considering the structural diversity of 12–16 as well as the in vitro activity data, we then selected 12b, 13c and 13d to achieve additional experiments to examine mGluR1 IC₅₀ values, human ether-à-go-go-related gene (hERG) inhibition, microsomal stability, and CYP inhibition. The results are summarized in Table 2. We observed the highest mGluR1 antagonistic activity with compound 13c (IC₅₀=2.16 µm). While three compounds displayed excellent metabolic stability in human liver microsomes, they moderately inhibited the activity of CYP isozymes at the concentration of 10 µm. Nevertheless, we decided to choose compound 13c as a lead compound for in vivo study on the basis of the overall pharmacological properties including in vitro potency, low hERG inhibition and microsomal stability. Thus, the in vivo efficacy of 13c was evaluated in the spinal nerve ligation (SNL) neuropathic pain model³⁵⁾ (Fig. 4). In order to induce a neuropathic pain state of rats, the L5 spinal nerve at a site distal to the DRG was tightly ligated. We executed two behavioral tests (mechanical allodynia and cold allodynia) after two weeks of surgical operation. The oral administration of the rats with 100 mg/kg of compound 13c was performed when gabapentin was used as a positive control. In case of mechanical allodynia, 13c exhibited only the half efficacy of pain reduction at 3 h in comparison with that of gabapentin. We also observed moderate paw withdrawal response at 3 h and 5 h after administration in the experiment of cold allodynia. This result indicated that the efficacy of compound 13c is not comparable to that of gabapentin yet. Thus, further optimization of compound 13c is necessary to develop 2,6-disubstituted quinoline derivatives as mGluR1 antagonists for treatment of neuropathic pain. In addition, evaluation of synthesized compounds for their selectivity against the other subtypes of the mGluR family should be considered for next lead optimization.

Table 2. The Results of IC50 (mGluR1 and hERG Channel), Microsomal Stability, and CYP Inhibition

Compounds	mGluR1 IC50 (µm)a)	hERG IC50 (µm)a)	HLM % remaining after 30 min	CYP (% remaining at 10 µm)
				1A2	2D6	2C9	3A4	2C19
12b	3.10±0.84	10.10±4.46	>99	15.72	44.42	26.11	6.85	39.83
13c	2.16±0.53	10.20±3.28	>99	15.27	35.80	25.59	9.73	17.20
13d	4.57±0.57	—	94.5	13.53	6.15	33.07	9.96	37.85

a) IC₅₀ value (±S.D.) was obtained from a dose–response curve.

Fig. 4. Effect on Mechanical Allodynia (A, B) and Cold Allodynia (C, D) after Oral Administration of Gabapentin (○, 100 mg/kg, n=4) and 13c (●, 100 mg/kg, n=6) to Neuropathic Pain-Induced Rats

Experimental time expressed as d for days after neuropathic injury (N) and h for hours after gabapentin or 13c administration, * p<0.05 (gabapentin), * p<0.05 (13c) vs. pre-administration value (paired t-test), ^♣ p<0.05 gabapentin vs. 13c (unpaired t-test).

Conclusion

In summary, we have synthesized a series of novel 2-amino or 2-methoxyquinoline 6-carboxamides 12–16 and evaluated their mGluR1 antagonistic activities. We found that 13 compounds in this series showed good potencies with inhibition values over 40% at the concentration of 10 µm. Based on the structure–activity relationship of those compounds, we recognized that a hydrophobic cyclic amine at R¹ and a small size of secondary amide comprising R² and R³ could have a significant impact on the inhibitory activity of quinoline derivatives against mGluR1. Examination of pharmacological properties of several hit compounds led to compound 13c as a potential lead, which was evaluated in rat SNL model of neuropathic pain. Oral administration of 13c was able to reduce both mechanical allodynia and cold allodynia, but its efficacy was not superior to that of gabapentin. Thus, the present study described herein could be further applied for the identification of more potent mGluR1 NAMs with high in vivo efficacy for treatment of neuropathic pain.

Experimental

Chemistry

All reactions were conducted using oven-dried glassware under an atmosphere of nitrogen (N₂). All commercially available reagents were purchased and used without further purification. Solvents Et₂O, CH₂Cl₂, and tetrahydrofuran (THF) were dried and distilled following usual protocols. Reactions were followed by TLC analysis using silica gel plates with fluorescent indicator (60F-254) using UV lamp and KMnO₄ solution with heat as visualizing agents. Flash chromatography was carried out using silica gel (60, particle size 0.040–0.063 mm). The ¹H-NMR spectra were measured with 400 MHz and ¹³C-NMR spectra were measured with 100 MHz using CDCl₃. ¹H-NMR chemical shifts are expressed in parts per million (δ) downfield to CHCl₃ (δ=7.26), ¹³C-NMR chemical shifts are expressed in parts per million (δ) relative to the central CDCl₃ resonance (δ=77.0). Coupling constants in ¹H-NMR are in Hz. The following abbreviations were used to designate multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, quint.=quintet, m=multiplet, br=broad. High resolution mass spectra (HR-MS) were obtained using positive electrospray ionization and mass/charge (m/z) ratios are reported as values in atomic mass units. Fourier transform infrared (FT-IR) analyses were obtained on a Perkin-Elmer Spectrum GX FT-IR system and were reported in cm⁻¹.

(E)-N-(4-Bromophenyl)-3-ethoxyacrylamide (7): Ethyl vinyl ether (10.0 mL, 104 mmol) was added dropwise to oxalyl chloride (13.5 mL, 157 mmol) at 0°C under nitrogen gas. After stirred for 1 h, the reaction was allowed to warm to room temperature and stirred for additional 12 h. Excessive oxalyl chloride was removed under reduce pressure with an aid of dichloromethane. Then, the reaction mixture was stirred at 120°C for 30 min. After cooled to room temperature, the reaction vessel was placed in vacuo to afford the corresponding acyl chloride (12.5 g, 89%) as a brown oil: ¹H-NMR (CDCl₃) δ: 1.37 (3H, t, J=7.1 Hz), 4.01 (2H, q, J=7.1 Hz), 5.48 (1H, d, J=12.1 Hz), 7.76 (1H, d, J=12.1 Hz); ¹³C-NMR (CDCl₃) δ: 168.1, 164.7, 102.9, 68.8, 14.4. To a mixture of 4-bromoaniline (8.80 g, 65.4 mmol) and pyridine (8.80 mL, 109 mmol) in CH₂Cl₂ was slowly added the above acryloyl chloride (9.34 g, 54.3 mmol). The reaction mixture was stirred for 3 h at room temperature, quenched with excess ice fragments, and filtered through a sintered glass funnel covered with a pad of Celite to afford crude solid. This material was washed with dichloromethane to give amide 7 (11.1 g, 80%) as a pink solid, which was used in the next step without further purification: ¹H-NMR (DMSO-d₆) δ: 9.82 (br s, 1H), 7.41 (m, 5H), 5.45 (d, J=11.9 Hz, 1H), 3.90 (q, J=6.3 Hz, 2H), 1.22 (t, J=7.0 Hz, 3H); ¹³C-NMR (DMSO-d₆) δ: 165.2, 160.3, 139.5, 131.9, 121.3, 114.6, 100.1, 67.0, 14.9; Purity: (HPLC) 98.81%.

6-Bromoquinolin-2(1H)-one (8): Sulfuric acid (29.0 mL, 544 mmol) was added to (E)-N-(4-bromophenyl)-3-ethoxyacrylamide (11.2 g, 45.9 mmol) at 0°C under N₂ (g). The mixture was stirred for 3 h at room temperature. The reaction was quenched by transferring the reaction mixture to ice fragments. At this point, the color of the reaction mixture turned to pink. After the ice melted, the quenched mixture was filtered and washed with sufficient H₂O to afford bromoquinoline 6 (9.10 g, 89%) as a pink solid: ¹H-NMR (DMSO-d₆) δ: 11.84 (br s, 1H), 7.91 (d, J=9.6 Hz, 1H), 7.86 (d, J=9.6 Hz, 1H), 7.62 (dd, J₁=2.2, J₂=8.8 Hz, 1H), 7.23 (d, J=8.8 Hz, 1H), 6.53 (d, J=9.6 Hz, 1H); ¹³C-NMR (DMSO-d₆) δ: 162.1, 139.6, 138.4, 133.3, 130.4, 123.6, 121.3, 117.7, 113.8; Purity: (HPLC) 95.39%.

6-Bromo-2-chloroquinoline (9): A mixture of 6-bromoquinolin-2(1H)-one 8 (804 mg, 3.59 mmol) and phosphorous oxychloride (3.00 mL, 3.22 mmol) was stirred at 120°C for 6 h. The reaction was quenched by transferring the reaction mixture to ice fragments. Then, 4 n NaOH solution was slowly added. The resulting solution was separated and extracted with EtOAc. The organic layer was separated, dried over MgSO₄, and filtered. The solvent was removed under reduced pressure to afford chloroquinoline 9 (863 mg, 99%) as a brownish solid, which was used in the next step without further purification: ¹H-NMR (DMSO-d₆) δ: 8.42 (1H, dd, J=2.5, 8.7 Hz), 8.35 (1H, d, J=2.0 Hz), 7.94 (1H, dt, J=2.3, 9.0 Hz), 7.89 (1H, dd, J=2.6, 8.7 Hz), 7.65 (1H, dd, J=2.5, 8.6 Hz); ¹³C-NMR (DMSO-d₆) δ: 150.8, 146.33, 139.7, 134.4, 130.6, 130.5, 128.5, 124.0, 120.6; Purity: (HPLC) 98.21%.

General Procedure for Preparing 2-Substituted-6-bromoquinolines (10a–e)

A mixture of 6-bromo-2-chloroquinoline 9 (2.50 mmol) and amines (for 10a–e) or sodium methoxide in MeOH (for 10g) was stirred at 90°C on an oil bath for 6–40 h. The reaction was quenched by excessive water and the resulting solution was extracted with EtOAc. The organic layer was separated, dried over MgSO₄, and filtered. The solvent was removed under reduced pressure to give the crude product, which was purified by column chromatography over silica gel (CH₂Cl₂–MeOH) to afford 2-aminoquinoline 10a–g.

6-Bromo-2-(pyrrolidin-1-yl)quinoline (10a): Yield 93%; ¹H-NMR (CDCl₃) δ: 7.73 (d, J=9.1 Hz, 1H), 7.69 (s, 1H), 7.55 (s, 2H), 6.72 (d, J=9.0 Hz, 1H), 3.60 (br s, 4H), 2.06–2.02 (m, 4H); ¹³C-NMR (CDCl₃) δ: 155.8, 147.3, 135.9, 132.4, 129.4, 127.9, 123.8, 113.7, 111.1, 46.8, 25.5. MS: m/z (electron ionization (EI)-MS) 276 (M⁺). Purity: (HPLC) 98.88%.

6-Bromo-2-(piperidin-1-yl)quinoline (10b): Yield 67%; ¹H-NMR (CDCl₃) δ: 7.70 (d, J=9.2 Hz, 1H), 7.67 (d, J=1.7 Hz, 1H), 7.54 (dd, J₁=8.9 Hz, J₂=2.0 Hz, 1H), 7.51 (d, J=8.9 Hz, 1H), 6.95 (d, J=9.2 Hz, 1H), 3.71–3.69 (m, 4H), 1.71–1.62 (m, 6H); ¹³C-NMR (CDCl₃) δ: 157.6, 146.9, 136.2, 132.4, 129.1, 128.2, 123.9, 114.6, 110.5, 46.1, 25.8, 24.8. MS: m/z (EI-MS) 290 (M⁺); Purity: (HPLC) 99.36%.

tert-Butyl 4-(6-Bromoquinolin-2-yl)piperazine-1-carboxylate (10c): Yield 80%; ¹H-NMR (CDCl₃) δ: 7.70 (d, J=9.2 Hz, 1H), 7.65 (d, J=2.0 Hz, 1H), 7.53 (dd, J₁=8.9 Hz, J₂=2.0 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 6.87 (d, J=9.2 Hz, 1H), 3.67–3.64 (m, 4H), 3.53–3.50 (m, 4H), 1.45 (s, 9H); ¹³C-NMR (CDCl₃) δ: 157.2, 154.8, 146.4, 136.5, 132.7, 129.2, 128.4, 124.3, 115.3, 110.3, 80.0, 44.9, 43.8, 28.5; Purity: (HPLC) 90.23%.

6-Bromo-2-(4-methylpiperazin-1-yl)quinoline (10d): Yield 67%; ¹H-NMR (CDCl₃) δ: 7.75 (d, J=9.2 Hz, 1H), 7.69 (d, J=1.5 Hz, 1H), 7.56 (dd, J₁=9.0 Hz, J₂=1.9 Hz, 1H), 7.52 (d, J=8.9 Hz, 1H), 6.95 (d, J=9.2 Hz, 1H), 3.74 (t, J=4.9 Hz, 4H), 2.53 (t, J=5.0 Hz, 4H), 2.34 (s, 3H); ¹³C-NMR (CDCl₃) δ: 157.3, 146.6, 136.3, 132.6, 129.2, 128.4, 124.2, 115.0, 110.2, 54.9, 46.2, 44.9; MS: m/z (EI-MS) 305 (M⁺); Purity: (HPLC) 99.32%.

6-Bromo-2-(4-ethylpiperazin-1-yl)quinoline (10e): Yield 66%; ¹H-NMR (CDCl₃) δ: 7.71 (d, J=9.2 Hz, 1H), 7.66 (d, J=1.4 Hz, 1H), 7.54 (dd, J₁=9.0 Hz, J₂=2.0 Hz, 1H), 7.51 (d, J=9.0 Hz, 1H), 6.91 (d, J=9.2 Hz, 1H), 3.72 (t, J=5.2 Hz, 4H), 2.53 (t, J=5.1 Hz, 4H), 2.43 (q, J=7.2 Hz, 2H), 1.10 (t, J=7.2 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 157.4, 146.6, 136.4, 132.6, 129.2, 128.4, 124.2, 115.0, 110.2, 52.7, 52.4, 44.9, 12.0; MS: m/z (EI-MS) 319 (M⁺); Purity: (HPLC) 99.35%.

4-(6-Bromoquinolin-2-yl)morpholine (10f): Yield 94%; ¹H-NMR (CDCl₃) δ: 7.72 (d, J=9.2 Hz, 1H), 7.70 (s, 1H), 7.53 (t, J=9.7 Hz, 2H), 6.87 (d, J=9.1 Hz, 1H), 3.80 (t, J=4.7 Hz, 4H), 3.65 (t, J=4.7 Hz, 4H).

6-Bromo-2-methoxyquinoline (10g): Yield 92%; ¹H-NMR (CDCl₃) δ: 7.86 (d, J=8.9 Hz, 1H), 7.84 (d, J=2.1 Hz, 1H), 7.71 (d, J=8.9 Hz, 1H), 7.67 (dd, J₁=8.9 Hz, J₂=2.1 Hz, 1H), 6.90 (d, J=8.8 Hz, 1H), 4.05 (s, 3H); ¹³C-NMR (CDCl₃) δ: 162.6, 145.3, 137.6, 132.7, 129.5, 129.0, 126.3, 117.2, 114.1, 53.5.

General Procedure for Preparing Compounds 11a–f

To a solution of 6-bromoquinolines 10 (5.00 mmol) in THF (35 mL) cooled to −78°C was slowly added n-BuLi (1.6 m in hexane, 7.50 mmol). After the resulting solution was stirred at −78°C for 20 min, CO₂ gas generated from dry ice was transferred to the reaction mixture via cannula. The reaction was slowly allowed to warm up to room temperature for 2 h and then poured into water. The addition of 1 n HCl (up to pH 5) led to formation of yellow solid, which was filtered through sintered glass to afford quinoline carboxylic acid 11a–f. The products were completely dried under vacuum and used in the next step without further purification.

2-(Pyrrolidin-1-yl)quinoline-6-carboxylic Acid (11a): Yield 62%; ¹H-NMR (DMSO-d₆) δ: 12.7 (br s, 1H), 8.32 (s, 1H), 8.10 (d, J=9.1 Hz, 1H), 7.95 (dd, J₁=8.8 Hz, J₂=1.6 Hz, 1H), 7.51 (d, J=8.7 Hz, 1H), 6.91 (d, J=9.1 Hz, 1H), 3.54 (br s, 4H), 1.97–1.96 (m, 4H); ¹³C-NMR (DMSO-d₆) δ: 167.9, 156.7, 151.0, 138.3, 130.9, 129.6, 125.9, 123.4, 121.7, 111.8, 47.0, 25.4; MS: m/z (EI-MS) 242 (M⁺); Purity: (HPLC) 91.36%.

2-(Piperidin-1-yl)quinoline-6-carboxylic Acid (11b): Yield 83%; ¹H-NMR (DMSO-d₆) δ: 12.7 (br s, 1H), 8.31 (d, J=1.7 Hz, 1H), 8.10 (d, J=9.3 Hz, 1H), 7.95 (dd, J₁=8.7 Hz, J₂=1.9 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 7.27 (d, J=9.3 Hz, 1H), 3.76–3.73 (m, 4H), 1.65–1.55 (m, 6H); ¹³C-NMR (DMSO-d₆) δ: 168.0, 158.2, 150.5, 138.7, 130.6, 129.7, 126.1, 124.2, 121.8, 111.0, 45.8, 25.8, 24.8; Purity: (HPLC) 99.02%.

2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)quinoline-6-carboxylic Acid (11c): Yield 37%; ¹H-NMR (DMSO-d₆) δ: 12.8 (br s, 1H), 8.38 (d, J=2.0 Hz, 1H), 8.21 (d, J=9.2 Hz, 1H), 8.01 (dd, J₁=8.7 Hz, J₂=2.0 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H), 7.31 (d, J=9.3 Hz, 1H), 3.78–3.76 (m, 4H), 3.48–3.46 (m, 4H), 1.44 (s, 1H); ¹³C-NMR (DMSO-d₆) δ: 180.1, 171.8, 158.0, 154.9, 139.0, 131.4, 130.0, 126.3, 123.1, 121.9, 110.1, 80.4, 44.8, 33.8, 28.4; MS: m/z (EI-MS) 356 [(M−H)⁺].

2-(4-Methylpiperazin-1-yl)quinoline-6-carboxylic Acid (11d): Yield 86%; ¹H-NMR (MeOD) δ: 8.25 (s, 1H), 8.08 (d, J=8.6 Hz, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.55 (d, J=8.6 Hz, 1H), 7.14 (d, J=9.2 Hz, 1H), 3.88–3.86 (m, 4H), 2.88–2.85 (m, 4H), 2.53 (s, 3H); ¹³C-NMR (MeOD) δ: 178.3, 157.7, 149.0, 138.6, 130.7, 130.0, 129.1, 125.2, 122.3, 109.8, 53.6, 43.6, 43.4; MS: m/z (EI-MS) 319 (M⁺).

2-(4-Ethylpiperazin-1-yl)quinoline-6-carboxylic acid (11e): Yield 44%; ¹H-NMR (CD₃OD) δ: 8.29 (s, 1H), 8.11 (d, J=8.5 Hz, 1H), 7.99 (d, J=8.8 Hz, 1H), 7.57 (d, J=8.6 Hz, 1H), 7.10 (d, J=8.6 Hz, 1H), 3.90 (bs, 4H), 2.98 (br s, 4H), 2.86 (q, J=7.1 Hz, 2H), 1.25 (t, J=7.2 Hz, 3H); ¹³C-NMR (MeOD) δ: 172.5, 157.6, 149.1, 138.7, 129.9, 129.6, 129.3, 125.3, 122.3, 109.8, 51.8, 51.5, 43.3, 9.3.

2-Methoxyquinoline-6-carboxylic Acid (11f): Yield 78%; ¹H-NMR (CD₃OD) δ: 8.49 (d, J=1.9 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 8.18 (d, J=8.8 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 6.99 (d, J=8.9 Hz, 1H), 4.06 (s, 3H); ¹³C-NMR (CD₃OD) δ: 167.8, 164.0, 147.4, 141.3, 130.5, 130.0, 126.7, 125.5, 124.1, 113.0, 53.7.

General Procedure for Preparing Compounds 12–16

To a N,N-dimethylformamide (DMF) solution of 2-morpholinoquinolone-6-carboxylic acid (0.05 mmol), HBTU (0.059 mmol), and amine (0.05 mmol) was added triethylamine (0.15 mmol). The mixture was stirred at room temperature for 12 h. The resulting mixture was poured into water, and extracted with EtOAc. The crude mixture was purified by column chromatography over silica gel (40 : 1 CH₂Cl₂–methanol+1% triethylamine) to give the products, which were recrystallized by CH₂Cl₂–hexane to afford the products 12–16.

2-(Pyrrolidin-1-yl)quinoline-6-carboxamide (12a): Yield 97%; ¹H-NMR (DMSO-d₆) δ: 8.20 (d, J=1.9 Hz, 1H), 7.99 (d, J=9.1 Hz, 1H), 7.92 (dd, J₁=8.8 Hz, J₂=2.9 Hz, 2H), 7.48 (d, J=8.7 Hz, 1H), 7.22 (br s, 4H), 6.99 (d, J=9.1 Hz, 1H), 3.38 (br s, 4H), 1.97–1.95 (m, 4H); ¹³C-NMR (DMSO-d₆) δ: 168.3, 156.5, 150.1, 138.1, 128.5, 128.4, 127.0, 125.6, 121.6, 111.8, 47.0, 25.5; MS: m/z (EI-MS) 241 (M⁺); Purity: (HPLC) 97.68%.

N-Ethyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12d): Yield 46%; ¹H-NMR (CDCl₃) δ: 8.06 (s, 1H), 7.84 (d, J=9.0 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 6.72 (d, J=9.1 Hz, 1H), 6.24 (br s, 1H), 3.62 (br s, 4H), 3.52 (q, J=6.7 Hz, 2H), 2.05–2.01 (m, 4H), 1.25 (t, J=7.2 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.3, 156.3, 150.2, 150.0, 137.8, 127.5, 127.2, 125.9, 121.7, 111.1, 47.0, 35.0, 25.5, 15.0; MS: m/z (EI-MS) 269 (M⁺); Purity: (HPLC) 91.27%.

N-Propyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12e): Yield 9%; ¹H-NMR (CD₃OD) δ: 8.13 (d, J=1.9 Hz, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.93 (dd, J₁=8.8 Hz, J₂=2.0 Hz, 1H), 7.67 (d, J=8.9 Hz, 1H), 6.93 (d, J=9.2 Hz, 1H), 3,65–3.61 (m, 4H), 3.36 (t, J=7.2 Hz, 2H), 2.10–2.05 (m, 4H), 1.72–1.60 (m, 2H), 0.99 (t, J=7.4 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.3, 155.8, 149.1, 138.2, 127.53, 127.49, 127.4, 125.3, 121.6, 111.2, 47.2, 41.9, 25.5, 23.0, 11.5; MS: m/z (EI-MS) 283 (M⁺); Purity: (HPLC) 95.05%.

N-Octyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12f): Yield 30%; ¹H-NMR (CDCl₃) δ: 8.05 (d, J=2.0 Hz, 1H), 7.85 (d, J=9.0 Hz, 1H), 7.81 (dd, J₁=8.8 Hz, J₂=2.1 Hz, 1H), 7.66 (d, J=8.7 Hz, 1H), 6.73 (d, J=9.1 Hz, 1H), 6.20 (s, 1H), 3.61 (br s, 4H), 3.45 (q, J=6.7 Hz, 2H), 2.06–2.00 (m, 4H), 1.65–1.58 (m, 2H), 1.41–1.36 (m, 2H), 1.33–1.24 (m, 8H), 0.86 (t, J=6.9 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.4, 156.4, 150.3, 137.6, 127.4, 127.1, 127.0, 126.1, 121.7, 111.1, 46.9, 40.2, 31.8, 30.0, 29.3, 29.2, 27.1, 25.5, 22.6, 14.1; MS: m/z (EI-MS) 353 (M⁺); Purity: (HPLC) 91.75%.

N-Phenyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12g): Yield 68%; ¹H-NMR (CDCl₃) δ: 8.17 (d, J=1.9 Hz, 1H), 7.94 (dd, J₁=8.8 Hz, J₂=5.0 Hz, 1H), 7.90 (d, J=9.0 Hz, 2H), 7.73 (d, J=8.8 Hz, 1H), 6.67 (d, J=7.7 Hz, 2H), 7.39 (d, J=7.6 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.14 (t, J=7.4 Hz, 1H), 6.78 (d, J=9.1 Hz, 1H), 3.65 (br s, 4H), 2.08–2.04 (m, 4H); ¹³C-NMR (CDCl₃) δ: 165.6, 156.6, 150.7, 138.2, 137.6, 129.1, 127.8, 127.1, 127.0, 126.4, 124.3, 121.8, 120.1, 111.3, 46.9, 25.5; MS: m/z (EI-MS) 317 (M⁺); Purity: (HPLC) 96.18%.

N-Benzyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12h): Yield 7%; ¹H-NMR (CDCl₃) δ: 8.11 (s, 1H), 7.87–7.85 (m, 2H), 7.76 (m, 1H), 7.40–7.29 (m, 5H), 6.74 (d, J=9.1 Hz, 1H), 6.63 (br s, 1H), 4.67 (d, J=5.6 Hz, 2H), 3.65 (br s, 4H), 2.07–2.04 (m, 4H); ¹³C-NMR (MeOD) δ: 168.4, 139.0, 138.2, 136.9, 128.7, 127.9, 127.7, 127.6, 127.4, 127.1, 126.7, 124.2, 121.6, 111.5, 43.2, 38.8, 25.0; MS: m/z (EI-MS) 331 (M⁺); Purity: (HPLC) 86.39%.

Morpholino(2-(pyrrolidin-1-yl)quinolin-6-yl)methanone (12i): Yield 74%; ¹H-NMR (CDCl₃) δ: 7.82 (d, J=9.1 Hz, 1H), 7.69–7.68 (m, 2H), 7.51 (dd, J₁=8.5 Hz, J₂=1.7 Hz, 1H), 6.74 (d, J=9.0 Hz, 1H), 3.70–3.62 (m, 12H), 2.05–2.02 (m, 4H); ¹³C-NMR (CDCl₃) δ: 170.8, 156.2, 149.4, 137.3, 128.2, 127.6, 127.5, 125.9, 121.8, 111.1, 77.2, 67.0, 47.0, 25.5; MS: m/z (EI-MS) 311 (M⁺); Purity: (HPLC) 93.15%.

(4-(4-Fluorophenyl)piperazin-1-yl)(2-(pyrrolidin-1-yl)quinolin-6-yl)methanone (12j): Yield 81%; ¹H-NMR (CDCl₃) δ: 7.84 (d, J=9.1 Hz, 1H), 7.72 (d, J=1.8 Hz, 1H), 7.70 (d, J=8.8 Hz, 1H), 7.55 (dd, J₁=8.6 Hz, J₂=1.9 Hz, 1H), 7.00–6.95 (m, 2H), 6.92–6.87 (m, 2H), 6.76 (d, J=9.1 Hz, 1H), 3.81 (m, 4H), 3.64 (m, 4H), 3.12 (m, 4H), 2.07–2.04 (m, 4H); ¹³C-NMR (CDCl₃) δ: 170.8, 157.6 (J=238 Hz), 156.3, 149.4, 147.7, 137.2, 128.2, 127.8, 127.5, 126.0, 121.9, 118.6 (J=7.6 Hz), 115.7 (J=22.1 Hz), 111.1, 50.8, 46.9, 45.8, 25.5; Purity: (HPLC) 96.24%.

(4-(2,4-Dimethylphenyl)piperazin-1-yl)(2-(pyrrolidin-1-yl)quinolin-6-yl)methanone (12k): Yield 23%; ¹H-NMR (CDCl₃) δ: 7.85 (d, J=9.1 Hz, 1H), 7.73 (d, J=1.7 Hz, 1H), 7.69 (d, J=8.6 Hz, 1H), 7.57 (dd, J₁=8.6 Hz, J₂=1.9 Hz, 1H), 7.02 (s, 1H), 6.99 (d, J=8.2 Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.75 (d, J=9.1 Hz, 1H), 3.81 (m, 4H), 3.63 (m, 4H), 2.90 (m, 4H), 2.30 (s, 3H), 2.28 (s, 3H), 2.07–2.04 (m, 4H); ¹³C-NMR (CDCl₃) δ: 170.8, 156.3, 149.4, 148.5, 137.2, 133.2, 132.7, 131.9, 128.3, 128.1, 127.4, 127.1, 126.0, 121.9, 119.2, 111.0, 52.3, 46.9, 30.9, 25.5, 20.7, 17.7; Purity: (HPLC) 92.40%.

N-Methyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13a): Yield 42%; ¹H-NMR (CDCl₃) δ: 8.04 (d, J=2.9 Hz, 1H), 7.83 (d, J=9.2 Hz, 1H), 7.82 (dd, J₁=8.7 Hz, J₂=2.1 Hz, 1H), 7.66 (d, J=8.6 Hz, 1H), 6.97 (d, J=9.3 Hz, 1H), 6.40 (s, 1H), 3.76–3.75 (m, 4H), 1.85 (d, J=9.1 Hz, 3H), 1.68–1.66 (m, 6H); ¹³C-NMR (CDCl₃) δ: 168.1, 157.8, 149.6, 138.2, 127.6, 127.2, 127.1, 126.1, 121.7, 110.4, 46.2, 26.9, 25.8, 24.7; MS: m/z (EI-MS) 269 (M⁺); Purity: (HPLC) 96.20%.

N,N-Dimethyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13b): Yield 29%; ¹H-NMR (CDCl₃) δ: 7.85 (d, J=9.2 Hz, 1H), 7.70 (d, J=1.7 Hz, 1H), 7.67 (d, J=8.2 Hz, 1H), 7.57 (dd, J₁=8.6 Hz, J₂=1.8 Hz, 1H), 7.01 (d, J=9.2 Hz, 1H), 3.77–3.76 (m, 4H), 3.10 (br s, 6H), 1.71 (br s, 6H); ¹³C-NMR (CDCl₃) δ: 171.8, 157.9, 137.5, 129.3, 128.3, 126.8, 126.2, 121.9, 115.2, 110.3, 46.1, 39.5, 35.6, 25.8, 24.8; MS: m/z (EI-MS) 283 (M⁺); Purity: (HPLC) 96.93%.

N-Ethyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13c): Yield 43%; ¹H-NMR (CDCl₃) δ: 8.03 (s, 1H), 7.83 (d, J=9.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 7.62 (d, J=8.6 Hz, 1H), 6.97 (d, J=9.3 Hz, 1H), 6.17 (br s, 1H), 3.74–3.73 (m, 4H), 3.55–3.46 (m, 2H), 1.66 (br s, 6H), 1.24 (t, J=7.2 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.4, 158.0, 149.8, 138.0, 127.6, 127.2, 127.1, 126.2, 121.7, 110.3, 46.0, 35.0, 25.8, 24.8, 14.9; MS: m/z (EI-MS) 283 (M⁺); Purity: (HPLC) 89.57%.

2-(Piperidin-1-yl)-N-propylquinoline-6-carboxamide (13d): Yield 38%; ¹H-NMR (CDCl₃) δ: 8.06 (d, J=2.0 Hz, 1H), 7.88 (d, J=9.2 Hz, 1H), 7.84 (dd, J₁=8.7 Hz, J₂=2.1 Hz, 1H), 7.66 (d, J=8.3 Hz, 1H), 7.01 (d, J=9.2 Hz, 1H), 6.24 (br s, 1H), 3.78–3.77 (m, 4H), 3.48–3.43 (m, 2H), 1.70–1.63 (m, 8H), 1.01 (t, J=7.4 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.4, 158.0, 149.8, 138.0, 127.7, 127.1, 126.8, 126.3, 121.8, 110.3, 46.1, 41.8, 25.8, 24.8, 23.0, 11.5; MS: m/z (EI-MS) 297 (M⁺); Purity: (HPLC) 89.26%.

2-(Piperidin-1-yl)-N,N-dipropylquinoline-6-carboxamide (13e): Yield 28%; ¹H-NMR (CDCl₃) δ: 7.83 (d, J=9.2 Hz, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.60 (d, J=1.8 Hz, 1H), 7.49 (dd, J₁=8.6 Hz, J₂=1.9 Hz, 1H), 7.00 (d, J=9.2 Hz, 1H), 3.74 (br s, 4H), 3.46 (br s, 2H), 3.24 (br s, 2H), 1.69 (br s, 8H), 1.56 (br s, 2H), 0.98 (br s, 3H), 4.34 (br s, 3H), 0.72 (br s, 3H); ¹³C-NMR (CDCl₃) δ: 172.0, 149.8, 148.4, 137.5, 130.6, 127.8, 126.3, 125.9, 122.0, 110.3, 46.1, 25.8, 24.8, 21.9, 20.8, 11.2; MS: m/z (EI-MS) 339 (M⁺); Purity: (HPLC) 92.84%.

N-Isopropyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13f): Yield 89%; ¹H-NMR (CDCl₃) δ: 8.04 (d, J=1.8 Hz, 1H), 7.86 (d, J=9.2 Hz, 1H), 7.82 (dd, J₁=8.7 Hz, J₂=2.0 Hz, 1H), 7.64 (d, J=8.7 Hz, 1H), 6.99 (d, J=9.2 Hz, 1H), 6.03 (d, J=7.2 Hz, 1H), 4.34–4.27 (m, 1H), 3.76–3.75 (m, 4H), 1.69 (br s, 6H), 1.28 (s, 3H), 1.27 (s, 3H); ¹³C-NMR (CDCl₃) δ: 166.5, 157.9, 149.6, 138.0, 127.9, 127.2, 127.0, 126.2, 121.7, 110.3, 46.1, 41.9, 25.8, 24.8, 22.9; MS: m/z (EI-MS) 297 (M⁺); Purity: (HPLC) 96.56%.

N-Cyclopropyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13g): Yield 52%; ¹H-NMR (CDCl₃) δ: 8.02 (d, J=2.0 Hz, 1H), 7.84 (d, J=9.3 Hz, 1H), 7.79 (dd, J₁=8.8 Hz, J₂=2.1 Hz, 1H), 6.65 (d, J=8.4 Hz, 1H), 6.97 (d, J=9.2 Hz, 1H), 6.41 (br s, 1H), 3.76–3.75 (m, 4H), 2.92 (octet, J=3.6 Hz, 1H), 1.68–1.64 (m, 6H), 0.88–0.84 (m, 2H), 0.65–0.61 (m, 2H); ¹³C-NMR (CDCl₃) δ: 168.8, 157.9, 149.8, 142.1, 138.1, 127.3, 127.1, 126.2, 121.7, 110.4, 46.1, 25.8, 24.8, 23.2, 6.8; MS: m/z (EI-MS) 295 (M⁺); Purity: (HPLC) 98.09%.

N-Phenyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13h): Yield 45%; ¹H-NMR (CDCl₃) δ: 8.14 (d, J=1.9 Hz, 1H), 7.93 (dd, J₁=8.8 Hz, J₂=2.2 Hz, 1H), 7.90 (d, J=10.2 Hz, 2H), 7.67 (d, J=7.9 Hz, 2H), 7.38 (d, J=7.5 Hz, 1H), 7.36 (d, J=6.7 Hz, 1H), 7.13 (t, J=6.4 Hz, 1H), 7.01 (d, J=9.3 Hz, 1H), 3.74 (br s, 4H), 0.98 (br s, 6H); ¹³C-NMR (CDCl₃) δ: 165.6, 158.1, 138.8, 138.2, 138.0, 129.1, 127.7, 127.4, 127.1, 126.5, 124.3, 121.8, 120.2, 110.5, 46.1, 25.8, 24.8; MS: m/z (EI-MS) 331 (M⁺); Purity: (HPLC) 95.67%.

N-Benzyl-2-(piperidin-1-yl)quinoline-6-carboxamide (13i): Yield 81%; ¹H-NMR (CDCl₃) δ: 8.08 (d, J=1.9 Hz, 1H), 7.86 (d, J=8.1 Hz, 1H), 7.85 (dd, J₁=8.7 Hz, J₂=2.2 Hz, 1H), 7.64 (d, J=7.3 Hz, 1H), 7.40–7.33 (m, 4H), 7.32–7.28 (m, 1H), 7.00 (d, J=9.3 Hz, 1H), 6.43 (br s, 1H), 4.68 (d, J=5.6 Hz, 2H), 3.76 (br s, 4H), 1.57 (br s, 6H); ¹³C-NMR (CDCl₃) δ: 167.2, 143.3, 143.1, 138.4, 138.2, 128.8, 128.6, 128.0, 127.6, 127.5, 127.3, 126.3, 121.7, 110.4, 46.2, 44.2, 25.8, 24.8; MS: m/z (EI-MS) 345 (M⁺).

Morpholino(2-(piperidin-1-yl)quinolin-6-yl)methanone (13j): Yield 93%; ¹H-NMR (CDCl₃) δ: 7.83 (d, J=9.2 Hz, 1H), 7.67 (d, J=1.8 Hz, 1H), 7.64 (d, J=8.6 Hz, 1H), 7.51 (dd, J₁=8.6 Hz, J₂=2.0 Hz, 1H), 6.99 (d, J=9.4 Hz, 1H), 3.75–3.70 (m, 12H), 1.68–1.66 (m, 6H); ¹³C-NMR (CDCl₃) δ: 170.7, 157.8, 148.8, 137.6, 128.18, 128.15, 127.1, 126.3, 121.9, 110.4, 67.0, 48.0, 46.2, 43.6, 25.8, 24.8; MS: m/z (EI-MS) 325 (M⁺); Purity: (HPLC) 97.55%.

(4-Phenylpiperazin-1-yl)(2-(piperidin-1-yl)quinolin-6-yl)methanone (13k): Yield 23%; ¹H-NMR (CDCl₃) δ: 7.85 (d, J=9.2 Hz, 1H), 7.71 (d, J=1.8 Hz, 1H), 7.68 (d, J=8.4 Hz, 1H), 7.56 (dd, J₁=8.6 Hz, J₂=1.9 Hz, 1H), 7.29 (d, J=7.3 Hz, 1H), 7.27 (dd, J₁=8.8 Hz, J₂=1.4 Hz, 1H), 7.01 (d, J=9.2 Hz, 1H), 6.94 (d, J=8.7 Hz, 2H), 6.91 (t, J=8.4 Hz, 1H), 3.77 (br s, 2H), 3.76 (s, 5H), 3.21 (br s, 4H), 1.70 (br s, 6H); ¹³C-NMR (CDCl₃) δ: 170.7, 158.0, 151.0, 137.5, 129.2, 128.8, 128.5, 128.2, 127.1, 126.4, 122.0, 120.6, 116.7, 110.4, 49.8, 49.8, 46.1, 25.8, 24.8; Purity: (HPLC) 95.74%.

N-Ethyl-2-(piperazin-1-yl)quinoline-6-carboxamide (14a): Yield 99%; ¹H-NMR (CDCl₃) δ: 8.08 (d, J=2.0 Hz, 1H), 7.91 (d, J=9.2 Hz, 1H), 7.85 (dd, J₁=8.7 Hz, J₂=2.1 Hz, 1H), 7.67 (d, J=8.7 Hz, 1H), 6.99 (d, J=9.2 Hz, 1H), 6.19 (br s, 1H), 3.76 (t, J=5.0 Hz, 4H), 3.56–3.49 (m, 2H), 3.01 (t, J=5.1 Hz, 4H), 1.27 (t, J=7.3 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.2, 158.1, 149.6, 138.1, 128.1, 127.13, 127.12, 126.6, 122.2, 110.1, 46.0, 45.9, 35.0, 15.0; MS: m/z (EI-MS) 284 (M⁺).

2-(Piperazin-1-yl)-N,N-dipropylquinoline-6-carboxamide (14b): Yield 54%; ¹H-NMR (CDCl₃) δ: 7.88 (d, J=9.2 Hz, 1H), 7.67 (d, J=8.6 Hz, 1H), 7.62 (d, J=1.7 Hz, 1H), 7.50 (dd, J₁=8.6 Hz, J₂=1.9 Hz, 1H), 6.98 (d, J=9.2 Hz, 1H), 3.76 (t, J=5.0 Hz, 4H), 3.46 (br s, 2H), 3.21 (br s, 2H), 3.03 (t, J=5.0 Hz, 4H), 1.68 (br s, 2H), 1.54 (br s, 2H), 0.98 (br s, 3H), 0.72 (br s, 3H); ¹³C-NMR (CDCl₃) δ: 171.9, 157.8, 148.1, 137.7, 131.2, 127.9, 126.6, 125.9, 122.5, 110.0, 46.68, 45.7, 29.7, 20.8, 11.2; MS: m/z (EI-MS) 340 (M⁺); Purity: (HPLC) 92.26%.

2-(Piperazin-1-yl)quinoline-6-carboxamide (14c): Yield 34%; ¹H-NMR (CDCl₃) δ: 8.10 (d, J=1.9 Hz, 1H), 7.90 (d, J=9.2 Hz, 1H), 7.85 (dd, J₁=8.7 Hz, J₂=2.0 Hz, 1H), 7.65 (d, J=8.7 Hz, 1H), 6.98 (d, J=9.2 Hz, 1H), 3.74 (t, J=5.0 Hz, 4H), 2.98 (t, J=5.0 Hz, 4H); ¹³C-NMR (MeOD) δ: 170.7, 158.3, 149.5, 138.4, 127.73, 127.70, 127.1, 125.8, 122.2, 110.3, 44.54, 44.47; MS: m/z (EI-MS) 256 (M⁺); Purity: (HPLC) 96.65%.

N-Methyl-2-(4-methylpiperazin-1-yl)quinoline-6-carboxamide (14d): Yield 27%; ¹H-NMR (CDCl₃) δ: 8.07 (d, J=1.8 Hz, 1H), 7.91 (d, J=9.3 Hz, 1H), 7.82 (dd, J₁=8.7 Hz, J₂=2.0 Hz, 1H), 7.66 (d, J=8.7 Hz, 1H), 6.99 (d, J=9.2 Hz, 1H), 6.17 (s, 1H), 3.82 (t, J=5.0 Hz, 4H), 3.03 (d, J=4.9 Hz, 3H), 2.57 (t, J=5.0 Hz, 4H), 2.37 (s, 3H); ¹³C-NMR (MeOD) δ: 169.1, 158.0, 149.3, 138.4, 128.0, 127.3, 127.0, 126.0, 122.3, 110.3, 54.1, 44.1, 43.7, 25.6; MS: m/z (EI-MS) 284 (M⁺); Purity: (HPLC) 95.49%.

N-Isopropyl-2-(4-methylpiperazin-1-yl)quinoline-6-carboxamide (14e): Yield 18%; ¹H-NMR (MeOD) δ: 8.13 (d, J=2.0 Hz, 1H), 8.05 (d, J=9.2 Hz, 1H), 7.92 (dd, J₁=8.8 Hz, J₂=2.1 Hz, 1H), 7.63 (d, J=8.8 Hz, 1H), 7.21 (d, J=9.2 Hz, 1H), 4.24–4.18 (m, 1H), 3.85 (br s, 4H), 2.77 (t, J=5.0 Hz, 4H), 2.49 (s, 3H), 1.25 (d, J=6.6 Hz, 6H); ¹³C-NMR (MeOD) δ: 167.8, 158.2, 149.3, 138.2, 128.2, 127.5, 127.0, 125.7, 122.1, 110.3, 54.4, 44.7, 44.2, 41.8, 21.2; MS: m/z (EI-MS) 312 (M⁺); Purity: (HPLC) 89.05%.

2-(4-Ethylpiperazin-1-yl)-N,N-dimethylquinoline-6-carboxamide (14f): Yield 28%; ¹H-NMR (CDCl₃) δ: 8.08 (d, J=2.0 Hz, 1H), 7.91 (d, J=9.1 Hz, 1H), 7.84 (dd, J₁=8.8 Hz, J₂=2.1 Hz, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.00 (d, J=9.2 Hz, 1H), 6.21 (d, J=4.0 Hz, 1H), 3.82 (t, J=5.1 Hz, 4H), 3.04 (d, J=4.9 Hz, 3H), 2.60 (t, J=5.1 Hz, 4H), 2.50 (q, J=7.2 Hz, 2H), 1.15 (t, J=7.2 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 170.6, 168.0, 158.0, 138.2, 127.9, 127.1, 126.7, 124.8, 122.2, 110.1, 52.7, 52.4, 44.8, 26.9, 11.9; MS: m/z (EI-MS) 312 (M⁺); Purity: (HPLC) 98.70%.

2-(4-Ethylpiperazin-1-yl)-N-isopropylquinoline-6-carboxamide (14g): Yield 23%; ¹H-NMR (MeOD) δ: 8.12 (d, J=2.0 Hz, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.92 (dd, J₁=8.8 Hz, J₂=2.1 Hz, 1H), 7.61 (d, J=8.8 Hz, 1H), 7.17 (d, J=9.2 Hz, 1H), 4.25–4.18 (m, 1H), 3.79 (t, J=5.1 Hz, 4H), 2.59 (t, J=5.1 Hz, 4H), 2.48 (q, J=7.2 Hz, 2H), 1.25 (d, J=6.6 Hz, 6H), 1.13 (t, J=7.2 Hz, 3H); ¹³C-NMR (MeOD) δ: 167.8, 158.2, 149.3, 138.2, 128.2, 127.5, 127.0, 125.7, 122.1, 110.3, 52.3, 52.0, 44.2, 41.8, 21.2, 10.4; MS: m/z (EI-MS) 326 (M⁺); Purity: (HPLC) 94.04%.

2-(4-Ethylpiperazin-1-yl)-N-methylquinoline-6-carboxamide (14h): Yield 5%; ¹H-NMR (MeOD) δ: 8.02 (d, J=9.2 Hz, 1H), 7.74 (s, 1H), 7.66 (d, J=8.6 Hz, 1H), 7.56 (dd, J₁=8.6 Hz, J₂=1.8 Hz, 1H), 7.20 (d, J=9.2 Hz, 1H), 3.80 (t, J=4.9 Hz, 4H), 3.11 (br s, 3H), 3.07 (br s, 3H), 2.61 (t, J=5.0 Hz, 4H), 2.50 (q, J=7.2 Hz, 2H), 1.15 (t, J=7.2 Hz, 3H); ¹³C-NMR (MeOD) δ: 172.4, 158.1, 148.3, 137.8, 129.4, 127.8, 126.5, 125.9, 122.3, 110.5, 52.3, 52.0, 44.3, 38.9, 34.5, 10.4; MS: m/z (EI-MS) 298 (M⁺); Purity: (HPLC) 94.59%.

2-Morpholinoquinoline-6-carboxamide (15a): Yield 97%; ¹H-NMR (CDCl₃) δ: 8.20 (s, 1H), 7.99 (d, J=9.3 Hz, 1H), 7.93 (dd, J₁=2.1 Hz, J₂=8.8 Hz, 1H), 7.73 (d, J=8.8 Hz, 1H), 7.03 (d, J=9.2 Hz, 1H), 3.80 (dt, J₁=4.6, J₂=25.1 Hz, 8H); MS: m/z (EI-MS) 257 (M⁺); Purity: (HPLC) 99.50%.

N-Methyl-2-morpholinoquinoline-6-carboxamide (15b): Yield 53%; ¹H-NMR (CDCl₃) δ: 8.09 (s, 1H), 7.92 (d, J=9.2 Hz, 1H), 7.85 (dd, J₁=2.1, J₂=8.7 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 6.96 (d, J=9.2 Hz, 1H), 6.30 (br s, 1H), 3.73 (dt, J₁=4.8 Hz, J₂=37.0 Hz, 8H), 3.03 (d, J=4.8 Hz, 3H); MS: (EI-MS) m/z 271 (M⁺); Purity: (HPLC) 96.59%.

N-Ethyl-2-morpholinoquinoline-6-carboxamide (15c): Yield 55%; ¹H-NMR (CDCl₃) δ: 8.10 (s, 1H), 7.92 (d, J=9.2 Hz, 1H), 7.85 (dd, J₁=2.1, J₂=8.8 Hz, 1H), 7.68 (d, J=8.6 Hz, 1H), 6.96 (d, J=9.2 Hz, 1H), 6.24 (br s, 1H), 3.74 (dt, J₁=4.7, J₂=37.7 Hz, 8H), 3.49 (m, 2H), 1.25 (t, J=7.3 Hz, 3H); MS: (EI-MS) m/z 285 (M⁺); Purity: (HPLC) 96.39%.

2-Morpholino-N-propylquinoline-6-carboxamide (15d): Yield 32%; ¹H-NMR (CDCl₃) δ: 8.10 (s, 1H), 7.85 (dd, J₁=2.1 Hz, J₂=8.7 Hz, 1H), 7.95 (d, J=6.9 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 6.97 (d, J=9.2 Hz, 1H), 6.24 (br s, 1H), 3.84 (t, J=4.7 Hz, 4H), 3.74 (t, J=4.8 Hz, 4H), 3.43 (q, J=6.7 Hz, 2H), 0.99 (t, J=7.4 Hz, 3H), 1.62 (sextet, 2H); MS: (EI-MS) m/z 299 (M⁺); Purity: (HPLC) 96.14%.

N-Isopropyl-2-morpholinoquinoline-6-carboxamide (15e): Yield 40%; ¹H-NMR (CDCl₃) δ: 8.09 (s, 1H), 7.95 (d, J=8.7 Hz, 1H), 7.84 (dd, J=1.9, 8.7 Hz, 1H), 7.70 (br s, 1H), 6.98 (d, J=9.2 Hz, 1H), 5.97 (br s, 1H), 4.28 (septet, 1H), 3.76 (m, 8H), 1.28 (d, J=6.6 Hz, 6H); MS: (EI-MS) m/z 299 (M⁺); Purity: (HPLC) 97.85%.

N,N-Dimethyl-2-morpholinoquinoline-6-carboxamide (15f): Yield 63%; ¹H-NMR (CDCl₃) δ: 7.89 (d, J=9.1 Hz, 1H), 7.71 (s, 1H), 7.67 (d, J=8.6 Hz, 1H), 7.56 (dd, J₁=1.9 Hz, J₂=8.6 Hz, 1H), 6.95 (d, J=9.2 Hz, 1H), 3.71 (dt, J₁=4.8 Hz, J₂=44.4 Hz, 8H), 3.05 (br s, 6H); MS: (EI-MS) m/z 285 (M⁺); Purity: (HPLC) 99.19%.

2-Morpholino-N,N-dipropylquinoline-6-carboxamide (15g): Yield 32%; ¹H-NMR (CDCl₃) δ: 7.90 (d, J=9.1 Hz, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.65 (s, 1H), 7.51 (dd, J₁=1.8 Hz, J₂=8.6 Hz, 1H), 6.97 (d, J=9.1 Hz, 1H), 3.72 (dt, J=4.5, 35.4 Hz, 8H), 3.47 (br s, 2H), 3.32 (br s, 2H), 1.66 (br s, 2H), 1.57 (br s, 2H), 0.99 (br s, 3H), 0.71 (br s, 3H); MS: (EI-MS) m/z 341 (M⁺); Purity: (HPLC) 97.54%.

N-Cyclohexyl-2-morpholinoquinoline-6-carboxamide (15h): Yield 89%; ¹H-NMR (CDCl₃) δ: 8.08 (s, 1H), 7.93 (d, J=9.1 Hz, 1H), 7.84 (dd, J₁=2.1 Hz, J₂=8.8 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 6.96 (d, J=9.2 Hz, 1H), 6.06 (d, J=7.8 Hz, 1H), 3.97 (m, 1H), 3.74 (dt, J₁=4.8 Hz, J₂=38.4 Hz, 8H), 2.04 (dd, J₁=3.3 Hz, J₂=12.3 Hz, 2H), 1.64 (dt, J₁=3.6 Hz, J₂=13.4 Hz, 4H), 1.46 (m, 2H), 1.30 (m, 2H); MS: (EI-MS) m/z 339 (M⁺); Purity: (HPLC) 95.82%.

(4-(4-Fluorophenyl)piperazin-1-yl)(2-morpholinoquinolin-6-yl)methanone (15i): Yield 77%; ¹H-NMR (CDCl₃) δ: 7.91 (d, J=9.2 Hz, 1H), 7.74 (s, 1H), 7.70 (d, J=8.6 Hz, 1H), 7.57 (dd, J₁=1,7 Hz, J₂=8.6 Hz, 1H), 6.97 (d, J=9.5 Hz, 2H), 6.95 (s, 1H), 6.86 (q, J=4.5 Hz, 2H), 3.83 (t, J=4.7 Hz, 6H), 3.73 (t, J=4.6 Hz, 6H), 3.11 (br s, 4H); ¹³C-NMR (CDCl₃) δ: 170.5, 158.8, 158.0, 156.5, 148.5, 147.6, 137.9, 129.3, 128.4, 127.1, 126.7, 122.5, 118.6, 115.8, 115.6, 110.0, 66.8, 50.8, 45.4; Purity: (HPLC) 90.99%.

(4-(2,4-Dimethylphenyl)piperazin-1-yl)(2-morpholinoquinolin-6-yl)methanone (15j): Yield 95%; ¹H-NMR (CDCl₃) δ: 7.92 (d, J=9.2 Hz, 1H), 7.76 (s, 1H), 7.71 (d, J=7.7 Hz, 1H), 7.60 (dd, J₁=1.6 Hz, J₂=8.6 Hz, 1H), 6.98 (m, 3H), 6.91 (d, J=8.0 Hz, 1H), 3.75 (m, 12H), 2.90 (br s, 4H), 2.28 (d, J=7.1 Hz, 6H); ¹³C-NMR (CDCl₃) δ: 170.5, 157.9, 148.5, 137.9, 133.3, 132.7, 131.3, 129.7, 128.5, 127.2, 127.0, 122.5, 119.2, 109.9, 66.8, 52.26, 45.4, 20.7, 17.7; Purity: (HPLC) 99.52%.

2-Methoxy-N-methylquinoline-6-carboxamide (16a): Yield 55%; ¹H-NMR (CDCl₃) δ: 8.16 (d, J=1.8 Hz, 1H), 7.96 (d, J=8.6 Hz, 1H), 7.93 (dd, J₁=8.5 Hz, J₂=2.1 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 6.90 (d, J=8.8 Hz, 1H), 6.47 (br s, 1H), 4.06 (s, 3H), 3.03 (d, J=4.8 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.9, 163.5, 148.2, 139.3, 130.1, 127.5, 127.2, 127.1, 124.4, 114.1, 53.7, 27.0; MS: m/z (EI-MS) 216 (M⁺); Purity: (HPLC) 96.29%.

2-Methoxy-N,N-dimethylquinoline-6-carboxamide (16b): Yield 46%; ¹H-NMR (CDCl₃) δ: 7.97 (d, J=8.8 Hz, 1H), 7.85 (d, J=8.6 Hz, 1H), 7.81 (d, J=1.7 Hz, 1H), 7.65 (dd, J=8.6 Hz, J=1.9 Hz, 1H), 6.93 (d, J=8.8 Hz, 1H), 4.07 (s, 3H), 3.14 (br s, 3H), 3.03 (br s, 3H); ¹³C-NMR (CDCl₃) δ: 171.3, 163.2, 147.1, 138.9, 131.8, 128.2, 127.3, 126.7, 124.4, 113.9, 53.5, 39.7, 35.5; Purity: (HPLC) 84.85%.

N-Ethyl-2-methoxyquinoline-6-carboxamide (16c): Yield 60%; ¹H-NMR (CDCl₃) δ: 8.17 (d, J=1.9 Hz, 1H), 7.98 (d, J=8.9 Hz, 1H), 7.95 (dd, J₁=8.7 Hz, J₂=2.0 Hz, 1H), 7.84 (d, J=8.7 Hz, 1H), 6.92 (d, J=8.8 Hz, 1H), 6.37 (br s, 1H), 4.07 (s, 3H), 3.57–3.50 (m, 2H), 1.28 (t, J=7.3 Hz, 3H); ¹³C-NMR (CDCl₃) δ: 167.1, 163.5, 148.2, 139.3, 130.2, 127.5, 127.2, 127.1, 124.4, 114.1, 53.6, 35.1, 14.9; MS: m/z (EI-MS) 230 (M⁺).

N-Isopropyl-2-methoxyquinoline-6-carboxamide (16d): Yield 51%; ¹H-NMR (CDCl₃) δ: 8.15 (d, J=1.8 Hz, 1H), 7.99 (d, J=8.8 Hz, 1H), 7.92 (dd, J₁=8.7 Hz, J₂=2.0 Hz, 1H), 7.84 (d, J=8.7 Hz, 1H), 6.91 (d, J=8.8 Hz, 1H), 6.08 (d, J=6.2 Hz, 1H), 4.36–4.24 (m, 1H), 4.06 (s, 3H), 1.28 (d, J=6.6 Hz, 6H); ¹³C-NMR (CDCl₃) δ: 166.3, 163.5, 148.2, 139.3, 130.4, 127.5, 127.2, 127.1, 124.4, 114.1, 53.6, 42.1, 22.9; MS: m/z (EI-MS) 244 (M⁺); Purity: (HPLC) 99.76%.

N-Cyclopropyl-2-methoxyquinoline-6-carboxamide (16e): Yield 56%; ¹H-NMR (CDCl₃) δ: 8.15 (s, 1H), 7.96 (d, J=8.8 Hz, 1H), 7.92 (d, J=8.6 Hz, 1H), 7.82 (d, J=8.6 Hz, 1H), 6.91 (d, J=8.8 Hz, 1H), 6.54 (br s, 1H), 2.96–2.92 (m, 1H), 0.90–0.85 (m, 2H), 0.68–0.64 (m, 2H); ¹³C-NMR (CDCl₃) δ: 168.6, 163.5, 148.3, 139.3, 139.2, 129.8, 127.5, 127.1, 124.3, 114.1, 53.6, 23.3, 6.8; MS: m/z (EI-MS) 242 (M⁺); Purity: (HPLC) 96.73%.

(2-Methoxyquinolin-6-yl)(pyrrolidin-1-yl)methanone (16f): Yield 33%; ¹H-NMR (CDCl₃) δ: 7.98 (d, J=8.8 Hz, 1H), 7.90 (d, J=1.5 Hz, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.76 (dd, J₁=8.6 Hz, J₂=1.7 Hz, 1H), 6.92 (d, J=8.8 Hz, 1H), 4.07 (s, 3H), 3.68 (t, J=6.9 Hz, 2H), 3.49 (t, J=6.5 Hz, 2H), 2.00–1.95 (m, 2H), 1.91–1.87 (m, 2H); ¹³C-NMR (CDCl₃) δ: 169.4, 163.3, 147.3, 139.0, 132.7, 128.2, 127.2, 126.8, 124.4, 113.9, 53.6, 49.8, 46.4, 26.5, 24.5; MS: m/z (EI-MS) 256 (M⁺); Purity: (HPLC) 96.47%.

(2-Methoxyquinolin-6-yl)(piperidin-1-yl)methanone (16g): Yield 73%; ¹H-NMR (CDCl₃) δ: 7.97 (d, J=8.8 Hz, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.78 (d, J=1.8 Hz, 1H), 7.62 (dd, J₁=8.5 Hz, J₂=1.8 Hz, 1H), 6.92 (d, J=8.8 Hz, 1H), 4.07 (s, 3H), 3.73 (br s, 2H), 3.40 (br s, 2H), 1.69 (br s, 4H), 1.57 (br s, 2H); ¹³C-NMR (CDCl₃) δ: 170.1, 163.1, 147.1, 138.9, 132.0, 128.0, 127.4, 126.4, 124.6, 113.9, 53.5, 48.9, 43.6, 26.3, 25.9, 24.6; MS: m/z (EI-MS) 270 (M⁺); Purity: (HPLC) 91.48%.

(2-Methoxyquinolin-6-yl)(4-methylpiperazin-1-yl)methanone (16h): Yield 59%; ¹H-NMR (CDCl₃) δ: 7.94 (d, J=8.9 Hz, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.76 (d, J=1.5 Hz, 1H), 7.59 (dd, J₁=8.5 Hz, J₂=1.7 Hz, 1H), 6.89 (d, J=8.8 Hz, 1H), 4.03 (s, 3H), 3.76 (br s, 2H), 3.53 (br s, 2H), 2.40 (br s, 4H), 2.29 (s, 3H); ¹³C-NMR (CDCl₃) δ: 170.1, 163.2, 147.1, 138.8, 131.2, 128.0, 127.4, 126.8, 124.5, 114.0, 55.0, 53.5, 47.8, 46.0, 42.2; MS: m/z (EI-MS) 285 (M⁺).

Procedures for Preparing Compounds 12b and 12c

2-(Pyrrolidin-1-yl)quinoline-6-carbonitrile (17): A mixture of 6-bromoquinoline 10a (1.26 g, 4.55 mmol) and tetrakis(triphenylphosphine)palladium(0) (527 mg, 0.456 mmol) and copper(I) cyanide (816 mg, 9.11 mmol) in DMF was stirred for 48 h at 120°C on an oil bath. After the reaction was finished (monitored by TLC), the mixture was filtered through Celite. The filtrate was diluted with water and extracted with EtOAc. The combined organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc–hexane 1 : 1) to afford the title compounds 17 (718 mg, 93%) as a yellow solid; ¹H-NMR (CDCl₃) δ: 7.87 (d, J=1.2 Hz, 1H), 7.78 (d, J=9.1 Hz, 1H), 7.66 (d, J=8.6 Hz, 1H), 7.60 (dd, J₁=8.7 Hz, J₂=1.6 Hz, 1H), 6.76 (d, J=9.1 Hz, 1H), 3.62 (br s, 4H), 2.05 (br s, 4H); ¹³C-NMR (DMSO-d₆) δ: 156.9, 150.5, 137.4, 134.2, 131.0, 127.0, 122.3, 120.2, 112.8, 102.6, 47.1, 25.4; MS: m/z (EI-MS) 223 (M⁺).

2-(Pyrrolidin-1-yl)quinoline-6-carboxamide (18): A mixture of 6-cyanoquinoline 17 (83.8 mg, 0.345 mmol) and potassium hydroxide (70 mg, 1.25 mmol) in tert-butanol (5 mL) was prepared. After the reaction mixture was stirred at 100°C for 2 h, it was quenched by excessive water and extracted with EtOAc (3×20 mL). The organic layer was separated, dried over MgSO₄, and concentrated under reduced pressure to give the title compound 18 (80.7 mg, 97%) as a yellow solid, which was used in the next step without further purification; ¹H-NMR (DMSO-d₆) δ: 8.20 (d, J=1.9 Hz, 1H), 7.99 (d, J=9.1 Hz, 1H), 7.92 (dd, J₁=8.8 Hz, J₂=2.9 Hz, 2H), 7.48 (d, J=8.7 Hz, 1H), 7.22 (br s, 4H), 6.99 (d, J=9.1 Hz, 1H), 3.38 (br s, 4H), 1.97–1.95 (m, 4H); ¹³C-NMR (DMSO-d₆) δ: 168.3, 156.5, 150.1, 138.1, 128.5, 128.4, 127.0, 125.6, 121.6, 111.8, 47.0, 25.5; MS: m/z (EI-MS) 241 (M⁺); Purity: (HPLC) 97.68%.

N-Methyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12b) and N,N-Dimethyl-2-(pyrrolidin-1-yl)quinoline-6-carboxamide (12c): To a solution of carboxamide 18 (28.1 mg, 117 µmol) in benzene was added 50% aq. NaOH (0.12 mL) and tetrabutyl ammonium hydrogen sulfate (3.97 mg, 11.7 µmol). After the reaction mixture was stirred at 100°C for 5 min, methyl bromde (3.0 mL, 54.7 mmol) was added. The resulting mixture was stirred at 100°C for additional 2.5 h and quenched with water. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3×15 mL). The combined organic layer was dried over MgSO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH₂Cl₂–MeOH 20 : 1) to afford the title compounds 12b (5.3 mg, 18%) as a white solid and 12c (22.3 mg, 71%) as a white solid; 12b: ¹H-NMR (CDCl₃) δ: 8.06 (d, J=2.0 Hz, 1H), 7.85 (d, J=9.1 Hz, 1H), 7.81 (dd, J₁=8.8 Hz, J₂=2.1 Hz, 1H), 7.72 (d, J=8.6 Hz, 1H), 6.72 (d, J=9.1 Hz, 1H), 6.31 (br s, 1H), 3.63 (br s, 4H), 3.02 (d, J=4.8 Hz, 3H), 2.05–2.00 (m, 4H); ¹³C-NMR (MeOD) δ: 169.1, 156.4, 149.5, 138.0, 127.4, 127.3, 126.8, 124.4, 121.6, 111.4, 29.3, 25.5, 25.0; Purity: (HPLC) 95.47%. 12c: ¹H-NMR (CDCl₃) δ: 7.79 (d, J=9.1 Hz, 1H), 7.66 (d, J=1.7 Hz, 1H), 7.64 (s, 1H), 7.52 (dd, J₁=8.6 Hz, J₂=1.8 Hz, 1H), 6.70 (d, J=9.0 Hz, 1H), 3.59 (br s, 4H), 3.05 (br s, 6H), 2.00 (t, J=6.5 Hz, 4H); ¹³C-NMR (CDCl₃) δ: 171.8, 156.1, 149.0, 137.3, 128.7, 128.4, 127.2, 125.7, 121.7, 111.0, 46.9, 40.0, 35.7, 25.5; MS: m/z (EI-MS) 269 (M⁺); Purity: (HPLC) 94.67%.

In Vitro Calcium Assay (Functional Drug Screening System (FDSS) Assay)

Chem-3 cells which stably express mGluR1 were purchased from Millipore Corporation (HTS145C). Cells were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL), and 1% (v/v) Chem-3 growth supplement at 37°C in a humid atmosphere of 5% CO₂ and 95% air.

For calcium assay, cells were harvested and dispensed into 96-well black wall clear bottom plates at a density of 100000 cells per a well. After 1 h of stabilization, cells were treated with Calcium-5 assay reagent, which is prepared by manufacturer’s instruction (Molecular Devices Corporation, California). During fluorescence-based FDSS6000 assay, mGluR1 was activated using a high concentration of l-glutamate (30 µm) in HBSS, and various concentrations of potential antagonists were treated to cells 75 s before mGluR activation. All data were collected and analyzed using FDSS6000 and related software (Hamamatsu, Japan).

In Vivo Behavioral Test

Two behavioral tests (mechanical allodynia and cold allodynia) were conducted for rats at 1 d prior to surgery and 14 d after surgery. After the postoperative behavioral test, the animals were treated orally with 100 mg/kg compound 13c or gabapentin. The tests were re-evaluated at 1 h, 3 h, and 5 h after administration.

Mechanical Allodynia: Testing was performed according to methods described previously.²⁵⁾ Rats were acclimated in a transparent plastic boxes permitting freedom of movement with a wire mesh floor to allow access to the planter surface of the hind paws. A von Frey filament (Stoelting, Wood Dale, IL, U.S.A.) was applied 5 times (once every 3–4 s) to hind paw. Von Frey filaments were used to assess the 50% mechanical threshold for paw withdrawal. The 50% withdrawal threshold was determined by using the up-down method and formula given by Dixon²⁶⁾: 50% threshold=10(X+kd)/10⁴, where X is the value of the final von Frey hair used (in log units), k is the tabular value for the pattern of positive/negative responses modified from Dixon²⁶⁾, and d is the mean difference between stimuli in log units (0.17).

Cold Allodynia: To quantify cold sensitivity of the paw, brisk paw withdrawal in response to acetone application was measured as described previously.²⁷⁾ The rat was placed under a transparent plastic box on a metal mesh floor and acetone was applied to the plantar surface of the hind paw. To do this, an acetone bubble was formed at the end of a small piece of polyethylene tubing which was connected to a syringe. The bubble was then gently touched to the heel. The acetone quickly spread over the proximal half of the plantar surface of the hind paw. The acetone was applied 5 times to hind paw at 2 min interval. The frequency of paw withdrawal was expressed as a percentage [(No. of trials accompanied by brisk foot withdrawal/total No. of trials)×l00].

Acknowledgment

This research was supported by the Korea Institute of Science and Technology (KIST, 2E23870, 2E24183, 2E23770) and by a Grant of the Korea Health technology R&D Project, the Ministry of Health and Welfare, Republic of Korea (HI11C0998).

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