Synthesis and antimalarial activity of new 4-aminoquinolines active against drug resistant strains

Abstract

In the present study we have synthesized a new class of 4-aminoquinoline derivatives via replacement of the diethylamine functionality of chloroquine (CQ) with acyclic and/or cyclic amine groups containing basic tertiary terminal nitrogen and bioevaluated them for antimalarial activity against Plasmodium falciparum in vitro (CQ-sensitive strain-3D7 & CQ-resistant strain-K1) and Plasmodium yoelii in vivo (N-67 strain). Among the series, thirteen compounds showed superior antimalarial activity against K1 strain as compared to CQ. In addition, all these analogs showed 100% suppression of parasitaemia on day 4 in the in vivo mouse model against N-67 strain when administered orally. Further, biophysical studies suggest that these compounds act on the heme polymerization target.

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1. Introduction

Despite several advances in drug development over a few decades, malaria still remains a serious health problem in the world because of its high mortality and morbidity burden, as well as its influence on the socio-economic development of the countries where it is endemic.¹ Malaria is caused by a parasitic protozoan of the genus Plasmodium. Among the five species of Plasmodium (P. falciparum, vivax, ovale, malariae and knowlesi) known to infect humans P. falciparum, is the most virulent and is responsible for 300–500 million clinical cases each year, and 3 million deaths annually.² Over the last 60 years the use of quinine has declined owing to the development of synthetic 4-aminoquinolines such as chloroquine (CQ)³ (Fig. 1). Further, CQ and other aminoquinoline derivatives (Fig. 1) exert activity against all plasmodial species in the erythrocytic stage and also they are believed to function by inhibiting the formation of crystalline hemozoin in the P. falciparum digestive vacuole (DV).⁴ The immense success of CQ and other aminoquinoline derivatives is due to their ability to interfere with the heme polymerization process and the ability to accumulate in biochemically relevant concentrations at the site of drug action.⁵ However, their clinical efficacy for the treatment of malaria has been seriously eroded in recent years, due to the emergence and wide-spread of drug resistant parasites. Therefore, there is an urgent need for the development of alternative drugs with unique modes of action that can circumvent the problem of P. falciparum resistance. The CQ scaffold still could be a front line pharmacophore for chemical modification despite the emergence of resistance.⁶

Fig. 1 Structures of some 4-aminoquinolines having antimalarial activity.

Based on this premise a number of research groups have developed a number of 4-aminoquinoline derivatives either by shortening (up to 3 carbons) or by lengthening (up to 10 carbon) the side chain length, and chemoselective modifications at diethylamino functionality of CQ.⁷ Among all CQ analogues, those having shorter chain length consistently show good inhibition against CQ-resistant strain of P. falciparum in the in vitro studies. This observation holds good only for quinoline derivatives that contain a diethyl substituent on the terminal nitrogen.⁸ However, shortening the chain length had no effect on the dealkylation of the distal amino group when administered in vivo, which is the major metabolic pathway of CQ.⁹

However, substitution of bulkier groups at the distal amino side chain led to the compounds that showed better in vivo activity and decrease in the cross resistance with CQ, presumably by circumventing metabolic dealkylation.¹⁰ However, Guy et al. synthesized a side chain modified 4-aminoquinolines (Fig. 1A) by incorporating bulkier substituent including aromatic and heterocyclic ring systems at the lateral part of CQ. Consequently, increased efficacy has been observed against both 3D7 and W2 strains of P. falciparum.¹¹ Furthermore, to overcome the problem of metabolic instability, Chibale et al. synthesized a series of 4-aminoquinolines (Fig. 1B) by incorporating bulkier substituent such as aromatic and tetrazole rings while varying the length of the alkyl side chain and obtained good anti-plasmodial activity in both in vitro (3D7 & K1) and in vivo (P. berghei) models.¹²

Inspired by these encouraging results and in continuation of our efforts to develop effective antimalarial agents, earlier we have reported the design, synthesis and antimalarial activity of some side chain modified 4-aminoquinolines.¹³ Subsequently, we have synthesized a series of 4-aminoquinolines with a variety of heterocyclic ring substituted thiourea functionality in the side chain (Fig. 2A). These analogues showed good activity against CQ-resistant strain (Dd2) of P. falciparum in vitro.¹⁴ Furthermore, we have demonstrated that modification of 4-aminoquinoline lateral side chain with thiazolidine-4-one ring substitution (Fig. 2B) has led to compounds with improved antimalarial activity, and more importantly some of these compounds were found to be more effective than CQ.¹⁵ Further, we introduced biologically privileged thiazolidine heterocyclic ring system (Fig. 2C) at lateral part of the side chain in order to enhance the lipophilic nature this could help the molecule to show better antimalarial activity against resistant strains of parasite. These analogs exhibited promising activity against CQ-sensitive strain of P. falciparum NF-54 in vitro and CQ-resistant strain N-67 of P. yoelii in vivo.¹⁶ Recently we reported a new series of side chain modified 4-aminoquinolines constructed by amide bond, led to the compounds with promising antiplasmodial activity against 3D7 & K1 strains of P. falciparum (Fig. 2D).

Fig. 2 Some lead molecules of 4-aminoquinoline derived antimalarials developed from this laboratory.

Taking a note on above statement, earlier we have reported the synthesis of chiral chloroquine and its analogues obtained by modifying amine side chain derived from various amino acids (Fig. 2E). These chloroquine analogues showed promising activity against P. falciparum (3D7 & K1) in vitro and P. yoelii (N-67 strain) in vivo.¹⁷ Based on the above observations herein we have rationally designed the target compounds (Fig. 2F) with special emphasis on circumventing metabolic N-dealkylation by incorporating bulkier substituents such as piperidinyl, substituted piperidinyl, pyrrolidinyl, morpholinyl and alkyl amines in the side chain. The results are described in the present communication.

2. Results and discussion

2.1 Chemistry

The strategy for the synthesis of the target compounds (10e–10m, 11e–11m and 12e–12j) is outlined in Scheme 1. The reaction of amino acids viz 4–6 with Boc anhydride in the presence of sodium hydroxide in dioxane at 0 °C for 2–3 h with continuous stirring afforded 4a, 5a and 6a in excellent yields. The intermediates obtained were used for the next reaction without further purification. Subsequently, the Boc protected amino acids (4a, 5a and 6a) were subjected to esterification with methyl iodide in the presence of potassium carbonate in DMF at 0 °C to afford 4b, 5b and 6b in good yields. Subsequently, methyl esters were converted to the corresponding alcohols (4c, 5c, and 6c) by NaBH₄ mediated reduction in THF at 65 °C.¹⁸ The obtained alcohols were subjected to mesylation using 3 equiv. of mesyl chloride in THF at 0 °C to give 4d, 5d and 6d in excellent yields, followed by nucleophilic substitution with corresponding amines in CH₃CN afforded 4e–4m, 5e–5m and 6e–6j. Then Boc group was finally deprotected using 15% HCl/dioxane to obtain the corresponding amine hydrochloride salts (7e–7m), (8e–8m) and (9e–9j), which were finally treated with 4,7-dichloroquinoline in phenol at 160 °C to obtain the target compounds in good yields.

Scheme 1 Synthesis of compounds (10e–10m, 11e–11m and 12e–12j); reagents and conditions: (a) (Boc)₂O, NaOH/water, dioxane, 0 °C, 1 h; (b) MeI/K₂CO₃, DMF, 0 °C, 6–8 h; (c) NaBH₄, CH₃OH, THF, 65 °C, 1 h; (d) MsCl/TEA, anhydrous THF, N₂ atm, 0 °C, 1 h; (e) amine, CH₃CN/TEA, N₂ atm, rt, 40 h; (f) 15% HCl/dioxane, 1 h; (g) 4,7-DCQ, phenol, 160 °C, 4–6 h.

2.2 In vitro antiplasmodial activity

The final compounds synthesized (10e–10m, 11e–11m, and 12e–12j) in the present study were evaluated for antiplasmodial activity against CQ-sensitive (3D7) & CQ-resistant (K1) strains of P. falciparum in vitro according to the procedure reported by Kondaparla et al. and the results are shown in Table 1.¹⁹ Some of the potent molecules, which have shown better activity than CQ against K1 strain, were evaluated for in vivo activity against the innately chloroquine resistant P. yoelii (N-67 strain) in Albino mice of Swiss strain (Table 2). The in vitro activity data (IC₅₀) showed that these derivatives, having bulky cyclic substituent in the side chain as well as diversity in the amino acid, showed very good antiplasmodial activity. Suggesting that modification at the side chain nitrogen atom is very well tolerated for antimalarial activity.

Table 1 Biological and biophysical data of the synthesized compounds

Cmpd no.	IC50^a (nM)		SI^b	CC50^c (nM)	log K^d	IC50^e (μM)
	3D7	K1
a IC50 (nM): concentration corresponding to 50% growth inhibition of the parasite.b Selectivity index (SI): (CC50 for cytotoxicity to vero cells/IC50(K1) for antiplasmodial activity).c Cytotoxicity (CC50): SI multiply with IC50 (K1).d 1 : 1 (compound : hematin) complex formation in 40% aqueous DMSO, 20 mM HEPES buffer, pH 7.5 at 25 °C (data are expressed as mean ± SD from at least three different experiments in triplicate).e The IC50 represents the millimolar equivalents of test compounds, relative to hemin, required to inhibit β-hematin formation by 50% (data are expressed as mean ± SD from at least three different experiments in triplicate): N.D stands for not done.
10e	945	395	173	68 260	5.22 ± 0.02	0.78 ± 0.05
10f	1439	147	688	101 250	4.92 ± 0.02	0.75 ± 0.02
10g	1911	141	614	86 510	4.96 ± 0.02	0.73 ± 0.01
10h	1313	58	1421	82 430	4.43 ± 0.01	0.62 ± 0.05
10i	1845	86	1033	88 830	4.26 ± 0.02	0.43 ± 0.02
10j	2783	613	220	134 810	6.12 ± 0.02	1.02 ± 0.07
10k	>5000	99	396	39 160	4.73 ± 0.01	0.48 ± 0.02
10l	>5000	89	1227	109 180	4.69 ± 0.02	0.49 ± 0.03
10m	41	2693	11	29 120	6.43 ± 0.01	0.96 ± 0.12
11e	>5000	395	93	36 760	5.13 ± 0.01	0.78 ± 0.02
11f	>5000	46	1591	73 180	5.25 ± 0.02	0.76 ± 0.02
11g	7	938	37	35 010	5.83 ± 0.01	0.82 ± 0.02
11h	578	93	419	38 970	4.83 ± 0.02	0.63 ± 0.03
11i	1134	1610	65	104 830	5.96 ± 0.01	0.92 ± 0.02
11j	112	78	466	36 360	4.74 ± 0.02	0.53 ± 0.03
11k	12	183	543	99 430	4.72 ± 0.02	0.59 ± 0.02
11l	>5000	118	763	90 030	5.32 ± 0.02	0.79 ± 0.05
11m	>5000	>5000	>10	53 850	7.13 ± 0.01	1.10 ± 0.18
12e	80	>5000	>14	71 120	5.41 ± 0.02	0.81 ± 0.03
12f	14	21	4803	100 860	4.19 ± 0.02	0.25 ± 0.02
12g	77	>5000	>28	144 810	6.83 ± 0.02	1.11 ± 0.03
12h	>5000	>5000	>24	123 580	7.26 ± 0.02	1.24 ± 0.15
12i	>5000	164	692	113 460	6.53 ± 0.01	1.08 ± 0.05
12j	74	4580	26	116 940	6.85 ± 0.01	1.13 ± 0.02
CQ	5	255 ± 65	490	125 000	5.52 ± 0.02	0.17 ± 0.02
Artemether	1.37	1.32 ± 0.11	N.D	N.D	N.D	N.D

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Table 2 In vivo antimalarial activity of selected compounds against CQ resistant (N-67) in Albino mice of Swiss strain

Cmpd no.	Dose	Route of administration	Percent suppression on day 4 postinfection	Survival^a	Cure^b
a Number of mice that survived till day 28 post-infection/total mice in the group.b Number of mice without parasitaemia (cured) till day 28 post-infection.c Route of administration intramuscular.
10f	100 mg kg^−1 × 4 days	Oral	100	5/5	3/5 cured
10g	100 mg kg^−1 × 4 days	Oral	100	4/5	4/5 cured
10h	100 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	50 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	25 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	12.5 mg kg^−1 × 4 days	Oral	100	4/5	2/5 cured
10i	100 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	50 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	25 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	12.5 mg kg^−1 × 4 days	Oral	100	0/5	None cured
10k	100 mg kg^−1 × 4 days	Oral	100	2/2 (3 died during treatment)	2/2 cured
10l	100 mg kg^−1 × 4 days	Oral	100	0/5	None cured
11f	100 mg kg^−1 × 4 days	Oral	100	0/5	None cured
11g	100 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	50 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	25 mg kg^−1 × 4 days	Oral	100	2/5	1/5 cured
11h	100 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	50 mg kg^−1 × 4 days	Oral	100	4/5	4/5 cured
11j	100 mg kg^−1 × 4 days	Oral	100	4/5	3/5 cured
11k	100 mg kg^−1 × 4 days	Oral	100	0/5	None cured
11l	100 mg kg^−1 × 4 days	Oral	100	0/5	None cured
12f	100 mg kg^−1 × 4 days	Oral	100	5/5	5/5 cured
	50 mg kg^−1 × 4 days	Oral	100	3/5	None cured
12i	100 mg kg^−1 × 4 days	Oral	100	0/5	None cured
CQ	20 mg kg^−1 × 4 days	Oral	99.0	5/5	0/5
Arteether	5 mg kg^−1 × 4 days (i.m)^c	i.m	100	5/5	5/5

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Among the 24 compounds tested, four compounds showed IC₅₀ range between 7–41 nM, three compounds had IC₅₀ ranging between 74–80 nM, three compounds exhibited IC₅₀ ranging between 112–945 nM, and IC₅₀ ranging between 1134–2783 nM was observed in six compounds. The remaining 8 compounds have shown IC₅₀ values above 5000 nM, when screened against chloroquine sensitive (3D7) strain of P. falciparum. Further, out of these 24 compounds, 13 compounds were found to be more active (with IC₅₀ between 21–183 nM) against the chloroquine resistant (K1) strain as compared to CQ (IC₅₀ = 255 nM). Two compounds (10e and 11e) showed IC₅₀ values of 395 and 395 nM which were comparable to CQ. The differences in the IC₅₀ values can be attributed to factors such as ring size, diversity in the amino acid side chain, position of methyl group on the piperidyl ring and type of dialkyl amine in the side chain.

The compounds 10f and 12f having methyl substitution at 3^rd position on piperidyl ring exhibited mild inhibition against 3D7 strain and superior potency against K1 strain, as compared to CQ. Whereas compound 11f was found to be inactive against 3D7 strain but found to be second most potent molecule in the series against K1 strain with IC₅₀ value 46 nM. On the other hand 4-methyl piperidyl analogue (10g, IC₅₀ = 141 nM) showed good activity against K1 strain than corresponding phenylalanine (11g, IC₅₀ = 938 nM) and alanine (12g, IC₅₀ = >5000 nM) derivatives. Among these analogues compound 11g (IC₅₀ = 7 nM) exhibited very akin antiplasmodial activity against 3D7 strain as compared to CQ. It may be appropriate to mention here that in the case of leucine (10e, 10f and 10g) improved activity was observed against K1 strain when the methyl group was change to 2^nd, 3^rd and 4^th position on piperidyl ring.

Further, in the case of an unsubstituted piperidyl compound 10h (IC₅₀ = 58 nM, leucine analogue), the activity increased significantly against the K1 strain when compared to its methyl substituted compounds. Whereas compound 11h (IC₅₀ = 93 nM, phenylalanine analogue) with similar moiety was found to be more active against K1 strain than its 2-methyl piperidyl (11e, IC₅₀ = 395 nM) and 4-methyl piperidyl (11g, IC₅₀ = 938 nM) analogues but it showed slightly less potency than 3-methyl piperidyl compound (11f, IC₅₀ = 46 nM) against K1 strain. This activity difference may be due to variation in the hydrophobic nature of the amino acid side chain. Moreover, in the case of leucine slight reduction in the antiplasmodial activity was observed in both strains (3D7 & K1) of P. falciparum when the size of the ring reduced to pyrrolidino (compound 10i, IC₅₀ = 1845 & 86 nM). On the other hand pyrrolidino analogue 11j (IC₅₀ = 112 & 78 nM) of phenylalanine was found to be slightly more active in both strains (3D7 & K1) of P. falciparum as compared to compound 11h (IC₅₀ = 578 & 93 nM). Whereas coming to alanine pyrrolidino analogue 12i (IC₅₀ = 164 nM) showed very good activity against K1 strain than piperidyl derivative 12h (IC₅₀ = >5000 nM). It is important to note that compounds (10i, 11j and 12i) exhibited 2.9 fold, 3.2 fold and 1.5 fold superior activities against K1 strain as compared to CQ. Further, introduction of morpholino in the side chain led the compounds 10j and (IC₅₀ = 2783 & 613 nM) 11i (IC₅₀ = 1134 & 1610 nM) with many fold decrease in the activity in both 3D7 & K1 strains of P. falciparum. This difference in the activity may be due to diversity in their lipophilic nature.

Further, with a view to modify the lipophilic nature of the side chain, another modification was performed in the series i.e. the replacement of cyclic amine with acyclic amines (dimethyl, diethyl and di-n-propylamine) at the side chain of 4-aminoquinoline to furnish compounds 10k–10m, 11k–11m and 12j respectively. However, in the case of leucine compounds 10k and 10l were found to be less active against 3D7 strain of P. falciparum, when compared to its cyclic derivatives. Nevertheless, some of the compounds (10k, 10l, 11k and 11l) exhibited much better activity (with IC₅₀ values 99 nM, 89 nM, 183 nM and 118 nM) against K1 strain as compared to CQ. Furthermore, the activity of the compounds (10k, 10l and 10m) against K1 strain is directly proportional to the branching of the alkyl amine side chain. The present results suggest that these 4-aminoquinolines appear to be promising candidates for further lead optimization to obtain compounds active against CQ-resistant parasites.

2.3 In vitro cytotoxicity

The cytotoxicity of all the synthesized molecules was determined against VERO cell line using MTT assay (Table 1). Most of the compounds in the series showed high selectivity indices (SI) values. Out of the 24 compounds, 17 compounds showed high selectivity indices (SI) ranging between 65 and 4803, rest of molecules showed selectivity index < 50. Compounds 10h, 10i, 10k, 10l and 11f which were most potent against K1 strain also showed good selectivity index 1421, 1033, 396, 1227 and 1591 respectively (Table 1). Further, compound 12f which was most potent in the series against resistant strain K1, also showed good selectivity index of 4803. In general, most of the compounds of the series exhibited greater potency than CQ against K1 strain, less cytotoxic effect with fairly high selectivity index, and therefore these 4-aminoquinoline derivatives appear to be good candidate for further lead optimization.

2.4 In vitro inhibition of β-hematin polymerization

The mode of action of these 4-aminoquinoline derivatives (10e–10m, 11e–11m, and 12e–12j) was investigated by the reported method and the results are shown in (Table 1). The results of heme binding assay showed that all the synthesized compounds form complex with hematin and the range of log K was found to be 4.19–7.26. Among all the compounds reported, compounds 11f and 12f have shown good binding to hematin. This result is concurrent with the previous results reported in the literature. The data suggest that the principle interaction may be hydrophobic as well as electrostatic between the 4-aminoquinoline ring and the porphyrin ring system that plays a role in hematin binding. The binding data suggest that the mechanism of action is similar to chloroquine class of molecules and there is no linear correlation between the heme binding and the in vivo activity because of PK/ADME considerations that influence the in vivo activity. Therefore in the present study heme binding data is used only to reiterate the mechanism of action.

All the synthesized 4-aminoquinoline derivatives (10e–10m, 11e–11m, and 12e–12j) inhibited the β-hematin formation in a concentration dependent manner (Table 1). The IC₅₀ obtained was in the range of 0.25–1.24 μM and the most active compound 12f in the series against K1 strain exhibited IC₅₀ (0.25 ± 0.02 μM).

2.5 In vivo antimalarial activity

Selected compounds that were significantly active in vitro (10f–10i, 10k, 10l, 11f–11h, 11j–11l, 12f and 12i) were evaluated for in vivo activity against innately chloroquine resistant P. yoelii (N-67 strain) in Albino mice of Swiss strain. Initially, the in vivo activity of selected compounds were determined through oral route at the dose of 100 mg kg⁻¹ administered once daily for four consecutive days and monitored for parasitaemia reduction, and survival of mice until day 28 post-infection (Table 2).

Compound 10l, 11f, 11k, 11l and 12i showed 100% parasitaemia suppression on day 4 at a dose of 100 mg kg⁻¹, but none of the mice survived up to day 28. Further, compounds 10g and 11j displayed 100% parasitaemia suppression on day 4 with 80% survival rate up to day 28 of treatment at a dose of 100 mg kg⁻¹. Among these compound 10g cured 4 mice out of 5 whereas compound 11j showed 60% curative rate only. Further, compounds 11g, 11h and 12f administered at two different doses 100 and 50 mg kg⁻¹. At a dose of 100 mg kg⁻¹ all three compounds showed 100% parasitaemia suppression on day 4 with 100% survival and curative rates as well. While, at lower dose 50 mg kg⁻¹ compound 11h displayed 100% parasitaemia suppression on day 4 with 80% survival rate up to day 28 and four mice out of five were cured. Whereas, compound 12f exhibited 100% parasitaemia suppression on day 4, but none of the mice were cured. However, compound 11g showed 100% parasitaemia suppression on day 4 with 100% curative rate at a dose of 50 mg kg⁻¹, but at lower dose 25 mg kg⁻¹ one mouse out of five was cured with 100% parasitaemia suppression on day 4. Furthermore, most active compounds (10h and 10i) against K1 strain, tested initially at a dose of 100 mg kg⁻¹, showed 100% survival as well as curative rates up to day 28. Further, these two compounds were administered at lower doses such as 50, 25 and 12.5 mg kg⁻¹. They displayed 100% parasitaemia suppression on day 4 with all mice were cured up to day 28 at doses of 50 and 25 mg kg⁻¹. Whereas, at a dose of 12.5 mg kg⁻¹ the compound 10h exhibited 100% parasitaemia suppression on day 4 with 80% survival rate up to day 28 and 2 mice were cured out of 5. Whereas, compound 10i at 12.5 mg kg⁻¹ showed 100% parasitaemia on day 4 but none of the mice survived up to day 28 of the treatment.

3. Conclusion

In summary, a novel series of side chain modified 4-aminoquinoline derivatives were prepared in an attempt to search for more potent molecule that can be effective against both CQ sensitive and resistant strains of P. falciparum. The in vitro antimalarial result of these compounds indicate that in comparison to reference drug CQ, 13 compounds (10f–10i, 10k–10l, 11f, 11h, 11j, 11k, 11l, 12f and 12i) showed superior activity against K1 strain of P. falciparum. Further, the in vivo results on selected compounds revealed that these compounds also show good antimalarial activity in the mouse model. The biochemical studies confirm that the mechanism of action is similar to that of CQ, as most of the compounds form an association complex with hematin and thereby inhibit hemozoin formation. In summary results discussed in the manuscript suggest that the presence of bulky cyclic substituent in the side chain and less hindred amino acids at 4^th position of quinoline exhibit potent activity against K1 strain and require further lead optimization.

4. Experimental procedures

4.1 General information

Melting points (mp) were taken in open capillaries on Complab melting point apparatus and are uncorrected. The ¹H NMR (300, 400 MHz) and ¹³C NMR (100, 125 MHz) spectra were recorded in CDCl₃ solvent on DPX-300, and 400 Bruker FT-NMR spectrometers. All chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane. The splitting pattern abbreviations are as follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), br s (broad singlet) and m (multiplet). Coupling constants are given in hertz. Mass spectra (ESI-MS), high resolution mass spectra HRMS (ESI-HRMS) were recorded on Jeol (Japan)/SX-102 and Agilent 6520 QTOF (ESI-HRMS) spectrometers respectively. Analytical thin-layer chromatography (TLC) was carried out on Merck's pre-coated silica-gel plates 60 F₂₅₄ and spots were visualized by irradiation with UV light (254 nm). Iodine was used as developing agent and/or by spraying with Dragendorff's reagent. Column chromatographic purification was performed over neutral alumina and silica gel (60–120, 100–200 and 230–400 mesh) using a gradient solvent system (n-hexane/EtOAc, DCM/hexane or chloroform/methanol as the eluent unless otherwise specified). All chemicals and reagents were obtained from Sigma Aldrich (St. Louis, MO, USA), Lancaster (Port of Heysham Industrial Park, Lancashire LA3 2XY, UK) and Spectrochem (Anand Bhavan, Princess Street, Mumbai, India) and were used without further purification.

4.2 General procedure for the synthesis of 4a, 5a and 6a

To a suspension of amino acid (leucine, phenylalanine and alanine) (1.0 equiv.) in dioxane–water mixture (1 : 1, 100 mL) at room temperature was added 2 N NaOH with stirring. The reaction mixture was then cooled to 0 °C and stirred for 15 min. Finally, (Boc)₂O (1.1 equiv.) in dioxane (10 mL) was added and the reaction mixture was allowed to stir at 0 °C for 45 min. The ice bath was then removed and the temperature of the reaction mixture was allowed to rise to the room temperature. On completion of the reaction as monitored by the TLC reaction mixture was concentrated under reduced pressure. The aqueous layer was acidified with citric acid (pH 2–3) and extracted with EtOAc. The combined organic layer was washed with brine, dried over Na₂SO₄ and concentrated under reduced pressure. The yields were quantitative. These intermediates were used for the next step without further purification.

4.3 General procedure for the synthesis of 4b, 5b and 6b

To a suspension of 4a, 5a and 6a (1.0 equiv.) in DMF (40 mL) at 0 °C, after 15 min K₂CO₃ (2.5 equiv.) was added. Then after 15 min, methyl iodide (1.5 equiv.) was added drop wise and the reaction mixture stirred (open air) from 0 °C to room temperature over 6–8 h. The reaction mixture was diluted with EtOAc (300 mL) and the organic layer was washed twice with 5% sodium bicarbonate and finally with brine. The organic layer was dried over Na₂SO₄, and concentrated to a gummy substance to get 4b, 5b and 6b. The yields were quantitative. This was used for the next step without further purification.

4.4 General procedure for the synthesis of 4c, 5c and 6c

Methanol (0.8 mL) was added drop wise over a period of 20 min to a mixture of N-protected amino ester (1 mmol) and NaBH₄ (2 mmol) in THF at 65 °C. The mixture was stirred for 1 h, and then water was added to the reaction mixture. Organic solvent was concentrated under reduced pressure and brine was added and the mixture was extracted with EtOAc. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue obtained was purified on the silica gel column chromatography using hexane:EtOAc as eluant.

4.4.1 (S)-tert-Butyl 1-hydroxy-4-methylpentan-2-ylcarbamate (4c). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (300 MHz, CDCl₃): δ 0.93 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.05 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.39 (s, 9H, C(CH₃)₃), 1.61–1.55 (m, 2H, CH₂CH(CH₃)₂), 1.75–1.70 (m, 1H, CH(CH₃)₂), 3.39–3.42 (dd, J = 5.4, 6.9 Hz, 2H, CH₂OH), 4.43 (brs, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 23.4, 25.4, 28.3, 42.6, 55.3, 61.4, 78.5, 155.4; ESI-MS: (m/z) 218.2 (M + H⁺); HRMS calcd for [C₁₁H₂₄NO₃ + H]⁺ 218.1751, found 218.1757.

4.4.2 (S)-tert-Butyl 1-hydroxy-3-phenylpropan-2-ylcarbamate (5c). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (300 MHz, CDCl₃): δ 1.42 (s, 9H, C(CH₃)₃), 2.84 (d, J = 7.0 Hz, 2H, CH₂C₆H₅), 3.49–3.70 (m, 2H, CH₂OH), 3.87 (brs, 1H, NHCH), 4.69 (t, J = 7.2 Hz, 1H, CH₂OH), 7.20–7.30 (m, 5H, CH₂C₆H₅); ¹³C NMR (100 MHz, CDCl₃): δ 28.3, 36.6, 58.6, 65.4, 78.4, 125.4, 127.7, 129.3, 137.7, 155.3; ESI-MS: (m/z) 252.3 (M + H⁺); HRMS calcd for [C₁₄H₂₂NO₃ + H]⁺ 252.1594, found 252.1597.

4.4.3 (S)-tert-Butyl 1-hydroxypropan-2-ylcarbamate (6c). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (300 MHz, CDCl₃): δ 1.26 (d, J = 6.4 Hz, 3H, CHCH₃), 1.46 (s, 9H, C(CH₃)₃), 3.30 (d, J = 4.8 Hz, 2H, CH₂OH), 3.72 (m, 1H, CHCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 17.5, 28.5, 52.4, 65.4, 78.5, 155.4; ESI-MS: (m/z) 176.2 (M + H⁺); HRMS calcd for [C₈H₁₈NO₃ + H]⁺ 176.1281, found 176.1286.

4.5 General procedure for the synthesis of 4d, 5d and 6d

To a suspension of 4c, 5c and 6c (1.0 equiv.) in anhydrous THF under nitrogen atmosphere was added triethylamine (3.0 equiv.). The mixture was cooled to 0 °C. Methanesulfonyl chloride (3.0 equiv.) was added slowly, keeping the temperature below 5 °C, and the reaction mixture was stirred in an ice bath for 1 h. After dilution with saturated NaHCO₃ solution, the aqueous layer was extracted with DCM. The combined organic extracts dried over Na₂SO₄ and concentrated under reduced pressure to afford 4d, 5d and 6d. These intermediates were used for the next step without further purification.

4.6 General procedure for the synthesis of 4e–4m, 5e–5m and 6e–6j

To a suspension of 4d, 5d and 6d (1.0 equiv.) in acetonitrile under nitrogen atmosphere was added triethylamine (2.0 equiv.) and amine (4.0 equiv.). The reaction mixture was stirred for 40 h at room temperature. After completion of the reaction (as monitored by TLC), the solvent was concentrated under reduced pressure and the residue dissolved in DCM, the organic layer was washed with 10% citric acid, finally the aqueous layer was basified with NaHCO₃ and extracted with DCM. The combine organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue obtained was purified on the silica gel (60–120 mesh) column chromatography using hexane:EtOAc as eluant.

4.6.1 tert-Butyl (2S)-4-methyl-1-(2-methylpiperidin-1-yl)pentan-2-ylcarbamate (4e). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.94 (d, J = 6.3 Hz, 3H, CH₂CHCH₃CH₃), 1.07 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.17 (d, J = 6.3 Hz, 3H, N(CH₂)₄CHCH₃), 1.29 (s, 9H, C(CH₃)₃), 1.34–1.46 (m, 6H, NCH₂(CH₂)₃CHCH₃), 1.56–1.78 (m, 5H, CH₂NCH₂(CH₂)₃CHCH₃), 2.37–2.52 (m, 1H, CH₂CH(CH₃)₂), 2.74–2.90 (m, 2H, NHCHCH₂N), 3.57–3.64 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 18.3, 22.2, 22.7, 23.6, 25.1, 26.3, 28.3, 34.3, 42.3, 54.4, 57.5, 63.2, 68.5, 78.5, 155.3; ESI-MS: (m/z) 299.2 (M + H⁺); HRMS calcd for [C₁₇H₃₅N₂O₂ + H]⁺ 299.2693, found 299.2696.

4.6.2 tert-Butyl (2S)-4-methyl-1-(3-methylpiperidin-1-yl)pentan-2-ylcarbamate (4f). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.92 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.09 (d, J = 6.3 Hz, 3H, CH₂CHCH₃CH₃), 1.17 (d, J = 6.4 Hz, 3H, N(CH₂)₄CHCH₃), 1.31 (s, 9H, C(CH₃)₃), 1.38–1.48 (m, 6H, NCH₂(CH₂)₃CHCH₃), 1.63–1.76 (m, 5H, CH₂NCH₂(CH₂)₃CHCH₃), 2.36–2.54 (m, 1H, CH₂CH(CH₃)₂), 2.74–2.89 (m, 2H, CH₂CH(CH₃)₂), 3.61–3.64 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 18.4, 22.3, 22.6, 23.6, 25.3, 27.3, 28.3, 34.2, 42.3, 53.4, 57.6, 63.7, 68.3, 78.2, 155.2; ESI-MS: (m/z) 299.2 (M + H⁺); HRMS calcd for [C₁₇H₃₅N₂O₂ + H]⁺ 299.2693, found 299.2699.

4.6.3 (S)-tert-Butyl 4-methyl-1-(4-methylpiperidin-1-yl)pentan-2-ylcarbamate (4g). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.87 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 0.96 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.06 (d, J = 6.3 Hz, 3H, N(CH₂)₄CHCH₃), 1.22–1.30 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 1.38 (s, 9H, C(CH₃)₃), 1.46–1.53 (m, 1H, N(CH₂)₂(CH₂)₂CHCH₃), 1.63–1.84 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 1.92–1.99 (m, 1H, CHC(H) (H)CH(CH₃)₂), 2.18–2.26 (m, 1H, CHC(H)(H)CH(CH₃)₂), 2.58 (d, J = 6.6 Hz, 2H, NHCHCH₂N), 2.68 (d, J = 11.4 Hz, 1H, CHC(H)(H)N), 2.89 (d, J = 11.3 Hz, 1H, CHC(H)(H)N), 3.64–3.71 (m, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 19.4, 23.3, 23.8, 24.3, 28.3, 33.2, 34.7, 42.3, 48.4, 51.6, 63.7, 78.2, 155.2; ESI-MS: (m/z) 299.3 (M + H⁺); HRMS calcd for [C₁₇H₃₅N₂O₂ + H]⁺ 299.2693, found 299.2692.

4.6.4 (S)-tert-Butyl 4-methyl-1-(piperidin-1-yl)pentan-2-ylcarbamate (4h). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.95 (d, J = 6.6 Hz, 3H, CH₂CHCH₃CH₃), 1.06 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.33 (s, 9H, (CH₃)₃), 1.41–1.59 (m, 6H, piperidine), 1.68–1.74 (m, 2H, CH₂CHCH₃CH₃), 1.77–1.84 (m, 1H, CH₂CHCH₃CH₃), 2.43–2.46 (m, 4H, piperidine), 2.58–2.63 (m, 2H, NHCHCH₂N), 3.66–3.73 (m, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 23.2, 23.6, 24.3, 24.7, 25.9, 28.4, 42.4, 52.2, 58.3, 62.8, 79.4, 155.4; ESI-MS: (m/z) 285.3 (M + H⁺); HRMS calcd for [C₁₆H₃₃N₂O₂ + H]⁺ 285.2537, found 285.2539.

4.6.5 (S)-tert-Butyl 4-methyl-1-(pyrrolidin-1-yl)pentan-2-ylcarbamate (4i). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.93 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.04 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.52–1.59 (m, 1H, CH₂CHCH₃CH₃), 1.64–1.69 (m, 2H, CH₂CHCH₃CH₃), 1.73–1.82 (m, 4H, pyrrolidine), 2.57–2.61 (m, 4H, pyrrolidine), 2.69–2.74 (q, J = 12.1 Hz, 1H, NHCHC(H)(H)N), 2.77–2.83 (q, J = 12.3 Hz, 1H, NHCHC(H)(H)N), 3.66–3.77 (m, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 23.3, 23.5, 24.6, 25.2, 28.3, 43.4, 50.4, 56.3, 62.7, 78.6, 155.4; ESI-MS: (m/z) 271.3 (M + H⁺); HRMS calcd for [C₁₅H₃₁N₂O₂ + H]⁺ 271.2380, found 271.2383.

4.6.6 (S)-tert-Butyl 4-methyl-1-morpholinopentan-2-ylcarbamate (4j). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.92 (d, J = 6.2 Hz, 3H, CH₂CHCH₃CH₃), 1.08 (d, J = 6.6 Hz, 3H, CH₂CHCH₃CH₃), 1.29 (s, 9H, C(CH₃)₃), 1.39–1.47 (m, 2H, CH₂CHCH₃CH₃), 1.71–1.80 (m, 1H, CH₂CHCH₃CH₃), 2.46 (t, J = 4.7 Hz, 4H, morpholine), 2.62 (d, J = 6.4 Hz, 2H, NHCHCH₂N), 3.69 (t, J = 4.5 Hz, 4H, morpholine), 3.90–4.12 (m, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 23.6, 24.8, 28.8, 43.5, 51.2, 56.6, 64.5, 66.6, 78.4, 155.4; ESI-MS: (m/z) 287.2 (M + H⁺); HRMS calcd for [C₁₅H₃₁N₂O₃ + H]⁺ 287.2329, found 287.2331.

4.6.7 (S)-tert-Butyl 1-(diethylamino)-4-methylpentan-2-ylcarbamate (4k). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.96 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.02 (t, J = 7.1 Hz, 6H, N(CH₂)₂(CH₃)₂), 1.08 (d, J = 6.3 Hz, 3H, CH₂CHCH₃CH₃), 1.38 (s, 9H, C(CH₃)₃), 1.44–1.52 (m, 1H, CH₂CHCH₃CH₃), 1.67–1.72 (m, 2H, CH₂CHCH₃CH₃), 1.74–1.85 (m, 2H, NHCHCH₂N), 2.48–2.61 (m, 4H, N(CH₂)₂(CH₃)₂), 2.69–2.73 (dd, J = 5.8, 13.0 Hz, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 14.3, 23.4, 24.8, 28.6, 42.4, 48.8, 51.2, 63.5, 78.8, 155.3; ESI-MS: (m/z) 273.2 (M + H⁺); HRMS calcd for [C₁₅H₃₃N₂O₂ + H]⁺ 273.2537, found 273.2539.

4.6.8 (S)-tert-Butyl 1-(dipropylamino)-4-methylpentan-2-ylcarbamate (4l). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.86 (t, J = 7.4 Hz, 6H, N(CH₂)₂(CH₂)₂(CH₃)₂), 0.93 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.06 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.33 (s, 9H, C(CH₃)₃), 1.41–1.54 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 1.59–1.77 (m, 2H, CH₂CHCH₃CH₃), 2.03–2.08 (m, 1H, CH₂CHCH₃CH₃), 2.34–2.45 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.56–2.69 (m, 2H, NHCHCH₂N), 3.53–3.59 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 11.4, 11.8, 23.6, 24.7, 28.4, 42.6, 49.5, 58.4, 63.6, 79.6, 155.6; ESI-MS: (m/z) 301.2 (M + H⁺); HRMS calcd for [C₁₇H₃₇N₂O₂ + H]⁺ 301.2850, found 301.2854.

4.6.9 (S)-tert-Butyl 1-(dimethylamino)-4-methylpentan-2-ylcarbamate (4m). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.93 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.07 (d, J = 6.3 Hz, 3H, CH₂CHCH₃CH₃), 1.34 (s, 9H, C(CH₃)₃), 1.49–1.57 (m, 1H, CH₂CHCH₃CH₃), 1.65–1.72 (m, 2H, CH₂CHCH₃CH₃), 2.27 (s, 6H, CH₂N(CH₃)₂), 2.48–2.51 (dd, J = 5.6, 12.3 Hz, 1H, NHCHC(H)(H)N), 2.54–2.59 (dd, J = 7.6, 12.3 Hz, 1H, NHCHC(H)(H)N), 3.64–3.70 (m, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 23.2, 23.7, 24.8, 28.6, 42.5, 46.2, 48.4, 63.6, 78.6, 155.6; ESI-MS: (m/z) 245.2 (M + H⁺); HRMS calcd for [C₁₃H₂₉N₂O₂ + H]⁺ 245.2224, found 245.2229.

4.6.10 tert-Butyl (2S)-1-(2-methylpiperidin-1-yl)-3-phenylpropan-2-ylcarbamate (5e). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.87 (d, J = 6.6 Hz, 3H, N(CH₂)₄CHCH₃), 1.36 (s, 9H, C(CH₃)₃), 1.46–1.67 (m, 6H, NCH₂CH₂CH₂CH₂CHCH₃), 2.36–3.17 (m, 7H, PhCH₂ (CH)CH₂NCH₂(CH₂)₃CHCH₃), 3.83–3.90 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 14.4, 23.2, 26.2, 28.3, 33.2, 38.6, 55.3, 57.2, 61.4, 65.6, 79.1, 125.6, 128.3, 128.6, 129.6, 130.1, 135.4, 155.5; ESI-MS: (m/z) 333.2 (M + H⁺); HRMS calcd for [C₂₀H₃₃N₂O₂ + H]⁺ 333.2537, found 333.2539.

4.6.11 tert-Butyl (2S)-1-(3-methylpiperidin-1-yl)-3-phenylpropan-2-ylcarbamate (5f). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.84 (d, J = 6.2 Hz, 3H, N(CH₂)₃CHCH₃), 1.36 (s, 9H, C(CH₃)₃), 1.76–2.29 (m, 7H, piperidine), 2.54–3.18 (m, 6H, PhCH₂(CH)CH₂NCH₂CH), 3.82–3.86 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 18.8, 23.8, 27.6, 28.4, 32.5, 39.1, 54.6, 57.7, 62.4, 65.5, 78.8, 125.4, 128.2, 128.4, 129.4, 130.3, 137.4, 155.4; ESI-MS: (m/z) 333.4 (M + H⁺); HRMS calcd for [C₂₀H₃₃N₂O₂ + H]⁺ 333.2537, found 333.2541.

4.6.12 (S)-tert-Butyl 1-(4-methylpiperidin-1-yl)-3-phenylpropan-2-ylcarbamate (5g). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.89 (d, J = 6.2 Hz, 3H, N(CH₂)₂(CH₂)₂CHCH₃), 1.03–1.09 (m, 1H, N(CH₂)₂(CH₂)₂CHCH₃), 1.29 (s, 9H, C(CH₃)₃), 1.36–1.55 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 1.86–1.91 (m, 1H, NCH₂C(H)(H)(CH₂)₂CHCH₃), 2.13–2.19 (m, 1H, NCH₂C(H)(H)(CH₂)₂CHCH₃), 2.44–2.48 (dd, J = 6.2, 12.4 Hz, 1H, NHCHC(H)(H)N), 2.56–2.61 (dd, J = 8.4, 12.6 Hz, 1H, NHCHC(H)(H)N), 2.66–2.69 (m, 1H, NC(H)(H)CH₂(CH₂)₂CHCH₃), 2.84–2.89 (m, 1H, NC(H)(H)CH₂(CH₂)₂CHCH₃), 2.95–2.99 (dd, J = 7.2, 13.7 Hz, 1H, NHCHC(H)(H)Ph), 3.15–3.19 (dd, J = 4.2, 13.6 Hz, 1H, NHCHC(H)(H)Ph), 3.86 (s, 1H, NHCHCH₂Ph); ¹³C NMR (100 MHz, CDCl₃): δ 21.4, 28.4, 32.1, 34.3, 34.6, 38.6, 47.6, 54.5, 61.8, 78.6, 125.3, 128.2, 128.6, 129.7, 130.5, 137.7, 155.1; ESI-MS: (m/z) 333.3 (M + H⁺); HRMS calcd for [C₂₀H₃₃N₂O₂ + H]⁺ 333.2537, found 333.2538.

4.6.13 (S)-tert-Butyl 1-phenyl-3-(piperidin-1-yl)propan-2-ylcarbamate (5h). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.38 (s, 9H, C(CH₃)₃), 1.47–1.58 (m, 6H, piperidine), 2.37–2.42 (m, 4H, piperidine), 2.48–2.54 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.61–2.66 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.89–2.94 (q, J = 13.7 Hz, 1H, NHCHC(H)(H)Ph), 3.14–3.18 (q, J = 13.7 Hz, 1H, NHCHC(H)(H)Ph), 3.86 (s, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 24.3, 25.8, 28.3, 38.6, 51.8, 56.3, 61.8, 78.7, 125.7, 128.6, 128.1, 129.3, 130.4, 137.8, 155.4; ESI-MS: (m/z) 319.3 (M + H⁺); HRMS calcd for [C₁₉H₃₁N₂O₂ + H]⁺ 319.2380, found 319.2383.

4.6.14 (S)-tert-Butyl 1-morpholino-3-phenylpropan-2-ylcarbamate (5i). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.39 (s, 9H, C(CH₃)₃), 3.23–3.26 (m, 4H, morpholine), 3.37–3.42 (m, 1H, NHCH), 3.66–3.72 (m, 4H, morpholine), 3.89 (m, 2H, CH₂-morpholine), 3.94–3.99 (m, 2H, CH₂Ph); ¹³C NMR (100 MHz, CDCl₃): δ 28.4, 38.7, 54.6, 56.2, 65.4, 79.2, 125.5, 128.4, 128.5, 129.6, 130.6, 137.4, 155.2; ESI-MS: (m/z) 321.4 (M + H⁺); HRMS calcd for [C₁₈H₂₉N₂O₃ + H]⁺ 321.2173, found 321.2177.

4.6.15 (S)-tert-Butyl 1-phenyl-3-(pyrrolidin-1-yl)propan-2-ylcarbamate (5j). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.39 (s, 9H, C(CH₃)₃), 1.74–1.78 (m, 4H, pyrrolidine), 2.49–2.52 (m, 4H, pyrrolidine), 2.58–2.64 (q, J = 12.6 Hz, 1H, NHCHC(H)(H)N), 2.77–2.83 (q, J = 12.6 Hz, 1H, NHCHC(H)(H)N), 2.95–3.01 (q, J = 13.6 Hz, 1H, NHCHC(H)(H)Ph), 3.10–3.16 (q, J = 13.6 Hz, 1H, NHCHC(H)(H)Ph), 3.88–3.92 (m, 1H, NHCHCH₂N); ¹³C NMR (100 MHz, CDCl₃): δ 23.4, 28.6, 38.7, 53.8, 55.2, 61.1, 78.5, 125.2, 128.6, 128.6, 129.6, 130.4, 137.7, 155.2; ESI-MS: (m/z) 305.2 (M + H⁺); HRMS calcd for [C₁₈H₂₉N₂O₂ + H]⁺ 305.2224, found 305.2228.

4.6.16 (S)-tert-Butyl 1-(dimethylamino)-3-phenylpropan-2-ylcarbamate (5k). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.36 (s, 9H, C(CH₃)₃), 2.24 (s, 6H, CH₂N(CH₃)₂), 2.39–2.44 (q, J = 12.2 Hz, 1H, CHC(H)(H)N(CH₃)₂), 2.53–2.59 (q, J = 12.2 Hz, 1H, CHC(H)(H)N(CH₃)₂), 2.91–2.96 (q, J = 13.4 Hz, 1H, CHC(H)(H)Ph), 3.10–3.16 (q, J = 13.4 Hz, 1H, CHC(H)(H)Ph), 3.84–3.89 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 28.4, 38.1, 46.8, 51.2, 63.4, 78.4, 125.4, 128.5, 128.7, 129.3, 130.6, 137.4, 155.4; ESI-MS: (m/z) 279.3 (M + H⁺); HRMS calcd for [C₁₆H₂₇N₂O₂ + H]⁺ 279.2067, found 279.2069.

4.6.17 (S)-tert-Butyl 1-(diethylamino)-3-phenylpropan-2-ylcarbamate (5l). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.93 (t, J = 7.2 Hz, 6H, N(CH₂)₂(CH₃)₂), 1.35 (s, 9H, C(CH₃)₃), 2.44–2.57 (m, 4H, N(CH₂)₂(CH₃)₂), 2.59 (d, J = 6.9 Hz, 2H, NHCHCH₂N), 2.85–2.89 (dd, J = 7.1, 13.9 Hz, 1H, NHCHC(H)(H)Ph), 3.12–3.18 (dd, J = 4.3, 14.0 Hz, 1H, NHCHC(H)(H)Ph), 3.76–3.84 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 12.1, 28.4, 38.6, 47.2, 55.4, 62.4, 78.4, 125.1, 128.3, 128.5, 129.6, 130.2, 137.6, 155.1; ESI-MS: (m/z) 307.3 (M + H⁺); HRMS calcd for [C₁₈H₃₁N₂O₂ + H]⁺ 307.2380, found 307.2384.

4.6.18 (S)-tert-Butyl 1-(dipropylamino)-3-phenylpropan-2-ylcarbamate (5m). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.82 (t, J = 7.4 Hz, 6H, N(CH₂)₂(CH₂)₂(CH₃)₂), 1.27 (s, 9H, C(CH₃)₃), 1.34–1.45 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.33–2.45 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.61–2.65 (m, 2H, CH₂N(CH₂)₂(CH₂)₂(CH₃)₂), 2.84–2.92 (m, 1H, CHCH(H)Ph), 3.11–3.17 (m, 1H, CHC(H)HPh), 3.74–3.81 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 11.6, 20.6, 28.4, 38.6, 55.6, 58.4, 62.3, 78.5, 125.4, 128.7, 128.3, 129.5, 130.4, 137.7, 155.3; ESI-MS: (m/z) 335.3 (M + H⁺); HRMS calcd for [C₂₀H₃₅N₂O₂ + H]⁺ 335.2693, found 335.2696.

4.6.19 tert-Butyl (2S)-1-(2-methylpiperidin-1-yl)propan-2-ylcarbamate (6e). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.04 (d, J = 6.5 Hz, 3H, N(CH₂)₄CHCH₃), 1.15 (d, J = 6.2 Hz, 3H, NHCHCH₃), 1.34 (s, 9H, C(CH₃)₃), 1.43–1.76 (m, 6H, NCH₂(CH₂)₃CHCH₃), 2.32–2.48 (m, 4H, CH₂NCH₂(CH₂)₃CHCH₃), 2.86–2.91 (m, 1H, CH₂NCH₂(CH₂)₃CHCH₃), 3.68 (s, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 14.6, 19.4, 22.9, 26.1, 28.3, 33.6, 47.8, 56.3, 59.2, 64.7, 78.4, 155.3; ESI-MS: (m/z) 257.2 (M + H⁺); HRMS calcd for [C₁₄H₂₉N₂O₂ + H]⁺ 257.2224, found 257.2227.

4.6.20 tert-Butyl (2S)-1-(3-methylpiperidin-1-yl)propan-2-ylcarbamate (6f). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.86 (d, J = 6.3 Hz, 3H, piperidine), 1.29 (d, J = 6.2 Hz, 3H, NHCHCH₃), 1.36 (s, 9H, C(CH₃)₃), 1.68–1.76 (m, 2H, piperidine), 1.83–1.91 (m, 3H, piperidine), 2.46–2.69 (m, 4H, piperidine), 2.77–2.83 (m, 2H, NHCHCH₂N), 3.62–3.69 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 18.6, 19.4, 24.3, 28.4, 29.2, 32.6, 47.6, 59.4, 64.4, 65.2, 78.6, 155.2; ESI-MS: (m/z) 257.3 (M + H⁺); HRMS calcd for [C₁₄H₂₉N₂O₂ + H]⁺ 257.2224, found 257.2225.

4.6.21 (S)-tert-Butyl 1-(4-methylpiperidin-1-yl)propan-2-ylcarbamate (6g). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.88 (d, J = 4.7 Hz, 6H, CH₃CHCH₂N(CH₂)₂(CH₂)₂CHCH₃), 1.36 (s, 9H, C(CH₃)₃), 1.68–1.72 (m, 1H, N(CH₂)₂(CH₂)₂CHCH₃), 1.94–2.06 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 2.23–2.26 (m, 1H, NCH(H)CH₂(CH₂)₂CHCH₃), 2.40–2.46 (m, 1H, NC(H)HCH₂(CH₂)₂CHCH₃), 2.53–2.58 (m, 1H, N(CH₂)₂CH(H)CH₂CHCH₃), 2.66–2.72 (m, 1H, N(CH₂)₂C(H)HCH₂CHCH₃), 2.77–2.82 (m, 1H, NHCHCH(H)N), 2.91–2.99 (m, 1H, NHCHC(H)HN), 3.72–3.78 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 18.5, 21.4, 28.4, 32.3, 34.5, 45.5, 49.5, 64.3, 78.8, 155.5; ESI-MS: (m/z) 257.2 (M + H⁺); HRMS calcd for [C₁₄H₂₉N₂O₂ + H]⁺ 257.2224, found 257.2229.

4.6.22 (S)-tert-Butyl 1-(piperidin-1-yl)propan-2-ylcarbamate (6h). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.29 (d, J = 6.3 Hz, 3H, NHCHCH₃), 1.37 (s, 9H, C(CH₃)₃), 1.44–1.64 (m, 6H, piperidine), 2.39 (s, 2H, NHCHCH₂N), 2.46–2.60 (m, 4H, piperidine), 3.62–3.73 (m, 1H, NHCHCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 18.1, 24.6, 25.7, 28.3, 46.8, 56.4, 64.3, 78.6, 155.3; ESI-MS: (m/z) 244.3 (M + H⁺); HRMS calcd for [C₁₃H₂₇N₂O₂ + H]⁺ 243.2067, found 243.2070.

4.6.23 (S)-tert-Butyl 1-(pyrrolidin-1-yl)propan-2-ylcarbamate (6i). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 1.34 (d, J = 6.4 Hz, 3H, NHCHCH₃), 1.39 (s, 9H, C(CH₃)₃), 1.68–1.75 (m, 4H, pyrrolidine), 1.86 (s, 2H, NHCHCH₂N), 2.52–2.57 (m, 4H, pyrrolidine), 2.83–2.86 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 18.4, 23.4, 28.4, 48.8, 55.2, 63.5, 78.5, 155.3; ESI-MS: (m/z) 229.3 (M + H⁺); HRMS calcd for [C₁₂H₂₅N₂O₂ + H]⁺ 229.1911, found 229.1915.

4.6.24 (S)-tert-Butyl 1-(dipropylamino)propan-2-ylcarbamate (6j). The compound was obtained as a gummy substance in quantitative yield; ¹H NMR (400 MHz, CDCl₃): δ 0.85 (t, J = 7.3 Hz, 6H, N(CH₂)₂(CH₂)₂(CH₃)₂), 1.27 (d, J = 6.2 Hz, 3H, NHCHCH₃), 1.37 (s, 9H, C(CH₃)₃), 1.41–1.49 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.34–2.45 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.63 (d, J = 7.4 Hz, 2H, NHCHCH₂N), 3.56–3.64 (m, 1H, NHCH); ¹³C NMR (100 MHz, CDCl₃): δ 11.5, 17.8, 21.2, 28.3, 46.7, 58.6, 62.3, 78.3, 155.4; ESI-MS: (m/z) 259.3 (M + H⁺); HRMS calcd for [C₁₄H₃₁N₂O₂ + H]⁺ 259.2380, found 259.2385.

4.7 General procedure for the synthesis of 7e–7m, 8e–8m and 9e–9j

To a suspension of 4e–4m, 5e–5m and 6e–6j (1.0 equiv.) dissolved in 15% HCl/dioxane and stirred for 1 h at room temperature. After completion of the reaction, the solvent was concentrated under reduced pressure. The product was purified by trituration with diethyl ether and finally the hydrochloride salt basified with triethylamine. These intermediates were used for the next step without further purification.

4.8 General procedure for the synthesis of 10e–10m, 11e–11m and 12e–12g, 12h²⁰ 12i²¹ and 12j

The free amines 7e–7m, 8e–8m and 9e–9j (2.0 equiv.) were added to 4,7-dichloroquinoline (1.0 equiv.) and heated the reaction mixture in phenol. After completion of the reaction as monitored by the TLC, chloroform (100 mL) was added to the reaction mixture. The organic layer was washed with 10% NaOH solution and finally with brine. The organic layer was dried over anhydrous Na₂SO₄, concentrated under reduced pressure and the product obtained was purified by column chromatography by using methanol–chloroform–triethylamine as eluent.

4.8.1 7-Chloro-N-((2S)-4-methyl-1-(2-methylpiperidin-1-yl)pentan-2-yl)quinolin-4-amine (10e). The compound was obtained as a gummy substance in 79% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.95 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.05 (d, J = 6.2 Hz, 3H, CH₂CHCH₃CH₃), 1.12 (d, J = 6.2 Hz, 3H, N(CH₂)₄CHCH₃), 1.25–1.45 (m, 6H, NCH₂(CH₂)₃CHCH₃), 1.58–1.77 (m, 5H, CH₂NCH₂(CH₂)₃CHCH₃), 2.36–2.51 (m, 1H, CH₂CH(CH₃)₂), 2.72–2.89 (m, 2H, CH₂CH(CH₃)₂), 3.59–3.67 (m, 1H, NHCH), 6.42 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.35–7.38 (dd, J = 2.2, 8.8 Hz, 1H, Ar–H quinoline), 7.70 (d, J = 8.8 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 18.6, 22.3, 22.8, 23.6, 25.0, 26.1, 34.5, 42.5, 55.6, 56.5, 56.8, 57.5, 99.4, 121.2, 125.2, 128.8, 134.7, 149.3, 149.7, 150.0, 152.0; ESI-MS: (m/z): 360.2 (M + H)⁺; HRMS calcd for [C₂₁H₃₁ClN₃ + H]⁺ 360.2201, found 360.2203.

4.8.2 7-Chloro-N-((2S)-4-methyl-1-(3-methylpiperidin-1-yl)pentan-2-yl)quinolin-4-amine (10f). The compound was obtained as a gummy substance in 76% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.93 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.12 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.19 (d, J = 6.2 Hz, 3H, piperidine), 1.26–1.48 (m, 6H, piperidine), 1.60–1.78 (m, 5H, piperidine), 2.35–2.53 (m, 1H, CH₂CH(CH₃)₂), 2.72–2.89 (m, 2H, CH₂CH(CH₃)₂), 3.60–3.67 (m, 1H, NHCH), 6.45 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.35–7.38 (dd, J = 2.2, 8.8 Hz, 1H, Ar–H quinoline), 7.73 (d, J = 8.8 Hz, 1H, Ar–H quinoline), 7.93 (d, J = 2.2 Hz, 1H, Ar–H quinoline), 8.54 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 18.6, 22.5, 22.9, 23.6, 25.5, 26.1, 34.8, 42.5, 55.7, 56.6, 56.8, 57.8, 99.4, 117.2, 125.2, 127.8, 133.7, 149.3, 149.9, 150.0, 152.2; ESI-MS: (m/z): 360.1 (M + H)⁺; HRMS calcd for [C₂₁H₃₁ClN₃ + H]⁺ 360.2201, found 360.2198.

4.8.3 (S)-7-Chloro-N-(4-methyl-1-(4-methylpiperidin-1-yl)pentan-2-yl)quinolin-4-amine (10g). The compound was obtained as a gummy substance in 83% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.89 (d, J = 6.3 Hz, 3H, CH₂CHCH₃CH₃), 0.93 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.03 (d, J = 6.3 Hz, 3H, N(CH₂)₄CHCH₃), 1.25–1.33 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 1.43–1.52 (m, 1H, N(CH₂)₂(CH₂)₂CHCH₃), 1.62–1.76 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 1.92–1.93 (m, 1H, CHC(H) (H)CH(CH₃)₂), 2.15–2.21 (m, 1H, CHC(H)(H)CH(CH₃)₂), 2.56 (d, J = 6.8 Hz, 2H, NHCHCH₂N), 2.69 (d, J = 11.7 Hz, 1H, NHCHC(H)(H)N), 2.87 (d, J = 11.2 Hz, 1H, NHCHC(H)(H)N), 3.64–3.66 (m, 1H, NHCH), 6.41 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.35–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.72 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.0 Hz, 1H, Ar–H quinoline), 8.50 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 21.7, 22.4, 23.4, 24.9, 30.6, 34.4, 34.6, 42.6, 48.3, 53.3, 55.2, 61.6, 99.2, 117.9, 121.2, 125.2, 128.7, 134.7, 149.3, 149.8, 152.0; ESI-MS: (m/z): 360.2 (M + H)⁺; HRMS calcd for [C₂₁H₃₁ClN₃ + H]⁺ 360.2201, found 360.2207.

4.8.4 (S)-7-Chloro-N-(4-methyl-1-(piperidin-1-yl)pentan-2-yl)quinolin-4-amine (10h). The compound was obtained as a white solid in 85% yield; mp 104–106 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.94 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.04 (d, J = 6.3 Hz, 3H, CH₂CHCH₃CH₃), 1.40–1.60 (m, 6H, piperidine), 1.67–1.71 (m, 2H, CH₂CHCH₃CH₃), 1.75–1.82 (m, 1H, CH₂CHCH₃CH₃), 2.41–2.44 (m, 4H, piperidine), 2.56–2.58 (m, 2H, NHCHCH₂N), 3.62–3.69 (m, 1H, NHCHCH₂N), 6.42 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.35–7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.74 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 22.4, 23.4, 24.2, 24.9, 26.1, 42.6, 48.1, 54.8, 61.9, 99.2, 117.9, 121.2, 125.2, 128.7, 134.7, 149.2, 149.9, 151.9; ESI-MS: (m/z): 346.1 (M + H)⁺; HRMS calcd for [C₂₀H₂₉ClN₃ + H]⁺ 346.2045, found 346.2037.

4.8.5 (S)-7-Chloro-N-(4-methyl-1-(pyrrolidin-1-yl)pentan-2-yl)quinolin-4-amine (10i). The compound was obtained as a white solid in 80% yield; mp 100–102 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.94 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.03 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.50–1.57 (m, 1H, CH₂CHCH₃CH₃), 1.63–1.68 (m, 2H, CH₂CHCH₃CH₃), 1.69–1.83 (m, 4H, pyrrolidine), 2.55–2.59 (m, 4H, pyrrolidine), 2.67–2.72 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.75–2.80 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 3.65–3.73 (m, 1H, NHCH), 6.43 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.34–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.71 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.4 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 22.5, 23.1, 23.6, 25.0, 42.5, 49.9, 54.5, 59.2, 99.1, 117.7, 121.3, 125.2, 128.6, 134.8, 149.2, 149.7, 151.9; ESI-MS: (m/z): 332.1 (M + H)⁺; HRMS calcd for [C₁₉H₂₇ClN₃ + H]⁺ 332.1888, found 332.1877.

4.8.6 (S)-7-Chloro-N-(4-methyl-1-morpholinopentan-2-yl)quinolin-4-amine (10j). The compound was obtained as a off-white solid in 79% yield; mp 97–99 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.93 (d, J = 6.0 Hz, 3H, CH₂CHCH₃CH₃), 1.04 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.37–1.44 (m, 2H, CH₂CHCH₃CH₃), 1.74–1.83 (m, 1H, CH₂CHCH₃CH₃), 2.48 (t, J = 4.3 Hz, 4H, morpholine), 2.59 (d, J = 6.7 Hz, 2H, NHCHCH₂N), 3.66 (t, J = 4.1 Hz, 4H, morpholine), 3.89–4.01 (m, 1H, NHCH), 6.43 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.37–7.39 (dd, J = 2.0, 8.8 Hz, 1H, Ar–H quinoline), 7.69 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.97 (d, J = 1.8 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 23.2, 25.1, 42.6, 44.4, 48.1, 50.9, 53.9, 61.9, 67.0, 70.6, 99.2, 117.6, 120.9, 125.4, 128.7, 134.9, 149.1, 149.6, 151.8; ESI-MS: (m/z): 348.2 (M + H)⁺; HRMS calcd for [C₁₉H₂₇ClN₃O + H]⁺ 348.1837, found 348.1811.

4.8.7 (S)-N²-(7-Chloroquinolin-4-yl)-N¹,N¹-diethyl-4-methylpentane-1,2-diamine (10k). The compound was obtained as a gummy substance in 71% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.94 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.00 (t, J = 7.0 Hz, 6H, N(CH₂)₂(CH₃)₂), 1.04 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.43–1.50 (m, 1H, CH₂CHCH₃CH₃), 1.65–1.69 (m, 2H, CH₂CHCH₃CH₃), 1.72–1.83 (m, 2H, NHCHCH₂N), 2.47–2.62 (m, 4H, N(CH₂)₂(CH₃)₂), 2.67–2.72 (dd, J = 5.6, 13.0 Hz, 1H, NHCHCH₂N), 6.42 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.34–7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar–H quinoline), 7.68 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 11.9, 22.5, 23.3, 25.0, 42.4, 47.2, 48.8, 56.6, 99.3, 117.9, 121.2, 125.2, 128.7, 134.7, 149.2, 149.8, 151.9; ESI-MS: (m/z): 334.1 (M + H)⁺; HRMS calcd for [C₁₉H₂₉ClN₃ + H]⁺ 334.2045, found 334.2049.

4.8.8 (S)-N²-(7-Chloroquinolin-4-yl)-4-methyl-N¹,N¹-dipropylpentane-1,2-diamine (10l). The compound was obtained as a gummy substance in 70% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.83 (t, J = 7.3 Hz, 6H, N(CH₂)₂(CH₂)₂(CH₃)₂), 0.94 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.04 (d, J = 6.4 Hz, 3H, CH₂CHCH₃CH₃), 1.39–1.51 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 1.59–1.77 (m, 2H, CH₂CHCH₃CH₃), 2.02–2.04 (m, 1H, CH₂CHCH₃CH₃), 2.35–2.45 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.61–2.67 (m, 2H, NHCHCH₂N), 3.58–3.59 (m, 1H, NHCHCH₂N), 6.42 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.33–7.36 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.67 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 11.1, 11.8, 20.3, 22.0, 22.5, 23.4, 25.0, 42.3, 48.8, 49.5, 56.2, 58.0, 99.3, 117.9, 121.2, 125.1, 128.7, 134.7, 149.2, 149.8, 152.0; ESI-MS: (m/z): 362.1 (M + H)⁺; HRMS calcd for [C₂₁H₃₃ClN₃ + H]⁺ 362.2358, found 362.2354.

4.8.9 (S)-N²-(7-Chloroquinolin-4-yl)-N¹,N¹,4-trimethylpentane-1,2-diamine (10m). The compound was obtained as a gummy substance in 76% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.94 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.04 (d, J = 6.5 Hz, 3H, CH₂CHCH₃CH₃), 1.47–1.54 (m, 1H, CH₂CHCH₃CH₃), 1.63–1.70 (m, 2H, CH₂CHCH₃CH₃), 2.25 (s, 6H, CH₂N(CH₃)₂), 2.45–2.49 (dd, J = 5.8, 12.4 Hz, 1H, NHCHC(H)(H)N), 2.52–2.57 (dd, J = 7.8, 12.4 Hz, 1H, NHCHC(H)(H)N), 3.59–3.68 (m, 1H, NHCHC(H)(H)N), 6.43 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.34–7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar–H quinoline), 7.70 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 22.5, 23.2, 25.0, 42.3, 45.8, 48.8, 62.8, 99.1, 117.7, 121.3, 125.2, 128.6, 134.8, 149.2, 149.7, 151.9; ESI-MS: (m/z): 306.1 (M + H)⁺; HRMS calcd for [C₁₇H₂₅ClN₃ + H]⁺ 306.1732, found 306.1723.

4.8.10 7-Chloro-N-((2S)-1-(2-methylpiperidin-1-yl)-3-phenylpropan-2-yl)quinolin-4-amine (11e). The compound was obtained as a off-white solid in 82% yield; mp 112–114 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.89 (d, J = 6.8 Hz, 3H, NCHCH₃), 1.47–1.69 (m, 6H, NCH₂CH₂CH₂CH₂CHCH₃), 2.34–3.18 (m, 7H, PhCH₂CHCH₂NCH₂(CH₂)₃CHCH₃), 3.82–3.87 (m, 1H, NHCHCH₂Ph), 6.48 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.20–7.24 (m, 3H, Ar–H Ph), 7.27–7.29 (m, 2H, Ar–H Ph), 7.35–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.67 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.97 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.53 (d, J = 5.4 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 14.1, 22.6, 26.0, 31.9, 38.7, 50.4, 51.5, 55.7, 56.4, 99.5, 114.0, 121.1, 121.3, 125.3, 125.4, 126.6, 128.5, 128.7, 129.4, 134.8, 149.2, 149.5, 149.8, 151.8; ESI-MS: (m/z): 394.6 (M + H)⁺; HRMS calcd for [C₂₄H₂₉ClN₃ + H]⁺ 394.2045, found 394.2043.

4.8.11 7-Chloro-N-((2S)-1-(3-methylpiperidin-1-yl)-3-phenylpropan-2-yl)quinolin-4-amine (11f). The compound was obtained as a off-white solid in 84% yield; mp 112–114 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.86 (d, J = 6.4 Hz, 3H, piperidine), 1.78–2.47 (m, 7H, piperidine), 2.53–3.16 (m, 6H, CH₂NCH₂CHCH₂Ph), 3.84–3.88 (m, 1H, NHCH), 6.51 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.18–7.20 (m, 2H, Ar–H Ph), 7.27–7.31 (m, 3H, Ar–H Ph), 7.35–7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.67 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.54 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 19.5, 25.4, 25.5, 32.7, 38.3, 54.9, 60.5, 60.9, 62.7, 99.6, 117.9, 121.2, 125.3, 126.6, 128.5, 128.8, 129.5, 134.8, 137.3, 149.3, 149.6, 152.0; ESI-MS: (m/z): 394.2 (M + H)⁺; HRMS calcd for [C₂₄H₂₉ClN₃ + H]⁺ 394.2045, found 394.2046.

4.8.12 (S)-7-Chloro-N-(1-(4-methylpiperidin-1-yl)-3-phenylpropan-2-yl)quinolin-4-amine (11g). The compound was obtained as a off-white solid in 81% yield; mp 111–113 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.88 (d, J = 6.2 Hz, 3H, N(CH₂)₂(CH₂)₂CHCH₃), 1.01–1.04 (m, 1H, N(CH₂)₂(CH₂)₂CHCH₃), 1.22–1.52 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 1.86–1.87 (m, 1H, NCH₂C(H)(H)(CH₂)₂CHCH₃), 2.11–2.16 (m, 1H, NCH₂C(H)(H)(CH₂)₂CHCH₃), 2.43–2.47 (dd, J = 6.0, 12.6 Hz, 1H, NHCHC(H)(H)N), 2.54–2.59 (dd, J = 8.6, 12.6 Hz, 1H, NHCHC(H)(H)N), 2.65–2.68 (m, 1H, NC(H)(H)CH₂(CH₂)₂CHCH₃), 2.83–2.87 (m, 1H, NC(H)(H)CH₂(CH₂)₂CHCH₃), 2.88–2.93 (dd, J = 7.1, 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.10–3.15 (dd, J = 4.1, 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.87 (s, 1H, NHCHCH₂Ph), 6.50 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.18–7.20 (m, 2H, Ar–H Ph), 7.23–7.24 (m, 1H, Ar–H Ph), 7.27–7.31 (m, 2H, Ar–H Ph), 7.36–7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.67 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 21.7, 30.7, 34.4, 34.6, 38.3, 50.4, 52.8, 54.9, 60.3, 99.5, 117.9, 121.2, 125.3, 126.6, 128.5, 128.8, 129.6, 134.8, 137.3, 149.3, 149.6, 152.0; ESI-MS: (m/z): 394.1 (M + H)⁺; HRMS calcd for [C₂₄H₂₉ClN₃ + H]⁺ 394.2045, found 394.2033.

4.8.13 (S)-7-Chloro-N-(1-phenyl-3-(piperidin-1-yl)propan-2-yl)quinolin-4-amine (11h). The compound was obtained as a white solid in 79% yield; mp 114–116 °C; ¹H NMR (400 MHz, CDCl₃): δ 1.48–1.57 (m, 6H, piperidine), 2.35–2.40 (m, 4H, piperidine), 2.44–2.48 (q, J = 12.6 Hz, 1H, NHCHC(H)(H)N), 2.55–2.60 (q, J = 12.6 Hz, 1H, NHCHC(H)(H)N), 2.88–2.93 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.11–3.16 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.88 (s, 1H, NHCHCH₂N), 6.50 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.18–7.20 (m, 2H, Ar–H Ph), 7.22–7.24 (m, 1H, Ar–H Ph), 7.27–7.31 (m, 2H, Ar–H Ph), 7.36–7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.70 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.97 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 24.1, 26.0, 31.6, 38.3, 50.3, 54.4, 60.5, 99.5, 115.9, 117.8, 121.3, 123.4, 123.9, 125.5, 126.7, 128.5, 129.6, 135.0, 137.2, 149.0, 149.8, 151.7; ESI-MS: (m/z): 380.2 (M + H)⁺; HRMS calcd for [C₂₂H₂₇ClN₃ + H]⁺ 380.1888, found 380.1893.

4.8.14 (S)-7-Chloro-N-(1-morpholino-3-phenylpropan-2-yl)quinolin-4-amine (11i). The compound was obtained as a off-white solid in 75% yield; mp 117–119 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.20–3.24 (m, 4H, morpholine), 3.38–3.41 (m, 1H, NHCH), 3.64–3.71 (m, 4H, morpholine), 3.88 (m, 2H, NHCHCH₂N), 3.96–3.99 (m, 2H, NHCHCH₂Ph), 6.52 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.16–7.23 (m, 2H, Ar–H Ph), 7.26–7.32 (m, 3H, Ar–H Ph), 7.38–7.40 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.65 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 8.01 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.54 (d, J = 5.4 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 48.5, 52.5, 53.5, 60.4, 66.6, 66.8, 66.9, 99.3, 115.5, 119.9, 121.0, 125.9, 126.9, 127.8, 128.6, 129.5, 135.6, 136.7, 149.9, 150.3, 151.1, 156.6; ESI-MS: (m/z): 382.2 (M + H)⁺; HRMS calcd for [C₂₂H₂₅ClN₃ + H]⁺ 382.1681, found 382.1686.

4.8.15 (S)-7-Chloro-N-(1-phenyl-3-(pyrrolidin-1-yl)propan-2-yl)quinolin-4-amine (11j). The compound was obtained as a white solid in 75% yield; mp 111–113 °C; ¹H NMR (400 MHz, CDCl₃): δ 1.73–1.76 (m, 4H, pyrrolidine), 2.53–2.56 (m, 4H, pyrrolidine), 2.57–2.62 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.75–2.81 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.92–2.97 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.09–3.13 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.86–3.90 (m, 1H, NHCH), 6.50 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.18–7.20 (m, 2H, Ar–H Ph), 7.21–7.24 (m, 1H, Ar–H Ph), 7.27–7.31 (m, 2H, Ar–H Ph), 7.34–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.66 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 23.6, 38.2, 52.2, 54.1, 57.9, 99.4, 117.8, 121.3, 125.3, 126.6, 128.5, 128.7, 129.5, 134.9, 137.3, 149.2, 149.4, 151.9; ESI-MS: (m/z): 366.1 (M + H)⁺; HRMS calcd for [C₂₂H₂₅ClN₃ + H]⁺ 366.1732, found 366.1703.

4.8.16 (S)-N²-(7-Chloroquinolin-4-yl)-N¹,N¹-dimethyl-3-phenylpropane-1,2-diamine (11k). The compound was obtained as a off-white solid in 72% yield; mp 109–111 °C; ¹H NMR (400 MHz, CDCl₃): δ 2.22 (s, 6H, CH₂N(CH₃)₂), 2.36–2.40 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N(CH₃)₂), 2.51–2.57 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N(CH₃)₂), 2.90–2.95 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.08–3.13 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.83–3.90 (m, 1H, NHCHC(H)(H)Ph), 6.51 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.17–7.19 (m, 2H, Ar–H Ph), 7.22–7.25 (m, 1H, Ar–H Ph), 7.28–7.31 (m, 2H, Ar–H Ph), 7.34–7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar–H quinoline), 7.65 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.54 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 37.9, 45.5, 50.9, 61.5, 99.5, 117.8, 121.3, 125.3, 126.7, 128.5, 128.8, 129.6, 134.9, 137.2, 149.3, 149.4, 152.0; ESI-MS: (m/z): 340.6 (M + H)⁺; HRMS calcd for [C₂₀H₂₃ClN₃ + H]⁺ 340.1575, found 340.1580.

4.8.17 (S)-N²-(7-Chloroquinolin-4-yl)-N¹,N¹-diethyl-3-phenylpropane-1,2-diamine (11l). The compound was obtained as a gummy substance in 76% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.96 (t, J = 7.1 Hz, 6H, N(CH₂)₂(CH₃)₂), 2.43–2.58 (m, 4H, N(CH₂)₂(CH₃)₂), 2.62 (d, J = 7.1 Hz, 2H, NHCHCH₂N), 2.86–2.92 (dd, J = 7.2, 13.9 Hz, 1H, NHCHC(H)(H)Ph), 3.10–3.15 (dd, J = 4.4, 14.0 Hz, 1H, NHCHC(H)(H)Ph), 3.76–3.84 (m, 1H, NHCH), 6.50 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.19–7.31 (m, 5H, Ar–H Ph), 7.35–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.65 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.96 (d, J = 2.0 Hz, 1H, Ar–H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 11.8, 38.5, 46.8, 51.1, 55.7, 99.6, 118.0, 121.2, 125.3, 126.6, 128.5, 128.8, 129.4, 134.8, 137.5, 149.3, 149.6, 152.0; ESI-MS: (m/z): 368.6 (M + H)⁺; HRMS calcd for [C₂₂H₂₇ClN₃ + H]⁺ 368.1888, found 368.1895.

4.8.18 (S)-N²-(7-Chloroquinolin-4-yl)-3-phenyl-N¹,N¹-dipropylpropane-1,2-diamine (11m). The compound was obtained as a gummy substance in 72% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.80 (t, J = 7.3 Hz, 6H, N(CH₂)₂(CH₂)₂(CH₃)₂), 1.31–1.43 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.31–2.42 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.60–2.64 (m, 2H, NHCHCH₂N), 2.85–2.91 (m, 1H, NHCHCH(H)Ph), 3.10–3.15 (m, 1H, NHCHC(H)HPh), 3.75–3.82 (m, 1H, NHCH), 6.50 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.19–7.25 (m, 3H, Ar–H Ph), 7.27–7.31 (m, 2H, Ar–H Ph), 7.34–7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar–H quinoline), 7.64 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 11.8, 20.1, 38.5, 51.2, 55.9, 57.1, 99.6, 118.0, 121.2, 125.3, 126.6, 128.5, 128.7, 129.4, 134.8, 137.6, 149.2, 149.7, 151.9; ESI-MS: (m/z): 396.6 (M + H)⁺; HRMS calcd for [C₂₁H₃₁ClN₃ + H]⁺ 396.2201, found 396.2202.

4.8.19 7-Chloro-N-((2S)-1-(2-methylpiperidin-1-yl)propan-2-yl)quinolin-4-amine (12e). The compound was obtained as a gummy substance in 78% yield; ¹H NMR (400 MHz, CDCl₃): δ 1.06 (d, J = 6.5 Hz, 3H, N(CH₂)₄CHCH₃), 1.18 (d, J = 6.2 Hz, 3H, NHCHCH₃), 1.41–1.73 (m, 6H, NCH₂(CH₂)₃CHCH₃), 2.35–2.50 (m, 4H, CH₂NCH₂(CH₂)₃CHCH₃), 2.84–2.89 (m, 1H, CH₂NCH₂(CH₂)₃CHCH₃), 3.66 (s, 1H, NHCH), 6.44 (d, J = 5.3 Hz, 1H, Ar–H quinoline), 7.37–7.39 (dd, J = 2.2, 8.9 Hz, 1H, Ar–H quinoline), 7.76 (d, J = 9.1 Hz, 1H, Ar–H quinoline), 7.97 (d, J = 2.0 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 19.1, 21.6, 25.5, 32.9, 44.7, 46.0, 56.4, 57.9, 58.5, 99.5, 115.6, 125.5, 128.0, 129.5, 135.1, 148.6, 150.1, 151.4; ESI-MS: (m/z): 318.1 (M + H)⁺; HRMS calcd for [C₁₈H₂₅ClN₃ + H]⁺ 318.1732, found 318.1738.

4.8.20 7-Chloro-N-((2S)-1-(3-methylpiperidin-1-yl)propan-2-yl)quinolin-4-amine (12f). The compound was obtained as a gummy substance in 76% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.88 (d, J = 6.4 Hz, 3H, piperidine), 1.30 (d, J = 6.0 Hz, 3H, NHCHCH₃), 1.65–1.75 (m, 2H, piperidine), 1.81–1.90 (m, 3H, piperidine), 2.45–2.67 (m, 4H, piperidine), 2.75–2.79 (m, 2H, NHCHCH₂N), 3.60–3.67 (m, 1H, NHCH), 6.43 (d, J = 5.2 Hz, 1H, Ar–H quinoline), 7.35–7.37 (dd, J = 1.3, 8.9 Hz, 1H, Ar–H quinoline), 7.72 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 1.8 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 18.9, 19.5, 25.4, 31.0, 32.7, 45.0, 60.5, 62.9, 63.3, 99.7, 118.1, 121.3, 125.2, 128.7, 134.7, 149.2, 149.9, 152.0; ESI-MS: (m/z): 318.6 (M + H)⁺; HRMS calcd for [C₁₈H₂₄ClN₃ + H]⁺ 318.1732, found 318.1745.

4.8.21 (S)-7-Chloro-N-(1-(4-methylpiperidin-1-yl)propan-2-yl)quinolin-4-amine (12g). The compound was obtained as a gummy substance in 79% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.89 (d, J = 4.7 Hz, 6H, CH₃CHCH₂N(CH₂)₂(CH₂)₂CHCH₃), 1.66–1.70 (m, 1H, N(CH₂)₂(CH₂)₂CHCH₃), 1.92–2.07 (m, 4H, N(CH₂)₂(CH₂)₂CHCH₃), 2.21–2.27 (m, 1H, NCH(H)CH₂(CH₂)₂CHCH₃), 2.38–2.42 (m, 1H, NC(H)HCH₂(CH₂)₂CHCH₃), 2.51–2.55 (m, 1H, N(CH₂)₂CH(H)CH₂CHCH₃), 2.65–2.71 (m, 1H, N(CH₂)₂C(H)HCH₂CHCH₃), 2.77–2.80 (m, 1H, NHCHCH(H)N), 2.90–2.94 (m, 1H, NHCHC(H)HN), 3.70–3.76 (m, 1H, NHCH), 6.45 (d, J = 5.6 Hz, 1H, Ar–H quinoline), 7.35–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.76 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 8.01 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.50 (d, J = 5.5 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 18.7, 21.7, 29.7, 34.0, 34.3, 45.0, 52.2, 55.2, 62.9, 99.4, 115.6, 119.8, 121.6, 125.7, 127.4, 129.5, 150.9, 156.8; ESI-MS: (m/z): 318.1 (M + H)⁺; HRMS calcd for [C₁₈H₂₅ClN₃ + H]⁺ 318.1732, found 318.1729.

4.8.22 (S)-7-Chloro-N-(1-(piperidin-1-yl)propan-2-yl)quinolin-4-amine (12h). The compound was obtained as a gummy substance in 78% yield; ¹H NMR (400 MHz, CDCl₃): δ 1.30 (d, J = 6.1 Hz, 3H, NHCHCH₃), 1.41–1.60 (m, 6H, piperidine), 2.36 (s, 2H, NHCHCH₂N), 2.42–2.58 (m, 4H, piperidine), 3.59–3.68 (m, 1H, NHCH), 6.43 (d, J = 5.3 Hz, 1H, Ar–H quinoline), 7.36–7.39 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.73 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.95 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 18.9, 24.2, 26.1, 45.0, 54.3, 63.6, 99.7, 118.1, 121.3, 125.3, 128.7, 134.7, 149.2, 150.0, 152.0; ESI-MS: (m/z): 304.1 (M + H)⁺; HRMS calcd for [C₁₇H₂₃ClN₃ + H]⁺ 304.1575, found 304.1575.

4.8.23 (S)-7-Chloro-N-(1-(pyrrolidin-1-yl)propan-2-yl)quinolin-4-amine (12i). The compound was obtained as a gummy substance in 81% yield; ¹H NMR (400 MHz, CDCl₃): δ 1.32 (d, J = 6.2 Hz, 3H, NHCHCH₃), 1.72–1.79 (m, 4H, pyrrolidine), 1.88 (s, 2H, NHCHCH₂N), 2.54–2.58 (m, 4H, pyrrolidine), 2.81–2.87 (m, 1H, NHCHCH₂N), 6.44 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.34–7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.72 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 18.8, 23.6, 47.0, 54.1, 61.0, 99.6, 117.9, 121.4, 125.2, 128.6, 134.7, 149.2, 149.7, 151.9; ESI-MS: (m/z): 290.1 (M + H)⁺; HRMS calcd for [C₁₆H₂₁ClN₃ + H]⁺ 290.1419, found 290.1430.

4.8.24 (S)-N²-(7-Chloroquinolin-4-yl)-N¹,N¹-dipropylpropane-1,2-diamine (12j). The compound was obtained as a gummy substance in 83% yield; ¹H NMR (400 MHz, CDCl₃): δ 0.83 (t, J = 7.3 Hz, 6H, N(CH₂)₂(CH₂)₂(CH₃)₂), 1.29 (d, J = 6.0 Hz, 3H, NHCHCH₃), 1.39–1.51 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.33–2.49 (m, 4H, N(CH₂)₂(CH₂)₂(CH₃)₂), 2.61 (d, J = 7.3 Hz, 2H, NHCHCH₂N), 3.54–3.60 (m, 1H, NHCH), 6.44 (d, J = 5.4 Hz, 1H, Ar–H quinoline), 7.34–7.36 (dd, J = 2.1, 8.9 Hz, 1H, Ar–H quinoline), 7.68 (d, J = 8.9 Hz, 1H, Ar–H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar–H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar–H quinoline); ¹³C NMR (100 MHz, CDCl₃): δ 11.8, 18.6, 20.2, 45.6, 55.9, 59.7, 99.8, 118.1, 121.3, 125.2, 128.6, 134.7, 149.1, 149.9, 151.9; ESI-MS: (m/z): 320.1 (M + H)⁺; HRMS calcd for [C₁₈H₂₇ClN₃ + H]⁺ 320.1888, found 320.1890.

4.9 Biological methods

4.9.1 In vitro antimalarial assay. The compounds were evaluated for antimalarial activity against 3D7 (CQ-sensitive) and K1 (CQ-resistant) strains of Plasmodium falciparum using Malaria SYBR Green I nucleic acid staining dye based fluorescence (MSF) assay as mentioned by Singh et al.²² The stock (10 mM) solution was prepared in DMSO and test dilutions were prepared in culture medium (RPMI-1640-FBS). The final concentration of DMSO in Plasmodium cultures was <1%. Chloroquine-diphosphate (SIGMA) was used as a reference drug. For assessment of antimalarial activity 50 μL of culture medium was dispensed in 96 well plate followed by addition of 50 μL of highest concentration (containing less than 0.5% of DMSO) of test compounds (in duplicate wells) in row B. Subsequent two-fold serial dilutions were prepared in culture medium and finally 50 μL of 2.0% parasitized cell suspension containing 0.8% parasitaemia (asynchronous culture containing more than 80% ring stages) was added to each well except 4 wells in row ‘A’ received non parasitized erythrocyte suspension. The plates were incubated at 37 °C in CO₂ incubator in an atmosphere of 5% CO₂ and air mixture and 72 hours later 100 μL of lysis buffer containing 2× concentration of SYBR Green-I (Invitrogen) was added to each well and incubated for one hour at 37 °C.²³ The plates were examined at 485 ± 20 nm of excitation and 530 ± 20 nm of emission for relative fluorescence units (RFUs) per well using the fluorescence plate reader (FLX800, BIOTEK). Data were transferred into a graphic programme (EXCEL) and IC₅₀ values were obtained by Logit regression analysis of dose response curves using pre-programmed Excel spreadsheet.

4.9.2 In vitro assay for evaluation of cytotoxic activity. Cytotoxicity of the compounds was carried out using Vero cell line (C1008; Monkey kidney fibroblast) following the method as mentioned in Mosmann.²⁴ The cells were incubated with compound-dilutions for 72 h and MTT was used as reagent for detection of cytotoxicity, 50% cytotoxic concentration (CC₅₀) was determined using nonlinear regression analysis of the dose response curves using pre-programmed Excel spreadsheet. Selectivity Index (SI) was calculated as SI = CC₅₀/IC₅₀

4.9.3 In vivo antimalarial assay. The in vivo drug response was evaluated in Swiss mice infected with P. yoelii (N-67 strain) which is innately resistant to CQ.^13,25,26 The study received ethical approval from CSIR-Central Drug Research Institute's ‘Institutional Animal Care and Use Committee’ recognized by ‘Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)’, Government of India. The mice (22 ± 2 g) were inoculated with 1 × 10⁶ parasitized RBC on day 0 and treatment was administered to a group of five mice from day 0–3, once daily. The aqueous suspensions of compounds were prepared with a few drops of Tween 80. The final concentration of Tween 80 was 0.5 vol%. The efficacy of test compounds was evaluated at 100 mg per kg per day and the required daily dose was administered in 0.5 mL volume via oral route. Parasitemia levels were recorded from thin blood smears at regular intervals of four days throughout the period of experiment. The mean value determined for a group of five mice was used to calculate the percent suppression of parasitemia with respect to the untreated control group. Mice treated with CQ served as reference controls. Whereas, in the case of arteether, the drug was dissolved in neutralized ground nut oil and administered via intramuscular route.

4.9.4 Determination of hematin 4-aminoquinoline derivatives association constant. Association constant for hematin and 4-aminoquinoline derivative complex formation were determined by spectrometric titration procedure in aqueous DMSO at pH-7.5.²⁷ In this assay condition, hematin is strictly in monomeric state and interpretation of the results is not complicated by the need to consider hematin disaggregation process. Association constant calculated in this technique is a good reflection of the interaction that would occur in the acidic food vacuole of the parasite and pH-7.5 improves the stability of hematin solutions and quality of the data.

4.9.5 In vitro inhibition of β-hematin formation assay. The ability of the 4-aminoquinoline derivatives to inhibit β-hematin formation induced by 1-oleoyl-rac-glycerol. Spectroscopic measurements were done using UV spectrophotometer wave length 405 nm and at pH 5.²⁸ The IC₅₀ values obtained from the assay are expressed as percent inhibition relative to β-hematin formation in a drug free control. The 50% inhibitory concentration values for the compounds were obtained from the sigmoidal dose–response curves using non-linear regression curve fitting analyses with GraphPad Prism v.3.00 software.²⁹

Acknowledgements

One of the authors (K. S. R.) thanks the CSIR, New Delhi, for Senior Research Fellowship. Authors thank the Director, CDRI, for the support, and the SAIF division for the spectral data. The CDRI Communication No. is 9354.

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR, ¹³C NMR and HRMS data of selected compounds. See DOI: 10.1039/c6ra14016e

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